Response of rat bone marrow cells to commercially pure titanium submitted to different surface treatments

OBJECTIVES: Alterations in the commercially pure titanium (cpTi) surface may be undertaken to improve its biological properties. The aim of this study is to investigate the biocompatibility of cpTi submitted to different surface treatments. METHODS: The cpTi surfaces were prepared so that machined and blasted surfaces, either acid etched or not, were compared using rat bone marrow cells cultured to differentiated into osteoblast. For attachment evaluation, cells were cultured for 4 and 24h. Cell morphology was evaluated after 3 days. After 7, 14, and 21 days cell proliferation was evaluated. Total protein content and alkaline phosphatase (ALP) activity were evaluated after 14 and 21 days. For bone-like nodule formation, cells were cultured for 21 days. Data were compared by analysis of variance. RESULTS: Cell attachment, cell morphology, cell proliferation, and ALP activity were not affected by surface treatments. Total protein content was reduced by blasted and acid etched surface. Bone-like nodule formation was significantly reduced by blasted, acid etched, and a combination of both blasted and acid etched surfaces. CONCLUSIONS: Based on these results, it can be suggested that cpTi surfaces that were submitted only to machining treatment favor the final event of osteoblastic differentiation of the rat bone marrow cells, evidenced by increased bone-like nodule formation.     

Nitric acid passivation does not affect in vitro biocompatibility of titanium

PURPOSE: In general, both chemical composition and surface features of implants affect cell response. The aim of this study was to evaluate the effect of titanium (Ti) passivation on the response of rat bone marrow cells, considering cell attachment, cell morphology, cell proliferation, total protein content, alkaline phosphatase (ALP) activity, and bonelike nodule formation. MATERIALS AND METHODS: Cells were cultured on both commercially pure titanium (cpTi) and titanium-aluminium-vanadium alloy (Ti-6Al-4V) discs, either passivated or not. For attachment evaluation, cells were cultured for 4 and 24 hours. Cell morphology was evaluated after 4 days. After 7, 14, and 21 days, cell proliferation, total protein content, and ALP activity were evaluated. Bonelike nodule formation was evaluated after 21 days. Data were compared by analysis of variance and the Duncan multiple range test. RESULTS: Cell attachment, cell morphology, cell proliferation, total protein content, ALP activity, and bonelike nodule formation all were unaffected by Ti composition or passivation. DISCUSSION AND CONCLUSION: Although the protocol for passivation used here could interfere with the pattern of ions released from Ti-6Al-4V and cpTi surfaces, the present study did not show any effect of this surface treatment on in vitro biocompatibility of Ti as evaluated by osteoblast attachment, proliferation, and differentiation.     

Osteoblastic differentiation of cultured rat bone marrow cells on hydroxyapatite with different surface topography

Hydroxyapatite (HA) has been used in orthopedic, dental, and maxillofacial surgery as a bone substitute. OBJECTIVE: The aim of this investigation was to study the effect of surface topography produced by the presence of microporosity on the response of the rat bone marrow cells, evaluating: cell attachment, proliferation, total protein content, alkaline phosphatase (ALP) activity, and bone-like nodule formation. METHODS: Cells were cultured on HA discs manufactured by a combination of uniaxial powder pressing and different sintering conditions, with different percentage of microporosity (<5%-HA5, 15%-HA15, and 30%-HA30). For attachment evaluation, cells were cultured for 2 h. Proliferation was evaluated after 7 and 14 days. After 14 days, total protein content and ALP activity were measured. For bone-like nodule formation, cells were cultured for 21 days. Data were compared by ANOVA and Duncan's multiple range test when appropriate. RESULTS: Cell attachment was not affected by surface topography (p=0.37). Proliferation (p=0.001), total protein content (p=0.039), ALP activity (p=0.050), and bone-like nodule formation (p=0.00001) were all significantly decreased by the most irregular surface (HA30). SIGNIFICANCE. These results suggest that initial cell events were not affected by the surface topography of the HA. However, intermediary and final events such as proliferation, protein synthesis, ALP activity, and bone-like nodule formation favored surfaces with a more regular topography, such as that presents in HA with 15% or less of microporosity.     

