|
1
|
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.
Collapse
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.
| |
Collapse
|
|
2
|
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.
Collapse
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
| |
Collapse
|
|
3
|
Collier CA, Salikhova A, Sabir S, Foncerrada S, Raghavan SA. Crisis in the gut: navigating gastrointestinal challenges in Gulf War Illness with bioengineering. Mil Med Res 2024; 11:45. [PMID: 38978144 PMCID: PMC11229309 DOI: 10.1186/s40779-024-00547-2] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/21/2023] [Accepted: 06/26/2024] [Indexed: 07/10/2024] Open
Abstract
Gulf War Illness (GWI) is characterized by a wide range of symptoms that manifests largely as gastrointestinal symptoms. Among these gastrointestinal symptoms, motility disorders are highly prevalent, presenting as chronic constipation, stomach pain, indigestion, diarrhea, and other conditions that severely impact the quality of life of GWI veterans. However, despite a high prevalence of gastrointestinal impairments among these veterans, most research attention has focused on neurological disturbances. This perspective provides a comprehensive overview of current in vivo research advancements elucidating the underlying mechanisms contributing to gastrointestinal disorders in GWI. Generally, these in vivo and in vitro models propose that neuroinflammation alters gut motility and drives the gastrointestinal symptoms reported in GWI. Additionally, this perspective highlights the potential and challenges of in vitro bioengineering models, which could be a crucial contributor to understanding and treating the pathology of gastrointestinal related-GWI.
Collapse
Affiliation(s)
- Claudia A Collier
- Department of Biomedical Engineering, Texas A&M University, College Station, TX, 77843, USA
| | - Aelita Salikhova
- Department of Biomedical Engineering, Texas A&M University, College Station, TX, 77843, USA
| | - Sufiyan Sabir
- Department of Biomedical Engineering, Texas A&M University, College Station, TX, 77843, USA
| | - Steven Foncerrada
- Department of Biomedical Engineering, Texas A&M University, College Station, TX, 77843, USA
| | - Shreya A Raghavan
- Department of Biomedical Engineering, Texas A&M University, College Station, TX, 77843, USA.
| |
Collapse
|
|
4
|
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.
Collapse
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.
| |
Collapse
|
|
5
|
Kim M, Oh BY, Lee JS, Yoon D, Kim YR, Chun W, Kim JW, Son IT. Differentiation of Adipose-Derived Stem Cells into Smooth Muscle Cells in an Internal Anal Sphincter-Targeting Anal Incontinence Rat Model. J Clin Med 2023; 12:jcm12041632. [PMID: 36836167 PMCID: PMC9959483 DOI: 10.3390/jcm12041632] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/16/2022] [Revised: 02/05/2023] [Accepted: 02/16/2023] [Indexed: 02/22/2023] Open
Abstract
OBJECTIVE Studies on development of an anal incontinence (AI) model targeting smooth muscle cells (SMCs) of the internal anal sphincter (IAS) have not been reported. The differentiation of implanted human adipose-derived stem cells (hADScs) into SMCs in an IAS-targeting AI model has also not been demonstrated. We aimed to develop an IAS-targeting AI animal model and to determine the differentiation of hADScs into SMCs in an established model. MATERIALS AND METHODS The IAS-targeting AI model was developed by inducing cryoinjury at the inner side of the muscular layer via posterior intersphincteric dissection in Sprague-Dawley rats. Dil-stained hADScs were implanted at the IAS injury site. Multiple markers for SMCs were used to confirm molecular changes before and after cell implantation. Analyses were performed using H&E, immunofluorescence, Masson's trichrome staining, and quantitative RT-PCR. RESULTS Impaired smooth muscle layers accompanying other intact layers were identified in the cryoinjury group. Specific SMC markers, including SM22α, calponin, caldesmon, SMMHC, smoothelin, and SDF-1 were significantly decreased in the cryoinjured group compared with levels in the control group. However, CoL1A1 was increased significantly in the cryoinjured group. In the hADSc-treated group, higher levels of SMMHC, smoothelin, SM22α, and α-SMA were observed at two weeks after implantation than at one week after implantation. Cell tracking revealed that Dil-stained cells were located at the site of augmented SMCs. CONCLUSIONS This study first demonstrated that implanted hADSc restored impaired SMCs at the injury site, showing stem cell fate corresponding to the established IAS-specific AI model.
Collapse
Affiliation(s)
- Minsung Kim
- Department of Surgery, Hallym Sacred Heart Hospital, College of Medicine, Hallym University, Anyang 14068, Republic of Korea
| | - Bo-Young Oh
- Department of Surgery, Hallym Sacred Heart Hospital, College of Medicine, Hallym University, Anyang 14068, Republic of Korea
| | - Ji-Seon Lee
- Burn Institute, Hangang Sacred Heart Hospital, College of Medicine, Hallym University, Seoul 07247, Republic of Korea
| | - Dogeon Yoon
- Burn Institute, Hangang Sacred Heart Hospital, College of Medicine, Hallym University, Seoul 07247, Republic of Korea
| | - You-Rin Kim
- Burn Institute, Hangang Sacred Heart Hospital, College of Medicine, Hallym University, Seoul 07247, Republic of Korea
| | - Wook Chun
- Burn Institute, Hangang Sacred Heart Hospital, College of Medicine, Hallym University, Seoul 07247, Republic of Korea
- Department of Surgery, Hangang Sacred Heart Hospital, College of Medicine, Hallym University, Seoul 07247, Republic of Korea
| | - Jong Wan Kim
- Department of Surgery, Dontan Sacred Heart Hospital, College of Medicine, Hallym University, Hwaseong-si 18450, Republic of Korea
| | - Il Tae Son
- Department of Surgery, Hallym Sacred Heart Hospital, College of Medicine, Hallym University, Anyang 14068, Republic of Korea
- Institute for Regenerative Medicine, Hallym Sacred Heart Hospital, College of Medicine, Hallym University, Anyang 14068, Republic of Korea
| |
Collapse
|
|
6
|
Elia E, Brownell D, Chabaud S, Bolduc S. Tissue Engineering for Gastrointestinal and Genitourinary Tracts. Int J Mol Sci 2022; 24:ijms24010009. [PMID: 36613452 PMCID: PMC9820091 DOI: 10.3390/ijms24010009] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/01/2022] [Revised: 12/10/2022] [Accepted: 12/14/2022] [Indexed: 12/24/2022] Open
Abstract
The gastrointestinal and genitourinary tracts share several similarities. Primarily, these tissues are composed of hollow structures lined by an epithelium through which materials need to flow with the help of peristalsis brought by muscle contraction. In the case of the gastrointestinal tract, solid or liquid food must circulate to be digested and absorbed and the waste products eliminated. In the case of the urinary tract, the urine produced by the kidneys must flow to the bladder, where it is stored until its elimination from the body. Finally, in the case of the vagina, it must allow the evacuation of blood during menstruation, accommodate the male sexual organ during coitus, and is the natural way to birth a child. The present review describes the anatomy, pathologies, and treatments of such organs, emphasizing tissue engineering strategies.
