Flavonoids from Dalbergia cochinchinensis: Impact on osteoclastogenesis.

Background/purpose: Dalbergia cochinchinensi has been widely used in traditional medicine because of its flavonoids. This study examined which components in D. cochinchinensis were capable of reducing or even stimulating the formation of bone-resorbing osteoclasts. Materials and methods: We have isolated subfamilies of chalcones (isoliquiritigenin, butein), flavones (7-hydroxy-6-methoxyflavone) and neoflavanoids (5-methoxylatifolin), and performed an in vitro bioassay on osteoclastogenesis. The flavonoids were tested for their potential to change the expression of tartrate-resistant acid phosphatase (TRAP) and cathepsin K (CTSK) in murine bone marrow cultures being exposed to RANKL, M-CSF and TGF-ß1 using RT-PCR, histochemistry and immunoassay. Results: We could confirm that isoliquiritigenin and butein significantly lower the expression of TRAP and CTSK in this setting. Moreover, histochemistry supported the decrease of TRAP by the chalcones. We further observed a trend towards an increase of osteoclastogenesis in the presence of 5-methoxylatifolin and 7-hydroxy-6-methoxyflavone, particular in bone marrow cultures being exposed to RANKL and M-CSF. Consistently, the anti-inflammatory activity was restricted to isoliquiritigenin and butein in murine RAW 264.7 inflammatory macrophages stimulated by lipopolysaccharide (LPS). With respect to osteoblastogenesis, neither of the flavonoids but butyrate, a short chain fatty acid, increased the osteogenic differentiation marker alkaline phosphatase activity in ST2 murine mesenchymal cells. Conclusion: We have identified two flavonoids from D. cochinchinensis with a potential pro-osteoclastogenic activity and confirm the anti-osteoclastogenic activity of isoliquiritigenin and butein.

Brief communication: Effects of conditioned media from human platelet lysate cultured MSC on osteogenic cell differentiation in vitro.

Culturing mesenchymal stromal cells (MSC) in human platelet lysate (HPL) supplemented media can enhance their osteogenic differentiation potential. The objective of this study was to test the hypothesis that conditioned media (CM) derived from HPL-cultured MSC also have pro-osteogenic effects. Pooled CM was prepared from HPL-cultured human bone marrow MSC (BMSC) of multiple donors and applied on BMSC of different donors (than those used for CM preparation), with or without additional supplementation [HPL, fetal bovine serum (FBS)] and osteogenic stimulation. At various time-points, cell proliferation, alkaline phosphatase (ALP) activity, osteogenic gene expression and in vitro mineralization were assessed. BMSC in standard unstimulated growth media served as controls. After 3-7 days, CM alone did not promote BMSC proliferation or ALP activity; supplementation of CM with HPL slightly improved these effects. After 2 and 7 days, CM alone, but not CM supplemented with HPL, promoted osteogenic gene expression. After 14 days, only CM supplemented with FBS and osteogenic stimulants supported in vitro BMSC mineralization; CM alone and CM supplemented with HPL did not support mineralization, regardless of osteogenic stimulation. In summary, CM from HPL-cultured BMSC promoted osteogenic gene expression but not in vitro mineralization in allogeneic BMSC even when supplemented with HPL and/or osteogenic stimulants. Future studies should investigate the role and relevance of supplementation and osteogenic induction in in vitro assays using CM from MSC.

Dalbergiones lower the inflammatory response in oral cells in vitro.

OBJECTIVES: Periodontitis is a global health burden that underlines the demand for anti-inflammatory treatment. Dalbergia melanoxylon being a rich source of flavonoids has been widely used in traditional medicine but the potential anti-inflammatory activity of its dalbergiones remains to be shown. MATERIAL AND METHODS: We have isolated 3'-hydroxy-4,4'-dimethoxydalbergione, 4-methoxydalbergione, and 4'-hydroxy-4-methoxydalbergione from Dalbergia melanoxylon and tested their potential anti-inflammatory activity. RESULTS: All dalbergiones are potent inhibitors of an LPS-induced inflammatory response of RAW 264.7 macrophages. This is specified by IL1ß and IL6 production, and the p65 nuclear translocation. Consistently, in primary macrophages, the dalbergiones caused an M1-to-M2 polarization switch indicated by the decreased ration of IL1ß and IL6 versus arginase 1 and YM1 expression. To implement oral cells, we have used gingival fibroblasts exposed to IL1ß and TNFα. Consistently, all dalbergiones reduced the expression of IL6 and IL8 as well as the nuclear translocation of p65. CONCLUSION: These findings increase the accumulating knowledge on dalbergiones and extend it towards its capacity to lower the inflammatory response of oral cells. CLINICAL RELEVANCE: These findings are another piece of evidence that supports the use of herbal medicine to potentially lower inflammatory events related to dentistry.

