The effect of light compressive and tensile mechanical forces on SOST and POSTN expressions in human periodontal ligament cells: an in vitro study.

Periodontal ligament (PDL) cells play an important role in mechanosensing and secretion of signaling molecules during bone remodeling. However, the regulatory mechanism is unknown. The aim of the present study is to investigate the expression pattern of periostin and sclerostin in response to orthodontic forces in periodontal ligament cells in vitro. PDL cells were isolated from extracted teeth and treated with compressive forces of 25 gr/cm2 or equiaxial tension forces at frequency 1 Hz for 0, 24, 48, and 72 h. qRT-PCR was applied to evaluate the gene expressions. The secretion of sclerostin and periostin was assessed using ELISA. DAPI staining was used to evaluate apoptosis. The expression of sclerostin elevated significantly at protein and gene levels under compression forces after 24 h, while the application of tensile forces induced the expression of periostin and its upstream regulator RUNX2 (p < 0.05). Gene expression up-regulation was significant for POSTN and RUNX2 after 48 and 72 h tensile forces. Also, the gene expression of sclerostin reduced in a time-dependent manner after application of tensile force. The compression forces enhanced apoptosis to 7.5 ± 3.5% and induced gene expression of apoptotic markers of CASP9, and BCL2 within 72 h of exposure. Periostin and sclerostin play an important role in orthodontic loads and their expressions are affected oppositely by compressive and tensile forces that might be suggested as a biomarker for assessment of bone remodeling during orthodontic treatment.

Changes in Dickkopf-1, but Not Sclerostin, in Gingival Crevicular Fluid Are Associated with Peroral Statin Treatment in Patients with Periodontitis.

Background and Objectives: Periodontitis is marked by the destruction of alveolar bone. Sclerostin (SOST) and dickkopf-1 (DKK-1) act as inhibitors of the Wingless-type (Wnt) signaling pathway, a key regulator of bone metabolism. Recent studies have suggested that statins play a role in bone resorption and formation by influencing Wnt signaling. The aim of this study was to determine the levels of SOST and DKK-1 in periodontal patients with and without peroral statins treatment in their therapy. Materials and Methods: A total of 79 patients with diagnosed periodontitis were divided into two groups: 39 patients on statin therapy (SP group) and 40 patients without statin therapy as a control group (P group). The periodontal clinical examination probing (pocket) depth (PD) and gingival recession (GR) were measured, and approximal plaque was detected, while vertical and horizontal bone resorption was measured using a panoramic radiograph image. Clinical attachment loss (CAL) values were calculated using PD and GR values. Gingival crevicular fluid (GCF) was collected and used for measuring SOST and DKK-1 levels. A questionnaire was used to assess lifestyle habits and statin intake. Patients' medical records were used to obtain biochemical parameters. Results: There was no significant difference in sclerostin concentration between the SP and P group. DKK-1 values were significantly higher in the SP group compared to the control group (p = 0.04). Also, PD (p = 0.001) and GR (p = 0.03) were significantly higher in the SP group. The level of DKK-1 had a positive relationship with the PD, the greater the PD, the higher the level of DKK-1 (Rho = 0.350), while there was no significant association with other parameters. Conclusions: Peroral statins in periodontal patients are associated with GCF levels of DKK-1 but not with sclerostin levels.

Romosozumab in osteoporosis: yesterday, today and tomorrow.

Osteoporosis is a systemic bone disease characterized by low bone mass, microarchitectural deterioration, increased bone fragility, and fracture susceptibility. It commonly occurs in older people, especially postmenopausal women. As global ageing increases, osteoporosis has become a global burden. There are a number of medications available for the treatment of osteoporosis, categorized as anabolic and anti-resorptive. Unfortunately, there is no drugs which have dual influence on bone, while all drugs have limitations and adverse events. Some serious adverse events include jaw osteonecrosis and atypical femoral fracture. Recently, a novel medication has appeared that challenges this pattern. Romosozumab is a novel drug monoclonal antibody to sclerostin encoded by the SOST gene. It has been used in Japan since 2019 and has achieved promising results in treating osteoporosis. However, it is also accompanied by some controversy. While it promotes rapid bone growth, it may cause serious adverse events such as cardiovascular diseases. There has been scepticism about the drug since its inception. Therefore, the present review comprehensively covered romosozumab from its inception to its clinical application, from animal studies to human studies, and from safety to cost. We hope to provide a better understanding of romosozumab for its clinical application.

