Differentiation Of Osteoblast Precursor Cells Research Articles

Enhancing the biological functionality of poly (lactic‐co‐glycolic acid) cage‐like structures through surface modification with micro‐ and nano‐sized hydroxyapatite particles

AbstractBiomaterials with exceptional performance are crucial for addressing the challenges of complex bone regeneration. Compared with traditional three‐dimensional scaffolds, injectable microspheres enable new strategies for the treatment of irregular bone defects. Biodegradable poly (lactic‐co‐glycolic acid) has found widespread applications as microcarriers of drugs, proteins, and other active macromolecules. Applied to the surface of poly (lactic‐co‐glycolic acid) cage‐like structures (PLGA‐CAS), hydroxyapatite (HA) effectively reduces inflammation while enhancing biological effects. In this study, we loaded the surface of PLGA‐CAS with micro‐ and nano‐hydroxyapatite particles, referred to as μHA/PLGA‐CAS and nHA/PLGA‐CAS, respectively. Subsequently, their material characteristics and biological effects were assessed. The incorporation of hydroxyapatite onto PLGA‐CAS resulted in enhanced surface roughness and hydrophilicity, coupled with improved thermal stability and delayed degradation. Furthermore, μHA/PLGA‐CAS induced osteogenic differentiation of osteoblast precursor cells, while nHA/PLGA‐CAS improved endothelial cell adhesion and stimulated angiogenic differentiation in vitro. Collectively, these findings suggest that μHA/PLGA‐CAS and nHA/PLGA‐CAS, each with distinct characteristics, hold significant potential for application as microcarriers in various biomedical contexts.

CRP inhibits the osteoblastic differentiation of OPCs via the up-regulation of primary cilia and repression of the Hedgehog signaling pathway.

Inflammation disrupts bone metabolism and leads to bone damage. C-reactive protein (CRP) is a typical inflammation marker. Although CRP measurement has been conducted for many decades, how osteoblastic differentiation influences molecular mechanisms remains largely unknown. The present study attempted to investigate the effects of CRP on primary cultured osteoblast precursor cells (OPCs) while elucidating the underlying molecular mechanisms. OPCs were isolated from suckling Sprague-Dawleyrats. Fewer OPCs were observed after recombinant C-reactive protein treatment. In a series of experiments, CRP inhibited OPC proliferation, osteoblastic differentiation, and the OPC gene expression of the hedgehog (Hh) signaling pathway. The inhibitory effect of CRP on OPC proliferation occurred via blockade of the G1-S transition of the cell cycle. In addition, the regulation effect of proto cilium on osteoblastic differentiation was analyzed using the bioinformatics p. This revealed the primary cilia activation of recombinant CRP effect on OPCs through in vitro experiments. A specific Sonic Hedgehog signaling agonist (SAG) rescued osteoblastic differentiation inhibited by recombinant CRP. Moreover, chloral hydrate, which removes primary cilia, inhibited the Suppressor of Fused (SUFU) formation and blocked Gli2 degradation. This counteracted osteogenesis inhibition caused by CRP. Therefore, these data depict that CRP can inhibit the proliferation and osteoblastic differentiation of OPCs. The underlying mechanism could be associated with primary cilia activation and Hh pathway repression.

P-15 promotes chondrocyte proliferation in osteoarthritis by regulating SFPQ to target the Akt-RUNX2 axis

