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Mechanical Properties of Collagen Biomimetic Films Formed in the Presence of Calcium, Silica and Chitosan
Journal of Bionic Engineering 5 (2008) 149í158
Mechanical Properties of Collagen Biomimetic Films Formed in the Presence of Calcium, Silica and Chitosan Mihai Chirita Medical Bioengineering Faculty, Medicine and Pharmacy University “Gr.T.Popa”, Iasi 7000115, Romania
Abstract Using eucollagen solutions from ox hide, we cast collagen films to assess the influence of calcium and silica on the reconstitution of the fibrous structure of collagen. The tensile strength and the breaking elongation of the reconstituted collagen films were measured and analysed. Significant differences were observed between reconstituted collagen films with and without calcium and silica. The breaking elongation of the films obtained in the presence of silica was significantly greater, and the degradation was lower than other films of reconstituted collagen. Collagen and chitosan do not exist together as blends in nature, but the specific properties of each may be used to produce in biomimetic way man-made blends with biomedical applications, that confer unique structural, mechanical (detail) and in vivo properties. Keywords: modified collagen biomimetic films, chitosan, biomaterials, biopolymers, mechanical properties Copyright © 2008, Jilin University. Published by Elsevier Limited and Science Press. All rights reserved.
1 Introduction The advantages of using collagen products[1] in medicine are their very low antigenicity, excellent biocompatibility, ease of association with other biologically active species such as glycose- aminoglycans, and their polyelectrolytic behavior. The reconstitution of collagen[2] from solution into native fibers is of interest because of applications in products such as threads for sutures, sponges for oral surgery, films for burn treatment, regenerative and rehydrating layers, semipermeable membranes and artificial skin, and controlled drug delivery systems. Calcium is an essential metabolic component. As an important coagulation factor, it forms the solid part of the vertebrate skeleton together with phosphorus. In its circulating state it supports normal nervous and muscular activities. As a cofactor it plays an essential role in many enzymic reactions. More than 95% of bone minerals are provided by calcium, orthophosphates and carbonates. Calcium represents 27.5% of degreased bone dried at 100 ˚C. Silicon is one of the fundamental elements of living or nonliving materials. It can be found in all organisms Corresponding author: Mihai Chirita E-mail: [email protected]
and is an indispensable element. The majority of organisms with high content of silicon come from species of lower animals and plants, most of which live in the sea. When living matter appeared, siliceous compounds probably played an essential role[3]. A common theory is that silica and the silicates have the capacity for surface adsorbing organic molecule complexes, and aligning them on a surface, at a sufficient concentration for them to interact. The asymmetry of organic substances, characteristics of living organisms, would then be an outcome of their synthesis on the asymmetric surface of quartz crystals. Silicates adsorb organic compounds such as glycols, amines, sugars and nucleic acids. Ovalbumins and other proteins are adsorbed readily on silica gel and quartz. The proteins are very easily adsorbed also by clay minerals. These adsorption phenomena on silica minerals are due to electrostatic forces, Van der Waals forces and hydrogen bonds. Silicon and its compounds are indispensable for the normal functions of epithelial and connective tissues, giving them strength and elasticity. Silica encourages the synthesis of collagen and the formation of bone tissue. When a bone fractures, there is not only the formation of
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collagen fibrils and intense cellular growth, but also a 50-fold increase in concentration of silicon. Silicon compounds are important in growth of keratins and bone, such as human hair and nails, animal fur, antlers and hoofs, and feathers. It appears that silicon provides crosslinks between the macromolecules of keratin. The addition of SiO2 to food allows children to develop normally, with proper growth and increase in weight. But we still have insufficient data to say whether silica influences general development. Silicon is active in the human body in illness. Many pathological problems, such as cancer, arteriosclerosis, tuberculosis, diabetes, goitre, some dermatitis, calculus on the renal system, are somehow related to upset in silicon metabolism. For example, tumors can contain considerable quantity of silicon compared with healthy tissues. In regions of the world rich in silicon, cancer is rare, while in regions richer in calcium cancer is more common. Perhaps the protective role of silicon in cancer is because this element is the most significant mineralisation and stimulatory factor of the connective tissue, which is an important protection of the body against cancer. The metabolic pathways of silicon and calcium compounds are related. Aging is accompanied by changes in the balance between these elements: the reduction of silicon concentration and the increase in calcium in the connective tissue. This makes these tissues weaker, because their elasticity is due to the silica. SiO2 appears in decalcified tissues, especially in osteoporosis and in some cases of atherosclerosis. In connective tissue there are several forms of collagen. Tropocollagen appears during the formation of the quarternary structure of the collagen fiber. It can be dissolved in dilute acid, that is why it is often called acid soluble collagen. To a lesser extent it can also be extracted with neutral salt solution. The remaining insoluble collagen can be dissolved with suitable chemical and enzyme treatment. The technique is to destroy intermolecular bridges, at the same time trying up to retain the molecular structure of the collagen triple helix. To distinguish the product from tropocollagen, even if its physical-chemical behaviour is close to that of tropocollagen, it has been called eucollagen. Researches resulted in the use of biosynthetic ma-
terials and tissue engineered living skin replacement. The advantages of collagen products for medical purposes are determined by its very low antigenicity, excellent biocompatibility, absence of immunoreactions, the ease of associations with other biological active species like glyco-amino-glycans and the polyelectrolyte behavior. The reconstitution of collagen from collagen solutions into native fibers is of considerable biomedical interest since the the final products are as threads (suture), sponges (oral surgery), films (pellicle for burn scorch treatment, regenerative and rehydrating layers, semi permeable membranes, bio-artificial skin) and controlled drug delivery systems. Chitosan is a linear polysaccharide, composed of glucosamine and N-acetyl glucosamine units linked by ȕ (1–4) glycosidic bonds. Chitosans represent a family of biocompatible natural biopolymers from renewable resources, obtained from shell of shellfish and the wastes of the seafood industry. Chitosan, fully absent in mammals, corresponds to an interesting series of natural glycosaminoglycans possessing the rare property of bioactivity. Chitosan activates cellular reconstruction, regenerating the skin surface by collagen fibres growth, stops haemorrhaging and provides an anesthetic property[4]. Chitosans have been combined with calcium phosphates for use as bone graft substitutes. It has novel properties such as biocompatibility, biodegradability, antibacterial, and wound-healing activity[5]. Bio-inspired bi-layered hydrogels made from chitosan and water were processed and applied to the treatment of full-thickness burn injuries. Three types of skin substitutes can be considered: those consisting only in epidermal equivalents, those encompassing dermal components from processed skin and those possessing distinct dermal and epidermal components, referred to as composite skins. Chitosan provides many advantages for the treatment of burns, wounds and other injuries of the skin. Chitosan activates the skin healing without scar, stimulates conjunctiva tissue and epidermal collagen growth. The major goal is to achieve a permanent skin regeneration with both dermal and epidermal tissues with good functional and aesthetic characteristics.