Collapse
Affiliation(s)
- Elissa Elia
- Centre de Recherche en Organogénèse Expérimentale/LOEX, Regenerative Medicine Division, CHU de Québec-Université Laval Research Center, Québec, QC G1J 1Z4, Canada
| | - David Brownell
- Centre de Recherche en Organogénèse Expérimentale/LOEX, Regenerative Medicine Division, CHU de Québec-Université Laval Research Center, Québec, QC G1J 1Z4, Canada
| | - Stéphane Chabaud
- Centre de Recherche en Organogénèse Expérimentale/LOEX, Regenerative Medicine Division, CHU de Québec-Université Laval Research Center, Québec, QC G1J 1Z4, Canada
| | - Stéphane Bolduc
- Centre de Recherche en Organogénèse Expérimentale/LOEX, Regenerative Medicine Division, CHU de Québec-Université Laval Research Center, Québec, QC G1J 1Z4, Canada
- Department of Surgery, Faculty of Medicine, Université Laval, Québec, QC G1V 0A6, Canada
- Correspondence: ; Tel.: +1-418-525-4444 (ext. 42282)
| |
Collapse
|
|
7
|
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.
Collapse
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
| |
Collapse
|
|
8
|
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.
Collapse
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.)
| |
Collapse
|
|
9
|
Dadhich P, Bohl JL, Tamburrini R, Zakhem E, Scott C, Kock N, Mitchell E, Gilliam J, Bitar KN. BioSphincters to treat Fecal Incontinence in Nonhuman Primates. Sci Rep 2019; 9:18096. [PMID: 31792260 PMCID: PMC6888838 DOI: 10.1038/s41598-019-54440-3] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/08/2019] [Accepted: 11/12/2019] [Indexed: 02/07/2023] Open
Abstract
Loss of anorectal resting pressure due to internal anal sphincter (IAS) dysfunctionality causes uncontrolled fecal soiling and leads to passive fecal incontinence (FI). The study is focused on immediate and long-term safety and potential efficacy of bioengineered IAS BioSphincters to treat passive FI in a clinically relevant large animal model of passive FI. Passive FI was successfully developed in Non-Human Primates (NHPs) model. The implantation of autologous intrinsically innervated functional constructs resolved the fecal soiling, restored the resting pressure and Recto Anal Inhibitory Reflex (RAIR) within 1-month. These results were sustained with time, and efficacy was preserved up to 12-months. The histological studies validated manometric results with the regeneration of a well-organized neuro-muscular population in IAS. The control groups (non-treated and sham) remained affected by poor anal hygiene, lower resting pressure, and reduced RAIR throughout the study. The pathological assessment of implants, blood, and the vital organs confirmed biocompatibility without any adverse effect after implantation. This regenerative approach of implanting intrinsically innervated IAS BioSphincters has the potential to offer a better quality of life to the patients suffering from FI.
Collapse
Affiliation(s)
- Prabhash Dadhich
- Wake Forest Institute for Regenerative Medicine, Wake Forest School of Medicine, Winston Salem, NC, USA
- Program in Neuro-Gastroenterology and Motility, Wake Forest School of Medicine, Winston Salem, NC, USA
| | - Jaime L Bohl
- Department of Surgery, Wake Forest School of Medicine, Winston Salem, NC, USA
| | - Riccardo Tamburrini
- Department of Surgery, Wake Forest School of Medicine, Winston Salem, NC, USA
| | - Elie Zakhem
- Wake Forest Institute for Regenerative Medicine, Wake Forest School of Medicine, Winston Salem, NC, USA
- Program in Neuro-Gastroenterology and Motility, Wake Forest School of Medicine, Winston Salem, NC, USA
| | - Christie Scott
- Wake Forest Institute for Regenerative Medicine, Wake Forest School of Medicine, Winston Salem, NC, USA
| | - Nancy Kock
- Department of Pathology, Section on Comparative Medicine, Wake Forest School of Medicine, Winston Salem, NC, USA
| | - Erin Mitchell
- Animal Resources Program, Wake Forest Baptist Health, Winston Salem, NC, USA
| | - John Gilliam
- Section on Gastroenterology, 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.
- Program in Neuro-Gastroenterology and Motility, Wake Forest School of Medicine, Winston Salem, NC, USA.
- Section on Gastroenterology, Wake Forest School of Medicine, Winston Salem, NC, USA.
- Virginia Tech-Wake Forest School of Biomedical Engineering and Sciences, Wake Forest School of Medicine, Winston Salem, NC, USA.
| |
Collapse
|
|
10
|
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.
Collapse
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
| |
Collapse
|
|
11
|
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.
