Small Molecule Cyclic Adenosine Monophosphate Analogues for Bone Regenerative Engineering
1, 2
Lo K. W.-H., 1, 2 Kan, H.M., & + 1, 2, 3, 4 Laurencin, C.T.
Institute for Regenerative Engineering, 2Department of Orthopaedic Surgery, 3Connecticut Institute for Clinical and Translational Science, University
of Connecticut Health Center, Farmington, CT; 4Chemical, Materials and Biomolecular Engineering Department, University of Connecticut, Storrs, CT
+Senior author (Cato T. Laurencin, M.D., Ph.D.): [email protected]
1
INTRODUCTION
Bone regenerative biodegradable scaffolds, often in combination
with cells and/or growth factors, have been heavily studied and applied
to certain clinical situations. The use of growth factors has become a
common strategy to enhance bone formation within tissue engineered
scaffolds. For instance, bone morphogenetic proteins (BMPs) have
shown great potential for use in bone regeneration and repair. While
these proteinacious growth factors have shown the potential to facilitate
bone formation within these scaffolds, the resulting tissue often
localizes to the surface of the scaffold rather than ingrow due to poor
vascularization and limited oxygen supply. To combat this issue,
vascular endothelial growth factor (VEGF) has been extensively
researched as a cofactor to be delivered into the scaffolds in order to
enhance angiogenesis. While tissue engineering constructs employing
both osteoinductive and angioinductive protein growth factors have
shown promise, unfortunately, protein instability, low solubility, high
cost, contamination, supra-physiologic dose and immunogenicity have
limited these protein-based therapeutic strategies. Therefore, an
alternative form of growth factors is needed to obviate the drawbacks.
Small molecules that carry the capacity to induce osteoblast
differentiation, have recently gained more spotlight in the bone
regenerative field. It is believed that these small molecules have the
intrinsic physical properties that allow them to overcome the issues
observed with protein growth factors1. We previously demonstrated
that the small molecule, 6-Bnz-cAMP, which is a target-specific
cAMP-analog that interacts via the protein kinase A (PKA) signaling
pathway, was able to induce initial cell adhesion 2, differentiation, and
mineralization of early passage MC3T3 osteoblast cells on
biodegradable polymeric scaffolds 1. Interestingly, as of recent, it has
been shown that cAMP signaling is also important for regulating VEGF
expression3. As a result, this observation has prompted us to test
whether the cAMP analogues could likewise induce angiogenesis by
increasing the production of VEGF.
Since human mescenchymal stem cells (hMSCs) demonstrate
the ability to differentiate into the osteogenic lineages and provide a
closer link between the laboratory and preclinical studies, we found it
appropirate to utlilize these cells in our experiments. In this study, we
evaluated the in vitro osteoinductivity potential of the small molecule
6-Bnz-cAMP in hMSCs. We characterized the in vitro cellular
behavior of hMSCs cultured on polylactic-co-glycolic acid (PLAGA)
thin films in the presence of 6-Bnz-cAMP. We evaluated the cells for
initial cell adhesion, proliferation, differentiation, and matrix
mineralization. Our data indicated that 6-Bnz-cAMP promoted in vitro
osteogenesis of hMSCs. We further explored the angioinductive
potential of different cAMP analogues in osteoblasts by monitoring the
protein expression of VEGF. It was found that target non-specific
cyclic AMP analog, 8-Br-cAMP, significantly induced the expression of
VEGF in MC3T3 osteoblast cells. The hypothesis of our work was
that cAMP analogs play an important role in controlling osteoblast
function on biomaterial surfaces.
METHODS
Reagents. 6-Bnz-cAMP and 8-Br-cAMP were purchased from Alexis
Biochemicals; Biodegradable PLAGA polymer (85:15) was purchased
from Lakeshore Biomaterials.
Two-dimensional (2D) thin films of PLAGA fabrication. 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
PLAGA films were sterilized by 70% ethanol.
Cell cultures. hMSCs (Lonza) (passage 2) were used for in vitro
osteogenesis experiments. Osteoblast-like MC3T3 cells (passage 22 to
30) (ATCC) were used for VEGF expression studies.
Alkaline phosphatase (ALP) activity and mineralization assays. The
assays were performed as previously described 1.
Immunocytochemistry. Cells were rinsed in PBS and blocked with
normal goat serum. Cells were then incubated with monoclonal
anti-VEGF antibody (Santa Cruz) diluted in 1% BSA blocking buffer
for overnight. Anti-mouse fluorescein isothiocyanate antibody (Santa
Cruz) was used for secondary fluorescence staining. Images of cells
were acquired with a confocal microscope (Zeiss LSM 780).
Photoshop 7.0 was used to process all images.
ELISA. Media were collected after 2d of incubation. ELISA was
performed using a kit (Mouse VEGF Immunoassay) according to the
manufacturer’s instructions (R&D Systems).
RESULTS
6-Bnz-cAMP at 100 M promoted ALP activities at day 14
(fig.1) and matrix calcification at day 20 (fig.2) of hMSCs on PLAGA
thin films. For calcification study, cells cultured in osteogenic medium
served as a positive control while cells cultured in regular medium
“Control” served as the baseline. To facilitate the comparison of
different experimental settings, the “control” values were set to one
relative unit. In addition, 6-Bnz-cAMP promoted not only hMSC
initial adhesion, but also supported hMSC proliferation on PLAGA thin
film (data not shown).
8-Br-cAMP at 100 M but not 6-Bnz-cAMP significantly
promoted extracellular production of VEGF in MC3T3 cells after 2d of
incubation on PLAGA as revealed by the ELISA experiment (fig.3).
Fig. 3
Consistent with the data in fig. 3, immunocytochemistry results
(Fig. 4A-C) revealed that only 8-Br-cAMP promoted production of
VEGF (green) in MC3T3 cells after 2d of incubation. Red color
(propidium iodide) represents chromatin. The magnification is 20X.
8-Br-cAMP
6-Bnz-cAMP
Control
DISCUSSION
Signaling of cAMP/PKA has been known to regulate numerous
biological processes including osteoblast adhesion and maturation 1, 2.
In this study, our results provide a clear connection between cAMP
signaling and VEGF expression in osteoblast MC3T3 cells. It is
interesting to point out that the PKA specific agonist, 6-Bnz-cAMP, did
not significantly enhance the expression of VEGF, but rather the target
non-specific cAMP analog, 8-Br-cAMP, greatly enhanced VEGF
expression (fig. 3 & 4). Interestingly, this suggests that
cAMP/PKA-independent signaling(s) is/are involved in the process. The
detailed underlying mechanism will be investigated by our group.
Our studies suggest that 6-Bnz-cAMP and 8-Br-cAMP may
serve promising roles as bio-factors for bone repair and regeneration.
We propose that the delivery of these synthetic small molecules,
6-Bnz-cAMP and 8-Br-cAMP, within the polymeric scaffold constructs,
when released in a controlled manner, may greatly improve the success
of tissue-engineered bone grafts. The data from this study will
facilitate the development of such scaffold construct.
SIGNIFICANT: Development of inexpensive stable small molecules
with biodegradable polymeric scaffolds for engineering bone tissue.
REFERENCES
1. Lo K.W.et al., J. Tissue Eng. Regen. Med. 2011 Epub ahead of print.
2. Lo K.W. et al., J. Orthop. Res. p602-8, 2011.
3. Namkoong, S. et al., Cell Signal. p906-15, 2009.
Poster No. 1649 • ORS 2012 Annual Meeting