Polymethylmethacrylate particles inhibit osteoblastic differentiation of MC3T3‐E1 osteoprogenitor cells

Orthopedic wear debris has been implicated as a significant inhibitory factor of osteoblast differentiation. Polymethylmethacrylate (PMMA) particles have been previously shown to inhibit the differentiation of osteoprogenitors in heterogeneous murine marrow stromal cell cultures, but the effect of PMMA particles on pure osteoprogenitor populations remains unknown. In this study, we challenged murine MC3T3‐E1 osteoprogenitor cells with PMMA particles during their initial differentiation in osteogenic medium. MC3T3‐E1 cultures challenged with PMMA particles showed a gradual dose‐dependent decrease in mineralization, cell number, and alkaline phosphatase activity at low particle doses (0.038–0.150% v/v) and complete reduction of these outcome parameters at high particle doses (≥0.300% v/v). MC3T3‐E1 cultures challenged with a high particle dose (0.300% v/v) showed no rise in these outcome parameters over time, whereas cultures challenged with a low particle dose (0.075% v/v) showed a normal or reduced rate of increase compared to controls. Osteocalcin production was not significantly affected by particles at all doses tested. MC3T3‐E1 cells grown in conditioned medium from particle‐treated MC3T3‐E1 cultures showed a significant reduction in mineralization only. These results indicate that direct exposure of MC3T3‐E1 osteoprogenitors to PMMA particles results in suppression of osteogenic proliferation and differentiation. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 26:932–936, 2008     

Relationship between the expression of parathyroid hormone receptors and hormonal effect during rat osteoprogenitor cell differentiation

The number of parathyroid hormone (PTH) receptors in rat calvaria (RC) cells increased with culture duration, reached a maximum on day 13 when the cells had not yet differentiated into osteoblasts, and then gradually decreased by day 21. However, short exposure (48h) of RC cells to PTH-(1–34) before day 12, but not after day 15, did not decrease alkaline phosphatase (ALP) activity of the cells. Bone-like nodule (BN) formation was also suppressed only when PTH-(1–34) was present during the late culture period (days 14–21). Pretreating the cells with staurosporine, a protein kinase C inhibitor, on day 7 resulted in augmentation of PTH-(1–34)-induced cyclic adenosine monophosphate (cAMP) accumulation and inhibition of ALP activity. PTH-(7–34), which only activates PKC, however, had no effect on RC cell differentiation under any conditions used.     

The role of adsorbed endotoxin in particle-induced stimulation of cytokine release.

Numerous in vitro models have demonstrated the capacity of wear particles to stimulate the release of soluble pro-inflammatory products with the ability to induce local bone resorption. Recent observations have demonstrated that binding of lipopolysaccharide (LPS) to particulate wear debris can significantly modulate the pattern of cell response in the in vitro models. These findings raise concerns over the possible role of LPS in the pathogenesis of aseptic loosening after total joint replacements, and also indicates the importance of controlling for possible confounding effects of LPS contamination in the in vitro models used to study the reactive nature of wear debris. Our studies were undertaken to rigorously analyze the effects of particle-associated LPS on cell responses and to assess the efficacy of different treatment protocols to inactivate LPS associated with different particulate materials. Particles of cobalt-chrome alloy, titanium-6-aluminum-4-vanadium, titanium nitride and silica were pretreated with LPS and exposed to multiple treatment protocols. When cells were treated with "as-received" particles prepared by washing in ethanol, small amounts of TNF-alpha, IL-1beta. and IL-1alpha were detected. In contrast, all particle species pretreated with LPS produced marked increases in TNF-alpha, IL-1alpha, and IL-1beta release, as well as upregulation of corresponding mRNA levels even after ethanol washing. Boiling the LPS-pretreated particles in 1% acetic acid or autoclaving and baking the particles also markedly reduced and in some instances abolished the effect of the LPS-pretreatment. This indicates that LPS binds to the surface of particles of diverse composition and that the bound LPS is biologically active. Treatment protocols to inactivate particle-associated LPS demonstrated significant differences in efficacy. When the most rigorous treatments were utilized, essentially all LPS activity could be eliminated. Particles treated with these methods retained some capacity to stimulate cytokine release, but activities were markedly reduced. These results provide further evidence indicating that LPS contamination of particulate materials can markedly enhance their biological activity. This potential confounding effect needs to be carefully monitored and controlled in the in vitro model systems used to evaluate wear particles. Furthermore, the presence of particle-associated endotoxin at the bone-implant interface in vivo could markedly enhance the adverse biological activity of particulate wear debris.     

