A comparative study of zirconium and titanium implants in rat: osseointegration and bone material quality

Permanent metal implants are widely used in human medical treatments and orthopedics, for example as hip joint replacements. They are commonly made of titanium alloys and beyond the optimization of this established material, it is also essential to explore alternative implant materials in view of improved osseointegration. The aim of our study was to characterize the implant performance of zirconium in comparison to titanium implants. Zirconium implants have been characterized in a previous study concerning material properties and surface characteristics in vitro, such as oxide layer thickness and surface roughness. In the present study, we compare bone material quality around zirconium and titanium implants in terms of osseointegration and therefore characterized bone material properties in a rat model using a multi-method approach. We used light and electron microscopy, micro Raman spectroscopy, micro X-ray fluorescence and X-ray scattering techniques to investigate the osseointegration in terms of compositional and structural properties of the newly formed bone. Regarding the mineralization level, the mineral composition, and the alignment and order of the mineral particles, our results show that the maturity of the newly formed bone after 8 weeks of implantation is already very high. In conclusion, the bone material quality obtained for zirconium implants is at least as good as for titanium. It seems that the zirconium implants can be a good candidate for using as permanent metal prosthesis for orthopedic treatments.     

Early tissue response to titanium implants inserted in rabbit cortical bone

The tissue response to screw-shaped implants of commercially pure titanium was studied by light microscopy 3–180 days after insertion in the rabbit tibia. The implant site in the tibial metaphysis consisted mainly of cortical bone. Three days after implantation, osteoblasts, producing osteoid, were observed at the endosteal surface and elongated mesenchymal cells were present in the injury area. Some macrophages but rather few other inflammatory cells were identified. Multinuclear giant cells were in direct contact with the implant and formed an almost continuous layer along the surface from the 7th day. The number of giant cells decreased with time and with increased bone-titanium contact. Bone formation was never seen direct on the implant surface but was first observed at day 7 as a woven trabecular bone formed at the endosteal surface and extending towards the implant and as a solitary formation of woven bone close to the implant. The solitary bone matrix served as a base for surface osteoblasts which produced osteoid in a lamellar arrangement. With time the two types of newly formed bone fused and more bone filled the threads and became remodelled by bone remodelling units. Light microscopic morphometry in ground sections demonstrated that the bone/titanium contact and bone area in the threads increased with time up to 6 months after implantation     

Assesment of the Biological Activity of Chemically Immobilized rhBMP‐2 on Titanium surfaces in vivo

Previously it has been shown that recombinant human bone morphogenetic protein (rhBMP‐2) can be chemically immobilized by “anchor molecules” on titanium surfaces for serving as a drug delivery device. This opened the question of whether the insoluble immobilized rhBMP‐2 retained its activity in comparison to the same amount of soluble rhBMP‐2 included with the implant samples. Electropolished titanium miniplates (10 × 6 × 0.8 mm) were “surface‐enhanced” by a novel treatment with chromosulfuric acid and then coated with a total amount of 150–200 ng rhBMP‐2 prepared by recombinant technology. Periosteal flaps (7 × 20 mm) were detached and isolated from the anterior surface of the tibiae of adult rabbits and wrapped around the titanium sample plates which were then implanted in the M. gastrocnemius. In the first experimental group various controls without rhBMP‐2 were combined (n = 12). In the second experimental group implants with chemically immobilized rhBMP‐2 (n = 8) were compared with implants to which non‐immobilized soluble rhBMP‐2 was added (n = 8). Animals were sacrificed after 28 days and a quantitative evaluation was carried out by means of serial sections. Untreated control plates showed bone formation in 2/12 implants, rhBMP‐2 coated implants in 6/8 and implants with free rhBMP‐2 administered subperiostally in 8/8 cases. In the case of rhBMP‐2 coated implants the induced bone had direct contact to the implant in all cases while in the group with free administered rhBMP‐2 the bone had no contact to the implant in two cases, but was separated by a fibrous capsule. Bone volume, bone surface area, and trabecular number displayed no difference between the two rhBMP‐2‐groups. However, in the biocoated group a tendency to an increase in the bone‐implant contact area was evident. No differences in osteoid area, osteoid perimeter and eroded perimeter were detected. We conclude that in the case of non‐immobilized rhBMP‐2 there is the danger for formation of fibrous tissue between the implant and the newly formed bone and in addition the generation of ectopic bone at inappropriate places. In contrast chemically immobilized rhBMP‐2 does not have these drawbacks and at the same time displays a biological activity on surfaces similar to that of soluble rhBMP‐2 demonstrating that biomaterial surfaces can be tailored for a selective and specific interaction with the target tissue.     

