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Son I, Kim M, Lee JS, Yoon D, Kim YR, Park JH, Oh BY, Chun W, Kang SB. 3D spheroids versus 2D-cultured human adipose stem cells to generate smooth muscle cells in an internal anal sphincter-targeting cryoinjured mouse model. Stem Cell Res Ther 2024; 15:360. [PMID: 39396044 PMCID: PMC11470548 DOI: 10.1186/s13287-024-03978-9] [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: 04/17/2024] [Accepted: 10/06/2024] [Indexed: 10/14/2024] Open
Abstract
BACKGROUND The efficacy of cell implantation via 3D-spheroids to treat basal tone in fecal incontinence remains unclear. To address this, in this study, we aimed to identify cell differentiation and assess the development of a contractile phenotype corresponding to smooth muscle cells (SMCs) following implantation of 3D-spheroid and 2D-cultured human adipose stem cells (hASCs) in an in vivo internal anal sphincter (IAS)-targeted mouse model. METHODS We developed an IAS-targeted in vivo model via rapid freezing (at - 196 °C) of the dorsal layers of the region of interest (ROI) of the IAS ring posterior quarter, between the submucosal and muscular layers, following submucosal dissection (n = 60 rats). After implantation of tetramethylindocarbocyanine perchlorate (Dil)-stained 3D and 2D-cells into randomly allocated cryoinjured rats, the entire sphincter ring or only the cryoinjured ROI was harvested. Expression of SMC markers, RhoA/ROCKII and its downstream molecules, and fibrosis markers was analyzed. Dil, α-smooth muscle actin (α-SMA), and RhoA signals were used for cell tracking. RESULTS In vitro, 3D-spheroids exhibited higher levels of SMC markers and RhoA/ROCKII-downstream molecules than 2D-hASCs. The IAS-targeted cryoinjured model exhibited substantial loss of SMC layers of the squamous epithelium lining of the anal canal, as well as reduced expression of SMC markers and RhoA-related downstream molecules. In vivo, 3D-spheroid implantation induced SMC markers and contractile molecules weakly at 1 week. At 2 weeks, the mRNA expression of aSma, Sm22a, Smoothelin, RhoA, Mypt1, Mlc20, Cpi17, and Pp1cd increased, whereas that of fibrosis markers reduced significantly in the 3D-spheroid implanted group compared to those in the sham, non-implanted, and 2D-hASC implanted groups. Protein levels of RhoA, p-MYPT1, and p-MLC20 were higher in the 3D-spheroid-implanted group than in the other groups. At 2 weeks, in the implanted groups, the cryoinjured tissues (which exhibited Dil, α-SMA, and RhoA signals) were restored, while they remained defective in the sham and non-implanted groups. CONCLUSIONS These findings demonstrate that, compared to 2D-cultured hASCs, 3D-spheroids more effectively induce a contractile phenotype that is initially weak but subsequently improves, inducing expression of RhoA/ROCKII-downstream molecules and SMC differentiation associated with IAS basal tone.
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Affiliation(s)
- Iltae Son
- Department of Surgery, Hallym Sacred Heart Hospital, Hallym University College of Medicine, 22 Gwanpyeong-Ro 170 Beon-Gil, Pyeongan-Dong, Dongan-Gu, Anyang, Gyeonggi-Do, Republic of Korea.
- Institute for Regenerative Medicine, Hallym Sacred Heart Hospital, Hallym University College of Medicine, Anyang, Republic of Korea.
| | - Minsung Kim
- Department of Surgery, Hallym Sacred Heart Hospital, Hallym University College of Medicine, 22 Gwanpyeong-Ro 170 Beon-Gil, Pyeongan-Dong, Dongan-Gu, Anyang, Gyeonggi-Do, Republic of Korea
| | - Ji-Seon Lee
- Burn Institute, Hangang Sacred Heart Hospital, Hallym University College of Medicine, Seoul, Republic of Korea
| | - Dogeon Yoon
- Burn Institute, Hangang Sacred Heart Hospital, Hallym University College of Medicine, Seoul, Republic of Korea
| | - You-Rin Kim
- Burn Institute, Hangang Sacred Heart Hospital, Hallym University College of Medicine, Seoul, Republic of Korea
| | - Ji Hye Park
- Burn Institute, Hangang Sacred Heart Hospital, Hallym University College of Medicine, Seoul, Republic of Korea
| | - Bo-Young Oh
- Department of Surgery, Hallym Sacred Heart Hospital, Hallym University College of Medicine, 22 Gwanpyeong-Ro 170 Beon-Gil, Pyeongan-Dong, Dongan-Gu, Anyang, Gyeonggi-Do, Republic of Korea
| | - Wook Chun
- Department of Surgery, Hangang Sacred Heart Hospital, Hallym University College of Medicine, Seoul, Republic of Korea
| | - Sung-Bum Kang
- Department of Surgery, Seoul National University Bundang Hospital, 166 Gumi-Ro, Bundang-Gu, 463-707, Seongnam, Republic of Korea.
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Mainali BB, Yoo JJ, Ladd MR. Tissue engineering and regenerative medicine approaches in colorectal surgery. Ann Coloproctol 2024; 40:336-349. [PMID: 39228197 PMCID: PMC11375227 DOI: 10.3393/ac.2024.00437.0062] [Citation(s) in RCA: 1] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/18/2024] [Revised: 07/26/2024] [Accepted: 07/26/2024] [Indexed: 09/05/2024] Open
Abstract
Tissue engineering and regenerative medicine (TERM) is an emerging field that has provided new therapeutic opportunities by delivering innovative solutions. The development of nontraditional therapies for previously unsolvable diseases and conditions has brought hope and excitement to countless individuals globally. Many regenerative medicine therapies have been developed and delivered to patients clinically. The technology platforms developed in regenerative medicine have been expanded to various medical areas; however, their applications in colorectal surgery remain limited. Applying TERM technologies to engineer biological tissue and organ substitutes may address the current therapeutic challenges and overcome some complications in colorectal surgery, such as inflammatory bowel diseases, short bowel syndrome, and diseases of motility and neuromuscular function. This review provides a comprehensive overview of TERM applications in colorectal surgery, highlighting the current state of the art, including preclinical and clinical studies, current challenges, and future perspectives. This article synthesizes the latest findings, providing a valuable resource for clinicians and researchers aiming to integrate TERM into colorectal surgical practice.
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Affiliation(s)
- Bigyan B Mainali
- Department of General Surgery, Atrium Health Wake Forest Baptist, Winston-Salem, NC, USA
| | - James J Yoo
- Wake Forest Institute for Regenerative Medicine, Winston-Salem, NC, USA
- Department of Biomedical Engineering, Wake Forest University, Winston-Salem, NC, USA
| | - Mitchell R Ladd
- Department of General Surgery, Atrium Health Wake Forest Baptist, Winston-Salem, NC, USA
- Wake Forest Institute for Regenerative Medicine, Winston-Salem, NC, USA
- Department of Biomedical Engineering, Wake Forest University, Winston-Salem, NC, USA
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Lu P, Lifshitz LM, Bellve K, ZhuGe R. TMEM16A in smooth muscle cells acts as a pacemaker channel in the internal anal sphincter. Commun Biol 2024; 7:151. [PMID: 38317010 PMCID: PMC10844222 DOI: 10.1038/s42003-024-05850-1] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/13/2022] [Accepted: 01/23/2024] [Indexed: 02/07/2024] Open
Abstract
Maintenance of fecal continence requires a continuous or basal tone of the internal anal sphincter (IAS). Paradoxically, the basal tone results largely from high-frequency rhythmic contractions of the IAS smooth muscle. However, the cellular and molecular mechanisms that initiate these contractions remain elusive. Here we show that the IAS contains multiple pacemakers. These pacemakers spontaneously generate propagating calcium waves that drive rhythmic contractions and establish the basal tone. These waves are myogenic and act independently of nerve, paracrine or autocrine signals. Using cell-specific gene knockout mice, we further found that TMEM16A Cl- channels in smooth muscle cells (but not in the interstitial cells of Cajal) are indispensable for pacemaking, rhythmic contractions, and basal tone. Our results identify TMEM16A in smooth muscle cells as a critical pacemaker channel that enables the IAS to contract rhythmically and continuously. This study provides cellular and molecular insights into fecal continence.
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Affiliation(s)
- Ping Lu
- Department of Microbiology and Physiological Systems, University of Massachusetts Chan Medical School, Worcester, MA, USA
| | - Lawrence M Lifshitz
- Program in Molecular Medicine, University of Massachusetts Chan Medical School, Worcester, MA, USA
| | - Karl Bellve
- Program in Molecular Medicine, University of Massachusetts Chan Medical School, Worcester, MA, USA
| | - Ronghua ZhuGe
- Department of Microbiology and Physiological Systems, University of Massachusetts Chan Medical School, Worcester, MA, USA.
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Jafari A, Behjat E, Malektaj H, Mobini F. Alignment behavior of nerve, vascular, muscle, and intestine cells in two- and three-dimensional strategies. WIREs Mech Dis 2023; 15:e1620. [PMID: 37392045 DOI: 10.1002/wsbm.1620] [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: 07/31/2022] [Revised: 02/28/2023] [Accepted: 05/23/2023] [Indexed: 07/02/2023]
Abstract
By harnessing structural hierarchical insights, plausibly simulate better ones imagination to figure out the best choice of methods for reaching out the unprecedented developments of the tissue engineering products as a next level. Constructing a functional tissue that incorporates two-dimensional (2D) or higher dimensions requires overcoming technological or biological limitations in order to orchestrate the structural compilation of one-dimensional and 2D sheets (microstructures) simultaneously (in situ). This approach enables the creation of a layered structure that can be referred to as an ensemble of layers or, after several days of maturation, a direct or indirect joining of layers. Here, we have avoided providing a detailed methodological description of three-dimensional and 2D strategies, except for a few interesting examples that highlight the higher alignment of cells and emphasize rarely remembered facts associated with vascular, peripheral nerve, muscle, and intestine tissues. The effective directionality of cells in conjunction with geometric cues (in the range of micrometers) is well known to affect a variety of cell behaviors. The curvature of a cell's environment is one of the factors that influence the formation of patterns within tissues. The text will cover cell types containing some level of stemness, which will be followed by their consequences for tissue formation. Other important considerations pertain to cytoskeleton traction forces, cell organelle positioning, and cell migration. An overview of cell alignment along with several pivotal molecular and cellular level concepts, such as mechanotransduction, chirality, and curvature of structure effects on cell alignments will be presented. The mechanotransduction term will be used here in the context of the sensing capability that cells show as a result of force-induced changes either at the conformational or the organizational levels, a capability that allows us to modify cell fate by triggering downstream signaling pathways. A discussion of the cells' cytoskeleton and of the stress fibers involvement in altering the cell's circumferential constitution behavior (alignment) based on exposed scaffold radius will be provided. Curvatures with size similarities in the range of cell sizes cause the cell's behavior to act as if it was in an in vivo tissue environment. The revision of the literature, patents, and clinical trials performed for the present study shows that there is a clear need for translational research through the implementation of clinical trial platforms that address the tissue engineering possibilities raised in the current revision. This article is categorized under: Infectious Diseases > Biomedical Engineering Neurological Diseases > Biomedical Engineering Cardiovascular Diseases > Biomedical Engineering.
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Affiliation(s)
- Amir Jafari
- Laboratório de Neurofisiologia, Instituto de Biologia Roberto Alcantara Gomes, Centro Biomédico, Universidade do Estado do Rio de Janeiro, Rio de Janeiro, Brazil
| | - Erfan Behjat
- Department of Biomaterials, School of Metallurgy & Materials Engineering, Iran University of Science and Technology, Tehran, Iran
| | - Haniyeh Malektaj
- Department of Materials and Production, Aalborg University, Aalborg, Denmark
| | - Faezeh Mobini
- Molecular Simulation Research Laboratory, Department of Chemistry, Iran University of Science and Technology, Tehran, Iran
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Sarveazad A, Yari A, Imani F, Fayyaz F, Mokhtare M, Babaei-Ghazani A, Yousefifard M, Sarveazad S, Assar S, Shamseddin J, Bahardoust M. The effect of Trolox on the rabbit anal sphincterotomy repair. BMC Gastroenterol 2023; 23:209. [PMID: 37337166 DOI: 10.1186/s12876-023-02842-z] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 11/03/2022] [Accepted: 05/31/2023] [Indexed: 06/21/2023] Open
Abstract
INTRODUCTION Fecal incontinence (FI) is caused by external anal sphincter injury. Vitamin E is a potential strategy for anal sphincter muscle repair via its antioxidant, anti-inflammatory, anti-fibrotic, and protective properties against myocyte loss. Thus, we aimed to evaluate the water-soluble form of vitamin E efficacy in repairing anal sphincter muscle defects in rabbits. METHODS Twenty-one male rabbits were equally assigned to the intact (without any intervention), control (sphincterotomy), and Trolox (sphincterotomy + Trolox administration) groups. Ninety days after sphincterotomy, the resting and squeeze pressures were evaluated by manometry, and the number of motor units in the sphincterotomy site was calculated by electromyography. Also, the amount of muscle and collagen in the injury site was investigated by Mallory's trichrome staining. RESULTS Ninety days after the intervention, the resting and squeeze pressures in the intact and Trolox groups were significantly higher than in the control group (P = 0.001). Moreover, the total collagen percentage of the sphincterotomy site was significantly lower in the Trolox group than in the control group (P = 0.002), and the total muscle percentage was significantly higher in the Trolox group compared to the control group (P = 0.001). Also, the motor unit number was higher in the Trolox group than in the control group (P = 0.001). CONCLUSION Trolox administration in the rabbit sphincterotomy model can decrease the amount of collagen and increase muscle, leading to improved anal sphincter electromyography and manometry results. Therefore, Trolox is a potential treatment strategy for FI.
