Osteogenic differentiation of periodontal ligament fibroblasts inhibits osteoclast formation.

One of the deficits of knowledge on bone remodelling, is to what extent cells that are driven towards osteogenic differentiation can contribute to osteoclast formation. The periodontal ligament fibroblast (PdLFs) is an ideal model to study this, since they play a role in osteogenesis, and can also orchestrate osteoclastogenesis.when co-cultured with a source of osteoclast-precursor such as peripheral blood mononuclear cells (PBMCs). Here, the osteogenic differentiation of PdLFs and the effects of this process on the formation of osteoclasts were investigated. PdLFs were obtained from extracted teeth and exposed to osteogenic medium for 0, 7, 14, or 21 out of 21 days. After this 21-day culturing period, the cells were co-cultured with peripheral blood mononuclear cells (PBMCs) for an additional 21 days to study osteoclast formation. Alkaline phosphatase (ALP) activity, calcium concentration, and gene expression of osteogenic markers were assessed at day 21 to evaluate the different stages of osteogenic differentiation. Alizarin red staining and scanning electron microscopy were used to visualise mineralisation. Tartrate-resistant acid phosphatase (TRAcP) activity, TRAcP staining, multinuclearity, the expression of osteoclastogenesis-related genes, and TNF-α and IL-1ß protein levels were assessed to evaluate osteoclastogenesis. The osteogenesis assays revealed that PdLFs became more differentiated as they were exposed to osteogenic medium for a longer period of time. Mineralisation by these osteogenic cells increased with the progression of differentiation. Culturing PdLFs in osteogenic medium before co-culturing them with PMBCs led to a significant decrease in osteoclast formation. qPCR revealed significantly lower DCSTAMP expression in cultures that had been supplemented with osteogenic medium. Protein levels of osteoclastogenesis stimulator TNF-α were also lower in these cultures. The present study shows that the osteogenic differentiation of PdLFs reduces the osteoclastogenic potential of these cells. Immature cells of the osteoblastic lineage may facilitate osteoclastogenesis, whereas mature mineralising cells may suppress the formation of osteoclasts. Therefore, mature and immature osteogenic cells may have different roles in maintaining bone homeostasis.

The Effect of Sclerostin and Monoclonal Sclerostin Antibody Romosozumab on Osteogenesis and Osteoclastogenesis Mediated by Periodontal Ligament Fibroblasts.

Sclerostin is a bone formation inhibitor produced by osteocytes. Although sclerostin is mainly expressed in osteocytes, it was also reported in periodontal ligament (PDL) fibroblasts, which are cells that play a role in both osteogenesis and osteoclastogenesis. Here, we assess the role of sclerostin and its clinically used inhibitor, romosozumab, in both processes. For osteogenesis assays, human PDL fibroblasts were cultured under control or mineralizing conditions with increasing concentrations of sclerostin or romosozumab. For analyzing osteogenic capacity and alkaline phosphatase (ALP) activity, alizarin red staining for mineral deposition and qPCR of osteogenic markers were performed. Osteoclast formation was investigated in the presence of sclerostin or romosozumab and, in PDLs, in the presence of fibroblasts co-cultured with peripheral blood mononuclear cells (PBMCs). PDL-PBMC co-cultures stimulated with sclerostin did not affect osteoclast formation. In contrast, the addition of romosozumab slightly reduced the osteoclast formation in PDL-PBMC co-cultures at high concentrations. Neither sclerostin nor romosozumab affected the osteogenic capacity of PDL fibroblasts. qPCR analysis showed that the mineralization medium upregulated the relative expression of osteogenic markers, but this expression was barely affected when romosozumab was added to the cultures. In order to account for the limited effects of sclerostin or romosozumab, we finally compared the expression of SOST and its receptors LRP-4, -5, and -6 to the expression in osteocyte rich-bone. The expression of SOST, LRP-4, and LRP-5 was higher in osteocytes compared to in PDL cells. The limited interaction of sclerostin or romosozumab with PDL fibroblasts may relate to the primary biological function of the periodontal ligament: to primarily resist bone formation and bone degradation to the benefit of an intact ligament that is indented by every chew movement.

Compressed Prostate Cancer Cells Decrease Osteoclast Activity While Enhancing Osteoblast Activity In Vitro.

