Inhibition of TGFβ signaling decreases osteogenic differentiation of fibrodysplasia ossificans progressiva fibroblasts in a novel in vitro model of the disease

Fibrodysplasia ossificans progressiva is a rare genetic disorder characterized by progressive heterotopic ossification. FOP patients develop soft tissue lumps as a result of inflammation-induced flare-ups which leads to the irreversible replacement of skeletal muscle tissue with bone tissue. Classical FOP patients possess a mutation (c.617G > A; R206H) in the ACVR1-encoding gene which leads to dysregulated BMP signaling. Nonetheless, not all FOP patients with this mutation exhibit equal severity in symptom presentation or disease progression which indicates a strong contribution by environmental factors. Given the pro-inflammatory role of TGFβ, we studied the role of TGFβ in the progression of osteogenic differentiation in primary dermal fibroblasts from five classical FOP patients based on a novel method of platelet lysate-based osteogenic transdifferentiation. During the course of transdifferentiation the osteogenic properties of the cells were evaluated by the mRNA expression of Sp7/Osterix, Runx2, Alp, OC and the presence of mineralization. During transdifferentiation the expression of osteoblast markers Runx2 (p < 0.05) and Alp were higher in patient cells compared to healthy controls. All cell lines exhibited increase in mineralisation. FOP fibroblasts also expressed higher baseline Sp7/Osterix levels (p < 0.05) confirming their higher osteogenic potential. The pharmacological inhibition of TGFβ signaling during osteogenic transdifferentiation resulted in the attenuation of osteogenic transdifferentiation in all cell lines as shown by the decrease in the expression of Runx2 (p < 0.05), Alp and mineralization. We suggest that blocking of TGFβ signaling can decrease the osteogenic transdifferentiation of FOP fibroblasts.     

Osteogenesis by human osteoblastic cells in diffusion chamber In vivo

Osteogenic potential of osteoblastic cells isolated from human bone was evaluated by a diffusion chamber method. Cells placed in diffusion chambers were implanted intraperitcneally into the athymic mice. The diffusion chambers cultured in vivo were harvested and examined after implantation for 6–8 weeks. The content of the chamber was proved by a soft roentogenogram to contain the radioopaque area. Light microscopic study revealed that this area was made up of bone-like nodule. Within the diffusion chambers, the cell layers were observed alongside the interior surface of the membrane filters, and mineralized nodules were formed among the cell layers. The mineralized nodules were confirmed by staining with the von Kossa technique for calcium mineral deposits. In electron microscopic study, the osteoblastic cells had a relatively large nuclei, and an abundant rough endoplasmic reticulum produced numerous extracellular matrix. In the bone-like nodules, the osteoblastic cells were surrounded by a heavily mineralized matrix with nonmineralized matrix separating the osteoblastic cells from the mineralized matrix. The mineralized matrix contained well-banded collagen fibrils. Matrix vesicles and collagen fibrils which were closely associated with mineral deposition were observed at the mineralizing front. These results together with our previous report [13] indicate that isolated human osteoblastic cells possessed osteogenic potential in vivo as well as mineralization activity in vitro.     

Femoral strength in osteoporotic women treated with teriparatide or alendronate

To gain insight into the clinical effect of teriparatide and alendronate on the hip, we performed non-linear finite element analysis of quantitative computed tomography (QCT) scans from 48 women who had participated in a randomized, double-blind clinical trial comparing the effects of 18-month treatment of teriparatide 20 μg/d or alendronate 10 mg/d. The QCT scans, obtained at baseline, 6, and 18 months, were analyzed for volumetric bone mineral density (BMD) of trabecular bone, the peripheral bone (defined as all the cortical bone plus any endosteal trabecular bone within 3 mm of the periosteal surface), and the integral bone (both trabecular and peripheral), and for overall femoral strength in response to a simulated sideways fall. At 18 months, we found in the women treated with teriparatide that trabecular volumetric BMD increased versus baseline (+ 4.6%, p < 0.001), peripheral volumetric BMD decreased (− 1.1%, p < 0.05), integral volumetric BMD (+ 1.0%, p = 0.38) and femoral strength (+ 5.4%, p = 0.06) did not change significantly, but the ratio of strength to integral volumetric BMD ratio increased (+ 4.0%, p = 0.04). An increase in the ratio of strength to integral volumetric BMD indicates that overall femoral strength, compared to baseline, increased more than did integral density. For the women treated with alendronate, there were small (< 1.0%) but non-significant changes compared to baseline in all these parameters. The only significant between-treatment difference was in the change in trabecular volumetric BMD (p < 0.005); related, we also found that, for a given change in peripheral volumetric BMD, femoral strength increased more for teriparatide than for alendronate (p = 0.02). We conclude that, despite different compartmental volumetric BMD responses for these two treatments, we could not detect any overall difference in change in femoral strength between the two treatments, although femoral strength increased more than integral volumetric BMD after treatment with teriparatide.     

