Preparation of Melatonin-Loaded Chitosan/Calcium Phosphate Ceramics Complex Particles by Ph-Mediated Precipitation Method for Guiding Bone Regeneration

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Project title

Code/計畫編號

MOST104-2221-E019-017-MY3

Translated Name/計畫中文名

利用pH介導沈澱法製備攜帶褪黑激素之幾丁聚醣/磷酸鈣陶瓷複合微粒引導骨再生

Project Coordinator/計畫主持人

Yi-Cheng Huang

Funding Organization/主管機關

National Science and Technology Council

Department/Unit

Department of Food Science

Website

https://www.grb.gov.tw/search/planDetail?id=12019739

Year

2017

Start date/計畫起

01-08-2017

Expected Completion/計畫迄

01-07-2018

Bugetid/研究經費

850千元

ResearchField/研究領域

醫學工程

"本計畫書擬以pH值介導沈澱法(pH-mediated precipitation method) 將褪黑激素(melatonin) 與幾丁聚醣(chitosan) 共沈澱於磷酸鈣陶瓷(calcium phosphate ceramics) 微粒，製備攜帶褪黑激素之幾丁聚醣/磷酸鈣陶瓷複合材料，開發成具有骨誘導(osteo-inductive) 特性的骨填補材料，探討其對於骨細胞生長、分化潛能的影響，分析成骨蛋白的基因表現，並以動物實驗評估此材料在臨床應用的可行性。申請者藉由過去的國科會補助計畫：以幾丁聚醣製備奈米級傳輸載體，應用於釋放基質細胞衍生因子調控幹細胞的遷移、釋放鹼性纖維母細胞生長因子幫助神經組織修復，得到令人雀躍的結果，成果亦持續發表中。然而，由於幾丁聚醣的機械強度不足，在硬組織的修復常受到限制。磷酸鈣陶瓷是一個大量存在生物體的骨頭與牙齒內的無機物，已廣泛應用於骨再生的研究，但因僅具有骨傳導(osteo-conductive) 的特性而限制其在臨床上的應用。本計畫製備幾丁聚醣/磷酸鈣陶瓷複合微粒，結合兩種材料的特性，並透過簡易的製備流程使其攜帶褪黑激素，開發具有骨誘導特性的骨填材。本計畫結合申請者先前的研究成果及骨組織修復、骨誘導特性等問題的探討，以期對臨床醫學中骨組織的修復及再生有所助益。在此專題研究計畫中，第一年：攜帶褪黑激素之幾丁聚醣/磷酸鈣陶瓷複合微粒的製備及性質探討。以逆微乳化法合成奈米級/微米級的磷酸鈣陶瓷微粒，再利用磷酸鈣陶瓷及幾丁聚醣溶解度隨pH值改變的特性，以磷酸鈣陶瓷微粒為基板，將幾丁聚醣及褪黑激素共同沉澱於磷酸鈣陶瓷微粒表面，形成具有骨誘導特性的骨填補材料。將尋找最佳的實驗參數，製備穩定的幾丁聚醣/磷酸鈣陶瓷複合微粒，分析複合微粒對褪黑激素之包覆率、裝載量及釋放曲線，並討論攜帶及釋放的可能機制。第二年：延續第一年的實驗結果，探討攜帶褪黑激素之幾丁聚醣/磷酸鈣陶瓷複合微粒對細胞的影響。以小鼠的成骨前驅細胞(Pre-osteoblast) MT3C3E1 細胞株為細胞模式，進行生物相容性測試；以鹼性磷酸酶(Alkaline phosphatase) 活性測試、礦化測試(Mineralization)、Alizarin red S 染色及掃描式電子顯微鏡觀察等，評估此材料誘導骨分化的潛能；透過real-time PCR 評估此材料是否能有效刺激成骨蛋白的基因表現。第三年：建立SD (Sprague Dawley) 大鼠下頷骨損傷人工材料植入的動物實驗模式，作骨再生的評估。透過大鼠進食、體重變化、組織切片分析及活體動物斷層掃描儀(Micro-CT) 等，分析骨損傷修復的情況。預期此研究成果，能引導骨再生並加速骨組織修復的成效，並實際應用於臨床醫學。" "In the current proposal, we will co-precipitate melatonin (Mel) and chitosan (CS) on calcium phosphate ceramics (CPC) particles using pH-mediated precipitation method. Try to develop Mel-loaded CS/CPC complex as osteo-inductive bone-filling material. This study will evaluate the effect of Mel-loaded CS/CPC complex on cells, including cell proliferation, cell differentiation, and the gene expression of osteogenic proteins. The clinic application will also be assessed by in vivo animal experiments. In the post investigations, the applicant has developed CS nanoparticles as nano-carriers. The stromal cell-derived factor-1 released from CS nanoparticles can mobilize mesenchymal stem cells. The basic fibroblast growth factor released from CS nanoparticles is effective on neurite extension. However, using CS for hard tissue repair is limited by its weak mechanical strength. Calcium phosphate ceramics, inorganic compounds abounded in bone and teeth of organisms, have been widely used in bone regeneration. However, the clinical useage of CPC is limited because CPC has only osteo-conductive property. This proposal is to create CS/CPC complex particles by combining the properties of these two materials and then load them with Mel for developing osteo-inductive bone-filling material. Combination the previous experimental results with the challenge of bone regeneration and osteo-induction, we hope the results of this project would be helpful for clinical treatment of bone repair and regeneration. This project includes three-year research program. In the first year, we will discuss the preparation and physico-chemical properties of CS/CPC particles. Nano-/micro- scale CPC particles will by synthesized by reverse micro-emulsion method. Due to the effect of pH value on dissolubility of CS and CPC, we use CPC particles as templates and co-precipitate CS and Mel on them. We will try to fabricate stable CS/CPC complex carriers by optimizing the experimental parameters and conditions. Then, analyze and discuss the release behavior of Mel from CS/CPC carriers, such as encapsulation efficiency, loading content, and release profile and release mechanism. In the second year, the prepared Mel-loaded CS/CPC particles of the first year will be used to observe their effects on cells. The Pre-osteoblast MT3C3E1 cells will be used as a cell model. Cell biocompatibility, cell osteogenic potential and the gene expression of osteogeinc proteins will be examined by MTT assay, alkaline phosphatase assay, Alizarin red S staining and real-time PCR. In the third year, we will establish the bone-filling animal mandible model of SD (Sprague Dawley) rat for bone regeneration evaluation. The rehabilitation will be analyzed by rat eating, rat weight, histological analysis and micro-CT images. We expect that osteo-inductive Mel-loaded CS/CPC complex material could be effective in bone regeneration."

Keyword(s)

幾丁聚醣
磷酸鈣陶瓷
褪黑激素
pH-介導沈澱法
骨組織再生
chitosan
calcium phosphate ceramics
melatonin
pH-mediated precipitation method
bone regeneration

DSpace CRIS

Preparation of Melatonin-Loaded Chitosan/Calcium Phosphate Ceramics Complex Particles by Ph-Mediated Precipitation Method for Guiding Bone Regeneration

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