磷酸鈣之顯微結構對牙髓細胞與骨細胞反應之影響

Abstract

在哺乳類動物的硬組織中，磷酸鹽是主要的無機物成分，而氫氧基磷灰石(hydroxyapatite, HAP）是主要的結構，且不同的組織中的氫氧基磷灰石有不同的次級結構，而非晶性磷酸鈣（amorphous calcium phosphate, ACP）被認為是氫氧基磷灰石的前驅物，兩者皆以奈米的大小存在於生物體內。在過去的研究中發現，人工合成的氫氧基磷灰石具有良好的生物相容性，而非晶性磷酸鈣則表現有較好的溶解度，生物降解性及生物活性，所以皆被廣泛應用在生醫材料的用途上。另外動物體內的鈣化組織除了有磷酸鹽之外，還包含有機物的膠原蛋白基質來做為礦化物的支架，也因此在組織工程的觀念裡面，除了氫氧基磷灰石之外，支架的材料也是研發的重點，而幾丁聚醣具有良好的生物相容性且其結構與膠原蛋白基質類似，所以利用幾丁聚醣（Chitosan, CS）與磷酸鈣進行結合，也是具有生醫潛力開發的材料。本研究團隊利用直接沉澱法成功研發出奈米級磷酸鈣(例如：HAP, ACP)與幾丁聚醣膜結合的製程，但是這些磷酸鈣的產物在膜上的分布情形、材料的生物相容性以及與細胞的交互作用行為目前仍不清楚。因此本研究的目的是分析奈米磷酸鈣顆粒於磷酸鈣-幾丁聚醣複合膜的分佈情況與材料性質，並且評估磷酸鈣結構對於牙髓細胞與骨細胞行為的影響。在磷酸鈣-幾丁聚醣複合膜製備方面，藉由改變磷酸根離子溶液與鈣離子溶液的反應順序以及反應環境條件來控制HAP與ACP產物的生成，先與磷酸根離子反應的製程為P-Ca系統，而先與鈣離子溶液反應的製程為Ca-P系統。使用X光繞射分析(XRD)與傅立葉轉換紅外線光譜儀(FTIR)來鑑定分析製備產物的結晶與官能基結構，使用SEM來觀察磷酸鈣的分佈與滲透情形，以萃取液的方式評估材料的毒性，並以直接接觸法評估材料對於骨細胞與牙髓細胞的細胞行為反應的評估。研究結果顯示，P-Ca系統在開放環境有CO2存在的情況下，可於幾丁聚醣膜上生成大小約100~200nm均勻分布的ACP(P-Ca)顆粒，而在密閉環境下則生成HAP(P-Ca)顆粒，滲透深度約為5~6μm。Ca-P系統所得的產物為HAP(Ca-P)，磷酸鈣滲透深度為7~9μm，Ca-P系統有較顯著的滲透深度，此結果可能與鈣離子比磷酸根離子小有關。在材料毒性測試的部分，幾丁聚醣膜以及磷酸鹽-幾丁聚醣膜皆沒有細胞毒性，都具有良好的生物相容性。在細胞貼附的部分，U2OS、MRPC-1及DPC在HAP組別有最好的表現，其細胞貼附數量與形態都略好於ACP組別；而CS組別則有最差的表現，細胞貼附數量最少且細胞都成現圓球狀。在細胞增生行為方面， CS組的細胞在第一天與第五天的生長數量沒有明顯的變化，增生倍率皆不到一倍，而HAP與ACP組的細胞增生速率都明顯比CS組快，其中HAP組又略優於ACP組，HAP組增生倍率約為一到兩倍，ACP組則約為一倍。此外，相較於細胞在細胞培養盤進行生長增生的行為，骨源性細胞（U2OS）在HAP組的細胞生長速率明顯較快，而牙髓細胞（MRPC-1, DPC）在HAP組的增生速率則有較低的現象。顯示磷酸鈣對於不同細胞對的增生行為反應有不同的影響效果，對於生長較緩慢的U2OS細胞，HAP具有促進生長的效果，而對於生長速度較快的DPC與MRPC-1細胞，HAP則有抑制其生長的效果。在細胞分化行為分析上， RT-PCR分析結果顯示HAP與ACP組都會促進U2OS細胞表現ALP與OPN，而有誘導細胞分化的效果。綜合本研究之結果，不同磷酸鈣結構的ACP與HAP所構成的磷酸鈣-幾丁聚醣複合膜都擁有良好的生物相容性，不具備細胞毒性。同時，ACP與HAP 均能促進牙髓細胞與骨細胞的貼附、增生與分化行為；其中HAP比起ACP，對牙髓細胞與骨細胞的行為功能有較為顯著的影響。

