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1.  Biomolecular surface coating to enhance orthopaedic tissue healing and integration 
Biomaterials  2007;28(21):3228-3235.
Implant osseointegration is a prerequisite for clinical success in orthopaedic and dental applications, many of which are restricted by loosening. Biomaterial surface modification approaches, including calcium-phosphate ceramic coatings and macro/microporosity, have had limited success in promoting integration. To improve osseointegration, titanium surfaces were coated with the GFOGER collagen-mimetic peptide, selectively promoting α2β1 integrin binding, a crucial event for osteoblastic differentiation. Titanium surfaces presenting GFOGER triggered osteoblastic differentiation and mineral deposition in bone marrow stromal cells, leading to enhanced osteoblastic function compared to unmodified titanium. Furthermore, this integrin-targeted coating significantly improved in vivo peri-implant bone regeneration and osseointegration, as characterized by bone-implant contact and mechanical fixation, compared to untreated titanium in a rat cortical bone-implant model. GFOGER-modified implants also significantly enhanced osseointegration compared to surfaces modified with full-length type I collagen, highlighting the importance of presenting specific biofunctional domains within the native ligand. In addition, this biomimetic implant coating is generated using a simple, single-step procedure that readily translates to a clinical environment with minimal processing and cytotoxicity concerns. Therefore, this study establishes a biologically active and clinically relevant implant coating strategy that enhances bone repair and orthopaedic implant integration.
PMCID: PMC2034748  PMID: 17448533
biomimetic material; cell adhesion; collagen; osseointegration; integrin
2.  Multivalent Integrin-Specific Ligands Enhance Tissue Healing and Biomaterial Integration 
Science translational medicine  2010;2(45):45ra60.
Engineered biointerfaces covered with biomimetic motifs, including short bioadhesive ligands, are a promising material-based strategy for tissue repair in regenerative medicine. Potentially useful coating molecules are ligands for the integrins, major extracellular matrix receptors that require both ligand binding and nanoscale clustering for maximal signaling efficiency. We prepared coatings consisting of well-defined multimer constructs with a precise number of recombinant fragments of fibronectin (monomer, dimer, tetramer, and pentamer) to assess how nanoscale ligand clustering affects integrin binding, stem cell responses, tissue healing, and biomaterial integration. Clinical-grade titanium was grafted with polymer brushes that presented monomers, dimers, trimers, or pentamers of the α5β1 integrin–specific fibronectin III (7 to 10) domain (FNIII7–10). Coatings consisting of trimers and pentamers enhanced integrin-mediated adhesion in vitro, osteogenic signaling, and differentiation in human mesenchymal stem cells more than did surfaces presenting monomers and dimers. Furthermore, ligand clustering promoted bone formation and functional integration of the implant into bone in rat tibiae. This study establishes that a material-based strategy in which implants are coated with clustered bioadhesive ligands can promote robust implant-tissue integration.
PMCID: PMC3128787  PMID: 20720217
3.  Simple application of fibronectin-mimetic coating enhances osseointegration of titanium implants 
Integrin-mediated cell adhesion to biomolecules adsorbed onto biomedical devices regulates device integration and performance. Because of the central role of integrin-fibronectin (FN) interactions in osteoblastic function and bone formation, we evaluated the ability of fibronectin-inspired biomolecular coatings to promote osteoblastic differentiation and implant osseointegration. Notably, these biomolecular coatings relied on physical adsorption of FN-based ligands onto biomedical-grade titanium as a simple, clinically-translatable strategy to functionalize medical implants. Surfaces coated with a recombinant fragment of FN spanning the central cell binding domain enhanced osteoblastic differentiation and mineralization in bone marrow stromal cell cultures and increased implant osseointegration in a rat cortical bone model compared to passively adsorbed RGD peptides, serum proteins, and full-length FN. Differences in biological responses correlated with integrin binding specificity and signaling among surface coatings. This work validates a simple, clinically-translatable, surface biofunctionalization strategy to enhance biomedical device integration.
