In vivo evaluation of plasma sprayed hydroxyapatite coatings having different crystallinity

In this paper, hydroxyapatite (HA) coatings having the crystallinities of 56% and 98% were deposited by the plasma spraying and vapor-flame treatment process. The phase composition and crystallinity of the coatings were investigated by X-ray diffraction and infrared spectra. The dissolution behavior of the coatings in tris-buffer solutions was examined. The results obtained indicated that the coating having the high crystallinity showed the lower dissolution as compared to the low crystallinity coating. The bone bonding ability of HA coatings were observed in vivo by implanted in dog's femur. After 3 months implantation, the high crystallinity coating showed the higher shear strengths and remained integrated, whereas the separation of the coating fragments was clearly observed in the coating having low crystallinity.     

In vivo behaviour of hydroxyapatite coatings on titanium implants: a quantitative study in the rabbit

The aim of this study was to evaluate quantitatively the behaviour of in vivo hydroxyapatite coated implants (HA) in the rabbit over time, and to compare the results with observations made on titanium plasma spray implants (TPS). Results were analysed according to the percentage of bone contact. Eighteen HA cylindrical implants (3.25 x 8 mm) and 6 TPS cylindrical implants from Steri-Oss were placed in the epiphysis of the femur in 24 white rabbits. Each rabbit received one implant. Three rabbits with one HA implant (n = 3) and 1 rabbit with one TPS implant (n = 1) were sacrificed after implantation periods of 2, 4, 6, 8, 10 and 12 months. Implants were cut along the long axis and prepared for histological and histomorphometrical evaluations. Measurements of coating thickness and percentage of bone contact were performed with scanning electron microscopy analysis on the sides of the implant, in 3 different types of bone, namely cortical, trabecular and marrow. In cortical bone, dense bone was apposed to the HA implants: from 92.3 +/- 5.5% at 2 months to 89.6 +/- 6.5% at 1 year, with no significant regression of HA thickness (P = 0.37). TPS coating showed less bone contact, but thickness was stable (P = 0.46). In trabecular zone, where bone contact was less pronounced, a significant regression of HA coatings thickness (P < 0.05) was observed. Nevertheless TPS coatings were stable (P = 0.81). Histomorphometrical results demonstrated that a highly significant regression (P < 0.0001) of HA thickness was observed in the marrow area, where the bone-to-implant contact never exceeded 7.6% from 2 to 12 months. TPS coating did not reveal any sign of resorption (P = 0.88), despite a rare bone contact. Histological analysis revealed inflammatory and giant cells, principally in the marrow area in contact with HA coating, but always in restrictive numbers. We conclude that bone contact protected the HA coating from resorption.     

In vitro dissolution behaviour of two morphologically different thermally sprayed hydroxyapatite coatings

Two types of hydroxyapatite coating on titanium substrates were produced by vacuum plasma spraying and detonation gun spraying. The response of the coatings to immersion in Ringer's solution at pH 7.2 and held at 37 degrees C for periods of one, two, four and eight weeks was compared. X-ray diffraction was used to measure changes in crystallinity and residual stresses. Porosity and coating thickness were measured using optical microscopy combined with image analysis. Surface morphology was characterised using scanning electron microscopy and Talysurf measurements. The DGUN coatings were found initially to have a lower crystallinity and porosity but higher surface roughness and residual stress level than the VPS coatings. This resulted in a higher rate of dissolution compared to the more stable VPS coatings.     

