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J Dent Res 86(1):84-89, 2007
© 2007 International and American Associations for Dental Research

RESEARCH REPORT
Biomaterials & Bioengineering

Delivery Mode and Efficacy of BMP-2 in Association with Implants

Y. Liu1,2, R.O. Huse1,4, K. de Groot3, D. Buser4, and E.B. Hunziker1,*

1 ITI Research Institute for Dental and Skeletal Biology, University of Bern, Switzerland;
2 ACTA, Section of Oral Implantology and Prosthetic Dentistry, Department of Oral Function, Amsterdam, The Netherlands;
3 Department of Science and Technology, University of Twente, The Netherlands; and
4 Department of Oral Surgery and Stomatology, School of Dental Medicine, University of Bern, Switzerland

* corresponding author, ernst.hunziker{at}iti.unibe.ch


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Bone healing may be improved in implant patients by the administration of osteogenic agents, such as bone morphogenetic protein 2 (BMP-2). But the efficacy of BMP-2 depends upon its mode of application. We hypothesized that BMP-2 is capable of a higher osteogenic efficacy when delivered physiologically, viz., when incorporated into a calcium-phosphate carrier that mimics mineralized bone matrix, than when administered via simple pharmacological modes, such as by adsorption onto a carrier surface. Using an ectopic rat model, we compared the osteoinductive efficacies of calcium-phosphate implant-coatings bearing either incorporated, adsorbed, or incorporated and adsorbed BMP-2. When adsorbed directly onto the naked implant surface, BMP-2 was not osteogenic. When adsorbed onto a calcium-phosphate coating, it was osteoinductive, but not highly efficacious. When BMP-2 was incorporated into calcium-phosphate coatings, it was a potent bone-inducer, whose efficacy was compromised, not potentiated, by the additional deposition of an adsorbed pool.

KEY WORDS: osteoinduction • drug-delivery mode • biomimetic • implants • coating • bone morphogenetic protein 2 (BMP-2)


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Concerted efforts have been and still are being made to accelerate and augment bone formation around dental and orthopedic implants, with a view to expediting the establishment of, as well as to improving, mechanical stability.

The osteoconductivity of titanium implants can be improved either if their surface properties are modified (Buser et al., 1991; Meffert, 1997; Cochran et al., 1998; Brunette and Chehroudi, 1999), or if they are coated with a layer of calcium phosphate (de Groot, 1989; Klein et al., 1994).

However, the implants can be rendered osteoinductive only by the introduction of an osteogenic agent, such as bone morphogenetic protein-2 (BMP-2). The BMP-2-carrier potential of numerous materials has been tested at both ectopic and orthotopic sites (Ono et al., 1995; Hollinger and Leong, 1996; Reddi, 2000). In all cases, the adsorbed agent was liberated too rapidly to induce a sustained osteogenic response.

Hitherto, it has not been possible for investigators to do otherwise than deposit osteogenic agents superficially upon pre-formed calcium-phosphate layers, owing to the extremely unphysiological conditions under which these coatings are prepared. But now, a technique is available for depositing calcium-phosphate layers upon implant surfaces under physiological conditions of temperature (37°C) and pH (7.4). With this so-called biomimetic technology (Kokubo et al., 1990; Liu et al., 2001, 2003a, Liu et al., 2003b, 2004), bioactive agents can be incorporated into the three-dimensional crystal latticework, from which they are released gradually in vivo as the layer undergoes degradation. BMP-2 has been incorporated into biomimetic calcium-phosphate coatings, and has been shown to induce and sustain bone formation at an ectopic implantation site in rats (Liu et al., 2005).

In the present study, the osteoinductive efficacies of biomimetic calcium-phosphate coatings bearing either adsorbed, incorporated, or incorporated and adsorbed BMP-2 were compared at an ectopic (subcutaneous) ossification site in rats.


   MATERIALS & METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Preparation of Implant Coatings and Mode of BMP-2 Application
Titanium-alloy (Ti6Al4V) discs (1 cm in diameter) were coated biomimetically with a layer of calcium phosphate as previously described (Barrere et al., 1999; Liu et al., 2001, 2003a, 2004; see also APPENDIX A). BMP-2 was adsorbed onto or incorporated into coatings as detailed in APPENDIX A. The amount of BMP-2 passively adsorbed onto or incorporated into the coatings was quantified by ELISA (see APPENDIX B).

