In vitro engineering of cartilage: effects of serum substitutes, TGF-beta, and IL-1alpha.
OBJECTIVE
Cartilage is avascular and relatively homogeneous, making it an attractive tissue for in vitro histogenesis and surgical use in patients. We developed novel platform technologies in order to define the requirements for optimal in vitro chondrogenesis by isolated cells. In this series of studies, we tested alternatives to fetal bovine serum (FBS) and the effects of growth factors on formation of cartilage in 3D porous collagen sponges.


METHODS
We used porous collagen sponges to assess the effects of serum substitutes and exogenous TGF-beta1 and IL-1alpha on chondrocytes (bovine articular chondrocytes, bACs) and on chondroinduced human dermal fibroblasts (hDFs). We determined the effects of low concentrations of FBS and two serum substitutes, Nutridoma and ITS(+3), on cellularity and matrix production. After culture for intervals, sponges were harvested for histological and biochemical measurement of cartilage-specific chondroitin 4-sulfate proteoglycan (C 4-S PG).


RESULTS
Cultured bACs showed equivalent growth in Nutridoma (1%) and 10% FBS. Both TGF-beta1 and IL-1alpha significantly stimulated accumulation of C 4-S PG by bACs in 3D porous collagen sponges. Many endogenous growth factors were upregulated in hDFs cultured with chondroinductive DBP. Addition of TGF-beta1 and IL-1alpha for 11 days significantly stimulated accumulation of C 4-S PG by hDFs cultured in DMEM with 1% Nutridoma.


CONCLUSIONS
Porous collagen sponges are supportive of chondrogenesis and of chondroinduction by DBP. Optimization of serum-free culture conditions, including growth factors, matrix components, and mechanical stimuli will expedite translation to wider clinical applications. Use of autogenous dermal fibroblasts pre-cultured with DBP and induced to chondrocytes offers an alternative to autogenous chondrocytes.
Glowacki
J
J
Orthopedic Research, Brigham and Women's Hospital, Harvard Medical School and Harvard School of Dental Medicine, Boston, MA 02115, USA. jglowacki@rics.bwh.harvard.edu
Yates
K E
KE
Maclean
R
R
Mizuno
S
S
D000818:Animals; D001802:Blood Substitutes / Q000494:pharmacology*; D001857:Bone Matrix; D002356:Cartilage / Q000187:drug effects* / Q000502:physiology; D002417:Cattle; D002465:Cell Movement / Q000187:drug effects; D002478:Cells, Cultured; D019902:Chondrocytes / Q000187:drug effects; D020219:Chondrogenesis / Q000187:drug effects; D011508:Chondroitin Sulfate Proteoglycans / Q000032:analysis; D002809:Chondroitin Sulfates / Q000032:analysis; D003094:Collagen / Q000737:chemistry; D003470:Culture Media; D016207:Cytokines / Q000032:analysis; D005109:Extracellular Matrix / Q000187:drug effects; D005347:Fibroblasts / Q000187:drug effects; D006801:Humans; D007375:Interleukin-1 / Q000494:pharmacology*; D016062:Porosity; D023822:Tissue Engineering / Q000379:methods*; D016212:Transforming Growth Factor beta / Q000494:pharmacology*
D001802:Blood Substitutes; D011508:Chondroitin Sulfate Proteoglycans; D003470:Culture Media; D016207:Cytokines; D007375:Interleukin-1; D016212:Transforming Growth Factor beta; D002809:Chondroitin Sulfates; D003094:Collagen