The role of TRPM7 in vascular calcification: Comparison between phosphate and uremic toxin.

AIMS: Vascular calcification is a common complication in patients with chronic kidney disease and associated with increased morbidity and mortality. The role of TRPM7 in vascular smooth muscle cell (VSMC) transformation during vascular calcification is not clear. We aim to investigate the effects of phosphate and indoxyl sulphate on the expression of TRPM7 and calcification-related molecules in VSMC. MAIN METHODS: Human aortic smooth muscle cells (HASMC) were treated with phosphate (3.3 mM) or indoxyl sulphate (500 µM and 1000 µM). 2-APB, a channel blocker of TRPM7 was added simultaneously in blocking experiment. Cells were then examined grossly and alizarin red solution was employed for calcification assessment. Lastly, cells were harvested for gene expression and protein abundance analysis. KEY FINDINGS: Phosphate treatment induced significant increase in BMP2, RUNX2, BMP7, vitamin D receptor (VDR), calcium sensing receptor (CaSR) and TRPM7, but 1-alpha hydroxylase, klotho, DKK1 and sclerostin were not changed. The addition of 2-APB prevented increase of BMP2, RUNX2, BMP7, VDR, CaSR and TRPM7. Indoxyl sulphate treatment was associated with decrease in TRPM7 and DKK1, but increase in RUNX2, BMP2 and VDR were noted. There were no significant alterations in BMP7, CaSR, klotho,1-alpha hydroxylase and sclerostin. Co-treatment with 2-APB reversed the increase in VDR. SIGNIFICANCE: Both phosphate and indoxyl sulphate induced calcification in VSMC but it was more prominent in phosphate. TRPM7 was upregulated by phosphate but downregulated in indoxyl sulphate treatment. Vascular calcification was reduced by blocking TRPM7 with 2-APB and there was partial anti-calcification effect in indoxyl sulphate.

Growth factor-mediated augmentation of long bones: evaluation of a BMP-7 loaded thermoresponsive hydrogel in a murine femoral intramedullary injection model.

BACKGROUND: Due to our aging population, an increase in proximal femur fractures can be expected, which is associated with impaired activities of daily living and a high risk of mortality. These patients are also at a high risk to suffer a secondary osteoporosis-related fracture on the contralateral hip. In this context, growth factors could open the field for regenerative approaches, as it is known that, i.e., the growth factor BMP-7 (bone morphogenetic protein 7) is a potent stimulator of osteogenesis. Local prophylactic augmentation of the proximal femur with a BMP-7 loaded thermoresponsive hydrogel during index surgery of an osteoporotic fracture could be suitable to reduce the risk of further osteoporosis-associated secondary fractures. The present study therefore aims to test the hypothesis if a BMP-7 augmented hydrogel is an applicable carrier for the augmentation of non-fractured proximal femurs. Furthermore, it needs to be shown that the minimally invasive injection of a hydrogel into the mouse femur is technically feasible. METHODS: In this study, male C57BL/6 mice (n = 36) received a unilateral femoral intramedullary injection of either 100 µl saline, 100 µl 1,4 Butan-Diisocyanat (BDI)-hydrogel, or 100 µl hydrogel loaded with 1 µg of bone morphogenetic protein 7. Mice were sacrificed 4 and 12 weeks later. The femora were submitted to high-resolution X-ray tomography and subsequent histological examination. RESULTS: Analysis of normalized CtBMD (Cortical bone mineral density) as obtained by X-ray micro-computed tomography analysis revealed significant differences depending on the duration of treatment (4 vs 12 weeks; p < 0.05). Furthermore, within different anatomically defined regions of interest, significant associations between normalized TbN (trabecular number) and BV/TV (percent bone volume) were noted. Histology indicated no signs of inflammation and no signs of necrosis and there were no cartilage damages, no new bone formations, or new cartilage tissues, while BMP-7 was readily detectable in all of the samples. CONCLUSIONS: In conclusion, the murine femoral intramedullary injection model appears to be feasible and worth to be used in subsequent studies that are directed to examine the therapeutic potential of BMP-7 loaded BDI-hydrogel. Although we were unable to detect any significant osseous effects arising from the mode or duration of treatment in the present trial, the effect of different concentrations and duration of treatment in an osteoporotic model appears of interest for further experiments to reach translation into clinic and open new strategies of growth factor-mediated augmentation.

Laminin associated with BMP7 as potential secondary astrocytic glioma fiber differentiation targets.