Ureterovesical junction obstruction causes increment in smooth muscle contractility,and cholinergic and adrenergic activity in distal ureter of rabbits

Background/Purpose

The controversy in management of primary obstructed megaureter necessitates further elucidation of the underlying pathophysiology. We evaluated smooth muscle contractility, and cholinergic, adrenergic and serotonergic activity of rabbit distal ureters after ureterovesical junction (UVJ) obstruction.

Methods

Sham (SH) operation, partial obstruction (PO) and complete obstruction (CO) of the right UVJ were performed in rabbits. Three weeks later, distal ureters were isolated; spontaneous contractions (SC), contractile responses to electrical field stimulation (EFS), high KCl, carbachol, phenylephrine and serotonin were recorded.

Results

SC amplitudes increased in CO compared to PO and SH (p < 0.001). SC frequency was higher in CO (p < 0.05). EFS-induced contraction amplitudes were greater in CO than other groups (p < 0.05). High KCl-induced contractions were greater in CO (p < 0.001) and PO (p < 0.01). Carbachol-induced contractility was enhanced in CO and PO (p < 0.05). Contractile response to phenylephrine was greater in CO than other groups (p < 0.05). Serotonin induced contractile responses in CO and PO, greater in CO (p < 0.05). UVJ obstruction also increased spontaneous contractility in contralateral PO and CO ureters.

Conclusions

UVJ obstruction increased spontaneous and neurotransmitter-induced contractions in an obstruction grade-dependent manner. Obstruction also altered contractility of the contralateral ureters. Our findings may serve to provide further understanding of the pathophysiology of megaureter.     

Titanium particles suppress expression of osteoblastic phenotype in human mesenchymal stem cells.

Long-term stability of arthroplasty prosthesis depends on the integration between osseous tissue and the implant biomaterial. Integrity of the osseous tissue requires the contribution of mesenchymal stem cells and their continuous differentiation into an osteoblastic phenotype. This study aims to investigate the hypothesis that exposure to wear debris particles derived from orthopaedic biomaterials affects the osteoblastic differentiation of human mesenchymal stem cells (hMSC). Upon in vitro culture in the presence of osteogenic supplements (OS), we observe that cultures of hMSCs isolated from femoral head bone marrow are capable of osteogenic differentiation, expressing alkaline phosphatase, osteocalcin, and bone sialoprotein (BSP), in addition to producing collagen type I and BSP accompanied by extracellular matrix mineralization. Exposure of OS-treated hMSCs to submicron commercially pure titanium (cpTi) particles suppresses BSP gene expression, reduces collagen type I and BSP production, decreases cellular proliferation and viability, and inhibits matrix mineralization. In comparison, exposure to zirconium oxide (ZrO2) particles of similar size did not alter osteoblastic gene expression and resulted in only a moderate decrease in cellular proliferation and mineralization. Confocal imaging of cpTi-treated hMSC cultures revealed patchy groups of cells displaying disorganized cytoskeletal architecture and low levels of extracellular BSP. These in vitro findings suggest that chronic exposure of marrow cells to titanium wear debris in vivo may contribute to decreased bone formation at the bone/implant interface by reducing the population of viable hMSCs and compromising their differentiation into functional osteoblasts. Understanding the nature of hMSC bioreactivity to orthopaedic wear debris should provide additional insights into mechanisms underlying aseptic loosening.     

