Effect of surface chemical modification of bioceramic on phenotype of human bone-derived cells.

In the search for methods to improve the biocompatibility of prosthetic materials, attention has recently been directed toward the potential use of surface chemical modification and its influence on cellular behavior. This in vitro study investigates the effect of surface chemistry modification of bioceramics on human bone-derived cells (HBDCs) grown on biomaterial surfaces for 2 weeks. Cells were cultured on either alumina (Al2O3), alumina doped with magnesium ions ([Mg]-Al2O3), or hydroxyapatite (HAP), as well as tissue culture polystyrene (TCPS). Expression of alkaline phosphatase (ALP), thrombospondin (Tsp), osteopontin (OP), osteocalcin (OC), osteonectin (ON/SPARC), type I collagen (Col I), and bone sialoprotein (BSP) were determined in terms of mRNAs and proteins. Protein levels for ALP, OP, OC, and BSP were significantly (p < 0. 05) greater at day 5 in HBDCs cultured on [Mg]-Al2O3 compared to those cells grown on Al2O3. At day 14 the levels of ALP, Tsp, Col I, OP, ON/SPARC, and BSP rose significantly (p < 0.05) above those occurring in HBDCs grown on Al2O3, HAP, and TCPS. This suggests that HBDCs from the same patient respond to differences in the surface chemical groups. This study confirms that the chemistry of a substratum, which facilitates cellular adhesion, will enhance cellular differentiation.     

Activation of human leukocytes on tantalum trabecular metal in comparison to commonly used orthopedic metal implant materials

We analyzed leukocyte functions and cytokine response of human leukocytes toward porous tantalum foam biomaterial (Trabecular Metaltrade mark, TM) in comparison to equally sized solid orthopedic metal implant materials (pure titanium, titanium alloy, stainless steel, pure tantalum, and tantalum coated stainless steel). Isolated peripheral blood mononuclear cells (PBMC) and polymorphonuclear neutrophil leukocytes (PMN) were cocultured with equally sized metallic test discs for 24 h. Supernatants were analyzed for cytokine content by enzyme-linked immunosorbent assay. Compared to the other used test materials there was a significant increase in the release of IL (interleukin)-1ra and IL-8 from PMN, and of IL-1ra, IL-6, and TNF-alpha from PBMC in response to the TM material. The cytokine release correlated with surface roughness of the materials. In contrast, the release of IL-2 was not induced showing that mainly myeloid leukocytes were activated. In addition, supernatants of these leukocyte/material interaction (conditioned media, CM) were subjected to whole blood cell function assays (phagocytosis, chemotaxis, bacterial killing). There was a significant increase in the phagocytotic capacity of leukocytes in the presence of TM-conditioned media. The chemotactic response of leukocytes toward TM-conditioned media was significantly higher compared to CM obtained from other test materials. Furthermore, the bactericidal capacity of whole blood was enhanced in the presence of TM-conditioned media. These results indicate that leukocyte activation at the surface of TM material induces a microenvironment, which may enhance local host defense mechanisms.     

Surface modification and evaluation of some commonly used catheter materials. I. Surface properties

Double catheter systems consisting of a stiff outer catheter and a flexible, buoyant, flow-directed, inner catheter which is often balloon-tipped have been employed with increasing frequency recently in both therapeutic and diagnostic procedures. Their use, however, has been restricted because of the excessive friction generated between the two catheters. In an attempt to decrease friction between polymers commonly used as catheter materials, oxidation of polyethylene, fluorinated ethylene-propylene copolymer, poly(vinyl chloride), silicone rubber, and polystyrene surfaces was induced by exposing the polymers to radio frequency glow discharge (RFGD) in a helium environment. All polymers were surface characterized utilizing x-ray photoelectron spectroscopy (XPS), scanning electron microscopy (SEM), and contact angle measurements before and after oxidation. This article describes the materials and methods used to fabricate and characterize the polymer surfaces and the results of the characterization. The results indicate that increases in oxygen concentration at the surface of the polymers and decreases in air-water contact angles occur with increased RFGD exposure time. Plateau values were usually obtained after 5-30 s exposure time, yet no apparent changes in surface topography were noted by scanning electron microscopy. The hydrophilic surfaces produced were stable for up to three months storage time in air.     

