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Kasmi N, Pieruccioni L, Pitot E, Fourquaux I, Wodrinski A, Gibot L, Fitremann J. The potential of carbohydrate supramolecular hydrogels for long-term 3D culture of primary fibroblasts. J Mater Chem B 2025; 13:4386-4405. [PMID: 40084972 DOI: 10.1039/d4tb02658f] [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: 03/16/2025]
Abstract
N-Alkyl-galactonamides, which are small synthetic molecules derived from galactose, self-assemble to give fibrous hydrogels. These molecules are biocompatible and, in a previous study, the cell culture of human neural stem cells was performed for 7 days on a gel of N-heptyl-D-galactonamide. With the objective of broadening the scope of these molecules as scaffolds for cell culture, in the present study, the culture of primary human dermal fibroblasts has been carried out on N-nonyl-D-galactonamide hydrogels. These supramolecular fibrillar hydrogels have a sufficient mechanical strength to withstand cell culture (≈50 kPa) and they are resistant enough on the long term to carry out the cell culture over at least 3 weeks. In contrast to N-heptyl-D-galactonamide, N-nonyl-D-galactonamide is insoluble in the culture medium. It avoids its dissolution at each renewal of the culture medium. The molecule is only slowly eliminated by other mechanisms (1/3rd in 3 weeks), which did not impair the cell culture on a monthly scale. The hydrogel's microstructure and how the cells organize on this scaffold have been studied using electron and two-photon microscopies. The gel is made of a quite homogeneous network with a width of ≈180 nm and hundreds of micrometer long fibers, except at the surface where a dense mat of heterogeneous fibers is formed. We focused on methods able to colocalize the cells and the gel fibers. Many cell clusters have elongated and multidirectionnal shapes, guided by the fibers. Chains of single cells are also found following the fibers from one cluster to another. N-Nonyl-D-galactonamide fibers, which have the advantage of not being autofluorescent, do not mask the fluorescence of cells. But interestingly, they give a strong second harmonic generation (SHG) signal, due to their well-organized lamellar structure. We also made a special effort to visualize the penetration of cells within the depth of the hydrogels, in 3D, notably by sectioning the hydrogels, despite their softness. It was found that most of the cells stayed at the surface, but several cells grew within the supramolecular fiber network between 50 and 100 μm depth.
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Affiliation(s)
- Nadia Kasmi
- Laboratoire Softmat, Université de Toulouse, CNRS UMR 5623, Université Toulouse III - Paul Sabatier, Toulouse, France.
| | - Laetitia Pieruccioni
- RESTORE Research Center, Université de Toulouse, INSERM 1301, CNRS 5070, EFS, ENVT, Toulouse, France
| | - Eve Pitot
- Cytometry and Imaging Core facility, Institute of Pharmacology and Structural Biology (IPBS), University of Toulouse, CNRS, Toulouse, France
| | - Isabelle Fourquaux
- Centre de Microscopie Electronique Appliquée à la Biologie (CMEAB), Faculté de Médecine Rangueil, Université de Toulouse III - Paul Sabatier, Toulouse, France
| | - Alexandre Wodrinski
- Laboratoire Softmat, Université de Toulouse, CNRS UMR 5623, Université Toulouse III - Paul Sabatier, Toulouse, France.
| | - Laure Gibot
- Laboratoire Softmat, Université de Toulouse, CNRS UMR 5623, Université Toulouse III - Paul Sabatier, Toulouse, France.
| | - Juliette Fitremann
- Laboratoire Softmat, Université de Toulouse, CNRS UMR 5623, Université Toulouse III - Paul Sabatier, Toulouse, France.
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Donderwinkel I, Tuan RS, Cameron NR, Frith JE. A systematic investigation of the effects of TGF-β3 and mechanical stimulation on tenogenic differentiation of mesenchymal stromal cells in a poly(ethylene glycol)/gelatin-based hydrogel. J Orthop Translat 2023; 43:1-13. [PMID: 37929240 PMCID: PMC10622696 DOI: 10.1016/j.jot.2023.09.006] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/07/2023] [Revised: 08/31/2023] [Accepted: 09/20/2023] [Indexed: 11/07/2023] Open
Abstract
Background High post-surgical failure rates following tendon injury generate high medical costs and poor patient recovery. Cell-based tendon tissue engineering has the potential to produce fully functional replacement tissue and provide new strategies to restore tendon function and healing. In this endeavour, the application of mesenchymal stromal cells (MSCs) encapsulated in biomaterial scaffolds has shown great promise. However, a consensus on optimal promotion of tenogenic differentiation of MSCs has yet to be reached, although growth factors and mechanical cues are generally acknowledged as important factors. Methods In this study, we prepared a hydrogel cell culture system consisting of methacrylated poly(d,l-lactic acid-ethylene glycol-d,l-lactic acid) (P(LA-EG-LA)) and gelatin methacrylate (GelMA) to encapsulate human bone marrow-derived MSCs (hBMSCs). We further systematically investigated the influence of static and intermittent cyclic uniaxial strain mechanical stimulation, in combination with transforming growth factor-β3 (TGF-β3) supplementation, on tenogenic differentiation of hBMSCs. Results Increased TGF-β3 concentration upregulated the tenogenic genes Scleraxis (SCX) and collagen type I (COL1A1) but showed no effects on tenascin-c (TNC) and collagen type III (COL3A1) expression. Mechanical stimulation had no observable effect on gene expression, but intermittent cyclic uniaxial strain stimulation improved matrix deposition. Together, these data provide new insights into how TGF-β3 and mechanical stimulation regulate MSC tenogenesis, with TGF-β3 promoting the expression of key tenogenic genes whilst mechanical stimulation aided matrix deposition in the engineered tissue. Furthermore, intermittent cyclic uniaxial strain at 3% elongation and 0.33 Hz for 1 h/day showed improved matrix effects compared to static strain. Conclusion Together, the most promising result for tenogenic differentiation of hBMSCs was identified as treatment with 5 ng/ml TGF-β3 under intermittent cyclic uniaxial strain (3% strain; 0.33 Hz; 1 h/day). This knowledge is of importance for the development of an improved protocol for tenogenic differentiation of MSCs and thereby for tendon tissue engineering. The translational potential of this article Tissue-engineered strategies for tendon repair require a consensus on the differentiation of mesenchymal stromal cells to tenocytes, which is currently lacking. This article provides a systematic investigation of two main tenogenic differentiation conditions to further development of a tenogenic differentiation protocol.
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Affiliation(s)
- Ilze Donderwinkel
- Department of Materials Science and Engineering, Monash University, Clayton, VIC, 3800, Australia
| | - Rocky S. Tuan
- Institute for Tissue Engineering and Regenerative Medicine, The Chinese University of Hong Kong, Hong Kong SAR, China
| | - Neil R. Cameron
- Department of Materials Science and Engineering, Monash University, Clayton, VIC, 3800, Australia
- School of Engineering, University of Warwick, Coventry, CV4 7AL, United Kingdom
- Australian Research Council Training Centre for Cell and Tissue Engineering Technologies, Monash University, Clayton, VIC, 3800, Australia
| | - Jessica E. Frith
- Department of Materials Science and Engineering, Monash University, Clayton, VIC, 3800, Australia
- Australian Research Council Training Centre for Cell and Tissue Engineering Technologies, Monash University, Clayton, VIC, 3800, Australia
- Australian Regenerative Medicine Institute, Monash University, Clayton, VIC, 3800, Australia
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Machour M, Hen N, Goldfracht I, Safina D, Davidovich‐Pinhas M, Bianco‐Peled H, Levenberg S. Print-and-Grow within a Novel Support Material for 3D Bioprinting and Post-Printing Tissue Growth. ADVANCED SCIENCE (WEINHEIM, BADEN-WURTTEMBERG, GERMANY) 2022; 9:e2200882. [PMID: 36261395 PMCID: PMC9731703 DOI: 10.1002/advs.202200882] [Citation(s) in RCA: 9] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Received: 03/15/2022] [Revised: 08/20/2022] [Indexed: 06/16/2023]
Abstract
3D bioprinting holds great promise for tissue engineering, with extrusion bioprinting in suspended hydrogels becoming the leading bioprinting technique in recent years. In this method, living cells are incorporated within bioinks, extruded layer by layer into a granular support material followed by gelation of the bioink through diverse cross-linking mechanisms. This approach offers high fidelity and precise fabrication of complex structures mimicking living tissue properties. However, the transition of cell mass mixed with the bioink into functional native-like tissue requires post-printing cultivation in vitro. An often-overlooked drawback of 3D bioprinting is the nonuniform shrinkage and deformation of printed constructs during the post-printing tissue maturation period, leading to highly variable engineered constructs with unpredictable size and shape. This limitation poses a challenge for the technology to meet applicative requirements. A novel technology of "print-and-grow," involving 3D bioprinting and subsequent cultivation in κ-Carrageenan-based microgels (CarGrow) for days is presented. CarGrow enhances the long-term structural stability of the printed objects by providing mechanical support. Moreover, this technology provides a possibility for live imaging to monitor tissue maturation. The "print-and-grow" method demonstrates accurate bioprinting with high tissue viability and functionality while preserving the construct's shape and size.
