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De Mori A, Aydin N, Lostia G, Manca A, Blunn G, Roldo M. Influence of Cell Seeding Density and Material Stiffness on Chondrogenesis of Human Stem Cells Within Soft Hydrogels, Without the Use of Exogenous Growth Factors. Gels 2025; 11:213. [PMID: 40136918 PMCID: PMC11941925 DOI: 10.3390/gels11030213] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/03/2025] [Revised: 03/13/2025] [Accepted: 03/14/2025] [Indexed: 03/27/2025] Open
Abstract
Mesenchymal stem cells (MSCs) can differentiate into chondrocytes provided with the appropriate environmental cues. In this study, we loaded human adipose-derived stem cells (hAdMSCs) into collagen/alginate hydrogels, which have been shown to induce chondrogenesis in ovine bone marrow stem cells without the use of any exogenous chondrogenic growth factors. We examined the influence of hydrogel stiffness (5.75 and 6.85 kPa) and cell seeding density (1, 2, 4, and 16 × 106 cells/mL) on the chondrogenic induction of hAdMSCs, without exogenous differentiation growth factors. Over time, the behaviour of the hAdMSCs in the scaffolds was investigated by analysing the amount of DNA; their morphology; their cell viability; the expression of chondrogenic genes (RT-qPCR); and the deposition of collagen I, collagen II, and aggrecan. The results showed that all scaffolds supported the acquisition of a rounded morphology and the formation of cell aggregates, which were larger with higher cell seeding densities. Furthermore, the cells were viable within the hydrogels throughout the experiment, indicating that high cell density did not have a detrimental effect on viability. All the conditions supported the upregulation of chondrogenic genes (SOX9, COL2A1, SOX5, and ACAN). By comparison, only the highest cell seeding density (16 × 106 cells/mL) promoted a superior extracellular matrix deposition composed of collagen II and aggrecan with limited production of collagen I. These molecules were deposited in the pericellular space. Furthermore, no histological difference was noted between the two stiffnesses.
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Affiliation(s)
- Arianna De Mori
- School of Medicine, Pharmacy and Biomedical Sciences, University of Portsmouth, St. Michael’s Building, White Swan Road, Portsmouth PO1 2DT, UK; (A.D.M.); (G.B.)
| | - Nadide Aydin
- School of Medicine, Pharmacy and Biomedical Sciences, University of Portsmouth, St. Michael’s Building, White Swan Road, Portsmouth PO1 2DT, UK; (A.D.M.); (G.B.)
| | - Giada Lostia
- School of Medicine, Pharmacy and Biomedical Sciences, University of Portsmouth, St. Michael’s Building, White Swan Road, Portsmouth PO1 2DT, UK; (A.D.M.); (G.B.)
| | - Alessia Manca
- School of Medicine, Pharmacy and Biomedical Sciences, University of Portsmouth, St. Michael’s Building, White Swan Road, Portsmouth PO1 2DT, UK; (A.D.M.); (G.B.)
- Department of Biomedical Science, University of Sassari, Viale San Pietro 43/B, 07100 Sassari, Italy
| | - Gordon Blunn
- School of Medicine, Pharmacy and Biomedical Sciences, University of Portsmouth, St. Michael’s Building, White Swan Road, Portsmouth PO1 2DT, UK; (A.D.M.); (G.B.)
| | - Marta Roldo
- School of Medicine, Pharmacy and Biomedical Sciences, University of Portsmouth, St. Michael’s Building, White Swan Road, Portsmouth PO1 2DT, UK; (A.D.M.); (G.B.)
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Ao R, Liang W, Wang Z, Li Q, Pan X, Zhen Y, An Y. Delivery Strategies of Growth Factors in Cartilage Tissue Engineering. TISSUE ENGINEERING. PART B, REVIEWS 2024. [PMID: 39345121 DOI: 10.1089/ten.teb.2024.0158] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 10/01/2024]
Abstract
Cartilage plays an important role in supporting soft tissues, reducing joint friction, and distributing pressure. However, its self-repair capacity is limited due to the lack of blood vessels, nerves, and lymphatic systems. Tissue engineering offers a potential solution to promote cartilage regeneration by combining scaffolds, seed cells, and growth factors. Among these, growth factors play a critical role in regulating cell proliferation, differentiation, and extracellular matrix remodeling. However, their instability, susceptibility to degradation and potential side effects limit their effectiveness. This article reviews the main growth factors used in cartilage tissue engineering and their delivery strategies, including affinity-based delivery, carrier-assisted delivery, stimuli-responsive delivery, spatial structure-based delivery, and cell system-based delivery. Each method shows unique advantages in enhancing the delivery efficiency and specificity of growth factors but also faces challenges such as cost, biocompatibility, and safety. Future research needs to further optimize these strategies to achieve more efficient, safe, and economical delivery of growth factors, thereby advancing the clinical application of cartilage tissue engineering.
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Affiliation(s)
- Rigele Ao
- Department of Plastic Surgery, Peking University Third Hospital, No. 49 North Garden Road, Haidian District, Beijing, 100191China
| | - Wei Liang
- Department of Plastic Surgery, Peking University Third Hospital, No. 49 North Garden Road, Haidian District, Beijing, 100191China
| | - Zimo Wang
- Department of Plastic Surgery, Peking University Third Hospital, No. 49 North Garden Road, Haidian District, Beijing, 100191China
| | - Qiaoyu Li
- Department of Plastic Surgery, Peking University Third Hospital, No. 49 North Garden Road, Haidian District, Beijing, 100191China
| | - Xingyi Pan
- Department of Plastic Surgery, Peking University Third Hospital, No. 49 North Garden Road, Haidian District, Beijing, 100191China
| | - Yonghuan Zhen
- Department of Plastic Surgery, Peking University Third Hospital, No. 49 North Garden Road, Haidian District, Beijing, 100191China
| | - Yang An
- Department of Plastic Surgery, Peking University Third Hospital, No. 49 North Garden Road, Haidian District, Beijing, 100191China
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Singer J, Knezic N, Layne J, Gohring G, Christiansen J, Rothrauff B, Huard J. Enhancing Cartilage Repair: Surgical Approaches, Orthobiologics, and the Promise of Exosomes. Life (Basel) 2024; 14:1149. [PMID: 39337932 PMCID: PMC11432843 DOI: 10.3390/life14091149] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/28/2024] [Revised: 08/22/2024] [Accepted: 08/30/2024] [Indexed: 09/30/2024] Open
Abstract
Treating cartilage damage is challenging as its ability for self-regeneration is limited. Left untreated, it can progress to osteoarthritis (OA), a joint disorder characterized by the deterioration of articular cartilage and other joint tissues. Surgical options, such as microfracture and cell/tissue transplantation, have shown promise as techniques to harness the body's endogenous regenerative capabilities to promote cartilage repair. Nonetheless, these techniques have been scrutinized due to reported inconsistencies in long-term outcomes and the tendency for the defects to regenerate as fibrocartilage instead of the smooth hyaline cartilage native to joint surfaces. Orthobiologics are medical therapies that utilize biologically derived substances to augment musculoskeletal healing. These treatments are rising in popularity because of their potential to enhance surgical standards of care. More recent developments in orthobiologics have focused on the role of exosomes in articular cartilage repair. Exosomes are nano-sized extracellular vesicles containing cargo such as proteins, lipids, and nucleic acids, and are known to facilitate intercellular communication, though their regenerative potential still needs to be fully understood. This review aims to demonstrate the advancements in cartilage regeneration, highlight surgical and biological treatment options, and discuss the recent strides in understanding the precise mechanisms of action involved.
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Affiliation(s)
- Jacob Singer
- Linda and Mitch Hart Regenerative and Personalized Medicine, Steadman Philippon Research Institute, Vail, CO 81657, USA
| | - Noah Knezic
- Linda and Mitch Hart Regenerative and Personalized Medicine, Steadman Philippon Research Institute, Vail, CO 81657, USA
| | - Jonathan Layne
- Linda and Mitch Hart Regenerative and Personalized Medicine, Steadman Philippon Research Institute, Vail, CO 81657, USA
| | - Greta Gohring
- Linda and Mitch Hart Regenerative and Personalized Medicine, Steadman Philippon Research Institute, Vail, CO 81657, USA
| | - Jeff Christiansen
- Linda and Mitch Hart Regenerative and Personalized Medicine, Steadman Philippon Research Institute, Vail, CO 81657, USA
| | - Ben Rothrauff
- Linda and Mitch Hart Regenerative and Personalized Medicine, Steadman Philippon Research Institute, Vail, CO 81657, USA
| | - Johnny Huard
- Linda and Mitch Hart Regenerative and Personalized Medicine, Steadman Philippon Research Institute, Vail, CO 81657, USA
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Bordbar S, Li Z, Lotfibakhshaiesh N, Ai J, Tavassoli A, Beheshtizadeh N, Vainieri L, Khanmohammadi M, Sayahpour FA, Baghaban Eslaminejad M, Azami M, Grad S, Alini M. Cartilage tissue engineering using decellularized biomatrix hydrogel containing TGF-β-loaded alginate microspheres in mechanically loaded bioreactor. Sci Rep 2024; 14:11991. [PMID: 38796487 PMCID: PMC11127927 DOI: 10.1038/s41598-024-62474-5] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/29/2023] [Accepted: 05/17/2024] [Indexed: 05/28/2024] Open
Abstract
Physiochemical tissue inducers and mechanical stimulation are both efficient variables in cartilage tissue fabrication and regeneration. In the presence of biomolecules, decellularized extracellular matrix (ECM) may trigger and enhance stem cell proliferation and differentiation. Here, we investigated the controlled release of transforming growth factor beta (TGF-β1) as an active mediator of mesenchymal stromal cells (MSCs) in a biocompatible scaffold and mechanical stimulation for cartilage tissue engineering. ECM-derived hydrogel with TGF-β1-loaded alginate-based microspheres (MSs) was created to promote human MSC chondrogenic development. Ex vivo explants and a complicated multiaxial loading bioreactor replicated the physiological conditions. Hydrogels with/without MSs and TGF-β1 were highly cytocompatible. MSCs in ECM-derived hydrogel containing TGF-β1/MSs showed comparable chondrogenic gene expression levels as those hydrogels with TGF-β1 added in culture media or those without TGF-β1. However, constructs with TGF-β1 directly added within the hydrogel had inferior properties under unloaded conditions. The ECM-derived hydrogel group including TGF-β1/MSs under loading circumstances formed better cartilage matrix in an ex vivo osteochondral defect than control settings. This study demonstrates that controlled local delivery of TGF-β1 using MSs and mechanical loading is essential for neocartilage formation by MSCs and that further optimization is needed to prevent MSC differentiation towards hypertrophy.
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Affiliation(s)
- Sima Bordbar
- Tissue Engineering Department, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran, Iran
- Department of Anatomy, School of Medicine, Tehran Medical Sciences Branch, Islamic Azad University, Tehran, Iran
- Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran
- AO Research Institute Davos, Davos, Switzerland
| | - Zhen Li
- AO Research Institute Davos, Davos, Switzerland
| | - Nasrin Lotfibakhshaiesh
- Tissue Engineering Department, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran, Iran.
| | - Jafar Ai
- Tissue Engineering Department, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran, Iran
| | - Amin Tavassoli
- Division of Biotechnology, Faculty of Veterinary Medicine, Ferdowsi University of Mashhad, Mashhad, Iran
| | - Nima Beheshtizadeh
- Department of Tissue Engineering, Faculty of Advanced Medical Sciences, Tabriz University of Medical Sciences, Tabriz, Iran
- Regenerative Medicine Group (REMED), Universal Scientific Education and Research Network (USERN), Tehran, Iran
| | | | - Mehdi Khanmohammadi
- Tissue Engineering Department, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran, Iran
- Biomaterials Group, Materials Design Division, Faculty of Materials Science and Engineering, Warsaw University of Technology, Wołoska 141, 02-507, Warsaw, Poland
| | | | - Mohamadreza Baghaban Eslaminejad
- Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran.
| | - Mahmoud Azami
- Tissue Engineering Department, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran, Iran
- Regenerative Medicine Group (REMED), Universal Scientific Education and Research Network (USERN), Tehran, Iran
| | | | - Mauro Alini
- AO Research Institute Davos, Davos, Switzerland.
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Dixit A, Mahajan A, Saxena R, Chakraborty S, Katti DS. Engineering sulfated polysaccharides and silk fibroin based injectable IPN hydrogels with stiffening and growth factor presentation abilities for cartilage tissue engineering. Biomater Sci 2024; 12:2067-2085. [PMID: 38470831 DOI: 10.1039/d3bm01466e] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 03/14/2024]
Abstract
The extracellular matrix (ECM) presents a framework for various biological cues and regulates homeostasis during both developing and mature stages of tissues. During development of cartilage, the ECM plays a critical role in endowing both biophysical and biochemical cues to the progenitor cells. Hence, designing microenvironments that recapitulate these biological cues as provided by the ECM during development may facilitate the engineering of cartilage tissue. In the present study, we fabricated an injectable interpenetrating hydrogel (IPN) system which serves as an artificial ECM and provides chondro-inductive niches for the differentiation of stem cells to chondrocytes. The hydrogel was designed to replicate the gradual stiffening (as a biophysical cue) and the presentation of growth factors (as a biochemical cue) as provided by the natural ECM of the tissue, thus exemplifying a biomimetic approach. This dynamic stiffening was achieved by incorporating silk fibroin, while the growth factor presentation was accomplished using sulfated-carboxymethyl cellulose. Silk fibroin and sulfated-carboxymethyl cellulose (s-CMC) were combined with tyraminated-carboxymethyl cellulose (t-CMC) and crosslinked using HRP/H2O2 to fabricate s-CMC/t-CMC/silk IPN hydrogels. Initially, the fabricated hydrogel imparted a soft microenvironment to promote chondrogenic differentiation, and with time it gradually stiffened to offer mechanical support to the joint. Additionally, the presence of s-CMC conferred the hydrogel with the property of sequestering cationic growth factors such as TGF-β and allowing their prolonged presentation to the cells. More importantly, TGF-β loaded in the developed hydrogel system remained active and induced chondrogenic differentiation of stem cells, resulting in the deposition of cartilage ECM components which was comparable to the hydrogels that were treated with TGF-β provided through media. Overall, the developed hydrogel system acts as a reservoir of the necessary biological cues for cartilage regeneration and simultaneously provides mechanical support for load-bearing tissues such as cartilage.
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Affiliation(s)
- Akansha Dixit
- Department of Biological Sciences and Bioengineering, Indian Institute of Technology-Kanpur, Kanpur-208016, Uttar Pradesh, India.
- The Mehta Family Centre for Engineering in Medicine, Indian Institute of Technology-Kanpur, Kanpur-208016, Uttar Pradesh, India
| | - Aman Mahajan
- Department of Biological Sciences and Bioengineering, Indian Institute of Technology-Kanpur, Kanpur-208016, Uttar Pradesh, India.
- The Mehta Family Centre for Engineering in Medicine, Indian Institute of Technology-Kanpur, Kanpur-208016, Uttar Pradesh, India
| | - Rakshita Saxena
- Department of Biological Sciences and Bioengineering, Indian Institute of Technology-Kanpur, Kanpur-208016, Uttar Pradesh, India.
- The Mehta Family Centre for Engineering in Medicine, Indian Institute of Technology-Kanpur, Kanpur-208016, Uttar Pradesh, India
| | - Saptomee Chakraborty
- Department of Biological Sciences and Bioengineering, Indian Institute of Technology-Kanpur, Kanpur-208016, Uttar Pradesh, India.
- The Mehta Family Centre for Engineering in Medicine, Indian Institute of Technology-Kanpur, Kanpur-208016, Uttar Pradesh, India
| | - Dhirendra S Katti
- Department of Biological Sciences and Bioengineering, Indian Institute of Technology-Kanpur, Kanpur-208016, Uttar Pradesh, India.