Once prostate cancer cells metastasize to bone, they perceive approximately 2 kPa compression. We hypothesize that 2 kPa compression stimulates the epithelial-to-mesenchymal transition (EMT) of prostate cancer cells and alters their production of paracrine signals to affect osteoclast and osteoblast behavior. Human DU145 prostate cancer cells were subjected to 2 kPa compression for 2 days. Compression decreased expression of 2 epithelial genes, 5 out of 13 mesenchymal genes, and increased 2 mesenchymal genes by DU145 cells, as quantified by qPCR. Conditioned medium (CM) of DU145 cells was added to human monocytes that were stimulated to differentiate into osteoclasts for 21 days. CM from compressed DU145 cells decreased osteoclast resorptive activity by 38% but did not affect osteoclast size and number compared to CM from non-compressed cells. CM was also added to human adipose stromal cells, grown in osteogenic medium. CM of compressed DU145 cells increased bone nodule production (Alizarin Red) by osteoblasts from four out of six donors. Compression did not affect IL6 or TNF-α production by PC DU145 cells. Our data suggest that compression affects EMT-related gene expression in DU145 cells, and alters their production of paracrine signals to decrease osteoclast resorptive activity while increasing mineralization by osteoblasts is donor dependent. This observation gives further insight in the altered behavior of PC cells upon mechanical stimuli, which could provide novel leads for therapies, preventing bone metastases.

Shear loaded osteocyte-like-cells affect epithelial and mesenchymal gene expression in DU145 prostate cancer cells, while decreasing their invasion in vitro.

Once prostate cancer (PC) metastasizes towards bone the 5-year survival rates drop with 70%, but it is largely unknown why. Bone is continuously mechanically loaded, which likely modulates the paracrine signaling from osteocytes towards PC cells to affect tumor behavior. We hypothesize that shear loaded osteocytes affect PC cell proliferation, invasion and epithelial and mesenchymal-related gene and protein expression. We cultured human DU145 cells, a commonly used cell line for prostate cancer metastases, in the conditioned medium (CM) from shear loaded or unloaded human osteocyte-like-cells (OCYLCs) for 1 and 3 days and assessed their number by staining nuclei with DAPI, their invasion by performing an invasion assay, and epithelial-to-mesenchymal (EMT)-related gene and protein expression by qPCR and immunocytochemistry. CM of shear loaded OCYLCs did not affect DU145 cell number compared to CM of static cultured OCYLCs, but decreased their invasion 1.34-fold. CM of shear loaded OCYLCs enhanced expression of epithelial genes: SYND1 and CDH1 after day 1, while it also enhanced CDH1 after day 3. CM of shear loaded osteocytes enhanced mesenchymal genes: VMN, Snail and MIP2 after day 1, while it decreased expression of mesenchymal CYR61 after day 3. We conclude that CM of shear loaded OCYLCs does not affect DU145 cell proliferation, but decreases their invasion, and differentially affects their EMT-related gene expression. Identifying paracrine signals from shear loaded osteocytes that decrease PC cell invasion may provide novel leads in developing treatments for bone metastases from PC.

Biologically Relevant In Vitro 3D-Model to Study Bone Regeneration Potential of Human Adipose Stem Cells.

Standard cell cultures may not predict the proliferation and differentiation potential of human mesenchymal stromal cells (MSCs) after seeding on a scaffold and implanting this construct in a bone defect. We aimed to develop a more biologically relevant in vitro 3D-model for preclinical studies on the bone regeneration potential of MSCs. Human adipose tissue-derived mesenchymal stromal cells (hASCs; five donors) were seeded on biphasic calcium phosphate (BCP) granules and cultured under hypoxia (1% O2) for 14 days with pro-inflammatory TNFα, IL4, IL6, and IL17F (10 mg/mL each) added during the first three days, simulating the early stages of repair (bone construct model). Alternatively, hASCs were cultured on plastic, under 20% O2 and without cytokines for 14 days (standard cell culture). After two days, the bone construct model decreased total DNA (3.9-fold), COL1 (9.8-fold), and RUNX2 expression (19.6-fold) and metabolic activity (4.6-fold), but increased VEGF165 expression (38.6-fold) in hASCs compared to standard cultures. After seven days, the bone construct model decreased RUNX2 expression (64-fold) and metabolic activity (2.3-fold), but increased VEGF165 (54.5-fold) and KI67 expression (5.7-fold) in hASCs compared to standard cultures. The effect of the bone construct model on hASC proliferation and metabolic activity could be largely mimicked by culturing on BCP alone (20% O2, no cytokines). The effect of the bone construct model on VEGF165 expression could be mimicked by culturing hASCs under hypoxia alone (plastic, no cytokines). In conclusion, we developed a new, biologically relevant in vitro 3D-model to study the bone regeneration potential of MSCs. Our model is likely more suitable for the screening of novel factors to enhance bone regeneration than standard cell cultures.

