Biomaterials
Volume 24, Issue 15 , July 2003, Pages 2623-2629

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

J. G. C. Wolke^, , J. P. C. M. van der Waerden, H. G. Schaeken and J. A. Jansen

Department of Biomaterials, College of Dental Science, University Medical Center Nijmegen, PO Box 9101, Nijmegen 6500 HB, Netherlands

Received 2 May 2002;  accepted 20 January 2003. ; Available online 6 March 2003.

1. Abstract

RF magnetron sputter deposition was used to produce 0.1, 1.0 and 4.0 
m thick Ca-P coatings on TiO₂-blasted titanium discs. Half of the as-sputtered coated specimens were subjected to an additional infrared heat treatment for 30 s at 425-475°C. X-ray diffraction demonstrated that infrared radiation changed the amorphous 4 
m sputtered coatings into an amorphous-crystalline structure, while the amorphous 0.1 and 1 
m 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 
m 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 
m thick amorphous coatings had disappeared within 1 week of implantation. On the other hand, the 4 
m 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 
m thick coatings could be detected. Occasionally, a granular precipitate was deposited on the heat-treated 4 
m thick coating. Fourier transform infrared spectroscopy showed the formation of carbonate apatite (CO₃-AP) on the 4 
m thick amorphous coating and on the heat-treated specimens.

On basis of our findings, we conclude that 1 
m 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.

Author Keywords: Calcium phosphate coating; RF magnetron sputtering; In vivo; Roughness

2. Article Outline

1. Introduction

2. Materials and methods

2.1. Implant materials

2.2. Characterization of the sputter coating

2.3. Animal model and implantation procedure

2.4. Histological and physicochemical procedures

3. Results

3.1. Characterization of Ca/P coatings before implantation

3.2. Descriptive light microscopic evaluation

3.3. Physicochemical analysis of the retrieved implants

3.3.1. X-ray diffraction

3.3.2. Fourier transform infrared spectroscopy

3.3.3. Scanning electron microscopy

3.3.4. Energy dispersive spectroscopy

4. Discussion and conclusions

Acknowledgements

References

3. 1. Introduction

Calcium phosphate (Ca-P) materials have been clinically applied in many areas of dentistry and orthopedics. Bulk material, available in dense and porous forms, is used for augmentation of the jaw, in maxillofacial reconstruction and artificial middle ear implants. However, because Ca-P bulk materials are mechanically weak, they cannot be subjected to loads other than compressive ones. Therefore, Ca-P coatings on metallic substrates have been developed, which are currently used in loaded situations, like total joint replacements and dental root implants. In this way the mechanical strength of the titanium implants and the biocompatibility of the Ca-P coating are combined [1]. As demonstrated in various publications, Ca-P coatings show a favorable bone response compared with non-coated titanium implants [2, 3, 4, 5, 6, 7 and 8]. Over the past few years, in our laboratory we have been investigating the characteristics and biocompatibility of RF magnetron-sputtered Ca-P coatings on titanium substrates [9 and 10]. This method allows: (1) the exclusion of other substrate parameters, like surface (micro) topography as additional experimental factor; and (2) the inclusion of additional substrate parameters, which only deal with the structure or composition of Ca-P ceramic. Previous experiments in our laboratory showed that the sputter deposited films had a uniform and dense structure, especially on complex implant designs, like threaded implants. In cell culture experiments, we demonstrated that magnetron-sputtered Ca-P coatings can indeed stimulate extracellular matrix and induce apatite formation. In addition, no severe degradation of the amorphous and crystalline coatings was observed [11]. In contrast, subcutaneous implantation of magnetron-sputtered Ca-P coatings in the back of rabbits proved that the in vivo dissolution behavior of sputter coatings is determined by their degree of crystallinity. Amorphous coatings disappeared completely or for a greater part, which was followed by the precipitation of carbonate apatite. Highly crystalline coatings were partially or completely detached from the as-machined titanium substrates [12]. It can be assumed that stresses developed as a result of the heat treatment (HT) at 500°C, causes the coating detachment. In view of this we have to emphasize that the success or failure of an implant surface coating has been shown to be related to morphology of the implant surface, including microgeometry and roughness. In several in vitro studies, rough surfaces were already found to produce less coating detachment than smooth surfaces. Also it has been suggested that surface roughness increased the amount of bone formation in osseous sites due to surface enlargement. In non-osseous sites, ectopic bone formation was regulated by the geometry of the substratum [13 and 14]. Ripamonti et al. demonstrated that porous hydroxylapatite with a defined size, structure and shape can induce bone formation when implanted intramuscular in baboons [15 and 16].

