Development of Combinatorial Therapeutics for Spinal Cord Injury using Stem Cell Delivery

In this study, nerve-mimetic composite hydrogels were developed and characterized that can be utilized to investigate and capitalize on the pro-regenerative behavior of adipose-derived stem cells for spinal cord injury repair.

Traumatic spinal cord injury (SCI) induces permanent sensorimotor deficit below the site of injury. It affects approximately a quarter million people in the US, and it represents an immeasurable public health concern. Research has been conducted to provide effective therapy; however, SCI is still considered incurable due to the complex nature of the injury site. A variety of strategies, including drug delivery, cell transplantation, and injectable biomaterials, are investigated, but one strategy alone limits their efficacy for regeneration. As such, combinatorial therapies have recently gained attention that can target multifaceted features of the injury. It has been shown that extracellular matrices (ECM) may increase the efficacy of cell transplantation for SCI. To this end, 3D hydrogels consisting of decellularized spinal cords (dSCs) and sciatic nerves (dSNs) were developed at different ratios and characterized. Histological analysis of dSCs and dSNs confirmed the removal of cellular and nuclear components, and native tissue architectures were retained after decellularization. Afterward, composite hydrogels were created at different volumetric ratios and subjected to analyses of turbidity gelation kinetics, mechanical properties, and embedded human adipose-derived stem cell (hASC) viability. No significant differences in mechanical properties were found among the different ratios of hydrogels and decellularized spinal cord matrices. Human ASCs embedded in the gels remained viable throughout the 14-day culture. This study provides a means of generating tissue-engineered combinatorial hydrogels that present nerve-specific ECM and pro-regenerative mesenchymal stem cells. This platform can provide new insights into neuro-regenerative strategies after SCI with future investigations.

Approximately 296,000 people are suffering from traumatic SCI, and every year there are about 18,000 new SCI cases occurring in the U.S.A.¹. Traumatic SCI is commonly caused by falls, gunshot wounds, vehicle accidents, and sports activities and often causes permanent loss of sensorimotor function below the site of injury. The estimated lifetime expenses for SCI treatment range between one to five million dollars per individual with significantly lower life expectancies¹. Yet, SCI is still poorly understood and largely incurable, mainly due to complex pathophysiological consequences after the injury². Various strategies have been investigated, including cell transplantation and biomaterials-based scaffolds. While transplantation of cells and biomaterials has demonstrated potential, the multifaceted nature of SCI suggests that combinatorial approaches may be more beneficial³. As a result, many combinatorial strategies have been investigated and demonstrated better therapeutic efficacy than individual components. However, further studies are needed to provide novel biomaterials for delivering cells and drugs³.

One promising approach to fabricating natural hydrogels is tissue decellularization. The process of decellularization utilizes ionic, non-ionic, physical, and combinatorial methods to remove all or most cellular and nucleic materials while preserving ECM components. By removing all or most of the cellular components, ECM-derived hydrogels are less immunoreactive to the host after implantation/injection⁴. There are several parameters to measure in order to assess the quality of decellularized tissues: removal of cellular/nucleic contents, mechanical properties, and ECM preservation. The following criteria have been established to avoid adverse immune responses: 1) less than 50 ng double-stranded DNA (dsDNA) per mg ECM dry weight, 2) less than 200 bp DNA fragment length, and 3) almost or no visible nuclear material stained with 4'6-diamidino-2-phenuylindole (DAPI)⁵. Mechanical properties can be quantified by tensile, compression, and/or rheology tests, and they should be similar to the original tissue⁶. In addition, protein preservation can be evaluated by proteomics or quantitative assays focusing on the main components of decellularized tissues, for instance, laminin, glycosaminoglycan (GAG), and chondroitin sulfate proteoglycan (CSPG) for the spinal cord^7,8. Verified ECM-derived hydrogels can be recellularized with different types of cells to aid cell-based therapy⁹.

