Serum-free media for periosteal chondrogenesis in vitro.
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 microg/ml growth hormone), and/or ITS+ (2.08 microg/ml each of insulin, transferrin, and selenious acid, plus 1.78 microg/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 10% FBS) was achieved simply by the addition of 2.0 microg/ml insulin in 0.1% 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.
Fitzsimmons
James S
JS
The Cartilage and Connective Tissue Research Laboratory, Mayo Clinic College of Medicine, Mayo Foundation, Rochester, MN 55905, USA.
Sanyal
Arunik
A
Gonzalez
Carlos
C
Fukumoto
Takumi
T
Clemens
Victoria R
VR
O'Driscoll
Shawn W
SW
Reinholz
Gregory G
GG
D000818:Animals; D002356:Cartilage / Q000166:cytology / Q000187:drug effects; D002417:Cattle; D002455:Cell Division / Q000187:drug effects; D019902:Chondrocytes / Q000187:drug effects / Q000378:metabolism; D020219:Chondrogenesis / Q000187:drug effects* / Q000502:physiology; D016895:Culture Media, Serum-Free / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D004338:Drug Combinations; D006133:Growth Substances / Q000494:pharmacology; D007328:Insulin / Q000494:pharmacology; D009924:Organ Culture Techniques / Q000379:methods*; D010521:Periosteum / Q000187:drug effects* / Q000502:physiology; D011817:Rabbits; D012710:Serum Albumin, Bovine / Q000494:pharmacology; D013977:Tibia; D016212:Transforming Growth Factor beta / Q000494:pharmacology
D016895:Culture Media, Serum-Free; D004338:Drug Combinations; D006133:Growth Substances; D007328:Insulin; D016212:Transforming Growth Factor beta; D012710:Serum Albumin, Bovine