Early Bone Healing on Hydroxyapatite-Coated and Chemically-Modified Hydrophilic Implant Surfaces in an Ovine Model.

Implant topography affects early peri-implant bone healing by changing the osteoconduction rate in the surrounding biological environment. Implant surfaces have been designed to promote faster and stronger bone formation for rapid and stable prosthesis loading. Early peri-implant bone healing has been observed with a sandblasted, acid-etched implant that was chemically modified to be hydrophilic (cmSLA). The present study investigates whether early peri-implant bone healing extends to a rough surface implant with a high crystalline hydroxyapatite surface (TSV MP-1 HA). Three implants were randomly placed in porous trabecular bone within both medial femoral condyles of 10 sheep. Early peri-implant bone stability was measured at 3- and 6-weeks healing time following implant insertion. Results indicated a similar implant stability quotient between the implants at insertion and over time. The significant increase over time of reverse torque values with respect to insertion torque (p < 0.001) did not differ between the implants. However, the bone-to-implant contact of TSV MP-1 HA was significantly higher than that of cmSLA implants at 6 weeks (p < 0.01). These data validate previous findings of a hydrophilic implant surface and extend the observation of early osseointegration to a rough surface implant in porous trabecular bone.

Comparison of the Effects of Tissue Processing on the Physicochemical Properties of Bone Allografts.

Purpose: To address the hypothesis that the tissue processing methods of solvent dehydration and freeze-drying would differentially affect the physicochemical characteristics of four commercially available bone allografts and the adhesion and differentiation of human bone marrow-derived mesenchymal stromal cells (hBMSCs) on such substrates in vitro. Materials and Methods: The surface morphology, surface area, and elemental composition of four commercially available cancellous bone allografts were examined using SEM, Brunauer-Emmett-Teller (BET) gas adsorption, and inductively coupled plasma (ICP) analyses. SEM was also employed to compare the allograft surfaces to that of human bone exposed by in vitro osteoclastic resorption. The allografts were seeded with hBMSCs, and the number of adhered cells was assessed at 3 and 7 days. Alkaline phosphatase (ALP) activity was quantified as a measure of osteogenic differentiation after 21 days. Results: Marked differences were seen between the physicochemical characteristics of the solvent-dehydrated and freeze-dried allografts, as well as between their resulting bone microarchitectures and that of osteoclast-resorbed human bone. Increased hBMSC adhesion and differentiation were observed on the solvent-dehydrated allografts compared to freeze-dried allografts, which suggests a higher putative osteogenic potential. The latter was attributed to better preservation of the bone collagen microarchitecture integrity, which may provide not only a more complex substrate architecture, but also a more favorable microenvironment to allow nutrients and oxygen to flow to the adhered cells. Conclusion: Commercially available cancellous bone allografts significantly differ in their physicochemical characteristics, stemming from differences in tissue processing and sterilization methods undertaken by tissue banks. These differences impact the response of MSCs in vitro and may alter the biologic performance of the grafts in vivo. Therefore, it is important to consider these characteristics when choosing a bone substitute for clinical application, as the physicochemical properties of the grafts play a crucial role in their interactions with the biologic environment and subsequent incorporation into the native bone.

Formation of OTS self-assembled monolayers at chemically treated titanium surfaces.

Enhanced biocompatibility of titanium implants highly depends on the possibility of achieving high degrees of surface functionalization for a low immune response and/or enhanced mineralization of bioactive minerals, such as hydroxyapatite. In this respect, surface modification with Self Assembled Monolayers (SAMs) has a great potential in delivering artificial surfaces of improved biocompatibility. Herein, the effectiveness of common chemical pre-treatments, i.e. hydrogen peroxide (H(2)O(2)) and Piranha (H(2)SO(4) + H(2)O(2)), in facilitating surface decontamination and hydroxylation of titanium surfaces to promote further surface functionalization by SAMs is investigated. The quality of the octadecyltrichlorosilane (OTS) based SAM appeared to strongly depend upon the surface morphology, the density and nature of surface hydroxyl sites resulting from the oxidative pre-treatments. Contrary to common belief, no further hydroxylation of the titanium substrate was observed after the selected chemical pre-treatments, but the number of hydroxyl groups available on the surface was decreased as a result of the formation of a titanium oxide layer with a gel-type structure. Further examinations by atomic force microscopy, infrared spectroscopy and X-ray photoelectron spectroscopy also revealed that mild oxidizing conditions were sufficient to remove surface contamination without detrimental effects on surface hydroxylation state and surface roughness. Furthermore, the adsorption of the alkylsiloxane molecules forming the SAM film is believed to proceed through hydrolysis at surface acidic hydroxyl groups rather than randomly. This site dependent adsorption process has significant implications for further functionalization of titanium based implants. It also highlights the difficulty of achieving an OTS based SAM at the surface of titanium and question the quality of SAMs reported at titanium surfaces so far.