Sclerostin deficiency effectively promotes bone morphogenetic protein-2-induced ectopic bone formation.

BACKGROUND AND OBJECTIVE: Severe periodontitis causes alveolar bone resorption, resulting in tooth loss. Developments of tissue regeneration therapy that can restore alveolar bone mass are desired for periodontal disease. The application of bone morphogenetic protein-2 (BMP-2) has been attempted for bone fractures and severe alveolar bone loss. BMP-2 reportedly induces sclerostin expression, an inhibitor of Wnt signals, that attenuates bone acquisition. However, the effect of sclerostin-deficiency on BMP-2-induced bone regeneration has not been fully elucidated. We investigated BMP-2-induced ectopic bones in Sost-knockout (KO) mice. METHODS: rhBMP-2 were implanted into the thighs of C57BL/6 (WT) and Sost-KO male mice at 8 weeks of age. The BMP-2-induced ectopic bones in these mice were examined on days 14 and 28 after implantation. RESULTS: Immunohistochemical and quantitative RT-PCR analyses showed that BMP-2-induced ectopic bones expressed sclerostin in osteocytes on days 14 and 28 after implantation in Sost-Green reporter mice. Micro-computed tomography analysis revealed that BMP-2-induced ectopic bones in Sost-KO mice showed a significant increased relative bone volume and bone mineral density (WT = 468 mg/cm3 , Sost-KO = 602 mg/cm3 ) compared with those in WT mice on day 14 after implantation. BMP-2-induced ectopic bones in Sost-KO mice showed an increased horizontal cross-sectional bone area on day 28 after implantation. Immunohistochemical staining showed that BMP-2-induced ectopic bones in Sost-KO mice had an increased number of osteoblasts with osterix-positive nuclei compared with those in WT mice on days 14 and 28 after implantation. CONCLUSION: Sclerostin deficiency increased bone mineral density in BMP-2-induced ectopic bones.

Evaluating the relationship between ankylosing spondylitis and periodontal disease: a case-control study.

OBJECTIVES: This study aimed to determine the possible relationship between periodontal disease and ankylosing spondylitis (AS) by evaluating clinical periodontal measurements and gingival crevicular fluid (GCF) levels of sclerostin, interleukin-1ß (IL-1ß), and matrix metalloproteinase-8 (MMP-8) levels. MATERIALS AND METHODS: Twenty-eight patients with AS (AS group) and 28 systemically healthy controls (C group) were enrolled in this study. Full-mouth periodontal measurements: plaque index, bleeding on probing (BOP), probing pocket depth (PPD), and clinical attachment level (CAL) measurements were obtained from all patients. AS-related parameters were included in the data analyses. An enzyme-linked immunosorbent assay determined GCF IL-1ß, MMP-8, and sclerostin levels. RESULTS: There were no significant differences in the clinical periodontal measurements between the two groups (p > 0.05). Interestingly, patients with AS had significantly lower GCF sclerostin levels than the C group (p < 0.05). But there were no statistical differences in the GCF levels of IL-1ß and MMP-8 between the two groups (p > 0.05). Serum C-reactive protein (CRP) levels strongly correlated with both BOP (r = 0.497, p < 0.05) and PPD (r = 0.570, p < 0.05) in the AS group. Bath AS Metrology Index (BASMI) also positively correlated with both BOP (r = 0.530, p < 0.05) and CAL (r = 0.568, p < 0.05). Similarly, Maastricht Ankylosing Spondylitis Enthesis Score (MASES) strongly correlated with both BOP (r = 0.487, p < 0.05) and CAL (r = 0.522, p < 0.05). CONCLUSION: These results suggest that the patient's systemic condition may influence local sclerostin levels in GCF, and the strong correlations between periodontal measurements and AS-related parameters may indicate an interrelationship between inflammatory periodontal disease and AS. CLINICAL RELEVANCE: The present study provides important information concerning the relationship between periodontal disease and ankylosing spondylitis. TRIAL REGISTRATION: Thai Clinical Trials.gov (TCTR20200908001) (08. September 2020).

Sclerostin is a promising therapeutic target for oral inflammation and regenerative dentistry.

