IGF-I and TGF-beta 1 incorporated in a poly(D,L-lactide) implant coating maintain their activity over long-term storage-cell culture studies on primary human osteoblast-like cells

Biodegradable coating of osteosynthetic materials with poly(D,L-lactide) (PDLLA) and incorporated growth factors has been used successfully as drug carrier to stimulate fracture healing in several rat and porcine models. A cold coating technique was used to incorporate growth factors without loss of activity during the coating process. The aim of this study was to investigate the activity of incorporated insulin like growth factor-I and transforming growth factor-beta 1 (TGF-beta1) after long-time storage (5 and 14 months at -20 degrees C). Primary human osteoblast-like cells (HOB) were cultured in a non-contact manner with titanium wires coated with PDLLA and IGF-I (33 microg) and TGF-beta1 (6 microg) for 0, 5, 10 and 15 days. Osteoblast culture without wires, with titanium wires or wires with the PDLLA coating served as control ( n=3 each time point and group). Cell vitality, cell proliferation and the production of procollagen 1 were measured. No differences in cell count and vitality were accessed in the two growth factor treated groups compared to the control groups at the same time point. Independently from the storage duration, the incorporated growth factors significantly stimulated the production of osteoblast specific type I collagen (CICP) compared to the controls. The results indicate, that the growth factors stimulated osteoblast to an enhanced collagen 1 production and that the coating method meets a major requirement for clinical use of growth factor-coated implants: biological activity of the incorporated growth factors for at least 14 months.     

The effect of zoledronic acid incorporated in a poly(D,L-lactide) implant coating on osteoblasts in vitro

Bisphosphonates such as zoledronic acid (ZOL) are used in diseases associated with osteoclast-mediated bone loss. However, their antiresorptive activity is partly due to their effect on osteoblasts. Local application might increase the therapeutical fence and their local efficiency and reduce systemic side effects. Aim of the study was to investigate the effect of ZOL on human osteoblasts like cells in vitro with special focus on the synthesis of factors mediating osteoclast differentiation (RANKL, OPG). ZOL was incorporated in an implant coating based on poly(D,L-lactide) (PDLLA) in different concentrations (10-150 microM). Control groups were treated with uncoated implants, PDLLA-coated implants, and ZOL pure substance in corresponding concentrations. After an experimental period of 144 h, primary human osteoblasts were stained with alamar blue and cell viability was measured. Procollagen I synthesis, osteoprotegerin (OPG) secretion, and soluble receptor activator of nuclear factor-kappaB ligand (sRANKL) were analyzed. Results showed that cell viability was not affected when treated with doses equivalent up to 100 microM ZOL-coated implants (ZOL-CI). Procollagen I synthesis was highest when treated with 50 microM ZOL-CI. OPG increased significantly in the 10 microM ZOL-CI group, whereas sRANKL decreased significantly with different concentrations of ZOL-CI. Higher concentrations or exposure to the pure substance showed a decrease in cell viability, collagen I, OPG, and sRANKL synthesis. In conclusion, exposure to specific concentrations of ZOL-CI showed a beneficial effect on osteoblast differentiation and protein synthesis without influencing their proliferation. Changes in sRANKL and OPG production may contribute to the inhibition of osteoclastic bone resorption. This local antiresorptive effect might be clinically useful in osseous implant integration and fracture healing.     

Regulation of osteoblast growth by interactions between transforming growth factor-beta and 1alpha,25-dihydroxyvitamin D3

Osteoblast growth and differentiation encompass a series of events including proliferation, changes in cell shape, and expression of the markers specific for osteoblast phenotype. Both transforming growth factor-beta (TGF-beta) and 1alpha,25-dihydroxyvitamin D3 (1alpha,25[OH]2D3) are effective in regulating osteoblast proliferation, differentiation, bone matrix maturation and cell-specific gene expression. Although there is some degree of controversy regarding the influences on osteoblasts in vitro, it is generally agreed that TGF-beta stimulates osteoblast proliferation and growth, and inhibits the expression of the markers characteristic of the osteoblast phenotype such as osteocalcin. In contrast, 1alpha,25(OH)2D3 causes inhibition of the proliferation of osteoblasts, arrests their growth, and stimulates expression of specific markers. In many studies, complex interactions have been demonstrated between TGF-beta and 1alpha,25(OH)2D3 modulating their receptor expression, synthesis, and effects on osteoblast-specific gene expression. The cooperative actions of TGF-beta and 1alpha,25(OH)2D3 can be synergistic or antagonistic. It has recently been established that Smad proteins that transduce signals downstream the TGF-beta stimulation may mediate the crosstalk between TGF-beta and 1alpha,25(OH)2D3 signaling. Future studies should focus on the explanation of the molecular basis of these interactions and the in vivo consequences of the regulation of osteoblast growth and differentiation by TGF-beta and 1alpha,25(OH)2D3.     