Evaluation of osteogenic cell differentiation in response to bone morphogenetic protein or demineralized bone matrix in a critical sized defect model using GFP reporter mice

We evaluated the osteoprogenitor response to rhBMP‐2 and DBM in a transgenic mouse critical sized defect. The mice expressed Col3.6GFPtopaz (a pre‐osteoblastic marker), Col2.3GFPemerald (an osteoblastic marker) and α‐smooth muscle actin (α‐SMA‐Cherry, a pericyte/myofibroblast marker). We assessed defect healing at various time points using radiographs, frozen, and conventional histologic analyses. GFP signal in regions of interest corresponding to the areas of new bone formation was quantified using a novel computer assisted algorithm. All defects treated with rhBMP‐2 healed. In contrast, the majority of the defects in the DBM (27/30) and control (28/30) groups did not heal. Quantitation of pre‐osteoblasts demonstrated a maximal response (% GFP+ cells/TV) in the Col3.6GFPtopaz mice at day 7 (7.2% ± 6.0, p < 0.05 compared to days 14, 21, 28, and 56). The maximal response of the Col2.3GFP cells was seen at days 14 (8.04% ± 5.0) and 21 (8.31% ± 4.32), p < 0.05. In contrast, DBM and control groups showed a limited osteogenic response at all time points. In conclusion, we demonstrated that the BMP and DBM induce vastly different osteogenic responses which should influence their clinical application as bone graft substitutes. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 32:1120–1128, 2014.     

Fluid flow stimulates expression of osteopontin and bone sialoprotein by bone marrow stromal cells in a temporally dependent manner

Bone marrow stromal cells (BMSCs) are multipotent progenitor cells with a capacity to form bone tissue in vivo, and to differentiate into the osteoblastic lineage in vitro. Drawing on evidence that bone is mechanosensitive and mechanical stimuli are anabolic, we postulate that proliferation and osteoblastic differentiation of BMSCs may be stimulated by mechanical forces. In this study, BMSCs cultured in the presence of osteogenic factors (dexamethasone, β-glycerophosphate, and ascorbate) were stimulated repeatedly (every second day) with shearing flow (1.6 dyn/cm²) for 5, 30, or 120 min, and assayed for systematic changes in cell number and phenotypic markers of osteoblastic differentiation. Cells exposed to fluid flow on days 2 and 4 after the addition of osteogenic factors and assayed at day 6 exhibited a modest decrease in cell number and increase in normalized alkaline phosphatase activity, suggesting the detachment of a non-osteogenic subpopulation. Cells exposed to fluid flow on days 6, 8, 10, and 12 and assayed at day 20 demonstrated maximal expression of osteopontin and bone sialoprotein mRNA with 30 min duration of flow. Concurrently, at day 20 expression of the adipogenic marker, lipoprotein lipase, was minimal with a 120-min duration of flow. These results indicate that repeated application of shear stress stimulates late phenotypic markers of osteoblastic differentiation of BMSCs in a manner that depends on the duration of stimulus. Finally, accumulation of prostaglandin E₂ in culture medium in response to shearing flow systematically decreased with repeated exposure to 30 and 120 min of shear stress (from day 6 to day 12), suggesting an adaptation of the cells to fluid flow.     