Sclerostin is the protein product of the SOST gene and is known for its inhibitory effects on bone formation. The monoclonal antibody against sclerostin has been approved as a novel treatment method for osteoporosis. Oral health is one of the essential aspects of general human health. Hereditary bone dysplasia syndrome caused by sclerostin deficiency is often accompanied by some dental malformations, inspiring the therapeutic exploration of sclerostin in the oral and dental fields. Recent studies have found that sclerostin is expressed in several functional cell types in oral tissues, and the expression level of sclerostin is altered in pathological conditions. Sclerostin not only exerts similar negative outcomes on the formation of alveolar bone and bone-like tissues, including dentin and cementum, but also participates in the development of oral inflammatory diseases such as periodontitis, pulpitis, and peri-implantitis. This review aims to highlight related research progress of sclerostin in oral cavity, propose necessary further research in this field, and discuss its potential as a therapeutic target for dental indications and regenerative dentistry.

Effects of periodontal and bisphosphonate treatment on the gingival crevicular levels of sclerostin and dickkopf-1 in postmenopausal osteoporosis with and without periodontitis.

OBJECTIVE AND BACKGROUND: Both periodontitis and osteoporosis are associated with osteoclast-related bone resorption. Bone metabolism is regulated by wingless-type MMTV integration site family (WNT), and WNT/ß-catenin signals are controlled by physiological antagonists including dickkopf-1 (DKK-1) and sclerostin (SOST). This study examined the effects of periodontal and bisphosphonate (BP) treatment on the gingival crevicular fluid (GCF) sclerostin (SOST) and dickkopf-related protein-1 (DKK-1) levels in osteoporotic and systemically healthy postmenopausal women with and without periodontitis. MATERIALS AND METHODS: A total of 48 postmenopausal women were divided into 4 groups (n = 12) according to periodontal health and osteoporosis status, as follows: Group OP/P: subjects with both osteoporosis and periodontitis; Group P: systemically healthy subjects with periodontitis; Group OP: periodontally healthy subjects with osteoporosis; Group H: systemically and periodontally healthy controls. Clinical data and GCF SOST and DKK-1 levels of the participants were collected at baseline and at 6 and 12 months following the initiation of periodontal and/or BP treatment in the experimental groups. GCF SOST and DKK-1 data were obtained by ELISA. RESULTS: Clinical improvements were observed in all experimental groups. GCF SOST and DKK1 baseline levels varied significantly between groups due to periodontal disease (p < .001). Following treatment, significant increases in SOST and DKK-1 concentrations and significant decreases in total amounts of SOST were observed in both periodontitis groups (OP/P, P). However, while total amounts of DKK-1 decreased in Group OP/P, in Group P, these amounts had significantly increased at 12 months post-treatment (p < .05). At both 6 and 12 months post-treatment, SOST and DDK1 total amounts in Groups OP/P, OP, and H were similar (p > .05), whereas significant differences were observed between Groups H and P, indicating a deviation from periodontal health in Group P (p < .01). CONCLUSIONS: Significant changes in GCF SOST and DKK-1 levels were observed among women with osteoporosis who received both periodontal and BP treatment. A more detailed examination of how these treatment protocols can be combined may lead to new therapeutic approaches towards periodontal disease.

Sclerostin in periodontal ligament: Homeostatic regulator in biophysical force-induced tooth movement.

AIM: To study the role of sclerostin in periodontal ligament (PDL) as a homeostatic regulator in biophysical-force-induced tooth movement (BFTM). MATERIALS AND METHODS: BFTM was performed in rats, followed by microarray, immunofluorescence, in situ hybridization, and real-time polymerase chain reaction for the detection and identification of the molecules. The periodontal space was analysed via micro-computed tomography. Effects on osteoclastogenesis and bone resorption were evaluated in the bone-marrow-derived cells in mice. In vitro human PDL cells were subjected to biophysical forces. RESULTS: In the absence of BFTM, sclerostin was hardly detected in the periodontium except in the PDL and alveolar bone in the furcation region and apex of the molar roots. However, sclerostin was up-regulated in the PDL in vivo by adaptable force, which induced typical transfiguration without changes in periodontal space as well as in vitro PDL cells under compression and tension. In contrast, the sclerostin level was unaffected by heavy force, which caused severe degeneration of the PDL and narrowed periodontal space. Sclerostin inhibited osteoclastogenesis and bone resorption, which corroborates the accelerated tooth movement by the heavy force. CONCLUSIONS: Sclerostin in PDL may be a key homeostatic molecule in the periodontium and a biological target for the therapeutic modulation of BFTM.

