Study on Synthesis of Calcium Phosphate Biomaterials Containing SMB M.H. Li 1,2, a, S.X.Qu 1, b, R. Shen 1, N. Yao 1,2, P.D. Ren 2 and J.

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1 Key Engineering Materials Online: ISSN: , Vols , pp doi: / Trans Tech Publications, Switzerland Study on Synthesis of Calcium Phosphate Biomaterials Containing SMB M.H. Li 1,2, a, S.X.Qu 1, b, R. Shen 1, N. Yao 1,2, P.D. Ren 2 and J. Weng 1, c 1 Key Lab of Advanced Technologies of Materials, Ministry of Education, School of Material Science and Engineering, Southwest Jiaotong University, Chengdu, China, 2 School of Pharmacy, Southwest Jiaotong University, Emei, China, a sclimaohong@sohu.com, b qushuxin@home.swjtu.edu.cn, C jweng@home.swjtu.edu.cn Keyword: Salvia Miltiorrhiza Bunge(SMB), ph, UV-VIS, calcium phosphate, effect Abstract. Calcium phosphate (CaP) biomaterials containing Chinese medicine, Danshen (Salvia Miltiorrhiza Bunge, SMB), have been synthesized in our previous study via the wet chemical method. However, CaP biomaterials were generally synthesized in the alkaline solution. The purpose of the present study was to investigate the effect of ph on SMB and the influence of SMB on the synthesis of CaP biomaterials. The SMB solutions of different ph from 5.0 to 10.0, were scanned with the UV-VIS spectrophotometer (UV-VIS) in the wavelength ranged from to nm. CaP biomaterials containing SMB were synthesized from (NH 4 ) 2 HPO 4, Ca(NO 3 ) 2, NH 4 OH and SMB. The filtrates of the wet synthesis of CaP biomaterials containing SMB were measured by UV-VIS. The synthesized CaP biomaterials containing SMB were characterized by the X-ray diffraction (XRD) and Fourier-transform infrared spectroscopy (FTIR). The results showed that the maximum absorption peak appeared at nm for solutions of ph 5.0, 6.0 and 7.0. Although there were a new absorption peaks as the ph were 8.0 and 9.0, the absorption curves of SMB became similar to that of SMB as ph at 7.0 after the ph were readjusted to 7.0. The absorption peak appeared an Einstein shift to nm at ph 10.0, which did not return to nm when the ph of SMB solution was readjusted to 7.0. The absorption peak of filtrates containing SMB of CaP biomaterials reaction system was still at nm when their ph was 7.0 and 8.0. Moreover, SMB had no obvious effect on the phase component and functional groups of products. Hence, it could be predicted that calcium phosphate biomaterials containing SMB, such as DCPD and TCP containing SMB, which could be prepared at the ph ranged from 5.0 to 9.0. Introduction CaP biomaterials have been proven to have excellent biocompatibility and bioactivity, which could form bone bonding with bone. They have been used to repair bone defects and augment osseous tissue in clinic. However, they only could offer osteoconductive scaffolds for bone ingrowth. Therefore, to improve the concrescence of bone fraction, it was used to add some growth factors or some drugs in the study of some researchers. In traditional Chinese medicine, some Chinese medicines have clinically been applied for treating bone disease and bone fracture. Danshen (Salvia Miltiorrhiza Bunge, SMB) is one of these Chinese medicines. It was reported that SMB could promote the blood circulation, remove the blood stasis, and improve the union of bone fraction [1]. However, it was difficult for Chinese medicine to reach the bone defect by oral or injection due to being lack of the neoformative blood vessel in the bone fraction site. The local delivery drug system, which CaP biomaterials were used as one of delivery materials, was attained attention. F.H. Lin reported that Chinese medicine, Gu-Sui-Bu, was immobilized onto calcium hydrogen-phosphate by a coupling agent [2]. In our previous study, SMB parenteral solution was added into hydroxyapatite by a spray dryer [3]. Subsequently, SBM parenteral solution was added into the wet synthesis of CaP solution to obtain CaP biomaterials containing SMB [4]. However, CaP biomaterials were generally synthesized in the alkaline solution while the SMB was an acidic solution. The effect of ph and calcium phosphate reaction system on the SMB has still been unclear. Meanwhile, the effect of SMB on the formation of CaP biomaterials All rights reserved. No part of contents of this paper may be reproduced or transmitted in any form or by any means without the written permission of Trans Tech Publications, (ID: , Pennsylvania State University, University Park, USA-10/05/16,02:11:03)