Heartwood of Dalbergia cochinchinensis: 4,7,2'-Trihydroxy-4'-methoxyisoflavanol and 6,4'-Dihydroxy-7-methoxyflavane Reduce Cytokine and Chemokine Expression In Vitro.

Dalbergia cochinchinensis has been widely used in traditional medicine because of its flavonoids; however, the impact of the flavonoids to modulate the inflammatory response to oral cells remains to be described. For this aim, we isolated 4,7,2'-trihydroxy-4'-methoxyisoflavanol (472T4MIF) and 6,4'-dihydroxy-7-methoxyflavane (64D7MF) from the heartwood of D. cochinchinensis and confirmed the chemical structure by nuclear magnetic resonance. We show here that both flavonoids are inhibitors of an inflammatory response of murine RAW 264.7 inflammatory macrophages stimulated by LPS. This is indicated by interleukin (IL)1, IL6, and chemokine CCL2 production besides the phosphorylation of p65. Consistently, in primary murine macrophages, both flavonoids decreased the inflammatory response by lowering LPS-induced IL1 and IL6 expression. To introduce oral cells, we have used human gingival fibroblasts and provoked the inflammatory response by exposing them to IL1ß and TNFα. Under these conditions, 472T4MIF, but not 64D7MF, reduced the expression of chemokines CXCL1 and CXCL2. Taken together, we identified two flavonoids that can reduce the expression of cytokines and chemokines in macrophages and fibroblastic cells.

Effect of recombinant PDGF-BB on bone formation in the presence of ß-tricalcium phosphate and bovine bone mineral matrix: a pilot study in rat calvarial defects.

BACKGROUND: Supplementation of bone substitutes with recombinant platelet-derived growth factor-BB (PDGF-BB) can enhance bone regeneration. The aim of the study was to evaluate the effect of PDGF-BB on bone formation in the presence of ß-tricalcium phosphate and bovine bone mineral matrix in a rat calvaria defect model. METHODS: The authors examined 5 mm rat calvarial defects treated with ß-tricalcium phosphate (TCP) or demineralized bovine bone mineral (DBBM) with and without 0.3 mg/ml recombinant PDGF-BB. Calvaria defects were randomly divided into the following treatment groups (n = 5); TCP; TCP plus PDGF-BB; DBBM; DBBM plus PDGF-BB; and untreated empty control. After 45 days, bone formation was evaluated by histomorphometry and fluorescence microscopy. RESULTS: The authors report that the area of newly formed bone was similar between the empty controls and the two bone substitutes, TCP and DBBM. Supplementation of TCP and DBBM with PDGF-BB had no significant impact on bone formation. Fluorochrome staining revealed no visible changes in the pattern of bone formation in defects filled with TCP and DBBM, irrespective of PDGF-BB. Furthermore, supplementation with PDGF-BB did not influence biomaterial degradation. CONCLUSIONS: The authors concluded that PDGF-BB had no impact on bone formation and degradation of bone substitutes in the respective rodent models. Thus, possible beneficial effects of PDGF-BB may require other model situations.

Bone substitute materials supplemented with prolyl hydroxylase inhibitors decrease osteoclastogenesis in vitro.