The influence of implant design on the kinetics of osseointegration and bone anchorage homeostasis.

Titanium implants have shown considerable success in terms of achieving quick and long-lasting stability in bone through the process of osseointegration. Further work aims to improve implant success rates by modifying implant design on the nano-, micro-, and macro- scales with the goal of achieving higher levels of bone anchorage more quickly. However, the most frequently used methods of analysis do not investigate bone anchorage as a whole but as a series of discrete points, potentially missing relevant insight which could inform the effects of topography on these 3 scale ranges. Herein we utilize an asymptotic curve fitting method to obtain a biologically relevant description of reverse torque data and compare the anchorage of 12 different implant groups. Implant surface topography had a significant effect on the rate and degree of anchorage achieved during the initial bone formation period of osseointegration but was not found to influence the relative change in anchorage during bony remodeling. Threaded implants significantly decreased the time required to reach peak anchorage compared to non-threaded implants and implants with micro-topographically complex surfaces required greater torque to be removed than implants without such features. Nanotopography increased overall anchorage and decreased the time required to reach peak anchorage but to a lesser degree than microtopography or macrogeometry respectively. The curve fitting method utilized in the present study allows for a more integrated analysis of bone anchorage and permits investigation of osseointegration with respect to time, which may lead to a more targeted approach to implant design.

Tau (τ): A New Parameter to Assess the Osseointegration Potential of an Implant Surface.

PURPOSE: Osseointegration has been defined in many ways, from both basic science and clinical perspectives, but generally represents the restoration of bony homeostasis following implant placement and is usually judged by some form of bone/implant disruption test. In this study, bone anchorage to two different implant surfaces, in tensile and shear modes, was compared to investigate the relation between implant surface topography and osseointegration over time. The purpose was to determine if mathematical parameters could be derived that would reflect the biologic relevance of the implant surface design. MATERIALS AND METHODS: Rectangular titanium implants (n = 244) were placed in the distal femora of 122 male Wistar rats proximal to the knee joint. Implants were either microsurfaced (MS) or nanosurfaced (NS). Animals were euthanized at one of six time points ranging from 5 days to 6 months, and the force required to disrupt the bone implant interface, in either shear or tension, was measured using an Instron machine. Data were analyzed by fitting the function F = C (1-e-x/τ), where F is the measured disruption force, C is the predicted average maximum disruption force, x is the time postimplantation, and τ is a time constant defined as the time required for F to reach 63.2% of C. RESULTS: Analysis showed that shear testing resulted in significantly larger values of C than seen in tension, but no significant difference was observed when comparing the values of C for NS and MS implants in shear (P = .7). Thus, in accord with clinical reports, both implants performed equivalently at longer implantation periods. The differences in C were significant in tension (P < .05). Importantly, NS implants had a significantly smaller τ than the MS implants (P < .01, in shear), but no significant differences were observed in τ due to mechanical testing vector. The disruption force values reached a plateau with time, representing bony homeostasis as a result of osseointegration. With time, both implant surfaces reached the same maximum (C) values, in shear. However, the value of τ was smaller in NS compared to MS implants, which represented a higher rate of osseointegration. CONCLUSION: Thus, τ emerges as a measureable and biologically relevant parameter that can be employed to compare the osseointegration potential of putative implant surfaces.

Early bone anchorage to micro- and nano-topographically complex implant surfaces in hyperglycemia.