Fluoridated hydroxyapatite coatings on titanium obtained by electrochemical deposition

Hydroxyapatite (HA) and fluoridated hydroxyapatite (FHA) coatings were deposited on titanium substrates using an electrochemical technique. Different concentrations of F^? ions were incorporated into the apatite structure by adding NaF into the electrolyte. Typical apatite structures were obtained for all the coatings after electrodeposition and subsequent post-treatment, including alkaline immersion and vacuum calcination. The coatings were uniform and dense, with a thickness of ～5 μm. When the F-concentration was higher than 0.012 M in the electrolyte, a saturation of F in the coating occurred and the F/Ca ratio in the coatings became almost constant (F/Ca ratio = 0.125). The FHA coatings showed higher bonding strength and lower dissolution rate than HA coating, particularly for those with a fluoridation level of 0.5–0.625. Compared with pure Ti, FHA and HA coatings exhibited higher biological affinity like cell proliferation and alkaline phosphatase activity. Regarding clinical application, it is suggested that a moderate content of F, such as Ca₅(PO₄)₃(OH)_0.375?0.5F_0.5?0.625, be most suitable as a compromise among cell attachment, cell proliferation, apatite deposition and dissolution resistance.     

Structural analysis of hydroxyapatite coatings on titanium

Hydroxyapatite from two sources was electrophoretically deposited onto flat titanium plate material. Depending upon the deposition conditions various changes in the structure of the ceramic were identified. A well-adhering Ti-P compound was present at the interface. Hydroxyapatite oxygenated to various degrees and tetracalcium phosphate were reproducibly formed in the coating.     

Early bone apposition in vivo on plasma-sprayed and electrochemically deposited hydroxyapatite coatings on titanium alloy

Three different implants, bare Ti-6Al-4V alloy, Ti-6Al-4V alloy coated with plasma-sprayed hydroxyapatite (PSHA), and Ti-6Al-4V alloy coated with electrochemically deposited hydroxyapatite (EDHA), were implanted into canine trabecular bone for 6 h, 7, and 14 days, respectively. Environmental scanning electron microscopy study showed that PSHA coatings had higher bone apposition ratios than those exhibited by bare Ti-6Al-4V and EDHA coatings after 7 days; however, at 14 days after implantation, EDHA and PSHA coatings exhibited similar bone apposition ratios, much higher than that for bare Ti-6Al-4V. The ultrastructure of the bone/implant interface observed by transmission electron microscope showed that the earliest mineralization (6 h-7 days) was in the form of nano-ribbon cluster mineral deposits with a Ca/P atomic ratio lower than that of hydroxyapatite. Later-stage mineralization (7-14 days) resulted in bone-like tissue with the characteristic templating of self-assembled collagen fibrils by HA platelets. Though adhesion of EDHA coatings to Ti-6Al-4V substrate proved problematical and clearly needs to be addressed through appropriate manipulation of electrodepositon parameters, the finely textured microstructure of EDHA coatings appears to provide significant advantage for the integration of mineralized bone tissue into the coatings.     

Porous titanium and silicon-substituted hydroxyapatite biomodification prepared by a biomimetic process: Characterization and in vivo evaluation

Porous titanium with a pore size of 150–600 μm and a porosity of 67% was prepared by fiber sintering. The porous titanium had a complete three-dimensional (3D) interconnected structure and a high yield strength of 100 MPa. Si-substituted hydroxyapatite (Si-HA) was coated on the surface by a biomimetic process to improve the surface bioactivity. X-ray diffraction results showed that Si-HA coating was not well crystallized. New bone tissue was found in the uncoated porous titanium after 2 weeks of implantation and a significant increase (p < 0.05) in the bone ingrowth rate (BIR) was found after 4 weeks of implantation, indicating the good osteoconductivity of the porous structure. The HA-coated and Si-HA-coated porous titanium exhibited a significantly higher BIR than the uncoated titanium at all intervals, highlighting the better surface bioactivity and osteoconductivity of the HA- and Si-HA coatings. Also, the Si-HA-coated porous titanium demonstrated a significantly higher BIR than the HA-coated porous titanium, showing that silicon plays an active role in the surface bioactivity. For Si-HA-coated porous titanium, up to 90% pore area was covered by new bone tissue after 4 weeks of implantation in cortical bone. In the bone marrow cavity, the pore spaces were filled with bone marrow, displaying that the interconnected pore structure could provide a channel for body fluid. It was concluded that both the 3D interconnected pore structure and the Si-HA coating contributed to the high BIR.     