Therefore, the objective of the present study was to characterize the surface features and dissolution properties of blasted implants provided with various types of magnetron-sputtered Ca-P coatings after subcutaneous implantation in rabbits.

4. 2. Materials and methods

2.1. Implant materials

For the experiments commercially pure titanium discs with a diameter of 8 mm and thickness of 1 mm were used. All discs were TiO₂-blasted on both sides. The discs were left uncoated (Ti-un) or provided with the following coatings:

• Ca-P coating, with thickness of 0.1 
m (CaP-0.1);

• Ca-P coating, with thickness of 1 
m (CaP-1);

• Ca-P coating, with thickness of 4 
m (CaP-4).

The coating procedure was performed using a commercially available RF magnetron sputter unit (Edwards ESM 100), as described earlier. After deposition, half of the coated specimens were subjected to an additional infrared HT for 30 s at 425-475°C.

In total 144 discs were prepared: 24 CaP-0.1, 24 CaP-0.1HT, 24 CaP-1 and 24 CaP-1HT, 24 CaP-4 and 24 CaP-4HT.

2.2. Characterization of the sputter coating

Before implantation the deposited films were characterized using the following techniques:

• The crystallographic structure of each film was determined by thin film X-ray diffraction (XRD) using a Philips
−2
diffractometer using a CuK
-radiation.

• The infrared spectra of the films on the substrates were obtained by reflection Fourier transform infrared spectroscopy (FTIR)(Perkin-Elmer).

• The surface topology of the films was examined using scanning electron microscopy (SEM) using a Jeol JSM-35.

• The elemental composition of the films was determined with an energy dispersive spectroscopy (EDS).

• The surface topography was measured using a non-contacting laser profilometer (Rodenstock Metrology RM 600^®).

2.3. Animal model and implantation procedure

Twelve female New Zealand white rabbits, 3-4 months old, were used in this study.

Surgery was performed under general anesthesia by intramuscular injection of Hypnorm^® (0.5 ml/kg) and atropine (0.5 mg/animal). After oro-tracheal intubation, anesthesia was maintained by ethrane 2-3% through a constant volume ventilator. To reduce the perioperative infection risk, prophilactic antibiotic (Terramycine^®) was administered postoperatively by a subcutaneous injection.

In two surgical sessions the implants were placed subcutaneously into the back. Before surgery the skin was shaved, washed and disinfected with iodine. During each surgical session six longitudinal incisions of about 1.5 cm were made parallel to the spinal column. Lateral to these incisions small subcutaneous pockets were created by blunt dissection with scissors. The implants were inserted into these pockets and the wounds were closed using Vicryl 3-0 intracutaneously.

Each rabbit received 12 discs, six in the left and six in the right side of the spinal column. The position of the various implants into the back was based on the method of Latin squares. Each side of the back represented one implantation period. The implantation periods were 1, 4, 8 and 12 weeks. Consequently, at the end of the experiment, six specimens of each coating and implantation time were present.

Postoperatively the animals were placed in cages (five rabbits sharing one big cage, i.e. 1.33×1.10 m²). They were provided with water and rabbit chow ad libitum and were allowed to move unrestricted at all times.

At end of the implantation the animals were killed by injecting an overdose of pentobarbitalsodium (Nembutal^®) peritoneally.

