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Evaluating the Feasibility of Small Molecule Phenamil as a Novel Osteogenic Growth Factor for Bone Regenerative Engineering Applications 1 Lo K. W.-H., 2Ashe, K.M., 1Kan, H.M. and + 1, 2Laurencin, C.T. University of Connecticut Health Center, Farmington, CT, 2University of Connecticut, Storrs, CT Senior author (Cato T. Laurencin, M.D., Ph.D.): [email protected] METHODS Reagents. Phenamil was purchased from Sigma-Aldrich; PLAGA (85:15) was purchased from Lakeshore Biomaterials. Two-dimensional (2D) thin films of PLAGA fabrication. The thin films of PLAGA were fabricated using a solvent casting method. Briefly, PLAGA was dissolved in methylene chloride (Fisher), poured into a teflon-coated dish, and placed at −20°C. Once the solvent evaporated, the 2D thin film discs were formed by cutting the polymer sheet into circle films. All films were sterilized by UV light. Cell culture. Osteoblast-like MC3T3E1 cells (passage 22 to 32) (ATCC) were used for our cell studies. The cells were maintained in alpha minimal essential medium (Invitrogen) supplemented with 10 % FBS and 1 % of antibiotic (100U/ml penicillin G and 100mg/ml streptomycin). The culture medium and phenamil (10 M) were replaced every 3 to 4 days. Cell Adhesion Assay. Cell adhesion was evaluated using crystal violet stain solution (Sigma-Aldrich). Briefly, cells were allowed to adhere on PLAGA thin films in the presence or absence of phenamil at 37 °C for 3h. Subsequently, the non-adherent cells were removed by washing with PBS three times. Crystal violet stain solution was added followed by 30 minutes incubation at room temperature. The plates were washed extensively with Milli-Q water. Stained cells were lysed by using 1 % SDS. The amount of dye taken up by the cells was quantified in a spectrophotometer at the wavelength of 590 nm. Cell proliferation assay. Cell proliferation study was performed using PicoGreen dsDNA assay kit (Molecular Probes). The procedures of the assay were performed according to the manufacturer’s instructions. Briefly, 5 × 104 cells ml-1 were seeded in the presence or absence of phenamil. Cells were collected at day 7 and 14 for the proliferation assays. Alkaline phosphatase activity (ALP). Alkaline phosphatase activity was measured according to the manufacturers’ instructions (Bio-Rad). Cells were collected at day 14 and 21 for the assays. . The ALP activities were normalized to the cellular DNA. RESULTS As shown in Fig. 1, the small molecule phenamil at 10 M promoted initial cell adhesion of osteoblast-like MC3T3-E1cells on PLAGA thin films after 3h incubation. 6 Relative Adhesion INTRODUCTION Growth factors for bone regenerative engineering technology have been extensively investigated in the field because of their inherent osteoinductive potential. Traditionally, growth factors are large recombinant proteins that have been shown to be osteoinductive. For instance, bone morphogenetic proteins (BMPs), platelet-derived growth factor (PDGF), transforming growth factor beta (TGF-), and fibroblast growth factor (FGF-2) have been shown potential for use in bone regeneration and repair. Unfortunately, there are several shortcomings of using protein growth factors for bone tissue engineering. Protein instability, low solubility, high cost, supra-physiologic dose and immunogenicity are the common limitations in these protein-based therapeutic strategies. Therefore, an alternative form of bone growth factors is needed to obviate the drawbacks. Small molecules with osteoinductive capacities have recently gained a lot of attention in the regenerative engineering field because their intrinsic physical properties. For instance, Park et al1 recently demonstrated that the small molecule phenamil, a derivative of the diuretic amiloride, was able to induce osteoblast differentiation and mineralization of mouse mesenchymal stem cells on tissue culture plates. Compared to the recombinant BMPs, phenamil is an inexpensive stable small molecule. More importantly, Park et al1 showed that in contrast to the required high dosages of recombinant BMP, low concentrations of phenamil was sufficient to induce bone formation via the BMP-Smad signaling pathway. These observations prompted us to test whether the small molecule phenamil could likewise induce bone formation when used in conjunction with biodegradable regenerative scaffolds. In this study, we evaluated the feasibility of using phenamil as a novel growth factor for bone repair and regeneration using biodegradable poly(lactide-co-glycolide acid) (PLAGA) thin films and osteoblast-like MC3T3-E1 cells. We characterized the in vitro cellular behaviors of MC3T3-E1 cells cultured on PLAGA thin films in the presence of phenamil with regard to initial cell adhesion, proliferation, differentiation marker expression, and matrix mineralization; all of which constitute relevant cell fates for bone regenerative engineering. Our data indicated that phenamil not only promoted initial MC3T3-E1 cell attachment, but also supported cell proliferation. Furthermore, it induced osteoblast-related marker up-regulation of the osteoblast-like MC3T3-E1 cells seeded on the PLAGA thin films. Fig. 1 * 5 4 3 2 1 0 Control Phenamil For cell proliferation studies (Fig. 2), both phenamil treated and untreated control cells showed significant increase in cell proliferation from day 7 to day 14. There were no statistically significant differences in cell proliferation among the phenamil treated cells as compared to the untreated control cells at the time points evaluated. These observations suggested that phenamil at 10M supports cell proliferation and it does not induce cytotoxicity to MC3T3-E1 cells. Fig. 2 Fluorescence (A. U.) 1 140 Day 7 Day 14 120 100 80 60 40 20 0 Control Phenamil Control Phenamil For ALP activities, at Day 14 and 21, cells cultured in regular medium with 10M of phenamil were found to significantly increase ALP activities as compared to the control untreated cells. (Fig.3) Fig. 3 DISCUSSION It has been reported that cells grown on different biomaterial surfaces will exhibit varying cellular behaviors2. Thus, the goal of this study is to investigate whether the proposed biodegradable polymer, i.e. PLAGA, would support or counteract the osteoinductive effect of phenamil on MC3T3-E1 cells. Our results demonstrated that phenamil supported not only the proliferation, but also promoted the differentiation of osteoblast-like MC3T3-E1 cells cultured on 2D-PLAGA films. This indicated that the PLAGA does not counteract the effects of phenamil. Moreover, in this study we uncovered that phenamil promoted initial cell adhesion on 2D-PLAGA. This novel property is of great interest since cell adhesion is an important pre-requisite for subsequent cell specialization. Therefore, the increased cell adhesion may also contribute to downstream cellular responses important for osteoblast differentiation and mineralization. In summary, the small molecule phenamil supported or promoted all relevant cellular events of MC3T3-E1 cells for bone regenerative engineering. The data from this study will aid in designing a novel bone grafting material system comprising PLAGA matrices with small molecule phenamil. REFERENCES 1. Park K.W. et al., Mol. Cell Biol. p3905-14, 2009. 2. Calvert J.W. et al., Plast. Reconstr. Surg. p567-76, 2005 Poster No. 1861 • ORS 2011 Annual Meeting