Enhanced Osteoblast Response Research Articles

Effect of Dehydrothermal Treatment on the Structure and Properties of a Collagen-Based Heterogeneous Bilayer Membrane

The use of the guided bone regeneration technique in oral surgery has been proven to be an effective method for repairing alveolar bone defects. However, the success of this technique heavily relies on the barrier membrane used. Collagen is the most frequently used raw material to fabricate a resorbable barrier membrane, which has some drawbacks during clinical application, such as poor mechanical properties and rapid biodegradation. To address these issues, a new approach was developed for the preparation of collagen-based heterogeneous bilayer membranes, which involves a combination of physical treatment and biocompatible chemical cross-linking. The dialdehyde carboxymethyl cellulose was used to cross-link collagen to fabricate the membrane, and dehydrothermal (DHT) treatment was applied to enhance its properties. The results showed that DHT treatment apparently improved the structure stability and compression strength in both dry and swollen states. The biodegradation rate of the bilayer membrane was depressed, and the porosity was improved by dehydrogenation. The bilayer membrane was found to have good cytocompatibility. Moreover, the compact lower layer of the bilayer membrane possessed a strong barrier function to fibroblasts while the loose upper layer was able to enhance the adhesion, proliferation, and osteogenic differentiation of osteoblasts. Overall, the collagen-based heterogeneous bilayer membrane has great potential for the application in guided bone regeneration. For the membrane, the improved mechanical properties, reduced biodegradation rate, and enhanced osteoblast response make it a promising material for oral surgery applications.

Nanostructured titanium alloy surfaces for enhanced osteoblast response: A combination of morphology and chemistry

This study is part of a new trend in the metallic biomaterials field that searches to use the titanium beta alloys with recognized bulk and surface properties for better biological response. Ti15Mo alloy (wt%) had it surface modified by TiO2 nanotubes growth using the anodic oxidation method and grafted with poly(sodium styrene sulfonate) (PNaSS). The motivation behind the combination of these techniques was to promote a surface, which enables better osteointegration associated with antimicrobial behavior. Surfaces were characterized and subjected to cell culture assays and bacterial colonization to evaluate their in vitro response. The results showed that the surfaces were completely covered with TiO2 nanotubes, which was successfully grafted with PNaSS. Compared with untreated Ti 15Mo surfaces, PNaSS grafted nanotubes surfaces showed better osteoblast cell response in terms of cell attachment and mineralization and weaker bacterial adhesion and colonization. The results suggest that the final product of the combine treatment – nanotube plus PNaSS grafting - represents a promising option to improve commercial metallic implants.

Review Article: Zirconia surface bone graft implant with Ultraviolet Stimulation works toward Accelerating Bone Healing

Biomedical zirconia was introduced in 1969 into medicine to solve the problem of alumina brittleness in hip replacement procedures and has since been used for various joint replacement appliances in orthopedic surgery. The most frequently-studied material is yttrium-stabilized zirconia, which is also known as tetragonal zirconia polycrystal (TZP). Y-TZP presents various interesting characteristics, such as low porosity, high density and high bending and compression strength, proving that it is suitable for biomedical application. UV-treated zirconia surfaces exhibited an enhanced osteoblast response, which was characterized by an accelerated and augmented cell attachment, accelerated cell spread and cytoskeletal development with increased proliferation. The purpose of this paper is to identify which method of treatment of zirconia material implant & ultraviolet stimulation effect for bone healing is the most effective and efficient based on literature review. Bone grafts are available in a variety of substances. These bone substitutes can be biological (natural) or synthetic. Re-absorption is also essential for bone growth. Specific cells continuously break down bones and rebuild them. Substitutes that break down too quickly are not suitable for bone grafts, as they do not allow enough time for the new bone to grow. From our literature review, Zirconia is one of the biomaterials that have a bright future because of its high mechanical strength and fracture toughness. Zirconia ceramics have several advantages over other ceramic materials due to the transformation toughening mechanisms operating in their microstructure that can be expressed in components made out of them. UV treatment substantially enhances the osteogenesis process, resulting in a greater amount of peri-implant bone, as well as an increased strength of bone-zirconia integration.