BackgroundThe disruption of chondrocyte proliferation and differentiation is a critical event during the process of joint injury in osteoarthritis (OA). P-15 peptides could bind to integrin receptors on various precursor cells, promote cell adhesion, release growth factors, and promote the differentiation of osteoblast precursor cells. However, the role of P-15 in OA, particularly in chondrocyte proliferation, is not fully understood.MethodsThe activity of SFPQ and RUNX2 in the bone tissue of patients with osteoarthritis was analyzed using quantitative real-time polymerase chain reaction (qRT-PCR). Interleukin-1β (IL-1β) inducer was performed to establish an in vitro model of OA. Cell proliferation was measured by CCK-8 assay. The expressions of COL2a1, ACAN, COMP, SOX9, and BMP2 related to cartilage differentiation were detected using qRT-PCR. In addition, the expression levels of SFPQ, AKT, p-AKT, and RUNX2 were detected using Western blotting.ResultsThe results showed that the expression of SFPQ was significantly decreased and the expression of RUNX2 was significantly increased in osteoarthritis cartilage tissue. P-15 peptide reversed IL-1β-induced cell proliferation obstruction and alleviated chondrocyte damage. Furthermore, P-15 polypeptide increased the expression levels of cartilage differentiation genes COL2a1, ACAN, and BMP2, while decreasing the expression of COMP and SOX9 in an inverse dose-dependent manner. Then specific interfering RNA proved that P-15 maintains chondrocyte stability and is associated with the SFPQ gene. Finally, we confirmed that P-15 inhibited the Akt-RUNX2 pathway, which is regulated in the expression of SFPQ.ConclusionsP-15 can mitigate chondrocyte damage and osteoarthritis progression by inhibiting cell death and modulating SFPQ-Akt-RUNX2 pathway, offering an opportunity to develop new strategies for the treatment of osteoarthritis.

Effect of Vancomycin Applied to the Surgical Site on Fracture Healing in a Diabetic Rat Model.

Prophylactic vancomycin treatment decreases the prevalence of surgical site and deep infections by >70% in diabetic patients undergoing reconstructive foot and ankle surgery. Thus, determining whether clinically relevant local vancomycin doses affect diabetic fracture healing is of medical interest. We hypothesized that application of vancomycin powder to the fracture site during surgery would not affect healing outcomes, but continuous exposure of vancomycin would inhibit differentiation of osteoblast precursor cells and their osteogenic activity in vitro. The vancomycin dose used to treat the diabetic rats was a modest increase to routine surgical site vancomycin application of 1 to 2 g for a 70-kg adult (21 mg/kg). After femur fracture in BB-Wistar type 1 diabetic rats, powdered vancomycin (25 mg/kg) was administered to the fracture site. Bone marrow and periosteal cells isolated from diabetic bones were cultured and treated with increasing levels of vancomycin (0, 5, 50, 500, or 5000 µg/mL). Radiographic scoring, micro-computed tomography (µCT) analysis, and torsion mechanical testing failed to identify any statistical difference between the vancomycin-treated and the untreated fractured femurs 6 weeks postfracture. Low to moderate levels of vancomycin treatment (5 and 50 µg/mL) did not impair cell viability, osteoblast differentiation, or calcium deposition in either the periosteum or bone marrow-derived cell cultures. In contrast, high doses of vancomycin (5000 µg/mL) did impair viability, differentiation, and calcium deposition. In this diabetic rodent fracture model, vancomycin powder application at clinically relevant doses did not affect fracture healing or osteogenesis.

Spheroid culture for chondrocytes triggers the initial stage of endochondral ossification.

Endochondral ossification is the process of bone formation derived from growing cartilage duringskeletal development. In previous studies, we provokedthe osteocyte differentiation of osteoblast precursor cells under a three-dimensional (3D) culture model. To recapitulate the endochondral ossification, the present study utilized the self-organized scaffold-free spheroid model reconstructed by pre-chondrocyte cells. Within 2-day cultivation in the absence of the chemically induced chondrogenesis supplements, the chondrocyte marker was greatly expressed in the inner region of the spheroid, whereas the hypertrophic chondrocyte marker was strongly detected in the surface region of the spheroid. Notably, we found out that the gene expression levels of osteocyte markers were also greatly upregulated compared to the conventional 2D monolayer. Moreover, after long-term cultivation for 28 days, it induced morphological changes in the spheroid, such as cellular hypertrophy and death. In this study, in order to recapitulate the initial stage of the endochondral ossification, we highlighted the potentials of the 3D culture method to drive the hypertrophic chondrocyte differentiation of the pre-chondrocyte cells.

Urine-derived stem cells-extracellular vesicles ameliorate diabetic osteoporosis through HDAC4/HIF-1α/VEGFA axis by delivering microRNA-26a-5p.

