Local inhibition of 5-lipoxygenase enhances bone formation in a rat model

Article focus

Investigation into whether pharmacological inhibition of the 5-lipoxygenase (5-LO) pathway can enhance bone regeneration

Key messages

Inhibition of 5-LO using A-79175 increased bone formation in a dose-related manner

Strengths and limitations

The effects of A-79175 on osteogenesis were measured at multiple doses

The model of cortical defects in rat femora provides for proof of concept but lacks direct clinical applicability

The mechanism by which 5-LO inhibition increased osteogenesis was not analysed in these experiments

Introduction

Recent studies have shown that reducing 5-lipoxygenase (5-LO) activity accelerates fracture healing.^1,2 In rats treated with oral doses of AA-861, a 5-LO inhibitor, femur fractures healed faster and had greater mechanical properties than placebo controls.¹ Similarly, mice homozygous for a targeted mutation in Alox5, the 5-LO gene, were found to heal fractures of the femur faster and with greater mechanical integrity than controls matched by age and background genotype.² Fracture healing was characterised by accelerated bone formation within the fracture callus, but it occurred concomitant with accelerated chondrogenesis in the 5-LO deficient animals. Thus, it is unclear whether loss or inhibition of 5-LO can enhance bone formation in the absence of endochondral ossification.

The principal function of 5-LO is to convert arachidonic acid into leukotriene A₄, which is used to synthesise leukotriene B₄ (LTB₄) and the cysteinyl leukotrienes.^3,4 LTB₄ and the cysteinyl leukotrienes are lipid signalling molecules that affect many physiological processes, including inflammation, which is an early physiological response to fracture. Arachidonic acid is also the substrate for cyclooxygenase-1 and cyclooxygenase-2 (COX-2), which convert arachidonic acid into prostaglandin (PG)-H₂. PGH₂ is used to synthesise thromboxane A₂, PGD₂, PGE₂, PGF_2α, and PGI₂. These prostaglandins also are lipid signalling molecules that affect many physiological processes including inflammation.⁵

Several studies have demonstrated that non-steroidal anti-inflammatory drugs can impair fracture healing and that this impairment is due to inhibition of COX-2.^1,6-9 In other studies, reduced 5-LO activity was associated with enhanced fracture healing, including elevated callus chondrogenesis, increased bone formation and elevated COX-2 expression.^1,2 These data were interpreted to be an acceleration of endochondral ossification that ultimately led to accelerated healing. Thus, 5-LO acts as a negative regulator of fracture healing while COX-2 is a positive regulator of fracture healing. However, many orthopaedic procedures rely upon direct bone formation, and not endochondral ossification, for healing. For example, stems used in arthroplasty often require the formation of bone in order to secure the implant in place.^10-12 Also, fractures treated by rigid surgical fixation have reduced capacity for endochondral ossification, and rely more upon direct bone formation and remodelling to heal.^13,14

The purpose of this study was to determine if a locally applied 5-LO inhibitor, A-79175, could enhance bone regeneration in an animal model of direct bone formation.^15,16 For this study, a unicortical bone defect model was used in rat femora, in which the defect site was filled with a polycaprolactone (PCL) scaffold containing 5-LO inhibitor or carrier.^17,18

Materials and Methods

Results

The surgical procedure was easily accomplished and well-tolerated by the rats. Slight variations in scaffold diameter sometimes necessitated creation of a burr hole slightly larger than 3 mm. This slight variation was accounted for in data analysis by creating a cylindrical volume of interest of ≥ 3 mm diameter, and was taken into account for the total volume of the cylinder when determining the bone volume to total volume ratio (BV/TV) and bone mineral density (BMD).