K-Carrageenan Stimulates Pre-Osteoblast Proliferation and Osteogenic Differentiation: A Potential Factor for the Promotion of Bone Regeneration?

Current cell-based bone tissue regeneration strategies cannot cover large bone defects. K-carrageenan is a highly hydrophilic and biocompatible seaweed-derived sulfated polysaccharide, that has been proposed as a promising candidate for tissue engineering applications. Whether κ-carrageenan can be used to enhance bone regeneration is still unclear. In this study, we aimed to investigate whether κ-carrageenan has osteogenic potential by testing its effect on pre-osteoblast proliferation and osteogenic differentiation in vitro. Treatment with κ-carrageenan (0.5 and 2 mg/mL) increased both MC3T3-E1 pre-osteoblast adhesion and spreading at 1 h. K-carrageenan (0.125-2 mg/mL) dose-dependently increased pre-osteoblast proliferation and metabolic activity, with a maximum effect at 2 mg/mL at day three. K-carrageenan (0.5 and 2 mg/mL) increased osteogenic differentiation, as shown by enhanced alkaline phosphatase activity (1.8-fold increase at 2 mg/mL) at day four, and matrix mineralization (6.2-fold increase at 2 mg/mL) at day 21. K-carrageenan enhanced osteogenic gene expression (Opn, Dmp1, and Mepe) at day 14 and 21. In conclusion, κ-carrageenan promoted MC3T3-E1 pre-osteoblast adhesion and spreading, metabolic activity, proliferation, and osteogenic differentiation, suggesting that κ-carrageenan is a potential osteogenic inductive factor for clinical application to enhance bone regeneration.

The novel endolysin XZ.700 effectively treats MRSA biofilms in two biofilm models without showing toxicity on human bone cells in vitro.

In this in vitro study the effect of XZ.700, a new endolysin, on methicillin resistant Staphylococcus aureus (MRSA) biofilms grown on titanium was evaluated. Biofilms of S. aureus USA300 were grown statically and under flow, and treatment with XZ.700 was compared with povidone-iodine (PVP-I) and gentamicin. To evaluate the cytotoxic effects of XZ.700 and derived biofilm lysates, human osteocyte-like cells were exposed to biofilm supernatants, and metabolism and proliferation were quantified. XZ.700 showed a significant, concentration dependent reduction in biofilm viability, compared with carrier controls. Metabolism and proliferation of human osteocyte-like cells were not affected by XZ.700 or lysates, unlike PVP-I and gentamicin lysates which significantly inhibited proliferation. Using time-lapse microscopy, rapid biofilm killing and removal was observed for XZ.700. In comparison, PVP-I and gentamicin showed slower biofilm killing, with no apparent biofilm removal. In conclusion, XZ.700 reduced MRSA biofilms, especially under flow condition, without toxicity for surrounding bone cells.

Diabetes Medication Metformin Inhibits Osteoclast Formation and Activity in In Vitro Models for Periodontitis.

