Liposomal clodronate inhibition of osteoclastogenesis and osteoinduction by submicrostructured beta-tricalcium phosphate

Bone graft substitutes such as calcium phosphates are subject to the innate inflammatory reaction, which may bear important consequences for bone regeneration. We speculate that the surface architecture of osteoinductive β-tricalcium phosphate (TCP) stimulates the differentiation of invading monocyte/macrophages into osteoclasts, and that these cells may be essential to ectopic bone formation. To test this, porous TCP cubes with either submicron-scale surface architecture known to induce ectopic bone formation (TCPs, positive control) or micron-scale, non-osteoinductive surface architecture (TCPb, negative control) were subcutaneously implanted on the backs of FVB strain mice for 12 weeks. Additional TCPs samples received local, weekly injections of liposome-encapsulated clodronate (TCPs + LipClod) to deplete invading monocyte/macrophages. TCPs induced osteoclast formation, evident by positive tartrate resistant acid phosphatase (TRAP) cytochemical staining and negative macrophage membrane marker F4/80 immunostaining. No TRAP positive cells were found in TCPb or TCPs + LipClod, only F4/80 positive macrophages and foreign body giant cells. TCPs stimulated subcutaneous bone formation in all implants, while no bone could be found in TCPb or TCPs + LipClod. In agreement, expression of bone and osteoclast gene markers was upregulated in TCPs versus both TCPb and TCPs + LipClod, which were equivalent. In summary, submicron-scale surface structure of TCP induced osteoclastogenesis and ectopic bone formation in a process that is blocked by monocyte/macrophage depletion.     

The size of surface microstructures as an osteogenic factor in calcium phosphate ceramics

The microporosity of calcium phosphate (CaP) ceramics has been shown to have an essential role in osteoinduction by CaP ceramics after ectopic implantation. Here we show that it is not the microporosity but the size of surface microstructural features that is the most likely osteogenic factor. Two tricalcium phosphate (TCP) ceramics, namely TCP-S and TCP-B, were fabricated with equivalent chemistry and similar microporosity but different sizes of surface microstructural features. TCP-S has a grain size of 0.99 ± 0.20 μm and a micropore size of 0.65 ± 0.25 μm, while TCP-B displays a grain size of 3.08 ± 0.52 μm and a micropore size of 1.58 ± 0.65 μm. In vitro, both cell proliferation and osteogenic differentiation were significantly enhanced when human bone marrow stromal cells were cultured on TCP-S without any osteogenic growth factors, compared to TCP-B ceramic granules. The possible involvement of direct contact between cells and the TCP ceramic surface in osteogenic differentiation is also shown with a trans-well culture model. When the ceramic granules were implanted in paraspinal muscle of dogs for 12 weeks, abundant bone was formed in TCP-S (21 ± 10% bone in the available space), whereas no bone was formed in any of the TCP-B implants. The current in vitro and in vivo data reveal that the readily controllable cue, i.e. the size of the surface microstructure, could be sufficient to induce osteogenic differentiation of mesenchymal stem cells, ultimately leading to ectopic bone formation in calcium phosphate ceramics.     

Inhibition of foreign body giant cell formation by 4- hexylresorcinol through suppression of diacylglycerol kinase delta gene expression

Grafted macromolecules often induce granuloma formation with foreign body giant cell (FBGC) infiltration, and this is the main reason for graft failure. Diacylglycerol kinase (DAGK) is an important intracellular mediator of FBGC formation in macrophages. In this study, 4-hexylresorcinol (4HR) inhibited DAGKδ in a macrophage cell line (RAW264.7 cells). As a result of DAGK-δ inhibition by 4HR, FBGC formation was significantly inhibited in RAW264.7 cells. Silk fibroin is a well-known natural macromolecule, and when it is grafted into bone defects, it results in granuloma formation with massive FBGC formation. 4HR-incorporating silk graft materials displayed significant reduction of granuloma formation and increases in the extent of new bone formation in a rabbit calvarial defect model. In conclusion, 4HR could inhibit foreign body reaction via a DAGK-mediated pathway.     