Intramedullary bone formation after polylactic acid wire implantation

Semi-crystalline poly-L-lactic acid (PLLA) wire was implanted intramedullary in rat tibiae to evaluate the fracture healing processes and the tissue reaction on the PLLA in a fractured bone. The fracture healing after PLLA implantation was compared to a sham-operated group of animals. In all animals with intramedullary PLLA wire implantation newly formed bone was seen immediately against the implant 2 and 6 months after implantation. None of the shamoperated animals showed newly formed intramedullary bone formation other than fracture healing callus and normal trabecular bone.     

Evaluation of the biocompatibility of two magnesium alloys as degradable implant materials in comparison to titanium as non‐resorbable material in the rabbit

The aim of this study is to compare the biocompatibility of the two magnesium based alloys LAE442 and LANd442 with that of titanium. For this purpose, cylindrical implants were introduced into the medullary cavity of rabbit's tibiae for 4 and 8 weeks. Animals without any implant served as a control. In the follow-up, clinical, X-ray and μCT-investigations were performed to evaluate the reactions of the bone towards the implanted materials. After euthanasia, ex vivo μCT- and histological investigations were performed to verify the results of the in vivo tests. It could be shown that all materials induce changes in the bone. Whereas LANd442 caused the most pronounced reactions, such as increasing bone volume and bone porosity and decreasing bone density, titanium showed the most bone–implant contact by forming trabeculae. The tibiae of rabbits without implants also reacted by forming cavities, it is therefore assumed that the surgery method itself influences the bone.     

Osseointegration of bioactive microarc oxidized amorphous phase/TiO2 nanocrystals composited coatings on titanium after implantation into rabbit tibia

The amorphous phase/TiO₂ nanocrystals (APTN) composited coatings were prepared on Ti implants for biomedical applications. The Ti implants without and with the APTN composited coatings both do not cause any adverse effects after implantation into the rabbit tibia. The osseointegration of Ti implants after covering the APTN coatings is improved pronouncedly, greatly increasing the interface bonding strength between the implants and newly formed bones. In addition, it is interesting that the newly formed bone tissues appear in the micro-pores of the APTN coatings, promoting the interface bonding between the implants and new bones by the mechanical interlock. Moreover, the Ti implant with the APTN coatings formed at higher applied voltage exhibit higher shear strength and displacement during the pushing out experiment probably due to its better osseointegration.     

A histological and histomorphometrical evaluation of screw-type calciumphosphate (Ca-P) coated implants; an in vivo experiment in maxillary cancellous bone of goats

The bone response to different calcium phosphate (Ca-P) coated implants was evaluated in a goat animal model. Two types of plasma spray coatings were applied to a commercially pure titanium (cpTi) tapered, conical screw-design implant (BioComp^®); hydroxyapatite (HA-PS) and a dual coating, consisting of FA and HA (FA/HA-PS). In addition an amorphous RF magnetron sputter coating (Ca-P-a) and uncoated implants were investigated. Forty-eight implants were inserted in the maxilla of 12 adult female goats. After implantation periods of 3 and 6 months, the bone implant interface was evaluated histologically and histomorphometrically. After both implantation periods all plasma spray coated implants were maintained. On the other hand three Ca-P-a and two cpTi implants were lost. Histological examination revealed a better bone response to both plasma spray coated implants. Histomorphometrical evaluation confirmed this finding. At 3 and 6 months significantly higher percentages of bone contact (p<0.001, ANOVA) were measured for both plasma spray coated implants than for the cpTi and Ca-P-a implants, while no significant difference (p<0.05) existed between both implantation periods. Degradation of both plasma spray coatings was observed. Supported by the results, it is concluded that, although Ca-P coatings can improve the performance of dental implants, the presence of a Ca-P coating is not the only important factor for bone healing around implants placed in low density trabecular bone.     