体外诱导家猪骨髓间充质干细胞向成骨细胞分化

目的观察家猪骨髓间充质干细胞(marrow mesenchymal stem cells,MSCs)在体外诱导条件下成骨分化的特征及相关基因的表达。方法选取3月龄雌性长白猪6头,无菌条件下于胫骨近端抽取骨髓15ml。采用密度梯度离心法并根据细胞贴壁特性对MSCs进行分离、纯化,倒置相差显微镜观察原代细胞生长情况,于培养第7天计算MSCs百分比含量及群体倍增值。将第1代细胞置于含1×10-8mmol/L地塞米松(dexamethasone,Dex)、10mmol/Lβ-磷酸甘油(β-glycerophosphate,β-GP)和82μg/ml抗坏血酸(ascorbic acid,Asc)的成骨诱导培养液中培养21d,作为实验组;DMEM培养液中培养作为对照组。分别行细胞形态学观察、碱性磷酸酶(alkaline phosphatase,ALP)组织化学染色、钙沉积和细胞增殖测定,采用实时定量PCR分析成骨分化的相关基因表达。结果原代MSCs特征:培养第1天,有核细胞大部分由悬浮的圆形血源性细胞组成;第3天换液弃除非贴壁细胞,MSCs克隆开始形成,细胞呈成纤维细胞样生长;第7天,镜下观察到大小不一的克隆。细胞在培养后12～14d基本长满,原代细胞群体倍增值平均为13。MSCs成骨分化:诱导培养14d实验组细胞形态由成纤维细胞样变成立方体样,而对照组细胞始终保持成纤维细胞样。培养5d,对照组细胞计数为11723±4040,实验组为10276±5513,二者差异无统计学意义(P>0.05)。与对照组相比,实验组诱导培养14d,ALP染色呈强阳性,21d钙沉积明显增加(P<0.01)。实验组MSCs成骨相关基因:核心结合因子α1(corebinding factorα1,Cbfα1)、osterix、ALP、型胶原、骨连接素(osteonectin,ON)、骨钙素(osteocalcin,OC)表达逐渐增强;Cbfα1、ALP、ON在分化早期增加明显;与第7天比较,第21天osterix和OC基因表达明显上调(P<0.05);第14天,型胶原表达也上调(P<0.05)。结论密度梯度离心法分离的猪MSCs,在体外诱导条件下能通过上调分化特异基因表达向成骨细胞分化。     

High-resolution morphometric analysis of human osteoblastic cell adhesion on clinically relevant orthopedic alloys.

Understanding the cellular basis of osteoblastic cell-biomaterial interaction is crucial to the analysis of the mechanism of osseointegration, a requirement of long-term orthopedic implant stability. Clinically, the amount of bone ingrowth is variable, and cellular parameters that influence ingrowth have yet to be clearly determined. In this study, two clinically relevant orthopedic alloys, titanium Ti6A14V (Ti) and cobalt-chrome-molybdenum (CC), were used for a comparative analysis of primary human osteoblastic cell adhesion and spreading, where cell adhesion represents the initial interaction between cellular elements and the biomaterial surface. The kinetic profile of adhesion revealed enhanced cell attachment upon rough Ti surfaces relative to rough CC. Using confocal laser scanning microscopy (CLSM), we observed that, during the first 12 h of contact with the substratum, osteoblastic cells were relatively less spread on rough Ti, whereas cells appeared elongated with multiple cellular extensions on rough CC. Focal adhesion contacts, as indicated by vinculin immunostaining, were distributed throughout the cells adhering to Ti, but were relatively sparse and localized to cellular processes on CC. Furthermore, three-dimensional CLSM reconstruction analysis indicated the presence of vinculin at all membrane-to-surface contact points on both Ti and CC. On Ti, these contact points closely followed the surface contour, whereas, on CC, they were restricted to relative topographic peaks only. Actin cytoskeletal reorganization was prominent in cells cultured on Ti, with stress fibers arranged throughout the cell body, whereas, on CC, actin filaments were sparse and localized primarily to cellular extensions. Because cell attachment mechanisms are likely to influence signal transduction and regulation of gene expression, these early differential responses of osteoblastic cells on Ti and CC may have functional implications on subsequent extracellular matrix mineralization and bone ingrowth at the cell-biomaterial interface.     

Covalent attachment of P15 peptide to titanium surfaces enhances cell attachment,spreading, and osteogenic gene expression

P15, a synthetic 15 amino acid peptide, mimics the cell‐binding domain within the alpha‐1 chain of human collagen is being tested in clinical trials to determine if it enhances bone formation in spinal fusions. We hypothesize that covalent attachment of P15 to titanium implants may also serve to promote osseointegration. To test this hypothesis, we measured osteoblast and mesenchymal cell adhesion, proliferation, and maturation on P15 tethered to a titanium (Ti‐P15) surface. P15 peptide was covalently bonded to titanium alloy surfaces and incubated with osteoblast like cells. Cell toxicity, adhesion, spreading, and differentiation was then evaluated. Real‐time quantitative PCR, Western blot analysis, and fluorescent immunohistochemistry was performed to measure osteoblast gene expression and differentiation. There was no evidence of toxicity. Significant increases in early cell attachment, spreading, and proliferation were observed on the Ti‐P15 surface. Increased filapodial attachments, α₂ integrin expression, and phosphorylated focal adhesion kinase immunostaining indicated activation of integrin signaling pathways. qRT‐PCR analysis indicated there was significant increase in osteogenic differentiation markers in cells grown on Ti‐P15 compared to control‐Ti. Western blotting confirmed these findings. Surface modification of titanium with P15 significantly increased cell attachment, spreading, osteogenic gene expression, and differentiation. Results of this study suggest that Ti‐P15 has the potential to safely enhance bone formation and promote osseointegration of titanium implants. © 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 30:1626–1633, 2012     