OBJECTIVE: To investigate the activation of the BMP7 and laminin pathway is associated with glioma cell proliferation and differentiation. PATIENTS AND METHODS: We enrolled 65 patients with primary operable glioma. Laminin and BMP7 protein expression and its subcellular localization were studied by immunofluorescence. RESULTS: We detected a higher level of BMP7 expression in glioma tissue in patients with a lower grade of glioma who had a lower eosinophil count. Compared to patients with a higher grade of glioma, we observed a lower level of laminin expression in patients with a lower grade of glioma. CONCLUSIONS: Our data indicated a potential link between eosinophil counts and the expression levels of laminin and BMP7 in glioma differentiation.

Local wound healing biomarkers for real-time assessment of periodontal regeneration: pilot study.

BACKGROUND AND OBJECTIVES: Within the same surgical procedure, a great variability on achievement of clinical outcomes exists and may be associated to different molecular factors related to tissue healing. The aim of the present study was to assess the distribution of clinical success separately in regenerative therapy (REG) and open flap debridement (OFD) to evaluate if factors related with healing of epithelium, connective tissue and bone may be associated to the clinical outcome within each surgical procedure. MATERIAL AND METHODS: Sixteen patients underwent periodontal REG and nine patients underwent OFD. Periodontal wound fluid was collected at baseline, 3-5, 7, 14 and 21 d after surgery, and expression of wound healing proteins was assessed. Pocket depth and clinical attachment level were taken at baseline and at 6 mo of follow-up. Percentage pocket depth reduction and percentage clinical attachment level gain were computed. Patients were regarded as better or worse responders depending on their percentage pocket depth reduction or percentage clinical attachment level gain. RESULTS: Higher percentage of better responders was observed in the REG group (68.7%) compared to the OFD group (22.2%). At 21 d, no difference in the profile of most of the proteins emerged, with two exceptions, both regarding REG treatment. Bone morphogenetic protein-7 tended to increase in better responders and to decrease in worse responders. Matrix metalloproteinase-1 increased in worse responders and remained substantially unchanged in better responders. CONCLUSION: Local expression of matrix metalloproteinase-1 and bone morphogenetic protein-7 during wound healing is associated with the clinical performance of periodontal regenerative surgery. The use of local biomarkers offers the potential for real-time assessment of the periodontal healing process.

Bone Marrow Mesenchymal Stem Cells Expressing Baculovirus-Engineered Bone Morphogenetic Protein-7 Enhance Rabbit Posterolateral Fusion.

Previous studies have suggested that bone marrow-derived mesenchymal stem cells (BMDMSCs) genetically modified with baculoviral bone morphogenetic protein-2 (Bac-BMP-2) vectors could achieve successful fusion in a femur defect model or in a spinal fusion model. In this study, BMDMSCs expressing BMP-7 (Bac-BMP-7-BMDMSCs) were generated. We hypothesized that Bac-BMP-7-BMDMSCs could secrete more BMP-7 than untransduced BMDMSCs in vitro and achieve spinal posterolateral fusion in a rabbit model. Eighteen rabbits underwent posterolateral fusion at L4-5. Group I (n = 6) was implanted with collagen-ß-tricalcium phosphate (TCP)-hydroxyapatite (HA), Group II (n = 6) was implanted with collagen-ß-TCP-HA plus BMDMSCs, and Group III (n = 6) was implanted with collagen-ß-TCP-HA plus Bac-BMP-7-BMDMSCs. In vitro production of BMP-7 was quantified with an enzyme-linked immunosorbent assay (ELISA). Spinal fusion was examined using computed tomography (CT), manual palpation, and histological analysis. ELISA demonstrated that Bac-BMP-7-BMDMSCs produced four-fold to five-fold more BMP-7 than did BMDMSCs. In the CT results, 6 fused segments were observed in Group I (50%, 6/12), 8 in Group II (67%, 8/12), and 12 in Group III (100%, 12/12). The fusion rate, determined by manual palpation, was 0% (0/6) in Group I, 0% (0/6) in Group II, and 83% (5/6) in Group III. Histology showed that Group III had more new bone and matured marrow formation. In conclusion, BMDMSCs genetically transduced with the Bac-BMP-7 vector could express more BMP-7 than untransduced BMDMSCs. These Bac-BMP-7-BMDMSCs on collagen-ß-TCP-HA scaffolds were able to induce successful spinal fusion in rabbits.

BMP-7 attenuates left ventricular remodelling under pressure overload and facilitates reverse remodelling and functional recovery.

