in vivo experimental study of implant-bone...

IN VIVO EXPERIMENTAL STUDY OF IMPLANT-BONE INTERACTION

J. NEAMŢU1*, I. MĂNDRILĂ1, P.R. MELINTE1, O.M. MĂRGINEAN1, CĂTĂLINA PISOSCHI1, ADELINA IANCULESCU2, I.N. MIHĂILESCU3

1University of Medicine and Pharmacy, Craiova, Romania 2Polytechnic University of Bucharest, Romania

3National Institute for Lasers Plasma and Radiation Physics, Bucharest, Romania

(Received March 4, 2008)

In this study we present an in vivo experimental animal model for testing some titanium structures covered with hydroxylapatite. The model can be applied for testing biomaterials used in manufacturing components of orthopedics and dental implants. We used 15/1.5 mm disc shaped titanium structures that were covered with hydroxylapatite by the pulsed laser deposition method (PLD) and implanted on rabbit tibia for six weeks. All the international regulations regarding animal care were respected. After extraction, the implanted structures were prepared for mechanical testing on a traction machine. We also performed SEM analysis by a scanning electronic microscope (SEM) with an EDAX device, type HITACHI S2600N. The medium force for loosening the implants covered with HA deposited by PLD was 16 N, and 7.5 N for the uncovered ones. After implant extraction, bone fragments were often left on its surface, which was confirmed by FTIR microspectroscopic analysis achieved by reflecting on these fragments. On certain lots of the implanted probes we found evidence of time degradation of the HA deposited layer. The results of the extraction test demonstrate that the nanostructured calcium phosphate based depositions obtained by PLD are a good choice regarding the increase of bioactivity and stimulation of implant fixation to bone. We encountered HA layers biodegradation in a lot of samples characterized by a low crystalline component that presented areas of intense osseointegration together with areas of intense HA biodegradation. Our analyzing methods can be used to characterize the bone-implant interface. Without replacing the classical histological and histomorphometric methods, our techniques represent a complementary study of this interaction. In order to diminish some of the disadvantages of our experimental model, encapsulating the implants in biocompatible resins and assuring a better contact to the underneath bone tissue should be considered by us in future research.

Key words: in vivo testing, bone, implant, micro spectroscopy FTIR.

INTRODUCTION

Biomaterials are more frequently used for the reconstruction and rebuilding of damaged bones and parts of vertebrates muscle and skeletal system (1–3). In the

* Corresponding author (E-mail: [email protected]; Tel./fax: +40 251 523 929)

ROM. J. BIOCHEM., 46, 1, 25–36 (2009)

J. Neamţu et al. 2 26

past, these types of materials were called biocompatible if they presented a minimum biologic response, in other words, biocompatibility was associated with the absence of adverse effects. In our days, biocompatibility is described as the ability of biomaterials to act satisfactorily in the above described conditions (4–5).

The process of covering metallic implants with thin layers of different biomaterials combines the mechanical characteristics of the implant with the excellent biocompatibility of the deposited biomaterial. Deposition of hydro-xylapatite on some parts of orthopedic implants has been used for more than 15 years in uncemented total hip arthroplasty (6–9). There are numerous studies regarding hydroxylapatite covering of hip prosthesis components that have shown there is an increase of mechanical resistance at the bone-implant interface, while acetabular cup migration is diminished. The hydroxylapatite-prosthesis interface remains the weak point of these coverings, as it may delaminate after a period of time depending on the deposition method.

There is a great interest for physics, biology and medical sciences in finding a method to change the surfaces of titanium alloy implants by deposition of different layers formed of calcium phosphate, proteins and other drugs that would favor cellular adhesion leading to a shortening of osteointegration time (10–12). An important parameter for the optimal functioning of an osseous implant is obtaining the best mechanical stability of the biomaterial at the so-called extraction test. In fact, this mechanical evaluation consists in measuring the extraction force of an implant at different time moments after implantation, by different tests (push and pull-out test, removal torque), and it provides information regarding the osteointegration process (13–15).