Osteocyte Dysfunction in Joint Homeostasis and Osteoarthritis.

Structural disturbances of the subchondral bone are a hallmark of osteoarthritis (OA), including sclerotic changes, cystic lesions, and osteophyte formation. Osteocytes act as mechanosensory units for the micro-cracks in response to mechanical loading. Once stimulated, osteocytes initiate the reparative process by recruiting bone-resorbing cells and bone-forming cells to maintain bone homeostasis. Osteocyte-expressed sclerostin is known as a negative regulator of bone formation through Wnt signaling and the RANKL pathway. In this review, we will summarize current understandings of osteocytes at the crossroad of allometry and mechanobiology to exploit the relationship between osteocyte morphology and function in the context of joint aging and osteoarthritis. We also aimed to summarize the osteocyte dysfunction and its link with structural and functional disturbances of the osteoarthritic subchondral bone at the molecular level. Compared with normal bones, the osteoarthritic subchondral bone is characterized by a higher bone volume fraction, a larger trabecular bone number in the load-bearing region, and an increase in thickness of pre-existing trabeculae. This may relate to the aberrant expressions of sclerostin, periostin, dentin matrix protein 1, matrix extracellular phosphoglycoprotein, insulin-like growth factor 1, and transforming growth factor-beta, among others. The number of osteocyte lacunae embedded in OA bone is also significantly higher, yet the volume of individual lacuna is relatively smaller, which could suggest abnormal metabolism in association with allometry. The remarkably lower percentage of sclerostin-positive osteocytes, together with clustering of Runx-2 positive pre-osteoblasts, may suggest altered regulation of osteoblast differentiation and osteoblast-osteocyte transformation affected by both signaling molecules and the extracellular matrix. Aberrant osteocyte morphology and function, along with anomalies in molecular signaling mechanisms, might explain in part, if not all, the pre-osteoblast clustering and the uncoupled bone remodeling in OA subchondral bone.

WNT-5a and SOST Levels in Gingival Crevicular Fluid Depend on the Inflammatory and Osteoclastogenic Activities of Periodontal Tissues.

Background and Objectives: Wnt signaling leads to stimulation of osteoblasts and it reduces osteoclastogenesis and bone resorption via the regulation of the osteprotegrin and receptor activator of nuclear factor kappa-Β ligan (RANKL). Wnt signaling pathways are regulated by their physiological antagonists such as sclerostin (SOST) as well as WNT-5a. The aim of this study was to determine the total amount of Sclerostin and WNT-5a in the gingival crevicular fluid (GCF) in sites with a continuum from a healthy to diseased periodontium. Materials and Methods: In this cross-sectional study, a total of 20 patients with generalized periodontitis, 10 subjects with gingivitis as well as 14 individuals with a healthy periodontium were recruited upon clinical and radiographic periodontal examination. In patients diagnosed with periodontitis, GCF samples were collected from periodontitis, gingivitis and healthy sites, while gingivitis patients provided samples from gingivitis and healthy sites. In healthy patients, only healthy sites were sampled. Protein total amount of SOST and WNT-5a were quantified by sandwich enzyme-linked immunosorbent assay (ELISA). Results: A total of 108 GCF samples were collected from a total of 44 individuals. When all periodontitis (n = 51), gingivitis (n = 12) and healthy (n = 45) sites were analyzed regardless of the patient diagnosis, periodontitis sites demonstrated significantly elevated WNT-5a total amounts (p = 0.03) when compared to gingivitis sites. Gingivitis sites demonstrated a trend of more total SOST (p = 0.09) when compared to periodontitis and healthy sites. Within each patient diagnostic category, sites showed similar SOST and WNT-5a total amounts (p > 0.05). Conclusions: WNT-5a levels in GCF depend on the stage of periodontitis sites. SOST trended higher in the GCF of gingivitis sites but similar in chronic periodontitis and healthy sites. WNT-5a and SOST play a crucial role in periodontal tissue remodeling and depend on the inflammatory and osteoclastogenic activities.

