Injectable Microcarriers as Human Mesenchymal Stem Cell Support and their Application for Cartilage and Degenerated Intervertebral Disc Repair

Abstract

Degeneration of the intervertebral disc (IVD) is a progressive and chronic process, and the high incidence of discogenic disorders calls for new therapeutic approaches, such as cell-based therapies using three dimensional cultures and mesenchymal stem cells (MSC), which can differentiate to chondrogenic-and IVD-lineages. Here, we investigated the growth and differentiation of human MSC culture on biodegradable collagen scaffolds in order to obtain an injectable suspension. Commercially available wound dressings were downsized to dimensions between 100 and 1500 μm and seeded with freshly isolated or early passages MSC. Proliferation rate and chondrogenic differentiation potential was tested at oxygenation levels of 2 %, 5 %, 10 % and 21 % in static and dynamic cultures. Evaluation methods included cell viability test, disc marker genes expression (aggrecan, collagen type I and type II), histological detection of proteoglycans and immunohistochemical analysis. On microcarriers, freshly isolated MSC had lower proliferation rate and chondrogenic differentiation potential compared with early passages MSC. Proliferation of MSC was significantly increased 1.7-fold at 5 % oxygen level and in combination with dynamic culture was further increased to 2.3-fold, with respect to normoxia. Chondrogenesis was positively affected by 2 % and 5 % hypoxia, as shown by increased transcription levels and protein expression of collagen type II and proteoglycan accumulation in static cultures, while it was inhibited in dynamic cultures. Collagen type I and aggrecan expression were not affected by hypoxia. In conclusion, collagen based microcarriers are a suitable support for in vitro MSC growth and chondrogenesis especially when cultured at 5 % oxygen level.

Growth Factors Cross-Linked to Collagen Microcarriers Promote Expansion and Chondrogenic Differentiation of Human Mesenchymal Stem Cells

“…Preparation of microcarriers particles was achieved by homogenization in Dispomix Drive system (Axonlab) of collagen sponge pad soaked in PBS (16).…”

“…Cells were expanded and differentiated on a microcarrier system, based on a mix of collagen particles of various size (16). Chondrogenesis was assessed by quantification of gene expression and protein accumulation.…”

“…For personal use only.DISCUSSIONSince its introduction in 1967 by Van Wezel, microcarrier culture has been applied successfully for growing primary cells and anchorage-dependent cell lines either for the production of vaccines or pharmaceuticals, or for cell expansion(19). In this study, we tested if covalent binding of growth factors to collagen microcarriers(16) with EDC/NHS and RB/UV could support cell expansion (e.g. bFGF) or differentiation (e.g.…”

Small leucine-rich proteoglycans (SLRPs): characteristics and function in the intervertebral disc

“…MSCs have been used to promote skeletal repair, such as in disc degeneration as well as cartilage degeneration and osteochondral defects, owing to their potential of differentiating into chondrogenic and IVD cell‐like lineages (Bertolo et al ., ; Tang et al ., ; Nam et al ., ; Lee et al ., ). It has been reported that autologous implanted MSCs proliferate and increase the expression of type II collagen, keratan sulphate and chondroitin sulphate after transplantation into degenerative IVDs (Sakai et al ., ).…”

Global Gene Expression Profiling and Alternative Splicing Events during the Chondrogenic Differentiation of Human Cartilage Endplate-Derived Stem Cells

“…Their capacity of expansion, self-renewal, and multilineage differentiation has been broadly validated [ 16 – 19 ]. In terms of intervertebral disc research, MSCs have been applied for IVD repair and regeneration in plenty of studies [ 20 – 23 ]. Except for exogenous MSCs, the stem cells in situ in IVDs are also an optional source.…”