Chirita: Mechanical Properties of Collagen Biomimetic Films Formed in the Presence of Calcium, Silica and Chitosan
Chitosan in the form of a chitosan-cotton blend was found to be an accelerator of wound healing by the activation and infiltration of polymorphonuclear cells at the wound site[6]. Chitosan has been shown to degrade in vivo, which is mainly by enzymatic hydrolysis. The degradability of a scaffold plays a crucial role for the long-term performance of tissue-engineered cell/material constructs because it effects many cellular process, including cell growth, tissue regeneration, and the host response. Many studies have been reported on the use of chitosan as a skin substitute material in skin tissue engineering due to its many advantages for wound healing such as hemostasis, acceleration of the tissue regeneration and stimulation of synthesis of collagen by fibroblast. Collagen and chitosan are amongst the most abundant natural polymers in life. Both have intrinsic properties that provide a strong but manipulable scaffolding structure in many multi-cellular organisms. Collagen and chitosan do not exist together as blends in nature, but the specific properties of each may be used to produce in biomimetic way man-made blends that confer unique structural and mechanical properties.
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2
K = ao + a1c + a2c + ... (Fig. 1). Using a rheoviscometer, we determined the apparent dynamic viscosity ( K W / J ) of the eucollagen solution over a range of concentrations at a pH of 3.5 and at a constant shear rate ( J = 145.8 sí1) at temperatures of 4 ˚C and 20 ˚C. (Fig. 2).
Fig. 1 The dependence of firmness concentration coeficient K.
2 Material and method 2.1 First experiment series We prepared eucollagen by dissolution of the collagen from ox skin dermis. 1 kg of skin was cut into 5 cm × 5 cm pieces and treated with 3 L of 10% NaOH, 3% NaCl and 3% Na2SO4 solution for 48 hours at 20 ˚C. Then it was washed in distilled water, neutralized with 10% HCl and washed again. The collagen was then dissolved in 10 L of 1% acetic acid for 24 hours to 48 hours and filtered. The eucollagen solution is rheologically nonnewtonian, described by the model of Ostwald de Waele
W
KJ n ,
where W is cutting tension; J is shear rate; n is a function of the parameters that influence the viscosity of collagen solutions (concentration, pH, time of maturation, temperature, the nature of the material, solvent, etc.); K = firmness coefficient, which depends on concentration, by a polynomial function of the form:
Fig. 2 The apparent dynamic viscosity variation with concentration at different temperatures.
The time of maturation was 16 hours at 4 ˚C. As the temperature is increased the slope decreases, showing that the viscosity is an inverse function of temperature. This collagen solution contained 3%í4% dry weight. It is heterogeneous, containing collagen molecules, other molecular fragments and amino acids, showing that the alkaline treatment produces an energetic hydrolysis of the collagen. From this we prepared collagen solutions with 0.6% and 0.8% dry matter respectively, which was poured onto flat glass plates lined with polyethylene to obtain a film through forced evaporation of the solvent in air at 25 ˚C. These films were brittle, so it was necessary to add a plasticizer, glycerine proving to be most adequate. For establishing the optimum quantity of glycerine we added different
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quantities, related to the dry weight of collagen, and determined the tensile strength and the breaking elongation of the films. Mechanical tests were performed on small pieces of 10 mm × 30 mm from each sample in an Instron mechanical testing machine. All samples fractured around the central area. Figs. 3 and 4 show the traction strength and breaking elongation of collagen, respectively.
Fig. 5 Traction strength of the 0.6% collagen films at different calcium acetate quantities and 120% glycerine.
Fig. 3 Traction strength of the collagen films at different glycerine quantities.
Fig. 6 Breaking elongation of the 0.6% collagen films at different calcium acetate quantities and 120% glycerine.
Fig. 4 Breaking elongation of the collagen films at different glycerine quantities.
The collagen concentrations of 0.6% and 0.8% were chosen so that the films would not be porous. The films when cast contained 1 g of collagen, were irrespective of the concentration used. The films were conditioned for 24 hours in an atmosphere of 66% RH at 20 ˚C. Figs. 5 and 6 show the traction strength and breaking elongation of the collagen film, respectively with calcium acetate content, obtained from collagen solutions of c = 0.6% and in which glycerine is added that is indicated by the ratio of dry substances of glycerine to collagen in percentage (120%).
Calcium was added to the solutions as 1% calcium acetate, since acetate was already present in the reaction medium used for the extraction. The films were cast from 0.6% collagen. The solubility of the collagen films, with different quantities of glycerine and with added calcium, was studied in physiological saline (0.9% NaCl in double-distilled water, brought to pH 7 with drops of 3% NaOH solution), in acid solution (0.5 M acetic acid at pH 3.5) and in alkaline solution of NaOH at pH 11. The films were maintained at 50% RH and 25 ˚C for 30 days. After these treatments, the losses of dry matter were determined. Figs. 7 and 8 show the dry substances losses from collagen films at different glycerine quantities in physiological serum and solution of 0.5 M acetic acid. Fig. 9 shows the dry substances losses from collagen films realized from 0.6% collagen solution, with 120%
Chirita: Mechanical Properties of Collagen Biomimetic Films Formed in the Presence of Calcium, Silica and Chitosan
glycerine and different quantities of Ca(CH3COO)2, in physiological serum medium, alkaline solution of NaOH at pH = 11, after 30 days at 25 ˚C. 0.6% collagen solutions were prepared, containing 0.1 or 0.2 ml sodium silicate and 1, 5, 10, 20 ml of 1% sodium acetate solution and a film containing 0.3 ml of 1% sodium acetate solution. Four films were cast from each solution. All quantities are quoted relative to the dry weight of collagen.
