Synthesis and in vivo evaluation of a novel hydroxyapatite/

collagen–alginate as a bone filler and a drug delivery

carrier of bone morphogenetic protein

指導教授指導教授 :: 林鴻儒 博林鴻儒 博士士學生學生 :: 徐楓茜徐楓茜日期日期 :98/12/15:98/12/15

Materials Science and Engineering C 24 (2004) 341–347Shinichi Sotome, Toshimasa Uemura, Masanori Kikuchic, Jiani Chen,Soichiro Itoh, Junzo Tanaka, Tetsuya Tateishi, Kenichi Shinomiya*

前言前言•硬骨具有良好的再生能力，但需要有適當的治療方式，較嚴重患者通常是使用自身移植，例如：骨癌。

•自身移植通常都使用腸骨或小腿骨，但是通常都會造成捐贈部位疼痛且數量又有限。

•基於這些原因，在骨組織有顯著的研究，像是骨組織成長因素或間葉幹細胞的發展來取代自身移植。

實驗步驟實驗步驟 - - HAp/col-alginate

HAp/col powder 0.5g 3ml 0.9% NaCl 35μl of 5M CaCO3

mixture 1.5ml 3% alginate

HAp/col-alginate mixture D-gluconic acid lactone

Injected into a mold 45min, room temperture

HAp/col-alginate was incubated in100 Mm CaCl2 30min

Cut to 2*2*3 and 2*2*5

實驗步驟實驗步驟 - - Implantation into bone holesImplantation into bone holes

Wistar rats(10weeks, 280-320g)

Femur by a lateral approach, and a 3mm hole using electric drill

The drill hole rinsed with 0.9% NaCl solution

The implant was placed into the hole

The fascia and skin were sutured

實驗步驟實驗步驟 - - Ectopic bone formation modelEctopic bone formation model

HAp/col-alginate rh-BMP2 (0, 4, 20, 100 μg/ml)

sample

Atelo-collagen rh-BMP2 (0, 4, 20, 100 μg/ml)

sample

Implanted into the bilateral side of dorsal muscle (5weeks, male, Wistar rat)

結果討論結果討論

Fig. 1. Photograph of HAp/Col–alginate. (A) Dry implant. (B) Wet implant.

Fig. 2. Implant site 2 weeks after implantation. (A, B) HAp/Col– alginate. [(B) Higher magnification of rectangular area in panel A.] (C) Alginate. (D) Porous HA. Black arrowheads indicate implant. White arrowheads indicate newly formed bone. The borders of HAp/Col–alginate and newly formed bone were not clear and bone tissue invaded the implant.

Fig. 3. Implant site of HAp/Col–alginate 4 weeks after implantation. (B) Higher magnification of rectangular area of panel A. Tissue invasion and bone formation around the HAp/Col– alginate implants increased.

Fig. 4. Implant site 8 weeks after implantation. (A–C) HAp/Col alginate. (D) Alginate. (E) Porous HA. (A, B) Fragmentation of HAp/Col– alginate and increase of tissue invasion and bone formation were observed. (C) TRAP-positive multinuclear cells attached to the implant were observed. (White arrowheads indicate TRAP-positive cells.) (D) Tissue invasion of the implants was not observed except at tears. (E) Most of the pores were not filled with bone tissue.

Fig. 5. Ectopic bone formation induced by BMP2 (100 g/ml), 5 weeks after implantation. (A–C) HAp/Col–alginate with BMP. (A) HE section of the whole implant. (B) Immunostaining section with antiosteocalcin. (C) Higher magnification. Bone formation extended throughout almost the entire implant and bone marrow-like tissue was also observed. White arrows indicate bone marrow-like tissue. (D) Collagen sponge with BMP. The area of bone formation was lessthan that of HAp/Col– alginate and occupied only a part of the implant. Black arrowheads indicate soft tissue which invaded the remnant of the collagen sponge. White arrowheads indicate bone.

結論結論• 由組織切片圖可看出， HAp/Col-alginate 可作為藥物載體且順利地使骨增生。

• 由組織切片圖可看出， HAp/Col-alginate 可作為人造骨材料