TGFα, EGF Recruit NGFR- and Nestin-Expressing Cells from TMJ Synovium

Objectives: Mandibular condyle articular cartilage heals ineffectively resulting in TMJ degeneration. Our objectives were to 1) identify growth factors that stimulate strong chemotaxis and proliferation responses by stem cells in the synovial membrane and 2) characterize the subpopulation of cells recruited by the chemotactic signals. An optimized signaling molecule panel and delivery schedule are necessary to orchestrate the key tissue regeneration stages of stem cell chemotaxis, proliferation, and differentiation.

Methods: Synovial cells were isolated from TMJ and knee synovium of 3 New Zealand white rabbits by enzymatic digestion. Six growth factors (TGFα, EGF, hbEGF, FGF-2, IGF-1, TGFβ-3) were evaluated at 1, 5, 10, and 20 ng/ml. Chemotaxis was assayed using Fluroblok 96-well transwell inserts (BD Biosciences). Proliferation was assayed in 96-well plates using the CyQuant kit (Invitrogen). Gene expression of recruited cells was evaluated using custom qPCR arrays (Qiagen) for markers associated with enhanced chondrogenesis.

Results: TGFα and EGF at 1 ng/ml stimulated the greatest chemotactic responses by knee synovial cells (p<0.001). FGF-2 at 10 ng/ml stimulated the greatest proliferation response (3.2X control). TGFα, EGF, and FGF-2 also stimulated strong responses in TMJ cells (Fig. 1). TGFβ-3 at 10 ng/ml inhibited chemotaxis in TMJ (p<0.001, Fig. 1) cells and proliferation in both knee and TMJ cells (p≤0.021). Simultaneous delivery of TGFα or EGF with FGF-2 did not inhibit responses. Gene expression for NGFR, nestin, and CD14 was greater for recruited cells than for controls.

Conclusions: Inhibition of chemotaxis and proliferion by TGFβ-3 indicates that a 2-stage delivery strategy is needed. Higher expression of NGFR and nestin suggests that TGFα and EGF selectively recruit a subpopulation of synovial cells. Higher expression of CD14, i.e., macrophages, suggests that off-target recruitment may also occur. This growth factor panel is a key step toward engineering a biomimetic in vivo mandibular cartilage healing response.