COMPUTATIONAL ANALYSIS OF POLYETHER-POLYOLS TYPE
POLYGLYCEROLS FOR TISSUE ENGINEERING APPLICATIONS
Passos, E. D.1, de Queiroz, A. A. A.2
1, 2
Center for Research and Innovation in Advanced Materials biofunctional – Federal University of
Itajubá – Unifei, Itajubá (MG), Brazil
esdrasdp@gmail.com
ABSTRACT
The development of scaffolds for tissue engineering applications has recently
focused on the design of biomimetic materials that are able to interact with surrounding
tissues by biomolecular recognition. When scaffolds are exposed to biological
environments, extracellular matrix (ECM) proteins are adsorbed on the surface and then
cells indirectly interact with the synthetic surface through the adsorbed ECM proteins.
A synthetic cardiovascular graft can be used for the replacement of damaged native
tissues in cardiac or peripheral bypass surgery when there is limited supply of
autologous tissues. However, synthetic vascular grafts generally exhibit a lower patency
rate than natural tissues mainly due to thrombus formation following the implantation.
Computational methods for design scaffolds with improved haemocompatibility has
become of growing importance as alternative approaches to animal experimentation. In
this work molecular dynamics (MD) was used to simulate the interaction between
human serum albumin (HSA) and human serum fibrinogen (HFb) and polyether-polyols
type poly(glycerol)´s, (PGL). The simulation was made by using GROMACS and the
media were aqueous solution, 37ºC and 1 atmosphere of pressure. Our results show that
the more effective interactions occur between PGL and HSA than HFb. MD
symulations indicates that PGL would be a class of consistent, economical, and high
performance multivalent scaffold 3D tissue constructs for soft-tissue engineering and
regenerative medicine.
Key words: Molecular Dynamics Simulation, Tissue Engineering, Aliphatic Polyether’s,
Scaffolds.
1.
INTRODUCTION.
The twentieth century was a watershed for the development of lifesaving
surgical procedures and techniques that also vastly improved the quality of human life.
The clinical surgeries were informed by the extraordinary explosion in knowledge of
human anatomy, physiology, and biology. All the knowledge converged to a complex
array of pre-existing lines of work from the interdisciplinary between the clinical
medicine, engineering, and science. Actually, an intense research has been devoted to
the development of scaffolds in the area of medicine that has come to known as tissue
engineering [1-5].
The success of polymeric scaffolds is determined by the response it elicits from
the surrounding biological environment. This response is governed, to a large extent, by
the surface properties of the scaffold. Multiple approaches have been developed to
provide micrometer to nanometer scale alterations in polymer scaffolds to enable
improved protein and cell interactions. Chemical modification of polymeric scaffold by
physical or chemical methods is one of the upcoming approaches that have been
employed successfully to providing cell adhesion and growth on polymeric scaffolds [612].
Over the past twenty years, our laboratory has developed expertise in the
fabrication of synthetic scaffolds with tailored chemistries for a variety of tissue
engineering applications. These novel materials are based on hyperbranched and
dendritic poly(glycerols) (PGL) [13-15] (Figure 1). We have demonstrated these
materials to be non-toxic to surrounding cells and tissues [16-18]. Additionally, as these
materials are based on naturally-occurring compounds, they degrade over time in the
body to natural products which can be safely and efficaciously excreted. Thus, after
implantation of PGL-based scaffolds, no further surgery would be needed for removal
of the material, thereby reducing discomfort to the patient and cost of the procedure.
Figure 1- Illustration of poly(glycerol) (PGL) (A), Hyperbranched poly(glycerol)
(HPGL) (B) and Dendritic poly(glycerol) (PGLD) (C).
One of the important criteria for a scaffold is that it should be able to serve as an
anchor for stem cells to be retained near the injury site, instead of allowing cells to be
attracted and migrate to healthy regions. Protein absorption on substrates is one of the
critical factors to control cell adhesion [19-21]. Such strong affinity to protein
molecules may be an indicative to the cell adhesion capability of scaffold.
The focus of this work is the study of PGL – protein interactions. It is based on
two concepts: (i) that an atomic level of detail will be required if the true effects of
polymers on protein behavior are to be completely elucidated and (ii) that in place of
direct experimental advances, the best way to approach this problem is through the use
of molecular simulation techniques, replacing animal experimentation . The use of
molecular dynamics (MD) simulations allows the physicochemical nature of molecular
interactions to be observed at a level of resolution that is currently beyond reach
experimentally.