A variety of cell types, such as Schwann cells, olfactory ensheathing cells, bone-marrow-derived mesenchymal stem cells (MSCs), and neural stem/progenitor cells, have been studied for SCI repair^10,11,12. However, clinical use of these cells is limited due to ethical concerns, sparse integration with neighboring cells/tissues, lack of tissue sources for high yield, inability to self-renew, and/or limited proliferative capacity^13,14,15. Unlike these cell types, human adipose-derived MSCs (hASCs) are an attractive candidate because they are easily isolated in a minimally invasive manner using lipoaspirates, and a large number of cells can be obtained¹⁶. In addition, hASCs have the ability to secrete growth factors and cytokines that have the potential of neuroprotective, angiogenetic, wound healing, tissue regeneration, and immunosuppression^{17,18,19,20,21}.

As was described, multiple studies have been conducted^22,23,24, and a lot has been learned from them, but heterogeneous characteristics of SCI have limited their efficacy in promoting functional recovery. As such, combinatorial approaches have been proposed to increase treatment efficacy for SCI. In this study, composite hydrogels were developed by combining decellularized spinal cords and sciatic nerves for a three-dimensional (3D) hASC culture. Successful decellularization was confirmed by histological and DNA analyses, and different ratios of nerve composite hydrogels were characterized by gelation kinetics and compression tests. The viability of hASCs in the nerve composite hydrogels was investigated to prove that this hydrogel can be utilized as a 3D cell culture platform.

The porcine tissues were commercially obtained, so approval was not required by the animal ethics committee.

1. Decellularization of porcine spinal cords (Estimated time: 5 days)

NOTE: Perform the decellularization using previously established protocols with modifications^25,26. All procedures should be done in a sterile biosafety cabinet at room temperature unless stated otherwise. All solutions should be sterile filtered using a bottle top filter (0.2 µm pore size) into autoclaved bottles. Procedures to be carried out at 37 °C can be done inside an incubator or a clean oven set to 37 °C.

1. Preparation of decellularization solutions
   NOTE: All solutions are calculated for 1 L. Users may need to adjust the final required volume according to their experimental needs.
   1. Dilute 500 mL of 0.05% trypsin/ ethylenediaminetetraacetic acid (EDTA) with 500 mL of phosphate-buffered saline (PBS) to make 0.025% trypsin/EDTA.
   2. Dilute 300 mL of 10% Triton X-100 with 700 mL of PBS to make 3% Triton X-100. Mix 0.56 g of NaCl, 1.31 g of NaH₂PO₄H₂O, and 10.85 g of HNa₂O₄P·7H₂O in 1 L of deionized water to make 100 mM Na/50 mM phos buffer.
   3. Mix 32.4 g of sucrose in 1 L of deionized water to make 1 M sucrose. Mix 40 g of sodium deoxycholate (SD) in 1 L of deionized water to make 4% SD solution. Dilute 6.7 mL of 15% peracetic acid with 993.3 mL of 4% ethanol.
2. Preparation of porcine spinal cord
   NOTE: Spinal cords were shipped frozen without any solution and kept at -80 °C until use.
   1. Thaw spinal cord at 4 °C in the fridge for 18-24 h before decellularization. Use sterile scissors to remove dura mater carefully.
   2. Cut the spinal cord into small pieces (approximately 1 cm long). Place one piece into a 15 mL tube or a maximum of three pieces into a 50 mL tube.
3. Decellularization of spinal cord
   NOTE: After each step, decellularization solutions are manually poured into a large beaker to be discarded. Small autoclavable stainless-steel strainer can be used to help discard decellularization solutions without losing the tissues needed for each step. Spinal cord was agitated at 83 rpm unless stated otherwise.
   1. Rinse the spinal cord with deionized water for 18-24 h at 4 °C and 60 rpm.
   2. Rinse the spinal cord with 0.025% trypsin/EDTA for 1 h at 37 °C and 40 rpm. Then, rinse the spinal cord with PBS for 15 min, 2x.
   3. Rinse the spinal cord with 3% Triton X-100 for 2 h. Rinse the spinal cord with 100 mM Na/50 mM phos buffer for 15 min, 2x.
   4. Rinse the spinal cord with 1 M sucrose for 1 h. Then, rinse the spinal cord with deionized water for 1 h.
   5. Rinse the spinal cord with 4% SD for 2 h. Then, rinse the spinal cord with 100 mM Na/50 mM phos buffer for 15 min, 2x.
   6. Rinse the spinal cord with 0.1% peracetic acid in 4% ethanol for 4 h. Then, rinse the spinal cord with PBS for 1 h.
   7. Rinse the spinal cord with deionized water for 1 h, 2x. Then, rinse the spinal cord with PBS for 1 h.
   8. Lyophilize the spinal cord at 0.01 mbar and -56 °C for 3 days and store dry until use.

