Biomacromolecules 2004, 5, 1575-1579
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Evaluation of a Novel Poly(E-caprolactone)-Organosiloxane Hybrid Material for the Potential Application as a Bioactive and Degradable Bone Substitute Sang-Hoon Rhee,* Yong-Keun Lee, and Bum-Soon Lim Department of Dental Materials Science and Dental Research Institute, College of Dentistry, Seoul National University, Seoul 110-749, Korea
Jeong Joon Yoo and Hee Joong Kim Department of Orthopedic Surgery, College of Medicine, Seoul National University Hospital, Seoul 110-744, Korea Received February 24, 2004; Revised Manuscript Received April 16, 2004
A novel poly(-caprolactone)-organosiloxane hybrid material containing calcium salt (Si-O-PCL) was prepared and evaluated as a bioactive and degradable bone substitute material. The Si-O-PCL hybrid was synthesized by the end-capping of 3-isocyanatopropyl triethoxysilane with R,ω-hydroxyl PCL following sol-gel reaction with calcium nitrate tetrahydrate. Its tensile mechanical properties were evaluated, and additional specimens were exposed to simulated body fluid (SBF) for the time range from 3 h to 7 days. The SBF exposure led to the deposition of a layer of apatite crystals on the surface of the Si-O-PCL hybrid within 9 h of soaking. The tensile strength was around 18 MPa, Young’s modulus was around 200 MPa, and the strain at break was around 290%. This material is likely to have a potential application as a bioactive and degradable bone substitute because of its apatite forming ability, biodegradability, and mechanical properties comparable to those of human cancellous bone. 1. Introduction Recently, a new organic/inorganic nanocomposite has been developed through the use of a sol-gel method. There is a good possibility that this new material will be able to answer the demand of an ideal bone substitute because it exhibits apatite-forming ability as well as mechanical properties comparable to those of human cancellous bone.1-9 In addition, when the biodegradable polymer is used as an organic part, the biodegradability can be also given to this nanocomposite. Indeed, a poly(-caprolactone)/silica nanocomposite has been developed,8,9 and it showed apatiteforming ability in simulated body fluid (SBF)10 as well as biodegradability in the phosphate buffered saline.8 However, the shortcoming of this bioactive and degradable poly(-caprolactone) (PCL)/silica nanocomposite was that it had a potential to leave behind the silica particles after the preferential degradation of PCL in vivo.8 Lai et al. has reported that silica granules are slowly excreted through urine following the dissolution in vivo.11 However, the implant material, which produces the decomposition products that are involved in the metabolic cycles of bio-organism, is desirable. Therefore, the attempt to diminish the content of the silica phase without deteriorating its apatite forming ability and mechanical properties has already been carried out in poly(-caprolactone)/silica nanocomposite.9 However, unfortunately, there was a limitation to reduce the content * To whom correspondence should be addressed. Tel: +82-2-740-8696. Fax: +82-2-740-8694. E-mail:
[email protected].
of the silica phase through the current method. Therefore, a new material design, which does not leave behind the silica particles after the preferential degradation of the biodegradable polymer in vivo while not loosing other properties, is demanded. In this investigation, a novel PCL-organosiloxane hybrid material was developed and its apatite-forming ability in the SBF and tensile mechanical properties were evaluated. The advantage of the design of this novel hybrid material is that it does not contain silica particles at all so it will not produce them even after the preferential degradation of PCL in vivo. 2. Materials and Methods 2.1. Preparation of Specimens. The polymer precursor triethoxysilane end-capped PCL (Si-PCL) was prepared by the reaction with R,ω-hydroxyl PCL (Aldrich Chem. Co. Inc., WI) and 3-isocyanatopropyl triethoxysilane (IPTS; Aldrich Chem. Co. Inc.) with 1,4-diazabicyclo-[2,2,2]-octane (DABCO; Aldrich Chem. Co. Inc.) as a catalyst and dry toluene as a solvent.12 The molar ratios of the reactants were PCL 1, IPTS 3, and DABCO 2. The reaction was carried out at 70 °C for 24 h with constant stirring under dry Ar gas using a three-necked round-bottom flask connected with a condenser, a thermometer, and a gas inlet/outlet port. Following the reaction for 24 h, it was purified via repeated precipitation in cold methanol 3 times and then dried under vacuum at room temperature for 24 h. The average molecular weight
10.1021/bm049885n CCC: $27.50 © 2004 American Chemical Society Published on Web 06/04/2004
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Figure 1. FT-IR spectra of R,ω-hydroxyl PCL, Si-PCL, and Si-OPCL, respectively.