Bone response to a titanium aluminium nitride coating on metallic implants

The design, surface characteristics and strength of metallic implants are dependant on their intended use and clinical application. Surface modifications of materials may enable reduction of the time taken for osseointegration and improve the biological response of bio-mechanically favourable metals and alloys. The influence of a titanium aluminium nitride (TAN) coating on the response of bone to commercially pure titanium and austenitic 18/8 stainless steel wire is reported. TAN coated and plain rods of stainless steel and commercially pure titanium were implanted into the mid-shaft of the femur of Wistar rats. The femurs were harvested at four weeks and processed for scanning electron and light microscopy. All implants exhibited a favourable response in bone with no evidence of fibrous encapsulation. There was no significant difference in the amount of new bone formed around the different rods (osseoconduction), however, there was a greater degree of shrinkage separation of bone from the coated rods than from the plain rods (p = 0.017 stainless steel and p = 0.0085 titanium). TAN coating may result in reduced osseointegration between bone and implant.     

A new composite made of polyurethane and glass ceramic in a loaded implant model: a biomechanical and histological analysis

The biocompatibility and osseous integration of a new composite material made of polyurethane and a calcium silicophosphate ceramic was investigated in a loaded implant model in sheep and compared to that of commercially pure titanium. Six months after implantation, interfacial shear strength was higher between the titanium and bone than between the composite and bone. After 2 years, however, the shear strength was significantly higher in the composite group. Histologically, both implants were surrounded by bone and fibrous tissue and there were no signs of inflammation. Direct contact of bone on the composite surface increased significantly with time, whereas there was no time-dependent increase of bone contact on titanium. It can be concluded that the biocompatibility and osseous integration of the composite was very good in the loaded implant model used. It is therefore suggested that the new composite is a promising biomaterial for orthopaedic implants.     

Studies of the healing of bone grafts, and the incorporation of titanium implants in grafted bone: an experimental animal model

An insufficient quality and amount of bone often necessitate the clinical use of implants together with bone transplants. The present study describes an experimental animal model for the study of implants in bone grafts. Circular defects were made bilaterally in the tibia of 36 rabbits. The defects received either autologous cortical bone (control), demineralized bone matrix (DBM), plasma-augmented DBM or were left empty (without bone graft). In all defects a titanium implant was centrally placed and anchored in the opposite cortex. Evaluation with light microscopic morphometry showed that the insertion of a threaded titanium implant centrally in a cortical defect was followed by a spontaneous healing of the defect after 6 mon. After 6 wk, all implants in cortical grafts were well integrated with a significantly higher bone-to-implant contact than in the DBM and plasma-augmented groups. After 6 mon, all experimental groups had a mean bone area within the threads ranging between 69% and 80% and a mean bone-to-implant contact between 31% and 42%. The results from the present study indicate that the model allows comparative studies on the early formation, resorption and remodelling of bone around implants after modification of implant, graft and host properties.     

The effect of biological environment on the surface of titanium and plasma-sprayed layer of hydroxylapatite

The solubility of titanium samples with different surface coatings, i.e., hydroxylapatite (HA) powders, a two-layer coating of ZrO₅+HA on a titanium substrate in solution and of tooth implants after long-term functioning in the human organism, was studied. A minimum difference in solubility of titanium samples with different surface finishes (polished or grit blasted) was established. For the HA powders and coatings, the lowest solubility was observed with a coarse-grained HA–B powder and a coating made of that powder. Clinical tests of tooth implants after long implantation times were performed. A titanium implant (implantation 12 y), a titanium implant with a two-layer coating of ZrO₅+HA–A (implantation time 4 y) and a titanium implant with a two-layer coating of Al₅O₃+3% TiO₂)+ HA–A (implantation time 6 y) were studied. The results show that the titanium surface and HA–A layers were dissolved. Nevertheless, after 6 y implantation, total removal of HA–A coating from that part of implant set into the bone, was not observed.     

