Numerous scaffold formulations have been investigated to support the regeneration of nucleus pulposus (NP) tissue for use as an early-stage therapy for intervertebral disc degeneration. Particular attention has focused on recreating the biochemical and mechanical properties of the native NP via the incorporation of exogenous extracellular matrix (ECM) components or synthetic surrogates. In the present study, we describe a novel approach to develop a tissue engineering (TE) scaffold comprised acellular porcine NP ECM. Complete decellularization of porcine NP was successfully achieved using a combination of chemical detergents, ultrasonication, and treatment with nucleases. Resulting NP scaffolds were devoid of host-cell remnants and the porcine antigen alpha-Gal. Native NP ECM components including aggrecan/chondroitin-6-sulfate and collagens types II, IX, and XI were found in physiologically relevant ratios within the NP scaffold. NP scaffold swelling capacity and unconfined mechanical properties were not significantly different from porcine NP tissue. Furthermore, NP scaffolds were conducive to repopulation with human adipose-derived stem cells as cell viability and proliferative capacity were maintained. These results demonstrate the successful decellularization of porcine NP and the resultant formation of a biomimetic scaffold exhibiting potential utility for TE the human NP.

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Nucleus pulposus (NP) tissue regeneration has been proposed as an early stage interventional therapy to combat intervertebral disc degeneration. We have previously reported on the development and characterization of a novel biomimetic acellular porcine NP (APNP) hydrogel. Herein, we aimed to evaluate this material for use as a suitable scaffold for NP tissue regeneration. Human-adipose-derived stem cells (hADSCs) were cultured for 14 days on APNP hydrogels in chemically defined differentiation media and were analyzed for an NP-cell-like mRNA expression profile, evidence of hydrogel remodeling including hydrogel contraction measurements, extracellular matrix production, and compressive dynamic mechanical properties. The innate capacity of the hydrogel itself to induce stem cell differentiation was also examined via culture in media lacking soluble differentiation factors. Additionally, the in vivo biocompatibility of non-crosslinked and ethyldimethylaminopropyl carbodiimide/N-hydroxysuccinimide and pentagalloyl glucose crosslinked hydrogels was evaluated in a rat subdermal model. Results indicated that hADSCs expressed putative NP-cell-positive gene transcript markers when cultured on APNP hydrogels. Additionally, glycosaminoglycan and collagen content of hADSC-seeded hydrogels was significantly greater than nonseeded controls and cell-seeded hydrogels exhibited evidence of contraction and tissue inhibitors of metalloproteinase-1 production. The dynamic mechanical properties of the hADSC-seeded hydrogels increased with time in culture in comparison to noncell-seeded controls and approached values reported for native NP tissue. Immunohistochemical analysis of explants illustrated the presence of mononuclear cells, including macrophages and fibroblasts, as well as blood vessel infiltration and collagen deposition within the implant interstices after 4 weeks of implantation. Taken together, these results suggest that APNP hydrogels, in concert with autologous ADSCs, may serve as a suitable scaffold for NP tissue regeneration.

Intervertebral disc (IVD) degeneration (IDD) and herniation (IDH) can result in low back pain and impart significant socioeconomic burden. These pathologies involve detrimental alteration to the nucleus pulposus (NP) either via biochemical degradation or extrusion from the IVD, respectively. Thus, engineering living NP tissue utilizing biomaterial scaffolds that recapitulate native NP microarchitecture, biochemistry, mechanical properties and which support cell viability represents an approach to aiding patients with IDD and IDH. To date, an ideal biomaterial to support NP regeneration has yet to be developed; however, one promising approach to generating biomimetic materials is to employ the decellularization (decell) of xenogeneic NP tissue to remove host DNA while maintaining critical native extracellular matrix (ECM) components. Herein, 13 different procedures were evaluated in an attempt to decell bovine caudal IVD NP tissue. An optimal method was identified which was confirmed to effectively remove bovine DNA, while maintaining physiologically relevant amounts of glycosaminoglycan (GAG) and type-II collagen. Unconfined static and dynamic compressive mechanical properties of scaffolds approached values reported for human NP and viability of human amniotic stem cells (hAMSCs) was maintained on non-crosslinked and EDC/NHS treated scaffolds for up to 14 days in culture. Taken together, NP tissue obtained from bovine caudal IVDs can be successfully decelled in order to generate a biomimetic scaffold for NP tissue regeneration.

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