In VitroCalcification of Immature Bovine Articular Cartilage

Abstract

Introduction:The zone of calcified cartilage (ZCC) anchors articular cartilage (AC) to subchondral bone through a layer of intermediate stiffness. The regulation and functional consequences of cartilage calcification may vary with depth from the articular surface. The hypothesis of this study was that the in vitro calcification of immature AC occurs selectively in the deep region and is associated with a local increase in stiffness.Methods:AC and growth plate cartilage (GPC) from calves were incubated in DMEM, 1% fetal bovine serum, 100 µg/mL ascorbate, and ±10 mM β-glycerophosphate (βGP) for up to 3 weeks. To assess the time course and effects of cell viability and βGP, full-depth strips of AC and GPC were analyzed by histology, indentation, and 45Ca++ uptake. To assess the effect of tissue zone, disks harvested from surface and deep zone AC and from reserve and hypertrophic zone of GPC were incubated independently and analyzed by compression and for 45Ca++ uptake and biochemical components.Results:The deep ~20% of immature AC calcified within 3 weeks, with calcification dependent on cell viability and βGP. Mineral was deposited continuously around cells in AC but only between cell columns in GPC. The deep zone of AC exhibited a compressive modulus of 0.53 MPa after βGP-induced calcification, ~4-fold stiffer than AC incubated without βGP.Conclusions:Cartilage explants exhibit inherent zone-specific calcification processes, resulting in an increase in stiffness associated with cartilage calcification. Such properties may be useful for engineering a biomimetic ZCC tissue to integrate cartilaginous tissue to bone, thereby forming a mechanically functional osteochondral unit.

Glycosylated superparamagnetic nanoparticle gradients for osteochondral tissue engineering

“…To ensure cartilage formation at the other end of the tissue, we included TGF-β3 alongside insulin-transferrin-selenium, ascorbic acid, dexamethasone and β-glycerophosphate in an optimized osteochondral differentiation medium based on similar compositions reported in the literature [ 13 , 32 , 33 ]. We used β-glycerophosphate at a concentration of 2 mM, a level that can provide a source of phosphate without initiating osteogenesis or nonspecific mineral precipitation [ 34 ]. Indeed, we showed that this osteochondral medium could support either osteogenic or chondrogenic differentiation of hMSCs in agarose, depending on the presence or absence of BMP-2 in the culture medium ( Supplementary Figure 6 ).…”

Mechanical evaluation of a tissue-engineered zone of calcification in a bone–hydrogel osteochondral construct

“…A cell-mediated technique was proposed using β-glycerophosphate during deep zone chondrocyte cell culture (Hwang et al 2010). In this work, we have adapted a technique based on a double diffusion system (Boskey 1989; Hunter et al 1996) to control the calcification at an interface between a gel and a piece of bone.…”

Buoyancy‐Driven Gradients for Biomaterial Fabrication and Tissue Engineering

“…We used these constructs for a 28 d course of osteochondral tissue engineering using a defined osteochondral differentiation medium that we have previously shown to be capable of supporting both osteogenesis and chondrogenesis (Figure 3A). [13] This medium included 2 × 10 −3 m β-glycerophosphate as a phosphate source, [32] transforming growth factor β3 (TGF-β3) to stimulate chondrogenesis, alongside supporting components such as insulin-transferrin-selenium, ascorbic acid, and dexamethasone.…”