Diabetes and periodontitis are comorbidities and may share common pathways. Several reports indicate that diabetes medication metformin may be beneficial for the periodontal status of periodontitis patients. Further research using appropriate cell systems of the periodontium, the tissue that surrounds teeth may reveal the possible mechanism. Periodontal ligament fibroblasts anchor teeth in bone and play a role in the onset of both alveolar bone formation and degradation, the latter by inducing osteoclast formation from adherent precursor cells. Therefore, a cell model including this type of cells is ideal to study the influence of metformin on both processes. We hypothesize that metformin will enhance bone formation, as described for osteoblasts, whereas the effects of metformin on osteoclast formation is yet undetermined. Periodontal ligament fibroblasts were cultured in the presence of osteogenic medium and 0.2 or 1 mM metformin. The influence of metformin on osteoclast formation was first studied in PDLF cultures supplemented with peripheral blood leukocytes, containing osteoclast precursors. Finally, the effect of metformin on osteoclast precursors was studied in cultures of CD14+ monocytes that were stimulated with M-CSF and receptor activator of Nf-κB ligand (RANKL). No effects of metformin were observed on osteogenesis: not on alkaline phosphatase activity, Alizarin red deposition, nor on the expression of osteogenic markers RUNX-2, Collagen I and Osteonectin. Metformin inhibited osteoclast formation and accordingly downregulated the genes involved in osteoclastogenesis: RANKL, macrophage colony stimulating factor (M-CSF) and osteoclast fusion gene DC-STAMP. Osteoclast formation on both plastic and bone as well as bone resorption was inhibited by metformin in M-CSF and RANKL stimulated monocyte cultures, probably by reduction of RANK expression. The present study unraveling the positive effect of metformin in periodontitis patients at the cellular level, indicates that metformin inhibits osteoclast formation and activity, both when orchestrated by periodontal ligament fibroblasts and in cytokine driven osteoclast formation assays. The results indicate that metformin could have a systemic beneficiary effect on bone by inhibiting osteoclast formation and activity.

LAMP-2 Is Involved in Surface Expression of RANKL of Osteoblasts In Vitro.

Lysosome associated membrane proteins (LAMPs) are involved in several processes, among which is fusion of lysosomes with phagosomes. For the formation of multinucleated osteoclasts, the interaction between receptor activator of nuclear kappa ß (RANK) and its ligand RANKL is essential. Osteoclast precursors express RANK on their membrane and RANKL is expressed by cells of the osteoblast lineage. Recently it has been suggested that the transport of RANKL to the plasma membrane is mediated by lysosomal organelles. We wondered whether LAMP-2 might play a role in transportation of RANKL to the plasma membrane of osteoblasts. To elucidate the possible function of LAMP-2 herein and in the formation of osteoclasts, we analyzed these processes in vivo and in vitro using LAMP-2-deficient mice. We found that, in the presence of macrophage colony stimulating factor (M-CSF) and RANKL, active osteoclasts were formed using bone marrow cells from calvaria and long bone mouse bone marrow. Surprisingly, an almost complete absence of osteoclast formation was found when osteoclast precursors were co-cultured with LAMP-2 deficient osteoblasts. Fluorescence-activated cell sorting FACS analysis revealed that plasma membrane-bound RANKL was strongly decreased on LAMP-2 deficient osteoblasts. These results suggest that osteoblastic LAMP-2 is required for osteoblast-induced osteoclast formation in vitro.

Chronic Exposure of Gingival Fibroblasts to TLR2 or TLR4 Agonist Inhibits Osteoclastogenesis but Does Not Affect Osteogenesis.

Chronic exposure to periodontopathogenic bacteria such as Porphyromonas gingivalis and the products of these bacteria that interact with the cells of the tooth surrounding tissues can ultimately result in periodontitis. This is a disease that is characterized by inflammation-related alveolar bone degradation by the bone-resorbing cells, the osteoclasts. Interactions of bacterial products with Toll-like receptors (TLRs), in particular TLR2 and TLR4, play a significant role in this chronic inflammatory reaction, which possibly affects osteoclastic activity and osteogenic capacity. Little is known about how chronic exposure to specific TLR activators affects these two antagonistic activities. Here, we studied the effect of TLR activation on gingival fibroblasts (GF), cells that are anatomically close to infiltrating bacterial products in the mouth. These were co-cultured with naive osteoclast precursor cells (i.e., monocytes), as part of the peripheral blood mononuclear cells (PBMCs). Activation of GF co-cultures (GF + PBMCs) with TLR2 or TLR4 agonists resulted in a weak reduction of the osteoclastogenic potential of these cultures, predominantly due to TLR2. Interestingly, chronic exposure, especially to TLR2 agonist, resulted in increased release of TNF-α at early time points. This effect, was reversed at later time points, thus suggesting an adaptation to chronic exposure. Monocyte cultures primed with M-CSF + RANKL, led to the formation of bone-resorbing osteoclasts, irrespective of being activated with TLR agonists. Late activation of these co-cultures with TLR2 and with TLR4 agonists led to a slight decrease in bone resorption. Activation of GF with TLR2 and TLR4 agonists did not affect the osteogenic capacity of the GF cells. In conclusion, chronic exposure leads to diverse reactions; inhibitory with naive osteoclast precursors, not effecting already formed (pre-)osteoclasts. We suggest that early encounter of naive monocytes with TLR agonists may result in differentiation toward the macrophage lineage, desirable for clearing bacterial products. Once (pre-)osteoclasts are formed, these cells may be relatively insensitive for direct TLR stimulation. Possibly, TLR activation of periodontal cells indirectly stimulates osteoclasts, by secreting osteoclastogenesis stimulating inflammatory cytokines.

