Higher Expression Of Osteocalcin Research Articles

Autogenous dentin combined with mesenchymal stromal cells as an alternative alveolar bone graft: an in vivo study.

Considering the chemical and structural properties of dentin, this study was aimed at evaluating the effect of dentin matrix alone or combined with mesenchymal stromal cells (MSC) on postextraction alveolar bone regeneration. Wistar rats were subjected to tooth extraction with osteotomy and allocated into groups according to the graft inserted: (1) Gelita-Spon®, (2) Bio-Oss®, (3) Dentin, (4) MSC, (5) Dentin/MSC, and (6) Control. Maxillae were analyzed by means of hematoxylin and eosin (H&E) staining, immunohistochemical (IHC) analysis, microcomputed tomography (micro-CT), and scanning electron microscopy (SEM). Serum levels of calcium and phosphorus were quantified. The Bio-Oss group showed less bone than Gelita-Spon and Dentin/MSC; no other significant differences were seen in H&E analysis. The Bio-Oss group showed higher expression of collagen type I compared to the Dentin and Dentin/MSC groups and also higher osteocalcin expression than the Dentin/MSC group. There was a tendency of higher expression of osteopontin in the MSC, Dentin, and Dentin/MSC groups and higher VEGF in the MSC group. On micro-CT analysis, the Bio-Oss and the Dentin/MSC groups exhibited greater bone volume than the Control. Serum calcium and phosphorus levels did not significantly differ between the groups. SEM analysis depicted particles of Bio-Oss and dentin in the respective groups, as well as significant cellularity in the MSC group. Autogenous nondemineralized dentin is an alternative for alveolar bone grafting, which can be improved by combination with MSC. This work provides support for the clinical applicability of dentin graft alone or combined with MSC.

Immunohistochemistry assay of osteocalcin on bone healing with CHA-10% propolis

Objective: To evaluate the effect of the application of carbonated hydroxyapatite-propolis 10% on open flap debridement towards osteocalcin expression of periodontitis induced Oryctolagus cuniculus.
 Material and Methods: Six rabbits (Oryctolagus cuniculus) were divided into three groups, which are carbonated hydroxyapatite-propolis 10% group, carbonated hydroxyapatite group, and open flap debridement group. Periodontitis on animal models was inducted with a combined technique using ligation and LPS injection of P.gingivalis. On each group, the animals were decapitated on day 7 and day 14 after the designated treatment. The immunohistochemistry assay was used to measure the osteocalcin expression. The data were analyzed statistically with Two Way Anova and continued with post hoc LSD.
 Results: The results showed that the carbonated hydroxyapatite-propolis 10% group had the highest osteocalcin expression on day 7, followed by carbonated hydroxyapatite group and the open flap debridement group (p<0,05). There was no significant difference in the expression of osteocalcin on day 7 and day 14 (p>0,05) on the carbonated hydroxyapatite-10% propolis group.
 Conclusion: The application of carbonated hydroxyapatite-propolis 10% could increase the osteocalcin expression on periodontitis induced Oryctolagus cuniculus.

Central giant cell granulomas of the jaws stromal cells harbour mutations and have osteogenic differentiation capacity, in vivo and in vitro.

Central giant cell granulomas (CGCG) of the jaws are osteolytic lesions that may behave aggressively and respond poorly to surgery. Microscopically, in addition to giant cells, there is a mononuclear cell population composed of macrophage/monocytic cells and spindle-shaped cells of mesenchymal origin. Seventy two percent of these tumours harbour mutually exclusive TRPV4, KRAS and FGFR1mutations. We aimed to assess the mutational status of mononuclear and giant cells and the osteogenic potential of stromal cells in vitro and in vivo. We screened CGCG for signature mutations and used laser-capture microdissection to demonstrate that the mutations are restricted to the mononuclear cells. Additionally, we established CGCG primary cell culture and observed that the cells retained the mutations throughout passages. By flow cytometry, we observed predominance of CD14- CD51- CD61- cells, consistent with the expected profile for stromal cells. Considering the mesenchymal origin of stromal cells, we assessed the osteogenic differentiation potential of CGCG cells in culture by cytochemistry (von Kossa and alizarin red staining), alkaline phosphatase (ALP) activity assay and gene expression of osteogenic markers. CGCG cells presented self-capacity to increase ALP levels in a time-dependent manner and under osteogenic induction presented increasing number of calcium deposits, and overall higher expression of osteocalcin, RUNX2, ALPL and osteopontin than cells without osteogenic induction. A patient-derived xenograft model for CGCG was established, and osteoid material deposition was observed. Collectively, the results confirm that the signature mutations are restricted to stromal cells in CGCG, and the in vitro and in vivo results support that these cells have the capacity to differentiate into osteoblasts, in line with the bone formation often observed in the stroma of these lesions.