Causes of titanium release from plate and screws implanted in rabbits

To investigate the mechanism behind the release of metal from titanium implants in vivo, bone-plate-screw sets consisting of pure titanium were implanted into the legs of rabbits for 48 weeks. Four groups of experiments containing control were conducted: (1) The tibia cut artificially was fixed by one set of bone plate and screws, (2) the same set was implanted separately into muscles in the leg, (3) the set was fixed on the tibia and immediately retrieved, and (4) no implantation was performed. The amounts of titanium in all tissues from knee to ankle were quantified using atomic adsorption spectrometry. The ratio of amounts of titanium detected in the groups (1), (2), and (3) was 100 : 10 : 43. No titanium was detected in the group (4). Causes of the release of titanium in the group (1) include that in the groups (2) and (3). Major causes of titanium release were surgical handling in implantation and wear and/or fretting during experimental-term for 48 weeks. Titanium was also released in the absence of wear. No morphological abnormality was observed around tissue of the implant by biopsy at post-operation week 48.     

Biomimetic CaP coating incorporated with parathyroid hormone improves the osseointegration of titanium implant

Parathyroid hormone (PTH) is a well-known therapeutic agent for osteoporosis treatment, however, the inconvenience of daily administration and side effect from systematic administration severely limits its application in clinic. PTH has been incorporated into a biomimetic calcium phosphate (CaP) coating via a co-precipitation method in a modified simulated body fluid. The aim of the current study is to evaluate the osseointegration response of PTH incorporated CaP coating on titanium implants. Implants with different doses of PTH were inserted into tibiae of mice and evaluated by X-ray, micro-CT, histology and back-scattered scanning electron microscopy. Improved osseointegration of the implants loaded with PTH was observed compared to CaP coating only after 28?days of implantation in mouse tibiae. Micro-CT analysis showed better bone integration around the implant incorporated with PTH. Bone area and bone contact evaluations have demonstrated that peri-implant bone regeneration is highly dependent on the dosage of PTH incorporated. The higher the PTH content, the more bone formed surrounding the implant. Therefore, our results suggest that biomimetic CaP coating could be a useful a carrier for PTH local delivery, which results in improved bone-to-implant integration.     

Osseointegration evaluation of laser-deposited titanium dioxide nanoparticles on commercially pure titanium dental implants

The nanotechnology field plays an important role in the improvement of dental implant surfaces. However, the different techniques used to coat these implants with nanostructured materials can differently affect cells, biomolecules and even ions at the nano scale level. The aim of this study is to evaluate and compare the structural, biomechanical and histological characterization of nano titania films produced by either modified laser or dip coating techniques on commercially pure titanium implant fixtures. Grade II commercially pure titanium rectangular samples measuring 35?×?12?×?0.25?mm length, width and thickness, respectively were coated with titania films using a modified laser deposition technique as the experimental group, while the control group was dip-coated with titania film. The crystallinity, surface roughness, histological feature, microstructures and removal torque values were investigated and compared between the groups. Compared with dip coating technique, the modified laser technique provided a higher quality thin coating film, with improved surface roughness values. For in vivo examinations, forty coated screw-designed dental implants were inserted into the tibia of 20 white New Zealand rabbits’ bone. Biomechanical and histological evaluations were performed after 2 and 4 weeks of implantation. The histological findings showed a variation in the bone response around coated implants done with different coating techniques and different healing intervals. Modified laser-coated samples revealed a significant improvement in structure, surface roughness values, bone integration and bond strength at the bone-implant interface than dip-coated samples. Thus, this technique can be an alternative for coating titanium dental implants.     