of the Si-PCL measured by gel permeation chromatography system (GPC; Waters 2690, Waters Co., Milford, MA) was 12 960. The Si-PCL was dissolved in ethanol with 10 wt % calcium nitrate tetrahydrate to the Si-PCL in dry Ar atmosphere. Following this, the HCl and water were added into the Si-PCL solution and reacted for 10 min. The molar ratio of the reactants Si-PCL:HCl:water was 1:0.01:6. The hydrolysis following the condensation reaction gelation was carried out for 1 week at 40 °C in a Teflon mold covered with Parafilm having a few pinholes. Following the gelation, the specimen was heat-treated at 60 °C for 2 days. Hereafter, the as-prepared specimen will be referred to as Si-O-PCL. The specimen was disk shape and its size was 200 mm in diameter by 2 mm in thickness. 2.2. Bioactivity Testing. The bioactivity of the specimen was assessed by evaluating its ability to form apatite on its surfaces in the SBF.10 The SBF was prepared by dissolving reagent grade NaCl, NaHCO3, KCl, K2HPO4‚3H2O, MgCl2‚ 6H2O, CaCl2, and Na2SO4 in ion exchanged distilled water. Their ionic concentrations were Na+, 142; K+, 5.0; Mg2+, 1.5; Ca2+, 2.5; Cl-, 147.8; HCO3-, 4.2; HPO42-, 1.0; SO42-, 0.5 (in mM). The solution was buffered at pH 7.4 with tris(hydroxymethyl) aminomethane ((CH2OH)3CNH2) and 1 M hydrochloric acid (HCl) at 36.5 °C. Nonsterilized disk shaped specimens 12 mm in diameter by 2 mm in thickness were cut, polished with #400 abrasive, washed with distilled water, dried in a vacuum at room temperature, and then soaked in 30 mL of the SBF at 36.5 °C for the time range from 3 h to 7 days. After soaking, the specimens were removed from the solution, gently rinsed with ion-exchanged distilled water and then dried at room temperature. 2.3. Mechanical Testing. The tensile mechanical properties of the specimen were evaluated using a general purposetesting machine (Instron 4482, Instron Co., Canton, MA) at
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Figure 2. SEM photographs of the specimen before and after soaking in the SBF for different periods of time.
Figure 3. TF-XRD patterns on the specimen before and after soaking in the SBF for different periods of time.
23 ( 2 °C and 50 ( 5% relative humidity conditions. Dumbbell-type specimens in conformity to ASTM D63882 (Type IV) were stamped out using a cutting die. The gage length was 25 mm, and crosshead speed was 5 mm/min. The thickness of the specimens was 2 mm. Seven specimens were tested and Young’s modulus was determined from the slope of the initial linear elastic portion of the stress-strain curve. 2.4. Characterizations. The change of functional groups during each step of the reactions were analyzed by Fourier transformed infrared spectrometry (FT-IR; Nexus, Thermo Nicolet Co., Madison, WI). For IR spectroscopy measurements, the pulverized specimens were diluted 150-fold with KBr powder and the background noise was corrected with pure KBr data. The microstructures of the specimens before and after the bioactivity testing were observed by scanning electron microscopy (SEM; JSM-840A, JEOL, Tokyo, Japan). The crystal phases present in the specimens before
Evaluation of a Novel Si-O-PCL Material
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Figure 4. Changes in element concentrations and pH of the SBF as a function of soaking time.
and after the bioactivity testing were analyzed by thin film X-ray diffractometry (TF-XRD; D8 Discover, Bruker, Germany) with an angle of 2° to the direction of incident X-ray beam. The atomic concentrations of calcium, phosphorus, and silicon in the SBF were measured by inductively coupled plasma atomic emission spectroscopy (ICP-AES; Ultima-C, Jovin-Yvon, France) after soaking the specimens for different periods of time in the SBF. Three samples were tested for each soaking time. The wettability of the specimens was determined by measuring the contact angle of water droplets on the surface of the specimen using the contact angle measurement system (SEO 300A, Surface and Electrooptics Co., Ansan, Korea). Unpolished five specimens were used for testing, and pure PCL was used as a control. 3. Results Figure 1 shows the FT-IR spectra of the R,ω-hydroxyl PCL, Si-PCL, and Si-O-PCL, respectively. After the reaction with IPTS (Si-PCL), the stretching mode of the hydroxyl peak at 3544 cm-1 which originated from the hydroxyl groups in the R,ω-hydroxyl PCL disappeared. On the contrary, the stretching mode of N-H and the bending mode of (N-H)+(C-N) peaks originating from the urethane linkage were newly occurred at 3393 and 1525 cm-1, respectively.13 It means that the R,ω-hydroxyl PCL was completely end-capped by IPTS through forming urethane linkage. New siloxane and silanol groups were observed to occur after the hydrolysis following the condensation reaction of triethoxysilane groups. The bands at 420, 452, 775, 1045, and 1105 cm-1 were assigned to Si-O-Si bending,14 O-Si-O bending,15 Si-O-Si symmetric stretching,15 and Si-O-Si asymmetric stretching16 modes, respectively. On the other hand, the band at 961 cm-1 was assigned to the Si-OH asymmetric stretching mode. The bands at 3434 and 1635 cm-1 were assigned to the stretching and bending modes of O-H bond, respectively, coming from water.15 It means that this new hybrid material has hydrophilic property. Indeed, the contact angle of the specimen obtained from the
Figure 5. Stress-strain curve of the specimen.
