This work was supported by the PhRMA Foundation and the National Institutes of Health through the award number P20GM139768 and R15NS121884&#160;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.

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 modifications25,26. All procedures should be done in a sterile biosafety cabinet at room temperature unless stated otherwise. All solutio...

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 efficacy28,29,30,31. Therefore, efforts to develop effective combinatorial therapeutics have increased. The nerve compo...

Approximately 296,000 people are suffering from traumatic SCI, and every year&#160;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&#39;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 cord7,8. 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 repair10,11,12. 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 capacity13,14,15. 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 immunosuppression17,18,19,20,21.
As was described, multiple studies have been conducted22,23,24, 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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>3-(Decyldimethylammonio)propane sulfonate inner salt</td>
			<td>Sigma-Aldrich</td>
			<td>D4266</td>
			<td>Used during sciatic nerve decellularization, SB-10</td>
		</tr>
		<tr>
			<td>3-(N,N-Dimethylpalmitylammonio)propane sulfonate</td>
			<td>Sigma-Aldrich</td>
			<td>H6883</td>
			<td>Used during sciatic nerve decellularization, SB-16</td>
		</tr>
		<tr>
			<td>AlamarBlue reagent</td>
			<td>Fisher Scientific</td>
			<td>DAL1100</td>
			<td>Used during AlamaBlue cell viabiiltiy test</td>
		</tr>
		<tr>
			<td>Chondroitinase ABC</td>
			<td>Sigma-Aldrich</td>
			<td>C3667</td>
			<td>Used during sciatic nerve decellularization</td>
		</tr>
		<tr>
			<td>Cryostat</td>
			<td>Leica</td>
			<td>CM1860</td>
			<td></td>
		</tr>
		<tr>
			<td>Deoxyribonuclase</td>
			<td>Sigma-Aldrich</td>
			<td>D4263</td>
			<td>Used during sciatic nerve decellularization</td>
		</tr>
		<tr>
			<td>Disodium hydrogen phosphate heptahydrate</td>
			<td>VWR</td>
			<td>BDH9296</td>
			<td>Chemical for 100 mM Na/50 mM phos and 50 mM Na/10 mM phos buffer</td>
		</tr>
		<tr>
			<td>DNeasy Blood &#38; Tissue kit</td>
			<td>Qiagen</td>
			<td>69506</td>
			<td>Used during DNA analysis</td>
		</tr>
		<tr>
			<td>Dpx Mountant for histology,slide mounting medium</td>
			<td>Sigma-Aldrich</td>
			<td>6522</td>
			<td>Used during H&#38;E staining</td>
		</tr>
		<tr>
			<td>Eosin</td>
			<td>Sigma-Aldrich</td>
			<td>HT110216</td>
			<td>Used during H&#38;E staining</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>VWR</td>
			<td>89125-172</td>
			<td></td>
		</tr>
		<tr>
			<td>Formaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>252549</td>
			<td>Used during H&#38;E staining</td>
		</tr>
		<tr>
			<td>Glutaraldehyde (GA)</td>
			<td>Sigma-Aldrich</td>
			<td>G6257</td>
			<td>Used during PDMS surface functionalization</td>
		</tr>
		<tr>
			<td>hASC growth media</td>
			<td>Lonza</td>
			<td>PT-4505</td>
			<td>Used to culture hASCs, containing of fetal bovine serum and penicilin/streptomycin</td>
		</tr>
		<tr>
			<td>Hematoxylin</td>
			<td>VWR</td>
			<td>26041-06</td>
			<td>Used during H&#38;E staining</td>
		</tr>
		<tr>
			<td>human adipose-derived stem cell</td>
			<td>Lonza</td>
			<td>PT-5006</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrochloric acid (HCl)</td>
			<td>Sigma-Aldrich</td>
			<td>320331</td>
			<td>Used to digest decellularizied tissues and adjust pregels solutions</td>
		</tr>
		<tr>
			<td>M199 media</td>
			<td>Sigma-Aldrich</td>
			<td>M0650</td>
			<td>Used to dilute pregels to desired concentration</td>
		</tr>
		<tr>
