Canine Sciatic Nerve Decellularization

Individual components of the ECM were examined through immunofluorescent staining of collagen I, collagen III, and collagen IV before and after decellularization (Figure 7A-F). Collagen I was seen both within the endoneurial and epineurial space before decellularization (Figure 7A) but was reduced by decellularization (Figure 7B). Little positive staining of collagen III was seen in both native (Figure 7C) and decellularized (Figure 7D) sciatic nerve tissue sections. Maintenance of the basal lamina was investigated through staining of collagen IV, a major constituent of the basal lamina. While immunofluorescent labeling showed that the content and organization of collagen I and III was affected by decellularization (Figure 7A–D), collagen IV within individual nerve bundles was strongly preserved (Figure 7E–F). Strong positive staining within the decellularized samples both within the endoneurial basal lamina and within the perineurium (Figure 7E–F) with a similar pattern of staining between the decellularized and native samples, suggests a strongly preserved basal lamina structure. The intensity of the staining within the endoneurium, however, was less than the perineurium and the architecture was slightly disrupted as compared to native tissue.

maintained. Enhanced magnification (1000X) of the same samples, native (Figure 7I) and decellularized (Figure 7J) endoneurial tissue. Images show the presence (Figure 7I) and removal of (Figure 7J) axons within the endoneurium. Biochemical assays for hydroxyproline (Figure 7K) and glycosaminoglycans (GAG) (Figure 7L) did not reveal any significant changes to either component after peripheral nerve decellularization (p = 0.57 n = 5, p = 0.26 n = 5). These components were compared to urinary bladder matrix (UBM) which had significantly more hydroxyproline (p = 0.001) (Figure 7K) but significantly less GAG (p = 0.001, Figure 7L) than PNM and native nerve. Different letters denote a significant difference (p < 0.05).

The solubilized PNM can be reconstituted into a stable hydrogel at concentrations as low as 8 mg/ml. This was visually confirmed by depositing the neutralized PNM solution within a stainless-steel ring and incubating at 37° C for 30 min (Figure 7M). Under SEM, the hydrogel was characterized by a highly porous network of fibular proteins (Figure 7N). Image shows macroscopic (Figure 7M) and SEM image (Figure 7N) of an 8 mg/ml equine PNM hydrogel

Figure 7: Characterization of remaining content within canine sciatic nerve after decellularization

(A–F) Immunofluorescent staining of collagen I, collagen III, and collagen IV before and after decellularization. Collagen I was seen both within the endoneurial and epineurial space before

staining was seen in the decellularized nerve tissue (F) suggesting a preservation of the basal lamina. (G, H) Low and (I, J) high magnification scanning electron microscopy (SEM) images of native and decellularized sciatic nerve. Cross-sectioned native (G) and decellularized (H) tissue at 150X magnification shows a dense epineurial space that it is largely cleared by decellularization but perineurium and endoneurium structure largely maintained. Enhanced magnification (1000X) of the same samples, native (I) and decellularized (J) endoneurial tissue. Images show the presence (I) and removal of (J) axons within the endoneurium. Biochemical assays for hydroxyproline (K) and glycosaminoglycans (GAG) (L) did not reveal any significant changes to either component after peripheral nerve decellularization (p=0.57 n=5, p=0.26 n=5). These components were compared to urinary bladder matrix (UBM) which had significantly more hydroxyproline but significantly less GAG than PNM and native nerve. Different letters denote a significant difference (p<0.05). Image shows macroscopic (E) and SEM image (F) of an 8 mg/ml equine PNS-ECM hydrogel. Reused with permission (4546560968632). [52]

2.3.3.1 Porcine sciatic nerve decellularization

The qualitative presence of tissue-specific extracellular matrix components was confirmed using immunofluorescent staining methods. Samples were stained with antibodies specific to collagen I, III, IV and laminin. Native and decellularized porcine nerve sections on the top and bottom rows respectively stained for collagen I (Figure 8A, B), collagen III (Figure 8C, D), collagen IV (Figure 8E, F), and laminin (Figure 8G, H). Collagen I was seen predominately within the epineurial space around the nerve bundles before decellularization ( 8A) but was completely

native ( 8C) and decellularized ( 8D) sciatic nerve tissue sections. Maintenance of the basal lamina was investigated through staining of collagen IV and laminin, a major constituent of the basal lamina. While immunofluorescent labeling showed that the content and organization of collagen I and III was affected by decellularization ( 8A–D), collagen IV and laminin within individual nerve bundles was strongly preserved ( 8E–H). Strong positive staining within the decellularized samples both within the endoneurial basal lamina and within the perineurium ( 8E–F) with a similar pattern of staining between the decellularized and native samples, suggests a strongly preserved basal lamina structure