- The Mehta Family Centre for Engineering in Medicine, Indian Institute of Technology-Kanpur, Kanpur-208016, Uttar Pradesh, India
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6
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Majumder N, Seit S, Bhabesh NS, Ghosh S. An Advanced Bioconjugation Strategy for Covalent Tethering of TGFβ3 with Silk Fibroin Matrices and its Implications in the Chondrogenesis Profile of Human BMSCs and Human Chondrocytes: A Paradigm Shift in Cartilage Tissue Engineering. Adv Healthc Mater 2024; 13:e2303513. [PMID: 38291832 DOI: 10.1002/adhm.202303513] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/13/2023] [Revised: 01/25/2024] [Indexed: 02/01/2024]
Abstract
The transforming growth factor-β class of cytokines plays a significant role in articular cartilage formation from mesenchymal condensation to chondrogenic differentiation. However, their exogenous addition to the chondrogenic media makes the protocol expensive. It reduces the bioavailability of the cytokine to the cells owing to their burst release. The present study demonstrates an advanced bioconjugation strategy to conjugate transforming growth factor-β3 (TGFβ3) with silk fibroin matrix covalently via a cyanuric chloride coupling reaction. The tethering and change in secondary conformation are confirmed using various spectroscopic analyses. To assess the functionality of the chemically modified silk matrix, human bone marrow-derived mesenchymal stem cells (hBMSCs) and chondrocytes are cultured for 28 days in a chondrogenic differentiation medium. Gene expression and histological analysis reveal enhanced expression of chondrogenic markers with intense Safranin-O and Alcian Blue staining in TGFβ3 conjugated silk matrices than where TGFβ3 is exogenously added to the media for both hBMSCs and chondrocytes. Therefore, this study successfully recapitulates the native niche of TGFβ3 and the role of the silk as a growth factor stabilizer. When cultured over TGFβ3 conjugated silk matrices, hBMSCs display increased proteoglycan secretion and maximum chondrogenic trait with attenuation of chondrocyte hypertrophy over human chondrocytes.
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Affiliation(s)
- Nilotpal Majumder
- Department of Textile and Fibre Engineering, Indian Institute of Technology Delhi, New Delhi, 110016, India
| | - Sinchan Seit
- Department of Textile and Fibre Engineering, Indian Institute of Technology Delhi, New Delhi, 110016, India
| | - Neel Sarovar Bhabesh
- International Centre for Genetic Engineering and Biotechnology (ICGEB), Transcription Regulation group, New Delhi, 110067, India
| | - Sourabh Ghosh
- Department of Textile and Fibre Engineering, Indian Institute of Technology Delhi, New Delhi, 110016, India
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7
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Mahajan A, Nengroo MA, Datta D, Katti DS. Converse modulation of Wnt/β-catenin signaling during expansion and differentiation phases of Infrapatellar fat pad-derived MSCs for improved engineering of hyaline cartilage. Biomaterials 2023; 302:122296. [PMID: 37696204 DOI: 10.1016/j.biomaterials.2023.122296] [Citation(s) in RCA: 6] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/16/2022] [Revised: 08/14/2023] [Accepted: 08/25/2023] [Indexed: 09/13/2023]
Abstract
Mesenchymal stem cells (MSCs) are potential candidates in cell-based therapy for cartilage repair and regeneration. However, during chondrogenic differentiation, MSCs undergo undesirable hypertrophic maturation. This poses a risk of ossification in the neo-tissue formed that eventually impedes the clinical use of MSCs for cartilage repair. TGF-β is a potent growth factor used for chondrogenic differentiation of MSCs, however, its role in hypertrophy remains ambiguous. In the present work, we decipher that TGF-β activates Wnt/β-catenin signaling through SMAD3 and increases the propensity of Infrapatellar fat pad derived MSCs (IFP-MSCs) towards hypertrophy. Notably, inhibiting TGF-β induced Wnt/β-catenin signaling suppresses hypertrophic progression and enhances chondrogenic ability of IFP-MSCs in plasma hydrogels. Additionally, we demonstrate that activating Wnt signaling during expansion phase, promotes proliferation and reduces senescence, while improving stemness of IFP-MSCs. Thus, conversely modulating Wnt signaling in vitro during expansion and differentiation phases generates hyaline-like cartilage with minimal hypertrophy. Importantly, pre-treatment of IFP-MSCs encapsulated in plasma hydrogel with Wnt modulators followed by subcutaneous implantation in nude mice resulted in formation of a cartilage tissue with negligible calcification. Overall, this study provides technological advancement on targeting Wnt/β-catenin pathway in a 3D scaffold, while maintaining the standard chondro-induction protocol to overcome the challenges associated with the clinical use of MSCs to engineer hyaline cartilage.
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Affiliation(s)
- Aman Mahajan
- Department of Biological Sciences and Bioengineering, Indian Institute of Technology-Kanpur, Kanpur, 208016, Uttar Pradesh, India; The Mehta Family Centre for Engineering in Medicine, Indian Institute of Technology-Kanpur, Kanpur, 208016, Uttar Pradesh, India
| | - Mushtaq A Nengroo
- Cancer Biology Division, CSIR-Central Drug Research Institute, Lucknow, 226031, Uttar Pradesh, India
| | - Dipak Datta
- Cancer Biology Division, CSIR-Central Drug Research Institute, Lucknow, 226031, Uttar Pradesh, India
| | - Dhirendra S Katti
- Department of Biological Sciences and Bioengineering, Indian Institute of Technology-Kanpur, Kanpur, 208016, Uttar Pradesh, India; The Mehta Family Centre for Engineering in Medicine, Indian Institute of Technology-Kanpur, Kanpur, 208016, Uttar Pradesh, India.
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8
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Uzieliene I, Bialaglovyte P, Miksiunas R, Lebedis I, Pachaleva J, Vaiciuleviciute R, Ramanaviciene A, Kvederas G, Bernotiene E. Menstrual Blood-Derived Stem Cell Paracrine Factors Possess Stimulatory Effects on Chondrogenesis In Vitro and Diminish the Degradation of Articular Cartilage during Osteoarthritis. Bioengineering (Basel) 2023; 10:1001. [PMID: 37760103 PMCID: PMC10525204 DOI: 10.3390/bioengineering10091001] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/18/2023] [Revised: 08/16/2023] [Accepted: 08/21/2023] [Indexed: 09/29/2023] Open
Abstract
Articular cartilage is an avascular tissue with a limited capacity for self-regeneration, leading the tissue to osteoarthritis (OA). Mesenchymal stem cells (MSCs) are promising for cartilage tissue engineering, as they are capable of differentiating into chondrocyte-like cells and secreting a number of active molecules that are important for cartilage extracellular matrix (ECM) synthesis. The aim of this study was to evaluate the potential of easily accessible menstrual blood-derived MSC (MenSC) paracrine factors in stimulating bone marrow MSC (BMMSCs) chondrogenic differentiation and to investigate their role in protecting cartilage from degradation in vitro. MenSCs and BMMSCs chondrogenic differentiation was induced using four different growth factors: TGF-β3, activin A, BMP-2, and IGF-1. The chondrogenic differentiation of BMMSCs was stimulated in co-cultures with MenSCs and cartilage explants co-cultured with MenSCs for 21 days. The chondrogenic capacity of BMMSCs was analyzed by the secretion of four growth factors and cartilage oligomeric matrix protein, as well as the release and synthesis of cartilage ECM proteins, and chondrogenic gene expression in cartilage explants. Our results suggest that MenSCs stimulate chondrogenic response in BMMSCs by secreting activin A and TGF-β3 and may have protective effects on cartilage tissue ECM by decreasing the release of GAGs, most likely through the modulation of activin A related molecular pathway. In conclusion, paracrine factors secreted by MenSCs may turn out to be a promising therapeutical approach for cartilage tissue protection and repair.
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Affiliation(s)
- Ilona Uzieliene
- Department of Regenerative Medicine, State Research Institute Centre for Innovative Medicine, LT-08406 Vilnius, Lithuania; (P.B.); (R.M.); (I.L.); (J.P.); (R.V.); (E.B.)
| | - Paulina Bialaglovyte
- Department of Regenerative Medicine, State Research Institute Centre for Innovative Medicine, LT-08406 Vilnius, Lithuania; (P.B.); (R.M.); (I.L.); (J.P.); (R.V.); (E.B.)
| | - Rokas Miksiunas
- Department of Regenerative Medicine, State Research Institute Centre for Innovative Medicine, LT-08406 Vilnius, Lithuania; (P.B.); (R.M.); (I.L.); (J.P.); (R.V.); (E.B.)
| | - Ignas Lebedis
- Department of Regenerative Medicine, State Research Institute Centre for Innovative Medicine, LT-08406 Vilnius, Lithuania; (P.B.); (R.M.); (I.L.); (J.P.); (R.V.); (E.B.)
| | - Jolita Pachaleva
- Department of Regenerative Medicine, State Research Institute Centre for Innovative Medicine, LT-08406 Vilnius, Lithuania; (P.B.); (R.M.); (I.L.); (J.P.); (R.V.); (E.B.)
| | - Raminta Vaiciuleviciute
- Department of Regenerative Medicine, State Research Institute Centre for Innovative Medicine, LT-08406 Vilnius, Lithuania; (P.B.); (R.M.); (I.L.); (J.P.); (R.V.); (E.B.)
| | - Almira Ramanaviciene
- Department of Immunology, State Research Institute Centre for Innovative Medicine, LT-08406 Vilnius, Lithuania;
- NanoTechnas—Center on Nanotechnology and Materials Sciences, Faculty of Chemistry and Geosciences, Vilnius University, LT-03225 Vilnius, Lithuania
| | - Giedrius Kvederas
- The Clinic of Rheumatology, Traumatology Orthopaedics and Reconstructive Surgery, Institute of Clinical Medicine of the Faculty of Medicine, Vilnius University, 03101 Vilnius, Lithuania;
| | - Eiva Bernotiene
- Department of Regenerative Medicine, State Research Institute Centre for Innovative Medicine, LT-08406 Vilnius, Lithuania; (P.B.); (R.M.); (I.L.); (J.P.); (R.V.); (E.B.)
- Department of Chemistry and Bioengineering, Faculty of Fundamental Sciences, VilniusTech, Vilnius Gediminas Technical University, 10223 Vilnius, Lithuania
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9
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Vaca-González JJ, Culma JJS, Nova LMH, Garzón-Alvarado DA. Anatomy, molecular structures, and hyaluronic acid - Gelatin injectable hydrogels as a therapeutic alternative for hyaline cartilage recovery: A review. J Biomed Mater Res B Appl Biomater 2023. [PMID: 37178328 DOI: 10.1002/jbm.b.35261] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/06/2023] [Revised: 04/24/2023] [Accepted: 05/03/2023] [Indexed: 05/15/2023]
Abstract
Cartilage damage caused by trauma or osteoarthritis is a common joint disease that can increase the social and economic burden in society. Due to its avascular characteristics, the poor migration ability of chondrocytes, and a low number of progenitor cells, the self-healing ability of cartilage defects has been significantly limited. Hydrogels have been developed into one of the most suitable biomaterials for the regeneration of cartilage because of its characteristics such as high-water absorption, biodegradation, porosity, and biocompatibility similar to natural extracellular matrix. Therefore, the present review article presents a conceptual framework that summarizes the anatomical, molecular structure and biochemical properties of hyaline cartilage located in long bones: articular cartilage and growth plate. Moreover, the importance of preparation and application of hyaluronic acid - gelatin hydrogels for cartilage tissue engineering are included. Hydrogels possess benefits of stimulating the production of Agc1, Col2α1-IIa, and SOX9, molecules important for the synthesis and composition of the extracellular matrix of cartilage. Accordingly, they are believed to be promising biomaterials of therapeutic alternatives to treat cartilage damage.
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Affiliation(s)
- Juan Jairo Vaca-González
- Escuela de Pregrado, Dirección Académica, Vicerrectoría de Sede, Universidad Nacional de Colombia, Sede de La Paz, Cesar, Colombia
- Biomimetics Laboratory, Biotechnology Institute, Universidad Nacional de Colombia, Bogotá, Colombia
| | - Juan José Saiz Culma
- Biomimetics Laboratory, Biotechnology Institute, Universidad Nacional de Colombia, Bogotá, Colombia
| | | | - Diego Alexander Garzón-Alvarado
- Biomimetics Laboratory, Biotechnology Institute, Universidad Nacional de Colombia, Bogotá, Colombia
- Numerical Methods and Modeling Research Group (GNUM), Universidad Nacional de Colombia, Bogotá, Colombia
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10
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Zhou Z, Wang W, Wang J, Wang H, Xia Y, Zhang W, Lai Y, Lin X, Huang Y, Zou X, Stoddart MJ, Li Z, Tian W, Liu S, Wu X, Gao M, Li J, Yang L, Chen D. Function-oriented design: A novel strategy for advanced biomedical materials. JOURNAL OF MATERIALS SCIENCE & TECHNOLOGY 2023; 145:197-209. [DOI: 10.1016/j.jmst.2022.09.066] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 03/07/2025]
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11
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Honarpardaz A, Daliri Joupari M, Tavakkoli S. In Vitro Chondrogenic Differentiation of Human Adipose-Derived Stem Cells by Diacerein. IRANIAN JOURNAL OF PHARMACEUTICAL RESEARCH : IJPR 2023; 22:e137803. [PMID: 38444710 PMCID: PMC10912900 DOI: 10.5812/ijpr-137803] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Received: 05/23/2023] [Revised: 08/30/2023] [Accepted: 09/02/2023] [Indexed: 03/07/2024]
Abstract
Background Tissue engineering is the application system that tries to restore damaged tissues by different approaches, such as cellular therapy, application of cell differential factors, and various materials. One of the important goals in tissue engineering is to guide stem cells directly to the desired tissue, and researchers tried to utilize different molecules as effective factors to improve this technique. Objectives This study aims to demonstrate the effects of diacerein, a slow-acting drug for the treatment of osteoarthritis, on mesenchymal stem cell proliferation and evaluate its potential in the chondrogenesis process. Methods Stem cells were isolated from adipose tissue, characterized by flow cytometry, and cells were treated with 10-5M diacerein for three weeks. Chondrogenic gene expression of SOX9, COL2A1, ACAN, and TGFB1 were analyzed by qRT-PCR and immunocytochemistry techniques. Results Our results showed that diacerein increased the expression of the following genes involved in chondrogenesis: SOX9 (2.9-fold, P < 0.00), COL2A1 (2.2-fold, P < 0.00), ACAN (2.7-fold, P < 0.00), and TGFB1 (2.6-fold, P < 0.00). Immunocytochemistry results also showed increased production of collagen type II as the main protein marker for chondrocytes. Conclusions We observed that diacerein alone could initiate and enhance chondrogenesis, and it can be used as a differentiation factor for stem cells to chondrocyte besides its ability to inhibit IL-1β. Knowing the actual function of diacerein, it could be a good candidate for the treatment of osteoarthritis.
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Affiliation(s)
- Ali Honarpardaz
- Department of Animal Biotechnology, National Institute of Genetic Engineering and Biotechnology, Tehran, Iran
| | - Morteza Daliri Joupari
- Department of Animal Biotechnology, National Institute of Genetic Engineering and Biotechnology, Tehran, Iran
| | - Sajjad Tavakkoli
- Gastroenterology and Liver Diseases Research Center, Research Institute for Gastroenterology and Liver Diseases, Shahid Beheshti University of Medical Sciences, Tehran, Iran
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12
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Xu X, Sui B, Liu X, Sun J. A bioinspired and high-strengthed hydrogel for regeneration of perforated temporomandibular joint disc: Construction and pleiotropic immunomodulatory effects. Bioact Mater 2022; 25:701-715. [PMID: 37056268 PMCID: PMC10086766 DOI: 10.1016/j.bioactmat.2022.07.006] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/27/2022] [Revised: 06/24/2022] [Accepted: 07/05/2022] [Indexed: 11/18/2022] Open
Abstract
Due to the lack of an ideal material for TMJ (temporomandibular joint) disc perforation and local inflammation interfering with tissue regeneration, a functional TGI/HA-CS (tilapia type I gelatin/hyaluronic acid-chondroitin sulfate) double network hydrogel was constructed in this paper. It was not only multiply bionic in its composition, structure and mechanical strength, but also endowed with the ability to immunomodulate microenvironment and simultaneously induce in situ repair of defected TMJ discs. On the one hand, it inhibited inflammatory effects of inflammasome in macrophages, reduced the extracellular matrix (ECM)-degrading enzymes secreted by chondrocytes, reversed the local inflammatory state, promoted the proliferation of TMJ disc cells and induced fibrochondrogenic differentiation of synovium-derived mesenchymal stem cells (SMSCs). On the other hand, it gave an impetus to repairing a relatively-large (6 mm-sized) defect in mini pigs' TMJ discs in a rapid and high-quality manner, which suggested a promising clinical application.