TGF-beta1 secretion of ROS-17/2.8 cultures on NiTi implant material

The biocompatibility of an orthopedic implant depends on the effect of the implant on bone-forming cells, osteoblasts. Changes in osteoblastic proliferation, maturation and differentiation are important events in ossification that enable monitoring the effect of the implant. Transforming growth factor-beta (TGF-beta) is known to suppress osteoblast proliferation and, on the other hand, to induce the maturation and differentiation of osteoblasts. Moreover, osteoblasts produce TGF-beta, which is embedded in the bone matrix and activated by bone-resorbing osteoclasts. TGF-beta inhibits osteoclastic activity. Here, we show for the first time the effect of nickel titanium shape memory metal (NiTi) on osteoblastic cytokine expression. In this study, we measured the levels of TGF-beta with enzyme-linked immunosorbent assay (ELISA) from a ROS-17/2.8 osteosarcoma cell line cultured on different metal alloy discs. ELISA results were proportioned to total DNA content of the samples. We compared NiTi, to stainless steel (Stst), pure titanium (Ti) and pure nickel (Ni). The TGF-beta1/DNA value in the NiTi group (0.0007 +/- 0.0003) was comparable with those seen in the Stst (0.0008 +/- 0.0001) and Ti (0.0007 +/- 0.0001) groups. The concentration in the Ni group was lower (0.0006 +/- 0.0003), though not statistically significantly so. In addition, the effect of surface roughness on TGF-beta1 production was studied. We compared three different grades of roughness in three differently hot-rolled alloys: NiTi. hot-rolled at 950 degrees C. Ti alloy hot-rolled at 850 degrees C (TiI) and the same Ti alloy hot-rolled at 1,050 degrees C (TiII). We found that increasing roughness of the NiTi surface increased the TGF-beta1 concentration. On the other hand, all roughness groups of TiII showed low levels of TGF-beta1. while a rough TiI surface induced similar TGF-beta1, expression as rough NiTi. Further, these same measurements made with interleukine 6 (IL-6) were found to be under the detection limit in these cultures. We conclude that a rough NiTi surface promotes TGF-beta1 expression in ROS-17/2.8 cells.     

Synergistic effects of 1,25-Dihydroxyvitamin D3 and TGF-beta1 on the production of insulin-like growth factor binding protein 3 in human bone marrow stromal cell cultures

1,25-Dihydroxyvitamin D3 (calcitriol), transforming growth factor-beta (TGF-beta), and insulin-like growth factors (IGFs) are all important bone regulatory factors known to affect proliferation and differentiation of human bone-forming cells (osteoblasts). We have previously shown that TGF-beta1 increased IGF-I and IGF-binding protein (IGFBP)-3 production in human bone marrow stromal (hMS) osteoblast progenitors and calcitriol stimulated IGFBP-3 and IGFBP-4 production. As interaction between signaling pathways of these factors has been reported, the present study aimed at examining the concerted actions on components of the IGF-system. We report that co-treatment with TGF-beta1 and calcitriol resulted in a synergistic increase in IGFBP-3 production, thereby suggesting that the effects of these factors on hMS osteoblast differentiation may involve the observed increase in IGFBP-3.     

