Investigation of Changes in Phases and Properties of a TTCP/DCPA/CSH Cement Immersed in Hanks Solution

Transcription

1 Materials Transactions, Vol. 52, No. 10 (2011) pp to 1953 #2011 The Japan Institute of Metals Investigation of Changes in Phases and Properties of a TTCP/DCPA/CSH Cement Immersed in Hanks Solution Jiin-Huey Chern Lin 1, Szu-Yao Yu 1; * 1, Chang-Keng Chen 1; * 1, Wei-Luen Chen 1; * 1, Jing-Wei Lee 2, Ruey-Mo Lin 3 and Chien-Ping Ju 1; * 2 1 Department of Materials Science and Engineering, National Cheng-Kung University, Tainan, Taiwan, R. O. China 2 Division of Plastic Surgery, Department of Surgery, National Cheng-Kung University Hospital, Tainan, Taiwan, R. O. China 3 Department of Orthopedics, National Cheng Kung University College of Medicine and Hospital, Dou-Liou Branch, Yunlin, R. O. China Investigated in this study are the changes in structure and properties of a tetracalcium phosphate/dicalcium phosphate anhydrous/calcium sulfate hemihydrate (TTCP/DCPA/CSH) cement immersed in Hanks solution. Experimental results show that the phase transition involving the hydration of CSH and formation of calcium sulfate dihydrate (CSD) continues up to 7 d of immersion. The phase transition from TTCP/ DCPA to hydroxyapatite (HA) is substantially completed after 14 d; after that, both CSH and CSD phase largely diminished, whereas HA becomes and remains to be the only dominant phase throughout 42 d of immersion. A maximum compressive strength is reached; after that, the cement gradually decreases in strength. After 42 d, its CS value is down to 8 MPa. The long-term ph value of the Hanks solution wherein the cement is immersed remains in the range between 5.4 and 7.0. The cytotoxicity test reveals that the viability value of the cells incubated with conditioned medium of cement extraction is 85% that of Al 2 O 3 control and 84% that of blank medium for an extraction ratio of 0.2; and 90% that of Al 2 O 3 control and 93% that of blank medium for an extraction ratio of 0.1. [doi: /matertrans.m ] (Received May 23, 2011; Accepted July 8, 2011; Published September 25, 2011) Keywords: calcium phosphate, calcium sulfate, bone cement, medical implant 1. Introduction The resorption rate of a bioresorbable bioceramic often depends on such material parameters as chemical composition, crystallinity, microstructure, etc. 1 5) When its resorption rate is adjusted to be similar to the growth rate of natural bone, the implanted material may be gradually replaced by new bone. 6) Calcium sulfates as well as some of calcium phosphates, such as -TCP, OCP and carbonate-apatite, 7 9) are typical such bioresorbable materials. 10,11) Furthermore, due to its unique injectable feature, calcium phosphate cement (CPC) may be used in bonding, filling, and repairing damaged natural bone in orthopedic, dental, maxillofacial and other medical applications via minimally invasive procedures ) Despite their many advantages, such as excellent biocompatibility, osteoconductivity, being non-exothermic and X- ray detectable, etc., most currently-used calcium phosphate and calcium sulfate materials have their respective disadvantages. For example, as being used as a bone substitute material, most popularly-used calcium phosphates exhibit relatively low bioresorption rates, 12) while most calcium sulfates demonstrate relatively low mechanical strengths and dissolution rates being too high, 11) that may not allow new bone cells to effectively grow into a bone cavity. 15) It seems logical to combine calcium phosphate and calcium sulfate into a composite formula and, if an appropriate process can be developed, expect the inherent advantages from each component. (For example, the creation * 1 Graduate Student, National Cheng-Kung University * 2 Corresponding author, cpju@mail.ncku.edu.tw of pores due to the fast dissolution of calcium sulfate may enhance the resorption rate of the implant material) For this purpose, the present work is devoted to investigating the phases and properties, as well as their changes when immersed in Hanks solution, of a TTCP/DCPA/CSH cement recently developed in the present authors laboratory. 2. Materials and Methods The TTCP powder was fabricated in-house using the method suggested by Brown and Epstein. 16) Appropriate amounts of in-house prepared TTCP and commercial DCPA powders were uniformly mixed, followed by mixing with an appropriate amount of CSH powder. The resultant TTCP/ DCPA/CSH powder mixture with a weight ratio of 0:89 : 0:33 : 1 for each constituent (or a weight ratio of 55 : 45 for TTCP/DCPA: CSH) was mixed uniformly with a 0.6 M (NH 4 ) 2 HPO 4 setting solution at a liquid/powder (L/P) ratio of 0.35 cc/g to form a cement. To evaluate the immersion effects on various properties of the cement, the hardened cement was immersed in Hanks physiological solution (Table 1) for different periods of time. The solution was maintained at 37 C and refreshed daily. The immersed hardened cement samples remained mechanically stable in the solution, as shown in Fig. 1. For compressive strength (CS) testing, after mixing for 1 min, the cement paste was packed into a 6 mm dia., 12 mm deep cylindrical stainless steel mold under a pressure of 1.4 MPa. The CS testing was conducted according to ASTM a method using a desk-top mechanical tester (Shimadzu AGS-500D, Tokyo, Japan) at a crosshead speed of 1.0 mm/min.