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Affiliation(s)
| | | | | | - Jiao Sun
- Corresponding author. No. 427, Ju-men Road, Shanghai, 200023, PR China.
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13
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Jofré DM, Hoffman DK, Cervino AS, Hahn GM, Grundy M, Yun S, Amrit FRG, Stolz DB, Godoy LF, Salvatore E, Rossi FA, Ghazi A, Cirio MC, Yanowitz JL, Hochbaum D. The CHARGE syndrome ortholog CHD-7 regulates TGF-β pathways in Caenorhabditis elegans. Proc Natl Acad Sci U S A 2022; 119:e2109508119. [PMID: 35394881 PMCID: PMC9169646 DOI: 10.1073/pnas.2109508119] [Citation(s) in RCA: 6] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/02/2021] [Accepted: 02/24/2022] [Indexed: 11/18/2022] Open
Abstract
CHARGE syndrome is a complex developmental disorder caused by mutations in the chromodomain helicase DNA-binding protein-7 (CHD7) and characterized by retarded growth and malformations in the heart and nervous system. Despite the public health relevance of this disorder, relevant cellular pathways and targets of CHD7 that relate to disease pathology are still poorly understood. Here we report that chd-7, the nematode ortholog of Chd7, is required for dauer morphogenesis, lifespan determination, stress response, and body size determination. Consistent with our discoveries, we found chd-7 to be allelic to scd-3, a previously identified dauer suppressor from the DAF-7/ tumor growth factor-β (TGF-β) pathway. Epistatic analysis places CHD-7 at the level of the DAF-3/DAF-5 complex, but we found that CHD-7 also directly impacts the expression of multiple components of this pathway. Transcriptomic analysis revealed that chd-7 mutants fail to repress daf-9 for execution of the dauer program. In addition, CHD-7 regulates the DBL-1/BMP pathway components and shares roles in male tail development and cuticle synthesis. To explore a potential conserved function for chd-7 in vertebrates, we used Xenopus laevis embryos, an established model to study craniofacial development. Morpholino-mediated knockdown of Chd7 led to a reduction in col2a1 messenger RNA (mRNA) levels, a collagen whose expression depends on TGF-β signaling. Both embryonic lethality and craniofacial defects in Chd7-depleted tadpoles were partially rescued by overexpression of col2a1 mRNA. We suggest that Chd7 has conserved roles in regulation of the TGF-β signaling pathway and pathogenic Chd7 could lead to a defective extracellular matrix deposition.
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Affiliation(s)
- Diego M. Jofré
- Departamento de Biodiversidad y Biología Experimental, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, C1053 Buenos Aires, Argentina
| | | | - Ailen S. Cervino
- Instituto de Fisiología, Biología Molecular y Neurociencias, Consejo Nacional de Investigaciones Científicas y Técnicas de Argentina, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, C1053 Buenos Aires, Argentina
| | - Gabriella M. Hahn
- Interdisciplinary Biomedical Graduate Program, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213
| | | | - Sijung Yun
- Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20814
| | - Francis R. G. Amrit
- Department of Pediatrics, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213
| | - Donna B. Stolz
- Center for Biologic Imaging, University of Pittsburgh Medical School, Pittsburgh, PA 15213
| | - Luciana F. Godoy
- Departamento de Biodiversidad y Biología Experimental, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, C1053 Buenos Aires, Argentina
| | - Esteban Salvatore
- Departamento de Biodiversidad y Biología Experimental, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, C1053 Buenos Aires, Argentina
| | - Fabiana A. Rossi
- Instituto de Investigaciones en Medicina Traslacional, Consejo Nacional de Investigaciones Científicas y Técnicas de Argentina, Universidad Austral, B1630 Pilar, Argentina
| | - Arjumand Ghazi
- Department of Pediatrics, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213
- Department of Developmental Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213
- Department of Cell Biology & Physiology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213
| | - M. Cecilia Cirio
- Instituto de Fisiología, Biología Molecular y Neurociencias, Consejo Nacional de Investigaciones Científicas y Técnicas de Argentina, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, C1053 Buenos Aires, Argentina
| | - Judith L. Yanowitz
- Magee-Womens Research Institute, Pittsburgh, PA 15213
- Department of Obstetrics, Gynecology & Reproductive Sciences, University of Pittsburgh, Pittsburgh, PA 15213
- Hillman Cancer Center, University of Pittsburgh, Pittsburgh, PA 15213
| | - Daniel Hochbaum
- Departamento de Biodiversidad y Biología Experimental, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, C1053 Buenos Aires, Argentina
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14
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Duan M, Liu Y, Guo D, Kan S, Niu Z, Pu X, Bai M, Zhang D, Du W, Xie J. TGF-β2 increases cell-cell communication in chondrocytes via p-Smad3 signalling. BIOCHIMICA ET BIOPHYSICA ACTA. MOLECULAR CELL RESEARCH 2022; 1869:119175. [PMID: 34863793 DOI: 10.1016/j.bbamcr.2021.119175] [Citation(s) in RCA: 9] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 08/08/2021] [Revised: 11/18/2021] [Accepted: 11/18/2021] [Indexed: 02/08/2023]
Abstract
Connexin 43 (Cx43)-mediated gap junction intercellular communication (GJIC) plays a crucial role in the pathology and physiology of joint tissues. Transforming growth factor-β2 (TGF-β2), one of the potent regulatory factors in chondrocytes, plays a key role in the regulation of cell cycle and development of joint diseases. However, it is still unknown how TGF-β2 mediates GJIC in chondrocytes. The aim of this study was to explore the potential mechanism by which TGF-β2 regulates GJIC in chondrocytes. CCK-8 assays and scratch assays were performed to define the role of TGF-β2 on cell proliferation and migration. The scrape loading/dye transfer assay and scanning electron microscopy (SEM) were used to verify the effect of TGF-β2 on GJIC between chondrocytes. qPCR was performed to analyse the expression of genes in the gap junction protein family in chondrocytes. The expression of the Cx43 protein and phosphorylated Smad3 (p-Smad3) was evaluated by western blot assay. Immunofluorescence staining was used to explore p-Smad3 signalling pathway activation and Cx43 distribution. From these experiments, we found that the Cx43 protein was the most highly expressed member of the gap junction protein family in chondrocytes. We also found that TGF-β2 facilitated cell-to-cell communication in chondrocytes by upregulating Cx43 expression in chondrocytes. Finally, we found that TGF-β2 activated Smad3 signalling and promoted the nuclear aggregation of p-Smad3. Inhibition experiments by SIS3 also confirmed that TGF-β2-mediated GJIC through p-Smad3 signalling. For the first time, this study confirmed that TGF-β2 could regulate the formation of Cx43-mediated GJIC in chondrocytes via the canonical p-Smad3 signalling pathway.
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Affiliation(s)
- Mengmeng Duan
- State Key Laboratory of Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu, China
| | - Yang Liu
- State Key Laboratory of Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu, China
| | - Daimo Guo
- State Key Laboratory of Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu, China
| | - Shiyi Kan
- State Key Laboratory of Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu, China
| | - Zhixing Niu
- State Key Laboratory of Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu, China
| | - Xiaohua Pu
- State Key Laboratory of Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu, China
| | - Mingru Bai
- State Key Laboratory of Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu, China; National Clinical Research Center for Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu, China; Department of Cariology and Endodontics, West China Hospital of Stomatology, Sichuan University, Chengdu, Sichuan 610064, China
| | - Demao Zhang
- State Key Laboratory of Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu, China
| | - Wei Du
- State Key Laboratory of Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu, China; National Clinical Research Center for Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu, China; Department of Cariology and Endodontics, West China Hospital of Stomatology, Sichuan University, Chengdu, Sichuan 610064, China.
| | - Jing Xie
- State Key Laboratory of Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu, China.
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15
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Ghandforoushan P, Hanaee J, Aghazadeh Z, Samiei M, Navali AM, Khatibi A, Davaran S. Novel nanocomposite scaffold based on gelatin/PLGA-PEG-PLGA hydrogels embedded with TGF-β1 for chondrogenic differentiation of human dental pulp stem cells in vitro. Int J Biol Macromol 2022; 201:270-287. [PMID: 34998887 DOI: 10.1016/j.ijbiomac.2021.12.097] [Citation(s) in RCA: 25] [Impact Index Per Article: 8.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/01/2021] [Revised: 12/15/2021] [Accepted: 12/16/2021] [Indexed: 12/20/2022]
Abstract
In the current study, a novel nanocomposite hydrogel scaffold comprising of natural-based gelatin and synthetic-based (poly D, L (lactide-co-glycolide) -b- poly (ethylene glycol)-b- poly D, L (lactide-co-glycolide) (PLGA-PEG-PLGA) triblock copolymer was developed and loaded with transforming growth factor- β1 (TGF-β1). Synthesized scaffolds' chemical structure was examined by 1H NMR and ATR-FTIR. Scanning electron microscopy (SEM) confirmed particle size and morphology of the prepared nanoparticles as well as the scaffolds. The morphology analysis revealed a porous interconnected structure throughout the scaffold with a pore size dimension of about 202.05 µm. The swelling behavior, in vitro degradation, mechanical properties, density, and porosity were also evaluated. Phalloidin/DAPI staining was utilized for confirming the extended cytoskeleton of the chondrocytes. Alcian blue staining was conducted to determine cartilaginous matrix sulfated glycosaminoglycan (sGAG) synthesis. Eventually, over a period of 21 days, a real-time RT-PCR analysis was applied to measure the mRNA expression of chondrogenic marker genes, type-II collagen, SOX 9, and aggrecan, in hDPSCs cultured for up to 21 days to study the influence of gelatin/PLGA-PEG-PLGA-TGF-β1 hydrogels on hDPSCs. The findings of the cell-encapsulating hydrogels analysis suggested that the adhesion, viability, and chondrogenic differentiation of hDPSCs improved by gelatin/PLGA-PEG-PLGA-TGF-β1 nanocomposite hydrogels. These data supported the conclusion that gelatin/PLGA-PEG-PLGA-TGF-β1 nanocomposite hydrogels render the features that allow thein vitrofunctionality of encapsulated hDPSCs and hence can contribute the basis for new effective strategies for the treatment of cartilage injuries.
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Affiliation(s)
- Parisa Ghandforoushan
- Stem Cell Research Center, Tabriz University of Medical Sciences, Tabriz, Iran; Department of Medicinal Chemistry, Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran
| | - Jalal Hanaee
- Department of Medicinal Chemistry, Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran; Pharmaceutical Analysis Research Center, Tabriz University of Medicinal Science, Tabriz, Iran
| | - Zahra Aghazadeh
- Stem Cell Research Center, Oral Medicine department, Tabriz University of Medical Sciences, Tabriz, Iran
| | - Mohammad Samiei
- Department of Endodontics, Faculty of Dentistry, Tabriz University of Medical Sciences, Tabriz, Iran
| | | | - Ali Khatibi
- Department of biotechnology, Alzahra University, Tehran, Iran
| | - Soodabeh Davaran
- Stem Cell Research Center, Tabriz University of Medical Sciences, Tabriz, Iran; Department of Medicinal Chemistry, Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran; Applied Drug Research Center, Tabriz University of Medical Sciences, Tabriz, Iran.
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16
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Shear Stress Alterations Activate BMP4/pSMAD5 Signaling and Induce Endothelial Mesenchymal Transition in Varicose Veins. Cells 2021; 10:cells10123563. [PMID: 34944071 PMCID: PMC8700678 DOI: 10.3390/cells10123563] [Citation(s) in RCA: 17] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/08/2021] [Revised: 12/01/2021] [Accepted: 12/02/2021] [Indexed: 11/17/2022] Open
Abstract
Chronic venous diseases, including varicose veins, are characterized by hemodynamic disturbances due to valve defects, venous insufficiency, and orthostatism. Veins are physiologically low shear stress systems, and how altered hemodynamics drives focal endothelial dysfunction and causes venous remodeling is unknown. Here we demonstrate the occurrence of endothelial to mesenchymal transition (EndMT) in human varicose veins. Moreover, the BMP4-pSMAD5 pathway was robustly upregulated in varicose veins. In vitro flow-based assays using human vein, endothelial cells cultured in microfluidic chambers show that even minimal disturbances in shear stress as may occur in early stages of venous insufficiency induce BMP4-pSMAD5-based phenotype switching. Furthermore, low shear stress at uniform laminar pattern does not induce EndMT in venous endothelial cells. Targeting the BMP4-pSMAD5 pathway with small molecule inhibitor LDN193189 reduced SNAI1/2 expression in venous endothelial cells exposed to disturbed flow. TGFβ inhibitor SB505124 was less efficient in inhibiting EndMT in venous endothelial cells exposed to disturbed flow. We conclude that disturbed shear stress, even in the absence of any oscillatory flow, induces EndMT in varicose veins via activation of BMP4/pSMAD5-SNAI1/2 signaling. The present findings serve as a rationale for the possible use of small molecular mechanotherapeutics in the management of varicose veins.
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17
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Azami M, Beheshtizadeh N. Identification of regeneration-involved growth factors in cartilage engineering procedure promotes its reconstruction. Regen Med 2021; 16:719-731. [PMID: 34287065 DOI: 10.2217/rme-2021-0028] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/08/2023] Open
Abstract
Aim: To fabricate mature cartilage for implantation, developmental biological processes and proteins should be understood and employed. Methods: A systems biology study of all protein-coding genes participating in cartilage regeneration resulted in a network graph with 11 nodes and 28 edges. Gene ontology and centrality analysis were performed based on the degree index. Results: The four most crucial biological processes along with the seven most interactive proteins involved in cartilage regeneration were identified. Some proteins, which are under serious discussion in cartilage developmental and disease processes, are included in regeneration. Conclusions: Findings positively correlate with the literature, supporting the use of the four most impressive proteins as growth factors applicable to cartilage tissue engineering, including COL2A1, SOX9, CTGF and TGFβ1.
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Affiliation(s)
- Mahmoud Azami
- Department of Tissue Engineering & Applied Cell Sciences, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran, Iran.,Regenerative Medicine group (REMED), Universal Scientific Education & Research Network (USERN), Tehran, Iran
| | - Nima Beheshtizadeh
- Department of Tissue Engineering & Applied Cell Sciences, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran, Iran.,Regenerative Medicine group (REMED), Universal Scientific Education & Research Network (USERN), Tehran, Iran
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18
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De Angelis E, Saleri R, Martelli P, Elviri L, Bianchera A, Bergonzi C, Pirola M, Romeo R, Andrani M, Cavalli V, Conti V, Bettini R, Passeri B, Ravanetti F, Borghetti P. Cultured Horse Articular Chondrocytes in 3D-Printed Chitosan Scaffold With Hyaluronic Acid and Platelet Lysate. Front Vet Sci 2021; 8:671776. [PMID: 34322533 PMCID: PMC8311290 DOI: 10.3389/fvets.2021.671776] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/24/2021] [Accepted: 05/14/2021] [Indexed: 11/13/2022] Open
Abstract
Three-dimensional (3D) printing has gained popularity in tissue engineering and in the field of cartilage regeneration. This is due to its potential to generate scaffolds with spatial variation of cell distribution or mechanical properties, built with a variety of materials that can mimic complex tissue architecture. In the present study, horse articular chondrocytes were cultured for 2 and 4 weeks in 3D-printed chitosan (CH)-based scaffolds prepared with or without hyaluronic acid and in the presence of fetal bovine serum (FBS) or platelet lysate (PL). These 3D culture systems were analyzed in terms of their capability to maintain chondrocyte differentiation in vitro. This was achieved by evaluating cell morphology, immunohistochemistry (IHC), gene expression of relevant cartilage markers (collagen type II, aggrecan, and Sox9), and specific markers of dedifferentiated phenotype (collagen type I, Runx2). The morphological, histochemical, immunohistochemical, and molecular results demonstrated that the 3D CH scaffold is sufficiently porous to be colonized by primary chondrocytes. Thereby, it provides an optimal environment for the colonization and synthetic activity of chondrocytes during a long culture period where a higher rate of dedifferentiation can be generally observed. Enrichment with hyaluronic acid provides an optimal microenvironment for a more stable maintenance of the chondrocyte phenotype. The use of 3D CH scaffolds causes a further increase in the gene expression of most relevant ECM components when PL is added as a substitute for FBS in the medium. This indicates that the latter system enables a better maintenance of the chondrocyte phenotype, thereby highlighting a fair balance between proliferation and differentiation.