PMCID: PMC2819599  PMID: 18752639
fibronectin; osseointegration; coating; integrins; biomimetic; implant
4.  The Effect of a Keratin Hydrogel Coating on Osseointegration: An Histological Comparison of Coated and Non-coated Dental Titanium Implants in an Ovine Model 
Bioactive substances may be used to enhance the rate and quantity of bone healing during osseointegration of titanium dental implants. A pilot observational study was undertaken to assess a novel keratin hydrogel in six adult sheep utilising the femoral condyles as the surgical site to assess osseointegration. Implants and osteotomy sites were coated with the keratin gel prior to implant placement (test implants) whereas the opposite knee received unmodified control implants in each animal. Fifty 3.5 mm × 7 mm Neoss dental implants were surgically implanted with a range of 3–5 Neoss dental implants placed per surgical site in each knee and allowed to heal for 5 days or 2, 4, 8, 12 and 16 weeks prior to the sheep being sacrificed. Of the 50 placed implants, 24 were used for this study and analysed via resin-embedded, undemineralised sections from test and control implants to assess the range of healing around the unloaded dental implants. These dental implants were analysed using histomorphometric methods for the best 3 consecutive threads on each side and the percentage of bone to implant contact (%BIC) was used to determine the degree of osseointegration between test and control dental implants at each time point. All implants appeared osseointegrated at the time of sacrifice. One each of the pairs of control implants at 2, 4 and 12 weeks demonstrated minimal integration histologically, with %BIC <10 %. No test implants had %BIC <35 % at any time point. Mean %BIC for test implants was higher than controls at all time points except 5 days and 2 weeks. The range from 2 to 16 weeks healing was 39.7 % [SD 25.5 %] to 85.4 % [14.2 %] for test implants and 35.6 % [43.4 %] to 46.6 % [23.1 %] for controls. %BIC appeared to increase earlier in the test implants (from 4 weeks onwards) compared to controls. After 16 weeks, %BIC was almost twice as great in test implants as controls. This pilot observational study suggests that keratin hydrogel may promote earlier osseointegration around titanium dental implants. Further cross-sectional studies with larger sample sizes are warranted. The most marked difference between test and control implants was seen after 4 weeks. It is recommended that future studies in this model focus on healing after 4 weeks.
PMCID: PMC4016387  PMID: 24822008
Dental implants; Bioactive substances; Osseointegration; Ovine model
5.  Synthetic osteogenic extracellular matrix formed by coated silicon dioxide nanosprings 
The design of biomimetic materials that parallel the morphology and biology of extracellular matrixes is key to the ability to grow functional tissues in vitro and to enhance the integration of biomaterial implants into existing tissues in vivo. Special attention has been put into mimicking the nanostructures of the extracellular matrix of bone, as there is a need to find biomaterials that can enhance the bonding between orthopedic devices and this tissue.
We have tested the ability of normal human osteoblasts to propagate and differentiate on silicon dioxide nanosprings, which can be easily grown on practically any surface. In addition, we tested different metals and metal alloys as coats for the nanosprings in tissue culture experiments with bone cells.
Normal human osteoblasts grown on coated nanosprings exhibited an enhanced rate of propagation, differentiation into bone forming cells and mineralization. While osteoblasts did not attach effectively to bare nanowires grown on glass, these cells propagated successfully on nanosprings coated with titanium oxide and gold. We observed a 270 fold increase in the division rate of osteoblasts when grow on titanium/gold coated nanosprings. This effect was shown to be dependent on the nanosprings, as the coating by themselves did not alter the growth rate of osteoblast. We also observed that titanium/zinc/gold coated nanosprings increased the levels of osteoblast production of alkaline phosphatase seven folds. This result indicates that osteoblasts grown on this metal alloy coated nanosprings are differentiating to mature bone making cells. Consistent with this hypothesis, we showed that osteoblasts grown on the same metal alloy coated nanosprings have an enhanced ability to deposit calcium salt.
We have established that metal/metal alloy coated silicon dioxide nanosprings can be used as a biomimetic material paralleling the morphology and biology of osteogenic extracellular matrix. The coated nanosprings enhance normal human osteoblasts cellular behaviors needed for improving osseointegration of orthopedic materials. Thus, metal-coated nanosprings represent a novel biomaterial that could be exploited for improving success rates of orthopedic implant procedures.
PMCID: PMC3276422  PMID: 22284364
nanosprings; nanomaterials; osteoblasts; osseointegration; calcification; bone regeneration
6.  Osteoblast response to titanium surfaces functionalized with extracellular matrix peptide biomimetics 
Clinical oral implants research  2011;22(8):865-872.
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.
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.
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.
PMCID: PMC4287399  PMID: 21244501
KRSR; KSSR; microstructure; non-fouling; osteoblast differentiation; PLL-g-PEG; RGD; surface modification; surface roughness; titanium
7.  Extracellular matrix-mimetic adhesive biomaterials for bone repair 
Limited osseointegration of current orthopaedic biomaterials contributes to the failure of implants such as arthroplasties, bone screws and bone grafts, which present a large socioeconomic cost within the United States. These implant failures underscore the need for biomimetic approaches that modulate host cell-implant material responses to enhance implant osseointegration and bone formation. Bioinspired strategies have included functionalizing implants with ECM proteins or ECM-derived peptides or protein fragments which engage integrins and direct osteoblast adhesion and differentiation. This review discusses 1) bone ECM composition and key integrins implicated in osteogenic differentiation, 2) the use of implants functionalized with ECM-mimetic peptides/protein fragments, and 3) growth-factor derived peptides to promote the mechanical fixation of implants to bone and to enhance bone healing within large defects.
