The aim of this study was to describe the mechanical and sorption features of homogeneous and composite membranes which consist of microcrystalline chitosan (MCCh) and fibrin (Fb) in various proportions as well as the
The tissue engineering approach to repair and regeneration is founded upon the use of polymer scaffolds which serve to support, reinforce, and in some cases organize the tissue [
The prevention of infection occurrence and pathological lesions resulting from membrane implantation inside an organism still remains an important problem, crucial during the guided bone regeneration procedures. For these reasons, many laboratories were established to develop infection-preventing membranes after implantation. The objective of previous study [
The importance of natural polymers, such as microcrystalline chitosan, and minerals, such as hydroxyapatite (HAp) and tricalcium phosphate (TCP), has grown significantly due to their renewable and biodegradable source, increasing the knowledge and functionality of composites in technological and biomedical applications. The excellent biocompatibility, biofunctionality, and nonantigenic property make the chitosan (Ch) and its derivatives, such as a microcrystalline chitosan (MCCh), an ideal material for tissue regeneration [
Our interest in the use of microcrystalline chitosan (MCCh) and fibrin (Fb) as a carrier (scaffold) of PDGF-BB was first roused by the wide range of practical applications of membranes in tissue engineering described in the work of Cartmell [
In our study, we measured the rate of release of PDGF-BB from membranes containing MCCh, Fb, or mixtures of the two with various polymer contents.
The natural polymers are preferred because the synthetic origin lacks cell-recognition signals [
Chitosan was chosen as a biomaterial because of its biocompatibility, biodegradability, and nontoxicity [
Polysaccharides, such as chitosan and its derivatives, have some excellent properties for medical applications: nontoxicity (monomer residues are not harmful to health), water solubility or high swelling ability after simple chemical modification, stability to pH variations, biocompatibility, high adhesiveness, and extensive chemical reactivity. Moreover, these materials evidence a strong ability to create hydrogen and ionic bonds and biostimulation of natural resistance by controlling and improving bioactivity [
In recent years, there has been increasing interest in the pharmaceutical field in discovering new excipient materials of natural origin. One such material is chitosan, a monograph (chitosan hydrochloride) on which was included in the fourth edition of the European Pharmacopoeia in 2002 and sixth edition of the Handbook of Pharmaceutical Excipients in 2009. This nontoxic, biodegradable, and biocompatible material has attained great interest in pharmaceutical applications, as versatile drug delivery agent. Chitosan has been investigated as an excipient in the pharmaceutical industry to be used in direct tablet compression, as a tablet disintegrant for the production of controlled release of solid dosage forms or for the improvement of drug dissolution. Chitosan membranes to provide an alternative means of evaluating transdermal drug delivery systems were developed and evaluated. Chitosan has been used as an aid to transdermal drug delivery both
Both chitin and chitosan possess properties characteristic of polycations. Like other natural polysaccharides, chitin and chitosan are biocompatible and can therefore be used as bioengineering material, are biodegradable by lysozymes, and show low toxicity. The properties and applications of amino saccharides have been described in the books of Muzzarelli [
A valuable physicochemical modification of chitosan, used among others as excipient in drug formulations, is microcrystalline chitosan (MCCh), obtained in the form of a suspension, powder, or granules [
The most important and extraordinary property of MCCh seems to be its direct-film-forming behaviour just from aqueous dispersion. Film obtained in this process shows excellent adhesion to different types of surface and water resistance [
Fibrinogen [
In our previous research Fb, MCCh, and methylcellulose (MC) hydrogels for bFGF in the presence of ketoprofen [
In this paper, an assessment of the impact of the physicochemical (sorption) and mechanical properties of MCCh-Fb composite membranes consisting of MCCh and Fb at different proportions on the release of PDGF-BB in the presence or absence of Am
Fibrinogen, fraction I, F-8630, type I-S from bovine plasma (9001-32-5), was supplied by Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). Microcrystalline chitosan MCCh/LA 0171 (weight-average molecular weight
Platelet-derived growth factor-BB (PDGF-BB) and Quantikine Human PDGF-BB Immunoassay ELISA Kit were supplied by R&D System, Inc., 614 McKinley Place NE (Minneapolis, MN 55413, USA). Thrombin EC 3.4.4.13 was supplied by Biomed (Lublin, Poland). Factor XIIIa (Fb-stabilizing factor, FSF), fragment 72–97, aprotinin from bovine lung 5 TI U mg−1 protein, amoxicillin A 8523, propylene glycol, and phosphate buffered saline, PBS pH 7.4, were supplied by Sigma-Aldrich Chemical Co. (St. Louis, MO, USA).