Collapse
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
| |
Collapse
|
|
12
|
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]
|
|
13
|
The extracellular matrix of the gastrointestinal tract: a regenerative medicine platform. Nat Rev Gastroenterol Hepatol 2017; 14:540-552. [PMID: 28698662 DOI: 10.1038/nrgastro.2017.76] [Citation(s) in RCA: 53] [Impact Index Per Article: 6.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/22/2022]
Abstract
The synthesis and secretion of components that constitute the extracellular matrix (ECM) by resident cell types occur at the earliest stages of embryonic development, and continue throughout life in both healthy and diseased physiological states. The ECM consists of a complex mixture of insoluble and soluble functional components that are arranged in a tissue-specific 3D ultrastructure, and it regulates numerous biological processes, including angiogenesis, innervation and stem cell differentiation. Owing to its composition and influence on embryonic development, as well as cellular and organ homeostasis, the ECM is an ideal therapeutic substrate for the repair of damaged or diseased tissues. Biologic scaffold materials that are composed of ECM have been used in various surgical and tissue-engineering applications. The gastrointestinal (GI) tract presents distinct challenges, such as diverse pH conditions and the requirement for motility and nutrient absorption. Despite these challenges, the use of homologous and heterologous ECM bioscaffolds for the focal or segmental reconstruction and regeneration of GI tissue has shown promise in early preclinical and clinical studies. This Review discusses the importance of tissue-specific ECM bioscaffolds and highlights the major advances that have been made in regenerative medicine strategies for the reconstruction of functional GI tissues.
Collapse
|
|
14
|
Walthers CM, Lyall CJ, Nazemi AK, Rana PV, Dunn JCY. Collagen and heparan sulfate coatings differentially alter cell proliferation and attachment in vitro and in vivo. TECHNOLOGY 2016; 4:159-169. [PMID: 28713850 PMCID: PMC5507618 DOI: 10.1142/s2339547816400033] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 06/07/2023]
Abstract
Tissue engineering is an innovative field of research applied to treat intestinal diseases. Engineered smooth muscle requires dense smooth muscle tissue and robust vascularization to support contraction. The purpose of this study was to use heparan sulfate (HS) and collagen coatings to increase the attachment of smooth muscle cells (SMCs) to scaffolds and improve their survival after implantation. SMCs grown on biologically coated scaffolds were evaluated for maturity and cell numbers after 2, 4 and 6 weeks in vitro and both 2 and 6 weeks in vivo. Implants were also assessed for vascularization. Collagen-coated scaffolds increased attachment, growth and maturity of SMCs in culture. HS-coated implants increased angiogenesis after 2 weeks, contributing to an increase in SMC survival and growth compared to HS-coated scaffolds grown in vitro. The angiogenic effects of HS may be useful for engineering intestinal smooth muscle.
Collapse
Affiliation(s)
- Christopher M Walthers
- Department of Bioengineering and Department of Surgery, University of California, Los Angeles, 10833 Le Conte Avenue, Los Angeles, CA 90095, USA
| | - Chase J Lyall
- Department of Bioengineering and Department of Surgery, University of California, Los Angeles, 10833 Le Conte Avenue, Los Angeles, CA 90095, USA
| | - Alireza K Nazemi
- Department of Bioengineering and Department of Surgery, University of California, Los Angeles, 10833 Le Conte Avenue, Los Angeles, CA 90095, USA
| | - Puneet V Rana
- Department of Bioengineering and Department of Surgery, University of California, Los Angeles, 10833 Le Conte Avenue, Los Angeles, CA 90095, USA
| | - James C Y Dunn
- Department of Bioengineering and Department of Surgery, University of California, Los Angeles, 10833 Le Conte Avenue, Los Angeles, CA 90095, USA
| |
Collapse
|
|
15
|
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.
Collapse
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
| |
Collapse
|
|
16
|
|
|
17
|
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.
Collapse
|
|
18
|
Rego SL, Zakhem E, Orlando G, Bitar KN. Bioengineered Human Pyloric Sphincters Using Autologous Smooth Muscle and Neural Progenitor Cells. Tissue Eng Part A 2015; 22:151-60. [PMID: 26563426 DOI: 10.1089/ten.tea.2015.0194] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/19/2022] Open
Abstract
Gastroparesis leads to inadequate emptying of the stomach resulting in severe negative health impacts. Appropriate long-term treatments for these diseases may require pyloric sphincter tissue replacements that possess functional smooth muscle cell (SMC) and neural components. This study aims to bioengineer, for the first time, innervated human pylorus constructs utilizing autologous human pyloric sphincter SMCs and human neural progenitor cells (NPCs). Autologous SMCs and NPCs were cocultured in dual-layered hydrogels and formed concentrically aligned pylorus constructs. Innervated autologous human pylorus constructs were characterized through biochemical and physiologic assays to assess the phenotype and functionality of SMCs and neurons. SMCs within bioengineered human pylorus constructs displayed a tonic contractile phenotype and maintained circumferential alignment. Neural differentiation within bioengineered constructs was verified by positive expression of βIII-tubulin, neuronal nitric oxide synthase (nNOS), and choline acetyltransferase (ChAT). Autologous bioengineered innervated human pylorus constructs generated a robust spontaneous basal tone and contracted in response to potassium chloride (KCl). Contraction in response to exogenous neurotransmitter acetylcholine (ACh), relaxation in response to vasoactive intestinal peptide (VIP), and electrical field stimulation (EFS) were also observed. Neural network integrity was demonstrated by inhibition of EFS-induced relaxation in the presence of a neurotoxin or nNOS inhibitors. Partial inhibition of ACh-induced contraction and VIP-induced relaxation following neurotoxin treatment was observed. These studies provide a proof of concept for bioengineering functional innervated autologous human pyloric sphincter constructs that generate a robust basal tone and contain circumferentially aligned SMCs, which display a tonic contractile phenotype and functional differentiated neurons. These autologous constructs have the potential to be used as (1) functional replacement organs and (2) physiologically relevant models to investigate human pyloric sphincter disorders.
Collapse
Affiliation(s)
- Stephen Lee Rego
- 1 Wake Forest Institute for Regenerative Medicine, Wake Forest School of Medicine , Winston-Salem, North Carolina
| | - Elie Zakhem
- 1 Wake Forest Institute for Regenerative Medicine, Wake Forest School of Medicine , Winston-Salem, North Carolina.,2 Department of Molecular Medicine and Translational Sciences, Wake Forest School of Medicine , Winston-Salem, North Carolina
| | - Giuseppe Orlando
- 3 Department of General Surgery, Wake Forest School of Medicine , Winston-Salem, North Carolina
| | - Khalil N Bitar
- 1 Wake Forest Institute for Regenerative Medicine, Wake Forest School of Medicine , Winston-Salem, North Carolina.,2 Department of Molecular Medicine and Translational Sciences, Wake Forest School of Medicine , Winston-Salem, North Carolina.,4 Virginia Tech-Wake Forest School of Biomedical Engineering and Sciences , Winston-Salem, North Carolina
| |
Collapse
|
|
19
|
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.
