Maintenance of "stem cell" features of cartilage cell sub-populations during in vitro propagation.

BACKGROUND: The discovery of mesenchymal stem cells (MSCs) or MSC-like cells in cartilage tissue does not tie in well with the established view that MSCs derive from a perivascular niche. The presence of MSCs may raise concerns about specificity and application safety, particularly in terms of the regulatory site. The aim of the present study was to investigate the benefits or possible risks of the MSC-like properties of cells isolated from cartilage in the context of autologous chondrocyte implantation. METHODS: Chondrocytic cells were isolated from cartilage or intervertebral disc tissue. Flow cytometry was used to analyze the expression of cell surface antigens. MSC-like cells were either enriched or depleted by means of magnetic cell sorting (MACS) involving the monoclonal antibodies W5C5/SUSD2 and W8B2/MSCA-1. We addressed the issues of prolonged expansion of such cells as well as the influence of culture medium as a trigger for selecting a single cell type. Established protocols were used to study in vitro differentiation. In addition to histological and biochemical assessment, the acquired phenotypes were also evaluated on the mRNA transcript level. RESULTS: In the studied cells, we found strongly analogous expression of antigens typically expressed on MSCs, including CD49e, CD73, CD90, CD105, CD140b and CD166. The expression of W5C5 and W8B2 antigens in cartilage cell sub-populations did not correlate with multi-potency. We demonstrated that a chondroid precursor, but not a bona fide multipotent mesenchymal, cell type can be obtained under established in vitro culture conditions. The culture media used for expansion influenced the cell phenotype. CONCLUSIONS: The risk of adverse adipose or osseous differentiation is not posed by expanded chondrocyte cultures, even after enrichment of putative MSC-like cell populations by MACS. It is possible that this limited "stemness" in chondrocytes, expanded for use in ACI, may instead be beneficial as it allows re-differentiation under appropriate conditions despite prolonged times in culture.

Intervertebral disc cell- and hydrogel-supported and spontaneous intervertebral disc repair in nucleotomized sheep.

PURPOSE: Regenerative repair is a promising new approach in treating damaged intervertebral discs. An experimental scheme was established for autologous and/or allogenic repair after massive disc injury. METHODS: Disc healing was promoted in 11 animals by injecting in vitro expanded autologous/homologous disc cells 2 weeks after stab injury of lumbar discs L1-2. The following control discs were used in our sheep injury model: L2-3, vehicle only; L3-4, injury only; L4-5, undamaged; and lumbar discs from four non-experimental animals. Disc cells were suspended in a biologically supportive albumin/hyaluronan two-component hydrogel solution that polymerizes when inserted in order to anchor cells at the injection site. The parameters studied were MRI, DNA, glycosaminoglycan, collagen content, histology, immunohistology for collagens type I, II and aggrecan, and mRNA expression of GAPDH, ß-actin, collagen type I, II, X, aggrecan, lubricin, and IL-1ß. RESULTS: All parameters demonstrated almost complete healing of the injured discs after 6 months, when compared with data from both the endogenous non-injured controls as well as from the healthy animals. CONCLUSION: Sheep experience spontaneous recovery from disc injury. The process of endogenous repair can be enhanced by means of hydrogel-supported cells.

Rheological and biological properties of a hydrogel support for cells intended for intervertebral disc repair.

BACKGROUND: Cell-based approaches towards restoration of prolapsed or degenerated intervertebral discs are hampered by a lack of measures for safe administration and placement of cell suspensions within a treated disc. In order to overcome these risks, a serum albumin-based hydrogel has been developed that polymerizes after injection and anchors the administered cell suspension within the tissue. METHODS: A hydrogel composed of chemically activated albumin crosslinked by polyethylene glycol spacers was produced. The visco-elastic gel properties were determined by rheological measurement. Human intervertebral disc cells were cultured in vitro and in vivo in the hydrogel and their phenotype was tested by reverse-transcriptase polymerase chain reaction. Matrix production and deposition was monitored by immuno-histology and by biochemical analysis of collagen and glycosaminoglycan deposition. Species specific in situ hybridization was performed to discriminate between cells of human and murine origin in xenotransplants. RESULTS: The reproducibility of the gel formation process could be demonstrated. The visco-elastic properties were not influenced by storage of gel components. In vitro and in vivo (subcutaneous implants in mice) evidence is presented for cellular differentiation and matrix deposition within the hydrogel for human intervertebral disc cells even for donor cells that have been expanded in primary monolayer culture, stored in liquid nitrogen and re-activated in secondary monolayer culture. Upon injection into the animals, gels formed spheres that lasted for the duration of the experiments (14 days). The expression of cartilage- and disc-specific mRNAs was maintained in hydrogels in vitro and in vivo, demonstrating the maintenance of a stable specific cellular phenotype, compared to monolayer cells. Significantly higher levels of hyaluronan synthase isozymes-2 and -3 mRNA suggest cell functionalities towards those needed for the support of the regeneration of the intervertebral disc. Moreover, mouse implanted hydrogels accumulated 5 times more glycosaminoglycans and 50 times more collagen than the in vitro cultured gels, the latter instead releasing equivalent quantities of glycosaminoglycans and collagen into the culture medium. Matrix deposition could be specified by immunohistology for collagen types I and II, and aggrecan and was found only in areas where predominantly cells of human origin were detected by species specific in situ hybridization. CONCLUSIONS: The data demonstrate that the hydrogels form stable implants capable to contain a specifically functional cell population within a physiological environment.