2 1034 Bioceramics 19 has not yet been understood. In the present study, the effects of ph and reaction systems of synthesizing CaP biomaterials, which included DCPD and TCP, on the SMB were studied in the present research. Meanwhile, the reaction products were characterized by XRD and FTIR to investigate the effect of SMB on DCPD and TCP. Materials and Methods The effect of ph on SMB. The SMB parenteral solution was diluted 320 multiples and was divided into six parts. Then they were adjusted to ph 5.0, 6.0, 7.0, 8.0, 9.0 and 10.0 by diluted NH 4 OH or HCl, respectively. The ph of solutions was measured by the ph meter. As-prepared solutions were scanned with UV-VIS in the wavelength ranged from to nm. Moreover, as-prepared solutions were again scanned with UV-VIS to investigate the reversibility of effect of ph after their ph was readjusted to ph 7.0, respectively. The synthesis of CaP biomaterials containing SMB. DCPD and TCP were synthesized by the wet chemical method according to following equations. Ca(NO 3 ) 2 +(NH 4 ) 2 HPO 4 =CaHPO 4 +2NH 4 NO 3 (1) 3Ca(NO 3 ) 2 +2(NH 4 ) 2 HPO 4 +2NH 4 OH+(n-2)H 2 O=Ca 3 (PO 4 ) 2.nH 2 O +6NH 4 NO 3 (2) The (NH 4 ) 2 HPO 4 solution was slowly added to the stirred Ca(NO 3 ) 2 solution to synthesize DCPD and TCP, respectively. Correspondingly, the ph of reaction solutions was adjusted to 7.0 and 8.0. SMB parenteral solution was added to the wet synthesis solutions to prepare DCPD containing SMB and TCP containing SMB, named as DCPD-Z and TCP-Z. DCPD and TCP without SMB, noted as DCPD and TCP, were used as the controls. The effect of CaP biomaterials reaction system on the SMB. The as-prepared sediments were aged for 24 hours and filtrated. The filtrated solutions were diluted comparative to that of the effect of ph on SMB. Then diluted solutions were scanned with UV-VIS in the wavelength ranged from to nm. The distilled water was used as the control. The effect of SMB on synthesized calcium phosphate. The CaP powders were characterized by the XRD and FT-IR. Results The Effect of ph on UV-VIS Spectrophotometer of SMB. The results of UV-VIS showed that the absorption curves of SMB solutions, of which ph were from 5.0 to 7.0, were similar in the wavelength ranged from to nm. There was only an absorption peak at nm in every absorption curve. However, the new absorption peaks appeared in the absorption curves at nm and nm besides the maximum peak at nm as the ph of SMB solution was 8.0 and 9.0, respectively. There was only an absorption peak at nm as the ph of SMB solution was After these ph (>7.0) of SMB solutions were readjusted to 7.0, the UV-VIS results (Table 1) showed that the absorption curves of SMB solutions, of which ph had been 8.0 and 9.0, were similar to that of the SMB solution of ph 7.0. But the Einstein shift occurred in the absorbency curve of the SMB solution, which ph was readjusted from 10.0 to 7.0. Table 1. The absorption peak of SMB solutions ph readjusted to 7.0 ph(initial) ph(final) λ[nm] reversibility yes yes no The Effect of CaP Biomaterials Reaction System on SMB. The UV-VIS results showed that there was only an absorption peak at nm in the absorption curve of the filtered solution from