Calcium phosphate is the main inorganic component of mammals’ hard tissues and hydroxyapatite (Meleti et al.) is its primary structure. There are several substructures of hydroxyapatite in different kinds of hard tissues and amorphous calcium phosphate (ACP) is regarded as the primer of hydroxyapatite. Both biological HAP and ACP are in nano-scale. According to previous researches, artificial hydroxyapatite was biocompatible and amorphous calcium phosphate presented good solubility, biodegradability and bioactivity. Hence, ACP and HAP are widely used as biomaterials. In addition, the design of scaffold to mimic the collagen matrix for precipitation of mineral contents in biological hard tissue also play important role in tissue engineering. Since chitosan (CS) has good biocompatibility and similar structure to protein matrix, calcium phosphate combined with chitosan is considered as a potential biomaterial in bone regeneration. Therefore, our research team has successfully developed a new biomaterials composed of chitosan membrane and nano-sized ACP (CS-ACP) and HAP (CS-HAP) via direct precipitation method. However, the disperse pattern of the calcium phosphate products on the CS membrane, the biocompatibility of these materials and their interaction with cells are unclear. The purpose of this study is to investigate the distribution of nano-scaled ACP and HAP in CS membrane, and to evaluate the biocompatibility of CS-ACP and CS-HAP membranes and their influences on the behavior of bone and pulpal cells. The calcium phosphates with different morphology were produced in 2% chitosan (CS) membrane via direct precipitation method. The synthetic process of P-Ca was the CS membrane soaking in solution with phosphate ions first and then in solution with calcium ions. The synthetic process of Ca-P was the reaction via different order of calcium and phosphate solutions. Using XRD, FTIR and SEM as tools, we found ACP (P-Ca) and HAP (P-Ca) particles with the size of 100~200 nm were produced and distributed evenly on CS membrane surface with the depth of 5~6m in close and open system via P-Ca synthetic process, respectively. By Ca-P synthetic process, only HAP (Ca-P) particles were produced but with deeper in depth (7~9m) on CS membrane surface, which may be due to the smaller calcium ions have better ability to penetrate through CS membrane. All of Cs, CS-ACP and CS-HAP membranes presented no cytotoxity and good biocompatibility. In addition, U2OS, MRPC-1 and DPC all presented bested cell adhesion and proliferation behaviors in CS-HAP group and worst in CS group. Compared to the cells growing on culture plate, U2OS, with slow growth in culture plate, presented a higher cell growth rate in CS-HAP group. However, MRPC-1 and DPC, with faster growth in culture plate, presented different behavior with a lower cell growth rate in CS-HAP group. These findings implies that HAP regulates cells functions differently according to their ability in growth. By RT-PCR, we found CS-HAP and CS-ACP could enhance ALP and OCN expression of U2OS cells, which indicates both HAP and ACP may enhance differentiation and mineralization of bone cells. In general, both CS-ACP and CS-HAP presented good biocompability and could enhance the cell adhesion, proliferation and differentiation of pulpal and bone cells. Furthermore, HAP presented better effects on cell functions of pulpal and bone cells than ACP in vitro.