Variability of BMP-2 content in DBM products derived from different long bone.

Demineralized bone matrix (DBM) has been regarded as an ideal bone substitute as a native carrier of bone morphogenetic proteins (BMPs) and other growth factors. However, the osteoinductive properties diverse in different DBM products. We speculate that the harvest origin further contributing to variability of BMPs contents in DBM products besides the process technology. In the study, the cortical bone of femur, tibia, humerus, and ulna from a signal donor were prepared and followed demineralizd into DBM products. Proteins in bone martix were extracted using guanidine-HCl and collagenase, respectively, and BMP-2 content was detected by sandwich enzyme-linked immunosorbent assay (ELISA). Variability of BMP-2 content was found in 4 different DBM products. By guanidine-HCl extraction, the average concentration in DBMs harvested from ulna, humerus, tibia, and femur were 0.613 ± 0.053, 0.848 ± 0.051, 3.293 ± 0.268, and 21.763 ± 0.344, respectively (p < 0.05), while using collagenase, the levels were 0.089 ± 0.004, 0.097 ± 0.004, 0.330 ± 0.012, and 1.562 ± 0.008, respectively (p < 0.05). In general, the content of BMP-2 in long bones of Lower limb was higher than that in long bones of upper limb, and GuHCl had remarkably superior extracted efficiency for BMP-2 compared to collagenase. The results suggest that the origin of cortical bones harvested to fabricate DBM products contribute to the variability of native BMP-2 content, while the protein extracted method only changes the measured values of BMP-2.

Incremental effects of diabetes mellitus and chronic kidney disease in medial arterial calcification: Synergistic pathways for peripheral artery disease progression.

Diabetes mellitus (DM) and chronic kidney disease (CKD) separately are known to facilitate the progression of medial arterial calcification (MAC) in patients with symptomatic peripheral artery disease (PAD), but their combined effect on MAC and associated mediators of calcification is not well studied. The association of MAC and calcification inducer bone morphogenetic protein (BMP-2) and inhibitor fetuin-A, with PAD, is well known. Our aim was to investigate the association of MAC with alterations in BMP-2 and fetuin-A protein expression in patients with PAD with DM and/or CKD. Peripheral artery plaques (50) collected during directional atherectomy from symptomatic patients with PAD were evaluated, grouped into no-DM/no-CKD (n = 14), DM alone (n = 10), CKD alone (n = 12), and DM+CKD (n = 14). MAC density was evaluated using hematoxylin and eosin, and alizarin red stain. Analysis of inflammation, neovascularization, BMP-2 and fetuin-A protein density was performed by immunohistochemistry. MAC density, inflammation grade and neovessel content were significantly higher in DM+CKD versus no-DM/no-CKD and CKD (p < 0.01). BMP-2 protein density was significantly higher in DM+CKD versus all other groups (p < 0.01), whereas fetuin-A protein density was significantly lower in DM+CKD versus all other groups (p < 0.001). The combined presence of DM+CKD may be associated with MAC severity in PAD plaques more so than DM or CKD alone, as illustrated in this study, where levels of calcification mediators BMP-2 and fetuin-A protein were related most robustly to DM+CKD. Further understanding of mechanisms involved in mediating calcification and their association with DM and CKD may be useful in improving management and developing therapeutic interventions.

Protective Mechanism of Adipose-Derived Stem Cells in Remodelling of the Skin Stem Cell Niche During Photoaging.

