THE EFFECT OF POLYHYDROXYBUTYRATE ON MICROSTRUCTURE … · THE EFFECT OF POLYHYDROXYBUTYRATE ON...

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Powder Metallurgy Progress, Vol.10 (2010), No 3 157 THE EFFECT OF POLYHYDROXYBUTYRATE ON MICROSTRUCTURE AND PROPERTIES OF CALCIUM PHOSPHATE CEMENT Ľ.Medvecký, R.Štulajterová, S.V. Kutsev, H. Bruncková, T. Sopčák ABSTRACT The calcium phosphate –polyhydroxybutyrate (PHB) composites were prepared by biopolymer precipitation in PHB-calcium phosphate cement powder mixture suspension and adding hardening liquid to the resulting composite powder mixture. Composites had more porous microstructures with a fraction of macropores (size of up to 50 μm) than in pure cement. The specific areas and mesopore volumes decreased with soaking time in simulated body fluid (SBF) because of the dissolution/recrystallization processes of the original calcium phosphates and the formation of new apatite-like phase. Low PHB degradation was found after soaking in SBF only, which was finished after 1 week. In composite microstructures, the large globular agglomerates of starting cement phases were surrounded by the PHB membranes composed of fibers, which were coated by hydroxyapatite phase. The compressive strengths and diametral tensile strengths in composites were about 20 MPa and 1.5-4 MPa respectively. Keywords: calcium phosphate cement - biopolymer composite; mechanical properties; microstructure; porosity; INTRODUCTION Poly–(R)-3–hydroxybutyrate (PHB) represents natural biodegradable polyesters that could release acidic degradation products (3-hydroxybutyric and crotonic acid) during degradation in vitro and in vivo. Developed acidosis can cause the damage tissue around the applied PHB implant. Agrawal et al. and W. Linhart et al. [1,2] solved above problem by the preparation of composites with basic calcium phosphates, which allows one to compensate the lower pH. Another possibility is the utilization of carbonated calcium phosphates as it has been shown by C. Schiller et al. [3,4]. Generally, the PHB-calcium phosphate composites were prepared by various methods such as the solvent method, wet, hot and injection moulding. The basic requirement for a bone implant is keep the pH body liquid close to implant within the narrow cell physiological range (7.2 – 7.6). Excellent cell proliferation was found on the composites, which contained PHB as one of the components [5]. Wang et al. [6] showed that the addition of more soluble and resorbable calcium phosphates to PHB improved the composite bioactivity and induced the formation of a bone-like apatite layer. Doyle et al. [7] established that implants with the PHB produced a consistent favourable bone tissue adaptation response, with no evidence of an undesirable chronic inflammatory response. Ni et al. [8] studied the effect of hydroxyapatite addition on the mechanical properties and bioactivity of HA/PHB composites. Chen et al. [9] found that loading the PHB-PHV (polyhydroxyvalerate) copolymer with hydroxyapatite reduces the Ľ. Medvecký, R.Štulajterová , H. Bruncková, T. Sopčák, a Institute of Materials Research of SAS, Kosice, Slovakia S.V. Kutsev, A.A.Baikov Institute of Metallurgy and Materials Science of RAS, Leninski Avenue 49, 119991 Moscow, Russia

Transcript of THE EFFECT OF POLYHYDROXYBUTYRATE ON MICROSTRUCTURE … · THE EFFECT OF POLYHYDROXYBUTYRATE ON...

Powder Metallurgy Progress, Vol.10 (2010), No 3 157

THE EFFECT OF POLYHYDROXYBUTYRATE ON MICROSTRUCTURE AND PROPERTIES OF CALCIUM PHOSPHATE CEMENT

Ľ.Medvecký, R.Štulajterová, S.V. Kutsev, H. Bruncková, T. Sopčák

ABSTRACT The calcium phosphate –polyhydroxybutyrate (PHB) composites were prepared by biopolymer precipitation in PHB-calcium phosphate cement powder mixture suspension and adding hardening liquid to the resulting composite powder mixture. Composites had more porous microstructures with a fraction of macropores (size of up to 50 μm) than in pure cement. The specific areas and mesopore volumes decreased with soaking time in simulated body fluid (SBF) because of the dissolution/recrystallization processes of the original calcium phosphates and the formation of new apatite-like phase. Low PHB degradation was found after soaking in SBF only, which was finished after 1 week. In composite microstructures, the large globular agglomerates of starting cement phases were surrounded by the PHB membranes composed of fibers, which were coated by hydroxyapatite phase. The compressive strengths and diametral tensile strengths in composites were about 20 MPa and 1.5-4 MPa respectively. Keywords: calcium phosphate cement - biopolymer composite; mechanical properties; microstructure; porosity;

