Background: Porous tantalum has been shown to be effective in achieving bone ingrowth. However, in some circumstances, bone quality or quantity may be insufficient to allow adequate bone ingrowth. We hypothesized that local delivery of alendronate from porous tantalum would enhance the ability of the tantalum to achieve bone ingrowth when there is a gap between the implant and bone. We evaluated the effect of alendronate-coated porous tantalum on new bone formation in an animal model incorporating a gap between the implant and bone.
Methods: A cylindrical porous tantalum implant was implanted in the distal part of each femur in eighteen rabbits (a total of thirty-six implants) and left in situ for four weeks. Three types of porous tantalum implants were inserted: those with no coating (the control group), those with microporous calcium phosphate coating, and those coated with microporous calcium phosphate and alendronate. Subcutaneous fluorescent labeling was used to track new bone formation. Bone formation was analyzed with backscattered electron microscopy and fluorescent microscopy of undecalcified samples.
Results: The relative increases in the mean volume of gap filling, bone ingrowth, and total bone formation in the group treated with the porous tantalum implants coated with calcium phosphate and alendronate were 143% (p < 0.001), 259% (p < 0.001), and 193% (p < 0.001), respectively, compared with the values in the control group treated with the uncoated porous tantalum implants. The percentage of the length of the implant that was in contact with new bone in the group treated with the calcium phosphate and alendronate coating was increased by an average of 804% compared with the percentage in the group treated with the uncoated implants.
Conclusions: The study demonstrated significantly enhanced filling of the bone-implant gap and bone ingrowth in association with the porous tantalum implants coated with calcium phosphate and alendronate.
Clinical Relevance: The addition of an alendronate-delivery surface coating would enhance biological fixation of a tantalum implant and promote the healing of bone defects and thus address the clinical problem of revision joint replacement in the face of bone loss.
1Division of Adult Lower Limb Reconstruction and Oncology, Department of Orthopaedics, University of British Columbia, Room 3114, 910 West 10th Avenue, Vancouver, BC V5Z 4E3, Canada. E-mail address for D.S. Garbuz: firstname.lastname@example.org. E-mail address for W.Y. Kim: email@example.com. E-mail address for B.A. Masri: firstname.lastname@example.org. E-mail address for C.P. Duncan: Clive.Duncan@vch.ca
2Department of Materials Engineering, University of British Columbia, 309-6350 Stores Road, Vancouver, BC V6T 1Z4, Canada. E-mail address for Y. Hu: email@example.com. E-mail address for K. Duan: firstname.lastname@example.org. E-mail address for R. Wang: email@example.com
3Division of Orthopaedic Engineering Research, Departments of Orthopaedics and Mechanical Engineering, 556-828 West 10th Avenue, Vancouver, BC V5Z 1L8, Canada. E-mail address: firstname.lastname@example.org
4Faculty of Pharmaceutical Sciences, University of British Columbia, 2146 East Mall, Vancouver, BC V6T 1Z3, Canada. E-mail address: email@example.com
The management of bone stock deficiency and the reliable achievement of durable implant-host bone fixation remain substantial challenges in revision joint replacement. Use of traditional uncemented acetabular implants (porous-coated and titanium fiber mesh) in the revision setting has had good clinical results1. However, one study has suggested that when there is <50% contact with the host bone, implant fixation is less predictable2. The properties of porous tantalum, including a high coefficient of friction, interconnecting pores, and high porosity by volume, make it an excellent material for use in revisions in the setting of compromised host bone3,4.
The concept of an implant used as a drug-delivery system in reconstructive orthopaedics is well established5, and the use of bisphosphonates as a means to promote early and enhanced bone ingrowth has been investigated in several previous studies6-8. Recently, a third-generation bisphosphonate, zoledronic acid, bound to hydroxyapatite-coated porous tantalum was found to increase bone formation within the medullary canal and within the pores of the porous tantalum in a canine model9. The zoledronic acid used in the study was 250 times more potent than alendronate, but it is toxic to bone at concentrations higher than 2.5 × 10−10 M as reported by Li and Davis10.