Critical roles of stem cell-extracellular vesicles (EVs) in the management of osteoporosis have been documented. Here, this study was designed to enlarge the research of the specific effects and underlying mechanism of urine-derived stem cells-EVs (USCs-EVs) on osteoporosis in diabetes rats. Firstly, miR-26a-5p and histone deacetylase 4 (HDAC4) expression in USCs of rats after diabetic osteoporosis (DOP) modeling induced by streptozotocin injection was determined, followed by study of their interaction. Then, USCs-EVs were co-cultured with osteogenic precursor cells, the effects of miRNA-26a-5p (miR-26a-5p) on osteoblasts, osteoclasts, bone mineralization deposition rate were evaluated. Meanwhile, the effect of USCs-EVs carrying miR-26a-5p on DOP rats was assessed. Elevated miR-26a-5p was seen in USCs-EVs which limited HDAC4 expression. Moreover, USCs-EVs delivered miR-26a-5p to osteogenic precursor cells, thereby promoting their differentiation, enhancing the activity of osteoblasts, and inhibiting the activity of osteoclasts, thereby preventing DOP through the activation of hypoxia inducible factor 1 subunit alpha (HIF-1α)/vascular endothelial growth factor A (VEGFA) pathway by repressing HDAC4. In a word, USCs-EVs-miR-26a-5p is a promising therapy for DOP by activating HIF-1α/VEGFA pathway through HDAC4 inhibition. 1. USCs-EVs-miR-26a-5p targeted HDAC4 and limited HDAC4 expression. 2. miR-26a-5p was delivered by USCs-EVs into osteoblast precursor cells. 3. USCs-EVs-miR-26a-5p promoted the differentiation of osteoblast precursor cells into osteoblasts. 4. miR-26a-5p delivered by USCs-EVs could inhibit HDAC4. 5. USCs-EVs-miR-26a-5p could prevent the pathogenesis of DOP via HIF-1α/VEGFA aix.

2021 J. Leonard Goldner Award Winner: Vancomycin Topically Applied at the Surgical Site Does Not Impair Diabetic Fracture Healing and Dose-Dependently Inhibits Calcified Tissue Formation by Osteoblast Precursors Cells

Category:Diabetes; Basic Sciences/Biologics; TraumaIntroduction/Purpose:Prophylactic vancomycin treatment decreases the rates of surgical site and deep infections by >70% for diabetic patients undergoing reconstructive foot and ankle surgery. We aimed to identify whether local vancomycin at a clinically relevant dose impaired fracture healing in diabetic rats. Our hypothesis was that local vancomycin powder to the fracture site would not affect long term healing outcomes, but continuous exposure of vancomycin would inhibit differentiation of osteoblast precursor cells and their osteogenic activity in vitro. The 25 mg/kg vancomycin was a modest increase to routine surgical site vancomycin application of 1-2 grams for a 70 kg. adult (21.42 mg/kg). Determining how vancomycin affects diabetic fracture healing is of clinical interest, particularly considering its prophylactic use in foot and ankle patients.Methods:Following induction of a femur fracture in male BB Wistar type 1 diabetic rats, a longitudinal incision was made in the lateral aspect of the femur. Powdered vancomycin (25mg/kg) was administered to fracture site of treated (n=5), and Sham surgery (n=6) control rats. Femurs harvested at 6 weeks after fracture, were X-ray scored, µCT scanned and tested to failure in torsion. Bone marrow and periosteal cells isolated from diabetic bones and MC3T3 cells were plated for MTT survival and osteogenic assays. After 4 days, beta glycerol phosphate, ascorbic acid and either 0, 50, 500, or 5,000 µg/mL vancomycin were added to the media (n=3 per group). Cells were fixed at 7 and 14 days for alkaline phosphatase (ALP) staining and at 28 days for Alizarin Red S staining. Parametric data were analyzed using student t-tests. Non-parametric data were analyzed using a Kruskal-Wallis ANOVA on RANKs and Tukey post-hoc tests.Results:Radiographic scoring did not show differences between the control (2.28+-1.57) and treatment (3.67+-0.75) groups (p=0.093). BV/TV was similar between the control (61.2+-8.4 %) and treatment (56.5+-5.2 %) groups (p=0.081). Mechanical testing found similar values in normalized torque to failure (69.56+-36.80 vs 50.05+-28.82 %N*mm, p=0.356), and torsional rigidity (105.97+-103.62 vs 48.68+-32.95 %Nmm2, p=0.291), for control and treatment groups, respectively. ALP staining was comparable between groups at either 7 (p=0.809) or 14 days (p=0.343) in bone marrow or MC3T3 cells. Alizarin Red S staining found dose- dependent decreases in mineralized nodule formation between the untreated (10.16+-5.01 nodules per 3.8cm2), and 500µg/mL vancomycin (3.16+-2.26 nodules per 3.8cm2) bone marrow groups at 28 days (p=0.026). Periosteal and MC3T3 cell viability were only impaired at the 5000µg/mL vancomycin dose (p=0.001).Conclusion:Our study argues that local application of vancomycin does not affect diabetic fracture healing at clinically relevant doses. We studied a nearly 2-fold higher vancomycin dose than the 'standard' dose of 14.3mg/kg and less than the 143.5mg/kg dose that did not impair rat spine fusion. Although in vitro vancomycin did not affect cell viability and osteogenic staining in periosteal and MC3T3 cells, mineralized nodule formation was dose-dependently inhibited at day 28 in bone marrow cells. Our results bring novel insight into the effects of vancomycin in diabetic fracture healing and the how long-term dosing impacts cell viability and osteoblastogenesis.