Implantation and Histomorphometric Evaluation
Animal experiments were conducted with the permission of, and in accordance with, the regulations laid down by the Animal Protection Commission of the State of Bern (Switzerland). We used 165 young adult male Wistar rats (each weighing from 185 to 250 g) for this study. A total of 330 implants was distributed among the 16 experimental and control groups (TableGo), with n = 6 per group and time-point. The samples were inserted subcutaneously in the dorsal region of each rat (2 samples per animal), under conditions of general anesthesia, as previously described (Liu et al., 2005). They were retrieved 1, 2, 3, and (in selected groups) 5 wks after surgery, and were processed for histologic and histomorphometric evaluation by chemical fixation in formaldehyde and embedding in methylmethacrylate (see APPENDIX C). Six saw-cuts were prepared from each embedded implant, polished, and surface-stained according to a published protocol (Schenk et al., 1984; see APPENDIX C).


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  		Table. Experimental and Control Groups
 
The mean volume of bone tissue per unit area of implant and the mean volume of coating per unit area of implant were determined from two-dimensional light micrographs, by the application of a systematic random sampling strategy (Gundersen and Jensen, 1987), and by the application of a cycloid test system (Baddeley et al., 1986) of curves and points (Maye et al., 1997; see APPENDIX D for a description of the mathematical expressions used).

Statistical Analyses
All numerical data are presented as mean values, together with either the standard deviation (SD) or the standard error of the mean (SEM). Differences within the same group at each sampling time were analyzed by the one-way ANOVA test. Differences between the various groups at a particular sampling time were analyzed by the two-way ANOVA test, the level of significance being set at p < 0.05. SAS statistical software (version 8.2) was used for this purpose. Post hoc comparisons were then made with Bonferroni corrections.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Coatings
Calcium-phosphate coatings had a thickness of 22 ± 6.4 (SD) µm. With the 3 different bathing concentrations of BMP-2—namely, 0.1 µg, 1 µg, and 10 µg per mL (groups 11, 12, and 13 in the TableGo)—the amounts of the drug incorporated into the coatings were estimated by ELISA to be 0.56 ± 0.03 µg, 0.61 ± 0.05 µg, and 1.70 ± 0.24 µg per implant, respectively. Expressed per unit volume of coating, these amounts were 12.5 µg, 17.2 µg, and 32.6 µg per mm3 of coating, respectively. The immersion of coated discs in PBS containing BMP-2 at 10 µg per mL (group 7 in the TableGo) for 48 hrs at 37°C resulted in the passive adsorption of 1.0 ± 0.1 µg of the drug per implant. The immersion of naked (uncoated) discs in a similar medium (group 3 in the TableGo) for the same time resulted in no passive adsorption of BMP-2. The amounts of BMP-2 deposited on discs by direct adsorption (i.e., by the evaporation of applied drops of a standard solution) were pre-determined and therefore not estimated by ELISA (see groups 4, 8, 9, and 10 in the TableGo).

Coatings Bearing Adsorbed BMP-2
One wk after implantation, ectopic bone formation was observed only in association with coatings bearing the highest initial loading dose of adsorbed BMP-2 (7.5 µg per implant). At this high dose, the volume of bone laid down increased continuously during the second and third wks (Fig. 1AGo). At the lowest and intermediate doses of BMP-2, bone was first observed after 2 wks. The volume of osseous tissue deposited at each juncture increased as a function of the adsorbed dose of BMP-2.


Figure 1
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  		Figure 1A. Bone formation in association with coatings bearing adsorbed BMP-2, expressed as a function of time and the amount of BMP-2 adsorbed. Mean values ± SEM (n = 6) are represented.

 
Coatings Bearing Incorporated BMP-2
At the lowest and intermediate doses of incorporated BMP-2, no ectopic bone formation was observed during the five-week monitoring period. At the highest dose, osseous tissue first appeared after the 2nd wk. Between the 2nd and the 5th wks, the volume of bone deposited increased significantly (p < 0.001), from 5.83 mm3 per implant to 10.34 mm3 per implant (Fig. 1BGo). During this time-span, the coating volume decreased significantly (p < 0.05), from 9.2 mm3 per implant to 5.2 mm3 per implant (data not presented).


Figure 1
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  		Figure 1B. Bone formation in association with coatings bearing incorporated BMP-2, 2 and 5 wks after implantation, expressed as a function of the amount of BMP-2 incorporated. Mean values ± SEM (n = 6) are represented. *P < 0.001.

 
Coatings Bearing Incorporated and Adsorbed BMP-2
In contrast to coatings that bore only incorporated BMP-2 (Fig. 1BGo), those that additionally bore a fixed amount of adsorbed BMP-2 were osteoinductive at both the lowest and the intermediate incorporated doses (Fig. 2AGo). The bone volume laid down after 2 wks increased significantly as a function of the incorporated dose.


