研發治療退化性關節炎之可關節內注射之控制釋放型PTH(1-34)玻尿酸水膠

Development of the intra-articular injectable controlled-release PTH(1-34)/HA-MA hydrogel for osteoarthritis treatment

王雪君(Hsueh-Chun Wang)

指導教授 ： 何美泠

高雄醫學大學/醫學院/生理及分子醫學研究所/碩士(2011年)

摘要

中文摘要
引言
退化性關節炎(osteoarthritis, OA)是一種容易發生在負重關節的退化性疾病。當關節炎發生時，關節軟骨逐漸流失細胞外間質(extracellular matrix)，主要為第II型膠原蛋白與葡萄糖胺聚醣(glycosaminoglycan, GAG)。由於造成退化性關節炎的原因以及機制很複雜，故截至目前為止，對於退化性關節炎的治療著重於消炎及解除症狀，但若從致病機制作早期治療，臨床上尚未具有效的治療辦法。目前在細胞學的研究上發現，在退化性關節炎發生時，成熟的軟骨細胞會走向終止分化(terminal differentiation)，同時表現出第X型膠原蛋白並逐漸走向凋亡，此一現象與胚胎期或生長板之軟骨細胞進行軟骨內骨化的情況類似。研究指出副甲狀腺素相關胜肽(parathyroid hormone related peptide, PTHrP)於軟骨內骨化過程中具有抑制軟骨細胞肥大，抑制終止分化，並維持軟骨細胞軟骨化 (chondrogenesis) 的能力。副甲狀腺素(parathyroid hormone, PTH)與PTHrP兩者均可透過PTH接受器1型(PTHR1)調控細胞內訊息活化蛋白激酶A (protein kinase A, PKA)及蛋白激酶C (protein kinase C, PKC)的訊息傳遞路徑。根據本實驗室先前的研究指出，每三天關節內施打一次PTH(1-34)，能有效抑制關節軟骨細胞走向終止分化，以及抑制退化性關節炎進程。然而，每三天施打一次藥劑之注射次數仍太頻繁，為了減少注射頻率，本實驗室先前已研發每15天注射一次之可關節內注射的PTH(1-34)微小球，可抑制油papain 所誘發的退化性關節炎之進程。但PLGA為一種合成的聚合物，本研究欲研發一種天然衍生物玻尿酸作為基質，玻尿酸水膠為軟骨細胞外基質內最主要的醣類並且富含於滑膜液(synovial fluid)當中，而關節內注射玻尿酸已在臨床上被使用來治療OA的症狀。基於以上的理由，我們假設經過交聯後的玻尿酸聚合物可當作藥物載體並可持續釋放PTH(1-34)來治療退化性關節炎。

方法
活體外研究：PTH(1-34)載體為以高分子量的玻尿酸(Hyaluronic acid)聚合水膠做為基材，利用光交聯法，製成HA-MA(HA-Methacrylate)，將PTH(1-34)包覆於HA-MA玻尿酸水膠中(PTH/HA-MA水膠)，觀察其物理化學特性：(1)使用掃描式電子顯微鏡(Scanning Electron Microscope)觀察水膠內部構造；(2)測試水膠的膨脹率(Swelling ratio)了解水膠的親水性質；(3)利用PTH ELISA來測試水膠包覆藥物的能力(encapsulation efficiency)；(4)並於活體外模擬體液的條件 (0.9% NaCl+10unit/ml hyaluronidase)下，觀察此水膠之藥物釋放曲線，隨後測試藥物於釋放後之活性。活性測試方法：將自 PTH(1-34)/HA-MA水膠中釋放出之PTH(1-34)處理於骨母細胞(MC3T3E1)，並以ELISA定量MC3T3E1所生成之環單磷酸腺甘 (cAMP)。
活體內研究：測試PTH/HA-MA水膠對於退化性關節炎的治療效果，是利用12週齡的大鼠 (Sprague Dawley Rat) 膝關節給予Papain注射進入膝關節腔中以誘發退化性膝關節炎，注射頻率為每三天一次，共三次；隨後給予PTH(1-34) 10nM每三天一次及PTH/HA-MA水膠 (PTH:0.4ug)每10天一次共四次，實驗週數為5週。評估方式是利用Safranin-O-Fast green 染色，觀察老鼠膝關節軟骨之氨基葡萄糖 (glycosaminoglycans, GAG)；利用免疫染色標定法觀察第二型膠原蛋白 (collagen type II, COL. II) 及第十型膠原蛋白 (collagen type X, COL. X) 在組織定位之變化。