Laboratory experimental models should replicate clinical scenarios as closely as possible. The presence of gaps or defects severely compromises the ability to achieve initial stability and secondary fixation by bone ingrowth in cementless acetabular reconstruction1,2. The objectives of this study were to determine the effects of porous tantalum coated with calcium phosphate and alendronate on gap filling and bone ingrowth and the resultant pattern and location of bone formation in a rabbit model.
Materials and Methods
Cylindrical porous tantalum (Trabecular Metal) implants, 3.18 mm in diameter and 8 mm in length, were provided by Zimmer (Warsaw, Indiana). The pore size was about 400 to 500 μm. The average volumetric porosity was approximately 75%.
Alendronate (monosodium 4-amino-1-hydroxybutylidene-1,1-diphosphonate trihydrate) was chosen as the bisphosphonate to be used in the study because of its efficacy, availability, and clinical record11. The method for binding the bisphosphonate to the porous tantalum involved electrolytic coating of calcium phosphate12 followed by alendronate immobilization onto the substrate. The electrolytic deposition technique avoided the line-of-sight effect of plasma spraying of calcium phosphate, with which only the superficial portion of the porous tantalum is coated.
Electrolytic coating of calcium phosphate was carried out in an aqueous solution of 5.25 mM calcium nitrate (Ca[NO3]2), 10.5 mM ammonium dihydrogen phosphate (NH4H2PO4), and 150 mM sodium chloride (NaCl). The pH of the solution was adjusted to 5.2 by adding 1 M sodium hydroxide (NaOH) under the monitoring of a calibrated pH meter (Thermo Orion 410, Beverly, Massachusetts). A two-electrode system was used for the electrolytic deposition. The distance between the cathode (porous tantalum implant) and the anode (a 25 × 25-mm platinum plate) was 9 mm. A DC voltage of 2.5 V was applied for three hours to deposit the coating. A thin layer (approximately 5 μm thick) of microporous (200 to 500-nm pore size) calcium phosphate coating was applied. The coating was a composite of octacalcium phosphate and apatite. The detailed structural characterizations of the coatings were previously described by one of us (Y.H.)13. Alendronate immobilization was carried out by soaking the calcium phosphate-coated implants in 3.2 mL of 10−4-M alendronate in phosphate-buffered saline solution at 37°C for seven days. The soaked implants were rinsed with distilled water five times. The rinsing steps ensured that only the immobilized (i.e., chemically adsorbed) alendronate remained.
All implants were beta-ray sterilized (25 to 27 kGy; Iotron Technologies, Port Coquitlam, British Columbia, Canada) before the implantation. In a separate experiment, five alendronate-immobilized and sterilized porous tantalum implants were assayed with a high-performance liquid chromatography assay adapted from the work of Fleisch and Hauffe14, to determine the dose of alendronate loaded on each porous tantalum implant. The average dose was determined to be 1.37 μg.
The gap model was achieved by attaching polymethylmethacrylate bone cement caps (outer diameter, 4.37 mm; height, 2 mm) at both ends of each implant (total length, 9 mm), creating a gap distance of 0.6 mm and a gap volume of 35.3 mm3 (Fig. 1). The implants were implanted, bilaterally, at the epiphyseal-metaphyseal junction of the distal part of the femur. The location enabled the assessment of gap filling and bone ingrowth in cancellous bone.
Eighteen New Zealand White adult female rabbits weighing 3.5 to 5.0 kg were randomly divided into three groups: six were treated with uncoated tantalum implants; six, with calcium phosphate-coated tantalum implants; and six, with tantalum implants coated with calcium phosphate and alendronate. The surgical procedure was performed in an animal operating room with the rabbits under general anesthesia and with use of standard sterile techniques. A 3-cm incision was made on the lateral aspect of the distal part of the femur, and the vastus lateralis was split along its fibers to expose the underlying bone. One 4.37-mm drill hole was made perpendicular to the distal femoral condyle bilaterally through sequential drilling (1.95, 3.18, and 4.37 mm) under saline solution irrigation, and the depth was confirmed with a depth gauge. The porous implant with the polymethylmethacrylate bone-cement caps was gently inserted into the hole. The wound was irrigated and was closed in a uniform manner. Two implants of the same type were implanted in each rabbit, to avoid the confounding influence of the presence or absence of different implant coatings. The anesthesia, operative procedures, and animal care were performed in compliance with university and federal guidelines. The study was approved by our ethical committee. All of the distal parts of the femora were harvested four weeks after the surgery.