Human osteoblast response to pulsed laser deposited calcium phosphate coatings

Octacalcium phosphate (OCP) and Mn(2+)-doped carbonate hydroxyapatite (Mn-CHA) thin films were deposited on pure, highly polished and chemically etched Ti substrates with pulsed laser deposition. The coatings exhibit different composition, crystallinity and morphology that might affect their osteoconductivity. Human osteoblasts were cultured on the surfaces of OCP and Mn-CHA thin films, and the cell attachment, proliferation and differentiation were evaluated up to 21 days. The cells showed a normal morphology and a very good rate of proliferation and viability in every experimental time. Alkaline phosphatase activity was always higher than the control and Ti groups. From days 7 to 21 collagen type I production was higher in comparison with control and Ti groups. The level of transforming growth factor beta 1 (TGF-beta1) was lower at 3 and 7 days, but reached the highest values during following experimental times (14 and 21 days). Our data demonstrate that both calcium phosphate coatings favour osteoblasts proliferation, activation of their metabolism and differentiation.     

Transforming growth factor-beta 1 (TGF-beta1) prevents the age-dependent decrease in bone formation in human osteoblast/implant cultures

Titanium implants have been extensively used in orthopedic surgery and dentistry. Most of the patients who receive such implants are elderly with a compromised ability to heal and form new bone. By using an in vitro osteoblast/implant culture system, the potency of TGF-beta1 in enhancing mineralization of human osteoblast cultures from elderly subjects was investigated in this study. Primary human osteoblast (HOB) cells obtained from different age group human subjects [Young (Y), Middle (M), and Old (O)] were cultured on Ti alloy (Ti-6Al-4V) disks with or without continuous administration of 0.2 ng/mL TGF-beta1 in the medium for 2 or 4 weeks. TGF-beta1 significantly (p < 0.05) increased calcium content and the size of calcified nodules on implant disks in the O group, but had no effect on the Y or M groups. The number of calcified nodules was not different with or without TGF-beta1 in all age groups. As measured by Northern blot analysis and RT-PCR, TGF-beta1 significantly increased the expression of bone-specific extracellular matrix proteins, including alkaline phosphatase, Type I collagen, bone sialoprotein and osteocalcin, after both 2 and 4 weeks in the O group but not in the Y group. In conclusion, TGF-beta1 enhances mineralization on implant materials of osteoblast cultures from elderly human subjects.     

The effect of growth factor treatment on meniscal chondrocyte proliferation and differentiation on polyglycolic acid scaffolds

The aim of this investigation was to determine the effect of growth factor treatment on ovine meniscal chondrocyte (OMC) proliferation in vitro and on the production of matrix proteins by OMCs grown within a polyglycolic acid (PGA) scaffold. Analysis of 72-h monolayer cultures using the mean transit time (MTT) assay revealed a greater increase in OMC numbers in the presence of platelet-derived growth factor (PDGF)-AB, PDGF-BB, insulin-like growth factor (IGF)-I, transforming growth factor-beta1 (TGF-beta1) and basic fibroblast growth factor (bFGF) than in untreated controls. In contrast, IGF-II and bone morphogenetic protein-2 had no effect on OMC proliferation at the concentrations tested. The growth factors that elicited the greatest proliferative response (PDGF-AB, PDGF-BB, TGF-beta1, and IGF-I) were subsequently tested for their ability to enhance OMC proliferation and differentiation within PGA scaffolds. Biochemical analysis revealed less glycosaminoglycan (GAG) production in the presence of all growth factors tested compared to untreated control samples. In contrast, all of the growth factors increased collagen type I production by OMCs within the scaffolds at day 20, and all except PDGF-BB resulted in an increase at day 39, when compared to appropriate control samples. With the exception of IGF-I, none of the growth factors tested had any significant effect on collagen type II production. Histological staining of sections from OMC-PGA scaffolds did not reveal any difference in GAG or collagen production between the treatment groups. However, immunohistochemical analysis demonstrated an increase in collagen type I expression and a decrease in collagen type II at day 39 in all growth factortreated constructs, concomitant with a high infiltration of cells. This suggests that PDGF-AB, PDGF-BB, TGF-beta1, and IGF-1 may be useful in future tissue engineering studies for promoting meniscal cell proliferation and differentiation within scaffolds.     