Novel highly biodegradable biphasic tricalcium phosphates composed of alpha-tricalcium phosphate and beta-tricalcium phosphate

Novel biodegradable biphasic tricalcium phosphates (BTCP) composed of alpha-tricalcium phosphate (alpha-TCP) and beta-tricalcium phosphate (beta-TCP) were successfully synthesized by heating amorphous calcium phosphate precursors with different structures at 800 degrees C for 3 h. The ratio of alpha-TCP and beta-TCP in the calcium phosphate particle can be controlled by aging time and pH value during synthesis of the amorphous precursor.     

Flow perfusion culture of human fetal bone cells in large beta-tricalcium phosphate scaffold with controlled architecture

One unsolved problem in bone tissue engineering is how to enable the survival and proliferation of osteoblastic cells in large scaffolds. In this work, large beta-tricalcium phosphate scaffolds with tightly controlled channel architectures were fabricated and a custom-designed perfusion bioreactor was developed. Human fetal bone cells in third passage were seeded onto the scaffolds and cultured in static or flow perfusion conditions for up to 16 days. Compared with nonperfused constructs, flow perfused constructs demonstrated improved cells proliferation and differentiation according to cell viability, glucose consumption, alkaline phosphatase activity, and osteopontin. Moreover, after 16 days of perfusion culture, a homogenous layer composed of cells and mineralized matrix throughout the whole scaffold was observed by scanning electron microscopy and histological study. In contrast, cells were located only along the scaffold perimeter in static culture. These results demonstrated the feasibility and benefit of perfusion culture in conjunction with well-defined three-dimensional environment for large bone graft construction. Porous scaffold with controlled architecture can be a potential tool to evaluate the effects of scaffold specific geometry on fluid flow configuration and cell behavior under perfusion culture.     

Osteoclast resorption of thermal spray hydoxyapatite coatings is influenced by surface topography

Coating characteristics such as composition, crystallite features and topography collectively impact the cell response. The influence from splats has not yet been assessed for hydroxyapatite (HAp) thermal spray coatings. The objective of this work is to (a) survey the topography on commercial implants, (b) ascertain topography formation from single splats, and (c) determine the osteoclast resorption pattern on a topographically refined coating compared to dentine. Coatings on dental implants, an orthopedic screw, a femoral stem and a knee implant were studied for reference. The effects of substrate pre-heat, roughness, spray distance and particle size on the coating roughness and topography were studied. Human-derived osteoclasts were placed on a coating with refined topography and compared to dentine, a polished coating and polished sintered HAp. A pre-heat of at least 200°C on titanium was required to form rounded splats. The greatest influence on coating roughness and topography arose from particle size. A 2-fold increase in the mean particle size from 30 to 72 μm produced a significant difference (P<0.001) in roughness from 4.8 and 9.7 μm. A model is shown to illustrate topography formation, nanostructure evolution on single splats, and the topography as seen in commercial implants. Osteoclasts showed a clear preference for activity on coatings with refined topography. A one-way ANOVA test revealed a significantly greater pit depth (P=0.022) for dentine (14 μm) compared to the as-sprayed and polished coating (5 μm). Coatings with topography display a similar number of resorption pits with dentine, but a 10-fold greater number than polished coatings, emphasizing the importance of flattened droplet topography on implant surfaces.     

Formation of osteoclast-like cells on HA and TCP ceramics

An essential property of bone substitute materials is that they are integrated into the natural bone remodelling process, which involves the resorption by osteoclast cells and the formation by osteoblast cells. If monocyte cells adhere to a calcium phosphate surface (bone or bone substitute material), they can fuse together and form multinucleated osteoclast cells. In this study we show that osteoclast-like cells derived from a human leukoma monocytic lineage responded in a different way to tricalciumphosphate (TCP) than to hydroxyapatite (HA) ceramics. Both ceramics were degraded by resorbing cells; however, HA enhanced the formation of giant cells. The osteoclast-like cells on HA formed a more pronounced actin ring, and larger lacunas could be observed. TCP ceramics are medically used as bone substitute materials because of their high dissolution rate. On the other hand, highly soluble calcium phosphate ceramics like TCP seem to be inappropriate for osteoclast resorption because they produce a high calcium concentration in the osteoclast interface and in the environment.     

