The regeneration potential of stem cells is dependent on their microenvironment. In this study, we investigated the effect of the microenvironment of dental pulp stem cells (DPSCs), including 3D structure of a macroporous and nanofibrous scaffold, the inflammatory stimulus lipopolysaccharide (LPS) and a biological molecule simvastatin, on their regenerative potential of mineralized dentin tissue. The results demonstrated that LPS upregulated inflammatory mediators and suppressed the odontogenic potential of DPSCs. Known as a lipid-lowing agent, simvastatin was excitingly found to repress the expression of pro-inflammatory mediators, up-regulate odontoblastic markers, and exert a pro-angiogenic effect on endothelial cells, resulting in enhanced vascularization and mineralized dentin tissue regeneration in a biomimetic 3D tissue engineering scaffold. This novel finding is significant for the fields of stem cells, inflammation and dental tissue regeneration.

Improving the structural integrity of bone reduces fracture risk and development of osteoporosis later in life. Exercise can increase the mechanical properties of bone, and this increase is often attributed to the dynamic loading created during exercise. However, the increase in systemic PTH levels during exercise gives reason to hypothesize that PTH signaling also regulates bone adaptation in response to exercise. Therefore, the first aim of this study was to establish the impact PTH signaling has on bone adaptation during exercise by inhibiting PTH signaling with PTH(7-34) and the second aim was to determine if increasing PTH levels during exercise with PTH(1-34) can augment bone adaptation. Thirty minutes after a single bout of running on a treadmill, mice exhibited a two-fold increase in systemic PTH levels. Under the same exercise regimen, the influence of PTH signaling on bone adaptation during exercise was then evaluated in mice after 21 consecutive days of exercise and treatment with PTH(7-34), PTH(1-34), or vehicle. Exercise alone caused a significant increase in trabecular bone volume with adaptation to a more plate-like structure, which was inhibited with PTH(7-34) during exercise. Changes in structural and tissue-level mechanical properties during exercise occurred in the absence of significant changes to cortical bone geometry. Inhibition of PTH signaling during exercise attenuated the changes in structural-level mechanical properties, but not tissue-level properties. Enhanced PTH signaling during exercise with PTH(1-34) increased trabecular and cortical bone volume, but had little effect on the structural and tissue-level mechanical properties compared to exercise alone. Our study is the first to demonstrate that bone adaptation during exercise is not only a function of the dynamic loading, but also PTH release, and that PTH signaling contributes differently at the structural and tissue-levels.

Discoidin Domain Receptor 2 (DDR2) is a collagen-activated receptor kinase that, together with integrins, is required for cells to respond to the extracellular matrix. Ddr2 loss-of-function mutations in humans and mice cause severe defects in skeletal growth and development. However, the cellular functions of Ddr2 in bone are not understood. Expression and lineage analysis showed selective expression of Ddr2 at early stages of bone formation in the resting zone and proliferating chondrocytes and periosteum. Consistent with these findings, Ddr2+ cells could differentiate into hypertrophic chondrocytes, osteoblasts, and osteocytes and showed a high degree of colocalization with the skeletal progenitor marker, Gli1. A conditional deletion approach showed a requirement for Ddr2 in Gli1-positive skeletal progenitors and chondrocytes but not mature osteoblasts. Furthermore, Ddr2 knockout in limb bud chondroprogenitors or purified marrow-derived skeletal progenitors inhibited chondrogenic or osteogenic differentiation, respectively. This work establishes a cell-autonomous function for Ddr2 in skeletal progenitors and cartilage and emphasizes the critical role of this collagen receptor in bone development.