健骨颗粒促进成骨细胞增殖的分子机制

背景：ERK是依赖于Ras途径激活的一个蛋白激酶,在成骨细胞增殖和分化过程中发挥重要作用。而PD98059是MEK特异性抑制剂,它可通过抑制MEK的活性来抑制ERK的磷酸化,从而起到阻断ERK信号通路的作用。目前有关ERK在大鼠成骨细胞增殖和分化过程中的作用研究甚少。ERK在健骨颗粒促进大鼠成骨细胞增殖和分化过程中调控的作用更未被阐明。 目的：观察ERK信号转导通路在健骨颗粒促成骨细胞增殖和分化过程中的作用。 方法：取第3代SD大鼠头盖骨成骨细胞,空白组加入生理盐水血清;中药组加入最佳浓度的健骨颗粒含药血清;阻滞剂组添加PD98059阻断剂,中药加阻断剂组添加PD98059阻断剂与健骨颗粒含药血清。用MTT法测定细胞的增殖能力,比色法测碱性磷酸酶、羟脯氨酸水平。收集细胞实时荧光定量PCR-SYBR GREEN法检测Cbfa1、Ⅰ型胶原、OSX mRNA的表达,Westren blot法检测成骨细胞ERK的表达情况。 结果与结论：添加PD98059阻断剂后,阻滞剂组和中药加阻滞剂组中ERK的表达显著低于空白组和中药组。阻断剂组的成骨细胞内碱性磷酸酶、羟脯氨酸的表达以及Cbfa1、Ⅰ型胶原、OSX mRNA表达显著低于空白组和中药组。ERK在健骨颗粒促进大鼠成骨细胞增殖和分化过程中发挥重要作用,ERK信号通路可能是健骨颗粒促进成骨细胞增殖和分化的主要途径。     

Biodegradable poly(D,L-lactide) coating of implants for continuous release of growth factors.

Local application of growth factors like insulin like growth factor-I (IGF-I) and transforming growth factor-beta 1 (TGF-beta1) from a biodegradable thin layer of poly(D,L-lactide) (PDLLA) coated implants could stimulate fracture healing. A new "cold coating technique" for metallic implants was established to produce a biodegradable coating with a high mechanical stability that provides a continuous release of incorporated growth factors. The properties of this bioactive coating were investigated in vitro and in vivo. Scanning electron microscope analysis revealed a coating thickness of in average 14.8 microm on titanium and 10.7 microm on steel wires. Intramedullary implantation and extraction experiments depicted a loss of PDLLA coating from titanium and steel implants of less than 5%. After explantation of the implants, the coating displayed a complete and regular layer without any defects of PDLLA uncovering the metallic surface. Smear tests demonstrate that the coating can be performed under sterile conditions. The PDLLA depicted a reduction of about 8% within 6 weeks in vitro and in vivo. The growth factors were incorporated in a stable form and demonstrated a loss of stability of less than 3% within 42 days and less than 5% within one year. In an elution experiment, 54% IGF-I and 48% TGF-beta1 were released within the first 48 h. After 42 days, 76% of IGF-I and 71% of TGF-beta1 were detected in the elution fluid by ELISA. Comparable results were obtained in the in vivo experiments after 42 days.     

Transforming growth factor beta1 immobilized adsorptively on Ti6Al4V and collagen type I coated Ti6Al4V maintains its biological activity

Titanium and titanium alloys are often used for orthopedic and dental implants. Osseointegration of Ti6Al4V may be improved not only by precoating of the surface with extracellular matrix proteins like collagen type I but also by additional immobilization of growth factors. In the present study, transforming growth factor beta1 (TGF-beta1) which is known as an inducer of collagen synthesis was immobilized adsorptively on uncoated and collagen type I coated Ti6Al4V surfaces. TGF-beta1 was found immobilized slightly faster to collagen type I coated than to uncoated Ti6Al4V and released slower from the collagen coated material. Immobilized TGF-beta1 is biologically active for at least 3 weeks storage at 4 degrees C. Sterilization by ethylene oxide inactivates immobilized TGF-beta1. In osteoblasts cultured on implants with adsorptively immobilized TGF-beta1, mRNA level and specific catalytic activity of alkaline phosphatase as well as accumulation of calcium and phosphate were found reduced, whereas procollagen alpha1(I) mRNA level and the rate of collagen synthesis were increased.     