The harvested femora were fixed, dehydrated, and embedded in epoxy resin (Spurr; Canemco, Canton de Gore, Quebec, Canada) with use of a standard histological procedure15. Each embedded sample was cut longitudinally, ground, and polished into three sections, 200, 850, and 1500 μm deep from the tangent surface of the implant. Thirty-six histological sections were obtained from each of the three implant groups. The polished samples were sputtered with gold-palladium alloy and then studied with a scanning electron microscope (model S3000N; Hitachi, Tokyo, Japan) operated under the backscattered electron mode. The backscattered electron microscopy generated different gray scales for tantalum, bone, and epoxy.
Histomorphometric analyses of the backscattered electron micrographs were performed with use of image analysis software (Clemex Vision PE 3.5; Clemex Technologies, Longueuil, Quebec, Canada). Gray-level discrimination was used to identify epoxy, new bone formation, and the tantalum implant. The backscattered electron micrographs of the three sections generated quantitative information regarding total gap volume, total volume available for bone ingrowth, and total volume available for new bone formation. The volume of bone-gap filling (new bone formation in the gap region between the tantalum implant and host bone), bone ingrowth into the porous tantalum (bone formation within the pores of the tantalum implants), and total bone formation (the sum of the two) were measured. The total available length of the tantalum implant and the length of the contact between the new bone and the implant were also measured on the three longitudinal sections.
The bone-formation front was labeled with fluorescence markers to track the pattern of new bone formation with time. Solutions of two commonly used calcium-seeking fluorescence markers16, alizarin complexone (absorption, 530 to 560 nm; emission, 624 to 645 nm; concentration, 30 mg/mL) and calcein (absorption, 494 nm; emission, 517 nm; concentration, 10 mg/mL), were prepared by dissolving the chemicals (Sigma, St. Louis, Missouri) in 1.4 wt% isotonic sodium bicarbonate (NaHCO3) solutions. The pH of the solutions was adjusted to 7.4 by adding hydrochloric acid. Within a Type-IIA biosafety cabinet, these solutions were filtration sterilized with 0.2-μm pore-size filters (Millex-GS; Millipore, Carrigtwohill, County Cork, Ireland) and placed in 10-mL sealed syringes. The fluorescent markers were injected subcutaneously at one week (calcein, 10 mg/kg), two weeks (calcein, 10 mg/kg), three weeks (alizarin complexone, 30 mg/kg), and four weeks (one day before the animal was killed) (alizarin complexone, 30 mg/kg) after the surgery. The labeling permitted qualitative information on the location, direction, rate, and mechanism of bone formation.
A fluorescent microscope (Eclipse E600; Nikon, Melville, New York) with a fluorescein isothiocyanate (FITC) and tetramethylrhodamine isothiocyanate (TRITC) dual exciter and dual emitter filter block (excitation, 475 to 495 nm and 540 to 575 nm; emission, 500 to 535 nm and 580 to 620 nm) was used to study the fluorescence labels and the new bone formation. The fluorescent microscopy was performed before sputtering of gold-palladium alloy for scanning electron microscopy. Under the fluorescent microscope, the bone-formation fronts in the first two weeks were marked by two green calcein labels, and the red alizarin complexone lines marked bone formation in the third and fourth weeks.
The results for the two implants in the same animal were averaged. The averaged results were tested for normality with use of a Shapiro-Wilk test performed with SPSS software (version 14.0; Chicago, Illinois) with the criteria of skewness/standard error <2, kurtosis/standard error <2, and p > 0.05. All results passed the normality test. Then, the results were analyzed with analysis of variance and the Tukey post hoc test performed with SPSS software (version 14.0). A p value of <0.05 was considered to be significant.