The development of Cu-incorporated micro/nano-topographical bio-ceramic coatings for enhanced osteoblast response

In order to take advantages of the structural complexity of hierarchical topography and benefits of Cu, novel Cu-incorporated micro/nano-topographical coatings were developed on titanium (Ti) substrates using micro-arc oxidation (MAO) and hydrothermal treatment. A post heat treatment was employed to alter the morphology of the nanostructures and modulate the release of Cu2+. The structural evolution, phase composition, surface wettability, ion release and protein adsorption of the coatings were investigated. The results showed that the MAO-fabricated coating (denoted as MAO surface) was composed of porous TiO2 matrix distributed with CaO, SiO2 and CaSiO3 amorphous compounds. After hydrothermal treatment, a thin layer of nano-particles (20 nm in diameter) composed of CuO and calcium silicate hydrate formed on MAO surface (denoted as MAO-HT surface). These nano-particles grew to larger ones (65 nm in diameter) composed of crystallized CaO·3CuO·4TiO2 and CaO·TiO2·SiO2 after post heat treatment (denoted as MAO-HT2 surface). Moreover, the ion release rate and surface hydrophilicity of various coatings followed the trend: MAO surface > MAO-HT surface > MAO-HT2 surface. The amounts of adsorbed proteins were comparable on all surfaces. Furthermore, SaOS-2 cell response to various surfaces was investigated. The results showed that the viability of cells was significantly inhibited on MAO-HT surface due to the cyto-toxicity of high concentration Cu2+ release. However, the attachment, proliferation and differentiation of SaOS-2 cells were enhanced on MAO-HT2 surface compared to MAO surface. The results indicate that modifying Ti surface with hierarchical micro/nano-topography could lead to enhanced osteoblast response.

Construction of Ag-incorporated coating on Ti substrates for inhibited bacterial growth and enhanced osteoblast response.

In orthopedic fields, effective anti-infection property and promotive biocompatibility on surface of titanium implants are two crucial factors for long-term successful implants. Herein, Ag nanoparticles (NPs) loaded TiO2 nanotubes (TNT) arrays were fabricated on Ti substrates with assistance of ultraviolet irradiation. Then, bioactive multilayer films of chitosan (CHI) and dialdehyde alginate (ADA) pair were deposited onto the Ag-loaded TNT arrays via a layer-by-layer (LBL) self-assembly technique, which could effectively achieve the impactful antibacterial ability of titanium and endow the substrates with favorable biocompatibility. The driving force of the assembling of multilayer films came from two sources, electrostatic interaction and covalent interaction of Schiff-bonds between CHI and ADA. The surface topography and wettability of different samples were characterized by field emission scanning electron microscopy, transmission electron microscopy and contact angle measurements, respectively. In addition, Ag ions release from TNT-Ag and LBL substrate was measured via inductively coupled plasma atomic emission spectroscopy (ICP-AES). The results of a series of biological behaviors of osteoblasts on different substrates in vitro, including lactate dehydrogenase activity assay, cytoskeleton observation and cell viability measurement, confirmed that LBL substrates coated with (ADA-CHI)10 multilayer films have negligible cytotoxicity and promote osteoblast growth compared with TNT-Ag substrates, which could ascribe to the slow-release of Ag ions and the biocompatibility of (ADA-CHI)10 multilayer. More importantly, owing to the release of Ag ions, the LBL samples still exhibited a prominent antibacterial activity for S.aureus and E.coli. Characteristics of bacterial adhesion and viability measurement proved that the fabricated Ag-incorporated platform was capable of obviously inhibiting the adhesion and growth of bacteria. Therefore, this approach of surface modification for Ti substrates presented here may provide an alternative strategy to simultaneously meet the desirable osteoblast growth and reduce bacterial infection for implants in clinical application.

Significantly enhanced osteoblast response to nano-grained pure tantalum

Tantalum (Ta) metal is receiving increasing interest as biomaterial for load-bearing orthopedic applications and the synthetic properties of Ta can be tailored by altering its grain structures. This study evaluates the capability of sliding friction treatment (SFT) technique to modulate the comprehensive performances of pure Ta. Specifically, novel nanocrystalline (NC) surface with extremely small grains (average grain size of ≤20 nm) was fabricated on conventional coarse-grained (CG) Ta by SFT. It shows that NC surface possessed higher surface hydrophilicity and enhanced corrosion resistance than CG surface. Additionally, the NC surface adsorbed a notably higher percentage of protein as compared to CG surface. The in vitro results indicated that in the initial culture stages (up to 24 h), the NC surface exhibited considerably enhanced osteoblast adherence and spreading, consistent with demonstrated superior hydrophilicity on NC surface. Furthermore, within the 14 days culture period, NC Ta surface exhibited a remarkable enhancement in osteoblast cell proliferation, maturation and mineralization as compared to CG surface. Ultimately, the improved osteoblast functions together with the good mechanical and anti-corrosion properties render the SFT-processed Ta a promising alternative for the load-bearing bone implant applications.