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Affiliation(s)
- Arash Sarveazad
- Colorectal Research Center, Iran University of Medical Sciences, Tehran, Iran
- Nursing Care Research Center, Iran University of Medical Sciences, Tehran, Iran
| | - Abazar Yari
- Department of Anatomy, Faculty of Medicine, Alborz University of Medical Sciences, Karaj, Iran
| | - Farnad Imani
- Pain Research Center, Department of Anesthesiology and Pain Medicine, Iran University of Medical Sciences, Tehran, Iran
| | - Farimah Fayyaz
- Colorectal Research Center, Iran University of Medical Sciences, Tehran, Iran
| | - Marjan Mokhtare
- Colorectal Research Center, Iran University of Medical Sciences, Tehran, Iran
| | - Arash Babaei-Ghazani
- Neuromusculoskeletal Research Center, Department of Physical Medicine and Rehabilitation, Iran University of Medical Sciences, Tehran, Iran
- Department of Physical Medicine and Rehabilitation, University of Montreal Health Center, Montreal, Canada
| | - Mahmoud Yousefifard
- Physiology Research Center, Iran University of Medical Sciences, Tehran, Iran
| | - Shahriar Sarveazad
- Colorectal Research Center, Iran University of Medical Sciences, Tehran, Iran
| | - Siavash Assar
- Department of Anesthesiology, kerman university of medical sciences, kerman, Iran
| | - Jebreil Shamseddin
- Infectious and Tropical Diseases Research Center, Hormozgan Health Institute, Hormozgan University of Medical Sciences, Bandar Abbas, Shahid Chamran Boulevard, Iran.
| | - Mansour Bahardoust
- Department of Epidemiology, School of Public Health, Shahid Beheshti University of Medical Sciences, Velenjak 7th Floor, Bldg No.2 SBUMS, Arabi Ave, Tehran, 19839-63113, Iran.
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Corti A, Shameen T, Sharma S, De Paolis A, Cardoso L. Biaxial testing system for characterization of mechanical and rupture properties of small samples. HARDWAREX 2022; 12:e00333. [PMID: 35795084 PMCID: PMC9251720 DOI: 10.1016/j.ohx.2022.e00333] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Received: 02/02/2022] [Revised: 06/21/2022] [Accepted: 06/25/2022] [Indexed: 06/15/2023]
Abstract
The study of damage and rupture of soft tissues using a tensile testing system is essential to understand the limits of mechanical behavior and loss of function in diseased tissues. However, commercial material testing systems are often expensive and may not be fully suitable for rupture tests of small samples. While several research laboratories have developed custom, less expensive, uniaxial or biaxial devices, there is a need for an open source, inexpensive, accurate and easy to customize biaxial material testing system to perform rupture tests in small soft samples. We designed a testing system (BiMaTS) that (a) was shown able to perform uniaxial and biaxial tests, (b) offers a large travel range for rupture tests of small samples, (c) maintains a centered field of view for effective strain mapping using digital image correlation, (d) provides a controlled temperature environment, (e) utilize many off-the-shelve components for easy manufacture and customization, and it is cost effective (∼$15 K). The instrument performance was characterized using 80%-scaled down, ASTM D412-C shaped PDMS samples. Our results demonstrate the ability of this open source, customizable, low-cost, biaxial materials testing system to successfully characterize the mechanical and rupture properties of small samples with high repeatability and accuracy.
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Corti A, Shameen T, Sharma S, De Paolis A, Cardoso L. Tunable elastomer materials with vascular tissue-like rupture mechanics behavior. Biomed Phys Eng Express 2022; 8. [PMID: 35863160 DOI: 10.1088/2057-1976/ac82f6] [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: 03/18/2022] [Accepted: 07/21/2022] [Indexed: 11/11/2022]
Abstract
Purpose:Laboratory models of human arterial tissues are advantageous to examine the mechanical response of blood vessels in a simplified and controllable manner. In the present study, we investigated three silicone-based materials for replicating the mechanical properties of human arteries documented in the literature.Methods:We performed uniaxial tensile tests up to rupture on Sylgard184, Sylgard170 and DowsilEE-3200 under different curing conditions and obtained their True (Cauchy) stress-strain behavior and Poisson's ratios by means of digital image correlation (DIC). For each formulation, we derived the constitutive parameters of the 3-term Ogden model and designed numerical simulations of tubular models under a radial pressure of 250mmHg.Results:Each material exhibits evident non-linear hyperelasticity and dependence on the curing condition. Sylgard184 is the stiffest formulation, with the highest shear moduli and ultimate stresses at relative low strains (µ184=0.52-0.88MPa, σ184=15.90-16.54MPa, ε184=0.72-0.96). Conversely, Sylgard170 and DowsilEE-3200 present significantly lower shear moduli and ultimate stresses that are closer to data reported for arterial tissues (µ170=0.33-0.7MPa σ170=2.61-3.67MPa, ε170=0.69-0.81; µdow=0.02-0.09MPa σdow=0.83-2.05MPa, εdow=0.91-1.05). Under radial pressure, all formulations except DowsilEE-3200 at 1:1 curing ratio undergo circumferential stresses that remain in the elastic region with values ranging from 0.1 to 0.18MPa. Conclusion: Sylgard170 and DowsilEE-3200 appear to better reproduce the rupture behavior of vascular tissues within their typical ultimate stress and strain range. Numerical models demonstrate that all three materials achieve circumferential stresses similar to human common carotid arteries (Sommer et al. 2010), making these formulations suited for cylindrical laboratory models under physiological and supraphysiological loading.
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Affiliation(s)
- Andrea Corti
- The City College of New York, 275 Convent Ave, New York, New York, 10031-9101, UNITED STATES
| | - Tariq Shameen
- The City College of New York, 275 Convent Ave, New York, New York, 10031-9101, UNITED STATES
| | - Shivang Sharma
- The City College of New York, 275 Convent Ave, New York, New York, 10031-9101, UNITED STATES
| | - Annalisa De Paolis
- The City College of New York, 275 Convent Ave, New York, New York, 10031-9101, UNITED STATES
| | - Luis Cardoso
- Biomedical Engineering, The City College of New York, 275 Convent Ave, New York, New York, New York, 10031-9101, UNITED STATES
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Kim M, Oh BY, Lee JS, Yoon D, Chun W, Son IT. A systematic review of translation and experimental studies on internal anal sphincter for fecal incontinence. Ann Coloproctol 2022; 38:183-196. [PMID: 35678021 PMCID: PMC9263305 DOI: 10.3393/ac.2022.00276.0039] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/21/2022] [Accepted: 05/10/2022] [Indexed: 12/05/2022] Open
Abstract
The complexity in the molecular mechanism of the internal anal sphincter (IAS) limits preclinical or clinical outcomes of fecal incontinence (FI) treatment. So far, there are no systematic reviews of IAS translation and experimental studies that have been reported. This systematic review aims to provide a comprehensive understanding of IAS critical role in FI. Previous studies revealed the key pathway for basal tone and relaxation of IAS in different properties as follows; calcium, Rho-associated, coiled-coil containing serine/threonine kinase, aging-associated IAS dysfunction, oxidative stress, renin-angiotensin-aldosterone, cyclooxygenase, and inhibitory neurotransmitters. Previous studies have reported improved functional outcomes of cellular treatment for regeneration of dysfunctional IAS, using various stem cells, but did not demonstrate the interrelationship between those results and basal tone or relaxation-related molecular pathway of IAS. Furthermore, these results have lower specificity for IAS-incontinence due to the included external anal sphincter or nerve injury regardless of the cell type. An acellular approach using bioengineered IAS showed a physiologic response of basal tone and relaxation response similar to human IAS. However, in both cellular and acellular approaches, the lack of human IAS data still hampers clinical application. Therefore, the IAS regeneration presents more challenges and warrants more advances.
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Affiliation(s)
- Minsung Kim
- Department of Surgery, Hallym University Sacred Heart Hospital, Hallym University College of Medicine, Anyang, Korea
| | - Bo-Young Oh
- Department of Surgery, Hallym University Sacred Heart Hospital, Hallym University College of Medicine, Anyang, Korea
| | - Ji-Seon Lee
- Burn Institute, Hangang Sacred Heart Hospital, Hallym University College of Medicine, Seoul, Korea
| | - Dogeon Yoon
- Burn Institute, Hangang Sacred Heart Hospital, Hallym University College of Medicine, Seoul, Korea
| | - Wook Chun
- Burn Institute, Hangang Sacred Heart Hospital, Hallym University College of Medicine, Seoul, Korea.,Department of Surgery, Hangang Sacred Heart Hospital, Hallym University College of Medicine, Seoul, Korea
| | - Il Tae Son
- Department of Surgery, Hallym University Sacred Heart Hospital, Hallym University College of Medicine, Anyang, Korea.,Institute for Regenerative Medicine, Hallym University Sacred Heart Hospital, Hallym University College of Medicine, Anyang, Korea
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Balaphas A, Meyer J, Meier RPH, Liot E, Buchs NC, Roche B, Toso C, Bühler LH, Gonelle-Gispert C, Ris F. Cell Therapy for Anal Sphincter Incontinence: Where Do We Stand? Cells 2021; 10:2086. [PMID: 34440855 PMCID: PMC8394955 DOI: 10.3390/cells10082086] [Citation(s) in RCA: 8] [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: 06/30/2021] [Revised: 08/05/2021] [Accepted: 08/06/2021] [Indexed: 12/12/2022] Open
Abstract
Anal sphincter incontinence is a chronic disease, which dramatically impairs quality of life and induces high costs for the society. Surgery, considered as the best curative option, shows a disappointing success rate. Stem/progenitor cell therapy is pledging, for anal sphincter incontinence, a substitute to surgery with higher efficacy. However, the published literature is disparate. Our aim was to perform a review on the development of cell therapy for anal sphincter incontinence with critical analyses of its pitfalls. Animal models for anal sphincter incontinence were varied and tried to reproduce distinct clinical situations (acute injury or healed injury with or without surgical reconstruction) but were limited by anatomical considerations. Cell preparations used for treatment, originated, in order of frequency, from skeletal muscle, bone marrow or fat tissue. The characterization of these preparations was often incomplete and stemness not always addressed. Despite a lack of understanding of sphincter healing processes and the exact mechanism of action of cell preparations, this treatment was evaluated in 83 incontinent patients, reporting encouraging results. However, further development is necessary to establish the correct indications, to determine the most-suited cell type, to standardize the cell preparation method and to validate the route and number of cell delivery.
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Affiliation(s)
- Alexandre Balaphas
- Division of Digestive Surgery, University Hospitals of Geneva, 1205 Geneva, Switzerland; (J.M.); (E.L.); (N.C.B.); (B.R.); (C.T.); (F.R.)
- Department of Surgery, Geneva Medical School, University of Geneva, 1205 Geneva, Switzerland
| | - Jeremy Meyer
- Division of Digestive Surgery, University Hospitals of Geneva, 1205 Geneva, Switzerland; (J.M.); (E.L.); (N.C.B.); (B.R.); (C.T.); (F.R.)
| | - Raphael P. H. Meier
- Department of Surgery, University of Maryland School of Medicine, Baltimore, MD 21201, USA;
| | - Emilie Liot
- Division of Digestive Surgery, University Hospitals of Geneva, 1205 Geneva, Switzerland; (J.M.); (E.L.); (N.C.B.); (B.R.); (C.T.); (F.R.)
| | - Nicolas C. Buchs
- Division of Digestive Surgery, University Hospitals of Geneva, 1205 Geneva, Switzerland; (J.M.); (E.L.); (N.C.B.); (B.R.); (C.T.); (F.R.)
| | - Bruno Roche
- Division of Digestive Surgery, University Hospitals of Geneva, 1205 Geneva, Switzerland; (J.M.); (E.L.); (N.C.B.); (B.R.); (C.T.); (F.R.)
| | - Christian Toso
- Division of Digestive Surgery, University Hospitals of Geneva, 1205 Geneva, Switzerland; (J.M.); (E.L.); (N.C.B.); (B.R.); (C.T.); (F.R.)
| | - Leo H. Bühler
- Faculty of Science and Medicine, University of Fribourg, 1700 Fribourg, Switzerland; (L.H.B.); (C.G.-G.)
| | - Carmen Gonelle-Gispert
- Faculty of Science and Medicine, University of Fribourg, 1700 Fribourg, Switzerland; (L.H.B.); (C.G.-G.)
| | - Frédéric Ris
- Division of Digestive Surgery, University Hospitals of Geneva, 1205 Geneva, Switzerland; (J.M.); (E.L.); (N.C.B.); (B.R.); (C.T.); (F.R.)