Quantitative assessment of ECM components hydroxyproline (Figure 8I) and glycosaminoglycans was determined through direct spectrophotometric assays (Figure 8J). No significant differences were observed in either hydroxyproline or glycosaminoglycan content between native and decellularized porcine sciatic nerve tissues.

Assessment of nerve specific growth factors including brain derived neurotrophic factor (BDNF) ( 9A), neurotrophic factor 3 (NT-3) ( 9B), ciliary neurotrophic factor (CNTF) ( 9C), and nerve growth factor (NGF) ( 9D) in PNM as compared to native tissues and decellularized tissues from other anatomic locations (UBM- urinary bladder, SIS- small intestine submucosa) was assessed using ELISA kits. Some similarities between PNM and both SIS and UBM were observed. PNM had similar levels as SIS of BDNF and similar levels to UBM for NGF. In addition to these similarities, both SIS and UBM showed significantly higher concentrations of NT-3 (p < 0.05, n = 5). However, in all four nerve-specific growth factors surveyed PNM most closely

Figure 8: Structural ECM components of porcine sciatic nerve

(A–F) Immunofluorescent staining of collagen I, collagen III, collagen IV, and laminin before and after decellularization. Collagen I was seen both within the endoneurial and epineurial space before decellularization (A) but was reduced by decellularization (B). Little positive staining of collagen III was seen in both native (C) and decellularized (D) sciatic nerve tissue sections. Collagen IV, a major component of the basal lamina had strong staining around the individual nerve fibers, in the endoneurial space, and in the perineurium in native nerve (E). The same pattern of collagen IV and laminin staining was seen in the decellularized nerve tissue (F) suggesting a preservation of the basal lamina. Biochemical assays for hydroxyproline (I) and glycosaminoglycans (GAG) (J) did not reveal any significant changes to either component after peripheral nerve decellularization (p=0.6 n=5, p=0.4 n=5).

Figure 9: Bioactive components in porcine PNM

Assessment of nerve specific growth factors including brain derived neurotrophic factor (BDNF)(A), neurotrophic factor 3 (NT-3) (B), ciliary neurotrophic factor (CNTF)(C), and nerve growth factor (NGF)(D) in PNM as compared to native tissues and decellularized tissues from other anatomic locations (UBM- urinary bladder, SIS- small intestine). PNM was found to have similar levels of nerve specific growth factors including brain derived neurotrophic factor (BDNF)(A), neurotrophic factor 3 (NT-3)(B), ciliary neurotrophic factor (CNTF)(C), and nerve

2.3.4 Maintenance of Extracellular Matrix Ultrastructure and Components

Maintenance of the basal lamina was investigated through staining of collagen IV, a major constituent of the basal lamina. While immunofluorescent labeling showed that the content and organization of collagen I and III was affected by decellularization (Figure 8A-D), collagen IV within individual nerve bundles was strongly preserved (Figure 8E-H). Strong positive staining within the decellularized samples both within the endoneurial basal lamina and within the perineurium (Figure 8E-H), suggests a strongly preserved basal lamina structure. The intensity of the staining within the endoneurium, however, was less than the perineurium and the architecture was slightly disrupted as compared to native tissue.

PNM was found to have similar levels of nerve specific growth factors including brain derived neurotrophic factor (BDNF) (Figure 9A), neurotrophic factor 3 (NT-3) (Figure 9B), ciliary neurotrophic factor (CNTF) (Figure 9C), and nerve growth factor (NGF) (Figure 9D) as compared to native tissues. In most cases, PNM had significantly increased levels as compared to decellularized tissues from other anatomic locations (UBM- urinary bladder, SIS- small intestine) except for NT-3 where both native nerve and PNM had significantly less than UBM and SIS.

2.3.5 Assessment of Mechanical and Morphometric Characteristics of Resultant PNM