Antoni Niekraszewicz, Magdalena Kucharska, Maria Wiśniewska-Wrona, Danuta Ciechańska, Maria Ratajska, Krzysztof Haberko 1

Transcription

1 SURGICAL BIOCOMPOSITES WITH CHITOSAN Antoni Niekraszewicz, Magdalena Kucharska, Maria Wiśniewska-Wrona, Danuta Ciechańska, Maria Ratajska, Krzysztof Haberko 1 Institute of Biopolymers and Chemical Fibres, M. Skłodowskiej-Curie19/27, Łódź, Poland 1 AGH - University of Science and Technology, Al. Mickiewicza 30, Kraków, Poland Abstract Reported are investigations in the formulation and preparation method of hydroxyapatite composites in form of glue, sponge and shaped products. Osteosynthesis ability, mechanical properties susceptiblity to biodegradation and biocompatibility of the prepared materials were assessed. It was found that, based on chitosan and hydroxyapatite, a material can be prepared with good gluing properties. Crucial is the useful form of chitosan and its physicalchemical properties like ph, polymer content and viscosity. 3 days from the application of the glue are required to complete osteosynthesiss both for porous and smooth bones which, a time acceptable in surgery for the fusion of bones. The glue joints in form of a film undergo enzymatic degradation in the presence of lysozyme. After 28 days action of the enzyme at a concentration of 200 mg/ cm3, the mass loss of the glue was about. 21%. From the compressed chitosan hydrroxyapatite sponge, elastic shaped products can be prepared presenting the potential use as defect fillers in maxillofacial surgery. Key words: hydroxyapatite composites, binding agents, bone glues, enzymatic biodegradation, bone tissue substitutes. 167

2 A. Niekraszewicz, M. Kucharska, M. Wiśniewska-Wrona, D. Ciechańska, M. Ratajska, K. Haberko 1. Introduction Osteoporosis, bone tumors and accident injuries are the prevalent diseases of the skeletal system. Fillers are frequently applied in the therapy to repair bone defects [1]. To this end various materials are used like mineral bones, bioactive glass, fibrous tissue and metal. Such materials, metal in particular, show an insufficient biocompatibility and, in consequence, are isolated from the surrounding bones remaining as foreign bodies frequently causing inflammations. In the presented research, hydroxyapatite (HAp) and various processed forms o chitosan were used for the construction of bone repair materials. Hydroxyapatite resembles in behavior to a large extent that of biological apatite which plays an important role in the interaction between cells and the biomaterial. Owing to such properties, it can be used in the joining of prostheses with the bone. However, HAp in its pure form when combined with normal saline or the patient s blood is prone to migration which may be the cause of injuries of the sound tissue [2]. The deficiency can be negotiated by combining of the HAp with synthetic or natural polymers. Chitosan, amongst the multitude of biomaterials, is the substance which arouses much interest [3]. Specific processed forms of chitosan such as its microcrystalline or gel form manifest outstanding properties like biocompatibility, biodegradability, non-toxicity, excellent ability to form film and good miscibility with other substances inclusive polymers, The molecular and supermolecular structure of the chitosan can be adequately tailored in the course of preparation [4]. The polymer also stimulates the forming of monocytes and inhibits the growth of bacteria and fungi thus lowering the risk of wound inflammation [5]. Unlike hydroxyapatite, chitosan and its derivatives undergo a stepwise biodegradation delivering degradation products which are successively absorbed. Excellent bioactivity makes the polymer a suitable material for the construction of various medical devices inclusive implants. The presented results are a continuation of earlier investigations in the formulation and method to prepare advanced surgical glues or implants (3-D sponge) based on hydroxyapatite and selected chitosan forms. 2. Materials and methods 2.1. Chitosan Initial chitosan used was delivered by Primex Co, Norway. It was characterized by average polymerization degree of `Mv = 344 kd and deacetylation degree of 83.0% Hydroksyapatite In the research, hydroxyapatite HAp 700 (deca-calcium di-hydroxy hexaphosphate) calcinated at 700 C was used. It was prepared by the Faculty of Material Engineering and Ceramics of the University of Science and Technology, Kraków Binding agent and plasticizer Analytically pure glutaric aldehyde by Aldrich was used as binder, and analytically pure glycerol by Fluka Co as plasticizer. 168