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Affiliation(s)
- Majd Machour
- Department of Biomedical EngineeringTechnion – Israel Institute of TechnologyHaifa32000Israel
| | - Noy Hen
- Department of Chemical EngineeringTechnion – Israel Institute of TechnologyHaifa32000Israel
- The Norman Seiden Multidisciplinary Program for Nanoscience and NanotechnologyTechnion – Israel Institute of TechnologyHaifa32000Israel
| | - Idit Goldfracht
- Department of Biomedical EngineeringTechnion – Israel Institute of TechnologyHaifa32000Israel
| | - Dina Safina
- Department of Biomedical EngineeringTechnion – Israel Institute of TechnologyHaifa32000Israel
| | - Maya Davidovich‐Pinhas
- Department of Biotechnology and Food EngineeringTechnion – Israel Institute of TechnologyHaifa32000Israel
| | - Havazelet Bianco‐Peled
- Department of Chemical EngineeringTechnion – Israel Institute of TechnologyHaifa32000Israel
| | - Shulamit Levenberg
- Department of Biomedical EngineeringTechnion – Israel Institute of TechnologyHaifa32000Israel
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Liu Q, Dai W, Gao Y, Dong L, Jia H, Li S, Guo L, Fan Y, Zhang X. The synergistic regulation of chondrogenesis by collagen-based hydrogels and cell co-culture. Acta Biomater 2022; 154:194-211. [PMID: 36309191 DOI: 10.1016/j.actbio.2022.10.017] [Citation(s) in RCA: 11] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/04/2022] [Revised: 09/19/2022] [Accepted: 10/07/2022] [Indexed: 01/24/2023]
Abstract
The suitable seeding cells and scaffolds are very important for tissue engineering to create functional cartilage. Although the physicochemical properties of scaffold and co-culture system of mesenchymal stem cells (MSCs) and chondrocytes could affect functional properties of engineered cartilage tissues respectively, the combined effects of them on chondrogenesis is currently unknown. Herein, methacrylated collagen (CMA30 and CMA80) hydrogels with different degradation rate and stiffness were prepared. The MSCs and chondrocytes were co-cultured or monocultured in collagen, CMA30 and CMA80 hydrogels in vitro or in vivo. The results demonstrated that cell spreading and proliferation was regulated by degradation rate and stiffness of hydrogels. Compared to single MSCs culture, co-culture cells in all collagen-based hydrogels significantly improved chondrogenesis. CMA30 hydrogel with moderate degradation rate and low storage modulus was the most effective for co-culture system to promote chondrogenesis compared to Col and CMA80 hydrogel in vitro culture, while there was no obvious difference between CMA30 and CMA80 hydrogel in vivo. Furthermore, the intercellular substance exchange was very important for co-culture system to maintain the positive effect on chondrogenesis. Overall, the current study highlights the synergistic effects of the physicochemical properties of collagen-based hydrogel and co-culture system on cartilage formation. STATEMENT OF SIGNIFICANCE: Scaffolds and cells play a key role in cartilage tissue engineering. The combined effects of physicochemical properties of collagen hydrogels and co-culture system (MSCs and chondrocytes) on chondrogenesis is unknown. In contrast to the studies that investigated the effect of single factor (scaffolds or cells) on cartilage formation, this manuscript explored the synergistic regulation of both scaffold properties and biological factors on chondrogenesis, and provided a promising strategy for cartilage tissue engineering.
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Affiliation(s)
- Qingli Liu
- National Engineering Research Center for Biomaterials, Sichuan University, 29 Wangjiang Road, Chengdu, Sichuan 610064, PR China
| | - Wenling Dai
- National Engineering Research Center for Biomaterials, Sichuan University, 29 Wangjiang Road, Chengdu, Sichuan 610064, PR China
| | - Yongli Gao
- National Engineering Research Center for Biomaterials, Sichuan University, 29 Wangjiang Road, Chengdu, Sichuan 610064, PR China
| | - Longpeng Dong
- National Engineering Research Center for Biomaterials, Sichuan University, 29 Wangjiang Road, Chengdu, Sichuan 610064, PR China
| | - Hengxing Jia
- National Engineering Research Center for Biomaterials, Sichuan University, 29 Wangjiang Road, Chengdu, Sichuan 610064, PR China
| | - Shikui Li
- National Engineering Research Center for Biomaterials, Sichuan University, 29 Wangjiang Road, Chengdu, Sichuan 610064, PR China
| | - Likun Guo
- National Engineering Research Center for Biomaterials, Sichuan University, 29 Wangjiang Road, Chengdu, Sichuan 610064, PR China.
| | - Yujiang Fan
- National Engineering Research Center for Biomaterials, Sichuan University, 29 Wangjiang Road, Chengdu, Sichuan 610064, PR China
| | - Xingdong Zhang
- National Engineering Research Center for Biomaterials, Sichuan University, 29 Wangjiang Road, Chengdu, Sichuan 610064, PR China
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Zhang Y, Zhou K, Feng Z, Feng K, Ji Y, Li C, Huang Z. Viscoelastic properties' characterization of corneal stromal models using non-contact surface acoustic wave optical coherence elastography (SAW-OCE). JOURNAL OF BIOPHOTONICS 2022; 15:e202100253. [PMID: 34713598 DOI: 10.1002/jbio.202100253] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 08/16/2021] [Revised: 10/20/2021] [Accepted: 10/27/2021] [Indexed: 06/13/2023]
Abstract
Viscoelastic characterization of the tissue-engineered corneal stromal model is important for our understanding of the cell behaviors in the pathophysiologic altered corneal extracellular matrix (ECM). The effects of the interactions between stromal cells and different ECM characteristics on the viscoelastic properties during an 11-day culture period were explored. Collagen-based hydrogels seeded with keratocytes were used to replicate human corneal stroma. Keratocytes were seeded at 8 × 103 cells per hydrogel and with collagen concentrations of 3, 5 and 7 mg/ml. Air-pulse-based surface acoustic wave optical coherence elastography (SAW-OCE) was employed to monitor the changes in the hydrogels' dimensions and viscoelasticity over the culture period. The results showed the elastic modulus increased by 111%, 56% and 6%, and viscosity increased by 357%, 210% and 25% in the 3, 5 and 7 mg/ml hydrogels, respectively. To explain the SAW-OCE results, scanning electron microscope was also performed. The results confirmed the increase in elastic modulus and viscosity of the hydrogels, respectively, arose from increased fiber density and force-dependent unbinding of bonds between collagen fibers. This study reveals the influence of cell-matrix interactions on the viscoelastic properties of corneal stromal models and can provide quantitative guidance for mechanobiological investigations which require collagen ECM with tuneable viscoelastic properties.
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Affiliation(s)
- Yilong Zhang
- School of Science and Engineering, University of Dundee, Dundee, UK
| | - Kanheng Zhou
- School of Science and Engineering, University of Dundee, Dundee, UK
| | - Zhengshuyi Feng
- School of Science and Engineering, University of Dundee, Dundee, UK
| | - Kairui Feng
- School of Science and Engineering, University of Dundee, Dundee, UK
| | - Yubo Ji
- School of Science and Engineering, University of Dundee, Dundee, UK
| | - Chunhui Li
- School of Science and Engineering, University of Dundee, Dundee, UK
| | - Zhihong Huang
- School of Science and Engineering, University of Dundee, Dundee, UK
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Yu Z, Liu KK. Soft Polymer-Based Technique for Cellular Force Sensing. Polymers (Basel) 2021; 13:2672. [PMID: 34451211 PMCID: PMC8399510 DOI: 10.3390/polym13162672] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/21/2021] [Revised: 08/05/2021] [Accepted: 08/06/2021] [Indexed: 01/03/2023] Open
Abstract
Soft polymers have emerged as a vital type of material adopted in biomedical engineering to perform various biomechanical characterisations such as sensing cellular forces. Distinct advantages of these materials used in cellular force sensing include maintaining normal functions of cells, resembling in vivo mechanical characteristics, and adapting to the customised functionality demanded in individual applications. A wide range of techniques has been developed with various designs and fabrication processes for the desired soft polymeric structures, as well as measurement methodologies in sensing cellular forces. This review highlights the merits and demerits of these soft polymer-based techniques for measuring cellular contraction force with emphasis on their quantitativeness and cell-friendliness. Moreover, how the viscoelastic properties of soft polymers influence the force measurement is addressed. More importantly, the future trends and advancements of soft polymer-based techniques, such as new designs and fabrication processes for cellular force sensing, are also addressed in this review.
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Affiliation(s)
| | - Kuo-Kang Liu
- School of Engineering, University of Warwick, Coventry CV4 7AL, UK;
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7
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Significance of Crosslinking Approaches in the Development of Next Generation Hydrogels for Corneal Tissue Engineering. Pharmaceutics 2021; 13:pharmaceutics13030319. [PMID: 33671011 PMCID: PMC7997321 DOI: 10.3390/pharmaceutics13030319] [Citation(s) in RCA: 21] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/21/2020] [Revised: 02/19/2021] [Accepted: 02/24/2021] [Indexed: 02/07/2023] Open
Abstract
Medical conditions such as trachoma, keratoconus and Fuchs endothelial dystrophy can damage the cornea, leading to visual deterioration and blindness and necessitating a cornea transplant. Due to the shortage of donor corneas, hydrogels have been investigated as potential corneal replacements. A key factor that influences the physical and biochemical properties of these hydrogels is how they are crosslinked. In this paper, an overview is provided of different crosslinking techniques and crosslinking chemical additives that have been applied to hydrogels for the purposes of corneal tissue engineering, drug delivery or corneal repair. Factors that influence the success of a crosslinker are considered that include material composition, dosage, fabrication method, immunogenicity and toxicity. Different crosslinking techniques that have been used to develop injectable hydrogels for corneal regeneration are summarized. The limitations and future prospects of crosslinking strategies for use in corneal tissue engineering are discussed. It is demonstrated that the choice of crosslinking technique has a significant influence on the biocompatibility, mechanical properties and chemical structure of hydrogels that may be suitable for corneal tissue engineering and regenerative applications.
