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

Summary

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.

Abstract

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.

Introduction

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.

Protocol

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…

Representative Results

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&E staining (Figure 2A). To quantitatively assess the decellularizatio…

Discussion

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 compo…

Materials

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