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Affiliation(s)
- Elena De Angelis
- Department of Veterinary Science, University of Parma, Parma, Italy
| | - Roberta Saleri
- Department of Veterinary Science, University of Parma, Parma, Italy
| | - Paolo Martelli
- Department of Veterinary Science, University of Parma, Parma, Italy
| | - Lisa Elviri
- Food and Drug Department, University of Parma, Parma, Italy
| | | | - Carlo Bergonzi
- Food and Drug Department, University of Parma, Parma, Italy
| | - Marta Pirola
- Department of Veterinary Science, University of Parma, Parma, Italy
| | - Roberta Romeo
- Department of Veterinary Science, University of Parma, Parma, Italy
| | - Melania Andrani
- Department of Veterinary Science, University of Parma, Parma, Italy
| | - Valeria Cavalli
- Department of Veterinary Science, University of Parma, Parma, Italy
| | - Virna Conti
- Department of Veterinary Science, University of Parma, Parma, Italy
| | | | | | | | - Paolo Borghetti
- Department of Veterinary Science, University of Parma, Parma, Italy
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19
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Regenerative Potential of Blood-Derived Products in 3D Osteoarthritic Chondrocyte Culture System. Curr Issues Mol Biol 2021; 43:665-675. [PMID: 34287259 PMCID: PMC8929075 DOI: 10.3390/cimb43020048] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/09/2021] [Revised: 07/06/2021] [Accepted: 07/08/2021] [Indexed: 12/19/2022] Open
Abstract
Intra-articular injection of different types of blood-derived products is gaining popularity and clinical importance in the treatment of degenerative cartilage disorders such as osteoarthritis. The regenerative potential of two types of platelet-rich plasma (PRP), prepared in the presence of EDTA (EPRP) and citrate (CPRP) and an alternative blood product-hyperacute serum (hypACT) was evaluated using a 3D osteoarthritic chondrocyte pellet model by assessing the metabolic cell activity, cartilage-related gene expression and extracellular matrix deposition within the pellets. Chondrocyte viability was determined by XTT assay and it revealed no significant difference in metabolic activity of OA chondrocyte pellets after supplementation with different blood products. Nevertheless, the selection of blood products influenced the cartilage-related genes expression, ECM morphology and the tissue quality of pellets. Both PRP types had a different biological effect depending upon concentration and even though CPRP is widely used in clinics our assessment did not reveal good results in gene expression either tissue quality. HypACT supplementation resulted in superior cartilage-related genes expression together with tissue quality and seemed to be the most stable product since no remarkable changes were observed between the two different concentrations. All in all, for successful regenerative therapy, possible molecular mechanisms induced by blood-derived products should be always carefully investigated and adapted to the specific medical indications.
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20
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Stampoultzis T, Karami P, Pioletti DP. Thoughts on cartilage tissue engineering: A 21st century perspective. Curr Res Transl Med 2021; 69:103299. [PMID: 34192658 DOI: 10.1016/j.retram.2021.103299] [Citation(s) in RCA: 36] [Impact Index Per Article: 9.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/09/2020] [Revised: 04/11/2021] [Accepted: 05/26/2021] [Indexed: 12/15/2022]
Abstract
In mature individuals, hyaline cartilage demonstrates a poor intrinsic capacity for repair, thus even minor defects could result in progressive degeneration, impeding quality of life. Although numerous attempts have been made over the past years for the advancement of effective treatments, significant challenges still remain regarding the translation of in vitro cartilage engineering strategies from bench to bedside. This paper reviews the latest concepts on engineering cartilage tissue in view of biomaterial scaffolds, tissue biofabrication, mechanobiology, as well as preclinical studies in different animal models. The current work is not meant to provide a methodical review, rather a perspective of where the field is currently focusing and what are the requirements for bridging the gap between laboratory-based research and clinical applications, in light of the current state-of-the-art literature. While remarkable progress has been accomplished over the last 20 years, the current sophisticated strategies have reached their limit to further enhance healthcare outcomes. Considering a clinical aspect together with expertise in mechanobiology, biomaterial science and biofabrication methods, will aid to deal with the current challenges and will present a milestone for the furtherance of functional cartilage engineering.
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Affiliation(s)
| | - Peyman Karami
- Laboratory of Biomechanical Orthopedics, EPFL, Lausanne, Switzerland.
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21
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Lee MS, Stebbins MJ, Jiao H, Huang HC, Leiferman EM, Walczak BE, Palecek SP, Shusta EV, Li WJ. Comparative evaluation of isogenic mesodermal and ectomesodermal chondrocytes from human iPSCs for cartilage regeneration. SCIENCE ADVANCES 2021; 7:eabf0907. [PMID: 34138734 PMCID: PMC8133756 DOI: 10.1126/sciadv.abf0907] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 10/05/2020] [Accepted: 03/29/2021] [Indexed: 05/12/2023]
Abstract
Generating phenotypic chondrocytes from pluripotent stem cells is of great interest in the field of cartilage regeneration. In this study, we differentiated human induced pluripotent stem cells into the mesodermal and ectomesodermal lineages to prepare isogenic mesodermal cell-derived chondrocytes (MC-Chs) and neural crest cell-derived chondrocytes (NCC-Chs), respectively, for comparative evaluation. Our results showed that both MC-Chs and NCC-Chs expressed hyaline cartilage-associated markers and were capable of generating hyaline cartilage-like tissue ectopically and at joint defects. Moreover, NCC-Chs revealed closer morphological and transcriptional similarities to native articular chondrocytes than MC-Chs. NCC-Ch implants induced by our growth factor mixture demonstrated increased matrix production and stiffness compared to MC-Ch implants. Our findings address how chondrocytes derived from pluripotent stem cells through mesodermal and ectomesodermal differentiation are different in activities and functions, providing the crucial information that helps make appropriate cell choices for effective regeneration of articular cartilage.
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Affiliation(s)
- Ming-Song Lee
- Laboratory of Musculoskeletal Biology and Regenerative Medicine, Department of Orthopedics and Rehabilitation, University of Wisconsin-Madison, Madison, WI 53705, USA
- Department of Biomedical Engineering, University of Wisconsin-Madison, Madison, WI 53706, USA
| | - Matthew J Stebbins
- Department of Chemical and Biological Engineering, University of Wisconsin-Madison, Madison, WI 53706, USA
| | - Hongli Jiao
- Laboratory of Musculoskeletal Biology and Regenerative Medicine, Department of Orthopedics and Rehabilitation, University of Wisconsin-Madison, Madison, WI 53705, USA
| | - Hui-Ching Huang
- Laboratory of Musculoskeletal Biology and Regenerative Medicine, Department of Orthopedics and Rehabilitation, University of Wisconsin-Madison, Madison, WI 53705, USA
- Department of Chemical and Biological Engineering, University of Wisconsin-Madison, Madison, WI 53706, USA
| | - Ellen M Leiferman
- Laboratory of Musculoskeletal Biology and Regenerative Medicine, Department of Orthopedics and Rehabilitation, University of Wisconsin-Madison, Madison, WI 53705, USA
| | - Brian E Walczak
- Laboratory of Musculoskeletal Biology and Regenerative Medicine, Department of Orthopedics and Rehabilitation, University of Wisconsin-Madison, Madison, WI 53705, USA
| | - Sean P Palecek
- Department of Chemical and Biological Engineering, University of Wisconsin-Madison, Madison, WI 53706, USA
| | - Eric V Shusta
- Department of Chemical and Biological Engineering, University of Wisconsin-Madison, Madison, WI 53706, USA
- Department of Neurological Surgery, University of Wisconsin-Madison, Madison, WI 53792, USA
| | - Wan-Ju Li
- Laboratory of Musculoskeletal Biology and Regenerative Medicine, Department of Orthopedics and Rehabilitation, University of Wisconsin-Madison, Madison, WI 53705, USA.
- Department of Biomedical Engineering, University of Wisconsin-Madison, Madison, WI 53706, USA
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22
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Bianchi VJ, Parsons M, Backstein D, Kandel RA. Endoglin Level Is Critical for Cartilage Tissue Formation In Vitro by Passaged Human Chondrocytes. Tissue Eng Part A 2021; 27:1140-1150. [PMID: 33323019 DOI: 10.1089/ten.tea.2020.0120] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/13/2022] Open
Abstract
Transforming growth factor beta (TGFβ) signaling is required for in vitro chondrogenesis. In animal models of osteoarthritis (OA), TGFβ receptor alterations are detected in chondrocytes in severe OA cartilage. It is not known whether such changes are dependent on the grade of human OA and if they affect chondrogenesis. Thus, the purpose of this study was to determine if human OA chondrocytes obtained from low-grade or high-grade disease could form cartilage tissue and to assess the role of the co-receptors, endoglin (ENG) and TGFβ receptor 3 (TGFBRIII), in the regulation of this tissue generation in vitro. We hypothesized that the grade of OA disease would not affect the ability of cells to form cartilage tissue and that the TGFβ co-receptor, ENG, would be critical to regulating tissue formation. Chondrocytes isolated from low-grade OA or high-grade OA human articular cartilage (AC) were analyzed directly (P0) or passaged in monolayer to P2. Expression of the primary TGFβ receptor ALK5, and the co-receptors ENG and TGFβRIII, was assessed by image flow cytometry. To assess the ability to form cartilaginous tissue, cells were placed in three-dimensional culture at high density and cultured in chondrogenic media containing TGFβ3. ENG knockdown was used to determine its role in regulating tissue formation. Overall, grade-specific differences in expression of ALK5, ENG, and TGFβRIII in primary or passaged chondrocytes were not detected; however, ENG expression increased significantly after passaging. Despite the presence of ALK5, P0 cells did not form cartilaginous tissue. In contrast, P2 cells derived from low-grade and high-grade OA AC formed hyaline-like cartilaginous tissues of similar quality. Knockdown of ENG in P2 cells inhibited cartilaginous tissue formation compared to controls indicating that the level of ENG protein expression is critical for in vitro chondrogenesis by passaged articular chondrocytes. This study demonstrates that it is not the grade of OA, but the levels of ENG in the presence of ALK5 that influences the ability of human passaged articular chondrocytes to form cartilaginous tissue in vitro in 3D culture. This has implications for cartilage repair therapies. Impact statement These findings are important clinically, given the limited availability of osteoarthritis (OA) cartilage tissue. Being able to use cells from all grades of OA will increase our ability to obtain sufficient cells for cartilage repair. In addition, it is possible that endoglin (ENG) levels, in the presence of ALK5 expression, may be suitable to use as biomarkers to identify cells able to produce cartilage.
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Affiliation(s)
- Vanessa J Bianchi
- Lunenfeld-Tanenbaum Research Institute, Toronto, Canada.,Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, Canada
| | | | - David Backstein
- Division of Orthopaedic Surgery, Mount Sinai Hospital, Toronto, Canada
| | - Rita A Kandel
- Lunenfeld-Tanenbaum Research Institute, Toronto, Canada.,Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, Canada.,Department of Pathology and Laboratory Medicine, Mount Sinai Hospital, Toronto, Canada.,Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Canada
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23
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Tekari A, Egli RJ, Schmid V, Justiz J, Luginbuehl R. A Novel Bioreactor System Capable of Simulating the In Vivo Conditions of Synovial Joints. Tissue Eng Part C Methods 2020; 26:617-627. [PMID: 33267725 PMCID: PMC7759289 DOI: 10.1089/ten.tec.2020.0161] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022] Open
Abstract
Any significant in vitro evaluation of cartilage tissue engineering and cartilage repair strategies has to be performed under the harsh conditions encountered in vivo within synovial joints. To this end, we have developed a novel automated physiological robot reactor system (PRRS) that is capable of recapitulating complex physiological motions and load patterns within an environment similar to that found in the human knee. The PRRS consists of a mechanical stimulation unit (MSU) and an automatic sample changer (ASC) within an environment control box in which the humidity, temperature, and gas composition are tightly regulated. The MSU has three linear (orthogonal) axes and one rotational degree of freedom (around the z-axis). The ASC provides space for up to 24 samples, which can be allocated to individual stimulation patterns. Cell-seeded scaffolds and ex vivo tissue culture systems were established to demonstrate the applicability of the PRRS to the investigation of the effect of load and environmental conditions on engineering and maintenance of articular cartilage in vitro. The bioreactor is a flexible system that has the potential to be applied for culturing connective tissues other than cartilage, such as bone and intervertebral disc tissue, even though the mechanical and environmental parameters are very different.
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Affiliation(s)
- Adel Tekari
- Group for Bone Biology and Orthopaedic Research, Department for Biomedical Research, University of Bern, Bern, Switzerland.,Graduate School for Cellular and Biomedical Sciences, University of Bern, Bern, Switzerland.,Laboratory of Molecular and Cellular Screening Processes, Centre of Biotechnology of Sfax, University of Sfax, Sfax, Tunisia
| | - Rainer J Egli
- Group for Bone Biology and Orthopaedic Research, Department for Biomedical Research, University of Bern, Bern, Switzerland.,RMS Foundation, Bettlach, Switzerland.,Department of Diagnostic, Interventional and Pediatric Radiology, Inselspital, Bern University Hospital, University of Bern, Bern, Switzerland
| | - Veit Schmid
- Institute for Human-Centered Engineering (HuCE) BME Lab, Bern University of Applied Sciences, Biel, Switzerland
| | - Joern Justiz
- Institute for Human-Centered Engineering (HuCE) BME Lab, Bern University of Applied Sciences, Biel, Switzerland
| | - Reto Luginbuehl
- Group for Bone Biology and Orthopaedic Research, Department for Biomedical Research, University of Bern, Bern, Switzerland.,RMS Foundation, Bettlach, Switzerland.,Blaser Swisslube AG, Hasle-Ruegsau, Switzerland
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24
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Kisiday JD, Liebig BE, Goodrich LR. Adult ovine chondrocytes in expansion culture adopt progenitor cell properties that are favorable for cartilage tissue engineering. J Orthop Res 2020; 38:1996-2005. [PMID: 32222117 PMCID: PMC8442064 DOI: 10.1002/jor.24671] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/01/2019] [Revised: 02/18/2020] [Accepted: 03/06/2020] [Indexed: 02/04/2023]
Abstract
Human chondrocytes in expansion culture can become progenitor-like in their ability to proliferate extensively and secrete neocartilage in chondrogenic culture. Sheep are used as a large animal model for cartilage tissue engineering, although for testing progenitor-like chondrocytes it is important that ovine chondrocytes resemble human in the ability to adopt progenitor properties. Here, we investigate whether ovine chondrocytes can adopt progenitor properties as indicated by rapid proliferation in a colony-forming fashion, and high levels of neocartilage secretion in chondrogenic culture. In conditions known to promote expansion of mesenchymal stromal cells, ovine chondrocytes proliferated through approximately 12 population doublings in 10 days. Time-lapse imaging indicated rapid proliferation in a colony-forming pattern. Expanded ovine chondrocytes that were seeded into agarose and cultured in chondrogenic medium accumulated neocartilage over 2 weeks, to a greater extent than primary chondrocytes. These data confirm that ovine chondrocytes resemble human chondrocytes in their ability to acquire progenitor properties that are important for cartilage tissue engineering. Given the broad interest in using progenitor cells to heal connective tissues, next we compared proliferation and trilineage differentiation of ovine chondrocytes, meniscus cells, and tenocytes. Meniscus cells and tenocytes experienced more than 13 population doublings in 10 days. In chondrogenic culture, cartilage matrix accumulation, and gene expression were largely similar among the cell types. All cell types resisted osteogenesis, while expanded tenocytes and meniscal cells were capable of adipogenesis. While ovine connective tissue cells demonstrated limited lineage plasticity, these data support the potential to promote certain progenitor properties with expansion.
