Biomolecular surface coating to enhance orthopaedic tissue healing and integration

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 glycine-phenylalanine-hydroxyproline-glycine-glutamate-arginine (GFOGER) collagen-mimetic peptide, selectively promoting alpha2beta1 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.     

Bone healing of commercial oral implants with RGD immobilization through electrodeposited poly(ethylene glycol) in rabbit cancellous bone

Immobilization of RGD peptides on titanium (Ti) surfaces enhances implant bone healing by promoting early osteoblastic cell attachment and subsequent differentiation by facilitating integrin binding. Our previous studies have demonstrated the efficacy of RGD peptide immobilization on Ti surfaces through the electrodeposition of poly(ethylene glycol) (PEG) (RGD/PEG/Ti), which exhibited good chemical stability and bonding. The RGD/PEG/Ti surface promoted differentiation and mineralization of pre-osteoblasts. This study investigated the in vivo bone healing capacity of the RGD/PEG/Ti surface for biomedical application as a more osteoconductive implant surface in dentistry. The RGD/PEG/Ti surface was produced on an osteoconductive implant surface, i.e. the grit blasted micro-rough surface of a commercial oral implant. The osteoconductivity of the RGD/PEG/Ti surface was compared by histomorphometric evaluation with an RGD peptide-coated surface obtained by simple adsorption in rabbit cancellous bone after 2 and 4 weeks healing. The RGD/PEG/Ti implants displayed a high degree of direct bone apposition in cancellous bone and achieved greater active bone apposition, even in areas of poor surrounding bone. Significant increases in the bone to implant contact percentage were observed for RGD/PEG/Ti implants compared with RGD-coated Ti implants obtained by simple adsorption both after 2 and 4 weeks healing (P<0.05). These results demonstrate that RGD peptide immobilization on a Ti surface through electrodeposited PEG may be an effective method for enhancing bone healing with commercial micro-rough surface oral implants in cancellous bone by achieving rapid bone apposition on the implant surface.     

In vivo evaluation of micro-rough and bioactive titanium dental implants using histometry and pull-out tests

We report on the in vivo histological and mechanical performance of titanium dental implants with a new surface treatment (2Step) consisting of an initial grit-blasting process to produce a micro-rough surface, followed by a combined chemical and thermal treatment that produces a potentially bioactive surface, i.e., that can form an apatitic layer when exposed to biomimetic conditions in vitro. Our aim was to assess the short- and mid-term bone regenerative potential and mechanical retention of 2Step implants in mandible and maxilla of minipigs and compare them with micro-rough grit-blasted, micro-rough acid-etched, and smooth as-machined titanium implants. The percent of bone-to-implant contact after 2, 4, 6, and 10 weeks of implantation as well as the mechanical retention after 4, and 6 weeks of implantation were evaluated with histometric and pull-out tests, respectively, as a measure of the osseointegration of the implants. We also aimed to assess the bioactive nature of 2Step surfaces in vivo. Our results demonstrated that the 2Step treatment produced micro-rough and bioactive implants that accelerated bone tissue regeneration and increased mechanical retention in the bone bed at short periods of implantation in comparison with all other implants tested. This was mostly attributed to the ability of 2Step implants to form in vivo a layer of apatitic mineral that coated the implant and could rapidly stimulate (a) bone nucleation directly on the implant surface, and (b) bone growing from the implant surface. We also proved that roughness values of Ra≈4.5 μm favoured osseointegration of dental implants at short- and mid-term healing periods, as grit-blasted implants and 2Step implants had higher retention values than as machined and acid-etched implants. The surface quality resulting from the 2Step treatment applied on cpTi provided dental implants with a unique combination of rapid bone regeneration and high mechanical retention.     