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Zakhem E, Raghavan S, Suhar RA, Bitar KN. Bioengineering and regeneration of gastrointestinal tissue: where are we now and what comes next? Expert Opin Biol Ther 2019; 19:527-537. [PMID: 30880502 DOI: 10.1080/14712598.2019.1595579] [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] [Indexed: 01/04/2023]
Abstract
INTRODUCTION The field of tissue engineering and regenerative medicine has been applied to the gastrointestinal (GI) tract for a couple decades. Several achievements have been accomplished that provide promising tools for treating diseases of the GI tract. AREAS COVERED The work described in this review covers the traditional aspect of using cells and scaffolds to replace parts of the tract. Several studies investigated different types of biomaterials and different types of cells. A more recent approach involved the use of gut-derived organoid units that can differentiate into all gut cell layers. The most recent approach introduced the use of organ-on-a-chip concept to understand the physiology and pathophysiology of the GI system. EXPERT OPINION The different approaches tackle the diseases of the GI tract from different perspectives. While all these different approaches provide a promising and encouraging future for this field, the translational aspect is yet to be studied.
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Affiliation(s)
- Elie Zakhem
- a Wake Forest Institute for Regenerative Medicine , Wake Forest School of Medicine , Winston Salem , NC , USA.,b Section on Gastroenterology , Wake Forest School of Medicine , Winston Salem , NC , USA
| | - Shreya Raghavan
- c Department of Materials Science and Engineering , University of Michigan , Ann Arbor , MI , USA
| | - Riley A Suhar
- d Department of Materials Science and Engineering , Stanford University , Stanford , CA , USA
| | - Khalil N Bitar
- a Wake Forest Institute for Regenerative Medicine , Wake Forest School of Medicine , Winston Salem , NC , USA.,b Section on Gastroenterology , Wake Forest School of Medicine , Winston Salem , NC , USA.,e Virginia Tech-Wake Forest School of Biomedical Engineering Sciences , Winston-Salem , NC , USA
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Son IT, Lee HS, Ihn MH, Lee KH, Kim DW, Lee KW, Kim JS, Kang SB. Isolation of internal and external sphincter progenitor cells from the human anal sphincter with or without radiotherapy. Colorectal Dis 2019; 21:38-47. [PMID: 30047583 DOI: 10.1111/codi.14351] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/26/2018] [Accepted: 07/16/2018] [Indexed: 02/08/2023]
Abstract
AIM We aimed to isolate and propagate internal and external anal sphincter progenitor cells from the human anal sphincter, with or without radiotherapy, for tailored cell therapy of faecal incontinence. METHODS Sphincter progenitor cells were isolated from normal internal and external anal sphincters collected from 10 patients with rectal cancer who had undergone abdominoperineal resection with (n = 6) or without (n = 4) preoperative chemoradiotherapy. The isolated cells and differentiated muscle fibres were identified using immunofluorescence assay, western blotting and reverse transcription polymerase chain reaction (RT-PCR). The proliferation of progenitor cells with and without radiotherapy was compared by quantitative 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. RESULTS The immunofluorescence assay before differentiation confirmed that the internal anal sphincter progenitor cells expressed CD34 and neural-glial antigen 2 (NG2), whereas the external anal sphincter progenitor cells expressed CD34 and PAX7. After differentiation, the internal anal sphincter progenitor cells expressed desmin, calponin and α-smooth muscle actin, whereas the external anal sphincter progenitor cells expressed desmin, myogenic factor 4 and myosin heavy chain. The differential expression profiles of both cell types were confirmed by western blotting and RT-PCR. MTT assays showed that the viability of internal and external anal sphincter progenitor cells was significantly lower in the radiotherapy group than that in the nonradiotherapy group. CONCLUSIONS This study describes the differential harvest internal and external sphincter muscle progenitor cells from human anal sphincters. We confirm that radiotherapy decreases the viability of internal and external anal sphincter progenitor cells.
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Affiliation(s)
- I T Son
- Department of Surgery, Seoul National University Bundang Hospital, Seongnam, South Korea
| | - H S Lee
- Department of Surgery, Uijeongbu St Mary's Hospital, Catholic University, Uijeongbu-si, South Korea
| | - M H Ihn
- Department of Pathology, Seoul National University College of Medicine, Seoul National University Bundang Hospital, Seongnam, South Korea
| | - K H Lee
- Department of Radiology, Seoul National University College of Medicine, Seoul National University Bundang Hospital, Seongnam, South Korea
| | - D-W Kim
- Department of Pathology, Seoul National University College of Medicine, Seoul National University Bundang Hospital, Seongnam, South Korea
| | - K-W Lee
- Department of Hemato-Oncology, Seoul National University College of Medicine, Seoul National University Bundang Hospital, Seongnam, South Korea
| | - J-S Kim
- Department of Radiation Oncology, Seoul National University College of Medicine, Seoul National University Bundang Hospital, Seongnam, South Korea
| | - S-B Kang
- Department of Pathology, Seoul National University College of Medicine, Seoul National University Bundang Hospital, Seongnam, South Korea
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Trébol J, Carabias-Orgaz A, García-Arranz M, García-Olmo D. Stem cell therapy for faecal incontinence: Current state and future perspectives. World J Stem Cells 2018; 10:82-105. [PMID: 30079130 PMCID: PMC6068732 DOI: 10.4252/wjsc.v10.i7.82] [Citation(s) in RCA: 17] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/21/2018] [Revised: 06/26/2018] [Accepted: 06/30/2018] [Indexed: 02/06/2023] Open
Abstract
Faecal continence is a complex function involving different organs and systems. Faecal incontinence is a common disorder with different pathogeneses, disabling consequences and high repercussions for quality of life. Current management modalities are not ideal, and the development of new treatments is needed. Since 2008, stem cell therapies have been validated, 36 publications have appeared (29 in preclinical models and seven in clinical settings), and six registered clinical trials are currently ongoing. Some publications have combined stem cells with bioengineering technologies. The aim of this review is to identify and summarise the existing published knowledge of stem cell utilization as a treatment for faecal incontinence. A narrative or descriptive review is presented. Preclinical studies have demonstrated that cellular therapy, mainly in the form of local injections of muscle-derived (muscle derived stem cells or myoblasts derived from them) or mesenchymal (bone-marrow- or adipose-derived) stem cells, is safe. Cellular therapy has also been shown to stimulate the repair of both acute and subacute anal sphincter injuries, and some encouraging functional results have been obtained. Stem cells combined with normal cells on bioengineered scaffolds have achieved the successful creation and implantation of intrinsically-innervated anal sphincter constructs. The clinical evidence, based on adipose-derived stem cells and myoblasts, is extremely limited yet has yielded some promising results, and appears to be safe. Further investigation in both animal models and clinical settings is necessary to drawing conclusions. Nevertheless, if the preliminary results are confirmed, stem cell therapy for faecal incontinence may well become a clinical reality in the near future.
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Affiliation(s)
- Jacobo Trébol
- General and Digestive Tract Surgery Department, Salamanca University Healthcare Centre, Salamanca 37007, Spain
| | - Ana Carabias-Orgaz
- Anaesthesiology Department, Complejo Asistencial de Ávila, Ávila 05004, Spain
| | - Mariano García-Arranz
- New Therapies Laboratory, Instituto de Investigación Sanitaria-Fundación Jiménez Díaz, Madrid 28040, Spain
| | - Damián García-Olmo
- General and Digestive Tract Surgery Department, Quiron-Salud Hospitals, Madrid 28040, Spain
- Surgery Department, Universidad Autónoma, Madrid 28040, Spain
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13
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Continence technologies whitepaper: Informing new engineering science research. Proc Inst Mech Eng H 2018; 233:138-153. [DOI: 10.1177/0954411918784073] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/19/2022]
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Sarveazad A, Newstead GL, Mirzaei R, Joghataei MT, Bakhtiari M, Babahajian A, Mahjoubi B. A new method for treating fecal incontinence by implanting stem cells derived from human adipose tissue: preliminary findings of a randomized double-blind clinical trial. Stem Cell Res Ther 2017; 8:40. [PMID: 28222801 PMCID: PMC5320771 DOI: 10.1186/s13287-017-0489-2] [Citation(s) in RCA: 38] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/28/2016] [Revised: 12/23/2016] [Accepted: 01/24/2017] [Indexed: 12/17/2022] Open
Abstract
BACKGROUND Anal sphincter defects are a major cause of fecal incontinence causing negative effects on daily life, social interactions, and mental health. Because human adipose-derived stromal/stem cells (hADSCs) are easier and safer to access, secrete high levels of growth factor, and have the potential to differentiate into muscle cells, we investigated the ability of hADSCs to improve anal sphincter incontinence. METHODS The present randomized double-blind clinical trial was performed on patients with sphincter defects. They were categorized into a cell group (n = 9) and a control group (n = 9). Either 6 × 106 hADSCs per 3 ml suspended in phosphate buffer saline (treatment) or 3 ml phosphate buffer saline (placebo) was injected. Two months after surgery, the Wexner score, endorectal sonography, and electromyography (EMG) results were recorded. RESULTS Comparing Wexner scores in the cell group and the control group showed no significant difference. In our EMG and endorectal sonography analysis using ImageJ/Fiji 1.46 software, the ratio of the area occupied by the muscle to total area of the lesion showed a 7.91% increase in the cell group compared with the control group. CONCLUSION The results of the current study show that injection of hADSCs during repair surgery for fecal incontinence may cause replacement of fibrous tissue, which acts as a mechanical support to muscle tissue with contractile function. This is a key point in treatment of fecal incontinence especially in the long term and may be a major step forward. TRIAL REGISTRATION Iranian Registry of Clinical Trials IRCT2016022826316N2 . Retrospectively registered 7 May 2016.
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Affiliation(s)
- Arash Sarveazad
- Colorectal Research Center, Iran University of Medical Sciences, Tehran, Iran
| | - Graham L Newstead
- Sydney Colorectal Associates, Prince of Wales Hospital, Randswick, NSW, Australia
| | - Rezvan Mirzaei
- Colorectal Research Center, Iran University of Medical Sciences, Tehran, Iran
| | - Mohammad Taghi Joghataei
- Cellular and Molecular Research Center, Faculty of Medicine, Iran University of Medical Sciences, Tehran, Iran
| | - Mehrdad Bakhtiari
- Cellular and Molecular Research Center, Faculty of Medicine, Iran University of Medical Sciences, Tehran, Iran
| | - Asrin Babahajian
- Liver and Digestive Research Center, Kurdistan University of Medical Sciences, Sanandaj, Iran
| | - Bahar Mahjoubi
- Colorectal Research Center, Iran University of Medical Sciences, Tehran, Iran.
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15
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Zakhem E, El Bahrawy M, Orlando G, Bitar KN. Biomechanical properties of an implanted engineered tubular gut-sphincter complex. J Tissue Eng Regen Med 2016; 11:3398-3407. [PMID: 27882697 DOI: 10.1002/term.2253] [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: 12/15/2015] [Revised: 04/13/2016] [Accepted: 07/03/2016] [Indexed: 12/26/2022]
Abstract
Neuromuscular diseases of the gut alter the normal motility patterns. Although surgical intervention remains the standard treatment, preservation of the sphincter attached to the rest of the gut is challenging. The present study aimed to evaluate a bioengineered gut-sphincter complex following its subcutaneous implantation for 4 weeks in rats. Engineered innervated human smooth muscle sheets and innervated human sphincters with a predefined alignment were placed around tubular scaffolds to create a gut-sphincter complex. The engineered complex was subcutaneously implanted in the abdomen of the rats for 4 weeks. The implanted tissues were vascularized. In vivo manometry revealed luminal pressure at the gut and the sphincter zone. Tensile strength, elongation at break and Young's modulus of the engineered complexes were similar to those of native rat intestine. Histological and immunofluorescence assays showed maintenance of smooth muscle circular alignment in the engineered tissue, maintenance of smooth muscle contractile phenotype and innervation of the smooth muscle. Electrical field stimulation induced relaxation of the smooth muscle of both the sphincter and the gut parts. Relaxation was partly inhibited by nitric oxide inhibitor indicating nitrergic contribution to relaxation. The present study has demonstrated for the first time a successfully developed and subcutaneously implanted a tubular human-derived gut-sphincter complex. The sphincteric part of Tubular Gut-Sphincter Complex (TGSC) maintained the basal tone characteristic of a native sphincter. The gut part also maintained its specific neuromuscular characteristics. The results of this study provide a promising therapeutic approach to restore gut continuity and motility. Copyright © 2016 John Wiley & Sons, Ltd.
