Necroptotic TNFα-Syndecan 4-TNFα Vicious Cycle as a Therapeutic Target for Preventing Temporomandibular Joint Osteoarthritis

Temporomandibular joint osteoarthritis (TMJOA) is a chronic degenerative disease for which the underlying mechanism still remains unclear. Compared with apoptosis and autophagy, necroptosis causes greater harm to tissue homeostasis by releasing damage-associated molecular patterns (DAMPs). However, the role of necroptosis and downstream key DAMPs in TMJOA is unknown. Here, rodent models of TMJOA were established by the unilateral anterior crossbite (UAC). Transmission electron microscopy (TEM) and immunohistochemistry of receptor interacting protein kinase 3 (RIPK3)/phosphorylation of mixed lineage kinase domain-like protein (pMLKL) were conducted to evaluate the occurrence of necroptosis in vivo. The therapeutic effects of blocking necroptosis were achieved by intra-articularly injecting RIPK3 or MLKL inhibitors and using RIPK3 or MLKL knockout mice. In vitro necroptosis of condylar chondrocyte was induced by combination of tumor necrosis factor alpha (TNFα), second mitochondria-derived activator of caspases (SMAC) mimetics and carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]- fluoromethylketone (z-VAD-fmk). The possible DAMPs released by necroptotic chondrocytes were screened by quantitative proteomics and blocked by specific antibody. Translucent cytosol, swollen organelles, and ruptured cell membranes, features of necroptosis, were frequently manifested in chondrocytes at the early stage of condylar cartilage degeneration in TMJOA, which was accompanied by upregulation of RIPK3/pMLKL. Inhibiting or knocking out RIPK3/MLKL significantly prevented cartilage degeneration. DAMPs released by necroptotic condylar chondrocytes, such as syndecan 4 (SDC4) and heat shock protein 90 (HSP90), were verified. Furthermore, blocking the function of SDC4 significantly attenuated the expression of TNFα in cartilage and synovium, and accordingly increased cartilage thickness and reduced synovial inflammation. Thus, the necroptotic vicious cycle of TNFα-SDC4-TNFα contributes to cartilage degeneration and synovitis, and can serve as a potential therapeutic target for treating TMJOA. © 2022 American Society for Bone and Mineral Research (ASBMR).     

Articular Cartilage Degradation and Aberrant Subchondral Bone Remodeling in Patients with Osteoarthritis and Osteoporosis

Osteoarthritis (OA) and osteoporosis (OP) are two skeletal disorders associated with joint structures. Occasionally, OA and OP occur in the same patient. However, the effect of OP changes on OA progression in patients with osteoporotic OA (OP-OA) has not been reported, especially the potential association between subchondral bone and articular cartilage. Thus we investigated the alterations in the microstructure, biomechanical properties, and remodeling of subchondral bone as well as their association with cartilage damage in the hip joint of patients with OP-OA. Thirty-nine femoral head specimens were obtained from patients who underwent total hip arthroplasty (OA group, n = 19; OP-OA group, n = 20), and healthy specimens from cadaver donors were used (control group, n = 10). The microstructure and biomechanical properties of subchondral bone were evaluated by micro–computed tomography and micro–finite-element analysis. Histology, histomorphometric measurements, and immunohistochemistry were used to assess subchondral bone remodeling and cartilage damage. Linear regression analysis was performed to elucidate the relationship between subchondral bone and articular cartilage. In the subchondral bone of the OP-OA group, compared with that of the OA group, aberrant bone remodeling leads to an inferior microstructure and worsening biomechanical properties, potentially affecting transmission of loading stress from the cartilage to the subchondral bone, and then resulting in accelerated OA progression in patients with OP-OA. The results indicate that changes in subchondral bone could affect OA development and the improvement in subchondral bone with bone-metabolism agents may help mitigate OA progression when OP and OA coexist in the same patients. © 2019 American Society for Bone and Mineral Research.     

A Collagen–Poly(Vinyl Alcohol) Nanofiber Scaffold for Cartilage Repair

Articular cartilage has a limited capacity for self-repair. Untreated injuries of cartilage may lead to osteoarthritis. This problem demands new effective methods to reconstruct articular cartilage. Mesenchymal stem cells (MSCs) have the proclivity to differentiate along multiple lineages giving rise to new bone, cartilage, muscle, or fat. This study was an animal model for autologous effects of transplantation of MSCs with a collagen–poly(vinyl alcohol) (PVA) scaffold into full-thickness osteochondral defects of the stifle joint in the rabbit as an animal model. A group of 10 rabbits had a defect created experimentally in the full thickness of articular cartilage penetrated into the subchondral space in the both stifle joints. The defect in the right stifle was filled with MSCs/collagen–PVA scaffold (group I), and in the left stifle, the defect was left without any treatment as the control group (group II). Specimens were harvested at 12 weeks after implantation, examined histologically for morphologic features, and stained immunohistochemically for type-II collagen. Histology observation showed that the MSCs/collagen–PVA repair group had better chondrocyte morphology, continuous subchondral bone, and much thicker newly formed cartilage compared with the control group at 12 weeks post operation. There was a significant difference in histological grading score between these two groups. The present study suggested that the hybrid collagen–PVA scaffold might serve as a new way to keep the differentiation of MSCs for enhancing cartilage repair.     