INTRODUCTION Poly–(R)-3–hydroxybutyrate (PHB) represents natural biodegradable polyesters

that could release acidic degradation products (3-hydroxybutyric and crotonic acid) during degradation in vitro and in vivo. Developed acidosis can cause the damage tissue around the applied PHB implant. Agrawal et al. and W. Linhart et al. [1,2] solved above problem by the preparation of composites with basic calcium phosphates, which allows one to compensate the lower pH. Another possibility is the utilization of carbonated calcium phosphates as it has been shown by C. Schiller et al. [3,4]. Generally, the PHB-calcium phosphate composites were prepared by various methods such as the solvent method, wet, hot and injection moulding. The basic requirement for a bone implant is keep the pH body liquid close to implant within the narrow cell physiological range (7.2 – 7.6). Excellent cell proliferation was found on the composites, which contained PHB as one of the components [5]. Wang et al. [6] showed that the addition of more soluble and resorbable calcium phosphates to PHB improved the composite bioactivity and induced the formation of a bone-like apatite layer. Doyle et al. [7] established that implants with the PHB produced a consistent favourable bone tissue adaptation response, with no evidence of an undesirable chronic inflammatory response. Ni et al. [8] studied the effect of hydroxyapatite addition on the mechanical properties and bioactivity of HA/PHB composites. Chen et al. [9] found that loading the PHB-PHV (polyhydroxyvalerate) copolymer with hydroxyapatite reduces the

Ľ. Medvecký, R.Štulajterová , H. Bruncková, T. Sopčák, aInstitute of Materials Research of SAS, Kosice, Slovakia

S.V. Kutsev, A.A.Baikov Institute of Metallurgy and Materials Science of RAS, Leninski Avenue 49, 119991 Moscow, Russia

Powder Metallurgy Progress, Vol.10 (2010), No 3 158 thermal stability of biopolymer and both the crystallinity and mechanical properties changed with the calcium phosphate content in the copolymer. No remarkable change on degradation of PHB or PHB hexanoate blended with HA, respectively, was observed in simulated body fluid [10]. The fibre reinforced CPC and the influence of bioresorbable fibres biodegradation on flexural strength and toughness of composites was studied [11]. Intensively studied were CPC-chitosan composites, (poly lactic acid) PLA or poly (lacticcoglycolic acid) (PLGA)–CPC composites [12] and dental composites composed of CPC and various resin types (methacrylate [13], polymethyl vinyl ether-maleic anhydride [14], carboxylated hydrophilic resins [15], polycarboxylic acid polymers [16, 17]. CPC – biopolymer composites can be effectively utilized for filling bone defects of any shape because composites are applied in the form of cement paste. The PHB represents hydrophobic biopolymers and its addition to CPC can actively increase the hydrophobicity of composites or control the hydrophilic/hydrophobic ratio of composites. It is known that the cell adhesion to the implant and cell activity are affected by implant surface properties such as e.g. surface tension, roughness, etc [36, 37].

In our work, the influence of the PHB addition to CPC´s on final composite mechanical properties and microstructure has been evaluated. The PHB degradation kinetics in tetracalcium phosphate water suspension was studied in relation to the cement matrix - biopolymer boundary formation during composite hardening in SBF.

EXPERIMENTAL

Materials Tetracalcium phosphate (Ca4(PO4)2O, TTCP) was prepared by annealing an

equimolar mixture of calcium carbonate (CaCO3 (analytical grade), Sigma Aldrich) and dicalcium phosphate anhydrous (DCPA) (CaHPO4 (Ph.Eur.), Fluka) at 1450°C for 5 hours. After cooling, the product was kept in a desiccator at room temperature. The TTCP phase purity was determined using the X-ray powder diffraction (XRD) analysis. The TTCP was crushed by milling in a planetary ball mill (Fritsch) for 2 hours. The particle size distributions of the TTCP and DCPA were measured in methanol by laser scattering particle size analyzer (SYMPATEC HELOS). The particle size distribution of TTCP characterizes an almost monomodal distribution curve (only a small fraction with sizes below 2μm) with d50 equals 5.4 μm and a bimodal distribution curve was measured in the case of DCPA powder with two maxima around 2 and 20 μm (d50 = 2.5 μm). The cement powder mixtures were composed of TTCP and DCPA in an equimolar ratio. The PHB – cement powder mixture suspensions were prepared by the PHB dissolution in chloroform (5% solution) and adding the mixture to solution using continuous stirring on magnetic stirrer at 300 rpm and temperature of 50 °C. The composite was obtained after the PHB precipitation in suspension with diethylether, filtration, drying at 80 °C for 4 hours and milling in the planetary ball mill (agate balls and vessel, Fritsch 5, 830 rpm). 1.5 M KH2PO4 (analytical grade, Merck) was applied as a hardening liquid and the powder/liquid ratio equaled 2.