AIMS: TGF-ß regulates tissue fibrosis: TGF-ß promotes fibrosis, whereas bone morphogenetic protein (BMP)-7 is antifibrotic. To demonstrate that (i) left ventricular (LV) remodelling after pressure overload is associated with disequilibrium in the signalling mediated by these cytokines, and (ii) BMP-7 exerts beneficial effects on LV remodelling and reverse remodelling. METHODS AND RESULTS: We studied patients with aortic stenosis (AS) and mice subjected to transverse aortic constriction (TAC) and TAC release (de-TAC). LV morphology and function were assessed by echocardiography. LV biopsies were analysed by qPCR, immunoblotting, and histology. Pressure overload reduced BMP-7 and pSmad1/5/8 and increased TGF-ß and pSmad2/3 in AS patients and TAC mice. BMP-7 correlated inversely with collagen, fibronectin, and ß-MHC expressions, and with hypertrophy and diastolic dysfunction, and directly with the systolic function. Multiple linear regression disclosed BMP-7 and TGF-ß as hypertrophy predictors, negative and positive, respectively. BMP-7 prevented TGF-ß-elicited hypertrophic program in cardiomyocytes, and Col1A1 promoter activity in NIH-3T3 fibroblasts. The treatment of TAC mice with rBMP-7 attenuated the development of structural damage and dysfunction, and halted ongoing remodelling. The reverse remodelling after pressure overload release was facilitated by rBMP-7, and hampered by disrupting BMP-7 function using a neutralizing antibody or genetic deletion. CONCLUSION: The disequilibrium between BMP-7 and TGF-ß signals plays a relevant role in the LV remodelling response to haemodynamic stress in TAC mice and AS patients. Our observations may provide new important insights aimed at developing novel therapies designed to prevent, halt, or reverse LV pathological remodelling in pressure overload cardiomyopathy.

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.

Quantitative expression of bone-related cytokines induced by mechanical tension-stress during distraction osteogenesis in a rabbit mandible.

AIM: The aim of the present study was to investigate the temporal and spatial gene expression of bone morphogenetic proteins (BMP)-2, -4, and -7, and transforming growth factor-ß (TGF-ß), during the distraction process of the rabbit mandible. METHODS: Twenty rabbits each had an osteotomy on the left mandibular body, and distraction devices were fixed. After a delay of 3 days, distraction was started at a rate of 0.5 mm/12 h for 10 days, followed by a 3-week consolidation phase. Four rabbits were killed 5 and 10 days of distraction, and 1, 2, and 3 weeks after the completion of distraction. The clinical, histological, and radiographic appearances were evaluated and analyzed with the concomitant BMP expression pattern at each interval. RESULTS: After the distraction was started, the fibrous interzone developed between the osteotomy fragments, where intramembranous ossification was noted. The quantitative expression of BMP-2, -4, and -7, and TGF-ß, were increased immediately after active distraction before a gradual decline to normal levels after the third week of consolidation. CONCLUSION: These results suggest that BMP and TGF-ß play an important role in the induction of bone formation during distraction osteogenesis. The selective expression of each bone-related cytokine could provide useful insight into accelerated bone maturation and the treatment of poorly-healing fractures in clinical cases.

Are bone morphogenetic protein-7 (BMP-7) serum levels correlated with development of hepatic fibrosis?

INTRODUCTION: Bone morphogenetic protein-7 (BMP-7) is a key protein in organogenesis and liver development. The protein has been studied in the context of liver fibrosis and regeneration. The aim of the present study was to explore any possible association between fibrosis levels (as revealed by liver biopsy) and serum BMP-7 levels. METHODOLOGY: A total of 189 patients with chronic hepatitis B and 51 healthy controls were enrolled in the study. RESULTS: The study group contained 120 (63.5%) males and 69 (36.5%) females, and the control group contained 25 males (49.0%) and 26 females (51%). In general, serum BMP-7 values of patients were higher than those of controls (p = 0.001). Serum BMP-7 values of patients with liver fibrosis of stages 1, 2, 3, or 4 were higher than control values (all p values = 0.01), but the serum BMP-7 levels of patients with stage 5 fibrosis were similar to that of controls. Associations between fibrosis stage and the serum levels of BMP-7, ALT, HBVDNA, platelets, and albumin were all statistically significant (p = 0.001). The AUROC for the BMP-7 level in advanced stage fibrosis was found to be 0.23. The data were analyzed using the binary logistic regression analysis (backward stepwise method) and BMP-7, HBVDNA, and platelet levels were found to be risk factors associated with fibrosis (p values 0.031, 0.040, and 0.001, respectively). CONCLUSIONS: BMP-7 may play anti-inflammatory and anti-fibrogenic roles in the pathogenesis of chronic hepatitis B infection.

