Most attempts to tissue-engineer cartilage have involved seeding of cultured cells into a biological or synthetic scaffold. We have developed a novel two-step culture approach that makes possible the in vitro formation of cartilaginous-like tissue by mature adult bovine chondrocytes without the aid of a synthetic matrix. The first step consists of culturing chondrocytes under conditions that maintain their rounded shape and their molecular phenotype as assessed by type I1 collagen and aggrecan production. This step was accomplished by culturing the isolated chondrocytes in alginate beads until the cells have reestablished a proteoglycan-rich cellassociated matrix (CM). The second step consists of culturing the cells with their CM, after recovery from the beads, on a tissue culture insert with a porous membrane. In this study, young adult bovine articular chondrocytes were cultured in alginate beads in the presence of 10% or 20%) fetal bovine serum (FBS). After 7 days of culture, the alginate beads were dissolved by incubating the beads for 20 min in sodium citrate buffer, a calcium chelator. Following a brief centrifugation, the cells with their CM were recovered, resuspended in medium containing 10% or 20% FBS and seeded onto a tissue culture insert. After 1 week of culture on the insert, the individual cells with their CM progressively became incorporated into a mass of cartilaginous tissue. Culture with 20% FBS resulted in the best formation of tissues. These tissues, easily recovered from the insert, were then subjected to biochemical and histological analyses. The biochemical results showed that the chondrocytes remain phenotypically stable in the tissues. The de novo tissue has a relatively high ratio of PGkollagen. Histological examination of the tissue revealed it contained a cartilage-like matrix strongly stained with toluidine blue. This scaffold-free system appears ideal to study, in vitro, the development of transplantable cartilaginous tissue.
Limb salvage for bone tumors has become the standard method of treatment. This technique involves removal of large segments of bone, most commonly around the hip and knee. Various types of reconstructive options are currently available, including osteoarticular allograft arthroplasty, modular oncology prosthetic arthroplasty, allograft prosthetic composite (APC) arthroplasty, and arthrodesis. Compared to other techniques, APC arthroplasty has many advantages, including restoration of bone stock, customization with conventional implant components, soft tissue attachment of tendons and ligaments, and preservation of the medullary canal of the host bone. The disadvantages of this technique include slow healing in the presence of chemotherapy, the possibility of disease transmission, and availability. The technique is suited either for aggressive benign tumors or for low‐grade sarcomas where chemotherapy is not necessary. Further, it represents a good alternative for a failed modular oncology prosthesis, and also for the failed osteoarticular allograft because it restores bone stock. Good functional results have been reported with APC replacements, but long‐term follow‐up is needed to determine their durability. Semin. Surg. Onc. 13:18–24. © 1997 Wiley‐Liss, Inc.
The experiments described herein tested the effects of CaCl2 and ZnCl2, added at various concentrations in the culture medium, upon the synthesis of collagen and proteoglycan by adult and fetal (articular, epiphyseal and hypertrophic) bovine chondrocytes maintained in high density multilayer cultures. CaCl2 concentrations below 0.5 mM or the addition of 1-50 microM ZnCl2 to the medium selectively promoted the production of collagen by all four populations of chondrocytes but had no effect on fibroblasts. Further, these changes had no statistically significant effect on the incorporation of 35S-sulfate into macromolecules or on the synthesis of gelatinase A, measured by gelatin zymography. The addition of CaCl2 and ZnCl2 at these concentrations did not result in a change in the relative proportion of non-crosslinked 3H-collagen molecules (synthesized in the presence of beta-aminopropionitrile) partitioning in the cell layer and medium compartments, and did not appreciably alter the pattern of collagens synthesized by any of the cell populations. The hypertrophic cells synthesized high levels of collagen type X in the presence as well as absence of exogenously added cations. However, CaCl2 at 10 mM caused a marked upregulation of collagen type X synthesis by a preparation of chondrocytes derived from the entire growth plate, consistent with the view that calcium at that concentration stimulated the differentiation of some of the cells into hypertrophic chondrocytes.
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