Zinc in calcium phosphate mediates bone induction: In vitro and in vivo model

Zinc-containing tricalcium phosphate (Zn-TCP) was synthesized to investigate the role of zinc in osteoblastogenesis, osteoclastogenesis and in vivo bone induction in an ectopic implantation model. Zinc ions were readily released in the culture medium. Zn-TCP with the highest zinc content enhanced the alkaline phosphatase activity of human bone marrow stromal cells and tartrate-resistant acid phosphatase activity, as well as multinuclear giant cell formation of RAW264.7 monocyte/macrophages. RAW264.7 cultured with different dosages of zinc supplements in medium with or without zinc-free TCP showed that zinc could influence both the activity and the formation of multinuclear giant cells. After a 12-week implantation in the paraspinal muscle of canines, de novo bone formation and bone incidence increased with increasing zinc content in Zn-TCP – up to 52% bone in the free space. However, TCP without zinc induced no bone formation. Although the observed bone induction cannot be attributed to zinc release alone, these results indicate that zinc incorporated in TCP can modulate bone metabolism and render TCP osteoinductive, indicating to a novel way to enhance the functionality of this synthetic bone graft material.     

复合同种异体成骨细胞的β-磷酸三钙人工骨修复桡骨缺损

背景：作为骨支架材料,β-磷酸三钙具有良好的生物相容性、骨诱导性及生物力学性能。 目的：探讨β-磷酸三钙复合同种异体成骨细胞修复兔桡骨节段性骨缺损的效果。 方法：建立45只兔单侧桡骨骨缺损模型,随机分为3组,实验组缺损区植入复合同种异体成骨细胞的β-磷酸三钙,对照组缺损区植入β-磷酸三钙,空白对照组缺损区不植入任何材料。植入后4,8,16周观察新骨生成情况,通过形态学、X射线等观察指标客观评价各组成骨及骨缺损修复能力。 结果与结论：随着修复时间的延长,实验组骨缺损逐渐修复,至16周时X射线见支架与宿主骨之间的骨痂已完全骨化,骨缺损完全修复;组织学见缺损区皮质骨连续,髓腔再通,并且不同时间点实验组修复效果明显优于对照组与空白对照组(P < 0.01)。表明复合同种异体成骨细胞的β-磷酸三钙人工骨具有良好的成骨能力,有引导组织再生、防止骨不连的作用。     

可注射聚富马酸丙二醇酯/β-磷酸三钙骨水泥的体外生物活性及其降解性

BACKGROUND: Poly(propylene fumarate) (PPF) can crosslink at room temperature, and β-tricalcium phosphate (β-TCP) has good biocompatibility, but PPF/β-TCP composite bone cement has not yet been systematically studied. OBJECTIVE: To prepare PPF/β-TCP composite bone cement and to explore its in vitro bioactivity and degradability. METHODS: β-TCP and PPF were respectively synthesized by liquid-phase precipitation and a two-step method, and PPF/β-TCP composite bone cement was prepared through mixing PPF with β-TCP. The    in vitro bioactivity of PPF/β-TCP was compared with the commercial poly(methyl methacrylate) (PMMA) through the ability of forming hydroxyapatite after immersed in simulated body fluid for 7 days. The in vitro degradability of PPF/β-TCP was studied via investigating the transformation of pH values, water uptake and mass loss, compressive strength and morphology at each time point. RESULTS AND CONCLUSION: There were hydroxyapatites formed on the PPF/β-TCP material, but none on the commercial PMMA material. The pH values of the PPF/β-TCP were stable in PBS for 63 days, indicating its degradation is moderate; the mass loss was up to 13.5% after 84 days. Scanning electron microscope displayed the degraded PPF/β-TCP surface, and its compressive strength was decreased gradually, which good for the integrity and sustainability of mechanical properties during degradation. These results suggest that PPF/β-TCP bone cement holds mineralization and degradability in vitro.     

