Abstract
Introduction: Autologous chondrocyte implantation is routinely used for the repair of articular cartilage defects. A similar method may be employed to treat degenerate intervertebral discs or other connective tissues. A system in which cells could not only be delivered, but also retained would offer advantages compared to ACI. Such a vehicle would also allow a homogenous distribution of cells throughout the defect and enhance nutrient penetration to the seeded cells.
Methods: Bovine nucleus cells were isolated via enzyme digestion and expanded in number to passage 3. The cells were resuspended in 0.8% alginate and loaded into poly vinyl alcohol (PVA) cubes. These constructs were placed into a solution of calcium chloride to ‘gel’ the alginate. Constructs were cultured in DMEM+10% FBS within 15ml conical tubes rotated at 37°C for up to 28 days. Cell distribution/morphology and proliferation were assessed on H& E and Ki-67 stained sections, respectively. The re-expression of a disc cell phenotype was assessed using toluidine blue staining and immunohistochemistry (with antibodies to collagen types I, II, IIA, VI and X, and to the glycosaminoglycans, chondroitin-4- and -6-sulphate and keratan sulphate. RT-PCR was performed using oligonucleotide primers to collagen types I, II and X, aggrecan, link protein, and small leucine-rich PGs.
Results: H& E staining of 10μm-thick cryosections revealed an even distribution of loaded cells throughout the scaffold at day 1 being maintained through to day 28. Toluidine blue staining revealed the presence of GAGs, increasing with time. Ki-67 revealed approximately 5% of cells were proliferating at all time points. Immunohistochemistry demonstrated the production of collagen types I, II, IIA, VI and X and the glycosaminoglycans, chondroitin-4-, -6 and keratan sulphate. RT-PCR results showed mRNA expression of fibromodulin throughout the experiment, lumican at days 14, 21 and 28. Types II and X collagen were present at days 21 and 28.
Conclusions: Combining 0.8% alginate with PVA retained 100% of the seeded cells and allowed an even distribution of cells throughout the scaffold. The immunohistochemistry and RT-PCR demonstrated that the system allowed the bovine nucleus cells to express phenotypic markers expressed by disc cells in vivo. These preliminary results indicate that the PVA/alginate system could act as a suitable delivery device for cells during autologous repair of the intervertebral disc or other connective tissues such as meniscus.
Correspondence should be addressed to Mr Carlos Wigderowitz, Honorary Secretary BORS, University Dept of Orthopaedic & Trauma Surgery, Ninewells Hospital & Medical School, Dundee DD1 9SY.