In vitro release of A-79175 from the implants

There was an initial release (at day one) of A-79175 from the implants into the PBS (Fig. 1, Table III). However, after the initial release, the remaining A-79175 stayed within the implant with very little additional A-79175 released over the following eight days (Fig. 1). On day ten the implant was crushed in methanol in order to break apart the solidified calcium sulphate and extract the remaining A-79175. As drug release from solidified calcium sulphate depends upon surface erosion,²³ it was unlikely that this procedure would extract all the remaining A-79175 still held in the small particles of solidified calcium sulphate. However, the large increase in surface area caused by the crushing enabled extraction of additional A-79175 from the implants, demonstrating that A-79175 was still present within the CaSO₄ matrix even after nine days incubation in PBS (Table III).

Table III

Summary of A-79175 release in vitro (PBS, phosphate buffered saline)

Treatment group	Mean drug per implant (µg)	Maximum total released into PBS (%)	Mean (sd) drug recovered from implant (µg)	Total recovered from implant on day ten (%)
Control	0.0	-	0.0 (0.0)	-
Low dose (0.01%)	1.5	66	0.30 (0.11)	20
Medium dose (0.05%)	6.4	31	1.88 (0.88)	29
High dose (0.25%)	40.4	12	6.89 (3.47)	17

Fig. 1

Graph showing the mean cumulative A-79175 release from implants in vitro. Error bars show the standard deviation and lines of best fit are given for each group, including the controls (CaSO₄-only).

Radiological analysis

Radiographs were made for each animal immediately after surgery in order to confirm defect site and placement. After death the excised femora were also examined by radiology. Visual evidence of healing was apparent in most of the radiographs compared with those made immediately post-operatively. Beyond the identification of complications, such as fractures or infections, the radiographs were not scored or in any other way correlated to the experimental treatments. Example radiographs of the defects in each group from the ten-day endpoint are shown in Figure 2.

Fig. 2

Sample radiographs from each group after ten days of healing in a) an open defect, b) a polycaprolactone (PCL)-only defect, c) a CaSO₄-only defect, d) a low-dose A-79175 defect, e) a medium-dose A-79175 defect, and f) a high-dose A-79175 defect.

Histological analysis of the healing defect

Histological examination of the defects sites showed bone deposition around and in the scaffold for control and treatment groups (Fig. 3). No cartilage was present in the sites suggesting that direct bone formation occurred in this model. Histomorphometry to determine the percentages of mineralised tissue was not conducted, as a previous study showed that histomorphometry and micro-CT analyses yield similar results in this bone defect model.¹⁸

Fig. 3

Histological images of femoral defects at ten days post-operatively in a) the control group (CaSO₄-only) and b) the medium-dose group treated with A-79175, and c) magnified to show newly formed bone (b), the void left by the polycaprolactone scaffold (s) and artifactual air bubbles from the embedding process (x).

Micro-CT analysis

New bone (BV/TV) and bone mineral density (BMD) in the defect site of each femur was measured by micro-CT. Representative 3D reconstructions of the defect from each of the treatment and control groups at the ten- and 30-day endpoints are shown in Figure 4. The CaSO₄-only controls in each treatment group from the ten-day and 30-day time points were compared using ANOVA. No significant difference in BV/TV or BMD were found at either the ten-day (p = 0.84 and 0.19, respectively) or 30-day (p = 0.61 and 0.66, respectively) time points between the CaSO₄ only scaffolds in each treatment group (Tables II, III, and IV). This indicates that no systemic drug effect or other healing variations occurred between groups. A two-way ANOVA showed a significant difference in BMD between the ten- and 30-day endpoints after accounting for differences in treatment (p < 0.001) (Table IV).