A Collagen Sponge Incorporating a Hydroxyapatite/Chondroitinsulfate Composite as a Scaffold for Cartilage Tissue Engineering

Because cartilage has limited potential for self-repair, tissue engineering is expected to replace the present therapies for damaged cartilage, such as total knee arthroplasty. However, scaffolds suitable for cartilage tissue engineering have not been established. We synthesized a novel porous scaffold, a collagen sponge incorporating a hydroxyapatite/chondroitinsulfate composite (pCol-HAp/ChS), containing materials which resemble extracellular matrices in bone and cartilage tissues. In this report, the physical, mechanical and biological properties of the scaffold are compared with those of a collagen sponge (pCol) and pCol incorporating a hydroxyapatite composite (pCol-HAp). HAp/ChS had smaller crystals and a larger total surface area than HAp. SEM images of the three materials showed pCol-HAp/ChS to have the roughest surface. The mechanical properties suggest that pCol-HAp/ChS and pCol/HAp are similar, and superior to pCol. Seeding experiments showed a uniform distribution of mesenchymal stem cells (MSCs) in pCol-HAp/ChS and pCol/HAp. Histochemical staining after 2 weeks of culture revealed pCol-HAp/ChS to be the most chondrogenic. From these results, pCol-HAp/ChS is expected to be a candidate for a scaffold for cartilage tissue engineering in place of collagen sponge.     

Runx2 Regulates Endochondral Ossification Through Control of Chondrocyte Proliferation and Differentiation

Synthesis of cartilage by chondrocytes is an obligatory step for endochondral ossification. Global deletion of the Runx2 gene results in complete failure of the ossification process, but the underlying cellular and molecular mechanisms are not fully known. Here, we elucidated Runx2 regulatory control distinctive to chondrocyte and cartilage tissue by generating Runx2 exon 8 floxed mice. Deletion of Runx2 gene in chondrocytes caused failure of endochondral ossification and lethality at birth. The limbs of Runx2^ΔE8/ΔE8 mice were devoid of mature chondrocytes, vasculature, and marrow. We demonstrate that the C‐terminus of Runx2 drives its biological activity. Importantly, nuclear import and DNA binding functions of Runx2 are insufficient for chondrogenesis. Molecular studies revealed that despite normal levels of Sox9 and PTHrP, chondrocyte differentiation and cartilage growth are disrupted in Runx2^ΔE8/ΔE8 mice. Loss of Runx2 in chondrocytes also impaired osteoprotegerin‐receptor activator of NF‐κB ligand (OPG‐RANKL) signaling and chondroclast development. Dwarfism observed in Runx2 mutants was associated with the near absence of proliferative zone in the growth plates. Finally, we show Runx2 directly regulates a unique set of cell cycle genes, Gpr132, Sfn, c‐Myb, and Cyclin A1, to control proliferative capacity of chondrocyte. Thus, Runx2 is obligatory for both proliferation and differentiation of chondrocytes. © 2014 American Society for Bone and Mineral Research.     

Identification of the Vitamin D Receptor in Osteoblasts and Chondrocytes But Not Osteoclasts in Mouse Bone

Bone is clearly a target of vitamin D and as expected, the vitamin D receptor (VDR) is expressed in osteoblasts. However, the presence of VDR in other cells such as osteocytes, osteoclasts, chondroclasts, and chondrocytes is uncertain. Because of difficulties in obtaining sections of undecalcified adult bone, identification of the site of VDR expression in adult bone tissue has been problematic. In addition, the antibodies to VDR used in previous studies lacked specificity, a property crucial for unambiguous conclusions. In the present study, VDR in the various cells from neonatal and adult mouse bone tissues was identified by a highly specific and sensitive immunohistochemistry method following bone decalcification with EGTA. For accurate evaluation of weak immunosignals, samples from Demay VDR knockout mice were used as negative control. Molecular markers were used to identify cell types. Our results showed that EGTA‐decalcification of bone tissue had no detectable effect on the immunoreactivity of VDR. VDR was found in osteoblasts and hypertrophic chondrocytes but not in the multinucleated osteoclasts, chondroclasts, and bone marrow stromal cells. Of interest is the finding that immature osteoblasts contain large amounts of VDR, whereas the levels are low or undetectable in mature osteoblasts including bone lining cells and osteocytes. Proliferating chondrocytes appear devoid of VDR, although low levels were found in the hypertrophic chondrocytes. These data demonstrate that osteoblasts and chondrocytes are major targets of 1α,25‐dihydroxyvitamin D, but osteoclasts and chondroclasts are minor targets or not at all. A high level of VDR was found in the immature osteoblasts located in the cancellous bone, indicating that they are major targets of 1α,25‐dihydroxyvitamin D. Thus, the immature osteoblasts are perhaps responsible for the vitamin D hormone signaling resulting in calcium mobilization and in osteogenesis. © 2014 American Society for Bone and Mineral Research.     