The histomorphometric results are summarized in Table I. Gap filling and bone ingrowth were seen to the greatest extent in association with the implants coated with calcium phosphate and alendronate (Fig. 2). Compared with the group treated with the uncoated implants, the group treated with the calcium phosphate-coated implants had a relative increase of 32% (p = 0.389) in the volume of gap filling, 50% (p = 0.327) in bone ingrowth, and 39% (p = 0.324) in total new bone formation. These increases were not significant, with the number of samples available. However, there were significant differences between the group treated with the implants coated with calcium phosphate and alendronate and the other two groups (Fig. 3). Compared with the group treated with the uncoated implants, the group treated with the implants coated with calcium phosphate and alendronate had a relative increase of 143% (p < 0.001) in the volume of gap filling, 259% (p < 0.001) in bone ingrowth, and 193% (p < 0.001) in total bone formation. Compared with the group treated with the calcium phosphate-coated implants, the group treated with the implants coated with calcium phosphate and alendronate had a relative increase of 84% in the volume of gap filling (p < 0.001), 140% (p < 0.001) in bone ingrowth, and 111% (p < 0.001) in total new bone formation.
Compared with the group treated with the uncoated implants, the group treated with the calcium phosphate-coated implants had a relative increase in the percentage of the length of the implant that was in contact with new bone (Table I, Fig. 4) of 66% (p = 0.516) in section 1, 127% (p = 0.563) in section 2, and 114% (p = 0.678) in section 3. These increases were not significant, with the number of samples available. However, there were significant differences between the group treated with the implants coated with calcium phosphate and alendronate and the other two groups. Compared with the group treated with the uncoated implants, the group treated with the implants coated with calcium phosphate and alendronate had a relative increase of 557% (p < 0.001) in section 1, 1005% (p < 0.001) in section 2, and 849% (p < 0.001) in section 3 (an average of 804% for the three sections). Compared with the group treated with the calcium phosphate-coated implants, the group treated with the implants coated with calcium phosphate and alendronate had a relative increase of 295% (p < 0.001) in section 1, 386% (p < 0.001) in section 2, and 343% (p < 0.001) in section 3.
The fluorescent labeling and the subsequent histomorphometric analyses indicated very different locations and patterns of bone ingrowth among the three groups. In the group treated with the uncoated implants, there was bone growth into the tantalum pores with little attachment to the surface of the tantalum itself. Early bone formation occurred in the center of the pores and expanded outward into the tantalum itself, as tracked by the fluorescent labels (Fig. 5-A). In contrast, in the group treated with the implants coated with calcium phosphate and alendronate, early bone ingrowth occurred predominantly on the surface of the tantalum (Fig. 5-B), followed by bone expansion toward the porous space. The group treated with the calcium phosphate-coated implants showed a pattern of bone formation that was similar to that in the group treated with the uncoated implants.
Biological fixation of porous-coated implants occurs by micro-interlock with ingrown tissue17. Autopsy studies have demonstrated that bone ingrowth occurs in only 30% to 40% of the surface area of uncemented acetabular implants used in primary total hip arthroplasty18,19. Although that is satisfactory for biological fixation of primary joint replacements, in revision arthroplasty, bone loss may reduce the host interface available to achieve bone ingrowth and implant fixation. Host-bone deficiencies compromise the surgeon's ability to achieve initial and secondary stability of implants used for cementless acetabular reconstruction. In many complex acetabular revisions, <50% of the host bone is available for bone growth into the newly implanted cementless acetabular component. Improved implant material that would enhance secondary biological bone ingrowth and fixation when the implant contact is suboptimal would be desirable.