2. Decellularization of porcine sciatic nerve (Estimated time: 5 days)

NOTE: Perform the decellularization using a previously established protocol²⁷. All procedures should be done in a sterile biosafety cabinet at room temperature unless stated otherwise. All solutions should be sterile filtered using a bottle top filter (0.2 µm pore size) into autoclaved bottles. Procedures to be carried out at 37 °C can be done inside an incubator or a clean oven set to 37°C.

1. Preparation of decellularization solutions
   NOTE: All solutions are calculated for 1 L. Users may need to adjust the final required volume according to their experimental needs.
   1. Prepare 50 mM Na/10 mM phos buffer by mixing 1.86 g of NaCl, 0.262 g of d NaH₂PO₄H₂O, and 2.17 g of HNa₂O₄P·7H₂O in 1 L of deionized water.
   2. Prepare 125 mM sulfobetaine-10 (SB-10) solution by mixing 38.4 g of SB-10 in 1 L of 50 mM Na/10 mM phos buffer.
   3. Prepare 3 % SD/0.6 mM sulfobetaine-16 (SB-16) solution by mixing 30 g of SD and 0.24 g of SB-16 in 1 L of 50 mM Na/10 mM phos buffer.
2. Preparation of porcine sciatic nerve
   NOTE: Sciatic nerves were shipped frozen with PBS and kept at -80 °C until use.
   1. Thaw the sciatic nerve at 4 °C in the fridge 18-24 h before decellularization.
   2. Cut sciatic nerve into small pieces (approximately 1 cm long). Place one piece into a 15 mL tube or a maximum of three pieces into a 50 mL tube.
3. Decellularization of the sciatic nerve
   NOTE: After each step, decellularization solutions are manually poured into a large beaker to be discarded, and a small autoclavable stainless-steel strainer can be used to help discard decellularization solutions without losing the tissues needed for each step. The sciatic nerve was agitated at 15 rpm unless stated otherwise.
   1. Rinse the sciatic nerve with deionized water for 7 h. Then, rinse the sciatic nerve with 125 mM sulfobetaine-10 (SB-10) in 50 mM Na/10 mM phos buffer for 18 h.
   2. Rinse the sciatic nerve with 100 mM Na/50 mM phos buffer for 15 min. Then, rinse the sciatic nerve with 3% SD/0.6 mM sulfobetaine-16 (SB-16) in 50 mM Na/10 mM phos buffer for 2 h.
   3. Rinse the sciatic nerve with 100 mM Na/50 mM phos buffer for 15 min, 3x. Then, rinse the sciatic nerve with 125 mM SB-10 in 50 mM Na/10 mM phos buffer for 7 h.
   4. Rinse the sciatic nerve with 100 mM Na/50 mM phos buffer for 15 min. Then, rinse the sciatic nerve with 3% SD/0.6 mM SB-16 in 50 mM Na/10 mM phos buffer for 1.5 h.
   5. Rinse the sciatic nerve with 50 mM Na/10 mM phos buffer for 15 min, 3x. Then, rinse the sciatic nerve with 75 U/mL of deoxyribonuclease (DNase) for 3 h without agitation.
   6. Rinse the sciatic nerve with 50 mM Na/10 mM phos buffer for 1 h, 3x. Then, rinse the sciatic nerve with 0.2 U/mL of Chondroitinase ABC for 16 h without agitation at 37 °C.
   7. Rinse the sciatic nerve with PBS for 3 h, 3x. Lyophilize the sciatic nerve at 0.01 mbar and -56 °C for 3 days and store dry until use.