Surface modification and characterization of some commonly used catheter materials. II. Friction characterization

The effects of the modification of polystyrene (PS), polyethylene (PE), poly(vinyl chloride) (PVC), silicone rubber (SR), and fluorinated ethylene propylene (FEP) copolymer by radio frequency glow discharge in a helium environment were presented in part I. The hydrated polymer surfaces were characterized by XPS, SEM, visual microscopy, and by contact angle measurements. In general, exposure of the polymers to RFGD produced an oxidized hydrophilic surface, yet the roughness of the surface was unaltered by the relatively mild plasma conditions used. In this article, the frictional behavior of oxidized and unoxidized SR, PE, and FEP in distilled water, isotonic saline, and blood plasma environments is examined experimentally. The results are discussed in relation to the properties generally believed to affect frictional phenomena and to the surface properties as determined in part I. Results indicate that RFGD-treated SR generates less friction than untreated SR when dragged across all untreated and treated polymer surfaces, whether the medium is distilled water or an isotonic saline solution. Friction is consistently lower in a blood plasma medium between all surfaces investigated, most probably because of the presence of adsorbed proteins at the polymer interfaces.     

假肢常用材料与人体皮肤摩擦学及其生物相容性

背景：目前主要采用强度高,质量轻的高分子材料来制造假肢零部件,其中热塑性塑料板材、树脂基复合材料、低温热塑材料采用最广泛。 目的：分析假肢常用高分子材料与人体皮肤的摩擦学及生物学相容性。 方法：由作者检索1990/2008万方数据库有关假肢的常用材料及其与皮肤摩擦学和生物学相容性等方面的文献。 结果与结论：聚乙烯、聚丙烯以及改性聚酯等热塑板材,低温热板材料,硅橡胶,钛合金等均与人体皮肤具有良好的生物相容性,但与人体皮肤摩擦学方面各有优缺点,今后应以分子生物学研究和毒理学研究为基础,不断改进假体材料的组织相容性,更进一步探讨假体材料在人体内生理环境下的摩擦行为,找到更为确实可靠的理论依据进行体外实验,以便更好的设计假体模型,达到仿生效果。     

Isolation and characterization of human bone-derived endothelial cells.

Historically, the etiology of local bone pathologies, such as avascular necrosis, has been related to intravascular occlusion. Recent reports have highlighted the occlusion of arteries, venules, and/or capillaries in bone tissue. Endothelium of bone presumably participates locally in the formation of the microvascular thrombosis. It is also known that endothelial cells (ECs) play a central role in angiogenesis, a process seen in osteosarcoma, amongst other bone diseases. Given the well-recognized heterogeneity of ECs throughout the body, investigations of local bone disease related to endothelium processes may be more appropriately targeted on bone ECs rather than other primary ECs or an immortalized EC line. In the current study, mechanical and enzymatic methods are described to isolate ECs from cancellous human bone tissue followed by immunomagnetic bead separation to purify the cell populations. The human bone-derived endothelial cells (hBDECs) were characterized based on endothelial cell antigen expression and functional assays. This study is the first report of isolation and expansion of ECs from human bone tissue. Isolation of hBDECs in human vascular bone diseases may facilitate the study of the molecular and/or genetic abnormalities in the vasculature system that contributes to the initiation and/or progression of the disease.     

Differentiation of human bone-derived cells grown on GRGDSP-peptide bound titanium surfaces