wettability testing with deionized water was 54.4 ( 0.3°, whereas that of pure PCL was 81.9 ( 1.5°. Figure 2 shows the microstructural evolutions of the surface of Si-O-PCL specimen after soaking in the SBF for the time range from 3 h to 7 days. Many grooves were observed on the surface of the as-prepared specimen because of the abrasive polishing. After soaking for 9 h, hemisphericshaped apatite granules containing numerous flakelike small apatite crystals were observed to occur. When the soaking time was increased, those apatite granules grew to a bigger size and an almost flat surface of the apatite layer was formed after 7 days of soaking (Figure 2f). Figure 3 shows the TF-XRD patterns on the surface of the Si-O-PCL specimen before and after soaking in the SBF for the time range from 3 h to 7 days. Several PCL peaks denoted by “0” symbols were only observed until 6 h of soaking because PCL had semicrystalline structure. The peaks found at around 21.5, 22.1, and 23.8 degrees were assigned to (110), (111), and (200) planes of the PCL,17 respectively. After soaking for 9 h, apatite peaks denoted by “b” symbols were observed to occur in addition to the PCL peaks. The decrease of the peak heights of PCL phase was also observed with the occurrence of apatite peaks. Figure 4 shows the change in pH, elemental concentrations of calcium, phosphorus, and silicon in the SBF after soaking
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Scheme 1. Scheme of a PCL/Silica Nanocomposite
Scheme 2. Scheme of a PCL-Organosiloxane Hybrid Material
the specimens for the different periods of time. The ionic activity product of apatite as a function of soaking time in the SBF was calculated by using the ICP-AES results. The concentration of calcium reached its maximum value at 1 day of soaking and then decreased gradually with soaking time. The concentration of phosphorus gradually decreased with soaking time. However, the concentration of silicon in the SBF was zero all through the testing period. The pH of the SBF showed its maximum value at 1 day of soaking and then decreased gradually with soaking time. The ionic activity product of apatite in the SBF calculated by using the ICP-AES data of calcium, phosphorus, and pH showed its maximum value at 1 day of soaking and then decreased abruptly. Figure 5 shows the stress-strain curve obtained from the tensile testing of the specimen. It showed typical polymerlike ductile-tough fracture behavior. Its yield and tensilefracture strengths were at around 5 and 18 MPa, respectively, whereas the strain at failure was around 290%. The Young’s modulus, which was obtained by the slope of the initial linear elastic portion of the stress-strain curve, was around 200 MPa. 4. Discussion The organic/inorganic nanocomposite has been developed as an ideal bone substitute material because it has an ability to produce an apatite layer on its surface in the SBF and its mechanical properties are comparable to those of human cancellous bone. By using a biodegradable polymer as an organic phase, it can achieve a biodegradable property.8 However, the drawback of this bioactive and degradable organic/inorganic nanocomposite is that it has a potential to leave behind the inorganic phase such as silica particles after the preferential degradation of the polymer phase in vivo.