Exosomes released from osteogenically differentiating SHEDs carry miRNAs that promote osteogenic differentiation of osteoblast precursor cells

Exosomes released from osteogenically differentiating SHEDs carry miRNAs that promote osteogenic differentiation of osteoblast precursor cells

Combined Fluorescence-Based in Vitro Assay for the Simultaneous Detection of Cell Viability and Alkaline Phosphatase Activity during Osteogenic Differentiation of Osteoblast Precursor Cells.

Novel bone substitute materials need to be evaluated in terms of their osteogenic differentiation capacity and possible unwanted cytotoxic effects in order to identify promising candidates for the therapy of bone defects. The activity of alkaline phosphatase (ALP) is frequently quantified as an osteogenic marker, while various colorimetric assays, like MTT assay, are used to monitor cell viability. In addition, the DNA or protein content of the samples needs to be quantified for normalization purposes. As this approach is time consuming and often requires the analysis of multiple samples, we aimed to simplify this process and established a protocol for the combined fluorescence-based quantification of ALP activity and cell viability within one single measurement. We demonstrate that the fluorogenic substrate 4-methylumbelliferone-phosphate (4-MUP) and the commonly used para-nitrophenylphosphate (p-NPP) produce comparable and highly correlating results. We further show that fluorescein–diacetate (FDA) can be used to quantify both cell viability and cell number without interfering with the quantification of ALP activity. The measurement of additional normalization parameters is, therefore, unnecessary. Therefore, the presented assay allows for a time-efficient, simple and reliable analysis of both ALP activity and cell viability from one sample and might facilitate experiments evaluating the osteogenic differentiation of osteoblast precursor cells.

EphB4/ TNFR2/ERK/MAPK signaling pathway comprises a signaling axis to mediate the positive effect of TNF-\u03b1 on osteogenic differentiation

BackgroundLow concentrations of tumor necrosis factor-alpha (TNF-α) and its receptor TNFR2 are both reported to promote osteogenic differentiation of osteoblast precursor cells. Moreover, low concentrations of TNF-α up-regulate the expression of EphB4. However, the molecular mechanisms underlying TNF-α-induced osteogenic differentiation and the roles of TNFR2 and EphB4 have not been fully elucidated.ResultsThe ALP activity, as well as the mRNA and protein levels of RUNX2, BSP, EphB4 and TNFR2, was significantly elevated in MC3T3-E1 murine osteoblast precursor cells when stimulated with 0.5 ng/ml TNF-α. After TNFR2 was inhibited by gene knockdown with lentivirus-mediated shRNA interference or by a neutralizing antibody against TNFR2, the pro-osteogenic effect of TNF-α was partly reversed, while the up-regulation of EphB4 by TNF-α remained unchanged. With EphB4 forward signaling suppressed by a potent inhibitor of EphB4 auto-phosphorylation, NVP-BHG712, TNF-α-enhanced expressions of TNFR2, BSP and Runx2 were significantly decreased. Further investigation into the signaling pathways revealed that TNF-α significantly increased levels of p-JNK, p-ERK and p-p38. However, only the p-ERK level was significantly inhibited in TNFR2-knockdown cells. In addition, the ERK pathway inhibitor, U0126 (10 μM), significantly reversed the positive effect of TNF-α on the protein levels of RUNX2 and BSP.ConclusionsThe EphB4, TNFR2 and ERK/MAPK signaling pathway comprises a signaling axis to mediate the positive effect of TNF-α on osteogenic differentiation.