水热合成法制备羟基磷灰石生物涂层的体内及体外实验研究

目的对水热合成法制备羟基磷灰石生物涂层材料的细胞相容性及动物体内植入体与骨界面的状况进行研究.方法水热合成法制备羟基磷灰石涂层,采用细胞培养和动物实验评估涂层材料的生物相容性.结果成骨细胞在涂层材料表面具有良好的细胞增殖率,碱性磷酸酶活性逐渐增加.植入动物体内1月后即有骨组织与涂层材料结合,3月后骨结合量增加.涂层材料在体内稳定,未见溶解脱落.结论水热合成法制备的羟基磷灰石生物涂层材料具有良好的生物效应.     

Effect of three distinct treatments of titanium surface on osteoblast attachment, proliferation, and differentiation

Cell-titanium interactions are crucial to the clinical success of bone and dental implants. The physico-chemical characteristics of the substrates surface influence osteoblast proliferation, differentiation, and activity as well. The osteoblast behavior was analyzed on three different titanium surfaces: ground with an abrasive 600 grit SiC paper, blasted with alumina particles (65 microm diameter) and alumina blasted followed by a double chemical etch (4% HF+4% HF/8% H2O2). Scanning electron microscopy (SEM) and profilometry showed distinct microtopographies. Ground samples showed parallel-groove orientation. The Al2O3-blasted surface presented the roughest microtopography with aluminum-rich particles incrusted in the titanium surface. Osteoblasts cells from femora of Balb/c mice were seeded onto the substrates tested. Cell morphology and initial attachment were evaluated by SEM. Osteoblasts adhered to and spread on all samples tested. However, on rough surfaces, osteoblasts did not spread completely and acquired a polygonal morphology. Besides, the cell proliferation rate was diminished at the beginning of incubation on rough surfaces. Our results suggest a delay, rather than an impairment, in osteoblast viability and alkaline phosphatase activity when cells are cultured on rough surfaces, inducing a distinct osteoblast phenotype, rather than blocking its activity. At least in the culture conditions used in this work, alumina particles did not affect osteoblast behavior.     

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目的 探讨应用等离子体电解氧化(plasma electrolytic oxidation,PEO)技术处理钛表面后对成骨细胞(人成骨肉瘤Saos-2细胞)早期增殖与分化的影响.方法 应用PEO技术在纯钛表面制备多孔氧化钛陶瓷膜(PEO组),并以钛表面机械抛光处理(机械抛光组)及喷砂酸蚀处理(喷砂酸蚀组)作为对照,应用场发射扫描电镜、粗糙度测试仪分析表面形貌.将Saos-2细胞接种于3组样品表面,通过对样品表面细胞形态、细胞增殖及碱性磷酸酶(ALP)活性的检测,分析3组样品时Saos-2早期生物学行为的影响.结果 应用PEO法可以在纯钛表面形成多孔结构.Saos-2在3组样品表面的黏附与增殖差异无统计学意义(P＞0.05);PEO组的ALP活性高于机械抛光组与喷砂酸蚀组(P＜0.05).结论 PEO处理后的多孔钛表面能促进Saos-2的早期分化功能.     

Osteoblast attachment on titanium disks after laser irradiation

PURPOSE: Osteoblast attachment on titanium surfaces is necessary to achieve new bone formation and osseointegration. The purpose of this study was to examine osteoblast attachment on irradiated titanium disks. MATERIALS AND METHODS: Machined, hydroxyapatite (HA)-coated, sandblasted, and titanium plasma-sprayed (TPS) surfaces were irradiated with either a carbon dioxide (CO2) or an Er,Cr:YSGG laser. A control group of nonirradiated disks was also examined. Osteoblast cultures were cultivated on the titanium disks and examined with scanning electron microscopy. RESULTS: The findings demonstrated that osteoblasts could be grown on all of the surfaces. Pseudopodia and a spread of cells that demonstrated maturation were observed on the lased irradiated titanium disks. CONCLUSIONS: The data show that laser irradiation of titanium surfaces may promote osteoblast attachment and further bone formation.     