Fig. 7 The dry substances losses from collagen films in physiological serum.
Fig. 8 The dry substances losses from collagen films in solution of 0.5 M acetic acid.
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air stream. After treatment, the breaking resistance and elongation of the films were determined. Figs. 10 and 11 show traction strength and breaking elongation vary with the quantities of Na2SiO3 and Ca(CH3COO)2. Fig. 12 describes the dry substances losses from collagen films in physiological serum. Fig. 13 shows the dry substances losses from collagen films in solution of NaOH at pH = 11.
Fig. 10 Traction strength of the 0.6% collagen films added calcium acetate, sodium silicate and 120% glycerine.
Fig. 11 Breaking elongation of the 0.6% collagen films added calcium acetate, sodium silicate and 120% glycerine.
Fig. 9 The dry substances losses from collagen films.
Some films were made with extra calcium acetate and sodium silicate. The films were dried at 25 ˚C in an
Fig. 12 The dry substances losses from collagen films in physiological serum.
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Table 2 Traction strength and elongation of the collagen films realised with different quantities of glycerine as plasticizer and 0.25% (w/w) chitosan Ratio of glycerine to collagen (%)
60
120
180
240
300
360
V (10 MPa)
c = 0.6 % c = 0.8 %
0.79 1.25
0.93 1.42
0.67 1.39
0.64 1.30
0.38 0.85
0.30 0.65
H (%)
c = 0.6 % c = 0.8 %
8.8 12.5
9.8 20.0
10.5 19.7
11.7 21.4
12.0 21.9
12.4 22.2
Table 3 Traction strength and elongation of the collagen films realised with different quantities of glycerine as plasticizer and 0.5% (w/w) chitosan Ratio of glycerine to collagen (%)
V (10 MPa)
Fig. 13 The dry substances losses from collagen films in solution of NaOH at pH = 11.
2.2 Second experiment series We used the same sort of collagen solution with 0.6% or 0.8% dry substance, with different quantities of glycerine, calcium acetate and sodium silicate, mixed with chitosan 0.1%, 0.25%, 0.5% (w/w) solution, which was poured onto flat glass plates lined with polyethylene to obtain a film, followed by forced evaporation of the solvent in air at 25 ˚C. Chitosan solution was obtained by mixing distilled water with a pharmaceutical chitin-chitosan product from the Central Institute of Fisheries Technology, Kochi, India, (chitin-15%, chitosan -85%), at 25 ˚C, filtered three times with quantitative filtering paper and then diluted again to generate solutions with the following concentrations: 0.1%, 0.25% and 0.5% chitosan solution obtained in the final mixture to be dried. Mechanical tests were performed on an Instron mechanical testing machine with small pieces of samples in the size of 10 mm × 30 mm. All samples fracture around the central area. Tables 1 to 3 show the values of traction strength and elongation of collagen films realised with different Table 1 Traction strength and elongation of the collagen films realised with different quantities of glycerine as plasticizer and 0.1% (w/w) chitosan Ratio of glycerine to collagen (%) c = 0.6 % V (10 MPa) c = 0.8 %
60
120
180
240
300
360
0.78 1.24
0.85 1.37
0.65 1.16
0.61 0.98
0.31 0.63
0.28 0.38
c = 0.6 % c = 0.8 %
8.6 12.0
8.9 19.4
9.9 19.6
10.3 22.5
11.1 23.9
12.4 25.7
H (%)
H (%)
c = 0.6 % c = 0.8 % c = 0.6 % c = 0.8 %
60
120
180
240
300
360
0.79 1.25 8.8 12.5
0.91 1.40 9.6 19.8
0.62 1.27 9.9 19.6
0.55 1.22 10.6 21.2
0.41 0.70 12.3 22.6
0.33 0.57 12.7 23.1
quantities of glycerine as plasticizer. The biggest values for films traction was obseved for 120% glycerine content and especialy in the case of solutions with 0.25% chitosan (Fig. 14).
Fig. 14 Traction strength of the 0.6% and 0.8% collagen films at different glycerine quantities and 0.25% (w/w) chitosan.
3 Results and discussion The tensile stiffness of films which contain calcium (Fig. 6) is greater than that of films without calcium (Fig. 4), which shows that the collagen is more stable in the presence of calcium. Also, as the calcium content increases the strength decreases. The breaking strain is greater at lower calcium concentration, and as the amount of calcium increases the elongation reduces. This correlates to that the fragility increases with calcium .
Chirita: Mechanical Properties of Collagen Biomimetic Films Formed in the Presence of Calcium, Silica and Chitosan
The results of Fig. 5 show that the traction resistances of films which contain calcium are superior to that without calcium presented Fig. 3, which proves a stronger reconstruction of the collagen takes place in the presence of calcium. It can be observed, also, that the resistances decreases with the increase in the quantity of calcium. It was mentioned that the experiments included a large number of tests, which are not presented here. These tests were performed with the increase in the quantity of calcium added to the collagen solutions for the films, until the combination limit with the reactive groups of the collagen, but were obtained friable films. The breaking elongations are bigger at lower calcium quantities, and after, if the amount of calcium increases, the elongation will reduce visibly. This behaviour correlates with the tendency to be friable as the amount of added calcium increases. Loss of dry weight from the films which contain glycerine increases as the glycerine content increase except in alkaline medium. In physiological saline, 50% to 80% of the films are dissolved, but in the alkaline medium the film is dissolved completely (Figs. 7 and 8). The films that contain calcium lose less dry weight. In the physiological saline, as calcium acetate increases, less substance is dissolved. These films resist the alkaline solution longer as the calcium content increases (Fig. 9).
