Junjun Jing

Chondrocytes Directly Transform into Bone Cells in Mandibular Condyle Growth

For decades, it has been widely accepted that hypertrophic chondrocytes undergo apoptosis prior to endochondral bone formation. However, very recent studies in long bone suggest that chondrocytes can directly transform into bone cells. Our initial in vivo characterization of condylar hypertrophic chondrocytes revealed modest numbers of apoptotic cells but high levels of antiapoptotic Bcl-2 expression, some dividing cells, and clear alkaline phosphatase activity (early bone marker). Ex vivo culture of newborn condylar cartilage on a chick chorioallantoic membrane showed that after 5 d the cells on the periphery of the explants had begun to express Col1 (bone marker). The cartilage-specific cell lineage–tracing approach in triple mice containing Rosa 26tdTomato (tracing marker), 2.3 Col1GFP (bone cell marker), and aggrecan CreERT2 (onetime tamoxifen induced) or Col10-Cre (activated from E14.5 throughout adult stage) demonstrated the direct transformation of chondrocytes into bone cells in vivo. This transformation was initiated at the inferior portion of the condylar cartilage, in contrast to the initial ossification site in long bone, which is in the center. Quantitative data from the Col10-Cre compound mice showed that hypertrophic chondrocytes contributed to ~80% of bone cells in subchondral bone, ~70% in a somewhat more inferior region, and ~40% in the most inferior part of the condylar neck (n = 4, P < 0.01 for differences among regions). This multipronged approach clearly demonstrates that a majority of chondrocytes in the fibrocartilaginous condylar cartilage, similar to hyaline cartilage in long bones, directly transform into bone cells during endochondral bone formation. Moreover, ossification is initiated from the inferior portion of mandibular condylar cartilage with expansion in one direction.

Adverse Effects of Osteocytic Constitutive Activation of ß‐Catenin on Bone Strength and Bone Growth

The activation of the canonical Wnt/β‐catenin signaling pathway in both mesenchymal stem cells and osteoblasts has been demonstrated to increase bone mass, showing promise for the treatment of low bone volume conditions such as osteoporosis. However, the possible side effects of manipulating this pathway have not been fully addressed. Previously, we reported that the constitutive activation of ß‐catenin in osteoblasts impaired vertebral linear growth. In the present study, β‐catenin was constitutively activated in osteocytes by crossing Catnb+/lox(exon 3) mice with dentin matrix protein 1(DMP1)‐Cre transgenic mice, and the effects of this activation on bone mass, bone growth and bone strength were then observed. DMP1‐Cre was found to be predominantly expressed in osteocytes, with weak expression in a small portion of osteoblasts and growth plate chondrocytes. After the activation, the cancellous bone mass was dramatically increased, almost filling the entire bone marrow cavity in long bones. However, bone strength decreased significantly. Thinner and more porous cortical bone along with impaired mineralization were responsible for the decrease in bone strength. Furthermore, the mice showed shorter stature with impaired linear growth of the long bones. Moreover, the concentration of serum phosphate decreased significantly after the activation of ß‐catenin, and a high inorganic phosphate (Pi) diet could partially rescue the phenotype of decreased mineralization level and impaired linear growth. Taken together, the constitutive activation of β‐catenin in osteocytes may increase cancellous bone mass; however, the activation also had adverse effects on bone strength and bone growth. These adverse effects should be addressed before the adoption of any therapeutic clinical application involving adjustment of the Wnt/β‐catenin signaling pathway.

Complete pulpodentin complex regeneration by modulating the stiffness of biomimetic matrix