Various surface modifications have been applied to titanium alloy (Ti-6Al-4V) implants, in an attempt to enhance osseointegration; crucial for ideal prosthetic fixation. Despite the numerous studies demonstrating that peptide-modified surfaces influence in vitro cellular behavior, there is relatively little data reporting their effects on bone remodeling. The objective of this article was to examine the effects of chemically modifying Ti-6Al-4V surfaces with a common RGD sequence, a 15-residue peptide containing GRGDSP (glycine-arginine-glycine-aspartate-serine-proline), on the modulation of bone remodeling. The expression of proteins known to be associated with osseous matrix and bone resorption were studied during the growth of human bone-derived cells (HBDC) on these peptide-modified surfaces. HBDC grown for 7 days on RGD surfaces displayed significantly increased levels of osteocalcin, and pro-collagen Ialpha1 mRNAs, compared with the production by HBDC grown on the native Ti-6Al-4V. A pattern that was also reflected at the protein levels for osteocalcin, type I collagen, and bone sialoprotein. Moreover, HBDC grown for 7 and 14 days on RGD-modified Ti-6Al-4V expressed significantly higher level of osteoclast differentiation factors and lower levels of osteoprotegerin and IL-6 proteins compared with other surfaces tested. These results suggest that different chemical treatments of implant material (Ti-6Al-4V) surface result in differential bone responses, not only their ability to form bone but also to stimulate osteoclastic formation.     

人牙髓干细胞在三种支架材料上黏附与增殖的研究

目的研究人牙髓干细胞(hDPSCs)在羟基磷灰石磷酸三钙(HA-TCP)、聚乙醇酸(PGA)和聚乳酸-聚羟基乙酸共聚物(PLGA)三种支架材料上的黏附与增殖情况。方法体外分离培养hDPSCs并进行鉴定。将hDPSCs分别接种于HA-TCP、PGA和PLGA支架上,通过扫描电镜和细胞计数观察细胞在支架上的黏附、增殖及基质分泌情况,采用方差分析对细胞计数结果进行统计学分析。结果 PGA和PLGA支架材料上黏附的细胞数量显著高于HA-TCP组(0.05),且PLGA组高于PGA组。细胞在三种支架材料上的增殖能力无显著差异(0.05),三种支架上均有丰富的基质形成,细胞的生长状态和基质分泌无明显差别,但PGA组可见支架降解产物。结论 PLGA是较适宜hDPSCs黏附和增殖的支架材料。     

The functional expression of human bone-derived cells grown on rapidly resorbable calcium phosphate ceramics

The use of biodegradable bone substitutes is advantageous for alveolar ridge augmentation, since it avoids second-site surgery for autograft harvesting. This study examines the effect of novel, rapidly resorbable calcium phosphates on the expression of bone-related genes and proteins by human bone-derived cells (HBDC) and compares this behavior to that of tricalciumphosphate (TCP). Test materials were alpha-TCP, and four materials which were created from beta-Rhenanite and its derivatives: R1-beta-Rhenanite (CaNaPO(4)); R1/M2 composed of CaNaPO(4) and MgNaPO(4); R1+SiO(2) composed of CaNaPO(4) and 9% SiO(2) (wt%); and R17-Ca(2)KNa(PO(4))(2). HBDC were grown on the substrata for 3, 5, 7, 14 and 21 days, counted and probed for various mRNAs and proteins (Type I collagen, osteocalcin, osteopontin, osteonectin, alkaline phosphatase and bone sialoprotein). All substrata supported continuous cellular growth for 21 days. At day 21, surfaces of R1+SiO(2) and R17 had the highest number of HBDC. At 14 and 21 days, cells on R1 and on R1+SiO(2) displayed significantly enhanced expression of all osteogenic proteins. Since all novel calcium phosphates supported cellular proliferation together with expression of bone-related proteins at least as much as TCP, these ceramics can be regarded as potential bone substitutes. R1 and R1+SiO(2) had the most effect on osteoblastic differentiation, thus suggesting that these materials may possess a higher potency to enhance osteogenesis than TCP.     

Effect of procyanidolic oligomers on cultured mesenchymal cells. I. Effect on attachment, proliferation and detachment of cells

The effect of procyanidolic oligomers (OPC) was studied on mesenchymal cells in culture: human skin fibroblasts (FB) and porcine aorta smooth muscle cells (CML). In presence of OPC part of the freshly seeded FB did not attach. There was no significant effect on the attachment of CML. Proliferation of FB was also slowed down in a dose-dependent manner. Proliferation of CML-s was also decreased, but much less than for FB-s. The detachment of the cells was also studied by adding trypsin to previously attached cells. Detachment of FB-s was strongly inhibited in presence of OPC in a dose-dependent manner. Much less effect was seen on CML. It appears therefore that OPC may interact with some components of the FB cell membrane and modify the attachment, proliferation and detachment of these cells. The only cell kinetic parameter significantly influenced by OPC for CML was their rate of proliferation. This may be due to the different constitution of the CML cell surface as compared to the FB cell surface.     