Figure 2
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  		Figure 2A. Bone formation in association with coatings bearing different amounts of incorporated BMP-2 and a fixed dose of adsorbed BMP-2 (7.5 µg per implant), 2 wks after implantation, expressed as a function of the incorporated dose of BMP-2. Mean values ± SEM (n = 6) are represented. *P < 0.05; **P < 0.001.

 
Control Groups
At no time during the five-week follow-up course was bone formation observed in association either with naked (uncoated) discs that bore no BMP-2 (groups 1, 2, and 3 in the TableGo), with naked discs that bore directly adsorbed BMP-2 (group 4 in the TableGo), with coated discs that bore no BMP-2 (groups 5 and 6 in the TableGo), or with coated discs that bore passively adsorbed BMP-2 (group 7 in the TableGo).

Coating Degradation and the Efficacy of BMP-2
The percentage degradation of coating material during the first 2 wks of implantation was highest for discs that bore adsorbed BMP-2, either alone or in combination with incorporated BMP-2, with mean values ranging between 32% and 47% (Fig. 2BGo). Coatings that bore only incorporated BMP-2 underwent relatively little degradation at the lowest and intermediate drug doses. But at the highest incorporated dose of BMP-2, the percentage degradation of coating material increased dramatically, to 43%, which falls within the range registered for implants bearing adsorbed BMP-2 (Fig. 2BGo).


Figure 2
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  		Figure 2B. Coating degradation in the various experimental groups, 2 wks after implantation.

 
To yield information regarding the efficacy of BMP-2 as a function of its delivery mode, we calculated the total amount of the incorporated drug that had been liberated, together with the degraded coating material, by the two-week juncture, and related it to the total volume of bone tissue that had been formed by this time. Since BMP-2 is known to be homogeneously incorporated into coatings (Liu et al., 2001, 2004), this calculation is valid. The initial loading dose of adsorbed BMP-2 was assumed to have been completely released by the two-week juncture (Ruhe et al., 2005). The largest volume of bone laid down per implant and per microgram of liberated BMP-2 was achieved for implants bearing incorporated BMP-2 alone at the highest initial loading dose. This bone volume (4.19 ± 0.23) was two-fold greater than that laid down in association with hybrid coatings bearing the same initial loading dose of incorporated BMP-2 (1.96 ± 0.4 mm3). The osteoinductive efficacy of the hybrid coatings increased as a function of the initially incorporated dose of BMP-2. The lowest osteoinductive efficacies were registered for the coatings that bore only an adsorbed depot of BMP-2. Likewise, in this latter category, the efficacy index increased as a function of the initial loading dose of BMP-2 (TableGo).

General Histological Findings
A mild inflammatory response involving foreign-body giant cells, macrophages, lymphocytes, and granulocytes was observed around all implants after the 1st wk. By the 2nd wk, the inflammatory response had abated. Each implant was mantled with a vascularized connective tissue capsule (Fig. 3CGo). In the bone-forming groups, osseous tissue was laid down directly (Figs. 3A, 3BGo), not indirectly (via an endochondral process).


Figure 3
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  		Figure 3. Light micrographs of titanium-alloy discs 2 weeks after implantation. (A) Low-magnification light micrograph of an implanted titanium-alloy disc (Ti) bearing a layer of calcium phosphate (C) into which BMP-2 has been incorporated. At this two-week juncture, the disc is surrounded by a growing mass of woven bone (B). (B) High-magnification view of the specimen illustrated in Fig. 3A. The coating (C) is being degraded by osteoclasts (arrows). This degradative activity occurs concomitantly with the deposition of woven bone (B). A = artefactual space between the coating (C) and the titanium-alloy disc (Ti), generated by shrinkage during tissue processing. (C) Low-magnification light micrograph of a naked (uncoated) titanium-alloy disc (Ti) bearing no BMP-2, 2 wks after implantation. The disc is surrounded by a connective tissue capsule (S) from which it has become artefactually separated (arrows) during tissue processing. No bone formation has occurred.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In the present study, we investigated the osteoinductive efficacies of BMP-2-bearing calcium-phosphate coatings as a function of different drug-delivery modes, using a well-established ectopic ossification model in rats (Liu et al., 2005).