Effect of cpTi surface roughness on human bone marrow cell attachment,proliferation, and differentiation

There is general agreement that rough surfaces improve both biologic and biomechanical responses to titanium (Ti) implants. The aim of this investigation was to study the effect of Ti surface roughness on the response of human bone marrow cell culture evaluating: cell attachment, cell proliferation, total protein content, alkaline phosphatase (ALP) activity, and bone-like nodule formation. Cells were cultured on commercially pure titanium (cpTi) discs with fourdifferent average roughnesses (Ra). For attachment evaluation, cells were cultured for 4 h. After 21 days, cell proliferation, total protein content, and ALP activity were evaluated. For bone-like nodule formation, cells were cultured for 28 days. Data were compared by ANOVA and Duncan's multiple range test. Cell attachment was not affected by surface roughness. For cells cultured on Ti with Ra ranging from 0.80 microm to 1.90 microm, proliferation was reduced while total protein content, and ALP activity were increased. There was a non-statistically significant increase of bone-like nodule formation on a surface with Ra near 0.80 microm. These results suggest that for Ti an Ra ranging from 0.80 microm to 1.90 microm would optimize both intermediary and final cellular responses but not affect the initial response, and a smoother surface would not favor any evaluated response.     

纯钛表面吸附硫酸软骨素A对成骨细胞生长的影响

目的 在体外研究纯钛表面吸附硫酸软骨素A(Chondroitin Sulfate—A,CS—A)后对成骨细胞生物学行为的影响。方法 在吸附不同浓度的CS—A钙处理钛片表面培养成骨细胞，检测其生长情况、碱性磷酸酶活性及细胞层钙含量。结果 钙处理钛片在吸附CS—A后促进了成骨细胞的增殖。增加了成骨细胞层钙聚集，促进成骨细胞的矿化，同时还增高了成骨细胞的碱性磷酸酶活性，促进了细胞的分化。结论 钛吸附CS—A后提高了其生物相容性。     

Corrosion resistance and biocompatibility of a new porous surface for titanium implants

Alterations of the commercially pure titanium (cpTi) surface may be undertaken to improve its biological properties. The aim of this study was to investigate the biocompatibility of cpTi when submitted to a new, porous titanium, surface treatment (porous Ti). Five types of surface treatments, namely sintered microspheres porous titanium (porous Ti), titanium plasma spray (TPS), hydroxyapatite (HA), sandblasted and acid etched (SBAE), and resorbable blast medium, sandblasted with hydroxyapatite (RBM) were made. In the experimental methods, the corrosion potentials were measured over time, and then a linear sweep voltammetric analysis measured the polarization resistances and corrosion currents. For biocompatibility evaluation, MG63 osteoblast-like cells were used. Cell morphology, cell proliferation, total protein content, and alkaline phosphatase (ALP) activity were evaluated after 2 h, and after 2, 4 and 7 d. Porous Ti and SBAE showed a better corrosion resistance, with a weak corrosion current and a high polarization resistance, than the other surfaces. Cell attachment, cell morphology, cell proliferation, and ALP synthesis were influenced by the surface treatments, with a significant increase observed of the activity of osteoblast cells on the porous coating (porous Ti). Based on these results, it is suggested that the porous Ti surface has a significantly better biocompatibility than the other surface treatments and an excellent electrochemical performance.     

The Effect of Surface Processing of Titanium Implants on the Behavior of Human Osteoblast‐Like Saos‐2 Cells

Background: The surface qualities of dental implants appear to modulate osteoblasts’ growth and differentiation, affecting bone healing. During manufacturing of implants, the surface quality is affected by industrial processes. Purpose: To examine the effect of manufacturing procedures on the growth and differentiation of human osteoblast‐like cells, Saos‐2. Materials and Methods: Saos‐2 cells were cultured on titanium (Ti) disks. Cell growth was examined using the XTT assay, and cell differentiation was tested by alkaline phosphatase (ALP) activity and osteocalcin secretion. The following variables were examined: roughening of the surface by sandblasting and acid‐etching, aging of the acid used for etching, fluoride modification of the surface, and the type of the packaging material. Results: An inverse relationship was noted between Saos‐2 growth and ALP activity on the tested surfaces. Roughening of the surface tended to decrease cell proliferation and to increase differentiation. Immersion of up to 200 cycles in acid decreased proliferation and increased differentiation. Cells grown on fluoride‐modified surfaces exhibited more ALP activity as compared to the unmodified surfaces. No difference was noted between the three packaging materials tested. Conclusions: The data suggests that industrial processes may affect the behavior of osteoblast‐like cells around titanium implants and should be monitored carefully by bioassays.     

