Serum‐free media for periosteal chondrogenesis in vitro

Abstract

Organ culture studies involving whole explants of periosteum have been useful for studying chondrogenesis, but to date the standard culture model for these explants has required the addition of fetal bovine serum to the media. Numerous investigators have succeeded in culturing chondrocytes and embryonic cells in serum-free conditions but there have been no studies focused on achieving a defined, serum-free media for culturing periosteal explants. The purpose of the present investigation was to determine if whole periosteal explants can be grown and produce cartilage in serum-free conditions, and to define the minimum media supplements that would be conducive to chondrogenesis. 321 periosteal explants were obtained from the medial proximal tibiae of 31 two month-old NZ white rabbits and cultured using a published agarose suspension organ culture model and DMEM for six weeks. The explants were cultured with and without fetal bovine serum or bovine serum albumin and exposed to transforming growth factor beta alone, a combination of growth factors we call ChondroMix (10 ng/ml transforming growth factor beta, 50 ng/ml basic fibroblast growth factor, and 5 pg/ml growth hormone), and/or ITS+ (2.08 pg/ml each of insulin, transferrin, and selenious acid, plus 1.78 pg/ml linoleic acid and 0.42 mg/ml BSA). Maximal chondrogenic stimulation in this study was observed with the combination of ChondroMix and ITS+. However, the minimal requirement to match or exceed the level of chondrogenic stimulation seen in the standard model (TGF-1 in 100/0 FBS) was achieved simply by the addition of 2.0 pg/ml insulin in 0.10/0 BSA-containing medium (p < 0.05). Therefore, based on our results, it would be reasonable to assume that insulin is the component in ITS+ responsible for the observed increase in total cartilage growth. Lower concentrations of insulin were not effective, suggesting that the observed effect of insulin requires activation of the IGF-1 receptor.

Influence of three-dimensional scaffold on the expression of osteogenic differentiation markers by human dermal fibroblasts

“…Such serum factors may counteract the effects of the additional media additives (i.e., vitamin D 3 ) on osteogenic differentiation of dermal fibroblasts. This is in agreement with other studies in which chondrogenic or osteogenic differentiation of dermal fibroblasts, periosteal-derived cells, and marrow-derived cells was enhanced when cultured in serum-free medium [33][34][35]50,51]. In addition, bone formation by bone marrow stromal cell/calcium phosphate composites implanted subcutaneously was significantly increased when cells were maintained in serum-free medium prior to implantation [52].…”

The role of recombinant proteins in the development of serum-free media

“…The options then are either to replace proteins with small molecular weight intermediates, to use recombinant alternatives or to engineer the cells to remove the requirement for protein additives. Two of the most common supplements added to basal media for serum-free development are insulin and transferrin (Fitzsimmons et al 2004;Barnes 1987).…”

“…BSA has been incorporated into a variety of SFM (Jäger et al 1988;Liu et al 2001;Fitzsimmons et al 2004) as a lipid transport system, source of intermediary metabolites and protection from shear and oxidation. Naturally bound ligands of albumin include bilirubin, fatty acids, vitamins, amino acids, hormones, globulins and metal ions of which, longchain fatty acids are quantitatively the most important (especially linoleic, arachidonic, palmitic, linolenic and oleic acids).…”

Methodological models for in vitro amplification and maintenance of human articular chondrocytes from elderly patients

“…Efforts to maintain differentiative features of articular chondrocytes expanded in vitro included reduction of oxygen tension (Kurz et al 2004, Murphy andPolak 2004;Murphy and Sambanis 2001), cultures in serum free conditions Mandl et al 2002) and at low cell density (Mandl et al 2004). Also the addition of agents capable to induce chondrogenic differentiation (Fitzsimmons et al 2004;Fukumoto et al 2003), the use of 3D systems, such as pellet (Yang et al 2004), gel (Mandl et al 2004;De Ceuninck et al 2004) and 3D scaffolds (Grigolo et al 2002;Yang et al 2004), and the introduction of engineered models of autologous implants (Guo et al 2004;Westreich et al 2004), acted to support the chondrogenic phenotype in different culture conditions. Several methods have been proposed for the use of materials biocompatible, nonimmunogenic and devoid of inflammatory stimuli, such as Alginate-Polylysine-Alginate (APA) (Hasse et al 2000;Lee et al 2003;Hauselmann et al 1996;Homicz et al 2003;Huch et al 2002) to be used subsequently to monolayer chondrocyte expansion (Homicz et al 2003;Mandl et al 2004).…”