BONITex® coating significantly increased bone implant contact rates between 14 and 30 days

Summary

This article discusses a study on the accelerated healing of dental implants using a coating called BONITex®. The success of dental implants depends on the attachment of bone to the implant, and the surface characteristics of the implant play a significant role in this process. The study aimed to evaluate the bone implant contact rates of implants with the BONITex® coating compared to other coatings.

The study was conducted on adult pigs, chosen for their similarity to humans in terms of bone healing. Implants were inserted in the pigs' calvaria, and the bone implant contact was evaluated at different time points. The results showed that implants with the BONITex® coating had significantly increased bone implant contact rates between 14 and 30 days compared to other coatings.

The findings suggest that the BONITex® coating promotes faster healing and integration of implants. This may allow for early loading of the implants after 30 days, leading to quicker masticatory and aesthetic rehabilitation of patients. The study concludes that further research is needed to investigate the effects of early loading on bony remodeling and functional capacity.

Animal experimental study of the healing of endosteal implants with vacuum titanium spray and calcium phosphate coating

Author:  Rainer Lutz, Safwan Srour, Peter Kessler, Emeka Nkenke, Karl Andreas Schlegel, Germany

Dental implants are often the only possibility for incorporating a functional denture in patients with marked jaw atrophy or following cancer surgery of the oral cavity. A crucial factor for the success of endosteal implants is the degree of bone deposition on the implant. This is considerably influenced by the bone density in the local tissue. The cortex is thinner in the maxilla compared with the mandible and the cancellous pattern is finer. Long-term clinical and experimental investigations have demonstrated statistically significantly that the success rate of implants is much poorer in the maxilla compared with the mandible. 

Fig. 1 Diagram of the bone-implant contact regions

The long-term success of oral endosteal implants requires both osseointegration and a permanent close bond with the soft tissues. This biological behaviour is influenced to a very great extent by the surfaces of the introduced biomaterials, as characterised by the macro-, micro- and nanostructure, along with the chemical composition. When the implant site is of good bone quality and the patient is healthy, treatment with oral endosteal implants has a high rate of long-term success so that this form of therapy is now regarded as a scientifically accepted part of dental management.

Since the introduction of oral endosteal implants, an unloaded healing period of three to six months has become established, depending on the quality of the implant bed. Modified surface structures, which enable osseointegration to be accelerated, might contribute to the early functional loading capacity and are therefore an important aspect of clinical research. The possible influence of the surface on the long-term success of oral endosteal implants is apparent when the surfaces of failed explanted implants are examined. Apart from modification of implant insertion, modified surfaces are recommended in difficult implant sites in augmented or very spongy bone and are provided by manufacturers. 

Fig. 2 Example of analysis of BIC(bone-implant contact) using the Bio-quant method 

Scientific investigation to detect a positive correlation between certain surface characteristics and healing behaviour and the time the implants remain in place in vivo is so far lacking. Because of the greater fracture strength with a simultaneously relatively low weight and excellent corrosion resistance, and because of the spontaneous formation of a passivating oxide layer, titanium has become established as a material for implants. Although the exact mechanism of the bone-titanium bond has not yet been fully explained, it can be assumed that the titanium oxide layer has a decisive influence on bony healing and epithelial adhesion. Implant healing can be further improved by modifications of their surface structure and advantages including earlier loading can be achieved by topographical changes.At every implantation there is initially adsorption of proteins, platelets and other macromolecules to the implant surface. 

Osseointegration of the implants takes place through the “conditioning film” of this protein layer. Cytokines released at the implant surface (mitogens and morphogens) play an important part in attracting osteogenic cells, which colonize the implant surface as osteoblasts and through this process, which takes place immediately after implantation, they enable contact osteogenesis of the implants to take place. The direct contact between the bone and the implant correlates significantly with its surface. The surface structure appears to be more important than an increase in diameter. The precise mechanisms are not known at present but previous authors have suggested that the choice of implant material and its surface structure has a profound influence on erythrocyte agglomeration and on the number and degree of activation of the platelets.It has been shown that platelet adhesion takes place through GPIIb/IIIa integrin binding to fibrinogen, which is adsorbed on the implant surface. The implant surface structure thus has a fundamental influence on osteoconduction not only because of the development of a concentration gradient for chemotactic cytokines and growth factors through the degree of platelet activation but it also represents a fixation surface for a three dimensional biological matrix, along which cells can migrate to the implant surface. The bone remodelling cascade can be divided into a multistage process. 

First, differentiating osteogenic cells secrete a collagen-free organic matrix, which provides germ centres for calcium phosphate mineralisation. Beginning in these germ centres, crystal growth and the formation of a collagen fibre scaffolding take place. Finally, the collagen scaffolding is mineralised.

The newly formed collagen-containing bone is separated from the implant surface by a layer of collagen-free calcified tissue. Formation of this layer can be simulated by a calcium phosphate coating. In contrast to uncoated metal oxide surfaces, this adsorbs more proteins on its surface. It can be assumed that greater platelet activation and thus faster peri-implant wound healing takes place because of the increased binding of fibrinogen. In addition, faster formation of the three-dimensional matrix with accelerated cell migration to the implant surface can take place because of the increased protein adsorption.