RANKL is an essential cytokine mediator of polymethylmethacrylate particle-induced osteoclastogenesis.

RANKL is a TNF superfamily member and an essential cytokine mediator of developmental osteoclastogenesis. We examined the role of RANKL in PMMA particle-induced osteoclastogenesis in vitro. In murine whole bone marrow cultures, PMMA particles stimulate a 2.5 fold increase in secreted RANKL, a 5-8 fold increase in osteoclast number and induce the formation of giant multinuclear osteoclasts. RANKL and TNF, potential cytokine mediators of PMMA, had similar osteoclastogenic effects. The RANKL inhibitor OPG was utilized to define the role of RANKL in mediating the PMMA response and was found to inhibit basal and PMMA particle-induced osteoclastogenesis. Additionally, particles stimulate osteoclast formation in RANKL-primed osteoclast precursor cells (devoid of supporting stromal cells) while RANKL untreated osteoclast precursors demonstrate no osteoclastogenic response to particles. Since TNF can potentiate RANKL action and is thought to mediate implant osteolysis we analyzed TNF(-/-) whole bone marrow cultures to elucidate the role of this cytokine. In TNF(-/-) cultures basal osteoclastogenesis remains intact, yet the PMMA effect is blunted. Finally, we show that PMMA, RANKL and TNF all activate the NF-kB and c-jun/AP-1 signaling pathways which are both fundamental to osteoclast formation and are potential sites of signal convergence in RANKL-mediated particle osteoclastogenesis.     

MCP-1-CCR2抑制剂治疗人工关节无菌性松动的研究进展

人工关节磨损颗粒诱导全身的巨噬细胞迁移到关节假体周围,并刺激巨噬细胞释放多种趋化因子,其中巨噬细胞趋化性蛋白-1(MCP-1)在调节全身的单核/巨噬细胞趋化迁移和慢性炎症过程中起关键作用。单核/巨噬细胞是破骨细胞的前体细胞,减少单核/巨噬细胞的迁移和活化,有可能减轻与磨损颗粒相关的假体周围骨溶解。该文介绍磨损颗粒分类及其生物学特征,以及MCP-1-CCR2信号通路与磨损颗粒诱导的全身性巨噬细胞迁移、最终导致假体周围骨溶解的研究进展情况。     

辛伐他汀对人牙髓干细胞增殖和分化的影响

目的:研究辛伐他汀对人牙髓干细胞(dental pulpstem cells,DPSCs)增殖和成骨分化的影响。方法:将第3代人DPSCs在矿化培养液中诱导培养,同时加入不同浓度的辛伐他汀(1×10-5mol/L、1×10-6mol/L、1×10-7mol/L、1×10-8mol/L),噻唑蓝(methyl thiazolyl tetrazolium,MTT)法检测细胞增殖情况,碱性磷酸酶试剂盒检测碱性磷酸酶(alkaline phosphatase,ALP)活性,茜素红染色鉴定成骨分化。结果:各浓度辛伐他汀均抑制人DPSCs增值,辛伐他汀浓度为1×10-5mol/L时,抑制作用最明显。适宜浓度辛伐他汀(1×10-6mol/L、1×10-7mol/L、1×10-8mol/L)促进人DPSCs向成骨细胞分化,其中,1×10-7mol/L的辛伐他汀促进ALP活性的作用最明显。结论:辛伐他汀抑制人DPSCs的增值,适宜浓度的辛伐他汀可有效促进人DPSCs的成骨分化。     

Particulate endocytosis mediates biological responses of human mesenchymal stem cells to titanium wear debris.