Gaps and defects that occur between the surface of the implant and the host bone in an acetabular reconstruction are usually filled with bone graft or a bone-graft substitute. In the present study, porous tantalum implants coated with calcium phosphate and alendronate were compared with controls in a rabbit model incorporating a gap between the porous tantalum and the bone. The gap model was produced by placing caps on both ends of the porous tantalum implant, drilling with progressively larger reamers, and transcortical implantation of the device. One limitation of this model is the relatively small gap distance. In a clinical study, Macheras et al.20 found that most gaps between porous tantalum acetabular cups and the adjacent bone were 1 to 5 mm immediately after the implantation. In several studies, peri-implant gaps of 0.75 to 5 mm were evaluated in canine models21-24. We chose a gap size of 0.6 mm on the basis of a pilot study on bone growth into porous tantalum. In that study, we compared two gap sizes, 0.6 and 1.5 mm, and found measurable gap filling and ingrowth in the 0.6-mm gap but undetectable gap filling in the 1.5-mm gap at four weeks postimplantation. Therefore, we believed that a gap size of 0.6 mm would enable us to study the effects of the local delivery of alendronate on total bone formation and on the pattern and location of new bone formation. In addition, a 0.6-mm gap was chosen because the size of the rabbit femur limited the size of the implant and therefore the gap distance that was possible. An implant with a larger diameter may have caused a higher risk of bone fracture and damage to the cartilage at the knee joint. Although a gap size of 0.6 mm is smaller than many gaps in reports in the literature, it is still within the range of clinical observations20. The dramatic improvement observed in this study warrants future studies with the use of wider gaps.
Correlation analyses (least-square-root linear regression) (Fig. 6, a) showed that the increases in ingrowth were clearly correlated with the increases in gap filling in the three implant groups. The slopes of the regression lines were similar, and the regression lines seemed to be relatively close. The correlation results suggested that the coating with calcium phosphate and alendronate increased both the ingrowth and the gap filling by a common mechanism and further supported the statistical conclusions. The percentage of the length of the implant that was in contact with new bone in the group treated with the calcium phosphate and alendronate coating was also significantly increased compared with the value in the control group. However, the correlations between the average bone-implant contact length and total bone formation showed different trends in the different groups. The slopes were relatively flat in the groups treated with the uncoated implants and the calcium phosphate-coated implants (Fig. 6, b), whereas the slope was much steeper in the group treated with the calcium phosphate and alendronate coating. The difference suggested that the new bone tended to “spread” on the surface of the implants coated with calcium phosphate and alendronate. Therefore, the histomorphometric and correlation analyses both indicated enhanced bioactivity of the porous implant surface in the presence of the alendronate coating.
The increases in gap filling and bone ingrowth in the group treated with the implants coated with calcium phosphate and alendronate may have been due to alterations in the bone remodeling. Under normal conditions, bone undergoes continuous and balanced osteoclastic resorption and osteoblastic formation. Two previous studies of long-bone healing in rats25 and sheep26 showed that osteoclastic activities began as early as two weeks after injury, indicating that the new bone tissue (i.e., callus) underwent resorption and remodeling in the early phase of bone-healing. Therefore, it may be reasonable to suggest that, in the present study, the locally delivered alendronate disrupted the osteoclastic activity and caused a net gain of osteoblastic activity, thus producing a larger volume of bone tissue compared with that in the control groups. However, since very limited amounts of new bone formed in the groups treated with the uncoated implants and the calcium phosphate-coated implants, the reduced bone resorption of the new osseous tissue might not be the only mechanism by which alendronate increases gap filling and bone ingrowth. Recent in vitro cell-culture studies showed that aminobisphosphonates also enhanced proliferation and bone-forming activities of osteoblasts, suggesting a direct anabolic effect of bisphosphonates27-29. In addition, Kubota et al. reported that osteoclasts inhibited differentiation and osteoblastogenesis of osteoblast precursor-like cells, suggesting an indirect anabolic effect30. The relative roles of the anticatabolic and potential anabolic effects in the present study were unknown and need to be differentiated in future studies.
We also examined the effects of locally delivered alendronate on the location and pattern of bone formation with use of subcutaneous fluorescent labeling and histomorphometric analysis. Alendronate strongly modulated the pattern and site of bone formation compared with those in the group treated with the uncoated porous tantalum. These findings were consistent with the increased length of contact between the new bone and the implants coated with calcium phosphate and alendronate and with the results of the correlation analyses (Fig. 6, b) and therefore confirmed the enhanced bioactivity of the surface of the implants with the calcium phosphate-alendronate coating. Additional studies are required to assess the mechanical properties of the bone-implant interface and changes to bone mineral density associated with implants coated with calcium phosphate and alendronate.
