Microstructural characteristics of calcium hydroxyapatite/poly-L-lactide based composites

Journal of Microscopy, Vol. 196, Pt 2, November 1999, pp. 243–248. Received 9 June 1998; accepted 15 April 1999

Microstructural characteristics of calcium hydroxyapatite/poly-L-lactide based composites N. L. IGNJATOVIC,* M. PLAVSIC,† M. S. MILJKOVIC,‡ L. M. ZIVKOVIC§ & D. P. USKOKOVIC* *Institute of Technical Sciences of the Serbian Academy of Sciences and Arts, K. Mihajlova 35/IV, 11000 Belgrade, Yugoslavia †Faculty of Technology and Metallurgy, Karnegijeva 4, 11000 Belgrade, Yugoslavia ‡University Center for Electron Microscopy, University of Nis, 18000 Nis, Yugoslavia §Faculty of Electronic Engineering, University of Nis, 18000 Nis, Yugoslavia

Key words. Biomaterials, bone, Ca-hydroxyapatite, composite, interface, microstructure, phases, poly-L-lactide, pressing temperature.

Summary Besides its high osteoinductive properties, hydroxyapatite (HAp) exhibits a relatively low mechanical strength. In order to improve the mechanical properties and reliability of HAp based composites, the addition of selected polymers is highly recommended. The main objective of this work is to study the microstructural characteristics of HAp/poly-Llactide (PLLA) composites obtained by cold or hot processing. The composites were prepared from a mixture of a chloroform solution of poly-L-lactide with granulated HAp. After elimination of chloroform by vacuum evaporation, dense compacts were obtained by cold or hot pressing. The pressing pressure ranged from 98·10 to 294·3 MPa for both cold and hot pressing. The hot pressing was performed in the temperature region 293–457 K for a time period of 15–60 min. Depending on the PLLA amount and the pressing procedure it is possible to obtain highly porous or nearly fully dense composites. The scanning electron microscopy examination of fracture as well as of free surfaces revealed that the final porosity and wetting are affected to a great extent by the synthesis conditions and amount of polymer added. An increase in temperature to 457 K for a longer period of time results in fully dense compacts. The formation of a nearly continuous polymer network that leads to the hardening of HAp has also been observed. However, it should be pointed out that some layers of HAp may be free of polymer film since PLLA penetrates more deeply into the porous HAp.

Introduction Osteoinductive properties of synthesized calcium hydroxyapatite (HAp) as a substitute for bone tissue have been Correspondence to: Professor Dr Dragan Uskokovic. Tel: þ 381 11 636994; fax: þ 381 11 185263; e-mail: uskok>itn.sanu.ac.yu q 1999 The Royal Microscopical Society

known for some years (McIntyre et al., 1991). Use of HAp as an implant is limited to some extent because of its poor mechanical properties compared with those of the bone tissue. To improve its properties and broaden the spectrum of its applications, biocomposites consisting of HAp and ceramic, polymer or metal as the other phase are produced. Composites with HAp and a polymer such as polyethylene (Huang et al. 1997), polyethyl ester (Liu et al., 1997) or polyphosphasone (Reed et al., 1996) as a constituent phase are used as potential materials for bone tissue substitution. Special attention has been focused on the composites with polylactide (Verheyen et al., 1993) as the polymer phase owing to their good bioresorbable (Rodrigues-Lorenzo et al., 1996) and biodegradable (Cho et al., 1997) properties. Synthesis of poly-L-lactide (PLLA) using nontoxic initiator enables application of this polymer as a biocompatible and nontoxic biomaterial. In this study, characterization of the microstructure of a HAp/PLLA biocomposite, synthesized in our laboratory, was performed using scanning electron microscopy. Also, the microstructure of the constituent phases HAp and PLLA was analysed. The effect of synthesis parameters and the design of the biomaterial on its properties was determined by analysing the microstructure of the fracture surface as well as the porosity and compression strength of the composite. The influence of pressing temperature, pressure and time on the biomaterial microstructure and interface properties as well as on the potential phenomena that may appear at interfaces (distance between phases, adhesion between phase surface layers, phase penetration, etc.) was studied.