BACKGROUND AND OBJECTIVE: Inhibition of prolyl hydroxylases stimulates bone regeneration. Consequently, bone substitute materials were developed that release prolyl hydroxylase inhibitors. However, the impact of prolyl hydroxylase inhibitors released from these carriers on osteoclastogenesis is not clear. We therefore assessed the effect of bone substitute materials that release prolyl hydroxylase inhibitors on osteoclastogenesis. MATERIAL AND METHODS: Dimethyloxalylglycine, desferrioxamine, and l-mimosine were lyophilized onto bovine bone mineral and hydroxyapatite, and supernatants were generated. Osteoclastogenesis was induced in murine bone marrow cultures in the presence of the supernatants from bone substitute materials. The formation of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells and TRAP activity were determined. To test for possible effects on osteoclast progenitor cells, we measured the effect of the supernatants on proliferation and viability. In addition, experiments were performed where prolyl hydroxylase inhibitors were directly added to the bone marrow cultures. RESULTS: We found that prolyl hydroxylase inhibitors released within the first hours from bone substitute materials reduce the number and activity of TRAP-positive multinucleated cells. In line with this, addition of prolyl hydroxylase inhibitors directly to the bone marrow cultures dose-dependently reduced the number of TRAP-positive multinucleated cells and the overall resorption activity. Moreover, the released prolyl hydroxylase inhibitors decreased proliferation but not viability of osteoclast progenitor cells. CONCLUSION: Our results show that prolyl hydroxylase inhibitors released from bone substitute materials decrease osteoclastogenesis in murine bone marrow cultures.

Alveolar bone regeneration in response to local application of calcitriol in vitamin D deficient rats.

AIM: Vitamin D deficiency is considered to diminish bone regeneration. Yet, raising the serum levels takes months. A topic application of the active vitamin D metabolite, calcitriol, may be an effective approach. Thus, it becomes important to know the effect of vitamin D deficiency and local application on alveolar bone regeneration. MATERIAL AND METHODS: Sixty rats were divided into three groups; two vitamin depletion groups and a control group. Identical single defects (2 mm diameter) were created in the maxilla and mandible treated with calcitriol soaked collagen in one deficiency group while in the other two groups not. Histomorphometric analysis and micro CTs were performed after 1 and 3 weeks. Serum levels of 25(OH)D3 and PTH were determined. RESULTS: Bone formation rate significantly increased within the observation period in all groups. Bone regeneration was higher in the maxilla than in the mandible. However, bone regeneration was lower in the control group compared to vitamin depletion groups, with no significant effects by local administration of calcitriol (micro CT mandible p = 0.003, maxilla p < 0.001; histomorphometry maxilla p = 0.035, mandible p = 0.18). CONCLUSION: Vitamin D deficiency not necessarily impairs bone regeneration in the rat jaw and a single local calcitriol application does not enhance healing.

Prolyl hydroxylase inhibitors increase the production of vascular endothelial growth factor in dental pulp-derived cells.

INTRODUCTION: Prolyl hydroxylase (PHD) inhibitors can induce a proangiogenic response that stimulates regeneration in soft and hard tissues. However, the effect of PHD inhibitors on the dental pulp is unclear. The purpose of this study was to evaluate the effects of PHD inhibitors on the proangiogenic capacity of human dental pulp-derived cells. METHODS: To test the response of dental pulp-derived cells to PHD inhibitors, the cells were exposed to dimethyloxalylglycine, desferrioxamine, L-mimosine, and cobalt chloride. To assess the response of dental pulp cells to a capping material supplemented with PHD inhibitors, the cells were treated with supernatants from calcium hydroxide. Viability, proliferation, and protein synthesis were assessed by formazan formation, (3)[H]thymidine, and (3)[H]leucine incorporation assays. The effect on the proangiogenic capacity was measured by immunoassays for vascular endothelial growth factor (VEGF). RESULTS: We found that all 4 PHD inhibitors can reduce viability, proliferation, and protein synthesis at high concentrations. At nontoxic concentrations and in the presence of supernatants from calcium hydroxide, PHD inhibitors stimulated the production of VEGF in dental pulp-derived cells. When calcium hydroxide was supplemented with the PHD inhibitors, the supernatants from these preparations did not significantly elevate VEGF levels. CONCLUSIONS: These results show that PHD inhibitors can stimulate VEGF production of dental pulp-derived cells, suggesting a corresponding increase in their proangiogenic capacity. Further studies will be required to understand the impact that this might have on pulp regeneration.

Impact of dietary vitamin D on osseointegration in the ovariectomized rat.

