Platelet-rich plasma stimulates cytokine expression and alkaline phosphatase activity in osteoblast-derived osteosarcoma cells

Objective: The aim of this study was to investigate the effects of PRP on SAOS-2 cells in terms of cytokine expression, cell activity and oxidative stress. Design: Cell line SAOS-2 (1 x 10(5) cells/mL) were grown in culture medium alpha-MEM with 10% FBS for 24 h and stimulated (or not) with PRP at concentrations of 3, 10 and 20%, LPS (E. coli, 10 g/mL) and IL-1 beta (1 mg/mL) for 24 h. The supernatant was collected and analyzed for the expression of cytokines in a panel array, ALP using a commercial kit and NO2- with Griess reaction method. Also, the cells were analyzed using Western blot for RANKL and slot blotting for nitrotyrosine expression. Result: There were no significant differences amongst the groups in terms of NO2-, protein nitrotyrosine content and RANKL expression. However, all stimuli increased ALP activity and in case of PRP, it was in a dose-dependent manner (p < 0.001). Also, all stimuli induced an increase in cytokines and chemokines expression, but only PRP promoted an increase of component C5, sICAM-1 and RANTES expression. Whilst IL-1 receptor antagonist (IL-1ra) expression was down-regulated by PRP, both LPS and IL-1 beta caused up-regulation of this cytokine. Conclusions: PRP can stimulate osteoblast activity and cytokine/chemokine release, as well as indicate some of the mediators that can (and cannot) be involved in this activation. (C) 2012 Elsevier Ltd. All rights reserved.

Repression of osteocyte Wnt/beta-catenin signaling is an early event in the progression of renal osteodystrophy

Chronic kidney diseasemineral bone disorder (CKD-MBD) is defined by abnormalities in mineral and hormone metabolism, bone histomorphometric changes, and/or the presence of soft-tissue calcification. Emerging evidence suggests that features of CKD-MBD may occur early in disease progression and are associated with changes in osteocyte function. To identify early changes in bone, we utilized the jck mouse, a genetic model of polycystic kidney disease that exhibits progressive renal disease. At 6 weeks of age, jck mice have normal renal function and no evidence of bone disease but exhibit continual decline in renal function and death by 20 weeks of age, when approximately 40% to 60% of them have vascular calcification. Temporal changes in serum parameters were identified in jck relative to wild-type mice from 6 through 18 weeks of age and were subsequently shown to largely mirror serum changes commonly associated with clinical CKD-MBD. Bone histomorphometry revealed progressive changes associated with increased osteoclast activity and elevated bone formation relative to wild-type mice. To capture the early molecular and cellular events in the progression of CKD-MBD we examined cell-specific pathways associated with bone remodeling at the protein and/or gene expression level. Importantly, a steady increase in the number of cells expressing phosphor-Ser33/37-beta-catenin was observed both in mouse and human bones. Overall repression of Wnt/beta-catenin signaling within osteocytes occurred in conjunction with increased expression of Wnt antagonists (SOST and sFRP4) and genes associated with osteoclast activity, including receptor activator of NF-?B ligand (RANKL). The resulting increase in the RANKL/osteoprotegerin (OPG) ratio correlated with increased osteoclast activity. In late-stage disease, an apparent repression of genes associated with osteoblast function was observed. These data confirm that jck mice develop progressive biochemical changes in CKD-MBD and suggest that repression of the Wnt/beta-catenin pathway is involved in the pathogenesis of renal osteodystrophy. (C) 2012 American Society for Bone and Mineral Research.