Homogeneous (M1, M2) and composite (M3–M5) membranes with growth factor PDGF-BB and additionally with amoxicillin (M1′–M5′) were prepared from biodegradable microcrystalline chitosan (MCCh) and fibrin (Fb) polymers at the following ratios MCCh : Fb (2 : 1, 1 : 1, and 1 : 2) in aseptic conditions. For comparison, one set of polymer membranes was prepared with MCCh (M1) in the absence of Fb, whereas another was prepared with Fb (M2) in the absence of MCCh. The method of film preparation was modified in comparison with our previous publication [
Component of fibrin, microcrystalline chitosan, and fibrin-microcrystalline chitosan membrane systems.
Components | Systems | |||||||||
---|---|---|---|---|---|---|---|---|---|---|
M1 | M1′ | M2 | M2′ | M3 | M3′ | M4 | M4′ | M5 | M5′ | |
Microcrystalline chitosan (mg) | 30.0 | 30.0 |
|
|
40.0 | 40.0 | 30.0 | 30.0 | 20.0 | 20.0 |
Fibrinogen (mg) |
|
− | 30.0 | 30.0 | 20.0 | 20.0 | 30.0 | 30.0 | 40.0 | 40.0 |
Amoxicillin (110 |
|
+ |
|
+ | − | + | − | + | − | + |
PDGF-BB (0.025 |
+ | + | + | + | + | + | + | + | + | + |
Aprotinin (20 |
|
− | + | + | + | + | + | + | + | + |
Thrombin (4.0 NIH) |
|
− | + | + | + | + | + | + | + | + |
FSF (1.0 |
|
− | + | + | + | + | + | + | + | + |
In all systems: CaCl2 (1.11 mg), propylene glycol (50 mg).
Homogeneous MCCh film (M1) (Table
To prepare a complex carrier (MCCh-Fb) containing MCCh and Fb, a mixture of these polymers in the form of a microcrystalline chitosan hydrogel (2.57 wt%) and fibrinogen solution (15.0 mg mL−1) was used (Table
Composite MCCh-Fb membranes (M3, M3′, M4, M4′, M5, and M5′) were prepared by adding fibrinogen solution in PBS buffer (0.01 mol L−1, pH 7.4) (1.33 mL, 2.0 mL, or 2.77 mL containing adequately 20.0 mg, 30.0 mg, or 40.0 mg of fibrin) to MCCh hydrogel (1.60 g, 1.20 g, or 0.80 g containing adequately 40.0 mg, 30.0 mg, or 20.0 mg of chitosan) with 20
An Fb film (M2) (Table
The sorption capacity of homogeneous MCCh (M1), Fb (M2), and composite at different ratios MCCh : Fb = 2 : 1 (M3), 1 : 1 (M4), and 1 : 2 (M5) membranes was determined by making the membranes swell in phosphate buffer solution, pH 7.40, at
After removing the membranes from medium (phosphate buffer solution, pH 7.40), the pH of the solution was measured at
The mechanical properties of the membranes prepared were determined at the Accredited Metrological Laboratory, Institute of Biopolymers and Chemical Fibres (Łódź, Poland), which holds an accreditation certificate number AB 338. The basic mechanical parameters of the polymer materials, which were in the form of dry and wet membranes, were estimated according to appropriate standards: thickness of the film (mm): PN-EN ISO 4593: 1999, mechanical properties: breaking strength (MPa) and elongation at break (%) according to PN-EN ISO 527-3: 1998.
The film samples were tested using Instron 5544 tensile tester. The film samples tested were 10 mm long and 15 mm wide, and the elongation rate was 10 mm min−1. The thickness of dry samples, measured by a micrometer screw, was in the range of 0.024 mm Fb (M2) to 0.077 mm MCCh : Fb = 2 : 1 (M3). For selected samples, the elastic modulus (Young’s modulus,
Scanning electron micrographs (surface and cross-section of the dry and wet membrane) were taken for the membranes studied using an ESEM type Quanta 200 (W) scanning electron microscope (SEM) from FEI Co. (Hillsboro, OR, USA).