BACKGROUND/AIMS: Skin photoaging is primarily caused by the functional attrition of skin stem cells. The skin stem cell niche plays an important role in maintaining stem cell survival and behaviour. In our study, we hypothesized that UVB irradiation induces skin photoaging by changing skin stem cell niches and that transferred adipose-derived stem cells (ADSCs) can remodel the niches by affecting the BMP signalling pathway and transdifferentiating into skin stem cells. METHODS: Sixty-four C57BL/6J mice were divided into the following groups: a control group, the UVB group and the UVB+ADSCs group. Western blot assays, immunofluorescence analysis and real-time PCR were used to measure differences in the expression of niche components among the three groups. Furthermore, we tested whether transplanted ADSCs express skin stem cell markers, such as p63, α6-integrin and CD34. RESULTS: The expression levels of Bmp4, its downstream factors Smad1 and MAPK1 and a regulatory factor of the niche, i.e., NFATc1, were lower in the UVB group than were those in the control group (P< 0.05) but higher in the UVB+ADSCs group than were those in the UVB group (P< 0.05). Compared with Bmp4, Nanog (a downstream factor of Bmp4), and MMP13 (a regulatory factor of the niche), ICAM-1 (a proinflammatory gene), p63 (a basal transcription factor), ß1-integrin, Mtnr1a and Tyr (melanogenesis-related factors) showed the opposite expression trends (P< 0.05). Bmp2 and Collagen IV levels did not significantly change among the three groups (P> 0.05). Skin stem cell markers, such as p63, α6-integrin and CD34, were coexpressed in the ADSCs, which suggested the ADSCs may transdifferentiate into skin stem cells. CONCLUSION: We found that UVB irradiation results in typical photoaging signs by altering skin stem cell niches and that Bmp4 was a key factor in BMP signalling in hair follicles. ADSCs reversed these typical photoaging signs by remodelling skin stem cell niches through BMP4 pathway modulation and transdifferentiation into skin stem cells.

Imaging of growth factors on a human tooth root canal by surface-enhanced Raman spectroscopy.

Endodontic treatment of immature permanent teeth with necrotic pulp poses several clinical challenges and is one of the most demanding interventions in endodontics. Recently, with new discoveries in the field of tissue engineering, novel treatment protocols have been established. The most promising treatment modality is revascularization, whose integral part is the exposure of collagen matrix and embedded growth factors. However, optimization of the treatment protocol requires a development of analytical procedures able to analyze growth factors directly on the sample surface. In this work, method based on surface-enhanced Raman spectroscopy (SERS) was developed to investigate the influence of the time of the medical treatment using EDTA on exposure and accessibility of the growth factors, namely TGF-ß1, BMP-2, and bFGF on the dentine surface. The nanotags, which consist of magnetic Fe3O4@Ag nanocomposite covalently functionalized by tagged antibodies (anti-TGF-ß1-Cy3, anti-BMP-2-Cy5, and anti-bFGF-Cy7), were employed as a SERS substrate. Each antibody was coupled with a unique label allowing us to perform a parallel analysis of all three growth factors within one analytical run. Developed methodology presents an interesting alternative to a fluorescence microscopy and in contrary allows evaluating a chemical composition and thus minimizing possible false-positive results. Graphical abstract.

Altered DNA Methylation of Long Noncoding RNA H19 in Calcific Aortic Valve Disease Promotes Mineralization by Silencing NOTCH1.

BACKGROUND: Calcific aortic valve disease is characterized by an abnormal mineralization of the aortic valve. Osteogenic activity in the aortic valve is under the control of NOTCH1, which regulates the expression of key pro-osteogenic genes such as RUNX2 and BMP2. Long noncoding RNAs (lncRNAs) may reprogram cells by altering the gene expression pattern. METHODS: Multidimensional genomic profiling was performed in human aortic valves to document the expression of lncRNAs and the DNA methylation pattern in calcific aortic valve disease. In-depth functional assays were carried out to document the impact of lncRNA on the mineralization of the aortic valve. RESULTS: We documented that lncRNA H19 (H19) was increased in calcific aortic valve disease. Hypomethylation of the promoter region was observed in mineralized aortic valves and was inversely associated with H19 expression. Knockdown and overexpression experiments showed that H19 induces a strong osteogenic phenotype by altering the NOTCH1 pathway. Gene promoter analyses showed that H19 silenced NOTCH1 by preventing the recruitment of p53 to its promoter. A knockdown of H19 in valve interstitial cells (VICs) increased the expression of NOTCH1 and decreased the level of RUNX2 and BMP2, 2 downstream targets repressed by NOTCH1. In rescue experiments, the transfection of a vector encoding for the active Notch intracellular domain prevented H19-induced mineralization of valve interstitial cells. CONCLUSIONS: These findings indicate that a dysregulation of DNA methylation in the promoter of H19 during calcific aortic valve disease is associated with a higher expression of this lncRNA, which promotes an osteogenic program by interfering with the expression of NOTCH1.

Expression of BMP-2 in Vascular Endothelial Cells of Recipient May Predict Delayed Graft Function After Renal Transplantation.