2.4. Histological and physicochemical procedures

After killing the animals, the skin was shaved and the implant with the surrounding tissues were excised immediately. A rectangular patch of the skin, encompassing each implant, was removed. Subsequently, three implants of each group per implantation period were retrieved out of the surrounding tissue capsules. After removal, the implants were dehydrated and examined by XRD, FTIR, SEM and EDS.

The other three specimens of each group per implantation period were left in their surrounding tissue capsule, dehydrated by alcohol series and embedded in methylmetacrylate (MMA). After polymerization, non-decalcified thin (10 
m) sections were prepared using a modified diamond blade sawing microtome technique. These sections were stained with methylene blue and basic fuchsin and examined with a light microscope. Finally, the remaining part of each PMMA tissue block was polished, ultrasonically cleaned with 100% aethylalcohol for 5 min and carbon coated for additional SEM and EDS evaluation.

5. 3. Results

3.1. Characterization of Ca/P coatings before implantation

The XRD patterns of the as-sputtered coatings showed an amorphous structure without specific reflection lines. Infrared HT for 30 s at 425-475°C changed the amorphous sputtered CaP-4 coating into an amorphous-crystalline apatite structure. HT of the CaP-0.1 and CaP-1 specimens resulted in the appearance of tetracalciumphosphate as a second phase.

FTIR measurements showed for all the amorphous coatings two clusters of bands from 900 to 1150 and from 550 to 600 cm⁻¹ attributed to the major absorption modes associated with the presence of phosphate. HT resulted for all coatings in the emergence of the hydroxyl band at 630 cm⁻¹, characteristic for hydroxylapatite and the appearance of the various P-O bands at a wavelength of 567, 587, 948, 965, 1009, 1083, and 1124 cm⁻¹.

SEM examination of the sputtered coatings showed an excellent coverage of the substrate surface. HT of the 4 
m thick coatings resulted in the appearance of a few cracks.

EDS analysis revealed that Ca/P ratio of the coatings varied between 1.8 and 2.0.

The values of the roughness measurements are presented in Table 1.

Table 1. Roughness values of the implant material

3.2. Descriptive light microscopic evaluation

In all cases, wound healing was uneventful. The experimental animals appeared to be in good health throughout the test period and showed no wound complications. At the time of the sacrifice the surgical sites showed no macroscopically signs of infections.

Evaluation of the prepared sections showed that the tissue reaction to the various materials after 1 week of implantation was mainly characterized by an inflammatory response (Fig. 1A). A moderately thick loose connective tissue capsule, containing many inflammatory cells and blood vessels surrounded the implants.

(61K)

Fig. 1. A light microscopical section of a coated specimen after 1 week of implantation showing a moderately thick loose connective tissue capsule, containing inflammatory cells and blood vessels surrounded the implant (A). After 4 weeks the capsule was almost free of inflammatory cells (B). At 12 weeks of implantation the implants were surrounded with a thin connective tissue capsule (C).

After 4 weeks of implantation, light microscopy showed a transition of the capsule. All specimens were surrounded by a thin to medium-thin fibrous tissue capsule. The capsule was almost free of inflammatory cells and contained fibroblasts, collagen and blood vessels (Fig. 1B).

At 8 and 12 weeks of implantation, the tissue response became very uniform. A very thin connective tissue capsule surrounded all implants. At the interface, there was a close contact between the capsule and implant surface and no inflammatory cells were seen (Fig. 1C).

3.3. Physicochemical analysis of the retrieved implants

3.3.1. X-ray diffraction

XRD measurements revealed that after 1 week of implantation the amorphous CaP-0.1 and CaP-1 coatings had disappeared and that on the CaP-4 specimens still coating could be detected. The amorphous phase disappeared during the implantation periods and an increase of crystallinity could be observed (Fig. 2).

(8K)

Fig. 2. XRD patterns of a 4 
m amorphous Ca-P sputter coating after 1-12 weeks of implantation showing an increase of crystallinity in time.