微弧氧化技术与钛合金表面成骨细胞增殖及骨向分化能力

文题释义：微弧氧化技术：是金属材料表面处理的一种技术，将金属材料(如，镁、铝、钛等)置于特定电解液中，通过弧光放电过程中瞬时的高温和高压条件产生结合于金属表面的陶瓷膜层，该种方法制备的涂层材料具有高硬度、耐磨的特点。  种植体：是针对于人体自身牙齿缺失而植入上下颌骨内的人工牙根。性能优良的种植体需要同时具备高强度、耐降解、生物相容性好的特性。目前复合材料类如金属表面添加陶瓷涂层材料，具有上述多种材料特性而被临床选择。 背景：采用普通电化学法可在钛及其合金表面制备纳米级羟基磷灰石涂层，但该涂层吸收降解缓慢，需要8-12周的时间。而微弧氧化可在复杂表面形成均匀薄膜，有利于细胞黏附和骨组织长入。 目的：探索微弧氧化羟基磷灰石涂层钛合金对成骨细胞增殖及骨向分化能力的影响。 方法：采用电化学法与微弧氧化法分别制备羟基磷灰石涂层钛合金材料，检测两种材料表面的接触角。将成骨细胞系hFOB1.19接种于两组羟基磷灰石涂层钛合金材料表面，培养48 h时，采用扫描电镜观察细胞在材料上的形态变化；培养1，12，24，48，72 h时，采用MTT法检测细胞增殖；培养1，3，5 d时，比较两组材料表面细胞计数与碱性磷酸活性；培养第5天，采用Western Blotting检测细胞内骨形态发生蛋白2和骨形态发生蛋白4表达。 结果与结论：①微弧氧化组材料表面的接触角小于电化学组[(66.5±2.2)°，(52.8±2.1)°，P=0.001 5)]；②扫描电镜显示，电化学组成骨细胞形态不规则，胞体皱缩不饱满，与材料贴合较差；微弧氧化组细胞充分伸展，形态扁平，与材料贴合紧密；③微弧氧化组12-72 h的细胞增殖快于电化学组(P < 0.05)，培养3，5 d的细胞计数多于电化学组(P < 0.05)；④微弧氧化组培养1，3，5 d的细胞碱性磷酸酶活性高于电化学组(P < 0.05)；⑤微弧氧化组骨形态发生蛋白2和骨形态发生蛋白4表达均高于电化学组(P < 0.05)；⑥结果表明，微弧氧化羟基磷灰石涂层钛合金材料可促进成骨细胞的增殖及骨向分化能力。 ORCID: 0000-0003-2787-4667(王艳玲) 中国组织工程研究杂志出版内容重点：生物材料；骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性；组织工程     

Rat osteoblast functions on the o-carboxymethyl chitosan-modified poly(D,L-lactic acid) surface

In this study, the functions of rat osteoblasts on o-carboxymethyl chitosan-modified poly(D,L-lactic acid) (PDLLA) films were investigated in vitro. The surface characterization was measured by contact angle and electron spectroscopy for chemical analysis (ESCA). Cell adhesion and proliferation were used to assess cell behavior on the modified surface and control. The MTT assay was used to determined cell viability and alkaline phosphatase (ALP) activity was performed to evaluate differentiated cell function. Compared to the control films, cell adhesion of osteoblasts on o-carboxymethyl chitosan-modified PDLLA films was significantly higher (p < 0.05) after 6 and 8 h culture, and osteoblast proliferation was also significantly higher (p < 0.01) between 4 and 7 days. The MTT assay suggested cell viability of osteoblasts cultured on o-carboxymethyl chitosan modified PDLLA films was significantly greater (p < 0.05) than that seeded on control one, and the ALP activity of cells cultured on modified PDLLA films was significantly higher (p < 0.01) than that found on control. These results give the first evidence that o-carboxymethyl chitosan could be used to modify PDLLA surface for improving biocompatibility.     

Synergistic effect of titanium alloy and collagen type I on cell adhesion, proliferation and differentiation of osteoblast-like cells