Burst, Short, and Sustained Vitamin D3 Applications Differentially Affect Osteogenic Differentiation of Human Adipose Stem Cells.

Incorporation of 1,25(OH)2 vitamin D3 (vitD3) into tissue-engineered scaffolds could aid the healing of critical-sized bone defects. We hypothesize that shorter applications of vitD3 lead to more osteogenic differentiation of mesenchymal stem cells (MSCs) than a sustained application. To test this, release from a scaffold was mimicked by exposing MSCs to exactly controlled vitD3 regimens. Human adipose stem cells (hASCs) were seeded onto calcium phosphate particles, cultured for 20 days, and treated with 124 ng vitD3, either provided during 30 min before seeding ([200 nM]), during the first two days ([100 nM]), or during 20 days ([10 nM]). Alternatively, hASCs were treated for two days with 6.2 ng vitD3 ([10 nM]). hASCs attached to the calcium phosphate particles and were viable (~75%). Cell number was not affected by the various vitD3 applications. VitD3 (124 ng) applied over 20 days increased cellular alkaline phosphatase activity at Days 7 and 20, reduced expression of the early osteogenic marker RUNX2 at Day 20, and strongly upregulated expression of the vitD3 inactivating enzyme CYP24. VitD3 (124 ng) also reduced RUNX2 and increased CYP24 applied at [100 nM] for two days, but not at [200 nM] for 30 min. These results show that 20-day application of vitD3 has more effect on hASCs than the same total amount applied in a shorter time span.

Polymethyl methacrylate does not adversely affect the osteogenic potential of human adipose stem cells or primary osteoblasts.

Custom-made polymethyl methacrylate (PMMA) bone cement is used to treat cranial bone defects but whether it is cytotoxic is still unsure. Possible PMMA-induced adverse effects in vivo affect mesenchymal stem cells and osteoblasts at the implant site. We aimed to investigate whether PMMA affects osteogenic and osteoclast activation potential of human mesenchymal stem cells and/or osteoblasts. Immediately after polymerization, PMMA was added to cultured human adipose stem cells (hASCs) or human osteoblasts (hOBs). Medium lactate dehydrogenase was measured (day 1), metabolic activity, proliferation, osteogenic and osteoclast-activation marker expression (day 1 and 7), and mineralization (day 14). PMMA did not affect lactate dehydrogenase, KI67 gene expression, or metabolic activity in hASCs and hOBs. PMMA transiently decreased DNA content in hOBs only. PMMA increased COL1 gene expression in hASCs, but decreased RUNX2 in hOBs. PMMA did not affect osteocalcin or alkaline phosphatase (ALP) expression, ALP activity, or mineralization. Only in hOBs, PMMA decreased RANKL/OPG ratio. In conclusion, PMMA is not cytotoxic and does not adversely affect the osteogenic potential of hASCs or hOBs. Moreover, PMMA does not enhance production of osteoclast factors by hASCs and hOBs in vitro. Therefore, PMMA bone cement seems highly suitable to treat patients with cranial bone defects.

IL-6 counteracts the inhibitory effect of IL-4 on osteogenic differentiation of human adipose stem cells.

