Optimized peripheral nerve decellularization protocol with enhanced extracellular matrix integrity, mechanical strength, and biocompatibility vs Hudson’s standard method

Objective(s): Decellularized nerve scaffolds are promising biomaterials for peripheral nerve regeneration. This study compared an optimized decellularization protocol with Hudson’s standard method to assess structural, mechanical, biochemical, and biological performance.Materials and Methods: Rat nerve tissues underwent native and optimized decellularization protocols. Morphological evaluation was performed using H&E and DAPI staining, while biomechanical properties and degradation kinetics were assessed over ۲۸ days. DNA content, S۱۰۰B, NEFL, collagen, laminin, and glycosaminoglycans (GAGs) were quantified to evaluate extracellular matrix (ECM) preservation and cellular removal. Neurotrophic factors, including NGF, BDNF, and GDNF, were measured to assess regenerative potential. Biocompatibility was evaluated after transplantation of rat bone marrow-derived mesenchymal stem cells (BM-MSCs) using MTT and Caspase-۳ assays. All experiments used n=۵ specimens per group, with triplicate measurements.Results: The optimized protocol achieved near-complete decellularization while preserving ECM architecture and significantly outperformed Hudson’s method (P<۰.۰۰۱). Optimized scaffolds exhibited intermediate stiffness (۱۳.۷۶ MPa), higher ultimate strength (۲.۳ MPa vs ۱.۶۲ MPa; P<۰.۰۱), and slower degradation (۴۸.۳% reduction; P<۰.۰۰۱). DNA content decreased by ۹۶.۹% in the optimized group compared with ۹۰.۵% in Hudson’s group. Neurotrophic factors were better preserved, with NGF reaching ۵۶.۲ pg/mg (P<۰.۰۰۱), and ECM components such as collagen and laminin improved significantly (P<۰.۰۵). Optimized scaffolds enhanced cell viability (OD ۰.۵۸ vs ۰.۴۳; P<۰.۰۰۱) and reduced Caspase-۳ activity (P<۰.۰۱).Conclusion: The optimized decellularization protocol surpasses Hudson’s method by achieving effective decellularization while preserving ECM structure, mechanical integrity, and BM-MSC viability, thereby supporting its potential application in peripheral nerve tissue engineering.

کلیدواژه ها:

Biocompatibility ، Caspase-۳ ، Decellularization ، Extracellular matrix ، Hudson’s method ، Optimized protocol ، Sciatic nerve