Continual loading and articulation cycles undergone by metallic (e.g., titanium) alloy arthroplasty prostheses lead to liberation of a large number of metallic debris particulates, which have long been implicated as a primary cause of periprosthetic osteolysis and postarthroplasty aseptic implant loosening. Long-term stability of total joint replacement prostheses relies on proper integration between implant biomaterial and osseous tissue, and factors that interfere with this integration are likely to cause osteolysis. Because multipotent mesenchymal stem cells (MSCs) located adjacent to the implant have an osteoprogenitor function and are critical contributors to osseous tissue integrity, when their functions or activities are compromised, osteolysis will most likely occur. To date, it is not certain or sufficiently confirmed whether MSCs endocytose titanium particles, and if so, whether particulate endocytosis has any effect on cellular responses to wear debris. This study seeks to clarify the phenomenon of titanium endocytosis by human MSCs (hMSCs), and investigates the influence of endocytosis on their activities. hMSCs incubated with commercially pure titanium particles exhibited internalized particles, as observed by scanning electron microscopy and confocal laser scanning microscopy, with time-dependent reduction in the number of extracellular particles. Particulate endocytosis was associated with reduced rates of cellular proliferation and cell-substrate adhesion, suppressed osteogenic differentiation, and increased rate of apoptosis. These cellular effects of exposure to titanium particles were reduced when endocytosis was inhibited by treatment with cytochalasin D, and no significant effect was seen when hMSCs were treated only with conditioned medium obtained from particulate-treated cells. These findings strongly suggest that the biological responses of hMSCs to wear debris are triggered primarily by the direct endocytosis of titanium particulates, and not mediated by secreted soluble factors. In this manner, therapeutical approaches that suppress particle endocytosis could reduce the bioreactivity of hMSCs to particulates, and enhance long-term orthopedic implant prognosis by minimizing wear-debris periprosthethic osteolysis.     

Role of epidermal growth factor receptor in osteoblastic differentiation of rat bone marrow stromal cells

In an attempt to understand the role of epidermal growth factor (EGF) and its receptor (EGF-R) in osteoblastic cell differentiation, the changes in [¹²⁵I]-EGF binding capacity, synthesis of EGF-R protein, and expression of EGF-R mRNA were investigated during osteoblastic differentiation of cultured bone marrow stromal cells which were collected from the femora of young adult rats. In addition, the ability of EGF to suppress osteoblastic differentiation was also studied. Dexamethasone at a concentration of 0.1 mM increased the expression of osteoblastic markers by bone marrow stromal cells cultured in alpha-modified minimum essential medium (-MEM) con taining 1% fetal bovine serum (FBS), 50 mg/ml ascorbic acid, and 10 mM -glycerophosphate, as revealed by elevated alkaline phosphatase activity, an increase in osteopontin mRNA expression, and bone nodule formation. This osteoblastic differentiation was accompanied by a decreased expression of EGF-R mRNA, decreased synthesis of EGF-R protein, and a decreased number of EGF-binding sites without any change in affinity. When these cells were incubated with dexamethasone and EGF in combination throughout the culture, they exhibited significantly lower levels of all osteoblastic markers than did dexamethasonetreated cells, indicating suppression of osteoblastic differentiation by EGF. In contrast, EGF treatment of the cells induced expression of EGF-R mRNA. Thus, a decrease in EGF binding associated with osteoblastic differentiation could lead to decreased responsiveness of bone marrow cells to EGF, whereas the EGF-induced increase in expression of EGF-R could facilitate the inhibition of cell differentiation by EGF. These findings suggested that upregulation of EGF-R on bone marrow stromal cells antagonizes their differentiation, and thus possibly functions as a negative regulator of osteoblastic differentiation.     