Characterization of dental pulp stem cells isolated from a patient diagnosed with Crouzon syndrome.

Stem cells isolated from patients with rare diseases are important to elucidate their pathogeny and mechanisms to enable regenerative therapy. However, the mechanisms underlying tissue regeneration using patient‐derived dental pulp stem cells (DPSCs) are unclear. In this study, we investigated the levels of mRNA and protein expression related to cellular differentiation of Crouzon syndrome patient‐derived DPSCs (CS‐DPSCs) with a Gly338Arg fibroblast growth factor receptor 2 mutation. Multipotency‐related gene expression levels were equivalent in both healthy donor DPSCs and CS‐DPSCs. CS‐DPSCs showed higher osteocalcin (OCN) expression than healthy donor DPSCs. CS‐DPSCs showed a lower increase in the rate of OCN expression among phorbol 12‐myristate 13‐acetate (PMA)‐treated cells than healthy donor DPSCs compared with untreated control cells. CS‐DPSCs showed a lower phosphorylation rate of p38 and p44/42 in PMA‐treated cells than healthy donor DPSCs compared with untreated control cells. These results demonstrate that CS‐DPSCs have higher OCN expression and lower PMA stimulation‐responsiveness than healthy donor DPSCs.

Low magnitude high frequency vibrations expedite the osteogenesis of bone marrow stem cells on paper based 3D scaffolds.

Anabolic effects of low magnitude high frequency (LMHF) vibrations on bone tissue were consistently shown in the literature in vivo, however in vitro efforts to elucidate underlying mechanisms are generally limited to 2D cell culture studies. Three dimensional cell culture platforms better mimic the natural microenvironment and biological processes usually differ in 3D compared to 2D culture. In this study, we used laboratory grade filter paper as a scaffold material for studying the effects of LHMF vibrations on osteogenesis of bone marrow mesenchymal stem cells in a 3D system. LMHF vibrations were applied 15 min/day at 0.1 g acceleration and 90 Hz frequency for 21 days to residing cells under quiescent and osteogenic conditions. mRNA expression analysis was performed for alkaline phosphatase (ALP) and osteocalcin (OCN) genes, Alizarin red S staining was performed for mineral nodule formation and infrared spectroscopy was performed for determination of extracellular matrix composition. The highest osteocalcin expression, mineral nodule formation and the phosphate bands arising from the inorganic phase was observed for the cells incubated in osteogenic induction medium with vibration. Our results showed that filter paper can be used as a model scaffold system for studying the effects of mechanical loads on cells, and LMHF vibrations induced the osteogenic differentiation of stem cells.

Investigation of The Cellular Response to Bone Fractures: Evidence for Flexoelectricity

The recent discovery of bone flexoelectricity (strain-gradient-induced electrical polarization) suggests that flexoelectricity could have physiological effects in bones, and specifically near bone fractures, where flexoelectricity is theoretically highest. Here, we report a cytological study of the interaction between crack stress and bone cells. We have cultured MC3T3-E1 mouse osteoblastic cells in biomimetic microcracked hydroxyapatite substrates, differentiated into osteocytes and applied a strain gradient to the samples. The results show a strong apoptotic cellular response, whereby mechanical stimulation causes those cells near the crack to die, as indicated by live-dead and caspase staining. In addition, analysis two weeks post-stimulation shows increased cell attachment and mineralization around microcracks and a higher expression of osteocalcin –an osteogenic protein known to be promoted by physical exercise. The results are consistent with flexoelectricity playing at least two different roles in bone remodelling: apoptotic trigger of the repair protocol, and electro-stimulant of the bone-building activity of osteoblasts.

