以透明質酸攜帶膠原蛋白酶添加氫氧基磷灰石和半水硫酸鈣作為支架應用於齒槽骨再生之研究

Abstract

牙周病是現代社會普及的口腔疾病，若無正確的潔牙方式和定期檢查是很難控制牙菌斑的成長，進一步會侵犯牙齦下方的齒槽骨和牙周韌帶，隨著被破壞的程度加深，使牙齒漸漸鬆脫。臨床醫生希望藉由病患齒槽骨的修復，提高植牙的成功效率。近年來，利用生醫材料包覆生長因子的相關研究也發展快速，但生長因子的不穩定性以及價格仍是使用率不高的原因。因此我們希望藉由初級培養上常用的膠原蛋白酶短時間的作用，並且使組織間的正常細胞游離出來。 本研究以透明質酸攜帶膠原蛋白酶並添加氫氧基磷灰石和半水硫酸鈣的複合材料作為實驗主要材料。材料特性方面，分別測量硬化時間、抗壓強度和膠原蛋白酶釋放曲線。體外測試方面，首先會以纖維母細胞測試材料的生物相容性並於活體外測試，用新生大鼠頭蓋骨器官培養方式觀察短時間內骨組織與材料之間的作用關係。體內測試方面，則是於大鼠齒槽骨缺損部位植入複合材料，評估實際骨缺損的修復效果。複合材料的初始硬化時間為21分鐘，在臨床上有足夠的操作時間。浸泡在磷酸緩衝溶液中的複合材料七天內的抗壓強度均可維持在6.5 MPa左右，並在兩週後有下降的趨勢。膠原蛋白酶的釋放在30分鐘內可測得60 %的釋放量，達到初期大量釋放的效果，符合一般初級培養上膠原蛋白酶的所需的作用時間。生物相容性測試結果顯示複合材料並不會對纖維母細胞株的增生或存活造成影響。而動物實驗方面，攜帶膠原蛋白酶的複合材料於十二週後，相對於其他組別包括無膠原蛋白酶組別和市售材料Bio-ossR皆有明顯良好的骨新生作用。因此，由結果顯示膠原蛋白酶確實能使細胞從細胞外基質游離出參予修復。而未來可以更進一步以犬隻作為動物實驗的模型，確認此複合材料對於齒槽骨再生的效果。

With the global prevalence of periodontitis which leads to irreversible bone loss, the regeneration of bone tissue poses a challenge to engineers and clinicians. The importance of alveolar bone cannot be overemphasized since it is one of the tooth-supporting structures. In the past few years, the research of growth factor for alveolar bone regeneration is blooming, but still remains controversial due to its instability and high cost. Instead of using growth factors, we hypothesized that cells could be recruited from extracellular matrix for bone repair by collagenase treatment without any autologous grafts. Hence, the purpose of this study is to evaluate the bone regenerative capacity after the implantation of hydroxyapatite and calcium sulfate (gypsum) with hyaluronic acid carrying collagenase in the rat model. In the study, the setting time, compressive strength, release characteristics and biocompatibility of Hyaluronic acid/Collagenase/Hydroxyapatite/Calcium sulfate were evaluated. The potential for reconstruction of destroyed alveolar bone by the implanted composite was also tested in rat model. The initial setting time of the composite was 21 minutes which is adequate for clinicians to handle. The compressive strength of composites after immersion in phosphate buffered saline solution in 7 days was 6.5MPa, and decreased after 2 weeks. The release profile shows 60 % of collagenase incorporated in the composite was released in 30 minutes that coincides with the time of collagenase digestion normally used in primary culture. The good biocompatibility of the composite was also confirmed by WST-1 test which indicates no significant difference between control group and experimental group. In animal study, the result of the composite HA/Collagenase/HAP/CS in 12-week after surgery was superior to other groups including commercial product Bio-OssR. Evidence of obvious regeneration in bone defects treated with HA/Collagenase /HAP/CS can be observed in micro-CT images histological results. Then we can conclude that it might be feasible for collagenase treatment to recruit more cells during healing process. In the future, the study can be applied to canines as an animal model for further confirmation.