Engineering cell adhesive surfaces that direct integrin alpha5beta1 binding using a recombinant fragment of fibronectin

Integrin receptors mediate cell adhesion to extracellular matrices and trigger signals that direct cell function. While many integrins bind to the arginine-glycine-aspartic acid (RGD) motif present in numerous extracellular proteins, integrin alpha(5)beta(1) requires both the PHSRN synergy site in the 9th and the RGD site in the 10th type III repeat of fibronectin (FN). Binding of alpha(5)beta(1) to FN is critical to many cellular processes, including osteoblast and myoblast differentiation. This work focused on engineering integrin-specific bioadhesive surfaces by immobilizing a recombinant FN fragment (FNIII(7-10)) encompassing the alpha(5)beta(1) binding domains of FN. Model hybrid surfaces were engineered by immobilizing FNIII(7-10) onto passively adsorbed, non-adhesive albumin. Homo- and hetero-bifunctional crosslinkers of varying spacer-arm length targeting either the cysteine or lysine groups on FNIII(7-10) were investigated in ELISA and cell adhesion assays to optimize immobilization densities and activity. FN-mimetic surfaces presenting controlled densities of FNIII(7-10) were generated by varying the concentration of FNIII(7-10) in the coupling solution at a constant crosslinker concentration. Cells adhered to these functionalized surfaces via integrin alpha(5)beta(1) and blocking with integrin-specific antibodies completely eliminated adhesion. In addition, adherent cells spread and assembled focal adhesions containing alpha(5)beta(1), vinculin, and talin. This biomolecular engineering strategy represents a robust approach to increase biofunctional activity and integrin specificity of biomimetic materials.     

Osteoblast culture on polished titanium disks modified with phosphonic acids

Titanium is widely used in dental implants due to its suitable physical properties and its good biocompatibility. However, it is integrated into bone only passively, and the resulting fixation in the bone, which is necessary for the function, is mainly mechanical in nature. With the objective of increasing the chemical interaction between the implant and the bone tissue, several phosphonic acids were synthesized and grafted onto titanium disks. Here we report on the proliferation, differentiation, and protein production of rat osteoblastic cells (CRP10/30) on phosphonic-acid-modified titanium surfaces studied in vitro. No statistical differences were found in osteoblast proliferation among the phosphonic-acid-modified titanium, unmodified titanium, and tissue culture plastic (used as a positive control), indicating that the phosphonic acids used were not cytotoxic to the osteoblasts used. For all surfaces (modified or not), the alkaline phosphatase activity was at least as good as it was on tissue culture plastic. However, the total amount of protein, and especially the collagen type I synthesis, was sensitive to surface modification. On titanium modified with ethane-1,1,2-triphosphonic acid, the total amount of synthesized protein was significantly higher than it was on unmodified titanium surfaces. A significant increase (up to 16%) of collagen type I production was observed on titanium surfaces modified with this acid or with methylenediphosphonic acid compared to unmodified titanium surfaces.     

Ectopic bone formation in rat marrow stromal cell/titanium fiber mesh scaffold constructs: effect of initial cell phenotype

Titanium fiber mesh scaffolds have been shown to be a suitable material for culture of primary marrow stromal cells in an effort to create tissue engineered constructs for bone tissue replacement. In native bone tissue, these cells are known to attach to extracellular matrix molecules via integrin receptors for specific peptide sequences, and these attachments can be a source of cell signaling, affecting cell behaviors such as differentiation. In this study, we examined the ability of primary rat marrow stromal cells at two different stages of osteoblastic differentiation to further differentiate into osteoblasts both in vitro and in vivo when seeded on titanium fiber mesh scaffolds either with or without RGD peptide tethered to the surface. In vitro, the tethered RGD peptide resulted in reduced initial cell proliferation. In vivo, there was no effect of tethered RGD peptide on ectopic bone formation in a rat subcutaneous implant model. Scaffold/cell constructs exposed to dexamethasone for 4 days prior to implantation (+dex constructs) resulted in significant bone formation whereas no bone formation was observed in--dex constructs. These results show that the osteoblastic differentiation of marrow stromal cells was not dependent on surface tethered RGD peptide, and that the initial differentiation stage of implanted cells plays an important role in bone formation in titanium fiber mesh bone tissue engineering constructs.     