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Affiliation(s)
- Elie Zakhem
- Wake Forest Institute for Regenerative Medicine, Wake Forest School of Medicine, Winston Salem, NC, USA.,Department of Molecular Medicine and Translational Sciences, Wake Forest School of Medicine, Winston Salem, NC, USA
| | - Mostafa El Bahrawy
- Wake Forest Institute for Regenerative Medicine, Wake Forest School of Medicine, Winston Salem, NC, USA
| | - Giuseppe Orlando
- Department of General Surgery, Wake Forest School of Medicine, Winston-Salem, NC, USA
| | - Khalil N Bitar
- Wake Forest Institute for Regenerative Medicine, Wake Forest School of Medicine, Winston Salem, NC, USA.,Department of Molecular Medicine and Translational Sciences, Wake Forest School of Medicine, Winston Salem, NC, USA.,Virginia Tech-Wake Forest School of Biomedical Engineering and Sciences, Winston Salem, NC, USA
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16
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Abstract
Functions of the gastrointestinal tract include motility, digestion and absorption of nutrients. These functions are mediated by several specialized cell types including smooth muscle cells, neurons, interstitial cells and epithelial cells. In gastrointestinal diseases, some of the cells become degenerated or fail to accomplish their normal functions. Surgical resection of the diseased segments of the gastrointestinal tract is considered the gold-standard treatment in many cases, but patients might have surgical complications and quality of life can remain low. Tissue engineering and regenerative medicine aim to restore, repair, or regenerate the function of the tissues. Gastrointestinal tissue engineering is a challenging process given the specific phenotype and alignment of each cell type that colonizes the tract - these properties are critical for proper functionality. In this Review, we summarize advances in the field of gastrointestinal tissue engineering and regenerative medicine. Although the findings are promising, additional studies and optimizations are needed for translational purposes.
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Affiliation(s)
- Khalil N Bitar
- Wake Forest Institute for Regenerative Medicine, Wake Forest School of Medicine, 391 Technology Way NE, Winston Salem, North Carolina 27101, USA.,Department of Molecular Medicine and Translational Sciences, Wake Forest School of Medicine, 1 Medical Center Blvd, Winston Salem, North Carolina 27157, USA.,Virginia Tech-Wake Forest School of Biomedical Engineering and Sciences, 391 Technology Way NE, Winston Salem, North Carolina 27101, USA
| | - Elie Zakhem
- Wake Forest Institute for Regenerative Medicine, Wake Forest School of Medicine, 391 Technology Way NE, Winston Salem, North Carolina 27101, USA.,Department of Molecular Medicine and Translational Sciences, Wake Forest School of Medicine, 1 Medical Center Blvd, Winston Salem, North Carolina 27157, USA
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17
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Gräs S, Tolstrup CK, Lose G. Regenerative medicine provides alternative strategies for the treatment of anal incontinence. Int Urogynecol J 2016; 28:341-350. [PMID: 27311602 DOI: 10.1007/s00192-016-3064-y] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/16/2016] [Accepted: 06/06/2016] [Indexed: 12/17/2022]
Abstract
INTRODUCTION AND HYPOTHESIS Anal incontinence is a common disorder but current treatment modalities are not ideal and the development of new treatments is needed. The aim of this review was to identify the existing knowledge of regenerative medicine strategies in the form of cellular therapies or bioengineering as a treatment for anal incontinence caused by anal sphincter defects. METHODS PubMed was searched for preclinical and clinical studies in English published from January 2005 to January 2016. RESULTS Animal studies have demonstrated that cellular therapy in the form of local injections of culture-expanded skeletal myogenic cells stimulates repair of both acute and 2 - 4-week-old anal sphincter injuries. The results from a small clinical trial with ten patients and a case report support the preclinical findings. Animal studies have also demonstrated that local injections of mesenchymal stem cells stimulate repair of sphincter injuries, and a complex bioengineering strategy for creation and implantation of an intrinsically innervated internal anal sphincter construct has been successfully developed in a series of animal studies. CONCLUSION Cellular therapies with myogenic cells and mesenchymal stem cells and the use of bioengineering technology to create an anal sphincter are new potential strategies to treat anal incontinence caused by anal sphincter defects, but the clinical evidence is extremely limited. The use of culture-expanded autologous skeletal myogenic cells has been most intensively investigated and several clinical trials were ongoing at the time of this report. The cost-effectiveness of such a therapy is an issue and muscle fragmentation is suggested as a simple alternative.
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Affiliation(s)
- Søren Gräs
- Department of Obstetrics and Gynecology, Copenhagen University Hospital Herlev, Herlev Ringvej 75, DK-2730, Herlev, Denmark.
| | - Cæcilie Krogsgaard Tolstrup
- Department of Obstetrics and Gynecology, Copenhagen University Hospital Herlev, Herlev Ringvej 75, DK-2730, Herlev, Denmark
| | - Gunnar Lose
- Department of Obstetrics and Gynecology, Copenhagen University Hospital Herlev, Herlev Ringvej 75, DK-2730, Herlev, Denmark
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18
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Angelopoulos I, Southern P, Pankhurst QA, Day RM. Superparamagnetic iron oxide nanoparticles regulate smooth muscle cell phenotype. J Biomed Mater Res A 2016; 104:2412-9. [PMID: 27176658 PMCID: PMC5006844 DOI: 10.1002/jbm.a.35780] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/11/2015] [Revised: 03/21/2016] [Accepted: 05/11/2016] [Indexed: 01/12/2023]
Abstract
Superparamagnetic iron oxide nanoparticles (SPION) are used for an increasing range of biomedical applications, from imaging to mechanical actuation of cells and tissue. The aim of this study was to investigate the loading of smooth muscle cells (SMC) with SPION and to explore what effect this has on the phenotype of the cells. Adherent human SMC were loaded with ∼17 pg of unconjugated, negatively charged, 50 nm SPION. Clusters of the internalized SPION particles were held in discrete cytoplasmic vesicles. Internalized SPION did not cause any change in cell morphology, proliferation, metabolic activity, or staining pattern of actin and calponin, two of the muscle contractile proteins involved in force generation. However, internalized SPION inhibited the increased gene expression of actin and calponin normally observed when cells are incubated under differentiation conditions. The observed change in the control of gene expression of muscle contractile apparatus by SPION has not previously been described. This finding could offer novel approaches for regulating the phenotype of SMC and warrants further investigation. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 2412–2419, 2016.
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Affiliation(s)
- Ioannis Angelopoulos
- Applied Biomedical Engineering Group, Division of Medicine, University College London, WC1E 6DD, UK
| | - Paul Southern
- UCL Institute of Biomedical Engineering, University College London, London, WC1E 6BT, UK
| | - Quentin A Pankhurst
- UCL Institute of Biomedical Engineering, University College London, London, WC1E 6BT, UK
| | - Richard M Day
- Applied Biomedical Engineering Group, Division of Medicine, University College London, WC1E 6DD, UK
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19
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Small bowel in vivo bioengineering using an aortic matrix in a porcine model. Surg Endosc 2016; 30:4742-4749. [PMID: 26902616 DOI: 10.1007/s00464-016-4815-z] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/17/2015] [Accepted: 02/03/2016] [Indexed: 12/13/2022]
Abstract
OBJECTIVE To evaluate the feasibility of an in vivo small bowel bioengineering model using allogeneic aortic grafts in pigs. BACKGROUND The best treatment for short bowel syndrome is still unclear. Intestinal transplantation, as well as lifelong parenteral nutrition is associated with a 5-year survival rate of less than 50 %. We have already used allogeneic arterial segments to replace the upper airway in sheep. The results were encouraging with an induced transformation of the aortic wall into tracheo-bronchial bronchial-type tissue. METHODS Seven young mini-pigs were used. A 10-cm-diameter, allogeneic, aortic graft was interposed in an excluded small bowel segment and wrapped by the neighboring omentum. Animals were autopsied at 1 (n = 2), 3 (n = 3), and 6 months (n = 2), respectively. Specimens were examined macroscopically and microscopically. RESULTS The overall survival rate of the animals was 71.4 %. No anastomotic leak occurred. Histologic analysis revealed intestinal-like wall transformation of the aortic graft in the surviving animals. CONCLUSION Aortic-enteric anastomosis is feasible in a porcine model. Moreover, in vivo, bioengineered, intestinal-like transformation of the vascular wall was identified.
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20
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Customizable engineered blood vessels using 3D printed inserts. Methods 2015; 99:20-7. [PMID: 26732049 DOI: 10.1016/j.ymeth.2015.12.015] [Citation(s) in RCA: 31] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/29/2015] [Revised: 12/15/2015] [Accepted: 12/24/2015] [Indexed: 11/21/2022] Open
Abstract
Current techniques for tissue engineering blood vessels are not customizable for vascular size variation and vessel wall thickness. These critical parameters vary widely between the different arteries in the human body, and the ability to engineer vessels of varying sizes could increase capabilities for disease modeling and treatment options. We present an innovative method for producing customizable, tissue engineered, self-organizing vascular constructs by replicating a major structural component of blood vessels - the smooth muscle layer, or tunica media. We utilize a unique system combining 3D printed plate inserts to control construct size and shape, and cell sheets supported by a temporary fibrin hydrogel to encourage cellular self-organization into a tubular form resembling a natural artery. To form the vascular construct, 3D printed inserts are adhered to tissue culture plates, fibrin hydrogel is deposited around the inserts, and human aortic smooth muscle cells are then seeded atop the fibrin hydrogel. The gel, aided by the innate contractile properties of the smooth muscle cells, aggregates towards the center post insert, creating a tissue ring of smooth muscle cells. These rings are then stacked into the final tubular construct. Our methodology is robust, easily repeatable and allows for customization of cellular composition, vessel wall thickness, and length of the vessel construct merely by varying the size of the 3D printed inserts. This platform has potential for facilitating more accurate modeling of vascular pathology, serving as a drug discovery tool, or for vessel repair in disease treatment.
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21
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Parmar N, Kumar L, Emmanuel A, Day RM. Prospective regenerative medicine therapies for obstetric trauma-induced fecal incontinence. Regen Med 2015; 9:831-40. [PMID: 25431918 DOI: 10.2217/rme.14.56] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/21/2022] Open
Abstract
Fecal incontinence is a major public health issue that has yet to be adequately addressed. Obstetric trauma and injury to the anal sphincter muscles are the most common cause of fecal incontinence. New therapies are emerging aimed at repair or regeneration of sphincter muscle and restoration of continence. While regenerative medicine offers an attractive option for fecal incontinence there are currently no validated techniques using this approach. Although many challenges are yet to be resolved, the advent of regenerative medicine is likely to offer disruptive technologies to treat and possibly prevent the onset of this devastating condition. This article provides a review on regenerative medicine approaches for treating fecal incontinence and a critique of the current landscape in this area.
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Affiliation(s)
- Nina Parmar
- Applied Biomedical Engineering Group, University College London, 21 University Street, London, WC1E 6JJ, UK
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22
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Zakhem E, Rego SL, Raghavan S, Bitar KN. The appendix as a viable source of neural progenitor cells to functionally innervate bioengineered gastrointestinal smooth muscle tissues. Stem Cells Transl Med 2015; 4:548-54. [PMID: 25873745 DOI: 10.5966/sctm.2014-0238] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/17/2014] [Accepted: 02/23/2015] [Indexed: 12/31/2022] Open
Abstract
UNLABELLED Appendix-derived neural progenitor cells (NPCs) have both neurogenic and gliogenic potential, but use of these cells for enteric neural cell therapy has not been addressed. The objective of this study was to determine whether NPCs obtained from the appendix would differentiate into enteric neural subsets capable of inducing neurotransmitter-mediated smooth muscle cell (SMC) contraction and relaxation. NPCs were isolated from the appendix and small intestine (SI) of rabbits. Bioengineered internal anal sphincter constructs were developed using the same source of smooth muscle and innervated with NPCs derived from either the appendix or SI. Innervated constructs were assessed for neuronal differentiation markers through Western blots and immunohistochemistry, and functionality was assessed through force-generation studies. Expression of neural and glial differentiation markers was observed in constructs containing appendix- and SI-derived NPCs. The addition of acetylcholine to both appendix and SI constructs caused a robust contraction that was decreased by pretreatment with the neural inhibitor tetrodotoxin (TTX). Electrical field stimulation caused relaxation of constructs that was completely abolished in the presence of TTX and significantly reduced on pretreatment with nitric oxide synthase inhibitor (Nω-nitro-l-arginine methyl ester hydrochloride [l-NAME]). These data indicate that in the presence of identical soluble factors arising from intestinal SMCs, enteric NPCs derived from the appendix and SI differentiate in a similar manner and are capable of responding to physiological stimuli. This coculture paradigm could be used to explore the nature of the soluble factors derived from SMCs and NPCs in generating specific functional innervations. SIGNIFICANCE This study demonstrates the ability of neural stem cells isolated from the appendix to differentiate into mature functional enteric neurons. The differentiation of neural stem cells from the appendix is similar to differentiation of neural stem cells derived from the gastrointestinal tract. The appendix is a vestigial organ that can be removed with minimal clinical consequence through laparoscopy. Results presented in this paper indicate that the appendix is a potential source of autologous neural stem cells required for cell therapy for the gastrointestinal tract.
