Probes for INS

The Temporomandibular joint (TMJ) is one of the most complex joints in the human body. TMJ is composed of the temporal bone, a disc and a movable mandibular condyle with abundant tendon attachments. Tendon has been thought to play the sole function of transmitting muscle forces to stabilize joints, yet it is largely unclear why tendon undergoes ectopic ossification in trauma or diseases, and whether it has any direct contribution to skeletal formation. This study aimed to investigate the full biological significance of tendon in TMJ growth. We first discovered that the TMJ condyle is composed of a well-established cartilage head and an overlooked “bony head” that grows after birth and continuously expands throughout the lifespan with little signs of remodeling. Mouse X-ray images (Fig.1a) showed little change in the cartilage head’s volume but a continuous expansion in the bony head’s mass with a low mineral content from 1 to 5 months (Fig.1b). Toluidine blue staining showed TMJ condyle had a large area of tendon attachment extending down to ramus (Fig.1c, white dotted line in lower magnification), defined by regions of tendon, interface, and TFB (Fig.1c1). The TFB morphology was distinct from endosteum-formed bone (EFB, Fig.1c1), cartilage-formed bone (CFB, Fig.1c2, rich in cartilage residual), or periosteum-formed bone (PFB, Fig.1c3) in cell shape and distribution, and ECM. TEM images further revealed that the osteocytes in the TFB were large in size, irregular in shape, had small nuclei but numerous ERs and Golgi complexes, and were embedded in ECM rich in fibropositors. In contrast, the osteocytes in EFB, CFB or PFB were spindle-shaped with larger nuclei but fewer ERs and Golgi complexes (Fig.1d). To reveal the cell source of the bony head, cell lineage tracing were used. Tracing data showed that most CFB cells originate from Col10a1+ hypertrophic chondrocytes, whereas the interface and TFB were derived from Scx+ cells (Fig.1e). RNAscope displayed high levels of Thbs4 (Thrombospondin-4, a tendon marker) and SOST (a potent inhibitor of Wnt signaling secreted by osteocytes) mRNA in TFB at bony head (Fig.1f). The Scx-CreERT2 tracing combined with IHC staining showed TFB maintained a mixed ECM of bone (Col1), cartilage (Aggrecan) and tendon (Periostin, Fig.1g). To further determine the role of tendon lineage in condyle expansion, we generated Scx-CreERT2; R26RDTA (carrying a loxP-flanked stop cassette associated with an attenuated diphtheria toxin fragment A, DTA, for the ablation of cells when Cre is active). Deletion of Scx+ cells greatly reduced the size of bony head (Fig.1h) and the thickness of interface with few Scx+/Col1+ bone cells in P28 DTA mice (Fig.1i); In conclusion, our study tendon cells, beyond their conventional role in joint movement, are key players for the postnatal growth and expansion of TMJ condyle (Fig.1j).

Although failure to establish a vascular network has been associated with many skeletal disorders, little is known about what drives development of vasculature in the intracortical bone compartments. Here, we show that intracortical bone resorption events are coordinated with development of the vasculature. We investigated the prevalence of vascular structures at different remodeling stages as well as their 3D organization using proximal femoral cortical bone from 5 girls and 6 boys (aged 6-15 years). A 2D analysis revealed that non-quiescent intracortical pores contained more vascular structures than quiescent pores (p < 0.0001). Type 2 pores, i.e., remodeling of existing pores, had a higher density of vascular structures than type 1 pores, i.e., de novo created pores (p < 0.05). Furthermore, pores at the eroded-formative remodeling stage, had more vascular structures than pores at any other remodeling stage (p < 0.05). A 3D reconstruction of an intracortical remodeling event showed that osteoclasts in the advancing tip of the cutting cone as well as preosteoclasts in the lumen expressed vascular endothelial growth factor-A (VEGFA), while VEGFA-receptors 1 and 2 mainly were expressed in endothelial cells in the adjacent vasculature. Consequently, we propose that the progression of the vascular network in intracortical remodeling events is driven by osteoclasts expressing VEGFA. Moreover, the vasculature is continuously reconfigured according to the demands of the remodeling events at the surrounding bone surfaces.

Relevance of mineralized nodules in two-dimensional (2D) osteoblast/osteocyte cultures to bone biology, pathology, and engineering is a decades old question, but a comprehensive answer appears to be still wanting. Bone-like cells, extracellular matrix (ECM), and mineral were all reported but so were non-bone-like ones. Many studies described seemingly bone-like cell-ECM structures based on similarity to few select bone features _in vivo_, yet no studies examined multiple bone features simultaneously and none systematically studied all types of structures coexisting in the same culture. Here, we report such comprehensive analysis of 2D cultures based on light and electron microscopies, Raman microspectroscopy, gene expression, and _in situ_ mRNA hybridization. We demonstrate that 2D cultures of primary cells from mouse calvaria do form _bona fide_ bone. Cells, ECM, and mineral within it exhibit morphology, structure, ultrastructure, composition, spatial-temporal gene expression pattern, and growth consistent with intramembranous ossification. However, this bone is just one of at least five different types of cell-ECM structures coexisting in the same 2D culture, which vary widely in their resemblance to bone and ability to mineralize. We show that the other two mineralizing structures may represent abnormal (disrupted) bone and cartilage-like formation with chondrocyte-to-osteoblast trans differentiation. The two non-mineralizing cell-ECM structures may mimic periosteal cambium and pathological, non-mineralizing osteoid. Importantly, the most commonly used culture conditions (10 mM β-glycerophosphate) induce artificial mineralization of all cell-ECM structures, which then become barely distinguishable. We therefore discuss conditions and approaches promoting formation of _bona fide_ bone and simple means for distinguishing it from the other cell-ECM structures. Our findings may improve osteoblast differentiation and function analyses based on 2D cultures and extend applications of these cultures to general bone biology and tissue engineering research.