3. Digestion of decellularized tissues and fabrication of composite hydrogels (Estimated time: 4 days)

1. Chop or grind the decellularized tissues into powder by using scissors or homogenizer. Sterilize the tools to chop or grind the tissues using an autoclave at 121 °C for 45 min. The ethylene oxide method is also applicable.
2. Digest the tissues separately in 0.01 N hydrochloric acid (HCl) solution containing 1 mg/mL pepsin at a concentration of 15 mg/mL. The estimated weights of decellularized spinal cord and sciatic nerve are 50-100 mg and 100-150 mg per piece, respectively.
3. Place the magnetic bar and stir at 500 rpm and 4 °C for at least 4 days to generate pregel solutions.
4. Mix sciatic nerve and spinal cord pregel at following volumetric ratios: 2:1, 1:1, and 1:2. Adjust pH 7.4 using 1 N sodium hydroxide (NaOH) and HCl and dilute to the desired concentration using M199 media and 1x PBS. Incubate at 37 °C for 30 min.
   NOTE: Add NaOH 1 µL at a time. M199 media can be used as a pH indicator since it turns light pink when the pH is 7.4, but pH strips should be used to confirm the pH level.

4. Verification of decellularization

1. Hematoxylin and Eosin staining (H&E; Estimated time: 8 days)
   NOTE: After each rinse step, the solutions are manually poured into a large beaker to be discarded.
   1. Fix fresh and decellularized spinal cord and sciatic nerve in 3.7% formaldehyde at 4 °C for 18 h. Place one piece in a 15 mL tube.
   2. Remove formaldehyde and place the tissues in 10% sucrose for 1 day. Remove 10% sucrose and place the tissues in 30% sucrose for 6 days.
   3. Fill an appropriately sized cryomold with optimal cutting temperature (OCT) halfway. Place the decellularized tissues, cover them with OCT, and allow them to absorb the OCT for 1 day.
   4. After 1 day, freeze the tissues overnight at -80 °C. Cryosection the tissues at 10 µm thickness using a cryostat.
   5. Rinse with 1x PBS for 5 min, 2x. Then, rinse with tap water for 5 min.
   6. Stain with hematoxylin solution for 1 min. Rinse with tap water for 1 min, 3x.
   7. Stain with eosin solution for 1 min. Rinse with 95 % ethanol for 1 min, 2x and with 100% ethanol for 1 min, 3x.
   8. Rinse with xylene for 1 min, 2 min, and then 1 min. Let the slides dry for 5 min.
   9. Use a cotton swab to drip 3 - 4 drops of dibutylphthalate polystyrene xylene (DPX) mount solution onto the slides. Place the coverslips on top of DPX-covered slides. Let the slides dry overnight.
2. DNA analysis (Estimated time: 1 h)
   1. Weigh decellularized and lyophilized tissues. Isolate and quantify DNA using commercially available kits according to the manufacturer's instructions.

5. Characterization of composite hydrogels

1. Gelation turbidity test (Estimated time: 1 h)
   1. Place 100 µL of pregel solutions in each well of a 96-well plate on ice. Read absorbance at 405 nm every 2 min for 45 min using a plate reader.
   2. Calculate normalized absorbance using the following equation:
      (Absorbance - Absorbance_Initial) / (Absorbance_Maximum - Absorbance_Initial)
   3. Calculate the slope of the curve and the time to achieve 50% and 95% gelation, t_1/2 and t₉₅, respectively.
2. Compression test (Estimated time: 1 min per hydrogel)
   1. Fabricate composite hydrogels at the concentration of 12 mg/mL with 8 mm diameter and 2 mm height.
   2. Use a rheometer to compress the samples with a load of 250 N in between stainless-steel parallel plates at a strain rate of 10% of sample height/min. The software that provides rheometer readings will provide a stress-strain curve by applying the force and measuring the strain until the hydrogels break.
   3. Calculate Young's modulus from the slope of the linear region of the stress-strain curves.