Effect of storage temperature and antibiotic impregnation on the quantity of bone morphogenetic protein seven in human bone grafts.

PURPOSE: The aim of this study was to quantify the amount of bone morphogenic protein 7 (BMP-7) in bone samples in different storage and treatment conditions used in bone banks and thereby evaluate the benefit of this test as a routine measure before bone grafting. METHODS: Fresh as well as frozen bone chips, each with and without antibiotic impregnation, were screened for their BMP-7 content. Human bone chips were produced from femoral heads of two female donors who had undergone total hip replacement surgery. The amount of BMP-7 was detected using a commercially available enzyme-linked immunosorbent assay (ELISA) test. RESULTS: There were no significant differences between groups in samples obtained from the first femoral head. Bone-chip samples derived from the second femoral head showed significant differences between groups. The actual amount of these differences was small and most likely biologically irrelevant. It is important to note that there was a significant difference between groups when comparing both femoral heads, reflecting donor-to-donor variability. CONCLUSION: ELISA testing for BMP-7 as a qualitative measurement of bone grafts should be considered a routine quality-control test for bone banks.

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.

A pilot trial on the molecular pathophysiology of traumatic temporomandibular joint bony ankylosis in a sheep model. Part II: The differential gene expression among fibrous ankylosis, bony ankylosis and condylar fracture.

OBJECTIVE: The purpose of the study was to preliminarily explore the differential expressions of a series of genes regulating bone formation in temporomandibular joint (TMJ) fibrous ankylosis, bony ankylosis and condylar fracture healing. METHODS: The cDNA from either the bony ankylosed callus or fracture callus of the 6 sheep, as described in the part I, were both used in the study. The differences of gene expressions between bony ankylosis and condylar fracture at 1, 3, and 6 months postoperatively were measured by real-time PCR, with 2 samples at each time point. In addition, another 2 sheep were added to have fibrous ankylosis induced on the right TMJ, and 1 sheep was sacrificed at 3 and 6 months after surgery, respectively. The differences of gene expressions between fibrous and bony ankylosis at 3 and 6 months postoperatively were measured by real-time PCR. RESULTS: Bony ankylosis showed higher mRNA expression trends in Wnt2b, Wnt5a, ß-Catenin, Lef1, CyclinD1, Runx2, Osterix, Sox9, Col10a1, Alp, Ocn, Bmp2, and Bmp7 compared to fibrous ankylosis, although no statistical analysis was performed due to the very small sample size. Whereas bony ankylosis showed a significant lower expression of Wnt5a, ß-Catenin, Lef1, Runx2, Osterix, Sox9, Col10a1, Alp, Ocn and Bmp4 compared to condylar fracture at several time points (P < 0.05). CONCLUSION: Our data provided a preliminary molecular evidence for the hypothesis that the development of traumatic TMJ bony ankylosis was the course of delayed bone healing or hypertrophic nonunion, and deserved to be further studied.

Effect of F68, F127, and P85 pluronic block copolymers on odontogenic differentiation of human tooth germ stem cells.

INTRODUCTION: The major challenge in dental pulp engineering is to make a successful combination of stem cells and biomaterials with the aim of providing the differentiation of stem cells into odontogenic cell types. Among biomaterials, some types of pluronics have been reported to increase bone formation of stem cells. The effect of these pluronics on odontogenic differentiation has not been addressed yet. This study aimed to examine the effect of pluronics F68, F127, and P85 on odontogenic differentiation of stem cells derived from third molar tooth germs of young adults. METHODS: Human tooth germ stem cells (hTGSCs) were induced to differentiate into odontogenic cells in the presence of different concentrations of pluronics. Differentiation efficiency was assessed by quantitative real-time polymerase chain reaction for determining expression messenger RNA levels and by immunocytostaining for determining the protein expression of odontogenic markers (ie, dentin sialoprotein, dentin matrix protein 1, bone morphogenic protein 2, bone morphogenic protein 7) by measuring alkaline phosphatase enzyme activity and lastly by von Kossa staining for determining mineralization. RESULTS: The results revealed for the first time that F68 has a great potential to boost odontogenic differentiation of hTGSCs. P85 was found to reduce cell viability during differentiation. F127 was nontoxic to hTGSCs but did not have any effect on differentiation. CONCLUSIONS: The positive effect of F68 on odontogenic differentiation might enable more efficient pulp regeneration. Yet, the exact mechanism of how F68 alters the differentiation pattern of hTGSCs remains to be investigated in the future studies.