The roles of osteocytes in alveolar bone destruction in periodontitis.

Periodontitis, a bacterium-induced inflammatory disease that is characterized by alveolar bone loss, is highly prevalent worldwide. Elucidating the underlying mechanisms of alveolar bone loss in periodontitis is crucial for understanding its pathogenesis. Classically, bone cells, such as osteoclasts, osteoblasts and bone marrow stromal cells, are thought to dominate the development of bone destruction in periodontitis. Recently, osteocytes, the cells embedded in the mineral matrix, have gained attention. This review demonstrates the key contributing role of osteocytes in periodontitis, especially in alveolar bone loss. Osteocytes not only initiate physiological bone remodeling but also assist in inflammation-related changes in bone remodeling. The latest evidence suggests that osteocytes are involved in regulating bone anabolism and catabolism in the progression of periodontitis. The altered secretion of receptor activator of NF-κB ligand (RANKL), sclerostin and Dickkopf-related protein 1 (DKK1) by osteocytes affects the balance of bone resorption and formation and promotes bone loss. In addition, the accumulation of prematurely senescent and apoptotic osteocytes observed in alveolar bone may exacerbate local destruction. Based on their communication with the bloodstream, it is noteworthy that osteocytes may participate in the interaction between local periodontitis lesions and systemic diseases. Overall, further investigations of osteocytes may provide vital insights that improve our understanding of the pathophysiology of periodontitis.

Role of intracellular Ca2+-based mechanotransduction of human periodontal ligament fibroblasts.

Human periodontal ligament (hPDL) fibroblasts are thought to receive mechanical stress (MS) produced by orthodontic tooth movement, thereby regulating alveolar bone remodeling. However, the role of intracellular calcium ([Ca2+]i)-based mechanotransduction is not fully understood. We explored the MS-induced [Ca2+]i responses both in isolated hPDL fibroblasts and in intact hPDL tissue and investigated its possible role in alveolar bone remodeling. hPDL fibroblasts were obtained from healthy donors' premolars that had been extracted for orthodontic reasons. The oscillatory [Ca2+]i activity induced by static compressive force was measured by a live-cell Ca2+ imaging system and evaluated by several feature extraction method. The spatial pattern of cell-cell communication was investigated by Moran's I, an index of spatial autocorrelation and the gap junction (GJ) inhibitor. The Ca2+-transporting ionophore A23187 was used to further investigate the role of [Ca2+]i up-regulation in hPDL cell behavior. hPDL fibroblasts displayed autonomous [Ca2+]i responses. Compressive MS activated this autonomous responsive behavior with an increased percentage of responsive cells both in vitro and ex vivo. The integration, variance, maximum amplitude, waveform length, and index J in the [Ca2+]i responses were also significantly increased, whereas the mean power frequency was attenuated in response to MS. The increased Moran's I after MS indicated that MS might affect the pattern of cell-cell communication via GJs. Similar to the findings of MS-mediated regulation, the A23187-mediated [Ca2+]i uptake resulted in the up-regulation of receptor activator of NF-κB ligand (Rankl) and Sost along with increased sclerostin immunoreactivity, suggesting that [Ca2+]i signaling networks may be involved in bone remodeling. In addition, A23187-treated hPDL fibroblasts also showed the suppression of osteogenic differentiation and mineralization. Our findings suggest that augmented MS-mediated [Ca2+]i oscillations in hPDL fibroblasts enhance the production and release of bone regulatory signals via Rankl/Osteoprotegerin and the canonical Wnt/ß-catenin pathway as an early process in tooth movement-initiated alveolar bone remodeling.-Ei Hsu Hlaing, E., Ishihara, Y., Wang, Z., Odagaki, N., Kamioka, H. Role of intracellular Ca2+-based mechanotransduction of human periodontal ligament fibroblasts.

Sclerostin inhibits odontogenic differentiation of human pulp-derived odontoblast-like cells under mechanical stress.

