TMJ Stem Cells Regenerate Intramembranous Bone and Recruit Blood Vessel Formation

TMJ Stem Cells Regenerate Intramembranous Bone and Recruit Blood Vessel Formation

J.M. Nathan¹, S. Pylawka¹, G. Casanovas¹, C. Shawber², J. Kitajewski², M. Embree¹

¹College of Dental Medicine, Columbia University, New York, NY

²OBGYN, Columbia University College of Physician and Surgeons, New York, NY

Objective: Regenerating vascular supply is a critical step for bone repair (Wegmann et al., 2013). Inadequate perfusion limits bone regeneration and leads to tissue necrosis (Bamias et al., 2005). Our lab identified a novel source of stem cells from the TMJ (TMJ- SCs) that spontaneously form endochondral bone, cartilage, and fat. Here, we propose that TMJ-SCs form intramembranous bone and promote angiogenesis when transplanted in a calvarial defect. Our goal is to define the interactions between TMJ-SCs and endothelial cells in mediating angiogenesis.

Methods: TMJ-SCs were derived from GFP rats and endothelial cells (ECs) were isolated from human umbilical veins and transfected with RFP. Angiogenesis was evaluated in vitro using a fibrin gel bead assay (FIBA) (n=12 experiments) and in vivo using a mouse calvarial defect model (n=38 mice) with TMJ- SCs. New vessel formation was evaluated by confocal microscopy, immunohistochemistry, and histomorphometry. TMJ-SC ossification was evaluated in vitro using TMJ-SC and EC co-cultures.

Results: FIBA showed TMJ-SC feeder layer secreted factors to support EC blood vessel formation. Histology and immunohistochemistry of mouse calvarial defect model showed that GFP+TMJ-SCs formed intramembranous bone that integrated into host bone defect with recruited blood vessel formation.  Cell to cell contact between ECs and TMJ-SCs enhance TMJ-SC mineralization.

Figure 1: (a) Superior view of nude mouse parietal bone (PB) defect (defect=yellow dashed circle; FB=frontal bone; OB=occipital bone) with transplanted cell/collagen sponge at 8 weeks. FITC microscopy and x-rays demonstrated GFP+ TMJ-PCs and radio-opaque tissue formed within transplants in calvarial defect. (b) Superior view of nude mouse calvarial bone defect model transplanted with empty sponge negative control at 8 weeks.

Figure 2: (a,e) Coronal H&E sections of TMJ-PC transplants (DB=de novo bone; HB=host bone, CS=collagen sponge) and (b-c) immunohistochemistry showed donor TMJ-PCs formed bone-like tissue (GFP⁺, osteocalcin⁺) that integrated into host calvaria bone. Green=TMJ-PCs; red=osteocalcin; blue=dapi. (c) Higher magnification of orange square box in (b). (f-h) immunohistochemistry showed blood vessel formation adjacent to de novo bone.

 

 

 

 

 

Figure 3: TMJ-PCs and HUVECs were cultured in osteogenic media for 3 weeks at various ratios (TMJ:HUVEC=1:0, 2:1, 1:1, 1:2). Relative to TMJ-PCs, TMJ-PC/HUVEC co-cultures had higher (a) alizarin red staining, (b) significant increases in alkaline phosphatase activity (****p² 0.0001, ***p²0.001, *p²0.05; 1:0 vs 2:1, 1:1, 1:2. ##p²0.01, ###p²0.001; 2:1 vs 1:1, 1:2. ~p²0.01, 1:1 vs 1:2), (c) and higher levels of bone-related genes (bsp, ocn). Fibrin bead assay showed that (d) TMJ-PC feeder layer supports HUVEC blood vessel formation after 6 days (red, arrows) (e) dermal fibroblast feeder layer supports HUVEC blood vessel formation after 6 days (f) HUVECs without cell feeder layer do not form blood vessels after 6 days. 

Conclusions: TMJ-SCs promote angiogenesis via secreted signaling molecules and EC contact signaling enhances TMJ-SC mineralization. Our on-going mechanistic studies are aimed toward identifying both secreted and contact-dependent factors mediating TMJ-SC and EC communication. A better understanding of these complex interactions could enhance bone healing and regenerative therapies.

References

Bamias, A., Kastritis, E., Bamia, C., Moulopoulos, L. A., Melakopoulos, I., Bozas, G., ... Dimopoulos, M. A. (2005). Osteonecrosis of the Jaw in Cancer After Treatment With Bisphosphonates: Incidence and Risk Factors. Journal of Clinical Oncology, 23(34), 8580–8587. http://doi.org/10.1200/JCO.2005.02.8670

Wegmann, K., Burkhart, K. J., Koslowsky, T. C., Koebke, J., Neiss, W. F., & MŸller, L. P. (2013). Arterial supply of the distal humerus. Surgical and Radiologic Anatomy: SRA. http://doi.org/10.1007/s00276-013-1240-z