The osteointegration process is best evaluated by histological and histomorpho-metric techniques for determining bone density and for evaluating the structural changes inside osseous trabecula around implants. Preparing samples for these techniques is a very difficult and time consuming process (16–17). After the extraction of bone-implant complex, the surrounding soft tissues are removed and the samples are fixed, dehydrated and finally included in polymeric resins (epoxy, methylmetacrylate). These resins penetrate deeply inside the tissue and harden the sample as they form a tough, solid compound containing bone and implant that can be later cut with an ultramicrotome. The resulting sections can be analyzed by histological and histomorphometric methods in optic and electron microscopy.

The process of simultaneously cutting the implant and the adjacent bone implies using diamanté knives that could liberate metallic debris and interfere to the histological analysis results. At the same time, there is a risk for delaminating the hydroxylapatite layer on the implant surface. Keeping intact the implant-bone interface remains a difficult procedure and quite often necessitates appealing to experimental cutting techniques that are not present in usual histology laboratories. In order to avoid these complications, we proposed ourselves to evaluate an

3 In vivo implant-bone interaction 27

experimental model for in vivo testing of titanium alloy implants covered with hydroxyapatite. Extracting these structures from rabbit tibia surface allowed us to consider the mechanical retention force and to analyze the physical and chemical properties of implant surface.

Even if we do not use histological and histomorphometric techniques, the interface analysis methods, we propose, are rapid, do not require supplemental preparing of the implant surface but still allow for qualitative evaluation of the processes that take place at the bone-implant interface. We pointed out the process of osseous matrix formation on the implant surface, the mineralization and the interaction with the biologic fluids by means of micro spectroscopy FTIR, scanning electron microscopy (SEM) and energy dispersive X ray spectroscopic EDX. In this way we can follow implant biodegradation.

MATERIAL AND METHODS

MATERIAL

We implanted disc shaped titanium structures with a diameter of 15 mm and a thickness of 1.5 mm; they were covered with hydroxylapatite by pulsed laser deposition method (PLD) (18).

As a support for deposition we used titanium type G4 disc offered and mechanically manufactured by Dentarum Gmbh. Prior to pulsed laser deposition, the samples were chemically corroded in order to induce a microporous surface. After hydroxylapatite deposition, the samples were investigated physically and chemically (X ray diffraction, infrared spectroscopy, and electron microscopy, EDX analysis) to confirm the hydroxylapatite deposition. Prior to in vivo experiments the structures have been tested in solutions that simulate biologic fluids.

SURGICAL PROCEDURE

The model we proposed for the study of the implant mechanic adhesion was a rabbit tibia (2500–3000 g rabbit). The instruments and the implants themselves were sterilized in a special PeraSafe solution; we created specially designed fixing devices out of 0.1 and 0.9 mm remanium wire for ensuring the contact between tibia bone and the studied implant. The animals were handled according to the official regulations concerning medical experiments; they were anesthetized with intramuscular ketamine (250 mg/5 ml) in a dose of 15–20 mg/kg, ten minutes before surgery; in selected cases the general anesthesia was augmented by local injection of 2–3 ml lidocaine; antibiotic prophylaxis was achieved by 500 mg intramuscular injection with oxacillin. The surgery protocol started as with every surgical


intervention, with the disinfection and isolation of the incision site using antiseptic solution and single use sterile drapes. We performed a 6 cm incision on the anterior lateral side of tibia; after precise dissection of the subcutaneous, fascia and muscle layers we exposed the tibial cortical bone. We sectioned and removed the periosteum with a rasp and smoothened the anterior side of tibia bone in order to provide a flat contact surface area for the implants that were then applied and secured with the 0.9 mm wire fixing devices and tided with the 0.1 mm remanium wire (Fig. 1). Afterwards, we sutured all layers with slow absorbable 4–0 polyglycolic acid wire. Antibiotic prophylaxis was finally accomplished by administering the final dose of 500 mg oxacillin. The implants were kept in for 6 weeks; all the international regulations regarding living environment and feeding of the animals were respected.

Fig. 1. – Implant fixed on rabbit tibia.

At the end of the experiment the rabbits were euthanasiated by intravenous injection of 1 ml fentanyl and 2 ml/kg pentobarbital.