The calcium phosphate coating used in our experimental design consists mainly of brushite (CaHPO4 x 2 H2O) with traces of hydroxyapatite and it is applied to the implant surface by an electrochemical process. Brushite is one of the most soluble calcium phosphate phases and is converted to hydroxyapatite in aqueous solutions. In vitro the coating demonstrated highly promising osteoblast activation. Osteoconductive properties were found in vivo.50 In this study, the healing of differently coated CTD-alphatech® implants with VTPS, VTPS + BONIT® and BONITex® coating was investigated in 24 domestic pigs. The adult domestic pig was chosen as experimental animal as it is particularly suitable for studies of bone healing and bone remodelling. Tissue perfusion, circulatory processes and fracture healing are very similar to conditions in humans. The bone new production rates in adult pigs correlate closely with those in humans (pigs: 1.2–1.5 μm/d; humans 1.0–1.5μm/d). The pig is therefore regarded as a very reliable model with regard to meaningfulness, reproducibility and applicability of experimental results.

Material and methods

After the animal study was approved by the relevant animal experiment committee of the Mittel-franken government, Ansbach, Germany (animal experiment proposal 54-2531.31-7/06), 24 pigs were included in the study. The following study groups were formed:

1. VTPS group (vacuum titanium plasma spray) 

Pure titanium was sprayed on the implant surface in an argon gas atmosphere under negative pressure conditions.

2. BONITex® group

Following previous Hydroxyapatite radiation and acid etching of the implant surface, the implants were coated by means of an electrochemical process in an aqueous solution containing calcium and phosphate ions. This coating was approx. 2 μm thick and consists of the two calcium phosphate phases Hydroxyapatite and Brushite (~ 5% HA, ~ 95 % Brushite).

3. VTPS + BONIT® group

Pure titanium layer in combination with an approx. 15 μm layer of electrochemically deposited CaP (~5 % HA, ~ 95 % Brushite).

Fig. 3 Histology of the BONITex®  surface 21 days postoperatively, 400x magnification (N=newly formedbone, O=osteocytes, A = artificial gap due to specimen preparation)

Nature, method and duration of the procedures

For all surgical procedures, the animals were anaesthetised by an intravenous injection of Ketamine HCl (Ketavets, Ratiopharm, Ulm, Germany). Following application of a local anaesthetic in the frontal region of the skull (Ultracain DS forte, Hoechst GmbH, Frankfurt/Main, Germany), a sagittal incision was made and the soft tissue and periosteum were mobilized. Three implants each per experimental group were then inserted randomly. Three animals were available for each experimental time. Nine implants were placed in each animal, and these were inserted in the skull according to a randomized selection process. Therefore, a total of nine implants could be followed up per group and experiment time. On the first three days postoperatively, the animals were given streptomycin (0.5g/day; Grünenthal GmbH, Stolberg, Germany) to reduce the risk of infection. Finally, the periosteum and skin were closed over the defects with absorbable Vicryl sutures (Vicryl® 3.0; Vicryl® 1.0; Ethicon GmbH & Co. KG, Norderstedt, Germany). The animals planned healing time of the implants was reached after 3, 7, 14, 21, 30, 56 and 84 days and after six months.

Removal and processing of the specimens

The animals’ frontal bones were removed and the samples were fixed with 1.4 % Paraformaldehyde solution to render the organic matrix insoluble. The samples were then dehydrated at room temperature in an ascending alcohol series in a dehydration unit (Shandon Citadel 1000, Shandon GmbH, Frankfurt/Main, Germany). Xylol was used for intermediate fixation. The samples were then embedded in Technovit 9100® (Heraeus Kulzer, Wehrheim, Germany).

Histology

The thin sections were then reduced to 30 μm and stained with toluidine blue O solution. The stained sections could then be examined light microscopically. The samples were inputted into a Pentium 5 computer by a Zeiss light microscope (Axio Imager A1; Zeiss, Jena, Germany) with a video camera. The boneimplant contact was then determined in the histological sections using Bioquant Osteo® software. This was followed by histopathological analysis of the samples.

Fig. 5  Histology of the BONITex® surface 30 days postoperatively,100x magnification

Fig. 6  Histology of the BONITex® surface 180 days postoperatively, 100x magnification

Statistics

Each histology sample was analysed by two investigators and the values for each sample were aggregated. A two-sided t-test was subsequently performed for verification. A significant difference in the compared results was assumed at p < 0.05.

