Download Incorporation of Strontium into Amorphous Calcium Polyphosphate

Incorporation of Strontium into Amorphous Calcium Polyphosphate Matrices
Improves the Local Tuneability of Vancomycin Delivery
Patricia Comeau, Mark Filiaggi.
School of Biomedical Engineering, Dalhousie University, Halifax, NS, Canada.
Disclosures:
P. Comeau: None. M. Filiaggi: None.
Introduction: An ideal scaffold material for treatment of chronic bone infection should provide a matrix for bone regeneration
and locally deliver relevant therapeutics in a controllable manner. Unfortunately, though calcium phosphates meet many of the
criteria to support bone growth, processing conditions have to date limited their use in local delivery. Recent progress in the
development of low-temperature fabrication protocols for inclusion of thermally labile therapeutic agents in amorphous
Calcium Polyphosphate (CPP) has shown strong potential for controllable delivery1-3. However, a need to properly harness the
delivery potential of CPP through novel fabrication strategies and in-depth material characterization is still required to improve
the structural integrity of the CPP matrices and the local delivery of therapeutics. The chemical similarity of strontium (Sr) to
calcium, as well as a body of evidence espousing the therapeutic potential of strontium salts for bone regeneration, makes
strontium a particularly appealing element to incorporate in tissue engineering scaffolds designed for treatment of
compromised bone tissue. In addition, the introduction of various ions such as strontium into the calcium phosphates may allow
for improved management of scaffold degradation4-6. The guiding hypothesis of this study was, therefore, that the addition of
strontium to amorphous CPP would reduce the degradation of the matrices in vitro without adversely modifying their ability to
release an antibiotic drug. We studied this hypothesis by observing the ability of the matrices to gel, to load and release an
antibiotic drug, as well as to degrade in vitro.
Methods: Amorphous Sr-incorporated CPP containing 0, 5, or 10mol% SrO was produced by modifying the protocol developed
by Pilliar et al (2001) for standard CPP (i.e. 0mol% SrO). In short, reagent grade powders of strontium phosphate (SrHPO 4),
ammonium phosphate (NH4H2PO4), and calcium monobasic monohydrate (CPMM) at the requisite ratios were mixed for 1 hour
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 ability of the Sr-incorporated CPP to gel and form suitable
delivery matrices was assessed using a protocol developed by our group1. Briefly, distilled water was mixed with amorphous CPP
powder to form a paste (150mg CPP: 0.0602mL distilled water). The resulting paste was transferred into disk-shaped
polyvinylsiloxane molds, and placed in a sealed vessel maintained at 37°C and ~100% relative humidity. After 2 hours the
samples were then left to dry at 37°C in atmospheric air for ~48 hours. The appearance of these G1 disks at different gelling time
points and during drying was noted. For in vitro study the disks (n=3) were added to glass scintillation vials with 15mL of 0.1M
TBS before placing the vials on a horizontally rotating plate in a room maintained at 37°C for a period of 6 days. At given time
points 7mL of elution media was removed for measurement of the calcium, phosphate, and strontium ions with ICP-OES
(PerkinElmer Optima8000). To maintain the original volume 7mL of fresh 0.1M TBS was then added to the vials. The impact of
strontium incorporation on loading and release of an antibiotic was studied using a 2-step gelling protocol2. Here, 7.5mg of
vancomycin (VCM) was added to the G1 CPP-water paste prior to 2 hours of gelling and 48 hours of drying. The resulting disks
were then milled and sieved to obtain <45µm G1 powders. Approximately 135mg of the VCM-loaded amorphous G1 powder
was then placed in individual 8mm diameter chambers of a punch-die system and a 113MPa stress applied with the Carver
manual, 25 ton, 2-column hydraulic press for a total contact time of 5 minutes. Subsequently, the die containing the compacted
powder was returned to the sealed vessel for 3 hours of additional gelling prior to drying. These G2 disks were studied in vitro in
accordance with the protocol previously mentioned. The release of calcium, phosphate, and strontium ions during G2 disk (n=4)
degradation in vitro was measured with ICP-OES, while the release of VCM was detected using ultraviolet-visible
spectrophotometer (BioTek Instruments Synergy HT) at a wavelength of 235nm. The loading and elution data was analyzed
using Minitab15.0, a statistics software program, 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: The presence of up to 10mol% SrO in the amorphous CPP did not significantly impact the ability of the material to gel, a
requisite for drug incorporation, nor did it significantly impact the loading efficiency of this amorphous system (p>0.05). With
the exception of the 2-hr time point, there was no significant difference in the percentage of total available calcium,
phosphorus, or strontium released from these G1 disks. However, standard CPP G1 disks appeared less stable in vitro, with
structural integrity quickly lost, and there appeared to be a trend towards a lower fraction of calcium and phosphorus released
after 96 hours compared to 5 or 10mol% SrO CPP G1 disks (p > 0.05). The 10mol% SrO CPP G1 disks did release significantly
greater strontium than 5mol% SrO CPP disks over the 6 day study as expected (p< 0.001; Fig.1). The incorporation of 10mol%
SrO into the amorphous CPP within G2 disks significantly decreased the burst release of VCM at 8 hours (p<0.001) and
significantly increased the cumulative amount of VCM released after 6 days in vitro(p<0.001) (Fig.2).
Discussion: The results of the present study demonstrate that the incorporation of strontium into amorphous CPP did not
significantly impact the gelling or antibiotic loading ability of the G1 disks, nor the fraction of available calcium or phosphorus
released from the G1 disks in vitro. The release of VCM from G2 disks was, however, significantly affected by the presence of
10mol% SrO within the amorphous CPP system. Upon addition of 10mol% SrO both the burst release and extent of VCM
delivered by the G2 disks were significantly improved.
Significance: In clinical practice the treatment of bone infection is very challenging and frequently unsuccessful. The results of
the present study suggest that a strontium-modified calcium polyphosphate local delivery matrix could provide a multifunctional device capable of eradicating infection and encouraging bone regeneration in a tuneable manner.
Acknowledgments: We are grateful to the Natural Sciences and Engineering Research Council for funding of this project and for
support in the form of an Alexander Graham Bell Canadian Graduate scholarship.
References: [1.] Dion et al. Biomater. 2005, 26, (21), 4486-4494.; [2.] Petrone et al. Acta Biomater. 2008, 4, (2), 403-413.; [3.]
Dion et al. Biomater. 2005, 16, (35), 7276-7285. ; [4.] Hoppe et al. Biomater. 2011, 32, 2757 - 2774.; [5.] Song et al. J Biomed
Mater Res Part B:Appl Biomater. 2009, 89B, 430 - 438.; [6.] Song et al. J Biomed Mater Res Part B: Appl Biomater. 2011, 98B, 255
- 262.; [7.] Pilliar et al. Biomater. 2001, 22, 963-972.
ORS 2014 Annual Meeting
Poster No: 0665