MICROSCOPY AND ANALYSIS TECHNIQUES

After tibia extraction we removed all the surrounding tissues and the implanted structures were prepared for mechanical testing. The pull-out of the implant was achieved by fixing the tibia in the locking device of a traction machine. On the implant surface that has not been in contact with the bone we glued a metallic disc with a screw. We tied a metallic wire to the screw and we applied elongation forces at a speed of 1 mm/min. A very important aspect that was taken into consideration was the perpendicular alignment between the elongation force and the implant surface. The morphology of the implanted structures and the ions distribution on the implant surface were studied by an electronic microscope (SEM) with an EDAX device, type HITACHI S2600N.


In the past ten years researchers have shown a great interest in infrared vibration spectroscopy (FTIR) as a way for non-invasive diagnostic in rapid characterization of biological tissues and fluids. The method is essentially based on obtaining a vibration spectrum that could help identify the biochemical constituents such as nucleic acids, proteins and lipids (19–21).

After coupling a FTIR spectroscope to an optical microscope we could get spectrums with a spatial resolution up to 10 microns. Applications of this FTIR microspectroscopy technique in the study of malign tumor pathology are quite in fashion and the term of “spectral marker” is more often used in characterizing some forms of cancer (prostate, skin, breast, etc.). Recent studies demonstrate the possibility of using this technique in analyzing the bone tissue mineral and organic components.

The chemical analysis of the interface has been done in microspectroscopy FTIR iN Nicolet; we used reflexion mode on a spectral interval 4000–650 cm–1.

RESULTS

The medium force for loosening the implants covered with HA deposited by PLD was 16 N, while the uncovered titanium implants were taken off at a medium force of 7.5 N. As one can see in optical microscopy revealed in Fig. 2, after implant extraction, there are often bone fragments left on its surface; the bony nature of these fragments was confirmed by FTIR microspectroscopic analysis achieved by reflecting on these fragments. Observing FTIR spectrum in Fig. 3, one can notice the characteristic bands of the mineral and of the bone organic components.

Fig. 2. – Implant surface after extraction viewed in optical microscopy. After

extraction, one can see bone fragments on the implant surface.


carbonate

Phosphate

Amide III

carbonate

Amide IIAmide I

water

0,0

0,1

0,2

0,3

0,4

0,5

0,6

0,7

0,8

0,9

1,0

Abs

1000 1500 2000 2500 3000 3500 cm-1

Fig. 3. – FTIR spectrum of bony fragments left on the implant surface.

At the same time, on certain lots of the implanted probes, besides the bony fragments remained on the implant surfaces, there is evidence of time degradation of the HA deposited layer. This phenomenon is also observed in SEM microscopy in Fig. 4, as craters from the erosion process after in vivo testing, even if the EDAX cartography achieved before implantation, Fig. 5, showed a uniform distribution of calcium and phosphorus ions on the implant surface, suggesting a uniform deposition.

Fig. 4. – SEM image of the implant surface after extraction, in a sample

characterized by a low crystalline grade.

Titanium


Fig. 5. – EDAX ionic distribution on the implant surface before in vivo implantation. The uniform calcium and phosphorus ions distribution demonstrates a uniform deposition.

On the same lot, the erosion process was pretty intense and the HA was completely degraded on some parts of the implant surface. The biodegradation process of HA is observed after EDAX cartography in Fig. 6, where we represented the spatial distribution of titanium ions on the implant surface. Practically, there are areas with more

Fig. 6. – SEM image: (left) – titanium ions distribution on implant surface after EDAX analysis;

(right) – after in vivo testing.


phosphateAmide III

carbonate

Amide II

Amide Iwater

a

0,0

0,2

0,4

0,6

0,8

1,0

Abs

phosphate

waterb

0,00

0,05

0,10

0,15

Abs

1000 2000 3000 4000 cm-1

calcium and phosphorus ions that exist in the crystalline structure of HA and/or in the newly formed mineral component on the implant surface. At the same time, on the same image one can notice areas where the HA layer was completely degraded and there are more titanium ions.