3 2.4. Phosphate-citric buffer Surgical Biocomposites with Chitosan Degradability of the prepared materials was assessed in the medium of a phosphatecitric buffer with ph = 7.23 prepared according to standard PN-81C Preparation of buffer solutions Lysozyme Lysozyme of Merck Co (EC ) with U/mg activity was used. It was derived from chicken albumen. The enzyme was applied in the biodegradability assessment of the chitosan/hap complex in form of film and sponge. 2.6.Preparation of the useful forms of chitosan Microcrystalline chitosan (MCCh) was prepared by agglomeration of chitosan according to a continuous process proposed by IBCHF [6]. A suitably adapted flow reactor Dispax Reactor Labor-Pilot 2000/4 was used to this end. 2% chitosanu lactate (ChL) was obtained by dissolution of the initial polymer in an 1% aqueous lactic acid. Modified chitosan lactate (MS/MCCh) was prepared by the dissolution of the MCCh suspension at ph = Preparation of the hydroxyapatite-chitosan composite in form of glue. The hydroxyapatite-chitosan composite was prepared in the T-50 homogenizer made by IKA-WERKE. The glue components were blended at r.p.m. of the agitator at ºC Preparation of the hydroxyapatite-chitosan composite in form of sponge The hydroxyapatite-chitosan composite was prepared in the T-50 homogenizer made by IKA-WERKE. The glue components were blended at r.p.m. of the agitator at ºC. The chitosan-hydroxyapatite sponges were prepared by freeze-drying using the laboratory liophylizing cabinet ALFA 1-4 made by Christ Co. The freeze drying proceeded in the temperature range of (-20) to 10 ºC and vacuum mbar. The drying time was hours Assessment of the degradability of the hydroxsyapatite- chitosan preparations in form of sponge and film. Chitosan-hydroxyapatite composites in both film and sponge form as well as the HAp/MCCh composite in sponge form were assessed in respect of degradability. The tested biocomposites were put into a citric-phosphate buffer at ph = 7.23 and a 1:300 bath module and sterilized in an autoclave at 121 C for 15 minutes. The susceptibility of the biocomposites to biodegradation was assessed in the buffer solution with an addition of lysozyme in the amount of 100 and 200 mg/cm 3.. The tests were carried out in an incubator at 37 C under static conditions. 169

4 A. Niekraszewicz, M. Kucharska, M. Wiśniewska-Wrona, D. Ciechańska, M. Ratajska, K. Haberko Chitosan/hydroxyapatite biocomposites in form of film were taken out of the bath after 3, 7, 14, 21 and 28 days, while those in sponge form still after 35 and 56 days. The samples were filtered off in a Buchner funnel, washed with distilled water at 50 C and poured over with 70 % ethanol, filtered off after 10 minutes and eventually dried at 105 C to constant weight. The progress of the hydrolytic and enzymatic degradation of the tested preparations was estimated based on the change of ph, measurement of aminosaccharides concentration ( products of degradation) in the bath and mass loss of the samples Preparation of animals bones for mechanical tests. Bovine bones were boiled for 6 hours in water with a small amount of ethanol. The remaining amount of soft tissue was next removed. The purified bones were cut into rectangles with dimensions of 2 4 cm and 3-5 mm thickness Analytical methods The viscosimetric average molecular weight of chitosan was calculated from the limiting viscosity number [h]. Viscosity of chitosan solutions was measured with the dilution viscometer using capillary No 1, K 0.01 [7]. The deacetylation degree (DD) of chitosan was estimated by a spectrophotometric method which consists in the determination of the maximum of the first derivative curve of the UV spectrum and computation of the DD according to procedure [8]. A colorimetric method with 3-5 dinitrosalicylic acid (DNS) was applied in the determination of the amount of amino-sugars in the supernatant liquid after the hydrolytic and enzymatic degradation of the hydroxyapatite-chitosan biocomposites [9]. Mechanical properties of the chitosan/hap sponges were tested in the Laboratory of Metrology of IBChF according to adequate standards [10-12]. The laboratory bears the PCA accreditation certificate AB 388. The strength of the glue joints was measured on the INSTRON tester model 5544 in the accredited Laboratory of Metrology of IBChF according to Polish ISO standards [13-15]. Scanning microscopy was applied to inspect the surface structure and crosssections of the prepared chitosan/hap composites. The inspection was made with the SEM apparatus Quanta 200 (FEI Co., USA) at magnification of 100, 200, 500, Results and discussion 3.1. Assesment of the mechanical properties of the joint provided by the hydroxyapatite-chitosan glue Two hydroxyapatite-chitosan glue preparations marked K9 and K10 were tested in respect of ability to join bones. The composition of the preparations was: 170