2. MATERIALS AND METHODS
2.1. Molecular Dynamics Simulation Details
All molecular dynamics (MD) simulations were carried out using GROMACS 4
software [21]. In each simulated system, water was modeled with the TIP3P model [22]
and proteins were modeled with the OPLS-AA force field [23] implemented in
GROMACS; each system also included 100 mM NaCl with literature parameters used
to describe the ions [24-25] at a system pH of 7.0. NaCl was used because they were the
predominate ions used in biological human organism. The system size of each
simulation was dependent on the specific volume of each protein, determined by
HYDROPRO [26]; the final volume of the box was adjusted so that each system
contained 100, 200 or 300 mg/mL PGL 3,350 with 6, 12 and 18 PGL molecules,
respectively. All simulations used a dodecahedral box with approximately an 80 Å
image distance – the distance from the center of one system to the next – and applied
with periodic boundary conditions.
All PGL structures were first energy minimized using the steepest descent
algorithm for 1000 steps and then incrementally heated to 310 K (37 ºC) over 500 ps in
the NPT ensemble; during this initial period, a pressure of 1 atm was maintained with
the Berendsen barostat [58] and temperature was maintained with the Berendsen
thermostat [27]. Directly following, each system was equilibrated for a further 10.5 ns
in the NPT ensemble, switching to the canonical coupling schemes of the ParrinelloRahman barostat [28] at 1 atm pressure and the Nosé-Hoover thermostat [29-30] at 310
K. Each production simulation lasted 500 ns in the canonical coupling scheme. Cutoffs
for both the short-range electrostatic and van der Waals interactions were set to 10 Å,
and the long-range electrostatic interactions utilized the Particle Mesh Ewald (PME)
method [31]. A 2.5 fs time step was employed with all covalent bonds constrained using
the LINCS algorithm [32]. All atomic coordinates were saved at 1 ps intervals for
subsequent analysis.
Two different proteins were simulated; Human Serum Albumin (HSA) and
Human Serum Fibrinogen (HFb), each was simulated alone in aqueous solution and
with three different concentrations of PGL 3,350 (100, 200 and 300 mg/mL, again
corresponding to 6, 12 and 18 monomers of PGL 3,350, respectively). All protein
structures were obtained from the Protein Data Bank [29].
3.
RESULTS E DISCUSSION
3.1. Structural Stability of the Systems
In order to show the reliability of MD experiments results obtained from our
simulation we followed up temperature changes of the system during simulation (Figure
2). The constancy of temperature at 310K indicates the stability of the system. Changes
in total and kinetic energy also confirmed the satiability of our system. In this context,
the ratio of Kinetic/Total energy is a bit better index for the prevalence of stability in
system. This ratio in our system is completely constant and it did not exceed 0.05 which
in turn show the conservation of energy during simulation (data not shown).
Figure 2- Changes in system temperature during simulation around 310K. The data
were obtained in aqueous solution, 310K and 1 atmosphere of pressure.
From misalliances data obtained from trajectory analysis that could be easily
interpreted are surface accessible area, gyration radius and RMSD changes of protein
during simulation. The first parameter, the accessible surface area (ASA), is the surface
area in Å2 of PGL that is accessible to a non-aqueous solvent [17]. Any change in ASA
during simulation indicates structural changes in PGL structure (tertiary).
Figure 3 show that PGL with n= 2 in contrast to n >2 more effectively decreases
surface accessible area of polyglycerol. This finding indicates that the hydrogen
bonding in poly(glycerols) with n<10 probably acts via increaseing polymer
compactness that in turn leads to decrease in disturbance of polymer structure and
necessarily induces increase in random coil proportion.
Figure 3- The surface accessible area of PGL in the presence of different inhibitors. The
data were obtained in aqueous solution, 310K and 1 atmosphere of pressure.
The next parameter we studied was the radius of gyration of PGL´s or Rg. The
size of a polymer molecule can be described by the radius of gyration which is the trace
of the tensor of gyration given as:
where rα is the position of site α, rCM is the position of the molecular centre of mass, mα
is the mass of site α and the angle brackets denote an ensemble average.
The radius of gyration (Rg) is thus the average distance from the centre of
gravity to the chain segment. It can be measured experimentally using different
techniques and is dependent on molecular weight. Polymers with higher degree of
polymerization normally have larger radius of gyration. Furthermore for a given value
of molecular weight, depending on the branching topology, the value of the radius of
gyration for different branched polymers can vary.