PMCID: PMC3059117  PMID: 21105174
8.  Effects of Coating a Titanium Alloy with Fibronectin on the Expression of Osteoblast Gene Markers in the MC3T3 Osteoprogenitor Cell Line 
A number of environmental and patient-related factors contribute to implant failure. A significant fraction of these failures can be attributed to limited osseointegration resulting from poor bone healing responses. The overall goal of this study was to determine whether surface treatment of a titanium-aluminum-vanadium alloy (Ti-6Al-4V) implant material with a biomimetic protein coating could promote the differentiation of attached osteoblastic cells. The specific aims of the study were to investigate whether osteoprogenitor cells cultured on a rigorously cleaned implant specimen showed a normal pattern of differentiation and whether preadsorbed fibronectin accelerated or enhanced osteoblast differentiation.
Materials and Methods
Ti-6Al-4V disks were rigorously cleaned, passivated in nitric acid, and dry heat–sterilized; some of the disks were then coated with 1 nmol/L fibronectin. MC3T3 osteoprogenitor cells were then cultured on the pretreated disks for several weeks. Quantitative real-time polymerase chain reaction was performed to measure changes over time in the mRNA levels of osteoblast genes.
Fibronectin increased the peak expression of all analyzed osteoblast gene markers. “Early” genes that normally mark the proliferative phase (0 to 10 days) of osteoblastic development showed peak expression within the first 10 days after cell attachment to the titanium alloy. In contrast, “late” genes that normally mark the differentiation (10 to 20 days) and mineralization (20 to 36 days) phases of osteoblastogenesis achieved peak expression only after approximately 3 to 4 weeks of culture.
Osteoprogenitors cultured on a rigorously cleaned Ti-6Al-4V alloy were found to demonstrate a normal pattern of osteoblast differentiation. Preadsorbed fibronectin was observed to stimulate osteoblast differentiation during the mineralization phase of osteoblastogenesis.
PMCID: PMC3810407  PMID: 23057020
coatings; differentiation; fibronectin; metal oxides; osteoblast; real-time polymerase chain reaction
9.  Comparative in vitro study regarding the biocompatibility of titanium-base composites infiltrated with hydroxyapatite or silicatitanate 
The development of novel biomaterials able to control cell activities and direct their fate is warranted for engineering functional bone tissues. Adding bioactive materials can improve new bone formation and better osseointegration. Three types of titanium (Ti) implants were tested for in vitro biocompatibility in this comparative study: Ti6Al7Nb implants with 25% total porosity used as controls, implants infiltrated using a sol–gel method with hydroxyapatite (Ti HA) and silicatitanate (Ti SiO2). The behavior of human osteoblasts was observed in terms of adhesion, cell growth and differentiation.
The two coating methods have provided different morphological and chemical properties (SEM and EDX analysis). Cell attachment in the first hour was slower on the Ti HA scaffolds when compared to Ti SiO2 and porous uncoated Ti implants. The Alamar blue test and the assessment of total protein content uncovered a peak of metabolic activity at day 8–9 with an advantage for Ti SiO2 implants. Osteoblast differentiation and de novo mineralization, evaluated by osteopontin (OP) expression (ELISA and immnocytochemistry), alkaline phosphatase (ALP) activity, calcium deposition (alizarin red), collagen synthesis (SIRCOL test and immnocytochemical staining) and osteocalcin (OC) expression, highlighted the higher osteoconductive ability of Ti HA implants. Higher soluble collagen levels were found for cells cultured in simple osteogenic differentiation medium on control Ti and Ti SiO2 implants. Osteocalcin (OC), a marker of terminal osteoblastic differentiation, was most strongly expressed in osteoblasts cultivated on Ti SiO2 implants.
The behavior of osteoblasts depends on the type of implant and culture conditions. Ti SiO2 scaffolds sustain osteoblast adhesion and promote differentiation with increased collagen and non-collagenic proteins (OP and OC) production. Ti HA implants have a lower ability to induce cell adhesion and proliferation but an increased capacity to induce early mineralization. Addition of growth factors BMP-2 and TGFβ1 in differentiation medium did not improve the mineralization process. Both types of infiltrates have their advantages and limitations, which can be exploited depending on local conditions of bone lesions that have to be repaired. These limitations can also be offset through methods of functionalization with biomolecules involved in osteogenesis.