The hardening liquid was added to the powder mixture and resulting pastes were packed in stainless cylindrical form (6 mm D × 12 mm H). Samples were hardened in 100 % humidity at 37 °C for 10 min and consequently soaked in simulated body fluid (SBF, prepared according to [18], VSBF = 100 ml) at 37°C for 48 h, 1 week and 2 weeks. The discs with dimensions of 6 mm in diameter and 12 mm in length were dried at 80 °C for 2 hours.

The diametral tensile strength of samples was measured according to Dickens-Venz et al. [15]. The discs had dimensions of 6 mm in diameter and 3 mm in length. For each experimental group, the compressive strength and diametral tensile strengths were measured

Powder Metallurgy Progress, Vol.10 (2010), No 3 159 for 10 samples on a universal testing machine (LR5K Plus, Lloyd Instruments ltd.) at a crosshead speed of 1 mm/min and 10 mm/min respectively. The mean value and standard deviation of each measured property were calculated for each group of ten specimens. The analysis of variance (ANOVA) was used. Statistical significance was considered at P > 0.05.

The phase composition of samples was analyzed by X-ray diffraction analysis (Philips X´ PertPro, using Cu Kα radiation), infrared spectroscopy (SPECORD M80, 400 mg KBr + 1 mg sample) and differential scanning calorimetry (DSC), thermogravimetric analysis (TG, Mettler 2000C). The microstructures of fractured surfaces of samples were observed by field emission scanning electron microscopy (JEOL FE SEM JSM-7000F).

The sample mesoporosity (3 samples for each state) was analyzed by N2 adsorption at 77 K in a TriStar 3000 system. The sample preparations included degassing at 105°C for 2 hours to eliminate the naturally physisorbed water and other species on the surface, as well as the humidity due to the immersion in SBF. Densities of samples were calculated from their weights and dimensions.

The degradation of reprecipitated PHB (form chloroform solution) in saturated pure TTCP-water suspension and PHB degradation in composites after immersion into SBF at 37 °C for various times were analyzed from the amount of 3-hydroxybutyrate acid (3HB), which is one of the PHB degradation products created during hydrolysis under alkaline conditions [19]. The monomer concentrations were determined by the enzyme method using 3HBdehydrogenase (3HB kit, Roche). The PHB content in composites after immersion into SBF solution was determined by TG analysis.

RESULTS AND DISCUSSION XRD diffraction patterns of composites with 10 wt% and 20 wt% PHB addition

after 2 weeks of soaking in SBF are shown in Fig.1. Remains of the starting tetracalcium phosphate (JCPDS 25-1137) and monetite (JCPDS 09-0080) phases were observed in patterns. The formation of new nanocrystalline hydroxyapatite phase (JCPDS 9-432) can be visible in patterns resulting from the comparison of XRD patterns (1) and (2) or (3). The low intensive lines from reflections of the PHB planes were found in XRD patterns because of the partially amorphous character of the precipitated PHB. The contents of starting phases in nanocomposites were slowly changed during the 2 weeks soaking in SBF only.

Fig.1. XRD diffraction patterns of the original cement mixture (1), composites with 10 wt % (2) and 20 wt % (3) of PHB after 2 weeks hardening in SBF at 37 °C (● TTCP (JCPDS

25-1137), ○DCPA (JCPDS 09-0080), ▲ hydroxyapatite (JCPDS 9-432), ■ PHB).