Table IV

Mean bone mineral densities at ten and 30 days post-operatively (PCL, polycaprolactone)

			Mean (sd) bone mineral density (mg/cm3)
			Day ten					Day 30
Treatment group	Experimental vs control		n	Experimental	Control	p-value		n	Experimental	Control	p-value
Scaffold	Empty vs PCL		6	0.52 (0.10)	0.52 (0.07)	0.96		5	0.59 (0.10)	0.58 (0.04)	0.79
CaSO4	PCL vs PCL+CaSO4		6	0.54 (0.04)	0.49 (0.05)	0.07		3	0.54 (0.05)	0.64 (0.12)	0.30
Low dose	A-79175 vs PCL+CaSO4		6	0.51 (0.05)	0.54 (0.06)	0.46		6	0.67 (0.04)	0.70 (0.06)	0.23
Medium dose	A-79175 vs PCL+CaSO4		5	0.54 (0.04)	0.55 (0.11)	0.91		3	0.64 (0.08)	0.66 (0.04)	0.76
High dose	A-79175 vs PCL+CaSO4		4	0.55 (0.06)	0.53 (0.06)	0.56		3	0.62 (0.02)	0.67 (0.07)	0.29

Fig. 4

Representative three-dimensional micro-CT reconstructions of the femoral defect sites, with the ten-day samples in the left column and the 30-day samples in the right. Within each panel are (top left) a two-dimensional slice of the defect, (bottom left) the analysed volume shown from top-down and side views, and (right) the highlighted defect within the femur.

The experimental design enabled a paired comparison of bone formation between experimental (scaffolds with CaSO₄ and A-79175) and control (scaffolds with CaSO₄ only) defects with Student’s t-tests. There was no significant difference in total volume (TV) between experimental and control defects for each group (data not shown). The data indicate that defects treated with medium and high doses of A-79175 had significantly more bone (BV/TV) after ten days of healing (Table I, Fig. 5). There was a mean increase in bone volume of 14.3% (sem 8.3) for the low-dose A-79175 treated defects compared with the CaSO₄-only controls (6.6% (sem 8.4)) at the ten-day endpoint, although this was not statistically significant (p = 0.5). The mean change in BV/TV for the medium and high-dose groups was 27.5 (sem 8.3) and 28.3 (sem 8.6), respectively, both of which were significantly greater than the controls with CaSO₄ only (p = 0.011 and p = 0.024, respectively). No statistically significant differences were found in the 30-day endpoint data (Table II). However, all A-79175 treated defects had a greater BV/TV ratio than their CaSO₄-only controls at the 30-day time point.

Table II

Effects of A-79175 on the bone volume/total volume (BV/TV) ratio at 30 days post-operatively

						Mean (sd) BV/TV
Treatment group	Implant (mg)	Drug dose (w/w) (%)	Approximate dose (µg)	Group size (n)		Experimental	Control	p-value (t-test)
Scaffold	-	0	0	5		0.33 (0.092)	0.31 (0.057)	0.630
CaSO4-only	14.7	0	0	3		0.40 (0.138)	0.38 (0.043)	0.767
Low dose	17.8	0.01	1.5	6		0.39 (0.101)	0.34 (0.095)	0.328
Medium dose	14.6	0.05	6.3	3		0.43 (0.042)	0.42 (0.046)	0.372
High dose	18.9	0.25	41.3	3		0.43 (0.103)	0.38 (0.087)	0.138

Fig. 5

Graph showing the mean percentage change in the bone volume/total volume (BV/TV) of the ten-day endpoint (as bar chart, with error bars denoting standard error) compared with the A-79175 dosage depicted by the open circles, for each of the groups.

Discussion

We found the local release of A-79175 from CaSO₄ infused into PCL scaffolds to enhance bone formation in a cortical defect. The data demonstrated that local treatment with A-79175 at doses of 6.4 µg and 40.4 µg led to significantly more bone in the defect site by ten days post-operatively when compared with the contralateral femur defect treated with CaSO₄-infused scaffolds without the drug. By 30 days after surgery, the differences in bone formed between controls and A-79175 treated defects were not significant, although the A-79175 treated defects had consistently higher BV/TV values. This result was expected as the control groups were expected to heal at a normal rate and achieve almost full healing by 30 days. The results support previous fracture healing studies, in which loss or inhibition of 5-LO was associated with accelerated bone regeneration.^1,2