Cell Condensation Triggers the Differentiation of Osteoblast Precursor Cells to Osteocyte-Like Cells.

Though the three-dimensional (3D) in vitro culture system has received attention as a powerful tool for conducting biological research, in vitro bone formation and osteocyte differentiation studies have mostly been based on results obtained using two-dimensional (2D) culture systems. Here, we introduced a rotatory culture system to fabricate 3D spheroids, using mouse osteoblast precursor cells. These spheroids, incubated for 2 days without chemical induction by osteogenic supplements, exhibited notably up-regulated osteocyte marker levels; osteoblast marker levels were down-regulated, as compared to those of the conventional 2D monolayer model. The cell condensation achieved with the 3D spheroid structure triggered a greater level of differentiation of osteoblast precursor cells into osteocyte-like cells than that observed during chemical induction. Our study might imply that osteoblasts proliferate and become condensed at the targeted bone remodeling site, because of which osteoblasts achieved the capability to differentiate into osteocytes in vivo.

Inhibiting effect of microRNA-187-3p on osteogenic differentiation of osteoblast precursor cells by suppressing cannabinoid receptor type 2

Induction of osteoblast differentiation is an effective approach in promoting osteoblastogenesis and bone formation. MicroRNA (miR) is a kind of small regulatory RNA molecules that control both physiological and pathological processes. The purpose of this research was to explore the function of miR-187-3p in proliferation and osteogenic differentiation in human osteoblastic precursor cells (hFOB 1.19). Our results showed a significant promotion of cell proliferation by miR-187-3p in hFOB 1.19 cell accompanied by increased proliferating cell nuclear antigen (PCNA) and Ki67 expression, whereas miR-187-3p knockdown led to an inhibition of cell proliferation. Moreover, our data revealed that miR-187-3p was decreased in hFOB 1.19 cells undergoing osteoblastic differentiation. Silencing of miR-187-3p dramatically accelerated hFOB 1.19 osteoblastic differentiation, as evidenced by the increase of alkaline phosphatase (ALP) activity and calcium deposition, as well as elevated osteopontin (OPN), collagen type I alpha 1 (COL1A1), and bone sialoprotein (BSP) gene expression, whereas overexpression of miR-187-3p suppressed osteoblastic differentiation. Furthermore, we demonstrated that miR-187-3p could inhibit cannabinoid receptor type 2 (CNR2) expression by targeting its 3’ untranslated region (UTR). Upregulation of CNR2 inversed the inhibiting influence of miR-187-3p on hFOB 1.19 osteogenic differentiation. Collectively, our results showed a pivotal role of miR-187-3p/CNR2 axis in osteoblastic differentiation, indicating that miR-187-3p may serve as a promising target in the therapy of osteoporosis.

Deletion of Estrogen Receptor Beta in Osteoprogenitor Cells Increases Trabecular but Not Cortical Bone Mass in Female Mice.