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8
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Prager J, Adams CF, Delaney AM, Chanoit G, Tarlton JF, Wong LF, Chari DM, Granger N. Stiffness-matched biomaterial implants for cell delivery: clinical, intraoperative ultrasound elastography provides a 'target' stiffness for hydrogel synthesis in spinal cord injury. J Tissue Eng 2020; 11:2041731420934806. [PMID: 32670538 PMCID: PMC7336822 DOI: 10.1177/2041731420934806] [Citation(s) in RCA: 18] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/11/2020] [Accepted: 05/21/2020] [Indexed: 12/14/2022] Open
Abstract
Safe hydrogel delivery requires stiffness-matching with host tissues to avoid
iatrogenic damage and reduce inflammatory reactions. Hydrogel-encapsulated cell
delivery is a promising combinatorial approach to spinal cord injury therapy,
but a lack of in vivo clinical spinal cord injury stiffness
measurements is a barrier to their use in clinics. We demonstrate that
ultrasound elastography – a non-invasive, clinically established tool – can be
used to measure spinal cord stiffness intraoperatively in canines with
spontaneous spinal cord injury. In line with recent experimental reports, our
data show that injured spinal cord has lower stiffness than uninjured cord. We
show that the stiffness of hydrogels encapsulating a clinically relevant
transplant population (olfactory ensheathing cells) can also be measured by
ultrasound elastography, enabling synthesis of hydrogels with comparable
stiffness to canine spinal cord injury. We therefore demonstrate
proof-of-principle of a novel approach to stiffness-matching hydrogel-olfactory
ensheathing cell implants to ‘real-life’ spinal cord injury values; an approach
applicable to multiple biomaterial implants for regenerative therapies.
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Affiliation(s)
- Jon Prager
- Bristol Veterinary School, University of Bristol, Bristol, UK.,The Royal Veterinary College, University of London, Hatfield, UK
| | - Christopher F Adams
- Cellular and Neural Engineering Group, Institute for Science and Technology in Medicine, Keele University, Keele, UK
| | - Alexander M Delaney
- Cellular and Neural Engineering Group, Institute for Science and Technology in Medicine, Keele University, Keele, UK
| | | | - John F Tarlton
- Bristol Veterinary School, University of Bristol, Bristol, UK
| | | | - Divya M Chari
- Cellular and Neural Engineering Group, Institute for Science and Technology in Medicine, Keele University, Keele, UK
| | - Nicolas Granger
- The Royal Veterinary College, University of London, Hatfield, UK
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Isolation and Culture of Corneal Stromal Stem Cells. Methods Mol Biol 2020. [PMID: 32542596 DOI: 10.1007/978-1-0716-0599-8_1] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/08/2023]
Abstract
An increasing body of evidence authenticates the benefit of corneal stroma-derived stem cells (CSSCs) in tissue engineering and regeneration oriented research, and potentially in the development of clinically relevant cellular therapies. Postmortem corneal tissue obtained from otherwise discarded material after keratoplasties is oftentimes the source of the cells for ex vivo research. Relatively easy to isolate and cultivate as well as inexpensive to culture, CSSCs now represent a well-described cell type with attributes of mesenchymal stem cells (MSCs). These include differentiation- and immunosuppressive potential, as well as a favorable capacity to expand in vitro. Here, we in detail describe two straightforward methods to isolate and establish CSSC cultures ex vivo.
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Wang K, Man K, Liu J, Liu Y, Chen Q, Zhou Y, Yang Y. Microphysiological Systems: Design, Fabrication, and Applications. ACS Biomater Sci Eng 2020; 6:3231-3257. [PMID: 33204830 PMCID: PMC7668566 DOI: 10.1021/acsbiomaterials.9b01667] [Citation(s) in RCA: 38] [Impact Index Per Article: 7.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/08/2023]
Abstract
Microphysiological systems, including organoids, 3-D printed tissue constructs and organ-on-a-chips (organ chips), are physiologically relevant in vitro models and have experienced explosive growth in the past decades. Different from conventional, tissue culture plastic-based in vitro models or animal models, microphysiological systems recapitulate key microenvironmental characteristics of human organs and mimic their primary functions. The advent of microphysiological systems is attributed to evolving biomaterials, micro-/nanotechnologies and stem cell biology, which enable the precise control over the matrix properties and the interactions between cells, tissues and organs in physiological conditions. As such, microphysiological systems have been developed to model a broad spectrum of organs from microvasculature, eye, to lung and many others to understand human organ development and disease pathology and facilitate drug discovery. Multiorgans-on-a-chip systems have also been developed by integrating multiple associated organ chips in a single platform, which allows to study and employ the organ function in a systematic approach. Here we first discuss the design principles of microphysiological systems with a focus on the anatomy and physiology of organs, and then review the commonly used fabrication techniques and biomaterials for microphysiological systems. Subsequently, we discuss the recent development of microphysiological systems, and provide our perspectives on advancing microphysiological systems for preclinical investigation and drug discovery of human disease.
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Affiliation(s)
- Kai Wang
- Department of Biomedical Engineering, University of North Texas, Denton, Texas 76207, United States
| | - Kun Man
- Department of Biomedical Engineering, University of North Texas, Denton, Texas 76207, United States
| | - Jiafeng Liu
- Department of Biomedical Engineering, University of North Texas, Denton, Texas 76207, United States
| | - Yang Liu
- North Texas Eye Research Institute, Department of Pharmacology & Neuroscience, University of North Texas Health Science Center, Fort Worth, Texas 76107, United States
| | - Qi Chen
- The Key Laboratory of Innate Immune Biology of Fujian Province, Biomedical Research Center of South China, College of Life Sciences, Fujian Normal University, Fuzhou 350117, China
| | - Yong Zhou
- Department of Emergency, Xinqiao Hospital, Chongqing 400037, China
| | - Yong Yang
- Department of Biomedical Engineering, University of North Texas, Denton, Texas 76207, United States
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Abstract
Decellularized corneal scaffolds have the potential to be used as alternatives to donor corneas during keratoplasty. Here a decellularization technique is described that involves the use of sodium dodecyl sulfate, Triton-X100, DNAse and RNAse to remove cells and cellular constituents. We have previously found that this combination of chemicals and enzymes to be effective at removing cells while retaining extracellular matrix proteins. In addition, different methods for assessing if the decellularization process has been successful are discussed. These include techniques to identify and quantify the presence of cells, DNA and extracellular matrix components as well as methods to examine the collagen fibril organization and scaffold transparency.
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Liu C, Tang S, Niu G, Zhang J, Huang X, Zhang Y, Bi Y. Ex vivo construction of rabbit corneal endothelial cell sheets on a porcine descemet membrane graft. Exp Ther Med 2019; 18:242-252. [PMID: 31258659 PMCID: PMC6566242 DOI: 10.3892/etm.2019.7573] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/30/2018] [Accepted: 03/26/2019] [Indexed: 11/10/2022] Open
Abstract
The aim of the present study was to investigate the feasibility of a new graft construction method using rabbit corneal endothelial cells (RCECs) and a porcine descemet membrane (DM) carrier. RCECs were isolated and the experimental group was treated with Y-27632, whereas the control group were cultured in medium without Y-27632. RCEC morphology was observed using an inverted microscope, and cell proliferation and apoptosis were detected by flow cytometry. To confirm the presence of RCECs, reverse transcription-quantitative PCR was used to detect gene expression levels of Na+-K+-ATPase, aquaporin 1, collagen α2 (IV), collagen α1 (VIII) and keratin-12. Histocompatibility testing was used to detect porcine DM antigenicity. A DM-RCEC graft was constructed, and morphology was observed using alizarin red-trypan blue and haematoxylin and eosin staining. Cell membrane potential was measured to evaluate the physical function of the DM-RCEC graft. Complex graft tension was measured using a modified tension detector and compared with fresh porcine DM-endothelium complex. In vitro-cultured RCECs formed a monolayer with a polygon morphology and cobblestone-like arrangement. In vitro-cultured RCECs exhibited typical RCEC characteristics before and after transplantation. The proliferation rates of the experimental and control groups were 62.68 and 34.50%, respectively (P<0.05); the apoptosis rates of the experimental and control groups were 8.99 and 35.68%, respectively (P<0.05). There was no antigenicity observed with the porcine DM. The action potential amplitude of the experimental and control groups was over −80 mV, reflecting normal RCEC physiological function. The tension measurements of the experimental and control groups were 20.0248±1.048 and 20.5013±0.657 g, respectively (P>0.05). Taken together, the results of the present study demonstrated that Y-27632 enhanced RCEC proliferation. In addition, the findings revealed the successful ex vivo construction of a RCEC sheet on a porcine DM graft.