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Affiliation(s)
- John D. Kisiday
- Department of Clinical Sciences, Orthopaedic Reserch CenterC. Wayne McIlwraith Translational Medicine Institute Fort Collins Colorado
| | - Bethany E. Liebig
- Department of Clinical Sciences, Orthopaedic Reserch CenterC. Wayne McIlwraith Translational Medicine Institute Fort Collins Colorado
| | - Laurie R. Goodrich
- Department of Clinical Sciences, Orthopaedic Reserch CenterC. Wayne McIlwraith Translational Medicine Institute Fort Collins Colorado
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25
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Laguette MJN, Barrow K, Firfirey F, Dlamini S, Saunders CJ, Dandara C, Gamieldien J, Collins M, September AV. Exploring new genetic variants within COL5A1 intron 4-exon 5 region and TGF-β family with risk of anterior cruciate ligament ruptures. J Orthop Res 2020; 38:1856-1865. [PMID: 31922278 DOI: 10.1002/jor.24585] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 11/15/2019] [Accepted: 01/03/2020] [Indexed: 02/04/2023]
Abstract
Variants within genes encoding structural and regulatory elements of ligaments have been associated with musculoskeletal soft tissue injury risk. The role of intron 4-exon 5 variants within the α1 chain of type V collagen (COL5A1) gene and genes of the transforming growth factor-β (TGF-β) family, TGFBR3 and TGFBI, was investigated on the risk of anterior cruciate ligament (ACL) ruptures. A case-control genetic association study was performed on 210 control (CON) and 249 participants with surgically diagnosed ruptures (ACL), of which 147 reported a noncontact mechanism of injury (NON). Whole-exome sequencing data were used to prioritize variants of potential functional relevance. Genotyping for COL5A1 (rs3922912 G>A, rs4841926 C>T, and rs3124299 C>T), TGFBR3 (rs1805113 G>A and rs1805117 T>C), and TGFBI (rs1442 G>C) was performed using Taqman SNP genotyping assays. Significant overrepresentation of the G allele of TGFBR3 rs1805113 was observed in CON vs ACL (P = .014) and NON groups (P = .021). Similar results were obtained in a female with the G allele (CON vs ACL: P = .029; CON vs NON: P = .016). The TGFBI rs1442 CC genotype was overrepresented in the female ACL vs CON (P = .013). Associations of inferred allele combinations were observed in line with the above results. COL5A1 intron 4-exon 5 genomic interval was not associated with the risk of ACL ruptures. Instead, this novel study is the first to use this approach to identify variants within the TGF-β signaling pathway to be implicated in the risk of ACL ruptures. A genetic susceptibility interval was identified to be explored in the context of extracellular matrix remodeling.
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Affiliation(s)
- Mary-Jessica N Laguette
- Division of Exercise Science and Sports Medicine (ESSM), University of Cape Town, Cape Town, South Africa.,International Federation of Sports Medicine (FIMS) Collaborative Centre of Sports Medicine, ESSM, University of Cape Town, Cape Town, South Africa.,Research Centre for Health Through Physical Activity and Sport, University of Cape Town, Cape Town, South Africa
| | - Kelly Barrow
- Department of Human Genetics, University of Cape Town, Cape Town, South Africa
| | - Firzana Firfirey
- Division of Exercise Science and Sports Medicine (ESSM), University of Cape Town, Cape Town, South Africa.,International Federation of Sports Medicine (FIMS) Collaborative Centre of Sports Medicine, ESSM, University of Cape Town, Cape Town, South Africa.,Research Centre for Health Through Physical Activity and Sport, University of Cape Town, Cape Town, South Africa
| | - Senanile Dlamini
- Division of Exercise Science and Sports Medicine (ESSM), University of Cape Town, Cape Town, South Africa.,International Federation of Sports Medicine (FIMS) Collaborative Centre of Sports Medicine, ESSM, University of Cape Town, Cape Town, South Africa.,Research Centre for Health Through Physical Activity and Sport, University of Cape Town, Cape Town, South Africa
| | - Colleen J Saunders
- South African National Bioinformatics Institute/MRC Unit for Bioinformatics Capacity, University of the Western Cape, Cape Town, Bellville, South Africa.,Division of Emergency Medicine, Department of Surgery, University of Cape Town, Cape Town, South Africa
| | - Collet Dandara
- Department of Human Genetics, University of Cape Town, Cape Town, South Africa
| | - Junaid Gamieldien
- South African National Bioinformatics Institute/MRC Unit for Bioinformatics Capacity, University of the Western Cape, Cape Town, Bellville, South Africa
| | - Malcolm Collins
- Division of Exercise Science and Sports Medicine (ESSM), University of Cape Town, Cape Town, South Africa.,International Federation of Sports Medicine (FIMS) Collaborative Centre of Sports Medicine, ESSM, University of Cape Town, Cape Town, South Africa.,Research Centre for Health Through Physical Activity and Sport, University of Cape Town, Cape Town, South Africa
| | - Alison V September
- Division of Exercise Science and Sports Medicine (ESSM), University of Cape Town, Cape Town, South Africa.,International Federation of Sports Medicine (FIMS) Collaborative Centre of Sports Medicine, ESSM, University of Cape Town, Cape Town, South Africa.,Research Centre for Health Through Physical Activity and Sport, University of Cape Town, Cape Town, South Africa
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26
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Abstract
PURPOSE OF REVIEW The decreased contact area, edge loading, and increased stress in the adjacent area cartilage resulting from chondral defects are believed to predispose this tissue to degenerative changes that have significant economic implications, especially when considering its progression to osteoarthritis of the knee. Growth factors are considered therapeutic possibilities to enhance healing of chondral injuries and modify the progression to degenerative arthritis. Thus, the purposes of this review are to first to summarize important points for defect preparation and recent advances in techniques for marrow stimulation and second, and to identify specific growth factors and cytokines that have the capacity to advance cartilage regeneration and the treatment of osteoarthritis in light of recent laboratory and clinical studies. RECENT FINDINGS TGF-β, BMP-2, BMP-7, IGF-1, as IL-1 receptor antagonist, and recombinant human FGF-18 are some of the promising growth factor/cytokine treatments with pioneering and evolving clinical developments. The bulk of the review describes and discusses these developments in light of fundamental basic science. It is crucial to also understand the other underlying advances made in the surgical management of cartilage defects prior to onset of OA. These advances are in techniques for defect preparation and marrow stimulation, a common cartilage repair procedure used in combination with growth factor/cytokine augmentation. Multiple growth factor/cytokine modulation therapies are currently undergoing clinical trial investigation including Invossa (currently in phase III study), Kineret (currently in phase I study), and Sprifermin (currently in phase II study) for the treatment of symptomatic osteoarthritis.
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27
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Zieba J, Munivez E, Castellon A, Jiang MM, Dawson B, Ambrose CG, Lee B. Fracture Healing in Collagen-Related Preclinical Models of Osteogenesis Imperfecta. J Bone Miner Res 2020; 35:1132-1148. [PMID: 32053224 DOI: 10.1002/jbmr.3979] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 11/06/2019] [Revised: 01/27/2020] [Accepted: 02/11/2020] [Indexed: 12/17/2022]
Abstract
Osteogenesis imperfecta (OI) is a genetic bone dysplasia characterized by bone deformities and fractures caused by low bone mass and impaired bone quality. OI is a genetically heterogeneous disorder that most commonly arises from dominant mutations in genes encoding type I collagen (COL1A1 and COL1A2). In addition, OI is recessively inherited with the majority of cases resulting from mutations in prolyl-3-hydroxylation complex members, which includes cartilage-associated protein (CRTAP). OI patients are at an increased risk of fracture throughout their lifetimes. However, non-union or delayed healing has been reported in 24% of fractures and 52% of osteotomies. Additionally, refractures typically go unreported, making the frequency of refractures in OI patients unknown. Thus, there is an unmet need to better understand the mechanisms by which OI affects fracture healing. Using an open tibial fracture model, our study demonstrates delayed healing in both Col1a2 G610c/+ and Crtap -/- OI mouse models (dominant and recessive OI, respectively) that is associated with reduced callus size and predicted strength. Callus cartilage distribution and chondrocyte maturation were altered in OI, suggesting accelerated cartilage differentiation. Importantly, we determined that healed fractured tibia in female OI mice are biomechanically weaker when compared with the contralateral unfractured bone, suggesting that abnormal OI fracture healing OI may prime future refracture at the same location. We have previously shown upregulated TGF-β signaling in OI and we confirm this in the context of fracture healing. Interestingly, treatment of Crtap -/- mice with the anti-TGF-β antibody 1D11 resulted in further reduced callus size and predicted strength, highlighting the importance of investigating dose response in treatment strategies. These data provide valuable insight into the effect of the extracellular matrix (ECM) on fracture healing, a poorly understood mechanism, and support the need for prevention of primary fractures to decrease incidence of refracture and deformity in OI patients. © 2020 American Society for Bone and Mineral Research.
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Affiliation(s)
- Jennifer Zieba
- Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA
| | - Elda Munivez
- Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA
| | - Alexis Castellon
- Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA
| | - Ming-Ming Jiang
- Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA
| | - Brian Dawson
- Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA
| | - Catherine G Ambrose
- Department of Orthopaedic Surgery, University of Texas Health Science Center at Houston, Houston, TX, USA
| | - Brendan Lee
- Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA
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28
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Kikuchi T, Shimizu T. Thickness-wise growth technique for human articular chondrocytes to fabricate three-dimensional cartilage grafts. Regen Ther 2020; 14:119-127. [PMID: 32055650 PMCID: PMC7005340 DOI: 10.1016/j.reth.2019.12.001] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/02/2019] [Revised: 11/02/2019] [Accepted: 12/03/2019] [Indexed: 11/14/2022] Open
Abstract
INTRODUCTION Cutting the cost of manufacturing is important for extending the use of tissue-engineered therapeutic products. The present study aimed to develop a simple method for fabrication of cartilaginous tissues for regenerative therapy, utilizing the phenomenon where human articular chondrocytes grow thickness-wise and spontaneously form three-dimensionally thick tissues. METHODS Normal human articular chondrocytes (NHACs) were cultured with varying concentrations of transforming growth factor beta 1 (TGF-β1) and/or fibroblast growth factor-2 (FGF-2) to optimize the culture condition for thickness-wise growth of chondrocytes. Next, the tissues grown in the optimal condition were subjected to re-differentiation culture in attached and detached states to assess differentiation capacity by evaluating secreted factors, histological analysis, and a gene expression assay. RESULTS NHACs grew thickness-wise efficiently in the presence of 1 ng/mL TGF-β1 and 10 ng/mL FGF-2. After two weeks of culture, NHACs grew with 11-fold higher thickness and 16-fold higher cell number compared to cells which were neither treated with TGF-β1 nor with FGF-2. These thickness-wise-grown chondrocytes could be re-differentiated by a differentiation medium according to the increase in melanoma inhibitory activity (MIA) and positive safranin-O staining. Interestingly, the cartilaginous gene expression was considerably different between the attached and detached conditions even in the same culture medium, indicating the necessity of detachment and shrinkage to achieve further differentiation. CONCLUSIONS Spontaneous thickness-wise growth might provide a simple tissue-engineering method for manufacturing cartilaginous 3D tissues.
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Affiliation(s)
| | - Tatsuya Shimizu
- Institute of Advanced Biomedical Engineering and Science, Tokyo Women's Medical University (TWIns), 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162-8666, Japan
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29
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Semba JA, Mieloch AA, Rybka JD. Introduction to the state-of-the-art 3D bioprinting methods, design, and applications in orthopedics. ACTA ACUST UNITED AC 2020. [DOI: 10.1016/j.bprint.2019.e00070] [Citation(s) in RCA: 33] [Impact Index Per Article: 6.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/05/2023]
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30
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Vieira JS, Cunha EJ, de Souza JF, Chaves LHK, de Souza JL, Giovanini AF. Alendronate disturbs femoral growth due to changes during immunolocalization of transforming growth factor-β1 and bone morphogenetic protein-2 in epiphyseal plate. World J Exp Med 2020; 10:1-9. [PMID: 31942441 PMCID: PMC6960019 DOI: 10.5493/wjem.v10.i1.1] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/05/2019] [Revised: 11/26/2019] [Accepted: 12/15/2019] [Indexed: 02/06/2023] Open
Abstract
BACKGROUND The epiphyseal growth plate is an important anatomical segment localized on the ends of a long bone. Despite the abovementioned atractive reasons for alendronate’s use, few data on the effect of alendronate during epiphyseal growth exist.
AIM Verify the effect of alendronate on the growth epiphyseal plate, and compare its effect with the size of the femur during the double-staining of the immunolocalization of transforming growth factor-β1 (TGF-β1) and bone morphogenetic protein-2 (BMP2) in endochondral ossifing in specimens that have received alendronate.
METHODS Forty newborn rats were randomly divided into two groups: a control group (were given applications of 1 mg/kg physiologic saline) and a group that received Alendronate (a dose of 2.5 mg/kg). These groups were then divided into two subgroups for euthanasia in two and 12 d of life. After euthanasia, the femurs were removed, and the femoral bones were measured linearly between the apex of the greater trochanter until the lower intercondylar midlle face to verify the probable bone growth between 3 and 12 d in control and alednroanto treated rats. Posteriorly, the surgical pieces were also sent to the histopathology laboratory to produce histological slides. The obtained slides were stained with hematoxylin and eosin to measure each of the cartilage zones in endochondral development. and other slides were immunohistochemically tested for anti- TGF-β1 and BMP-2 antibodies to investigate the immunolocalization of these proteins in the epiphyseal plaque area.
RESULTS On the third day, some diferences between the control group and specimens treated with alendronate were verified. Macroscopiccaly, we found similarities in size between the femoral bones when we compared the control group with the specimens that received alendronate. On the 12th day, the bone size of the mice receiving the drug was significantly smaller than those of the control group. These results coincide with changes in the TGF-β1 and BMP-2 expression. In the specimens that received alendronate, the TGF-β1 was expressed in some sites of trabecular bone that was neoformed, peripherally to the bone marrow area. The BMP-2 was also positive in proliferative chondrocytes and hypertrofic chondrocytes. On the 12th day, all layers of chondrocytes exhibited positivity for BMP-2 in the specimens that received alendronate. In the interface between the trabecular bone and cartilage, an area of disorganized bone deposition was evident. Neoformed bone also appeared to be different at 12 d. In the control group, BMP-2 was positive in an intense area of bone trabeculae, whereas the alendronate-treated group showed TGF-β1 positive trabeculae and a greater bone area.
CONCLUSION Alendronate alters the immunolocalization of TGF-β1 and BMP-2 simultaneously, a condition that changes the usual histological aspects of the cartilage zone and impairs epiphysis growth and femur growth.
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31
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Dasargyri A, Reichmann E, Moehrlen U. Bio-engineering of fetal cartilage for in utero spina bifida repair. Pediatr Surg Int 2020; 36:25-31. [PMID: 31576465 DOI: 10.1007/s00383-019-04573-3] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Accepted: 09/12/2019] [Indexed: 10/25/2022]
Abstract
PURPOSE During in utero surgical spina bifida repair, a multi-layer closure is used to cover the defect. These soft tissues, however, might be not sufficient to protect the spinal cord during the future life. Our goal is to develop a more rigid protective tissue construct consisting of bioengineered cartilage and skin. METHODS Ovine fetal chondrocytes were tested for their in vitro chondrogenic potential in three-dimensional cultures. Scaffolds based on natural biopolymers (collagen I, fibrin glue) were loaded with varying amounts of fetal chondrocytes and assessed for their ability to support cartilage formation in vitro. The bioengineered constructs were analyzed using cartilage-specific histology stainings and compared to native fetal cartilage. RESULTS Fetal chondrocytes actively produced cartilage extracellular matrix in three-dimensional cultures, even at high passages. Among all bioengineered scaffolds, only the collagen I-based hydrogels loaded with high densities of fetal chondrocytes showed cartilage-like structure in vitro but also extensive shrinking. CONCLUSION Fetal chondrocytes represent a good cell source for cartilage bioengineering. Collagen I scaffolds support cartilage formation in vitro, but the construct shrinking constitutes a major limitation. Future steps include the identification of suitable bioprintable materials which maintain their shape and size, as well as the analysis of the interphase between bioengineered cartilage and skin.
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Affiliation(s)
- Athanasia Dasargyri
- Tissue Biology Research Unit, Department of Surgery, University Children's Hospital Zurich, Zurich, Switzerland
| | - Ernst Reichmann
- Tissue Biology Research Unit, Department of Surgery, University Children's Hospital Zurich, Zurich, Switzerland
| | - Ueli Moehrlen
- Tissue Biology Research Unit, Department of Surgery, University Children's Hospital Zurich, Zurich, Switzerland. .,Pediatric Surgery, University Children's Hospital Zurich, Steinwiesstrasse 75, 8032, Zurich, Switzerland. .,Zurich Center for Fetal Diagnosis and Therapy, Zurich, Switzerland.