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The strength of films that contain silica is greater than those films that contain equivalent amounts of calcium (Fig. 10). In all cases, as the content of calcium increases the strength decreases due to increased fragility. As the silica content increases there is a slight decrease in strength, but there appears to be a more complex interaction as the result of the solubility differences between calcium and silica, so that the silica has a lower solubility due to the presence of calcium. The breaking elongation of the films with silica is higher than films that contain only calcium (Fig. 11). The loss of dry weight from the films that contain silica and calcium combined is slightly less than from those that contain only calcium (Fig. 12). Regarding the traction strength and elongation realised with different quantities of glycerine as plasticizer and 0.1%í0.5% (w/w) chitosan from Tables 1 to 3 we can observe that the maximum strength was obtained at 120% plasticizer and both 0.6% and 0.8% collagen films at 0.25% chitosan content. We used the following concentrations of glycerine and chitosan, 120% and 0.25%, respectively, to execute all other experiments. Table 4 and Fig. 15 show a better traction strength with 0.25% chitosan mixed films compared to data in Fig. 5.
Table 4 Traction strength and breaking elongation with amount of calcium acetate, obtained from collagen solutions with c = 0.6%, 120% glycerine and 0.25% chitosan 1% Ca(CH3COO)2 solution (ml)
V (10 MPa) H (%)
1 1.21 11.5
2 1.28 10.6
Fig. 15 Traction strength of the 0.6% collagen films at different calcium acetate quantities, 120% glycerine and 0.25% chitin.
3
4
5
6
7
8
1.11
0.91
0.85
0.77
0.74
0.63
9.3
7.6
6.4
6.3
6.0
10.1
The traction resistances and breaking elongations of collagen films, with added calcium acetate and sodium silicate, obtained from collagen solutions with concentration of 0.6 %, with 120 % glycerine substance and 0.25% chitosan demonstrate the superior values compared to the data in Figs. 10 and 11, especially for the films with 0.1 and 0.2 ml of Na2SiO3 (shown in Figs. 16 and 17). A one-way ANOVA statistic was used to analyse data. Typically, the algorithm is used for comparing the means of two or more columns of data in an m-by-n
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matrix (X), where each column represents an independent sample (m) containing mutually independent observations. The function returns the p-value for the null hypothesis that all samples in X are drawn from the same population (or from different populations with the same mean).
If the p-value is near zero, this casts doubt on the null hypothesis and suggests that the mean of at least one sample is significantly different from that of other samples. Figs. 18 and 19 show the distributions of traction resistance and breaking elongation, respectively.
Fig. 18 Boxplot depicting the distributions of traction resistance values for Fig. 10 (groups 1–3) and for Fig. 16 (groups 4–6).
Fig. 16 Traction resistance of 6% collagen films with 120% glycerine and 0.25% chitosan at different calcium acetate quantities.
Fig. 19 Boxplot depicting the distributions of breaking elongation percentage values for Fig. 10 (groups 1–3) and for Fig. 16 (groups 4–6).
Fig. 17 Breaking elongation of 0.6% collagen films with 120 % glycerine and 0.25% chitosan at different calcium acetate quantities.
In ANOVA test, a very small p value, p<<0.005 for example, suggests that small differences are highly significant between the analyzed measurement groups. Therefore, it can be concluded that the mechanical property of traction resistance formed in the presence of chitosan of the biomimetic films outperform the one formed in the presence of calcium-silica. In contrast, a rather high p-value, p<0.5 for example, suggests that no significant differences are present in the data. Thus, we can conclude that the mechanical property of breaking elongation formed in the presence of chitosan of the biomimetic films is similar to the one formed in the presence of calcium-silica.
Chirita: Mechanical Properties of Collagen Biomimetic Films Formed in the Presence of Calcium, Silica and Chitosan
Finally we examined the films with optical microscopy, (Fig. 20). Those films that contain a small
(a) 0.1 ml Na2SiO3 and 1 ml calcium acetate
157
amount of calcium and a large amount of silica are more uniform (Figs. 20g, h, i).
(b) 0.1 ml Na2SiO3 and 5 ml calcium acetate
(d) 0.1 ml Na2SiO3 and 20 ml calcium acetate
(e) 0.2 ml Na2SiO3 and 1 ml calcium acetate
(g) 0.2 ml Na2SiO3 and 10 ml calcium acetate
(h) 0.2 ml Na2SiO3 and 20 ml calcium acetate
(c) 0.1 ml Na2SiO3 and 10 ml calcium acetate
(f) 0.2 ml Na2SiO3 and 5 ml calcium acetate
(i) 0.3 ml Na2SiO3 and 3 ml calcium acetate
Fig. 20 Optical microscopy of the samples with Na2SiO3 and calcium acetate (×150).
4 Conclusions Silicon plays an important role in macromolecular architecture of collagen during its reconstitution. In order to make the collagen films less brittle a plasticizer, glycerine, has to be added. The strength of collagen films is increased by adding small amounts of calcium but is decreased as further calcium is added. Similarly the breaking elongation of the films increases with small amounts of calcium and then decreases as the film becomes more friable. Films with calcium lose less substance during their treatment with physiological saline, but also in alkaline medium, as the amount of calcium is increased. The tensile strength and breaking elongation of
films with added silica is greater than those with calcium. The dry weight loss, in physiological saline as in alkaline solution, of the silica and calcium films is slightly less than the ones that contain only calcium. Chitosan 0.25% (w/w) at dry collagen improved the traction resistance and the breaking elongation of 0.6% collagen films. It’s possible that both bio-polymers interact to form a more compact 3D structure, more “crystalline”, more ordered that the collagen can do on reconstruction stage[7í12]. Optical microscopy shows that those films that contain a lower amount of calcium and a higher content of silica are more uniform in appearance. Research must be continued to study different kinds of response of human cell growth on these films to determine their utility within an in vivo model.
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Chirita M, Oniscu C, Vlad M D, Veisa G. Contributions at the biomaterials base development. The collagen hydrolyse
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[10] Shanmugasundaram N, Ravichandran P, Neelakanta P R. Nalini R & Collagen–chitosan polymeric scaffolds for the in vitro culture of human epidermoid carcinoma cells. Biomaterials, 2001, 22, 1943–1951. [11] Taravel M N, Domard A. Collagen and its interaction with chitosan II. Influence of the physicochemical characteristics of collagen. Biomaterials, 1995, 16, 865–871.