Fracture repair is characterized by cytokine production and hypoxia. To better predict cytokine modulation of mesenchymal stem cell (MSC)-aided bone healing, we investigated whether interleukin 4 (IL-4), IL-6, and their combination, affect osteogenic differentiation, vascular endothelial growth factor (VEGF) production, and/or mammalian target of rapamycin complex 1 (mTORC1) activation by MSCs under normoxia or hypoxia. Human adipose stem cells (hASCs) were cultured with IL-4, IL-6, or their combination for 3 days under normoxia (20% O 2 ) or hypoxia (1% O 2 ), followed by 11 days without cytokines under normoxia or hypoxia. Hypoxia did not alter IL-4 or IL-6-modulated gene or protein expression by hASCs. IL-4 alone decreased runt-related transcription factor 2 (RUNX2) and collagen type 1 (COL1) gene expression, alkaline phosphatase (ALP) activity, and VEGF protein production by hASCs under normoxia and hypoxia, and decreased mineralization of hASCs under hypoxia. In contrast, IL-6 increased mineralization of hASCs under normoxia, and enhanced RUNX2 gene expression under normoxia and hypoxia. Neither IL-4 nor IL-6 affected phosphorylation of the mTORC1 effector protein P70S6K. IL-4 combined with IL-6 diminished the inhibitory effect of IL-4 on ALP activity, bone nodule formation, and VEGF production, and decreased RUNX2 and COL1 expression, similar to IL-4 alone, under normoxia and hypoxia. In conclusion, IL-4 alone, but not in combination with IL-6, inhibits osteogenic differentiation and angiogenic stimulation potential of hASCs under normoxia and hypoxia, likely through pathways other than mTORC1. These results indicate that cytokines may differentially affect bone healing and regeneration when applied in isolation or in combination.

Mechanical Loading Differentially Affects Osteocytes in Fibulae from Lactating Mice Compared to Osteocytes in Virgin Mice: Possible Role for Lacuna Size.

Hormonal changes during lactation are associated with profound changes in bone cell biology, such as osteocytic osteolysis, resulting in larger lacunae. Larger lacuna shape theoretically enhances the transmission of mechanical signals to osteocytes. We aimed to provide experimental evidence supporting this theory by comparing the mechanoresponse of osteocytes in the bone of lactating mice, which have enlarged lacunae due to osteocytic osteolysis, with the response of osteocytes in bone from age-matched virgin mice. The osteocyte mechanoresponse was measured in excised fibulae that were cultured in hormone-free medium for 24 h and cyclically loaded for 10 min (sinusoidal compressive load, 3000 µÎµ, 5 Hz) by quantifying loading-related changes in Sost mRNA expression (qPCR) and sclerostin and ß-catenin protein expression (immunohistochemistry). Loading decreased Sost expression by ~ threefold in fibulae of lactating mice. The loading-induced decrease in sclerostin protein expression by osteocytes was larger in lactating mice (55% decrease ± 14 (± SD), n = 8) than virgin mice (33% decrease ± 15, n = 7). Mechanical loading upregulated ß-catenin expression in osteocytes in lactating mice by 3.5-fold (± 0.2, n = 6) which is significantly (p < 0.01) higher than the 1.6-fold increase in ß-catenin expression by osteocytes in fibulae from virgin mice (± 0.12, n = 4). These results suggest that osteocytes in fibulae from lactating mice with large lacunae may respond stronger to mechanical loading than those from virgin mice. This could indicate that osteocytes residing in larger lacuna show a stronger response to mechanical loading.

Hypothermia reduces VEGF-165 expression, but not osteogenic differentiation of human adipose stem cells under hypoxia.