Comparison of plasma-sprayed hydroxyapatite coatings and zirconia-reinforced hydroxyapatite composite coatings: in vivo study

The clinical use of plasma-sprayed hydroxyapatite (HA) coatings on metal implants has been widely adopted because the HA coating can achieve the firmly and directly biological fixation with the surrounding bone tissue. However, the long-term mechanical properties of HA coatings has been concern for the long-term clinical application. Previous research showed that the concept of adding ZrO2 as second phase to HA significantly increased the bonding strength of plasma-sprayed composite material. The present work aimed to explore the biological properties, including the histological responses and shear strength, between the plasma-sprayed HA and HA/ZrO2 coating, using the transcortical implant model in the femora of canines. After 6 and 12 weeks of implantation, the HA coating revealed the direct bone-to-coating contact by the backscattered electron images (BEIs) of scanning electron microscope (SEM), but the osseointegration was not observed at the surface of HA/ZrO2 coating. For new bone healing index (NBHI) and apposition index (AI), the values for HA implants were significantly higher than that for HA/ZrO2 coatings throughout all implant periods. After push-out test, the shear strength of HA-coated implants were statistically higher than HA/ZrO2 coated implants at 6- and 12-week implantation, and the failure mode of HA/ZrO2 coating was observed at the coating-bone interface by SEM. The results indicate that the firm fixation between bone and HA/ZrO2 has not been achieved even after 12-week implantation. Consequently, the addition of ZrO2 could improve the mechanical properties of coatings, while the biocompatibility was influenced by the different material characteristics of HA/ZrO2 coating compared to HA coatings.     

Silver/hydroxyapatite composite coatings on porous titanium surfaces by sol-gel method

Hydroxyapatite (HA) coatings loaded with nanosilver particles is an attractive method to impart the HA coating with antibacterial properties. Producing Ag/HA coatings on porous Ti substrates have been an arduous job since commonly used line-of-sight techniques are not able to deposit uniform coatings on the inner pore surfaces of the porous Ti. In this study, porous Ti scaffolds with high porosity and interconnected structures were prepared by polymer impregnating method. A sol-gel process was used to produce uniform Ag/HA composite coatings on the surfaces of porous Ti substrates. Ca(NO(3) )(2) ·4H(2) O and P(2) O(5) in an ethyl alcohol based system was selected to prepare the sol, which ensured the homogeneous distribution of Ag in the sol. The characterization revealed that silver particles uniformly distributed in the coatings without agglomeration. High antibacterial ratio (>95%), against E. coli and S. albus was expressed by the silver-containing coatings (Ag/HA 0.8 and 1.6 wt %). The biocompatibility of the Ag/HA 0.8 surfaces was as good as that of pure HA surface, as revealed by culturing osteoblasts on them. The results indicated that Ag/HA 0.8 had the good balance between the biocompatibility and antibacterial properties of the coatings.     

Stress influence on interface in plasma-sprayed hydroxyapatite coatings on titanium alloy

During clinical use of hydroxyapatite-coated implants, mechanical stresses are added to pre-existing residual stresses. The magnitude of these stresses affects the coating's performance. In this work we studied, by neutron diffraction and conventional X-ray diffraction methods, the macrostresses induced by a plasma-spraying process in the coating and at the interface. Neutron diffraction is one of the most suitable techniques for studying the strain distribution in a bulk material. X-ray diffraction was used to determine residual stress in the hydroxyapatite coating. Using a neutron diffraction method at the nearest point to the interface, we saw no real difference between the stress observed on coated and that on non-coated samples. With the X-ray diffraction method, it appeared that the stress level was compressive on every sample. The major advantage of the neutron diffraction method is that measurements can be made on a thick coating in a nondestructive way. The disadvantage is the large-gauge volume that we had to use because of the relatively low intensity of the neutron beam. Polishing is necessary for measurements inside the material when using the X-ray method. This destructive method may alter the stress field of the deposit.     

