Early stage differentiation response of pre-osteoblastic MC3T3-E1 cells to therapeutically-loaded calcium polyphosphate bead degradation extracts

摘要

Introduction: Osteomyelitis, or bone infection, is a disease that affects patients of any age, impacts any bone and can lead to persistent morbidity. The main objective of this study was to provide an initial assessment of the clinical applicability of calcium polyphosphate (CPP) beads for localized osteomyelitis treatment. It was hypothesized that doping CPP with 10 mol% strontium would improve pre-osteoblastic cell response to degradation extracts, while vancomycin (VCM) would not hinder this same response relative to the study controls. Materials and Methods: A paste mixed at a ratio of 150mg xSrO-CPP glass powder (x=0,10 mol%): 0.0602mL distilled water: 7.5mg VCM (or 0mg for blanks) was added to disk molds and gelled at 37°C under high humidity for 2h. The freshly gelled disks were then dried and milled to obtain ＜45μm powders. Approximately 85mg of this powder was placed in bead molds prior to vacuum bagging and placing in the cold isostatic pressing chamber at 113MPa for 5 min (Avure Technologies). The beads were then gelled under humidity at 37°C for 3h before drying. For this study the final gelled CPP beads were added to 37°C 0.1M TBS for 12 days. Degradation extracts were collected at predetermined time points and 0.2μm sterile-filtered before being added to the cells at 10 vol% of media. The cell study positive control was differentiation media only, while a positive reagent control was differentiation media and 10% 0.1M TBS. A 12-day cell study was performed using MC3T3-E1 cells (ATCC® CRL-2593). The cells were maintained in 75cm~2 tissue culture flasks with α-modified minimum essential medium supplemented with 10% fetal bovine serum within a humidified 5% CO_2 balanced air incubator at 37°C. Cells at passage 25 were removed from the flasks and seeded at 3×10~4cells/mL/well in a 24-well plate. After incubating for 24h (t=0h), media was exchanged for differentiation medium containing 10mM sodium β-glycerophosphate, 50μg/mL ascorbic acid, and 100U/mL. penicillin/streptomycin. Alkaline phosphate (ALP) activity was evaluated with a Sigma APF-1kt kit at 3,6, and 12d. Mineralization was assessed at 6d and 12d using light microscopy and a staining protocol of 40mM Alizarin Red solution (ARS) and 0.2% SF Yellowish solution (SFYS) (for calcium deposit and collagen detection, respectively). Mineralization was quantified by de-staining with cetylpyridinium chloride solution and measuring OD540 absoibance on a BIOTEK microplate reader. To normalize ALP and mineralization values, the total cell protein was measured using a Sigma QuantiPro BCA kit. Data was analyzed with two-way analysis of variance and post-hoc pairwise Tukey comparison (Minitab15.0, p=0.05, n=6). Results and Discussion: The presence of strontium and lack of VCM in the CPP-based bead extracts increased the ALP and mineralization activity of the cells at 12d (Fig 1 and Fig 2). In addition, the reported cell activity at each time point was comparable or greater for VCM-loaded extracts relative to the study controls. Conclusion: Early-stage pre-osteoblast functionality assays demonstrated the capacity for these strontium-doped CPP beads to enhance osteoblast differentiation, as evident by substantive increases to ALP expression and mineralization markers. This study provided early evidence for the enhanced clinical applicability of the CPP glass-based beads for localized osteomyelitis therapy.