The water dispersion of MCCh (M1) and a solution of coagulated fibrinogen (M2) or a coagulation mixture of these polymers (M3, M4, and M5) were placed on a Teflon plate and left to dry at room temperature (
Platelet-derived growth factor (PDGF-BB) release was performed both in the presence and absence of 100
Amoxicillin (Am) was released from the membranes into 1 mL of PBS buffer (0.01 mol L−1, pH 7.40) (Figure
The study was repeated two times. The measurement error was less than 5%. Statistical analysis was performed using the Microsoft Excel Analysis Tool Pak in Microsoft Office Excel 2010 and Statistica 10.
Water absorption ability and retaining this ability are an important factor in determining the usefulness of the biomaterials. The ability of swelling is an essential characteristic to analyze this kind of formulations. The swelling of the polymeric membranes is shown in Figure
The absorption ability of the MCCh-Fb membranes was measured in terms of degree of swelling at equilibrium. It was found that the degree of swelling of the membranes was in range of 260–100% of their dry weight and relatively correlated with the MCCh content (Figure
Diagram of the membrane preparation.
Diagram of the release of PDGF-BB and Am from membrane.
Swelling percentage of homogeneous MCCh (M1), Fb (M2), and composite: MCCh : Fb = 2 : 1 (M3), 1 : 1 (M4), and 1 : 2 (M5) membranes in phosphate buffer solution. pH 7.40 at 37°C from 24 h. The values are an average of three determinations.
The composite MCCh : Fb = 1 : 2 (M5) membrane (of a higher concentration of fibrin) shows less swelling ability (150% after 120 min) and stability. The smallest swelling capacity was found for homogeneous Fb (M2) membrane (Figure
Measurements of pH of the acceptor fluid after swelling of membranes were conducted in order to check the influence of the soluble membrane components on the acceptor fluid. Aqueous extracts of membranes M1, M3, and M4 remain clear and transparent. The studies show that membranes have a slight impact on the change in physiological pH value (7.40) of phosphate buffer (M1 pH 7.38, M2 pH 7.48, M3 pH 7.41, M4 pH 7.44, and M5 pH 7.45).
Two kinds of biomaterials (dry and wet samples) were tested in order to evaluate their mechanical properties such as breaking strength (Bs), elongation at break (Eb), and elastic modulus (Young’s modulus,
Propylene glycol and calcium chloride were added to the hydrogel of MCCh (Table
Influence of MCCh and Fb content on breaking strength (Bs) and elongation at break (Eb) of membrane systems.
From the results presented in Figure
Besides the main properties, namely, thickness, breaking strength (Bs), and elongation at break (Eb), another parameter, Young’s modulus (elasticity modulus,
Influence of MCCh and Fb content on Young’s modulus (
From the results shown in Figure
The porous surface of a membrane is crucial for the diffusion of growth factors and free oxygen penetration; these processes are essential in enhancing regeneration. Pictures of homogeneous MCCh (M1), Fb (M2), and composite MCCh-Fb (M4) membranes obtained by the SEM (Quanta 200 SEM) are shown in Figure
SEM images of MCCh : Fb = 1 : 1 composite membrane (M4). (a) Dry membrane surface – ×5000, (b) wet membrane surface—×5000 and (c) dry cross-section—×2000, (d) wet cross-section—×2000.
The FTIR spectra of homogeneous MCCh (M1), Fb (M2) and of related composite MCCh-Fb (M3, M4, and M5) membranes without the presence of Am are shown in Figure
FTIR spectra obtained in thin polymer films of homogeneous: MCCh, Fb, and composite. MCCh : Fb = 1 : 1 membranes with (M1′, M4′) and without (M1, M2, and M4) Am. Comparative spectrum for Am was taken in KBr pellets.
The spectra of MCCh from thin films (Figure
The FTIR spectrum of MCCh-Fb composite (Figure
Analysis of the FTIR spectroscopy for the MCCh-Fb composite (Figure
The release profile of PDGF-BB was determined for ten kinds of carriers (with M1′, M2′, M3′, M4′, and M5′ and without M1, M2, M3, M4, and M5 amoxicillin), which contain MCCh and Fb in various proportions (Table
Release profile of PDGF-BB from homogeneous membranes, with amoxicillin M1′ MCCh, M2′ Fb and without amoxicillin M1 MCCh, M2 Fb.