2 1950 J.-H. C. Lin et al. (a) (c) (e) (b) (d) (f) Fig. 1 Mechanical stability of the cement immersed in Hanks solution for (a) 1 d; (b) 3 d; (c) 7 d; (d) 14 d; (e) 28 d; (f) 42 d. Table 1 Composition of Hanks solution. 17Þ Component Concentration (g/l) NaCl 8.00 Na 2 HPO 4. 2H2 O 0.06 CaCl NaHCO KCl 0.40 Glucose 1.00 MgCl 2. 6H2 O 0.10 MgSO 4. 7H2 O 0.06 KH 2 PO The working time of cement paste was determined by the time after that the cement paste was no longer workable. The setting time of cement paste was measured according to the standard method set forth in ISO 1566 for dental zinc phosphate cements. The cement is considered set when a 400 gm weight loaded onto a Vicat needle with a 1 mm diameter tip fails to make a perceptible circular indentation on the surface of the cement. The present TTCP/DCPA/CSH cement paste exhibited a working time of 8.5 min and setting time of 10.3 min. Such working time and setting time are suitable for most orthopedic and dental surgeries. The early stage (during hardening process) variation in ph value was determined using a ph meter (Suntex Instruments SP2000, Taipei, Taiwan) that was buried in the cement paste immediately after the powder and setting liquid were mixed. The ph value of the Hanks solution wherein the cement paste sample was subsequently immersed was monitored using the same ph meter. In so doing, 2 g readily-mixed cement paste was immersed in 20 ml Hanks solution with a ph value of The solution was refreshed daily and maintained at 37 C throughout the test. The various phases of the cement were analyzed using a Rigaku D-MAX B X-ray diffractometer (XRD) (Tokyo, Japan) with Ni-filtered CuK radiation operated at 30 kv and 20 ma at a scanning speed of 1 /min. Each phase was identified by matching its characteristic peaks with data compiled in the JCPDS files. Scanning electron microscopy was conducted using a field-emission scanning electron microscope (FE-SEM) (XL- 40, Philips, Holland) operated at 10 kv. The fracture surface being examined was coated with a thin layer of gold using an ion sputtering system (JFC-1100, JEOL, Japan) to facilitate conducting the sample. The cytotoxicity test was performed according to ISO , wherein an extraction method was used for this study. NIH/3T3 fibroblasts with a seeding density of 5000 per well were pre-cultured for 24 h in Dulbecco s modified essential medium (DMEM) supplemented with bovine serum (10%) and PSF (1%). The extract was prepared by immersing hardened cement in the culture medium at a ratio of 0.1 or 0.2 g/ml at 37 C for 24 h, followed by the collection of the liquid by centrifugation. The extract was added to a 96 well microplate (100 ml per well) incubated in a 5% CO 2 humidified atmosphere at 37 C. After 24 h, the extract was removed from the microplate and then a mixture of the culture medium (100 ml) and WST-1 (10 ml) was added to the wells and incubated for 1 h at 37 C. Cell viability was measured by using the WST-1 assay, which is a colorimetric assay of mitochondrial dehydrogenase activity where the absorbance at 450 nm is proportional to the amount of dehydrogenase activity in the cell. After 1 h incubation, the mixture of medium and WST-1 was transferred to a 96 well microplate and the absorbance at 450 nm was measured with an ELISA reader. Due to its nontoxic and bioinert nature, alumina (Al 2 O 3 ) powder was assayed as a control. 18,19) 3. Results and Discussion 3.1 Changes in cement phases and microstructure during immersion Figure 2 demonstrates typical XRD patterns of the various

3 Investigation of Changes in Phases and Properties of a TTCP/DCPA/CSH Cement Immersed in Hanks Solution Fig. 2 XRD patterns of the cement being hardened and immersed in Hanks solution. Fig starting powders and the hardened cement immersed in Hanks solution for different periods of time. The XRD peaks of pure TTCP, DCPA, CSH, CSD and HA phases are also given as a reference. The XRD patterns indicate that the phase transition involving the hydration of CSH and formation of CSD continues up to 7 d of immersion. The TTCP and DCPA peaks had largely diminished in 1 h sample, indicating a quick dissolution process of TTCP and DCPA in the solution. The phase transition from TTCP/DCPA to hydroxyapatite (HA) is substantially completed after the cement is immersed for 14 d; after that, both CSH and CSD phase largely diminished, whereas HA became and remained to be the only dominant phase of the cement throughout 42 d of immersion. Figure 3 represents typical scanning electron micrographs of the cement immersed in Hanks solution for different periods of time. The overall SEM morphology of the cement immersed for different periods of time was not much different, except for the clear appearance of the relatively large-sized crystals within 8 h-7 d time frame. Although the various phases as identified in the XRD patterns could not be distinguished in these SEM micrographs, such relatively large-sized, faceted crystals were mostly likely CSD crystals.20) In addition, the appearance (in 8 h micrograph) and disappearance (in 14 d micrograph) of such believed CSD crystals are consistent with the XRD patterns. Scanning electron micrographs of the cement immersed in Hanks solution for different periods of time.