AIM: Vitamin D deficiency is highly prevalent in the population and associated with impaired peri-implant bone regeneration. Yet, there is a gap in understanding the impact of vitamin D supplementation on the process of osseointegration. In this study, the effect of vitamin D supplementation on peri-implant bone regeneration was investigated. METHODS: Fifty ovariectomized Sprague-Dawley rats were divided into three groups. The depletion group was fed a vitamin D-free diet for 8 weeks. The repletion group received vitamin D-free diet for 6 weeks, before animals were switched to standard diet containing 2400 IU/kg vitamin D. The control group was fed the standard diet. Two titanium mini-implants were placed in the tibia. All groups remained on their previous diet until sacrifice. Blood sample testing and histomorphometric analysis were performed. RESULTS: Vitamin D depletion caused a significant reduction in 25-hydroxvitamin D in rat serum that returned to control levels in the repletion group. This vitamin deficiency was associated with a decrease in bone-to-implant contact in the cortical area, which was leveled to controls in the repletion group. No significant changes by vitamin D depletion were noticed in the medullar compartment. Moreover, also the peri-implant bone area and the mineral apposition rate remained unchanged upon vitamin D depletion. CONCLUSION: These results indicate that vitamin D deficiency has a negative impact on cortical peri-implant bone formation in ovariectomized rats, which can be compensated by vitamin D supplementation. This study provides first insight into the potential beneficial effect of vitamin D supplementation in implant dentistry.

The effect of BMP-2 on the osteoconductive properties of ß-tricalcium phosphate in rat calvaria defects.

Bone formation in critical-sized calvaria defects is strongly dependent on the osteoconductive properties of grafts. It remains a matter of controversy whether biomaterials can replace autografts and whether the supplementation of biomaterials with Bone Morphogenetic Proteins (BMPs) is necessary to enhance bone formation. We examined rat calvaria critical-sized defects (5-mm-diameter) treated with ß-tricalcium phosphate (TCP; Cerasorb® M), polylactic and polyglycolic acid gel (PLA/PGA; Fisiograft®) and calcium phosphate cement (CPC; Norian® CRS®), either alone or in the presence of 5 µg of BMP-2 after 45 days. Autografts and untreated defects served as controls. Bone formation was evaluated based on µCT analysis, histomorphometric analysis and fluorescence analysis. We report that TCP supported bone formation more efficiently than did autografts. Bone formation in the presence of TCP alone reached a maximal level, as BMP-2 supplementation failed to enhance bone formation. By contrast, no significant difference in bone formation was observed when PLA/PGA and CPC were compared to autografts. Moreover, the presence of BMP-2 did not substantially change the osteoconductive properties of PLA/PGA or CPC. We conclude that the osteoconductive properties of TCP are superior to those of autografts and that TCP does not require BMP-2 supplementation. Our findings also show that the decreased osteoconductive properties of PLA/PGA and CPC cannot be overcome by BMP-2 supplementation in rat calvaria defects.

Paste-like inorganic bone matrix: preclinical testing of a prototype preparation in the porcine calvaria.