Differences in BV/TV between the A-79175 dose groups at the ten-day endpoint were compared using ANOVA, but no significant difference was found between the three doses (p = 0.125). However, the experiment was designed to allow paired comparisons between defects treated with A-79175 and their corresponding contralateral CaSO₄-only controls, and, as a consequence, the ANOVA comparison between the dosage groups was statistically underpowered (power = 0.249). Given the large observed differences between the A-79175 low-dose group and the medium- and high-dose groups, larger sample sizes would be likely to show a significant dose-dependent effect. This potential difference between the low-, medium- and high-dose groups may reflect the in vitro burst release of over half of the A-79175 from the low-dose implants (Table III). In contrast, the medium- and high-dose groups retained over 40% of the initial A-79175 after nine days of incubation in vitro, suggesting that these doses provided for sustained release of A-79175 in vivo as the calcium sulphate matrix is released.

Any osteoconductive effects of the PCL scaffold or the infused CaSO₄ was controlled for in the scaffold group, in which an empty defect was compared with a PCL scaffold containing no CaSO₄, and the CaSO₄-only group in which a PCL scaffold was compared with a PCL scaffold infused with CaSO₄. The micro-CT analysis found no significant differences between the PCL scaffold and empty defect groups at ten days (p = 0.836) or at 30 days (p = 0.630). Additionally, no significant differences were found when the PCL scaffold was compared with PCL infused with CaSO₄ at ten days (p = 0.745) or at 30 days (p = 0.767).

The paired design of this study controlled for local effects of A-79175 within each rats. However, a risk associated with this design is that systemic distribution of A-79175 could influence bone formation in the control defect. As the absolute dose of A-79175 per animal was very low and was further tempered by nominally slow-release from CaSO₄, the potential for confounding effects of systemic A-79175 was considered to be low. A comparison of all treatment groups failed to identify any significant effects of drug treatment on BV/TV in the defects filled with CaSO₄-only scaffolds (ten-day endpoint, p = 0.19; 30-day endpoint, p = 0.66; both ANOVA) suggesting that, at the dose used and in the CaSO₄ carrier, the A-79175 had no systemic effect on bone formation.

Local treatment with A-79175 increased direct bone formation in this rat femur defect model. Bone healing in this model occurs by direct formation and not endochondral ossification, and therefore suggests that 5-LO inhibition can stimulate osteoblasts to synthesise bone.¹⁸ Previous studies have shown accelerated bone formation during fracture repair when 5-LO was absent in mice homozygous for a targeted mutation in Alox5 or in rats treated with oral doses of AA-861, a 5-LO inhibitor.^1,2 The accelerated fracture repair was associated with enhanced chondrogenesis, suggesting that inhibition of 5-LO accelerates endochondral bone formation. However, these previous studies also showed early increases in peripheral fracture site bone formation, which is initially mediated by direct bone formation from the periosteum and would be consistent with the current findings.^1,2

Bone healing by direct bone formation occurs by osteoclast resorption of the damaged bone and subsequent new bone formation by osteoblasts.²⁴ Studies have shown that 5-LO inhibition can inhibit the activity of osteoclasts and osteoblasts.^25,26 However, cathepsin K and osteocalcin mRNA expression, indicators of osteoclast and osteoblast activity, respectively, were elevated during fracture repair in animals treated with 5-LO inhibitor.¹ Similarly, others have found that 5-LO metabolites can inhibit osteoblast or osteosarcoma cell line proliferation and function.^27,28 The inconsistent findings between studies likely relates to differences between in vitro and in vivo findings, differences in cell preparations, and differences in outcome measurements. Therefore, whether A-71975 stimulation of bone formation is occurring by mediating osteoclast activity, osteoblast activity, a combination of osteoclast and osteoblast activity, or is mediated through another cell type remains to be resolved.

