Abstract
BackgroundCell-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.MethodsA 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.ResultsThe 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.ConclusionsThe data demonstrate that the hydrogels form stable implants capable to contain a specifically functional cell population within a physiological environment.
Highlights
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
The underlying philosophy of the experiments was to provide a continuous data set for the behavior of the intervertebral disc cells related to the hydrogel environment, as displayed during the cell expansion, after harvest into secondary culture as a monolayer or as an in vitro 3D culture, and isolated in the ectopic environment of the subcutaneous location in the mouse
Thirteen measurements were performed with freshly prepared hydrogel components, five measurements were performed after storage gel components at 4°C for 24 h and six measurements were performed after storing the gel components at 4°C for 72 h
Summary
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. The injection of a liquid suspension of cells into the disc tissue is not safe enough without any measures to warrant the cells to remain within the disc and not be ejected during initial loading of the disc upon patient motion. Beyond this safety aspect, the introduction of fresh cells may not deliver success without simultaneously restoring the nutritional requirements and the moderation or prevention of inflammatory insults. A limited number has already been applied in animal experiments, either in ectopic or intradiscal administrations, with so far none reported to generate local or systemic adverse reactions
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