Download Evaluating the Feasibility of Small Molecule Phenamil as a Novel

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 10M 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 10M 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