The Role of Strontium Enriched Hydroxyapatite and Tricalcium Phosphate Biomaterials in Osteoporotic Bone Regeneration

Background: Strontium (Sr) enriched biomaterials have been used to improve bone regeneration in vivo. However, most studies provide only two experimental groups. The aim of our study was to compare eleven different bone sample groups from osteoporotic and healthy rabbits’ femoral neck, as it is the most frequent osteoporotic fracture in humans. Methods: Osteoporotic bone defects were filled with hydroxyapatite 30% (HA) and tricalcium phosphate 70% (TCP), 5% Sr-enriched HA30/TCP70, HA70/TCP30, or Sr-HA70/TCP30 granules and were compared with intact leg, sham surgery and healthy non-operated bone. Expression of osteoprotegerin (OPG), nuclear factor kappa beta 105 (NFkB-105), osteocalcin (OC), bone morphogenetic protein 2/4 (BMP-2/4), collagen I (Col-1α), matrix metalloproteinase 2 (MMP-2), tissue inhibitor of matrix metalloproteinase 2 (TIMP-2), interleukin 1 (IL-1) and interleukin 10 (IL-10) was analyzed by histomorphometry and immunohistochemistry. Results: Our study showed that Sr-HA70/TCP30 induced higher expression of all above-mentioned factors compared to intact leg and even higher expression of OC, MMP-2 and NFkB-105 compared to Sr-HA30/TCP70. HA70/TCP30 induced higher level of NFkB-105 and IL-1 compared to HA30/TCP70. Conclusion: Sr-enriched biomaterials improved bone regeneration at molecular level in severe osteoporosis and induced activity of the factors was higher than after pure ceramic, sham or even healthy rabbits.

Loading deproteinized bovine bone with strontium enhances bone regeneration in rat calvarial critical size defects

ObjectiveTo evaluate the effect of grafting with strontium (Sr)-loaded deproteinized bovine bone (DBB) on bone healing in calvarial critical size defects (CSD) in rats.Material and methodsTwo circular bone defects (5 mm in diameter) were created in the calvaria of 42 rats. One of the defects, randomly chosen, was grafted with (a) DBB, (b) DBB loaded with 19.6 μg/g of Sr (DBB/Sr1), or (c) DBB loaded with 98.1 μg/g of Sr (DBB/Sr2). The other defect was left empty as negative control. Groups of seven animals from each of the groups were euthanized 15 and 60 days post-op. Bone healing in the CSD was evaluated by micro-CT and histology/histomorphometry and immunohistochemistry.ResultsDBB/Sr2-grafted sites showed statistically significantly shorter radiographic residual defect length compared with DBB/Sr1- and DBB-grafted sites, and with empty controls at 60 days. Further, the amount of new bone formation in the DBB/Sr1- and DBB/Sr2-grafted sites was significantly higher compared with that in the DBB-grafted sites at 60 days. A larger number of DBB/Sr1- and DBB/Sr2-grafted sites presented with no- or only limited to mild inflammation, compared with the DBB-grafted sites, especially at 60 days. Higher expression of osteocalcin was observed in DBB/Sr1- and DBB/Sr2-grafted sites as compared to DBB-grafted sites.ConclusionGrafting with Sr-loaded DBB enhanced bone formation in CSD in rats, when compared with grafting with non-loaded DBB.Clinical relevanceGrafting with Sr-loaded DBB may enhance bone formation in bone defects.

An innovative Mg/Ti hybrid fixation system developed for fracture fixation and healing enhancement at load-bearing skeletal site