BACKGROUND/AIMS: Delayed graft function (DGF) is associated with adverse outcomes after renal transplantation. Bone morphogenetic protein-2 (BMP-2) is involved in both endothelial function and immunological events. We compared expression of BMP-2 in epigastric artery of renal transplant recipients with immediate graft function (IGF) and DGF. METHODS: 79 patients were included in this prospective study. Patients were divided in IGF group (64 patients) and DGF group (15 patients). BMP-2 expression in intima media (BMP2m) and endothelium (BMP2e) of epigastric artery was assessed by immunohistochemistry. RESULTS: Lower intensity of BMP2e staining was recorded in DGF compared to IGF. In DGF patients, 93% had no expression of BMP2e and 7% had 1st grade expression, compared to 45% and 41% in IGF group, respectively (P=0.001) (P<0.001 for no expression and P = 0.015 for 1st grade expression). Patients who had BMP2e staining positive had lower odds for DGF (OR 0.059 [0.007, 0.477]) and this remained significant even after adjustment for donor and recipient variables, cold ischemia time, and immunological matching (OR 0.038 [0.003, 0.492]). CONCLUSIONS: Our results demonstrate that BMP-2 expression in endothelial cells of epigastric arteries may predict development of DGF.

Differences in calcification and osteogenic potential of herniated discs according to the severity of degeneration based on Pfirrmann grade: a cross-sectional study.

BACKGROUND: Herniated discs may exhibit calcification, and calcified discs may complicate surgical treatment. However, the osteogenic potential and expression of osteogenic markers in degenerative discs of different degenerative grades are still unclear. Our purposes are to study the differences in calcification rate and osteogenic potential of herniated discs according to different degenerative grades. METHODS: Fifty-eight lumbar intervertebral discs were removed from 41 patients. After grading according to the Pfirrmann scale, calcification was analyzed by micro computed tomography (µ-CT), and expression of osteogenic markers was analyzed by immunohistochemistry and real-time quantitative polymerase chain reaction (qPCR). Data from µ-CT scans were compared with the Kruskal-Wallis test. The Mann-Whitney test was applied to compare data between any two groups. Differences in osteogenic mRNA expression in different regions of the removed discs (posterior vs. anterior) were analyzed by paired t tests. Differences in the posterior portion of removed discs of different Pfirrmann grades were analyzed by one-way analysis of variance (ANOVA), and comparisons of data between discs of any two grades were completed with least significant difference (LSD) tests. RESULTS: Significant differences in calcification according to µ-CT scanning were observed between discs of different degenerative grades. Nearly half of the discs of Pfirrmann grade V showed the highest degree of calcification compared to Pfirrman grade II discs. Bone morphogenetic protein (BMP)-2, Osterix, and Osteocalcin were detected histologically in discs of Pfirrmann grades III-V. Alkaline phosphatase (ALP) expression was observed in discs showing evidence of calcification. The qPCR analysis showed that BMP-2, Osterix, and Osteocalcin were expressed in most degenerated discs. We also observed greater expression of these osteogenic markers in the posterior portion of removed discs than in the anterior portion. CONCLUSIONS: The osteogenic potential of degenerated intervertebral discs appears to increase with the severity of degeneration and to be greater in the tissue near the spinal canal than in tissue in the inner portion of the disc.

Growth Factors in Bone Marrow Blood of the Mandible With Application of Extracorporeal Shock Wave Therapy.

INTRODUCTION: Enhancement of bone regeneration is crucial to dental implantology. Growth factors play a significant role during osteogenesis and angiogenesis. Extracorporeal shock wave therapy (ESWT) enhances bone healing; however, no studies have yet been performed in oral implantology. MATERIALS AND METHODS: Twenty patients who underwent bilateral mandibular wisdom tooth removal were included. ESWT was applied to 1 side of the jaw. Blood samples were collected from the peripheral vein (PB), mandibular bone marrow without and with ESWT (BM-/+SW). Quantity and quality of the growth factors bone morphogenetic protein (BMP)-2, BMP-4, insulin-like growth factor 1 (IGF-1), vascular endothelial growth factor (VEGF), and transforming growth factor-beta (TGF-ß) were investigated via ELISA and cell proliferation assay. RESULTS: ELISA revealed superior amounts of IGF-1 and VEGF in BM-/+SW compared to PB (P < 0.05). TGF-ß demonstrated no variance. Levels of BMP-2 and BMP-4 were too low for adequate detection in the ELISA. No difference was noticed upon ESWT. The cell proliferation assay did not identify any changes comparing PB versus BM-SW versus BM + SW. CONCLUSION: IGF-1 and VEGF are present at higher levels in mandibular bone marrow than in peripheral blood (PB). This study did not identify any benefits of extracorporeal shock wave therapy to increase the investigated growth factors.

