Establishment of Caprine Pancreas Derived Extracellular Matrix as a Novel Reservoir to Develop Xenotransplantable Organ and Scarless Wound Healing Hydrogel

Abstract

The quest for biomimetic scaffolds with enhanced regenerative potential in tissue engineering has led to the exploration of natural extracellular matrices (ECMs) derived from various sources. This thesis focuses on investigating the potential of the caprine pancreas-derived ECM as a promising biomaterial for tissue engineering and regenerative medicine applications. Commencing with the isolation and characterization of decellularized Caprine pancreatic ECM using several detergents, the aim was to identify the best decellularization method. Upon decellularization, the ECM of different tissues responds differently to the detergents used for decellularization at a physical and physiological level; hence the impact of decellularization by ionic (SDS and SDC), non-ionic (Triton X-100, and Tween-20), and zwitterionic detergents (CHAPS) was first analyzed via immersion and perfusion decellularization. The mode of decellularization (i.e., immersion and perfusion) was discovered to significantly impact the final scaffold quality, in addition to detergents. It was observed that perfusion decellularization yielded a significantly superior scaffold compared to immersion decellularization across all evaluated aspects, including residual DNA content (SDS, SDC, CHAPS, Tw-20, and TX-100 had 17.43 ± 1.18 ng/mg, 0.83 ± 0.31 ng/mg, 3.86 ± 0.32 ng/mg, 2.95 ± 0.11 ng/mg and 1.99 ± 0.37 ng/mg of DNA respectively), decellularization time (SDS, SDC, CHAPS, Tw-20, and TX-100 had taken 28h, 35h, 51h, 48h, and 35 h respectively ), tensile strength (SDS, SDC, CHAPS, Tw-20, and TX-100 had Young’s Modulus of 5.8 MPa, 5.6 MPa, 3.2 MPa, 6.01 MPa respectively), and overall physical properties. The caprine pancreas was decellularized and found suitable for use as a scaffold after the decellularization mode and detergents were verified. This was done using a thorough series of analytical methods, including histological inspection, biochemical tests, and scanning electron microscopy (SEM). The scaffold's mechanical properties and degradation kinetics were meticulously analyzed to ensure their stability and long-term functionality. The biological performance of this scaffold was assessed through in-vitro cell culture studies, elucidating its ability to support cell adhesion, proliferation, and differentiation just like native tissue. After the scaffold underwent successful evaluations, a decellularized ECM was created for Hydrogel. Its physical and biological compatibility were then assessed. The structural integrity, composition, and biocompatibility of hydrogel were rigorously evaluated to ascertain its suitability as a scaffold material. Additionally, in-vivo studies for the full-thickness wound model were conducted to investigate the regenerative potential of this hydrogel in the Wistar rat model. The hydrogel was able to heal the wound without scar formation due to its wet wound healing potential. We also demonstrated that the scaffold enabled the pancreatic cells to grow in a naturally occurring pattern resembling a grape bunch; however, the hydrogel lost its growth factors attached to the ECM due to frequent grinding and sterilization, consequently, pancreatic cells were sporadically multiplying within the hydrogel. Conclusively, the results obtained in the research showcase that this novel scaffold serve as a viable replacement of the porcine derived organ (which run the risk of developing zoonotic disease upon xenotransplantation). Overall, the findings of this thesis highlight the potential of this novel reservoir, i.e., caprine pancreas, as a versatile biomaterial for tissue engineering application, offering a promising avenue for developing innovative therapeutic strategies to address various clinical challenges in tissue repair and regeneration.