Effect of recombinant human bone morphogenetic protein-7 (rhBMP-7) on the viability, proliferation and differentiation of osteoblast-like cells cultured on a chemically modified titanium surface

Aim: The aim of the present study was to assess the influence of the chemical characteristics and roughness of titanium surfaces on the viability, proliferation and differentiation of osteoblast-like cells cultured in a medium supplemented with recombinant human bone morphogenetic protein-7 (rhBMP-7).
Material and methods: Osteo-1 cells were grown on titanium disks presenting with the following surfaces: (1) machined, (2) coarse grit-blasted and acid-attacked (SLA) and (3) chemically modified SLA (SLAmod) in the absence or presence of 20 ng/ml rhBMP-7 in culture medium. The viability and number of osteo-1 cells were evaluated after 24 h. Analyses of total protein content (TP) and alkaline phosphatase (AP) activity at 7, 14 and 21 days, collagen content at 7 and 21 days and mineralized matrix formation at 21 days were performed.
Results: Cell viability ( P =0.5516), cell number ( P =0.3485), collagen content ( P =0.1165) and mineralized matrix formation ( P =0.5319) were not affected by the different surface configurations or by the addition of rhBMP-7 to the medium. Osteo-1 cells cultured on SLA surfaces showed a significant increase in TP at 21 days. The ALPase/TP ratio ( P =0.00001) was affected by treatment and time.
Conclusion: The results suggest that the addition of rhBMP-7 to the culture medium did not exert any effect on the viability, proliferation or differentiation of osteoblast-like cells grown on the different surfaces tested. All titanium surfaces analyzed allowed the complete expression of the osteoblast phenotype such as matrix mineralization by osteo-1 cells.     

Titanium nitride oxide coating on rough titanium stimulates the proliferation of human primary osteoblasts

Objectives: Titanium is widely used in contemporary endosseous implantology and there is considerable thrust to further promote osseointegration by implant surface modifications. The aim of this study was to evaluate the effect of a titanium–nitride–oxide (TiNOx) coating on commercially pure microroughened titanium by assessing the proliferation and differentiation of human primary osteoblasts. Materials and methods: Cell proliferation, gene expression, alkaline phosphatase activity, osteoprotegerin and osteocalcin secretion were analyzed for a time course of 3 weeks, with or without additional stimulation by 1.25(OH)₂ vitamin D₃ 100 nM. Results: A 1.5‐fold increase in the proliferation rate of cells grown on TiNOx‐coated titanium as compared with uncoated surfaces was observed. SEM views indicated that the cells' normal morphology with their numerous extensions was maintained. The differentiation process on the TiNOx surface was only affected to a minor degree and translated into a slight delay in osteoblast maturation when compared to uncoated titanium. Conclusion: Pending confirmation of these results in vivo, TiNOx coatings could potentially accelerate and enhance osseointegration. To cite this article:
Durual S, Pernet F, Rieder P, Mekki M, Cattani‐Lorente M, Wiskott H. W. A. Titanium nitride oxide coating on rough titanium stimulates the proliferation of human primary osteoblasts.
Clin. Oral Impl. Res. 22 , 2011; 552–559
doi: 10.1111/j.1600‐0501.2010.02033.x     

Effects of fluoride-modified titanium surfaces on osteoblast proliferation and gene expression

