Studies Of Extracellular Matrix Composites For Biomimetic Synthesis Of Neural Tissue Engineering Scaffolds

There is a move towards the use of tissue engineering method to promote the repair efficacy of peripheral nerve injury in nerve repair. An ideal tissue engineering nerve has to posses three dimension structure to allow easy ingrowth of regenerating axon and participate in regulating axon guidance. Schwann cell arranged in the scaffold in a highly organized manner, as well as secreted nerve growth factor, cell adhesion molecule and extracellular matrix (ECM) with neuroprotective effects to guide axon growth. In this study, neural tissue engineering scaffolds were constructed to mimick the natural nerve in both composition and structure to imitate the function of original tissue.To imitate the composition of original tissue, we used cauda equine-derived extracellular matrix (ce-ECM) and Wharton’s jelly ECM (wj-ECM) as raw materials to prepare neural tissue engineering scaffolds. CE-ECM retains bioactive components which can improve nerve repair to the maximum degree. WJ-ECM with low immunogenicity can restore the original vitality of cells. Both of them can set foundation for application of ECM by mimicking composition. To imitate the structure of original tissue, the ce-ECM was blended with poly(1-lactide-coglycolide) (PLGA) to fabricate nanostructured scaffolds with aligned fibers using electrospinning, which can improve the mechanical properties of composite scaffold while maintaining biological activity of ECM. wj-ECM was blended with modified bacterial cellulose (BC) derivatives to prepare scaffolds with oriented and porous structure by oriented crystallization technique, which provides the microarchitecture required for tissue repair. Based on the above method, the ECM might be a promising material for use in neural tissue repair. The detailed contents of the research are as follows:Natural porcine cauda equina was decellularized using Triton X-100 and sodium deoxycholate, shattered physically, and made into a suspension by differential centrifugation. The analysis of microstructure revealed that the ECM had a nanofibrous structure. Nucleic acid staining of the ce-ECM and quantitative PicoGreen analysis of the residual DNA content demonstrated that the cellular contents were effectively removed to prevent undesirable immune rejection. Analysis of the biochemical components demonstrated that extracellular collagen and sulfated glycosaminoglycan (sGAG) were preserved. Histochemical staining and immunofluorescent staining confirmed that the CE-ECM was rich in natural ECM components of collagen type I, laminin, and fibronectin. The wj-ECM retains significant biological components that might be useful for nerve regeneration.The ce-ECM was blended with PLGA to fabricate scaffolds using electrospinning for neural tissue engineering. Scanning electron microscope (SEM) results showed that the scaffolds possessed a nanostructure that could be well oriented. Fourier transform infrared spectroscopy (FTIR) and multiphoton-induced autofluorescence images showed the presence of the ECM in the scaffolds. The results of contact angle measurement revealed that composite scaffolds possessed good hydrophilicity. ce-ECM/PLGA scaffolds supported Schwann cells (SCs) migration and maintained healthy cellular morphological features, and the aligned fibers could regulate cell morphologic features by modulating cellular orientation. To examine the growth of neural tissue-derived cells on the ce-ECM scaffolds, dorsal root ganglions (DRGs) were cultured on ce-ECM/PLGA scaffolds. Axons in DRG explants extended parallel to the direction of the aligned fibers.WJ-ECM was blended with dialdehyde BC (DBC) and carboxymethye BC (CMBC). respectively, and then prepared scaffolds with oriented and porous structure by oriented crystallization and freezing-dry technique. The morphology observations showed that the scaffolds possess interconnecting, micro-hole network structure. The porosity of the scaffolds was about 70%, which can satisfy the requirements of tissue engineering. Immunofluorescent staining confirmed that the ECV1 components were uniformly distributed in the scaffolds. Bioactive components such as collagen and laminin were retained. Bone marrow stem cells (BMSCs) were cultured on the scaffolds. BMSCs proliferated well on the scaffolds, which indicated that the scaffolds had good biocompatibility. Observations carried out by SEM showed that BMSCs could grow into the pore of the scaffolds and secrete particles like extracellular matrices.Results above showed that ce-ECM/PLGA fiber scaffolds, wj-ECM/DBC and wj-ECM/CMBC porous scaffolds with orientation could support cells growth and proliferation and guide the growth of cells and neural tissue. The biomimetic scaffolds were consist of the composition and structure like natural ECM. This study established theoretical and experimental basis for the application of ECM materials in neural tissue engineering.