聚醚醚酮基骨科植入物的改性技术研究

目的 结合聚醚醚酮（PEEK）、聚乳酸（PLA）和硫酸软骨素锶（SrCS）各自的优势,制备出易加工、与松质骨力学性能匹配且兼具生物活性的聚醚醚酮基生物材料。方法 将熔融的PLA充分地填充在PEEK二维编织结构孔隙中,通过热压制备PLA/PEEK复合材料,并对其表面形貌和结构、结晶度、宏观和微观力学强度进行研究。随后在最优力学性能的PLA/PEEK复合材料中引入SrCS并使其与细胞共培养,通过MTT法评价细胞在复合材料上的增殖情况。结果 在155℃和10 MPa的热压条件下,PLA与PEEK细丝结合致密并形成了互锁结构,所得PLA/PEEK复合材料具有与松质骨相似的抗断裂性能、弹性模量和多尺度力学特性。SrCS的引入对复合材料的力学特性进行了微调,并显著提升其生物活性。结论 PLA和SrCS的引入可以有效地改善PEEK基材料的可加工性和生物活性,且结构互锁的PLA/PEEK复合材料具有仿松质骨的力学特性,因此SrCS/PLA/PEEK复合材料有潜力作为新型的骨科修复材料。     

Mechanisms of magnesium-stimulated adhesion of osteoblastic cells to commonly used orthopaedic implants

Poor cell adhesion to orthopaedic and dental implants may result in implant failure. Cellular adhesion to biomaterial surfaces primarily is mediated by integrins, which act as signal transduction and adhesion proteins. Because integrin function depends on divalent cations, we investigated the effect of magnesium ions modified bioceramic substrata (Al(2)O(3)-Mg(2+)) on human bone-derived cell (HBDC) adhesion, integrin expression, and activation of intracellular signalling molecules. Immunohistochemistry, flow cytometry, cell adhesion, cell adhesion blocking, and Western blotting assays were used. Our findings demonstrated that adhesion of HBDC to Al(2)O(3)-Mg(2+) was increased compared to on the Mg(2+)-free Al(2)O(3). Furthermore, HBDC adhesion decreased significantly when the fibronectin receptor alpha5beta1- and beta1-integrins were blocked by functional blocking antibodies. HBDC grown on the Mg(2+)-modified bioceramic expressed significantly enhanced levels of beta1-, alpha5beta1-, and alpha3beta1-integrins receptors compared to those grown on the native unmodified Al(2)O(3). Tyrosine phosphorylation of intracellular integrin-dependent signalling proteins as well as the expression of key signalling protein Shc isoforms (p46, p52, p66), focal adhesion kinase, and extracellular matrix protein collagen type I were significantly enhanced when HBDC were grown on Al(2)O(3)-Mg(2+) compared to the native Al(2)O(3). We conclude that cell adhesion to biomaterial surfaces is probably mediated by alpha5beta1- and beta1-integrin. Cation-promoted cell adhesion depends on 5beta1- and beta1-integrins associated signal transduction pathways involving the key signalling protein Shc and results also in enhanced gene expression of extracellular matrix proteins. Therefore, Mg(2+) supplementation of bioceramic substrata may be a promising way to improve integration of implants in orthopaedic and dental surgery.     