PURPOSE: The objective of this study was to test the hypothesis that fluoride-modified titanium surfaces would enhance osteoblast differentiation. Osteoblast growth on a moderately rough etched fluoride-modified titanium surface (alteration in cellular differentiation) was compared to osteoblast growth on the same surface grit-blasted with titanium dioxide. The potential role of nanometer-level alterations on cell shape and subsequent differentiation was then compared. MATERIALS AND METHODS: Human embryonic palatal mesenchymal (HEPM) cultures were incubated on the respective surfaces for 1, 3, and 7 days, followed by analysis for cell proliferation, alkaline phosphatase (ALP) -specific activity, and mRNA steady-state expression for bone-related genes (ALP, type I collagen, osteocalcin, bone sialoprotein [BSP] II, Cbfa1, and osterix) by real-time polymerase chain reaction (PCR). RESULTS: The different surfaces did not alter the mRNA expression for ALP, type I collagen, osterix, osteocalcin, or BSP II. However, Cbfa1 expression on the fluoride-modified titanium surface was significantly higher (P < .001) at 1 week. The number of cells on this surface was 20% lower than the number of cells on the surface TiO2-blasted with 25-microm particles but not significantly different from the number of cells on the surface TiO2-blasted with 125-microm particles. Cells grown on all the titanium surfaces expressed similar levels of ALP activity. CONCLUSIONS: The results indicated that a fluoride-modified surface topography, in synergy with surface roughness, may have a greater influence on the level of expression of Cbfa1 (a key regulator for osteogenesis) than the unmodified titanium surfaces studied.     

Osteoblast response to titanium surfaces functionalized with extracellular matrix peptide biomimetics

Objective: Functionalizing surfaces with specific peptides may aid osteointegration of orthopedic implants by favoring attachment of osteoprogenitor cells and promoting osteoblastic differentiation. This study addressed the hypothesis that implant surfaces functionalized with peptides targeting multiple ligands will enhance osteoblast attachment and/or differentiation. To test this hypothesis, we used titanium (Ti) surfaces coated with poly‐l ‐lysine‐grafted polyethylene glycol (PLL‐g‐PEG) and functionalized with two peptides found in extracellular matrix proteins, arginine–glycine–aspartic acid (RGD) and lysine–arginine–serine–arginine (KRSR), which have been shown to increase osteoblast attachment. KSSR, which does not promote osteoblast attachment, was used as a control. Materials and methods: Sandblasted acid‐etched titanium surfaces were coated with PLL‐g‐PEG functionalized with varying combinations of RGD and KRSR, as well as KSSR. Effects of these surfaces on osteoblasts were assessed by measuring cell number, alkaline phosphatase‐specific activity, and levels of osteocalcin, transforming growth factor beta‐1 (TGF‐β1), and PGE₂. Results: RGD increased cell number, but decreased markers for osteoblast differentiation. KRSR alone had no effect on cell number, but decreased levels of TGF‐β1 and PGE₂. KRSR and RGD/KRSR coatings inhibited osteoblast differentiation vs. PLL‐g‐PEG. KSSR decreased cell number and increased osteoblast differentiation, indicated by increased levels of osteocalcin and PGE₂. Conclusions: The RGD and KRSR functionalized surfaces supported attachment but did not enhance osteoblast differentiation, whereas KSSR increased differentiation. RGD decreased this effect, suggesting that multifunctional peptide surfaces can be designed that improve peri‐implant healing by optimizing attachment and proliferation as well as differentiation of osteoblasts, but peptide combination, dose and presentation are critical variables. To cite this article:
Bell BF, Schuler M, Tosatti S, Textor M, Schwartz Z, Boyan BD. Osteoblast response to titanium surfaces functionalized with extracellular matrix peptide biomimetics.
Clin. Oral Impl. Res. 22 , 2011; 865–872.
doi: 10.1111/j.1600‐0501.2010.02074.x     

Formation of mineralizing osteoblast cultures on machined, titanium oxide grit-blasted, and plasma-sprayed titanium surfaces

Altering osseous responses at implant surfaces to enhance bone is a current goal of clinical therapy. Cell culture may be used to investigate surface-dependent responses of bone-forming cells. In this report, the ability of primary fetal bovine mandibular osteoblast cultures to form a mineralizing matrix on machined, titanium plasma-sprayed, and titanium oxide grit-blasted surfaces has been compared. Immunohistochemical markers associated with bone formation were used to define the differentiated state of the formed matrix using qualitative light microscopy, and von Kossa staining was used to demonstrate the presence of mineralization within this matrix. Compared to either titanium oxide grit-blasted or machined surfaces, titanium plasma-sprayed surfaces displayed a unique pattern of mineralized matrix formation. Scanning electron microscopy further revealed that each surface accumulated unique organic and inorganic deposits during matrix formation, suggesting that surface-dependent physicochemical and biochemical conditioning of implant surfaces takes place. Surface topographic features of commercially pure titanium substrates can alter cultured osteoblast extracellular matrix formation and mineralization. Similar molecular and cellular assessment of in vivo responses to implant surface topography may contribute to improved engineering of endosseous implants.     