Magnesium (Mg) is a potential biomaterial suitable for developing biodegradable orthopaedic implants, especially as internal fixators for fracture fixation at non-load bearing skeletal sites. However, Mg alone cannot provide sufficient mechanical support for stable fracture fixation at load bearing sites due to its rapid degradation in the early stage after implantation. In consideration of the strengths and weaknesses of Mg, we developed an innovative magnesium/titanium (Mg/Ti) hybrid fixation system for long bone fracture fixation and investigated the fixation efficacy. The finite element analysis (FEA) results indicated that the Mg/Ti hybrid fixation system provided sufficient mechanical support for fracture fixation at load-bearing skeletal site. As a proof-of-concept, we performed a “Z-shaped” open osteotomy at the mid-shaft of rabbit tibia. For comparison, the animals were divided into two groups: Mg/Ti group (fixated with Mg screws and Ti fixators) and Ti control group (fixated with Ti screws and Ti fixators). The radiographic, four-point bending mechanical test, histological and histomorphometric analysis were postoperatively performed in a temporal manner up to 12 weeks. Both X-ray and micro-CT images of the Mg/Ti group showed a larger callus (14.7% at 3rd week and 24.8% at 6th week, n = 5–7, p < 0.05) in the regions of interest (ROIs) over time, especially at the opposite cortex of the fixation plate. At the 12th week post-operation, the biomechanical test result indicated that the rabbit tibia in the Mg/Ti group healed better and the overall mechanical strength was approximately 3-fold higher (n = 8, p < 0.05) than that at 6th week. Furthermore, the FEA revealed that the Mg/Ti group had a higher mechanical strength (19.5% at week 6 and 31.5% at week 12) at the specified ROI and resulted in an earlier and faster endochondral ossification (68.0% at week 3 and 71.4% at week 6) with a higher expression of osteocalcin (54.0%) and collagen I (34.2%) than the Ti control group (n = 4, p < 0.05). Further evaluation suggested that a higher expression of calcitonin gene-related peptide (CGRP), a known osteogenic neuron peptide, in the fracture callus of the Mg/Ti group might be a major underlying mechanism of enhanced fracture healing attributed to the release of Mg ions during the degradation of Mg screws.

970 nm low-level laser affects bone metabolism in orthodontic tooth movement

ObjectiveDuring orthodontic tooth movement (OTM), the speed of movement depends on the rate of bone turnover. In this study, we used a rat model to investigate the effect of 970 nm low-level laser therapy (LLLT) on OTM under different dose and frequency protocols. MethodsWe first compared the OTM rates between the OTM only control and the OTM + LLLT group (1250 J/cm2) in Experiment 1 and showed that LLLT significantly increased OTM. In Experiment 2, we employed 3 different LLLT protocols: the low-dose group and the high-dose group receiving 5 doses of 750 J/cm2 and 15,000 J/cm2 of LLLT every 3 days, respectively, and the early high-dose group which received 5 daily doses at 15,000 J/cm2 at the beginning of the experiments. The OTM-only control group received no LLLT. Tooth movement rate was measured through sequential silicone impressions. MicroCT was also performed to evaluate bone de-mineralization rate. Bone histmorphometry was used to compare the bone turnover rate between LLLT group and control group. Finally, TRAP, Osteocalcin, and VEGF expression is evaluated by immunohistochemistry (IHC) in tissue sections. ResultsWhen LLLT treatment was given every three days, both the 1250 J/cm2 and 15,000 J/cm2 groups showed significantly increased OTM compared to the control group. No significant difference was observed in the 750 J/cm2 group, or in the early irradiation group, when compared with controls, although 750 J/cm2 showed the same trend of accelerating OTM. The MicroCT result of rat maxilla demonstrated that LLLT increased bone remodeling and showed decreased bone mineral density and bone volume/total volume in the furcation areas of the maxillary first molars at the end of experiment. LLLT without OTM increased bone turnover as evidenced by fluorochrome incorporation. Immunohistochemistry analyses revealed high osteocalcin expression at later stages of OTM in the LLLT group, while VEGF expression was highly induced in the LLLT + OTM group at an early stage. ConclusionOur results suggest that the 970 nm LLLT increases the rate of OTM in a dose-sensitive and frequency-dependent manner. Further animal and human studies are needed to determine the optimal timing and dosage of LLLT for OTM acceleration.

Effect of Avocado/Soybean Unsaponifiables (ASU) on Osseointegration in Rats with Experimental Arthritis.

To evaluate the influence of the use of avocado/soybean unsaponifiables (ASU) on osseointegration of implants in animals with experimental arthritis. One hundred twenty rats were randomly divided into four groups: CTR, healthy animals and saline solution administration; ASU, healthy animals and ASU administration; ART, arthritic animals and saline solution administration; and ART/ASU, arthritic animals and ASU administration. The solutions were administered daily by gavage, beginning 7 days before the surgical procedures until the completion of the experimental period (15, 30, and 60 days after the placement of the implants in the tibia). The osseointegration of the implants was evaluated by histometric analysis (bone-to-implant contact [% BIC], bone area between the threads [% BBT]) and biomechanical analysis. Microcomputed tomography (micro-CT) analysis was used to assess bone volume in the vicinity of the implant. Immunohistochemistry analysis was performed to assess the expression of osteocalcin and transforming growth factor beta 1 (TGF-β1). The ART/ASU group showed a decreased percentage of bone in the area around the implant compared with the ASU and ART groups (15 and 30 days). The ART/ASU group showed increased removal torque values (30 days) and % BIC and % BBT (30 to 60 days) compared with the ART group. The ASU group had increased % BIC values compared with the ART and CTR groups (60 days). The CTR group had the highest expression of osteocalcin, while the ASU group presented the highest expression of TGF-β1 at 60 days. The ASU administration improved the osseointegration, particularly in animals with induced arthritis.