PMCID: PMC4077223  PMID: 24987458
Implants; Porous titanium; Hydroxyapatite; Silicatitanate; Osteoblasts; Cell adhesion; Differentiation; Mineralization
10.  Nanotechnology and Dental Implants 
The long-term clinical success of dental implants is related to their early osseointegration. This paper reviews the different steps of the interactions between biological fluids, cells, tissues, and surfaces of implants. Immediately following implantation, implants are in contact with proteins and platelets from blood. The differentiation of mesenchymal stem cells will then condition the peri-implant tissue healing. Direct bone-to-implant contact is desired for a biomechanical anchoring of implants to bone rather than fibrous tissue encapsulation. Surfaces properties such as chemistry and roughness play a determinant role in these biological interactions. Physicochemical features in the nanometer range may ultimately control the adsorption of proteins as well as the adhesion and differentiation of cells. Nanotechnologies are increasingly used for surface modifications of dental implants. Another approach to enhance osseointegration is the application of thin calcium phosphate (CaP) coatings. Bioactive CaP nanocrystals deposited on titanium implants are resorbable and stimulate bone apposition and healing. Future nanometer-controlled surfaces may ultimately direct the nature of peri-implant tissues and improve their clinical success rate.
PMCID: PMC3021857  PMID: 21253543
11.  Biological response on a titanium implant-grade surface functionalized with modular peptides☆ 
Acta biomaterialia  2012;9(2):5341-5352.
Titanium (Ti) and its alloys are among the most successful implantable materials for dental and orthopedic applications. The combination of excellent mechanical and corrosion resistance properties makes them highly desirable as endosseous implants that can withstand a demanding biomechanical environment. Yet, the success of the implant depends on its osteointegration, which is modulated by the biological reactions occurring at the interface of the implant. A recent development for improving biological responses on the Ti-implant surface has been the realization that bifunctional peptides can impart material binding specificity not only because of their molecular recognition of the inorganic material surface, but also through their self-assembly and ease of biological conjugation properties. To assess peptide-based functionalization on bioactivity, the present authors generated a set of peptides for implant-grade Ti, using cell surface display methods. Out of 60 unique peptides selected by this method, two of the strongest titanium binding peptides, TiBP1 and TiBP2, were further characterized for molecular structure and adsorption properties. These two peptides demonstrated unique, but similar molecular conformations different from that of a weak binder peptide, TiBP60. Adsorption measurements on a Ti surface revealed that their disassociation constants were 15-fold less than TiBP60. Their flexible and modular use in biological surface functionalization were demonstrated by conjugating them with an integrin recognizing peptide motif, RGDS. The functionalization of the Ti surface by the selected peptides significantly enhanced the bioactivity of osteoblast and fibroblast cells on implant-grade materials.
PMCID: PMC4410049  PMID: 23159566
Implants; Titanium binding peptide; Molecular recognition; Biomaterial interface; Bioenabled surface modification
12.  Strain driven fast osseointegration of implants 
Although the bone's capability of dental implant osseointegration has clinically been utilised as early as in the Gallo-Roman population, the specific mechanisms for the emergence and maintenance of peri-implant bone under functional load have not been identified. Here we show that under immediate loading of specially designed dental implants with masticatory loads, osseointegration is rapidly achieved.
We examined the bone reaction around non- and immediately loaded dental implants inserted in the mandible of mature minipigs during the presently assumed time for osseointegration. We used threaded conical titanium implants containing a titanium2+ oxide surface, allowing direct bone contact after insertion. The external geometry was designed according to finite element analysis: the calculation showed that physiological amplitudes of strain (500–3,000 ustrain) generated through mastication were homogenously distributed in peri-implant bone. The strain-energy density (SED) rate under assessment of a 1 Hz loading cycle was 150 Jm-3 s-1, peak dislocations were lower then nm.
Bone was in direct contact to the implant surface (bone/implant contact rate 90%) from day one of implant insertion, as quantified by undecalcified histological sections. This effect was substantiated by ultrastructural analysis of intimate osteoblast attachment and mature collagen mineralisation at the titanium surface. We detected no loss in the intimate bone/implant bond during the experimental period of either control or experimental animals, indicating that immediate load had no adverse effect on bone structure in peri-implant bone.
In terms of clinical relevance, the load related bone reaction at the implant interface may in combination with substrate effects be responsible for an immediate osseointegration state.
PMCID: PMC1277014  PMID: 16270927
13.  Tuning Cell Adhesion on Titanium with Osteogenic Rosette Nanotubes 
Self-assembled rosette nanotubes (RNTs), obtained from a twin G∧C base functionalized with lysine-arginine-serine-arginine (KRSR–(G∧C)2), were designed and investigated as bioactive coatings on titanium. These results were compared to RNTs derived from Lysine G∧C (K G∧C), Arg-Gly-Asp G∧C (RGD G∧C) and aminobutane–(G∧C)2 (AB–(G∧C)2). The results from this study revealed that these materials had excellent cytocompatibility properties as they enhanced osteoblast (bone forming cell) adhesion when coated on titanium. In particular, KRSR and RGD functionalized RNTs coated on titanium promoted the greatest osteoblast densities relative to untreated titanium. Furthermore, KRSR functionalized RNTs selectively improved osteoblast adhesion relative to fibroblast (soft-tissue forming cell) and endothelial cell adhesion. In contrast with these results, RNTs obtained from an unfunctionalized twin base (AB–(G∧C)2), RGD G∧C co-assembled with K G∧C and K-G∧C significantly enhanced endothelial cell attachment, which may find applications in the vascularization of newly formed bone tissue. In summary, these studies suggest that the surface of orthopedic implant materials (such as titanium) could be tailored to promote selective cell adhesion using biologically-inspired nanotubular structures functionalized with osteogenic compounds.