A number of studies have demonstrated the pivotal role of collagen in modulating cell growth and differentiation. In bone, where the extracellular matrix is composed of approximately 85% type I collagen, cellular interaction with matrix components has been shown to be important in the regulation of the osteoblast phenotype. Preservation or enhancement of normal osteoblast function and appositional bone formation after implant placement represents a strategy that can be useful for the purpose of improving osseointegration. In order to further improve biocompatibility, we combined two known favorable compounds, namely the titanium alloy, Ti6A14V, with type I collagen. We assessed the in vitro behavior of primary osteoblasts grown on both fibrillar collagen-coated and tropocollagen-coated Ti6A14V in comparison with uncoated titanium alloy, using an improved adsorption procedure. As parameters of biocompatibility, a variety of processes, including cell attachment, spreading, cytoskeletal organization, focal contact formation, proliferation and expression of a differentiated phenotype, were investigated. Our results demonstrated for the first time that in comparison to uncoated titanium alloy, collagen-coated alloy enhanced spreading and resulted in a more rapid formation of focal adhesions and their associated stress fibers. Growing on collagen-coated Ti6A14V, osteoblasts had a higher proliferative capacity and the intracellular expression of osteopontin was upregulated compared to uncoated titanium alloy. Type I collagen-coated titanium alloy exhibits favorable effects on the initial adhesion and growth activities of osteoblasts, which is encouraging for its potential use as bone graft material. Moreover, collagen type I may serve as an excellent biocompatible carrier for osteotropic factors such as cell adhesion molecules (e.g. fibronectin) or bone-specific growth factors.     

Osteoblast culture on polished titanium disks modified with phosphonic acids

Titanium is widely used in dental implants due to its suitable physical properties and its good biocompatibility. However, it is integrated into bone only passively, and the resulting fixation in the bone, which is necessary for the function, is mainly mechanical in nature. With the objective of increasing the chemical interaction between the implant and the bone tissue, several phosphonic acids were synthesized and grafted onto titanium disks. Here we report on the proliferation, differentiation, and protein production of rat osteoblastic cells (CRP10/30) on phosphonic-acid-modified titanium surfaces studied in vitro. No statistical differences were found in osteoblast proliferation among the phosphonic-acid-modified titanium, unmodified titanium, and tissue culture plastic (used as a positive control), indicating that the phosphonic acids used were not cytotoxic to the osteoblasts used. For all surfaces (modified or not), the alkaline phosphatase activity was at least as good as it was on tissue culture plastic. However, the total amount of protein, and especially the collagen type I synthesis, was sensitive to surface modification. On titanium modified with ethane-1,1,2-triphosphonic acid, the total amount of synthesized protein was significantly higher than it was on unmodified titanium surfaces. A significant increase (up to 16%) of collagen type I production was observed on titanium surfaces modified with this acid or with methylenediphosphonic acid compared to unmodified titanium surfaces.     

Influence of recovering collagen with bioactive glass on osteoblast behavior

Bioactive ceramics have interesting properties from the biological standpoint, but their effects on cellular events remain partially unknown. In the current work, we investigated cellular viability, proliferation, and metabolic activity of rat primary osteoblasts in contact with four different samples: type I collagen, bioactive glass-coated collagen (GC), and both samples submitted to immersion for 5 days in a simulated body fluid. The bioactive glass coating was obtained from a sol-gel process. The cell viability, the alkaline phosphate, the collagen secretion, and the nitric oxide production by osteoblast were measured after 72 h of incubation in the presence of the samples. The GC that was immersed for 5 days in a simulated body fluid solution showed an increase in osteoblast viability and proliferation when it was compared with control and the other samples.     

Growth factors reduce the suppression of proliferation and osteogenic differentiation by titanium particles on MSCs

This study was conducted to test the hypothesis that growth factors can reduce the suppressive effect of titanium particles on MSCs. Cultured human MSCs at passage 3 were challenged with prepared cpTi particles at a concentration of 500 particles/cell along with one of the following growth factors: TGF-beta(1) (10 ng/mL), FGF-2 (10 ng/mL), IGF-I (100 ng/mL), and BMP-6 (50 ng/mL). After various periods of time, the treatment effects on cellular proliferation, viability, and osteogenic differentiation were measured. All the four growth factors positively promoted cell proliferation and viability to a varying extent. FGF-2 most effectively enhanced cell proliferation, whereas IGF-I was the most effective growth factor for enhancing cell viability. FGF-2, IGF-I, and BMP-6 reversed the titanium-mediated suppression of osteogenic differentiation, BMP-6 being the most effective one. Various growth factors can mitigate the suppressive effects of titanium particles on MSCs and enhance cell proliferation, viability, and osteogenic differentiation.     