5 Surgical Biocomposites with Chitosan K 9 - chitosan salt MS/MCCh 1.0 weight parts, HAp weight parts, K 10 - chitosan salt MS/MCCh 1.0 weight parts, HAp weight parts, glutaric aldehyde weight parts. Estimated was the strength of the glue joint in frontal and overlap joining of the bones as well as the adhesion strength of the film joint to the bone surface. After the application of the hydroxyapatite glue on the surface, the bones were joined and left for 24 hours at 37 C. Results of the measurements of the mechanical parameters of the joint made with the K 9 and K 10 preparations are presented in Table 1. In Figure 1 shown are photos which visualize the way the measurements were made. Table 1. Mechanical properties of the glue joints during frontal and overlap combining of the bones. Glue symbol Shearing force; N Smooth bone Overlap joint Porous bone Shear strenght, MPa Smooth bone Porous bone Adhesion strenght, N Smooth bone Frontal joint Peel strenght, MPa Smooth bone K 9 Measurement after 24 h K 9 Measurement after 72 h K 9 Measurement after 144 h K 10 Measurement after 24 h K 10 Measurement after 72 h K 10 Measurement after 144 h On the grounds of the obtained results shown in Table 1, it could be concluded that the K 9 and K 10 overlap joints reveal better shear strength with the porous than smooth bones. It may be expected that the porous surface provides a better penetration of the glue to the bone interior and increases the contact surface yielding a stronger joint between the combined elements. The K 10 compared with K 9 glue manifests better joining properties giving after 24 hours a strong integration of the combined bones both smooth and porous. The K10 preparation contains glutaric aldehyde which, cross-linking the chitosan, enhances the joint durability especially in the overlap version Depending upon the type of the bones and the joining mode, the joints based on the glutaric aldehyde-containing preparations reveal a strength higher by % than those without the cross-linking agent. The tenacity of spongy bones amounts to 1 2 MPa indicating that the K 10 preparation as a suitable material for the joining of such bones. The presented results show that both K 9 and K 10 joints increase strength with the passage of time. After about 3 days from application a full stabilization and maximum joining strength can be observed for both materials. The adhesion of the film joint to the bone surface was also determined. Peeling tests are seen as one of the possible ways to estimate the adhesion of film to the bone surface. It was found that the adhesion of the K 9 and K 10 preparations to the bone surface prevails over the tenacity of the film itself. It evidences a good affinity of the materials to the bone surface and excellent adhesive properties. The conclusion can be drawn that chitosan and hydroxyapatite 171