Cryotherapy is successfully used in the clinic to reduce pain and inflammation after musculoskeletal damage, and might prevent secondary tissue damage under the prevalent hypoxic conditions. Whether cryotherapy reduces mesenchymal stem cell (MSC) number and differentiation under hypoxic conditions, causing impaired callus formation is unknown. We aimed to determine whether hypothermia modulates proliferation, apoptosis, nitric oxide production, VEGF gene and protein expression, and osteogenic/chondrogenic differentiation of human MSCs under hypoxia. Human adipose MSCs were cultured under hypoxia (37°C, 1% O2), hypothermia and hypoxia (30°C, 1% O2), or control conditions (37°C, 20% O2). Total DNA, protein, nitric oxide production, alkaline phosphatase activity, gene expression, and VEGF protein concentration were measured up to day 8. Hypoxia enhanced KI67 expression at day 4. The combination of hypothermia and hypoxia further enhanced KI67 gene expression compared to hypoxia alone, but was unable to prevent the 1.2-fold reduction in DNA amount caused by hypoxia at day 4. Addition of hypothermia to hypoxic cells did not alter the effect of hypoxia alone on BAX-to-BCL-2 ratio, alkaline phosphatase activity, gene expression of SOX9, COL1, or osteocalcin, or nitric oxide production. Hypothermia decreased the stimulating effect of hypoxia on VEGF-165 gene expression by 6-fold at day 4 and by 2-fold at day 8. Hypothermia also decreased VEGF protein expression under hypoxia by 2.9-fold at day 8. In conclusion, hypothermia decreased VEGF-165 gene and protein expression, but did not affect differentiation, or apoptosis of MSCs cultured under hypoxia. These in vitro results implicate that hypothermia treatment in vivo, applied to alleviate pain and inflammation, is not likely to harm early stages of callus formation.

Mechanical Stimulation and IGF-1 Enhance mRNA Translation Rate in Osteoblasts Via Activation of the AKT-mTOR Pathway.

Insulin-like growth factor-1 (IGF-1) is anabolic for muscle by enhancing the rate of mRNA translation via activation of AKT and subsequent activation of the mammalian target of rapamycin complex 1 (mTOR), thereby increasing cellular protein production. IGF-1 is also anabolic for bone, but whether the mTOR pathway plays a role in the rate of bone matrix protein production by osteoblasts is unknown. We hypothesized that anabolic stimuli such as mechanical loading and IGF-1 stimulate protein synthesis in osteoblasts via activation of the AKT-mTOR pathway. MC3T3-E1 osteoblasts were either or not subjected for 1 h to mechanical loading by pulsating fluid flow (PFF) or treated with or without human recombinant IGF-1 (1-100 ng/ml) for 0.5-6 h, to determine phosphorylation of AKT and p70S6K (downstream of mTOR) by Western blot. After 4 days of culture with or without the mTOR inhibitor rapamycin, total protein, DNA, and gene expression were quantified. IGF-1 (100 ng/ml) reduced IGF-1 gene expression, although PFF enhanced IGF-1 expression. IGF-1 did not affect collagen-I gene expression. IGF-1 dose-dependently enhanced AKT and p70S6K phosphorylation at 2 and 6 h. PFF enhanced phosphorylation of AKT and p70S6K already within 1 h. Both IGF-1 and PFF enhanced total protein per cell by ∼30%, but not in the presence of rapamycin. Our results show that IGF-1 and PFF activate mTOR, thereby stimulating the rate of mRNA translation in osteoblasts. The known anabolic effect of mechanical loading and IGF-1 on bone may thus be partly explained by mTOR-mediated enhanced protein synthesis in osteoblasts.

Early activation of the beta-catenin pathway in osteocytes is mediated by nitric oxide, phosphatidyl inositol-3 kinase/Akt, and focal adhesion kinase.

Bone mechanotransduction is vital for skeletal integrity. Osteocytes are thought to be the cellular structures that sense physical forces and transform these signals into a biological response. The Wnt/beta-catenin signaling pathway has been identified as one of the signaling pathways that is activated in response to mechanical loading, but the molecular events that lead to an activation of this pathway in osteocytes are not well understood. We assessed whether nitric oxide, focal adhesion kinase, and/or the phosphatidyl inositol-3 kinase/Akt signaling pathway mediate loading-induced beta-catenin pathway activation in MLO-Y4 osteocytes. We found that mechanical stimulation by pulsating fluid flow (PFF, 0.7+/-0.3 Pa, 5 Hz) for 30 min induced beta-catenin stabilization and activation of the Wnt/beta-catenin signaling pathway. The PFF-induced stabilization of beta-catenin and activation of the beta-catenin signaling pathway was abolished by adding focal kinase inhibitor FAK inhibitor-14 (50 microM), or phosphatidyl inositol-3 kinase inhibitor LY-294002 (50 microM). Addition of nitric oxide synthase inhibitor L-NAME (1.0mM) also abolished PFF-induced stabilization of beta-catenin. This suggests that mechanical loading activates the beta-catenin signaling pathway by a mechanism involving nitric oxide, focal adhesion kinase, and the Akt signaling pathway. These data provide a framework for understanding the role of beta-catenin in mechanical adaptation of bone.

