Preliminary study on the preparation of lyophilized acellular nerve scaffold complexes from rabbit sciatic nerves with human umbilical cord mesenchymal stem cells

Figure 1 Comparison of DNA and collagen content among the various groups. A: DNA content in each group; B: Collagen content in each group. ^aP < 0.05 vs control group; n = 3.

Figure 2 Histological observation of acellular scaffolds. A: Hematoxylin and eosin (HE) staining shows the peripheral nerve fibers enveloped by the perineurium and a large amount of nuclear components may be seen in the peripheral nerves; B: After decellularization, the matrix structure of the HE-stained nerve fibers is preserved; however, the cell structure is no longer visible; C: Luxol fast blue (LFB) staining of peripheral nerve tissue reveals significant blue staining of myelin sheath tissue; D: LFB staining shows decreased staining of decellularized nerve tissue and myelin sheaths.

Figure 3 Mechanical properties of the acellular scaffolds. A: Comparison of the maximum load between acellular scaffolds and peripheral nerves; B: Comparison of elastic modulus between acellular scaffolds and peripheral nerves; C: Stress durability test of acellular scaffolds and peripheral nerves; D: Stress-time diagram of peripheral nerves; E: Stress-time relationship of acellular scaffolds. ^aP < 0.0001 (control vs acellular nerve scaffolds); n = 10.

Figure 4 Hematoxylin and eosin staining after acellular scaffold implantation. A: 3 days after surgery, a large number of inflammatory cells began to appear around the scaffold, including those surrounding the scaffold and spreading toward the middle; B: 7 days postoperatively, the number of inflammatory cells around the scaffold was further increased and the scaffold was further enveloped by the surrounding inflammatory cells; C: 3 weeks after surgery, the inflammatory cells around the scaffold began to fade; D: 6 weeks postoperatively, the inflammatory reaction further subsided and the shape of the scaffold was preserved.

Figure 5 Systemic compatibility testing. A: Changes in postoperative C-reactive protein levels; B: Postoperative erythrocyte sedimentation rate changes; C: Changes in postoperative IL-6 Levels; D: MTT detection of cytotoxicity. n = 10. CRP: C-reactive protein; ESR; Erythrocyte sedimentation rate.

Figure 6 Culture and identification of human umbilical cord mesenchymal stem cells. A: Morphological observation of human umbilical cord mesenchymal stem cells (hUC-MSCs) after 3 days of culture; B: Morphological observation of hUC-MSCs cultured for one week; C: Flow cytometry for measuring the levels of hUC-MSC surface markers CD44, CD29, and CD34; D: Alizarin red staining; E: Oil red O staining. APC: Adipocyte precursor cell.

Figure 7 Identification of Schwan-like cells after induced differentiation. A: Cell morphology after 3 days of induced differentiation and culture; B: Cell morphology after 7 days of induced differentiation and culture; C: Immunohistochemical fluorescence identification of differentiated cells.

Figure 8 In vitro assessment of myelination capacity of the cells after induction. A: MPZ expression in the induced cells; B: PC12 cells; C: Morphology of PC12 cells co-cultured with differentiated cells; D: Layered myelin structure suspected to be formed under scanning electron microscopy.