Murine model of prosthesis failure for the long-term study of aseptic loosening.

We examined a novel mouse model of wear debris-induced prosthesis instability and osteolysis, and its application for the evaluation of therapy. A stainless steel or titanium-alloy pin was implanted into the proximal tibia to form a contiguous surface with the articular cartilage. In some mice, titanium particles were injected into the tibial canal during the surgery, followed by monthly intraarticular injection. MicroCT scans revealed that the implants without particle challenge were stable without bone mineral density changes for 6 months. Histological analysis showed new bone formation around the implant at 6 weeks postsurgery. Periprosthetic soft tissue with inflammatory cells was a ubiquitous finding at the interface between the implant and surrounding bone in samples exposed to titanium particles, and expression of IL-1beta, TNFalpha, and CD68 was common in these joints. Pullout tests indicated that an average 5N load was required to pull out stable implants from surrounding bone. However, particle stimulation dramatically reduced the pullout force to less than 0.4 N. The feasibility of in vivo gene transfer on this model was confirmed by X-gal staining of synovial membrane and periprosthetic tissue after injection of AAV-LacZ in the prosthetic joint. This murine model of weight-bearing knee prosthesis provides an economical, reproducible, and easily obtained means to study joint arthroplasty failure. The ability to evaluate the biomechanical properties of the prosthetic joint, in addition to histological and biochemical examination, results in a useful model to investigate many of the properties of prosthetic joint components during the response to debris-associated osteolysis.     

肝卵圆细胞来源分化增殖的研究进展

卵圆细胞为肝内一种祖细胞，在肝细胞严重受损或分裂增生受抑制时可向肝细胞和胆管上皮细胞分化，是一种具有双向分化潜能的祖细胞。卵圆细胞的分化增殖受多种因素调控．新近研究认为SDF-1-CXCR4生物学轴在卵圆细胞增殖过程中起非常重要的作用。肝癌的发生与卵圆细胞的分化异常有一定的关系。卵圆细胞分化为肝细胞和胆管上皮细胞使其可能成为旰脏细胞移植的重要细胞来源，在治疗终末期肝病、生物人工肝及基因治疗的研究领域有着广阔的应用前景。     

阻断钙调磷酸酶/激活T细胞核因子通路对聚甲基丙烯酸甲酯颗粒抑制骨祖细胞向成骨细胞分化的影响

目的 探讨VIVIT肽阻断钙调磷酸酶(Cn)/激活T细胞核因子(NFAT)信号通路对聚甲基丙烯酸甲酯(PMMA)颗粒抑制骨祖细胞向成骨细胞分化的影响. 方法 体外分离培养Sprague-Drawley大鼠胎鼠颅骨原代细胞(包含大量骨祖细胞),根据处理条件不同分为4组:对照组、PMMA组、PMMA/VIVIT组和VIVIT组.细胞培养2、4、7和14 d用MTT法检测细胞增殖情况,7 d和14d用碱性磷酸酶(ALP)定量反映细胞分化;细胞培养14 d后菏素红染色观察细胞矿化,RT-PCR法观察ALP、骨钙素、Ⅰ型胶原、Fra-2(与成骨细胞分化有关的转录因子)、NFATc1的基因表达,Western Blot法检测细胞核和细胞质中NFATc1蛋白的表达. 结果 PMMA组较对照组NFATc1基因和蛋白表达增加,并伴有NFATc1蛋白转位入核明显增加,成骨细胞分化、矿化和相关基因表达明显降低.PMMA/VIVIT组较PMMA组细胞分化、矿化和相关基因表达增加,但低于VIVIT组,NFATc1基因表达降低,转位入核的NFATc1蛋白明显减少.各组细胞增殖差异均无统计学意义(P>0.05). 结论 PMMA颗粒抑制骨祖细胞向成骨细胞分化与Cn/NFAT信号通路激活有关,VIVIT肽阻断Cn/NFAT信号通路可促进PMMA颗粒抑制的骨祖细胞向成骨细胞分化.     

