Uncemented total joint replacements are standard procedures in orthopaedic surgery. After implantation, initial mechanical stability is achieved by press-fit implantation of the components.43 Secondary stability and long-term implant survival depend on osseointegration. Osseointegration is the direct structural and functional connection between ordered living bone and the surface of a load- bearing implant without intervening fibrous tissue.10,29 Porous surfaces encourage this bony ingrowth and, of the various materials investigated, titanium and titanium-alloy implants are the most biocompatible.21 Tissue integration requires adherence and proliferation of cells on the surface of the implant, which can be enhanced by coating with hydroxyapatite (HA).21 Hydroxyapatite is osteoconductive, that is, it can support the ingrowth of sprouting capillaries, perivascular tissues, and osteoprogenitor cells from the host into the three-dimensional structure of the implant.20
Bisphosphonates are known to have an affinity for bone mineral and therefore act specifically on bone.14 The ingestion of bisphosphonates during osteoclast-mediated bone resorption interferes with specific intracellular processes, which impair osteoclast function and cause apoptosis and cell death.34 Nitrogen-containing bisphosphonates are reported to inhibit enzymes in the mevalonate pathway, thereby preventing biosynthesis of essential compounds for protein prenylation and function of key regulatory proteins.2 Therefore, bisphosphonates inhibit osteoclast-mediated bone resorption more than osteoblast-mediated bone formation, possibly activate osteo- blasts,12,19 and apparently improve implant osseointegration.22 Experimental investigations have shown that the amount of bone attached to the surface can be increased by using a bisphosphonate after implantation of a cementless implant.9,28 There is an increase in the early bone formation rate around metal implants as a result of a bisphosphonate treatment,24 and clinical trials have shown increased periprosthetic bone mineral density (BMD).8,17,37
A possible problem for patients is the necessity of daily dosage. Repeated injections of bisphosphonates are related to side effects and may result in suboptimal adherence to therapy. Therefore, less frequent dosing is desirable. The nitrogen-containing bisphosphonate ibandronate is effective in daily and intermittent therapy in preventing bone and architectural deterioration.4,25
We, therefore, asked whether a daily dose and a dose- equivalent single injection of ibandronate compared with an untreated control would enhance the osseointegrated surface and improve bone volume to tissue volume around HA-coated and uncoated titanium implants.
MATERIALS AND METHODS
Fifty-five 6-month-old female Sprague-Dawley rats were used in accordance with the institutional guidelines for the care of laboratory animals. The rats were allowed free access to tap water and commercially standard rodent food (SSNIFF R/M-H®, SSNIFF Spezialdiäten, Soest, Germany). All animals were housed under identical conditions with an alternating day/night rhythm of 12 hours.
The animals were randomly assigned to two treatment groups and one control group (Table 1). For a treatment period of 28 days, the first treatment group (G1) received daily subcutaneous injections of 25 μg/kg body weight ibandronate starting the day of surgery. The second treatment group (G2) received a dose-equivalent single injection of 28 × 25 μg/kg body weight ibandronate the day of surgery. The chosen dosage of ibandronate was according to standard dosage for treatment of malignant bone disease and below toxicity level of the agent in daily and in dose-equivalent single injections. The control group (G0) was treated with daily subcutaneous injections of NaCl 0.9%.