[12] Salome Machado A A, Martins V C A, Plepis A M G. Thermal and rheological behaviour of collagen–chitosan blends. Journal of Thermal Analysis and Calorimetry, 2002, 67, 491–498.
Mechanical Properties of Collagen Biomimetic Films Formed in the Presence of Calcium, Silica and Chitosan Mihai Chirita Medical Bioengineering Faculty, Medicine and Pharmacy University “Gr.T.Popa”, Iasi 7000115, Romania
Abstract Using eucollagen solutions from ox hide, we cast collagen films to assess the influence of calcium and silica on the reconstitution of the fibrous structure of collagen. The tensile strength and the breaking elongation of the reconstituted collagen films were measured and analysed. Significant differences were observed between reconstituted collagen films with and without calcium and silica. The breaking elongation of the films obtained in the presence of silica was significantly greater, and the degradation was lower than other films of reconstituted collagen. Collagen and chitosan do not exist together as blends in nature, but the specific properties of each may be used to produce in biomimetic way man-made blends with biomedical applications, that confer unique structural, mechanical (detail) and in vivo properties. Keywords: modified collagen biomimetic films, chitosan, biomaterials, biopolymers, mechanical properties Copyright © 2008, Jilin University. Published by Elsevier Limited and Science Press. All rights reserved.
1 Introduction The advantages of using collagen products[1] in medicine are their very low antigenicity, excellent biocompatibility, ease of association with other biologically active species such as glycose- aminoglycans, and their polyelectrolytic behavior. The reconstitution of collagen[2] from solution into native fibers is of interest because of applications in products such as threads for sutures, sponges for oral surgery, films for burn treatment, regenerative and rehydrating layers, semipermeable membranes and artificial skin, and controlled drug delivery systems. Calcium is an essential metabolic component. As an important coagulation factor, it forms the solid part of the vertebrate skeleton together with phosphorus. In its circulating state it supports normal nervous and muscular activities. As a cofactor it plays an essential role in many enzymic reactions. More than 95% of bone minerals are provided by calcium, orthophosphates and carbonates. Calcium represents 27.5% of degreased bone dried at 100 ˚C. Silicon is one of the fundamental elements of living or nonliving materials. It can be found in all organisms Corresponding author: Mihai Chirita E-mail: [email protected]
and is an indispensable element. The majority of organisms with high content of silicon come from species of lower animals and plants, most of which live in the sea. When living matter appeared, siliceous compounds probably played an essential role[3]. A common theory is that silica and the silicates have the capacity for surface adsorbing organic molecule complexes, and aligning them on a surface, at a sufficient concentration for them to interact. The asymmetry of organic substances, characteristics of living organisms, would then be an outcome of their synthesis on the asymmetric surface of quartz crystals. Silicates adsorb organic compounds such as glycols, amines, sugars and nucleic acids. Ovalbumins and other proteins are adsorbed readily on silica gel and quartz. The proteins are very easily adsorbed also by clay minerals. These adsorption phenomena on silica minerals are due to electrostatic forces, Van der Waals forces and hydrogen bonds. Silicon and its compounds are indispensable for the normal functions of epithelial and connective tissues, giving them strength and elasticity. Silica encourages the synthesis of collagen and the formation of bone tissue. When a bone fractures, there is not only the formation of
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collagen fibrils and intense cellular growth, but also a 50-fold increase in concentration of silicon. Silicon compounds are important in growth of keratins and bone, such as human hair and nails, animal fur, antlers and hoofs, and feathers. It appears that silicon provides crosslinks between the macromolecules of keratin. The addition of SiO2 to food allows children to develop normally, with proper growth and increase in weight. But we still have insufficient data to say whether silica influences general development. Silicon is active in the human body in illness. Many pathological problems, such as cancer, arteriosclerosis, tuberculosis, diabetes, goitre, some dermatitis, calculus on the renal system, are somehow related to upset in silicon metabolism. For example, tumors can contain considerable quantity of silicon compared with healthy tissues. In regions of the world rich in silicon, cancer is rare, while in regions richer in calcium cancer is more common. Perhaps the protective role of silicon in cancer is because this element is the most significant mineralisation and stimulatory factor of the connective tissue, which is an important protection of the body against cancer. The metabolic pathways of silicon and calcium compounds are related. Aging is accompanied by changes in the balance between these elements: the reduction of silicon concentration and the increase in calcium in the connective tissue. This makes these tissues weaker, because their elasticity is due to the silica. SiO2 appears in decalcified tissues, especially in osteoporosis and in some cases of atherosclerosis. In connective tissue there are several forms of collagen. Tropocollagen appears during the formation of the quarternary structure of the collagen fiber. It can be dissolved in dilute acid, that is why it is often called acid soluble collagen. To a lesser extent it can also be extracted with neutral salt solution. The remaining insoluble collagen can be dissolved with suitable chemical and enzyme treatment. The technique is to destroy intermolecular bridges, at the same time trying up to retain the molecular structure of the collagen triple helix. To distinguish the product from tropocollagen, even if its physical-chemical behaviour is close to that of tropocollagen, it has been called eucollagen. Researches resulted in the use of biosynthetic ma-
terials and tissue engineered living skin replacement. The advantages of collagen products for medical purposes are determined by its very low antigenicity, excellent biocompatibility, absence of immunoreactions, the ease of associations with other biological active species like glyco-amino-glycans and the polyelectrolyte behavior. The reconstitution of collagen from collagen solutions into native fibers is of considerable biomedical interest since the the final products are as threads (suture), sponges (oral surgery), films (pellicle for burn scorch treatment, regenerative and rehydrating layers, semi permeable membranes, bio-artificial skin) and controlled drug delivery systems. Chitosan is a linear polysaccharide, composed of glucosamine and N-acetyl glucosamine units linked by ȕ (1–4) glycosidic bonds. Chitosans represent a family of biocompatible natural biopolymers from renewable resources, obtained from shell of shellfish and the wastes of the seafood industry. Chitosan, fully absent in mammals, corresponds to an interesting series of natural glycosaminoglycans possessing the rare property of bioactivity. Chitosan activates cellular reconstruction, regenerating the skin surface by collagen fibres growth, stops haemorrhaging and provides an anesthetic property[4]. Chitosans have been combined with calcium phosphates for use as bone graft substitutes. It has novel properties such as biocompatibility, biodegradability, antibacterial, and wound-healing activity[5]. Bio-inspired bi-layered hydrogels made from chitosan and water were processed and applied to the treatment of full-thickness burn injuries. Three types of skin substitutes can be considered: those consisting only in epidermal equivalents, those encompassing dermal components from processed skin and those possessing distinct dermal and epidermal components, referred to as composite skins. Chitosan provides many advantages for the treatment of burns, wounds and other injuries of the skin. Chitosan activates the skin healing without scar, stimulates conjunctiva tissue and epidermal collagen growth. The major goal is to achieve a permanent skin regeneration with both dermal and epidermal tissues with good functional and aesthetic characteristics.