Experimental model of tooth movement in mice: A standardized protocol for studying bone remodeling under compression and tensile strains

During orthodontic tooth movement (OTM), alveolar bone is resorbed by osteoclasts in compression sites (CS) and is deposited by osteoblasts in tension sites (TS). The aim of this study was to develop a standardized OTM protocol in mice and to investigate the expression of bone resorption and deposition markers in CS and TS. An orthodontic appliance was placed in C57BL6/J mice. To define the ideal orthodontic force, the molars of the mice were subjected to forces of 0.1 N, 0.25 N, 0.35 N and 0.5 N. The expression of mediators that are involved in bone remodeling at CS and TS was analyzed using a Real-Time PCR. The data revealed that a force of 0.35 N promoted optimal OTM and osteoclast recruitment without root resorption. The levels of TNF-alpha, RANKL, MMP13 and OPG were all altered in CS and TS. Whereas TNF-a and Cathepsin K exhibited elevated levels in CS. RUNX2 and OCN levels were higher in TS. Our results suggest that 0.35 N is the ideal force for OTM in mice and has no side effects. Moreover, the expression of bone remodeling markers differed between the compression and the tension areas, potentially explaining the distinct cellular migration and differentiation patterns in each of these sites. (C) 2012 Elsevier Ltd. All rights reserved.

Exploration of the Antiplatelet Activity Profile of Betulinic Acid on Human Platelets

Betulinic acid, a natural pentacyclic triterpene acid, presents a diverse mode of biological actions including antiretroviral, antibacterial, antimalarial, and anti-inflammatory activities. The potency of betulinic acid as an inhibitor of human platelet activation was evaluated, and its antiplatelet profile against in vitro platelet aggregation, induced by several platelet agonists (adenosine diphosphate, thrombin receptor activator peptide-14, and arachidonic acid), was explored. Flow cytometric analysis was performed to examine the effect of betulinic acid on P-selectin membrane expression and PAC-1 binding to activated platelets. Betulinic acid potently inhibits platelet aggregation and also reduced PAC-1 binding and the membrane expression of P-selectin. Principal component analysis was used to screen, on the chemical property space, for potential common pharmacophores of betulinic acid with approved antithrombotic drugs. A common pharmacophore was defined between the NMR-derived structure of betulinic acid and prostacyclin agonists (PGI2), and the importance of its carboxylate group in its antiplatelet activity was determined. The present results indicate that betulinic acid has potential use as an antithrombotic compound and suggest that the mechanism underlying the antiplatelet effects of betulinic acid is similar to that of the PGI2 receptor agonists, a hypothesis that deserves further investigation.

Exogenous Administration of 15d-PGJ(2)-Loaded Nanocapsules Inhibits Bone Resorption in a Mouse Periodontitis Model

The 15-deoxy-(Delta 12,14)-PG J(2) (15d-PGJ(2)) has demonstrated excellent anti-inflammatory results in different experimental models. It can be used with a polymeric nanostructure system for modified drug release, which can change the therapeutic properties of the active principle, leading to increased stability and slower/prolonged release. The aim of the current study was to test a nano-technological formulation as a carrier for 15d-PGJ(2), and to investigate the immunomodulatory effects of this formulation in a mouse periodontitis model. Poly (D, L-lactide-coglycolide) nanocapsules (NC) were used to encapsulate 15d-PGJ(2). BALB/c mice were infected on days 0, 2, and 4 with Aggregatibacter actinomycetemcomitans and divided into groups (n = 5) that were treated daily during 15 d with 1, 3, or 10 mu g/kg 15d-PGJ(2)-NC. The animals were sacrificed, the submandibular lymph nodes were removed for FACS analysis, and the jaws were analyzed for bone resorption by morphometry. Immunoinflammatory markers in the gingival tissue were analyzed by reverse transcriptase-quantitative PCR, Western blotting, or ELISA. Infected animals treated with the 15d-PGJ(2)-NC presented lower bone resorption than infected animals without treatment (p < 0.05). Furthermore, infected animals treated with 10 mu g/kg 15d-PGJ(2)-NC had a reduction of CD4(+)CD25(+)FOXP3(+) cells and CD4/CD8 ratio in the submandibular lymph node (p < 0.05). Moreover, CD55 was upregulated, whereas RANKL was downregulated in the gingival tissue of the 10 mu g/kg treated group (p < 0.05). Several proinflammatory cytokines were decreased in the group treated with 10 mu g/kg 15d-PGJ(2)-NC, and high amounts of 15d-PGJ(2) were observed in the gingiva. In conclusion, the 15d-PGJ(2)-NC formulation presented immunomodulatory effects, decreasing bone resorption and inflammatory responses in a periodontitis mouse model. The Journal of Immunology, 2012, 189: 1043-1052.