UNLABELLED: The aim of this work was to investigate the effect of implant surface design on early bone anchorage in the presence of hyperglycemia. 108 Wistar rats were separated into euglycemic (EG) controls and STZ-treated hyperglycemic (HG) groups, and received bilateral femoral custom rectangular implants of two surface topographies: grit blasted (GB) and grit-blast with a superimposed calcium phosphate nanotopography (GB-DCD). The peri-implant bone was subjected to a tensile disruption test 5, 7, and 9days post-operatively (n=28/time point); the force was measured; and the residual peri-implant bone was observed by scanning electron microscopy (SEM). Disruption forces at 5days were not significantly different from zero for the GB implants (p=0.24) in either metabolic group; but were for GB+DCD implants in both metabolic groups (p<0.001). Contact osteogenesis was greater on GB-DCD than the GB surface. The nano-and micro-surfaced implants showed significantly different disruption forces at all time points (e.g. >15N and <5N respectively at 9days). Such differences were not seen within the GB implants, as all values were very low (<5N). Even in hyperglycemia the GB-DCD surface outperformed the GB surfaces in both metabolic groups. Significantly, SEM of peri-implant bone showed compromised intra-fibrillar collagen mineralization in hyperglycemia, while inter-fibrillar and cement line mineralization remained unaffected. Enhanced bone anchorage to the implant surfaces was observed on the nanotopographically complex surface independent of metabolic group. The compromised intra-fibrillar mineralization observed provides a mechanism by which early bone mineralization is affected in hyperglycemia. STATEMENT OF SIGNIFICANCE: It is generally accepted that the hyperglycemia associated with diabetes mellitus compromises bone quality, although the mechanism by which this occurs is unknown. Uncontrolled hyperglycemia is therefore a contra-indication for bone implant placement. It is also known that nano-topographically complex implant surfaces accelerate early peri-implant healing. In this report we show that, in our experimental model, nano-topographically complex surfaces can mitigate the compromised bone healing seen in hyperglycemia. Importantly, we also provide a mechanistic explanation for compromised bone quality in hyperglycemia. We show that intra-fibrillar collagen mineralization is compromised in hyperglycemia, but that interfibrillar and cement line mineralization, remain unaffected.

An improved mechanical testing method to assess bone-implant anchorage.

Recent advances in material science have led to a substantial increase in the topographical complexity of implant surfaces, both on a micro- and a nano-scale. As such, traditional methods of describing implant surfaces - namely numerical determinants of surface roughness - are inadequate for predicting in vivo performance. Biomechanical testing provides an accurate and comparative platform to analyze the performance of biomaterial surfaces. An improved mechanical testing method to test the anchorage of bone to candidate implant surfaces is presented. The method is applicable to both early and later stages of healing and can be employed for any range of chemically or mechanically modified surfaces - but not smooth surfaces. Custom rectangular implants are placed bilaterally in the distal femora of male Wistar rats and collected with the surrounding bone. Test specimens are prepared and potted using a novel breakaway mold and the disruption test is conducted using a mechanical testing machine. This method allows for alignment of the disruption force exactly perpendicular, or parallel, to the plane of the implant surface, and provides an accurate and reproducible means for isolating an exact peri-implant region for testing.

Topographic scale-range synergy at the functional bone/implant interface.

We sought to explore the biological mechanisms by which endosseous implant surface topography contributes to bone anchorage. To address this experimentally, we implanted five groups of custom-made commercially pure titanium implants of varying surface topographical complexity in rat femora for 9 days; subjected them to mechanical testing; and then examined the interfacial bone matrix by electron microscopy. The five implant surfaces were prepared by combinations of dual acid etching and grit blasting the titanium substrates and, in some cases, modifying the created surfaces with the deposition of nanocrystals of calcium phosphate, which resulted in 10 samples per group. In parallel, we cultured rat bone marrow cells on surrogate implants constructed from polymer resin coated with the same calcium phosphate nanocrystals, and monitored the deposition of bone sialoprotein by transmission electron immunohisto-micrography. We found that implant samples modified with sub-micron scale crystals were bone-bonding, as described by the interdigitation of a mineralized cement line matrix with the underlying implant surface. The in vitro assay showed that bone sialoprotein could be deposited in the interstices between, and undercuts below, the nanocrystals. In addition, when mineralized, the cement line matrix globules occupied micron-sized pits in the implant surfaces, and in part obliterated them, creating an additional form of anchorage. Our results also showed that collagen, elaborated by the osteogenic cells, wrapped around the coarse-micron features, and became mineralized in the normal course of bone formation. This provided a mechanism by which coarse-micron implant features contributed to a functional interface, which we have previously described, that is capable of resisting the mechanical loading that increases as peri-implant bone matures. Thus, our findings provide mechanistic explanations for the biologically-relevant criteria that can be employed to assess the importance of implant surface topography at different scale-ranges.