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Sample microstructures of pure cement after 1 and 2 weeks hardening are shown in Fig.2. The small amount of irregular pores size up to 10 µm and the large fraction of micropores with 1-2 µm size can be found in the compact microstructure of the pure cement (Fig.2a). Apatite particles have rod and plate-like shape with length around 100 nm. The higher fraction of spherical agglomerates (1-3 µm size) composed of fine needle-like apatite particles were observed in larger pores in the cement microstructure (Fig.2b) after a 2 week hardening. The composite microstructures are not as compact as cement microstructures after 1 week of hardening. Large irregular pores with length of 20-30 µm (up to 50 µm in samples with 20 wt% PHB), and the high micropore fraction (size around 1 µm) was found in composite microstructures (Fig.3). In microstructures, the globular agglomerates with 30-40 µm in size composed of starting calcium phosphates are clearly visible (Fig.3a). Agglomerates were formed during the PHB precipitation in PHB-cement powder mixture suspensions. The number of agglomerates rises with PHB content in samples (Fig.3b). From detailed micrographs (Fig.3c.) it results that agglomerates are encapsulated and covered with PHB membranes, which consist of thin (~100 nm) PHB fibers. The phase separation of hydrophilic biopolymer and calcium phosphate particles with strong hydrophobic surfaces allows one to decrease the interphase mismatch caused by the different surface tensions of individual components in composite suspensions. The strong repulsive surface forces between surfaces of hardening liquid (water solution) and PHB are constrained from the diffluence of solution into the form of thin film on the surface of PHB phase and the achievement of system stability (by lowering the Gibbs interphase free energy), the liquid phase has to continuously fill the largest volume with the smallest surface area which is in the contact with hydrophobic PHB surface. This arrangement allows the creation of a higher fraction of macropores, which was observed in composite microstructures.

Fig.2. Microstructure of pure cement after 1 (a) and 2 (b) week hardening in SBF.

The biopolymer fibres are incorporated into the final cement matrix after the calcium phosphate cement components transformation to apatite-like phase in the form of spherical agglomerates (~1µm size) composed of fine plate-like particles. Besides this, the apatite-like phase can be tightly bonded to the PHB fibre surface as shown in Fig.3d. The number and size of agglomerates of the plate-like apatite particles rose after the 2 week hardening (Fig.3e).

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Fig.3. Microstructures of composites after 1 week hardening in SBF: 10 wt% PHB (a,d,e)

and 20 wt % PHB (b,c; large pores →).

The pH in pure cement (the same cement as in this work) is kept above 9.5 during the first 14 hours of hardening at 100% humidity caused by TTCP hydrolysis to Ca(OH)2 [20]. In Fig.4, the PHB (after PHB dissolution in chloroform and its precipitation in diethylether) degradation study via the determination of the 3-hydroxybutyric acid (3HB) as one of the hydrolysis products (the second product is the crotonic acid) in TTCP water suspension at 37 °C during the first 7 days are shown. The TTCP was added in an amount which corresponds to saturated Ca(OH)2 solution after full TTCP hydrolysis, which could arise during the cement hardening. The pH of the TTCP water suspension increased above

