The Study of Neointestinal Regeneration Using an Autologous Intestinal Smooth Muscle Cells-Seeded Tubular Scaffold Fabricated by Gelatin and Poly(Ε-Caprolactone)-Block-Poly(Γ-Glutamic Acid) Composite Hydrogel

Treatment of short bowel syndrome is challenging. Although intestinal adaptation is an effective bio-response after post-resection compensation, malnutrition associated with severe short bowel syndrome cannot always be fully compensated for by intestinal adaptation. The addition of small segments of autologous grafts fabricated by tissue engineering to enhance intestinal adaptation is a compelling idea. In the 21 th century, tissue engineering has enabled the use of biological replacements for reconstructing and repairing skin, urinary bladder, bone, cartilage and heart valve. However, animal studies on the reconstruction of small intestine are still in their early stage. A literature review of tissue engineering of the small intestine indicated that those animals had silicone stent survived in accompany with neointestinal regeneration along the stent direction. Unfortunately, slow-growing and incomplete muscle regeneration are 2 common drawbacks of previous designs. Naturally derived biomaterials with less favorable biomechanical property, such as small intestine submucosa or collagen, are known to degrade rapidly almost before a permanent extracellular matrix (ECM) is constituted. In such situations, the contribution of naturally derived biomaterials to neointestinal regeneration is not considered significant when compared to silicone stents. This speculation is also supported by our previous results of 2 individual novel models, which are published by BJS and WRR, wherein silicone tubes without degradable biomaterials were used for neointestinal regeneration. At early stage of regeneration, the silicone tubes were found to be covered with a thin ECM, which was prepared for subsequent regeneration by emerge cells derived from neighboring intestinal tissues, but it led to the formation of a slow-growing and incomplete muscle layer possibly owing to an insufficient amount of initial ECM for accelerating regeneration. Therefore, the identification of an optimal combination of naturally derived biomaterials and synthetic polymers with favorable biomechanical properties becomes the most important concern of small intestine tissue engineering. The main objective of the first year project is to complete the fabrication of tubular composite scaffolds by using gelatin (G) and poly (ε-caprolactone)-block-poly (γ-glutamic acid) (CG). Material identification by dilute solution viscometry, Fourier transform infrared spectroscopy, proton nuclear magnetic resonance spectroscopy, and gel permeation chromatography is performed to confirm the synthetic nature of polymer. Physical and chemical properties of the G/CG composite tubular scaffold are also identified using a scanning electron microscope, capillary flow porometer, rheometer, as well as by determining the in vitro degradation rate and swelling ratio. Intestinal smooth muscle cells (ISMCs) is isolated and identified for cell seeding within the G/CG composite tubular scaffold. Cell adhesion test and cell proliferation test are also performed to evaluate the in vitro biocompatibility of the G/CG composite tubular scaffold. The main objective in the second year is to accomplish the in vivo implantation of scaffolds acquired from the control, contrast, or experimental group. The effect of using autologous ISMCs seeded G/CG composite tubular scaffolds on neointestinal regeneration is also evaluated and compared among all groups at 4 and 12 weeks post-implantation by measuring the length, weight, circumference, villi height, crypt depth, muscle thickness, mucosa and muscle length, proliferation index, and phenotype change of muscular tissue. In functional studies, animals underwent neointestinal regeneration for 12 weeks are enrolled for end-to-end reanastomosis in an attempt to reconnect neointestine back to original intestine, and then followed-up to determine their survival rate at 1, 4, 12 weeks post-surgery; and neointestinal function including peristalsis using X-ray small bowel series and absorption by detecting changes in the serum levels of citrulline/I-FABP or tissue mRNA levels of SGLT-1. In the third year, we intend to examine different protein expression during neointestinal regeneration in 5 pathways (Wnt, Hedgehog, BMP-4, Notch, Hippo) and 6 cytokines (FGF, EGF, IGF-I, PDGF, TGF-β, VEGF) among individual groups by western blotting at 4 and 12 weeks post-surgery. Target genes are screened using cDNA microarray and bioinformatics software. Expression molecules with significant differences will be marked after biotechnical examination by either qualitative detection of genes by in situ hybridization and proteins by IHC, or quantitative detection of genes by real-time PCR and proteins by western blotting. Localization of marked molecules and their possible interaction within tissues were analyzed by immunofluorescence imagines obtained using confocal microscopy. The adjacent factors for suspicious protein-protein interaction were verified using co-immunoprecipitation (co-IP). While stimulatory/inhibitory testing in vitro, primary culture including smooth muscle cells, epithelial cells, and mesenteric mixed cells, are individually isolated from regenerated neointestine. Culture cells are either stimulated by the growth hormone and expression vectors reconstructed from marked molecules, or interfered with siRNA and chemical inhibitors. Localization and interaction of intracellular signaling factors is also observed and confirmed by confocal microscopy and co-IP. In summary, this 3-year project helps investigate the effects of G/CG composite scaffolds, ISMC seeded cells, and cell signaling on neointestinal regeneration, focusing on 3 key factors in tissue engineering. Based on deep learning, enhancing the efficiency of neointestinal regeneration and possessing actual function of neointestine, autologous transplantation with regenerated neointestine will become next chance to provide the best option for current clinical therapy of short bowel syndrome.

Project ID:PB10207-1921
External Project ID:NSC102-2320-B182-006