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Affiliation(s)
- Chunyu Liu
- Department of Ophthalmology, Tongji Hospital Affiliated with Tongji University School of Medicine, Shanghai 200333, P.R. China
| | - Shenfei Tang
- Department of Ophthalmology, Tongji Hospital Affiliated with Tongji University School of Medicine, Shanghai 200333, P.R. China
| | - Guozhen Niu
- Department of Ophthalmology, Tongji Hospital Affiliated with Tongji University School of Medicine, Shanghai 200333, P.R. China
| | - Juan Zhang
- Department of Ophthalmology, Tongji Hospital Affiliated with Tongji University School of Medicine, Shanghai 200333, P.R. China
| | - Xinyu Huang
- Department of Ophthalmology, Tongji Hospital Affiliated with Tongji University School of Medicine, Shanghai 200333, P.R. China
| | - Yushan Zhang
- Department of Ophthalmology, Tongji Hospital Affiliated with Tongji University School of Medicine, Shanghai 200333, P.R. China
| | - Yanlong Bi
- Department of Ophthalmology, Tongji Hospital Affiliated with Tongji University School of Medicine, Shanghai 200333, P.R. China
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Fish Collagen Surgical Compress Repairing Characteristics on Wound Healing Process In Vivo. Mar Drugs 2019; 17:md17010033. [PMID: 30625985 PMCID: PMC6357035 DOI: 10.3390/md17010033] [Citation(s) in RCA: 77] [Impact Index Per Article: 12.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/12/2018] [Revised: 11/16/2018] [Accepted: 11/20/2018] [Indexed: 12/21/2022] Open
Abstract
The development of biomaterials with the potential to accelerate wound healing is a great challenge in biomedicine. In this study, four types of samples including pepsin soluble collagen sponge (PCS), acid soluble collagen sponge (ACS), bovine collagen electrospun I (BCE I) and bovine collagen electrospun II (BCE II) were used as wound dressing materials. We showed that the PCS, ACS, BCE I and BCE II treated rats increased the percentage of wound contraction, reduced the inflammatory infiltration, and accelerated the epithelization and healing. PCS, ACS, BCE I, and BCE II significantly enhanced the total protein and hydroxyproline level in rats. ACS could induce more fibroblasts proliferation and differentiation than PCS, however, both PCS and ACS had a lower effect than BCE I and BCE II. PCS, ACS, BCE I, and BCE II could regulate deposition of collagen, which led to excellent alignment in the wound healing process. There were similar effects on inducing the level of cytokines including EGF, FGF, and vascular endothelial marker CD31 among these four groups. Accordingly, this study disclosed that collagens (PCS and ACS) from tilapia skin and bovine collagen electrospun (BCE I and BCE II) have significant bioactivity and could accelerate wound healing rapidly and effectively in rat model.
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Youngblood RL, Truong NF, Segura T, Shea LD. It's All in the Delivery: Designing Hydrogels for Cell and Non-viral Gene Therapies. Mol Ther 2018; 26:2087-2106. [PMID: 30107997 PMCID: PMC6127639 DOI: 10.1016/j.ymthe.2018.07.022] [Citation(s) in RCA: 52] [Impact Index Per Article: 7.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/13/2018] [Revised: 07/24/2018] [Accepted: 07/24/2018] [Indexed: 01/08/2023] Open
Abstract
Hydrogels provide a regenerative medicine platform with their ability to create an environment that supports transplanted or endogenous infiltrating cells and enables these cells to restore or replace the function of tissues lost to disease or trauma. Furthermore, these systems have been employed as delivery vehicles for therapeutic genes, which can direct and/or enhance the function of the transplanted or endogenous cells. Herein, we review recent advances in the development of hydrogels for cell and non-viral gene delivery through understanding the design parameters, including both physical and biological components, on promoting transgene expression, cell engraftment, and ultimately cell function. Furthermore, this review identifies emerging opportunities for combining cell and gene delivery approaches to overcome challenges to the field.
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Affiliation(s)
- Richard L Youngblood
- Department of Biomedical Engineering, University of Michigan, Ann Arbor, MI 48109, USA
| | - Norman F Truong
- Department of Chemical and Biomolecular Engineering, University of California, Los Angeles, Los Angeles, CA 90095, USA
| | - Tatiana Segura
- Department of Chemical and Biomolecular Engineering, University of California, Los Angeles, Los Angeles, CA 90095, USA; Department of Biomedical Engineering, Duke University, Durham, NC 27708, USA.
| | - Lonnie D Shea
- Department of Biomedical Engineering, University of Michigan, Ann Arbor, MI 48109, USA.
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El-Fiqi A, Buitrago JO, Yang SH, Kim HW. Biomimetically grown apatite spheres from aggregated bioglass nanoparticles with ultrahigh porosity and surface area imply potential drug delivery and cell engineering applications. Acta Biomater 2017; 60:38-49. [PMID: 28754647 DOI: 10.1016/j.actbio.2017.07.036] [Citation(s) in RCA: 16] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/22/2017] [Revised: 07/06/2017] [Accepted: 07/24/2017] [Indexed: 02/07/2023]
Abstract
Here we communicate the generation of biomimetically grown apatite spheres from aggregated bioglass nanoparticles and the potential properties applicable for drug delivery and cell/tissue engineering. Ion releasing nanoparticulates of bioglass (85%SiO2-15%CaO) in a mineralizing medium show an intriguing dynamic phenomenon - aggregation, mineralization to apatite, integration and growth into micron-sized (1.5-3μm) spheres. During the progressive ionic dissolution/precipitation reactions, nano-to-micro-morphology, glass-to-crystal composition, and the physico-chemical properties (porosity, surface area, and charge) change dynamically. With increasing reaction period, the apatite becomes more crystallized with increased crystallinity and crystal size, and gets a composition closer to the stoichiometry. The developed microspheres exhibit hierarchical surface nanostructure, negative charge (ς-potential of -20mV), and ultrahigh mesoporosity (mesopore size of 6.1nm, and the resultant surface area of 63.7m2/g and pore volume of 0.153cm3/g) at 14days of mineralization, which are even higher than those of its precursor bioglass nanoparticles. Thanks to these properties, the biomimetic mineral microspheres take up biological molecules effectively, i.e., loading capacity of positive-charged protein is over 10%. Of note, the release is highly sustainable at a constant rate, i.e., profiling almost 'zero-order' kinetics for 4weeks, suggesting the potential usefulness as protein delivery systems. The biomimetic mineral microspheres hold some remnant Si in the core region, and release calcium, phosphate, and silicate ions over the test period, implying the long-term ionic-related therapeutic functions. The mesenchymal stem cells favour the biomimetic spheres with an excellent viability. Due to the merit of sizes (a few micrometers), the spheres can be intercalated into cells, mediating cellular interactions in 3D cell-spheroid engineering, and also can stimulate osteogenic differentiation of cells when incorporated into cell-laden gels. The intriguing properties observed in this study, including biomimetic composition, high mesoporosity, release of therapeutic ions, effective loading and long-term release of proteins, and diverse yet favorable 3D cellular interactions, suggest great potential of the newly developed biomimetic microspheres in biomedical applications, such as drug delivery and cell/tissue engineering. STATEMENT OF SIGNIFICANCE This work reports the generation of apatite spheres with a few micrometers in size biomimetically grown from bioactive glass nanoparticles, through a series of intriguing yet unprecedented phenomenon involving aggregation of nanoparticles, mineralization and sphere growth. The mineral microspheres possess some unique physico-chemical properties including mesoporosity, ultrahigh surface area, and therapeutic ionic release. Furthermore, the spheres show excellent loading and delivery capacity of protein molecules, and mediate favorable cellular interactions in 2D and 3D culture conditions, demonstrating a future multifunctional microcarrier platform for the therapeutics delivery and cell/tissue engineering.
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16
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Ghezzi CE, Marelli B, Omenetto FG, Funderburgh JL, Kaplan DL. 3D Functional Corneal Stromal Tissue Equivalent Based on Corneal Stromal Stem Cells and Multi-Layered Silk Film Architecture. PLoS One 2017; 12:e0169504. [PMID: 28099503 PMCID: PMC5242458 DOI: 10.1371/journal.pone.0169504] [Citation(s) in RCA: 43] [Impact Index Per Article: 5.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/24/2016] [Accepted: 12/19/2016] [Indexed: 12/13/2022] Open
Abstract
The worldwide need for human cornea equivalents continues to grow. Few clinical options are limited to allogenic and synthetic material replacements. We hypothesized that tissue engineered human cornea systems based on mechanically robust, patterned, porous, thin, optically clear silk protein films, in combination with human corneal stromal stem cells (hCSSCs), would generate 3D functional corneal stroma tissue equivalents, in comparison to previously developed 2D approaches. Silk film contact guidance was used to control the alignment and distribution of hCSSCs on RGD-treated single porous silk films, which were then stacked in an orthogonally, multi-layered architecture and cultured for 9 weeks. These systems were compared similar systems generated with human corneal fibroblasts (hCFs). Both cell types were viable and preferentially aligned along the biomaterial patterns for up to 9 weeks in culture. H&E histological sections showed that the systems seeded with the hCSSCs displayed ECM production throughout the entire thickness of the constructs. In addition, the ECM proteins tested positive for keratocyte-specific tissue markers, including keratan sulfate, lumican, and keratocan. The quantification of hCSSC gene expression of keratocyte-tissue markers, including keratocan, lumican, human aldehyde dehydrogenase 3A1 (ALDH3A1), prostaglandin D2 synthase (PTDGS), and pyruvate dehydrogenase kinase, isozyme 4 (PDK4), within the 3D tissue systems demonstrated upregulation when compared to 2D single silk films and to the systems generated with the hCFs. Furthermore, the production of ECM from the hCSSC seeded systems and subsequent remodeling of the initial matrix significantly improved cohesiveness and mechanical performance of the constructs, while maintaining transparency after 9 weeks.
