Session VI - Fracture Repair

*Evaluation of the Effects of Exogenous Treatment with Osteoinductive Bone Protein on the Distraction Callus in a Rabbit Limb-Lengthening Model

Lisa J. Holt, MS; Kam Momi, MD; Christopher J. Damien, PhD; J. Russel Parson, PhD; Fred Behrens, MD, University of Medicine and Dentistry of New Jersey, Newark, NJ; Sulzer Orthopaedics Biologics, Inc., Wheat Ridge, CO

Purpose: The purpose of this study was to accelerate the healing of the distraction callus in a rabbit limb-lengthening model through the administration of osteoinductive factors at the distraction site. These osteoinductive bone proteins(BP) include a combination of bone morphogenetic proteins (BMP). The BMP's have been shown to induce the differentiation of mesenchymal stem cells into chondroblasts and osteoblasts during bone healing¹. Previously, Aronson, et al,² detected mesenchymal stem cells in the radiolucent zone of the distraction gap. Therefore, it is the hypothesis of this study that treatment with exogenous BP will increase the osteogenic activity in the distracted callus.

Scanning acoustic microscopy (SAM) is a novel method for investigating the spatial micromechanical properties of bone in both a nondestructive and quantitative manner. It uses reflected ultrasound to measure these properties. Others have shown that there is a good correlation between the acoustic properties and the mechanical elasticity of bone³. SAM will help demonstrate the effects of BP on the spatial micromechanical properties of the bony regenerate. A comparison will be made between SAM and standard mechanical testing, which only provides the gross mechanical properties of the entire specimen. In addition, to gain a better understanding of the early effect that the BP has on cellular activity in the bony regenerate, proliferating cell nuclear antigen (PCNA) analysis will be used. By tagging the PCNA antigens that are present during cellular proliferation, and then counting the cells in specific areas, the proliferative cellular activity can be quantified. Thus, this study evaluated the effects of osteoinductive bone protein on the distraction callus in a rabbit limb- lengthening model using three methods: cellular proliferation, mechanical testing and SAM.

Methods: Extraction and chromatographic purification were used to obtain a mixture of BP from bovine bone with verifiable osteoinductive activity (Sulzer Orthopedics Biologics, Inc., Wheat Ridge, CO). The BP was subsequently mixed with bovine type I tendon collagen carrier solution (14 mg carrier and 1 mg BP), lyophilized and refrigerated at 2-8 ^oC. Immediately prior to use, the materials were reconstituted with 0.5 ml of sterile water.

Fourteen skeletally mature, male New Zealand White Rabbits underwent unilateral tibial limb lengthenings as per previously reported procedures⁴. A midshaft osteotomy was created and reduced with an Orthofix M100 minilengthener. After a latency period of 10 days, the tibiae were distracted 0.35 mm twice daily, for twenty days. Immediately post-distraction, half of the animals were treated with carrier (controls) and the other half were treated with BP. Under fluoroscopic imaging, both treatment types were injected directly into the radiolucent zone of the distraction callus.

Cell Proliferation Six animals, two at each time point, were sacrificed at 2, 4, and 7 days post-distraction. At each time point there was one control and one treated animal. The tibiae were harvested, fixed in 4% paraformaldehyde and decalcified. The samples were embedded in paraffin and sectioned sagittally along the midline. Cell proliferation was quantified by staining for proliferating cell nuclear antigen (PCNA), an antigen that is present in the nucleus of cells when in the G₁ to S phases of the cell cycle. To mark this antigen, the sections were treated with Anti-PCNA/Clone PC10 antibody (DAKO, Denmark). A stable DAB solution (Research Genetics, Huntsville, AL) was used to aid in the development of the PCNA stain. Eleven standardized sites were analyzed at a fixed magnification of 40X: four at the periosteum and seven within the bony regenerate.

Mechanical Testing and Scanning Acoustic Microscopy Eight animals were sacrificed at six weeks post-distraction. Both lengthened and contralateral tibiae were harvested. All tibiae were nondestructively tested in tension at a rate of 0.05 mm/s using a servo-hydraulic testing machine (MTS) with an extensometer. The stiffness was calculated and normalized as the percentage of stiffness of the contralateral unlengthened limb. The specimens were then fixed in formalin, dehydrated, and embedded in hard polymethylmethacrylate. The SAM equipment includes a 50 MHz transducer (model V3204, Panameterics Inc., Waltham, MA), a pulser receiver (Matec, Northborough, MA), and an ultrasonic analyzer (Panameterics Inc.). Mid-sagittal sections were made and then placed in a bath of deionized water at room temperature for scanning. Materials of known impedance (Bakelite' and Plexiglas') were used to calibrate the images. The impedance, averaged over the area of the bony regenerate, was measured in Mrayls (10⁶xKg m^-2s^-1), the SI unit of acoustic impedance, and the area of the bone within the callus itself was measured in mm².

Results: PCNA Since there were only six specimens (n=1), and there was no statistically significant difference between the cellular activity at the different times of sacrifice, the animals treated with BP were grouped together and the animals treated with carrier were grouped together to now give an n=3 for each group. The number of PCNA positive stained cells was significantly greater(Scheffe's Post Hoc test: p<0.05) in both the periosteal and regenerate regions in BP-treated animals as compared with controls.

There was no statistically significant difference between the cellular activity in the periosteum and the bony regenerate.

Table 1: Average proliferative cell count of the carrier and BP-treated lengthened tibiae within the microscopic field. (* Statistically significant from carrier p<0.05)

Mechanical Testing and Scanning Acoustic Microscopy On average, the normalized stiffness of the BP-treated tibiae was greater than that of the control tibiae; however, the difference was not statistically significant (Student's T-test: p=0.16). Only six of the eight mechanically tested tibiae were used for acoustic microscopy due to processing problems. The area of bony regenerate formed with treatment with BP was significantly less than those treated with carrier alone (Student's T-test: p=0.02); however, more advanced remodeling of the cortex was evident. The mean value of the average impedance of the regenerate treated with BP was greater than the mean of those treated with carrier alone.

* p-0.16 ** Significantly different from BP area at the p<0.05 level.

Discussion: The results show that the treatment in the early consolidation phase of the distracted callus with BP is associated with increased proliferative activity in the osteogenic regions of the regenerate. This increase in cellular activity appears to result in the formation of new bone with increased mechanical stiffness shown by mechanical testing and advanced bone remodeling evidenced by a decrease in the bony regenerate area and the formation of a more mature cortical shell. The histology of the six-week specimens confirms this. The distraction callus of the tibiae that were treated with carrier were largely filled with immature woven bone interspersed with loose connective tissue that contained little normal marrow content and had periosteal bridging. Those treated with the BMP had less woven bone with periosteal bridging that remodeled into a thicker, more organized cortical shell-like structure filled with normal marrow cells. In either case, there was little evidence of any collagen carrier left in the distraction gap at six weeks post-distraction.

Conclusion: The results suggest that BP is an exogenous treatment that may advance the bone remodeling in limbs lengthened by distraction osteogenesis. An increase in the rate of bone healing could lead to a decrease in time until the fixator can be removed for the patient and a reduced risk of fracture once the fixator is removed. Still, it is necessary to evaluate the effects of the BP in longer-term experiments and higher animal models before clinical trials could be considered.

Acknowledgements: This project was funded by the OTA. BP was provided by Sulzer Orthopedics Biologics, Inc., Wheat Ridge, CO.

References: 1) Wozney, J. Prog in Growth Factor Research. 167, 1989. 2) Aronson, CORR, 241, 1989. 3) Harten, ORS. 411, 1997. 4) Harten, ORS. 349, 1996.