PMCID: PMC2943967  PMID: 20725961
rosette nanotubes; nanomaterials; biomimetic; osteogenic peptides; coating; orthopedic implant
14.  Biological Functionalization of Dental Implants with Fibronectin: A Scanning Electron Microscopic Study 
Early stages of peri-implant bone formation play an essential role in the osseointegration and long-term success of dental implants. Biological implant surface coatings are an emerging technology to enhance the attachment of the implant to the surrounding bone and stimulate bone regeneration. The purpose of this study was to determine the effect of coating the implant surface with fibronectin on osseointegration.
Material and methods
The experiment was conducted on a total of twelve New Zealand white mature male rabbits, weight between 2.5–4 kg. Twenty four pure titanium implants were used in this study. Each rabbits received two implants, one implant in each tibia; the implant in the right limb was coated with fibronectin (experimental group), whilst on the contralateral side the implants were placed without coating (control group). Six rabbits were sacrificed for Scanning Electron Microscopic evaluation after 4 and 8 week healing periods.
The results of the present study demonstrating the mean gap distance between the bone and implant was greater in the control group compared to fibronection group at both observation periods however, the difference between these two groups was not statistically significant.
Thus, it could be suggested that the biological functionalization of dental implants with fibronectin, may influence the integration or biocompatibility and bonding of the implant to the surrounding bone.
PMCID: PMC4039585  PMID: 24899880
Dental implant; Osseointegration; Biofunctionalization; Extracellular matrix; Fibronectin
15.  Effect of local zoledronate on implant osseointegration in a rat model 
An implant coating with poly(D, L-lactide) (PDLLA) releasing incorporated Zoledronic acid (ZOL) has already proven to positively effect osteoblasts, to inhibit osteoclasts and to accelerate fracture healing. Aim of this study was to investigate the release kinetics of the chosen coating and the effect of different concentrations of ZOL locally released from this coating on the osseointegration of implants.
For release kinetics the release of C14-labled ZOL out of the coating was monitored over a period of six weeks in vitro. For testing the osseointegration, titanium Kirschner wires were implanted into the medullary canal of right femurs of 100 Sprague Dawley rats. The animals were divided into five groups receiving implants either uncoated or coated with PDLLA, PDLLA/ZOL low (1.2% w/w) or PDLLA/ZOL high (2% w/w). Additionally, a group with uncoated implants received ZOL intravenously (i.v.). After 56 days animals were sacrificed, femurs dissected and either strength of fixation or histological bone/implant contacts and newly formed bone around the implants were determined.
Release kinetics revealed an initial peak in the release of C14-ZOL with a slight further progression over the following weeks. There was no significant enhancement of osseointegration for both groups who received ZOL-coated implants or ZOL i.v. compared to the controls in biomechanical or histological analyses, except for a significant raise in strength of fixation of ZOL i.v. versus PDLLA.
Even though the investigated local ZOL application did not enhance the osseointegration of the implant, the findings might support its application in fracture treatment, since fracture stabilization devices are often explanted after consolidation.
PMCID: PMC3323428  PMID: 22439827
16.  Evaluation of Biological Properties of Electron Beam Melted Ti6Al4V Implant with Biomimetic Coating In Vitro and In Vivo 
PLoS ONE  2012;7(12):e52049.
High strength porous titanium implants are widely used for the reconstruction of craniofacial defects because of their similar mechanical properties to those of bone. The recent introduction of electron beam melting (EBM) technique allows a direct digitally enabled fabrication of patient specific porous titanium implants, whereas both their in vitro and in vivo biological performance need further investigation.
In the present study, we fabricated porous Ti6Al4V implants with controlled porous structure by EBM process, analyzed their mechanical properties, and conducted the surface modification with biomimetic approach. The bioactivities of EBM porous titanium in vitro and in vivo were evaluated between implants with and without biomimetic apatite coating.
The physical property of the porous implants, containing the compressive strength being 163 - 286 MPa and the Young’s modulus being 14.5–38.5 GPa, is similar to cortical bone. The in vitro culture of osteoblasts on the porous Ti6Al4V implants has shown a favorable circumstance for cell attachment and proliferation as well as cell morphology and spreading, which were comparable with the implants coating with bone-like apatite. In vivo, histological analysis has obtained a rapid ingrowth of bone tissue from calvarial margins toward the center of bone defect in 12 weeks. We observed similar increasing rate of bone ingrowth and percentage of bone formation within coated and uncoated implants, all of which achieved a successful bridging of the defect in 12 weeks after the implantation.