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Affiliation(s)
- Chiara E. Ghezzi
- Department of Biomedical Engineering, Tufts University, Medford, Massachusetts, United States of America
| | - Benedetto Marelli
- Department of Biomedical Engineering, Tufts University, Medford, Massachusetts, United States of America
| | - Fiorenzo G. Omenetto
- Department of Biomedical Engineering, Tufts University, Medford, Massachusetts, United States of America
| | - James L. Funderburgh
- Department of Ophthalmology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, United States of America
| | - David L. Kaplan
- Department of Biomedical Engineering, Tufts University, Medford, Massachusetts, United States of America
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Ceccaldi C, Strandman S, Hui E, Montagnon E, Schmitt C, Hadj Henni A, Lerouge S. Validation and application of a nondestructive and contactless method for rheological evaluation of biomaterials. J Biomed Mater Res B Appl Biomater 2016; 105:2565-2573. [DOI: 10.1002/jbm.b.33797] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/25/2016] [Revised: 08/18/2016] [Accepted: 09/12/2016] [Indexed: 11/09/2022]
Affiliation(s)
- Caroline Ceccaldi
- Department of mechanical engineering; École de technologie supérieure (ÉTS), 1100 Notre-Dame Street West; Montreal QC H3C 1K3 Canada
- Laboratory of biomaterials and endovascular implants (LBEV); Centre de recherche du CHUM (CRCHUM), 900 St Denis; Montreal QC H2X 0A9 Canada
| | - Satu Strandman
- Rheolution Inc., 5333 Avenue Casgrain, suite 712; Montreal QC H2T1X3 Canada
| | - Eve Hui
- Department of mechanical engineering; École de technologie supérieure (ÉTS), 1100 Notre-Dame Street West; Montreal QC H3C 1K3 Canada
- Laboratory of biomaterials and endovascular implants (LBEV); Centre de recherche du CHUM (CRCHUM), 900 St Denis; Montreal QC H2X 0A9 Canada
| | - Emmanuel Montagnon
- Rheolution Inc., 5333 Avenue Casgrain, suite 712; Montreal QC H2T1X3 Canada
| | - Cédric Schmitt
- Rheolution Inc., 5333 Avenue Casgrain, suite 712; Montreal QC H2T1X3 Canada
| | - Anis Hadj Henni
- Rheolution Inc., 5333 Avenue Casgrain, suite 712; Montreal QC H2T1X3 Canada
| | - Sophie Lerouge
- Department of mechanical engineering; École de technologie supérieure (ÉTS), 1100 Notre-Dame Street West; Montreal QC H3C 1K3 Canada
- Laboratory of biomaterials and endovascular implants (LBEV); Centre de recherche du CHUM (CRCHUM), 900 St Denis; Montreal QC H2X 0A9 Canada
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18
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Law JX, Musa F, Ruszymah BHI, El Haj AJ, Yang Y. A comparative study of skin cell activities in collagen and fibrin constructs. Med Eng Phys 2016; 38:854-61. [DOI: 10.1016/j.medengphy.2016.05.017] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/01/2015] [Revised: 02/24/2016] [Accepted: 05/18/2016] [Indexed: 11/26/2022]
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Jin T, Li L, Siow RCM, Liu KK. Collagen matrix stiffness influences fibroblast contraction force. Biomed Phys Eng Express 2016. [DOI: 10.1088/2057-1976/2/4/047002] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022]
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Mousavi SJ, Doweidar MH. Numerical modeling of cell differentiation and proliferation in force-induced substrates via encapsulated magnetic nanoparticles. COMPUTER METHODS AND PROGRAMS IN BIOMEDICINE 2016; 130:106-117. [PMID: 27208526 DOI: 10.1016/j.cmpb.2016.03.019] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 11/20/2015] [Revised: 03/16/2016] [Accepted: 03/17/2016] [Indexed: 06/05/2023]
Abstract
BACKGROUND AND OBJECTIVE Cell migration, differentiation, proliferation and apoptosis are the main processes in tissue regeneration. Mesenchymal Stem Cells have the potential to differentiate into many cell phenotypes such as tissue- or organ-specific cells to perform special functions. Experimental observations illustrate that differentiation and proliferation of these cells can be regulated according to internal forces induced within their Extracellular Matrix. The process of how exactly they interpret and transduce these signals is not well understood. METHODS A previously developed three-dimensional (3D) computational model is here extended and employed to study how force-free substrates and force-induced substrate control cell differentiation and/or proliferation during the mechanosensing process. Consistent with experimental observations, it is assumed that cell internal deformation (a mechanical signal) in correlation with the cell maturation state directly triggers cell differentiation and/or proliferation. The Extracellular Matrix is modeled as Neo-Hookean hyperelastic material assuming that cells are cultured within 3D nonlinear hydrogels. RESULTS In agreement with well-known experimental observations, the findings here indicate that within neurogenic (0.1-1kPa), chondrogenic (20-25kPa) and osteogenic (30-45kPa) substrates, Mesenchymal Stem Cells differentiation and proliferation can be precipitated by inducing the substrate with an internal force. Therefore, cells require a longer time to grow and maturate within force-free substrates than within force-induced substrates. In the instance of Mesenchymal Stem Cells differentiation into a compatible phenotype, the magnitude of the net traction force increases within chondrogenic and osteogenic substrates while it reduces within neurogenic substrates. This is consistent with experimental studies and numerical works recently published by the same authors. However, in all cases the magnitude of the net traction force considerably increases at the instant of cell proliferation because of cell-cell interaction. CONCLUSIONS The present model provides new perspectives to delineate the role of force-induced substrates in remotely controlling the cell fate during cell-matrix interaction, which open the door for new tissue regeneration methodologies.
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Affiliation(s)
- Seyed Jamaleddin Mousavi
- Mechanical Engineering Department, School of Engineering and Architecture (EINA), University of Zaragoza, Zaragoza, Spain; Aragón Institute of Engineering Research (I3A), University of Zaragoza, Zaragoza, Spain; Biomedical Research Networking Center in Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), Zaragoza, Spain
| | - Mohamed Hamdy Doweidar
- Mechanical Engineering Department, School of Engineering and Architecture (EINA), University of Zaragoza, Zaragoza, Spain; Aragón Institute of Engineering Research (I3A), University of Zaragoza, Zaragoza, Spain; Biomedical Research Networking Center in Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), Zaragoza, Spain.
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21
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Zhang J, Zhang CW, Du LQ, Wu XY. Acellular porcine corneal matrix as a carrier scaffold for cultivating human corneal epithelial cells and fibroblasts in vitro. Int J Ophthalmol 2016; 9:1-8. [PMID: 26949602 DOI: 10.18240/ijo.2016.01.01] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/02/2015] [Accepted: 07/22/2015] [Indexed: 12/13/2022] Open
Abstract
AIM To investigate the feasibility of corneal anterior lamellar reconstruction with human corneal epithelial cells and fibroblasts, and an acellular porcine cornea matrix (APCM) in vitro. METHODS The scaffold was prepared from fresh porcine corneas which were treated with 0.5% sodium dodecyl sulfate (SDS) solution and the complete removal of corneal cells was confirmed by hematoxylin-eosin (HE) staining and 4', 6-diamidino-2-phenylindole (DAPI) staining. Human corneal fibroblasts and epithelial cells were cultured with leaching liquid extracted from APCM, and then cell proliferative ability was evaluated by MTT assay. To construct a human corneal anterior lamellar replacement, corneal fibroblasts were injected into the APCM and cultured for 3d, followed by culturing corneal epithelial cells on the stroma construction surface for another 10d. The corneal replacement was analyzed by HE staining, and immunofluorescence staining. RESULTS Histological examination indicated that there were no cells in the APCM by HE staining, and DAPI staining did not detect any residual DNA. The leaching liquid from APCM had little influence on the proliferation ability of human corneal fibroblasts and epithelial cells. At 10d, a continuous 3 to 5 layers of human corneal epithelial cells covering the surface of the APCM was observed, and the injected corneal fibroblasts distributed within the scaffold. The phenotype of the construction was similar to normal human corneas, with high expression of cytokeratin 12 in the epithelial cell layer and high expression of vimentin in the stroma. CONCLUSION Corneal anterior lamellar replacement can be reconstructed in vitro by cultivating human corneal epithelial cells and fibroblasts with an acellular porcine cornea matrix. This laid the foundation for the further transplantation in vivo.
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Affiliation(s)
- Ju Zhang
- Department of Ophthalmology, Qilu Hospital of Shandong University, Jinan 250012, Shandong Province, China; The Key Laboratory of Cardiovascular Remodeling and Function Research, Chinese Ministry of Education and Chinese Ministry of Health, Qilu Hospital of Shandong University, Jinan 250012, Shandong Province, China
| | - Can-Wei Zhang
- Department of Ophthalmology, Qilu Hospital of Shandong University, Jinan 250012, Shandong Province, China
| | - Li-Qun Du
- Department of Ophthalmology, Qilu Hospital of Shandong University, Jinan 250012, Shandong Province, China
| | - Xin-Yi Wu
- Department of Ophthalmology, Qilu Hospital of Shandong University, Jinan 250012, Shandong Province, China
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22
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Jin T, Li L, Siow RCM, Liu KK. A novel collagen gel-based measurement technique for quantitation of cell contraction force. J R Soc Interface 2016; 12. [PMID: 25977960 DOI: 10.1098/rsif.2014.1365] [Citation(s) in RCA: 23] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022] Open
Abstract
Cell contraction force plays an important role in wound healing, inflammation,angiogenesis and metastasis. This study describes a novel method to quantify single cell contraction force in vitro using human aortic adventitial fibroblasts embedded in a collagen gel. The technique is based on a depth sensing nano-indentation tester to measure the thickness and elasticity of collagen gels containing stimulated fibroblasts and a microscopy imaging system to estimate the gel area. In parallel, a simple theoretical model has been developed to calculate cell contraction force based on the measured parameters. Histamine (100 mM) was used to stimulate fibroblast contraction while the myosin light chain kinase inhibitor ML-7 (25 mM) was used to inhibit cell contraction. The collagen matrix used in the model provides a physiological environment for fibroblast contraction studies. Measurement of changes in collagen gel elasticity and thickness arising from histamine treatments provides a novel convenient technique to measure cell contraction force within a collagen matrix. This study demonstrates that histamine can elicit a significant increase in contraction force of fibroblasts embedded in collagen,while the Young's modulus of the gel decreases due to the gel degradation.