Non-inflammatory destructive periodontal disease: a clinical, microbiological, immunological and genetic investigation

Periodontitis comprises a group of multifactorial diseases in which periodontopathogens accumulate in dental plaque and trigger host chronic inflammatory and immune responses against periodontal structures, which are determinant to the disease outcome. Although unusual cases of non-inflammatory destructive periodontal disease (NIDPD) are described, their pathogenesis remains unknown. A unique NIDPD case was investigated by clinical, microbiological, immunological and genetic tools. The patient, a non-smoking dental surgeon with excessive oral hygiene practice, presented a generalized bone resorption and tooth mobility, but not gingival inflammation or occlusion problems. No hematological, immunological or endocrine alterations were found. No periodontopathogens (A. actinomycetemcomitans, P. gingivalis, F. nucleatum and T. denticola) or viruses (HCMV, EBV-1 and HSV-1) were detected, along with levels of IL-1 beta and TNF-alpha in GCF compatible with healthy tissues. Conversely ALP, ACP and RANKL GCF levels were similar to diseased periodontal sites. Genetic investigation demonstrated that the patient carried some SNPs, as well HLA-DR4 (*0404) and HLA-B27 alleles, considered risk factors for bone loss. Then, a less vigorous and diminished frequency of toothbrushing was recommended to the patient, resulting in the arrest of alveolar bone loss, associated with the return of ALP, ACP and RANKL in GCF to normality levels. In conclusion, the unusual case presented here is compatible with the previous description of NIDPD, and the results that a possible combination of excessive force and frequency of mechanical stimulation with a potentially bone loss prone genotype could result in the alveolar bone loss seen in NIDPD.

Expression Analysis of Wound Healing Genes in Human Periapical Granulomas of Progressive and Stable Nature

Introduction: Wound healing process involves the activation of extracellular matrix components, remodeling enzymes, cellular adhesion molecules, growth factors, cytokines and chemokines genes. However, the molecular patterns underlying the healing process periapical environment remain unclear. Here we hypothesized that endodontic infection might result in an imbalance in the expression of wound healing genes involved in the pathogenesis of periapical lesions. Furthermore, we suggest that differential expression of wound healing markers in active and latent granulomas could account for different clinical outcomes for such lesions. Methods: Study samples consisted of 93 periapical granulomas collected after endodontic surgeries and 24 healthy periodontal ligament tissues collected from premolars extracted for orthodontic purposes as control samples. Of these, 10 periapical granulomas and 5 healthy periapical tissues were used for expression analysis of 84 wound healing genes by using a pathway-specific real-time polymerase chain reaction array. The remaining 83 granulomas and all 24 control specimens were used to validate the obtained array data by real-time polymerase chain reaction. Observed variations in expression of wound healing genes were analyzed according to the classification of periapical granulomas as active/progressive versus inactive/stable (as determined by receptor activator for nuclear factor kappa B ligand/osteoprotegerin expression ratio). Results: We observed a marked increase of 5-fold or greater in SERPINE1, TIMP1, COL1A1, COL5A1, VTN, CTGF, FGF7, TGFB1, TNF, CXCL11, ITGA4, and ITGA5 genes in the periapical granulomas when compared with control samples. SERPINE1, TIMP1, COL1A1, TGFB1, and ITGA4 mRNA expression was significantly higher in inactive compared with active periapical granulomas (P < .001), whereas TNF and CXCL11 mRNA expression was higher in active lesions (P < .001). Conclusions: The identification of novel gene targets that curb the progression status of periapical lesions might contribute to a more accurate diagnosis and lead to treatment modalities more conducive to endodontic success. (J Endod 2012;38:185-190)

Adjunct effect of the antimicrobial photodynamic therapy to an association of non-surgical and surgical periodontal treatment in modulation of gene expression