When BMP-2 was adsorbed onto the uncoated metal surface of a titanium-alloy implant, it failed to elicit an osteogenic response. When adsorbed onto calcium-phosphate coatings at the same or lower doses, it was osteoinductive. However, the osteoinductive efficacy of an adsorbed depot of BMP-2 was five- to 70-fold lower than that of a coating-incorporated pool (TableGo). The osteogenic response was not potentiated in the hybrid system. Indeed, it was markedly compromised by the adsorbed depot (TableGo). At the same initial loading dose of incorporated BMP-2 (32.6 µg per mm3 of coating), the two-week efficacy index was two-fold higher for coatings that bore incorporated BMP-2 alone than for those that additionally bore an adsorbed depot of the drug (4.19 ± 0.23 mm3 vs. 1.95 ± 0.4 mm3) (TableGo). This lowering of the efficacy index for the hybrid coatings may be linked to the initial burst release of BMP-2 from the adsorbed pool. The transiently high local concentration of the osteogenic agent could promote the recruitment and activation of osteoclasts, thereby enhancing bone-resorption activity (Ruhe et al., 2005). It could also lead to the non-specific activation and expression of Noggin, and, hence, to the inactivation of BMP-2 and a depression of bone formation.

Two wks after implantation, a similar volume of bone (i.e., approximately 6 mm3 per implant) was associated with coatings that bore either adsorbed BMP-2 at an initial loading dose of 7.5 µg per implant (Fig. 1AGo) or incorporated BMP-2 at an initial loading dose of 1.70 µg per implant, namely, 32.6 µg per mm3 of coating (Fig. 1BGo). However, the amounts of BMP-2 actually liberated into the implant milieu at this two-week juncture were, respectively, 7.5 µg (i.e., the entire adsorbed pool) and 1.39 µg (see TableGo)—hence the superior efficacy of the coating-incorporated drug system.

Differences in the osteoinductive efficacy of BMP-2 primarily reflected differences in its release kinetics. Most of the adsorbed depot of BMP-2 was liberated in a single rapid burst of a few hours’ duration. The remaining portion was released more gradually over a period of several days or weeks. BMP-2 is water-soluble and susceptible to local protease activity within the interstitial fluid. Hence, when adsorbed at a low loading dose, most of the initially released BMP-2 either diffused away from the implantation site or underwent non-specific enzymatic inactivation before it was able to exert an osteoinductive effect.

However, when BMP-2 was adsorbed at a high loading dose, the local concentration transiently generated after its initial burst release might have been sufficiently high to promote non-specific secondary binding to local collagen fibrils (Hartman et al., 2005; Ruhe et al., 2005) within the subcutaneous tissue. Hence, the osteogenic response observed with this system was probably elicited by BMP-2 released (a) from such a secondary collagen-bound pool, and/or (b) from the small residual coating-adsorbed depot characterized by slow-release kinetics. Since a high loading dose is required to induce and sustain osteogenesis for several weeks at a substantial level, this system is inefficient. When BMP-2 was incorporated into the crystal latticework of a coating at a comparatively much lower dose, it was liberated gradually and at a more constant rate as this underwent cell-mediated degradation (Fig. 3Go) during the ensuing weeks. Hence, in this system, the rate of BMP-2 release was related to the rate of coating degradation; since the coatings had not been completely degraded by the end of the five-week monitoring period, the incorporated BMP-2 depot was presumably not exhausted at this juncture (Liu et al., 2005). The conditions operating in this system are thus conducive to a sustained and substantial osteogenic response with low pharmacological doses of BMP-2. Hence, it is highly efficient. Precisely why the osteoinductive efficacy is reduced in the hybrid system is not entirely clear, but an osteoclast-induced enhancement of bone-resorption activity may be implicated, as indicated above (Urist, 1992; Uludag et al., 1999). Our previous (Liu et al., 2005) and present findings indicate that foreign-body giant cells actively participate in the degradation of coatings. Hence, these cells may influence the rate of release of BMP-2 therefrom (Liu et al., 2005).

In conclusion, simple manipulations in the mode of drug delivery by biomimetic calcium-phosphate coatings can effect vast improvements in the osteoinductive efficacy of the system. With an expensive drug such as BMP-2, improvements of this kind are all-important in the development of marketable, functionalized prostheses, which are sorely needed to enhance and expedite osseointegration at compromised implantation sites.


   ACKNOWLEDGMENTS
 
This work was supported by grants from the Swiss National Science Foundation (to E.B. Hunziker) and the AO/ASIF Foundation (to Y. Liu). The authors would like to thank Dr. Xiaoshun Liu for his help with the statistical analysis, Wyeth (Cambridge, MA, USA) for its generous gift of human recombinant BMP-2, and Simon Nüssli for his technical assistance.


   FOOTNOTES
 
A supplemental appendix to this article is published electronically only at http://www.dentalresearch.org.

Received October 10, 2005; Last revision September 26, 2006; Accepted October 5, 2006


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 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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