Calcium Phosphate Microcarriers for Bone Regeneration:in-vitro Osteoproduction and Ex-vivo Biomechanical Assessment

Introduction: Bone loss as a result of trauma, tumor resection or other skeletal diseases is a serious clinical problem. Novel grafting materials to augment bone loss include calcium phosphates, bioactive glasses and polymers. The materials can be supplemented with growth factors or proteins such as bone morphogenetic proteins (BMPs) to accelerate bone ingrowth to the grafted site. Ideally, grafting materials would deliver growth factors in a controlled fashion to optimize bone formation and ingrowth. In addition to protein delivery, the graft material should have adequate mechanical integrity and resorbability. The research objectives were to evaluate osteogenic protein–1 (OP-1, Stryker Biotech, Hopkinton, MA) elution characteristics from HA (hydroxyapatite) and TCP (tri-calcium phosphate) ceramic microcarriers with a mean diameter of 750 μm (CaP Biotechnology, Golden, CO) and to characterize the mechanical strength of these microcarriers at one-week intervals during the 28-day elution period. Materials and Methods: OP-1 Reconstitution: The lyophilized OP-1 provided by Stryker Biotech was reconstituted and aliquoted to 15 ml conical vials containing 1 ml of microcarriers to initiate protein adsorption. A standard quantity of OP-1 was adsorbed onto the beads. Protein remaining in the vial after adsorption was quantified and accounted for when calculating total OP-1 within the bead samples. Elution Kinetics: CaP Biotechnology provided 750 μm diameter dense and porous HA and TCP microcarriers for the elution kinetics portion of the experiment. Twelve 1ml microcarrier samples were run for each combination of chemical composition and porosity with six samples from each combination receiving no protein to serve as negative controls. Treatment groups included HA porous (P) or dense (S) with (O) or without (X) OP-1 (HPO, HPX, HSO, HSX) and TCP porous (P) or dense (S) with (O) or without (X) OP-1 (TPO, TPX, TSO, TSX). The microcarriers were suspended in phosphate-buffered saline (PBS) and maintained at 37°C in a shaking water bath for the duration of the experiment. Samples were collected every 15 minutes for the first hour and then at increasing intervals over the 28-day elution period. The pH of the PBS was adjusted to mimic changes that occur over time during osseous wound healing. Elutant was collected from all treatment samples at each time point and stored in 5 ml cryovials at -70°C. Elutants were assayed in duplicate for protein concentration with a colorimetric protein assay (Bio-rad Laboratories, Hercules, CA) at a wavelength of 595 nm. Mechanical Testing: A customized mechanical testing system was designed and fabricated. Random samples of ten of the dense HA and TCP microcarriers in pH-adjusted PBS were removed immediately after hydration and at one-week intervals for 28 days and tested to failure in compression. Dry samples of the dense HA and TCP microcarriers were also tested to failure in compression. Statistical Analysis: Two-way ANOVA testing was used to determine differences between treatment and time in solution for protein elution kinetics and biomechanical data followed by a Tukey’s multiple comparison procedure (α = 0.05). Results: Elution Kinetics: The HA porous (HPO) treatment group released significantly more protein than the other three treatment groups (HSO, TSO, TPO) over the 28-day elution period (Figure 1). At one month, the HPO ceramic microcarriers had released a total of 253.4 μg of OP-1 (33.4%) whereas the HSO, TSO, and TPO treatment groups had released 10.5%, 6.4%, and 5.5% of the absorbed protein, respectively. Mechanical Testing: Strength profile data on the dense HA and TCP ceramic microcarriers have been collected and analyzed through 28 days and are presented in Figure 2 (data means with different letters are significantly different from each other). The dense HA microcarriers are significantly stronger than the dense TCP microcarriers at all time points. In addition, there is a trend of decreasing strength of the dense TCP microcarriers over time in solution, whereas the dense HA ceramic microcarriers maintained their strength over the 28 day test period. Stress analyses were also performed on the data and indicate a range of mean wall strengths from 616.1 MPa (89.4 ksi) to 418.5 MPa (60.7 ksi) for the HA carriers and 255.3 MPa (37.1 ksi) to 104.6 MPa (15.2 ksi) for the TCP carriers over the one-month time period. Discussion: Elution kinetics data suggests a much greater affinity of the OP-1 protein for the TCP ceramic composition as compared to the HA ceramic as evidenced by the greater cumulative protein released by the HA ceramic after 28 days in solution. This difference in cumulative release was the direct result of significantly higher rates of protein elution from the HPO microcarriers from 2 hours (16.5 μg/hr) through 36 hours (2.12 μg/hr) of elution. The porous HA microcarriers released more protein into the surrounding aqueous environment than the dense ceramic architecture. This same phenomenon is not seen with the TCP ceramic. The affinity of the OP-1 protein to the TCP matrix appeared to negate the effects of porosity. Compression testing data suggests that TCP strength significantly decreases over time in solution. TCP, unlike HA, has been shown to resorb in vivo and dissolve when placed in aqueous solution ex vivo. TCP dissolution over the sampling period may explain the decline in strength. The TCP microcarriers were significantly weaker than HA microcarriers at all sampling time points. The HA carriers maintained their strength despite the aqueous environment. Conclusions: This preliminary study indicates that HA and TCP microcarriers provide an effective delivery vehicle for sustained release of OP-1 and possibly other proteins or drugs. Future studies will optimize the composition and porosity of the microcarriers for specific skeletal applications and protein/drug delivery rates. Acknowledgements: The authors would like to thank the Colorado Commission of Higher Education as well as the National Science Foundation for financial support of this project and CaP Biotechnology and Stryker Biotech for providing microcarriers and protein, respectively. Also, the authors would like to thank Chad Lewis for his help with the mechanical testing system design and implementation.