6 A. Niekraszewicz, M. Kucharska, M. Wiśniewska-Wrona, D. Ciechańska, M. Ratajska, K. Haberko yield preparations which are featured by good gluing properties. The useful form of chitosan and its physical-chemical properties like ph, polymer content and viscosity are crucial for the application. The full binding time of both porous and smooth bones amounts to 3 days which is acceptable in bone surgery. It is, therefore, hoped that the elaborated preparations will find application in a step-wise combining of bones. Considering the composition it may be expected that the K 9 preparation (no cross-linking agent) could better contribute to the acceleration of accretion. This is the reason why the K 9 has been selected for further investigations (susceptibility to degradation and cytotxicity). Strenght measurement of the K 10 glue joint in form of film adhering to the bone surface Strenght meeasurement of the glue joint K 10 - bone overlap joining Figure 1. Photos presenting the measurements of mechanical parameters of the K10 bone joints 3.2 Assessment of the mechanical properties of the hydroxyapatite-chitosan biocomposite AHp/chit in sponge form The material in sponge form was prepared by freeze-drying. Two forms of chitosan were used for this purpose: microcrystalline chitosan (MCCh) with `Mv = 307 kd, polymer content = 2.75% and chitosan lactate (ChL) with `Mv = 285 kd, and polymer content = 2.0%. These were blended with hydroxyapatite- HAp 700 in adequate weight proportion to arrive at the content of dry components on the 3.0 ± 0.5% level. 172

7 Surgical Biocomposites with Chitosan The obtained sponges were tested in respect of mechanical properties like strength and elasticity qualities which are crucial in maxillofascial surgery for defects-filling implants. The impact of the hydroxyapatite upon the mechanical properties of the composite sponges was compared with the sponges made solely of MCCh and ChL with or without addition of a plasticizer. Results of the investigation are shown in Table 2. Table 2. Mechanical properties of the hydroxyapatite- chitosan (HAp/chit) composite in sponge form; * samples were compressed. Sample symbol Weight proportion of components Chit. : HAp : Glic. Sponge thickness, mm Elongation at break, % Tenacity, MPa Standard deviation of tenacity, MPa MCCh reference 1 : 0 : G1/MCCh/HAp700 3 : 1 : G1/MCCh/HAp700/S* 3 : 1 : G2/MCCh/HAp700 1 : 1 : G3/MCCh/Hap700 1 : 3 : G3/MCCh/Hap700/S* 1 : 3 : MCCh/Glic reference 1 : 0 : G4/MCCh/Hap700 3 : 1 : G5/MCCh/Hap700 1 : 1 : G6/MCCh/Hap700 1 : 3 : ML reference 1 : 0 : G7/ChL/Hap : 1 : G7/ChL/Hap700/S* 0.5 : 1 : G8/ChL/Hap700 1 : 1 : G9/ChL/Hap700 1 : 2 : G9/ChL/Hap700/S* 1 : 2 : ChL/Glic reference 1 : 0 : G10/ChL/Hap : 1 : G11/ChL/Hap700 1 : 1 : G12/ChL/Hap700 1 : 2 : The increase of the hydroxyapatite content in the blend with MCCh or ChL causes a deterioration of mechanical properties inclusive elasticity of the formed sponges while admixing of glycerol improves elasticity and lowers tenacity. In the shaped forms prepared by compression of the sponges (samples G1 i G-2/MCCh-2/HAp700/S and G7 and G9/ChL/HAp700/S), the highest tenacity and elasticity was attained. The tenacity increased by 16-20% after the compression of the MCCh sponges (G1-G1/S, G3-G3/S), while for the ChL sponges (G7-G7/S, G9-G9/S) the increase after compression amounted to about 25%. The sponge marked G2/MKCh/HAp 700 made of hydroxyapatite and MCCh in the weight proportion of 1 : 1 featured by best mechanical parameters qualifies for further investigations. The equal amounts of HAp and chitosan warrants the implementation of the material as an implant that fulfills two requirements : reconstruction of the bone tissue and acceleration of the joining process. 173