Sclerotic dentin is a natural self-protective barrier beneath non-carious cervical lesions (NCCLs), which are mainly induced by mechanical stress. Sclerostin is a mechanosensory protein and serves as an inhibitor of dentinogenesis. However, its function on mechanotransduction in dentine-pulp complex has not been elucidated yet. In this study, decreased sclerostin expression was detected in odontoblasts beneath NCCL-affected sclerotic dentin. Then human pulp-derived odontoblast-like cells (hOBs) were subjected to mechanical strain (MS) in vitro: the results showed that MS-induced upregulation of odontogenic differentiation markers (dentin sialophosphoprotein, osteopontin, osteocalcin, and runt-related transcription factor 2) in hOBs with downregulated sclerostin expression, and this inductive differentiation was attenuated when sclerostin was overexpressed. Additionally, MS activated ERK1/2 pathway and ERK1/2 inhibition restored MS-induced downregulation of sclerostin. Proteasome inhibitor MG132 could also rescue MS-induced decrease of sclerostin. Furthermore, MS suppressed STAT3 pathway, which could be reversed by sclerostin overexpression. STAT3 inhibition was shown to ameliorate the reduction of odontogenic markers induced by sclerostin overexpression. Taken together, we conclude that MS downregulates sclerostin expression via the ERK1/2 and proteasome signaling pathways to promote odontogenic differentiation of hOBs through the STAT3 signaling pathway. It can therefore be inferred that under mechanical stress, sclerostin inhibition promotes reactive dentin formation by enhancing odontogenic differentiation of odontoblasts, which might be one of potential forming mechanisms of sclerotic dentin beneath NCCLs.

Carbonic anhydrase III protects osteocytes from oxidative stress.

Osteocytes are master orchestrators of bone remodeling; they control osteoblast and osteoclast activities both directly via cell-to-cell communication and indirectly via secreted factors, and they are the main postnatal source of sclerostin and RANKL (receptor activator of NF-kB ligand), two regulators of osteoblast and osteoclast function. Despite progress in understanding osteocyte biology and function, much remains to be elucidated. Recently developed osteocytic cell lines-together with new genome editing tools-has allowed a closer look at the biology and molecular makeup of these cells. By using single-cell cloning, we identified genes that are associated with high Sost/sclerostin expression and analyzed their regulation and function. Unbiased transcriptome analysis of high- vs. low-Sost/sclerostin-expressing cells identified known and novel genes. Dmp1 (dentin matrix protein 1), Dkk1 (Dickkopf WNT signaling pathway inhibitor 1), and Phex were among the most up-regulated known genes, whereas Srpx2, Cd200, and carbonic anhydrase III (CAIII) were identified as novel markers of differentiated osteocytes. Aspn, Enpp2, Robo2, Nov, and Serpina3g were among the transcripts that were most significantly suppressed in high-Sost cells. Considering that CAII was recently identified as being regulated by Sost/sclerostin and capable of controlling mineral homeostasis, we focused our attention on CAIII. Here, we report that CAIII is highly expressed in osteocytes, is regulated by parathyroid hormone both in vitro and in vivo, and protects osteocytes from oxidative stress.-Shi, C., Uda, Y., Dedic, C., Azab, E., Sun, N., Hussein, A. I., Petty, C. A., Fulzele, K., Mitterberger-Vogt, M. C., Zwerschke, W., Pereira, R., Wang, K., Divieti Pajevic, P. Carbonic anhydrase III protects osteocytes from oxidative stress.

Metformin protects bone mass in ultra-high-molecular-weight polyethylene particle-induced osteolysis by regulating osteocyte secretion.

Metformin, an anti-hyperglycemic agent used for type 2 diabetes, has recently been found to have more effects apart from glucose regulation. We found that, in ultra-high-molecular-weight polyethylene particle-induced osteolysis mouse models, metformin had bone protect property and reduced the negative regulator of bone formation sclerostin (SOST) and Dickkopf-related protein 1 (DKK1), and increased osteoprotegerin (OPG) secretion and the ratio of OPG/Receptor Activator for Nuclear Factor-κB Ligand (RANKL). In vitro, we established a 3D co-culture system in which metformin affects osteoblasts and osteoclasts through mature osteocytes secretion. Metformin (50 µM) significantly decreased SOST and DKK1 mRNA expression, stimulating alkaline phosphatase activity and proliferation of osteoblast, and increased OPG secretion and the ratio of OPG/RANKL, inhibiting osteoclastogenesis. Moreover, the effect on OPG was reversed by adenosine 5'-monophosphate-activated protein kinase inhibitor, Compound C. Our finding suggests that metformin induces differentiation and mineralization of osteoblasts, while inhibits osteoclastogenesis via mature osteocytes secretion. Therefore, the drug might be beneficial for not only diabetes but also in other bone disorders by acting on mature osteocytes.