After 4 weeks of implantation the CaP-0.1HT was still present. Further, XRD revealed that at all implantation periods the CaP-1HT and CaP-4HT coatings could be detected. XRD also showed the disappearance of the tetraCP phase had disappeared (Fig. 3 and Fig. 4).

(11K)

Fig. 3. XRD patterns of a 1 
m heat-treated Ca-P sputter coating after 1-12 weeks of implantation. After 1 week the tretra Ca-P phase disappeared and the Ca-P coating could be detected at the end of the implantation period. (asterisk=tetracalcium phosphate)

(8K)

Fig. 4. XRD patterns of a 4 
m heat-treated Ca-P sputter coating after 1-12 weeks of implantation.

3.3.2. Fourier transform infrared spectroscopy

FTIR analysis of the amorphous coatings showed that 1 week after implantation on the CaP-0.1 specimens no PO-bands could be detected, while the 1 and 4 
m samples still showed the presence of PO-bands. In addition, on these specimens CaP-1 and CaP-4 specimens carbonate apatite (CO₃-AP) at 1454 and 1404 cm⁻¹ associated with an organic phase (NH₂-groups) at 1652 and 1545 cm⁻¹ was present. After 4 weeks of implantation, PO-bands and carbonate apatite could only be seen on the amorphous CaP-4 specimens. These PO- and CO₃-AP peaks were still present after 12 weeks of implantation.

On all heat-treated samples after 1 and 4 weeks of implantation PO, OH and CO₃-AP bands were found. After 8 weeks, all these bands were disappeared for the CaP-0.1HT samples. At this implantation time, the CaP-1 and CaP-4 heat-treated coatings, showed the presence of PO bands and small CO₃-AP bands. These PO- and CO₃-AP bands were still present after 12 weeks of implantation (Fig. 5).

(8K)

Fig. 5. A Fourier transform infrared spectrum of a 1 
m heat-treated Ca-P sputter coating after 12 weeks of implantation.

3.3.3. Scanning electron microscopy

SEM evaluation confirmed that already 1 week after implantation the amorphous CaP-0.1 coating had disappeared, while the CaP-1 coating showed signs of surface dissolution (Fig. 6A). Further, on the CaP-4 specimens a precipitate was observed and the coating surface showed a crackled appearance (Fig. 6B). Four weeks after implantation all CaP-1 amorphous coatings were dissolved completely. The CaP-4 amorphous coatings were still present, although they showed a low adhesion with the titanium surface ( Fig. 6C). After 8 and 12 weeks these coatings with precipitate were partly disappeared.

(70K)

Fig. 6. Scanning electron micrograph of an amorphous CaP-1 coating after 1 week of implantation. The coated surface showed surface dissolution (A). Scanning electron micrograph of amorphous CaP-4 coating after 1 week of implantation showing a crackled appearance and the formation of a Ca-P precipitate (B). Scanning electron micrograph of amorphous CaP-4 coating after 4 weeks of implantation showing dissolution of the coating (C). Scanning electron micrograph of heat-treated CaP-4HT coating after 12 weeks of implantation showing the formation of a granular Ca-P precipitate (D).

All heat-treated coatings were still present after 4 weeks of implantation.

After 8 weeks the CaP-0.1HT coatings were disappeared completely. Both other heat-treated coatings were maintained. Occasionally, a granular precipitate was deposited on the CaP-4HT coating (Fig. 6D).

3.3.4. Energy dispersive spectroscopy

EDS analysis showed that the Ca/P ratio of the CaP-4.0HT coatings decreased during implantation and varied after 12 weeks between 1.62 and 1.69. Further, EDS confirmed the Ca-P nature of the occasionally deposited precipitate. The Ca/P ratio of this precipitate varied between 1.00 and 1.45.

6. 4. Discussion and conclusions

The purpose of the present study was to investigate the in vivo dissolution properties of Ca-P coatings applied to TiO₂-blasted implants.

