2009 Advanced Technologies for Enhanced Quality of Life
Chitosan-Hydroxyapatite Composite Obtained by Biomimetic Method as New Bone Substitute C.E.Tanase, M.I.Popa Department of Chemical Physics, ”Gh.Asachi” Technical University, Faculty of Chemical Engineering and Environmental Protection, Iasi, Romania,
[email protected]
L.Verestiuc Department of Prosthetic Technology and Biotechnology, Gr.T.Popa” University of Medicine and Pharmacy, Faculty of Medical Bioengineering, Iasi, Romania,
[email protected] phosphates, mixed or not with biopolymers (collagen, chitosan) [3]. Composites based on chitosan (Cs) and calcium phosphates were found suitable in the treatment of tissue bone defects.
Abstract—Chitosan-Hydroxyapatite (Cs-Hap) composites were prepared through a biomimetic method by Hap precipitation from its precursors, CaCl2 and NaH2PO4, on the chitosan fibres. Materials composition and structure have been analyzed by usual analytical techniques, Fourier transform infrared spectroscopy (FT-IR) and scanning electron microscopy (SEM). FT-IR and SEM data have shown the formation of Hap onto Cs fibers and Cs acting as glue, bonding the Hap crystals. The Cs-Hap composites porosity, from SEM data analysis, increases by increasing the content of Hap. The biodegradation of materials was tested in buffered lysozyme solution and the degraded polysaccharide was measured; the SEM data, before and after degradation, revealed that composites morphology had not appreciable changed. ‘In vitro’ degradation studies indicate that these composite have slighter degradation rate which is coupled to the degree of N-deacetylation, hydrophilicity and crystallinity. Swelling properties measurements in simulated body fluids have shown that the swelling ratio of composites is decreased when the content of Hap is higher. The obtained results revealed that obtained Cs-Hap composites are promising materials as bone substitute due to their adequate swelling properties and controlled degradation rate.
Cs is a semicrystalline homopolymer made up of β (1→4) linked N—acetyl—D–glucosamine and D— glucosamine subunits. Cs can be obtained by deacetylation of chitin under alkaline conditions. Cs is a most abundant constituent of the exo-skeleton and because of its stable, crystalline structure, chitosan, is normally insoluble in aqueous solutions above pH 7. However, in dilute acids the free amino groups are protonated and the molecule becomes fully soluble below pH 5 and medical products in aqueous solution are possible to be obtained [4]. Due to its controlled biodegradability, biocompatible, bioresorbable, nontoxicity, antibacterial, haemostatic, fungistatic, antitumoral, minimizes local inflammation, anticholesteremic and gel-forming properties, Cs is one of the biopolymers that may be used for a wide number of biomedical applications, such as sutures, wound dressings, bone substitutes, tissue engineering and drug or gene delivery vehicles [5,6].
Keywords - composite biomaterials; biomimetics; chitosan; hydroxyapatite
I.
Cs is a natural material that can be degraded into physiologically tolerable compound and form threedimensional structures with potential of colonization by seeded cells [4].
INTRODUCTION
In recent years, in clinical application field, has been a significant interest for synthetic bone substitutes. Through the drawbacks of autograft that describe a high risk of complication (between 8.5-20%) and allograft that present a less osteoconductivity and weak mechanical properties, the synthetic bone substitutes offer an alternative for tissue engineering repair of bone defects [1,2].
From the ceramic family of biomaterials several calcium phosphates are at the moment on the market but the most used ceramic for bone tissue engineering is hydroxyapatite-Ca10(PO4)6(OH)2 [6]. Hydroxyapatite (Hap) has a chemical structure similar to mineral bone, can promote the formation of bone like apatite on his surface, is the most stable in fluids and human condition, it is a bioactive material and its osteoconductivity has been attractive in bone tissue repair or substitution applications [7-10]. All of these characteristics makes from Hap to be the suitable biomaterial for hard tissue repair.