Powder Metallurgy Progress, Vol.10 (2010), No 3 162 10 after 8 hours with following slowly decreased to 9. The hydrolysis rate of precipitated PHB (0.545 h-1, r=0.995) was approximately ten times lower than in ref. [19] (in 1N NaOH) (note the results suggest that the parallel formation of 3HB and crotonic acid by PHB hydrolysis under alkaline conditions follows the 0th-order kinetics). Note that the amount of 3HB formed after 1 week degradation in TTCP water suspension did not exceed the 1 wt % of the origin PHB amount. M.I.A. Majid et al. [21] found the rate PHB film hydrolysis constant values equal 0.48% week-1, 0.50% week-1 and 0.84% week-1 for pH 7.4, 10.0 and 13.0 at 37 °C. In the case of composite samples (samples used for DTS testing), the situation is more complicated because of the mutual interaction between hydrolyzed TTCP and DCPA, which are transformed to hydroxyapatite phase. The final PHB content in composites was determined from mass losses at the decomposition temperature around 300 °C with endo-effect on the DSC curve (Fig.5). The amount of PHB phase present in composites was approximately 91±1% of the origin PHB amount after 1 week of hardening in SBF and it was unchanged with soaking prolongation. This fact is in accordance with SEM observation, which showed the formation of the compact hydroxyapatite coating on PHB fibers. J.C. Skinner et al. [22] verified the strong effect of carbon chain length, acidity of carboxylic acid and the steric arrangement of the carboxylate group on carboxylate sorption at the hydroxyapatite surface. These interactions are responsible for a lower transformation degree of starting calcium phosphates to apatite phase. In IR spectra of composites after a 2 week soaking (Fig.5), changes in band intensity at wavenumbers of 1720-1750 cm-1 and decrease in intensity ratio of I1720/I1750, which are characteristic for C=O stretching vibration [23] of PHB, caused by the rise of noncrystalline fraction in PHB after hydrolysis. The wavenumber in the case C=O bond of carboxylate ions is shifted to lower value and split [19,14]. New low intensive bands at 1700 and 1650 cm-1, partially overlapped with bending vibrations of physisorbed water correspond to the formation of carboxylates on hydrolysed PHB surface. The peak from OH bending hydroxyapatie vibration at 630 cm-1, bands characterize vibrations of the phosphate groups at 1050, 960, 600 cm-1 [24] and vibrations of the remaining starting calcium phosphates were found. From TG and DSC analysis resulted that the PHB decomposition start is shifted from 250 °C to 180 °C after 1 and 2 weeks soaking in SBF (Fig.5). K. Csomorova et al. [25] showed that the addition of CaO or MgO to PHB in low amounts caused a decrease in the thermal decomposition temperature (Tmax) (crotonic acid and carboxylates) because of mutual interaction between basic ions and carboxylate end groups on the PHB surface. K.J. Kim et al. [26] confirmed that the trace amounts of Lewis acids (Ca2+ and Mg2+ ions) in PHB interact with the carboxyl group facilitating the formation of the double bond present in crotonyl unit. Moreover, the calcium carboxylates created by the interaction of Ca2+ ions and end carboxylate groups on PHB surfaces (originated in PHB hydrolysis), significantly influence the thermal decomposition temperature. No effect of a PHB addition on cement setting time (around 5 min.) was observed.

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Fig.4. Degradation of precipitated PHB to 3-hydroxybutyric acid in TTCP water suspension

(see text).(n=3, relative standard deviation < 5%).

Fig.5. DSC (a) and TG (b) analysis of starting composite mixture with 10 wt% PHB (1)

after 1 (2) and 2 (3) week hardening in SBF.

Fig.6. IR spectra of starting composite mixture with 10 wt% PHB (before hardening, a) and

after 2 week hardening in SBF (b). (■ vibrations of DCPA, ● vibrations of TTCP, ▲ vibrations of PHB groups, □ vibrations of hydroxyapatite,○ vibrations of carbonate group, ● bending vibrations of physisorbed water,♦ new formed vibrations of carboxylate group).

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The influence of the PHB addition on micro- and mesopore (according to IUPAC nomenclature rules) [27] size distribution after hardening is shown in Table 1 and Fig.7. The specific surface area was determined using the Brunauer–Emmett–Teller (BET) method [28]. Porosity less than 30 nm was analyzed by the Barrett–Joyner–Halenda [29] algorithm from the desorption branch of the isotherms, and the contribution of microporosity to the total porosity was estimated by the t-plot method employing the Harkins–Jura statistical thickness curve [30]. From Fig.7a it results that the measured N2 adsorption/desorption curves correspond with the curve of type IV with a hysteresis region typical for mesoporous materials. The analysis of curves showed that the specific area decreased with soaking time (Table 1) around 10 % after 2 weeks in all samples. The composite specific areas were about 50% larger than these in pure cements as well. The volume of mesopores decreased with soaking time (Table 1) and the composites had high mesopore fractions (the highest mesopore volumes). On pore size distribution curves (Fig.7 b,c), the one intensive maximum located at 3.4 nm can be seen and no other peaks were observed. The presence of micropores in samples was not found by the t-plot analysis (Harkins and Jura) after 48 hour soaking, but they were confirmed after 1 and 2 weeks soaking. Similarly, mocropore volumes in composites with 20 wt % PHB were lower than in composites with 10 wt % PHB, but the total pore volumes were higher. This fact verifies that the fraction of larger pores rises with the PHB content in composites. The micropore volumes correspond with the observed relationship between specific areas and total pore volumes, and they decreased with soaking time. The above dependences are due to the new apatite phase formation in microstructures by the precipitation from metastable SBF and the partial dissolution and recrystallization of nanoapatite particles or the transformation of original cement phases [20]. S. Sanchez-Salcedo et al. [31] found similar changes in porosities of biphasic calcium phosphates after their immersion in SBF at 37 °C. The evolution of N2 adsorption/desorption isotherms of samples verified the microstructure observations.

