Incorporating Strontium Improves Therapeutic Potential of Calcium Polyphosphate Delivery Matrices for Osteomyelitis Treatment

Comeau, Patricia A. (School of Biomedical Engineering, Dalhousie University, Halifax, Canada)
Filiaggi, Mark J. (Department of Applied Oral Sciences, Dalhousie University, Halifax, Canada)

Introduction

Predictable, localized therapeutic delivery is important for both treating chronic infection and assisting with subsequent bone regeneration. With recent progress in the development of low-temperature fabrication protocols for inclusion of thermal labile therapeutic agents, calcium polyphosphate (CPP) has shown strong potential as a delivery matrix [1,2]. However, there is still a need to examine the potential of our CPP matrix to deliver multiple therapeutic agents with distinct targets relevant to infection eradication and bone regeneration. The chemical similarity of strontium (Sr) to calcium and the body of evidence espousing the therapeutic potential of Sr salts for bone regeneration make this ion particularly appealing to incorporate in our CPP delivery matrix. For this study, our guiding hypothesis was that the addition of up to 10mol% Sr to our CPP matrix would not significantly alter the basic structural nature of the undoped CPP glass and hence the therapeutic potential of the delivery matrix. We studied this hypothesis by evaluating the doping efficiency of our fabrication protocol, the crystallization and melting temperatures of the resulting glasses as well as crystallization profiles, and the chain length. Furthermore, the extent to which these physical changes impact elution behaviour was also studied.

Materials and Methods

Amorphous Sr-incorporated CPP containing 0, 5, or 10mol% SrO was produced by modifying the protocol developed by Pilliar et al.[3] for undoped CPP (i.e. 0mol% SrO). In short, reagent grade powders of strontium phosphate (SrHPO4) or strontium carbonate (SrCO3), ammonium phosphate, and calcium phosphate monobasic monohydrate at the requisite ratios were mixed for 1hr and then placed in a Pt crucible to be calcined at 500°C for 10 hours. The calcined powder was then melted at 1100°C for 2hrs and quenched in distilled water to produce an amorphous frit. The composition of the resulting glasses was determined by ICP-OES (PerkinElmer Optima8000). Doping efficiency was calculated by comparing the expected Sr level to that detected in the dissolved glass. Thermal analysis of the CPP glasses was performed using DSC/TG (Netzsch Luxx 409 PC) and heating the glasses up to 1100°C at a rate of 10K/min. Using Proteus Thermal Analysis 5.2.0 software the crystallization (Tc) and melting temperatures (Tm) were determined. The CPP glasses were then heated to 50°C above Tc and 50°C below Tm for 2hrs before the samples were cooled to room temperature, then assessed with a Bruker D8 Advanced XRD system complete with a high speed LynxEyeTM detector as well as DIFFRACplus software with access to the latest PDF database. Chain length was evaluated using solid-state 31P NMR spectrometry (Bruker Avance) and Dmfit 2010 software. To assess impact of Sr-incorporation on elution behaviour, Sr-doped and undoped vancomycin – loaded CPP disks were fabricated following the protocol of Petrone et al [1]. Here, a 2-hr initial gelling phase (G1) for drug loading was observed, prior to compaction of resulting dried G1 powder (at 113MPa) and an additional gelling segment (G2) of 3hrs. For the one-week in vitro elution study, 15mL of 0.1M TBS was added to each G2 disk within a 20mL glass scintillation vial on a horizontally rotating plate in a 37°C room [3]. At predetermined time points 7mL of elution media was removed for measurement of the release of calcium, and phosphate by ICP-OES. The data was analyzed using Minitab15.0 by one-way analysis of variance with a significance value of p=0.05. In addition, a post-hoc pairwise Tukey analysis was performed.

Results

Our processing protocol enabled Sr doping efficiencies of at least 97%, with no significant dependence on Sr salt or concentration (p>0.05) (Table 1). There was some dependence of crystallization and melting temperatures on the amount of Sr present in the CPP, but no significant impact of Sr salt used. In general, higher Sr content yielded lower crystallization and melting temperatures. Upon XRD assessment all doped and undoped CPP crystalline phases above Tc and below Tm were confirmed as Beta-CPP (PDF 77-1953) without phase separation (Fig.1). NMR analysis found that the chain length of the amorphous 10mol% Sr-incorporated CPP was significantly greater than that of undoped CPP (Fig.2). In vitro analysis of the G2 disk elution behaviour revealed that there was little significant difference between ion release from CPP G2 matrices with and without 10mol% Sr incorporated (data not shown). However, the disks retained their original shape best in vitro when Sr was present. This improved disk stability coincides with our concurrent measurement of VCM release where it was found that the burst release of VCM at 8 hours was significantly decreased when Sr was present (data presented at the 2014 ORS Meeting [4]).

Discussion and Conclusion

The results of the present study demonstrate that the incorporation of up to 10mol% Sr into amorphous CPP had an impact on the physical nature of the base matrix without subsequently increasing structural instability in vitro. By further controlling the release of VCM the presence of up to 10mol% Sr in our amorphous CPP significantly improved the therapeutic potential of this delivery system. Future studies will consider the extent to which greater Sr-incorporation into our amorphous CPP system may further improve our targeting of infection eradication and subsequent bone regeneration.

Table 1: Compositional and Thermal Analysis of Strontium-incorporated CPP (n=6).

Figure 1: XRD Profiles of Strontium-incorporated CPP Crystallized (left) Above Tc, and (right) Below Tm.

Figure 2: Solid State 31P NMR Chain Length of Strontium-incorporated CPP with Varying SrO concentrations and salts. Data Reported as Average Values while Error Bars Represent One Standard Deviation (n=5). Horizontal Bars Represent Significant Difference.

Acknowledgements

We are grateful to the Natural Sciences and Engineering Research Council for funding of this study and for support in the form of an Alexander Graham Bell Canadian Graduate scholarship. In addition, we thank Dr. Werner-Zwanziger for NMR assistance and the Institute for Research in Materials at Dalhousie for access to characterization facilities.

References

1.Petrone, C, Hall, G, Langman, M, Filiaggi, M. Compaction strategies for modifying the drug delivery capabilities of gelled CPP matrices. Acta biomater. 2008, 4, 403-413. 2.Dion, A, Langman, M, Hall, G, Filiaggi, M. VCM release behaviour from amorphous CPP matrices intended for osteomyelitis treatment. Biomater. 2005, 26, 7276-7285. 3.Pilliar, R, Filiaggi, M, Wells, J, Grynpas, M, Kandel, R. Porous CPP scaffolds for bone substitute applications in vitro characterization. Biomater. 2001, 22, 963-972. 4.Comeau, P, Filiaggi, M. Incorporation of Sr into Amorphous CPP Matrices Improves the Local Tuneability of VCM Delivery. 2014 ORS Meeting.

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