8 A. Niekraszewicz, M. Kucharska, M. Wiśniewska-Wrona, D. Ciechańska, M. Ratajska, K. Haberko 3.3. Assessment of the degradation of the hydroxyapatite-chitosan preparation in form of gluing film A film preparation made of the K 9 glue composed of 1.0 weight part of the chitosan salt MS/MCCh and 0.33 weight part of HAp 700 was subjected to hydrolytic and enzymatic degradation. The following was estimated in the course of the degradation: ph, percentage of the mass loss of the preparation (calculated on chitosan contained in the biocomposite) and concentration of aminosugars ( products of degradation contained in the citric-phosphate buffer). Considering it that the preparations only assist in the joining of bones and their role in the organism is short lasting, the testing time was limited to 28 days. The results of the investigations are presented in Table 3. Table 3. The course of the hydrolytic degradation of the K9 preparation Degradation time, days ph Mass loss, % Concentration of aminosugars, mg/cm As can be seen, the film made of the K 9 glue undergoes a partial hydrolytic degradation. After 3 days of the film in the buffer medium at 37 C, the mass lost was only slightly above 15% without the forming of reductive aminosugars. No further mass change could be observed after 28 days. The enzymatic degradation of the biocomposite film was estimated by the same parameters as in the hydrolytic degradation. Results are compiled in Table 4. Table 4. The course of the enzymatic degradation of the K9 preparation. Degradation time, days Enzyme concentration, mg/cm 3 ph Mass loss, % Concentration of aminosugars, mg/cm

9 Surgical Biocomposites with Chitosan As can be seen, the tested preparation is susceptive to enzymatic degradation whose intensity depends on the applied lysozyme concentration. At 100 mg/cm 3 of the lysozyme concentration, the mass loss was over 20 % after 28 days with a mg/cm3 concentration of the delivered aminosugars. At 200 mg/cm3 concentration of the lysozyme, the increase of the mass loss was insignifican, while the amount of the formed oligomeric products climbed to 0.26 mg/cm3. It may be concluded that the enzymatic degradation of the film of hydroxyapatite glue proceeds rather intensively particularly in the initial phase Assessment of the degradation of the hydroxyapatite-chitosan preparation in form of sponge Degradation was analyzed of the sponge marked (G2/MKCh/HAp700) made of a blend of MCCh with hydroxyapatite Hap 700 in the weight proportion of 1 : 1. The progress of the degradation process was followed by the proceeding changes in ph, mass loss of the sponge (re-counted on the chitosan mass contained in the biocomposite) and concentration of aminosugars ( products of the degradation contained in the medium). Results of the analysis are compiled in Table 5. Table 5. The course of the hydrolytic degradation of the G2/MKCh/HAp700 sponge Degradation time, days ph Mass loss, % Concentration of aminosugars, mg/cm The mass loss gives evidence to that the chitosan component of the preparation undergoes a partial hydrolytic degradation. After 56 days, the mass loss of the sponge was 7.86 %, distinctly lower than for the glue films The degradation process was not accompanied by the formation of oligomeres like aminosugars. The run of the of the enzymatic degradation of the biocomposite was assessed the same way as for the hydrolytic degradation. Results are presented in Table 6. Photographic documentation was also made which visualizes the impact of the enzyme onto the appearance of the sponge preparation after 56 days (Figure 2). The degradation of the sponge preparations proceeds slower than that of the glue films evidenced by a lower mass loss, while the amount of the delivered aminosugars remains on the same level (slightly higher for the sponges). The results presented in Table 6 indicate that the enzymatic degradation is primarily governed by the concentration of the lysozyme which is most pronounced after 56 days of the process. The mass loss and the amount of the degradation products - aminosugars increase with the concentration of the enzyme The mass loss after 56 day amounted to 12.57% and 21.59% at 100 mg/cm 3 and 200 mg/cm 3 enzyme concentration accordingly. The increase of the enzyme concentration 175