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23
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Wilson SL, Sidney LE, Dunphy SE, Dua HS, Hopkinson A. Corneal Decellularization: A Method of Recycling Unsuitable Donor Tissue for Clinical Translation? Curr Eye Res 2015; 41:769-82. [PMID: 26397030 PMCID: PMC4926783 DOI: 10.3109/02713683.2015.1062114] [Citation(s) in RCA: 64] [Impact Index Per Article: 6.4] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022]
Abstract
Background: There is a clinical need for biomimetic corneas that are as effective, preferably superior, to cadaveric donor tissue. Decellularized tissues are advantageous compared to synthetic or semi-synthetic engineered tissues in that the native matrix ultrastructure and intrinsic biological cues including growth factors, cytokines and glycosaminoglycans may be retained. However, there is currently no reliable, standardized human corneal decellularization protocol. Methods: Corneal eye-bank tissue unsuitable for transplantation was utilized to systematically compare commonly used decellularization protocols. Hypertonic sodium chloride; an ionic reagent, sodium dodecyl sulphate; a non-ionic detergent, tert-octylphenol polyoxyethylene (Triton-X); enzymatic disaggregation using Dispase; mechanical agitation; and the use of nucleases were investigated. Decellularization efficacy, specifically for human corneal tissue, was extensively evaluated. Removal of detectable cellular material was evidenced by histological, immunofluorescence and biochemical assays. Preservation of macroscopic tissue transparency and light transmittance was evaluated. Retention of corneal architecture, collagen and glycosaminoglycans was assessed via histological, immunofluorescence and quantitative analysis. Biocompatibility of the resulting scaffolds was assessed using cell proliferation assays. Results: None of the decellularization protocols investigated successfully removed 100% of cellular components. The techniques with the least residual cellular material were most structurally compromised. Biochemical analysis of glycosaminoglycans demonstrated the stripping effects of the decellularization procedures. Conclusion: The ability to utilize, reprocess and regenerate tissues deemed “unsuitable” for transplantation allows us to salvage valuable tissue. Reprocessing the tissue has the potential to have a considerable impact on addressing the problems associated with cadaveric donor shortage. Patients would directly benefit by accessing greater numbers of corneal grafts and health authorities would fulfill their responsibility for the delivery of effective corneal reconstruction to alleviate corneal blindness. However, in order to progress, we may need to take a step back to establish a “decellularization” criterion; which should balance effective removal of immune reactive material with maintenance of tissue functionality.
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Affiliation(s)
- Samantha L Wilson
- a Academic Ophthalmology, Division of Clinical Neuroscience, Queen's Medical Centre Campus, University of Nottingham , Nottingham , UK
| | - Laura E Sidney
- a Academic Ophthalmology, Division of Clinical Neuroscience, Queen's Medical Centre Campus, University of Nottingham , Nottingham , UK
| | - Siobhán E Dunphy
- a Academic Ophthalmology, Division of Clinical Neuroscience, Queen's Medical Centre Campus, University of Nottingham , Nottingham , UK
| | - Harminder S Dua
- a Academic Ophthalmology, Division of Clinical Neuroscience, Queen's Medical Centre Campus, University of Nottingham , Nottingham , UK
| | - Andrew Hopkinson
- a Academic Ophthalmology, Division of Clinical Neuroscience, Queen's Medical Centre Campus, University of Nottingham , Nottingham , UK
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24
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Yeung CYC, Zeef LAH, Lallyett C, Lu Y, Canty-Laird EG, Kadler KE. Chick tendon fibroblast transcriptome and shape depend on whether the cell has made its own collagen matrix. Sci Rep 2015; 5:13555. [PMID: 26337655 PMCID: PMC4559659 DOI: 10.1038/srep13555] [Citation(s) in RCA: 25] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/22/2015] [Accepted: 07/30/2015] [Indexed: 12/03/2022] Open
Abstract
Collagen- and fibrin-based gels are extensively used to study cell behaviour. However, 2D–3D and collagen-fibrin comparisons of gene expression, cell shape and mechanotransduction, with an in vivo reference, have not been reported. Here we compared chick tendon fibroblasts (CTFs) at three stages of embryonic development with CTFs cultured in collagen- or fibrin-based tissue engineered constructs (TECs). CTFs synthesised their own collagen matrix in fibrin-based TECs and better recapitulated the gene expression, collagen fibril alignment and cell shape seen in vivo. In contrast, cells in 3D collagen gels exhibited a 2D-like morphology and expressed fewer of the genes expressed in vivo. Analysis of YAP/TAZ target genes showed that collagen gels desensitise mechanotransduction pathways. In conclusion, gene expression and cell shape are similar on plastic and 3D collagen whereas cells in 3D fibrin have a shape and transcriptome better resembling the in vivo situation. Implications for wound healing are discussed.
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Affiliation(s)
- Ching-Yan Chloé Yeung
- Wellcome Trust Centre for Cell-Matrix Research, Oxford Road, Manchester M13 9PT United Kingdom.,Faculty of Life Sciences, University of Manchester, Michael Smith Building, Oxford Road, Manchester M13 9PT United Kingdom
| | - Leo A H Zeef
- Faculty of Life Sciences, University of Manchester, Michael Smith Building, Oxford Road, Manchester M13 9PT United Kingdom
| | - Chloe Lallyett
- Wellcome Trust Centre for Cell-Matrix Research, Oxford Road, Manchester M13 9PT United Kingdom.,Faculty of Life Sciences, University of Manchester, Michael Smith Building, Oxford Road, Manchester M13 9PT United Kingdom
| | - Yinhui Lu
- Wellcome Trust Centre for Cell-Matrix Research, Oxford Road, Manchester M13 9PT United Kingdom
| | - Elizabeth G Canty-Laird
- Department of Musculoskeletal Biology, Institute of Ageing and Chronic Disease, Faculty of Health and Life Sciences, University of Liverpool, United Kingdom.,The MRC-Arthritis Research UK Centre for Integrated research into Musculoskeletal Ageing (CIMA), United Kingdom
| | - Karl E Kadler
- Wellcome Trust Centre for Cell-Matrix Research, Oxford Road, Manchester M13 9PT United Kingdom.,Faculty of Life Sciences, University of Manchester, Michael Smith Building, Oxford Road, Manchester M13 9PT United Kingdom
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25
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Słoniecka M, Le Roux S, Boman P, Byström B, Zhou Q, Danielson P. Expression Profiles of Neuropeptides, Neurotransmitters, and Their Receptors in Human Keratocytes In Vitro and In Situ. PLoS One 2015. [PMID: 26214847 PMCID: PMC4516240 DOI: 10.1371/journal.pone.0134157] [Citation(s) in RCA: 38] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/27/2022] Open
Abstract
Keratocytes, the quiescent cells of the corneal stroma, play a crucial role in corneal wound healing. Neuropeptides and neurotransmitters are usually associated with neuronal signaling, but have recently been shown to be produced also by non-neuronal cells and to be involved in many cellular processes. The aim of this study was to assess the endogenous intracellular and secreted levels of the neuropeptides substance P (SP) and neurokinin A (NKA), and of the neurotransmitters acetylcholine (ACh), catecholamines (adrenaline, noradrenaline and dopamine), and glutamate, as well as the expression profiles of their receptors, in human primary keratocytes in vitro and in keratocytes of human corneal tissue sections in situ. Cultured keratocytes expressed genes encoding for SP and NKA, and for catecholamine and glutamate synthesizing enzymes, as well as genes for neuropeptide, adrenergic and ACh (muscarinic) receptors. Keratocytes in culture produced SP, NKA, catecholamines, ACh, and glutamate, and expressed neurokinin-1 and -2 receptors (NK-1R and NK-2R), dopamine receptor D2, muscarinic ACh receptors, and NDMAR1 glutamate receptor. Human corneal sections expressed SP, NKA, NK-1R, NK-2R, receptor D2, choline acetyl transferase (ChAT), M3, M4 and M5 muscarinic ACh receptors, glutamate, and NMDAR1, but not catecholamine synthesizing enzyme or the α1 and β2 adrenoreceptors, nor M1 receptor. In addition, expression profiles assumed significant differences between keratocytes from the peripheral cornea as compared to those from the central cornea, as well as differences between keratocytes cultured under various serum concentrations. In conclusion, human keratocytes express an array of neuropeptides and neurotransmitters. The cells furthermore express receptors for neuropeptides/neurotransmitters, which suggests that they are susceptible to stimulation by these substances in the cornea, whether of neuronal or non-neuronal origin. As it has been shown that neuropeptides/neurotransmitters are involved in cell proliferation, migration, and angiogenesis, it is possible that they play a role in corneal wound healing.
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Affiliation(s)
- Marta Słoniecka
- Department of Integrative Medical Biology, Umeå University, Umeå, Sweden
- * E-mail:
| | - Sandrine Le Roux
- Department of Integrative Medical Biology, Umeå University, Umeå, Sweden
| | - Peter Boman
- Department of Integrative Medical Biology, Umeå University, Umeå, Sweden
| | - Berit Byström
- Department of Clinical Sciences, Ophthalmology, Umeå University, Umeå, Sweden
| | - Qingjun Zhou
- Department of Integrative Medical Biology, Umeå University, Umeå, Sweden
- Shandong Provincial Key Laboratory of Ophthalmology, Shandong Eye Institute, Qingdao, China
| | - Patrik Danielson
- Department of Integrative Medical Biology, Umeå University, Umeå, Sweden
- Department of Clinical Sciences, Ophthalmology, Umeå University, Umeå, Sweden
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26
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Ahearne M, Lynch AP. Early Observation of Extracellular Matrix-Derived Hydrogels for Corneal Stroma Regeneration. Tissue Eng Part C Methods 2015; 21:1059-69. [PMID: 25951055 DOI: 10.1089/ten.tec.2015.0008] [Citation(s) in RCA: 43] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022] Open
Abstract
A lack of healthy transplantable tissue to treat corneal blindness has led researchers to investigate the development and application of different scaffold materials for corneal tissue engineering and regeneration. In this study, hydrogels fabricated from decellularized corneal extracellular matrix were developed as a new approach to corneal stromal tissue regeneration. Porcine corneas were decellularized using a technique that combined freeze-thaw cycles with a nuclease treatment. The corneas were then freeze-dried, milled, and digested in an acidic pepsin solution that was used to form a hydrogel after adjusting the pH and gelation temperature. The resultant corneal matrix hydrogels (CMHs) were seeded with human corneal stromal cells and cultured for several days. When compared to collagens hydrogels, CMHs had superior optical transparency, similar mechanical properties, and were better able to retain the stromal cells native keratocyte phenotype. The CMHs also supported cell viability and proliferation and contained sulfated glycosaminoglycan, a vital constituent, for maintaining corneal transparency. These results suggest that the CMHs could provide an exceptional biomaterial for corneal stroma regeneration.
