A novel two‐step method for the formation of tissue‐engineered cartilage by mature bovine chondrocytes: The alginate‐recovered‐chondrocyte (ARC) method

Abstract

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.

Shaped, Stratified, Scaffold-free Grafts for Articular Cartilage Defects

“…The biochemical content of the shaped constructs in this study is consistent with previously described constructs [26,29,38]. Like many tissue-engineered cartilaginous constructs, the constructs here had lower extracellular matrix content, especially collagen, compared to that of native cartilage.…”

“…Cell source is an important consideration since these constructs were formed solely from the chondrocytes and the pericellular matrix they produced. While this study was performed with immature calf chondrocytes, which are metabolically active, this shaping technology would likely be applicable to chondrocytes from mature articular cartilage since such chondrocytes can be used to form scaffold-free disk constructs [29]. Additionally, the cell source limitation may be circumvented by expansion of the cell source followed by three-dimensional alginate bead culture [9,38].…”

“…To prepare for construct formation, the isolated chondrocytes were cultured in alginate beads for 7 days as described previously [25,29]. We isolated the chondrocytes from the distal femoral condyles of bovine calf knees (1-3 weeks old) by sequential digestion in 0.2% pronase for 1 hour and 0.019% collagenase P for 16 hours.…”

2010 Nicolas Andry Award: Multipotent Adult Stem Cells from Adipose Tissue for Musculoskeletal Tissue Engineering

“…To promote cartilage repair, several surgical techniques have been developed to induce bleeding and clot formation, including drilling or microfracture of the subchondral bone [1,8,29,31,43]. Numerous tissue engineering approaches have been proposed for enhancing cartilage regeneration in vitro or in vivo [2,12,14,30,34,41,55,56,70,77,79,81].…”

A cartilage growth mixture model for infinitesimal strains: solutions of boundary-value problems related to in vitro growth experiments

“…The 10 fluid velocity v w is solved from (41) and used in the diffusive force equation (42) 3 . Then, the stresses and diffusive force from (42) are substituted into (35). The resulting equations can be decoupled, yielding…”