Collapse
|
|
20
|
Rego SL, Zakhem E, Orlando G, Bitar KN. Bioengineering functional human sphincteric and non-sphincteric gastrointestinal smooth muscle constructs. Methods 2015; 99:128-34. [PMID: 26314281 DOI: 10.1016/j.ymeth.2015.08.014] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/02/2015] [Revised: 06/29/2015] [Accepted: 08/23/2015] [Indexed: 01/04/2023] Open
Abstract
Digestion and motility of luminal content through the gastrointestinal (GI) tract are achieved by cooperation between distinct cell types. Much of the 3 dimensional (3D) in vitro modeling used to study the GI physiology and disease focus solely on epithelial cells and not smooth muscle cells (SMCs). SMCs of the gut function either to propel and mix luminal contents (phasic; non-sphincteric) or to act as barriers to prevent the movement of luminal materials (tonic; sphincteric). Motility disorders including pyloric stenosis and chronic intestinal pseudoobstruction (CIPO) affect sphincteric and non-sphincteric SMCs, respectively. Bioengineering offers a useful tool to develop functional GI tissue mimics that possess similar characteristics to native tissue. The objective of this study was to bioengineer 3D human pyloric sphincter and small intestinal (SI) constructs in vitro that recapitulate the contractile phenotypes of sphincteric and non-sphincteric human GI SMCs. Bioengineered 3D human pylorus and circular SI SMC constructs were developed and displayed a contractile phenotype. Constructs composed of human pylorus SMCs displayed tonic SMC characteristics, including generation of basal tone, at higher levels than SI SMC constructs which is similar to what is seen in native tissue. Both constructs contracted in response to potassium chloride (KCl) and acetylcholine (ACh) and relaxed in response to vasoactive intestinal peptide (VIP). These studies provide the first bioengineered human pylorus constructs that maintain a sphincteric phenotype. These bioengineered constructs provide appropriate models to study motility disorders of the gut or replacement tissues for various GI organs.
Collapse
Affiliation(s)
- Stephen L Rego
- Wake Forest Institute for Regenerative Medicine, Wake Forest School of Medicine, Winston-Salem, NC, United States.
| | - Elie Zakhem
- Wake Forest Institute for Regenerative Medicine, Wake Forest School of Medicine, Winston-Salem, NC, United States; Department of Molecular Medicine and Translational Sciences, Wake Forest School of Medicine, Winston-Salem, NC, United States.
| | - Giuseppe Orlando
- Department of General Surgery, Wake Forest School of Medicine, Winston-Salem, NC, United States.
| | - Khalil N Bitar
- Wake Forest Institute for Regenerative Medicine, Wake Forest School of Medicine, Winston-Salem, NC, United States; Department of Molecular Medicine and Translational Sciences, Wake Forest School of Medicine, Winston-Salem, NC, United States; Virginia Tech-Wake Forest School of Biomedical Engineering and Sciences, Winston-Salem, NC, United States.
| |
Collapse
|
|
21
|
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.
Collapse
Affiliation(s)
- Nina Parmar
- Applied Biomedical Engineering Group, University College London, 21 University Street, London, WC1E 6JJ, UK
| | | | | | | |
Collapse
|
|
22
|
Rego SL, Raghavan S, Zakhem E, Bitar KN. Enteric neural differentiation in innervated, physiologically functional, smooth muscle constructs is modulated by bone morphogenic protein 2 secreted by sphincteric smooth muscle cells. J Tissue Eng Regen Med 2015; 11:1251-1261. [PMID: 25926098 DOI: 10.1002/term.2027] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/01/2014] [Revised: 02/09/2015] [Accepted: 03/19/2015] [Indexed: 01/01/2023]
Abstract
The enteric nervous system (ENS) controls gastrointestinal (GI) functions, including motility and digestion, which are impaired in ENS disorders. Differentiation of enteric neurons is mediated by factors released by the gut mesenchyme, including smooth muscle cells (SMCs). SMC-derived factors involved in adult enteric neural progenitor cells (NPCs) differentiation remain elusive. Furthermore, physiologically relevant in vitro models to investigate the innervations of various regions of the gut, such as the pylorus and lower oesophageal sphincter (LES), are not available. Here, neural differentiation in bioengineered innervated circular constructs composed of SMCs isolated from the internal anal sphincter (IAS), pylorus, LES and colon of rabbits was investigated. Additionally, SMC-derived factors that induce neural differentiation were identified to optimize bioengineered construct innervations. Sphincteric and non-sphincteric bioengineered constructs aligned circumferentially and SMCs maintained contractile phenotypes. Sphincteric constructs generated spontaneous basal tones. Higher levels of excitatory and inhibitory motor neuron differentiation and secretion of bone morphogenic protein 2 (BMP2) were observed in bioengineered, innervated, sphincteric constructs compared to non-sphincteric constructs. The addition of BMP2 to non-sphincteric colonic SMC constructs increased nitrergic innervations, and inhibition of BMP2 with noggin in sphincteric constructs decreased functional relaxation. These studies provide: (a) the first bioengineered innervated pylorus and LES constructs; (b) physiologically relevant models to investigate SMCs and adult NPCs interactions; and (c) evidence of the region-specific effects of SMCs on neural differentiation mediated by BMP2. Furthermore, this study paves the way for the development of innervated bioengineered GI tissue constructs tailored to specific disorders and locations within the gut. Copyright © 2015 John Wiley & Sons, Ltd.
Collapse
Affiliation(s)
- Stephen L Rego
- Wake Forest Institute for Regenerative Medicine, Wake Forest School of Medicine, Winston-Salem, NC, USA
| | - Shreya Raghavan
- Wake Forest Institute for Regenerative Medicine, Wake Forest School of Medicine, Winston-Salem, NC, USA
| | - Elie Zakhem
- Wake Forest Institute for Regenerative Medicine, 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
| |
Collapse
|
|
23
|
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.