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32
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Kelly DC, Raftery RM, Curtin CM, O'Driscoll CM, O'Brien FJ. Scaffold-Based Delivery of Nucleic Acid Therapeutics for Enhanced Bone and Cartilage Repair. J Orthop Res 2019; 37:1671-1680. [PMID: 31042304 DOI: 10.1002/jor.24321] [Citation(s) in RCA: 35] [Impact Index Per Article: 5.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/30/2018] [Accepted: 04/09/2019] [Indexed: 02/04/2023]
Abstract
Recent advances in tissue engineering have made progress toward the development of biomaterials capable of the delivery of growth factors, such as bone morphogenetic proteins, in order to promote enhanced tissue repair. However, controlling the release of these growth factors on demand and within the desired localized area is a significant challenge and the associated high costs and side effects of uncontrolled delivery have proven increasingly problematic in clinical orthopedics. Gene therapy may be a valuable tool to avoid the limitations of local delivery of growth factors. Following a series of setbacks in the 1990s, the field of gene therapy is now seeing improvements in safety and efficacy resulting in substantial clinical progress and a resurgence in confidence. Biomaterial scaffold-mediated gene therapy provides a template for cell infiltration and tissue formation while promoting transfection of cells to engineer therapeutic proteins in a sustained but ultimately transient fashion. Additionally, scaffold-mediated delivery of RNA-based therapeutics can silence specific genes associated with orthopedic pathological states. This review will provide an overview of the current state-of-the-art in the field of gene-activated scaffolds and their use within orthopedic tissue engineering applications. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:1671-1680, 2019.
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Affiliation(s)
- Domhnall C Kelly
- Tissue Engineering Research Group (TERG), Department of Anatomy, Royal College of Surgeons in Ireland (RCSI), Dublin, Ireland.,Trinity Centre of Bioengineering (TCBE), Trinity College Dublin (TCD), Dublin, Ireland.,Advanced Materials and Bioengineering Research Centre (AMBER), RCSI and TCD, Dublin, Ireland.,Centre for Research in Medical Devices (CÚRAM), Biomedical Sciences Building, National University of Ireland Galway (NUI, Galway), Galway, Ireland
| | - Rosanne M Raftery
- Tissue Engineering Research Group (TERG), Department of Anatomy, Royal College of Surgeons in Ireland (RCSI), Dublin, Ireland.,Trinity Centre of Bioengineering (TCBE), Trinity College Dublin (TCD), Dublin, Ireland.,Advanced Materials and Bioengineering Research Centre (AMBER), RCSI and TCD, Dublin, Ireland
| | - Caroline M Curtin
- Tissue Engineering Research Group (TERG), Department of Anatomy, Royal College of Surgeons in Ireland (RCSI), Dublin, Ireland.,Trinity Centre of Bioengineering (TCBE), Trinity College Dublin (TCD), Dublin, Ireland.,Advanced Materials and Bioengineering Research Centre (AMBER), RCSI and TCD, Dublin, Ireland
| | - Caitriona M O'Driscoll
- Centre for Research in Medical Devices (CÚRAM), Biomedical Sciences Building, National University of Ireland Galway (NUI, Galway), Galway, Ireland.,Pharmacodelivery Group, School of Pharmacy, University College Cork, Cork, Ireland
| | - Fergal J O'Brien
- Tissue Engineering Research Group (TERG), Department of Anatomy, Royal College of Surgeons in Ireland (RCSI), Dublin, Ireland.,Trinity Centre of Bioengineering (TCBE), Trinity College Dublin (TCD), Dublin, Ireland.,Advanced Materials and Bioengineering Research Centre (AMBER), RCSI and TCD, Dublin, Ireland.,Centre for Research in Medical Devices (CÚRAM), Biomedical Sciences Building, National University of Ireland Galway (NUI, Galway), Galway, Ireland
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Qu D, Zhu JP, Childs HR, Lu HH. Nanofiber-based transforming growth factor-β3 release induces fibrochondrogenic differentiation of stem cells. Acta Biomater 2019; 93:111-122. [PMID: 30862549 DOI: 10.1016/j.actbio.2019.03.019] [Citation(s) in RCA: 28] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/18/2018] [Revised: 03/06/2019] [Accepted: 03/07/2019] [Indexed: 12/21/2022]
Abstract
Fibrocartilage is typically found in regions subject to complex, multi-axial loads and plays a critical role in musculoskeletal function. Mesenchymal stem cell (MSC)-mediated fibrocartilage regeneration may be guided by administration of appropriate chemical and/or physical cues, such as by culturing cells on polymer nanofibers in the presence of the chondrogenic growth factor TGF-β3. However, targeted delivery and maintenance of effective local factor concentrations remain challenges for implementation of growth factor-based regeneration strategies in clinical settings. Thus, the objective of this study was to develop and optimize the bioactivity of a biomimetic nanofiber scaffold system that enables localized delivery of TGF-β3. To this end, we fabricated TGF-β3-releasing nanofiber meshes that provide sustained growth factor delivery and demonstrated their potential for guiding synovium-derived stem cell (SDSC)-mediated fibrocartilage regeneration. TGF-β3 delivery enhanced cell proliferation and synthesis of relevant fibrocartilaginous matrix in a dose-dependent manner. By designing a scaffold that eliminates the need for exogenous or systemic growth factor administration and demonstrating that fibrochondrogenesis requires a lower growth factor dose compared to previously reported, this study represents a critical step towards developing a clinical solution for regeneration of fibrocartilaginous tissues. STATEMENT OF SIGNIFICANCE: Fibrocartilage is a tissue that plays a critical role throughout the musculoskeletal system. However, due to its limited self-healing capacity, there is a significant unmet clinical need for more effective approaches for fibrocartilage regeneration. We have developed a nanofiber-based scaffold that provides both the biomimetic physical cues, as well as localized delivery of the chemical factors needed to guide stem cell-mediated fibrocartilage formation. Specifically, methods for fabricating TGF-β3-releasing nanofibers were optimized, and scaffold-mediated TGF-β3 delivery enhanced cell proliferation and synthesis of fibrocartilaginous matrix, demonstrating for the first time, the potential for nanofiber-based TGF-β3 delivery to guide stem cell-mediated fibrocartilage regeneration. This nanoscale delivery platform represents an exciting new strategy for fibrocartilage regeneration.
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Affiliation(s)
- Dovina Qu
- Biomaterials and Interface Tissue Engineering Laboratory, Department of Biomedical Engineering, Columbia University, 351 Engineering Terrace Building, MC 8904, 1210 Amsterdam Avenue, New York, NY 10027, United States
| | - Jennifer P Zhu
- Biomaterials and Interface Tissue Engineering Laboratory, Department of Biomedical Engineering, Columbia University, 351 Engineering Terrace Building, MC 8904, 1210 Amsterdam Avenue, New York, NY 10027, United States
| | - Hannah R Childs
- Biomaterials and Interface Tissue Engineering Laboratory, Department of Biomedical Engineering, Columbia University, 351 Engineering Terrace Building, MC 8904, 1210 Amsterdam Avenue, New York, NY 10027, United States
| | - Helen H Lu
- Biomaterials and Interface Tissue Engineering Laboratory, Department of Biomedical Engineering, Columbia University, 351 Engineering Terrace Building, MC 8904, 1210 Amsterdam Avenue, New York, NY 10027, United States.
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Schneider MC, Chu S, Randolph MA, Bryant SJ. An in vitro and in vivo comparison of cartilage growth in chondrocyte-laden matrix metalloproteinase-sensitive poly(ethylene glycol) hydrogels with localized transforming growth factor β3. Acta Biomater 2019; 93:97-110. [PMID: 30914256 DOI: 10.1016/j.actbio.2019.03.046] [Citation(s) in RCA: 42] [Impact Index Per Article: 7.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/01/2018] [Revised: 03/20/2019] [Accepted: 03/21/2019] [Indexed: 12/25/2022]
Abstract
While matrix-assisted autologous chondrocyte implantation has emerged as a promising therapy to treat focal chondral defects, matrices that support regeneration of hyaline cartilage remain challenging. The goal of this work was to investigate the potential of a matrix metalloproteinase (MMP)-sensitive poly(ethylene glycol) (PEG) hydrogel containing the tethered growth factor, transforming growth factor β3 (TGF-β3), and compare cartilage regeneration in vitro and in vivo. The in vitro environment comprised chemically-defined medium while the in vivo environment utilized the subcutaneous implant model in athymic mice. Porcine chondrocytes were isolated and expanded in 2D culture for 10 days prior to encapsulation. The presence of tethered TGF-β3 reduced cell spreading. Chondrocyte-laden hydrogels were analyzed for total sulfated glycosaminoglycan and collagen contents, MMP activity, and spatial deposition of aggrecan, decorin, biglycan, and collagens type II and I. The total amount of extracellular matrix (ECM) deposited in the hydrogel constructs was similar in vitro and in vivo. However, the in vitro environment was not able to support long-term culture up to 64 days of the engineered cartilage leading to the eventual breakdown of aggrecan. The in vivo environment, on the other hand, led to more elaborate ECM, which correlated with higher MMP activity, and an overall higher quality of engineered tissue that was rich in aggrecan, decorin, biglycan and collagen type II with minimal collagen type I. Overall, the MMP-sensitive PEG hydrogel containing tethered TGF-β3 is a promising matrix for hyaline cartilage regeneration in vivo. STATEMENT OF SIGNIFICANCE: Regenerating hyaline cartilage remains a significant clinical challenge. The resultant repair tissue is often fibrocartilage, which long-term cannot be sustained. The goal of this study was to investigate the potential of a synthetic hydrogel matrix containing peptide crosslinks that can be degraded by enzymes secreted by encapsulated cartilage cells (i.e., chondrocytes) and tethered growth factors, specifically TGF-β3, to provide localized chondrogenic cues to the cells. This hydrogel led to hyaline cartilage-like tissue growth in vitro and in vivo, with minimal formation of fibrocartilage. However, the tissue formed in vitro, could not be maintained long-term. In vivo this hydrogel shows great promise as a potential matrix for use in regenerating hyaline cartilage.
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Charlier E, Deroyer C, Ciregia F, Malaise O, Neuville S, Plener Z, Malaise M, de Seny D. Chondrocyte dedifferentiation and osteoarthritis (OA). Biochem Pharmacol 2019; 165:49-65. [PMID: 30853397 DOI: 10.1016/j.bcp.2019.02.036] [Citation(s) in RCA: 297] [Impact Index Per Article: 49.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/08/2019] [Accepted: 02/28/2019] [Indexed: 02/08/2023]
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36
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Vieira JS, Cunha EJ, de Souza JF, Sant'Ana RD, Zielak JC, Costa-Casagrande TA, Giovanini AF. Alendronate induces postnatal maxillary bone growth by stimulating intramembranous ossification and preventing premature cartilage mineralization in the midpalatal suture of newborn rats. Int J Oral Maxillofac Surg 2019; 48:1494-1503. [PMID: 31054875 DOI: 10.1016/j.ijom.2019.04.002] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/18/2018] [Revised: 03/25/2019] [Accepted: 04/01/2019] [Indexed: 12/21/2022]
Abstract
Cleft palate is a common malformation of craniofacial development, and postnatal deficiencies in palate formation may occur. The aim of this study was to determine whether alendronate treatment could induce maxillary mineralization and thus reduce the need for surgical procedures. The effects of alendronate on maxillary bone development, the midpalatal suture, and the levels of transforming growth factor beta-1 (TGF-β1), bone morphogenetic protein 2 (BMP-2), collagen I and II, and V-ATPase were evaluated in newborn rats. Thirty newborn rats were placed in a control group and 30 in a group that received intraperitoneal alendronate (2.5 mg/kg/day). The animals were euthanized on day 7 or 12, and the heads were subjected to histological and immunohistochemical analyses. Specimens from rats that received alendronate presented larger bone matrix deposition in areas of intramembranous ossification of the maxillary bone when compared to controls. Furthermore, higher levels of TGF-β1, BMP-2, and collagen I were observed, whereas osteoclasts showed no V-ATPase. The alendronate group also showed higher levels of TGF-β1 and collagen II in the midpalatal suture, whereas BMP-2 levels were lower than in controls. These results coincided with an expansion of the chondroid. In conclusion, alendronate increased the intramembranous ossification in the maxillary bone in association with increased expression of TGF-β1, BMP-2, and collagen I and decreased V-ATPase. The drug induced an expansion of chondrocytes and a decrease in mineral bone deposition despite the high levels of TGF-β1 in this area. Alendronate may therefore be useful in the treatment of diseases affecting bone growth.
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Affiliation(s)
- J S Vieira
- Graduate Programme in Clinical Dentistry, Positivo University, Curitiba, Paraná, Brazil
| | - E J Cunha
- Graduate Programme in Clinical Dentistry, Positivo University, Curitiba, Paraná, Brazil
| | - J F de Souza
- Department of Stomatology, School of Dentistry, Federal University of Paraná, UFPR, Paraná, Brazil
| | - R D Sant'Ana
- Graduate Programme in Clinical Dentistry, Positivo University, Curitiba, Paraná, Brazil
| | - J C Zielak
- Graduate Programme in Clinical Dentistry, Positivo University, Curitiba, Paraná, Brazil
| | - T A Costa-Casagrande
- Graduate Programme in Clinical Dentistry, Positivo University, Curitiba, Paraná, Brazil
| | - A F Giovanini
- Graduate Programme in Clinical Dentistry, Positivo University, Curitiba, Paraná, Brazil.
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Rathan S, Dejob L, Schipani R, Haffner B, Möbius ME, Kelly DJ. Fiber Reinforced Cartilage ECM Functionalized Bioinks for Functional Cartilage Tissue Engineering. Adv Healthc Mater 2019; 8:e1801501. [PMID: 30624015 DOI: 10.1002/adhm.201801501] [Citation(s) in RCA: 90] [Impact Index Per Article: 15.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/21/2018] [Indexed: 01/17/2023]
Abstract
Focal articular cartilage (AC) defects, if left untreated, can lead to debilitating diseases such as osteoarthritis. While several tissue engineering strategies have been developed to promote cartilage regeneration, it is still challenging to generate functional AC capable of sustaining high load-bearing environments. Here, a new class of cartilage extracellular matrix (cECM)-functionalized alginate bioink is developed for the bioprinting of cartilaginous tissues. The bioinks are 3D-printable, support mesenchymal stem cell (MSC) viability postprinting and robust chondrogenesis in vitro, with the highest levels of COLLII and ACAN expression observed in bioinks containing the highest concentration of cECM. Enhanced chondrogenesis in cECM-functionalized bioinks is also associated with progression along an endochondral-like pathway, as evident by increases in RUNX2 expression and calcium deposition in vitro. The bioinks loaded with MSCs and TGF-β3 are also found capable of supporting robust chondrogenesis, opening the possibility of using such bioinks for direct "print-and-implant" cartilage repair strategies. Finally, it is demonstrated that networks of 3D-printed polycaprolactone fibers with compressive modulus comparable to native AC can be used to mechanically reinforce these bioinks, with no loss in cell viability. It is envisioned that combinations of such biomaterials can be used in multiple-tool biofabrication strategies for the bioprinting of biomimetic cartilaginous implants.
