TGF-ß3 POTENTIATES TENOGENESIS AND NEOTISSUE FORMATION BY HUMAN MESENCHYMAL STEM CELLS IN 3D CULTURE

*Kuo, C K; *Taboas, J M; +*Tuan, R S +*Cartilage Biology and Orthopaedics Branch, National Institute of Arthritis, and Musculoskeletal and Skin Diseases, National Institutes of Health,

Department of Health and Human Services, Bethesda, MD tuanr@mail.nih.gov

Introduction Tenogenesis, differentiation down the tendon/ligament lineage, of adult mesenchymal stem cells (MSCs) has been demonstrated in three-dimensional (3D) systems cultured under static or dynamic mechanical strains [1,2]. Recently, the response of two-dimensionally (2D) cultured MSCs to potentially tenogenic growth factors has also been investigated [3]. Generally, differentiation in either 2D or 3D was assessed on the basis of proliferation, gene expression profile of collagen types I and III, cell and fibril morphology, and mechanical strength of MSC-laden collagen constructs. Our focus, instead, is to probe the biologic response of MSCs in these established 3D systems to elucidate potential mechanisms involved in tenogenesis. Our previous work demonstrated that 3D culture of MSCs under static uniaxial tension was required to upregulate scleraxis [4], a tendon-specific transcription factor expressed throughout embryogenesis and in the mature tendon [5]. Further, we showed that dynamic stimulation was necessary to maintain the increased transcript levels of scleraxis with time [6]. Interestingly, increased matrix accumulation in dynamically loaded constructs correlated with differential regulation of matrix metalloproteinase (MMP) expression levels [6]. We also previously identified TGF-βs as potentially tenogenic growth factors based on their protein expression patterns during embryonic tendon development [7]. Based on these results, we hypothesize that dynamic uniaxial tensile loading coupled with TGF-β3 treatment will drive tenogenic differentiation of MSCs in 3D and also neotissue formation, potentially via regulation of MMP expression. Methods Low passage, bone marrow-derived human MSCs were suspended in type I collagen gels and cast in Tissue Train culture plates (Flexcell International, Hillsborough, NC) to form linear gels anchored at two opposite ends. The cell-gel constructs were cultured for 24h prior to the first day of cyclic loading. Dynamically stimulated samples were subjected to cyclic, uniaxial tensile loading each day for 30 min/day at 1% elongation and 1Hz. Culture medium (DMEM, 0.1%FBS, 1%P/S, 100µM/L ascorbic acid, ±10ng/mL TGF-β3) was changed every two days. Statically and cyclically loaded constructs were harvested 24h after dynamic loading for 1, 3, 5 and 7 days. Paraffin-embedded samples were sectioned at 5µm thickness and stained with Hematoxylin-eosin (H&E) or Mallory’s trichrome. Gene expression was analyzed by real-time, quantitative RT-PCR with Sybr green Results Cyclic loading for 7 days resulted in greater matrix production, as demonstrated by H&E staining (not shown), and specifically collagen production, indicated by increased trichrome staining (Figure 1). When treated with TGF-ß3, collagen matrix production was significantly enhanced with both static and cyclic conditions (Figure 1).

StaticStatic CyclicCyclic

ControlControl

TGF-TGF-ßß33

Figure 1. Trichrome staining of constructs subjected to either static or cyclic loading for 7 days in medium supplemented ±10ng/mL TGF-β3. Original magnification, 10X. Under both static and cyclic conditions, one day of culture coupled with TGF-ß3 treatment upregulated scleraxis (Figure 2), elastin, wnt9a (a soluble signaling factor implicated in tendon/ligament development), and collagen type III expression levels, when compared to control medium conditions (without TGF-ß3). Seven days of loading coupled with TGF-ß3 treatment maintained increased mRNA levels of scleraxis (Figure 2), but downregulated decorin expression relative to control medium conditions. The upregulation and maintenance of higher scleraxis transcript levels under both static and cyclic conditions indicated tenogenic activity of TGF-ß3. To examine the potential role of MMPs in neotissue formation seen in response to cyclic loading and TGF-ß3 treatment, expression levels of MMP-1 and -9 were examined.

MMP-1 (Figure 3) and -9 expression levels were strongly downregulated (>3-fold and >2-fold, respectively, n=2) after just one day of loading with TGF-ß3 treatment. In contrast, loading with TGF-ß3 treatment for seven days upregulated MMP-1 (>3.5-fold, n=2) (Figure 3). Interestingly, the effect of TGF-ß3 was greater than the effect of cyclic loading for all genes, when compared to static conditions.

Figure 2. Fold change of scleraxis mRNA levels normalized to CT-N on days 1 and 7 (CT=control, T3=TGF-ß3, N=static, C=cyclic), n=2.

Figure 3. Fold change of MMP-1 mRNA levels normalized to CT-N on days 1 and 7 (CT=control, T3=TGF-ß3, N=static, C=cyclic), n=2. Discussion A challenge to differentiating MSCs down the tendon/ligament lineage has been the limited availability of unique markers for tendon and ligament. Instead, assays have relied upon parameters such as mechanical strength, cell and fibril alignment, and collagen type I:III expression level ratios [1,2]. In this work, the focus was not to tissue engineer a functional tendon or ligament, but rather to probe the biology of the system and the potential mechanisms behind tenogenesis of human MSCs when coupling mechanical and soluble stimulatory factors. Previously, we identified TGF-βs as potential tenogenic factors based on protein expression during tendon development [7]. In this work, we assessed the tenogenic potential of TGF-β3 in this dynamic 3D system by analyzing changes in gene expression levels of candidate markers including scleraxis and wnt9a. While the function of scleraxis is not known, the use of this molecule as a unique marker for tendon/ligament is based on studies using in situ hybridization to characterize tendon and ligament development during embryogenesis [5]. Wnt9a, a soluble signaling factor, has also been shown via in situ hybridization to be expressed during development, specifically in cells that form the ligament in the joint [8]. Here, treatment of both statically and cyclically loaded 3D cultures with TGF-β3 resulted in significant upregulation of wnt9a and scleraxis, with sustained upregulation of the latter. Previously, we demonstrated that MSCs responded to cyclic stimulation by differentially regulating MMP expression levels. We proposed that these enzymes were involved in increased neotissue formation when cyclically loaded. Here, we hypothesized that TGF-β3 would enhance tenogenesis as well as neotissue formation, and that this might occur in part via regulation of gene expression of MMPs. In this work, TGF-β3 did enhance neotissue formation, as indicated by increased collagen matrix accumulation, as well as regulate MMP-1 and -9 expression and upregulate tenogenic markers. Taken together, our results suggest that tenogenesis and neotissue formation by MSCs might be potentiated in part by TGF-ß3, perhaps via modulation of MMP expression. References [1]Awad+, J Biomed Mater Res 51:233-40,2000; [2]Altman+, FASEB J 10.1096/fj.01-0656fje,2001; [3]Moreau+, J Orthoped Res 23:164-74,2005; [4]Kuo & Tuan, ISL&T V,2005; [5]Schweitzer+, Development 128:3855-66,2001; [6]Kuo&Tuan, ISL&T VI,2006; [7]Kuo+, ORS2006; [8]Guo+, Genes and Development 18:2404-17,2004. Acknowledgements This research was supported by the Intramural Research Program of the NIH, National Institute of Arthritis and Musculoskeletal and Skin Diseases.

53rd Annual Meeting of the Orthopaedic Research Society

Poster No: 0487