This study did not explore the mechanisms by which 5-LO inhibition is enhancing osteogenesis. Although this model predominantly works through primary bone formation, it is unclear whether A-79175 is increasing bone formation or decreasing bone turnover. Future research will focus on understanding the mechanisms by which 5-LO and its downstream products effect the major cell types involved in primary and secondary bone healing. Our histological examination was consistent with primary bone formation as expected suggesting that 5-LO inhibition can affect primary bone formation as well as endochondral ossification as previously published.^1,2 Additional measures of bone formation were not attempted, as our previous studies have found a high degree of correlation between micro-CT measured bone volumes and bone areas measured by histomorphometry.¹⁸

The present study had some limitations. Differences in surgical creation of the defect sites required optimisation of micro-CT volume measurements for each defect. In order to account for these small differences, the amount of bone formed was normalised to the volume assayed (BV/TV). Surgical limitations were not limited to defect size variations. The implants used were PCL scaffolds infused with CaSO₄ and A-79175. The implants would crumble under light pressure or dissolve when exposed to any aqueous substance, such as saline or blood. Efforts were made to ensure the defect site was of adequate size and dry before attempting to insert the implant. However, scaffolds did break on occasion. In that event, the pieces of the scaffold were sufficiently moldable that the scaffold pieces were pushed into the defect site, much like a paste, rather than inserted as an entire solid scaffold. The paired experimental design allowed for statistical comparisons between an experimental scaffold and the control scaffold inserted in the defect in the contralateral femur. However, this also reduced the required number of animals in each group and reduced the statistical power for across group comparisons.

We are unaware of any approved pharmaceutical treatments to promote cortical bone defect healing or to promote direct bone formation into an arthroplastic device. Bone morphogenetic protein-2 is used to treat severe tibial fractures and is used in other application such as spinal arthrodesis.^29-31 Platelet derived growth factor is used in dental application to promote bone formation.^32-34 Presently, cortical bone defects are left to heal without intervention if the defect is small enough or may be filled with graft materials including autograft, allograft, xenograft, or synthetic materials to promote healing. Synthetic graft materials, such as calcium sulphate pellets and β-tricalcium phosphate particles, rely upon osteoconduction to promote healing.^35,36 Similarly, implants used in arthroplasty are sometimes coated with hydroxyapatite to promote osteoconduction.³⁷ However, no small molecule drug therapy is currently available as a systemic or local therapy for promoting bone formation. Several pharmaceutical approaches are being developed to address this clinical need. Potential therapies include use of locally applied bisphosphonates,^19,38 other inhibitors of osteoclast development or function,^18,39 use of parathyroid hormone or analogues,^40,41 Wnt pathway modifiers,^42,43 bone morphogenetic protein analogues,⁴⁴ prostaglandins analogues,^45-47 statins,^48,49 and as described here, drugs that modify the 5-lipoxygenase pathway. As there is no set standard pharmaceutical treatment for this model, and because the goal of the study was determine whether A-79175 would promote bone healing in a model of direct bone formation, no comparisons with other developing therapies was performed. Future studies that directly compare the efficacy of A-79175 therapy with other therapies in more clinically relevant models would aid in cost-benefit and clinical decisions regarding use of A-79175 or other 5-LO inhibitor therapies. However, additional experiments are needed to optimise drug-carrier, drug-release profiles and 5-LO inhibitor choice, in order to maximise bone formation before such comparisons are undertaken.

Conclusions

Local application of A-79175, a second-generation 5-LO inhibitor, effectively enhanced bone formation in a rat femoral defect model. Future studies with A-79175 or other 5-LO inhibitors are required in order to ascertain the ideal dose, carrier, and release profile for each clinical application. Studies involving larger mammals are currently underway to test a similar drug for efficacy as a cost-effective method for achieving accelerated bone healing.

The authors would like to thank K. Uhrich (Rutgers University, New Jersey) and J. Ricci (New York University College of Dentistry, New York) for their comments on the manuscript.