Release profile of PDGF-BB from composite membranes: with amoxicillin M3′ MCCh : Fb = 2 : 1, M4′ MCCh : Fb = 1 : 1, and M5′ MCCh : Fb = 1 : 2, and without M3 MCCh : Fb = 2 : 1, M4 MCCh : Fb = 1 : 1, and M5 MCCh : Fb = 1 : 2.
Figure
Figure
Values of correlation coefficient
Profiles of PDGF-BB release (Figures
In the first phase, this process is function of change in drug concentration in surface layer, of which the total release of particles is more easily accessible. The second phase corresponds to the effective delayed release of drug substance from the deeper layers of the polymer membranes. It can be assumed that in this phase there is diffusion of drug substances from the deeper layers of the membrane.
Interpretation of kinetics data for PDGF-BB release as zero order and first order as well as the assumed dependence of concentration changes with the square root of time did not result in a straight line relation. In the case of first order, the kinetics was expressed as a log function of the remaining factor concentration in relation to time, and curve lines corresponding to the initial phase of factor release were determined in all systems. Analysis of the diagrams depicted reveals that two different release phases may be found. The obtained data indicates (Figures
The values of
Rate constant values for the release process during the first phase
Constant values of kinetic equation describing
Type of membranes | Phase I | Phase II |
| ||||
---|---|---|---|---|---|---|---|
|
|
|
|
|
|
||
M1 | 2.43 ± 0.11 | 0.476 ± 0.086 | 1.46 | 4.45 ± 0.10 | 13.0 ± 0.76 | 53.3 | 0.9998 |
M1′ | 2.01 ± 0.07 | 0.416 ± 0.047 | 1.67 | 4.57 ± 0.06 | 13.9 ± 4.82 | 49.9 | 0.9999 |
M2 | 5.20 ± 0.11 | 0.792 ± 0.217 | 0.88 | 2.26 ± 0.11 | 10.4 ± 1.5 | 66.6 | 0.9998 |
M2′ | 4.56 ± 0.08 | 0.743 ± 0.128 | 0.93 | 2.30 ± 0.19 | 5.86 ± 1.29 | 118 | 0.9999 |
M3 | 0.244 ± 0.020 | 0.212 ± 0.045 | 3.27 | 1.20 ± 0.068 | 3.24 ± 0.043 | 214 | 0.9994 |
M3′ | 0.572 ± 0.020 | 0.0240 ± 0.0076 | 28.9 | 0.146 ± 0.014 | 3.00 ± 0.15 | 231 | 0.9973 |
M4 | 0.317 ± 0.035 | 0.174 ± 0.044 | 3.98 | 1.24 ± 0.038 | 5.50 ± 0.060 | 126 | 0.9992 |
M4′ | 0.352 ± 0.015 | 0.0378 ± 0.0014 | 18.3 | 0.518 ± 0.013 | 7.57 ± 0.29 | 91.5 | 0.9991 |
M5 | 0.132 ± 0.009 | 0.250 ± 0.045 | 2.77 | 0.650 ± 0.014 | 4.79 ± 0.321 | 145 | 0.9997 |
M5′ | 0.309 ± 0.022 | 0.259 ± 0.041 | 2.66 | 0.468 ± 0.020 | 11.2 ± 1.10 | 61.9 | 0.9993 |
In the first phase, the half-life of PDGF-BB release is 3.27 h, 3.98 h, and 2.77 h for composite membranes (M3, M4, and M5). For homogeneous (M1, M2) membranes
The analysis of the data from Figures
For each pair of membranes (M1 and M1′, M2 and M2′, M3 and M3′, M4 and M4′, and M5 and M5′) Student’s
Student’s
Membrane | Student’s | |
---|---|---|
Calculated | Theoretical | |
M1 | 0.271 | 2.178 |
M1′ | ||
M2 | −0.561 | |
M2′ | ||
M3 | 0.724 | |
M3′ | ||
M4 | 1.079 | |
M4′ | ||
M5 | −1.055 | |
M5′ |
Comparing the profiles of PDGF-BB release on the basis of the parameter determined (
The implantation of a membrane into a body carries the risk of inflammation or immunogenicity. Many side effects can be avoided by local antibiotic release from membranes what results in the systemic administration of antibiotics in large amounts [
Release profile of amoxicillin from membrane systems: M1′ MCCh, M2′ Fb, and M3′ MCCh : Fb = 2 : 1, M4′ MCCh : Fb = 1 : 1, and M5′ MCCh : Fb = 1 : 2.