			<td>Optimal cutting temperatue compound</td>
			<td>Tissue-Tek</td>
			<td>4583</td>
			<td></td>
		</tr>
		<tr>
			<td>Pepsin</td>
			<td>Sigma-Aldrich</td>
			<td>P7000</td>
			<td>Used to digest decellularized tissues</td>
		</tr>
		<tr>
			<td>Peracetic acid</td>
			<td>Lab Alley</td>
			<td>PAA1001</td>
			<td>Used during spinal cord decellularization</td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS)</td>
			<td>VWR</td>
			<td>97062-948</td>
			<td></td>
		</tr>
		<tr>
			<td>Plate reader</td>
			<td>BioTek Instruments</td>
			<td>Synergy Mx</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyethyleneimine (PEI)</td>
			<td>Sigma-Aldrich</td>
			<td>181978</td>
			<td>Used during PDMS surface functionalization</td>
		</tr>
		<tr>
			<td>Porcine sciatic nerve</td>
			<td>Tissue Source LLC</td>
			<td></td>
			<td>Live pigs, with no identifiable information and no traceability details</td>
		</tr>
		<tr>
			<td>Porcine spinal cord</td>
			<td>Tissue Source LLC</td>
			<td></td>
			<td>Live pigs, with no identifiable information and no traceability details</td>
		</tr>
		<tr>
			<td>QuantiFluor dsDNA system</td>
			<td>Promega</td>
			<td>E2670</td>
			<td>Used to analyze DNA contents</td>
		</tr>
		<tr>
			<td>Rheometer</td>
			<td>TA Instruments</td>
			<td>DHR 2</td>
			<td></td>
		</tr>
		<tr>
			<td>Rugged rotator</td>
			<td>Glas-co</td>
			<td>099A RD4512</td>
			<td>Used during spinal cord decellularization</td>
		</tr>
		<tr>
			<td>Sodium chloride (NaCl)</td>
			<td>VWR</td>
			<td>BDH9286</td>
			<td>Chemical for 100 mM Na/50 mM phos and 50 mM Na/10 mM phos buffer</td>
		</tr>
		<tr>
			<td>sodium deoxycholate</td>
			<td>Sigma-Aldrich</td>
			<td>D6750</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium dihydrogen phosphate monohydrate</td>
			<td>VWR</td>
			<td>BDH9298</td>
			<td>Chemical for 100 mM Na/50 mM phos and 50 mM Na/10 mM phos buffer</td>
		</tr>
		<tr>
			<td>Sodium hydroxide solution (NaOH)</td>
			<td>Sigma-Aldrich</td>
			<td>415443</td>
			<td>Used to adjust pregels solutions</td>
		</tr>
		<tr>
			<td>SU-8</td>
			<td>Kayaku advnaced materials</td>
			<td>SU8-100</td>
			<td>Used to coat silicon wafer</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma-Aldrich</td>
			<td>S8501</td>
			<td>Used during spinal cord decellularization</td>
		</tr>
		<tr>
			<td>Sylgard 184 silicone elastomer kit</td>
			<td>DOW</td>
			<td>1317318</td>
			<td>Polydimethylxiloxane (PDMS) base and curing agent</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>X100</td>
			<td>Used during spinal cord decellularization</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0.05%), phenol red</td>
			<td>Thermo Fisher</td>
			<td>25300062</td>
			<td>Used during hASC work and during spinal cord decellularization</td>
		</tr>
		<tr>
			<td>Tube revolver rotator</td>
			<td>Thermo Fisher</td>
			<td>88881001</td>
			<td>Used during sciatic nerve decellularization</td>
		</tr>
		<tr>
			<td>Xylene</td>
			<td>VWR</td>
			<td>MK866816</td>
			<td>Used during H&#38;E staining</td>
		</tr>
	</tbody>
</table>

development of combinatorial therapeutics for spinal cord injury using stem cell delivery

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.

Decellularized tissues were prepared using previously established protocols with slight modifications26,27. 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&#38;E staining (Figure 2A). To quantitatively assess the decellularizatio...

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.

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

Traumatic spinal cord injury (SCI) induces permanent sensorimotor deficit below the site of injury. It affects approximately a quarter million people in ...

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

Abstract