In vivo osseointegration of dental implants with an antimicrobial peptide coating

This study aimed to evaluate the in vivo osseointegration of implants with hydrophobic antimicrobial GL13K-peptide coating in rabbit femoral condyles by micro-CT and histological analysis. Six male Japanese Rabbits (4 months old and weighing 2.5?kg each) were included in this study. Twelve implants (3.75?mm wide, 7?mm long) were randomly distributed in two groups, with six implants in the experimental group coated with GL13K peptide and six implants in the control group without surface coating. Each implant in the test and the control group was randomly implanted in the left or right side of femoral condyles. On one side randomly-selected of the femur, each rabbit received a drill that was left without implant as control for the natural healing of bone. After 3?weeks of healing radiographic evaluation of the implant sites was taken. After 6?weeks of healing, rabbits were sacrificed for evaluation of the short-term osseointegration of the dental implants using digital radiography, micro-CT and histology analysis. To perform evaluation of osseointegration, implant location and group was double blinded for surgeon and histology/radiology researcher. Two rabbits died of wound infection in sites with non-coated implants 2?weeks after surgery. Thus, at least four rabbits per group survived after 6?weeks of healing. The wounds healed without suppuration and inflammation. No implant was loose after 6?weeks of healing. Radiography observations showed good osseointegration after 3 and 6?weeks postoperatively, which proved that the tissues followed a natural healing process. Micro-CT reconstruction and analysis showed that there was no statistically significant difference (P?>?0.05) in volume of bone around the implant between implants coated with GL13K peptide and implants without coating. Histomorphometric analysis also showed that the mineralized bone area was no statistically different (P?>?0.05) between implants coated with GL13K peptide and implants without coating. This study demonstrates that titanium dental implants with an antimicrobial GL13K coating enables in vivo implant osseointegration at similar bone growth rates than gold-standard non-coated dental implants up to 6?weeks of implantation in rabbit femurs.     

Distribution of cells in soft tissue and fluid space around hollow and solid implants in the rat

The development of the tissue surrounding an implanted material is anticipated to be regulated by the biological factors in the interface as well as the physicochemical properties of the implant material. In the present study light microscopic morphometry and transmission electron microscopy were used to evaluate the distribution of cells adjacent to the implant surface of different implant designs (hollow and solid implants) and materials (titanium and polytetrafluoroethylene). An increased number of leukocytes, predominantly PMN, was retrieved from the exudate inside hollow implants 1 and 9 days after surgery. In contrast, the increased cellularity in the soft tissue around the hollow implants was mainly due to an increased number of monocytes/macrophages and fibroblasts. The presence of a fluid space around both hollow and solid implants was revealed by the use of an electropolishing technique and ground sections. In the fluid space around solid titanium the concentration of leukocytes and the proportion of PMN decreased between 1 and 7 days. After 1 day the majority of leukocytes were freely suspended in the fluid and were rarely directly apposed to the implant surface. A majority of the monocytes/macrophages present in the fluid space after 7 days were attached to the fibrin matrix at the border between the fluid space and the reorganized tissue. Our studies demonstrate that hollow implants promote the influx and a persistence of PMN in the interior of the implant in comparison with the tissue surrounding the hollow and solid implants. Furthermore, during the first week after implantation inflammatory cells are not preferentially distributed directly on the titanium implant surface.     

Bone ingrowth behaviour of hydroxyapatite-coated,polyethylene-intruded and uncoated,sandblasted pure titanium implants in an infected implantation site: an experimental study in miniature pigs

We studied the dynamics of bone tissue ingrowth into the pores of hydroxyapatite-coated (plasma-spraying technique) and uncoated wire meshes of pure Ti in an infected implantation site. Samples of the test materials were implanted into the femora of 15 adult Göttingen minipigs. Just before implantation they were contaminated with Staphylococcus aureus. The pigs were killed after 4, 8, 12 or 24 weeks. Undecalcified ground sections of bone tissue were prepared and stained with toluidine blue for comparative histological evaluation. The hydroxyapatite-coated implants already demonstrated advanced new bone formation after 4 weeks. By 12 weeks most of the implant pores were filled with newly formed bone although all samples showed macro- as well as microscopic signs of persistent infection. Comparable reactions of the uncoated implants could be observed only after 24 weeks. Signs of degradation of the hydroxyapatite coating were seen in contact with soft tissue. This was more extensive in the infected than in the uninfected site. The results and possible clinical consequences are discussed.     