The animals were anesthetized by an intraperitoneal injection of ketamine (Ketanest®, Parke-Davis, Karlsruhe, Germany) with 75 mg/kg body weight and xylazine (Rompun 2%®, Bayer, Leverkusen, Germany) with 5 mg/kg body weight. Both hind limbs were shaved and cleaned with Betaisodona® solution (Mundipharma, Limburg, Germany). Under aseptic conditions, the extensor mechanism was exposed and a longitudinal medial parapatellar incision was made. The extensor mechanism with the patella was dislocated laterally. With the knee in 90° flexion, a 0.9-mm diameter reamer was passed manually with a twisting motion through the intercondylar notch and distal femoral metaphysis into the medullary canal of the femur diaphysis. After removing the reamer, the implant and a 0.96-mm diameter Kirschner wire were inserted into the medullary canal of the femur using a press-fit technique, cut to the length of the individual femur, and positioned to rest approximately 1 mm below the cortical surface of distal femur epiphysis (Fig 1). Two implants (Stryker Howmedica, Duisburg, Germany) were tested: a titanium implant for regular commercial purposes and a HA-coated titanium implant. The HA-coating (composition Ca5(PO4)3OH, crystallinity 80%) was applied by plasma spraying13 using the Biolox-osprovit® method (CeramTec, Plochingen, Germany). Hydroxyapatite-coated implants were used on the right femur and uncoated titanium implants were used on the left femur. The patella was relocated and the extensor mechanism was reconstructed using single stitches with 4.0 Vicryl (Ethicon, Norderstedt, Germany). The soft tissues and skin were closed using a single subcuticular continuous stitch with 4.0 Vicryl (Ethicon). The limbs were checked for normal postoperative motion of the knee. The rats were allowed unrestricted ambulation in their cages after recovery and were observed daily for activity and weightbearing. After 28 days of treatment, all animals were anesthetized with an intraperitoneal injection of ketamine (Ketanest®, Parke-Davis, Karlsruhe, Germany) with 75 mg/kg body weight and xylazine (Rompun 2%®, Bayer, Leverkusen, Germany) with 5 mg/kg body weight, and sacrificed in a carbon monoxide chamber.
After harvesting the specimen by removing all soft tissue from the femur, anteroposterior (AP) and lateral view radio- graphs were taken to find radiographic signs of periprosthetic infection or osteolysis. The specimens were prepared for quantitative histomorphometric examination by dehydration in progressively ascending ethyl alcohol solutions (40-100%) followed by embedding in xylol. Then they were undecalcified and embedded in methylmethacrylate (MMA®, Biomet Merck, Darmstadt, Germany). After curing, 10 cross sections of 500-μm thick slices were taken (Exakt Cutting-Grinding System, Exakt Apparatebau, Norderstedt, Germany) from distal to proximal. Contact radiographs of each section were taken, and the first metaphyseal cross section behind the epiphysis was identified on the radiographs. This identification procedure is repeatable and ensured comparability between the sampled cross sections of different specimens. The selected cross section was ground to approximately 300 μm, transferred to a high-power ultrasonic cleaning unit (Bandelin Sonorex Super RK 514 BH, Schalltec, Mörfelden, Germany), and stained using the Masson-Goldner technique.
We excluded specimens if there was clinical or histologic evidence of periprosthetic infection (n = 10), malpositioning of the implant (n = 9), or damage from sectioning (n = 1). One animal died before surgery, and three died because of perioperative or postoperative complications, therefore, eight specimens had to be excluded (Table 2).
Histomorphometric analysis was performed using semiautomatic image analysis software (Q-Win 2.31, Leica, Bensheim, Germany) and a high-precision motorized light microscope (Leica DMRXA, Leica, Bensheim, Germany). To describe changes in periprosthetic bone mineralization, the ratio of bone volume to tissue volume of the entire cross section excluding the cortical bone was calculated. The osseointegrated implant surface was determined to quantify the extent of bone ingrowth. This was defined as the ratio (percent) of the implant surface in direct contact with surrounding bone to the total implant surface. The osseointegrated implant surface is an accepted parameter to describe the status of osseointegration of a bone-surrounded implant.1,15 Interobserver variability and intraobserver variability of the histomorphometric findings were tested by two operators (CE, JB) assessing the same specimen on different days.
Data were expressed as mean and standard deviation (SD). Statistical analyses were performed using the statistics package SigmaStat™ (SPSS Inc, Chicago, IL). A Wilk-Shapiro test for normality was conducted on all parameters. To answer the research questions, a one-way analysis of variance (ANOVA) was done on all groups. A Tukey post hoc analysis was performed to determine which groups differed significantly. The level of significance was set at p < 0.05.
Intraobserver variability and interobserver variability of the histomorphometric findings were within 5%.
With HA-coated implants (Figs 2, 3) both treatment groups (Tables 3, 4) had enhanced (p < 0.05) osseointegration compared with controls (45.3% versus 29.4% for single injection and 46.1% versus 29.4% for daily injections). With uncoated implants (Fig 4), both treatment groups (Table 5) had enhanced osseointegration compared to the control (34.9% versus 23.7% for single injection and 40.3% versus 23.7% for daily injections), but only continuous application revealed a statistically significant (p < 0.05) difference, while single injection failed (Table 6).