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Affiliation(s)
- Elie Zakhem
- Wake Forest Institute for Regenerative Medicine and Department of Molecular Medicine and Translational Science, Wake Forest School of Medicine, Winston-Salem, North Carolina, USA; Virginia Tech-Wake Forest University School of Biomedical Engineering and Sciences, Winston-Salem, North Carolina, USA
| | - Stephen L Rego
- Wake Forest Institute for Regenerative Medicine and Department of Molecular Medicine and Translational Science, Wake Forest School of Medicine, Winston-Salem, North Carolina, USA; Virginia Tech-Wake Forest University School of Biomedical Engineering and Sciences, Winston-Salem, North Carolina, USA
| | - Shreya Raghavan
- Wake Forest Institute for Regenerative Medicine and Department of Molecular Medicine and Translational Science, Wake Forest School of Medicine, Winston-Salem, North Carolina, USA; Virginia Tech-Wake Forest University School of Biomedical Engineering and Sciences, Winston-Salem, North Carolina, USA
| | - Khalil N Bitar
- Wake Forest Institute for Regenerative Medicine and Department of Molecular Medicine and Translational Science, Wake Forest School of Medicine, Winston-Salem, North Carolina, USA; Virginia Tech-Wake Forest University School of Biomedical Engineering and Sciences, Winston-Salem, North Carolina, USA
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23
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Parmar N, Day RM. Appropriately sized bioengineered human external anal sphincter constructs. Surgery 2015; 157:177-8. [DOI: 10.1016/j.surg.2014.05.025] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/15/2014] [Accepted: 05/30/2014] [Indexed: 10/25/2022]
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Bitar KN, Zakhem E. Design strategies of biodegradable scaffolds for tissue regeneration. Biomed Eng Comput Biol 2014; 6:13-20. [PMID: 25288907 PMCID: PMC4147780 DOI: 10.4137/becb.s10961] [Citation(s) in RCA: 36] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/27/2014] [Revised: 04/07/2014] [Accepted: 04/08/2014] [Indexed: 02/07/2023] Open
Abstract
There are numerous available biodegradable materials that can be used as scaffolds in regenerative medicine. Currently, there is a huge emphasis on the designing phase of the scaffolds. Materials can be designed to have different properties in order to match the specific application. Modifying scaffolds enhances their bioactivity and improves the regeneration capacity. Modifications of the scaffolds can be later characterized using several tissue engineering tools. In addition to the material, cell source is an important component of the regeneration process. Modified materials must be able to support survival and growth of different cell types. Together, cells and modified biomaterials contribute to the remodeling of the engineered tissue, which affects its performance. This review focuses on the recent advancements in the designs of the scaffolds including the physical and chemical modifications. The last part of this review also discusses designing processes that involve viability of cells.
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Affiliation(s)
- Khalil N Bitar
- Wake Forest Institute for Regenerative Medicine, Wake Forest School of Medicine, Winston-Salem, NC, USA. ; Department of Molecular Medicine and Translational Science, Wake Forest School of Medicine, Winston-Salem, NC, USA. ; Virginia Tech-Wake Forest School of Biomedical Engineering and Sciences, Winston-Salem, NC, USA
| | - Elie Zakhem
- Wake Forest Institute for Regenerative Medicine, Wake Forest School of Medicine, Winston-Salem, NC, USA. ; Department of Molecular Medicine and Translational Science, Wake Forest School of Medicine, Winston-Salem, NC, USA
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25
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Gilmont RR, Raghavan S, Somara S, Bitar KN. Bioengineering of physiologically functional intrinsically innervated human internal anal sphincter constructs. Tissue Eng Part A 2014; 20:1603-11. [PMID: 24328537 DOI: 10.1089/ten.tea.2013.0422] [Citation(s) in RCA: 24] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/30/2023] Open
Abstract
Muscle replacement for patients suffering from extensive tissue loss or dysfunction is a major objective of regenerative medicine. To achieve functional status, bioengineered muscle replacement constructs require innervation. Here we describe a method to bioengineer functionally innervated gut smooth muscle constructs using neuronal progenitor cells and smooth muscle cells isolated and cultured from intestinal tissues of adult human donors. These constructs expressed markers for contractile smooth muscle, glial cells, and mature neuronal populations. The constructs responded appropriately to physiologically relevant neurotransmitters, and neural network integration was demonstrated by responses to electrical field stimulation. The ability of enteric neuroprogenitor cells to differentiate into neuronal populations provides enormous potential for functional innervation of a variety of bioengineered muscle constructs in addition to gut. Functionally innervated muscle constructs offer a regenerative medicine-based therapeutic approach for neuromuscular replacement after trauma or degenerative disorders.
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Affiliation(s)
- Robert R Gilmont
- 1 Institute for Regenerative Medicine, Wake Forest School of Medicine , Winston-Salem, North Carolina
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26
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Raghavan S, Miyasaka EA, Gilmont RR, Somara S, Teitelbaum DH, Bitar KN. Perianal implantation of bioengineered human internal anal sphincter constructs intrinsically innervated with human neural progenitor cells. Surgery 2013; 155:668-74. [PMID: 24582493 DOI: 10.1016/j.surg.2013.12.023] [Citation(s) in RCA: 27] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/08/2013] [Accepted: 12/19/2013] [Indexed: 12/14/2022]
Abstract
BACKGROUND The internal anal sphincter (IAS) is a major contributing factor to pressure within the anal canal and is required for maintenance of rectoanal continence. IAS damage or weakening results in fecal incontinence. We have demonstrated that bioengineered, intrinsically innervated, human IAS tissue replacements possess key aspects of IAS physiology, such as the generation of spontaneous basal tone and contraction/relaxation in response to neurotransmitters. The objective of this study is to demonstrate the feasibility of implantation of bioengineered IAS constructs in the perianal region of athymic rats. METHODS Human IAS tissue constructs were bioengineered from isolated human IAS circular smooth muscle cells and human enteric neuronal progenitor cells. After maturation of the bioengineered constructs in culture, they were implanted operatively into the perianal region of athymic rats. Platelet-derived growth factor was delivered to the implanted constructs through a microosmotic pump. Implanted constructs were retrieved from the animals 4 weeks postimplantation. RESULTS Animals tolerated the implantation well, and there were no early postoperative complications. Normal stooling was observed during the implantation period. At harvest, implanted constructs were adherent to the perirectal rat tissue and appeared healthy and pink. Immunohistochemical analysis revealed neovascularization. Implanted smooth muscle cells maintained contractile phenotype. Bioengineered constructs responded in vitro in a tissue chamber to neuronally evoked relaxation in response to electrical field stimulation and vasoactive intestinal peptide, indicating the preservation of neuronal networks. CONCLUSION Our results indicate that bioengineered innervated IAS constructs can be used to augment IAS function in an animal model. This is a regenerative medicine based therapy for fecal incontinence that would directly address the dysfunction of the IAS muscle.
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Affiliation(s)
- Shreya Raghavan
- Virginia Tech-Wake Forest University School of Biomedical Engineering and Sciences, Winston-Salem, NC; Wake Forest Institute for Regenerative Medicine, Wake Forest School of Medicine, Winston-Salem, NC
| | - Eiichi A Miyasaka
- Department of Surgery, University of Michigan Medical School, Ann Arbor, MI
| | - Robert R Gilmont
- Wake Forest Institute for Regenerative Medicine, Wake Forest School of Medicine, Winston-Salem, NC
| | - Sita Somara
- Wake Forest Institute for Regenerative Medicine, Wake Forest School of Medicine, Winston-Salem, NC
| | | | - Khalil N Bitar
- Virginia Tech-Wake Forest University School of Biomedical Engineering and Sciences, Winston-Salem, NC; Wake Forest Institute for Regenerative Medicine, Wake Forest School of Medicine, Winston-Salem, NC.
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WANG LU, CHEN YANNI, QIAN JUN, TAN YANYAN, HUANGFU SHAOHUA, DING YIJIANG, DING SHUQING, JIANG BIN. A BOTTOM-UP METHOD TO BUILD 3D SCAFFOLDS WITH PREDEFINED VASCULAR NETWORK. J MECH MED BIOL 2013. [DOI: 10.1142/s0219519413400083] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022]
Abstract
Tissue engineering is a promising approach to regenerate transplantable tissue or organ substitutes in vitro. However, the existing methods are based on seeding cells on macroscale polymer scaffolds, which are associated with several challenges including limited control over cell microenvironment, limited nutrient diffusion, directed cell alignment. The emerging bottom-up tissue engineering methods hold great potential to address these challenges by assembling building blocks into complex 3D tissue constructs. In this study, we developed a layer-by-layer assembly approach to recreate 3D cell-laden constructs. Our experiment showed the predefined channels form a vascular system and help the transplant cells to transport the requirement of culture cells in early case of cells attaching and growing up. It is an original concept to demonstrate the feasibility of forming a network with a vascular geometry in a biocompatible polymer and fabricated different scaffold with different cells. The concept was developed to create a complete branching vascular circulation in 3D on surface of mixture of chitosan and gelatin structures and pre-define the structure of channel for culturing smooth muscle for controlling the SMC growing up as smooth muscle fiber.
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Affiliation(s)
- LU WANG
- Nanjing University of Chinese Medicine, Nanjing 210001, P. R. China
| | - YANNI CHEN
- Nanjing Dachang Hospital, Nanjing 210035, P. R. China
| | - JUN QIAN
- The Third Affiliated Hospital, Nanjing University of Chinese Medicine, Nanjing 210001, P. R. China
| | - YANYAN TAN
- The Third Affiliated Hospital, Nanjing University of Chinese Medicine, Nanjing 210001, P. R. China
| | - SHAOHUA HUANGFU
- The Third Affiliated Hospital, Nanjing University of Chinese Medicine, Nanjing 210001, P. R. China
| | - YIJIANG DING
- The Third Affiliated Hospital, Nanjing University of Chinese Medicine, Nanjing 210001, P. R. China
| | - SHUQING DING
- The Third Affiliated Hospital, Nanjing University of Chinese Medicine, Nanjing 210001, P. R. China
| | - BIN JIANG
- The Third Affiliated Hospital, Nanjing University of Chinese Medicine, Nanjing 210001, P. R. China
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Bae SH. Recent achievements in stem cell therapy for pediatric gastrointestinal tract disease. Pediatr Gastroenterol Hepatol Nutr 2013; 16:10-6. [PMID: 24010100 PMCID: PMC3746046 DOI: 10.5223/pghn.2013.16.1.10] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/08/2013] [Revised: 03/12/2013] [Accepted: 03/14/2013] [Indexed: 12/24/2022] Open
Abstract
The field of stem cell research has been rapidly expanding. Although the clinical usefulness of research remains to be ascertained through human trials, the use of stem cells as a therapeutic option for currently disabling diseases holds fascinating potential. Many pediatric gastrointestinal tract diseases have defect in enterocytes, enteric nervous system cells, smooth muscles, and interstitial cells of Cajal. Various kinds of therapeutic trials using stem cells could be applied to these diseases. This review article focuses on the recent achievements in stem cell applications for pediatric gastrointestinal tract diseases.
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Affiliation(s)
- Sun Hwan Bae
- Department of Pediatrics, School of Medicine, Konkuk University, Seoul, Korea
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Koch KL, Bitar KN, Fortunato JE. Tissue engineering for neuromuscular disorders of the gastrointestinal tract. World J Gastroenterol 2012; 18:6918-25. [PMID: 23322989 PMCID: PMC3531675 DOI: 10.3748/wjg.v18.i47.6918] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/19/2012] [Revised: 11/19/2012] [Accepted: 11/24/2012] [Indexed: 02/06/2023] Open
Abstract
The digestive tract is designed for the optimal processing of food that nourishes all organ systems. The esophagus, stomach, small bowel, and colon are sophisticated neuromuscular tubes with specialized sphincters that transport ingested food-stuffs from one region to another. Peristaltic contractions move ingested solids and liquids from the esophagus into the stomach; the stomach mixes the ingested nutrients into chyme and empties chyme from the stomach into the duodenum. The to-and-fro movement of the small bowel maximizes absorption of fat, protein, and carbohydrates. Peristaltic contractions are necessary for colon function and defecation.