3 Key Engineering Materials Vols synthesized DCPD, which was corresponded to that of SMB of ph 7.0. Moreover, the results showed that UV-VIS absorption curve of the filtered solution from synthesized TCP was similar to that of SMB of ph 7.0. The present results indicated that the reaction system of preparing DCPD and TCP had no effect on the absorption peak of SMB solution. The effect of SMB on synthesizing calcium phosphate. Figure 1 presented the XRD patterns of DCPD and DCPD-Z. It was confirmed to be calcium phosphate dihydroxide (DCPD). All diffraction peaks of the as-prepared DCPD were corresponded with those of the PDF cards (090077). It indicated that the addition of SMB had no obvious effect on the synthesis of DCPD. The X-ray patterns of TCP and TCP-Z were shown in figure 2. It was identified to be Ca 3 (PO 4 ) 2 nh 2 O. All diffraction peaks were corresponded with those of the PDF cards (180303). Compared to the XRD of TCP and TCP-Z, it was inferred that the addition of SMB had no obvious effect on the synthesis of TCP. However, the broader peaks appeared in the XRD of TCP compared to those of DCPD showed that DCPD had finer grain size. TCP-Z DCPD-Z DCPD TCP theta Figure 1 Powder X-ray patterns of DCPD and. DCPD-Z. The main diffraction peaks of DCPD were indicated with( ) Figure 3 presented the FTIR spectra of DCPD and DCPD-Z. The FTIR spectra of DCPD was identical with that of DCPD reported in the other research [5]. Additionally, there was a weak adsorption peaks at 2925cm -1 in the FTIR spectra of DCPD-Z compared to that of DCPD, which has been reported to be one of the absorption peaks of SMB. Figure 4 presented the FTIR spectra of TCP and TCP-Z. In the FTIR spectrum of TCP, the bands at 566, 602, 960, 1039, 1637, 3136, and 3437 cm -1 were corresponded with the adsorption peaks of TCP. There was not nearly different in the absorption bands of TCP and TCP-Z. 2 theta Figure 2 Powder X-ray patterns of TCP and TCP-Z. The main diffraction peaks of TCP were indicated with( ). DCPD-Z TCP-Z DCPD TCP W avenumber(cm -1 ) Figure 3 FTIR spectra of DCPD and DCPD- Z Wavenumber(cm -1 ) Figure 4 FTIR spectra of TCP and TCP-Z Discussion The chemical constituents of SMB have been identified clearly in many studies. There are two chemical types: lipophilic diterpenoid and water-soluble phenolic acids. It has been confirmed that water-soluble phenolic acids were significant for biological activity of SMB parenteral solution,

4 1036 Bioceramics 19 which have been identified, including salvianolic acid A and B, Danshensu and so on. It was reported that the maximum peak of SMB solution (λmax) was at nm in UV-VIS absorption spectrum, which could be used for the marker of SMB [6]. The present results showed that the ph of the solution had no effect on the UV-VIS absorption curve and the maximum adsorption peak of SMB as the ph ranged from 5.0 to 7.0 due to the main constituents of SMB to be phenolic acids. The absorption curve and the maximum of SMB solution would take place a reversible change as the ph ranged from 8.0 to 9.0. However, there were different absorption curves and maximal adsorption peaks of SMB solution as the ph was 10.0 and readjusted to 7. Moreover, reaction system of preparing DCPD and TCP had no obvious effect on the absorption peaks of SMB. It was consistent with the mentioned stability of SMB in the other study [7]. When the ph of solution was more than 10.0, there might be a chemical bonding between NH 4 OH and the main component of SMB. The main constituents of SMB, salvianolic acids and Danshensu [8], possess the phenyl structure. It was speculated that NH 2 group bonded onto the phenyl resulting in the occurrence of n π* transition. Correspondingly, an Einstein shift was observed in the UV-VIS spectrum. As the starting agents,ca(no 3 ) 2 and (NH 4 ) 2 HPO 4 are almost neutral. They are neither oxidant nor reducer. Calcium ions could not chelated salvianolic acids and Danshensu in SMB. Therefore it was difficult for them to react with SMB. Based on the result of filtration solution s UV-VIS scanning, XRD and FTIR, it can be deduced that there was no chemical reaction between SMB and calcium phosphate reaction system. According to the FTIR spectra, it should be noticed that there was some quantity of adding SMB due to some SMB remaining in the filtrate. The effective quantity of SMB would be added to satisfy with the requirement for clinic. The present as-prepared DCPC and TCP are the main constituents of calcium phosphate cement (CPC). Furthermore, CPC containing Chinese medicine SMB would be developed based on the present study. Conclusion SMB can be added in the wet synthesis of CaP biomaterials as the ph ranged from 5.0 to 9.0. Acknowledgements The present study was supported by National Natural Science Foundation of China ( ) and Program for New Century Excellent Talents in University (NCET ). References [1] J.G.Wang, B.Cao: China J Orthop & Trauma Vol.16 (2003), p.637 [2] F.H. Lin, G.Ch. Dong and K.Sh. Chen: Biomaterials Vol.24 (2003), p.2413 [3] S.X. Qu, J. Weng and B. Feng: J Materials Science Vol.40 (2005), p.3035 [4] C. Huang, S.X. Qu and B. Feng: Key Engineering Materials Vol (2006), p.873 [5] J.W Xu, I.S. Butler and D.F. R: Spectrochimica Acta Part A Vol.55 (1999), p.2801 [6] Y.Q. Sun, Y.Zh. Hu and Zh.W. Li: Analytical Chemistry (Science Publishing Company, China 2003), p.256 [7] Y.S. Fu, J. Zhang: Pharmacy Research Vol.10 (2000), p.28 [8] Y.N. Pan, D.Yuan and W.W. Fu: Journal of Shenyang Pharmaceutical University Vol.21 (2004), p.196.

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