Ceramic and PMMA particles differentially affect osteoblast phenotype

There is increasing evidence that wear debris particles present in periprosthetic tissues have direct effects on osteoblasts. The nature of the cell response varies with the chemistry of the particle and the number of particles. Most studies have used Ti, Ti-6Al-4V, and ultrahigh molecular weight polyethylene (UHMWPE) particles since these materials are most frequently used in implants and as a result, these particles predominate in peri-prosthetic tissues. Ceramics have also been used successfully as load-bearing surfaces in implants for years, although it is unknown how wear debris from these surfaces may contribute to aseptic bone loss. Further, particles resulting from polymethylmethacrylate (PMMA) cements used for fixation may also be involved in aseptic loosening of implants, but how these particles may affect bone formation is unknown. In the present study, we examined whether aluminum oxide (Al2O3), zirconium oxide (ZrO2), and PMMA particles exert effects on osteoblast proliferation, phenotypic expression, and local factor production, and if so, whether the effects were specific to the particle type. ZrO2 particles were produced in a custom-made axial mixer in which ZrO2 containers were filled with ZrO2 bars and 95% ethanol and then rotated continuously at room temperature. PMMA particles were prepared in a ZrO2 roller mill. Al2O3 was produced and provided by Aesculap AG. Particles were endotoxin-free with equivalent circle diameters <3 microm; Al2O3 particles were significantly smaller than ZrO2 or PMMA particles. Particle suspensions were added to confluent cultures of MG63 osteoblast-like cells after diluting them 1:100, 1:10, and 1:1 with culture medium. Cells were incubated with the particles for 24 h. Transmission electron microscopy showed that MG63 cells phagocytosed Al2O3 particles and exhibited ultrastructural changes consistent with cytotoxicity. This was supported by biochemical changes as well. Proliferation, alkaline phosphatase activity, and TGF-beta1 levels were decreased. ZrO2 and PMMA particles increased proliferation and alkaline phosphatase specific activity. The effect of ZrO2 on alkaline phosphatase was targeted to matrix vesicles, the effect of PMMA was greater on the cells. All particles increased prostaglandin E2 production. These results show that Al2O3, ZrO2, and PMMA particles elicit direct effects on osteoblasts and that cell response depends on the particle type. None of the particles tested had the same effect as noted previously for UHMWPE: increased proliferation and decreased alkaline phosphatase. These results may indicate that the response of peri-prosthetic tissues to wear particles may be modulated by the relative contributions of the various particle types present.     

辛伐他汀对成骨细胞增殖分化及连接蛋白43表达的调控

背景：辛伐他汀对成骨细胞增殖分化的影响以及分子机制尚不完全明了,尤其对连接蛋白43的作用知之甚少。 目的：探讨辛伐他汀对成骨细胞增殖分化及成骨基因和连接蛋白43表达的调控作用。 方法：选取新生SD大鼠,采用颅盖骨消化法培养成骨细胞。采用不同浓度辛伐他汀(0.062 5,0.125,0.25,0.5和1.0 μmol/L)处理成骨细胞,MTT检测辛伐他汀对成骨细胞增殖作用的影响;碱性磷酸酶活性检测辛伐他汀对成骨细胞分化作用的影响;实时定量RT-PCR和免疫印迹检测细胞成骨基因和间隙连接蛋白43 mRNA和蛋白的表达。 结果与结论：细胞培养第3天,辛伐他汀组各浓度组MTT吸光度值比较差异无显著性意义(P > 0.05);但是培养第4天和第5天,辛伐他汀各浓度组MTT吸光度值低于对照组(P < 0.05)。通过不同浓度辛伐他汀处理成骨细胞后,与对照组比较,成骨细胞碱性磷酸酶活性增加(P < 0.05),且0.25 μmol/L 浓度组作用对成骨细胞碱性磷酸酶活性的影响最为显著。采用0.25 μmol/L辛伐他汀处理成骨细胞后,辛伐他汀组与对照组比较,成骨细胞骨钙蛋白、碱性磷酸酶、I型胶原和连接蛋白43 mRNA和蛋白表达均增加(P < 0.05)。提示辛伐他汀可能通过上调成骨基因和间隙连接蛋白43 mRNA和蛋白的表达来抑制成骨细胞增殖和促进其分化,这为他汀类药物治疗骨质疏松症提供新的干预靶点。 中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程全文链接：