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Singh J, Rattan S. Bioengineered human IAS reconstructs with functional and molecular properties similar to intact IAS. Am J Physiol Gastrointest Liver Physiol 2012; 303:G713-22. [PMID: 22790596 PMCID: PMC3468534 DOI: 10.1152/ajpgi.00112.2012] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/31/2023]
Abstract
Because of its critical importance in rectoanal incontinence, we determined the feasibility to reconstruct internal anal sphincter (IAS) from human IAS smooth muscle cells (SMCs) with functional and molecular attributes similar to the intact sphincter. The reconstructs were developed using SMCs from the circular smooth muscle layer of the human IAS, grown in smooth muscle differentiation media under sterile conditions in Sylgard-coated tissue culture plates with central Sylgard posts. The basal tone in the reconstructs and its changes were recorded following 0 Ca(2+), KCl, bethanechol, isoproterenol, protein kinase C (PKC) activator phorbol 12,13-dibutyrate, and Rho kinase (ROCK) and PKC inhibitors Y-27632 and Gö-6850, respectively. Western blot (WB), immunofluorescence (IF), and immunocytochemical (IC) analyses were also performed. The reconstructs developed spontaneous tone (0.68 ± 0.26 mN). Bethanechol (a muscarinic agonist) and K(+) depolarization produced contraction, whereas isoproterenol (β-adrenoceptor agonist) and Y-27632 produced a concentration-dependent decrease in the tone. Maximal decrease in basal tone with Y-27632 and Gö-6850 (each 10(-5) M) was 80.45 ± 3.29 and 17.76 ± 3.50%, respectively. WB data with the IAS constructs' SMCs revealed higher levels of RhoA/ROCK, protein kinase C-potentiated inhibitor or inhibitory phosphoprotein for myosin phosphatase (CPI-17), phospho-CPI-17, MYPT1, and 20-kDa myosin light chain vs. rectal smooth muscle. WB, IF, and IC studies of original SMCs and redispersed from the reconstructs for the relative distribution of different signal transduction proteins confirmed the feasibility of reconstruction of IAS with functional properties similar to intact IAS and demonstrated the development of myogenic tone with critical dependence on RhoA/ROCK. We conclude that it is feasible to bioengineer IAS constructs using human IAS SMCs that behave like intact IAS.
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Affiliation(s)
- Jagmohan Singh
- Division of Gastroenterology and Hepatology, Department of Medicine, Jefferson Medical College, Thomas Jefferson University, Philadelphia, Pennsylvania
| | - Satish Rattan
- Division of Gastroenterology and Hepatology, Department of Medicine, Jefferson Medical College, Thomas Jefferson University, Philadelphia, Pennsylvania
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Orlando G, Wood KJ, De Coppi P, Baptista PM, Binder KW, Bitar KN, Breuer C, Burnett L, Christ G, Farney A, Figliuzzi M, Holmes JH, Koch K, Macchiarini P, Mirmalek Sani SH, Opara E, Remuzzi A, Rogers J, Saul JM, Seliktar D, Shapira-Schweitzer K, Smith T, Solomon D, Van Dyke M, Yoo JJ, Zhang Y, Atala A, Stratta RJ, Soker S. Regenerative medicine as applied to general surgery. Ann Surg 2012; 255:867-80. [PMID: 22330032 PMCID: PMC3327776 DOI: 10.1097/sla.0b013e318243a4db] [Citation(s) in RCA: 70] [Impact Index Per Article: 5.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
Abstract
The present review illustrates the state of the art of regenerative medicine (RM) as applied to surgical diseases and demonstrates that this field has the potential to address some of the unmet needs in surgery. RM is a multidisciplinary field whose purpose is to regenerate in vivo or ex vivo human cells, tissues, or organs to restore or establish normal function through exploitation of the potential to regenerate, which is intrinsic to human cells, tissues, and organs. RM uses cells and/or specially designed biomaterials to reach its goals and RM-based therapies are already in use in several clinical trials in most fields of surgery. The main challenges for investigators are threefold: Creation of an appropriate microenvironment ex vivo that is able to sustain cell physiology and function in order to generate the desired cells or body parts; identification and appropriate manipulation of cells that have the potential to generate parenchymal, stromal and vascular components on demand, both in vivo and ex vivo; and production of smart materials that are able to drive cell fate.
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Affiliation(s)
- Giuseppe Orlando
- Wake Forest Institute for Regenerative Medicine, Winston Salem, NC, USA.
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Zakhem E, Raghavan S, Gilmont RR, Bitar KN. Chitosan-based scaffolds for the support of smooth muscle constructs in intestinal tissue engineering. Biomaterials 2012; 33:4810-7. [PMID: 22483012 DOI: 10.1016/j.biomaterials.2012.03.051] [Citation(s) in RCA: 63] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/22/2012] [Accepted: 03/13/2012] [Indexed: 02/08/2023]
Abstract
Intestinal tissue engineering is an emerging field due to a growing demand for intestinal lengthening and replacement procedures secondary to massive resections of the bowel. Here, we demonstrate the potential use of a chitosan/collagen scaffold as a 3D matrix to support the bioengineered circular muscle constructs maintain their physiological functionality. We investigated the biocompatibility of chitosan by growing rabbit colonic circular smooth muscle cells (RCSMCs) on chitosan-coated plates. The cells maintained their spindle-like morphology and preserved their smooth muscle phenotypic markers. We manufactured tubular scaffolds with central openings composed of chitosan and collagen in a 1:1 ratio. Concentrically aligned 3D circular muscle constructs were bioengineered using fibrin-based hydrogel seeded with RCSMCs. The constructs were placed around the scaffold for 2 weeks, after which they were taken off and tested for their physiological functionality. The muscle constructs contracted in response to acetylcholine (Ach) and potassium chloride (KCl) and they relaxed in response to vasoactive intestinal peptide (VIP). These results demonstrate that chitosan is a biomaterial possibly suitable for intestinal tissue engineering applications.
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Affiliation(s)
- Elie Zakhem
- Wake Forest Institute for Regenerative Medicine, Wake Forest School of Medicine, Winston-Salem, NC 27101, USA
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Bitar KN, Raghavan S. Intestinal tissue engineering: current concepts and future vision of regenerative medicine in the gut. Neurogastroenterol Motil 2012; 24:7-19. [PMID: 22188325 PMCID: PMC3248673 DOI: 10.1111/j.1365-2982.2011.01843.x] [Citation(s) in RCA: 34] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
Abstract
Functional tissue engineering of the gastrointestinal (GI) tract is a complex process aiming to aid the regeneration of structural layers of smooth muscle, intrinsic enteric neuronal plexuses, specialized mucosa, and epithelial cells as well as interstitial cells. The final tissue-engineered construct is intended to mimic the native GI tract anatomically and physiologically. Physiological functionality of tissue-engineered constructs is of utmost importance while considering clinical translation. The construct comprises of cellular components as well as biomaterial scaffolding components. Together, these determine the immune response a tissue-engineered construct would elicit from a host upon implantation. Over the last decade, significant advances have been made to mitigate adverse host reactions. These include a quest for identifying autologous cell sources like embryonic and adult stem cells, bone marrow-derived cells, neural crest-derived cells, and muscle derived-stem cells. Scaffolding biomaterials have been fabricated with increasing biocompatibility and biodegradability. Manufacturing processes have advanced to allow for precise spatial architecture of scaffolds to mimic in vivo milieu closely and achieve neovascularization. This review will focus on the current concepts and the future vision of functional tissue engineering of the diverse neuromuscular structures of the GI tract from the esophagus to the internal anal sphincter.
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Affiliation(s)
- Khalil N. Bitar
- Address Correspondence to: Khalil N. Bitar, PhD., AGAF, Wake Forest Institute for Regenerative Medicine, 391 Technology Way, Winston-Salem NC 27101, Phone: (336) 713-1470, FAX: (336) 713-7290,
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Khait L, Birla RK. Bypassing the Patient: Comparison of Biocompatible Models for the Future of Vascular Tissue Engineering. Cell Transplant 2012; 21:269-83. [DOI: 10.3727/096368910x564535] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/24/2022] Open
Abstract
The objective of vascular tissue engineering is to develop tissue-engineered, biocompatible, small-diameter vessels suitable to withstand in vivo systolic pressures as well as be immunologically compatible with the patient, in order to minimize graft rejection. In this study, we present and compare two models of biocompatible, tissue-engineered vascular grafts (TEVG), using chitosan and acellularized rat aortas as options for scaffolds. Human aortic adventitial smooth muscle cells and fibroblasts were seeded onto a fibrin gel and subsequently wrapped around either of the two scaffolds. After several weeks of maturation in standard culturing conditions, the graft models were analyzed and compared by mechanical testing, cell viability, and histology. Histological and viability data showed that both models were viable and developed similarly, with a scaffold surrounded by two layers of cells, the fibroblasts lying on top of the smooth muscle cells. Both models responded to 200 mM potassium chloride (KCl) (tensions of 38 ± 3, 78 ± 13, and 52 ± 7 μN) and 25 mM 8-bromo-cyclic AMP (tensions of −23 ± 4, −39 ± 10, and −31 ± 12 μN) stimulation by vasoconstriction and vasorelaxation ( n = 3), respectively; however, the chitosan model was unable to maintain the contracted and relaxed tension. Because the acellularized aorta TEVGs were able to maintain stimulated tension better than chitosan TEVGs, we concluded that the acellularized aorta model was better suited for further development.
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Affiliation(s)
- L. Khait
- Section of Cardiac Surgery, University of Michigan, Ann Arbor, MI, USA
| | - R. K. Birla
- Section of Cardiac Surgery, University of Michigan, Ann Arbor, MI, USA
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Somara S, Bashllari D, Gilmont RR, Bitar KN. Real-time dynamic movement of caveolin-1 during smooth muscle contraction of human colon and aged rat colon transfected with caveolin-1 cDNA. Am J Physiol Gastrointest Liver Physiol 2011; 300:G1022-32. [PMID: 21372166 PMCID: PMC3119117 DOI: 10.1152/ajpgi.00301.2010] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/31/2023]
Abstract
Caveolin-1 (cav-1) plays a key role in PKC-α and RhoA signaling pathways during acetylcholine (ACh)-induced contraction of colonic smooth muscle cells (CSMC). Aged rat CSMC showed sluggish contractility, concomitant with reduced expression of cav-1 with an associated reduction in activation of PKC-α and RhoA signaling pathway. Real-time monitoring of live human CSMC transfected with yellow fluorescent protein-tagged wild-type caveolin 1 cDNA (YFP-wt-cav-1) cDNA in the present study suggests that cav-1 cycles within and along the membrane in a synchronized, highly organized cytoskeletal path. These studies provide, for the first time, the advantages of real-time monitoring of the dynamic movement of caveolin in living cells. Rapid movement of cav-1 in response to ACh suggests its dynamic role in CSMC contraction. Human CSMC transfected with YFP-ΔTFT-cav-1 dominant negative cDNA show fluorescence in the cytosol of the CSMC and no movement of fluorescent cav-1 in response to ACh mimicking the response shown by aged rat CSMC. Transfection of CSMC from aged rat with YFP-wt-cav-1 cDNA restored the physiological contractile response to ACh as well as the dynamic movement of cav-1 along the organized cytoskeletal path observed in normal adult CSMC. To study the force generation by CSMC, three-dimensional colonic rings were bioengineered. Colonic bioengineered rings from aged CSMC showed reduced force generation compared with colonic bioengineered rings from adult CSMC. Colonic bioengineered rings from aged CSMC transfected with wt-cav-1 cDNA showed force generation similar to colonic bioengineered rings from adult rat CSMC. The data suggest that contraction in CSMC is dependent on cav-1 reorganization dynamics, which restores the physiological contractile response in aged CSMC. We hypothesize that dynamic movement of cav-1 is essential for physiological contractile response of colonic smooth muscle.
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Affiliation(s)
- Sita Somara
- Gastrointestinal Molecular Motors Laboratory, Department of Pediatrics, Gastroenterology, University of Michigan Medical Center, Ann Arbor, Michigan
| | - Daniela Bashllari
- Gastrointestinal Molecular Motors Laboratory, Department of Pediatrics, Gastroenterology, University of Michigan Medical Center, Ann Arbor, Michigan
| | - Robert R. Gilmont
- Gastrointestinal Molecular Motors Laboratory, Department of Pediatrics, Gastroenterology, University of Michigan Medical Center, Ann Arbor, Michigan
| | - Khalil N. Bitar
- Gastrointestinal Molecular Motors Laboratory, Department of Pediatrics, Gastroenterology, University of Michigan Medical Center, Ann Arbor, Michigan
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Schek R, Michalek A, Iatridis J. Genipin-crosslinked fibrin hydrogels as a potential adhesive to augment intervertebral disc annulus repair. Eur Cell Mater 2011; 21:373-83. [PMID: 21503869 PMCID: PMC3215264 DOI: 10.22203/ecm.v021a28] [Citation(s) in RCA: 102] [Impact Index Per Article: 7.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/31/2023] Open
Abstract
Treatment of damaged intervertebral discs is a significant clinical problem and, despite advances in the repair and replacement of the nucleus pulposus, there are few effective strategies to restore defects in the annulus fibrosus. An annular repair material should meet three specifications: have a modulus similar to the native annulus tissue, support the growth of disc cells, and maintain adhesion to tissue under physiological strain levels. We hypothesized that a genipin crosslinked fibrin gel could meet these requirements. Our mechanical results showed that genipin crosslinked fibrin gels could be created with a modulus in the range of native annular tissue. We also demonstrated that this material is compatible with the in vitro growth of human disc cells, when genipin:fibrin ratios were 0.25:1 or less, although cell proliferation was slower and cell morphology more rounded than for fibrin alone. Finally, lap tests were performed to evaluate adhesion between fibrin gels and pieces of annular tissue. Specimens created without genipin had poor handling properties and readily delaminated, while genipin crosslinked fibrin gels remained adhered to the tissue pieces at strains exceeding physiological levels and failed at 15-30%. This study demonstrated that genipin crosslinked fibrin gels show promise as a gap-filling adhesive biomaterial with tunable material properties, yet the slow cell proliferation suggests this biomaterial may be best suited as a sealant for small annulus fibrosus defects or as an adhesive to augment large annulus repairs. Future studies will evaluate degradation rate, fatigue behaviors, and long-term biocompatibility.
