Cell Tissue Res (2005) 320: 269–276DOI 10.1007/s00441-004-1075-3REGULAR ARTICLEIchiro Sekiya . Benjamin L. Larson . Jussi T. Vuoristo .Roxanne L. Reger . Darwin J. ProckopComparison of effect of BMP-2, -4, and -6 on in vitro cartilageformation of human adult stem cells from bone marrow stromaReceived: 30 August 2004 / Accepted: 15 December 2004 / Published online: 19 March 2005# Springer-Verlag 2005Abstract The human adult stem cells from bone marrowstroma referred to as mesenchymal stem cells or marrowstromal cells (MSCs) are of interest because they are easilyisolated and expanded and are capable of multipotentialdifferentiation. Here, we examined the ability of recombi-nant human bone morphogenetic protein (BMP)-2, -4, and-6 to enhance in vitro cartilage formation of MSCs. HumanMSCs were isolated from bone marrow taken from normaladult donors. The cells were pelleted and cultured for 21daysinchondrogenicmediumcontainingtransforminggrowthfactorβ3anddexamethasonewithorwithoutBMP-2,-4,or-6. All the BMPs tested increased chondrogenic differen-tiationasassayedbyimmunohistochemistry andbythesizeand weight of the cartilage synthesized. However, BMP-2wasthemosteffective.Microarrayanalysesofapproximately12,000 genes and reverse transcription-polymerase chainreaction assays established that the critical genes for car-tilage synthesis were expressed in the expected time se-quence in response to BMP-2. The tissue engineering ofautologous cartilage derived from MSCs in vitro for trans-plantation will be a future alternative for patients withcartilage injuries. To obtain large amounts of cartilage richinproteoglycans,theuseofBMP-2isrecommended,insteadof BMP-4 or -6.Keywords Marrow stromal cells . Chondrogenesis .Micromass culture . BMP-2 . Type II collagen . HumanIntroductionArticular cartilage has a limited capacity for healing aftertrauma. Numerous preclinical and clinical studies have ex-plored various methods of promoting cartilage repair withprocedures such as abrasion arthroplasty (Johnson 1986),microfracturechondroplasty(Steadmanetal.2001),periostealtransplantation (Homminga et al. 1990), autologous chondro-cyte transplantation (Brittberg et al. 1994), and osteochondralgrafts (Hangody et al. 2001). The tissue engineering of autolo-gouscartilageinvitrofortransplantationcouldprovetobeafeasiblefuturealternativeforpatientswithcartilageinjuries.The adult stem cells from human bone marrow stromareferred to as mesenchymal stem cells or marrow stromalcells (MSCs) are of interest because they are easily isolatedand expanded and are capable of multilineage differentia-tion (Prockop 1997; Sekiya et al. 2004). MSCs have beenshown to differentiate into cartilage in vitro (Pittenger et al.1999), but the technology is still in its infancy and is not yetat the level needed to provide cartilage for clinical re-placement therapy.Chondrogenesis of MSCs with micromass cultures indefinedmediacontainingtransforminggrowthfactor(TGF)-βanddexamethasone(DEX) was first described by Johnstoneet al. (1998). We have previously reported that the additionof bone morphogenetic protein (BMP)-6 to the medium en-hances chondrogenesis (Sekiya et al. 2001, 2002b). Here,we compare the effect of BMP-2, -4, and -6 on in vitrochondrogenesisofMSCsanddemonstrate,forthefirsttime,that BMP-2 is even more effective than BMP-6 in the pro-motion of chondrogenesis. Moreover, BMP-2 elicits theexpected time sequence of gene expression for cartilage-specificmatrixproteins,receptors,andtranscriptionfactors.Materials and methodsMaterialsThe recombinant BMP-6, -4 and -2 were purchased fromR&D, Minn., USA. BMP-6 and -4 were synthesized in theThis work was supported in part by NIH grants AR47796 andAR44210, the Oberkotter Foundation, HCA-the Health CareCompany, and the Louisiana Gene Therapy Research Consortiumto D.J.P.I. Sekiya . B. L. Larson . J. T. Vuoristo . R. L. Reger .D. J. Prockop (*)Center for Gene Therapy,Tulane University Health Sciences Center,SL99, 1324 Tulane Avenue,New Orleans, LA 70112-2699, USAe-mail: dprocko@tulane.eduTel.: 1-504-9887711Fax: 1-504-9887710