Figure 4 show the Rg change during simulation as a function of PGL
concentration. Rg describes the overall spread of the molecule and determines the PGL
structure compactness, the more Rg the less compactness of PGL structure. As it is
evident the decrease in polymerization degree (n) decreases the PGL radius during
simulation which is in confirmatory with surface accessible area changes (Fig. 3).
Figure 4- Dependence of gyration radius of PGL with different polymerization degree.
The data were obtained in aqueous solution at PGL concentration of 300 mg/mL, 310K
and 1 atmosphere of pressure.
We also examined the influence of n for a fixed polymer concentration of 300
mg/L as shown in Figure 5. This leads to Rg = 1.98n0.48, where the exponent of 0.48 is
in comparison to the predicted value of 1/2. The exponent of 0.48 suggests that the PGL
chains are quite flexible in the semidilute solution and is consistent with the single chain
density–density correlation function, modeled by the Debye structure factor. However,
more experiments are needed to confirm this result over a wider n range.
Figure 5- Dependence of Rg on degree of polymerization at PGL concentration of 300
mg/mL.
The development of biomaterials for tissue engineering applications has recently
focused on the design of biomimetic materials that are able to interact with surrounding
tissues by biomolecular recognition [33-35]. When biomaterials are exposed to
biological environments, extracellular matrix (ECM) proteins are non-specifically
adsorbed on the surface of nearly all the biomaterials, and then cells indirectly interact
with the biomaterial surface through the adsorbed ECM proteins. The design of
biomimetic materials is an attempt to make the materials such that they are capable of
eliciting specific cellular responses and directing new tissue formation mediated by
specific interactions, which can be manipulated by altering design parameters instead of
by non-specifically adsorbed ECM proteins.
A synthetic cardiovascular graft can be used for the replacement of damaged
native tissues in cardiac or peripheral bypass surgery when there is limited supply of
autologous tissues. However, synthetic vascular grafts generally exhibit a lower patency
rate than natural tissues mainly due to thrombus formation and intimal hyperplasia
following the implantation [36]. In addition, the failure rate of synthetic grafts increased
particularly in small diameter applications in which the internal diameter is less than 6
mm. Thus, the design of vascular grafts with improved biocompatibility is critical for
cardiovascular tissue engineering.
Generally, blood coagulates and causes blood clotting when it encounters
foreign synthetic surfaces. This phenomenon is assumed to begin with the initial
absorption of blood proteins, which is followed by platelet adhesion and activation of
the coagulation pathway, leading to thrombus formation. Studies have pointed that the
significantly reduction of thrombus formation onto synthetic polymers surfaces is a
specifically albumin adsorption. This specific absorption was postulated to increase the
amount of albumin on the surface and to decrease the number of platelets on the surface
available to aggregate into a thrombus [37].
Figure 6 show the Root Mean Square Displacement (RMSD) of PGL-HSA and
PGL-HFb complexes. RMSD changes in Figure 6 shows that the PGL – proteins
complexes reaches the equilibrium state over 1 ps of simulation. The more increase in
RMSD means the more interaction of protein with PGL during simulation as it seen for
HSA (Fig. 6) and this is reflected in more changes in PGL to proteins distance.
Figure 6- Root Mean Square Displacement (RMSD) changes of PGL in the presence of
HSA (A) and HFb (B). The data were obtained in aqueous solution, 310K and 1
atmosphere of pressure.
Figure 7 shows that the RMSDs of HAS in 300 mg/mL of PGL 3,350 are either
lower or equal to those in solutions without PGL 3,350. Not only does PGL 3,350
appear not to unfold the HSA, therefore, but decreased the RMSD of the protein
backbone.
Figure 7- RMSD analysis for HSA. The RMSD compare 0 mg/mL (gray) to 300 mg/mL
(red) PEG 3,350.
CONCLUSIONS
Overally in case of poly(glycerol) polymers we can conclude that HSA bind
preferentially to PGL increasing its compactness, increasing its random structures and
its surface accessible area. The decreasing gyration radius of PGL with polymerization
degree convert the native, relaxed and functional structure of polyglycerol to deformed.
The MD simulation results indicates that more effective interactions occur between
PGL and HSA than HFb. MD symulations suggests that PGL would be a class of
consistent, economical, and high performance multivalent scaffold 3D tissue constructs
for soft-tissue engineering and regenerative medicine.
Acknowledgments
The authors are grateful for funding from the Capes, Finep and CNPq by the
financial support.
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