6. Three-dimensional culture of human ASCs in nerve composite hydrogels

1. Preparation of three-dimensional (3D) platform (Estimated time: 3 h)
   1. Etch silicon wafer with SU-8 photoresist to generate circular patterns of 200 µm in depth and 4 mm in diameter.
   2. Mix polydimethylsiloxane (PDMS) base and curing agent at a ratio of 10:1. Pour the mixture onto the silicon wafer and let it sit for 20 min to remove all the bubbles that arise from mixing the PDMS base and curing agent.
   3. Place it in the oven and cure for 2 h at 70 °C. Demold the cured PDMS sheet and punch out using an 8 mm diameter biopsy punch to make PDMS microwells.
   4. Place microwells in a 96-well plate and put the well plate in an air-plasma cleaner to sterilize PDMS microwells.
   5. Functionalize PDMS microwells with 1% polyethyleneimine (PEI) and 0.1% glutaraldehyde (GA) for 10 min and 20 min, respectively. Wash microwells with distilled water, 2x.
2. Preparation of hASCs (Estimated time: 7 days)
   1. Culture passage cells for 2-5 passages in hASCs growth media (hASCs basal media supplemented with fetal bovine serum (FBS) and penicillin/streptomycin (Pen-strep)) until confluent. Passage and calculate the number of cells by using a hemacytometer or cell counter.
3. ASC-laden nerve composite hydrogels (Estimated time: 2 h)
   1. Mix sciatic nerve and spinal cord pregel at a volumetric ratio of 2:1, 1:1, 1:2, and prepare spinal cord-only hydrogel without mixing in any sciatic nerve pregel.
   2. Add M199 media and adjust pH 7.4 using 1 N NaOH and HCl. Resuspend hASCs with growth media in the pregel at a density of 1 x 10⁶ cells/mL.
      NOTE: The amount of M199 and cell suspension should be 10% of pregel.
   3. Dilute the pregel to 12 mg/mL using 1x PBS. Place the pregel onto microwells and incubate for 30 min. Culture ASC-laden hydrogels in hASCs growth media.
4. Cell viability test (Estimated time: 4 h per day)
   1. Prepare viability test solutions using commercially available reagents according to the manufacturer's instructions.
   2. Aspirate media on days 1, 4, 7, and 14. Add reagent to the wells and incubate at 37 °C for 3 h following the manufacturer's instructions.
   3. Read fluorescence at excitation/emission of 560/590 nm using a plate reader. Calculate the percentage difference using the following equation:
      [(fluorescence_sample- fluorescence_blank) / fluorescence_blank] x 100

Decellularized tissues were prepared using previously established protocols with slight modifications^26,27. After decellularization, lyophilization, and digestion, nerve composite hydrogels at ratios of SN:SC = 2:1, 1:1, 1:2, and spinal cord-only hydrogels were fabricated (Figure 1). Removal of nuclear components was confirmed by H&E staining (Figure 2A). To quantitatively assess the decellularization, residual DNA was measured within ECM. dsDNA content in the fresh dura mater was 132.6 ± 21.3 ng dsDNA/mg dry tissue, whereas fresh and decellularized spinal cord parenchyma contained 22.6 ± 8.3 ng dsDNA/mg dry tissue and 23.74 ± 6.69 ng dsDNA/mg dry tissue, respectively (Figure 2B). It indicated that the dura mater was to be removed prior to the decellularization. The amount of dsDNA of fresh and decellularized sciatic nerve were 222.5 ± 42.65 and 0.63 ng dsDNA/mg dry tissue, respectively (Figure 2C).