A new approach to graft bioactive polymer on titanium implants: Improvement of MG 63 cell differentiation onto this coating

Integration of titanium implants into bone is only passive and the resulting fixation is mainly mechanical in nature, with anchorage failure. Our objective, to increase the biointegration of the implant and the bone tissue, could be obtained by grafting a bioactive ionic polymer to the surface of the titanium by a covalent bond. In this paper, we report the grafting of an ionic polymer model poly(sodium styrene sulfonate) (polyNaSS), in a two-step reaction procedure. Treatment of the titanium surface by a mixture of sulfuric acid and hydrogen peroxide allows the formation of titanium hydroxide and titanium peroxide. In the second reaction step, heating of a metal implant, placed in a concentrated solution of sodium styrene sulfonate monomer (NaSS), induces the decomposition of titanium peroxides with the formation of radicals capable of initiating the polymerization of NaSS. Various parameters, such as temperature of polymerization and time of polymerization, were studied in order to optimize the yield of polyNaSS grafting. Colorimetry, Fourier-transformed infrared spectra recorded in an attenuated total reflection, X-ray photoelectron spectroscopy techniques and contact angle measurements were applied to characterize the surfaces. MG63 osteoblastic cell response was studied on polished, oxidized and grafted titanium samples. Cell adhesion, alkaline phosphatase activity and calcium nodules formation were significantly enhanced on grafted titanium samples compared to unmodified surfaces.     

A novel nano-porous alumina biomaterial with potential for loading with bioactive materials

Nano-porous alumina, with the potential for being loaded with bioactive materials, has been proposed as a novel material for coating implants. In this study, the shear strength of the interface between such nano-porous anodic aluminium oxide (AAO) coatings and titanium substrates, their biocompatibility, and their potential for pore loading have been investigated. An interface shear strength in excess of 29 MPa was obtained which is comparable with that of conventional plasma sprayed hydroxyapatite implant coatings. The viability and differentiation of MG63 osteoblastic cells co-cultured on the coating was found to be broadly comparable to that of similar cells co-cultured on conventional bioinert implant materials such as titanium and fully dense alumina. Extensive pore loading with silica nano-particles of different sizes and in different combinations was demonstrated throughout the thickness of AAO layers 1 microm and 60 microm thick. This work has demonstrated, that with suitable choice of pore filling materials, this novel coating might simultaneously combat infection, encourage bone regeneration, and secure fixation of the implant to bone.     

Integrin specificity and enhanced cellular activities associated with surfaces presenting a recombinant fibronectin fragment compared to RGD supports

Biomimetic strategies focusing on presenting short bioadhesive oligopeptides, including the arginine-glycine-aspartic acid (RGD) motif present in numerous adhesive proteins, on a non-fouling support have emerged as promising approaches to improve cellular activities and healing responses. Nevertheless, these bio-inspired strategies are limited by low activity of the oligopeptides compared to the native ligand due to the absence of complementary or modulatory domains. In the present analysis, we generated well-defined biointerfaces presenting RGD-based ligands of increasing complexity to directly compare their biological activities in terms of cell adhesion strength, integrin binding and signaling. Mixed self-assembled monolayers of alkanethiols on gold were optimized to engineer robust supports that present anchoring groups for ligand tethering within a non-fouling, protein adsorption-resistant background. Controlled bioadhesive interfaces were generated by tethering adhesive ligands via standard peptide chemistry. On a molar basis, biointerfaces functionalized with the FNIII7-10 recombinant fragment presenting the RGD and PHSRN adhesive motifs in the correct structural context exhibited significantly higher adhesion strength, FAK activation, and cell proliferation rate than supports presenting RGD ligand or RGD-PHSRN, an oligopeptide presenting these two sites separated by a polyglycine linker. Moreover, FNIII7-10-functionalized surfaces displayed specificity for alpha5beta1 integrin, while cell adhesion to supports presenting RGD or RGD-PHSRN was primarily mediated by alphavbeta3 integrin. These results are significant to the rational engineering of bioactive materials that convey integrin binding specificity for directed cellular and tissue responses in biomedical and biotechnological applications.     