Effects of commonly used immunosuppressants on graft-derived fibroblasts

In acute rejection of transplanted organs intragraft fibroblasts increase their production of hyaluronan. Hyaluronan has strong water binding capacity and an increased tissue content of hyaluronan thus contributes to the development of interstitial oedema. The present study examined the effects of commonly used immunosuppressants (prednisolone, cyclosporin, tacrolimus, mycophenolic acid and sirolimus) on fibroblast proliferation, hyaluronan production and cell surface receptor expression. Fibroblasts isolated from rejecting tissue and from normal, non-transplanted tissue were studied in parallel. All substances investigated, except tacrolimus, were found to affect fibroblasts in one way or another. The most striking effect was the almost total inhibition of fibroblast proliferation in the presence of mycophenolic acid. Cyclosporin reduced the proliferation by about 50% and prednisolone had an inhibiting effect on hyaluronan production (50% reduction). These effects were observed on fibroblasts isolated from rat cardiac allografts undergoing rejection as well as on fibroblasts obtained from normal heart tissue. In contrast, sirolimus was found to stimulate the proliferation of fibroblasts from rejecting tissue (100% increase), but not that of normal fibroblasts. The majority of the fibroblasts expressed the hyaluronan receptor CD44, with a more intense expression in cultures of fibroblasts derived at rejection. None of the immunosuppressants affected the staining pattern (number of positive cells or intensity). The inhibitory effects of prednisolone, cyclosporin and mycophenolic acid on fibroblasts may contribute to the overall beneficial effects of these drugs when used for prevention or treatment of rejection.     

Partial characterization of a bone-derived chemotactic factor for tumor cells.

Medium that has been bathing organ cultures of resorbing bone contains a factor that is chemotactic for cultured Walker carcinosarcoma cells, as assayed by the Boyden chamber technique. There is a positive correlation between the chemotactic activity released by the resorbing bones and the extent of resorption as measured by release of previously incorporated 45Ca from the bones. Generation of the chemotactic factor occurs independent of the humoral mediator of bone resorption. The tumor cell chemotactic factor has a steep dose-response curve, with a fall from maximal to minimal activity extending over a four-fold dilution. The chemotactic activity is stable to heating and has an estimated molecular weight of 6000 daltons, as determined by gel filtration chromatography and retention of activity following dialysis. The chemotactic activity has been distinguished from the tumor cell chemotactic factor derived from the fifth component of complement because the former is not inactivated by antiserum to C5 and because it is chemotactic for EL-4 lymphoma cells, whereas the latter is not chemotactically active for these cells.     

人胎骨髓MSCs的分离鉴定及其向肝细胞样细胞的分化

目的: 分离鉴定人胎骨髓中的间质干细胞(MSCs),探索其体外培养的生物学特性，并在化学因子作用下诱导其向肝细胞分化。方法: 利用细胞差速贴壁生长特性分离纯化人胎骨髓MSCs；利用流式细胞仪检测其细胞周期和表面标志；添加常规诱导液诱导其向脂肪、成骨方向分化;采用DMSO、β-Me和5-aza联合预诱导24 h,换用H-DMEM和 rh-HGF正式诱导人胎骨髓MSCs向肝细胞分化，并从形态学和特异性细胞化学染色等方面加以鉴定。结果: 从人胎骨髓中成功分离、纯化得到MSCs，P4代MSCs有92.3%的细胞处于G₀/G₁期；P5代MSCs有96.1%的细胞处于G₀/G₁期。流式细胞仪检测P3代MSCs结果显示：人胎骨髓MSCs表达CD29、CD44、CD105和CD106 ,不表达造血细胞标志CD34、CD45,不表达与GVHD相关的HLA-DR、CD80、CD86、CD40、CD40L。在经典的诱导条件下，人胎骨髓MSCs可快速向脂肪及成骨细胞分化；在上述诱导条件下，人胎肝MSCs可分化为类肝细胞,表达特异性抗原甲胎蛋白（AFP）和白蛋白（ALB）。结论: 人胎骨髓中含有丰富的MSCs,人胎骨髓来源的MSCs具有较强的多向分化潜能，经DMSO、β-Me和5-aza联合预诱导及rh-HGF、nictinion等化学因子的作用，易向肝细胞样细胞分化，且免疫原性弱,是组织工程（生物型人工肝）的较为理想的种子细胞。     

The effect of polymeric chemistry on the expression of bone-related mRNAs and proteins by human bone-derived cells in vitro

This study used human bone-derived cells (HBDC) grown on two defined polymeric substrata to examine the effect of substrata chemistry on the expression of mRNAs and proteins characteristic of the osteoblastic phenotype. The growth profile of cells grown on tissue culture polystyrene (TCP) was exponential whereas for those seeded on polyethyleneterephthalate (PET) there was a pronounced lag period before cellular multiplication. The temporal expression pattern of mRNAs in HBDC cultured on TCP was similar to that of cells on PET. On TCP, the levels of several mRNAs peaked at day 4, as cellular proliferation slowed. In contrast, the induction in mRNA levels in cells grown on PET corresponded to maximum mitotic activity. There appears to be sequential cascade in protein expression in cells grown on TCP with overlapping peaks of thrombospondin (Tsp), osteocalcin (OC) and osteopontin (OP) expression. In contrast, peak intracellular protein expression levels for Tsp, OC and OP did not overlap when cells were grown on PET.     