Monitoring bone morphogenetic protein-2 and -7, soluble receptor activator of nuclear factor-κB ligand and osteoprotegerin levels in the peri-implant sulcular fluid during the osseointegration of hydrophilic-modified sandblasted acid-etched and sandblasted acid-etched surface dental implants.

BACKGROUND AND OBJECTIVE: The implant surface plays a major role in the biological response to titanium dental implants. The aim of this study was to investigate levels of soluble receptor activator of nuclear factor-κB ligand (sRANKL), osteoprotegerin (OPG), bone morphogenetic protein-2 (BMP-2) and -7 (BMP-7) in the peri-implant crevicular fluid (PICF) of different implants during the osseointegration period. MATERIAL AND METHODS: Forty-seven patients (22 females and 25 males, mean age 47.34 ± 10.11) were included in this study. Forty-seven implants from two implant systems (group A1 (sandblasted acid-etched [SLA]-16), group A2 (hydrophilic-modified SLA [SLActive]-16), and group B (sandblasted acid-etched [SLA]-15) were placed using standard surgical protocols. PICF samples, plaque index, gingival index and probing depth measurements were obtained at 1 and 3 mo after surgery. PICF levels of sRANKL, OPG, BMP-2/-7 were analyzed by ELISA. RESULTS: No complications were observed during the healing period. No significant differences were observed in the PICF levels of sRANKL, OPG, BMP-2 and BMP-7 for all groups at any time point (p > 0.05). A significant decrease was observed in BMP-2 levels in group A1 (p < 0.05). A significant increase in BMP-7 levels was observed only for group A2 (p < 0.05). There was a strong negative correlation between OPG and gingival index and a negative correlation between BMP-7 and plaque index (p < 0.05). CONCLUSION: Considering the correlations between clinical and biochemical parameters, the levels of these cytokines in PICF during early healing of implants reflects the degree of peri-implant inflammation, rather than differences in the implant surfaces.

The Effect of Tumor Necrosis Factor-α at Different Concentrations on Osteogenetic Differentiation of Bone Marrow Mesenchymal Stem Cells.

Tumor necrosis factor-alpha (TNF-α) has been demonstrated to have a close relationship with inflammation in the body. Although most researchers confirmed that TNF-α can have an effect on the expression of osteoblast specific genes, they had not elucidated the regulation of inflammatory factors in osteogenic gene expression during the process of bone marrow mesenchymal stem cells (BMMSCs) differentiating to osteoblast. The aim of this study was to investigate the effect of TNF-α at different concentrations on osteogenetic differentiation of BMMSCs. In this study, BMMSCs proliferation was analyzed by using cell counting kit-8 assay, cell osteogenic differentiation was evaluated by means of alkaline phosphatase activity assay and Von Koaas staining and the messenger RNA (mRNA) expression of bone morphogenetic proteins-2 (BMP-2) and drosophila mothers against decapentaplegic protein 1 (Smad1) was measured through real-time polymerase chain reaction. The results indicated that a low concentration of TNF-α at short-term promotes the osteogenetic differentiation of BMMSCs and increases the mRNA expression of BMP-2 and Smad1, but inhibits the osteogenetic differentiation of BMMSCs and the expression of BMP-2 and Smad1 at long term. In addition, regardless of a short or long time, a high concentration of TNF-α inhibits the osteogenetic differentiation of BMMSCs and the expression of Smad1, but results in a high expression of BMP-2.

Age-related alterations in gene expression of gingival fibroblasts stimulated with Porphyromonas gingivalis.