The essential needed characteristics for a bone substitute are: osteogenesis (formation and development of bony tissue), osteoinduction (the act or process of stimulating osteogenesis), and osteoconduction (the growth of bony tissue into the structure of an implant or graft). or
A great attention has been directed towards synthetic natural-synthetic composites, such as calcium
978-0-7695-3753-5/09 $25.00 © 2009 IEEE DOI 10.1109/AT-EQUAL.2009.19
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Cs-Hap composite are able to improve the osteoblasts adhesion, migration, differentiation, proliferation and to enhance the bioactivity of bone tissue, higher cell growth, alkaline phosphatise activity and mineralization without the use of organic solvents [11, 12].
Ø 10 mm) that was attached to a micro syringe. The swelling ratio (SR,w/w) were was calculated by using following equation: SR= [(wt-w0)/w0] × 100%
The hydrophilic characteristic of Cs facilitates cell adhesion, proliferation and differentiation as the surface promotes cell adhesion due to its free energy and presence of hydroxyl group from Hap and Cs also promote a strong adsorption interaction with Hap [13]
(1)
where wt was the weight at time point t and w0 was the initial sample weight. Three samples of each composite were tested and the mean value was taken as the swelling ratio.
In the aim to obtain composites based on Cs and Hap the paper present a biomimetic method, which is mimicking the natural processes involved in the generation of hard tissues [14, 15].
c) Biodegradability study: The ability of Cs-Hap composites to degrade was studied in presence of lysozyme, the main enzyme responsible for Cs degradation in vivo, at similar concentration to those from living tissues. The degradation was evaluated by measuring the amount of Cs reducing ends after enzyme action, with the ferricyanide method. The initial sample were incubated in 0.01M/L PBS, pH 7.1; 25 mL, containing 30 mg lysozyme in a shaking water bath at 37ºC. On the days 2, 4, 6, 8, 10, 12, 14 filtered aliquots (1mL) withdrawn at determineed time and were cooled in a ice bath, mixed with alkaline potassium ferricyanide solution (4 mL; prepared form 0.25g potassium ferricyanide complex salt dissolved in sodium carbonate: 0.5M, 500 mL) and quenched in boiling water, for 15 min; following by a rapid cooling at room temperature within 5 minutes in a ice bath. Optical absorbance of the solution was measured at 420 nm in a UV-VIS spectrophotometer, (Shimadzu 1700, Japan). The value of the reduced chitosan was calculated from the calibration curve of Nacetyl-D-glucosamine.
Therefore this study is presenting synthesis, characterization and to evaluate the swelling and enzymatic degradation of composites based on Cs-Hap, obtained by biomimetic method, in the order to achieve a synthetic bone substitute with adequate properties. II. MATERIALS AND METHOD Materials Chitosan (Cs) with an average molecular weight of 755900 Da and a degree of N-deacetylation (DDA) of 72.9% was supplied by Vascon Co. Canada. Lysozyme (from chicken egg) was obtained from Fluka. All other chemicals, were of analytical grade and used without further purification. Preparation of Cs-Hap composites Composite materials have been obtained by precipitation of Hap from its precursors (CaCl2 and NaH2PO4) on the chitosan fibers, in presence of NH4OH. The process was accomplished at physiological temperature. An aqueous solution CaCl2 (40%, wt) and aqueous solutions NaH2PO4 (25 %, wt) were slowly added into solution of Cs (1 %, wt; obtained by dissolution of the biopolymer in 1.5% wt aqueous HCl) and homogenized; the pH was adjusted to 7 with an aqueous solution of NH4OH (25 %, wt). The obtained composite was maintained at room temperature for 24 h to establish the pH, thereto were extensively washed with distilled water and dried at 37ºC for 24 hours.
d) SEM observation: The cross-section morphology of Cs-Hap composites was examined with a scanning electron microscopy (SEM Tescan-Vega), at 30 kV after sputtering gold. III. RESULTS AND DISSCOSION A.