常用3种光固化复合树脂材料修复楔状缺损离体牙的组织学评价

背景：延长口腔生物材料的使用寿命,是当今口腔治疗学与材料学的关注点。 目的：评价3种光固化复合树脂修复牙体楔状缺损的效果。 方法：在体外模型中对离体牙进行统一造模,扫描电子显微镜观察3MZ350树脂、3M流体树脂及P90树脂3种修复材料充填后材料与牙体的结合界面。 结果与结论：3种材料都显示了与牙体组织基本良好的结合性能,3MZ350树脂微裂隙多出现在粘接剂层,可有空泡出现;P90组树脂材料侧有断裂纹;流体树脂微裂隙很小,这与其流动性有关系。说明3种材料都可以直接充填,良好的边缘封闭性能的粘接剂可有效减少复合树脂-牙齿界面的微渗漏,增加树脂修复的成功率。关键词：复合树脂;楔状缺损;光固化;结合界面;扫描电子显微镜 doi:10.3969/j.issn.1673-8225.2012.16.023     

The effect of different titanium and hydroxyapatite-coated dental implant surfaces on phenotypic expression of human bone-derived cells

Roughened titanium (Ti) surfaces have been widely used for dental implants. In recent years, there has been the tendency to replace Ti plasma-sprayed surfaces by sandblasted and acid-etched surfaces in order to enhance osseous apposition. Another approach has been the utilization of hydroxyapatite (HA)-coated implants. This study examines the effect of two roughened Ti dental implant surfaces on the osteoblastic phenotype of human bone-derived cells (HBDC) and compares this behavior to that for cells on an HA-coated surface. Test materials were an acid-etched and sandblasted Ti surface (Ti-DPS), a porous Ti plasma-sprayed coating (Ti-TPS), and a plasma-sprayed porous HA coating (HA). Smooth Ti machined surfaces served as control (Ti-ma). HBDC were grown on the substrata for 3, 7, 14, and 21 days, counted and probed for various bone-related mRNAs and proteins (type I collagen, osteocalcin, osteopontin, osteonectin, alkaline phosphatase, and bone sialoprotein). All dental implant surfaces significantly affected cellular growth and the temporal expression of an array of bone-related genes and proteins. HA-coated Ti had the most effect on osteoblastic differentiation inducing a greater expression of an array of osteogenic markers than recorded for cells grown on Ti-DPS and Ti-TPS, thus suggesting that the HA-coated surface may possess a higher potency to enhance osteogenesis. Furthermore, Ti-DPS surfaces induced greater osteoblast proliferation and differentiation than Ti-TPS.     

F+ ion implantation induced cell attachment on intraocular lens.

Cell attachment on the polymethylmethacrylate (PMMA) intraocular lens (IOL) was studied by ion implantation. F+ ion implantation was performed at an energy of 80 keV with fluences ranging from 5 x 10(12) to 5 x 10(15) ions/cm2 at room temperature. The cell attachment tests gave interesting results in that the number of the platelets, the neutral granulocytes, and the macrophages adhering on the surface of the IOLs was reduced significantly after F+ ion implantation. The optimal fluence was about 3 x 10(14) to 4 x 10(14) ions/cm2. The hydrophobicity imparted to the surface was also monitored. At the same time, no appreciable change in the tensile strength and the optical transmittance of the implanted samples was observed. X-ray photoelectron spectroscopy (XPS) and fourier transfer infrared (FTIR) analysis showed that F+ ion implantation caused the cleavage of some pendants, the oxidation of the surface, and the formation of some new F-containing groups. These results were responsible for the cell attachment changes.