Zinc loaded calcium sulphate enhances rhBMP-2 induced bone formation

Spatafora, Michael V (Oral Surgery, University of Toronto)
Crasto, Gazelle J (Oral Surgery, University of Toronto)
Peel, Sean A.F. (Induce Biologics Inc)

Introduction

Recombinant human bone morphogenetic protein 2 (BMP) bioimplants are used clinically to promote spinal fusion, fracture non-union and bone defect repair. Supraphysiological doses of BMP-2 are required for the bioimplants to be effective, impacting the cost-effectiveness of BMP bioimplants and increasing the risk of adverse events. Zinc compounds have been shown to stimulate osteogenesis. We hypothesized that addition of a zinc compound to a BMP bioimplant would require less BMP to be effective, thereby reducing the dose of BMP required potentially reducing the cost of BMP therapies.

Materials and Methods

To evaluate the effect of Zinc on BMP activity in vitro C2C12 cells were cultured in media containing varying concentrations of ZnCl2 with or without BMP-2. Cell extracts were assayed for alkaline phosphatase (ALP) activity as a measure of BMP potency. Scaffolds were prepared by combining calcium sulphate hemihydrate (CSH) with 0.01 to 10wt% ZnCl2 after which distilled water was added and the mixture was placed in molds and allowed to set. Setting time was evaluated using a dental probe, compressive strength was measured by an instron system and scanning electron microscopy (SEM) and X-ray diffraction (XRD) were used to further characterize the scaffolds. Several scaffolds were incubated in alpha MEM + 10% fetal bovine serum and, after a week, the media was assayed for zinc using a colorimetric assay. To evaluate the effect of zinc on BMP bioimplant activity gamma irradiated scaffolds with 0, 0.1 or 1wt% ZnCl2 were incubated with BMP and surgically implanted in a muscle pouch in the biceps femoris of a mouse. Matching scaffolds without BMP were used as controls in the contralateral limb. The mice were sacrificed 28 days post implantation. MicroCT was performed to quantitatively and qualitatively measure the amount of new bone induced by the implants.

Results

The addition of ZnCl2 to the cell culture media increased BMP induced ALP activity dose dependently (P<0.05). Addition of ZnCl2, to CSH reduced setting time and compressive strength and increased crystal size. However the effects on compressive strength were only significant at 0.5% wt% and above. XRD measurements showed no differences in the diffraction patterns with increasing concentrations of ZnCl2. Scaffolds with 0.05% wt% ZnCl2 and above released detectable amounts of zinc into media over 7 days. The in vivo studies revealed that the all scaffolds soaked with BMP produced bone, while control scaffolds did not. Those scaffolds with 0.1 or 1wt% ZnCl2 formed significantly larger ossicles with more bone than control scaffolds containing no zinc (Fig 1 & 2). While the total volume of the ossicles produced by the 0.1 and 1wt% BMP scaffolds was similar, the 0.1wt% scaffold contained significantly more bone within the ossicle.

Discussion and Conclusion

ZnCl2 enhances BMP-2 activity in vitro and when incorporated into calcium sulphate scaffolds increases the amount of bone formed with BMP in vivo, suggesting a ZnCl2-CaS scaffold may reduce the amount of BMP required for clinical efficacy.

Figure 1: Total Volume of ossicles produced by ZnCl2-calcium sulphate scaffolds. Scaffolds with ZnCl2 produced larger ossicles than the control scaffolds

Figure 2: Bone volume within ossicles produced by ZnCl2-calcium sulphate scaffolds. Scaffolds with ZnCl2 produced more bone than the control scaffolds.

Acknowledgements

Funding for this project was provided by a University of Toronto Dental Faculty Research Grant.

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