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Affiliation(s)
| | | | - J.C. Iatridis
- Address for correspondence: James C. Iatridis, Leni and Peter W. May Department of Orthopaedics, Mount Sinai School of Medicine, One Gustave L. Levy Place, Box 1188, New York, NY 10029-6574, USA, Telephone Number: 212-241-1517,
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Raghavan S, Lam MT, Foster LL, Gilmont RR, Somara S, Takayama S, Bitar KN. Bioengineered three-dimensional physiological model of colonic longitudinal smooth muscle in vitro. Tissue Eng Part C Methods 2011; 16:999-1009. [PMID: 20001822 DOI: 10.1089/ten.tec.2009.0394] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/13/2022] Open
Abstract
BACKGROUND The objective of this study was to develop a physiological model of longitudinal smooth muscle tissue from isolated longitudinal smooth muscle cells arranged in the longitudinal axis. METHODS Longitudinal smooth muscle cells from rabbit sigmoid colon were isolated and expanded in culture. Cells were seeded at high densities onto laminin-coated Sylgard surfaces with defined wavy microtopographies. A highly aligned cell sheet was formed, to which addition of fibrin resulted in delamination. RESULTS (1) Acetylcholine (ACh) induced a dose-dependent, rapid, and sustained force generation. (2) Absence of extracellular calcium attenuated the magnitude and sustainability of ACh-induced force by 50% and 60%, respectively. (3) Vasoactive intestinal peptide also attenuated the magnitude and sustainability of ACh-induced force by 40% and 60%, respectively. These data were similar to force generated by longitudinal tissue. (4) Bioengineered constructs also maintained smooth muscle phenotype and calcium-dependence characteristics. SUMMARY This is a novel physiologically relevant in vitro three-dimensional model of colonic longitudinal smooth muscle tissue. Bioengineered three-dimensional longitudinal smooth muscle presents the ability to generate force, and respond to contractile agonists and relaxant peptides similar to native longitudinal tissue. This model has potential applications to investigate the underlying pathophysiology of dysfunctional colonic motility. It also presents as a readily implantable band-aid colonic longitudinal muscle tissue.
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Affiliation(s)
- Shreya Raghavan
- Department of Pediatrics-Gastroenterology, University of Michigan Medical School, Ann Arbor, Michigan 48109-0658, USA
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Natesan S, Zhang G, Baer DG, Walters TJ, Christy RJ, Suggs LJ. A bilayer construct controls adipose-derived stem cell differentiation into endothelial cells and pericytes without growth factor stimulation. Tissue Eng Part A 2011; 17:941-53. [PMID: 21083419 DOI: 10.1089/ten.tea.2010.0294] [Citation(s) in RCA: 55] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/23/2022] Open
Abstract
This work describes the differentiation of adipose-derived mesenchymal stem cells (ASC) in a composite hydrogel for use as a vascularized dermal matrix. Our intent is that such a construct could be utilized following large-surface-area burn wounds that require extensive skin grafting and that are limited by the availability of uninjured sites. To develop engineered skin replacement constructs, we are pursuing the use of ASC. We have established that a PEGylated fibrin gel can provide a suitable environment for the proliferation of ASC over a 7 day time course. Further, we have demonstrated that PEGylated fibrin can be used to control ASC differentiation toward vascular cell types, including cells characteristic of both endothelial cells and pericytes. Gene expression analysis revealed strong upregulation of endothelial markers, CD31, and von Willebrand factor, up to day 11 in culture with corresponding evidence of protein expression demonstrated by immunocytochemical staining. ASC were not only shown to express endothelial cell phenotype, but a subset of the ASC expressed pericyte markers. The NG2 gene was upregulated over 11 days with corresponding evidence for the cell surface marker. Platelet-derived growth factor receptor beta gene expression decreased as the multipotent ASC differentiated up to day 7. Increased receptor expression at day 11 was likely due to the enhanced pericyte gene expression profile, including increased NG2 expression. We have also demonstrated that when cells are loaded onto chitosan microspheres and sandwiched between the PEGylated fibrin gel and a type I collagen gel, the cells can migrate and proliferate within the two different gel types. The matrix composition dictates the lineage specification and is not driven by soluble factors. Utilizing an insoluble bilayer matrix to direct ASC differentiation will allow for the development of both vasculature as well as dermal connective tissue from a single population of ASC. This work underscores the importance of the extracellular matrix in controlling stem cell phenotype. It is our goal to develop layered composites as wound dressings or vascularized dermal equivalents that are not limited by nutrient diffusion.
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Affiliation(s)
- Shanmugasundaram Natesan
- Regenerative Medicine Research Program, US Army Institute of Surgical Research, Fort Sam, Houston, Texas, USA
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Synthesis of embryonic tendon-like tissue by human marrow stromal/mesenchymal stem cells requires a three-dimensional environment and transforming growth factor β3. Matrix Biol 2010; 29:668-77. [PMID: 20736064 PMCID: PMC3611595 DOI: 10.1016/j.matbio.2010.08.005] [Citation(s) in RCA: 57] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/07/2008] [Revised: 08/06/2010] [Accepted: 08/06/2010] [Indexed: 01/05/2023]
Abstract
Tendon-like tissue generated from stem cells in vitro has the potential to replace tendons and ligaments lost through injury and disease. However, thus far, no information has been available on the mechanism of tendon formation in vitro and how to accelerate the process. We show here that human mesenchymal stem cells (MSCs) and bone marrow-derived mononuclear cells (BM-MNCs) can generate tendon-like tissue in 7days mediated by transforming growth factor (TGF) β3. MSCs cultured in fixed-length fibrin gels spontaneously synthesized narrow-diameter collagen fibrils and exhibited fibripositors (actin-rich, collagen fibril-containing plasma membrane protrusions) identical to those that occur in embryonic tendon. In contrast, BM-MNCs did not synthesize tendon-like tissue under these conditions. We performed real-time PCR analysis of MSCs and BM-MNCs. MSCs upregulated genes encoding type I collagen, TGFβ3, and Smad2 at the time of maximum contraction of the tendon-like tissue (7days). Western blot analysis showed phosphorylation of Smad2 at maximum contraction. The TGFβ inhibitor SB-431542, blocked the phosphorylation of Smad2 and stopped the formation of tendon-like tissue. Quantitative PCR showed that BM-MNCs expressed very low levels of TGFβ3 compared to MSCs. Therefore we added exogenous TGFβ3 protein to BM-MNCs in fibrin gels, which resulted in phosphorylation of Smad2, synthesis of collagen fibrils, the appearance of fibripositors at the plasma membrane, and the formation of tendon-like tissue. In conclusion, MSCs that self-generate TGFβ signaling or the addition of TGFβ3 protein to BM-MNCs in fixed-length fibrin gels spontaneously make embryonic tendon-like tissue in vitro within 7days.
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Raghavan S, Miyasaka EA, Hashish M, Somara S, Gilmont RR, Teitelbaum DH, Bitar KN. Successful implantation of physiologically functional bioengineered mouse internal anal sphincter. Am J Physiol Gastrointest Liver Physiol 2010; 299:G430-9. [PMID: 20558766 PMCID: PMC2928530 DOI: 10.1152/ajpgi.00269.2009] [Citation(s) in RCA: 24] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/09/2009] [Accepted: 06/09/2010] [Indexed: 01/31/2023]
Abstract
We have previously developed bioengineered three-dimensional internal anal sphincter (IAS) rings from circular smooth muscle cells isolated from rabbit and human IAS. We provide proof of concept that bioengineered mouse IAS rings are neovascularized upon implantation into mice of the same strain and maintain concentric smooth muscle alignment, phenotype, and IAS functionality. Rings were bioengineered by using smooth muscle cells from the IAS of C57BL/6J mice. Bioengineered mouse IAS rings were implanted subcutaneously on the dorsum of C57BL/6J mice along with a microosmotic pump delivering fibroblast growth factor-2. The mice remained healthy during the period of implantation, showing no external signs of rejection. Mice were killed 28 days postsurgery and implanted IAS rings were harvested. IAS rings showed muscle attachment, neovascularization, healthy color, and no external signs of infection or inflammation. Assessment of force generation on harvested IAS rings showed the following: 1) spontaneous basal tone was generated in the absence of external stimulation; 2) basal tone was relaxed by vasoactive intestinal peptide, nitric oxide donor, and nifedipine; 3) acetylcholine and phorbol dibutyrate elicited rapid-rising, dose-dependent, sustained contractions repeatedly over 30 min without signs of muscle fatigue; and 4) magnitudes of potassium chloride-induced contractions were 100% of peak maximal agonist-induced contractions. Our preliminary results confirm the proof of concept that bioengineered rings are neovascularized upon implantation. Harvested rings maintain smooth muscle alignment and phenotype. Our physiological studies confirm that implanted rings maintain 1) overall IAS physiology and develop basal tone, 2) integrity of membrane ionic characteristics, and 3) integrity of membrane associated intracellular signaling transduction pathways for contraction and relaxation by responding to cholinergic, nitrergic, and VIP-ergic stimulation. IAS smooth muscle tissue could thus be bioengineered for the purpose of implantation to serve as a potential graft therapy for dysfunctional internal anal sphincter in fecal incontinence.
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Affiliation(s)
- Shreya Raghavan
- Department of Pediatrics-Gastroenterology, University of Michigan Medical School, Ann Arbor, 48109-0658, USA
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Colombo A, Zahedmanesh H, Toner D, Cahill P, Lally C. A method to develop mock arteries suitable for cell seeding and in-vitro cell culture experiments. J Mech Behav Biomed Mater 2010; 3:470-7. [DOI: 10.1016/j.jmbbm.2010.04.003] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/24/2009] [Revised: 03/31/2010] [Accepted: 04/23/2010] [Indexed: 10/19/2022]
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Kang SB, Lee TG. Muscle Regeneration: Research for the Treatment of Fecal Incontinence. JOURNAL OF THE KOREAN SOCIETY OF COLOPROCTOLOGY 2010. [DOI: 10.3393/jksc.2010.26.1.1] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 10/19/2022]
Affiliation(s)
- Sung-Bum Kang
- Department of Surgery, Seoul National University Bundang Hospital, Seoul National University College of Medicine, Seongnam, Korea
| | - Taek-Gu Lee
- Department of Surgery, Seoul National University Bundang Hospital, Seoul National University College of Medicine, Seongnam, Korea
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Hashish M, Raghavan S, Somara S, Gilmont RR, Miyasaka E, Bitar KN, Teitelbaum DH. Surgical implantation of a bioengineered internal anal sphincter. J Pediatr Surg 2010; 45:52-8. [PMID: 20105579 PMCID: PMC3018766 DOI: 10.1016/j.jpedsurg.2009.10.010] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/26/2009] [Accepted: 10/06/2009] [Indexed: 11/26/2022]
Abstract
PURPOSE Fecal incontinence is a common disorder that can have devastating social and psychologic consequences. However, there are no long-term ideal solutions for such patients. Although loss of continence is multifactorial, the integrity of the internal anal sphincter (IAS) has particular significance. We previously described the development of 3-dimensional bioengineered constructs using isolated smooth muscle tissue from donor C57BL/6 IAS. We hypothesized that the bioengineered ring constructs would retain cellular viability and promote neovascularization upon implantation into a recipient mouse. METHODS Internal anal sphincter ring constructs were surgically implanted into the subcutaneous tissue of syngeneic C57BL/6 mice and treated with either fibroblastic growth factor 2 (0.26 microg daily) or saline controls using a microosmotic pump. Internal anal sphincter constructs were harvested after 25 days (range, 23-26 days) and assessed morphologically and for tissue viability. RESULT Gross morphology showed that there was no rejection. Rings showed muscle attachment to the back of the mouse with no sign of inflammation. Fibroblastic growth factor 2 infusion resulted in a significantly improved histologic score and muscle viability compared with the control group. CONCLUSIONS Three-dimensional bioengineered IAS rings can be successfully implanted into the subcutaneous tissue of recipient mice. The addition of fibroblastic growth factor 2 led to improved muscle viability, vascularity, and survival. This approach may become a feasible option for patients with fecal incontinence.
