William F. McKay

Short-term Osteoclastic Activity Induced by Locally High Concentrations of Recombinant Human Bone Morphogenetic Protein–2 in a Cancellous Bone Environment

Study Design. An experimental study investigating osteoclastic activity induced by rhBMP-2 in sheep. Objective. To examine the effects of increasing local rhBMP-2 concentration on osteoclastic response and peri-implant bone resorption. Summary of Background Data. Level I clinical studies have established the safe and effective volume and concentration of rhBMP-2 delivered on an absorbable collagen sponge. However, peri-implant bone resorption appearing as decreased mineral density has been observed radiographically in rare instances after implantation of rhBMP-2 on an absorbable collagen sponge (rhBMP-2/ACS). Methods. Bilateral corticocancellous defects were created in the distal femora of 30 adult sheep. Combinations of rhBMP-2/ACS implant volume (V) (1V = normal fill or 2V = overfilled) and rhBMP-2 solution concentration (×) (1× = normal concentration or 3.5× = hyperconcentrated) resulted in local rhBMP-2 concentrations of 0×, 1×, 2×, 3.5×, and 7× the estimated effective concentration for this model. Faxitron radiography, quantitative CT, histology, and quantitative histomorphometry were conducted in a blinded fashion to analyze the effect of the treatments. Results. At 1 week, the normal fill-normal concentration implants (1×) produced the least transient osteoclastic activity resulting in limited peri-implant resorption. Overfilled-hyperconcentrated implants (2×, 3.5×) demonstrated moderate resorption zones. Overfilled-hyperconcentrated implants (7×) demonstrated extensive osteoclastic activity and marked resorption. Results at 4 and 8 weeks revealed dense osteoid and bone in the voids with progressive bony healing. Control defects showed no osteoclastic activity with little to no bony healing. Conclusion. Increasing the local rhBMP-2 concentration by overfilling the defect with rhBMP-2/ACS or hyperconcen-trating the rhBMP-2 solution on the absorbable collagen sponge led to a concentration-dependent osteoclastic resorption of peri-implant bone. The osteoclastic effect was transient, and progressive healing took place over the 8-week survival period.

Effect of etanercept, a tumor necrosis factor-alpha inhibitor, on neuropathic pain in the rat chronic constriction injury model.

Study Design. The effects of a low, local dose of a tumor necrosis factor-alpha (TNF-&agr;) inhibitor on neuropathic pain behaviors in a rat chronic constriction injury model were evaluated. Objective. To investigate whether a peripherally implanted polymer drug depot can deliver a dose of etanercept sufficient to reduce thermal hyperalgesia and mechanical allodynia in a rat model of neuropathic pain. Summary of Background Data. TNF-&agr; inhibitors reduce pain-associated behavior in experimental models of neuropathic pain. Moreover, systemic injections of TNF-&agr; inhibitors have suggested some efficacy in treating sciatic pain in limited, off-label clinical studies. Improvements in these results may be obtained by optimal dosing via targeted, sustained delivery at the site of disc-induced inflammation. Methods. Unilateral chronic constriction injury was applied to the sciatic nerve of 56 male, Wistar rats. Four groups of animals (n = 7) received 0.5 mL phosphate-buffered saline every 3 days, 0.3 or 3 mg/kg etanercept every 3 days, or gabapentin (60 mg/kg) 1 hour before each behavioral test all via subcutaneous injection. Two groups of animals received 1.5 or 3.0 &mgr;g/h etanercept delivered by poly(lactic-co- glycolic acid) (PLGA) millicylinders (1 mm diameter × 10 mm long) implanted near the injured sciatic nerve. One group received a PLGA millicylinder implanted near the injured sciatic nerve. The final group received 3.0 &mgr;g/h etanercept via PLGA millicylinder implanted next to the uninjured, contralateral sciatic nerve. Results. A low, local dose of etanercept (∼3 &mgr;g/h) delivered by a polymer depot significantly reduced (P < 0.05) thermal hyperalgesia for 57 days as compared to polymer depot without drug or an etanercept-loaded depot implanted near the contralateral sciatic nerve, and equivalent to a 10-fold higher dose delivered by repeat subcutaneous injection. Conclusion. This preclinical study indicates that delivering TNF-&agr; inhibitors by means of a locally administered polymeric formulation provides long-lasting analgesia in an inflammatory neuropathic pain model.

Transient Soft-tissue Edema Associated with Implantation of Increasing Doses of rhbmp-2 on an Absorbable Collagen Sponge in an Ectopic Rat Model