The ratio of bone volume to tissue volume for periprosthetic bone mineralization showed increased (46.2% versus 31.5% for single injections and 53.4% versus 31.5% for daily injections) mineralization around all implants in both treatment groups (Table 7). Both treatment groups showed improvements (p < 0.05) compared with the control group. There were no differences between the two treatment groups (Table 8).
Early secondary stabilization of cementless implants is achieved by osseointegration and is critical to early rehabilitation and long-term survival of total joint replacements. Although using porous prostheses and various coatings,21 particularly HA,20 has been shown to enhance osseointegration, improvements can be made.
One possible improvement is administration of growth factors. Some animal studies have shown that local use of these agents, including TGF-β1,23 BMP-233 or BMP-3,16 has the potential to improve osseointegration of implants. Parathyroid hormone (PTH) has been suggested as a means to promote osseointegration because of its ability to increase bone formation.18 As with the growth factors, preclinical data with PTH are unconvincing. One study in rats showed bone ingrowth distance was not increased by PTH treatment.36 Because of the importance of ensuring early implant stabilization, there is an additional need to investigate treatments that enhance osseointegration.
We showed that bisphosphonate treatment with the 25 μg/kg body weight ibandronate daily or with a dose- equivalent single injection of 28 × 25 μg/kg body weight improves osseointegration of HA-coated titanium implants. In uncoated titanium implants, daily injections also improve osseointegration whereas a dose-equivalent single injection did not. The periprosthetic bone mineralization expressed by bone volume to tissue volume also was enhanced for both treatment regimens for all implants.
Preclinical findings support the hypothesis that bisphosphonates improve osseointegration of metal implants. Local administration of alendronate increases early bone formation around dental implants (with and without HA coating) in dogs, and increases bone-to-implant contact with uncoated implants.24 Alendronate improves the amount of bone growing into press-fit cup implants.42 Continuous application of ibandronate increases periprosthetic BMD.5,27 The antiresorptive properties of bisphosphonates and the less-pronounced inhibition of osteoblasts in comparison with osteoclasts, together with possible preosteoblastic effects, provide a convincing explanation for the accelerated osseointegration and enhancement of bone formation.19 These findings are consistent with our results, showing improvement of the osseointegrated surface of HA-coated and uncoated titanium implants for daily ibandronate injections and enhancement of periprosthetic bone mineralization for both therapy regimens.
However, daily injections of bisphosphonates is a problem in a clinical setting owing to the problem of noncompliance, therefore, the possibility of a single injection is desirable. Efficacy for an intermittent or single injection has been reported for treatment of malignant hypercalcemia31,40 and cancer-associated bone loss7,38 with increases of BMD.32,39 Daily and intermittent doses of ibandronate have been reported to improve BMD in patients with osteoporosis in well-designed clinical and preclinical studies.11,25,35 In our study, ibandronate in a dose-equivalent single injection was as effective as daily injections to improve the osseointegrated surface of HA-coated implants. For uncoated titanium implants, a single-dose injection revealed no statistical benefit to the control. Published data are inconsistent; some studies have shown better outcomes with HA-coated implants, whereas others have not.24 Hydroxyapatite-coated surfaces seem to encourage, whereas uncoated titanium implant materials discourage, the adherence of bone marrow cells.44 Also, a high affinity of bisphosphonates to mineralized bone14 and accumulation on HA-coated implant surfaces has been described.26,30 These high concentrations at the implant-bone interface might be an explanation for better outcomes with HA- coated implants with bisphosphonate treatment.
There are studies showing the potency of single-dose bisphosphonate to improve osseointegration of metal implants in cases of instability-induced or wear debris- induced loosening.3,41 All these findings suggest that the effect of bisphosphonate therapy depends on the total administered dose and not the choice of administration.6
Our results suggest the potency of a daily dose or dose- equivalent single injection of ibandronate equally improve the osseointegrated surface of HA-coated implants compared with an untreated control. This suggests that the long-term survival rate of such implants could be improved although animal data or clinical results to support this supposition are unavailable. In clinical practice, improved osseointegration of cementless implants and prolonged long-term survival rates are crucial for final outcome, and future studies should investigate the clinical and long-term effects of bisphosphonate on implant integration in humans.
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