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Affiliation(s)
- Mark Ahearne
- 1 Trinity Centre for Bioengineering, Trinity Biomedical Sciences Institute , Trinity College Dublin, Dublin, Ireland .,2 Department of Mechanical and Manufacturing Engineering, School of Engineering, Trinity College Dublin , Dublin, Ireland
| | - Amy P Lynch
- 1 Trinity Centre for Bioengineering, Trinity Biomedical Sciences Institute , Trinity College Dublin, Dublin, Ireland .,2 Department of Mechanical and Manufacturing Engineering, School of Engineering, Trinity College Dublin , Dublin, Ireland
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Ghezzi CE, Rnjak-Kovacina J, Kaplan DL. Corneal tissue engineering: recent advances and future perspectives. TISSUE ENGINEERING PART B-REVIEWS 2015; 21:278-87. [PMID: 25434371 DOI: 10.1089/ten.teb.2014.0397] [Citation(s) in RCA: 116] [Impact Index Per Article: 11.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
To address the growing need for corneal transplants two main approaches are being pursued: allogenic and synthetic materials. Allogenic tissue from human donors is currently the preferred choice; however, there is a worldwide shortage in donated corneal tissue. In addition, tissue rejection often limits the long-term success of this approach. Alternatively, synthetic homologs to donor corneal grafts are primarily considered temporary replacements until suitable donor tissue becomes available, as they result in a high incidence of graft failure. Tissue engineered cornea analogs would provide effective cornea tissue substitutes and alternatives to address the need to reduce animal testing of commercial products. Recent progress toward these needs is reviewed here, along with future perspectives.
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Affiliation(s)
- Chiara E Ghezzi
- 1Department of Biomedical Engineering, Tufts University, Medford, Massachusetts
| | - Jelena Rnjak-Kovacina
- 1Department of Biomedical Engineering, Tufts University, Medford, Massachusetts.,2Graduate School of Biomedical Engineering, UNSW Australia, Sydney, Australia
| | - David L Kaplan
- 1Department of Biomedical Engineering, Tufts University, Medford, Massachusetts
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Dunphy SE, Bratt JA, Akram KM, Forsyth NR, El Haj AJ. Hydrogels for lung tissue engineering: Biomechanical properties of thin collagen–elastin constructs. J Mech Behav Biomed Mater 2014; 38:251-9. [DOI: 10.1016/j.jmbbm.2014.04.005] [Citation(s) in RCA: 28] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/18/2013] [Revised: 04/07/2014] [Accepted: 04/09/2014] [Indexed: 12/13/2022]
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Abstract
The development of hydrogel-based biomaterials represents a promising approach to generating new strategies for tissue engineering and regenerative medicine. In order to develop more sophisticated cell-seeded hydrogel constructs, it is important to understand how cells mechanically interact with hydrogels. In this paper, we review the mechanisms by which cells remodel hydrogels, the influence that the hydrogel mechanical and structural properties have on cell behaviour and the role of mechanical stimulation in cell-seeded hydrogels. Cell-mediated remodelling of hydrogels is directed by several cellular processes, including adhesion, migration, contraction, degradation and extracellular matrix deposition. Variations in hydrogel stiffness, density, composition, orientation and viscoelastic characteristics all affect cell activity and phenotype. The application of mechanical force on cells encapsulated in hydrogels can also instigate changes in cell behaviour. By improving our understanding of cell-material mechano-interactions in hydrogels, this should enable a new generation of regenerative medical therapies to be developed.
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Affiliation(s)
- Mark Ahearne
- 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 , Ireland
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Wilson SL, Guilbert M, Sulé-Suso J, Torbet J, Jeannesson P, Sockalingum GD, Yang Y. A microscopic and macroscopic study of aging collagen on its molecular structure, mechanical properties, and cellular response. FASEB J 2013; 28:14-25. [PMID: 24025727 DOI: 10.1096/fj.13-227579] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/01/2023]
Abstract
During aging, collagen structure changes, detrimentally affecting tissues' biophysical and biomechanical properties due to an accumulation of advanced glycation end-products (AGEs). In this investigation, we conducted a parallel study of microscopic and macroscopic properties of different-aged collagens from newborn to 2-yr-old rats, to examine the effect of aging on fibrillogenesis, mechanical and contractile properties of reconstituted hydrogels from these collagens seeded with or without fibroblasts. In addition to fibrillogenesis of collagen under the conventional conditions, some fibrillogenesis was conducted alongside a 12-T magnetic field, and gelation rate and AGE content were measured. A nondestructive indentation technique and optical coherence tomography were used to determine the elastic modulus and dimensional changes, respectively. It was revealed that in comparison to younger specimens, older collagens exhibited higher viscosity, faster gelation rates, and a higher AGE-specific fluorescence. Exceptionally, only young collagens formed highly aligned fibrils under magnetic fields. The youngest collagen demonstrated a higher elastic modulus and contraction in comparison to the older collagen. We conclude that aging changes collagen monomer structure, which considerably affects the fibrillogenesis process, the architecture of the resulting collagen fibers and the global network, and the macroscopic properties of the formed constructs.
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Affiliation(s)
- Samantha L Wilson
- 1Institute for Science and Technology in Medicine, Keele University, Stoke-on-Trent, ST4 7QB, UK.
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Wilson SL, Yang Y, El Haj AJ. Corneal stromal cell plasticity: in vitro regulation of cell phenotype through cell-cell interactions in a three-dimensional model. Tissue Eng Part A 2013; 20:225-38. [PMID: 23895175 DOI: 10.1089/ten.tea.2013.0167] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022] Open
Abstract
In vivo, epithelial cells are connected both anatomically and functionally with stromal keratocytes. Co-culturing aims at recapturing this cellular anatomy and functionality by bringing together two or more cell types within the same culture environment. Corneal stromal cells were activated to their injury phenotype (fibroblasts) and expanded before being encapsulated in type I collagen hydrogels constructs. Three different epithelial-stromal co-culture methods were then examined: epithelial explant; transwell; and the use of conditioned media. The aim was to determine whether the native, inactivated keratocyte cell phenotype could be restored in vitro. Media supplementation with transforming growth factor beta-1 (TGF-β1) was then used to determine whether the inactivated stromal cells retained their plasticity in vitro and could be re-activated to the fibroblast phenotype. Finally, media supplementation with wortmannin was used to inhibit epithelial-stromal cell interactions. Two different nondestructive techniques, spherical indentation and optical coherence tomography, were used to reveal how epithelial-stromal co-culturing with TGF-β1, and wortmannin media supplementation, respectively, affect stromal cell behavior and differentiation in terms of construct contraction and elastic modulus measurement. Cell viability, phenotype, morphology, and protein expression were investigated to corroborate our mechanical findings. It was shown that activated stromal cells could be inactivated to a keratocyte phenotype via co-culturing and that they retained their plasticity in vitro. Activated corneal stromal cells that were fibroblastic in phenotype were successfully reverted to a nonactivated keratocyte cell lineage in terms of behavior and biological properties; and then back again via TGF-β1 media supplementation. It was then revealed that epithelial-stromal interactions can be blocked via the use of wortmannin inhibition. A greater understanding of stromal-epithelial interactions and what mediates them offers great pharmacological potential in the regulation of corneal wound healing, with the potential to treat corneal diseases and injury by which such interactions are vital.
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Affiliation(s)
- Samantha L Wilson
- Institute for Science and Technology in Medicine, School of Medicine, Keele University , Stoke-on-Trent, United Kingdom
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Hashmani K, Branch MJ, Sidney LE, Dhillon PS, Verma M, McIntosh OD, Hopkinson A, Dua HS. Characterization of corneal stromal stem cells with the potential for epithelial transdifferentiation. Stem Cell Res Ther 2013; 4:75. [PMID: 23800436 PMCID: PMC4058700 DOI: 10.1186/scrt226] [Citation(s) in RCA: 54] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/31/2013] [Accepted: 06/04/2013] [Indexed: 12/12/2022] Open
Abstract
INTRODUCTION The corneal stroma is being increasingly recognized as a repository for stem cells. Like the limbal and endothelial niches, stromal stem cells often reside in the peripheral cornea and limbus. These peripheral and limbal corneal stromal cells (PLCSCs) are known to produce mesenchymal stem cells in vitro. Recently, a common corneal stromal and epithelial progenitor was hinted at. This study aims to examine the stem cell potential of corneal stromal cells and to investigate their epithelial transdifferentiation ability. METHODS PLCSCs were grown in traditional Dulbecco modified Eagle medium (DMEM)-based keratocyte culture medium and an M199-based medium and analyzed for a profile of cell-surface markers by using flow cytometry and differentiated into mesenchymal phenotypes analyzed with quantitative polymerase chain reaction (qPCR) and histologic staining. PLCSCs in M199 were subsequently divided into subpopulations based on CD34 and CD105 expression by using fluorescence- activated cell sorting (FACS). Subpopulations were characterized by marker profile and mesenchymal differentiation ability. Both whole PLCSCs and subpopulations were also cultured for epithelial transdifferentiation. RESULTS Cells cultured in M199 demonstrated a more stem-like cell-surface marker profile, and the keratocyte marker CD34 was retained for several passages but absent in cells cultured in DMEM. Cells cultured in M199 also exhibited a greater mesenchymal differentiation potential, compared with DMEM. PLCSCs could be divided into CD34(+)CD105(+), CD34-CD105(+), and CD34-CD105- subpopulations, of which CD34(+)CD105(+) cells were the most stemlike with regard to marker expression and mesenchymal differentiation potential. Subpopulations of PLCSCs exhibited differing abilities to transdifferentiate into epithelial phenotypes. Cells that were initially CD34(+)CD105(+) showed the greatest differentiation potential, producing CK3(+) and CK19(+) cells, and expressed a range of both epithelial progenitor (HES1, FRZB1, DCT, SOD2, ABCG2, CDH1, KRT19) and terminally differentiated (DSG3, KRT3, KRT12, KRT24) genes. CONCLUSIONS Culture medium has a significant effect on the phenotype and differentiation capacity of PLCSCs. The stroma contains a heterogeneous cell population in which we have identified CD34(+) cells as a stem cell population with a capacity for mesenchymal and epithelial differentiation.