Chirita: Mechanical Properties of Collagen Biomimetic Films Formed in the Presence of Calcium, Silica and Chitosan
Chitosan in the form of a chitosan-cotton blend was found to be an accelerator of wound healing by the activation and infiltration of polymorphonuclear cells at the wound site[6]. Chitosan has been shown to degrade in vivo, which is mainly by enzymatic hydrolysis. The degradability of a scaffold plays a crucial role for the long-term performance of tissue-engineered cell/material constructs because it effects many cellular process, including cell growth, tissue regeneration, and the host response. Many studies have been reported on the use of chitosan as a skin substitute material in skin tissue engineering due to its many advantages for wound healing such as hemostasis, acceleration of the tissue regeneration and stimulation of synthesis of collagen by fibroblast. Collagen and chitosan are amongst the most abundant natural polymers in life. Both have intrinsic properties that provide a strong but manipulable scaffolding structure in many multi-cellular organisms. Collagen and chitosan do not exist together as blends in nature, but the specific properties of each may be used to produce in biomimetic way man-made blends that confer unique structural and mechanical properties.
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2
K = ao + a1c + a2c + ... (Fig. 1). Using a rheoviscometer, we determined the apparent dynamic viscosity ( K W / J ) of the eucollagen solution over a range of concentrations at a pH of 3.5 and at a constant shear rate ( J = 145.8 sí1) at temperatures of 4 ˚C and 20 ˚C. (Fig. 2).
Fig. 1 The dependence of firmness concentration coeficient K.
2 Material and method 2.1 First experiment series We prepared eucollagen by dissolution of the collagen from ox skin dermis. 1 kg of skin was cut into 5 cm × 5 cm pieces and treated with 3 L of 10% NaOH, 3% NaCl and 3% Na2SO4 solution for 48 hours at 20 ˚C. Then it was washed in distilled water, neutralized with 10% HCl and washed again. The collagen was then dissolved in 10 L of 1% acetic acid for 24 hours to 48 hours and filtered. The eucollagen solution is rheologically nonnewtonian, described by the model of Ostwald de Waele
W
KJ n ,
where W is cutting tension; J is shear rate; n is a function of the parameters that influence the viscosity of collagen solutions (concentration, pH, time of maturation, temperature, the nature of the material, solvent, etc.); K = firmness coefficient, which depends on concentration, by a polynomial function of the form:
Fig. 2 The apparent dynamic viscosity variation with concentration at different temperatures.
The time of maturation was 16 hours at 4 ˚C. As the temperature is increased the slope decreases, showing that the viscosity is an inverse function of temperature. This collagen solution contained 3%í4% dry weight. It is heterogeneous, containing collagen molecules, other molecular fragments and amino acids, showing that the alkaline treatment produces an energetic hydrolysis of the collagen. From this we prepared collagen solutions with 0.6% and 0.8% dry matter respectively, which was poured onto flat glass plates lined with polyethylene to obtain a film through forced evaporation of the solvent in air at 25 ˚C. These films were brittle, so it was necessary to add a plasticizer, glycerine proving to be most adequate. For establishing the optimum quantity of glycerine we added different
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quantities, related to the dry weight of collagen, and determined the tensile strength and the breaking elongation of the films. Mechanical tests were performed on small pieces of 10 mm × 30 mm from each sample in an Instron mechanical testing machine. All samples fractured around the central area. Figs. 3 and 4 show the traction strength and breaking elongation of collagen, respectively.
Fig. 5 Traction strength of the 0.6% collagen films at different calcium acetate quantities and 120% glycerine.
Fig. 3 Traction strength of the collagen films at different glycerine quantities.
Fig. 6 Breaking elongation of the 0.6% collagen films at different calcium acetate quantities and 120% glycerine.
Fig. 4 Breaking elongation of the collagen films at different glycerine quantities.
The collagen concentrations of 0.6% and 0.8% were chosen so that the films would not be porous. The films when cast contained 1 g of collagen, were irrespective of the concentration used. The films were conditioned for 24 hours in an atmosphere of 66% RH at 20 ˚C. Figs. 5 and 6 show the traction strength and breaking elongation of the collagen film, respectively with calcium acetate content, obtained from collagen solutions of c = 0.6% and in which glycerine is added that is indicated by the ratio of dry substances of glycerine to collagen in percentage (120%).