BACKGROUND AND OBJECTIVE: Elderly people exhibit increased susceptibility to a number of autoimmune and infectious diseases, such as periodontitis. Although aging is reportedly associated with a decline in immune function, age-related alterations in periodontal tissue have remained elusive. In the present study, we comprehensively analyzed the effect of aging on the expression of selected genes using mouse gingival fibroblasts. MATERIAL AND METHODS: Gingival fibroblasts derived from young (8 wk of age) and old (≥ 24 mo of age) C57BL/6 mice were stimulated with Porphyromonas gingivalis lipopolysaccharide or live P. gingivalis strain W83. Expression of cytokines/chemokines, innate immune receptors, growth factors, matrix metalloproteinases, tissue inhibitors of metalloproteinases and osteoclastogenesis-related molecules were evaluated using real-time polymerase chain reaction and ELISA for interleukin-6 and transforming growth factor-ß1. RESULTS: Gingival fibroblasts derived from old mice exhibited decreased gene expression of Il-6, Cxcl1, Tlr2, Tlr4, Irak3 (IRAK-M), Kgf, Timp1, Timp3 and Rankl under resting conditions, whereas the expression levels of Tgfß1, Mmp3, Mmp13 and Opg were increased. Age-related differences were also detected at the protein level. Although P. gingivalis W83 upregulated Vegf, Fgf-2 and Bmp2 expression in both young and old gingival fibroblasts, the stimulatory effect on these genes was significantly lower in old gingival fibroblasts. CONCLUSION: Our findings demonstrated that aging altered the expression of a number of genes in gingival fibroblasts. Thus, alterations in the balance of these molecules could play a critical role in periodontitis progression in the elderly.

Importance of CD44 in the proliferation and mineralization of periodontal ligament cells.

BACKGROUND AND OBJECTIVE: CD44 is a transmembrane glycoprotein that exhibits various biological functions. Histological studies have shown that CD44 is expressed in periodontal ligament (PDL). We investigated the role of CD44 in the in vitro biological functions of PDL cells. MATERIAL AND METHODS: Immunohistochemistry and immunocytochemistry were used to examine CD44 protein expression in mouse molars and human PDL cells, respectively. PDL cells isolated from human third molars were assayed for their proliferation and mineralization after stably silencing their expression of CD44 using the small hairpin RNA technique. An osteogenesis-associated quantitative polymerase chain reaction array was used to identify downstream molecules of CD44 signaling in osteogenic medium. The PDL cells, transduced with control small hairpin RNA, were used as controls in all assays. RESULTS: PDLs expressed CD44 in vitro and in vivo. When CD44 expression was stably suppressed in PDL cells, their proliferation and mineralization activities in both media were substantially decreased. The quantitative polymerase chain reaction array and Western blotting verified that bone morphogenetic protein-2 (BMP-2), fibroblast growth factor-1 (FGF-1) and intercellular adhesion molecule-1 (ICAM-1) were downregulated after CD44 was knocked down in PDL cells. Exogenous FGF-1 attenuated the proliferative suppression of CD44 knockdown cells. Exogenous BMP-2 rescued the suppressed mineralization of CD44 knockdown cells more than ICAM-1 did. CONCLUSION: The in vitro assays demonstrated that CD44 is crucial for the proliferation and mineralization of PDL cells and is possibly mediated by BMP-2, FGF-1 and ICAM-1. Further studies are required to verify the mediators and identify the physiological ligands of CD44 for possible application in periodontal regeneration.

Acemannan sponges stimulate alveolar bone, cementum and periodontal ligament regeneration in a canine class II furcation defect model.