仿生溶液法诱导钛表面形成钙磷涂层的研究

采用3种不同的方法即不作处理，酸和碱处理但不加热、酸和碱处理后600℃加热对商用纯钛进行表面处理，然后将样品浸泡于仿生溶液中2周后，经过处理的两组样品表面均形成一层薄的钙磷涂层，SEM和EDX分析表明：酸碱处理再经600℃加热组较单纯的酸碱处理组涂层晶体构型均一；XRD分析表明晶体组成主要为含有少量氯元素的羟基磷灰石。     

复合磷酸钙颗粒溶解性能测试研究

目的研究复合磷酸钙颗粒(HA∶TCP=3∶7)在TRIS缓冲溶液中的体外溶解特性。方法配制一系列浓度逐级递增的Ca~(2+)标准溶液,绘制Ca~(2+)离子溶液的工作曲线。将复合磷酸钙颗粒(HA∶TCP=3∶7)置于pH值为7.4的TRIS缓冲溶液中浸泡,每小时取样一次(初始阶段每3分钟取样一次),用高速离心技术提取上层清液,采用电感耦合等离子体质谱法测定各时间点提取液的Ca~(2+)浓度,绘制Ca~(2+)浓度对浸泡时间的溶解曲线。结果 Ca~(2+)离子溶液的工作曲线的线性方程为y=4043.9x+23783,相关系数R为0.9999,方法检出限为0.0063 g/m L,定量限为0.021 g/mL。浸泡后最初的0.5 h以内,Ca~(2+)浓度随时间延长急剧升高,0.5 h处有明显转折,0.5 h至20 h,Ca~(2+)浓度仍随时间增加而升高,但是增加的速率较前0.5 h明显下降,20 h以后溶解达到基本平衡。复合磷酸钙颗粒的溶解过程分为3个阶段,首先是Ca~(2+)离子浓度快速升高的溶解过程,第二阶段是Ca~(2+)离子溶解与HA或Ca HPO_4·2H_2O沉积的竞争过程,第三阶段是Ca~(2+)离子浓度不再继续增加的稳定过程。结论复合磷酸钙颗粒(HA∶TCP=3∶7)在TRIS缓冲溶液中的溶解具有一定的规律性,24 h后溶出的Ca~(2+)离子浓度在1200 g/L左右。     

Immersion behavior of gelatin-containing calcium phosphate cement

Calcium phosphate cements (CPCs) have many favorable properties that support their clinical use as bone defect repair. However, it is difficult to deliver to the required site and hard to compact adequately due to inherently low ductility of ceramics. The aim of this study focused on the effect of the gelatin content on properties of CPCs. The diametral tensile strength, morphology, and weight loss of gelatin cements were evaluated after immersion in physiological solution, in addition to setting time. The results indicated that the setting time significantly increased with increasing gelatin amount. The 2 wt.% gelatin could make CPCs attain the maximum strength value of 2.1 MPa at 15-day immersion, while 1.6 MPa for the cement without gelatin. It is concluded that the presence of gelatin improved mechanical properties of CPCs; in particular, 2 wt.% gelatin. CPCs containing 2 wt.% gelatin hardened in an acceptable time recommended for clinical applications.     

Targeting and activation of antigen-specific B-cells by calcium phosphate nanoparticles loaded with protein antigen

Cross-linking of the B-cell receptors of an antigen-specific B-cell is the initial signal for B-cell activation, proliferation, and differentiation into antibody secreting plasma cells. Since multivalent particulate structures are efficient activators of antigen-specific B-cells, we developed biodegradable calcium phosphate nanoparticles displaying protein antigens on their surface and explored the efficacy of the B-cell activation after exposure to these nanoparticles. The calcium phosphate nanoparticles were functionalized with the model antigen Hen Egg Lysozyme (HEL) to take advantage of a HEL-specific B-cell receptor transgenic mouse model. The nanoparticles were characterized by scanning electron microscopy and dynamic light scattering. The functionalized calcium phosphate nanoparticles were preferentially bound and internalized by HEL-specific B-cells. Co-cultivation of HEL-specific B-cells with the functionalized nanoparticles also increased surface expression of B-cell activation markers. Functionalized nanoparticles were able to effectively cross-link B-cell receptors at the surface of antigen-matched B-cells and were 100-fold more efficient in the activation of B-cells than soluble HEL. Thus, calcium phosphate nanoparticles coated with protein antigens are promising vaccine candidates for induction humoral immunity.     