This study demonstrated that the EBM porous Ti6Al4V implant not only reduced the stress-shielding but also exerted appropriate osteoconductive properties, as well as the apatite coated group. The results opened up the possibility of using purely porous titanium alloy scaffolds to reconstruct specific bone defects in the maxillofacial and orthopedic fields.
PMCID: PMC3525565  PMID: 23272208
17.  Early bone growth on the surface of titanium implants in rat femur is enhanced by an amorphous diamond coating 
Acta Orthopaedica  2011;82(4):499-503.
Background and purpose
Amorphous diamond (AD) is a durable and compatible biomaterial for joint prostheses. Knowledge regarding bone growth on AD-coated implants and their early-stage osseointegration is poor. We investigated bone growth on AD-coated cementless intramedullary implants implanted in rats. Titanium was chosen as a reference due to its well-known performance.
Materials and methods
We placed AD-coated and non-coated titanium implants (Ra ≈ 0.2 μm) into the femoral bone marrow of 25 rats. The animals were divided in 2 groups according to implant coating and they were killed after 4 or 12 weeks. The osseointegration of the implants was examined from hard tissue specimens by measuring the new bone formation on their surface.
4 weeks after the operation, the thickness of new bone in the AD-coated group was greater than that in the non-coated group (15.3 (SD 7.1) μm vs. 7.6 (SD 6.0) μm). 12 weeks after the operation, the thickness of new bone was similar in the non-coated group and in the AD-coated group.
We conclude that AD coating of femoral implants can enhance bone ongrowth in rats in the acute, early stage after the operation and might be an improvement over earlier coatings.
PMCID: PMC3237044  PMID: 21504369
18.  Decreased Fibroblast and Increased Osteoblast Functions on Ionic Plasma Deposited Nanostructured Ti Coatings 
Nanoscale Research Letters  2007;2(8):385-390.
Bioactive coatings are in high demand to control cellular functions for numerous medical devices. The objective of this in vitro study was to characterize for the first time fibroblast (fibrous scar tissue forming cells) adhesion and proliferation on an important polymeric biomaterial (silicone) coated with titanium using a novel ionic plasma deposition (IPD) process. Fibroblasts are one of the first anchorage-dependent cells to arrive at an implant surface during the wound healing process. Persistent excessive functions of fibroblasts have been linked to detrimental fibrous tissue formation which may cause implant failure. The IPD process creates a surface-engineered nanostructure (with features usually below 100 nm) by first using a vacuum to remove all contaminants, then guiding charged metallic ions or plasma to the surface of a medical device at ambient temperature. Results demonstrated that compared to currently used titanium and uncoated silicone, silicone coated with titanium using IPD significantly decreased fibroblast adhesion and proliferation. Results also showed competitively increased osteoblast (bone-forming cells) over fibroblast adhesion on silicone coated with titanium; in contrast, osteoblast adhesion was not competitively increased over fibroblast adhesion on uncoated silicone or titanium controls. In this manner, this study strongly suggests that IPD should be further studied for biomaterial applications in which fibrous tissue encapsulation is undesirable (such as for orthopedic implants, cardiovascular components, etc.).
PMCID: PMC3246386
Nanometer; Coatings; Fibroblasts; Osteoblasts; Orthopedic
19.  Titanium Implant Osseointegration Problems with Alternate Solutions Using Epoxy/Carbon-Fiber-Reinforced Composite 
Metals  2014;4(4):549-569.
The aim of the article is to present recent developments in material research with bisphenyl-polymer/carbon-fiber-reinforced composite that have produced highly influential results toward improving upon current titanium bone implant clinical osseointegration success. Titanium is now the standard intra-oral tooth root/bone implant material with biocompatible interface relationships that confer potential osseointegration. Titanium produces a TiO2 oxide surface layer reactively that can provide chemical bonding through various electron interactions as a possible explanation for biocompatibility. Nevertheless, titanium alloy implants produce corrosion particles and fail by mechanisms generally related to surface interaction on bone to promote an inflammation with fibrous aseptic loosening or infection that can require implant removal. Further, lowered oxygen concentrations from poor vasculature at a foreign metal surface interface promote a build-up of host-cell-related electrons as free radicals and proton acid that can encourage infection and inflammation to greatly influence implant failure. To provide improved osseointegration many different coating processes and alternate polymer matrix composite (PMC) solutions have been considered that supply new designing potential to possibly overcome problems with titanium bone implants. Now for important consideration, PMCs have decisive biofunctional fabrication possibilities while maintaining mechanical properties from addition of high-strengthening varied fiber-reinforcement and complex fillers/additives to include hydroxyapatite or antimicrobial incorporation through thermoset polymers that cure at low temperatures. Topics/issues reviewed in this manuscript include titanium corrosion, implant infection, coatings and the new epoxy/carbon-fiber implant results discussing osseointegration with biocompatibility related to nonpolar molecular attractions with secondary bonding, carbon fiber in vivo properties, electrical semiconductors, stress transfer, additives with low thermal PMC processing and new coating possibilities.