BACKGROUND AND OBJECTIVE: Periodontal disease is a common infectious disease, found worldwide, causing the destruction of the periodontium. The periodontium is a complex structure composed of both soft and hard tissues, thus an agent applied to regenerate the periodontium must be able to stimulate periodontal ligament, cementum and alveolar bone regeneration. Recent studies demonstrated that acemannan, a polysaccharide extracted from Aloe vera gel, stimulated both soft and hard tissue healing. This study investigated effect of acemannan as a bioactive molecule and scaffold for periodontal tissue regeneration. MATERIAL AND METHODS: Primary human periodontal ligament cells were treated with acemannan in vitro. New DNA synthesis, expression of growth/differentiation factor 5 and runt-related transcription factor 2, expression of vascular endothelial growth factor, bone morphogenetic protein-2 and type I collagen, alkaline phosphatase activity, and mineralized nodule formation were determined using [(3)H]-thymidine incorporation, reverse transcription-polymerase chain reaction, enzyme-linked immunoabsorbent assay, biochemical assay and alizarin red staining, respectively. In our in vivo study, premolar class II furcation defects were made in four mongrel dogs. Acemannan sponges were applied into the defects. Untreated defects were used as a negative control group. The amount of new bone, cementum and periodontal ligament formation were evaluated 30 and 60 d after the operation. RESULTS: Acemannan significantly increased periodontal ligament cell proliferation, upregulation of growth/differentiation factor 5, runt-related transcription factor 2, vascular endothelial growth factor, bone morphogenetic protein 2, type I collagen and alkaline phosphatase activity, and mineral deposition as compared with the untreated control group in vitro. Moreover, acemannan significantly accelerated new alveolar bone, cementum and periodontal ligament formation in class II furcation defects. CONCLUSION: Our data suggest that acemannan could be a candidate biomolecule for periodontal tissue regeneration.

Hypoxia induces osteogenesis in rabbit adipose-derived stem cells overexpressing bone morphogenic protein-2.

OBJECTIVE: Hypoxic culture potentiates mesenchymal stem cells (MSCs) to survive and secrete various growth factors. Genetically modified stem cells overexpressing bone morphogenic protein-2 (BMP-2) demonstrate strong osteogenic ability. Hence, we investigated the coeffect of hypoxic culture conditions and BMP-2 overexpression on the osteogenic ability of rabbit adipose-derived stem cells (rASCs) in vitro. MATERIALS AND METHODS: Rabbit adipose-derived stem cells with or without adenoviral-BMP-2 transduction were cultured in hypoxic (1%) and normoxic (21%) conditions. Cell viability, attachment, and proliferation were compared. Real-time PCR amplification of osteogenic and angiogenic genes including alkaline phosphatase (ALP), osteocalcin (OCN), HIF-1α, and vascular endothelial growth factor (VEGF) was performed. Moreover, ALP activity, immunofluorescent staining of OCN, and mineralization assay by alizarin red S quantification and von Kossa staining were conducted. RESULTS: Cells under hypoxic conditions attached better within 12 h and proliferated faster. While BMP-2 overexpression and hypoxic condition separately elevated the transcription of key osteogenic and angiogenic genes, a cooperative effect was observed to enhance the upregulation of osteogenic as well as angiogenic genes. Identical changes were observed in ALP activity, immunofluorescent staining of OCN, and mineralization assay. CONCLUSIONS: Hypoxic culture can enhance the osteogenic ability of BMP-2 gene-modified rASCs, which provides a strategy to improve the osteogenesis of rASCs for in vivo bone regeneration.

Osteogenic gene expression correlates with development of heterotopic ossification in war wounds.

BACKGROUND: Heterotopic ossification (HO) is a frequent complication of modern wartime extremity injuries. The biological mechanisms responsible for the development of HO in traumatic wounds remain elusive. QUESTION/PURPOSES: The aims of our study were to (1) characterize the expression profile of osteogenesis-related gene transcripts in traumatic war wounds in which HO developed; and (2) determine whether expression at the mRNA level correlated with functional protein expression and HO formation. METHODS: Biopsy specimens from 54 high-energy penetrating extremity wounds obtained at the initial and final surgical débridements were evaluated. The levels of selected osteogenic-related gene transcripts from RNA extracts were assessed by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. As a result of its key role in osteogenesis, the concentration of BMP-2 in the effluent of 29 wounds also was determined. RESULTS: The transcripts of 13 genes (ALPL [p = 0.006], BMP-2 [p < 0.001], BMP-3 [p = 0.06], COL2A1 [p < 0.001], COLL10A1 [p < 0.001], COL11A1 [p = 0.006], COMP [p = 0.02], CSF2 [p = 0.003], CSF3 [p = 0.012], MMP8 [p < 0.001], MMP9 [p = 0.014], SMAD1 [p = 0.024], and VEGFA [p = 0.017]) were upregulated greater than twofold in wounds in which HO developed compared with wounds in which it did not develop. Gene transcript expression of BMP-2 also correlated directly with functional protein expression in the wounds that formed HO (p = 0.029). CONCLUSIONS: Important differences exist in the osteogenic gene expression profile of wounds in which HO developed compared with wounds in which it did not develop. The upregulation of multiple osteogenesis-related gene transcripts indicates the presence of a proosteogenic environment necessary for ectopic bone formation in traumatic wounds. CLINICAL RELEVANCE: Understanding the osteogenic environment associated with war wounds may allow for the development of novel therapeutic strategies for HO.