4 1952 J.-H. C. Lin et al. Fig. 4 Variation in ph value of cement paste during hardening. Fig. 5 Variation in ph value of Hanks solution wherein the cement is immersed. 3.2 Changes in cement ph during hardening and solution ph during immersion As shown in Fig. 4, following an initial small decrease, the ph value of the cement paste slightly increased from its 5- min value of 7.4 to 30-min value of 7.6 during the hardening process, whereas a plateau was reached. The initial decrease in ph is possibly caused by a rapid dissolution of CSH. The subsequent increase in ph value is considered as a result of the dissolution of TTCP in the phosphate-containing setting solution. 21) This interpretation is supported by an early result of the present authors in their TTCP/DCPA-derived CPC system, wherein the early-stage dissolution rate of TTCP was found to be five times higher than that of DCPA in a phosphate-containing solution. 22) The early-stage mildly basic nature of the solution is considered a result of the compromise of the dissolution of TTCP that can cause the solution to turn basic 22) and the dissolution of CSH that can cause the solution to turn acidic. 8) Figure 5 shows long-term ph variation of the Hanks solution wherein the cement was immersed for different periods of time. As shown in the figure, the ph value of the solution increased from its 10-min value of 7.5 to 8-h value of 8.1, followed by a sharp decrease to 5.9 after 3 d. After 3 d, the ph value of the solution remained in the range of 5.4 to 6.2. This long-term decrease in ph value of the solution is probably due to a combined effect of the formation of HA, 21) the hydration of CSH 11) and the dissolution of CSD, 11) which could all cause the ph value of the solution to decrease. 3.3 Changes in cement strength during immersion Demonstrated in Fig. 6 is the variation in CS value of the cement immersed in Hanks solution for different periods of time. As shown in the figure, the CS value of the hardened cement continued to increase after the cement was immersed in Hanks solution. When the cement was immersed for 1 d, a maximum CS value (28 MPa) was obtained; after that, the cement gradually lost its strength. After being immersed for 42 d, its CS value was down to 8 MPa. The early-stage Fig. 6 Changes in CS value of cement immersed in Hanks solution for different periods of time. increase and the long-term decay in CS of the cement could be respectively explained by the continual TTCP/DCPA-HA phase transformation and the immersion-induced dissolution of the cement, as observed in many CPC systems ) 3.4 Viability of cultured cells Figure 7 demonstrates the viability values of cells incubated for 24 h in conditioned mediums adulterated with 24 h composite cement extraction, blank medium and Al 2 O 3 powder control groups, as measured according to the method described in Materials and Methods. As shown in the figure, the cells incubated with conditioned medium of Al 2 O 3 powder and with blank medium exhibit similar average viability values (0.73 and 0.74 for the extraction ratio of 0.2; and 0.74 and 0.72 for the extraction ratio of 0.1). Although the viability values of cells incubated with conditioned

5 Investigation of Changes in Phases and Properties of a TTCP/DCPA/CSH Cement Immersed in Hanks Solution 1953 control and 84% that of blank medium for an extraction ratio of 0.2; and 90% that of Al 2 O 3 control and 93% that of blank medium for an extraction ratio of 0.1. Acknowledgment The authors would like to acknowledge the support for this research by the Southern Taiwan Science Park (Kaohsiung Science Park), Taiwan, ROC under the Research Grant # BZ REFERENCES Fig. 7 Viability values of cells incubated for 24 h in conditioned mediums adulterated with cement extraction, blank medium and Al 2 O 3 powder control groups. medium of cement extractions (0.62 and 0.67) were lower than those of the control groups, they were still within an acceptable range (85% that of Al 2 O 3 control and 84% that of blank medium for the extraction ratio of 0.2; and 90% that of Al 2 O 3 control and 93% that of blank medium for the extraction ratio of 0.1). 