Collagen signaling is critical for proper bone and tooth formation. Discoidin domain receptor 2 (DDR2) is a collagen-activated tyrosine kinase receptor shown to be essential for skeletal development. Patients with loss of function mutations in DDR2 develop spondylo-meta-epiphyseal dysplasia (SMED), a rare, autosomal recessive disorder characterized by short stature, short limbs, and craniofacial anomalies. A similar phenotype was observed in Ddr2-deficient mice, which exhibit dwarfism and defective bone formation in the axial, appendicular, and cranial skeletons. However, it is not known if Ddr2 has a role in tooth formation. We first defined the expression pattern of Ddr2 during tooth formation using Ddr2-LacZ knock-in mice. Ddr2 expression was detected in the dental follicle/sac and dental papilla mesenchyme of developing teeth and in odontoblasts and the periodontal ligament (PDL) of adults. No LacZ staining was detected in wild-type littermates. This Ddr2 expression pattern suggests a potential role in the tooth and surrounding periodontium. To uncover the function of Ddr2, we used Ddr2 slie/slie mice, which contain a spontaneous 150-kb deletion in the Ddr2 locus to produce an effective null. In comparison with wild-type littermates, Ddr2 slie/slie mice displayed disproportional tooth size (decreased root/crown ratio), delayed tooth root development, widened PDL space, and interradicular alveolar bone defects. Ddr2 slie/slie mice also had abnormal collagen content associated with upregulation of periostin levels within the PDL. The delayed root formation and periodontal abnormalities may be related to defects in RUNX2-dependent differentiation of odontoblasts and osteoblasts; RUNX2-S319-P was reduced in PDLs from Ddr2 slie/slie mice, and deletion of Ddr2 in primary cell cultures from dental pulp and PDL inhibited differentiation of cells to odontoblasts or osteoblasts, respectively. Together, our studies demonstrate odontoblast- and PDL-specific expression of Ddr2 in mature and immature teeth, as well as indicate that DDR2 signaling is important for normal tooth formation and maintenance of the surrounding periodontium.

Mutations in the PHEX gene lead to X-linked hypophosphatemia (XLH), a form of inherited rickets featuring elevated fibroblast growth factor 23 (FGF23), reduced 1,25-dihydroxyvitamin D (1,25D), and hypophosphatemia. Hyp mutant mice replicate the XLH phenotype, including dentin, alveolar bone, and cementum defects. We aimed to compare effects of 1,25D versus FGF23-neutralizing antibody (FGF23Ab) monotherapies on Hyp mouse dentoalveolar mineralization. Male Hyp mice, either injected subcutaneously with daily 1,25D or thrice weekly with FGF23 blocking antibody from 2 to 35 d postnatal, were compared to wild-type (WT) controls and untreated Hyp mice. Mandibles were analyzed by high-resolution micro–computed tomography (micro-CT), histology, and immunohistochemistry. Both interventions maintained normocalcemia, increased serum phosphate levels, and improved dentoalveolar mineralization in treated versus untreated Hyp mice. 1,25D increased crown dentin volume and thickness and root dentin/cementum volume, whereas FGF23Ab effects were limited to crown dentin volume. 1,25D increased bone volume fraction, bone mineral density, and tissue mineral density in Hyp mice, whereas FGF23Ab failed to significantly affect these alveolar bone parameters. Neither treatment fully attenuated dentin and bone defects to WT levels, and pulp volumes remained elevated regardless of treatment. Both treatments reduced predentin thickness and improved periodontal ligament organization, while 1,25D promoted a more profound improvement in acellular cementum thickness. Altered cell densities and lacunocanalicular properties of alveolar and mandibular bone osteocytes and cementocytes in Hyp mice were partially corrected by either treatment. Neither treatment normalized the altered distributions of bone sialoprotein and osteopontin in Hyp mouse alveolar bone. Moderate improvements from both 1,25D and FGF23Ab treatment regimens support further studies and collection of oral health data from subjects receiving a newly approved anti-FGF23 therapy. The inability of either treatment to fully correct Hyp mouse dentin and bone prompts further experiments into underlying pathological mechanisms to identify new therapeutic approaches.

