In the present study, local release of antiresorptive and osteogenic drugs from hydrogel implants was used as an approach to enhance bone formation in a cortical defect as a preliminary test model for increasing bone formation around arthroplastic implants. The data demonstrated that local treatment with the bisphosphonate alendronate led to significantly more new bone at the rat femur defect site as compared to the controls (both blank and empty defect) and the other osteogenic drug treatments. Comparison of total new bone formation at the defect site between treatment groups found that both doses of alendronate led to significantly more bone formation as compared to omeprazole or lovastatin.
The paired design of this study controlled for biological differences between rats. With this method of local drug administration, a risk existed for systemic distribution of the agents that could influence the bone on the control (blank) side. However, a separate control group (empty defect-blank implant) was completed so that we could analyze the local effects as well as the systemic effects of each drug. MicroCT analysis of bone volume detected a positive systemic effect in the control side of the low-dose omeprazole group. However, no local effect on bone healing was found with omeprazole treatment. These results suggest that higher concentrations of omeprazole (such as those applied locally or that circulate systemically in the high-dose omeprazole group) do not increase bone formation while a low concentration (similar to the concentration circulating systemically in the low-dose omeprazole group) can increase bone formation. Additional studies are needed to confirm this apparent omeprazole effect. No systemic effect was detected for any other treatment groups.
We suspect that the surgical insult produces a significant bone regenerative response in the rats. This response likely includes a significant activation of periosteal osteoblasts and bone marrow preosteoblasts to form new bone. However, this flux of osteoblasts would have contributed to bone formation in all test groups and does not explain the approximate 2-fold increase in bone volume found in rats treated with the alendronate hydrogel implants.
The mechanism by which alendronate increased bone formation was not investigated in these studies. Alendronate is expected to prevent osteoclast mediated remodeling of the newly formed bone, which could in part account for the increased amount of mineralized tissue measured in the alendronate treatment groups [36
]. Alendronate and other nitrogen-containing bisphosphonates also can increase osteoblast proliferation [38
], prevent osteoblast apoptosis [41
], inhibit differentiation of mesenchymal stem cells into adipocytes [43
], and promote osteoblast differentiation and activity including enhancing expression of Runx2 and BMP-2 [39
]. Alendronate effects on osteoblasts appear to be mediated through ERK and JNK activation and alteration of the mevalonate pathway [38
]. Thus, alendronate can have both anabolic and anticatabolic effects on bone.
We suggest that local alendronate treatment enhanced bone formation and reduced osteoclast-mediated remodeling at the cortical defect site leading to a large increase in bone formation. Our results are similar to those of Jakobsen et al. who demonstrate that local alendronate treatment increased fixation of implants inserted in cancellous bone after 4 and 12 weeks in a canine model by increasing bone volume and density [17
]. These results contrast a rabbit femoral condyle study where alendronate had no positive effect when it was incorporated into bone cement and inserted into the defect. However, in this rabbit study, alendronate appeared to evoke a toxic response which may have contributed to the absence of a positive bone healing response [15
]. This suggests that alendronate effect on osteoblast activity may be more important for increasing bone formation during healing of bone defects than alendronate effects on osteoclast activity. Future studies to characterize the cell types and quantify osteoblast- and osteoclast-related factors at the bone defect site should provide a better understanding of how these drugs alter the local bone forming environment.
Since an overall aim of adjuvant therapies for arthroplasty is to increase early fixation of these implants and reduce instability [18
], local alendronate treatment could increase the long-term success of arthroplastic surgeries by preventing osteolysis and promoting osteogenesis around the implant [49
]. Treatment with lovastatin showed no positive effects on bone formation at the implant site. Lovastatin is expected to promote osteoblast activity by inducing BMP-2 expression but would not directly affect osteoclast activity [28
]. Previous studies have shown that large doses of orally administered lovastatin can stimulate bone formation [28
]. If lovastatin did induce a large bone formation response in this study, it is possible that subsequent osteoclast mediated remodeling may have destroyed most of the initial bone. The data indicate that this hypothesis is unlikely since bone remodeling would have likely led to increased bone mineral density and no such increase was observed (). Lovastatin concentration may have contributed to the lack of bone formation. The highest concentration of lovastatin incorporated per implant was approximately 0.01
mg/kg body weight. This is similar to the dose used by Gutierrez et al. which demonstrated that 5 consecutive days of transdermally applied lovastatin (0.01
mg/kg per day) to ovariectomized rats increased trabecular bone volume by 30–60% as well as increasing bone formation [27
]. However, based on the in vitro elution studies, the largest portion of the lovastatin dose in this model would have been released by day 1 (which is expected to be less than 0.01
mg/kg) with subsequent smaller daily releases of drug. Therefore, lovastatin concentration or length of release may have contributed to the absence of increased bone formation.
Experimentally, omeprazole produced no consistent effect on bone formation. Omeprazole is expected to inhibit proton pumps (H+K+ATPase) in osteoclasts and prevent bone resorption [52
]. The large amount of new bone volume in the blank-treated defects of the low-dose omeprazole treatment group as compared to the blank-treated defects from the empty (P
= .004), low-dose alendronate (P
= .020), and low-dose lovastatin (P
= .002) groups was unexpected (see and ). This elevation suggests a systemic effect of low-dose omeprazole, but a similar effect was not found in the high-dose omeprazole. One plausible explanation is that the local omeprazole concentrations as well as the systemic levels of omeprazole in the high-dose group were too high to promote formation. If this is true, lower doses of omeprazole may help promote bone formation. This hypothesis will require additional investigation.
Alendronate treatment was superior to lovastatin or omeprazole in this in vivo assay of bone formation. Peter et al. found that local zoledronate (another bisphosphonate) doses of 0.2 and 2.0
ug were not effective in promoting osteogenesis while doses of 8.5
ug and higher (maximum tested was 16
ug) all stimulated osteogenesis [53
]. In contrast, Bodde et al. found that a local alendronate dose of 8.2
mg inhibited bone formation in a rabbit femoral condyle defect model [15
]. Consequently we tested alendronate using 6.8 and 26.2
ug doses to identify any potential dose response effects in this dose range or any potential negative effect of the drug. Similar to Peter et al. [53
], we found that both bisphosphonate doses increased bone formation at the defect site. Nominally, bisphosphonates can be incorporated into bone, and since the defect site is actively producing new bone, it is likely that release of alendronate from the implant led to a locally high concentration of drug that remained at the site. In contrast, lovastatin and omeprazole are not known to be incorporated into the bone matrix. Thus the effective release kinetics between alendronate, lovastatin, and omeprazole are likely very different. In addition, preliminary in vitro studies indicated a sharp burst of drug release initially from the pellets which dropped off precipitously (within three days). Therefore, additional experiments to test implants that show a longer-lasting release of lovastatin or omeprazole may show that these compounds also are effective osteogenic compounds.
This study screened 3 drugs for potential use as agents to enhance local bone formation. As such, the study has significant limitations. First, the study design used only a single time point, which may not have captured early or later positive effects of the drugs. There is also the concern whether results from rodent models will translate to similar effects in humans. The rat femur lacks large amounts of trabecular bone that is typically found in the metaphyses of human long bones. Consequently, the drugs in this study appeared to primarily affect cortical bone rather than trabecular bone as would be expected in humans undergoing an arthroplastic procedure. The bilateral experimental design controlled for potential drug systemic effects; however, systemic and local drug levels were not measured in vivo. Drug release data would be helpful in designing future studies and for comparing drug effects when released from different carriers or when used in different models.