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     

Photofunctionalization and non-thermal plasma activation of titanium surfaces

Clinical Oral Investigations - The aim of this study was to compare UV light and non-thermal plasma (NTP) treatment regarding the improvement of physical material characteristics and cell reaction...     

Immunohistochemical study of bone sialoprotein and osteopontin in healthy and diseased root surfaces

BACKGROUND: Periodontal disease is marked by inflammation and damage to tooth-supporting tissues. In particular, damage occurs to factors present in cementum that are thought to have the ability to influence the regeneration of surrounding tissues. Bone sialoprotein and osteopontin are major non-collagenous proteins in mineralized connective tissues associated with precementoblast chemo-attraction, adhesion to the root surface, and cell differentiation. The purpose of this investigation was to determine whether the expression and distribution of bone sialoprotein and osteopontin on root surfaces affected by periodontitis are altered compared to healthy, non-diseased root surfaces. METHODS: Thirty healthy and 30 periodontitis-affected teeth were collected. Following fixation and demineralization, specimens were embedded in paraffin, sectioned, and exposed to antibodies against bone sialoprotein and osteopontin. Stained sections were assessed using light microscopy. RESULTS: Bone sialoprotein was not detected in the exposed cementum (absence of overlying periodontal ligament) of diseased teeth. In most areas where the periodontal ligament was intact, bone sialoprotein was detected for healthy and diseased teeth. For teeth reactive for bone sialoprotein, the matrix of the cementum just below the periodontal ligament was moderately stained. A similar immunoreactivity pattern for osteopontin was observed. CONCLUSIONS: The absence of bone sialoprotein and osteopontin staining along exposed cementum surfaces may be due to structural and compositional changes in matrix components associated with periodontal disease. This may influence the ability for regeneration and new connective tissue attachment onto previously diseased root surfaces.     

Characterization of sterilized CP titanium implant surfaces

Surface analysis techniques and in vitro biologic assays were used to characterize sterilized commercially pure titanium surfaces. Significant surface alterations were observed following sterilization treatments. These alterations led to decreased fibroblast cell attachment and altered cellular spreading phenomena compared to nonsterilized control surfaces.     

Effect of biomimetic deposition on anodized titanium surfaces

Surface energy and hydrophilicity of implant surfaces have been known to play an important role in subsequent cellular responses on the implant surface. The aim of the present study was to evaluate the effects of biomimetic deposition of anodized surfaces on surface wettability, surface energy, and osteoblast responses. Ti discs with 2 different surface topographies (machined and anodized) were immersed in Hanks' balanced salt solution (HBSS) and modified simulated body fluid (SBF) solution for 2 weeks at physiologic conditions of 37 °C, initial pH of 7.4, and p(CO(2)) of 0.05 atm. Scanning electron microscopic (SEM) observation and energy-dispersive spectroscopic (EDS) microanalysis showed the deposition of calcium phosphate (CaP) onto anodized Ti surfaces immersed in modified SBF. Surface energy, surface wettability, and osteoblast responses, including cell attachment capacity, cell proliferation rate, and cell differentiation level, significantly increased on anodized Ti surfaces immersed in modified SBF. The effects of biomimetic deposition with modified SBF on physiochemical surface characteristics and cell biological responses were greater on anodized surfaces than on machined surfaces. These results indicate that biomimetic deposition with effective SBF may enhance the interaction between anodized Ti surfaces and their biological environment, consequently improving bone healing of dental Ti implants.     

Cleaning and heat-treatment effects on unalloyed titanium implant surfaces

This study tested the following hypotheses: (1) acid-cleaned and passivated unalloyed titanium implants have higher surface energies (which are considered desirable for bone implants) than ethanol-cleaned titanium; (2) higher temperatures of heat treatment of unalloyed titanium result in higher surface energies; and (3) these changes can be related to changes in surface composition and roughness. Thus, unalloyed titanium specimens were either acid-cleaned and passivated (CP) or ethanol-cleaned (Et). Each set was then divided into 3 groups and heat-treated for 1 hour at 316 degrees C (600 degrees F), 427 degrees C (800 degrees F), and 538 degrees C (1,000 degrees F), respectively. Surface roughness values for each of these groups were determined using atomic force microscopy, while surface compositions were determined using Auger electron, x-ray photoelectron, and Raman spectroscopic techniques. Surface energies were estimated using a 2-liquid geometric mean technique and correlated with surface roughness, elemental composition, and elemental thickness. The CP surfaces were slightly rougher than the Et specimens, which had greater oxide thickness and hydrocarbon presence. The surface oxides were composed of TiO2, Ti2O3, and possibly titanium peroxide; those heat-treated at 427 degrees C or above were crystalline. The CP specimens had carbonaceous coverage that was of a different composition from that on Et specimens. The CP specimens had significantly higher surface energies, which showed statistically significant correlations with oxide thickness and carbonaceous presence. In conclusion, ethanol cleaning of unalloyed titanium dental implants may not provide optimal surface properties when compared to cleaning with phosphoric acid followed by nitric acid passivation.     

