In Vivo Study of Porous Calcium Silicate Bioceramic in Extra-osseous Sites

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1 23 À 3 Ó Ö Vol. 23, No Journal of Inorganic Materials May, 2008 ÍÒ : X(2008) ¼ Ä ÎÅ Ç Õ º 1, ± 1, 2, ² 1, 1, 2, 1, 3 (1. Ý ÜÆ ß «Û ; 2. Ð Æ Å «200050; 3. ÞĐÜÆ Æ «201620) Ñ Æ¾Õ Ñ ÌÃ ¹ (CS) β- Ø Ä¹ (β-tcp) Ó ºÌÁ Á «ÚÉ ÆÝ º ÁÝ Ö ¾ CS, β-tcp ÌÁ SPECT Micro-CT V-G Ê SEM EDX ß» Ý Ã Ý ½ Ì Ã ¹ Ì Á Æ«½ ÉÚÑ Á ÕØ Á Ð Ó Î Ý Ì Ñ Ì Á 4 SPECT È ½ CS β-tcp Ý ROI Î ¾ 53.95± ±3.64(p <0.01), ½ CS ÝÙ Æ Æ¼² β-tcp. ÌÁ 4 Micro-CT ½ CS β-tcp Ý ÓØ Ú ¾ (16.41±1.96)% (30.72±0.69)%(p <0.05), ÚÉÖ ½ CS Ý Ú Æ ² β-tcp(p <0.01), Ð CS Ý ³ Æ ² β-tcp. β- Ø Ä¹Ì ÌÃ ¹ Ó Ù ÚÉ Ì Ó ³ Á Æ Ð Ý ±ÇÆ ÌÃ ¹ ¹ Þ ÚÉ µ ÚÉ Æ Ó ² Å ÌÃ ¹Æ Æ ÐÆ ³ ÆÙ ØÊ ½ R318 Í Á A In Vivo Study of Porous Calcium Silicate Bioceramic in Extra-osseous Sites XU Song-Feng 1, HU Yun-Yu 1, LIN Kai-Li 2, WANG Zhen 1, WANG Lin 1, CHANG Jiang 2, BAI Feng 1, NI Si-Yu 3 (1. Institute of Orthopedic Surgery, Xijing Hospital, Fourth Military Medical University, Xi an , China; 2. Shanghai Institute of Ceramics, Chinese Academy of Sciences, Shanghai , China; 3. Institute of Biological Sciences and Biotechnology, Donghua University, Shanghai , China) Abstract: Porous calcium silicate (CS) and β-tricalcium phosphate (β-tcp) bioceramics were obtained by sintering polymeric sponges infiltrated with ceramic slurry. They were implanted in rabbit subcutaneous sites and the biological characteristics were investigated. After 1, 2 and 4 weeks implantation, specimens were harvested and analyzed by SPECT, Von Gieson staining, Micro-CT, SEM and EDX. There is no obvious toxic reaction in porous CS ceramics, showing the excellent biocompatibility of CS ceramics. In SPECT scanning, the ROI of CS and β-tcp is (53.95±15.14) and (9.81±3.64), respectively (p <0.01), showing higher vascularization for CS. In Micro-CT analysis, the percentage of residual material volume fraction of CS and β-tcp after 4 weeks implantation is 16.41%±1.96% and 30.72%±0.69% respectively (p <0.01). In semi-quantitative analysis of histological observation, the percentage of residual material in CS is obviously lower than that in β-tcp. These results show that the biodegradation of CS is higher than that of β-tcp. The deposition of the bone-like hydroxyapatite layer Â , Â« Å 973 Î (2005CB522704); ÍÌÔÉ Ò Î (07ZR14127) ÚÓ± Ó ± (1977 ), Þ Å È º Đ ÖÅ ortho1@fmmu.edu.cn; À ÖÅ wangzhen@fmmu.edu.cn

