Paper No. 36 • 54th Annual Meeting of the Orthopaedic Research Society

Inhibition of Terminal Chondrocyte Differentiation in an in vitro Hypertrophy Culture System of Human MSCs

Michael B. Mueller2,1, Lauren Statman1, Peter A. Angele2, Rocky S. Tuan1

1Cartlilage Biology and Orthopaedics Branch, NIAMS, NIH, Bethesda, MD; 2Department for Trauma Surgery, University of Regensburg,Regensburg, Germany

michaelbmueller@web.de

Introduction: Adult mesenchymal stem cells (MSCs) are a candidate cellsource for cartilage tissue engineering. The expression of chondrocyte hypertrophyassociated genes in MSCs undergoing chondrogenesis has been reported [1,2]. Inprevious work, we could show that a wide array of cell surface receptors and othergenes relevant to chondrocyte hypertrophy are regulated in MSCs in a typical pat-tern for terminal chondrocyte differentiation. In addition, chondrifying MSCsfunctionally behave like growth plate chondrocytes when exposed to distinct medi-um conditions. Thyroid hormone can induce hypertrophy and TGF-beta and highdoses of dexamethasone inhibit thyroid hormone induced hypertrophy [3]. Thisbiological behavior is a concern for the application of MSCs in articular cartilagerepair, because chondrocyte hypertrophy ultimately leads to apoptosis, vascularinvasion and ossification. The close biological proximity of chondrogenic differen-tiating MSCs to growth plate chondrocytes suggests that substances known toinhibit chondrocyte hypertrophy may be useful for avoiding the transition ofMSCs from an articular to a hypertrophic chondrogenic phenotype, e.g. parathy-roid hormone related peptide (PTHrP). During embryonic development and bonegrowth, it is part of a feedback loop that regulates chondrocyte maturation. PTHrPis secreted by periarticular chondrocytes, binds to its receptor (PTHrPR).PTHrPR in prehypertrophic and hypertrophic chondrocytes and slows downchondrocyte maturation [4]. In previous work, we could show that PTHrPR is notexpressed in undifferentiated MSCs and is upregulated during MSC chondrogen-esis [3]. In this study, we used an in vitro hypertrophy culture system of chondro-genic differentiating MSCs to test the suitability of PTHrP for the inhibition ofhypertrophy under defined culture conditions.

Materials and Methods: MSCs were isolated from proximal femora of patientsundergoing total hip replacement with IRB approval, and chondrogenically pre-differentiated for 2 weeks in pellet culture in chondrogenic medium (CM)(DMEM, 1% ITS+, 50 μg/ml ascorbate-2-phosphate, 40 μg/ml L-proline, 100nM dexamethasone, 10 ng/ml TGF-beta3) in the presence of 0, 1, 10 or 100 pMPTHrP(1-40). On day 14, the cultures were either kept in CM or exposed hyper-trophic medium (Hyp) (DMEM, 1% ITS+, 50 μg/ml ascorbate-2-phosphate, 40μg/ml L-proline, 1 nM dexamethasone, 10 mM beta-glycerophosphate, 1 nM tri-iodothyronine (T3)). Cultures were harvested on day 1, 14 and 28 for histology andbiochemical analysis. Alkaline phosphatase (ALP) activity was determined in themedium. Two independent experiments with cells of two different patients werecarried out.

Results: Histological evaluation shows chondrogenic differentiation in pelletson day 28 when cultured in CM PTHrP at 0 to 10 pM by collagen 2 (Fig. 1A,B,C) and alcian blue staining (Fig. 1 E,F,G). At 100 pM PTHrP, collagen 2 isnot detectable on day 28. In Hyp with 0 or 1 pM PTHrP, there are dedifferentiat-ed and hypertrophic areas. At 10 and 100 pM PTHrP, pellets are dedifferentiatedin Hyp (Fig. 1 D,H). Biochemical analysis confirmed the histological findings.Sulfated Glycosaminoglycan content (GAG) normalized to DNA content (Fig.2A) was higher in CM than uin Hyp and significantly reduced at 100 pM PTHrP.ALP activity in the medium (Fig. 2B) was significantly higher in Hyp comparedto CM. PTHrP at 1 pM did not significantly change ALP activity in either CMor Hyp, but significantly reduced ALP secretion at concentrations of 10 and 100pM under both medium conditions.

Discussion: In previous work, we could clearly show that under the hypertro-phy conditions used, chondrifying MSCs undergo a similar differentiation pro-gram as growth plate chondrocytes [3]. We now used a previously established invitro hypertrophy model to test a substance with the potential to inhibit cell mat-uration, PTHrP. PTHrP has a dose dependent inhibitory effect on both chondro-genesis of MSCs as indicated by histology and GAG-accumulation and on matu-ration to a hypertrophic phenotype as indicated by ALP activity. At 1 pM, PTHrPdoes not have significant effect on GAG accumulation or alkaline phosphataseactivity whereas it significantly inhibits both chondrogenic differentiation andALP secretion at 100 pM. At 10 pM, GAG accumulation is not affected whereas

ALP activity appears to be significantly reduced in CM and Hyp. At 10 pMPTHrP in Hyp however, pellets were entirely dedifferentiated. This indicates aninhibitory effect on chondrogenesis also at 10 pM. In summary, PTHrP seems tohave both positive (inhibition of hypertrophy) and negative (inhibition of chon-drogenesis) effects on MSC chondrogenesis. At 10 pM, the positive effect over-weighs the negative effect indicating that this concentration may be in a therapeu-tic range where hypertrophy may be inhibited significantly affecting chondrogen-esis. Further experiments are necessary to confirm these results and the examina-tion of additional hypertrophy and chondrogenic markers as well as in vivo exper-iments are necessary to finally decide whether PTHrP is a suitable medium addi-tive for the stabilization of an articular chondrogenic phenotype of MSCs.

References: 1] Johnstone B et al. Exp Cell Res. 238(1):265-272, 1998; [2]Sekiya I et al. Proc Natl Acad Sci U S A. 99(7): 4397-4402, 2002; [3] Mueller, M.B., Tuan, R. S. Trans Orthop Res Soc, 32: 0303, 2007M; [4] Kronenberg, H.M.,PTHrP and skeletal development. Ann N Y Acad. 1068: 1-13, 2006

Acknowledgements: Supported by NIH (Z01 AR41131) and DFG(MU2318-1).

Fig. 1. Histology day 28. (A-D) CM Collagen 2 IHC. (E-H) CM Alcian blue. (I-L) HypAlcian blue. (A,E,I) no PTHrP. (B,F,J) 1 pM PTHrP. (C,G,K) 10 pM PTHrP. (D,H,L) 100pM PTHrP.

Fig. 2. Biochemical evaluation. (A) GAG content normalized to DNA. (B) ALP activity in hemedium. *=significantly different from 0 pM PTHrP (t-test), error bars = standard deviation