Protein adsorption and osteoblast responses to heat-treated titanium surfaces

The clinical success of dental implants is governed in part by surface properties of implants and their interactions with the surrounding tissues. The objective of this study was to investigate the effect of heat-treated titanium (Ti) surfaces on protein adsorption and osteoblast responses in vitro. The passivated Ti samples used in this study were either nonheat-treated or heat-treated at 750 degrees C for 90 minutes. Using x-ray diffraction analyses, no oxide peaks were observed on the nonheat-treated surfaces, suggesting an amorphous oxide. Crystalline rutile TiO2 peaks were observed on the heat-treated Ti surfaces. The contact angles of water on heat-treated Ti surfaces (32.0 +/- 2.5 degrees) were statistically lower compared with the nonheat-treated Ti surfaces (47.7 +/- 2.3 degrees). In addition, the mean albumin concentration on the nonheat-treated Ti surfaces (3.57 +/- 0.33 micrograms/mL) was observed to be significantly different from the mean albumin concentration on heat-treated Ti surfaces (2.25 +/- 0.26 micrograms/mL). In the presence of an osteoblast precursor cell line, significantly different hexosaminidase activity, protein production, and alkaline phosphatase activity were observed for cells grown on heat-treated Ti surfaces compared with nonheat-treated Ti surfaces.     

纯钛表面含丹参钙磷涂层的制备及表征

目的研究在粗化纯钛表面采用仿生钙磷涂层法组装丹参的可行性。方法钛片表面粗化,采用仿生钙磷涂层技术,在纯钛表面建立不同浓度含丹参涂层,通过场发射扫描电子显微镜（FSEM）、傅立叶红外光谱（FTIR）、X射线衍射仪（XRD）和元素分析仪观察涂层晶体形貌、化学结构及组成,通过细胞培养验证其生物活性。结果 FSEM证实低浓度的丹参对钙磷涂层晶体的生长影响不大,高浓度的丹参对钙磷涂层晶体的生长影响较大;XRD和FTIR表明丹参并没有破坏涂层的化学结构和组成;元素分析结果表明实验组的碳元素质量比高于对照组;与对照组相比,实验组能够明显促进碱性磷酸梅（ALP）的表达。结论采用仿生钙磷涂层法可在粗化纯钛表面成功构建含丹参涂层。     

微弧氧化钛表面对成骨细胞形态及细胞骨架的影响

目的 研究微弧氧化钛表面对成骨细胞形态及细胞骨架的影响。方法 将直径15 mm、厚度1 mm的纯钛片根据表面处理方法不同分为4组：机械打磨（G）组、喷砂（SB）组、打磨微弧氧化（GMAO）组和喷砂微弧氧化（SBMAO）组。采用扫描电子显微镜（SEM）、激光共聚焦显微镜（LSCM）研究钛片表面成骨细胞生长情况及细胞骨架的改变。结果 成骨细胞接种12 h后,各组细胞均沿钛片表面铺展开,且GMAO组和SBMAO组细胞覆盖于火山口状微孔上。各组肌动蛋白纤维清晰可见,GMAO组和SBMAO组肌动蛋白纤维平行排列,汇聚成束伸向微孔内。结论 微弧氧化后的钛表面可以影响成骨细胞铺展的形态及细胞骨架的排列。     

Participation of extracellular signal‐regulated kinases 1/2 in osteoblast and adipocyte differentiation of mesenchymal stem cells grown on titanium surfaces

Osteoblasts and adipocytes coexist in the implantation site and affect the process of titanium (Ti) osseointegration. As extracellular signal‐regulated kinases 1/2 (ERK1/2) are involved in osteogenesis and adipogenesis, the aim of our study was to investigate if the effects of Ti surface topography on osteoblast and adipocyte differentiation are modulated by ERK1/2. The experiments were conducted based on the effect of the ERK1/2 inhibitor, PD98059, on mesenchymal stem cells (MSCs) grown under osteogenic and adipogenic conditions on Ti with nanotopography (Ti‐Nano) or on machined Ti (Ti‐Machined). The results showed that, in general, ERK1/2 inhibition favored osteoblast and adipocyte differentiation of MSCs grown on Ti‐Machined. In MSCs grown on Ti‐Nano, ERK1/2 inhibition upregulated the expression of alkaline phosphatase and osteocalcin and reduced extracellular matrix mineralization. In terms of adipocyte differentiation, ERK1/2 inhibition elicited similar MSC responses to Ti‐Nano and Ti‐Machined, upregulating gene expression of adipocyte markers without affecting lipid accumulation. Our results indicate that, under osteogenic and adipogenic conditions, the responses of MSCs to Ti surface topography in terms of osteogenesis and adipogenesis are dependent on ERK1/2. Thus, a precise modulation of ERK1/2 expression and activity induced by surface topography could be a good strategy to drive the process of implant osseointegration.     