Background: This study has evaluated the effect of antimicrobial photodynamic therapy (aPDT) used in conjunction with non-surgical and surgical periodontal treatment (PT) in modulating gene expression during periodontal wound healing. Methods: Fifteen patients with chronic periodontitis, presenting bilaterally lower molars with class III furcation lesions and scheduled for extraction, were selected. In initial therapy, scaling and root planing (SRP) was performed in the Control Group (CG), while SRP + aPDT were performed in the Test Group (TG). 45 days later, flap surgery plus SRP, and flap surgery plus SRP + aPDT were performed in the CG and TG, respectively. At 21 days post-surgery, the newly formed granulation tissue was collected, and Real-time PCR evaluated the expression of the genes: tumor necrosis factor-?, interleukin-1?, interleukin-4, interleukin-10, matrix metalloproteinase-2 (MMP-2), tissue inhibitor of metalloproteinase-2 (TIMP-2), osteoprotegerin (OPG), receptor activator of nuclear factor- ?B ligand (RANKL), type I collagen, alkaline phosphatase, osteopontin, osteocalcin, and bone sialoprotein. Results: There were statistically significant differences between the groups in relation to mRNA levels for MMP-2 (TG = 3.26 ± 0.89; CG = 4.23 ± 0.97; p = 0.01), TIMP-2/MMP-2 ratio (TG = 0.91 ± 0.34; CG = 0.73 ± 0.32; p = 0.04), OPG (TG = 0.84 ± 0.45; CG = 0.30 ± 0.26; p = 0.001), and OPG/RANKL ratio (TG = 0.60 ± 0.86; CG = 0.23 ± 0.16; p = 0.04), favoring the TG. Conclusion: The present data suggest that the aPDT associated to nonsurgical and surgical periodontal therapy may modulate the extracellular matrix and bone remodeling by up regulating the TIMP- 2/MMP-2 and OPG/RANKL mRNA ratio, but the clinical relevance needs to be evaluated in further studies.

The effect of CCL3 and CCR1 in bone remodeling induced by mechanical loading during orthodontic tooth movement in mice

Bone remodeling is affected by mechanical loading and inflammatory mediators, including chemokines. The chemokine (C–C motif) ligand 3 (CCL3) is involved in bone remodeling by binding to C–C chemokine receptors 1 and 5 (CCR1 and CCR5) expressed on osteoclasts and osteoblasts. Our group has previously demonstrated that CCR5 down-regulates mechanical loading-induced bone resorption. Thus, the present study aimed to investigate the role of CCR1 and CCL3 in bone remodeling induced by mechanical loading during orthodontic tooth movement in mice. Our results showed that bone remodeling was significantly decreased in CCL3−/− and CCR1−/− mice and in animals treated with Met-RANTES (an antagonist of CCR5 and CCR1). mRNA levels of receptor activator of nuclear factor kappa-B (RANK), its ligand RANKL, tumor necrosis factor alpha (TNF-α) and RANKL/osteoprotegerin (OPG) ratio were diminished in the periodontium of CCL3−/− mice and in the group treated with Met-RANTES. Met-RANTES treatment also reduced the levels of cathepsin K and metalloproteinase 13 (MMP13). The expression of the osteoblast markers runt-related transcription factor 2 (RUNX2) and periostin was decreased, while osteocalcin (OCN) was augmented in CCL3−/− and Met-RANTES-treated mice. Altogether, these findings show that CCR1 is pivotal for bone remodeling induced by mechanical loading during orthodontic tooth movement and these actions depend, at least in part, on CCL3.

