關節軟骨因缺乏神經纖維、血管及淋巴系統，自我修復及再生能力非常有限，而透過軟骨組織工程提供一暫時性支架，可以有效幫助其修復。水膠因含水率高有助於物質的代謝，且具有組織般的彈性，適合作為軟骨組織修復的支架，而作為組織工程的支架，必須具有良好的機械強度，避免植入體內後因無法承受外力而崩解，因此本實驗結合了高分子生物性材料及氫氧基磷灰石(Hydroxyapatite，HAP)形成複合材料，分別對軟、硬骨層做修復。HAP為一種天然磷灰石礦物，其化學性質及晶體結構和生物體的硬組織相似，具有良好的生物相容性、生物活性及骨傳導性，已廣泛應用於骨組織的修復。
為配合動物實驗之模型，達到軟、硬骨層分別修復的效果，本實驗將利用PEG、PCL形成三團共聚物PCL-PEG-PCL(命名為PEC)，並結合氫氧基磷灰石，設計出三層結構之材料：下層為HAP，對應硬骨層之修復；上層及中間層皆為PEC，上層對應軟骨層之修復，扮演細胞支架的角色，中間層則扮演幫助tidemark形成之角色，期望達到軟、硬骨層之間緊密結合但具有明顯區隔界線的目的。
由表面結構分析、體外降解、機械性質、基因表現、趨化性實驗等結果，決定出上層及中間層之PEC濃度以及下層之HAP濃度。結果顯示，PEC12.5%結構最致密、降解最慢，且對MC3T3有最佳的基因表現結果；PEC10%則對chondrocytes有最佳的基因表現結果。另外，PEC能夠改善HAP過於硬脆的特性，而HAP能夠提升整體材料之機械強度，其中以10%HAP有最顯著的效果。由HAP對MC3T3之趨化性實驗可得知，MC3T3在5%及10% nano-HAP的環境下，細胞遷移量最大，代表nano-HAP對MC3T3的趨化性佳。綜合以上結果，在動物實驗的材料選擇上，下層會選用機械強度最高且細胞趨化性最佳之10%HAP。中間層因期望區隔出軟、硬骨層間的界線，會選用降解較慢、孔洞結構較緻密、機械強度最高且對MC3T3基因表現較佳的PEC12.5%。而上層則會選用降解稍快、孔洞結構較鬆散卻又不失機械強度，且對軟骨細胞有較佳基因表現的PEC10%。
為了提高軟骨層的修復效率，並解決軟、硬骨層之修復速度不同的問題，本實驗結合了富含血小板之血漿(Platelet-Rich Plasma，PRP)，活化的血小板可以釋放出許多生長因子及其他生物活性分子，能夠有效促進組織癒合及調節OA病理學上之異常炎症過程。透過上層PEC10%與PRP之結合，並測試出具有最佳機械強度之成膠方式與混合比例，在植入動物體內時釋放出TGF-β1、EGF、PDGF-AB等生長因子，可以有效幫助軟骨修復。進一步以動物實驗12週後發現，三層結構並結合PRP之設計可以成功達到軟硬骨層同時修復並具有明顯區隔界線的目的。

Owing to the absence of nerve fiber, blood vessel and lymphatic system, articular cartilage cannot be repaired by itself like other tissue. Tissue engineering has been proposed for cartilage repair through a temporary scaffold. The cells can be encapsulated in the scaffold and gradually become tissue as the degradation of materials. Hydrogel is a suitable cartilage tissue engineering scaffold due to its high water content that can help nutrients transport. To withstand the external forces after implanting in the knee joint, the mechanical strength is important. Therefore, this experiment combines polymer biomaterials with hydroxyapatite (HAP) to form the composite materials which can repair the cartilage and osteochondral layer, respectively. HAP is a naturally occurring mineral form of calcium apatite whose chemical properties and crystal structure are similar to those of hard tissue in the living body. It has good biocompatibility, bioactivity and osteoconductivity, and is commonly used for bone repair.
In order to match the animal experiment that can repair the cartilage and osteochondral layer respectively, this experiment combines triblock copolymer, PCL-PEG-PCL (named PEC), with HAP. According to the concept of multilayer, the three layers structure is designed. The lower layer is HAP, corresponding to the repair of the osteochondral layer. The upper layer and the middle layer are pure PEC. Chondrocytes are encapsulated in the upper layer which plays the role of scaffold, and the middle layer will be expected to help to form the tidemark that cartilage and osteochondral layers are tightly bonded but have distinct boundaries.
PEC concentration of the upper and middle layers and the HAP concentration of the lower layer were determined by the results of in vitro degradation, mechanical properties and gene expression, etc. The results showed that the slowest degradation and the best gene expression for MC3T3 are PEC12.5% and PEC10% has the best gene expression for chondrocytes. In addition, PEC can improve the brittle characteristic of HAP, while HAP can enhance the mechanical strength of material. Among the three concentration of HAP, 10% HAP has the significant effect; moreover, 5% and 10% nano-HAP have better promoting chemotactic effects on MC3T3 in the chemotactic experiment. Based on the results, 10% HAP will be used in the lower layer. PEC12.5% will be used in the middle layer which has the most dense pore structure, the highest mechanical strength and the best gene expression of MC3T3. PEC10% will be used in the upper layer which has the better gene expression of chondrocytes and a certain level of mechanical strength.
PRP, which means platelet-rich plasma, will release many growth factors and other bioactive molecules by activated platelets. PRP can effectively promote tissue healing and regulate abnormal inflammatory processes in OA pathology. In order to improve the efficiency of cartilage repair and solve the problem of different repair efficiency between cartilage and subchondral bone layers, this experiment combines upper layer with PRP. The best gel forming methods and mixing ratio between PEC10% and PRP would be tested. By releasing TGF-β1, EGF, PDGF-AB and the other growth factors in vivo can help cartilage repair effectively. After 12 weeks of animal testing, the design of three-layer structure combined with PRP can successfully repair the cartilage and osteochondral layers at the same time and have distinct boundaries.

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