Sequential injection immunoassay for human bone morphogenic protein-7 using an immunoreactor immobilized with anti-human bone morphogenic protein-7 antibody--CdSe/ZnS quantum dot conjugates.

The detection of human bone morphogenic protein-7 (BMP-7) was achieved using a sequential injection immunoassay (SIIA) system. The SIIA system is based on the binding between BMP-7 and anti-human BMP-7 (AbBMP7)-CdSe/ZnS quantum dot (QD) conjugates immobilized onto a glass disk or an optical fiber, using fluorescence detection at excitation and emission wavelengths of 470 nm and 580 nm, respectively. The AbBMP7-QD conjugates were prepared by conjugating anti-human BMP-7 antibody (AbBMP7) to hydrophilic CdSe/ZnS core/shell quantum dots (QDs). The SIIA system was fully automated using software written in the LabVIEW™ development environment. The analytical performance of the SIIA system was characterized with a number of variables such as carrier flow rate and elution buffer. Under partially optimized operating conditions, the SIIA system had a linear calibration graph at up to 10.0 ng mL(-1) BMP-7 (R(2)≥0.975) and a sample frequency of two samples per hour. The SIIA system with an optical fiber immunosensor was used to detect and quantify BMP-7 in spiked real samples obtained from a biological process with recoveries in the range of 95-102%.

Effect of erythropoietin on mesenchymal stem cell differentiation and secretion in vitro in an acute kidney injury microenvironment.

We investigated the effect of erythropoietin (EPO) on differentiation and secretion of bone marrow-derived mesenchymal stem cells in an acute kidney injury microenvironment. Acute kidney injury mouse models were prepared. Both renal cortices were then immediately collected to produce the ischemia/reperfusion kidney homogenate supernatant. The morphological and ultrastructural changes in the cells were observed using an inverted microscope and a transmission electron microscope. Cytokeratin-18 was detected using flow cytometry. Bone morphogenetic protein-7 levels, hepatocyte growth factor, and vascular endothelial growth factor in the culture medium were detected using an enzyme-linked immunosorbent assay. The cells had high CD29 and CD44 expression, as well as low CD34 and CD45 expression. More round and oval cells with cobble-like appearances were observed after EPO treatment. In addition, an increase in the number of rough endoplasmic reticula, lysosomes, and mitochondria was observed in the cytoplasm; the intercellular junction peculiar to epithelial cells was also seen on the cell surface. After treatment with ischemia/reperfusion kidney homogenate supernatant, cytokeratin-18 expression increased significantly and EPO could magnify its expression. Bone morphogenetic protein-7 levels, hepatocyte growth factor, and vascular endothelial growth factor levels after treatment with ischemia/reperfusion kidney homogenate supernatant significantly decreased, whereas EPO increased the cytokine secretion. The acute kidney injury microenvironment can induce the bone marrow-derived mesenchymal stem cells to partially differentiate into renal tubular epithelium-shaped cells, but weaken their secretion function. EPO intervention can boost up their differentiation function and reverse their low secretion effect.

Bone graft and bone graft substitutes in spine surgery: current concepts and controversies.

Iliac crest bone graft has long been the standard adjunct used in spine fusion surgery. This graft provides osteogenic, osteoinductive, and osteoconductive elements that aid in creation of a fusion mass. However, morbidity associated with bone graft harvest has led surgeons to seek other potential adjuncts, including bone morphogenetic proteins, demineralized bone matrix, and graft expanders such as synthetic bone graft and allograft. Knowledge of fusion biology is required to understand the benefits and limitations of these agents, which promote fusion via one of four mechanisms: osteogenesis, osteoinduction, osteoconduction, and osteopromotion. Although bone morphogenetic proteins have shown a clear ability to aid in bone formation and successful fusion, recent concern regarding their safety has tempered enthusiasm regarding their use.

Effects of estrogen deficiency and/or caffeine intake on alveolar bone loss, density, and healing: a study in rats.