BACKGROUND Recombinant human bone morphogenetic protein-2 (rhBMP-2) is an osteoinductive protein. However, soft-tissue edema adjacent to the site of rhBMP-2 implantation has been reported. This animal study was designed to examine soft-tissue edema associated with increasing rhBMP-2 doses with implantation on an absorbable collagen sponge (ACS) and with injection directly into muscle. METHODS Thirty-six Lewis rats received intramuscular implantation of rhBMP-2 on an ACS (Part I) or intramuscular injection of rhBMP-2 solution (Part II). Part-I sites received rhBMP-2/ACS at doses of 0 μg, 30 μg (normal), 129 μg (mid), or 450 μg (high). Part-II sites received rhBMP-2/ACS or rhBMP-2 intramuscular injection at doses of 10 μg (normal) or 150 μg (mid). A previous rat model showed 10 μg to be 100% effective at inducing osseous spinal fusion. In our study, T2-weighted magnetic resonance imaging (MRI) was performed at two and seven days to assess edema volume, and statistical comparisons were carried out with analysis of variance (ANOVA). Cellular response, vascularity, and ossification were examined histologically. RESULTS Quantitative MRI demonstrated similar peri-implant edema volumes in the control (buffer on an ACS) and normal-dose rhBMP-2 groups. Higher doses resulted in increased edema volume. Edema decreased significantly from two to seven days. Similar capillary densities were observed in all rhBMP-2 groups at two days, and there was dose-dependent increased ossification at seven days. Compared with the rhBMP-2 injection, implantation of the rhBMP-2/ACS resulted in increased edema. This edematous response was transient in all groups. Minimal or no ossification occurred after the rhBMP-2 injections. CONCLUSIONS Transient peri-implant soft-tissue edema occurred in a dose-dependent manner following implantation of rhBMP-2/ACS in this rat model. The normal dose of rhBMP-2/ACS produced edema similar to that in the controls. Finally, rhBMP-2 solutions injected directly into muscle resulted in minimal soft-tissue edema.

Evaluating the effects of recombinant human bone morphogenetic protein-2 on pain-associated behaviors in a rat model following implantation near the sciatic nerve

OBJECTIVE It has been hypothesized that the recombinant human bone morphogenetic protein-2 (rhBMP-2) amplification of the host inflammatory response interacts with nerves in the spine and contributes to the occurrence of new, postoperative complaints of radiculitis. This in vivo rat study was conducted to assess the capacity for rhBMP-2/ACS (rhBMP-2 applied to absorbable collagen sponge [ACS]) to stimulate pain-associated behaviors in the rat chronic constriction injury (CCI) model. METHODS Rats were randomly assigned to one of 14 treatment groups. Half of the animals underwent a sham procedure in which the left sciatic nerve was exposed and manipulated but no ligature was placed (Sham cohort), while the remaining animals had chromic gut sutures tied around the sciatic nerve to induce CCI (CCI cohort). The following test articles were applied to the sciatic nerve in each cohort: saline alone, saline applied to ACS, 0.1 mg/ml rhBMP-2 applied to ACS, or 1.0 mg/ml rhBMP-2 applied to ACS. The ACS was either wrapped around the sciatic nerve or implanted adjacent to the nerve. Thermal withdrawal latency was assessed on Days 7, 14, 21, and 28 postoperatively. Isolated nerves from selected rats in each group were examined and assessed for histopathological changes on Days 3, 7, 14, and 28. RESULTS CCI produced a significant pain behavioral response for all treatment groups at all time points. In the Sham cohort, 0.1 mg/ml rhBMP-2/ACS wrapped around the nerve (WRP) decreased thermal withdrawal on Day 28, and 1.0 mg/ml rhBMP-2/ACS placed adjacent to the nerve (ADJ) decreased thermal withdrawal on Days 21 and 28. Conversely, in the CCI cohort, 0.1 mg/ml rhBMP-2/ACS ADJ increased thermal withdrawal latencies on Day 7; 1.0 mg/ml rhBMP-2/ACS ADJ increased thermal withdrawal latencies on Day 7; and 1.0 mg/ml rhBMP-2/ACS WRP increased thermal withdrawal on Days 7 and 14. Histologically, the effect of rhBMP-2 on nerve inflammation was unclear, as inflammatory cell infiltration was similar in the rhBMP-2/ACS and saline/ACS groups. rhBMP-2 was variably associated with bone formation within the epineurium at 14 days, and more prevalently at 28 days, with no clear relationship between dose or ACS positioning. CONCLUSIONS In this study, rhBMP-2/ACS did not appear to induce pain independent of grossly visible ectopic bone formation. At the earliest time points, rhBMP-2 appeared to have a neuroprotective effect as evidenced by decreased pain exhibited by the rhBMP-2-treated animals in the CCI cohort, but this effect diminished over time, and by Day 28, the pain behavioral responses in the rhBMP-2-treated group were comparable to those in the group in which saline was applied to the nerve. In the Sham cohort, there was a dose-independent induction of pain at later time points, presumably due to new bone formation mechanically irritating the nerve. Histological examination revealed nerve lesions that appeared to be caused by mechanical trauma associated with surgical manipulation of the nerve during placement of the ACS and/or CCI sutures.

Development of a novel compression-resistant carrier for recombinant human bone morphogenetic protein-2 (rhBMP-2) and preliminary clinical results

Recombinant human bone morphogenetic protein-2 (rhBMP-2) has been commercially available in the United States since July 2002 (INFUSE® Bone Graft/LTCAGE ®Lumbar Tapered Fusion Device, Medtronic, Inc., Memphis, TN). It was initially approved for use in interbody spinal fusions inside a threaded titanium interbody fusion device. Since then, it has been approved for two additional clinical indications: fresh tibial fractures and certain oral maxillofacial procedures (sinus elevation and extraction sockets). The approvals were based on Level I prospective, randomized clinical trials involving the absorbable collagen sponge (ACS) carrier and rhBMP-2 at a concentration of 1.5 mg/mL.