Runx1 dose-dependently regulates endochondral ossification during skeletal development and fracture healing

Runx1 is expressed in skeletal elements, but its role in fracture repair has not been analyzed. We created mice with a hypomorphic Runx1 allele (Runx1(L148A) ) and generated Runx1(L148A/-) mice in which >50% of Runx1 activity was abrogated. Runx1(L148A/-) mice were viable but runted. Their growth plates had extended proliferating and hypertrophic zones, and the percentages of Sox9-, Runx2-, and Runx3-positive cells were decreased. Femoral fracture experiments revealed delayed cartilaginous callus formation, and the expression of chondrogenic markers was decreased. Conditional ablation of Runx1 in the mesenchymal progenitor cells of the limb with Prx1-Cre conferred no obvious limb phenotype; however, cartilaginous callus formation was delayed following fracture. Embryonic limb bud-derived mesenchymal cells showed delayed chondrogenesis when the Runx1 allele was deleted ex vivo with adenoviral-expressed Cre. Collectively, our data suggest that Runx1 is required for commitment and differentiation of chondroprogenitor cells into the chondrogenic lineage.     

Misexpression of Dickkopf-1 in endothelial cells, but not in chondrocytes or hypertrophic chondrocytes, causes defects in endochondral ossification

Developing cartilage serves as a template for long-bone development during endochondral ossification. Although the coupling of cartilage and bone development with angiogenesis is an important regulatory step for endochondral ossification, the molecular mechanisms are poorly understood. One possible mechanism involves the action of Dickkopf (DKK), which is a family of soluble canonical Wnt antagonists with four members (DKK1-4). We initially observed opposite expression patterns of Dkk1 and Dkk2 during angiogenesis and chondrocyte differentiation: downregulation of Dkk1 and upregulation of Dkk2. We examined the in vivo role of Dkk1 and Dkk2 in linking cartilage/bone development and angiogenesis by generating transgenic (TG) mice that specifically express Dkk1 or Dkk2 in chondrocytes, hypertrophic chondrocytes, or endothelial cells. Despite specific expression pattern during cartilage development, chondrocyte- and hypertrophic chondrocyte-specific Dkk1 and Dkk2 TG mice showed normal developmental phenotypes. However, Dkk1 misexpression in endothelial cells resulted in defects of endochondral ossification and reduced skeletal size. The defects are caused by the inhibition of angiogenesis in developing bone and subsequent inhibition of apoptosis of hypertrophic chondrocytes and cartilage resorption.     

Assessment of Bone Fragility in Patients With Multiple Myeloma Using QCT‐Based Finite Element Modeling

Multiple myeloma (MM) is a malignant plasma cell disease associated with severe bone destruction. Surgical intervention is often required to prevent vertebral body collapse and resulting neurological complications; however, its necessity is determined by measuring lesion size or number, without considering bone biomechanics. Finite element (FE) modeling, which simulates the physiological loading, may improve the prediction of fragility. To test this, we developed a quantitative computed tomography (QCT)‐based FE model of the vertebra and applied it to a dataset of MM patients with and without prevalent fracture. FE models were generated from vertebral QCT scans of the T₁₂ (T₁₁ if T₁₂ was fractured) of 104 MM patients, 45 with fracture and 59 without, using a low‐dose scan protocol (1.5 mm slice thickness, 4.0 to 6.5 mSv effective dose). A calibration phantom enabled the conversion of the CT Hounsfield units to FE material properties. Compressive loading of the vertebral body was simulated and the stiffness, yield load, and work to yield determined. To compare the parameters between fracture and nonfracture groups, t tests were used, and standardized odds ratios (sOR, normalized to standard deviation) and 95% confidence intervals were calculated. FE parameters were compared to mineral and structural parameters using linear regression. Patients with fracture showed lower vertebral stiffness (–15.2%; p = 0.010; sOR = 1.73; 95% CI, 1.11 to 2.70), yield force (–21.5%; p = 0.002; sOR = 2.09; 95% CI, 1.27 to 3.43), and work to yield (–27.4%; p = 0.001; sOR = 2.28; 95% CI, 1.33 to 3.92) compared to nonfracture patients. All parameters correlated significantly with vBMD (stiffness: R² = 0.57, yield force: R² = 0.59, work to yield: R² = 0.50, p < 0.001), BV/TV (stiffness: R² = 0.56, yield force: R² = 0.58, work to yield: R² = 0.49, p < 0.001), and Tb.Sp (stiffness: R² = 0.51, yield force: R² = 0.53, work to yield: R² = 0.45, p < 0.001). FE modeling identified MM patients with compromised mechanical integrity of the vertebra. Higher sOR values were obtained for the biomechanical compared to structural or mineral measures, suggesting that FE modeling improves fragility assessment in these patients. © 2016 American Society for Bone and Mineral Research.