Applying a calcium phosphate layer on PEO/PBT copolymers affects bone formation in vivo

Recently a copolymer (Polyactive^R) has been introduced that combines elastomeric and bone-bonding properties. Since calcification of the copolymer is a prerequisite for bone bonding. Polyactive was precalcified in vitro in order to increase the bone-bonding rate. Precalcification was performed by subsequent incubation in Ca and P solutions and resulted in formation of a hydroxyapatite layer on the surface of the implant. Within one week after implantation this layer had disappeared from the surface and a new calcification zone was formed under the surface of the copolymer. Longer implantation periods showed that in precalcified implants bone was apposited along the walls of the pores, while in control implants new bone was first formed in the centre of the pores. Consequently, the percentage of bone contact was increased in precalcified implants, however, the amount of bone ingrowth was equal in both control and precalcified implants. Transmission electron microscopy showed the presence of an electron-dense layer at the bone implant interface, which was indicative for bone-bonding. It is concluded from these experiments that precalcification of PEO/PBT copolymers affected the direction of bone apposition and increased the bone-bonding rate.     

Structure of the interface between rabbit cortical bone and implants of gold, zirconium and titanium

The role of surface properties (chemical and structural) for the interaction between biomaterials and tissue is not yet understood. In the present study, implants made of titanium, zirconium (transition metals with surface oxides) and gold (metallic surface) were inserted into the rabbit tibia. Light microscopic (LM) morphometry showed that after 1 and 6 mo the gold implants had less amount of bone within the threads and a lower degree of bone-implant contact than the titanium and zirconium implants, which did not differ from each other. These quantitative differences were supported by LM and ultrastructural observations of the interface. The ultrastructural observations in addition demonstrated that the layer of non-collagenous amorphous material located between the implant and the calcified bone was appreciably thicker around zirconium than around titanium implants. The factors potentially responsible for the observed morphological differences in the bone around the different material surfaces are discussed.     

Bioactive glass granules and polytetrafluoroethylene membrane in the repair of bone defects adjacent to titanium and bioactive glass implants

An experimental animal model was used to investigate the effect of bioactive glass (BG) granules and nonresorbable polytetrafluoroethylene (PTFE) membrane on the repair of cortical bone defects adjacent to titanium and BG implants. Thirty-two Astra® (diameter 3.5 mm) dental implants were inserted bicortically and 42 conical BG implants (diameter 2.5–3.0 mm) monocortically, into fitted holes of rabbit tibia. Before implantation, a standardized bone defect was created by drilling an extra hole (diameter 3.0 mm) adjacent to each implant site. Twenty-eight defects were filled with BG granules (diameter 630–800 m) (BG group) and 28 defects were left empty but covered with PTFE membrane (PTFE group). No material was used in 18 control defects (control group). Morphometrical evaluation with a digital image analysis system was used to measure bone repair as percentages of the defect area on scanning electron microscopy (SEM) and light microscopy pictures. Bone–implant contact was measured as percentages of the thickness of the cortical bone. At 6 and 12 wk, bone repair in defects in connection with titanium implants was 23.2% and 36.6% in the BG group, 23.2% and 32.4% in the PTFE group, and 47.2% and 46.2% in control defects. Corresponding figures for BG implants were 33.2% and 40.1% in the BG group, 16.6% and 33.5% in the PTFE group, and 25.7% and 54.9% in control defects, BG granules and new bone together filled 82.7% and 68.5% of the defect area adjacent to titanium implants, and 75.9% and 74.4% of the defect adjacent to BG implants at 6 and 12 wk, respectively. Better bone–implant contact was achieved at the defect side with BG than titanium implants (77.0% versus 45.0% at 12 wk). The results indicate that BG granules are useful in treatment of bone defects adjacent to dental implants. BG coating of the implant seems to improve osseointegration in the defect area.