gms | German Medical Science

Objectives: The objective of the decellularization process is to maximize the removal of cellular material, while minimizing cell-associated immunogenicity and loss and damage of extracellular matrix (ECM) components. A cell- and antigen-free decellularized ECM could provide stimuli and a natural niche necessary for recellularization. Up to now there exist only few approaches utilizing intervertebral disc (IVD)-derived decellularized ECM for comparison of mesenchymal stromal cells (MSCs) and IVD cells simultaneously. Therefore, this study was to establish a suitable IVD-derived cell-free and biocompatible ECM as a scaffold for IVD tissue engineering.

Methods: IVDs were obtained during spinal surgery in 4 patients. MSCs and IVD cells were respectively isolated from 4 different human femoral heads and IVDs. The decellularization protocol was based on mechanical/physical treatment (6 freeze-thaw cycles), detergents (2% SDS and Triton X-100) and enzymatic decellularization (0.25% trypsin). Scaffolds were subsequently analyzed for effective cell-removal and ECM alterations by histology and biochemical assays. Moreover, we adopted an innovative way for recellularization preconditioning, utilizing bovine serum albumin (BSA) to bind residual SDS for decreasing its toxicity. Cyto-compatibility of the ECM and recellularization efficacy were compared after MSCs (chondrogenically induced or undifferentiated) and IVD cells were dynamically cultured on the ECM scaffolds for 14 days. Recellularized scaffolds were analyzed using vitality assays, histology and biochemical assays for measurements of total collagen, glycosaminoglycans (GAGs), and DNA content. Statistical analysis was performed using the Kruskal-Wallis test with Dunn's multiple comparisons test (n=4). P<0.05 was considered significant.

Results and Conclusion: Our modified protocol was effective to prepare mostly cell-free IVD-derived scaffolds, while hematoxylin- eosin Y (HE), Alcian blue (AB) and DAPI stainings showed no discernible nuclei compared to the native tissue. It allowed to produce a cyto-compatible biomaterial as cell vitality assay illustrated. Additionally, HE, AB and DAPI stainings indicated that the recolonized cells were more evenly distributed on the surface of the scaffolds than in the inner part for both MSCs and IVD cells. Analysis of total collagen revealed a significant difference between the chondrogenically induced MSCs group and the decellularized blank ECM (p<0.01)but not between other groups. GAGs content in the native tissue was significantly different compared to scaffolds reseeded with IVD cells (p<0.05). However, the scaffolds seeded with IVD cells had a significantly higher DNA content than the native tissue (p<0.01).

These decellularized IVD scaffolds are suitable for further tissue engineering applications, as they proved their cyto-compatibility by successful cell recolonization with MSCs and IVD cells while maintaining its typical three-dimensional histoarchitecture and conserving beneficial ECM contents.