Mutational analysis of FOXL2 p.C134W and expression of bone morphogenetic protein 2 in Japanese patients with granulosa cell tumor of ovary.

AIM: To assess whether FOXL2 p.C134W mutation may play a role in the development of human ovarian tumors in the Japanese, we investigated the FOXL2 codon 134 mutation and protein expression of inhibin-α, bone morphogenetic protein 2 (BMP2) and follistatin (FST) in Japanese patients with granulosa cell tumor (GCT) of the ovary and other ovarian tumors. METHODS: We analyzed 114 tumor tissues from ovarian tumors, including 44 adult-type and two juvenile-type GCT of the ovary and 68 ovarian tumors by DNA sequencing. Immunohistochemistry was also performed in the adult and juvenile GCT tissues by immunostaining inhibin-α, BMP2 and FST. RESULTS: We found the FOXL2 p.C134W mutation in 27 out of 44 (61.4%) adult-type GCT of the ovary, but none in other ovarian tumors. Histologically, all of the adult-type GCT sections were positive for inhibin-α, and the expression of BMP2 and FST was detected in 14 of 44 (31.8%) and zero of 47 (0%), respectively. No significant differences regarding the diagnosed age, preoperative serum carbohydrate antigen 125 levels, or BMP2 immunopositivity between the FOXL2 p.C134W mutation-positive and mutation-negative were found in the adult-type GCT patients. CONCLUSION: Our findings suggest that FOXL2 p.C134W mutation-positive adult-type GCT of the ovary may not be common in the Japanese as compared to the previous data.

Gingival crevicular fluid bone morphogenetic protein-2 release profile following the use of modified perforated membrane barriers in localized intrabony defects: a randomized clinical trial.

BACKGROUND: In guided tissue regenerative surgery, membrane perforations may serve as a mechanism for the passage of cells and biologic mediators from the periosteum and overlying gingival connective tissue into the periodontal defects. To test this assumption, this study was designed to evaluate levels of bone morphogenetic protein-2 (BMP-2) in gingival crevicular fluid (GCF) during the early stages of healing for sites treated with modified perforated membranes (MPMs) as compared with occlusive membranes (OMs). METHODS: Fifteen non-smoking patients with severe chronic periodontitis participated in this prospective, randomized and single-blinded clinical trial. Each patient contributed two interproximal contralateral defects that were randomly assigned to either an experimental modified perforated membrane group (15 sites) or a control occlusive membrane group (15 sites). Plaque index, gingival index, probing depth(PD), clinical attachment level (CAL) and the relative intrabony depth of the defect (rIBD) were measured at baseline and reassessed at three, six and nine months after therapy. Gingival crevicular fluid samples were collected on day 1 and 3, 7, 14, 21, and 30 days after therapy. RESULTS: The MPM-treated group showed a statistically significant improvement in PD reduction and clinical attachment gain compared to the OM control group. Similarly, rIBD was significantly reduced in MPM-treated sites as compared with those of the OM group. BMP-2 concentrations peaked in the MPM samples obtained during the early postoperative period (days 1, 3 and 7) with a statistically significant difference compared with OM-treated groups. BMP-2 levels decreased sharply in the samples obtained at days 14, 21 and 30 with non-significant higher levels in MPM samples as compared with those of OM sites. CONCLUSION: Within the limits of the present study, one can conclude that MPM coverage of periodontal defects is associated with a significant initial increase in GCF levels of BMP-2, a factor that could improve the clinical outcomes of guided tissue regenerative surgery.

Analysis of human alveolar osteoblast behavior on a nano-hydroxyapatite substrate: an in vitro study.