Results

The bone-implant contact (BIC) was determined as shown diagrammatically in Figure 1. The bone marrow contains not only mesenchymal precursor cells, which can differentiate into osteoblasts, but also shows high perfusion, which provides osteoclast precursor cells and also cells for neoangiogenesis. Cancellous bone therefore has a much higher remodelling rate than cortical bone. That is why much more marked effects of the different implant coatings can be expected in this region between the individual groups and also over time. To ensure successful osseointegration and long-term stability of the implants, contact osteogenesis of the implants is required. This requires osteoblasts to be deposited on the implant surface right at the start of the peri-implant bone remodelling. The results of the analysis of the bone-implant contact are shown in Table 1 as mean and standard deviation. There were major differences with regard to bone-implant contact. After seven days, bone-to-implant-contact was significantly increased in the VTPS + BONIT® group compared to the BONITex® and VTPS group (p = 0.025 and p = 0.0081). In the period between 14 and 30 days, significantly increased values (p = 0.0018 and p = 0) were demonstrated in the BONITex® group (86.53% + 8.55; 83.42% + 14.26; 87.96% + 7.90) compared with the other two groups. The results obtained here are well above the average results described in the literature and are in the region described by Schwarz et al. in 2007 for the modified SLA surface after 12 weeks. Over the further course of time, the values of the bone-implant contact in the framework of the bone remodelling became similar between the individual experimental groups and averaged 51.8%.

Discussion

In animal studies of bone regeneration, the choice of an animal model with the best possible analogy to the patient is crucial for applying experimental data to practice. This requirement was met by the chosen model. The pig’s skull consists of desmal bone, with cortical and cancellous parts, and is particularly good for investigations in the area of implantology because of its resemblance to human bone with regard to bone healing and bone remodelling.The implants were all inserted according to the manufacturer’s operation protocol. By securely covering the implants with the skin of the head, the risk of post-operative bacterial contamination in the form of peri-implantitis was reduced to a minimum and thus corresponded to the closed healing mode. According to Heimke et al, a definitive conclusion on the behaviour of trabecular bone around implants can only be drawn in absolutely load-free models in order to rule out the influence of different loads on bone remodelling.

Observation period	Coating	Bone-implant contact (%)
3 days	VTPS  BONITex® VTPS+ BONIT®	42.17±20.24 24.06±14.66 25.98±17.62
7 days	VTPS  BONITex® VTPS+ BONIT®	20.23±9.99 19.63±6.86 73.15±21.08
14 days	VTPS  BONITex® VTPS+ BONIT®	56.09±18.04 86.53±8.55 26.54±18.79
21 days	VTPS  BONITex® VTPS+ BONIT®	61.92±14.03 83.42±14.26 53.79±30.46
30 days	VTPS  BONITex® VTPS+ BONIT®	74.70±23.80 87.96±7.90 60.47±19.41
56 days	VTPS  BONITex® VTPS+ BONIT®	46.48±8.71 54.73±5.36 44.50±13.33
84 days	VTPS  BONITex® VTPS+ BONIT®	55.42±11.61 54.57±17.29 41.63±13.79
180 days	VTPS  BONITex® VTPS+ BONIT®	68.55±20.16 49.11±18.17 51.49±24.87

Table 1 Summary of the results of bone-implant contact.

This was the case in our experimental model because the extraoral location of the implants provided primary stability of the inserted implants. This also minimised the risk of micromovements during the healing period. The implants with BONITex® coating in particular showed high bone-implant contact rates in the cancellous bone at the early times, between 14 and 30 days (see Fig. 4). This result is in agreement with the results of other studies which also report significantly increased bone-implant contact or an improved ability to bridge peri-implant gaps in investigations of calcium phosphate-coated implants.

Calcium phosphate coatings that are applied by plasma spray and have a thickness of 50 to 100μm often demonstrate mechanically induced microfractures, as a result of which there are increased signs of degradation and resulting unfavourable tissue reactions after separation of the coating material. In recent years there has therefore been a switch to reducing the thickness of the calcium phosphate coatings and thus selecting an approach that corresponds more to bone biology. The BONITex® coating has a markedly reduced thickness of approx. 2 μm. As a result, the biological advantages of osteoconduction, cell attraction and improved attachment for the extracellular matrix can be utilised. The fate of the calcium phosphate coatings in vivo has not been adequately elucidated. At a neutral pH, the dissolution of the coating in vitro depends especially on phase composition and the crystallinity and crystal size of the coating. 

In the subsequent course the unloaded implants with BONITex® coating showed bone-implant contact rates during bone remodelling that were similar to the values described in the literature. The implants with VTPS and VTPS + BONIT® coating showed bone-implant contact rates during the investigated period as described in the literature. The VTPS coating can be regarded as standard. However, the chemical composition can alter in the course of the healing process. Because of this and because of an acid environment during the early period, the lifetime of the calcium phosphate coating can be impaired in vivo. In the present study, isolated parts of the coating were  observed in the peri-implant bone. However, negative effects of degradation of the coating were not found. Nagano et al. did not find a negative influence on the peri-implant bone after degradation of calcium phosphate coatings eithereither.

Conclusion

Accelerated implant healing with markedly increased bone-implant contact rates was found in the group with BONITex® coating between just 14 and 30 days. On the basis of these results, early loading of these implants after 30 days and the effect on bone remodelling would be interesting subjects for research. The possibility of very early functional loading could be evaluated, which would allow markedly faster functional and also aesthetic patient 

Acknowledgement

We would like to thank Prof. Dr. Dr. Karl Donath, Wiehenstr. 73, 32289 Rödinghausen, Germany, for his assistance in the histopathological analysis._ The article was first published in Dentale Implantologie, Flohr Verlag, November 2007. The literature list can be requested from the editorial office.