OBJECTIVE: To evaluate the osteoconductive properties and the volume stability of an injectable paste-like inorganic bone matrix (PBM) in porcine calvaria defects. MATERIAL AND METHODS: We created six circumferential defects in the calvaria of 12 adult iberico pigs. The defects were filled with either PBM, Bio-Oss((R)) of different particle size, carrier alone, or left empty. PBM was composed of Bio-Oss((R)) with a particle size ranging from 250 to 500 mum and a hydrogel-carrier of carboxymethylcellulose and collagen. After 6 and 12 weeks of healing, the animals were sacrificed and undecalcified ground sections were prepared and subjected to histologic and histomorphometric analysis. To quantify the osteoconductive properties of PBM, bone volume per tissue volume (BV/TV) in the defect area was determined. To determine the volume stability, bone substitute volume per tissue volume (BSV/TV) was measured. RESULTS: After 6 weeks, PBM particles in the center of the defect were surrounded by fibrous connective tissue, which was later replaced by bone. BV/TV in the PBM group increased from 29.7+/-12.7% (minimum 12.2%, maximum 43.7%) after 6 weeks to 43.9+/-14.9% (minimum 27.8%, maximum 63.9%) after 12 weeks (Mann-Whitney test; P=0.6). According to the Friedman test, BV/TV in groups containing Bio-Oss((R)) of different particle sizes, the carrier and the empty defects was similar to the results obtained with PBM (6 weeks P=0.8; 12 weeks P=0.22). BSV/TV in the PBM group was stable over time, with 10.1+/-9% (minimum 3.3%, maximum 27.6%) and 16.5+/-12.9% (minimum 1%, maximum 32.7%), after 6 and 12 weeks, respectively (P=0.72). BSV/TV in the PBM group was comparable to the results obtained with the Bio-Oss((R)) particles of different sizes (Friedman test; 6 weeks P=0.0503; 12 weeks P=0.56). CONCLUSIONS: The results of this preclinical study showed that the PBM is osteoconductive and maintains the augmented volume, similar to commercial Bio-Oss((R)). These data suggest that the osteoconductive properties of Bio-Oss((R)) are maintained at the smaller particle size and in the presence of the carrier.

Bone formation following sinus grafting with autogenous bone-derived cells and bovine bone mineral in minipigs: preliminary findings.

Bone formation in a sinus grafted with a cell-free scaffold requires the presence of local progenitor cells that differentiate into osteoblasts. The purpose of this study was to examine the effect of culture expanded autogenous bone-derived cells (ABC) with bovine bone mineral (BBM) on bone formation after single-stage sinus grafting in minipigs. Bone biopsies from the iliac crest were harvested 4 weeks prior to sinus grafting and ABC were culture expanded in vitro. The sinuses of five adult minipigs were grafted. In one sinus of each minipig the space between Schneider's membrane (SM) and the sinus wall was grafted with ABC (325,000 cells per sinus, on average) and BBM. In the other sinus, BBM alone was used. The animals were sacrificed after 12 weeks. One block of each grafted area was prepared by saw cutting and the amount of newly formed bone was analysed by micro-computed tomography (mu-CT). The addition of ABC to BBM significantly increased the amount of newly formed bone compared with BBM alone on mu-CT analysis (ABC+BBM: 29.86+/-6.45% vs. BBM: 22.51+/-7.28% (P=0.016)). Bone formation was increased near SM (ABC+BBM: 20.7+/-4.5% vs. BBM: 15.43+/-3.62% (P=0.009)) and in the middle zone of the grafting material (ABC+BBM: 31.63+/-7.74% vs. BBM: 22.5+/-7.91% (P=0.001)), but not near the local host bone (ABC+BBM: 37.23+/-8.23% vs. BBM: 28.42+/-12.54% (P=0.15)). These preliminary findings indicate that supplementation of cell-free grafting material with culture expanded ABC can stimulate bone formation in areas with low bone-forming capacity.

Platelet-released supernatants increase migration and proliferation, and decrease osteogenic differentiation of bone marrow-derived mesenchymal progenitor cells under in vitro conditions.

Platelet-rich plasma is currently promoted to serve as an adjuvant for bone grafts to enhance quantity and quality of newly forming bone; however, the underlying cellular mechanisms are not fully understood. We show here that supernatants of leukocyte-depleted thrombin-activated platelets increase migration and proliferation, and decrease osteogenic differentiation of bone marrow-derived mesenchymal progenitor cells under in vitro conditions. Using neutralizing antibodies raised against platelet-derived growth factor (PDGF), the observed effects of platelet-released supernatants were diminished. The mitogenic response was also decreased when extracellular signal-regulated protein kinase (ERK) signalling was inhibited by PD98059; however, PD98059 did not reverse the effects of platelet-released supernatants on migration and osteogenic differentiation. Consistent with an ERK-mediated mitogenic activity, incubation of serum-starved mesenchymal cell progenitors with platelet-released supernatants increased phosphorylation of the kinase. Together, these observations indicate that PDGF is a key factor released upon platelet activation that can increase migration and proliferation, and decreases osteogenic differentiation of mesenchymal progenitor cells under in vitro conditions. The results further suggest that ERK signalling is required to mediate the mitogenic response to platelet-released supernatants.