微弧氧化钛基种植材料对成骨细胞早期黏附的影响

目的：检测纯钛种植体材料微弧氧化（microarc oxidation，MAO）表面改性后的成骨细胞生物相容性，探讨微弧氧化技术在钛种植体表面改性中的价值。方法：在纯钛种植体材料表面用微弧氧化法制备羟基磷灰石陶瓷薄膜，将MAO改性钛种植体材料作为实验组Ⅰ，将纯钛表面阳极氧化改性处理的种植体材料作为实验组Ⅱ．并设立对照组Ⅰ（纯钛种植体材料）和对照组Ⅱ（即细胞直接生长在培养板上），分别进行扫描电镜（SEM）、能谱分析（EDS）、X射线衍射图谱分析（XRD）等检测，比较成骨细胞的黏附水平，对数据采用SPSS11．0统计软件包进行单因素方差分析。结果：MAO改性后生成粗糙、多孔的陶瓷薄膜层，与处理前电解液成分相比，钙磷比无显著改变。MAO改性钛组黏附的细胞密度显著高于其他组（P〈0．01），而对照组Ⅰ、Ⅱ的细胞密度无显著差异（P〉0．05）。结论：与阳极氧化表面改性材料相比，该钛基微弧氧化薄膜层能够显著促进成骨细胞的附着，具有良好的细胞相容性。     

Effects of dissolved calcium and phosphorous on osteoblast responses

The dissolution behavior of hydroxyapatite (HA) and its effect on the initial cellular response is of both fundamental and clinical importance. In this study, plasma-sprayed HA coatings were characterized by X-ray diffraction and Fourier transform infrared spectroscopy (FTIR). Calcium (Ca) and inorganic phosphorous (Pi) ions released from plasma-sprayed HA coatings within 3 weeks were measured by flame atomic absorption and colorimetrically molybdenum blue complex, respectively. To investigate the effect of dissolution of HA coatings on osteoblast response, additional Ca and Pi were added into the cell culture media to simulate the dissolution concentrations. Human embryonic palatal mesenchyme cells, an osteoblast precursor cell line, were used to evaluate the biological responses to enhanced Ca and Pi media over 2 weeks. Osteoblast differentiation and mineralization were measured by alkaline phosphatase-specific assay and 1,25 (OH)2 vitamin D3 stimulated osteocalcin production. The coatings exhibited an HA-type structure. FTIR indicated the possible presence of carbonates on the coatings. A dissolution study indicated a continual increase in Ca and Pi over time. In the cell culture study, enhanced osteoblast differentiation occurred in the presence of additional Ca concentration in the cell culture media. However, additional Pi concentration in the cell culture media was suggested to slow down osteoblast differentiation and mineralization.     

Rat bone marrow cell response to titanium and titanium alloy with different surface roughness

In general, cell response is affected by both chemical composition and surface roughness of implant materials. The aim of this study was to evaluate the effect of titanium (Ti) chemical composition and surface roughness on the response of rat bone marrow cells, examining cell attachment, cell proliferation, total protein content, alkaline phosphatase (ALP) activity, and bone-like nodule formation. Cells were cultured on both commercially pure titanium (cpTi) and titanium-6-aluminum-4-vanadium alloy (Ti-A) discs with four different average roughnesses (Ra). For attachment evaluation, cells were cultured for 2 h. After 14 days, cell proliferation, total protein content, and ALP activity were evaluated. Bone-like nodule formation was evaluated after 21 days. Data were compared by anova and Duncan's multiple range test when appropriate. Cell attachment and total protein content were affected by neither Ti chemical composition (P = 0.201, and P = 0.639, respectively) or surface roughness (P = 0.972, and P = 0.660, respectively). Proliferation, ALP activity, and bone-like nodule formation were affected only by Ti chemical composition (P = 0.0001, P = 0.064, and P = 0.0001, respectively). These results suggest that cpTi would optimize osteoblastic differentiation by rat bone marrow cells, including reduced cell proliferation, and increased ALP activity and bone-like nodule formation, while surface roughness, within the Ra parameters used, would not affect significantly the rat bone marrow cell response.