C反应蛋白激活原纤毛抑制颅骨成骨细胞的增殖和分化

目的 研究C反应蛋白(C-reactive protein,CRP)对原代培养SD大鼠乳鼠颅骨成骨细胞(rat calvarial osteoblasts,ROB)增殖及成骨分化的影响.方法 取SD乳鼠颅骨组织用胰蛋白酶和胶原蛋白酶Ⅰ消化得到原代ROB,随后将ROB用α-MEM培养基培养,传代3次后用于后续实验.在对照...     

Hypoxia promotes proliferation and osteogenic differentiation potentials of human mesenchymal stem cells

Mesenchymal stem cells (MSCs), which can be isolated from bone marrow and other somatic tissues, are residing in an environment with relative low oxygen tension. The purpose of this study is to investigate the effects of hypoxia on MSCs, and we hypothesize that oxygen concentration regulates the intricate balance between cellular proliferation and commitment towards differentiation. In this study, human bone marrow‐derived MSCs were cultured under hypoxia with 1% O₂. The proliferation ability of MSCs was increased after a 7‐day hypoxic culture period. Migration assay showed that hypoxia enhanced the migration capabilities of MSCs. Moreover, expression of stemness genes Oct4, Nanog, Sall4 and Klf4 was increased under hypoxia. Furthermore, the differentiation ability of MSCs under hypoxia favored osteogenesis while adipogenesis was inhibited during a 4‐week induction period. Cytokine antibody array analysis showed that a number of growth factors were up‐regulated after a 7‐day hypoxic incubation and the differential expression of growth factors may account for the increased proliferation and osteogenic potentials of MSCs under hypoxic condition. Taken together, hypoxia provides a favorable culture condition to promote proliferation as well as osteogenesis of MSCs through differential growth factor production. © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 30:260–266, 2012     

Effects of melatonin on the proliferation and differentiation of rat adipose-derived stem cells

Background:

Osteogenesis driven by adipose-derived stem cells (ADSCs) is regulated by physiological and pathological factors. Accumulating evidence from in vitro and in vivo experiments suggests that melatonin may have an influence on bone formation. However, little is known about the effects of melatonin on osteogenesis, which thus remains to be elucidated. This study was performed to determine whether melatonin at physiological concentrations (0.01-10 nM) could affect the in vitro proliferation and osteogenic differentiation of rat ADSCs.

Materials and Methods:

ADSCs were isolated from the fat of adult rats. After cell expansion in culture media and through three passages, osteogenesis was induced in a monolayer culture using osteogenic medium with or without melatonin at physiological concentrations (0.01-10 nM). After four weeks, the cultures were examined for mineralization by Alizarin Red S and von Kossa staining and for alkaline phosphatase (ALP) activity using an ALP kit. Cell viability and apoptosis were also assayed by 3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTT) assay and flow cytometry, respectively.

Results:

The results indicated that at physiological concentrations, melatonin suppressed proliferation and differentiation of ADSCs. These data indicate that ADSCs exposed to melatonin, had a lower ALP activity in contrast to the cells exposed to osteogenic medium alone. Similarly, mineral deposition (calcium level) also decreased in the presence of melatonin. Flow cytometry confirmed that cell growth had decreased and that the numbers of apoptotic cells had increased.

Conclusion:

These results suggest that the physiological concentration of melatonin has a negative effect on ADSC osteogenesis.     