Resorbability of rigid beta-tricalcium phosphate wedges in open-wedge high tibial osteotomy: a retrospective radiological study

The open-wedge high tibial osteotomy (OWHTO) is a well accepted treatment modality for patients with osteoarthritis of the medial compartment associated with genu varum. To fill in the osteotomy gap 30% macroporosity rigid beta-tricalcium phosphate (β-TCP) is frequently used as a stable resorbable bone substitute. However, the resorbability of these β-TCP wedges is not known. The aim of this study was to investigate this.

Twenty-one OWHTO procedures in seventeen patients were performed with the use of 30% macroporosity rigid β-TCP wedges. The osteotomies were fixed using an angle-stable locking plate. Conventional AP and lateral radiographs were examined in order to assess the resorbability of the 30% macroporosity rigid β-TCP wedges as a function of time. A radiological classification system consisting of five phases was used to monitor the resorption of the 30% macroporosity rigid β-TCP wedges. The mean duration of follow-up was 62 months (± 23 range of 28–99).

In all 21 cases, remnants of the 30% macroporosity rigid β-TCP wedges were still present at maximum follow-up. Although the boundaries between 30% macroporosity rigid β-TCP wedges and bone remained slightly visible, all osteotomies were completely consolidated and full osseointegration took place. In 16 out of 21 knees the fixation system was removed after a mean duration of 32 months (± 19 range of 6–62). In six out of 21 knees a conversion to a knee arthroplasty was performed after a mean duration of 56 months (± 18 range of 37–82). The OWHTO did not interfere with the placement of knee prostheses.

Complete resorption of 30% macroporosity rigid β-TCP wedges did not take place up to 8 years after operation.     

Foreign body-type multinucleated giant cell formation requires protein kinase C beta, delta, and zeta

Multinucleated giant cells are a classic cellular feature of chronic inflammation, although the mechanism of macrophage fusion leading to their formation is not well understood. Here, we investigate the participation of protein kinase C (PKC) in the interleukin (IL)-4-induced fusion of human monocyte-derived macrophages and foreign body giant cell (FBGC) formation in vitro. The PKC inhibitors H-7 and calphostin C attenuated macrophage fusion, whereas H-8, which is more selective for PKA and PKG, did not. Macrophage fusion was also prevented by the phospholipase C inhibitor, Et-18-OCH(3), the PKC isoform inhibitors GO6983 or rottlerin and by peptide inhibitors for PKC (20-28), PKCbeta, or PKCzeta but not by HBDDE or peptide inhibitors for PKCvarepsilon or PKA. In cultures of fusing macrophages/FBGC, we detected only PKCalpha, beta, delta, and zeta by immunoprecipitation and immunoblotting, and we also observed strong expression of these isoforms by immunocytochemistry. Our collective results suggest that the gamma, varepsilon, eta, mu, theta, or iota PKC isoforms are not required in the mechanism of IL-4-induced macrophage fusion; whether PKCalpha is required is unclear. However, new evidence is provided that FBGC formation is supported by PKCbeta, PKCdelta, and PKCzeta in combined diacylglycerol-dependent (PKCbeta and PKCdelta) and -independent (PKCzeta) signaling pathways.     

SDF-1 Increases Recruitment of Osteoclast Precursors by Upregulation of Matrix Metalloproteinase-9 Activity

Although chemokines play essential roles in the trafficking and homing of many circulating hematopoietic cell types, their potential influences on osteoclast (OC) recruitment or bone remodeling are not well known. Therefore, chemokine receptor expression was analyzed by RNase protection assay during OC formation induced by RANKL in a murine mononuclear cell line (RAW 264.7). Relatively high CXCR4 expression was detected in RAW cells (pre-OCs), whereas CXCR4 levels were downregulated during RAW-OC development. SDF-1, the unique ligand for CXCR4, stimulated RAW cell production of matrix metalloproteinase (MMP)-9 activity, a matrix-degrading enzyme essential for pre-OC migration into the developing bone marrow cavity. Induced MMP-9 activity in RAW cells was associated with their increased MMP-dependent transmigration through a collagen gel in response to SDF-1. We conclude that SDF-1 stimulation of MMP-9 activity in pre-OCs may be a key aspect of their recruitment to bone and migration within the marrow to sites for OC differentiation and bone resorption.     

