A collagen microencapsulation : assisted stem cell-based approach for treating degenerative disc disease

Abstract

Degenerative disc disease (DDD) is a medical condition whereby the intervertebral discs (IVD) of the human spine degenerates and may cause pain which significantly affects the quality of one掇 life. Its prevalence has sparked off much research in deciphering its causes and developing new treatments. Recently, attempts to treat this degenerative problem have turned to seeking answers from regenerative medicine. One approach is to deliver mesenchymal stem cells (MSCs) with or without carriers to the nucleus pulposus (NP) in degenerative disc to restore both its structural and functional properties. However, the optimal conditions and signals for inducing MSCs differentiation toward NP-like phenotype have not been identified. This work aimed to develop injectable microspheres with matrix microenvironment mimicking that of native NP tissue for MSCs delivery. Firstly, it was aimed to establish a collagen microencapsulation based 3D culture system for maintenance of the phenotype of nucleus pulposus cells (NPCs) and remodeling of the collagen matrix to one that was similar to the native NP. Secondly, it was aimed to optimize a decellularization protocol for complete removal of the encapsulated NPCs with minimal loss of remodeled extracellular matrix. Thirdly, it was aimed to investigate whether this acellular matrix produced by NPCs was inductive for MSCs discogenic differentiation. Finally, it was aimed to evaluate the efficacy of the MSC-seeded acellular matrix microspheres in a pilot rabbit disc degeneration model. It demonstrated that NPCs maintained their phenotype, survived within the collagen microspheres and produced NP-like ECM such as glycosaminoglycan (GAG) and collagen type II. GAG production of NPCs was found to positively correlate with the dosage of TGF-? within a short period, initial collagen concentration and cell seeding density. An optimized decellularization protocol with 50mM SB-10, 0.6mM SB-16 and 0.14% Triton X-200 was established to completely remove the encapsulated NPCs with partial retention of the GAG-rich matrix. The decellularized microspheres were able to be repopulated with human MSCs (hMSCs) or rabbit MSCs (rMSCs). Within the NPC-derived acellular matrix, the repopulated hMSCs were able to partially exhibit NPC phenotype with upregulated expression of a few NPC markers and NP-like ECM according to histological, biochemical, immunohistological and real-time PCR results. In the pilot in vivo evaluation study, preliminary results showed that intra-discal injection of MSCs reseeded acellular NPC-matrix microspheres maintained a better water content than the control MSC-microspheres without the NPC-derived acellular matrix. This work reconstituted in vitro a NP-like 3D matrix and provided preliminary evidence on discogenic differentiation of MSCs in such a matrix environment. This work laid down the foundation to future development of stem cell-based therapies for DDD. Further studies should focus on deciphering the soluble and insoluble composition of such a NP-like matrix environment and understanding the molecular mechanism of the cell-matrix interactions involved.