The gelation kinetics of nerve composite hydrogels were evaluated to determine the speed of gelation (S) and the times to reach 50% and 95% of the final turbidity (t_1/2 and t₉₅). The gelation kinetics showed a sigmoidal shape for all hydrogel groups (Figure 3A). The speed of gelation (S) in all hydrogel groups was not significantly different from one another. The time required to reach half of the final turbidity, t_1/2, was 11.32 ± 0.57 min, 13.33 ± 0.6 min, and 15.7 ± 0.92 min, and 17.23 ± 1.13 min for SN:SC= 2:1, 1:1, 1:2, and spinal cord-only hydrogels, respectively. The time required to reach 95% of the final turbidity, t₉₅, was 13.8 ± 0.83 min, 15.53 ± 0.83 min, 18.38 ± 0.79 min, and 19.62 ± 1.27 for SN:SC= 2:1, 1:1, 1:2 and spinal cord only hydrogels, respectively. These results indicate that hydrogels with more sciatic nerve content reach the steady state faster, and sciatic nerve ECM may promote hydrogel assembly. The compression test result showed a trend that hydrogels with more spinal cord content provide increased stiffness; nevertheless, it was not significantly different across all different ratios of composite hydrogels (Figure 3B).

Lastly, to determine the cell viability of hASCs in all hydrogel groups, 3D cultures were set up using microfabricated PDMS microwells, as shown in Figure 4. Throughout the 14-day culture, ASC viability was measured using a commercially available cell culture media-based colorimetric and fluorescence metabolic assay, where percentage differences from the readouts were calculated at four different time points: day 1, day 4, day 7, and day 14 (Figure 5). The value of the percentage difference increased as the time points increased, and that of spinal cord-only hydrogel was lower than the rest of the groups on days 1, 4, and 14. Taken together, these data suggest that hASC viability increased along the culture period due to cell proliferation and increment in cell metabolic activity.

Figure 1: Fabrication of nerve composite hydrogels. The porcine spinal cords and sciatic nerves were decellularized, lyophilized, and digested to obtain pregel solutions. Sciatic nerve and spinal cord pregel solutions were mixed at a ratio of 2:1, 1:1, and 1:2, and spinal cord-only pregel was also prepared. All pregel solutions were incubated at 37 °C to generate nerve composite hydrogels. Please click here to view a larger version of this figure.

Figure 2: Verification of decellularization. (A) H&E staining of fresh and decellularized porcine spinal cords and sciatic nerves. Arrows indicate the presence of cells in tissues. Scale bars = 1 mm for wide-view images and 500 µm for zoom-in images (200 µm only for zoom-in decellularized sciatic nerve images). (B) DNA analysis of fresh dura mater, parenchyma, and decellularized spinal cords (n=10, 16, and 6, respectively). (C) DNA analysis of fresh and decellularized sciatic nerves (n=8 and 3, respectively). Error bars represent standard deviations. Statistical analysis was done via one-way ANOVA with a Tukey's post-hoc test for (B) and a t-test for (C). Abbreviations: ns = not significant. Please click here to view a larger version of this figure.

Figure 3: Characterizations of nerve composite hydrogels. (A) Normalized absorbance of nerve composite hydrogels (n = 5). (B) Young's modulus of decellularized spinal cord and nerve composite hydrogels (n = 4 for decellularized spinal cord and 3 for each composite hydrogel group, respectively). Error bars represent standard deviations. Statistical analysis was done via one-way ANOVA with Tukey's post-hoc test. Abbreviations: ns = not significant. Please click here to view a larger version of this figure.

Figure 4: Schematic image of 3D culture. (A) PDMS microwells were fabricated on an SU-8-coated silicon wafer. Each microwell was punched out using an 8 mm biopsy punch. (B) Decellularized spinal cord and sciatic nerve at different ratios, adjust pH to 7.4, resuspend with hASCs, place the pregel on microwell, and incubate for 30 min at 37^°C. Please click here to view a larger version of this figure.

Figure 5: Cell viability assay. The percentage difference of hASCs cultured in nerve composite hydrogels at days 1, 4, 7, and 14 (n = 3). Error bars represent standard deviations. Statistical analysis was done via one-way ANOVA with Tukey's post-hoc test. * p<0.05; ** p<0.01; *** p<0.005; **** p<0.001; ns: not significant. Please click here to view a larger version of this figure.