Collapse
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
| |
Collapse
|
|
24
|
Smooth muscle strips for intestinal tissue engineering. PLoS One 2014; 9:e114850. [PMID: 25486279 PMCID: PMC4259486 DOI: 10.1371/journal.pone.0114850] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/15/2014] [Accepted: 11/14/2014] [Indexed: 01/04/2023] Open
Abstract
Functionally contracting smooth muscle is an essential part of the engineered intestine that has not been replicated in vitro. The purpose of this study is to produce contracting smooth muscle in culture by maintaining the native smooth muscle organization. We employed intact smooth muscle strips and compared them to dissociated smooth muscle cells in culture for 14 days. Cells isolated by enzymatic digestion quickly lost maturity markers for smooth muscle cells and contained few enteric neural and glial cells. Cultured smooth muscle strips exhibited periodic contraction and maintained neural and glial markers. Smooth muscle strips cultured for 14 days also exhibited regular fluctuation of intracellular calcium, whereas cultured smooth muscle cells did not. After implantation in omentum for 14 days on polycaprolactone scaffolds, smooth muscle strip constructs expressed high levels of smooth muscle maturity markers as well as enteric neural and glial cells. Intact smooth muscle strips may be a useful component for engineered intestinal smooth muscle.
Collapse
|
|
25
|
Bitar KN, Raghavan S, Zakhem E. Tissue engineering in the gut: developments in neuromusculature. Gastroenterology 2014; 146:1614-24. [PMID: 24681129 PMCID: PMC4035447 DOI: 10.1053/j.gastro.2014.03.044] [Citation(s) in RCA: 30] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/31/2013] [Revised: 03/17/2014] [Accepted: 03/20/2014] [Indexed: 12/13/2022]
Abstract
The complexity of the gastrointestinal (GI) tract lies in its anatomy as well as in its physiology. Several different cell types populate the GI tract, adding to the complexity of cell sourcing for regenerative medicine. Each cell layer has a specialized function in mediating digestion, absorption, secretion, motility, and excretion. Tissue engineering and regenerative medicine aim to regenerate the specific layers mimicking architecture and recapitulating function. Gastrointestinal motility is the underlying program that mediates the diverse functions of the intestines, as an organ. Hence, the first logical step in GI regenerative medicine is the reconstruction of the tubular smooth musculature along with the drivers of their input, the enteric nervous system. Recent advances in the field of GI tissue engineering have focused on the use of scaffolding biomaterials in combination with cells and bioactive factors. The ability to innervate the bioengineered muscle is a critical step to ensure proper functionality. Finally, in vivo studies are essential to evaluate implant integration with host tissue, survival, and functionality. In this review, we focus on the tubular structure of the GI tract, tools for innervation, and, finally, evaluation of in vivo strategies for GI replacements.
Collapse
Affiliation(s)
- Khalil N. Bitar
- Wake Forest Institute for Regenerative Medicine, Wake Forest School of Medicine, Winston-Salem NC 27101,Virginia Tech-Wake Forest School of Biomedical Engineering and Sciences, Winston-Salem NC 27101
| | - Shreya Raghavan
- Wake Forest Institute for Regenerative Medicine, Wake Forest School of Medicine, Winston-Salem NC 27101,Virginia Tech-Wake Forest School of Biomedical Engineering and Sciences, Winston-Salem NC 27101
| | - Elie Zakhem
- Wake Forest Institute for Regenerative Medicine, Wake Forest School of Medicine, Winston-Salem NC 27101,Virginia Tech-Wake Forest School of Biomedical Engineering and Sciences, Winston-Salem NC 27101
| |
Collapse
|
|
26
|
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: 29] [Impact Index Per Article: 2.6] [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.
Collapse
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
| |
Collapse
|
|
27
|
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.
Collapse
Affiliation(s)
- Robert R Gilmont
- 1 Institute for Regenerative Medicine, Wake Forest School of Medicine , Winston-Salem, North Carolina
| | | | | | | |
Collapse
|
|
28
|
Abstract
PURPOSE OF REVIEW To describe basic principles of tissue engineering with emphasis on the potential role of gastrointestinal endoscopy in regenerative medicine. RECENT FINDINGS Stricturing associated with endoscopic submucosal resection and circumferential endoscopic mucosal resection can be prevented through transplantation of autologous epidermal cell sheets or seeded decellularized biological scaffolds. Lower esophageal sphincter augmentation through injection of muscle-derived cells is a novel potential treatment for gastroesophageal reflux disease. Stem cell derived tissue has been used to repair injured colon in a mouse model of colitis. A bioengineered internal anal sphincter has been successfully implanted in mice and showed preserved functionality. SUMMARY The immediate foreseeable application of tissue engineering in gastrointestinal endoscopy is in the field of mucosal repair after acute injury. Tissue regeneration can be achieved through expansion of autologous somatic cells or by induction of multipotent or pluripotent stem cells. Advances in cellular scaffolding have made bioengineering of complex tissues a reality. Tissue engineering in endoscopy is also being pioneered by studies looking at enteral sphincter augmentation and regeneration. The availability of engineered tissue for endoscopic application will increase with advances in cell-culturing techniques.
Collapse
|
|
29
|
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.
Collapse
Affiliation(s)
- Sun Hwan Bae
- Department of Pediatrics, School of Medicine, Konkuk University, Seoul, Korea
| |
Collapse
|
|
30
|
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.
Collapse
|
|
31
|
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.
Collapse
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
| |
Collapse
|
|
32
|
PENFIELD JOSHUAD, GOROSPE EMMANUELC, WANG KENNETHK. Tissue-engineered cell sheets for stricture prevention: a new connection between endoscopy and regenerative medicine. Gastroenterology 2012; 143:526-529. [PMID: 22842059 PMCID: PMC3815668 DOI: 10.1053/j.gastro.2012.07.030] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/02/2022]
|
|
33
|
Wong VW, Wan DC, Gurtner GC, Longaker MT. Regenerative Surgery: Tissue Engineering in General Surgical Practice. World J Surg 2012; 36:2288-99. [DOI: 10.1007/s00268-012-1710-1] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022]
|
|
34
|
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.
Collapse
Affiliation(s)
- Giuseppe Orlando
- Wake Forest Institute for Regenerative Medicine, Winston Salem, NC, USA.
| | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | |
Collapse
|
|
35
|
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.