Atherogenic diet and minimally oxidized low density lipoprotein inhibit osteogenic and promote adipogenic differentiation of marrow stromal cells.

In osteoporosis, the bone marrow stroma osteogenic cell population declines and adipocyte numbers increase. We recently showed that oxidized lipids inhibit differentiation of preosteoblasts. In this report, we assess the effect of minimally oxidized low density lipoprotein (MM-LDL) on osteoblastic differentiation of murine marrow stromal cells, M2-10B4. MM-LDL, but not native LDL, inhibited stromal cell osteoblastic differentiation as demonstrated by inhibition of alkaline phosphatase activity, collagen I processing, and mineralization, through a mitogen-activated protein kinase-dependent pathway. In addition, marrow stromal cells from C57BL/6 mice fed a high fat, atherogenic diet failed to undergo osteogenic differentiation in vitro. The ability of MM-LDL to regulate adipogenesis was also assessed. Treatment of M2-10B4 as well as 3T3-L1 preadipocytes with MM-LDL, but not native LDL, promoted adipogenic differentiation in the presence of peroxisome proliferator-activated receptor (PPAR) gamma agonist thiazolidinediones, BRL49653 and ciglitizone. Based on promoter-reporter construct experiments, MM-LDL may be acting in part through activating PPARalpha. These observations suggest that LDL oxidation products promote osteoporotic loss of bone by directing progenitor marrow stromal cells to undergo adipogenic instead of osteogenic differentiation. These data lend support to the "lipid hypothesis of osteoporosis."     

Comparison of Osteogenic Ability of Rat Mesenchymal Stem Cells from Bone Marrow, Periosteum, and Adipose Tissue

Mesenchymal stem cells (MSCs) reside in many types of tissue and are able to differentiate into various functional cells including osteoblasts. Recently, adipose tissue–derived MSCs (AMSCs) have been shown to differentiate into many lineages, and they are considered a source for tissue regeneration. The purpose of this study was to compare the osteogenic differentiation capability of MSCs from bone marrow (BMSCs), MSCs from periosteum (PMSCs), and AMSCs using in vitro culture and in vivo implantation experiments. We harvested these MSCs from 7-week-old rats. The cells were seeded and cultured for 7 days in primary culture to assay a colony-forming unit. The frequency of the unit was the smallest in the BMSCs (P < 0.001). After primary culture, subculture was performed under osteogenic differentiation conditions for 1 and 2 weeks to detect mineralization as well as the bone-specific proteins of alkaline phosphatase and osteocalcin as osteogenic markers. BMSCs and PMSCs showed distinct osteogenic differentiation capability in comparison with other MSCs (P < 0.001). For the in vivo assay, composites of these cells and hydroxyapatite ceramics were subcutaneously implanted into syngeneic rats and harvested after 6 weeks. Micro-computed tomographic (CT) and histological analyses demonstrated that new bone formation was detected in the composites using BMSCs and PMSCs, although it was hard to detect in other composites. The CT analyses also demonstrated that the bone volume of BMSC composites was more than that of AMSC composites (P < 0.001). These results indicate that BMSCs and PMSCs could be ideal candidates for utilization in practical bone tissue regeneration.     