BACKGROUND: Nano-hydroxyapatite (nHA) is a potential ideal biomaterial for bone regeneration. However, studies have yet to characterize the behavior of human osteoblasts derived from alveolar bone on nHA. Thus, the aim of the present study was to evaluate the influence of nHA on the adhesion, proliferation and differentiation of these alveolar bone-derived cells. METHODS: Primary human alveolar osteoblasts were collected from the alveolar ridge of a male periodontal patient during osseous resective surgery and grown on culture plates coated with either polylysine or polylysine with nano-hydroxyapatite (POL/nHA) composite. The cells were grown and observed for 14 days, and then assessed for potential modifications to osteoblasts homeostasis as evaluated by quantitative reverse transcriptase-polymerase chain reaction (real time RT-PCR), scanning electron microscopy and atomic force microscopy. RESULTS: Real time PCR revealed a significant increase in the expression of the selected markers of osteoblast differentiation (bone morphogenetic protein (BMP)-2,-5,-7, ALP, COLL-1A2, OC, ON) in cells grown on the POL/nHA substrate. In addition, as compared with the POL surface, cells grown on the POL/nHA substrate demonstrated better osteoconductive properties, as demonstrated by the increase in adhesion and spreading, likely as a result of the increased surface roughness of the composite. CONCLUSIONS: The increased expression of BMPs and osteoinductive biomarkers suggest that nano-hydroxyapatite may stimulate the proliferation and differentiation of local alveolar osteoblasts and thus encourage bone regeneration at sites of alveolar bone regeneration.

Correlation of synovial cytokine expression with quality of cells used for autologous chondrocyte implantation in human knees.

The cell quality plays a decisive role in autologous chondrocyte implantation (ACI). Aim of the study was the analysis of in vivo interactions between synovial concentrations of cytokines and cell quality used for ACI. Knee lavage fluids of patients undergoing an ACI were examined for total protein content (TPC) and by ELISA for levels of basic fibroblast growth factor (bFGF), insulin-like growth factor 1, bone morphogenetic proteins 2 and 7 (BMP-2 and BMP-7). Cell quality following amplification for ACI was determined by surface expression of CD44, aggrecan, collagen type II and evaluation of cell characteristics. Data of 17 patients were supplemented by epidemiological parameters and clinical scores (IKDC, Lysholm, pain strength, subjective knee function). CD44 expression was positively associated with TPC and bFGF, and negatively linked to BMP-2 levels (p < 0.01). In contrast, expression of collagen type II did not show any statistically significant correlations with synovial protein concentrations. TPC was positively associated with intraarticular bFGF levels and pain strength (p < 0.01), both indicators for osteoarthritis (OA). Correlating with the negative relation of TPC and BMP-2, subjective knee function after 1 year was positively linked to intraarticular BMP-2 concentrations (p < 0.001). Similarly, expression of collagen type II indicated a favorable clinical result reaching statistical significance in case of pain strength (p < 0.01). Initially increased bFGF levels and CD44 expression indicated a worse clinical outcome after 1 year (IKDC, Lysholm Scores, pain strength). Surface expression of CD44 on chondrocytes used for ACI was negatively associated with synovial BMP-2 and positively to TPC and bFGF indicating catabolic synovial conditions. These correlations were also reflected by clinical outcome parameters.

In-vitro analysis of rhBMP-2 effects in human osteogenic cells.

This study evaluated the in vitro expression of bone-related proteins by osteoblasts in the presence of different concentrations of human recombinant bone morphogenetic protein-2 (rhBMP-2). Immortalized human fetal osteoblastic cell line 1.19 (hFOB) were exposed to different concentrations of rhBMP-2 (10, 50, or 100 ng/mL) for 72 h. Cell proliferation and viability (MTT assay), as well as the expression of fibronectin, osteonectin, and osteopontin were assessed by indirect immunofluorescence and Western blot. Neither of the 3 concentrations of rhBMP-2 caused statistically significant alterations in cell proliferation and viability, although the concentration of 100 ng/mL showed lower values for both assays after both 48 and 72 h of exposure. There was no alteration in the expression of noncollagenous proteins, as analyzed by immunofluorescence, when compared with the control group. Furthermore, in the Western blot assay we observed a statistically significant decrease in fibronectin and osteonectin at 100 ng rhBMP-2/mL (p < 0.05) by comparison with the medium alone. The expression of osteopontin decreased slightly in all 3 concentrations of rhBMP-2 tested; however, the change was not statistically significant (p > 0.05). In this in-vitro study, the tested concentrations of rhBMP-2 appeared to decrease the expression of important bone-related molecules in pre-osteoblast cells.