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Affiliation(s)
- Swetha Rathan
- Trinity Centre for Bioengineering, Trinity Biomedical Sciences Institute, Trinity College Dublin, Dublin 2, Ireland
- Department of Mechanical and Manufacturing Engineering, School of Engineering, Trinity College Dublin, Dublin 2, Ireland
| | - Léa Dejob
- Trinity Centre for Bioengineering, Trinity Biomedical Sciences Institute, Trinity College Dublin, Dublin 2, Ireland
- Ecole Nationale Supérieure de Chimie de Mulhouse, Université de Haute-Alsace, 68200, Mulhouse, France
| | - Rossana Schipani
- Trinity Centre for Bioengineering, Trinity Biomedical Sciences Institute, Trinity College Dublin, Dublin 2, Ireland
- Department of Mechanical and Manufacturing Engineering, School of Engineering, Trinity College Dublin, Dublin 2, Ireland
| | | | | | - Daniel J Kelly
- Trinity Centre for Bioengineering, Trinity Biomedical Sciences Institute, Trinity College Dublin, Dublin 2, Ireland
- Department of Mechanical and Manufacturing Engineering, School of Engineering, Trinity College Dublin, Dublin 2, Ireland
- Advanced Materials and Bioengineering Research Centre (AMBER), Royal College of Surgeons in Ireland and Trinity College Dublin, Dublin 2, Ireland
- Department of Anatomy, Royal College of Surgeons in Ireland, Dublin 2, Ireland
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38
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Silicate-based bioceramic scaffolds for dual-lineage regeneration of osteochondral defect. Biomaterials 2019; 192:323-333. [DOI: 10.1016/j.biomaterials.2018.11.025] [Citation(s) in RCA: 69] [Impact Index Per Article: 11.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/09/2018] [Revised: 11/12/2018] [Accepted: 11/14/2018] [Indexed: 01/26/2023]
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Jonitz-Heincke A, Klinder A, Boy D, Salamon A, Hansmann D, Pasold J, Buettner A, Bader R. In Vitro Analysis of the Differentiation Capacity of Postmortally Isolated Human Chondrocytes Influenced by Different Growth Factors and Oxygen Levels. Cartilage 2019; 10:111-119. [PMID: 28715962 PMCID: PMC6376569 DOI: 10.1177/1947603517719318] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/16/2022] Open
Abstract
OBJECTIVE In the present in vitro study, we analyzed the chondrogenic differentiation capacity of human chondrocytes postmortally isolated from unaffected knee cartilage by the addition of transforming growth factor-β1 (TGF-β1) and/or insulin-like growth factor-1 (IGF-1) and different oxygen levels. DESIGN After 14 and 35 days, DNA concentrations and protein contents of Col1, Col2, aggrecan as well as glycosaminoglycans (GAGs) of chondrocytes cultivated as pellet cultures were analyzed. Additionally, expression rates of mesenchymal stem cell (MSC)-associated differentiation markers were assessed in monolayer cultures. RESULTS All cultivated chondrocytes were found to be CD29+/CD44+/CD105+/CD166+. Chondrocytic pellets stimulated with TGF-β1 showed enhanced synthesis rates of hyaline cartilage markers and reduced expression of the non-hyaline cartilage marker Col1 under hypoxic culture conditions. CONCLUSIONS Our results underline the substantial chondrogenic potential of human chondrocytes postmortally isolated from unaffected articular knee cartilage especially in case of TGF-β1 administration.
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Affiliation(s)
- Anika Jonitz-Heincke
- Department of Orthopaedics, Biomechanics and Implant Technology Research Laboratory, University Medical Center Rostock, Rostock, Germany,Anika Jonitz-Heincke, Department of Orthopaedics, Biomechanics and Implant Technology Research Laboratory, University Medical Center Rostock, Doberaner Strasse 142, 18057 Rostock, Germany.
| | - Annett Klinder
- Department of Orthopaedics, Biomechanics and Implant Technology Research Laboratory, University Medical Center Rostock, Rostock, Germany
| | - Diana Boy
- Institute of Forensic Medicine, University Medical Center Rostock, Rostock, Germany
| | - Achim Salamon
- Department of Cell Biology, University Medical Center Rostock, Rostock, Germany
| | - Doris Hansmann
- Department of Orthopaedics, Biomechanics and Implant Technology Research Laboratory, University Medical Center Rostock, Rostock, Germany
| | - Juliane Pasold
- Department of Orthopaedics, Biomechanics and Implant Technology Research Laboratory, University Medical Center Rostock, Rostock, Germany
| | - Andreas Buettner
- Institute of Forensic Medicine, University Medical Center Rostock, Rostock, Germany
| | - Rainer Bader
- Department of Orthopaedics, Biomechanics and Implant Technology Research Laboratory, University Medical Center Rostock, Rostock, Germany
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40
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Weiss SG, Kuchar GO, Gerber JT, Tiboni F, Storrer CLM, Casagrande TC, Giovanini AF, Scariot R. Dose of alendronate directly increases trabeculae expansivity without altering bone volume in rat femurs. World J Orthop 2018; 9:190-197. [PMID: 30364827 PMCID: PMC6198290 DOI: 10.5312/wjo.v9.i10.190] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/05/2018] [Revised: 06/20/2018] [Accepted: 06/28/2018] [Indexed: 02/06/2023] Open
Abstract
AIM To evaluate the effects of sodium alendronate on bone repair in fractures created in appendicular bones.
METHODS Wistar rats (n = 36) were allocated into three distinct groups: group C (control), group B1 (received 1 mg/kg of alendronate), and group B2 (received 3 mg/kg of alendronate). The rats underwent femoral transversal linear fracture surgery using stable internal fixation with a 2.0 mm plate and screw system. Each animal randomly received intraperitoneal applications of sodium alendronate at a dose corresponding to group B1 or B2 three times a week, while the control group received a 0.9% saline solution. Drug administration was performed until euthanasia at 45 d. The femurs were removed and each surgical piece was sent for radiographic, tomographic and microtomographic analysis. Data were submitted to descriptive and inferential statistical analysis (95% confidence interval).
RESULTS Quantitative evaluations of bone neoformation did not show differences among the groups in the radiographic (P = 0.341), microtomographic (P = 0.581) and tomographic evaluations (P = 0.171). In the qualitative microtomographic analysis, a smaller distance was observed between the internal bone trabeculae in the groups that used alendronate (P = 0.05). On the other hand, group B2 had a higher amount of bone trabeculae per unit length when compared to the other groups (P = 0.04).
CONCLUSION It is likely that the use of alendronate did not have a direct influence on the amount of bone neoformation, however it did influence the bone quality in a dose-dependent manner, ultimately affecting the distance and quantity of the trabeculae.
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Affiliation(s)
- Suyany G Weiss
- School of Health Science, Department of Dentistry, Positivo University, Curitiba, Paraná 81280-330, Brazil
| | - Gabrielle O Kuchar
- School of Health Science, Department of Dentistry, Positivo University, Curitiba, Paraná 81280-330, Brazil
| | - Jennifer T Gerber
- School of Health Science, Department of Dentistry, Positivo University, Curitiba, Paraná 81280-330, Brazil
| | - Fernanda Tiboni
- School of Health Science, Department of Dentistry, Positivo University, Curitiba, Paraná 81280-330, Brazil
| | - Carmen Lucia M Storrer
- School of Health Science, Department of Dentistry, Positivo University, Curitiba, Paraná 81280-330, Brazil
| | - Thaís C Casagrande
- School of Health Science, Department of Biotecnology, Positivo University, Curitiba, Paraná 81280-330, Brazil
| | - Allan F Giovanini
- School of Health Science, Department of Dentistry, Positivo University, Curitiba, Paraná 81280-330, Brazil
| | - Rafaela Scariot
- School of Health Science, Department of Dentistry, Positivo University, Curitiba, Paraná 81280-330, Brazil
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41
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Global analysis of tissue-differential gene expression patterns and functional regulation of rapid antler growth. MAMMAL RES 2018. [DOI: 10.1007/s13364-018-0394-9] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/20/2022]
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42
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Wang D, Jiang X, Lu A, Tu M, Huang W, Huang P. BMP14 induces tenogenic differentiation of bone marrow mesenchymal stem cells in vitro. Exp Ther Med 2018; 16:1165-1174. [PMID: 30116367 PMCID: PMC6090266 DOI: 10.3892/etm.2018.6293] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/16/2017] [Accepted: 05/24/2018] [Indexed: 01/28/2023] Open
Abstract
Bone marrow mesenchymal stem cells (BMSCs) are pluripotent cells, which have the capacity to differentiate into various types of mesenchymal cell phenotypes, including osteoblasts, chondroblasts, myoblasts and tendon fibroblasts (TFs). The molecular mechanism for tenogenic differentiation of BMSCs is still unknown. The present study investigated the effects of bone morphogenetic protein (BMP) 14 on BMSC differentiation in vitro. It was revealed that BMP14 significantly increased the expression of tendon markers (scleraxis and tenomodulin) at the mRNA and protein level, which led to the upregulation of sirtuin 1 (Sirt1) expression. The gain or loss of Sirt1 function may promote or inhibit tenogenic differentiation by deacetylating the peroxisome proliferator-activated receptor (PPAR)-γ. BMP14 also triggered the phosphorylation of c-Jun N-terminal kinase (JNK) and Smad1; overexpression of Sirt1 significantly increased the phosphorylation and knockdown of Sirt1 significantly decreased the phosphorylation. The inhibition of JNK and Smad significantly increased the acetylation of PPARγ and inhibited the expression of tenogenic differentiation markers. These results suggest that BMP14 may induce the tenogenic differentiation of BMSCs via the Sirt1-JNK/Smad1-PPARγ signaling pathway. The present study provided a cellular and molecular basis for the development of novel therapeutic strategies for tendon healing.
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Affiliation(s)
- Dan Wang
- Department of Orthopedics, Jinmen No. 2 People's Hospital, Jingmen, Hubei 448000, P.R. China.,Department of Orthopedics, Jingchu Center Hospital Affiliated to The Institute of Technology, Jingmen, Hubei 448000, P.R. China
| | - Xinhao Jiang
- Department of Orthopedics, Jinmen No. 2 People's Hospital, Jingmen, Hubei 448000, P.R. China.,Department of Orthopedics, Jingchu Center Hospital Affiliated to The Institute of Technology, Jingmen, Hubei 448000, P.R. China
| | - Aiqing Lu
- Department of Orthopedics, Jinmen No. 2 People's Hospital, Jingmen, Hubei 448000, P.R. China.,Department of Orthopedics, Jingchu Center Hospital Affiliated to The Institute of Technology, Jingmen, Hubei 448000, P.R. China
| | - Min Tu
- Department of Orthopedics, Jinmen No. 2 People's Hospital, Jingmen, Hubei 448000, P.R. China.,Department of Orthopedics, Jingchu Center Hospital Affiliated to The Institute of Technology, Jingmen, Hubei 448000, P.R. China
| | - Wei Huang
- Department of Orthopedics, Jinmen No. 2 People's Hospital, Jingmen, Hubei 448000, P.R. China.,Department of Orthopedics, Jingchu Center Hospital Affiliated to The Institute of Technology, Jingmen, Hubei 448000, P.R. China
| | - Ping Huang
- Department of Orthopedics, Jinmen No. 2 People's Hospital, Jingmen, Hubei 448000, P.R. China.,Department of Orthopedics, Jingchu Center Hospital Affiliated to The Institute of Technology, Jingmen, Hubei 448000, P.R. China
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43
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Frauchiger DA, Heeb SR, May RD, Wöltje M, Benneker LM, Gantenbein B. Differentiation of MSC and annulus fibrosus cells on genetically engineered silk fleece-membrane-composites enriched for GDF-6 or TGF-β3. J Orthop Res 2018; 36:1324-1333. [PMID: 29058815 DOI: 10.1002/jor.23778] [Citation(s) in RCA: 19] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/03/2017] [Accepted: 10/13/2017] [Indexed: 02/04/2023]
Abstract
Intervertebral disc (IVD) repair is a high-priority topic in our active and increasingly ageing society. Since a high number of people are affected by low back pain treatment options that are able to restore the biological function of the IVD are highly warranted. Here, we investigated whether the feasibility of genetically engineered (GE)-silk from Bombyx mori containing specific growth factors to precondition human bone-marrow derived mesenchymal stem cells (hMSC) or to activate differentiated human annulus fibrosus cells (hAFC) prior transplantation or for direct repair on the IVD. Here, we tested the hypothesis that GE-silk fleece can thrive human hMSC towards an IVD-like phenotype. We aimed to demonstrate a possible translational application of good manufacturing practice (GMP)-compliant GE-silk scaffolds in IVD repair and regeneration. GE-silk with growth and differentiation factor 6 (GDF-6-silk) or transforming growth factor β3 (TGF-β3, TGF-β3-silk) and untreated silk (cSilk) were investigated by DNA content, cell activity assay and glycosaminoglycan (GAG) content and their differentiation potential by qPCR analysis. We found that all silk types demonstrated a very high biocompatibility for both cell types, that is, hMSC and hAFC, as revealed by cell activity, and DNA proliferation assay. Further, analyzing qPCR of marker genes revealed a trend to differentiation toward an NP-like phenotype looking at the Aggrecan/Collagen 2 ratio which was around 10:1. Our results support the conclusion that our GE-silk scaffold treatment approach can thrive hMSC towards a more IVD-like phenotype or can maintain the phenotype of native hAFC. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:1324-1333, 2018.
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Affiliation(s)
- Daniela A Frauchiger
- Institute for Surgical Technology and Biomechanics, University of Bern, Stauffacherstrasse 78, CH-3014 Bern, Switzerland
| | - Silvan R Heeb
- Institute for Surgical Technology and Biomechanics, University of Bern, Stauffacherstrasse 78, CH-3014 Bern, Switzerland.,Department of Hematology and Central Hematology Laboratory, University of Bern, Inselspital, Bern University Hospital, CH-3010 Bern, Switzerland.,Department for BioMedical Research, University of Bern, CH-3010 Bern, Switzerland
| | - Rahel D May
- Institute for Surgical Technology and Biomechanics, University of Bern, Stauffacherstrasse 78, CH-3014 Bern, Switzerland
| | - Michael Wöltje
- Institute of Textile Machinery and High Performance Material Technology, TU Dresden, DE-01069 Dresden, Germany
| | - Lorin M Benneker
- Department of Orthopaedic Surgery and Traumatology, University of Bern, Inselspital, Bern University Hospital, CH-3010 Bern, Switzerland
| | - Benjamin Gantenbein
- Institute for Surgical Technology and Biomechanics, University of Bern, Stauffacherstrasse 78, CH-3014 Bern, Switzerland
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44
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Wuest SL, Caliò M, Wernas T, Tanner S, Giger-Lange C, Wyss F, Ille F, Gantenbein B, Egli M. Influence of Mechanical Unloading on Articular Chondrocyte Dedifferentiation. Int J Mol Sci 2018; 19:ijms19051289. [PMID: 29693628 PMCID: PMC5983850 DOI: 10.3390/ijms19051289] [Citation(s) in RCA: 29] [Impact Index Per Article: 4.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/20/2018] [Revised: 04/13/2018] [Accepted: 04/18/2018] [Indexed: 01/10/2023] Open
Abstract
Due to the limited self-repair capacity of articular cartilage, the surgical restoration of defective cartilage remains a major clinical challenge. The cell-based approach, which is known as autologous chondrocyte transplantation (ACT), has limited success, presumably because the chondrocytes acquire a fibroblast-like phenotype in monolayer culture. This unwanted dedifferentiation process is typically addressed by using three-dimensional scaffolds, pellet culture, and/or the application of exogenous factors. Alternative mechanical unloading approaches are suggested to be beneficial in preserving the chondrocyte phenotype. In this study, we examined if the random positioning machine (RPM) could be used to expand chondrocytes in vitro such that they maintain their phenotype. Bovine chondrocytes were exposed to (a) eight days in static monolayer culture; (b) two days in static monolayer culture, followed by six days of RPM exposure; and, (c) eight days of RPM exposure. Furthermore, the experiment was also conducted with the application of 20 mM gadolinium, which is a nonspecific ion-channel blocker. The results revealed that the chondrocyte phenotype is preserved when chondrocytes go into suspension and aggregate to cell clusters. Exposure to RPM rotation alone does not preserve the chondrocyte phenotype. Interestingly, the gene expression (mRNA) of the mechanosensitive ion channel TRPV4 decreased with progressing dedifferentiation. In contrast, the gene expression (mRNA) of the mechanosensitive ion channel TRPC1 was reduced around fivefold to 10-fold in all of the conditions. The application of gadolinium had only a minor influence on the results. This and previous studies suggest that the chondrocyte phenotype is preserved if cells maintain a round morphology and that the ion channel TRPV4 could play a key role in the dedifferentiation process.
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Affiliation(s)
- Simon L Wuest
- Lucerne University of Applied Sciences and Arts, School of Engineering and Architecture, Institute of Medical Engineering, Space Biology Group, CH-6052 Hergiswil, Switzerland.
- University of Bern, Institute for Surgical Technology and Biomechanics, Tissue and Organ Mechanobiology, CH-3014 Bern, Switzerland.
| | - Martina Caliò
- Lucerne University of Applied Sciences and Arts, School of Engineering and Architecture, Institute of Medical Engineering, Space Biology Group, CH-6052 Hergiswil, Switzerland.