The effect of plasma chemical oxidation of titanium alloy on bone-implant contact in rats

Many different technologies have been used to enhance osseointegration in orthopaedic and dental implant surgery. Hydroxyapatite coatings, pure or in combination with growth factors or bisphosphonates, showed improved osseointegration of titanium alloy implants. We choose a different approach to enhance osseointegration: plasma chemical oxidation was used to modify the surface of titanium alloy implants. This technique converts the nm-thin natural occurring titanium oxide layer on an implant to a 4?μm thick ceramic coating (TiOB surface). Bioactive TiOB surfaces have a macroporous structure and were loaded with calcium and phosphorus, while bioinert TiOB surfaces are smooth. A rat tibial model with bilateral placement of titanium alloy implants was employed to analyze the bone response to TiOB surfaces in?vivo. 64 rats were randomly assigned to four groups of implants: (1) titanium alloy (control), (2) titanium alloy, type III anodization, (3) bioinert TiOB surface and (4) bioactive TiOB surface. Mechanical fixation, peri-implant-bone area and bone contact were evaluated by pull-out tests and histology at three and eight weeks. Shear strength and bone contact at eight weeks were significantly increased in the bioactive TiOB group compared to all other groups. The results of plasma chemical oxidation in a rat model showed that the bioactive TiOB surface has a positive effect on implant anchorage by enhancing the bone-implant contact in normal bone.     

Influence of titanium surfaces on attachment of osteoblast-like cells in vitro

Implant surface topography influences osteoblastic proliferation, differentiation and extracellular matrix protein expressions. Studies on preliminary interactions of osteoblast-like cells on implant interface through in vitro systems, can give lucid insights to osseo-integrative efficacies of when in vivo implants. In the present investigation two titanium surfaces of dental implants, a sandblasted and acid-etched surface and an experimental grooved surface were compared through in vitro systems. The titanium implants were seeded with osteoblast-like primary cells and maintained for a period of 1-7 days. Expressions of fibronectin and osteonectin were assessed through immunogold labelling by scanning electron microscopy. The grooved surface, supported better osteoblastic cell adhesion and proliferation than the rough surfaces. Further, osteoblastic cells on the grooved surfaces also displayed a strong labelling for fibronectin at the cytoplasmic extensions coupled with intense osteonectin expression in comparison to the rough surfaced implants. In conclusion, grooved surfaces offered better cell attachment and proliferation than the other rough surfaces studied.     

Experimental procedure for the evaluation of the mechanical properties of the bone surrounding dental implants.

The mechanical stability of the fixture in bone is one of the most important factors for the long-term reliability of dental implants. This paper focuses on an experimental procedure to evaluate the mechanical properties of the bone surrounding dental implants. The procedure is based on a surgical animal model followed by mechanical tests. The experimental mechanical testing has been used for preliminary investigations on the role played by different parameters such as the healing time and the surgical technique (standard or with regenerative material). The procedure has been evaluated in some preliminary tests on a few specimens. Microradiographic analyses have been performed on the bone surrounding the implants in order to give an interpretation of the bone properties on the basis of the bone morphology and to distinguish the newly formed bone from the pre-existing bone. The preliminary results relevant to 10 threaded titanium implants are presented and discussed. Our findings show that the mechanical properties of the bone surrounding the implant improve with the increase in the healing time from 24 to 45 days. The ultimate loads recorded during mechanical tests arise from 395 N to 2665 N in case of coronal defects filled with bone regenerative and from 2200 N to 5700 N in case of standard technique.     