兔骨髓间充质干细胞的分离、扩增及诱导分化

目的探讨兔骨髓间充质干细胞(bone marrow mesenchymal stem cells,BM-MSCs)体外培养、定向诱导分化为成骨细胞和成脂肪细胞的途径,为进一步的实验研究打下基础。方法抽取兔股骨骨髓,以全骨髓贴壁培养法进行体外培养,贴壁细胞传代,倒置显微镜下观察细胞形态。取第3代细胞向成骨细胞和成脂肪细胞诱导,14d后成骨细胞诱导组检测碱性磷酸酶,成脂肪细胞诱导组进行油红O染色。结果全骨髓贴壁培养法可获得BM-MSCs,原代和传代培养的BM-MSCs具有活跃的增殖能力。成骨细胞诱导组碱性磷酸酶检测表达阳性,成脂肪细胞诱导组油红O染色见胞浆内出现大量红染脂滴。结论全骨髓贴壁培养法可有效地分离和扩增BM-MSCs,分离培养的BM-MSCs生长稳定,增殖力强,可向成骨细胞和成脂肪细胞诱导分化。     

Review of periprosthetic osteolysis in total joint arthroplasty: an emphasis on host factors and future directions

Periprosthetic osteolysis is one of the leading causes of total joint revision procedures. If allowed to progress in the absence of radiographic diagnosis and/or proper medical treatment, osteolysis may result in aseptic loosening yielding failure of the implant and the need for complex revision arthroplasty. The purpose of this review was to assess the current understanding of periprosthetic osteolysis with an emphasis on host factors and future directions. A PubMed search was conducted using the following key words; osteolysis, periprosthetic osteolysis, osteolysis imaging. Pertinent articles, as it pertained to the outline of the review, were selected. Periprosthetic osteolysis stems from numerous risk factors. Osteolysis host characteristic risk factors include gender, body weight, and genetics. Current implant designs have reduced the incidence of this disease; however no current design has been able to replicate the in vivo characteristics and therefore development of wear particles continues to be seen. Advanced methods of imaging diagnosis are on the rise, however early imaging diagnosis is currently ineffective. Pharmacologic intervention appears to be a logical avenue for medical intervention, but no approved drug therapy to prevent or inhibit periprosthetic osteolysis is currently available. Although the rate of periprosthetic osteolysis seems to be decreasing with advances in implant design and increased knowledge of the biological process of wear particle induced osteolysis, the rapid increase in the total number of total joint arthroplasties over the next two decades means that better ways of detecting and treating periprosthetic osteolysis are greatly needed.     

Differential effects of biologic versus bisphosphonate inhibition of wear debris‐induced osteolysis assessed by longitudinal micro‐CT

Aseptic loosening of total joint replacements is caused by wear debris‐induced osteoclastic bone resorption, for which bisphosphonates (BPs) and RANK antagonists have been developed. Although BPs are effective in preventing metabolic bone loss, they are less effective for inflammatory bone loss. Because this difference has been attributed to the antiapoptotic inflammatory signals that protect osteoclasts from BP‐induced apoptosis, but not RANK antagonists, we tested the hypothesis that osteoprotegerin (OPG) is more effective in preventing wear debris‐induced osteolysis than zoledronic acid (ZA) or alendronate (Aln) in the murine calvaria model using in vivo micro‐CT and traditional histology. Although micro‐CT proved to be incompatible with titanium (Ti) particles, we were able to demonstrate a 3.2‐fold increase in osteolytic volume over 10 days induced by polyethylene (PE) particles versus sham controls (0.49 ± 0.23mm³ versus 0.15 ± 0.067mm³; p < 0.01). Although OPG and high‐dose ZA completely inhibited this PE‐induced osteolysis (p < 0.001), pharmacological doses of ZA and Aln were less effective but still reached statistical significance (p < 0.05). Traditional histomorphometry of the sagital suture area of calvaria from both Ti and PE‐treated mice confirmed the remarkable suppression of resorption by OPG (p < 0.001) versus the lack of effect by physiological BPs. The differences in drug effects on osteolysis were largely explained by the significant difference in osteoclast numbers observed between OPG versus BPs in both Ti‐ and PE‐treated calvaria; and linear regression analyses that demonstrated a highly significant correlation between osteolysis volume and sagittal suture area versus osteoclast numbers (p < 0.001). © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 26:1340–1346, 2008