Calcium was added to the solutions as 1% calcium acetate, since acetate was already present in the reaction medium used for the extraction. The films were cast from 0.6% collagen. The solubility of the collagen films, with different quantities of glycerine and with added calcium, was studied in physiological saline (0.9% NaCl in double-distilled water, brought to pH 7 with drops of 3% NaOH solution), in acid solution (0.5 M acetic acid at pH 3.5) and in alkaline solution of NaOH at pH 11. The films were maintained at 50% RH and 25 ˚C for 30 days. After these treatments, the losses of dry matter were determined. Figs. 7 and 8 show the dry substances losses from collagen films at different glycerine quantities in physiological serum and solution of 0.5 M acetic acid. Fig. 9 shows the dry substances losses from collagen films realized from 0.6% collagen solution, with 120%
Chirita: Mechanical Properties of Collagen Biomimetic Films Formed in the Presence of Calcium, Silica and Chitosan
glycerine and different quantities of Ca(CH3COO)2, in physiological serum medium, alkaline solution of NaOH at pH = 11, after 30 days at 25 ˚C. 0.6% collagen solutions were prepared, containing 0.1 or 0.2 ml sodium silicate and 1, 5, 10, 20 ml of 1% sodium acetate solution and a film containing 0.3 ml of 1% sodium acetate solution. Four films were cast from each solution. All quantities are quoted relative to the dry weight of collagen.
Fig. 7 The dry substances losses from collagen films in physiological serum.
Fig. 8 The dry substances losses from collagen films in solution of 0.5 M acetic acid.
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air stream. After treatment, the breaking resistance and elongation of the films were determined. Figs. 10 and 11 show traction strength and breaking elongation vary with the quantities of Na2SiO3 and Ca(CH3COO)2. Fig. 12 describes the dry substances losses from collagen films in physiological serum. Fig. 13 shows the dry substances losses from collagen films in solution of NaOH at pH = 11.
Fig. 10 Traction strength of the 0.6% collagen films added calcium acetate, sodium silicate and 120% glycerine.
Fig. 11 Breaking elongation of the 0.6% collagen films added calcium acetate, sodium silicate and 120% glycerine.
Fig. 9 The dry substances losses from collagen films.
Some films were made with extra calcium acetate and sodium silicate. The films were dried at 25 ˚C in an
Fig. 12 The dry substances losses from collagen films in physiological serum.
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Table 2 Traction strength and elongation of the collagen films realised with different quantities of glycerine as plasticizer and 0.25% (w/w) chitosan Ratio of glycerine to collagen (%)
60
120
180
240
300
360
V (10 MPa)
c = 0.6 % c = 0.8 %
0.79 1.25
0.93 1.42
0.67 1.39
0.64 1.30
0.38 0.85
0.30 0.65
H (%)
c = 0.6 % c = 0.8 %
8.8 12.5
9.8 20.0
10.5 19.7
11.7 21.4
12.0 21.9
12.4 22.2
Table 3 Traction strength and elongation of the collagen films realised with different quantities of glycerine as plasticizer and 0.5% (w/w) chitosan Ratio of glycerine to collagen (%)
V (10 MPa)
Fig. 13 The dry substances losses from collagen films in solution of NaOH at pH = 11.
2.2 Second experiment series We used the same sort of collagen solution with 0.6% or 0.8% dry substance, with different quantities of glycerine, calcium acetate and sodium silicate, mixed with chitosan 0.1%, 0.25%, 0.5% (w/w) solution, which was poured onto flat glass plates lined with polyethylene to obtain a film, followed by forced evaporation of the solvent in air at 25 ˚C. Chitosan solution was obtained by mixing distilled water with a pharmaceutical chitin-chitosan product from the Central Institute of Fisheries Technology, Kochi, India, (chitin-15%, chitosan -85%), at 25 ˚C, filtered three times with quantitative filtering paper and then diluted again to generate solutions with the following concentrations: 0.1%, 0.25% and 0.5% chitosan solution obtained in the final mixture to be dried. Mechanical tests were performed on an Instron mechanical testing machine with small pieces of samples in the size of 10 mm × 30 mm. All samples fracture around the central area. Tables 1 to 3 show the values of traction strength and elongation of collagen films realised with different Table 1 Traction strength and elongation of the collagen films realised with different quantities of glycerine as plasticizer and 0.1% (w/w) chitosan Ratio of glycerine to collagen (%) c = 0.6 % V (10 MPa) c = 0.8 %
60
120
180
240
300
360
0.78 1.24
0.85 1.37
0.65 1.16
0.61 0.98
0.31 0.63
0.28 0.38
c = 0.6 % c = 0.8 %
8.6 12.0
8.9 19.4
9.9 19.6
10.3 22.5
11.1 23.9
12.4 25.7
H (%)
H (%)
c = 0.6 % c = 0.8 % c = 0.6 % c = 0.8 %
60
120
180
240
300
360
0.79 1.25 8.8 12.5
0.91 1.40 9.6 19.8
0.62 1.27 9.9 19.6
0.55 1.22 10.6 21.2
0.41 0.70 12.3 22.6
0.33 0.57 12.7 23.1
quantities of glycerine as plasticizer. The biggest values for films traction was obseved for 120% glycerine content and especialy in the case of solutions with 0.25% chitosan (Fig. 14).
Fig. 14 Traction strength of the 0.6% and 0.8% collagen films at different glycerine quantities and 0.25% (w/w) chitosan.
3 Results and discussion The tensile stiffness of films which contain calcium (Fig. 6) is greater than that of films without calcium (Fig. 4), which shows that the collagen is more stable in the presence of calcium. Also, as the calcium content increases the strength decreases. The breaking strain is greater at lower calcium concentration, and as the amount of calcium increases the elongation reduces. This correlates to that the fragility increases with calcium .
Chirita: Mechanical Properties of Collagen Biomimetic Films Formed in the Presence of Calcium, Silica and Chitosan
The results of Fig. 5 show that the traction resistances of films which contain calcium are superior to that without calcium presented Fig. 3, which proves a stronger reconstruction of the collagen takes place in the presence of calcium. It can be observed, also, that the resistances decreases with the increase in the quantity of calcium. It was mentioned that the experiments included a large number of tests, which are not presented here. These tests were performed with the increase in the quantity of calcium added to the collagen solutions for the films, until the combination limit with the reactive groups of the collagen, but were obtained friable films. The breaking elongations are bigger at lower calcium quantities, and after, if the amount of calcium increases, the elongation will reduce visibly. This behaviour correlates with the tendency to be friable as the amount of added calcium increases. Loss of dry weight from the films which contain glycerine increases as the glycerine content increase except in alkaline medium. In physiological saline, 50% to 80% of the films are dissolved, but in the alkaline medium the film is dissolved completely (Figs. 7 and 8). The films that contain calcium lose less dry weight. In the physiological saline, as calcium acetate increases, less substance is dissolved. These films resist the alkaline solution longer as the calcium content increases (Fig. 9).