Although the role of ERα in regulating bone metabolism has been extensively studied, ERβ has been largely dismissed as a relevant modulator of bone mass. Previous studies examining ERβ utilized a germline knockout mouse expressing transcript variants of ERβ and displaying systemic hormonal changes that confounded interpretation of the skeletal phenotype. Thus, we used a conditional ERβ mouse model to achieve deletion of ERβ specifically in early osteoprogenitor cells using the Prx1-Cre driver. We observed marked increases in the trabecular bone volume fraction (of 58% [p < 0.003] and 93% [p < 0.0003] in 6- and 12-week-old female ERβ(Prx1-CKO) mice, respectively) but no changes in cortical bone. Serum estradiol and IGF-I levels were unaltered in ERβ(Prx1-CKO) mice. Bone formation and resorption indices by histomorphometry and serum assays were unchanged in these mice, suggesting that alterations in bone turnover may have occurred early in development. However, the ratio of colony-forming unit-osteoblasts (CFU-OBs) to CFU-fibroblasts (CFU-Fs) was increased in bone marrow cultures from ERβ(Prx1-CKO) compared with control mice, indicating increased differentiation of osteoblast precursor cells into osteoblasts in ERβ(Prx1-CKO) mice. Detailed quantitative polymerase chain reaction analyses of 128 genes in 16 prespecified pathways revealed significant downregulation of 11 pathways in ERβ(Prx1-CKO) mice. Thus, deletion of ERβ specifically in osteoblast lineage cells, in the absence of all splice variants, increases trabecular bone mass and modulates multiple pathways related to bone metabolism. These findings suggest that pharmacological inhibition of ERβ in bone may provide a novel approach to treat osteoporosis.

Local application of a proteasome inhibitor enhances fracture healing in rats.

The ubiquitin/proteasome system plays an important role in regulating the activity of osteoblast precursor cells. Proteasome inhibitors (PSIs) have been shown to stimulate the differentiation of osteoblast precursor cells and to promote bone formation. This raises the possibility that PSIs might be useful for enhancing fracture healing. In this study, we examined the effect of the local administration of PSI on fracture repair in rats. The effects of treatment on the healing of a fractured femur were assessed based on radiographs, micro-computed tomography (μCT) analysis, biomechanical testing, and histological analysis. PSI enhanced osteogenic differentiation in bone marrow- and periosteum-derived mesenchymal progenitor cells in vitro. Moreover, the local administration of PSI in vivo promoted fracture healing in rats, as demonstrated by an increased fracture callus volume in radiographs at 2 weeks post-fracture, and improved radiographic scores. By week 4, PSI treatment had enhanced biomechanical strength and mineral density in the callus as assessed using bending tests, and μCT, respectively. Histological sections demonstrated that PSI treatment accelerated endochondral ossification during the early stages of fracture repair. Although further investigations are necessary to assess its clinical use, the local administration of PSIs might be a novel, and effective therapeutic approach for fracture repair.

In vitro bio-functional performances of the novel superelastic beta-type Ti–23Nb–0.7Ta–2Zr–0.5N alloy

The materials used for internal fracture fixations and joint replacements are mainly made of metals which still face problems ranging from higher rigidity than that of natural bone to leaching cytotoxic metallic ions. Beta (β)-type titanium alloys with low elastic modulus made from non-toxic and non-allergenic elements are desirable to reduce stress shielding effect and enhance bone remodeling. In this work, a new β-type Ti–23Nb–0.7Ta–2Zr–0.5N alloy with a Young's modulus of approximately 50GPa was designed and characterized. The behavior of MC3T3-E1 pre-osteoblasts on the new alloy, including adhesion, proliferation and differentiation, was evaluated by examining the cytoskeleton, focal adhesion formation, metabolic activity and extracellular matrix mineralization. Results indicated that the pre-osteoblast cells exhibited a similar degree of attachment and growth on Ti–23Nb–0.7Ta–2Zr–0.5N and Ti–6Al–4V. However, the novel alloy proved to be significantly more efficient in sustaining mineralized matrix deposition upon osteogenic induction of the cells than Ti–6Al–4V control. Further, the analysis of RAW 264.7 macrophages cytokine gene and protein expression indicated no significant inflammatory response. Collectively, these findings suggest that the Ti–23Nb–0.7Ta–2Zr–0.5N alloy, which has an increased mechanical biocompatibility with bone, allows a better osteogenic differentiation of osteoblast precursor cells than Ti–6Al–4V and holds great potential for future clinical prosthetic applications.