4. Conclusions (1) The cement paste prepared by mixing TTCP/DCPA/ CSH composite cement powder with 0.6 M (NH 4 ) 2 HPO 4 setting solution at a L/P ratio of 0.35 cc/g exhibited an average working time and setting time of 8.5 min and 10.3 min, respectively, suitable for most orthopedic and dental surgeries. (2) Subsequent to an initial decrease, the average ph value of the cement paste slightly increased from its 5-min value of 7.4 to 30-min value of 7.6 during the hardening process, whereas a plateau was reached. The long-term (up to 42 d) ph value of the Hanks solution wherein the cement was immersed remained within the range between 5.4 and 7.0. (3) The phase transition involving the hydration of CSH and formation of CSD continued up to 7 d of immersion. The TTCP/DCPA-HA phase transition was substantially completed after 14 d; after that, both CSH and CSD phase diminished, whereas HA became and remained to be the only dominant phase of the cement throughout 42 d immersion. (4) The compressive strength of the immersed cement continued to increase up to 1 day, whereas a maximum CS value of 28 MPa was reached; after that, it gradually decayed. (5) Cells incubated with conditioned medium of Al 2 O 3 powder and with blank medium exhibited similar viability values. The viability value of cells incubated with conditioned medium of cement extraction was 85% that of Al 2 O 3 1) K. Ohura, M. Bohner, P. Hardouin, J. Lemaitre, G. Pasquier and B. Flautre: J. Biomed. Mater. Res. 30 (1996) ) E. P. Frankenburg, S. A. Goldstein, T. W. Bauer, S. A. Harris and R. D. Poser: J. Bone Joint Surg. Am. 80 (1998) ) D. Apelt, F. Theiss, A. O. El-Warrak, K. Zlinszky, R. Bettschart- Wolfisberger, M. Bohner, S. Matter, J. A. Auer and B. von Rechenberg: Biomaterials 25 (2004) ) D. Knaack, M. E. Goad, M. Aiolova, C. Rey, A. Tofighi, P. Chakravarthy and D. D. Lee: J. Biomed. Mater. Res. 43 (1998) ) S. Yamada, D. Heymann, J. M. Bouler and G. Daculsi: Biomaterials 18 (1997) ) W. C. Chen, C. P. Ju, Y. C. Tien and J. H. Chern Lin: Acta Biomater. 5 (2009) ) R. Z. LeGeros: Clin. Orthop. Relat. Res. 395 (2002) ) O. Suzuki, S. Kamakura, T. Katagiri, M. Nakamura, B. Zhao, Y. Honda and R. Kamijo: Biomaterials 27 (2006) ) M. Okazaki, H. Ohmae, J. Takahashi, H. Kimura and M. Sakuda: Biomaterials 11 (1990) ) M. Bohner: Injury 31 (2000) Suppl 4: ) M. V. Thomas and D. A. Puleo: J. Biomed. Mater. Res. B 88 (2009) ) H. Oda, K. Nakamura, T. Matsushita, S. Yamamoto, H. Ishibashi, T. Yamazaki and S. Morimoto: J. Orthop. Sci. 11 (2006) ) G. D. Brown, B. L. Mealey, P. V. Nummikoski, S. L. Bifano and T. C. Waldrop: J. Periodontol. 69 (1998) ) L. Comuzzi, E. Ooms and J. A. Jansen: Clin. Oral Implants Res. 13 (2002) ) K. A. Hing, L. E. Wilson and T. Buckland: Spine J. 7 (2007) ) W. E. Brown and E. F. Epstein: J. Res. Natl. Bur. Stand. Sect. A: Phys. Chem. 69 (1965) ) D. C. Mears: Int. Metals Rev. June (1977) ) P. S. Christel: Clin. Orthop. 282 (1992) ) H. Oonishi, L. L. Hench, J. Wilson, F. Sugihara, E. Tsuji, S. Kushitani and H. Iwaki: J. Biomed. Mater. Res. 44 (1999) ) E. Fernandez, M. D. Vlad, M. M. Gel, J. Lopez, R. Torres, J. V. Cauich and M. Bohner: Biomaterials 26 (2005) ) L. Xie and E. A. Monroe: Mat. Res. Soc. Symp. Proc. 179 (1991) ) W. C. Chen, J. H. Chern Lin and C. P. Ju: J. Biomed. Mater. Res. A 64 (2003) ) S. Takagi and L. C. Chow: J. Mater. Sci. Mater. Med. 12 (2001) ) Y. Miyamoto, K. Ishikawa, H. Fukao, M. Sawada, M. Nagayama, M. Kon and K. Asaoka: Biomaterials 16 (1995) ) S. Takagi, L. C. Chow and K. Ishikawa: Biomaterials 19 (1998) ) W. C. Chen, C. P. Ju and J. H. Chern Lin: J. Oral. Rehabil. 34 (2007) ) I. C. Wang, C. P. Ju and J. H. Chern Lin: Mater. Trans. 46 (2005)