Dental caries is one of the most prevalent chronic diseases in all populations. The regeneration of dentin-pulp tissues (pulpodentin) using a scaffold-based tissue engineering strategy is a promising approach to replacing damaged dental structures and restoring their biological functions. However, the current scaffolding design for pulpodentin regeneration does not take into account the distinct difference between pulp and dentin, therefore, is incapable of regenerating a complete tooth-like pulpodentin complex. In this study, we determined that scaffolding stiffness is a crucial biophysical cue to modulate dental pulp stem cell (DPSC) differentiation. The DPSCs on a high-stiffness three-dimensional (3D) nanofibrous gelatin (NF-gelatin) scaffold had more organized cytoskeletons and a larger spreading area than on a low-stiffness NF-gelatin scaffold. In the same differentiation medium, a high-stiffness NF-gelatin facilitated DPSC differentiation to form a mineralized tissue, while a low-stiffness NF-gelatin promoted a soft pulp-like tissue formation from the DPSCs. A facile method was then developed to integrate the low- and high-stiffness gelatin matrices into a single scaffold (S-scaffold) for pulpodentin complex regeneration. A 4-week in vitro experiment showed that biomineralization took place only in the high-stiffness peripheral area and formed a ring-like structure surrounding the non-mineralized central area of the DPSC/S-scaffold construct. A complete pulpodentin complex similar to natural pulpodentin was successfully regenerated after subcutaneous implantation of the DPSC/S-scaffold in nude mice for 4weeks. Histological staining showed a significant amount of extracellular matrix (ECM) formation in the newly formed pulpodentin complex, and a number of blood vessels were observed in the pulp tissue. Taken together, this work shows that modulating the stiffness of the NF-gelatin scaffold is a successful approach to regenerating a complete tooth-like pulpodentin complex.

BMP receptor 1A determines the cell fate of the postnatal growth plate

Bone morphogenic proteins (BMPs) are critical for both chondrogenesis and osteogenesis. Previous studies reported that embryos deficient in Bmp receptor (Bmpr)1a or Bmpr1b in cartilage display subtle skeletal defects; however, double mutant embryos develop severe skeletal defects, suggesting a functional redundancy that is essential for early chondrogenesis. In this study, we examined the postnatal role of Bmpr1a in cartilage. In the Bmpr1a conditional knockout (cKO, a cross between Bmpr1a flox and aggrecan-CreERT2 induced by a one-time-tamoxifen injection at birth and harvested at ages of 2, 4, 8 and 20 weeks), there was essentially no long bone growth with little expression of cartilage markers such as SOX9, IHH and glycoproteins. Unexpectedly, the null growth plate was replaced by bone-like tissues, supporting the notions that the progenitor cells in the growth plate, which normally form cartilage, can form other tissues such as bone and fibrous; and that BMPR1A determines the cell fate. A working hypothesis is proposed to explain the vital role of BMPR1A in postnatal chondrogenesis.

Measurement of hydroxyapatite density and Knoop hardness in sound human enamel and a correlational analysis between them

OBJECTIVE The aim of this study was to measure the hydroxyapatite (HAP) density and Knoop hardness (KHN) of enamel slabs and to analyse the relationship between them. DESIGN Twenty enamel slabs (10 lingual sides and 10 buccal sides) were prepared and scanned with micro-CT. Tomographic images of each slab from dental cusp to dentinoenamel junction (DEJ) were reconstructed. On these three-dimensional (3D) images, regions of interest (ROIs) were defined at an interval of 50 microm, and the HAP density for each ROI was calculated. Then the polished surfaces were indented from cusp to DEJ at intervals of 50 microm with a Knoop indenter. Finally, the data were analysed with one-way ANOVA, Students t-test, and linear regression analysis. RESULTS The HAP density and KHN decreased from the dental cusp to DEJ. Both HAP density and KHN in the outer-layer enamel were significantly higher than those in the middle- or inner-layer enamel (P<0.05). The HAP density showed no significant difference between the buccal and lingual sides for enamel in the outer, middle and inner layers, respectively (P>0.05). The KHN in the outer-layer enamel of the lingual sides was significantly lower than that of the buccal sides (P<0.05); there was no significant difference between the lingual and buccal sides in the middle or inner layer. Linear regression analysis revealed a linear relationship between the mean KHN and the mean HAP density (r=0.87). CONCLUSION Both HAP density and KHN decrease simultaneously from dental cusp to DEJ, and the two properties are highly correlated.