Hydrothermal crystallization of carbonate-containing hydroxyapatite coatings prepared by radiofrequency-magnetron sputtering method

Carbonate-containing hydroxyapatite (HA) films were prepared by low-temperature hydrothermal annealing from carbonate-containing calcium phosphate amorphous coatings on titanium substrates. The biocompatibility of the carbonate-containing HA layers was estimated by in vitro tests using simulated body fluid (SBF). Precursory amorphous coatings were deposited with rf-magnetron sputtering apparatus, using calcium phosphate glass target in Ar/CO2 atmosphere. The carbonate-containing HA coatings were successfully formed by the annealing at above 130 degrees C for 20 h. On the basis of SEM observation, about 2-microm thickness films coated rigidly were durable enough for the hydrothermal treatment. The coating layer was revealed to consist of single phase of PO4(-) and OH- partially carbonated HA by XRD and IR analyses. Overgrowing of bone-like apatite layers on the carbonate-containing HA surfaces in the SBF implied that the obtained films acquired a sufficient osteoconductivity, while it was still unclear that activity was enhanced, compared to pure HA coatings. The low-temperature hydrothermal annealing method was effective for preparation of rigid HA coatings on titanium as well as modification of their chemical compositions.     

In vitro and in vivo biological responses of plasma-sprayed hydroxyapatite coatings with posthydrothermal treatment

This study was undertaken to evaluate the effect of post-hydrothermal treatment on the biological responses of the plasma-sprayed hydroxyapatite (HA)-coated Ti-6Al-4V implant system both in vitro and in vivo. After hydrothermal treatment, the HA coating (HAC) shows the high mechanical strength and indices-of-crystallinity, denser microstructure, lower concentrations of amorphous and impurity phases, when compared with the as-sprayed HAC. The in vitro cell-culture studies, using UMR106 osteoblast-like cell, demonstrated no signifiacnt cell growth on both surface of as-sprayed and hydrothermal-treated HACs during 10-day culture. The in vivo studies, using the transcortical implant model in the femora of goats, evaluated the histological responses of two coatings. After 6 week of implantation, using backscattered electron images, no substantial histological variations in the extents of new bone apposition and new bone healing between the two HACs were observed. However, the as-sprayed HAC, owing to the dissolution induced the granular particles dissociated from the HAC, showed the statically lower extent of new bone apposition than hydrothermal-treated HAC at 12 weeks. The results suggest that hydrothermal treatment could be used to improve the mechanical strength, crystallinity, and phase composition of HAC, which are important factors of long-term fixation and stability of implant. Besides, the treated HAC could also achieve the initial fixation of implant in clinical use.     

Nanocrystalline hydroxyapatite/titania coatings on titanium improves osteoblast adhesion

Bulk hydroxyapatite (HA) and titania have been used to improve the osseointegration of orthopedic implants. For this reason, composites of HA and titania have been receiving increased attention in orthopedics as novel coating materials. The objective of this in vitro study was to produce nanophase (i.e., materials with grain size less than 100 nm) HA/titania coatings on titanium. The adhesion of bone forming cells (osteoblasts) on the composite coatings were also assessed and compared with single-phase nanotitania and nano-HA titanium coatings. Nanocrystalline HA powders were synthesized through wet chemistry and hydrothermal treatments at 200 degrees C. Nanocrystalline titania powders obtained commercially were mixed with the nanocrystalline HA powders at various weight ratios. The mixed powders were then deposited on titanium utilizing a room-temperature coating process called IonTite. The results of the present study showed that such coatings maintained the chemistry and crystallite size of the original HA and titania powders. Moreover, osteoblasts adherent on single-phase nanotitania coatings were well-spread whereas they became more round and extended distinct filopodia on the composite and single-phase HA coatings. Interestingly, the number of osteoblasts adherent on the nanotitania/HA composite coatings at weight ratios of 2/1 and 1/2 were significantly greater compared with single-phase nanotitania coatings, currently-used plasma-sprayed HA coatings, and uncoated titanium. These findings suggest that nanotitania/HA coatings on titanium should be further studied for improved orthopedic applications.     