Potential differentiation ability of gingiva originated human mesenchymal stem cell in the presence of tacrolimus.

The aim of the present study is to evaluate the potential differentiation ability of gingiva originated human mesenchymal stem cell in the presence of tacrolimus. Tacrolimus-loaded poly(lactic-co-glycolic acid) microspheres were prepared using electrospraying technique. In vitro release study of tacrolimus-loaded poly(lactic-co-glycolic acid) microspheres was performed in phosphate-buffered saline (pH 7.4). Gingiva-derived stem cells were isolated and incubated with tacrolimus or tacrolimus-loaded microspheres. Release study of the microspheres revealed prolonged release profiles of tacrolimus without any significant initial burst release. The microsphere itself did not affect the morphology of the mesenchymal stem cells, and cell morphology was retained after incubation with microspheres loaded with tacrolimus at 1 μg/mL to 10 μg/mL. Cultures grown in the presence of microspheres loaded with tacrolimus at 1 μg/mL showed the highest mineralization. Alkaline phosphatase activity increased with an increase in incubation time. The highest expression of pSmad1/5 was achieved in the group receiving tacrolimus 0.1 μg/mL every third day, and the highest expression of osteocalcin was achieved in the group receiving 1 μg/mL every third day. Biodegradable poly(lactic-co-glycolic acid)-based microspheres loaded with tacrolimus promoted mineralization. Microspheres loaded with tacrolimus may be applied for increased osteoblastic differentiation.

The effect of surface treatment of titanium with sand-blasting/acid-etching or hydroxyapatite-coating and application of bone morphogenetic protein-2 on attachment, proliferation, and differentiation of stem cells derived from buccal fat pad

This study was performed to evaluate the effect of surface treatment of titanium, including: machined (MA), sand-blasted and acid-etched (SA), and hydroxyapatite-coated (HA) surfaces, and application of bone morphogenetic protein-2 (BMP-2) on attachment, proliferation, and differentiation of stem derived from buccal fat pad. Stem cells were isolated from buccal fat pad. Stem cells were cultured on MA, SA, and HA titanium discs with and without BMP-2. Cellular attachment and alkaline phosphatase (ALP) activity were evaluated at day 3, 7, and 14. Osteocalcin expression was performed using immunofluorescent assay. The cells grown on MA surface demonstrated good attachment with spindle-like morphology, and cells grown on SA and HA surfaces demonstrated more stellate-like morphology. An increase of ALP activity was seen in the rough surface (SA and HA groups) and a statistically significant increase was seen in the HA group (p < 0.05). The addition of BMP-2 made an expression of osteocalcin, and the highest expression of osteocalcin was noted in the HA group. Within the limits of this study, surface modification of titanium surfaces by sand-blasting/acid-etching or hydroxyapatite-coating was not able to allow the stem cells to differentiate into osteoblasts without exogenous soluble factors. The addition of BMP-2 showed osteogenic differentiation of stem cells and HA-coated surfaces demonstrated the highest ALP and osteocalcin expression.

Ectopic study of calcium phosphate cement seeded with pBMP-2 modified canine bMSCs mediated by a non-viral PEI derivative