10 A. Niekraszewicz, M. Kucharska, M. Wiśniewska-Wrona, D. Ciechańska, M. Ratajska, K. Haberko results also in a higher amount of the aminosugars formed in the course of the degradation. After 56 days of the degradation, the amount of the oligosaccharides arrived at 0,291 mg/ cm3 for the 100 mg/cm3 enzyme concentration and was by 20% higher at the concentration of 200 mg/cm 3. Tabela 6. The course of the enzymatic degradation of the G2/MKCh/HAp700 sponge. Degradation time, days Enzyme concentration, mg/cm 3 ph Mass loss, % Concentration of aminosugars, mg/cm The comparison of the pictures before and after the degradation (Figure 2) indicates that under the action of the enzyme at 200 mg/cm3 concentration, the surface structure of the sponge undergoes changes with the forming of new and wider pores. 6. Conclusions 1. The investigation resulted in the elaboration of two chitosan-hydroxyapatite preparations designed for bone surgery. 2. The chitosan-hydroxyapatite glue is characterized by good adhesion to the bone surface. The strength of the glue joint surpasses that of a film prepared from the glue. 3. A maximum of the glue joint is attained after 2-3 days, which time is acceptable in bone surgery. The glue joint of the preparation containing glutaric acid manifests a tenacity by % (depending upon kind of the bones and joining method) higher than that of the glue without the cross-linking agent. 4. The glue joint in film form is susceptible to enzymatic degradation in the presence of lysozyme. Under the action of f the enzyme at a 200 mg/cm 3 concentration, the mass loss of the preparation amounted to about 21%. 5. The chitosan-hydroxyapatite sponges after compressing can be shaped into elastic forms which have thepotential to be used as fillers in maxillofascial surgery. 6. The chitosan-hydroxyapatite sponge undergoes a hydrolytic,and, to a higher degree, an enzymatic degradation. A mass loss of 10% and 21.5% after 28 and 56 days accordingly could be observed under the action of lysozyme at the concentration of 200 mg/ cm

11 Surgical Biocomposites with Chitosan SpongeG2/MKCh/HAp700 before degradation (magnification x100) SpongeG2/MKCh/HAp700 after 56 days of degradation (magnification x100) SpongeG2/MKCh/HAp700 before degradation (magnification x500) SpongeG2/MKCh/HAp700 after 56 days of degradation (magnification x500) Figure 2. SEM microphotos of the hydroxyapatite-chitosan preparation in sponge form before and after enzymatic degradation ( concentration of lysozyme -200 mg/cm3. 7. Acknowledgment The investigation were carried out within the research project No 3 T98E supported by the Ministry of Science and High Education. 8. References 1. Murugan R., Ramakrishna S.: Bioresorbable composite bone paste using polysaccharide based nano hydroxyapatite, Biomaterials, 25, (2004), pp Olmi E. et al: Hydroksyapatites alloyed with bone cement: physical and biological characterization. Ceramics in Surgery Proc. of 2nd Int. Symp. on Bioceramics, 1982, p

12 A. Niekraszewicz, M. Kucharska, M. Wiśniewska-Wrona, D. Ciechańska, M. Ratajska, K. Haberko 3. Viala S., Freche M., Lacout J. L.: Preparation of a new organic-mineral composite:chitosanhydroxyapatite, Ann. Chim. Mat., 1998, 23, pp Polish Patent P (1989). 5. Muzzarelli R. A. A.; Biochemical significance of exogenous chitins and chitosans in animals and patients, Carbohydrate Polym. 1993, 20, pp Polish Patent P (1989). 7. Procedure SPR/BPB/5 Viscometric estimation of the average molecular weight according to GLP Nr. G-016 (IBWCh). 8. Procedure SPR/BLF/21 Estimation of the deacetylation degree of chitosan by the determination of the maximum of the first derivative curve of the UV spectrum according to GLP Nr. G-016 (IBWCh). 9. Procedure SPR/BBP/5 Estimation of aminosugars by a colorimetric method with 3,5 dinitrosalicylic acid according to GLP Nr. G-016 (IBWCh). 10. Polish standard PN EN ISO Plastics Determination of mechanical properties at static extension. General Rules, Polish standard PN EN ISO Plastics Determination of mechanical properties at static extension. Conditions for the testing of film and sheets. 12. Polish standard PN EN ISO Plastics Film and sheets. Determination of thickness by mechanical scanning. 13. Polish Standard PN-EN ISO Inactive surgery implants Arthrosis replacing implants. Special requirements. 14. Polish Standard PN-EN ISO Inactive surgery implants Arthrosis replacing implants. Specific requirements concerning hip joint. 15. Cagle C. V. Poradnik inżyniera i technika Kleje i klejenie (Adviser for engineers and technicians Glues and gluing ) Wydawnictwo Naukowo-Techniczne, Warszawa