Collapse
Affiliation(s)
- Elie Zakhem
- Wake Forest Institute for Regenerative Medicine, Wake Forest School of Medicine, Winston-Salem, NC 27101, USA
| | | | | | | |
Collapse
|
|
36
|
Rattan S, Singh J. RhoA/ROCK pathway is the major molecular determinant of basal tone in intact human internal anal sphincter. Am J Physiol Gastrointest Liver Physiol 2012; 302:G664-75. [PMID: 22241857 PMCID: PMC3330775 DOI: 10.1152/ajpgi.00430.2011] [Citation(s) in RCA: 26] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/31/2023]
Abstract
The knowledge of molecular control mechanisms underlying the basal tone in the intact human internal anal sphincter (IAS) is critical for the pathophysiology and rational therapy for a number of debilitating rectoanal motility disorders. We determined the role of RhoA/ROCK and PKC pathways by comparing the effects of ROCK- and PKC-selective inhibitors Y 27632 and Gö 6850 (10(-8) to 10(-4) M), respectively, on the basal tone in the IAS vs. the rectal smooth muscle (RSM). Western blot studies were performed to determine the levels of RhoA/ROCK II, PKC-α, MYPT1, CPI-17, and MLC(20) in the unphosphorylated and phosphorylated forms, in the IAS vs. RSM. Confocal microscopic studies validated the membrane distribution of ROCK II. Finally, to confirm a direct relationship, we examined the enzymatic activities and changes in the basal IAS tone and p-MYPT1, p-CPI-17, and p-MLC(20), before and after Y 27632 and Gö 6850. Data show higher levels of RhoA/ROCK II and related downstream signal transduction proteins in the IAS vs. RSM. In addition, data show a significant correlation between the active RhoA/ROCK levels, ROCK enzymatic activity, downstream proteins, and basal IAS tone, before and after ROCK inhibitor. From these data we conclude 1) RhoA/ROCK and downstream signaling are constitutively active in the IAS, and this pathway (in contrast with PKC) is the critical determinant of the basal tone in intact human IAS; and 2) RhoA and ROCK are potential therapeutic targets for a number of rectoanal motility disorders for which currently there is no satisfactory treatment.
Collapse
Affiliation(s)
- Satish Rattan
- Dept. of Medicine, Division of Gastroenterology & Hepatology, Philadelphia, PA 19107, USA.
| | | |
Collapse
|
|
37
|
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: 33] [Impact Index Per Article: 2.5] [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.
Collapse
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,
| | | |
Collapse
|
|
38
|
Affiliation(s)
- Johann Peterson
- Department of Pediatrics, Stanford University School of Medicine
| | | |
Collapse
|
|
39
|
Singh J, Maxwell PJ, Rattan S. Immunocytochemical evidence for PDBu-induced activation of RhoA/ROCK in human internal anal sphincter smooth muscle cells. Am J Physiol Gastrointest Liver Physiol 2011; 301:G317-25. [PMID: 21566015 PMCID: PMC3154599 DOI: 10.1152/ajpgi.00084.2011] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/31/2023]
Abstract
Studies were performed to determine the unknown status of PKC and RhoA/ROCK in the phorbol 12,13-dibutyrate (PDBu)-stimulated state in the human internal anal sphincter (IAS) smooth muscle cells (SMCs). We determined the effects of PDBu (10(-7) M), the PKC activator, on PKCα and RhoA and ROCK II translocation in the human IAS SMCs. We used immunocytochemistry and fluorescence microcopy in the basal state, following PDBu, and before and after PKC inhibitor calphostin C (10(-6) M), cell-permeable RhoA inhibitor C3 exoenzyme (2.5 μg/ml), and ROCK inhibitor Y 27632 (10(-6) M). We also determined changes in the SMC lengths via computerized digital micrometry. In the basal state PKCα was distributed almost uniformly throughout the cell, whereas RhoA and ROCK II were located in the higher intensities toward the periphery. PDBu caused significant translocation of PKCα, RhoA, and ROCK II. PDBu-induced translocation of PKCα was attenuated by calphostin C and not by C3 exoenzyme and Y 27632. However, PDBu-induced translocation of RhoA was blocked by C3 exoenzyme, and that of ROCK II was attenuated by both C3 exoenzyme and Y 27632. Contraction of the human IAS SMCs caused by PDBu in parallel with RhoA/ROCK II translocation was attenuated by C3 exoenzyme and Y 27632 but not by calphostin C. In human IAS SMCs RhoA/ROCK compared with PKC are constitutively active, and contractility by PDBu is associated with RhoA/ROCK activation rather than PKC. The relative contribution of RhoA/ROCK vs. PKC in the pathophysiology and potential therapy for the IAS dysfunction remains to be determined.
Collapse
Affiliation(s)
- Jagmohan Singh
- Department of Medicine, Division of Gastroenterology and Hepatology, and Department of Surgery, Division of Colon and Rectal Surgery, Jefferson Medical College of Thomas Jefferson University, Philadelphia, Pennsylvania
| | - Pinckney J. Maxwell
- Department of Medicine, Division of Gastroenterology and Hepatology, and Department of Surgery, Division of Colon and Rectal Surgery, Jefferson Medical College of Thomas Jefferson University, Philadelphia, Pennsylvania
| | - Satish Rattan
- Department of Medicine, Division of Gastroenterology and Hepatology, and Department of Surgery, Division of Colon and Rectal Surgery, Jefferson Medical College of Thomas Jefferson University, Philadelphia, Pennsylvania
| |
Collapse
|
|
40
|
Huber A, Badylak SF. Phenotypic changes in cultured smooth muscle cells: limitation or opportunity for tissue engineering of hollow organs? J Tissue Eng Regen Med 2011; 6:505-11. [PMID: 21755602 DOI: 10.1002/term.451] [Citation(s) in RCA: 29] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/10/2010] [Accepted: 05/23/2011] [Indexed: 01/27/2023]
Abstract
Smooth muscle cells (SMCs) are typically used as a cell source for the reconstruction of hollow organs by conventional tissue engineering techniques. However, the necessity for and advantage of the use of tissue-specific SMCs are unknown. The present study investigated the phenotypic changes that occur following isolation and in vitro expansion of rat SMC populations isolated from three different tissues: the aorta, oesophagus and urinary bladder. rSMCs were isolated by enzymatic dispersion and expanded by conventional cell culture techniques, yielding microscopically homogeneous populations. SMC phenotypes were monitored according to their expression of marker proteins during the first two passages. Two of the three SMC populations (rSMC-a and rSMC-e) showed a marked change in their marker protein profiles during the first two passages, which resulted in a homogeneous phenotype that was neither fully contractile nor fully synthetic. SMCs from the urinary bladder did not show such a shift. Differences between the three rSMC populations were observed with regard to proliferative activity and gene expression patterns, suggesting the retention of some tissue-specific cell characteristics. In summary, phenotypic changes in SMCs occur as a result of conventional cell isolation and expansion techniques, thus questioning the necessity for a tissue-specific cell source for regenerative medicine applications.