Materials and methods HAp was synthesized by precipitation in a solution via reaction between Ca(NO3)2 and (NH4)3PO4. The precipitate 243

244

N. L . I G NJATOV I C ET AL .

Fig. 1. (a) SEM image of HAp; (b) SEM image of PLLA.

of Ca-HAp was obtained according to the reaction: 5CaðNO3 Þ2 þ 3ðNH4 Þ3 PO4 þ NH4 OH ¼ Ca5 ðPO4 Þ3 ðOHÞ þ 10NH4 NO3 Well-crystallized Ca-HAp phase, in the form of granules, was obtained by drying the gel and subsequent annealing at 1373 K for 6 h (Ignjatovic et al., 1999). Synthesis of PLLA of 400 000 g/mole was performed using L-lactide (Aldrich

Chemical Company, Wisconsin, U.S.A.) as a monomer and stannous octoate (Sigma Chemical Company, St. Louis, U.S.A.) as a nontoxic catalyst. The polymer structure was studied by a 1H-NMR Varian apparatus at 90 MHz in deuterated chloroform (CDCl3) at room temperature. HAp/PLLA biocomposite was prepared by dissolving PLLA in chloroform for 2 h at 293 K, and by adding 80 mass% HAp to the solution. After addition of HAp, q 1999 The Royal Microscopical Society, Journal of Microscopy, 196, 243–248

CA / H A P / P L L A CO M P O S I T E S

chloroform was removed by evaporation in a vacuum drying oven. Non-compact, very porous material obtained was shaped by pressing in cylindrical moulds (d ¼ 10 mm). Pressing was performed at temperatures of 293, 353 and 457 6 3 K and pressures of 98·1 and 294·3 MPa. Pressing time was 15, 30, 45, and 60 min. The microstructure of each phase and the biocomposite were studied by scanning electron microscopy (SEM) (JSM 5300). The density of the samples was determined geometrically from the ratio of the sample mass to its volume, and the compression strength using an INSTRON M 1185 instrument. Prior to SEM analysis, a thin gold film was deposited over the biocomposite samples and their fracture surfaces by evaporation.

Results and discussion Qualitative X-ray analysis of the dried precipitate revealed its amorphous structure and the beginning of the Ca-HAp

245

phase crystallization. The same analysis of the sample after thermal treatment showed the presence of a well-crystallized Ca-HAp phase. 1 H-NMR spectra of PLLA and L-lactide showed the same signal at d ¼ 5·0–5·3 ppm for methine hydrogen (quartet, J ¼ 6·96 Hz), and at about d ¼ 1·6 ppm for methyl hydrogen (doublets). The methyl hydrogen of the polymer is shifted slightly to the right in relation to the monomer due to the chain length effect. However, the 1H-NMR spectrum of the polymer gave signals at 0·5 and 1·03 ppm. It was assumed that these signals originate from the polymer end groups or initiator remaining after precipitation. Both polymer and monomer have low signals at d ¼ 2·56–2·66 ppm (polymer) and d ¼ 2·73–2·83 ppm (monomer). The microstructure of the ceramic phase (a HAp granule with a size of 1 mm) obtained by SEM is presented in Fig. 1(a). HAp particles are arranged in rounded grainy clusters, which continuously grow and become bonded into agglomerates. These mutually bonded agglomerates, with voids 0·1–3 mm in diameter between them, form the basis of

Fig. 2. (a) SEM image of HAp/PLLA obtained by pressing at 293 K and 98·10 MPa; (b) SEM image of HAp/PLLA with chloroform obtained by pressing at 293 K and 98·10 MPa; (c) SEM image of HAp/PLLA obtained by hot pressing at 353 K and 98·10 MPa for 15 min; (d) SEM image of HAp/PLLA obtained by hot pressing at 457 K and 98·10 MPa for 15 min. q 1999 The Royal Microscopical Society, Journal of Microscopy, 196, 243–248