This problem can be partly solved by decreasing the inorganic content up to the lowest limit which does not deteriorate its bioactivity and mechanical properties.9 However, the inorganic phase must be left behind even through this method. Therefore, a new material design which does not leave behind the inorganic phase after the preferential degradation of the polymer phase is required for solving this problem. The problem of current material design is the existence of inorganic phase which is chemically bonded to polymer phase through silane coupling agent like in Scheme 1 where R is (CH2)3. In Scheme 1, if the triethoxysilane end-capped polymer can be directly connected to each other without silica phase, it will be like in Scheme 2. New siloxane linkages can be formed by the condensation reaction among the silanol groups which originate from the silane coupling agent. Besides, some silanol groups which do not participate in the condensation reaction will be left. Therefore, even though this new hybrid material contains no silica phase in its structure at all, it can induce the formation of apatite crystals on its surface because it has silanol groups and soluble calcium salt together which act as nucleation sites and an accelerator for the formation of apatite crystals, respectively. Indeed, apatite crystals were successfully formed on the surface of the specimen in the SBF (Figure 2), and the silicon was not released from the specimen at all (Figure 4), whereas it was released from the PCL/silica nanocomposite.8,9 It means that the new PCLorganosiloxane hybrid material has apatite-forming ability even without silica phase. The strong bonding strength of the siloxane linkage could be speculated by its excellent mechanical properties, especially by its extraordinary high value of strain at failure (about 290%). Pure PCL film could not even be made by
Evaluation of a Novel Si-O-PCL Material
conventional hot pressing or solvent casting methods using similar average molecular weight PCL (Mw 10 000) to SiPCL (Mw 12 960). It means that siloxane linkage containing the silanol groups provides not only the apatite forming ability but also excellent mechanical properties to this hybrid material. The urethane groups are known to be more resistant to hydrolytic cleavage18 and not considered susceptible to hydrolysis under normal implant conditions.19 Therefore, if this new hybrid material is implanted into the body, it is conjectured that the hydrolysis is mainly occurred at the ester group exists in the PCL chain. No release of silicon from the specimen into the SBF until 1 week of soaking supports the belief. In vivo and biodegradability testings of this new hybrid material are under investigation and will be published soon. 5. Conclusion A new hybrid material, which is composed by biodegradable poly(-caprolactone), calcium salt, siloxane, and silanol groups, has been developed. The triethoxysilane end-capped poly(-caprolactone) segment containing calcium salt were linked together through the hydrolysis following condensation reaction, i.e., the sol-gel reaction. The apatite layer completely covered the surface within 9 h of soaking in the SBF. The un-reacted silanol groups and water soluble calcium salt were acted as nucleation sites and accelerator, respectively, for the formation of apatite crystals. In addition, the mechanical properties of this hybrid material were comparable to those of human cancelous bone. Therefore, it is likely applicable to bioactive and degradable bone substitute material. Acknowledgment. This work was supported by a Grantin Aid for International Science & Technology Cooperation
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Program from the Ministry of Science and Technology (MOST; No. 05000020-02A0100-01610) of Korean Government. The authors specially thank Prof. C. Ohtsuki for offering the program to calculate ionic activity product. References and Notes (1) Jones, S. M.; Friberg, S. E.; Sjoblom, J. J. Mater. Sci. 1994, 29, 4075-4080. (2) Tsuru, K.; Ohtsuki, C.; Osaka, A.; Iwamoto, T.; Mackenzie, J. D. J. Mater. Sci. Mater. Med. 1997, 8. (3) Chen, Q.; Miyaji, F.; Kokubo, T.; Nakamura, T. Biomaterials 1999, 20, 1127-32. (4) Chen, Q.; Miyata, N.; Kokubo, T.; Nakamura, T. J. Biomed. Mater. Res. 2000, 51, 605-11. (5) Chen, Q.; Miyata, N.; Kokubo, T.; Nakamura, T. J. Mater. Sci. Mater. Med. 2001, 12, 515-522. (6) Kamitakahara, M.; Kawashita, M.; Kokubo, T.; Nakamura, T. Biomaterials 2001, 22, 3191-6. (7) Miyata, N.; Fuke, K.; Chen, Q.; Kawashita, M.; Kokubo, T.; Nakamura, T. Biomaterials 2002, 23, 3033-40. (8) Rhee, S. H.; Choi, J. Y.; Kim, H. M. Biomaterials 2002, 23, 49154921. (9) Rhee, S. H. Biomaterials 2004, 25, 1167-1175. (10) Kokubo, T.; H., K.; Sakka, S.; Kitusgi, T.; Yamamuro, T. J. Biomed. Mater. Res. 1990, 24, 721-34. (11) Lai, W.; Garino, J.; Ducheyne, P. Biomaterials 2002, 23, 213-7. (12) Tian, D.; Dubois, P.; Grandfils, C.; Jerome, R.; Viville, P.; Lazzaroni, R.; Bredas, J. L. Chem. Mater. 1997, 9, 871-874. (13) Lamba, N. M. K.; Woodhouse, K. A.; Copper, S. L. Polyurethanes in biomedical applications; CRC Press: Boca Raton, FL, 1998. (14) Ro, J. C.; Chung, I. J. J. Non-Cryst. Solids 1991, 130, 8-17. (15) Ou, D. L.; Seddon, A. B. J. Non-Cryst. Solids 1997, 210, 187-203. (16) Dire, S.; Babonneau, F. J. Sol-Gel Sci. Tech. 1994, 2, 139-142. (17) Nojima, S.; Hashizume, K.; Rohadi, A.; Sasaki, S. Polymer 1997, 38, 2711-2718. (18) Lelah, M. D.; Cooper, S. L. Polyurethanes in Medicine; CRC Press: Boca Raton, FL, 1986. (19) Stokes, K.; McVenes, R.; Anderson, J. M. J. Biomater. Appl. 1995, 9, 321-54.
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