- University of Bern, Institute for Surgical Technology and Biomechanics, Tissue and Organ Mechanobiology, CH-3014 Bern, Switzerland.
| | - Timon Wernas
- Lucerne University of Applied Sciences and Arts, School of Engineering and Architecture, Institute of Medical Engineering, Space Biology Group, CH-6052 Hergiswil, Switzerland.
| | - Samuel Tanner
- Lucerne University of Applied Sciences and Arts, School of Engineering and Architecture, Institute of Medical Engineering, Space Biology Group, CH-6052 Hergiswil, Switzerland.
| | - Christina Giger-Lange
- Lucerne University of Applied Sciences and Arts, School of Engineering and Architecture, Institute of Medical Engineering, Space Biology Group, CH-6052 Hergiswil, Switzerland.
| | - Fabienne Wyss
- Lucerne University of Applied Sciences and Arts, School of Engineering and Architecture, Institute of Medical Engineering, Space Biology Group, CH-6052 Hergiswil, Switzerland.
| | - Fabian Ille
- Lucerne University of Applied Sciences and Arts, School of Engineering and Architecture, Institute of Medical Engineering, Space Biology Group, CH-6052 Hergiswil, Switzerland.
| | - Benjamin Gantenbein
- University of Bern, Institute for Surgical Technology and Biomechanics, Tissue and Organ Mechanobiology, CH-3014 Bern, Switzerland.
| | - Marcel Egli
- Lucerne University of Applied Sciences and Arts, School of Engineering and Architecture, Institute of Medical Engineering, Space Biology Group, CH-6052 Hergiswil, Switzerland.
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Hassan G, Bahjat M, Kasem I, Soukkarieh C, Aljamali M. Platelet lysate induces chondrogenic differentiation of umbilical cord-derived mesenchymal stem cells. Cell Mol Biol Lett 2018; 23:11. [PMID: 29568314 PMCID: PMC5859745 DOI: 10.1186/s11658-018-0080-6] [Citation(s) in RCA: 15] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/02/2017] [Accepted: 03/12/2018] [Indexed: 12/17/2022] Open
Abstract
Purpose Articular cartilage has a poor capacity for self-repair, and thus still presents a major challenge in orthopedics. Mesenchymal stem cells (MSCs) are multipotent stem cells with the potential to differentiate into chondrocytes in the presence of transforming growth factor beta (TGF-β). Platelet lysate (PL) contains a relatively large number of growth factors, including TGF-β, and has been shown to ameliorate cartilage repair. Here, we investigated the ability of PL to direct chondrogenic differentiation of MSCs along with other standard differentiation components in a pellet culture system. Methods We isolated and expanded MSCs from human umbilical cords using a PL-supplemented medium and characterized the cells based on immunophenotype and potential for differentiation to adipocytes and osteocytes. We further cultured MSCs as pellets in a chondrogenic-differentiation medium supplemented with PL. After 21 days, the pellets were processed for histological analysis and stained with alician blue and acridine orange. The expression of SOX9 was investigated using RT-PCR. Results MSCs maintained their stemness characteristics in the PL-supplemented medium. However, the distribution of cells in the pellets cultured in the PL-supplemented chondrogenic differentiation medium had a greater similarity to cartilage tissue-derived chondrocytes than to the negative control. The intense alician blue staining indicated an increased production of mucopolysaccharides in the differentiated pellets, which also showed elevated expression of SOX9. Conclusions Our data suggest that MSCs could be differentiated to chondrocytes in the presence of PL and absence of exogenous TGF-β. Further research needs to be conducted to understand the exact role and potential of PL in chondrogenic differentiation and chondrocyte regeneration.
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Affiliation(s)
- Ghmkin Hassan
- 1Faculty of Pharmacy, Damascus University, Damascus, Syria
| | | | - Issam Kasem
- 2Faculty of Sciences, Damascus University, Damascus, Syria.,National Commission for Biotechnology (NCBT), Damascus, Syria
| | - Chadi Soukkarieh
- 2Faculty of Sciences, Damascus University, Damascus, Syria.,National Commission for Biotechnology (NCBT), Damascus, Syria
| | - Majd Aljamali
- 1Faculty of Pharmacy, Damascus University, Damascus, Syria.,2Faculty of Sciences, Damascus University, Damascus, Syria.,National Commission for Biotechnology (NCBT), Damascus, Syria
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Wongin S, Waikakul S, Chotiyarnwong P, Siriwatwechakul W, Kino-Oka M, Kim MH, Viravaidya-Pasuwat K. Maintenance of human chondrogenic phenotype on a dendrimer-immobilized surface for an application of cell sheet engineering. BMC Biotechnol 2018. [PMID: 29540167 PMCID: PMC5853058 DOI: 10.1186/s12896-018-0426-1] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/10/2022] Open
Abstract
BACKGROUND Dedifferentiation of chondrocytes during cell expansion is one of the barriers in tissue construction for cartilage repair. To understand chondrocyte behavior and improve cell expansion in monolayer culture, this study investigated the effects of morphological changes and cellular aggregation on the maintenance of chondrogenic capacity by observing the expression patterns of chondrogenic (collagen type II and aggrecan) and dedifferentiation (collagen type I) markers. Primary human chondrocytes were cultured on either a polystyrene surface (PS) or a polyamidoamine dendrimer surface with a fifth-generation (G5) dendron structure to create a one-step process of cell expansion and the maintenance of chondrogenic activities prior to the construction of cell sheets. RESULTS During the first two passages (P0 - P2), the relative mRNA level of collagen type II decreased in all cultures, while that of collagen type I increased. Remarkably, the level of collagen type II was higher and aggrecan was retained in the chondrocytes, forming cell aggregates and showing some round-shaped cells with less production of stress fibers on the G5 surface compared to fibroblast-like chondrocytes with abundant stress fibers on the PS surface. The numbers of P2 chondrocytes on the G5 and PS surfaces were nearly the same and sufficient for construction of chondrocyte sheets using a temperature-responsive plate. Without a supporting material during cell sheet manipulation, chondrocyte sheets spontaneously detached and exhibited a honeycomb-like structure of stress fibers. Unlike the chondrocyte sheets constructed from cells on the PS surface, the chondrocyte sheets from cells on the G5 surface had higher chondrogenic activities, as evidenced by the high expression of chondrogenic markers and the low expression of dedifferentiation markers. CONCLUSIONS The one-step process of cell expansion and maintenance of chondrogenic activity could be obtained using the G5 surface. Human chondrocyte sheets were successfully constructed with high chondrogenic activity. These findings may lead to an alternative cultivation technique for human chondrocytes that offers high clinical potential in autologous chondrocyte implantation.
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Affiliation(s)
- Sopita Wongin
- Biological Engineering Program, Faculty of Engineering, King Mongkut's University of Technology Thonburi, Bangkok, 10140, Thailand
| | - Saranatra Waikakul
- Department of Orthopaedic Surgery, Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok, 10700, Thailand
| | - Pojchong Chotiyarnwong
- Department of Orthopaedic Surgery, Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok, 10700, Thailand
| | - Wanwipa Siriwatwechakul
- School of Bio-Chemical Engineering and Technology, Sirindhorn International Institute of Technology, Thammasat University, Pathum Thani, 12121, Thailand
| | - Masahiro Kino-Oka
- Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka, 565-0871, Japan
| | - Mee-Hae Kim
- Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka, 565-0871, Japan
| | - Kwanchanok Viravaidya-Pasuwat
- Biological Engineering Program, Faculty of Engineering, King Mongkut's University of Technology Thonburi, Bangkok, 10140, Thailand. .,Department of Chemical Engineering, Faculty of Engineering, King Mongkut's University of Technology Thonburi, Bangkok, 10140, Thailand.
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Liu C, Sun J, Zhang H, Li L. TGF β1 gene polymorphisms correlate with the susceptibility of osteoarthritis. INTERNATIONAL JOURNAL OF CLINICAL AND EXPERIMENTAL PATHOLOGY 2017; 10:8780-8785. [PMID: 31966743 PMCID: PMC6965479] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Subscribe] [Scholar Register] [Received: 01/06/2016] [Accepted: 03/20/2016] [Indexed: 06/10/2023]
Abstract
PURPOSE We investigated the potential role of transforming growth factor beta 1 (TGF β1) gene polymorphisms (rs1800470 and rs1800469) in the occurrence of osteoarthritis (OA). METHODS Genotypes of TGF β1 gene polymorphisms (rs1800470 and 1800469) were genotyped by TaqMan method in 111 OA patients and 129 healthy controls. The representativeness of case and control was inspected by Hardy-Weinberg equilibrium (HWE). Genotype and allele distribution differences between case and control groups were calculated by Chi-square test. Odds ratios (ORs) and their corresponding 95% confidence intervals (95% CIs) were utilized to emerge the relative risk of OA. RESULTS Genotype distributions of the two TGF β1 gene polymorphisms were according to HWE examination. TT genotype of rs1800470 was significantly associated with the occurrence of OA (P=0.046, OR=2.093, 95% CI=1.009-4.340). For rs1800469, both TT genotype and T allele had significant association with the susceptibility of OA (P=0.000, OR=3.650, 95% CI=1.759-7.575; P=0.000, OR=1.957, 95% CI=1.360-2.817). CONCLUSION TT genotype of rs1800470, TT genotype and T allele of rs1800469 were increased the risk of OA. We conjectured that the polymorphisms of TGF β1 gene might increase the individual susceptible of OA.
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Affiliation(s)
- Chang Liu
- Department of Orthopedics, Cangzhou Central Hospital, Hebei Medical UniversityCangzhou, Hebei, China
| | - Jian Sun
- Department of Orthopedics, Shanghai Tenth People’s HospitalShanghai, China
| | - Haisen Zhang
- Department of Orthopedics, Cangzhou Central Hospital, Hebei Medical UniversityCangzhou, Hebei, China
| | - Longjie Li
- Department of Orthopedics, Cangzhou Central Hospital, Hebei Medical UniversityCangzhou, Hebei, China
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Li Z, Fei H, Wang Z, Zhu T. Low‑dose halofuginone inhibits the synthesis of type I collagen without influencing type II collagen in the extracellular matrix of chondrocytes. Mol Med Rep 2017; 16:3290-3298. [PMID: 28713920 PMCID: PMC5547978 DOI: 10.3892/mmr.2017.7009] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/17/2016] [Accepted: 05/24/2017] [Indexed: 12/28/2022] Open
Abstract
Full‑thickness and large area defects of articular cartilage are unable to completely repair themselves and require surgical intervention, including microfracture, autologous or allogeneic osteochondral grafts, and autologous chondrocyte implantation. A large proportion of regenerative cartilage exists as fibrocartilage, which is unable to withstand impacts in the same way as native hyaline cartilage, owing to excess synthesis of type I collagen in the matrix. The present study demonstrated that low‑dose halofuginone (HF), a plant alkaloid isolated from Dichroa febrifuga, may inhibit the synthesis of type I collagen without influencing type II collagen in the extracellular matrix of chondrocytes. In addition, HF was revealed to inhibit the phosphorylation of mothers against decapentaplegic homolog (Smad)2/3 and promoted Smad7 expression, as well as decrease the synthesis of type I collagen synthesis. Results from the present study indicated that HF treatment suppressed the synthesis of type I collagen by inhibiting the transforming growth factor‑β signaling pathway in chondrocytes. These results may provide an alternative solution to the problems associated with fibrocartilage, and convert fibrocartilage into hyaline cartilage at the mid‑early stages of cartilage regeneration. HF may additionally be used to improve monolayer expansion or 3D cultures of seed cells for the tissue engineering of cartilage.
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Affiliation(s)
- Zeng Li
- Department of Respiratory, The General Hospital of Shenyang Military Region, Shenyang, Liaoning 110015, P.R. China
| | - Hao Fei
- Department of Orthopedics, The First Affiliated Hospital of Nanjing Medical University, Nanjing, Jiangsu 210029, P.R. China
| | - Zhen Wang
- Department of Orthopedics, The First Affiliated Hospital of Nanjing Medical University, Nanjing, Jiangsu 210029, P.R. China
| | - Tianyi Zhu
- Department of Respiratory, The General Hospital of Shenyang Military Region, Shenyang, Liaoning 110015, P.R. China
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Combined effects of oscillating hydrostatic pressure, perfusion and encapsulation in a novel bioreactor for enhancing extracellular matrix synthesis by bovine chondrocytes. Cell Tissue Res 2017; 370:179-193. [PMID: 28687928 DOI: 10.1007/s00441-017-2651-7] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/18/2016] [Accepted: 05/16/2017] [Indexed: 01/10/2023]
Abstract
The influence of combined shear stress and oscillating hydrostatic pressure (OHP), two forms of physical forces experienced by articular cartilage (AC) in vivo, on chondrogenesis, is investigated in a unique bioreactor system. Our system introduces a single reaction chamber design that does not require transfer of constructs after seeding to a second chamber for applying the mechanical forces, and, as such, biochemical and mechanical stimuli can be applied in combination. The biochemical and mechanical properties of bovine articular chondrocytes encapsulated in agarose scaffolds cultured in our bioreactors for 21 days are compared to cells statically cultured in agarose scaffolds in addition to static micromass and pellet cultures. Our findings indicate that glycosaminoglycan and collagen secretions were enhanced by at least 1.6-fold with scaffold encapsulation, 5.9-fold when adding 0.02 Pa of shear stress and 7.6-fold with simultaneous addition of 4 MPa of OHP when compared to micromass samples. Furthermore, shear stress and OHP have chondroprotective effects as evidenced by lower mRNA expression of β1 integrin and collagen X to non-detectable levels and an absence of collagen I upregulation as observed in micromass controls. These collective results are further supported by better mechanical properties as indicated by 1.6-19.8-fold increases in elastic moduli measured by atomic force microscopy.
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Witt A, Salamon A, Boy D, Hansmann D, Büttner A, Wree A, Bader R, Jonitz-Heincke A. Gene expression analysis of growth factor receptors in human chondrocytes in monolayer and 3D pellet cultures. Int J Mol Med 2017; 40:10-20. [PMID: 28534942 PMCID: PMC5466384 DOI: 10.3892/ijmm.2017.2994] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/02/2016] [Accepted: 04/10/2017] [Indexed: 11/06/2022] Open
Abstract
The main goal of cartilage repair is to create functional tissue by enhancing the in vitro conditions to more physiological in vivo conditions. Chondrogenic growth factors play an important role in influencing cartilage homeostasis. Insulin‑like growth factor (IGF)‑1 and transforming growth factor (TGF)‑β1 affect the expression of collagen type II (Col2) and glycosaminoglycans (GAGs) and, therefore, the targeted use of growth factors could make chondrogenic redifferentiation more efficient. In the present study, human chondrocytes were postmortally isolated from healthy articular cartilage and cultivated as monolayer or 3D pellet cultures either under normoxia or hypoxia and stimulated with IGF‑1 and/or TGF‑β1 to compare the impact of the different growth factors. The mRNA levels of the specific receptors (IGF1R, TGFBR1, TGFBR2) were analyzed at different time points. Moreover, gene expression rates of collagen type 1 and 2 in pellet cultures were observed over a period of 5 weeks. Additionally, hyaline‑like Col2 protein and sulphated GAG (sGAG) levels were quantified. Stimulation with IGF‑1 resulted in an enhanced expression of IGF1R and TGFBR2 whereas TGF‑β1 stimulated TGFBR1 in the monolayer and pellet cultures. In monolayer, the differences reached levels of significance. This effect was more pronounced under hypoxic culture conditions. In pellet cultures, increased amounts of Col2 protein and sGAGs after incubation with TGF‑β1 and/or IGF‑1 were validated. In summary, constructing a gene expression profile regarding mRNA levels of specific growth factor receptors in monolayer cultures could be helpful for a targeted application of growth factors in cartilage tissue engineering.
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Affiliation(s)
- Anika Witt
- Department of Orthopaedics, Biomechanics and Implant Technology Research Laboratory, University Medical Centre Rostock, D‑18057 Rostock, Germany
| | - Achim Salamon
- Department of Cell Biology, University Medical Centre Rostock, D‑18057 Rostock, Germany
| | - Diana Boy
- Institute of Forensic Medicine, University Medical Centre Rostock, D‑18057 Rostock, Germany
| | - Doris Hansmann
- Department of Orthopaedics, Biomechanics and Implant Technology Research Laboratory, University Medical Centre Rostock, D‑18057 Rostock, Germany
| | - Andreas Büttner
- Institute of Forensic Medicine, University Medical Centre Rostock, D‑18057 Rostock, Germany
| | - Andreas Wree
- Institute of Anatomy, University Medical Centre Rostock, D‑18057 Rostock, Germany
| | - Rainer Bader
- Department of Orthopaedics, Biomechanics and Implant Technology Research Laboratory, University Medical Centre Rostock, D‑18057 Rostock, Germany
| | - Anika Jonitz-Heincke
- Department of Orthopaedics, Biomechanics and Implant Technology Research Laboratory, University Medical Centre Rostock, D‑18057 Rostock, Germany
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