The biodegradation process was also revealed by FTIR microspectroscopy (Fig. 7); the absorption bands characteristic to the HA mineral component are very weak in the areas of intense biodegradation (Fig. 7b), while in the areas surrounding the bony fragments there is an intense process of osseous matrix synthesis and mineralization (Fig. 7a).

Fig. 7. – FTIR spectrum in two areas of the implant: a – around the bony fragments;

b – biodegradation area.

DISCUSSION

The measured values of the force responsible for extraction of the HA covered implants were two times higher than those of the uncovered implants. We must specify that the forces we measured by the traction test are a little bit higher than those reported in the specialty literature; this is due to our experimental model that allowed the interaction of the bone to the lateral uncovered side of the implant (22–23). Recent in vivo studies recommend the use of a polytetrafluorethylene capsule that surrounds the lateral side of the implant avoiding the supplemental mechanical retention that could lead to a false positive effect in estimating the interaction HA-bone. For these reasons, an important aspect in this kind of models is the complete removal of the newly formed bone around the implant.


The results of the extraction test demonstrate that the nanostructured calcium phosphate-based depositions obtained by PLD are a good choice in terms of increasing the bioactivity and stimulation of implant fixation to the bone. It is well known that PLD offers many advantages to other deposition methods and the greatest of all are the ability of growing pure and crystalline HA layers and the flexibility in controlling phase and chemical composition (Ca/P ratio) of other calcium phosphate bioceramics.

The advantage of this deposition method is proven by the existence of bony fragments remained on the implant surface (Fig. 2) after extraction, standing up for greater bonding force between HA-implant than between HA-bone.

Regarding the performance of biomaterials, it comprises two components: the response of the host to the implant and the behavior of the material in the host (24–26). The almost immediate event that occurs upon implantation of metals, as with other biomaterials, is adsorption of proteins (27) that first come from the blood and tissue fluids at the wound site and later from cellular activity in the region; once on the surface, proteins can desorb or remain to mediate tissue-implant interactions. At the same time, there is ample literature describing oxidation of metallic implants and surface analytic studies show that the chemical composition of the oxide film changes by incorporating Ca, P and S (26–27). Another consequence is the release of metal ions into surrounding tissues and in vitro studies revealed that these metal ions, even at sublethal doses, interfere with the differentiation of osteoblasts and osteoclasts. Thus, the host response to implants placed in bone involves a series of cell and matrix events, ideally culminating in intimate apposition of bone to biomaterial, i.e., osseointegration. Even more recent studies (25) during more than 15 years describe the cascade of de novo bone formation of solid surfaces as a four stage process comprising: the adsorption of non-collagenous bone proteins to the solid surface; the initiation of mineralization by the adsorbed proteins; continued mineralization due to crystal growth; and finally, the assembly of a collagen matrix overlying the interfacial matrix with mineralization within the collagen matrix.

The synthesis of the collagen matrix and its mineralization on the implant surface can be pointed out by FTIR microspectroscopy. We state that FTIR microspectroscopy cannot replace the histological and histomorphometric bone analyzing techniques but it is a complementary method for the rapid characterization of these tissues.

The spectrum illustrated in Fig. 3 shows the presence of all the bands of the mineral and organic components. The vibration bands in the region 900–1200 cm–1 are created by the bone mineral component. A greater number of narrow bands are characteristic for a highly crystallized bone. In fact, these bands are due to phosphate groups bonding vibrations inside HA and their presence is indicative for the bony tissue fragments that remain on the implant surface after extraction. In the same spectrum there are also the main absorption bands that correspond to collagen. They appear because of the peptides bonding vibrations around the values 1660, 1540,


1240 cm–1 and are encountered in the specialty literature as Amide I, II and III bands, respectively. The most important is the Amide I band and the ratio between the phosphate band and this band is successfully used in establishing bone mineralization.

Other important bands that appear in bone vibration spectrum correspond to the carbonate group that replaces the phosphate and hydroxyl from HA. As illustrated in Fig. 3, these bands appear in the intervals 850–900 and 1350–1470 cm–1. In this way, the ratio between the 850 cm–1 band and the phosphate or Amide I band is used in establishing the amount of carbonate inside bone tissue. At the same time, several studies demonstrate that these ratios increase with the age of the investigated tissue, offering clues about the maturation of the investigated bone.