PMCID: PMC4307950  PMID: 25635227
titanium; composite; bisphenol polymer; carbon fiber; osseointegration; corrosion; infection; estrogen; microbiocircuit; semiconductor
20.  Adherent Lipopolysaccharide Inhibits the Osseointegration of Orthopaedic Implants by Impairing Osteoblast Differentiation 
Bone  2012;52(1):93-101.
Osseointegration is the process by which an orthopaedic implant makes direct bone-to-implant contact and is crucial for the long-term function of the implant. Surface contaminants, such as bacterial debris and manufacturing residues, may remain on orthopaedic implants after sterilization and impair osseointegration. For example, specific lots of implants that were associated with impaired osseointegration and high failure rates were discovered to have contaminants including bacterial debris. Therefore, the goals of this study were to determine if bacterial debris exists on sterile orthopaedic implants and if adherent bacterial debris inhibits the osseointegration of orthopaedic implants. We found that debris containing lipopolysaccharide (LPS) from Gram-negative bacteria exists on both sterile craniofacial implants and wrist implants. Levels of bacterial debris vary not only between different lots of implants but within an individual lot. Using our murine model of osseointegration, we found that ultrapure LPS adherent to the implants inhibited bone-to-implant contact and biomechanical pullout measures. Analysis of osseointegration in knock-out mice demonstrated that adherent LPS inhibited osseointegration by signaling through its primary receptor, Toll-like receptor 4, and not by signaling through Toll-like receptor 2. Ultrapure LPS adherent to titanium alloy discs had no detectable effect on early stages of MC3T3-E1 osteogenesis in vitro such as attachment, spreading or growth. However, later stages of osteogenic differentiation and mineralization were inhibited by adherent LPS. Thus, LPS may inhibit osseointegration in part through cell autonomous effects on osteoblasts. These results highlight bacterial debris as a type of surface contaminant that can impair the osseointegration of orthopaedic implants.
PMCID: PMC3513552  PMID: 22995462
orthopaedic implants; osseointegration; osteoblasts; bacterial debris; lipopolysaccharide
21.  UV- Killed Staphylococcus aureus Enhances Adhesion and Differentiation of Osteoblasts on Bone-associated Biomaterials 
Titanium alloys (Ti) are the preferred material for orthopaedic applications. However, very often, these metallic implants loosen over a long period and mandate revision surgery. For implant success, osteoblasts must adhere to the implant surface and deposit a mineralized extracellular matrix. Here, we utilized UV-killed Staphylococcus aureus as a novel osteoconductive coating for Ti surfaces. S. aureus expresses surface adhesins capable of binding to bone and biomaterials directly. Furthermore, interaction of S. aureus with osteoblasts activates growth factor-related pathways that potentiate osteogenesis. While UV-killed S. aureus cells retain their bone-adhesive ability, they do not stimulate significant immune modulator expression. All of the above properties were utilized for a novel implant coating so as to promote osteoblast recruitment and subsequent cell functions on the bone-implant interface. In the present study, osteoblast adhesion, proliferation, and mineralized extracellular matrix synthesis were measured on Ti surfaces coated with fibronectin with and without UV-killed bacteria. Osteoblast adhesion was enhanced on Ti alloy surfaces coated with bacteria compared to uncoated surfaces while cell proliferation was sustained comparably on both surfaces. Osteoblast markers such as collagen, osteocalcin, alkaline phosphatase activity and mineralized nodule formation were increased on Ti alloy coated with bacteria compared to uncoated surfaces.
PMCID: PMC2943998  PMID: 20725968
Titanium implant surfaces; Osseointegration; Staphylococcus aureus; Calvarial osteoblasts; Osteoblast differentiation
22.  Self-assembled Monolayer Films of Phosphonates for Bonding RGD to Titanium 
Modification of the implant surface with the Arg-Gly-Asp tripeptide (RGD) putatively facilitates osteoblast attachment for improved implant fixation in the laboratory. We compared the histomorphometric and mechanical performance of titanium implants coated with RGD using a novel interface of self-assembled monolayers of phosphonates (RGD/SAMP) and implants coated with RGD using the more conventional thiolate-gold interface (RGD/thiolate-gold). We hypothesized RGD/SAMP-coated implants would show greater bone ongrowth and implant fixation than RGD/thiolate-gold-coated ones. We implanted an RGD/SAMP-coated implant in one femur and an RGD/thiolate-gold-coated in the contralateral femur of 60 rats. At 2, 4, and 8 weeks after implantation, 10 rats were sacrificed for histologic evaluation and another 10 for biomechanical testing. Bone-implant ongrowth and implant force-to-failure of the two implants were similar at all times. Although RGD/SAMP-coated implants did not show superior bone ongrowth and implant fixation, RGD/SAMP-coated implants have at least equally good histomorphometric and mechanical in vivo performance as RGD/thiolate-gold-coated ones. Additional in vivo characterization of self-assembled monolayer films of phosphonates as interface to bond RGD to titanium is needed to explore its full potential and seems justified based on the results of this study.