Enhanced Osteoblast Response to Porosity and Resolution of Additively Manufactured Ti-6Al-4V Constructs with Trabeculae-Inspired Porosity.

The addition of porosity to the traditionally used solid titanium metal implants has been suggested to more closely mimic the natural mechanical properties of bone and increase osseointegration in dental and orthopedic implants. The objective of this study was to evaluate cellular response to three-dimensional (3D) porous Ti-6Al-4V constructs fabricated by additive manufacturing using laser sintering with low porosity (LP), medium porosity (MP), and high porosity (HP) with low resolution (LR) and high resolution (HR) based on a computed tomography scan of human trabecular bone. After surface processing, construct porosity ranged from 41.0% to 76.1%, but all possessed micro-/nanoscale surface roughness and similar surface chemistry containing mostly Ti, O, and C. Biological responses (osteoblast differentiation, maturation, and local factor production) by MG63 osteoblast-like cells and normal human osteoblasts favored 3D than two-dimensional (2D) solid constructs. First, MG63 cells were used to assess differences in cell response to 2D compared to LR and HR porous 3D constructs. MG63 cells were sensitive to porosity resolution and exhibited increased osteocalcin (OCN), vascular endothelial growth factor (VEGF), osteoprotegerin (OPG), and bone morphogenetic protein 2 (BMP2) on HR 3D constructs than on 2D and LR 3D constructs. MG63 cells also exhibited porosity-dependent responses on HR constructs, with up to a 6.9-fold increase in factor production on LP-HR and MP-HR constructs than on HP-HR constructs. NHOsts were then used to validate biological response on HR constructs. NHOsts exhibited decreased DNA content and alkaline phosphatase activity and up to a 2.9-fold increase in OCN, OPG, VEGF, BMP2, and BMP4 on 3D HR constructs than on 2D controls. These results indicate that osteoblasts prefer a 3D architecture than a 2D surface and that osteoblasts are sensitive to the resolution of trabecular detail and porosity parameters of laser-sintered 3D Ti-6Al-4V constructs.

Mechanical and biological properties of photocurable oligolactide-HA composites investigated under accelerated degradation

The major concern related to biodegradable bone substitute materials is the loss of mechanical strength which can be undesirable when occurring too quickly before new bone formation. In this study, the multifunctional lactide oligomers having 2, 3, and 4 arms end capped with methacrylate groups were synthesized with the aim of improving the degradation properties. Their composites with hydroxyapatite (HA) were photopolymerized and subjected to accelerated degradation at 60 °C. The results showed that increasing number of arms significantly improved thermal and mechanical properties as well as biocompatibility of the composites. All composites although varying in number of arms had similar levels of bone-specific gene expression and calcification indicating their equal bioactivity in supporting bone formation. The high HA content in the composites was proposed to be responsible for enhanced osteoblast response, and this tended to suppress the effects of polymeric structure.

Second-phase-dependent grain refinement in Ti–25Nb–3Mo–3Zr–2Sn alloy and its enhanced osteoblast response

Ti–25Nb–3Mo–3Zr–2Sn (TLM) substrates, which consist of pure β phase and duplex α+β phases were achieved by different heat treatment. Different substrates with and without α phase were subjected to surface mechanical attrition treatment (SMAT) for 5 and 30min, respectively. Investigated by transmission electron microscopy (TEM), it is found that the content and morphology of α phase in the TLM substrates play crucial roles in nanocrystallization of the alloy. During SMAT, the substrates composed of duplex α+β phases are much easier to nanocrystallized than that composed of pure β phase, and the duplex-phase substrate containing 35vt.% α needles is more inclined to grain refinement than those substrates containing 27vt.% α cobbles and 31vt.% α needles. Accompanied with the nanocrystallization in the surface layers of the duplex-phase substrates, the stress induced α-to-β phase transition occurs. In addition, employing hFOB1.19 cells, the behaviors of osteoblasts on the unSMATed and as-SMATed surfaces were evaluated by examining the morphology and viability of the cells. It shows that the SMAT-induced grain refinement in the surface layer of the alloy can significantly improve the osteoblast response. Our study lays the foundation for nanostructuring β titanium alloys to be used as biomedical implants.