It is widely believed that the pathophysiology of SCI is complex and multifaceted. Even though single therapies such as cell transplantation, drug delivery, and biomaterials each have provided valuable insights into SCI, the complicated nature of SCI may limit their individual efficacy^28,29,30,31. Therefore, efforts to develop effective combinatorial therapeutics have increased. The nerve composite hydrogels described in this study may serve as an effective delivery vehicle for cells or drugs. With regards to cells, ASCs are one of the promising candidates for cell transplantation. ASCs secrete immunomodulatory factors such as interleukin-10 (IL-10) and transforming growth factor beta (TGF-β) as well as neurotrophic factors such as nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), glial-derived neurotrophic factor (GDNF), and neurotrophin-3 (NT-3) that lead to increased dorsal root ganglia neurite outgrowth in vitro and function recovery in vivo^{17,19,32,33,34}. ASCs delivered in aqueous solutions promoted functional recovery after SCI in rats and humans; however, challenges with mediating migration, differentiation, survival, and differentiation of ASCs were noted^35,36,37. In addition, there are conflicting reports on the tumorigenic potential of transplanted ASCs; while some studies indicated that ASCs may contribute to the proliferation and metastasis of different types of cancer, other studies have also shown anti-tumorigenic potentials^38,39,40,41Hence, a long-term safety profile of ASCs in nerve composite hydrogels needs to be investigated in the future. Here, decellularized spinal cords and sciatic nerves have been studied separately for SCI repair.

Decellularized spinal cords were injected into a rat model of SCI and stimulated axonal ingrowth into the lesion and elevated functional recovery; however, due to rapid degradation, a cyst was formed, and recovery diminished after 4-8 weeks of treatment^{4, 42}. Acellular decellularized sciatic nerve hydrogel was injected in SCI rats and promoted neural regeneration, but the efficacy of hydrogel was limited due to rapid degradation²⁴. Therefore, combinatorial approaches using decellularized tissues have recently gained attention. It was reported that decellularized spinal cord hydrogels promoted neural stem cell/progenitor cell proliferation, migration, and differentiation into neurons⁴. It also facilitated 3D neurite outgrowth from N1E-115 neuroblastoma cells, indicating that the decellularized spinal cord hydrogels might be an attractive scaffold to promote axonal regeneration²⁶. Decellularized sciatic nerves have been studied with Schwann encapsulation and showed potential as a nerve graft after SCI⁴³. Taken together, a combination of decellularized spinal cords and sciatic nerves is expected to promote both axonal regeneration and tissue restoration. The current study also does not include assessing the therapeutic effects of ASC-laden nerve hydrogels. Therefore, it is essential to evaluate regenerative potentials, such as pro-angiogenic and neurotrophic effects, of the platform via in vitro and in vivo studies. In addition, nerve composite hydrogels can be easily applied to encapsulate other cell types, such as Schwann cells, olfactory stem cells, and neural stem cells, to improve the therapeutic efficacy of cell transplantation. It would be intriguing to investigate the safety and efficacy of various cell types within the nerve composite hydrogel at different stages of SCI.

One of the critical steps in creating composite hydrogels is to prepare an adequate amount of tissue, especially spinal cords. The yield of decellularized spinal cords is significantly low, as noted in the protocol (50-100 mg spinal cord piece and 100-150 mg sciatic nerve piece); thus, harvesting or obtaining spinal cords from small animals such as mice and rats is not recommended. At least middle-sized animals, such as rabbits, are suggested to gain a substantial amount of spinal cord matrices. Also, it is important to check the pH when digesting decellularized tissues since the addition of tissue can shift the pH beyond the required pepsin activation range, which is around 1.5-2. Agitation times for spinal cord decellularization are longer than the previously established method²⁶ because agitation speed is slower in the current protocol. The rotator used in this protocol offers up to 83 rpm, whereas a maximum of 200 rpm was used in the previously established methods. Therefore, agitation times were increased twice to thoroughly wash all the cellular debris. To ensure all the decellularization solutions can penetrate the tissues and wash all the cellular and nuclear contents, agitation times and speed need to be optimized using histology or DNA analysis on the user's end. It is suggested that the ratio between the tissues and decellularization solution be a minimum of 1:15 [w/v]. Decellularized tissues in the previously established protocol²⁶ were digested/stirred at room temperature; however, in this protocol, they were digested with a magnetic bar on a stir plate at 4 °C and 500 rpm for 4 days. The stir plate motor could generate heat and introduce it to the pregel solution. Therefore, it is recommended to digest at low temperatures or use a temperature-controllable stir plate.