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Ahearne M, Kelly DJ. A comparison of fibrin, agarose and gellan gum hydrogels as carriers of stem cells and growth factor delivery microspheres for cartilage regeneration. Biomed Mater 2013; 8:035004. [DOI: 10.1088/1748-6041/8/3/035004] [Citation(s) in RCA: 47] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/01/2023]
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Lomas AJ, Webb WR, Han J, Chen GQ, Sun X, Zhang Z, El Haj AJ, Forsyth NR. Poly (3-hydroxybutyrate-co-3-hydroxyhexanoate)/collagen hybrid scaffolds for tissue engineering applications. Tissue Eng Part C Methods 2013; 19:577-85. [PMID: 23281705 DOI: 10.1089/ten.tec.2012.0457] [Citation(s) in RCA: 44] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/09/2023] Open
Abstract
The benefits associated with polyhydroxyalkanoates (PHA) in tissue engineering include high immunotolerance, low toxicity, and biodegradability. Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx), a molecule from the PHA family of biopolymers, shares these features. In this study, the applicability of human embryonic stem cells (hESCs), spontaneously differentiated hESCs (SDhESCs), and mesenchymal stem cells (hMSCs) in conjunction with PHBHHx and collagen as a biocompatible replacement strategy for damaged tissues was exploited. Collagen gel contraction was monitored by seeding cells at controlled densities (0, 10(3), 10(4), and 10(5) cells/mL) and measuring length and diameter at regular time intervals thereafter when cultured in a complete medium. Cell viability was measured by trypan blue exclusion assay. Porous PHBHHx tube scaffolds were prepared using a dipping method followed by salt leaching. PHBHHx/collagen composites were generated via syringe injection of collagen/cell mixtures into sterile PHBHHx porous tubes. Reverse transcription polymerase chain reaction was used to determine the fate of cells within PHBHHx/collagen scaffolds with tendon, bone, cartilage, and fat-linked transcript expression being explored at days 0, 5 10, and 20. The capacity of PHBHHx/collagen scaffolds to support differentiation was explored using a medium specific for osteogenic, chondrogenic, and adipogenic lineage generation. Collagen gel tube contraction required initial seeding densities of ≥10(5) hMSCs or SDhESCs in 1.5 mg/mL collagen gel tubes. Gels with a collagen concentration of 3 mg/mL did not display contraction across the examined cell seeding densities. Cell viability was ∼50% for SDhESC and 90% for hMSCs at all cell densities tested in porous PHBHHx tube/3 mg/mL collagen hybrid scaffolds after 20 days in vitro culture. Undifferentiated hESCs did not contract collagen gel tubes and were unviable after 20 days culture. In the absence of additional stimuli, SOX9 was sporadically found, while RUNX2 was not present in both hMSC and SDhESC. Hybrid scaffolds were shown to promote retention of osteogenic, chondrogenic, and adipogenic differentiation by expression of RUNX2, SOX9, and PPARγ genes, respectively, following exposure to the appropriate induction medium. PHBHHx/collagen scaffolds have been successfully used to culture hMSC and SDhESC over an extended period supporting the potential of this scaffold combination in future tissue engineering applications.
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Affiliation(s)
- Alex J Lomas
- Institute for Science and Technology in Medicine, Keele University, Guy Hilton Research Centre, Stoke-on-Trent, United Kingdom
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Lynch AP, Ahearne M. Strategies for developing decellularized corneal scaffolds. Exp Eye Res 2012; 108:42-7. [PMID: 23287438 DOI: 10.1016/j.exer.2012.12.012] [Citation(s) in RCA: 98] [Impact Index Per Article: 7.5] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/19/2012] [Revised: 12/17/2012] [Accepted: 12/18/2012] [Indexed: 12/01/2022]
Abstract
The main obstacle to successfully engineering corneal tissue has been the replication of the structural and biochemical composition of native cornea in a scaffold. In recent years decellularized corneas have been under investigation as an alternative scaffold source for use in engineering cornea. Several strategies for lysing cells and removing cellular material from corneas are discussed. The removal of such cellular components and antigen molecules whilst maintaining the corneal extracellular matrix components and architecture is required to generate scaffolds capable of generating functional tissue grafts suitable for transplantation. Different techniques to ascertain the degree of decellularization and the change in structural, mechanical and biological characteristics of the corneas after treatment are examined. In addition several in vitro and in vivo studies have been performed to ascertain the suitability of decellularized corneas as a scaffold for restoring vision.
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Affiliation(s)
- Amy P Lynch
- Trinity Centre for Bioengineering, Trinity Biomedical Sciences Institute, Trinity College Dublin, Dublin 2, Ireland
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Lomas AJ, Chen GG, El Haj AJ, Forsyth NR. Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) supports adhesion and migration of mesenchymal stem cells and tenocytes. World J Stem Cells 2012; 4. [PMID: 23193433 PMCID: PMC3507844 DOI: 10.4252/wjsc.v4.i9.94] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM To establish the potential of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) as a material for tendon repair. METHODS The biocompatibility of PHBHHx with both rat tenocytes (rT) and human mesenchymal stem cells (hMSC) was explored by monitoring adhesive characteristics on films of varying weight/volume ratios coupled to a culture atmosphere of either 21% O(2) (air) or 2% O(2) (physiological normoxia). The diameter and stiffness of PHBHHx films was established using optical coherence tomography and mechanical testing, respectively. RESULTS Film thickness correlated directly with weight/volume PHBHHx (r(2) = 0.9473) ranging from 0.1 mm (0.8% weight/volume) to 0.19 mm (2.4% weight/volume). Film stiffness on the other hand displayed a biphasic response which increased rapidly at values > 1.6% weight/volume. Optimal cell attachment of rT required films of ≥ 1.6% and ≥ 2.0% weight/volume PHBHHx in 2% O(2) and 21% O(2) respectively. A qualitative adhesion increase was noted for hMSC in films ≥ 1.2% weight/volume, becoming significant at 2% weight/volume in 2% O(2). An increase in cell adhesion was also noted with ≥ 2% weight/volume PHBHHx in 21% O(2). Cell migration into films was not observed. CONCLUSION This evaluation demonstrates that PHBHHx is a suitable polymer for future cell/polymer replacement strategies in tendon repair.
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Affiliation(s)
- Alex J Lomas
- Alex J Lomas, Alicia J El Haj, Nicholas R Forsyth, Guy Hilton Research Centre, Keele University, Stoke on Trent, ST4 7QB, United Kingdom
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O’Dea RD, Osborne JM, El Haj AJ, Byrne HM, Waters SL. The interplay between tissue growth and scaffold degradation in engineered tissue constructs. J Math Biol 2012; 67:1199-225. [DOI: 10.1007/s00285-012-0587-9] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/22/2012] [Revised: 08/31/2012] [Indexed: 01/21/2023]
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Simpson DL, Dudley SC. Modulation of human mesenchymal stem cell function in a three-dimensional matrix promotes attenuation of adverse remodelling after myocardial infarction. J Tissue Eng Regen Med 2011; 7:192-202. [PMID: 22095744 DOI: 10.1002/term.511] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/14/2010] [Revised: 06/01/2011] [Accepted: 07/26/2011] [Indexed: 12/31/2022]
Abstract
The application of tissue engineering (TE) practices for cell delivery offers a unique approach to cellular cardiomyoplasty. We hypothesized that human mesenchymal stem cells (hMSCs) applied to the heart in a collagen matrix would outperform the same cells grown in a monolayer and directly injected for cardiac cell replacement after myocardial infarction in a rat model. When hMSC patches were transplanted to infarcted hearts, several measures for left ventricle (LV) remodelling and function were improved, including fractional area change, wall thickness, -dP/dt and LV end-diastolic pressure. Neovessel formation throughout the LV infarct wall after hMSC patch treatment increased by 37% when compared to direct injection of hMSCs. This observation was correlated with increased secretion of angiogenic factors, with accompanying evidence that these factors enhanced vessel formation (30% increase) and endothelial cell growth (48% increase) in vitro. These observations may explain the in vivo observations of increased vessel formation and improved cardiac function with patch-mediated cell delivery. Although culture of hMSC in collagen patches enhanced angiogenic responses, there was no effect on cell potency or viability. Therefore, hMSCs delivered as a cardiac patch showed benefits above those derived from monolayers and directly injected. hMSCs cultured and delivered within TE constructs may represent a good option to maximize the effects of cellular cardiomyoplasty.
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