The kinetics of amoxicillin release from selected membranes was evaluated on the basis of the amount of drug substance released to phosphate buffer at pH 7.4 in the time of the experiment (Figure
The data obtained, shown in Figure
Constant values of kinetic equation describing
Type of membranes | Phase I | Phase II |
| ||||
---|---|---|---|---|---|---|---|
|
|
|
|
|
|
||
M1′ | — | — | — | 80.54 ± 7.25 | 85.0 ± 12.3 | 8.15 | 0.9924 |
M2′ | 51.97 ± 4.23 | 0.136 ± 0.021 | 5.09 | 47.99 ± 3.34 | 1.57 ± 0.027 | 441.4 | 0.9932 |
M3′ | — | — | — | 88.78 ± 3.31 | 11.44 ± 1.33 | 60.58 | 0.9911 |
M4′ | — | — | — | 89.76 ± 4.52 | 10.63 ± 1.63 | 65.19 | 0.9854 |
M5′ | — | — | — | 83.93 ± 2.73 | 9.27 ± 0.89 | 74.76 | 0.9944 |
In the first phase the half-life of amoxicillin release is 5.09 h for homogeneous Fb (M2′) membrane, while in the second phase
Different physicochemical properties of membranes have an effect on various release profiles of amoxicillin, especially the swelling ability and stability, as it was previously shown (Figure
Insertion of growth factors in the membranes might significantly support and modify tissue regeneration. The use of growth factors such as PDGF-BB with biocompatible matrices to promote tissue regeneration represents a promising approach in the disciplines of oral and maxillofacial surgery [
The varied release of growth factors (b-FGF, TGF-
The results of the PDGF-BB and amoxicillin release from homogeneous MCCh and Fb and composite MCCh-Fb membranes indicated a correlation between the level of release and composition of the membrane. The platelet growth factor was released in the highest amount from the Fb membrane (M2′) with the presence of amoxicillin. An optimal release of amoxicillin was observed in the case of the MCCh-Fb membrane (M3′ and M4′).
The process of the PDGF-BB growth factor release from the membranes studied was of a two-phase nature. The first phase was characterised by rapid release, while in the second phase it was much slower, which is positive from the point of view of the drug application assignment (prolonged therapeutic effect). The faster effect indicates rapid water uptake of polymer membrane and dissolution of the exposed PDGF-BB particles at the surface of the membrane. The second process is slower and it is caused by the swelling of the inner matrix layer and the acceptor fluid absorption, together with the active substance.
The microcrystalline chitosan (M1) and fibrin membranes (M2) have promising properties because of the fast release of PDGF-BB as a significant angiogenic growth factor for tissue regeneration. Moreover, the slow and gradual release of antibiotic (amoxicillin) from Fb membrane may have protective role during the regeneration process. Our results suggested that MCCh and Fb membranes may be beneficial to enhance periodontal bone regeneration.
The obtained results of the PDGF-BB and Am release from the membranes indicated a correlation between the level of the release and their composition. The platelet growth factor-BB was released in the highest amount from the MCCh and Fb membranes (M1 and M2). The profiles of PDGF-BB growth factor release from the homogeneous MCCh (M1) and Fb (M2) membranes are suitable for oral surgery. Connection of MCCh with Fb decreases the release of PDGF-BB and increases the release of Am. The results reveal that MCCh-Fb films demonstrate higher efficiency in binding PDGF-BB and, at the same time, are less effective in their release. The PDGF-BB release was lower with increasing of Fb concentrations in the membrane. The results indicate that factor PDGF-BB is released
Chitosan
Microcrystalline chitosan
Fibrin
Amoxicillin
Platelet-derived growth factor-BB
Fourier transform infrared
Membrane
Hydroxyapatite
Tricalcium phosphate
Water retention value
Weight-average molecular weight
Degree of deacetylation
Fb-stabilizing factor
Phosphate buffered saline
Swelling index
Equilibrium swelling
Young’s modulus
Breaking strength
Elongation at break
Scanning electron microscope.
The authors declare that there is no conflict of interests regarding the publication of this paper.
This work was supported by Grant nos. 503/3-021-01/503-01, 503/3-015-02/503-01, and 503/5-061-02/503-01 from the Medical University of Lodz, Poland.