As noted in DNA analysis (Figure 2B), it is critical to remove the dura mater of the spinal cord because a significant amount of DNA can be found within the dura mater. Dura mater, unlike spinal cord parenchyma, consists of fibrous tissue; therefore, different decellularization methods should be utilized as previously documented^44,45. H&E staining and DNA analysis of decellularized tissues have been utilized to verify the effectiveness of the decellularization process. The following criteria need to be satisfied to fulfill the goals of successful decellularization: 1. Less than 50 ng dsDNA/mg ECM dry weight, 2. Less than 200 bp DNA fragment length, 3. Lack of nuclear material in tissue sections or H&E⁵. Qualitative analysis of H&E staining after decellularization showed a lack of nuclei staining, suggesting successful removal of cells. In addition, components of ECM, such as collagen and other matrix proteins, were retained, and the staining intensity was reduced after decellularization, reflecting the removal of the cellular material. Following the decellularization, DNA contents were 0.63 ng in the sciatic nerve and 23.74 ± 6.69 ng in the spinal cord per mg ECM dry weight, which is below the acceptable threshold for in vivo application.

Rheological measurements of composite hydrogels need to be carefully designed, as rheometers with high compression force or tensile testers cannot be used to analyze the mechanical properties of soft materials such as nerve composite hydrogels created in this study. Samples will be torn/ripped apart when they are gripped on the tensile tester, and compression with high force will not provide data that is sensitive enough to analyze. In this study, a rheometer with a compression force of 250 N was utilized, and it is advised to find an apparatus with similar or proper force for users' purposes. As an alternative, different rheological measurements, such as oscillatory shear measurement, may help determine storage and loss moduli. Reduced mechanical properties after decellularization are a limitation in this study. Even though Young's modulus of all hydrogel groups is similar to that of decellularized spinal cord, it is significantly different from fresh spinal cords (data not shown). Neural stem cells and MSCs cultured on relatively soft materials showed increased neuronal differentiation and β-III tubulin expression, respectively^46,47. However, It is possible to reinforce or crosslink the decellularized materials with other materials such as alginate, poly(ethylene glycol), genipin, and glutaraldehyde^48,49,50,51. Optimization will be necessary to ensure that these strategies to enhance the mechanical properties of the hydrogels do not influence cellular behavior or viability.

Batch-to-batch variability of nerve composite hydrogels may be observed due in part to differences in animals' age, gender, weight, and species. Controlled and consistent use of the same type of animals may help to mitigate the variability. Pooling the tissue samples from different animals can also help reduce the variability between batches⁵². One of the potential challenges for translating this study to clinical applications is scalability. The low yield of spinal cord decellularization would hinder the production of a large amount of decellularized tissues. New methods would need to be developed/optimized to generate more tissues, or dura mater could be decellularized together to increase the yield after decellularization. Another potential challenge is regulatory clearance. In the US, the FDA is responsible for regulatory approval of cell-based therapies⁵³. The regulatory authority of the Republic of Korea approved the market entry of CARTISTEM, which is an MSC-based product consisting of allogeneic umbilical cord blood-derived MSCs and 4% of hyaluronate hydrogel for knee cartilage defect treatment⁵⁴. Precedent cases like these may help streamline the regulatory review process by providing safety and efficiency data.

The authors do not have anything to disclose.

This work was supported by the PhRMA Foundation and the National Institutes of Health through the award number P20GM139768 and R15NS121884 awarded to YS. We want to thank Dr. Kartik Balachandran and Dr. Raj Rao in the Department of Biomedical Engineering at the University of Arkansas for letting us use their equipment. Also, we want to thank Dr. Jin-Woo Kim and Mr. Patrick Kuczwara from the Department of Biological and Agricultural Engineering at the University of Arkansas for providing training on rheometer.