FT-IR spectroscopy
FT-IR spectroscopy is an appropriate technique to examine the interaction between Cs and Hap in the obtained composites and these results are revealed in Fig.1
The obtained composites were characterized by Fourier Transform Infrared Spectroscopy (FT-IR) and Scanning Electronic Microscopy (SEM). Swelling in buffered solutions was evaluated and in vitro enzymatic degradation was also studied.
The FT-IR spectrum of pure Cs reveals characteristic bands of peaks around 3437 cm-1 which represents the stretching vibration of νs(N-H), while 1651 cm-1 characteristic peaks of ν (-C=O-) amide I carbonyl stretching and the peaks from 1423 cm-1 corresponding to the δ (C-H) symmetrical deformation and 1074 cm-1 is related to νs(C-O-C) stretching vibration and related to the crystallinity of Cs [16, 17].
Physical-chemical properties a) FT-IR analysis: A Bio-Rad Win-IR was used for Fourier transform infrared (FT-IR) spectroscopy. The spectra were collected over the range 4000-400 cm-1.
The characteristic bands for Hap are representing by 3435 cm-1 for n(O-H) the peaks from 1034 cm-1 and 603 cm-1 belong to ν3 and ν4 PO34¯ and finally the peaks at 565 cm-1 related to ν4 PO34¯ stretching vibration and related to the crystallinity of Cs [16, 17].
b) Swelling behaivour: To assess the swelling behaviour of Cs-Hap composites was submerged at physiological temperature, in phosphate buffer saline (PBS) pH 7.1 under shaking conditions, for a period of 48h. The swelling properties were evaluated by determining the volume of buffer absorbed by each sample in a micro column (QIAquick® Spin Column 50, 43
The FTIR spectra of Cs-Hap composites reveal that characteristic bands of both Cs and Hap are presents, corresponding to different Cs-Hap ratios respectively Cs11%, Cs-25% and Cs-40%.
The mechanism of hydroxyapatite synthesis include two steps: the unstable brushite and amorphous calcium phosphate (ACP) are formed in the first stepand then crystalline Hap is developed according to the reactions [18]: 10CaHPO4 + 12OH- = Ca10(PO4)6(OH)2 + 10H2O + PO43PO43- + ACP + OH- = Ca10(PO4)6(OH)2 Studies on literature marking out that the c-axis of Hap crystals tends to align along the Cs fibers [19]. The formation of Hap into the Cs fibres could limit the ability of the Hap to migrate away from the Cs matrix which diminuate the break of composites in a future application opportunity as bone substitute. B.
Swelling behaviour in PBS
The results obtained on the swelling behaviour studies are shown in Table I, indicate that the swelling ratio of CsHap composites decreases at higher content of Hap, as was expected, indicate that the addition of Hap in Cs is useful in reducing the water absorption and goes to a decrease of composites hydrophilicity through the reduction of polar hydroxyl group and amino groups from Cs-Hap composites. Due to its swelling behaviour, the Cs-Hap composites have one important property that is desired to enlarge the pore size and facilitate afterwards a good cell attachment and growth. TABLE I. SWELLING DEGREE OF CS-HAP COMPOSITES FOR DIFFERENT AMOUNT OF CS AND HAP.
Figure 1. FTIR spectra of pure Chitosan (Cs), Hydroxyapatite (Hap), Cs-Hap 1(Cs-11%), Cs-Hap 2 (Cs40%), Cs-Hap 3 (25%).
The peaks from 3429 cm-1, 3437 cm-1 and 3441 cm-1, from Cs-Hap composites, represents the movement of polar groups of Cs suggesting that the hydroxyl ions on the surface of Hap, might interact with the amino and hydroxyl groups from Cs forming hydrogen bonds and also the hydrogen-bonding between Cs and Hap point out to a decrease of Cs crystallinity with the increase of Hap content from composites [16, 17].
C.