246

N. L . I G NJATOV I C ET AL .

the HAp structure. These voids represent the inner porosity of the HAp phase. Figure 1(b) shows a fracture surface of the PLLA phase of the HAp/PLLA biocomposite which has fibrous and layered structures, with fibres interconnected into a continuous phase in the form of a layer. The HAp unit cell is given in the literature (Katz & Harper, 1986). As evident, outer groups of the cell (mostly OH¹) are essential for interactions with other phases and for the appearance of interfaces and interactions at them. Such a structure enables not only chemical but also physical bonds with other phases to be established. Porosity of the HAp surface (Fig. 1a) enables penetration of potentially existing liquid phase into its structure, one of the phenomena on which the mechanical theory of phase adhesion is based (Skeist, 1977). Compacting of the material by pressing at room temperature and 98·1 MPa gives a composite with the surface illustrated in Fig. 2(a). High system porosity (37%) results in high roughness, making the SEM analysis at magnifications higher than 350× difficult. There is no intimate contact but rather a void between the HAp and PLLA phase (Fig. 2a). Pasty material was obtained after addition of 30 mass% chloroform and pressing of the mixture under the same conditions. Evaporation of chloroform, after pressing, hardens the material and results in its porosity of 24%. The fracture surface of the material is presented in Fig. 2(b). Spherical holes, 16–200 mm in diameter, are evident in the PLLA phase. Chloroform was present in these holes during pressing, and was removed afterwards by evaporation. A sample with a porosity of 22%, the fracture surface of which is presented in Fig. 2(c), was obtained by increasing the pressing temperature from 293 to 353 6 3 K at a pressure of 98·1 MPa for 15 min. A relatively spherical HAp granule, shown in Fig. 2(c), is coated by PLLA; the layer thickness is 12 mm. A system where PLLA is in the liquid state was obtained by increasing the pressing temperature from 353 to 457 6 3 K (the melting point of PLLA). Analysis of the system reveals its compaction by sintering in the presence of the liquid phase, because at 457 6 3 K PLLA is in the liquid state while HAp remains unchanged. Factors affecting the adhesion at the interfaces of such a system are wetting, contact angle of wetting and possible penetration of the liquid phase (German, 1996). A fracture surface of the composite prepared by hot pressing at 457 6 3 K and 98·1 MPa for 15 min is presented in Fig. 2(d). The porosity of the system is 4·0% and bonds formed at interfaces are probably of different origin: OH groups of HAp with carbonyl groups in the polymer chain can form hydrogen bonds, polymer end-groups can interact with HAp groups but also van der Waals interactions are possible (Fig. 2d). Contacts between HAp and PLLA in the form of tiny bridges

from PLLA to HAp are evident. During the SEM analysis of the biocomposite, an accelerating voltage higher than 10 kV and a magnification of 3500 × led to melting and degradation of the PLLA phase. Under given conditions it was not possible to obtain satisfactory images; higher magnifications therefore were not applied. Dependence of poly L-lactide density on temperature, in the range 341–445 K, according to (Witzake et al., 1997) can be expressed by the following equation: rðg cm¹3 Þ ¼ 1?145½1 þ ð0?0007391ðTð8CÞ ¹ 150ÞÞÿ¹1 ð1Þ An increase in temperature causes a decrease in polymer density according to Eq. (1), which reaches its lowest value at the melting point temperature. A decrease in PLLA density results in better wetting of HAp and PLLA (probably due to the high number of contacts between the polymer chains and HAp caused by an increase of chain flexibility provoked by temperature increase) and increased penetration of PLLA, which causes better compaction of the system. At 457 K, PLLA is in the liquid state so the least porous systems with maximum strength are expected to result. The effect of pressing temperature on porosity and compressive strength of the biocomposite is illustrated in Fig. 3.

Fig. 3. (a) Effect of hot pressing temperature on HAp/PLLA porosity (P ¼ 98·10 MPa, t ¼ 15 min). (b) Effect of hot pressing temperature on HAp/PLLA compressive strength (P ¼ 98·10 MPa, t ¼ 15 min). q 1999 The Royal Microscopical Society, Journal of Microscopy, 196, 243–248

CA / H A P / P L L A CO M P O S I T E S

As shown in Fig. 3(a), the porosity of the system decreases with an increase in hot pressing temperature, reaching its lowest value of 4% at 457 K. Adhesion is best with the samples obtained by pressing at 457 K (Fig. 3b) because the compressive strength during pressing abruptly increases when approaching the PLLA melting point temperature. At this temperature, besides a decrease in PLLA density, better wetting of HAp and penetration of

247

PLLA are observed, resulting in an abrupt increase in compressive strength. Also, rheological effects are significant. Higher thermal mobility of chains provides better structural conditions for hot pressing, mainly due to disentanglement of polymer chains. Higher mobility of chains also provides a better flow and pore penetration and decreases the chances of mechanochemical degradation of PLLA during hot pressing.

Fig. 4. (a) SEM image of HAp/PLLA obtained by hot pressing at 457 K and 294·3 MPa for 15min. (b) SEM image of HAp/PLLA obtained by hot pressing at 457 K and 98·10 MPa for 45 min. q 1999 The Royal Microscopical Society, Journal of Microscopy, 196, 243–248