2 612 Ó Ö 23 À after 2 week implantation show good bioactivity of CS in vivo. In conclusion, compared with β-tcp, porous CS bioceramics have superiority in vascularization, ingrowth of new tissue and degradation in early stage. Therefore, porous CS bioceramics may be potential candidates as biocompatible, bioactive and biodegradable scaffolds for hard tissue repair and tissue engineering applications. Key words: porous calcium silicate; bioceramics; animal experiment; degradability; vascularization 1 Ý½Þß ¾Å Ä º (CaSiO 3, CS) Ô Í Õ ³ Ï Þ Ò ØÊÜ ĐÖÙÐ (HAp) ÂÞ [1 7]. HAp ÂÞ Í ¼ ÕÞ Ø ¼ Õ Ã / Û Ê Í Ø ß [1,6]. Ä º ÏÞÀ Í [8]. ¾ÅÄ º ÒÅ Þ Â Þ Ò Ð Õ«³Ä º Õ ß ³ ÕÞ ³Ò À Í ß Å ÍÂ [9 11] Ã Ú «De Aza Ð ß Ò Ä º ÍÂÒÙ Â Õ¹ Þµ [9,10] ; Xue Ð ÚÒÄ º Â ÍÂ ÝÀÐ ÕÞµ [11]. Í ÕÅ ÏÞ Ò Õ ² ÛÊ«Ô Ý É Û Ê Þ Ð [12]. ¹ Ú Ê Õ Ó À Ó Đ Ð º ³ ÕÞ ³ ÛÊÞÈÂ ËÔ [13] [14] Å Ú Í Õ ¼ Í Â Þ ¼ «ß Ù Õ Þ Í ² µ ² ³ Û Ê Ð [15], ½ Í Ü Ê ¼ Û Ê È Â Þ Ü [16]. ³Ä º Í² Þß µ ² «³ ß ß Í Ä º Õ Í Â Å Â ÛÊ Þ Ø Ú ¹ Ä º Þ Ø ¼ ÂÞÒ «2 2.1 ØÊ ±Ä º [17] Û Õ β- Ù Å º (β-ca 3 (PO 4 ) 2, β-tcp) [18]. Ö Î² ±ÜÙ Í CS β-tcp Õ [8]. ± Ð 5mm 5mm 5mm, Ò Í Í µm, Í Ü 90%. ± Ï Þ ± Ö ßÂ Ð γ Ë 18h ± [19]. 2.2 ÆÏÖ À Õ Ã «Ò Ø Å Þ Ç Þ ¼ ÄÇÞ 3 Ú kg, Ê«ÅÙÞ ¾ Æ (1 2 4 ) Í 3 Û Û (CS) Û (β-tcp) 2 Í Â 2 Õ «Õ Â Õ Í Â «Ü Ü ± - (È «Ò Ø) Ý À Ý Ü Ð mL/kg. Þ Ä 3cm È ³ Î Â Â Ï ÕÑ Â Õ 1 ( 1(a) (c)), Â ² Â ³«Ü Ý 40 µ U/d 3d. 2.3 Ã (SPECT) Ö Á Ó Ù Â Ç (SPECT) Ú Ü Ò Æ Þ ³ «Ú Þ Ì ß [20]. Ü mCi 99m Tc-MDP 5h É Ç Á , Æ 0.15m/min ¼ Ä ß «ECT Ù (Region of Interest, ROI)/ ³ ÞµÏ ¼ Þ «2.4 Ë CT(Micro-CT) ÍÂ 2 4 ¼³ 1 ³É ½ (φ15mm) ¼ Micro-CT(eXplore Locus SP, GE) É É 80kVp-80mA. Å ÔÓÃ (Microview ABA 2.1.2, GE) Õ CT Ï Ó Ü Õ Ü Þ (Residual Material Volume Fraction, RMVF) ß Õ Ù ÜÞ µ ÕÞ ÕÞÅ [21]. 2.5 ÙÔ ÍÂ ĐÅ ( 1(d) (e)), Ë 10% Þ 3d. (70% 90% 100%) ² Å ß ( 1(f)), Leica SP 1600 ÛÊ Ù 200µm, È ß Á 50µm. Van-Gieson (V-G) Ë ¼ ÁØÇ¼ Ä (Leica DMLA) (Simple PCI). Õ Þ Ô ¼

3 Ò ß ÌÃ ¹ ÝÌÁÝ 613 1 ÕÛ ÞÊ ÌÁ 4 Ý Ï Fig. 1 The process of operation and the sample harvested after 4 weeks implantation Í «ÍÂ 2 Í CS Õ É Ú ÍÂ 4 ÒÐ Ú ÏÝÑ ( 3).