The effect of surface modification of a porous TiO2/perlite composite on the ingrowth of bone tissue in vivo

The porous TiO2/perlite composite Ecopore is a synthetic biomaterial with possible clinical application in bone substitution. In our previous work, we demonstrated that surface modification of Ecopore with fibronectin (FN) enhanced spreading and growth of human osteoblasts in vitro. In the present study, we implanted untreated, alkaline-etched and FN-coated Ecopore cylinders into critical size defects of rabbit femora and applied pulsed polychrome sequence staining. After 6 weeks, sections of the implants were investigated via conventional and fluorescence microscopy. A partial ingrowth of bone matrix into the pore system of the Ecopore implants was observed. At the contact zones, the bone appeared to be directly connected to the implant without detectable gaps. Defect healing was complete within 6 weeks, while fibrous tissue generation or inflammation were absent in the implant modification groups, demonstrating basic Ecopore biocompatibility. The mean bone apposition rates within the implant cross-section were 4.1+/-0.6 microm/day (p<0.001) in the FN-coated group and 3.3+/-0.5 microm/day (p<0.05) in the NaOH-etched group. In both treated Ecopore modification groups, the apposition rates were significantly higher than in the non-modified control (2.9+/-0.6 microm/day), indicating bone growth stimulation by pre-treatment. Energy-dispersive X-ray analysis confirmed that significantly more bone tissue was formed inside the pores of the FN-coated implants compared to the unmodified control. The cross-sectional areas identified as ingrown bone amounted to 18.5+/-6.1% (p<0.05) in the FN group, 13.4+/-5.1% (p>0.05) in the NaOH-etched group and 10.2+/-5.5% in the unmodified group. In summary, we conclude that bone tissue tolerates Ecopore well and that tissue ingrowth can be enhanced by etching and coating with FN.     

In vivo behavior and mechanical stability of surface-modified titanium implants by plasma spray coating and chemical treatments

The geometric design and chemical compositions of an implant surface may have an important part in affecting early implant stabilization and influencing tissue healing. In this study, in vivo behavior and mechanical stability in implants of three surface designs, which were smooth surface (SS), rough titanium (Ti) surface by plasma spray coating (PSC), and alkali- and heat-treated (AHT) Ti surface after plasma spray coating, were compared by histological and mechanical analyses. Surface morphologies of the implants were observed by optical microscopy and scanning electron microscopy. Chemical compositional surface changes were investigated by energy dispersive spectroscopy. The implants were inserted transversely in a dog thighbone and evaluated at 4 weeks of healing. At 4 weeks of healing after implantation in bone, the healing tissue was more extensively integrated with an AHT implant than with the implants of smooth (SS) and/or rough Ti surfaces (PSC). The bone bonding strength (pull-out force) between living bone and implant was observed by a universal testing machine. At 4 weeks' healing after implant placement in bone, the pull-out forces of the SS, PSC, and AHT implants were 235 (+/-34.25), 710 (+/-142.25), and 823 (+/-152.22) N, respectively. Histological and mechanical data demonstrate that appropriate surface design selection can improve early bone growth and induce an acceleration of the healing response, thereby improving the potential for implant osseointegration.     

Identification of a bioactive core sequence from human laminin and its applicability to tissue engineering

Finding bioactive short peptides derived from proteins is a critical step to the advancement of tissue engineering and regenerative medicine, because the former maintains the functions of the latter without immunogenicity in biological systems. Here, we discovered a bioactive core nonapeptide sequence, PPFEGCIWN (residues 2678–2686; Ln2-LG3-P2-DN3), from the human laminin α2 chain, and investigated the role of this peptide in binding to transmembrane proteins to promote intracellular events leading to cell functions. This minimum bioactive sequence had neither secondary nor tertiary structures in a computational structure prediction. Nonetheless, Ln2-LG3-P2-DN3 bound to various cell types as actively as laminin in cell adhesion assays. The in vivo healing tests using rats revealed that Ln2-LG3-P2-DN3 promoted bone formation without any recognizable antigenic activity. Ln2-LG3-P2-DN3-treated titanium (Ti) discs and Ti implant surfaces caused the enhancement of bone cell functions in vitro and induced faster osseointegration in vivo, respectively. These findings established a minimum bioactive sequence within human laminin, and its potential application value for regenerative medicine, especially for bone tissue engineering.     