Ion beam etching titanium for enhanced osteoblast response

As the demand for hip and knee replacements continues to grow, researchers look to increase the operational lifetimes of these implants. Many implant failures are attributed to aseptic loosening caused from the repeated loading of these joints. It is believed that by improving the interface between the implant and natural tissue, implant life could be extended. This study evaluates the effects of argon ion etching on Ti6Al4V titanium alloy and the resulting implications this etching has on living cells. Three ion energies (300, 700, and 1100 eV) were used to etch the as-received titanium substrates. Topographical changes were examined by scanning electron microscopy. Etching at 700 and 1100 eV resulted in the formation of a hierarchical structure of micro/nano-texturization of micron-sized depressions with nano-structured ripples. A rat mesenchymal stem cell population was differentiated to an osteoblastic phenotype to test the biocompatibility of the surfaces. It was found that ion etching the titanium results in an improvement of early cellular activity and may enhance osteoblast performance.

Zirconium oxide nanotube surface prompts increased osteoblast functionality and mineralization

Electrochemical formation of tunable nanoscale oxide layers on biomedical metallic surfaces has recently drawn much attention in biomaterials research. In this study, we report on the cellular response to a unique vertically aligned, laterally spaced nanotube nanostructure made of zirconium oxide (ZrO 2) fabricated by anodization. The growth, morphology, and functionality of osteoblasts cultured on ZrO 2 nanotubes have been investigated. The initial adhesion and spreading was considerably improved on the nanotube surface as compared to a flat zirconium (Zr) surface without a nanostructure. The morphology of the adhered cells on the nanotube surface elicited a highly organized cytoskeleton with crisscross patterned actin, which was lacking on the flat Zr. Increased alkaline phosphatase activity levels and the formation of calcified extracellular matrix implied improved osteoblast functionality and mineralization on the nanotube substrate. This in vitro study suggests that the ZrO 2 nanotubes provided an enhanced osteoblast response and demonstrated their apparent role in providing a platform for bone growth.

Initial biocompatibility and enhanced osteoblast response of Si doping in a porous BCP bone graft substitute

Granular shape biphasic calcium phosphate (BCP) bone grafts with and without doping of silicon cations were evaluated in regards to biocompatibility and MG-63 cellular response. To do this we studied Cellular cytotoxicity, cellular adhesion and spreading behavior and cellular differentiation with alizarin red S staining. Gene expression in MG-63 cells on the implanted bone substitutes was also examined at different time points using RT-PCR. In comparison, the Si-doped BCP granule showed more cellular viability than the BCP granule without doping in MTT assay. Moreover, cell proliferation was much higher when Si doping was employed. The cells grown on the silicon-doped BCP substitutes had more active filopodial growth with cytoplasmic webbing that proceeded to the flattening stage, which was indicative of well cellular adhesion. When these cells were exposed to Si-doped BCP granules for 14 days, well differentiated MG-63 cells were observed. Osteonectin and osteopontin genes were highly expressed in the late stage of differentiation (14 days), whereas collagen type I mRNA were found to be highly expressed during the early stage (day 3). These combined results of this study demonstrate that silicon-doped BCP enhanced osteoblast attachment/spreading, proliferation, differentiation and gene expression.