Í ÕÍÂ»Ú Ë ¹Í Â Þ ÐÍÂ µ Ð Ë 2 Ð ÌÁ 4 SPECT È Ï Fig. 2 SPECT scanning image of animals after 4 weeks implantation (a) CS and (b) β-tcp.

6 ¹ X ÍÂ 2 4 ¼³ 1 µå 5% 30% 70% 100% Ð²» ¹ ¼ É ÓÇ¼ (S-3400N, Hitachi, Japan) Ä «Í Â 1 2 Å ß ± 1 Á ¼ Ç É (EPMA, JXA-8100, JEOL, Japan) Ä EDX (INCA Energy, Oxford Instruments,

3 V-G staining image of porous CS after 4 weeks implantation. pores of the porous CS were filled with new fibrous tissues with new vessels ( ).

3 3 Ò ß ÌÃ ¹ ÝÌÁÝ ÕÛ ÞÊ ÌÁ 4 Ý Ï Fig. 1 The process of operation and the sample harvested after 4 weeks implantation Í «ÍÂ 2 Í CS Õ É Ú ÍÂ 4 ÒÐ Ú ÏÝÑ ( 3). ÍÂ 4 Í β-tcp Õº ÞÐ Þ Ú Å ² ÛÊ ÍÚ Ï ÜÑ «2 ÍÂ 4 ÕÞ ROI ²Â Þ CS β-tcp ± ±3.64, Ç Å (p <0.01). Û Ê Ø Ä SPECT ß Â¾Å CS Þ ¹Ú ÅÇ½³ Þ β-tcp. Ú Ò Û Ê Í Õ Í Þ [13]. Í ÕÍÂ»Ú Ë ¹Í Â Þ ÐÍÂ µ Ð Ë 2 Ð ÌÁ 4 SPECT È Ï Fig. 2 SPECT scanning image of animals after 4 weeks implantation (a) CS and (b) β-tcp. The average counting of ROI in CS and β-tcp is 53.95±15.14 and 9.81±3.64, respectively (p <0.01) ÛÊØ Ä ÕÞ Ð Õ µ = µ Ô Û 100%, ¾Â ÕÞ Ô Û [22]. 2.6 ¹ X ÍÂ 2 4 ¼³ 1 µå 5% 30% 70% 100% Ð²» ¹ ¼ É ÓÇ¼ (S-3400N, Hitachi, Japan) Ä «Í Â 1 2 Å ß ± 1 Á ¼ Ç É (EPMA, JXA-8100, JEOL, Japan) Ä EDX (INCA Energy, Oxford Instruments, UK) [22]. 2.7 É ¾ «Þ χ ± s ¾ «Ö Å Ô t «3 Æ 3.1 Ï Ð ÛÊØ Ä¾Å Í Â 1 Í CS Õ Đ ÄÙÒÐ ßÞ À É Đ ÅÇÀ CS ÏÞ 3 ÌÁ 4 Ì CS Ý V-G Ê Ï Í Ñ Ù ( ) Ý Ä ÚÉ Ì Ó (CS) ÊÎÈ Áº Ì º Fig. 3 V-G staining image of porous CS after 4 weeks implantation. pores of the porous CS were filled with new fibrous tissues with new vessels ( ). residual materials (CS) which became thinner were stained showing different layers 4 ÌÁ 4 Ì CS Ý V-G Ê Ï Ì CS Ó (CS) ÈÑ»Ì ( ) Ï Ä ÚÉ Fig. 4 V-G staining image of porous CS after 4 weeks implantation. Numerous micropores ( ) filled with new fibrous tissues were observed in the central part of porous CS (CS)

Í À Ï É Í Ø Ñ ( ½ Ë), Ö À ÞÐ ³ Å ² ÛÊ Õ Ë Ï É Â» Í Ô» ( 3). Õ É Ò Ð ¼ Í Ð Å Û Ê ( 4), Å Õ É É «Õ Ü ¼ Û Ê Ø Ð Ê Æ Æ CS Þ ÜÂÅÇ ³ β-tcp, Ç Å (p <0.01)( 5), Í Â 4 CS β-tcp Þ Õ Ü µ (36.49±1.81)% (57.98±2.