Collapse
Affiliation(s)
- Alexander Huber
- McGowan Institute for Regenerative Medicine, University of Pittsburgh, PA, USA
| | | |
Collapse
|
|
41
|
Raghavan S, Gilmont RR, Miyasaka EA, Somara S, Srinivasan S, Teitelbaum DH, Bitar KN. Successful implantation of bioengineered, intrinsically innervated, human internal anal sphincter. Gastroenterology 2011; 141:310-9. [PMID: 21463628 PMCID: PMC3129458 DOI: 10.1053/j.gastro.2011.03.056] [Citation(s) in RCA: 56] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/16/2010] [Revised: 02/25/2011] [Accepted: 03/15/2011] [Indexed: 12/15/2022]
Abstract
BACKGROUND & AIMS To restore fecal continence, the weakened pressure of the internal anal sphincter (IAS) must be increased. We bioengineered intrinsically innervated human IAS to emulate sphincteric physiology in vitro. METHODS We cocultured human IAS circular smooth muscle with immortomouse fetal enteric neurons. We investigated the ability of bioengineered innervated human IAS, implanted in RAG1-/- mice, to undergo neovascularization and preserve the physiology of the constituent myogenic and neuronal components. RESULTS The implanted IAS was neovascularized in vivo; numerous blood vessels were observed with no signs of inflammation or infection. Real-time force acquisition from implanted and preimplant IAS showed distinct characteristics of IAS physiology. Features included the development of spontaneous myogenic basal tone; relaxation of 100% of basal tone in response to inhibitory neurotransmitter vasoactive intestinal peptide (VIP) and direct electrical field stimulation of the intrinsic innervation; inhibition of nitrergic and VIPergic electrical field-induced relaxation (by antagonizing nitric oxide synthesis or receptor interaction); contraction in response to cholinergic stimulation with acetylcholine; and intact electromechanical coupling (evidenced by direct response to potassium chloride). Implanted, intrinsically innervated bioengineered human IAS tissue preserved the integrity and physiology of myogenic and neuronal components. CONCLUSIONS Intrinsically innervated human IAS bioengineered tissue can be successfully implanted in mice. This approach might be used to treat patients with fecal incontinence.
Collapse
Affiliation(s)
- Shreya Raghavan
- GI Molecular Motors Lab, Department of Pediatrics-Gastroenterology, University of Michigan Medical School, Ann Arbor MI
| | - Robert R. Gilmont
- GI Molecular Motors Lab, Department of Pediatrics-Gastroenterology, University of Michigan Medical School, Ann Arbor MI
| | - Eiichi A. Miyasaka
- Department of Surgery, University of Michigan Medical School, Ann Arbor MI
| | - Sita Somara
- GI Molecular Motors Lab, Department of Pediatrics-Gastroenterology, University of Michigan Medical School, Ann Arbor MI
| | | | | | - Khalil N. Bitar
- GI Molecular Motors Lab, Department of Pediatrics-Gastroenterology, University of Michigan Medical School, Ann Arbor MI
| |
Collapse
|
|
42
|
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.
Collapse
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
| |
Collapse
|
|
43
|
de Godoy MAF, Rattan S. Role of rho kinase in the functional and dysfunctional tonic smooth muscles. Trends Pharmacol Sci 2011; 32:384-93. [PMID: 21497405 DOI: 10.1016/j.tips.2011.03.005] [Citation(s) in RCA: 51] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/29/2010] [Revised: 03/05/2011] [Accepted: 03/11/2011] [Indexed: 01/13/2023]
Abstract
Tonic smooth muscles play pivotal roles in the pathophysiology of debilitating diseases of the gastrointestinal and cardiovascular systems. Tonic smooth muscles differ from phasic smooth muscles in the ability to spontaneously develop myogenic tone. This ability has been primarily attributed to the local production of specific neurohumoral substances that can work in conjunction with calcium sensitization via signal transduction events associated with the Ras homolog gene family, member A (RhoA)/Rho-associated, coiled-coil containing protein kinase 2 (ROCK II) pathways. In this article, we discuss the molecular pathways involved in the myogenic properties of tonic smooth muscles, particularly the contribution of protein kinase C vs the RhoA/ROCK II pathway in the genesis of basal tone, pathophysiology and novel therapeutic approaches for certain gastrointestinal and cardiovascular diseases. Emerging evidence suggests that manipulation of RhoA/ROCK II activity through inhibitors or silencing of RNA interface techniques could represent a new therapeutic approach for various gastrointestinal and cardiovascular diseases.