A common binding motif for various bacteria of the bacteria-binding peptide SRCRP2 of DMBT1/gp-340/salivary agglutinin.

Abstract Salivary agglutinin (DMBT1SAG) is identical to lung glycoprotein-340 and encoded by deleted in malignant brain tumors-1. It is a member of the scavenger receptor cysteine-rich (SRCR) superfamily, proteins that have one or more SRCR domains. Salivary agglutinin plays a role in oral innate immunity by the binding and agglutination of oral streptococci. S. mutans has been shown to bind to a 16-mer peptide (QGRVEVLYRGSWGTVC) located within the SRCR domains. Within this peptide, designated SRCR Peptide 2, residues VEVL and W were critical for binding. The aim of this study was to investigate binding of DMBT1SAG to other bacteria. Therefore, interaction between a series of bacteria and DMBT1(SAG), SRCR peptide 2 and its alanine substitution variants was studied in adhesion and agglutination assays. For different bacteria there was a highly significant correlation between adhesion to DMBT1SAG and adhesion to SRCR peptide 2 suggesting that SRCR peptide 2 is the major bacteria binding site. An alanine substitution scan showed that 8 amino acids were involved in binding (xRVEVLYxxSWxxxx). The binding motifs varied for different species were found, but the residues VxVxY and W were always present. In conclusion, a common binding motif (RVEVLYxxxSW) within the SRCR domains is responsible for the broad bacteria-binding spectrum of DMBT1SAG.

Histatin and lactoferrin derived peptides: antimicrobial properties and effects on mammalian cells.

In order to analyze the clinical potential of two antimicrobial peptides, human lactoferrin 1-11 (hLF1-11) and synthetic histatin analogue Dhvar-5, we measured the killing effect on bacteria, and the potential toxicity on erythrocytes and bone cells. The antimicrobial activity was determined in a killing assay on six strains, including methicillin resistant Staphylococcus Aureus. The effect on human erythrocytes and MC3T3 mouse bone cells was measured with a hemolysis assay and a viability assay, respectively. Both hLF1-11 and Dhvar-5 dose-dependently killed all bacterial strains, starting at concentrations of 6 microg/mL. hLF1-11 had no effect on mammalian cells at concentrations up to 400 microg/mL, but Dhvar-5 induced significant hemolysis (37% at 200 microg/mL) and bone cell death (70% at 400 microg/mL). This indicates that both peptides are able to kill various resistant and non-resistant bacteria, but Dhvar-5 may exert a cytotoxic effect on host cells at higher concentrations.

Bacteria binding by DMBT1/SAG/gp-340 is confined to the VEVLXXXXW motif in its scavenger receptor cysteine-rich domains.

The scavenger receptor cysteine-rich (SRCR) proteins form an archaic group of metazoan proteins characterized by the presence of SRCR domains. These proteins are classified in group A and B based on the number of conserved cysteine residues in their SRCR domains, i.e. six for group A and eight for group B. The protein DMBT1 (deleted in malignant brain tumors 1), which is identical to salivary agglutinin and lung gp-340, belongs to the group B SRCR proteins and is considered to be involved in tumor suppression and host defense by pathogen binding. In a previous study we used nonoverlapping synthetic peptides covering the SRCR consensus sequence to identify a 16-amino acid bacteria-binding protein loop (peptide SRCRP2; QGRVEVLYRGSWGTVC) within the SRCR domains. In this study, using overlapping peptides, we pinpointed the minimal bacteria-binding site on SRCRP2, and thus DMBT1, to an 11-amino acid motif (DMBT1 pathogen-binding site 1 or DMBT1pbs1; GRVEVLYRGSW). An alanine substitution scan revealed that VEVL and Trp are critical residues in this motif. Bacteria binding by DMBT1pbs1 was different from the bacteria binding by the macrophage receptor MARCO in which an RXR motif was critical. In addition, the homologous consensus sequences of a number of SRCR proteins were synthesized and tested for bacteria binding. Only consensus sequences of DMBT1 orthologues bound bacteria by this motif.