Tab.1. Results from analysis of N2 adsorption/desorption isotherms of samples after soaking in SBF at various times.

Cement Time 48 h 1 week 2 weeks

Bet surface area (m2g-1) 19.85±0.097 18.64±0.055 16.88±0.047 Micropore volume (cm3g-1) none 0.00023 0.0001 Pore volume (cm3g-1) 0.078 0.068 0.056 Nanocomposite (10 wt% PHB) Bet surface area (m2g-1) 22.4±0.4 22.7±0.6 22.6±0.15 Micropore volume (cm3g-1) 0.00136 0.00203 0.00207 Pore volume (cm3g-1) 0.081 0.083 0.082 Nanocomposite (20 wt% PHB) Bet surface area (m2g-1) 27.5±0.15 26.7±0.20 25.0±0.15 Micropore volume (cm3g-1) none 0.00037 0.00014 Pore volume (cm3g-1) 0.105 0.099 0.097

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Fig.7. N2 adsorption/desorption isotherms (a), desorption cummulative pore distribution (b)

and derivative (dV/rdr) pore distribution (c) of samples after 1 week hardening in SBF. ( pure cement, composite (10 wt % PHB), composite (20 wt % PHB)).

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In composites, a rapid fall (three-fold) in DTS with soaking time was found (Fig.8a). The DTS of the composite with 10 wt% PHB was around 4 MPa contrary to 1.5 MPa of the sample with 20 wt % PHB. Composite CS´s (Fig.8b) were not statistically different (around 23 MPa, p>0.01). Note, the decrease in PHB content by hydrolytic degradation could reduce composite strength but no PHB hydrolysis continuation was found after the 2 week hardening. The reason for significant lowering of mechanical properties of composites was high porous microstructure with macropores, which destabilize the strength of composite samples. Apart from this, the formation of globular agglomerates with a hydrophobic biopolymer membrane without mutual interconnection via apatite-like nanoparticles on boundaries, which was found in pure cements [20], does not increase the resistance of composite microstructure to loading. Composites with 40 wt% hydroxyaptite prepared by injection or compression moulding had CS´s around 50-60 MPa [32] or 62 MPa (30 wt% HAP) in the work of N.Galeo et.al [33]. In both references, the observed CS values were significantly higher than in nanocomposites in this paper because samples had lower porosity than in our samples and the filler was stable, not chemically active. The CPC-PHB nanocomposite properties can be compared with DTS or CS of CPCs prepared with mixing of various water soluble polymer types. For example, K. Miyazaki et al. [16] measured DTS (between 6-10 MPa) and CS (between 25-80 MPa) or Y. Matsuya et al. [14] found lower values of DTS (≈ 7 MPa) and CS (≈50 MPa) in CPCs with an addition of polymethyl vinyl ether-maleic anhydride polymer. CS in composites with 10 wt% microspheres prepared by X. Qi et al. [34,35] equals 25 MPa but strong hydrolysis of PLGA was observed in the microstructure after 42 days. Note, the measured mechanical properties are appropriate for the utilization of composites as bone defect fillers and the presence of macropores can allow the ingrowth of new formed tissues into the composite microstructure.

Fig.8. Diametral tensile strength (a) and compressive strength (b) of samples after 1 (empty

bars) and 2 week (full bars) hardening in SBF.

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CONCLUSIONS No additional thermal treatment or use of the complicated mixing method with the

special devices is needed in the preparation process of these composites. The nanocomposites were formed after the transformation of CPC powder components to nanoapatite phase. The mechanical properties and microstructure of calcium phosphate cements were strongly influenced by the PHB addition (precipitated from suspension). Composite microstructures were not so compact like pure cement sample microstructures, which was verified by higher values of specific areas and mesopore volumes. Both the specific areas and mesopore volumes decreased with soaking time in SBF as a result of dissolution/recrystalization processes. In composite microstructures, the globular agglomerates (30-40 µm) of starting calcium phosphate phases coated with PHB membranes and a rise in the fraction of macropores with the PHB content were found, which was the reason for lower composite mechanical properties (DTS ~4 MPa, CS ~20 MPa). The PHB degradation was stopped after 1 week of soaking in SBF because of the formation of apatite layer on the surfaces of the PHB fibres.

Acknowledgements This work was realized within the framework of the project „Advanced implants

seeded with stem cells for hard tissues regeneration and reconstruction“, which is supported by the Operational Program “Research and Development” financed through the European Regional Development Fund.

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