Bioactivity of xerogels as modulators of osteoclastogenesis mediated by connexin 43

In order to investigate the effects of different degrees of bioactivity of xerogels on connexin 43 (cx43) signaling of osteoclasts a cell culture approach was developed. Cells isolated from peripheral blood mononuclear cells were cultured in combination with the xerogels and were harvested for further investigations on day 1, day 5, and day 10. By means of quantitative PCR increased cx43 mRNA levels and coincident decreasing mRNA levels of the calcium sensing receptor, TRAP, and Cathepsin K were detected with increasing bioactivity of the xerogel samples. Additionally, osteoclasts cultured on tissue culture plates were used to perform principle investigations on cell differentiation by means of transmission electron microscopy, life cell imaging, and immunofluorescence, and the results demonstrated that cx43-signaling could be attributed to migration and fusion of osteoclast precursors. Therefore, the positive correlation of cx43 expression with high xerogel bioactivity was caused by proceeding differentiation of the osteoclasts. Finally, the presently observed pattern of cx43 signaling refers to strong effects regarding bioactivity on cx43-associated cell differentiation of osteoclasts influenced by extracellular calcium ions.     

蛇床子素对TCP颗粒诱导小鼠颅骨溶解的影响

 目的: 观察蛇床子素对磷酸三钙(tricalcium phosphate,TCP)颗粒诱导小鼠颅骨溶解的影响。方法: 采用TCP颗粒诱导小鼠颅骨溶解模型,于术后第2天颅顶局部注射蛇床子素(osthole)20 mg/kg,每周3次,持续干预2周。干预结束后处死动物取材,应用HE染色和抗酒石酸酸性磷酸酶(tartrate resistant acid phosphatase,TRACP)染色观察假体周围破骨细胞生成和骨溶解程度;ELISA法检测血清和骨组织中骨转换标志物骨钙素(osteocalcin)水平、碱性磷酸酶(alkaline phosphatase,ALP)和TRACP活性以及骨膜中肿瘤坏死因子α (tumor necrosis factor-α,TNF-α)、白细胞介素6(interleukin-6,IL-6)和白细胞介素1β(interleukin-1β,IL-1β)水平。Western blot法检测颅骨组织葡萄糖调节蛋白 78(glucose-regulated protein 78,GRP78)和CAAT/增强子结合蛋白同源蛋白(CAAT/ enhancer binding protein homologous protein,CHOP)的表达变化。结果: 蛇床子素可明显抑制TCP颗粒诱导的破骨细胞生成,减少骨溶解面积,显著增加ALP和osteoclacin水平,降低TRAP活性并阻断炎症因子TNF-α、IL-6和IL-1β释放。此外,蛇床子素干预能明显减弱TCP颗粒激活的内质网应激反应。结论: 蛇床子素可抑制TCP颗粒诱导的小鼠颅骨溶解,其机制可能与抑制TCP颗粒诱导的内质网应激反应有关。     

A case of pulmonary adenocarcinoma harboring osteoclast‐like giant cells: Its evaluation by immunohistochemical and genetic analyses

Tumors harboring osteoclast‐like giant cells (OGCs) at extraosseous site are extremely rare. These rare tumors have been detected most frequently in the pancreas and few pulmonary tumors harboring OGCs have been previously reported. In addition, the genetic profiles of these tumors have remained virtually unknown. Therefore, we report a case of pulmonary adenocarcinoma harboring OGCs in which k‐ras mutation and immunohistochemical study of proteins associated with OGCs were examined. The case was a 70‐year‐old man, who demonstrated a pulmonary mass associated with unusual radiological features. Histopathologically, three different cell types, mucinous adenocarcinoma cell, OGC and mononuclear cell were detected. OGCs were immunohistochemically negative for epithelial markers and positive for histiocytic markers but mononuclear cells were immunopositive for epithelial markers. In addition, both mononuclear and adenocarcinoma cells had the same k‐ras mutation profiles and mononuclear cells were immunohistochemically positive for macrophage colony‐stimulating factor (M‐CSF), one of the factors associated with OGC differentiation. Therefore, mononuclear cells were considered to be derived from neoplastic epithelium and OGCs could represent non‐neoplastic cells. In addition, M‐CSF locally produced could promote the differentiation of OGCs.