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.     

Interaction of chlorhexidine with smooth and rough types of titanium surfaces

BACKGROUND: Chlorhexidine (CHX) digluconate exerts plaque inhibitory efficacy in the natural dentition environment due to a superior degree of persistence at the tooth surface. The purpose of the present study was to assess the interaction of CHX with titanium surfaces to estimate its antiplaque potential in the peri-implant environment. METHODS: Saliva-coated machined smooth (S) and sand-blasted acid-etched rough (R) titanium disks were soaked in either 0.1% or 0.2% CHX solution. After 24 hours, CHX amounts that were adsorbed, washed out, and desorbed from the titanium surfaces were determined spectrophotometrically at 230 nm. The antibacterial activity of CHX-treated titanium disks was assessed by measuring bacterial inhibition zones on Streptococcus mutans lawns. RESULTS: Titanium disks adsorbed 3% to 8% of the available CHX, which was significantly higher with 0.2% CHX (P<0.001) than with 0.1% CHX and two-fold higher on the R titanium disks compared to S titanium surface (P<0.001). After rinsing with water, 2.2% of the adsorbed CHX was washed out. Over 24 hours, S- and R-type disks released 1.1% and 0.6% of the adsorbed agent, respectively. Larger bacterial inhibition zones were obtained with 0.2% CHX and in R disks compared to S disks. CONCLUSIONS: CHX displayed persistence at the titanium surface. The adsorption level and bacterial growth inhibition were affected by CHX concentration and titanium surface characteristics, with higher levels of adsorption and antibacterial activity with 0.2% CHX and rough titanium surface. The slow CHX release rate suggests persistence of this agent at the titanium-pellicle surface, which can provide a long-term antiplaque effect.     

Integrin mediated attachment of periodontal ligament to titanium surfaces

ObjectiveReducing the force between the implant and the bone by recapitulating a similar matrix has the potential to reduce implant failure. To begin to pursue the goal of creating a periodontal ligament interface between a dental implant and bone, the mechanism of cellular attachment to dental implant surfaces must be characterized.MethodsIn this study we examined the role of integrin receptors in the attachment of periodontal ligament fibroblasts to titanium surfaces utilized on dental implants; those surfaces included smooth polished titanium, acid pickled titanium, ground titanium, sandblasted and acid etched titanium, non-oxidized titanium that has been sandblasted and acid etched, hydroxyapatite coated titanium, titanium plasma sprayed or uncoated titanium. For these studies integrin mediated fibroblast attachment was blocked by the integrin blocking peptide GRGDSP or anti-integrin β1 antibody or a combination of the two. Quantitation of periodontal ligament fibroblast attachment was completed by counting cells on the various implant surfaces after culturing in vitro for 24 h with and without the integrin receptor blockers.ResultsAntibody and peptide treatment significantly reduced the number of fibroblasts cells attached to the various implant surfaces but this effect varied significantly depending on the surface. Moreover, increased levels of peptide further decreased fibroblasts attachment in a dose dependent manner.SignificanceBlocking studies suggest first, that integrin receptors function in periodontal ligament attachment to titanium surfaces and second, that different integrin subunits are important in attachment to a particular surface.     

Red blood cell and platelet interactions with titanium implant surfaces

The influence of the micro-roughened surface, produced by dual acid-etching (DAE) of machined commercially pure titanium, on initial blood cell/implant interactions was investigated by observing the blood components remaining at the implant surface following freeze-fracture of clotted, and fixed, human blood. Glass surfaces were also used for immunolabelling studies to identify fibrin and platelets. The interface comprised predominantly fibrin and red blood cells (RBCs). The difference in distribution of RBCs was statistically significant (P < 0.05) at 10 min of blood/implant contact, but diminished thereafter. Micro-roughened DAE implant surfaces showed, qualitative, more platelets than machined surfaces, while the textured glass surfaces demonstrated increased platelet aggregation. We believe that these early blood cell/implant interactions may play a key role in the osteoconduction stage of peri-implant bone healing response to micro-roughened implants.