Comparison of plasma-sprayed hydroxyapatite coatings and hydroxyapatite/tricalcium phosphate composite coatings: in vivo study

This study aimed to compare biological properties, including osteoconduction, osseointegration, and shear strength, between plasma-sprayed hydroxyapatite (HA) and HA/tricalcium phosphate (TCP) coatings, using a transcortical implant model in the femora of canines. After 3 and 12 weeks of implantation, the implants with surrounding bone were assessed histologically in undecalcified sections in backscattered electron images (BEIs) under a scanning electron microscope (SEM). After short-term (3 week) follow-up, both coatings conducted new bone formation and revealed direct bone-to-coating contact. The HA/TCP coating could not enhance early host-to-coating responses. At 12 weeks, serious dissolution of the HA/TCP coatings evidently occurred. By the new bone healing index (NBHI) and apposition index (AI), we found no significant difference between HA/TCP-coated implants and HA-coated implants throughout all implant periods. At 12 weeks of implantation, some particles dissociated from the HA/TCP coating were found within the remodeling canal. After push-out measurements, the shear strength and failure mode of HA/TCP-coated implants were similar to those of HA-coated implants, and no statistical differences were found between either coating. Consequently, this study indicates that HA/TCP coatings have excellent biological response and may be considered suitable bioactive ceramic coatings for short-term clinical use.     

Electrochemical preparation of chitosan/hydroxyapatite composite coatings on titanium substrates

Composite coatings containing brushite (CaHPO(4). 2H(2)O) and chitosan were prepared by electrochemical deposition. The brushite/chitosan composites were converted to hydroxyapatite/chitosan composites in aqueous solutions of sodium hydroxide. The coatings ranged from approximately 1 to 15% chitosan by weight. Qualitative assessment of the coatings showed adhesion significantly improved over that observed for electrodeposited coatings of pure HA.     

In vivo dissolution behavior of various RF magnetron-sputtered Ca-P coatings on roughened titanium implants

RF magnetron sputter deposition was used to produce 0.1, 1.0 and 4.0 microm thick Ca-P coatings on TiO(2)-blasted titanium discs. Half of the as-sputtered coated specimens were subjected to an additional infrared heat treatment for 30s at 425-475 degrees C. X-ray diffraction demonstrated that infrared radiation changed the amorphous 4 microm sputtered coatings into an amorphous-crystalline structure, while the amorphous 0.1 and 1 microm changed in a crystalline apatite structure with the presents of tetracalciumphosphate as a second phase. Scanning electron microscopically examination of the sputtered coatings revealed that annealing of the 4 microm thick coatings resulted in the appearance of small cracks. Subsequently, the discs were implanted subcutaneous into the back of rabbits. After 1, 4, 8 and 12 weeks of implantation, the implants were retrieved and prepared for histological and physicochemical evaluation. Histological evaluation revealed that the tissue response to all coated implants was very uniform. A very thin connective tissue capsule surrounded all implants. The capsule was usually free of inflammatory cells. At the interface, there was a close contact between the capsule and implant surface and no inflammatory cells were seen. Physicochemical evaluation showed that the 0.1 and 1 microm thick amorphous coatings had disappeared within 1 week of implantation. On the other hand, the 4 microm thick amorphous phase disappeared during the implantation periods, which was followed by the precipitation of a crystalline carbonate apatite. Further, at all implantation periods the heat-treated 1 and 4 microm thick coatings could be detected. Occasionally, a granular precipitate was deposited on the heat-treated 4 microm thick coating. Fourier transform infrared spectroscopy showed the formation of carbonate apatite (CO(3)-AP) on the 4 microm thick amorphous coating and on the heat-treated specimens. On basis of our findings, we conclude that 1 microm thick heat-treated Ca-P sputter coating on roughened titanium implants appear to be of sufficient thickness to show bioactive properties, under in vivo conditions.     