Immunohistochemical localization of osteoclastogenic cell mediators in feline tooth resorption and healthy teeth

Tooth resorption is among the most common and most challenging problems in feline dentistry It is a progressive disease eventually leading to tooth loss and often root replacement. The etiology of moth resorption remains obscure and to date no effective therapeutic approach is known. The present study is aimed at assessing the reliability of radiographic imaging and addressing the possible involvement of receptor activator of NF kappa B (RANK), its ligand (RANKL), and osteoprotegerin (OPG) in the process of tooth resorption. Teeth from 8 cats were investigated by means of radiographs and paraffin sections followed by immunolabeling. Six cats were diagnosed with tooth resorption based on histopathologic and radiographic findings. Samples were classified according to a four-stage diagnostic system. Radiologic assessment of tooth resorption correlated very strongly with histopathologic findings. Tooth resorption was accompanied by a strong staining with all three antibodies used, especially with anti-RANK and anti-RANKL antibodies. The presence of OPG and RANKL at the resorption site is indicative of repair attempts by fibroblasts and stromal cells. These findings should be extended by further investigations in order to elucidate the pathophysiologic processes underlying tooth resorption that might lead to prophylactic and/or therapeutic measures. J Vet Dent 27(2); 75 - 83, 2010

Impact of Bone Harvesting Techniques on Cell Viability and the Release of Growth Factors of Autografts

Background: Autogenous bone grafts obtained by different harvesting techniques behave differently during the process of graft consolidation; the underlying reasons are however not fully understood. One theory is that harvesting techniques have an impact on the number and activity of the transplanted cells which contribute to the process of graft consolidation. Materials and Methods: To test this assumption, porcine bone grafts were harvested with four different surgical procedures: bone mill, piezosurgery, bone drilling (bone slurry), and bone scraper. After determining cell viability, the release of molecules affecting bone formation and resorption was assessed by reverse transcription polymerase chain reaction and immunoassay. The mitogenic and osteogenic activity of the conditioned media was evaluated in a bioassay with isolated bone cells. Results: Cell viability and the release of molecules affecting bone formation were higher in samples harvested by bone mill and bone scraper when compared with samples prepared by bone drilling and piezosurgery. The harvesting procedure also affected gene expression, for example, bone mill and bone scraper samples revealed significantly higher expression of growth factors such as bone morphogenetic protein-2 and vascular endothelial growth factor compared with the two other modalities. Receptor activator of nuclear factor kappa B ligand expression was lowest in bone scraper samples. Conclusion: These data can provide a scientific basis to better understand the impact of harvesting techniques on the number and activity of transplanted cells, which might contribute to the therapeutic outcome of the augmentation procedure.

TNFα inhibits the development of osteoclasts through osteoblast-derived GM-CSF

Inflammatory cytokines such as tumor necrosis factor-alpha (TNFα) are potent stimulators of osteoclast formation and bone resorption and are frequently associated with pathologic bone metabolism. The cytokine exerts specific effects on its target cells and constitutes a part of the cellular microenvironment. Previously, TNFα was demonstrated to inhibit the development of osteoclasts in vitro via an osteoblast-mediated pathway. In the present study, the molecular mechanisms of the inhibition of osteoclastogenesis were investigated in co-cultures of osteoblasts and bone marrow cells (BMC) and in cultures of macrophage-colony stimulating factor (M-CSF) dependent, non-adherent osteoclast progenitor cells (OPC) grown with M-CSF and receptor activator of NF-κB ligand (RANKL). Granulocyte-macrophage colony stimulating factor (GM-CSF), a known inhibitor of osteoclastogenesis was found to be induced in osteoblasts treated with TNFα and the secreted protein accumulated in the supernatant. Dexamethasone (Dex), an anti-inflammatory steroid, caused a decrease in GM-CSF expression, leading to partial recovery of osteoclast formation. Flow cytometry analysis revealed that in cultures of OPC, supplemented with 10% conditioned medium (CM) from osteoblasts treated with TNFα/1,25(OH)(2)D(3), expression of RANK and CD11c was suppressed. The decrease in RANK expression may be explained by the finding, that GM-CSF and the CM from wt osteoblasts were found to suppress the expression of c-Fos, Fra-1, and Nfatc-1. The failure of OPC to develop into CD11c(+) dendritic cells suggests that cell development is not deviated to an alternative differentiation pathway, but rather, that the monocytes are maintained in an undifferentiated, F4/80(+), state. The data further implies possible interactions among inflammatory cytokines. GM-CSF induced by TNFα acts on early hematopoietic precursors, inhibiting osteoclastogenesis while acting as the growth factor for M-CSF independent inflammatory macrophages. These in turn may condition a microenvironment enhancing osteoclast differentiation and bone resorption upon migration of the OPC from circulation to the bone/bone marrow compartment.