The spectrum in Fig. 7 corresponds to an area on the implant surface situated close to the bony fragments. Even if on the interval 900–1200 cm–1, the phosphate characteristic bands are wider and fewer in number, the presence of the bands characteristic of collagen and carbonate shows that an osseous matrix synthesis and mineralization occurs at the implant surface. The presence of this structure on the implant surface suggests that there is no adverse reaction and that the implant osseointegration process has begun.

The process of HA layers biodegradation after in vivo implantation is frequently reported in the specialty literature even after a few weeks (27). We encountered a similar situation in a lot of probes characterized by a low crystalline component that presented areas of intense osseointegration together with areas of intense HA biodegradation. Taking into account that HA is an osteoconductive biomaterial and that our experimental model does not allow a uniform contact of the implant on tibia, it is obvious that in the regions where this contact was tighter, the osseointegration was faster. This aspect is suggested by the optical microscopy that shows the presence of some bony fragments remained on the implant surface after the extraction test.

Taking into account that testing of the same structures in solutions that simulate biologic fluids did not prove the HA degradation, it is obvious that degradation is not a simple hydrolytic process. In fact, in the areas where HA is not in tight contact to the bone surface, a large amount of biologic fluids and cells could determine a highly increased enzymatic activity that could lead to the HA biodegradation. These findings are in good accordance with literature data that reported a high resorption rate when the hip prostheses components came in contact to the bone marrow (28).

CONCLUSIONS

The model we propose allows for the study of the in vivo behavior of some biomaterials used as components of the orthopedics and dental implants. The results


of this study show that our analysis methods can be used to characterize the bone-implant interface. Without replacing the classical histological and histomorphometric methods, our techniques represent a complementary study of this interaction. With no further need for prior supplemental preparing techniques, the methods of electron microscopy and FTIR microspectroscopy allowed us to obtain a qualitative survey of the processes that take place at the bone-implant interface.

In order to diminish some of the disadvantages of our experimental model we should consider in the future encapsulating the implants in biocompatible resins and assuring a better contact to the underneath bone tissue.

REFERENCES

1. Hench L.L., Wilson J., Introduction, in: An introduction to bioceramics, Hench L.L., Wilson J., eds., World Scientific, Singapore, 1999, p. 1–24.

2. Porter J.A., von Fraunhofer J.A., Success or failure of dental implants? A literature review with treatment considerations, Gen. Dent., 53, 423–32 (2005).

3. Williams D., Introduction, in: Biocompatibility of clinical implant materials, Williams D., eds., CRC Press, 1981, p. 3–42.

4. von Recum A.F., Jenkins Michelle E., von Recum H., Introduction: Biomaterials and Biocompatibility, in: Handbook of biomaterials evaluation, Andreas F von Recum, ed., Taylor & Francis, 1999, p. 1–8.

5. Geesink R.G., Hoefnagels N.H., Six-year results of hydroxyapatite-coated total hip replacement, J. Bone Joint Surg. Br., 77, 534–47 (1995).

6. Tanzer M., Gollish J., Leighton R., Orrell K., Giacchino A., Welsh P., The effect of adjuvant calcium phosphate coating on a porous-coated femoral stem, Clin. Orthop. Relat. Res., 424, 153–60 (2004).

7. Ducheyne P., Qiu Q., Bioactive ceramics: the effect of surface reactivity on bone formation and bone cell function, Biomaterials, 20, 2287–303 (1999).

8. Dumbleton J., Manley M.T., Hydroxyapatite-coated prostheses in total hip and knee arthroplasty, J. Bone Joint Surg. Am., 86, 2526–40 (2004).

9. Garcia A.J., Keselowsky B.G., Biomimetic surfaces for control of cell adhesion to facilitate bone formation, Crit. Rev. Eukaryot. Gene Expr., 12, 151–62 (2002).

10. Hersel U., Dahmen C., Kessler H., RGD modified polymers: biomaterials for stimulated cell adhesion and beyond. Biomaterials, 24, 4385–415 (2003).