Strontium ranelate: the mechanism of its effect on the bone, prevention and treatment of fractures (a review of literature)

On the basis of literature data and their own studies, the authors present information about strontium ranelate. There is voluminous evidential base that strontium ranelate has a double mechanism of action in the influence on the process of bone remodelling – inhibition of processes of resorption and stimulation of osteogenesis. The key mechanism of the latter consists in stimulation of proliferation and differentiation of osteoblast precursor cells by the drug, increase of their viability and metabolism, directed to building of the bone tissue matrix and bone mineralization, these items being the basic link in the prevention of osteoporotic fractures and bone regeneration in fractures. The authors describe certain mechanisms of cell resorption and the role of programmed osteoblastic diferentiation cells in activation of osteoclustogenesis by means of the RANK/RANKL/OPG cytokine system, which is the key molecular target for the double effect of strontium ranelate on bone cells. For the first time in the assessment of osteotrophic preparations, attention is paid to the influence of strontium ranelate on osteocytes and its positive role in the differentiation of osteoblasts into osteocytes is shown. The efficacy of the drug is proved by a reduction of the absolute risk of development of vertebral and peripheral fractures as well as in the optimization of reparative osteogenesis, thereby enabling doctors to approach the prophylaxis and treatment of osteoporotic fractures differentially in each particular case.

Reactive oxygen species derived from Nox4 mediateBMP2gene transcription and osteoblast differentiation

BMP-2 (bone morphogenetic protein-2) promotes differentiation of osteoblast precursor cells to mature osteoblasts that form healthy bone. In the present study, we demonstrate a novel mechanism of BMP-2-induced osteoblast differentiation. The antioxidant NAC (N-acetyl-L-cysteine) and the flavoprotein enzyme NAD(P)H oxidase inhibitor DPI (diphenyleneiodonium) prevented BMP-2-stimulated alkaline phosphatase expression and mineralized bone nodule formation in mouse 2T3 pre-osteoblasts. BMP-2 elicited a rapid generation of ROS (reactive oxygen species) concomitant with increased activation of NAD(P)H oxidase. NAC and DPI inhibited BMP-2-induced ROS production and NAD(P)H oxidase activity respectively. NAD(P)H oxidases display structurally similar catalytic subunits (Nox1-5) with differential expression in various cells. We demonstrate that 2T3 pre-osteoblasts predominantly express the Nox4 isotype of NAD(P)H oxidase. To extend this finding, we tested the functional effects of Nox4. Adenovirus-mediated expression of dominant-negative Nox4 inhibited BMP-2-induced alkaline phosphatase expression. BMP-2 promotes expression of BMP-2 for maintenance of the osteoblast phenotype. NAC and DPI significantly blocked BMP-2-stimulated expression of BMP2 mRNA and protein due to a decrease in BMP2 gene transcription. Dominant-negative Nox4 also mimicked this effect of NAC and DPI. Our results provide the first evidence for a new signalling pathway linking BMP-2-stimulated Nox4-derived physiological ROS to BMP-2 expression and osteoblast differentiation.

Anti-DKK1 mAb (BHQ880) as a Potential Therapeutic for Multiple Myeloma.