Bone Sialoprotein (BSP) is a Crucial Factor for the Expression of Osteoblastic Phenotypes of Bone Marrow Cells Cultured on Type I Collagen Matrix

In this study, we demonstrated that type I collagen matrix induced the expression of osteoblastic phenotypes of bone marrow cells, and that antibone sialoprotein (BSP) monoclonal antibody suppressed the expression of these phenotypes. On the other hand, BSP accelerated the expression of osteoblastic phenotypes of bone marrow cells. The adherent bone marrow cells were harvested from rat femur and cultured on type I collagen matrix gels in medium containing 15% fetal calf serum, neither β-glycerophosphate nor glucocorticoid. Cells showed osteoblastic phenotypes (high alkaline phosphatase activity, osteocalcin synthesis, and responsiveness against parathyroid hormone) on collagen matrix gels at week 3 after the inoculation, and simultaneously, BSP was detected in the conditioned medium by Western blotting using an anti-BSP monoclonal antibody. However, cells in the conventional culture dishes did not show osteoblastic phenotypes during the experimental period. To investigate the physiological function of BSP in osteoblastic differentiation, bone marrow cells were cultured on collagen matrix with an anti-BSP monoclonal antibody for 3 weeks. This treatment suppressed the expression of the osteoblastic phenotypes, and the effect of the antibody was abolished by the addition of bovine bone BSP. Furthermore, bovine bone BSP stimulated the expression of osteoblastic phenotypes of bone marrow cells. Our results indicate that BSP plays a crucial role in the expression of osteoblastic phenotypes of bone marrow cells. Received: 17 February 1999 / Accepted: 14 December 1999     

脂联素通过调节BMP信号通路促进骨髓干细胞成骨分化

目的 研究脂联素（adiponectin, ApN）对骨髓间充质干细胞（bone marrow mesenchymal stem cells, BMSCs）成骨分化的作用,并探讨其可能的机制。方法 体外培养BMSCs,构建ApN过表达质粒及干扰质粒,转染至BMSCs中。将BMSCs随机分为5组：对照组、过表达组、过表达空载组、干扰组和干扰空载组。茜素红染色观察各组细胞钙化沉积。碱性磷酸酶（alkaline phosphatase, ALP）染色观察各组细胞成骨分化能力。qRT-PCR检测ApN受体、骨形态发生蛋白（bone morphogenetic protein, BMP）信号通路及成骨相关基因表达情况。结果 与对照组相比,过表达组BMSCs中钙化沉积和ALP阳性表达增多,AdipoR1、AdipoR2、BMP2、RUNX2、Smad1和Smad5 mRNA表达量显著升高（P<0.05）;干扰组BMSCs中钙化沉积和ALP阳性表达减少,AdipoR1、AdipoR2、BMP2、RUNX2、Smad1和Smad5 mRNA表达量显著降低（P<0.05）。结论 ApN可能通过上调BMP信号通路促进BMSCs成骨分化。     

Lennox gastaut syndrome, review of the literature and a case report

Background

Facial trauma or tumor surgery in the head and face area often lead to massive destruction of the facial skeleton. Cell-based bone reconstruction therapies promise to offer new therapeutic opportunities for the repair of bone damaged by disease or injury. Currently, embryonic stem cells (ESCs) are discussed to be a potential cell source for bone tissue engineering. The purpose of this study was to investigate various supplements in culture media with respect to the induction of osteogenic differentiation.

Methods

Murine ESCs were cultured in the presence of LIF (leukemia inhibitory factor), DAG (dexamethasone, ascorbic acid and β-glycerophosphate) or bone morphogenetic protein-2 (BMP-2). Microscopical analyses were performed using von Kossa staining, and expression of osteogenic marker genes was determined by real time PCR.

Results

ESCs cultured with DAG showed by far the largest deposition of calcium phosphate-containing minerals. Starting at day 9 of culture, a strong increase in collagen I mRNA expression was detected in the DAG-treated cells. In BMP-2-treated ESCs the collagen I mRNA induction was less increased. Expression of osteocalcin, a highly specific marker for osteogentic differentiation, showed a double-peaked curve in DAG-treated cells. ESCs cultured in the presence of DAG showed a strong increase in osteocalcin mRNA at day 9 followed by a second peak starting at day 17.

Conclusion

Supplementation of ESC cell cultures with DAG is effective in inducing osteogenic differentiation and appears to be more potent than stimulation with BMP-2 alone. Thus, DAG treatment can be recommended for generating ESC populations with osteogenic differentiation that are intended for use in bone tissue engineering.