11. Balasundaram G., Sato M., Webster T.J., Using hydroxyapatite nanoparticles and decreased crystallinity to promote osteoblast adhesion similar to functionalizing with RGD, Biomaterials, 27, 2798–805 (2006).

12. Berzins A., Summe D., Implant pushout and pullout tests, in: Mechanical testing of bone and the bone-implant interface, An Y.H., Draughn R.A., eds., Boca Raton, FL: CRC Press LCC, 1999, p. 463–76.

13. Ronold H.J., Ellingsen J.E., Lyngstadaas S.P., Tensile force testing of optimized coin-shaped titanium implant attachment kinetics in the rabbit tibiae, Journal of Materials Science: Materials in Medicine, 14, 843–849 (2003).

14. Ronold H.J. and Ellingsen J.E., Effect of micro-roughness produced by TiO2 blasting-tensile testing of bone attachment by using coin-shaped implants, Biomaterials, 23, 4211–4219 (2002).


15. Alava J.I., Marı’n F., Intxaurrandieta A., Braceras I., De Maeztu M.A., Simultaneous materialographic and histological thin section preparation: Investigation of bone integration on dental implants, Structure Struers J. Materialography, 6, 1–6 (2005).

16. Nuss K.M., Auer J.A., Boos A., von Rechenberg B., An animal model in sheep for biocompatibility testing of biomaterials in cancellous bones, BMC Musculoskelet Disord., 7, 67 (2006).

17. Aparicio S., Doty S.B., Camacho N.P., Paschalis E.P., Spevak L., Mendelsohn R., Boskey L.A., Optimal methods for processing mineralized tissues for fourier transformed infrared microspectroscopy, Calcif. Tissue, 70, 422–429 (2002).

18. Nelea V., Ristoscu C., Chiritescu C., Ghica C., Mihailescu I.N., Pelletier H. et al., Pulsed laser deposition of hydroxyapatite thin films on Ti-5Al-2.5Fe substrates with and without buffer layers, Applied Surface Science, 168, 127–1`31 (2007).

19. Boskey A., Pleshko C.N., FT-IR imaging of native and tissue-engineered bone and cartilage, Biomaterials, 28, 2465–2478 (2007).

20. Boskey A.L., Mendelsohn R., Infrared spectroscopic characterization of mineralized tissues, Vibrational spectroscopy, 38, 107–114 (2005).

21. Berzins A., Summe D., Implant pushout and pullout tests, in: Mechanical testing of bone and the bone–implant interface, An Y.H., Draughn R.A., eds., Boca Raton, FL: CRC Press LCC, 1999, p. 463–76.

22. Ronold H.J., Ellingsen J.E., Lyngstadaas S.P., Tensile force testing of optimized coin-shaped titanium implant attachment kinetics in the rabbit tibiae, Biomaterials, 23, 4211–4219 (2002).

23. Davies J.E., Bone bonding at natural and biomaterial surfaces, Biomaterials, 28, 5058–5067 (2007).

24. Puleo D.A., Nanci A., Understanding and controlling the bone implant interface, Biomaterials, 20, 2311–2321 (1999).

25. Takatsuka K., Yamamuro T., Nakamura T., Kokubo T., Bone bonding behavior of titanium alloy evaluated mechanically with detaching failure load, J. Biomed. Mater. Res., 29, 157–63 (1995).

26. Merolli A., Moroni A., Faldini C., Tranquilli Leali P., Giannini S., Histomorphological study of bone response to hydroxyapatite coating on stainless, Journal of Materials Science: Materials in Medicine, 14, 843–849 (2003).

27. Ronold H.J. and Ellingsen J.E., Effect of micro-roughness produced by TiO2 blasting-tensile testing of bone attachment by using coin-shaped implants steel, Journal of Materials Science: Materials in Medicine, 14, 327–3333 (2003).

28. Overgaard S., Soballe K., Lind M., Bunger C., Resorption of hydroxyapatite and fluoroapatite coatings in man, J. Bone Jt. Surg. Br., 79, 654–9 (1997).

in vivo experimental study of implant-bone...

Documents