奈米化之鈣劑預防骨質疏鬆症之保健功效與安全性評估

Efficiency and Safety Evaluation of Nanosizing Calcium to Prevent Osteoporosis

許晉瑋(Chin-Wei Hsu)

指導教授 ： 張恒雄

中原大學/工學院/醫學工程研究所/碩士(2007年)

https://doi.org/10.6840/CYCU.2007.00661

摘要

在人口年齡結構日趨老化的現代社會裡，骨質疏鬆症是日益嚴重的大眾健康問題，在眾多預防與治療骨質疏鬆症方法中，以增加食物中鈣質攝取量，為最常被建議所使用的方法之ㄧ。然而傳統鈣劑溶解度低，不易被人體所吸收，因此本研究之目的在於探討，利用研磨方式將鈣劑奈米化，並進行其物化鑑定、生物安全性試驗與功效評估試驗。
製備出之奈米鈣劑，分別利用動態光散射粒徑分析儀、掃描式電子顯微鏡、原子力顯微鏡與X光粉末繞射分析儀，且探討鈣質可吸收率與生物利用率，及奈米化鈣劑之物化性質。在安全性評估方面，則進行7天急毒性試驗與28天亞急毒性試驗。在體外評估試驗，利用MTT及TRAP染色法以評估骨細胞活性與形態。在動物評估試驗中，將小鼠卵巢切除後，再分別投予傳統鈣劑與奈米鈣劑並添加維生素D3，於實驗6個月後，利用雙能量X光測定儀測定小鼠骨質密度變化；並測定血清中鹼性磷酸酵素之活性；而後，再作小鼠股骨組織切片染色分析，觀察骨質生理之變化。
實驗結果顯示，傳統鈣劑經研磨後所形成之水相奈米微粒，其平均粒徑可由2000∼3000nm降至200∼300nm，且經掃描式電子顯微鏡與原子力顯微鏡觀測得知，其形態呈現圓形，由X光粉末繞射分析儀得知，其晶格結構不受影響。且發現奈米鈣劑可吸收率與生物利用率高於傳統鈣劑。在7天急毒性試驗與28天亞急毒性試驗中，發現未造成ICR小鼠死亡，只能求得最大耐受量為2.34g/Kg/day。
在體外評估試驗，發現粒徑為200∼300nm，濃度為0.5mM與1.5mM之鈣劑有促進造骨細胞之存活率，但在濃度為2.5mM與5mM，對於造骨細胞存活率有下降之趨勢。在蝕骨細胞方面，發現不論是傳統鈣劑或是奈米鈣劑，濃度在1.5mM、2.5mM與5mM則會降低蝕骨細胞之生成數。
在動物功效評估試驗，顯示出共同投予傳統鈣劑與奈米鈣劑併用維生素D3，在骨密度的改善與股骨組織切片染色及血清中鹼性磷酸酵素之活性方面上，且奈米鈣劑更優於傳統鈣劑。
利用研磨技術可將傳統鈣劑製備為穩定的奈米鈣劑，安全性評估證實奈米鈣劑不具有毒性，鈣劑確有調節骨質代謝的效果，可能是藉由抑制蝕骨細胞的活性來降低骨質流失，且可活化造骨細胞，促進骨質生成；奈米鈣劑與維生素D3併用可能有更好的效果。

關鍵字

骨質疏鬆症 ； 蝕骨細胞 ； 造骨細胞 ； 奈米鈣

並列摘要

Osteoporosis was a serious public health problem in an aging society. Among various preventions and cures for osteoporosis, to take more rich-calcium food was a common recommendation. However, the dissolution of the traditional calcium complexs was much lower than the nanoscaled calcium complexs. It was a little difficult to be absorbed by human being absorption. The aim of this study was to determine the feasibility of using milling technology to formulate the nanoscaled calcium and evaluate its physics chemistry, safety, and efficiency.
The physical properties of the nanoscaled calcium complexs were studied using dynamic light scattering (DLS), scanning electron microscope (SEM), atomic force microscopy (AFM), and X-ray diffraction examination (XRD). The chemical properties of the nanoscaled calcium complexs were studied by absorbability and bioavailability. To verify safety of 7-day acute toxicity and 28-day feeding toxicity in vivo of ICR mice were carried out. In vitro study, the activity and morphology of osteoblasts and osteoclasts were evaluated by the methods of MTT assay and TRAP stain. In animal study, we used ovariectomized (OVX) mice as experimental model and examined the therapeutic efficacy of the traditional calcium complexs, the nanoscaled calcium complexs, and the combination of vitamin D3. Each group had six ICR mice and treated for 6 month. Bone mineral density (BMD) were measured by using dual energy x-ray absorptiometry (DEXA). ICR mice serum alkaline phosphatase (ALP) were measure to detect osteoblasts activity. Bone sections by histomophometry were also employed to determine the bone-physiology.
The traditional calcium complexs (mean size＝2000∼3000nm) were processed using milling technology to form the nanoscaled calcium complexs with a mean particle size of 200∼300nm. The nanoscaled calcium complexs were analyzed by SEM and AFM to understand its morphology. Its morphology was round. In addition, by XRD analyses, it was found that the crystallization of the nanoscaled calcium complexs was not affected by using milling technology. The absorbability and bioavailability in the group of nanoscaled calcium complexs were significantly higher than that of the traditional calcium complexs. There were no adverse or toxic effects in 7-day acute toxicity and 28-day feeding toxicity test in vivo. No ICR mice were dead within the period of observation. The maximum dose for the mice given intragastrically was more than 2.34g/Kg per day.
In vitro study, osteoblasts viability rate was increased at the concentration of 1.5mM of the nanoscaled calcium (mean size＝2000∼3000nm). The cell viability rate was decreased at the concentration of 2.5mM, and 5mM. The results showed that the traditional calcium complexs and the nanoscaled calcium had a positive effect on the osteoporosis by inhibiting the activity of osteoclasts number at 1.5mM, 2.5mM, and 5mM.
In animal study, feeding the combination of traditional calcium complexs, the nanoscaled calcium complexs, and vitamin D3 could reduce the bone loss according to BMD analysis and bone histomophometry. On the other hand, it could increase ALP in osteoblasts. The efficiency of the nanoscaled calcium complexs was much better than the traditional calcium complexs.
In conclusion, the traditional calcium complexs could be formulated as stable the nanoscaled calcium complexs using milling technology. And in vivo safety evaluations indicated that no toxic responses were observed in this study. Our results show that it can prevent bone resorption diseases by the promotion of osteoblasts activation (bone formation) and the prevention of osteoclasts activation (bone resorption). Combining the nanoscaled calcium complexs with vitamin D3 even have better effects.

並列關鍵字

osteoblasts ； the nanoscaled calcium ； osteoporosis ； osteoclasts

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