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Affiliation(s)
- Mohamed Hashish
- Section of Pediatric Surgery, University of Michigan, Mott Children's Hospital F3970, Box 0245, Ann Arbor, MI 48109-0245, USA
| | - Shreya Raghavan
- GI Molecular Motors Lab, Department of Pediatrics, Gastroenterology, University of Michigan, Ann Arbor, MI 48109-0245, USA
| | - Sita Somara
- GI Molecular Motors Lab, Department of Pediatrics, Gastroenterology, University of Michigan, Ann Arbor, MI 48109-0245, USA
| | - Robert R. Gilmont
- GI Molecular Motors Lab, Department of Pediatrics, Gastroenterology, University of Michigan, Ann Arbor, MI 48109-0245, USA
| | - Eiichi Miyasaka
- Section of Pediatric Surgery, University of Michigan, Mott Children's Hospital F3970, Box 0245, Ann Arbor, MI 48109-0245, USA
| | - Khalil N. Bitar
- GI Molecular Motors Lab, Department of Pediatrics, Gastroenterology, University of Michigan, Ann Arbor, MI 48109-0245, USA
| | - Daniel H. Teitelbaum
- Section of Pediatric Surgery, University of Michigan, Mott Children's Hospital F3970, Box 0245, Ann Arbor, MI 48109-0245, USA,Corresponding author. Tel.: +1 734 936 8464; fax: +1 734 936 9784. (D.H. Teitelbaum)
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Khait L, Hodonsky CJ, Birla RK. Variable optimization for the formation of three-dimensional self-organized heart muscle. In Vitro Cell Dev Biol Anim 2009; 45:592-601. [PMID: 19756885 DOI: 10.1007/s11626-009-9234-1] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/18/2008] [Accepted: 08/18/2009] [Indexed: 11/27/2022]
Abstract
Cardiac tissue-engineering research is focused on the development of functional three-dimensional (3D) heart muscle in vitro. These models allow the detailed study of critical events in organogenesis, such as the establishment of cell-cell communication and construction and modification of the extracellular matrix. We have previously described a model for 3D heart muscle, termed cardioids, formed by the spontaneous delamination of a cohesive monolayer of primary cells in the absence of any synthetic scaffolding material. In an earlier publication, we have shown that, upon electrical stimulation, cardioids generate a twitch force in the range of 200-300 microN, generate a specific force (twitch force normalized to total cross-sectional area) of 2-4 kN/m(2), and can be electrically paced at frequencies of up to 10 Hz without any notable fatigue. We have two objectives for the current study: model development and model optimization. Our model development efforts are focused on providing additional characterization of the cardioid model. In this study, we show for the first time that cardioids show a pattern of gene expression comparable to that of cells cultured in two dimensions on tissue culture plastic and normal mammalian heart muscle. Compared with primary cardiac cells cultured on tissue culture plastic, the expression of alpha-myosin heavy chain (MHC), beta-MHC, SERCA2, and phospholamban was significantly higher in cardioids. Our second objective, model optimization, is focused on evaluating the effect of several cell culture variables on cardioid formation and function. Specifically, we looked at the effect of plating density (1.0-4.0 x 10(6) cells per cardioid), concentration of two adhesion proteins (laminin at 0.2-2.0 microg/cm(2) and fibronectin at 1-10 microg/cm(2)), myocyte purity (using preplating times of 15 and 60 min), and ascorbic acid stimulation (1-100 microl/ml). For our optimization studies, we utilized twitch force in response to electrical stimulation as our endpoint metric. Based on these studies, we found that cardioids formed with a plating density in the range 3-4 x 10(6) cells per cardioid generated the maximum twitch force, whereas increasing the surface adhesion protein (using either laminin or fibronectin) and increasing the myocyte purity both resulted in a decrease in twitch force. In addition, increasing the ascorbic acid concentration resulted in an increase in the baseline force of cardioids, which was recorded in the absence of electrical stimulation. Based on the model development studies, we have shown that cardioids do indeed exhibit a gene expression pattern similar to normal mammalian heart muscle. This provides further validity for the cardioid model. Based on the model optimization studies, we have identified specific cell culture regimes which support cardioid formation and function. These results are specific to the cardioid model; however, they may be translated and applied to other tissue-engineering models. Collectively, the work described in this study provides insight into the formation of functional 3D heart muscle and the effect of several cell culture variables on tissue formation and function.
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Affiliation(s)
- Luda Khait
- Division of Cardiac Surgery, Artificial Heart Laboratory, Ann Arbor, MI 48103, USA
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Ahmed TAE, Dare EV, Hincke M. Fibrin: a versatile scaffold for tissue engineering applications. TISSUE ENGINEERING PART B-REVIEWS 2009; 14:199-215. [PMID: 18544016 DOI: 10.1089/ten.teb.2007.0435] [Citation(s) in RCA: 613] [Impact Index Per Article: 38.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/19/2023]
Abstract
Tissue engineering combines cell and molecular biology with materials and mechanical engineering to replace damaged or diseased organs and tissues. Fibrin is a critical blood component responsible for hemostasis, which has been used extensively as a biopolymer scaffold in tissue engineering. In this review we summarize the latest developments in organ and tissue regeneration using fibrin as the scaffold material. Commercially available fibrinogen and thrombin are combined to form a fibrin hydrogel. The incorporation of bioactive peptides and growth factors via a heparin-binding delivery system improves the functionality of fibrin as a scaffold. New technologies such as inkjet printing and magnetically influenced self-assembly can alter the geometry of the fibrin structure into appropriate and predictable forms. Fibrin can be prepared from autologous plasma, and is available as glue or as engineered microbeads. Fibrin alone or in combination with other materials has been used as a biological scaffold for stem or primary cells to regenerate adipose tissue, bone, cardiac tissue, cartilage, liver, nervous tissue, ocular tissue, skin, tendons, and ligaments. Thus, fibrin is a versatile biopolymer, which shows a great potential in tissue regeneration and wound healing.
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Affiliation(s)
- Tamer A E Ahmed
- Department of Cellular and Molecular Medicine, University of Ottawa, Ontario, Canada
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Somara S, Gilmont RR, Dennis RG, Bitar KN. Bioengineered internal anal sphincter derived from isolated human internal anal sphincter smooth muscle cells. Gastroenterology 2009; 137:53-61. [PMID: 19328796 DOI: 10.1053/j.gastro.2009.03.036] [Citation(s) in RCA: 46] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/20/2008] [Revised: 02/02/2009] [Accepted: 03/17/2009] [Indexed: 12/15/2022]
Abstract
BACKGROUND & AIMS The internal anal sphincter (IAS) is a specialized circular smooth muscle that maintains rectoanal continence. In vitro models are needed to study the pathophysiology of human IAS disorders. We bioengineered sphincteric rings from human IAS smooth muscle cells (SMC) and investigated their response to cholinergic stimulation as well as investigated whether protein kinase C (PKC) and Rho kinase signaling pathways remain functional. METHODS 3-Dimensional bioengineered ring (3DBR) model of the human IAS was constructed from isolated human IAS SMC obtained from surgery. Contractile properties and force generation in response to acetylcholine, PKC inhibitor calphostin-C, Rho/ROCK inhibitor Y-27632, permeable Rho/ROCK inhibitor c3-exoenzyme, and PKC activator PdBU was measured. RESULTS The human IAS 3DBR has the essential characteristics of physiologically functional IAS; it generated a spontaneous myogenic basal tone, and the constructs were able to relax in response to relaxants and contract in response to contractile agents. The constructs generated dose-dependent force in response to acetylcholine. Basal tone was significantly reduced by calphostin-C but not with Y-27632. Acetylcholine-induced force generation was also significantly reduced by calphostin-C but not with Y-27632. PdBU generated force that was equal in magnitude to acetylcholine. Thus, calphostin-C inhibited PdBU-induced force generation, whereas Y-27632 and c3 exoenzyme did not. CONCLUSIONS These data indicate that basal tone and acetylcholine-induced force generation depend on signaling through the PKC pathway in human IAS; PKC-mediated force generation is independent of the Rho/ROCK pathway. This human IAS 3DBR model can be used to study the pathophysiology associated with IAS malfunctions.
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Affiliation(s)
- Sita Somara
- Department of Pediatrics-Gastroenterology University of Michigan, Ann Arbor, Michigan 48109, USA
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Dennis RG, Smith B, Philp A, Donnelly K, Baar K. Bioreactors for guiding muscle tissue growth and development. ADVANCES IN BIOCHEMICAL ENGINEERING/BIOTECHNOLOGY 2009; 112:39-79. [PMID: 19290497 DOI: 10.1007/978-3-540-69357-4_3] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/20/2022]
Abstract
Muscle tissue bioreactors are devices which are employed to guide and monitor the development of engineered muscle tissue. These devices have a modern history that can be traced back more than a century, because the key elements of muscle tissue bioreactors have been studied for a very long time. These include barrier isolation and culture of cells, tissues and organs after isolation from a host organism; the provision of various stimuli intended to promote growth and maintain the muscle, such as electrical and mechanical stimulation; and the provision of a perfusate such as culture media or blood derived substances. An accurate appraisal of our current progress in the development of muscle bioreactors can only be made in the context of the history of this endeavor. Modern efforts tend to focus more upon the use of computer control and the application of mechanical strain as a stimulus, as well as substrate surface modifications to induce cellular organization at the early stages of culture of isolated muscle cells.
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Affiliation(s)
- R G Dennis
- Department of Biomedical Engineering, University of North Carolina at Chapel Hill, Chapel Hill, USA
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Liang S, Kahlenberg MS, Rousseau DL, Hornsby PJ. Neoplastic conversion of human colon smooth muscle cells: No requirement for telomerase. Mol Carcinog 2008; 47:478-84. [PMID: 18085530 DOI: 10.1002/mc.20405] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/05/2022]
Abstract
The role of telomerase as an essential requirement for the neoplastic conversion of human cells has been controversial. In the model of conversion of normal human cells to cancer cells by the combination of simian virus 40 (SV40) early region genes and oncogenic Ras (H-Ras(G12V)), telomerase (hTERT) was originally described as essential in conjunction with these other genes. Here we used primary cultures of colon smooth muscle cells isolated from surgical specimens. SV40 large T antigen (TAg) and oncogenic Ras(G12V) were introduced into the cells by retroviral transduction and cells were rapidly transplanted into the subrenal capsule space in immunodeficient mice, without selection in culture. Malignant tumors were formed from transduced cells. Extensive invasion into the kidney occurred even when tumors were small; in contrast, at the same tumor size, oncogene-expressing fibroblasts did not show much invasion. Increased invasiveness was also observed in vitro. However, cells in these cancers showed morphological evidence of crisis, consistent with their lack of telomerase. These experiments on human colon smooth muscle cells support the concept that Ras(G12V) and SV40 TAg form a minimal set of genes that can convert normal human cells to cancer cells without a requirement for hTERT.
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Affiliation(s)
- Sitai Liang
- Department of Physiology, Sam and Ann Barshop Institute for Longevity and Aging Studies, University of Texas Health Science Center, San Antonio, Texas 78245, USA
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Abstract
Smooth muscle cells (SMC) make up the muscular portion of the gastrointestinal (GI) tract from the distal oesophagus to the internal anal sphincter. Coordinated contractions of these cells produce the motor patterns of GI motility. Considerable progress was made during the last 20 years to understand the basic mechanisms controlling excitation-contraction (E-C) coupling. The smooth muscle motor is now understood in great molecular detail, and much has been learned about the mechanisms that deliver and recover Ca2+ during contractions. The majority of Ca2+ that initiates contractions comes from the external solution and is supplied by voltage-dependent Ca2+ channels (VDCC). VDCC are regulated largely by the effects of K+ and non-selective cation conductances (NSCC) on cell membrane potential and excitability. Ca2+ entry is supplemented by release of Ca2+ from IP(3) receptor-operated stores and by mechanisms that alter the sensitivity of the contractile apparatus to changes in cytoplasmic Ca2+. Molecular studies of the regulation of smooth muscle have been complicated by the plasticity of SMC and difficulties in culturing these cells without dramatic phenotypic changes. Major questions remain to be resolved regarding the details of E-C coupling in human GI smooth muscles. New discoveries regarding molecular expression that give GI smooth muscle their unique properties, the phenotypic changes that occur in SMC in GI motor disorders, tissue engineering approaches to repair or replace defective muscular regions, and molecular manipulations of GI smooth muscles in animals models and in cell culture will be topics for exciting investigations in the future.
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Affiliation(s)
- K M Sanders
- Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, NV 89557, USA.
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Micro-Perfusion for Cardiac Tissue Engineering: Development of a Bench-Top System for the Culture of Primary Cardiac Cells. Ann Biomed Eng 2008; 36:713-25. [DOI: 10.1007/s10439-008-9459-2] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/04/2007] [Accepted: 01/31/2008] [Indexed: 10/22/2022]
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