Nanocrystalline hydroxyapatite coatings on titanium: a new fast biomimetic method

We obtained a fast biomimetic deposition of hydroxyapatite (HA) coatings on Ti6Al4V substrates using a slightly supersaturated Ca/P solution, with an ionic composition simpler than that of simulated body fluid (SBF). At variance with other fast deposition methods, which produce amorphous calcium phosphate coatings, the new proposed composition allows one to obtain nanocrystalline HA. Soaking in supersaturated Ca/P solution results in the deposition of a uniform coating in a few hours, whereas SBF, or even 1.5SBF, requires 14 days to deposit a homogeneous coating on the same substrates. The coating consists of HA globular aggregates, which exhibit a finer lamellar structure than those deposited from SBF. The extent of deposition increases on increasing the immersion time. Transmission electron microscope (TEM) images recorded on the material detached from the coating show that the deposition is constituted of thin nanocrystals. Electron diffraction (ED) patterns recorded from most of the crystals exhibit the presence of rings, which can be indexed as reflections characteristic of HA. Furthermore, several HA single-crystal spot ED images were obtained from individual crystals.     

In vivo degradability of hydrogels prepared from different gelatins by various cross-linking methods

This study is an investigation to evaluate the in vivo degradation of gelatin hydrogels in terms of their number of cross-links. Various hydrogels were prepared from acidic gelatin, extracted from bovine bone, porcine skin or fish scale, and basic gelatin, extracted from porcine skin, through four types of cross-linking methods, i.e., glutaraldehyde (GA) or dehydrothermal treatment and ultraviolet (UV) or electron beam irradiation. The water content of hydrogels and their number of cross-links, calculated from the tensile modulus of hydrogels, were evaluated as the measure of hydrogel cross-linking extent. Following subcutaneous implantation of ¹²⁵I-labeled gelatin hydrogels into mice, the radioactivity remaining was measured at different time intervals to assess the in vivo degradability of hydrogels. Irrespective of the gelatin type and cross-linking method, a good correlation was found between the in vivo degradability of hydrogels and their number of cross-links, which is different from the correlation to their water content. This finding indicates that the degradability of hydrogels is governed by their number of cross-links.     

In vivo degradability of hydrogels prepared from different gelatins by various cross-linking methods

This study is an investigation to evaluate the in vivo degradation of gelatin hydrogels in terms of their number of cross-links. Various hydrogels were prepared from acidic gelatin, extracted from bovine bone, porcine skin or fish scale, and basic gelatin, extracted from porcine skin, through four types of cross-linking methods, i.e., glutaraldehyde (GA) or dehydrothermal treatment and ultraviolet (UV) or electron beam irradiation. The water content of hydrogels and their number of cross-links, calculated from the tensile modulus of hydrogels, were evaluated as the measure of hydrogel cross-linking extent. Following subcutaneous implantation of 125I-labeled gelatin hydrogels into mice, the radioactivity remaining was measured at different time intervals to assess the in vivo degradability of hydrogels. Irrespective of the gelatin type and cross-linking method, a good correlation was found between the in vivo degradability of hydrogels and their number of cross-links, which is different from the correlation to their water content. This finding indicates that the degradability of hydrogels is governed by their number of cross-links.     

Characterization and stability of hydroxyapatite coatings prepared by an electrodeposition and alkaline-treatment process

Hydroxyapatite (HA) coatings on titanium alloy substrates were prepared by an alkaline treatment of electrodeposited precursors. The structure, residual stress, and bond strength of the coatings were investigated. Test results showed that the coatings processed in this study exhibited fairly low tensile residual stress, high crystallinity, and were free of an amorphous phase. The bond strength of the coatings increased with the decrease of current density in the range of 0.2-15 mA/cm(2), and reached 14 MPa at 0.2 mA/cm(2). Evaluation of the coatings was performed together with the evaluation of the plasma-sprayed HA coatings immersed in distilled water. It was revealed that the dissolution and bond strength degradation of the coatings were much lower than those of the plasma-sprayed HA coatings.