BACKGROUND: The aim of this study is to evaluate the effects of caffeine and/or estrogen deficiency on ligature-induced bone loss (BL), trabecular bone area (TBA), and postextraction bone healing (BH). METHODS: Rats were assigned into one of the following groups (15 each): 1) control = non-ingestion of caffeine/sham surgery; 2) caffeine = ingestion of caffeine/sham surgery); 3) ovariectomized (OVX) = non-ingestion of caffeine/ovariectomy; or 4) caffeine/OVX = ingestion of caffeine/ovariectomy. The rats were under caffeine administration for 65 days and/or estrogen deficiency for 51 days. On day 21 after ovariectomy, one first mandibular molar received a ligature and the contralateral tooth was not ligated. The first maxillary molars were extracted 8 days before sacrifice. BL, TBA, the positive cells for tartrate-resistant acid phosphatase (TRAP), receptor activator of nuclear factor-κB ligand (RANKL), and osteoprotegerin (OPG) were analyzed in the furcation area of mandibular molars. Histometric BH and gene expression of bone morphogenetic protein (BMP)-2, BMP-7, osteopontin, and bone sialoprotein were evaluated in alveolar sockets. RESULTS: The caffeine group presented the greatest BL and the OVX group the highest number of TRAP-positive (TRAP(+)) cells around ligated teeth (P <0.05). The control group presented higher TBA and BH than the other groups (P <0.05). All test groups presented higher RANKL/OPG(+) cells than the control group around ligated/unligated teeth. The OVX and caffeine/OVX groups presented a greater number of TRAP(+) cells around unligated teeth than the control group (P <0.05). There were no differences among groups for gene expression (P >0.05). CONCLUSIONS: Caffeine increased BL in ligated teeth. Caffeine and/or estrogen deficiency decreased TBA in the unligated teeth and reduced BH after tooth extraction.

Overexpression of PACEsol improves BMP-7 processing in recombinant CHO cells.

Bone morphogenetic protein-7 (BMP-7) is a member of the TGF-ß superfamily and plays a critical role in cartilage, bone, and kidney development. BMP-7 is synthesized as a large precursor and undergoes proteolytic cleavage by subtilisin-like proprotein convertase to secrete the functionally active mature dimer. When CHO cells producing recombinant human BMP-7 (rhBMP) (CHO-BMP-7) were cultivated in a serum-free suspension culture, a significant amount of unwanted precursor forms of rhBMP-7 (ca. 69% of total rhBMP-7), along with the mature form of rhBMP-7, was secreted into the culture medium, likely due to the insufficient amount of the proteolytic cleaving enzyme within the secretory pathway. In order to solve this problem, a soluble form of the paired basic amino acid cleaving enzyme (PACEsol), responsible for the majority of the processing events occurring in the constitutive secretory pathway in mammalian cells, was overexpressed in CHO-BMP-7 cells. Overexpression of PACEsol was effective in processing the precursor forms of BMP-7, while it did not significantly affect cell growth. As a result, the culture supernatants of CHO-BMP-7 cells overexpressing PACEsol contained almost 100% of the mature BMP-7 form. Taken together, the results show that PACEsol overexpression in CHO-BMP-7 cells is an efficient means of increasing the production of mature BMP-7 and facilitating the downstream purification steps by eliminating the need to remove the precursor forms.

Activity of bone morphogenetic protein-7 after treatment at various temperatures: freezing vs. pasteurization vs. allograft.

Insufficient bone union is the occasional complication of biomechanical reconstruction after malignant bone tumor resection using temperature treated tumor bearing bone; freezing, pasteurization, and autoclaving. Since bone morphogenetic protein (BMP) plays an important role in bone formation, we assessed the amount and activity of BMP preserved after several temperature treatments, including -196 and -73°C for 20 min, 60 and 100°C for 30 min, 60°C for 10h following -80°C for 12h as an allograft model, and 4°C as the control. The material extracted from the human femoral bone was treated, and the amount of BMP-7 was analyzed using an enzyme-linked immunosorbent assay. Then, the activity of recombinant human BMP-7 after the treatment was assessed using a bioassay with NIH3T3 cells and immunoblotting analysis to measure the amount of phospho-Smad, one of the signaling substrates that reflect the intracellular reaction of BMPs. Both experiments revealed that BMP-7 was significantly better preserved in the hypothermia groups. The percentages of the amount of BMP-7 in which the control group was set at 100% were 114%, 108%, 70%, 49%, and 53% in the -196, -73, 60, 100°C, and the allograft-model group, respectively. The percentages of the amount of phospho-Smad were 89%, 87%, 24%, 4.9%, and 14% in the -196, -73, 60, 100°C, and the allograft-model group, respectively. These results suggested that freezing possibly preserves osteoinductive ability than hyperthermia treatment.