Osterix Couples Chondrogenesis and Osteogenesis in Post-natal Condylar Growth

Osterix (Osx) is a transcription factor essential for osteoblast differentiation and bone mineralization. Although there are indications that Osx also plays a regulatory role in cartilage, this has not been well-studied. The goal of this study was to define the function of Osx in the post-natal growth of the secondary cartilage at the mandibular condyle. Conditional Osx knockout (cKO) mice that were missing Osx only in cartilage were generated by crossing Osx-loxP mice to Aggrecan-Cre mice. Cre activity was induced by tamoxifen injection twice a week from day 12 to 1 mo of age, and specimens were collected at 1 and 5 mo of age. At 1 mo of age, the condylar hypertrophic chondrocyte zone in the cKO-mice was > three-fold thicker than that in the age-matched control, with little sign of endochondral bone formation. Immunohistochemistry and analysis of histological data revealed a defect in the coupling of chondrogenesis and osteogenesis in the cKO mice. In five-month-old mice examined to address whether late-stage removal of the Cre-deletion event would alleviate the phenotype, the hypertrophic chondrocyte zone in the cKO condyles was considerably larger than in wild-type mice. There were large discrete areas of calcified cartilage in the hypertrophic zone, few signs of endochondral bone formation, and large regions of disorganized intramembranous bone. Analysis of these data further strengthens the notion that Osterix is essential for the coupling of terminal cartilage differentiation and endochondral ossification in mandibular condylar cartilage.

Roles of Chondrocytes in Endochondral Bone Formation and Fracture Repair

The formation of the mandibular condylar cartilage (MCC) and its subchondral bone is an important but understudied topic in dental research. The current concept regarding endochondral bone formation postulates that most hypertrophic chondrocytes undergo programmed cell death prior to bone formation. Under this paradigm, the MCC and its underlying bone are thought to result from 2 closely linked but separate processes: chondrogenesis and osteogenesis. However, recent investigations using cell lineage tracing techniques have demonstrated that many, perhaps the majority, of bone cells are derived via direct transformation from chondrocytes. In this review, the authors will briefly discuss the history of this idea and describe recent studies that clearly demonstrate that the direct transformation of chondrocytes into bone cells is common in both long bone and mandibular condyle development and during bone fracture repair. The authors will also provide new evidence of a distinct difference in ossification orientation in the condylar ramus (1 ossification center) versus long bone ossification formation (2 ossification centers). Based on our recent findings and those of other laboratories, we propose a new model that contrasts the mode of bone formation in much of the mandibular ramus (chondrocyte-derived) with intramembranous bone formation of the mandibular body (non-chondrocyte-derived).

Genetic Influences on Temporomandibular Joint Development and Growth

The temporomandibular joint (TMJ) is a small synovial joint at which the mandible articulates with the skull during movements involved in speaking and mastication. However, the secondary cartilage lining its joint surfaces is indicative of a very different developmental history than limb cartilages. This review summarizes our current knowledge of genes that regulate the formation of primary components of the TMJ, as well as genes that regulate postnatal growth of the TMJ. Although the TMJ is regulated by some of the same genes that are important in limb joints, others appear unique to the TMJ or have different actions. Runx2, Sox9, and members of the TGF-β/BMP family are critical drivers of chondrogenesis during condylar cartilage morphogenesis, and Indian hedgehog (Ihh) is important for formation of the articular disc and cavitation. Osterix (Osx) is a critical regulator of endochondral bone formation during postnatal TMJ growth.

Critical role of Bmpr1a in mandibular condyle growth.

Abstract The importance of Bone Morphogenetic Proteins (BMPs) in the regulation of cell fate, differentiation and proliferation in the growth plate is well-known. However, in secondary cartilages (such as that in the temporomandibular joint) that grow by proliferation of prechondrocytes and differ in their pattern of growth, the role of BMPs is largely unexplored. To examine this question, we ablated Bmpr1a in the condylar cartilage of neonatal mice and assessed the consequences for mandibular condyle growth and organization at intervals over the ensuing 4 weeks. Bmpr1a deficiency caused significant chondrodysplasia and almost eliminated the chondrocytic phenotype in the TMJ. Expression of Sox9, collagen II, proteoglycan were all greatly reduced, and cell proliferation as detected by BrdU was almost non-existent in the knockout mice. Primary bone spongiosa formation was also disturbed and was accompanied by reduced Osterix expression. These findings strongly suggest that Bmpr1a is critical for the development and growth of the mandibular condyle via its effect on proliferation of prechondroblasts and chondrocyte differentiation.

Bmpr1a Signaling in Cartilage Development and Endochondral Bone Formation

The type IA bone morphogenetic protein receptor (Bmpr1a), encoded by 11 exons and spanning about 40 kb on chromosome 14 in mice and chromosome 10 in human (Derynck & Feng, 1997; Mishina, Hanks, Miura, Tallquist, & Behringer, 2002), is an essential receptor for BMP signaling. This chapter focuses on the current understanding of the role of Bmpr1a in cartilage development and endochondral ossification, including formation of the mesenchymal condensation, chondrocyte differentiation and maturation, and endochondral bone development.