4 614 Ó Ö 23 À «³ÛÊ ¹Ï Ú Ê Í«ÑÞÒÐ Đ «Ú ÞË ÊÓÂ ÞÍ À Ó ¼Å ¼Ç ÛÊÞ ¹ÒÐ Í«3.2 Ï³µ ÛÊØ Ä¾Å ³³ß Þ Í CS Õ Ù ÏÉ ³¾ Þ Õ ÛÊ ÈÂÞË «ÍÂ 1 Í CS ¾ Å ² ÛÊ É ÅÒÐÖ À CS Ë ±ÓÀ ¹ CS Õ³¾² ¾ Å Í CS Õ É É «Í Â 2 Ú ³¾ Å ² ÛÊ Õ Ô (Í ²» ), Ö À ß» ßÍ Ñ ¼Å ¾Å CS Ë À ±ÓË ¹«ÍÂ 4 ÒÐ Ú ÏÝÑ Å ² ÛÊ Ç ÍÚ Í À Ï É Í Ø Ñ ( ½ Ë), Ö À ÞÐ ³ Å ² ÛÊ Õ Ë Ï É Â» Í Ô» ( 3). Õ É Ò Ð ¼ Í Ð Å Û Ê ( 4), Å Õ É É «Õ Ü ¼ Û Ê Ø Ð Ê Æ Æ CS Þ ÜÂÅÇ ³ β-tcp, Ç Å (p <0.01)( 5), Í Â 4 CS β-tcp Þ Õ Ü µ (36.49±1.81)% (57.98±2.67)%, ¾Å CS Þ Å Ç ³ β-tcp. Micro-CT ¾ Å 4 CS β-tcp Í Õ Þ Õ Ù Ü µ (16.41±1.96)% (30.72±0.69)%, Ç Å (p <0.05)( 6). ÕÅ ²È ( 7) Ë ĐÇ Ò CS Þ ÅÇ ³ β-tcp Hench [23] ÅÔ Õ Þ «ÅÔ Õ Ò ± «³ß ÞÛÊØ Ä Ç ÄÂ ¾Å ÕÍÂ 1 É É «Micro-CT ²ÈÇ ÍÂ 4 Í CS β-tcp Þ Õ Ù Ü µ 16.41% 30.72%. ² ÛÊØ ²È ÍÂ 4 Í CS β-tcp Þ Õ Ü µ 36.49% 57.98%, Ê ÍÄ ºÞ ÅÇ ³ β- Ù Åº«Ç ¼ CT(Micro-CT) ÀÐÛÊ Ç ÕÞÍ Ü Í² ÐÐ¾Â ÞÉ Û Ö Û ¾ Ð ÅÇÞ Ñ [24]. ³ß Micro-CT ¼ Ï ¾ ( Õ CT Ï Å Ç ½ ³ ), Õ CT Ï ÓÜ Ü Þ ÕÞ 5 V-G Ê Ó Fig. 5 Semiquantitative analysis of the residual material area after V-G staining The bar means the standard deviation. p < Ì CS β-tcp Ó Ú ÝÆ» CT Fig. 6 Residual material volume fraction of the porous CS and β-tcp after implantation using Micro-CT The bar means the standard deviation. p < ÌÁ 2 4 CS TCP ÓÝ Micro- CT Ä Ï Fig. 7 3D reconstruction images of the residual material of CS and β-tcp after 2 and 4 weeks implantation using Micro-CT

3 Ò ß ÌÃ ¹ ÝÌÁÝ 615 ÖË Þ Ê ÛÊ Í Õß «3.3 Ï SEM ¾Å ÍÂ 2 Í CS ² ² ÛÊ É ¾ É Ca-P ² ÂÊÜ ÍÂ 4 C-S ¾ ÊÜÒÅÂ Þ Ca-P ² Â ÐÍÚ ÏÉ Ò È ( 8).