Enhanced osteoblast response to hydrophilic strontium and/or phosphate ions‐incorporated titanium oxide surfaces

This study investigated the surface characteristics and in vitro biocompatibility of titanium (Ti) surfaces incorporated with strontium ions (Sr) and/or phosphate ions (P) produced by hydrothermal treatment for future applications as endosseous implant surfaces. Sr and/or P-incorporated Ti oxide surfaces were produced by hydrothermal treatment. The surface characteristics were evaluated by scanning electron microscopy, thin-film X-ray diffractometry, X-ray photoelectron spectroscopy, contact angle and surface energy measurements, inductively coupled plasma atomic emission spectroscopy, and profilometry. MC3T3-E1 pre-osteoblast cell attachment, morphology of spread cells, viability, and quantitative analysis of osteoblastic gene expression on grit-blasted microrough (RBM), P-incorporated (P), and P- and Sr-incorporated (SrP) Ti surfaces were evaluated. Microstructured P and SrP surfaces showed significantly higher wettability and surface energy compared with RBM surfaces (P<0.01). After immersion in Hank's balanced salt solution, considerable apatite deposition was observed on the P and SrP surfaces. Sr incorporation significantly increased cellular attachment and viability compared with other surfaces (P<0.05). Real-time polymerase chain reaction analysis showed markedly higher mRNA expressions of the osteoblast transcription factor gene (Runx2) and the osteoblast phenotype genes (alkaline phosphatase, osteopontin, bone sialoprotein, and osteocalcin) in cells grown on the P and SrP surfaces compared with those on the RBM surface. These results demonstrate that Sr- and P-incorporated Ti oxide surfaces may improve implant bone healing by enhancing attachment, viability, and differentiation of osteoblastic cells.

Enhanced osteoblast response to an equal channel angular pressing-processed pure titanium substrate with microrough surface topography

This study investigated the surface characteristics and in vitro biocompatibility of ultrafine-grain pure titanium substrates produced by equal channel angular pressing (ECAP) using MC3T3-E1 pre-osteoblast cells, compared with those of conventional coarse-grain pure titanium (CP) and Ti–6Al–4V (Ti64) substrates. All Ti surfaces were grit-blasted with hydroxyapatite particles to produce microrough surfaces. The surface characteristics were evaluated by electron back-scattered diffractometry, scanning electron microscopy, contact angle and surface energy measurements, and optical profilometry. The morphology of spread cells, cell attachment, viability, alkaline phosphatase (ALP) activity, quantitative analysis of osteoblastic gene expression and mineralization nodule formation on different surfaces were evaluated. ECAP-processed substrates showed a significantly lower water contact angle and higher surface energy compared with coarse-grain CP and Ti64 substrates ( p < 0.05). They also showed enhanced cell spreading, attachment, viability and ALP activity compared with the CP and Ti64 surfaces ( p < 0.05). Real-time polymerase chain reaction analysis showed notably higher ALP, osteopontin and osteocalcin mRNA levels in cells grown on the ECAP surfaces than on the CP and Ti64 surfaces, and the ECAP surfaces showed significantly greater mineralization nodule formation compared with the CP and Ti64 substrates ( p < 0.05). These results demonstrate the superior osteoblast cell compatibility of microroughened Ti surface made of ECAP-processed ultrafine-grain pure Ti substrates over coarse-grain pure Ti and Ti64 substrates.

Enhanced osteoblast response to a polymethylmethacrylate–hydroxyapatite composite

Hydroxyapatite (HA)-reinforced polymers have been proposed as a method of improving the biological properties of bone cements and implant materials. For example, bone cements based on polymethylmethacrylate (PMMA) have long been used to secure orthopedic implants to the skeleton. This composite could also be used as a polished coating on other materials or in bulk form, shaped or molded, to custom fit a specific clinical need. However, complications may occur as a result of the limited mechanical and biological properties of PMMA. The purpose of this investigation was to determine whether the incorporation of HA in a PMMA matrix would enhance the biological properties of osteoblast response as compared to PMMA alone. Fetal rat calvarial osteoblasts were plated on discs of PMMA, PMMA/HA, commercially pure titanium (CpTi) and tissue culture polystyrene (control). Osteoblast attachment and day 2 proliferation were similar on all implant materials, whereas, day 8 proliferation on PMMA/HA was significantly higher than on PMMA and similar to CpTi and control. Extracellular matrix production was examined by immunohistochemistry which indicated that osteoblasts cultured on PMMA/HA showed a more distinct networked pattern of organized fibronectin. Histochemical staining of mineralization was examined by confocal microscopy which demonstrated a higher degree of mineralization in nodules formed on PMMA/HA as compared to PMMA. Together, these results indicate that the addition of HA in a PMMA matrix improves osteoblast response as compared to PMMA alone. Therefore, the incorporation of HA into a PMMA matrix may be a useful method to provide PMMA materials with enhanced osteogenic properties.