248

N. L . I G NJATOV I C ET AL .

Samples with a porosity of 1·3% were obtained by increasing the pressure from 98·1 to 294·3 MPa at a temperature of 457 6 3 K for 15 min. An increase in pressure causes a decrease in the system porosity and more intimate contact of phases, as can be seen in Fig. 4(a). There is a PLLA layer with a mean thickness of about 10 mm between two HAp granules. Contact between the HAp and the PLLA phase is very close, with voids at certain positions. The sample porosity decreases with increasing hot pressing time. The lowest porosity of 0·4% was achieved at a pressure of 98·1 MPa and temperature of 457 6 3 K for 30 min pressing. The same porosity (0·4%) was achieved after a prolonged pressing time of 60 min. Figure 4(b) shows a fracture surface of the sample obtained by pressing for 45 min. Very intimate contact of HAp and PLLA phase, effecting the penetration of PLLA into the HAp surface pores, can be observed. Penetration depths up to 1 mm were observed. According to the mechanical theory on adhesion, penetration plays the main role in achieving good adhesion. The sample obtained exhibits a compressive strength of 92·7 MPa, the highest compressive value for a HAp/PLLA composite obtained in our experiments. The compressive strength of the HAp/PLLA composite of 92·7 MPa is significantly higher than that (3 MPa) obtained by Lu & Mikos (1996). Reproducibility of the composite mechanical properties using the same preparation procedure yielded the same or very similar composite microstructure, responsible for these properties, as shown.

Conclusion Phenomena occurring at the interface of a HAp/PLLA biocomposite, obtained under different conditions, were evaluated by scanning electron microscopy. Microstructure of interfaces is directly dependent on the pressing temperature, pressure and time. Phases obtained by pressing at 293 6 3 K and 98·1 MPa for 15 min are not in close contact. There are a lot of voids between them, as can be seen in SEM images. The system obtained under these conditions has the highest porosity of 37% and the lowest compressive strength (19 MPa). Close contact of phases is observed at a pressing temperature of 457 6 3 K, where PLLA phase penetrates the HAp pores (penetration depth up

to 1 mm). A porosity of 0·4% and a compressive strength of 92·7 MPa for the system confirm this close contact of HAp and PLLA phases.

References Cho, S., Kikuchi, M., Suetsugu, Y. & Tanaka, J. (1997) Novel calcium phosphate/polylactide composites: its in vitro evalution. Key Eng. Mater. 132–136, 802–805. German, R. (1996) Sintering Theory and Practice, John Wiley and Sons, New York, pp. 225–247. Huang, J., Di Silvio, L., Wang, M., Tanner, K. & Bonfield, W. (1997) In vitro mechanical and biological assessment of hydroxyapatitereinforced polyethylene composite. J. Mater. Sci: Mater. Med. 8, 775–779. Ignjatovic, N., Tomic, S., Dakic, M., Miljkovic, M., Plavsic, M. & Uskokovic, D. (1999) Synthesis and properties of hydroxyapatite/ poly-L-lactide composite biomaterials. Biomaterials, 20, 809–815. Katz, J. & Harper, R. (1986) Calcium phosphates and apatites. Encyclopedia of Materials Science and Engineering, (ed. by M. Bever), pp. 474–481. Pergamon Press, Oxford. Liu, Q., De Wijn, J. & Van Blitterswijk, C. (1997) Nano-apatite/ polymer composites: mechanical and physicochemical characteristics. Biomaterials, 18, 1263–1270. Lu, L. & Mikos, A. (1996) The importance of new processing techniques in tissue engineering. MRS Bull. 21, 28–31. McIntyre, J., Shackelford, J., Chapman, M. & Pool, R. (1991) Characterization of a bioceramic composite for repair of large bone defects. Ceram. Bull. 70, 1499–1503. Reed, C., TenHuisen, K., Brown, P. & Allcock, H. (1996) Thermal stability and compressive strength of calcium-deficient hydroxyapatite-poly(bis(carboxylatophenoxy)phosphazane) composites. Chem. Mater. 8, 440–447. Rodrigues-Lorenzo, L., Salinas, A., Valet-Regi, M. & San Roman, J. (1996) Composite biomaterials based on ceramic polymers. I. Reinforced system based on Al2O3/PMMA/PLLA. J. Biomed. Mater. Res. 30, 515–522. Skeist, I. (1977) Handbook of Adhesives. Van Nostrand Reinhold, New York, pp. 20–70. Verheyen, C., Klein, C., De Blieck, J., Wolke, J., Van Blitterswijk, C. & De Groot, K. (1993) Evalution of hydroxylapatite/poly (L-lactide) composites: physico-chemical properties. J. Mater. Sci. Mater. Med. 4, 58–65. Witzake, D., Narayan, R. & Kolstad, J. (1997) Reversible kinetics and thermodynamics: the homopolymerization of L-lactide with 2-ethylhexanoic acid tin (II) salt. Macromolecules. 30, 7075– 7085.

q 1999 The Royal Microscopical Society, Journal of Microscopy, 196, 243–248