Hap %
89
75
60
SR %
52.1
40.3
20.0
In vitro degradation study
The main aim of the degradation studies, using lysozyme is to simulate the physiological conditions. The biodegradation study of the material have an essential role on long term accomplishment of bone tissue engineering. The foremost benefit of biodegradable versus nonbiodegradable materials is the disappearance of implanted foreign material, which might draw out foreign body reactions from the host’s defence system during their long-term contact with a living tissue. Degradation of CsHap composites in PBS containing lysozyme was examined over a period of 14 days. From these studies we observed that was no significant mass loss, for different Cs-Hap ratio, detected during degradation.
Due to these facts the Cs network not only serves as a matrix to the Hap but also provided a bolting site for Hap in the structure. When Cs was mixed with Hap, the amide I carbonyl stretching, 1651 cm-1, were shifted to lower wave number, 1641 cm-1, 1645 cm-1 and 1649 cm-1. The peaks from 1402 cm-1, 1382 cm-1 and 1384 cm-1 represent the shifts of CH3 symmetrical deformation mode.
The low degradation observed along these studies, as shown in Fig.2, is likely due to the fact that Cs have a high DDA and crystallinity. Another fact is that the Cs fibers have a strong bonding with Hap which make difficult for lysozyme to tear off this linkage and as result the degradation of Cs matrix is slower. Through the hydrophilic nature of Cs the diffusion of water into Cs-
The bands from 1035 cm-1, 1033 cm-1, 1031 cm-1, 603 cm and 563 cm-1 of Cs-Hap composites, are related to ν 3 and ν 4 PO34¯ respectively ν 4 PO34¯. -1
The results obtained suggest a stronger hydrogen bonding interaction between Cs and Hap, through the decreasing of Cs crystallinity with the increase of Hap content. 44
Hap composites is faster than degradation and the composites are begins to swell before to degradation.
TABLE II. EFFECT OF HAP CONTENT ON THE WATER ABSORPTION RATIO AFTER DEGRADATION.
Therefore the DDA, hydrophilicity and crystallinity of Cs are important factors in designing new materials for bone substitutes. The swelling degree of the degraded materials, presented in Table II, revealed that the water absorption (WA%) ratio decreasing with the decrease of Hap content in the Cs-Hap composite. By comparison with nondegraded materials, the degraded Cs-Hap composites have a higher swelling capacity; this effect is considering being a result of the increased porosity after Cs degradation, and not an extended in the material hydrophilicity.
D.
Hap %
89
75
60
WA %
128.22
123.9
99.41
Scanning Electron Microscopy analysis
The morphology of Cs-Hap composites with 11% and 40% Cs, before (A-B) and after (C-D) degradation, in cross-section, was evaluated by using scanning electron microscopy (SEM), Fig 3. After analyses the SEM photographs of the composite, we can make an important remark, that the porosity is increasing with the rising of Hap content, more than that it’s obviously, from SEM pictures that the Cs is working like a matrix, providing an anchoring site for Hap, Fig. 3 A and B. After 14 days of degradation from SEM photos, it can be known that Cs from composite is degraded by lysozyme and the Hap crystals are not bonded through polymeric connections, revealed on Fig. 3 C and D. SEM images showed that the morphology of the CsHap composites had not appreciable changed after degradation. Using a co-precipitation method, we have produce Cs-Hap composites with Hap well integrated and distributed through Cs matrix.
Figure 2. Cinetic of degradation time vs. chitosan reducing rate
Figure 3. SEM (×5µm) photographs of the cross-section of Cs-Hap composites, before (A, B) and after degradation (C, D).
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IV. CONCLUSIONS [15]
Composites based on Cs and Hap has been obtained by a biomimetic method. FT-IR and SEM data have shown the forming of Hap crystals onto Cs fibres. Cs network not only serves as a matrix to the Hap but also provided an anchoring site for Hap in the structure due to strong chemical interaction via covalent bonding. From swelling and degradation studies, we can make an important conclusion that Cs-Hap composites are controlled swellable into physiological pH, the swelling degree being strongly dependent by the final composition and degradation studies shown that these composites are slowly degraded by enzymes fact that highlight the future use as a bone substitute.
[16]
[17]
[18]
[19]
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