Combined surface treatment of etching with hydrofluoric acid and phosphoric acid provides the highest bond strengths to porcelain. Also, PFC exhibited higher SBS than CEC did.

Temporomandibular joint (TMJ) disorders are often associated with development of osteoarthritis-like changes in the mandibular condyle. Discoidin domain receptor 2 (DDR2), a collagen receptor preferentially activated by type I and III collagen found in the TMJ and other fibrocartilages, has been associated with TMJ degeneration, but its role in normal joint development has not been previously examined. Using Ddr2 LacZ-tagged mice and immunohistochemistry, we found that DDR2 is preferentially expressed and activated in the articular zone of TMJs but not knee joints. To assess the requirement for Ddr2 in TMJ development, studies were undertaken to compare wild-type and smallie ( slie) mice, which contain a spontaneous deletion in Ddr2 to produce an effective null allele. Analysis of TMJs from newborn Ddr2 mice revealed a developmental delay in condyle mineralization, as measured by micro-computed tomography and histologic analysis. In marked contrast, knee joints of Ddr2 mice were normal. Analysis of older Ddr2 mice (3 and 10 mo) revealed that the early developmental delay led to a dramatic and progressive loss of TMJ articular integrity and osteoarthritis-like changes. Mutant condyles had a rough and flattened bone surface, accompanied by a dramatic loss of bone mineral density. Mankin scores showed significantly greater degenerative changes in the TMJs of 3- and 10-mo-old Ddr2 mice as compared with wild-type controls. No DDR2-dependent degenerative changes were seen in knees. Analysis of primary cultures of TMJ articular chondrocytes from wild-type and Ddr2 mice showed defects in chondrocyte maturation and mineralization in the absence of Ddr2. These studies demonstrate that DDR2 is necessary for normal TMJ condyle development and homeostasis and that these DDR2 functions are restricted to TMJ fibrocartilage and not seen in the hyaline cartilage of the knee.

Hypophosphatasia (HPP) is caused by loss-of-function mutations in the ALPL gene that encodes tissue-nonspecific alkaline phosphatase (TNAP), whose deficiency results in the accumulation of extracellular inorganic pyrophosphate (PP i ), a potent mineralization inhibitor. Skeletal and dental hypomineralization characterizes HPP, with disease severity varying from life-threatening perinatal or infantile forms to milder forms that manifest in adulthood or only affect the dentition. Enzyme replacement therapy (ERT) using mineral-targeted recombinant TNAP (Strensiq/asfotase alfa) markedly improves the life span, skeletal phenotype, motor function, and quality of life of patients with HPP, though limitations of ERT include frequent injections due to a short elimination half-life of 2.28 days and injection site reactions. We tested the efficacy of a single intramuscular administration of adeno-associated virus 8 (AAV8) encoding TNAP-D 10 to increase the life span and improve the skeletal and dentoalveolar phenotypes in TNAP knockout (Alpl À/À ) mice, a murine model for severe infantile HPP. Alpl À/À mice received 3 Â 10 11 vector genomes/body of AAV8-TNAP-D 10 within 5 days postnatal (dpn). AAV8-TNAP-D 10 elevated serum ALP activity and suppressed plasma PP i . Treatment extended life span of Alpl À/À mice, and no ectopic calcifications were observed in the kidneys, aorta, coronary arteries, or brain in the 70 dpn observational window. Treated Alpl À/À mice did not show signs of rickets, including bowing of long bones, enlargement of epiphyses, or fractures. Bone microstructure of treated Alpl À/À mice was similar to wild type, with a few persistent small cortical and trabecular defects. Histology showed no measurable osteoid accumulation but reduced bone volume fraction in treated Alpl À/À mice versus controls. Treated Alpl À/À mice featured normal molar and incisor dentoalveolar tissues, with the exceptions of slightly reduced molar enamel and alveolar bone density. Histology showed the presence of cementum and normal periodontal ligament attachment. These results support gene therapy as a promising alternative to ERT for the treatment of HPP.