Malignant melanoma and bone resorption

The cellular and humoral mechanisms accounting for osteolysis in skeletal metastases of malignant melanoma are uncertain. Osteoclasts, the specialised multinucleated cells that carry out bone resorption, are derived from monocyte/macrophage precursors. We isolated tumour-associated macrophages (TAMs) from metastatic (lymph node/skin) melanomas and cultured them in the presence and absence of osteoclastogenic cytokines and growth factors. The effect of tumour-derived fibroblasts and melanoma cells on osteoclast formation and resorption was also analysed. Melanoma TAMs (CD14+/CD51-) differentiated into osteoclasts (CD14-/CD51+) in the presence of receptor activator for nuclear factor kappaB ligand (RANKL) and macrophage-colony stimulating factor. Tumour-associated macrophage-osteoclast differentiation also occurred via a RANKL-independent pathway when TAMs were cultured with tumour necrosis factor-alpha and interleukin (IL)-1alpha. RT-PCR showed that fibroblasts isolated from metastatic melanomas expressed RANKL messenger RNA and the conditioned medium of cultured melanoma fibroblasts was found to be capable of inducing osteoclast formation in the absence of RANKL; this effect was inhibited by the addition of osteoprotegerin (OPG). We also found that cultured human SK-Mel-29 melanoma cells produce a soluble factor that induces osteoclast differentiation; this effect was not inhibited by OPG. Our findings indicate that TAMs in metastatic melanomas can differentiate into osteoclasts and that melanoma fibroblasts and melanoma tumour cells can induce osteoclast formation by RANKL-dependent and RANKL-independent mechanisms, respectively.

Immunohistochemical localization of RANK, RANKL and OPG in healthy and arthritic canine elbow joints

OBJECTIVE: To determine if the receptor activator of nuclear factor-kappaB-receptor activator of nuclear factor-kappaB ligand-osteoprotegerin (RANK-RANKL-OPG) system is active in bone remodeling in dogs and, if so, whether differences in expression of these mediators occur in healthy and arthritic joints. STUDY DESIGN: Experimental study. SAMPLE POPULATION: Fragmented processus coronoidei (n=20) were surgically removed from dogs with elbow arthritis and 5 corresponding healthy samples from dogs euthanatized for reasons other than elbow joint disease. METHODS: Bright-field immunohistochemistry and high-resolution fluorescence microscopy were used to investigate the distribution of RANK, RANKL, and OPG in healthy and arthritic joints. RESULTS: All 3 molecules were identified by immunostaining of canine bone tissue. In elbow dysplasia, the number of RANK-positive osteoclasts was increased. In their vicinity, cells expressing RANKL, a mediator of osteoclast activation, were abundant whereas the number of osteoblasts having the potential to limit osteoclastogenesis and bone resorption via OPG was few. CONCLUSIONS: The RANK-RANKL-OPG system is active in bone remodeling in dogs. In elbow dysplasia, a surplus of molecules promoting osteoclastogenesis was evident and is indicative of an imbalance between the mediators regulating bone resorption and bone formation. Both OPG and neutralizing antibodies against RANKL have the potential to counterbalance bone resorption. CLINICAL RELEVANCE: Therapeutic use of neutralizing antibodies against RANKL to inhibit osteoclast activation warrants further investigation.

Synthesis of NO-donor bisphosphonates and their in-vitro action on bone resorption

A new class of bisphosphonates containing nitrooxy NO-donor functions has been developed. The products proved to display affinity for hydroxyapatite. Injection of (99m)Tc-labeled derivatives 11 and 18 into male rats showed a preferential accumulation of the compounds in bone as compared to blood and muscles. The products were found to inhibit the differentiation of pre-osteoclasts to functional osteoclasts induced by receptor activator of NF-kappaB ligand (RANKL), through a prevalent NO-dependent mechanism.