The roles of different scale ranges of surface implant topography on the stability of the bone/implant interface.

We sought to deconvolute the effects of sub-micron topography and microtopography on the phenomena of bone bonding and interfacial stability of endosseous implants. To address this experimentally, we implanted custom-made titanium alloy implants of varying surface topographical complexity in rat femora, for 6, 9 or 12 days. The five surfaces were polished, machined, dual acid etched, and two forms of grit blasted and acid etched; each surface type was further modified with the deposition of nanocrystals of calcium phosphate to make a total of 10 materials groups (n = 10 for each time point; total 300 implants). At sacrifice, we subjected the bone-implant interface to a mechanical disruption test. We found that even the smoothest surfaces, when modified with sub-micron scale crystals, could be bone-bonding. However, as locomotor loading through bone to the implant increased with time of healing, such interfaces failed while others, with sub-micron features superimposed on surfaces of increasing microtopographical complexity remained intact under loading. We demonstrate here that higher order, micron or coarse-micron, topography is a requirement for longer-term interfacial stability. We show that each of these topographical scale-ranges represents a scale-range seen in natural bone tissue. Thus, what emerges from an analysis of our findings is a new means by which biologically-relevant criteria can be employed to assess the importance of implant surface topography at different scale-ranges.

Calcium phosphate growth at electropolished titanium surfaces.

This work investigated the ability of electropolished Ti surface to induce Hydroxyapatite (HA) nucleation and growth in vitro via a biomimetic method in Simulated Body Fluid (SBF). The HA induction ability of Ti surface upon electropolishing was compared to that of Ti substrates modified with common chemical methods including alkali, acidic and hydrogen peroxide treatments. Our results revealed the excellent ability of electropolished Ti surfaces in inducing the formation of bone-like HA at the Ti/SBF interface. The chemical composition, crystallinity and thickness of the HA coating obtained on the electropolished Ti surface was found to be comparable to that achieved on the surface of alkali treated Ti substrate, one of the most effective and popular chemical treatments. The surface characteristics of electropolished Ti contributing to HA growth were discussed thoroughly.

Functionalization of electropolished titanium surfaces with silane-based self-assembled monolayers and their application in drug delivery.

This work reports a novel and reproducible route for the successful modification of the surface of titanium (Ti) with self-assembled monolayers (SAMs). By electropolishing the surface of Ti, suitable physical/chemical surface properties were obtained for adequate growth of OctadecylTrichloroSilane (OTS) based SAM. Optimum conditions to achieve a well-organized and densely packed OTS film were also determined by monitoring the effect of different parameters including time, concentration, and temperature for OTS adsorption. The optimum conditions for the formation of an OTS-SAM were found to be upon immersion of the electropolished Ti substrate in a 10mM OTS solution at 10°C for 24h. Furthermore, multiple growth regimes for the formation of OTS-SAM on electropolished Ti surface were observed. The kinetics for the self-assembly were fast at the beginning of OTS adsorption, but rapidly slowed down after 10h of immersion, i.e. during the densification process of the film at the surface of Ti. In addition, the growth behavior was found to be random as opposed to the island growth behavior usually observed with OTS at the surface of silica. The successful implementation of OTS-SAM was further investigated through the immobilization and delivery of a model drug and the OTS monolayer showed clear abilities in drug delivery with an initial burst release up to 5 days followed by a sustained release up to 26 days.