Proliferation, Migration, and ECM Formation Potential of Human Annulus Fibrosus Cells Is Independent of Degeneration Status

Abstract

Objective The objective was to evaluate the proliferating, migratory and extracellular matrix (ECM) forming potential of annulus fibrosus cells derived from early (edAFC) or advanced (adAFC) degenerative tissue and their usability as a possible cell source for regenerative approaches for AF closure. Design EdAFC ( n = 5 Pfirrman score of 2-3) and adAFC (n = 5 Pfirrman score of 4-5) were isolated from tissue of patients undergoing spine stabilizing surgery. Cell migration on stimulation with human serum (HS), platelet-rich plasma (PRP), and transforming growth factor β-3 (TGFB3) was assessed by migration assay and proliferation was assessed on stimulation with HS. Induction of ECM synthesis was evaluated by gene expression analysis of AF-related genes in three-dimensional scaffold cultures that have been stimulated with 5% PRP or 10 ng/mL TGFB3 and histologically by collagen type I, type II, alcian blue, and safranin-O staining. Results EdAFC and adAFC were significantly attracted by 10% HS and 5% PRP. Additionally, both cell groups proliferated under stimulation with HS. Stimulation with 10 ng/mL TGFB3 showed significant induction of gene expression of collagen type II and aggrecan, while 5% PRP decreased the expression of collagen type I. Both cell groups showed formation of AF-like ECM after stimulation with TGFB3, whereas stimulation with PRP did not. Conclusions Our study demonstrated that AF cells retain their potential for proliferation, migration, and ECM formation independent of the degeneration status of the tissue. Proliferation, migration, and ECM synthesis of the endogenous AF cells can be supported by different supplements. Hence, endogenous AF cells might be a suitable cell source for a regenerative repair approaches.

Degeneration of Lumbar Intervertebral Discs: Characterization of Anulus Fibrosus Tissue and Cells of Different Degeneration Grades

“…These differentially expressed genes after 24 h of induction may indicate a lower capacity towards cell differentiation and a tendency to a higher inflammatory response in cells derived from severe AF tissue. Nevertheless, differentiation studies producing 3D cell cultures with TGF and BMP stimulation over several weeks demonstrated AF grafts with similar properties independent of AF degeneration grade [18]. This may indicate that our findings after 24 h demonstrate a delayed reaction of AF cells from severe degenerated tissues upon this stimulation with TGF and BMP.…”

“…Since our overall goal was a regenerative approach, we focused on cells with a chondrogenic potential. In previous experiments we were able to demonstrate that AF cells isolated with the used enzymatic procedures were able to show this chondrogenic potential [17][18][19].…”

“…Especially during degeneration, an ingrowth of blood vessels and nerve roots can occur. Due to cell isolation and culture conditions, the growth of cells with a chondrogenic differentiation potential is promoted [17][18][19]. That might explain the changes in monolayer gene expression.…”

“…Bone morphogenetic proteins (BMP) and other transforming growth factors (TGF) are prominent supplements in regenerative therapy research [14][15][16][17]. In addition, factors like chemokines or patient-derived blood and platelet compounds are potential candidates for regeneration [18,19].…”

“…Therefore, the focus of our study lies in revealing the differences and similarities of these two groups. For annular cell-based therapies, earlier studies indicated that cells derived from AF tissues of different degeneration states are able to form transplants of sufficient quality in vitro after several weeks [18]. However, differences between tissues of different degeneration statuses and isolated cells are not sufficiently characterized, and their response on regenerative stimulators depending on the grade of degeneration remains unknown.…”