Callus formation and remodeling at titanium implants

Titanium is biocompatible with bone tissue, and during the healing process bone makes intimate contact with the implant surface. Although much is known about the long-term healing of implants, less is known about the callus formation at implants. In this study, the histology of bone healing was studied during the period between 4 and 14 days. Incisions were made in rat tibia. Some incisions were simply left to heal, while in others titanium discs were implanted. Smooth implants as well as implants with different porosities were used. After 4, 7, and 14 days of healing, the sites of bone incisions were retrieved, decalcified, sectioned, and stained. The aim was to compare normal fracture healing with implant healing and to see whether implant properties influenced the short-term healing process. Similarities between fracture healing and implant healing were evident. In both cases, inflammation, soft and hard callus formation, and remodeling had taken place during the period investigated. Between 7 and 14 days substantial bone resorption occurred around the implants. While after 14 days the marrow was almost completely reconstituted during normal wound healing, at the implants a thin layer of bone remained in close contact with the surface. Results from bone-implant contact measurements indicate that the surface properties of the implants do not have a significant influence on the early bone formation, since there were no significant differences between the smooth surface and any of the porous surfaces.     

The effect of the surface modification of titanium using a recombinant fragment of fibronectin and vitronectin on cell behavior

The surface of titanium implants is in direct contact with host tissue and plays a critical role in determining biocompatibility. Fibronectin (FN) and vitronectin (VN) are major cell adhesive proteins found in the extracellular matrix (ECM) of various tissues, and in circulating blood. The aim of this study was to evaluate the engineered biomimetic surface of titanium by using recombinant fragment of FN(8-10) and VN(NTD) that contains the binding site for integrins. MC3T3-E1 cells seeded upon the FN(8-10)-coated titanium showed a marked increase in cell adhesion, proliferation, and differentiation over VN(NTD)-coated titanium. In addition, we confirmed that the surface properties of titanium prefer for FN(8-10) over VN(NTD) (p<0.05) in protein adhesion. These results suggest that the FN(8-10)-modified titanium surface can be used to improve the osseointegration of titanium implants by enhancing bone formation.     

The effect of ultraviolet functionalization of titanium on integration with bone

Titanium implants are used as a reconstructive anchor in orthopedic and dental diseases and problems. Recently, ultraviolet (UV) light-induced photocatalytic activity of titanium has earned considerable and broad interest in environmental and clean-energy sciences. This study determines whether UV treatment of titanium enhances its osteoconductive capacity. Machined and acid-etched titanium samples were treated with UV for various time periods up to 48 h. For both surfaces, UV treatment increased the rates of attachment, spread, proliferation and differentiation of rat bone marrow-derived osteoblasts, as well as the capacity of protein adsorption, by up to threefold. In vivo histomorphometry in the rat model revealed that new bone formation occurred extensively on UV-treated implants with virtually no intervention by soft tissue, maximizing bone–implant contact up to nearly 100% at week 4 of healing. An implant biomechanical test revealed that UV treatment accelerated the establishment of implant fixation 4 times. The rates of protein adsorption and cell attachment strongly correlated with the UV dose-responsive atomic percentage of carbon on TiO₂, but not with the hydrophilic status. The data indicated that UV light pretreatment of titanium substantially enhances its osteoconductive capacity, in association with UV-catalytic progressive removal of hydrocarbons from the TiO₂ surface, suggesting a photofunctionalization of titanium enabling more rapid and complete establishment of bone–titanium integration.