We have evaluated the ectopic new bone formation effects of CPC (calcium phosphate cement) seeded with pBMP-2 (plasmids containing bone morphogenetic protein-2 gene) transfected canine bMSCs (bone marrow stromal cells) mediated by a non-viral PEI (polyethylenimine) derivative (GenEscort™ II) in nude mice. Canine bMSCs were transfected with pBMP-2 or pEGFP (plasmids containing enhanced green fluorescent protein gene) mediated by GenEscort™ II in vitro, and the osteoblastic differentiation was explored by ALP (alkaline phosphatase) staining, ARS (alizarin red S) staining and RT-qPCR (real-time quantitative PCR) analysis. Ectopic bone formation effects of CPC/pBMP-2 transfected bMSCs were evaluated and compared with CPC/pEGFP transfected bMSCs or CPC/untransfected bMSCs through histological, histomorphological and immunohistochemical analysis 8 and 12 weeks post-operation in nude mice. Transfection efficiency was up ∼35% as demonstrated by EGFP (enhanced green fluorescent protein) expression. ALP and ARS staining were stronger with pBMP-2 gene transfection, and mRNA expression of BMP-2 (bone morphogenetic protein-2), Col 1 (collagen 1) and OCN (osteocalcin) in pBMP-2 group was significantly up-regulated at 6 and 9 days. Significantly higher NBV (new bone volume) was achieved in pBMP-2 group than in the control groups at 8 and 12 weeks (P<0.05). In addition, immunohistochemical analysis indicated higher OCN expression in pBMP-2 group (P<0.01). We conclude that CPC seeded with pBMP-2 transfected bMSCs mediated by GenEscort™ II could enhance ectopic new bone formation in nude mice, suggesting that GenEscort™ II mediated pBMP-2 gene transfer is an effective non-viral method and CPC is a suitable scaffold for gene enhanced bone tissue engineering.

Microporous Biphasic Calcium Phosphate Granules (MBCP®) Retain Immunological Properties of Bone Marrow-Derived Mesenchymal Stromal Cells and Promote Osteoblastic Differentiation

Abstract 1924Bone is among the most frequently transplanted tissue with about 1 million procedures annually in Europe. Allografts and autografts account for more than 80% of total graft volume, despite their considerable disadvantages, including the risk of disease transfer and immunologic rejection, limited supply of bone, costs and complications. Significant growth opportunities exist for synthetic bone grafts in association with mesenchymal stromal cells (MSC) from autologous or allogeneic sources as alternatives to biological bone grafts in orthopaedic and maxillofacial surgery. The objective of REBORNE is to perform clinical trials using advanced biomaterials and cells triggering bone healing in patients. To reach this goal, five phase I clinical studies with 20 patients have been planned in 12 clinical Centers spread in 8 European countries.Aim of the Immunological Unit of Reborne is to assess the MSC immunomodulatory properties in presence of the biomaterial used as scaffold for MSC delivery. All the functional experiments were performed in parallel, by comparing the effects of standard culture conditions and three-dimensional culture setting using MBCP (Biomatlante).Material and methods: Bone marrow MSC were provided from REBORNE Consortium Centers. To perform proliferation assays, different immune effector cells (T, B and NK cells) were stained with CFSE according to manufacturer's protocol. Active caspase-3 cell staining was used for survival quantification of immune effector cells after co-culture experiments. Differentiation potential was evaluated by culturing MSC with two different media containing either bone morphogenetic protein 4 (BMP4) or dexamethasone. After three weeks, osteogenic differentiation was quantified by qRT-PCR, alkaline phosphatase activity and alizarin red staining. Results:We found that primed MSC, pre-treated with the inflammatory cytokines IFNg and TNFa, displayed upregulation of HLA-ABC, CD54, CD106 and de novo expression of HLA-DR, both in standard culture conditions and in association with MBCP. Immune effector cells could be cultured and collected even in presence of MBCP and no significant differences were found between standard- (MSC + effector cells) and 3D-coculture conditions (MSC + MBCP + effector cells), in terms of immune effector cell proliferation. In both experimental conditions MSC suppressed T and NK cell proliferation (% suppression: MSC + T = 68.4; MSC + MBCP + T = 62.4; MSC + NK = 17.5; MSC + MBCP + NK = 20.2) and increased B cell proliferation (MSC + B = +13%; MSC + MBCP + B = +12.3%). In addition, immune effector cells viability was not affected by MBCP and MSC co-culture increased their survival even in presence of MBCP; in fact, in each culture condition the percentage of inhibition of T, B and NK cell apoptosis was higher than 20% in comparison to immune effector cells cultured without MSC.Dexamethasone and BMP4 were capable of inducing MSC differentiation into osteoblast-like cells, as confirmed by qRT-PCR analysis. We demonstrated that BMP4-based medium led to fully differentiated osteoblasts (Osterix+, RUNX2+, DLX5+ and alkaline-phosphatase+). Moreover, MBCP was more efficient in increasing osteoblastic differention as compared to standard culture conditions, as shown by the higher expression of Osteocalcin and Osterix.These data show that the association of MBCP and MSC does not affect MSC properties and suggest that it could be a treatment of choice of bone defects instead of allograft and autograft transplantation. Disclosures:No relevant conflicts of interest to declare.