Decreased activity of osteoblasts (OB) contributes to osteolytic lesions in multiple myeloma (MM). Dickkopf-1 (DKK1) is a soluble Wnt inhibitor produced by MM cells that inhibits osteoblastogenesis in the bone marrow (BM) microenvironment. DKK1, also present in MM patient plasma, has been shown to inhibit the differentiation of osteoblast precursor cells in vitro, and its plasma level correlates with focal bone lesions in MM. Therefore, we have evaluated DKK1 as a target in MM in the context of the BM microenvironment. We first analyzed the effects of a DKK1 neutralizing antibody (BHQ880) on OB differentiation. Anti-DKK1 Ab was able to increase differentiation of mesenchymal stem cells (MSCs) to OB and reduce IL-6 levels following MSC differentiation to OB. While OB activity in MSCs cultured with osteoblast differentiation media was reduced in the presence of INA-6 MM cells, treatment with DKK1 neutralizing antibody was able to restore OB activity in a dose-dependent manner, overcoming the negative effect of MM cells. We did not observe a direct effect of this Ab on growth or survival of human MM cell lines. However, when a panel of MM cell lines was cultured with BM stromal cells (BMSC), the Ab induced significant growth inhibitory effects on MM cells, associated with downregulation of IL-6 produced by MSCs. To evaluate the in vivo bone and antitumor effects of anti-DKK1 Ab treatment on MM cells in the human microenvironment, we used a SCID-hu murine model, using INA-6 MM cells. In this model we observed a direct correlation between the level of soluble human DKK1 in murine blood and the tumor growth. Following treatment with BHQ880, by bone histology analysis, we observed increased trabecular bone and numbers of OBs in the retrieved bone chip from BHQ880 treated mice compared to control mice, one month after initial dose. We also found an increase in human osteocalcin in the serum of BHQ880-treated mice compared to the controls, suggesting an increase in OB activity. Interestingly, we observed suppression of myeloma growth, measured by changes in serum level of soluble huIL6sR, 4 weeks following BHQ880 treatment of the mice. These results support DKK1 as an important therapeutic target in myeloma and provide the rationale for clinical evaluation of this molecule in MM to improve bone disease and to inhibit MM growth.

Inhibition of p38 MAPK signaling in chondrocyte cultures results in enhanced osteogenic differentiation of perichondral cells

Chondrocytes and osteoblasts originate from the same progenitor cell; however, both are characterized by distinct gene expression profiles once they are differentiated. Signals from differentiating chondrocytes, such as Indian hedgehog (Ihh), regulate the differentiation of osteoblast precursor cells. The MAPK pathways play important roles in controlling the differentiation of both chondrocytes and osteoblasts, with the p38 pathway being particularly relevant in skeletal cells. In the present study, we investigated the effects of p38 inhibition on osteoblastic marker gene expression in chondrocyte cultures. Using high-density micromass cultures of mesenchymal cells as well as chondrocytes that had differentiated in vivo and were maintained in short-term monolayer culture, we demonstrate elevated Runx2, Osterix and Osteocalcin transcript levels in chondrocyte cultures upon inhibition of p38 activity with the pharmacological inhibitor PD169316. Osteocalcin immunolocalization was restricted to perichondral/periosteal cells in micromass cultures, suggesting that inhibition of p38 results in increased periosteal osteogenesis. Coinciding with increased expression of these genes, we observed elevated levels of transcripts for Ihh and its target gene, Ptch, in response to p38 inhibition. Addition of recombinant hedgehog protein mimicked some effects of p38 inhibitors. We therefore suggest that p38 signaling regulates chondrocyte–perichondral cell communication during skeletal development, partially through increased Ihh signaling.

The mechanical phenotype of biglycan-deficient mice is bone- and gender-specific

Biglycan (bgn) is a small leucine-rich proteoglycan (SLRP) enriched in the extracellular matrix of skeletal tissues. While bgn is known to be involved in the growth and differentiation of osteoblast precursor cells and regulation of collagen fibril formation, it is unclear how these functions impact bone's geometric and mechanical properties, properties which are integral to the structural function of bone. Because the genetic control of bone structure and function is both local- and gender-specific and because there is evidence of gender-specific effects associated with genetic deficiencies, it was hypothesized that the engineered deletion of the gene encoding bgn would result in a cortical bone mechanical phenotype that was bone- and gender-specific. In 11-week-old C57BL6/129 mice, the cortical bone in the mid-diaphyses of the femora and tibiae of both genders was examined. Phenotypic changes in bgn-deficient mice relative to wild type controls were assayed by four-point bending tests to determine mechanical properties at the whole bone (structural) and tissue levels, as well as analyses of bone geometry and bone formation using histomorphometry. Of the bones examined, bgn deficiency most strongly affected the male tibiae, where enhanced cross-sectional geometric properties and bone mineral density were accompanied by decreased tissue-level yield strength and pre-yield structural deformation and energy dissipation. Because pre-yield properties alone were impacted, this implies that the gene deletion causes important alterations in mineral and/or the matrix/mineral ultrastructure and suggests a new understanding of the functional role of bgn in regulating bone mineralization in vivo.