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The strength of films that contain silica is greater than those films that contain equivalent amounts of calcium (Fig. 10). In all cases, as the content of calcium increases the strength decreases due to increased fragility. As the silica content increases there is a slight decrease in strength, but there appears to be a more complex interaction as the result of the solubility differences between calcium and silica, so that the silica has a lower solubility due to the presence of calcium. The breaking elongation of the films with silica is higher than films that contain only calcium (Fig. 11). The loss of dry weight from the films that contain silica and calcium combined is slightly less than from those that contain only calcium (Fig. 12). Regarding the traction strength and elongation realised with different quantities of glycerine as plasticizer and 0.1%í0.5% (w/w) chitosan from Tables 1 to 3 we can observe that the maximum strength was obtained at 120% plasticizer and both 0.6% and 0.8% collagen films at 0.25% chitosan content. We used the following concentrations of glycerine and chitosan, 120% and 0.25%, respectively, to execute all other experiments. Table 4 and Fig. 15 show a better traction strength with 0.25% chitosan mixed films compared to data in Fig. 5.
Table 4 Traction strength and breaking elongation with amount of calcium acetate, obtained from collagen solutions with c = 0.6%, 120% glycerine and 0.25% chitosan 1% Ca(CH3COO)2 solution (ml)
V (10 MPa) H (%)
1 1.21 11.5
2 1.28 10.6
Fig. 15 Traction strength of the 0.6% collagen films at different calcium acetate quantities, 120% glycerine and 0.25% chitin.
3
4
5
6
7
8
1.11
0.91
0.85
0.77
0.74
0.63
9.3
7.6
6.4
6.3
6.0
10.1
The traction resistances and breaking elongations of collagen films, with added calcium acetate and sodium silicate, obtained from collagen solutions with concentration of 0.6 %, with 120 % glycerine substance and 0.25% chitosan demonstrate the superior values compared to the data in Figs. 10 and 11, especially for the films with 0.1 and 0.2 ml of Na2SiO3 (shown in Figs. 16 and 17). A one-way ANOVA statistic was used to analyse data. Typically, the algorithm is used for comparing the means of two or more columns of data in an m-by-n
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matrix (X), where each column represents an independent sample (m) containing mutually independent observations. The function returns the p-value for the null hypothesis that all samples in X are drawn from the same population (or from different populations with the same mean).
If the p-value is near zero, this casts doubt on the null hypothesis and suggests that the mean of at least one sample is significantly different from that of other samples. Figs. 18 and 19 show the distributions of traction resistance and breaking elongation, respectively.
Fig. 18 Boxplot depicting the distributions of traction resistance values for Fig. 10 (groups 1–3) and for Fig. 16 (groups 4–6).
Fig. 16 Traction resistance of 6% collagen films with 120% glycerine and 0.25% chitosan at different calcium acetate quantities.
Fig. 19 Boxplot depicting the distributions of breaking elongation percentage values for Fig. 10 (groups 1–3) and for Fig. 16 (groups 4–6).
Fig. 17 Breaking elongation of 0.6% collagen films with 120 % glycerine and 0.25% chitosan at different calcium acetate quantities.
In ANOVA test, a very small p value, p<<0.005 for example, suggests that small differences are highly significant between the analyzed measurement groups. Therefore, it can be concluded that the mechanical property of traction resistance formed in the presence of chitosan of the biomimetic films outperform the one formed in the presence of calcium-silica. In contrast, a rather high p-value, p<0.5 for example, suggests that no significant differences are present in the data. Thus, we can conclude that the mechanical property of breaking elongation formed in the presence of chitosan of the biomimetic films is similar to the one formed in the presence of calcium-silica.
Chirita: Mechanical Properties of Collagen Biomimetic Films Formed in the Presence of Calcium, Silica and Chitosan
Finally we examined the films with optical microscopy, (Fig. 20). Those films that contain a small
(a) 0.1 ml Na2SiO3 and 1 ml calcium acetate
157
amount of calcium and a large amount of silica are more uniform (Figs. 20g, h, i).
(b) 0.1 ml Na2SiO3 and 5 ml calcium acetate
(d) 0.1 ml Na2SiO3 and 20 ml calcium acetate
(e) 0.2 ml Na2SiO3 and 1 ml calcium acetate
(g) 0.2 ml Na2SiO3 and 10 ml calcium acetate
(h) 0.2 ml Na2SiO3 and 20 ml calcium acetate
(c) 0.1 ml Na2SiO3 and 10 ml calcium acetate
(f) 0.2 ml Na2SiO3 and 5 ml calcium acetate
(i) 0.3 ml Na2SiO3 and 3 ml calcium acetate
Fig. 20 Optical microscopy of the samples with Na2SiO3 and calcium acetate (×150).
4 Conclusions Silicon plays an important role in macromolecular architecture of collagen during its reconstitution. In order to make the collagen films less brittle a plasticizer, glycerine, has to be added. The strength of collagen films is increased by adding small amounts of calcium but is decreased as further calcium is added. Similarly the breaking elongation of the films increases with small amounts of calcium and then decreases as the film becomes more friable. Films with calcium lose less substance during their treatment with physiological saline, but also in alkaline medium, as the amount of calcium is increased. The tensile strength and breaking elongation of
films with added silica is greater than those with calcium. The dry weight loss, in physiological saline as in alkaline solution, of the silica and calcium films is slightly less than the ones that contain only calcium. Chitosan 0.25% (w/w) at dry collagen improved the traction resistance and the breaking elongation of 0.6% collagen films. It’s possible that both bio-polymers interact to form a more compact 3D structure, more “crystalline”, more ordered that the collagen can do on reconstruction stage[7í12]. Optical microscopy shows that those films that contain a lower amount of calcium and a higher content of silica are more uniform in appearance. Research must be continued to study different kinds of response of human cell growth on these films to determine their utility within an in vivo model.
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