A novel form of electrical stimulation increases osteoblast activity: potential implications for enhanced fracture healing

Delayed facture repair and bony non-unions pose a clinical challenge. Understandably, novel methods to enhance bone healing have been studied by researchers worldwide. Electrical stimulation (ES) has shown to be effective in enhancing bone healing, however the best wave form and mechanism by which it stimulates osteoblasts remains unknown. Interestingly, it is considered that osteoblast activity depends on specific waveforms applied. Therefore, the aim of this study was to evaluate whether particular waveforms have a differential effect on osteoblast activity. An osteoblast cell line was electrically stimulated with either capacitive coupling (CC) or a novel degenerate wave (DW) using a unique in vitro ES system. Following application of both waveforms, the extent of cytotoxicity, proliferation, differentiation and mineralisation of the osteoblasts were assessed using various assays. Differentiation and mineralisation were further analysed using quantitative real-time PCR (qRT PCR) and immunocytochemistry (ICC). DW stimulation significantly enhanced the differentiation of the osteoblasts compared to CC stimulation, with increased protein and gene expression of alkaline phosphatase and type 1 collagen at 28 hours (p < 0.01). DW significantly enhanced the mineralisation of the osteoblasts compared to CC with greater Alizarin Red S staining and gene expression of osteocalcin, osteonectin, osteopontin and bone sialoprotein at 28 hours (p < 0.05). Moreover, immunocytochemical assays showed higher osteocalcin expression after DW stimulation compared to CC at 28 hours. In conclusion we have shown that ES waveforms enhanced osteoblast activity to different extent but importantly demonstrate for the first time that DW stimulation has a greater effect on differentiation and mineralisation of osteoblasts than CC stimulation. DW stimulation has potential to provide a secure, controlled and effective application for bone healing. These findings have significant implications in the clinical management of fracture repair and bone non-unions.

Influence of the proportion of particulate autogenous bone graft/platelet-rich plasma on bone healing in critical-size defects: An immunohistochemical analysis in rat calvaria

This immunohistochemical analysis evaluated the influence of the proportion of particulate autogenous bone (AB) graft/platelet-rich plasma (PRP) on bone healing in surgically created critical-size defects (CSD) in rat calvaria. 25 rats were divided into 5 groups: C (control), AB, AB/PRP-50, AB/PRP-100 and AB/PRP-150. A 5 mm diameter critical-size defect was created in the calvarium of each animal. In Group C, defect was filled by blood clot only. In Group AB, defect was filled with AB graft. In Groups AB/PRP-50, AB/PRP-100 and AB/PRP-150, defects were filled with AB graft combined with 50, 100 and 150 μl of PRP, respectively. Animals were euthanized at 30 days post-operative. Osteocalcin (OC) and osteopontin (OP) immunohistochemical staining were performed. OP-positive and OC-positive cells were quantified. The amount of vital bone (VBA) and remaining bone graft particles (RPA) were calculated as percentage of the total area of the original defect. Data were statistically analyzed (ANOVA, Tukey, p<0.05).Group AB/PRP-100 presented a significantly higher number of OP-positive cells than groups C, AB/PRP-50 and AB/PRP-150 (p<0.05) and also a significantly higher number of OC-positive cells than all of the other groups (p<0.05). The number of OP-positive cells in Group AB/PRP-150 was significantly lower than in Group AB (p<0.05). Statistically significant differences were not found between groups with regard to either VBA or RPA (p>0.05). However, clear trends were indicated by an increase in VBA in Group AB/PRP-100 when compared to groups C, AB and AB/PRP-150 and an increase in RPA in Group AB/PRP-150 when compared to groups AB, AB/PRP-50 and AB/PRP-100.The proportion AB graft/PRP seems to influence bone healing in CSD in rat calvaria. The highest expression of osteocalcin and osteopontin, and thus the most developed healing, was found in Group AB/PRP-100 indicating that there may be an optimal proportion beyond which increasing the concentration PRP is deleterious.

