Monomeric, porous type II collagen scaffolds promote chondrogenic differentiation of human bone marrow mesenchymal stem cells in vitro

Abstract

Osteoarthritis (OA) is a common cause of pain and disability and is often associated with the degeneration of articular cartilage. Lesions to the articular surface, which are thought to progress to OA, have the potential to be repaired using tissue engineering strategies; however, it remains challenging to instruct cell differentiation within a scaffold to produce tissue with appropriate structural, chemical and mechanical properties. We aimed to address this by driving progenitor cells to adopt a chondrogenic phenotype through the tailoring of scaffold composition and physical properties. Monomeric type-I and type-II collagen scaffolds, which avoid potential immunogenicity associated with fibrillar collagens, were fabricated with and without chondroitin sulfate (CS) and their ability to stimulate the chondrogenic differentiation of human bone marrow-derived mesenchymal stem cells was assessed. Immunohistochemical analyses showed that cells produced abundant collagen type-II on type-II scaffolds and collagen type-I on type-I scaffolds. Gene expression analyses indicated that the addition of CS - which was released from scaffolds quickly - significantly upregulated expression of type II collagen, compared to type-I and pure type-II scaffolds. We conclude that collagen type-II and CS can be used to promote a more chondrogenic phenotype in the absence of growth factors, potentially providing an eventual therapy to prevent OA.

Figure 1. Schematic of experimental procedure: hBMMSCs were isolated from human iliac crest and expanded in culture.

Type I and type II collagens were processed to produce afibrillar and reconstituted fibrillar scaffolds incorporating CS. The scaffolds were seeded with hBMMSCs and cell-scaffold interactions including cell viability, morphology and metabolic activity were examined. Cellular differentiation was studied using immunostaining and gene expression.

Figure 2

Scaffold microstructure and cell-scaffold interactions: (a) SEM micrographs showing the porous structure and internal features of collagen scaffolds. Red arrows indicate secondary pores, which are perforations in pore walls. (b) hBMMSCs viability by Live/Dead^® assay at day 1 and 14. Live cells are green and dead cells are red. (c) Actin cytoskeleton staining (red) and nuclei (blue) at day 1 and 14.

Figure 3

Cell proliferation, scaffold size changes and CS release: (a) Metabolic activity of cells on scaffolds determined by measuring alamarBlue activity (arbitrary unit: fluorescent intensity). One-way ANOVA followed by Tukey test was used to evaluate the alamarBlue data; *shows significantly lower metabolic activity compared to 1af at the same time point, *represents p = 0.05, **represents p = 0.005, *** represents p = 0.0003; + shows significantly lower metabolic activity compared to 2af at the same time point; ∆ significantly lower than 2af-CS, p = 0.002. (b) Changes in scaffold size upon contact with culture media. (c) Changes in scaffold size after 1 and 28 days in culture with cells (arbitrary unit: mm²/fluorescent activity). Whilst the size of type-I scaffolds decreased in the presence of cells, presumed matrix production mediated an increase in the size of type-II scaffolds. One-way ANOVA test with Fisher LSD was used to evaluate the scaffold size data; *shows significant difference compared to 2af (p = 0.02), 2af-CS (p = 0.03) and 2rf-CS (p = 0.004). (d) Initial amount of CS per dry scaffold and the amount released into cell culture media after seeding the scaffolds with cells.

Figure 4. Histology and immunostaining of scaffolds: immunostaining of collagen type I and type-II on scaffolds after 28 days in culture, blue: nuclei and green: type I or II collagen.

H&E, Picosirius Red, Safranin-O and Alizarin Red staining after 28 days in culture.

Figure 5. Assessment of cellular differentiation: relative expression of chondro- and osteogenic genes in hBMMSCs cultured on scaffolds for 28 days.

One-way ANOVA test with Fisher LSD was used to evaluate the gene expression data; *indicates a significant difference compared to pure scaffolds (1af and 2af) at p < 0.04.