結果
活體外之結果顯示PTH/HA-MA水膠經電子掃描式顯微鏡觀測後結果顯示，內部有似蜂窩狀的結構，並且有孔洞可以讓水分通透擴散，隨著時間的推移，玻尿酸水膠會逐漸產生降解的現象。致於HA-MA對PTH(1-34)之包覆率平均可達到90.05% (對BSA之包覆率平均可達到99.1%)，並能持續穩定釋放至少12天，其釋放濃度介於4.3~12.3nM；並且自PTH/HA-MA玻尿酸水膠中所釋放出的PTH(1-34)能有效的刺激MC3T3E1產生cAMP，因此證實PTH/HA-MA玻尿酸水膠中釋出的PTH(1-34)仍具有生物活性。
活體內之結果顯示，目前初步動物實驗已完成，並使用Safranin-O-Fast green 染色，觀察老鼠膝關節軟骨之氨基葡萄糖 (glycosaminoglycans, GAG)；利用免疫染色標定法觀察第二型膠原蛋白 (collagen type II， COL. II)及第十型膠原蛋白 (collagen type X， COL.X) 在組織定位之變化，並利用TUNEL stain觀察細胞凋亡的情形。

結論
本研究結果指出，利用光交聯法所製成的PTH(1-34)/HA-MA玻尿酸水膠，能有效作為PTH(1-34)的藥物載體，並已證實從PTH/HA-MA玻尿酸水膠中釋放出的PTH(1-34)仍具有生物活性；經由動物實驗結果顯示，每10天注射PTH(1-34)/HA-MA玻尿酸水膠共3次可以和每3天注射一次共11次PTH(1-34)達到類似的效果，未來還會繼續進行COL. II 、COL.X的免疫染色標定法及TUNEL stain進一步分析PTH(1-34)/HA-MA玻尿酸水膠對於經由papain所誘發的退化性關節大鼠身上的治療效果，並且已重複第二次動物實驗，來證實實驗的結果。

關鍵字

玻尿酸 ； 副甲狀腺素 ； 退化性關節炎

並列摘要

英文摘要
Objectives
Osteoarthritis (OA) is a degenerative joint disease and easily to decrease glycosaminoglycan (GAG) and collagen levels, but the mechanism remains unclear. During OA progession, articular chondrocytes undergo terminal differentiation and decrease level of GAG and COL. II, but increase level of COL. X, which are similar as those during endochondral ossification. Previous studies showed that parathyroid hormone-related peptide (PTHrP) can suppress hypertrophy and terminal differentiation of chondrocytes. Previous studies also indicated that PTHrP and parathyroid hormone (PTH) share the same receptor, PTHR1, and stimulate activities of adenylate cyclase (PKA pathway) and phospholipase-C (PKC pathway). Our previous finding emphasized that PTH(1-34) can suppress the progress of terminal differentiation in cultured human articular chondrocytes (HACs) and the papain-induced OA in rats. The previous results showed that intra-articular injection of PTH(1-34) once every 3 days suppressed chondrocyte terminal differentiation in OA cartilage and in turn suppressed the progression of OA; however, the treatment strategy is with too frequent injection. For increasing the injection interval, the drug carrier of PTH(1-34) has been investigated. We previously found that once in 15 days intra-articular injection of controlled-release PLGA microspheres carrying PTH(1-34) suppressed the progression of papain induced OA. Other than the synthetic polymer PLGA, hyaluronan (HA) is a major non-protein extra-cellular matrix of cartilage and is abundant in synovial fluid. Intra-articular injection of HA has been used for reducing OA symptom clinically. Accordingly, we hypothesized that coss-linked HA polymer may be used as a carrier to sustainably release PTH(1-34) for OA treatment.

Methods
In this study, we developed a photocrosslinked HA-methacrylate (HA-MA) hydrogel to carry PTH(1-34), and studied the effect of PTH(1-34)/HA-MA on papain induced OA rats. The morphology of HA-MA was observed by scanning electron microscopy (SEM). The PTH(1-34) encapsulation efficiency and release profile were examined. The bioactivity of released PTH(1-34) was tested by examining cAMP levels in MC3T3-E1 cells. In vivo study, we studied the effect of PTH(1-34)/HA-MA hydrogel on papain-induced OA and non-OA groups. The histologic changes in GAG, were evaluated with Safranin-O-Fast green stain .

Results
The SEM observation emphasized that there are porous in the inner structure of HA-MA hydrogel, indicating it encapable to encapsulate PTH(1-34), and can be diffused by fluid. The HA-MA hydrogel can efficiently encapsulate in average of 90.05% of PTH(1-34), and the PTH released from HA-MA hydrogel was detectable through 2 weeks at constant rate. The bioactivity data showed that the released PTH(1-34) from HA-MA hydrogel increase the cAMP level in MC3T3E1 cells, indicating the released PTH(1-34) was bioactive. In vivo results, in non-OA cartilages showed no significant differences in GAG expressions among the control group. In the papain induced OA cartilages, HA-MA and PTH(1-34)/HA-MA treatment were capable to papain-induced OA changes by increasing GAG levels in the knee cartilage of rats.

Conclusion
The result in this study showed that the feasibility of achieving controlled local delivery of PTH(1-34) and maintain their constant concentration during 12 days with bioactivity. In vivo study, the injection of PTH(1-34)/HA-MA hydrogel every 10 days has the similar effect with the injection of PTH(1-34) every 3 days. In future, we will keep finishing IHC stain for COL. II 、COL.X and TUNEL stain.

並列關鍵字

HA ； PTH(1-34) ； Osteoarthritis

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