Dietary vitamin mix levels influence the ossification process in European sea bass (Dicentrarchus labrax) larvae

The influence of dietary vitamins on growth, survival, and morphogenesis was evaluated until day 38 of posthatching life in European sea bass larvae (Dicentrarchus labrax). A standard vitamin mix (VM), at double the concentration of the U.S. National Research Council's recommendations, was incorporated into larval feeds at 0.5%, 1.5%, 2.5%, 4.0%, and 8.0% to give treatments VM 0.5, VM 1.5, VM 2.5, VM 4.0, and VM 8.0, respectively. The group fed the VM 0.5 diet all died before day 30. At day 38, the larvae group fed VM 1.5 had 33% survival, while the other groups, with higher vitamin levels, showed at least 50% survival. The higher the percentage VM in the diet, the lower the percentage of column deformities. High dietary vitamin levels positively influenced the formation of mineralized bone in larvae: the higher the dietary vitamin level, the higher the ossification status. In the larvae group fed at the highest vitamin levels, we observed a temporal sequence of coordinated growth factor expression, in which the expression of bone morphometric protein (BMP-4) preceded the expression of IGF-1, which stimulated the maturation of osteoblasts (revealed by high osteocalcin expression levels). In groups fed lower proportions of vitamins, elevated proliferator peroxisome-activated receptors (PPAR-gamma) expression coincided with low BMP-4 expression. Our results suggest that high levels of PPAR-gamma transcripts in larvae-fed diets with a low VM content converted some osteoblasts into adipocytes during the first two weeks of life. This loss of osteoblasts is likely to have caused skeletal deformities.

Bone regeneration using hyaluronic acid-based hydrogel with bone morphogenic protein-2 and human mesenchymal stem cells

Acrylated hyaluronic acid (HA) was used as a scaffold for bone morphogenic protein-2 (BMP-2) and human mesenchymal stem cells (hMSCs) for rat calvarial defect regeneration. HA was acrylated by two-step reactions: (1) introduction of an amine group using adipic acid dihydrazide (ADH); (2) acrylation by N-acryloxysuccinimide. Tetrathiolated poly(ethylene) glycol (PEG-SH 4) was used as a cross-linker by a Michael-type addition reaction and the hydrogel was formed within 10 min under physiological conditions. This hydrogel is degraded completely by 100 U/ml hyaluronidase in vitro. hMSCs and/or BMP-2 was added during gelation. Cellular viability in vitro was increased up to 55% in the hydrogels with BMP-2 compared with the control. For in vivo calvarial defect regeneration, five different samples (i.e., control, hydrogel, hydrogel with BMP-2, hydrogel with MSCs, and hydrogel with BMP-2 and MSCs) were implanted for 4 weeks. The histological results demonstrated that the hydrogels with BMP-2 and MSCs had the highest expression of osteocalcin and mature bone formation with vascular markers, such as CD31 and vascular endothelial growth factors, compared with the other samples. This study demonstrated that HA base hydrogel can be used for cell and growth factor carriers for tissue regeneration.

Combination of local and systemic parathyroid hormone enhances bone regeneration.

Parathyroid hormone is one of the most promising therapeutic agents for osteoporosis, but its use to facilitate bone regeneration in osseous defects is less clear. The purpose of the current study was to determine the effects of combining systematic parathyroid hormone and a local parathyroid hormone gene therapy in a critical-sized osteotomy model. Rats received bilateral femoral osteotomies followed by implantation of a gene-activated matrix encoding parathyroid hormone (1-34) on one side and a control gene-activated matrix on te opposite side. Systematic parathyroid hormone (1-34) or vehicle was injected daily and rats were sacrificed 6 weeks later. Systematic parathyroid hormone increased bone mineral density and bone mineral content measured by dual-energy xray absorptiometry analysis of tibias and vertebrae, and increased serum osteocalcin levels during healing of osteotomies. Furthermore, comparing osteotomy sites that received the same gene-activated matrices as vehicle-injected rats, parathyroid hormone-injected rats showed trends of greater bone areas via histomorphometric and microradiographic analyses and higher osteocalcin messenger ribonucleic acid expression via Northern blot analyses. The combination of systemic and local parathyroid hormone led to higher bone mineral density, bone mineral content, and bone area, a trend for greater radiographic-detected bone area and higher expression of osteocalcin in osteotomy sites when compared with the individual treatment or control groups. Local parathyroid hormone gene therapy enhanced the anabolic effect of systemic parathyroid hormone during osteotomy healing. This study supports the concept of a combined local and systemic approach for enhancing the repair of a fracture at risk for nonunion.