Þ ³ [1 5] À Đ [6,7] Â¾ÅÄ º Õ ÏÞ Ò Ê ¾ ØÊÜ HAp Â«³ß Þ SEM EDX ß ² È ¾ Å Í Â 2 Í CS ¾ Ê Ü Ò HAp Â «ß ¾ Å Ò A-W Ð Ò Õ ØÊÜ HAp Â ÊÜÞ HAp Â¹ ½ Í¼ Í Þ Ø «³ÊÜ Þ HAp Â¹ ½Å Í Þ Ø ß «Ò Ë Å HAp Â ½Å ÂÐÃÛÊ Í Þ Ø

9 Back-scattered SEM image (a) and EDX analyses (b) of the cross-section of CS after 2 weeks implantation The points are hydroxyapatite where the average ratio of Ca/P is about 1.

5 3 Ò ß ÌÃ ¹ ÝÌÁÝ 615 ÖË Þ Ê ÛÊ Í Õß «3.3 Ï SEM ¾Å ÍÂ 2 Í CS ² ² ÛÊ É ¾ É Ca-P ² ÂÊÜ ÍÂ 4 C-S ¾ ÊÜÒÅÂ Þ Ca-P ² Â ÐÍÚ ÏÉ Ò È ( 8). β- TCP ¾ Ä Ù Ca-P ² Â Ê Ü «Ç É Ê ÄÙ CS Õ¾ Þ Ð Õ ¹ Â Þ µ ( 9(a)), EDX ¾Å Ca-P ² ÊÜÂ ĐÖÙÐ (HAp), Ca/P ²ÂÏ 1.63( 9(b)), ½ÄÇ Ca/P(1.67). Þ ³ [1 5] À Đ [6,7] Â¾ÅÄ º Õ ÏÞ Ò Ê ¾ ØÊÜ HAp Â«³ß Þ SEM EDX ß ² È ¾ Å Í Â 2 Í CS ¾ Ê Ü Ò HAp Â «ß ¾ Å Ò A-W Ð Ò Õ ØÊÜ HAp Â ÊÜÞ HAp Â¹ ½ Í¼ Í Þ Ø «³ÊÜ Þ HAp Â¹ ½Å Í Þ Ø ß «Ò Ë Å HAp Â ½Å ÂÐÃÛÊ Í Þ Ø «9 ÌÁ 2 Ì CS Ý Æ È (a) ±Ç (b) Fig. 9 Back-scattered SEM image (a) and EDX analyses (b) of the cross-section of CS after 2 weeks implantation The points are hydroxyapatite where the average ratio of Ca/P is about 1.63 Õ¾ ØÊÜ ÍÞ HAp Â ³ ¼ Õ Þ Ø [1,6,11], ¼ Õ Ã / Û Ê Í Ø ß [1,6]. ³ ß ¾ Å CS Â ØÊÜ HAp Â ( 9), ¼Å Å ÍÄ º ÏÞ Ò «4 ÍÄ º ÍÂ ÂÛÊ ÅÇ ØÊÜ ĐÖÙÐ Â ¾Å Õ Ï Þ Í Ò «¹ β- Ù ÅºÍµ ÍÄ º ÍÂ Þ ¹Ú ÛÊÈÂ Õ Ð Â ÅÇ Ñ«ÍÄ º º ³ ÛÊÐ ÛÊ Ç Û»Ì 8 ÌÁ 2 (a) 4 (b) Ì CS È Ï Fig. 8 SEM images of porous CS after 2 weeks (a) and 4 weeks (b) implantation [1] De Aza P N, Luklinska Z B, Anseau M, et al. J. Microsc., 1996, 182 (Pt 1): [2] Tsuru K, Hayakawa S, Ohtsuki C, et al. J. Mater. Sci. Mater. Med., 1998, 9 (8): [3] Liu X, Ding C, Chu P K. Biomaterials, 2004, 25 (10):

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