In this study, alendronate was immobilized onto the calcium phosphate coating surface by chemical adsorption in 10−4-M alendronate solution for seven days. The alendronate concentration was chosen on the basis of rationale that alendronate exhibited toxicity for human osteoblasts31 in vitro when the concentration was above 10−4 M. According to the simple principle of chemical adsorption, we assumed that alendronate eluted in vitro by desorption would not exceed the 10−4-M level and would be safe for osteoblasts. The adsorption period of seven days was first used by Meraw and Reeve32 and was adopted in the present study without changes. Our study demonstrated the effect of the immobilization protocol, but its development involves simplification, and finding the optimal protocol remains the objective of future studies.
The results of this study are consistent with those in recent reports in the literature. The effect of a third-generation bisphosphonate, zoledronic acid, on bone growth into porous tantalum implants was examined in a canine model33. Zoledronic acid in saline solution was administered in a single postoperative intravenous injection at a dose of 0.1 mg/kg. At six weeks postoperatively, the mean relative increase in bone ingrowth was 85% compared with the bone ingrowth in controls. Subsequently, an investigation performed by the same group, in which zoledronic acid was bound to hydroxyapatite-coated porous tantalum, demonstrated an increase in peri-implant bone within the medullary canal (32.2% compared with 13.8% in controls, a relative difference of 134%) and in intra-implant bone formation (19.8% compared with 12.5%, a relative difference of 58%)9. We report new findings of bone formation in the presence of a gap and a greater relative increase in bone ingrowth compared with that in the previous, comparable study (259% compared with 134%)9. The differences in our findings may be related to several factors, including differences in the animal model, type of bisphosphonate used, duration of implantation, location of implantation, and design of the implants.
Other methods of augmenting the rate and extent of bone growth into porous implants have been described in the literature; these include the use of bone-like carbonated apatite coating34, bone matrix gel35, noninvasive low-intensity ultrasound36, and bone morphogenetic proteins (BMPs)21-24,37. BMPs effectively enhanced bone ingrowth in bone defects by stimulating the differentiation of skeletal progenitor cells toward osteoblasts38. Barrack et al. showed that when bone defects as large as 8 mm in diameter and 5 mm in depth, created in canine acetabula, were filled with BMP, they healed with a bone density equivalent to that in intact control hips in which an acetabular component had been implanted but no defect had been created24. However, recombinant BMPs are expensive, and special formulations must be developed to control the release kinetics39. The advantage of bisphosphonates over BMPs is their relatively low cost and their intrinsic affinity toward calcium, which ensures their slow release. Other advantages of bisphosphonates are their documented clinical record, which includes reduction of bone resorption secondary to stress shielding40 and wear particle-induced osteolysis41.
Improvements in the design and biomaterial properties of any implant used as a drug-delivery system can be made only with an understanding of the principles underlying the mechanism of action of the system. Understanding the mechanism by which porous tantalum coated with calcium phosphate and alendronate enhances bone ingrowth and osteoconductivity provides further insight into the underlying action of this drug-delivery system. The enhanced bone ingrowth and gap filling and increased osteoconductivity provided by this coating could have substantial advantages in complex revision joint arthroplasties associated with host bone defects by providing early and better biological fixation.
Disclosure: In support of their research for or preparation of this work, one or more of the authors received, in any one year, outside funding or grants in excess of $10,000 from the National Sciences and Engineering Research Council of Canada, Zimmer, Inc., Canada Research Chairs, and the Killam Trust. Neither they nor a member of their immediate families received payments or other benefits or a commitment or agreement to provide such benefits from a commercial entity. No commercial entity paid or directed, or agreed to pay or direct, any benefits to any research fund, foundation, division, center, clinical practice, or other charitable or nonprofit organization with which the authors, or a member of their immediate families, are affiliated or associated.
Investigation performed at the University of British Columbia, Vancouver, British Columbia, Canada
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