Pigment epithelium-derived factor (PEDF) reduced expression and synthesis of SOST/sclerostin in bone explant cultures: implication of PEDF-osteocyte gene regulation in vivo.

Mutations in Serpinf1 gene which encodes pigment epithelium-derived factor (PEDF) lead to osteogenesis imperfecta type VI whose hallmark is defective matrix mineralization. We reported previously that PEDF reduced expression and synthesis of Sost/Sclerostin as well as other osteocytes genes encoding proteins that regulate matrix mineralization [1]. To determine whether PEDF had an effect on osteocyte gene expression in bone, we used bone explant cultures. First, osteocytes were isolated from surgical waste of bone fragments obtained from patients undergoing elective foot surgeries under approved IRB protocol by Penn State College of Medicine IRB committee. Primary osteocytes treated with PEDF reduced expression and synthesis of Sost/Sclerostin and matrix phosphoglycoprotein (MEPE) as well as dentin matrix protein (DMP-1). On the whole, PEDF reduced osteocyte protein synthesis by 50% and by 75% on mRNA levels. For bone explants, following collagenase digestion, bone fragments were incubated in alpha-MEM supplemented with 250 ng/ml of PEDF or BSA. After 7 days of incubation in a medium supplemented with PEDF, analysis of mRNA by PCR and protein by western blotting of encoded osteocyte proteins showed reduced Sclerostin synthesis by 39% and MEPE by 27% when compared to fragments incubated in medium supplemented with BSA. mRNA expression levels of osteocytes in bone fragments treated with PEDF were reduced by 50% for both SOST and MEPE when compared to BSA-treated bone fragments. Taken together, the data indicate that PEDF has an effect on osteocyte gene expression in bone and encourage further studies to examine effect of PEDF on bone formation indices in animal models and its effect on osteocyte gene expression in vivo following PEDF administration.

Sclerostin and WNT-5a gingival protein levels in chronic periodontitis and health.

BACKGROUND AND OBJECTIVE: Wnt signaling pathways regulate osteoblast differentiation and bone formation and are associated with inflammatory responses driven by innate and adaptive immunity via the NF-κB pathway. The aim of this study was to compare the levels of sclerostin (SOST), WNT-5a, and TNF-α between chronic periodontitis and periodontally healthy sites and determine their value as diagnostic markers of chronic periodontitis. MATERIAL AND METHODS: In a cross-sectional assessment 25 chronic periodontitis cases and 25 periodontally healthy controls were selected upon clinical and radiographic periodontal evaluation. Gingival crevicular fluid (GCF) was collected cross-sectionally from diseased and healthy sites in periodontitis patients and from healthy sites in each control subject. In a subgroup analysis, ten patients with generalized moderate and severe chronic periodontitis and ten generalized periodontally healthy individuals were included. The protein levels of SOST, WNT-5a, and TNF-α in GCF were measured by sandwich ELISA. The Shapiro-Wilk test was utilized to assess the normality of the distribution and non-parametric comparisons were performed. RESULTS: The protein levels of SOST were significantly higher in the generalized moderate and severe chronic periodontitis subgroup when compared to the generalized healthy (P = 0.002), while the WNT-5a and TNF-α GCF total amounts were similar (P > 0.05). Diseased sites in the periodontitis patients exhibited significantly higher total protein levels of WNT-5a than in healthy sites (P = 0.017), whereas no differences were detected for SOST and TNF-α (P > 0.05). The total protein levels of SOST, WNT-5a, and TNF-α in GCF were similar in periodontitis and non-periodontitis patients (P > 0.05). CONCLUSIONS: Sclerostin and WNT-5a gingival protein levels demonstrated a high diagnostic value for generalized moderate and severe chronic periodontitis, while a low accuracy was detected for localized chronic periodontitis.

The Osteocyte as a Novel Key Player in Understanding Periodontitis Through its Expression of RANKL and Sclerostin: a Review.