The results of the XRD measurement showed that a rapid heating with infrared radiation of amorphous Ca-P coatings changed their structure into an amorphous/crystalline structure. This corroborates with the studies of Watanabe and Yoshinari, who investigated the influence of rapid heating with infrared radiation on thin Ca-P coatings [17 and 18]. They found an increase of the crystallinity and a decrease in the heating time from several hours to few minutes. Further, we observed that at end of the implantation period the heat-treated coating were still present and the amorphous coatings were completely dissolved. These results are in contrast with our in vivo findings using similar coating on as-machined titanium implants [12]. These experiments demonstrated coating detachment from the as-machined titanium disc at 12 weeks of implantation. We assume that residual film stress developed as a result of the HT, causes coating detachment. These residual stresses are attributed to grain-growth during the HT and differences in thermal expansion coefficients between Ca-P coating and substrate. These stresses influence the Ca-P coating/substrate properties. For example, high residual stresses can result in a loss of adhesion between the coating and substrate and consequently lead to a failure of the implant [19]. It is well known that precoating treatment, grit- and glass-bead blasting affect the final residual film stress [20 and 21]. A three-dimensional surface enlargement, as created by TiO₂-blasting, results in a decrease in the residual film stress. This will have a beneficial effect on the film adhesion to the substrate. Another remark has to be made about the difference in adhesion strength between the amorphous- and heat-treated coatings. SEM and FTIR inspections of the CaP-4 and CaP-4HT specimens revealed that dissolution/precipitation mediated events induces the precipitation of a stable Ca-P phase, which incorporates other ions (carbonate, magnesium, etc.) and organic macromolecules from the biological fluids [22]. This carbonated apatitic precipitate of the amorphous coating showed a low adhesion with the titanium surface, while the heat-treated coating appeared to be stable. This difference in both dissolution behavior and interfacial adhesion strength is due to the dissolution of the amorphous phase in the sputter coatings. In heat-treated coatings, apatite nuclei grow only on the surface of the Ca-P coating. In contrast, on amorphous coating apatite nuclei grow not only on the surface of the amorphous coating, but also inside the amorphous layer. Apparently, these apatite nuclei grow spontaneously by consuming the calcium and phosphate ions from the amorphous phase and the surrounding biological fluids. Further, we have to notice that this bone-like apatite formation is limited by the thickness of the amorphous layer. Furthermore, the weak adhesive strength of amorphous sputter coatings will hamper the long-term stability of the implants. Therefore, we conclude that the rationale for the use of thin amorphous sputter coatings can be questioned.

Finally, the histological evaluation demonstrated that there are no differences in soft tissue reactions to the various Ca-P coated implants. The implants at 1 week of implantation showed a mild tissue response, characterized by the presence of a thick fibrous capsule and the occurrence of many inflammatory cells at the implant-tissue interface. This capsule evolved during the implantation period into a very thin capsule without inflammatory cells and containing fibrocytes, collagen and blood vessels. This finding is in agreement with our previously performed studies using the same animal model. These experiments demonstrated that the fibrous tissue capsule around the implants is the result of the wound healing response to surgical trauma.

Finally, we have to emphasize that the results of the current study confirm one of our previous experiments, where we tested the biological behavior of Ca-P sputter coatings placed in trabecular femoral bone [23]. This bone study demonstrated that the initial bone response around TiO₂-grit blasted titanium implants provided with a 1 thick heat-treated Ca-P sputter coating enhances the bone response compared to just grit-blasted implants. Evidently, Ca-P sputter coatings have to possess a certain crystallinity combined with sufficient thickness to perform their claimed functions, i.e. (1) to establish reduced healing time before loading the implant, (2) to improve the integration of oral implants in bone of poor quality and quantity, and (3) to improve the healing response to other types of "bone" replacing devices, like for example orthopedic implants.

Based on the current results, we can conclude that 1 
m 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, while the titanium surface roughened by grit-blasting has a greater resistance to delamination. Of course, the final relevance and efficacy of Ca-P sputter coated implants has also to be proven in human trials.

7. Acknowledgements

This study was supported by the Dutch Technology Foundation (STW).

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