One major problem of current cartilage repair techniques is that three-dimensional encapsulated mesenchymal progenitor cells frequently differentiate into hypertrophic cells that express type X collagen and osteogenic marker genes. Studies on wild-type cells of murine mesenchymal C3H10T1/2 progenitor cells as well as on cells transfected with cDNA encoding for bone morphogenetic protein (BMP)-2 or -4 in alginate revealed that the formation o f markers for osteogenesis and chondrogenic hypertrophy apparently depended on the BMP-transfection. Cells were encapsulated in ultrahigh-viscosity, clinical grade alginate and differentiation was studied over a period of 17 days. Consistent with results published previously (Biomaterials, 2002;23:2003) staining with haematoxylin--eosin or Alcian blue. immunohistochemical analysis, and quantitative RT-PCR confirmed the expression of chondrogenic markers (chondroitin-4-and -6-sulfate as well as type I1 collagen). Production of chondrogenic markers was particularly high in BMP-4 transfected cells. Hypertrophic chondrogenesis did not occur in BMP-4 transfected cells, as revealed by measurement of type X collagen, but could be demonstrated for wild-type cells and to some extent for BMP-2 transfected cells. The osteogenic markers, type 1 collagen, alkaline phosphatase, and Cbfal were upregulated in all cell lines even though the levels and the time of upregulation differed significantly. In any case, the markers were less and only very shortly expressed in BMP-4 transfected cells as revealed quantitatively by real time RT-PCR. Thus, the in vitro results suggested that BMP-4 is a very promising candidate for suppressing chondrogenic hypertrophy, while simultaneously enhancing the production of chondrogenic components.
A simple procedure is described for the extraction and purification of alginate from the inner stipes of the kelp Laminaria pallida. Alginate yield was about 10-15% of the dry mass, with a 70:30 mannuronic/guluronic acid ratio. Analysis of the purified alginate revealed a low polyphenol content while proteins were below detection level. The purified alginate was highly viscous, with 10-15 mPa s and 281 mPa s for a 0.1% and 0.5% solution, respectively, indicating a very high molecular mass (larger than 250 kDa). Bead formation occurred in the presence of divalent cations, but also in the presence of artificial serum (FCSIII) without added divalent cations. The biocompatibility of the alginate was tested with the in vitro mice lymphocyte test as well as by implantation of Ba2+ cross-linked beads beneath the kidney capsule of BB/OK rats. There was no evidence for significant mitogenic activity or fibrotic reaction. Biocompatibility of the alginate was also demonstrated by the encapsulation of human chondrocytes into Ca2+ cross-linked alginate beads. Immobilized chondrocytes grew and remained functional (i.e. they produced collagen).
Permanent hypoparathyroidism is one of the most difficult of all endocrine disorders to treat medically. Because this deficiency syndrome rarely is a life-threatening condition, systemic immunosuppression for recipients of transgenic transplants is not justified. An alternative would be protecting the tissue to be transplanted from the immunologic response (immunoisolation) by coating it with a semipermeable membrane- microencapsulation. Unfortunately, prior to the first clinical use, further analysis of the coating substance (alginate) demonstrated that it has potential cancerogenic properties. Using a purified amitogenic alginate suitable for clinical use, we accomplished allotransplantation in a long-term animal model and reported the first clinical cases without postoperative immunosuppression recently. In view of the potential clinical use, we investigated the ability of the microencapsulation technology with the novel amitogenic alginate in experimental hypoparathyroidism (80 parathyroidectomized rats) to enable transgenic transplantation across the highest immunologic barrier (xenotransplantation: human to rat) to ensure intact transplant function without immunosuppression. In a controlled, long-term animal study, the effect of microencapsulation on xenotransplanted human parathyroid tissue was evaluated over a period of 30 weeks (microencapsulated and naked hyperplastic parathyroid tissue, respectively). Functionally, human parathyroid tissue was able to replace that of rats. More than 6 months after xenotransplantation 32 of 40 animals that had received microencapsulated transplants were normocalcemic. In contrast, serum calcium concentrations dropped to postparathyroidectomy levels within 3 weeks in the animals that had received naked tissue only. Correspondingly, normocalcemic animals showed vital parathyroid tissue inside the microcapsules, which were surrounded by a small rim of fibroblasts. When combining microencapsulation with an improved tissue culture method, xenotransplantation of human parathyroid tissue and maintenance of its physiologic function is reproducibly achieved over the highest transplantation barrier. Using the amitogenic alginate may be a crucial step toward the first clinical use of this technique for parathyroid xenotransplantation without immunosuppression.
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