PURPOSE OF REVIEW: Periodontitis is the inflammation-associated bone loss disease of the alveolar bone that surrounds teeth. Classically, the emphasis on the etiology of periodontitis has been on the products of periodontal pathogens that lead to an inflammatory response of the soft tissues of the periodontium, eventually leading to activation of osteoclasts that degrade the alveolar bone. Until recently, the response of osteocytes that populate the alveolar bone, and that are known for their regulatory role in bone anabolism and catabolism, has not been addressed. RECENT FINDINGS: This review demonstrates that osteocytes play a key contributing role in periodontitis progression in various experimental mouse and rat periodontitis models. Osteocytes are the key expressing cells of both osteoclast differentiation factor RANKL as well as osteoblast activity regulator sclerostin. Targeted deletion of RANKL in osteocytes prevents osteoclast formation, thereby impairing periodontitis, despite the pressure of periodontitis-associated bacteria. Antibodies against the osteocyte-derived protein sclerostin inhibit and partially revert periodontitis by stimulating bone formation. Experimental mouse and rat periodontitis models strongly indicate a key role for the bone-encapsulated osteocyte in understanding periodontitis etiology.

Osteocytes and mechanical loading: The Wnt connection.

Bone adapts to the mechanical forces that it experiences. Orthodontic tooth movement harnesses the cell- and tissue-level properties of mechanotransduction to achieve alignment and reorganization of the dentition. However, the mechanisms of action that permit bone resorption and formation in response to loads placed on the teeth are incompletely elucidated, though several mechanisms have been identified. Wnt/Lrp5 signalling in osteocytes is a key pathway that modulates bone tissue's response to load. Numerous mouse models that harbour knock-in, knockout and transgenic/overexpression alleles targeting genes related to Wnt signalling point to the necessity of Wnt/Lrp5, and its localization to osteocytes, for proper mechanotransduction in bone. Alveolar bone is rich in osteocytes and is a highly mechanoresponsive tissue in which components of the canonical Wnt signalling cascade have been identified. As Wnt-based agents become clinically available in the next several years, the major challenge that lies ahead will be to gain a more complete understanding of Wnt biology in alveolar bone so that improved/expedited tooth movement becomes a possibility.

Simvastatin attenuates tibial bone loss in rats with type 1 diabetes and periodontitis.

BACKGROUND: Diabetes induces long bone loss and aggravation of periodontitis-induced alveolar bone loss. Simvastatin (SIM), which is a lipid-lowering agent is known to have an anabolic effect on bone. Therefore, we investigated effect of SIM on tibial and alveolar bone loss in type 1 diabetic rats with periodontitis. METHODS: Rats were divided into control (C), diabetes with periodontitis (DP), and diabetes with periodontitis treated with SIM (DPS) groups. DP and DPS groups were intravenously injected with streptozotocin (50 mg/kg), and C group was injected with citrate buffer. Seven days later (day 0), periodontitis was induced by ligatures of mandibular first molars. DP and DPS groups were orally administered vehicle or SIM (30 mg/kg) from day 0 to days 3, 10, or 20. Alveolar and tibial bone loss was measured using histological and m-CT analysis alone or in combination. Osteoclast number and sclerostin-positive osteocytes in tibiae were evaluated by tartrate-resistant acid phosphatase and immunohistochemical staining, respectively. Glucose, triglyceride (TG), cholesterol (CHO), and low-density lipoprotein (LDL) were evaluated. RESULTS: Consistent with diabetes induction, the DP group showed higher glucose and TG levels at all timepoints and higher CHO levels on day 20 than C group. Compared to the DP group, the DPS group exhibited reduced levels of glucose (day 3), TG (days 10 and 20), CHO, and LDL levels (day 20). Bone loss analysis revealed that the DP group had lower bone volume fraction, bone mineral density, bone surface density, and trabecular number in tibiae than C group at all timepoints. Interestingly, the DPS group exhibited elevation of these indices at early stages compared to the DP group. The DPS group showed reduction of osteoclasts (day 3) and sclerostin-positive osteocytes (days 3 and 20) compared with the DP group. There was no difference in alveolar bone loss between DP and DPS groups. CONCLUSIONS: These results suggest that SIM attenuates tibial, but not alveolar bone loss in type 1 diabetic rats with periodontitis. Moreover, attenuation of tibial bone loss by SIM may be related to inhibition of osteoclast formation and reduction of sclerostin expression.