PMCID: PMC2504684  PMID: 18224380
23.  Enhanced osteoblast adhesion to drug-coated anodized nanotubular titanium surfaces 
Current orthopedic implants have functional lifetimes of only 10–15 years due to a variety of reasons including infection, extensive inflammation, and overall poor osseointegration (or a lack of prolonged bonding of the implant to juxtaposed bone). To improve properties of titanium for orthopedic applications, this study anodized and subsequently coated titanium with drugs known to reduce infection (penicillin/streptomycin) and inflammation (dexamethasone) using simple physical adsorption and the deposition of such drugs from simulated body fluid (SBF). Results showed improved drug elution from anodized nanotubular titanium when drugs were coated in the presence of SBF for up to 3 days. For the first time, results also showed that the simple physical adsorption of both penicillin/streptomycin and dexamethasone on anodized nanotubular titanium improved osteoblast numbers after 2 days of culture compared to uncoated unanodized titanium. In addition, results showed that depositing such drugs in SBF on anodized titanium was a more efficient method to promote osteoblast numbers compared to physical adsorption for up to 2 days of culture. In addition, osteoblast numbers increased on anodized titanium coated with drugs in SBF for up to 2 days of culture compared to unanodized titanium. In summary, compared to unanodized titanium, this preliminary study provided unexpected evidence of greater osteoblast numbers on anodized titanium coated with either penicillin/streptomycin or dexamethasone using simple physical adsorption or when coated with SBF; results which suggest the need for further research on anodized titanium orthopedic implants possessing drug-eluting nanotubes.
PMCID: PMC2527662  PMID: 18686785
anodization; titanium; adhesion; simulated body fluid; nanotubes; osteoblasts
24.  Osseointegration of zirconia implants compared with titanium: an in vivo study 
Head & Face Medicine  2008;4:30.
Titanium and titanium alloys are widely used for fabrication of dental implants. Since the material composition and the surface topography of a biomaterial play a fundamental role in osseointegration, various chemical and physical surface modifications have been developed to improve osseous healing. Zirconia-based implants were introduced into dental implantology as an altenative to titanium implants. Zirconia seems to be a suitable implant material because of its tooth-like colour, its mechanical properties and its biocompatibility. As the osseointegration of zirconia implants has not been extensively investigated, the aim of this study was to compare the osseous healing of zirconia implants with titanium implants which have a roughened surface but otherwise similar implant geometries.
Forty-eight zirconia and titanium implants were introduced into the tibia of 12 minipigs. After 1, 4 or 12 weeks, animals were sacrificed and specimens containing the implants were examined in terms of histological and ultrastructural techniques.
Histological results showed direct bone contact on the zirconia and titanium surfaces. Bone implant contact as measured by histomorphometry was slightly better on titanium than on zirconia surfaces. However, a statistically significant difference between the two groups was not observed.
The results demonstrated that zirconia implants with modified surfaces result in an osseointegration which is comparable with that of titanium implants.
PMCID: PMC2614983  PMID: 19077228
25.  Effects of Structural Properties of Electrospun TiO2 Nano-fiber Meshes on their Osteogenic Potential 
Acta Biomaterialia  2011;8(2):878-885.
Ideal outcomes in the field of tissue engineering and regenerative medicine involve biomaterials that can enhance cell differentiation and production of local factors for natural tissue regeneration without the use of systemic drugs. Biomaterials typically used in tissue engineering applications include polymeric scaffolds that mimic the 3-D structural environment of the native tissue, but these are often functionalized with proteins or small peptides to improve their biological performance. For bone applications, titanium (Ti) implants, or more appropriately the titania (TiO2) passive oxide layer formed on their surface, have been shown to enhance osteoblast differentiation in vitro and to promote osseointegration in vivo. In this study we evaluated the effect on osteoblast differentiation of pure TiO2 nano-fiber meshes with different surface micro-roughness and nano-fiber diameters, prepared by the electrospinning method. MG63 cells were seeded on TiO2 meshes, and cell number, differentiation markers and local factor production were analyzed. The results showed that cells grew throughout the entire surfaces and with similar morphology in all groups. Cell number was sensitive to surface micro-roughness, whereas cell differentiation and local factor production was regulated by both surface roughness and nano-fiber diameter. These results indicate that scaffold structural cues alone can be used to drive cell differentiation and create an osteogenic environment without the use of exogenous factors.
PMCID: PMC3309709  PMID: 22075122
nano structures; electrospinning; scaffold; titanium implant; tissue engineering; bone

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