Collapse
Affiliation(s)
- Márcio A F de Godoy
- Department of Medicine, Division of Gastroenterology and Hepatology, Jefferson Medical College of Thomas Jefferson University, Philadelphia, PA, USA
| | | |
Collapse
|
|
44
|
Miyasaka EA, Raghavan S, Gilmont RR, Mittal K, Somara S, Bitar KN, Teitelbaum DH. In vivo growth of a bioengineered internal anal sphincter: comparison of growth factors for optimization of growth and survival. Pediatr Surg Int 2011; 27:137-43. [PMID: 21046117 PMCID: PMC3022992 DOI: 10.1007/s00383-010-2786-z] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/30/2022]
Abstract
PURPOSE Our laboratory has developed and implanted a novel bioengineered internal anal sphincter (IAS) to treat anal incontinence. Fibroblast growth factor-2 (FGF-2) has been used in mice; however, the optimal growth factor for successful IAS implantation is unclear. This study compares several growth factors in order to optimize IAS viability and functionality. METHODS Bioengineered IAS rings were implanted subcutaneously into the dorsum of wildtype C57Bl/6 mice, with an osmotic pump dispensing FGF-2, vascular endothelial growth factor (VEGF), or platelet-derived growth factor (PDGF) (n = 4 per group). Control mice received IAS implants but no growth factor. The IAS was harvested approximately 25 days post-implantation. Tissue was subjected to physiologic testing, then histologically analyzed. Muscle phenotype was confirmed by immunofluorescence. RESULTS All implants supplemented with growth factors maintained smooth muscle phenotype. Histological scores, blood vessel density and muscle fiber thickness were all markedly better with growth factors. Neovascularization was comparable between the three growth factors. Basal tonic force of the constructs was highest with VEGF or PDGF. CONCLUSION All growth factors demonstrated excellent performance. As our ultimate goal is clinical implantation, our strong results with PDGF, a drug approved for use in the United States and the European Union, pave the way for translating bioengineered IAS implantation to the clinical realm.
Collapse
Affiliation(s)
- Eiichi A Miyasaka
- Section of Pediatric Surgery, Department of Surgery, University of Michigan, Mott Children's Hospital, F3970, Ann Arbor, MI 48109-0245, USA
| | | | | | | | | | | | | |
Collapse
|
|
45
|
Huang SC. Endothelin A receptors mediate relaxation of guinea pig internal anal sphincter through cGMP pathway. Neurogastroenterol Motil 2010; 22:1009-1, e264. [PMID: 20465591 DOI: 10.1111/j.1365-2982.2010.01513.x] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/08/2023]
Abstract
BACKGROUND Endothelin (ET) modulates motility of the internal anal sphincter through unclear receptor subtypes. METHODS We measured relaxation of guinea pig internal anal sphincter strips caused by ET-related peptides and binding of (125)I-ET-1 to cell membranes prepared from the internal anal sphincter muscle. Visualization of (125)I-ET-1 binding sites in tissue was performed by autoradiography. KEY RESULTS In the guinea pig internal anal sphincter, ET-1 caused a marked relaxation insensitive to tetrodotoxin, atropine, or omega-conotoxin GVIA. ET-2 was as potent as ET-1. ET-3 caused a mild relaxation. The relative potencies for ETs to cause relaxation were ET-1 = ET-2 > ET-3. The ET-1-induced relaxation was inhibited by BQ-123, an ET(A) antagonist, but not by BQ-788, an ET(B) antagonist. These indicate that ET(A) receptors mediate the relaxation. The relaxant response of ET-1 was attenuated by LY 83583, KT 5823, Rp-8CPT-cGMPS, tetraethyl ammonium, 4-aminopyridine and N(omega)-nitro-L-arginine, but not significantly affected by N(G)-nitro-L-arginine methyl ester, N(G)-methyl-L-arginine, charybdotoxin, apamin, KT 5720, and Rp-cAMPS. These suggest the involvement of cyclic guanosine 3',5'-cyclic monophosphate (cGMP), and potassium channels. Autoradiography localized (125)I-ET-1 binding to the internal anal sphincter. Binding of (125)I-ET-1 to the cell membranes prepared from the internal anal sphincter revealed the presence of two subtypes of ET receptors, ET(A) and ET(B) receptors. CONCLUSIONS & INFERENCES Taken together, these results demonstrate that ET(A) receptors mediate relaxation of guinea pig internal anal sphincter through the cGMP pathway.
Collapse
Affiliation(s)
- S-C Huang
- Department of Internal Medicine, Buddhist Tzu Chi General Hospital, Hualien and Tzu Chi University, Hualien, Taiwan.
| |
Collapse
|
|
46
|
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.
Collapse
Affiliation(s)
- Shreya Raghavan
- Department of Pediatrics-Gastroenterology, University of Michigan Medical School, Ann Arbor, 48109-0658, USA
| | | | | | | | | | | | | |
Collapse
|
|
47
|
Whitehead WE, Bharucha AE. Diagnosis and treatment of pelvic floor disorders: what's new and what to do. Gastroenterology 2010; 138:1231-5, 1235.e1-4. [PMID: 20176023 PMCID: PMC3924316 DOI: 10.1053/j.gastro.2010.02.036] [Citation(s) in RCA: 47] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/18/2022]
Affiliation(s)
- William E. Whitehead
- Center for Functional Gastrointestinal and Motility Disorders, Division of Gastroenterology and Hepatology and Department of Obstetrics and Gynecology, University of North Carolina at Chapel Hill, Chapel Hill, NC
| | - Adil E. Bharucha
- Clinical Enteric Neuroscience Translational and Epidemiological Research (C.E.N.T.E.R.) Program, Division of Gastroenterology and Hepatology, Mayo Clinic and Mayo Foundation, Rochester, Minnesota
| |
Collapse
|
|
48
|
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.
Collapse
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)
| |
Collapse
|
|
49
|
RATTAN SATISH, PHILLIPS BENJAMINR, MAXWELL PINCKNEYJ. RhoA/Rho-kinase: pathophysiologic and therapeutic implications in gastrointestinal smooth muscle tone and relaxation. Gastroenterology 2010; 138:13-8.e1-3. [PMID: 19931260 PMCID: PMC5599165 DOI: 10.1053/j.gastro.2009.11.016] [Citation(s) in RCA: 45] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/22/2023]
Affiliation(s)
- SATISH RATTAN
- Department of Medicine, Division of Gastroenterology & Hepatology, Jefferson Medical College of Thomas Jefferson University, Philadelphia, PA
| | - BENJAMIN R. PHILLIPS
- Department of Surgery, Division of Colon and Rectal Surgery, Jefferson Medical College of Thomas Jefferson University, Philadelphia, PA
| | - PINCKNEY J. MAXWELL
- Department of Surgery, Division of Colon and Rectal Surgery, Jefferson Medical College of Thomas Jefferson University, Philadelphia, PA
| |
Collapse
|