自癒性水膠搭配間葉幹細胞在骨修復之應用

Self healing Hydrogels Combined with Mesenchymal Stem Cells for Bone Repair

郭逸雯(Yi-Wen Kuo)

指導教授 ： 李文婷

中原大學/工學院/生物醫學工程研究所/碩士(2018年)

https://doi.org/10.6840/cycu201800794

摘要

骨骼是一種具有自我癒合能力的組織，然而，大面積的骨缺損、骨質疏鬆則須借助生物材料來幫助修復。本研究目的在製備可注射性的自癒性水膠，結合骨髓間葉幹細胞 (Mesenchymal stem cells, MSCs)，填補骨缺陷處，以提升骨修復治療的效果。首先，利用琥珀酸酐改質幾丁聚醣製備 N-琥珀酰幾丁聚醣 (N-succinyl chitosan, NSC)，並以高碘酸鈉氧化海藻酸鈉製備多醛基海藻酸鈉 (Oxidative alginate, OAlg) 做為交聯劑。將 NSC、OAlg 與 0、2.5 或 5.0 wt% 氫氧基磷灰石 (Hydroxyapatite, HAp) 混合，分別形成雙網絡水膠 NO、HA2.5 與 HA5。HA2.5 與 HA5 水膠都具有癒合效果，且可透過針筒注射出特定的形態。流變特性分析也證實三種水膠具有自癒性。掃描式電子顯微鏡 (Scanning electron microscopy, SEM) 影像分析顯示，水膠的孔徑大小 NO > HA2.5 > HA5。孔洞率量測結果顯示，HA5 > HA2.5 > NO。降解試驗結果顯示，降解率NO > HA2.5 > HA5。生物相容性部分，MSCs 培養於水膠上具有良好存活率；植入大鼠皮下組織切片觀察，此水膠生物相容性良好且具有降解性。硬骨分化方面，鹼性磷酸酶 (Alkaline phosphatase, ALP) 檢測結果顯示 HA5 > HA2.5 > NO，添加 HAp 可促進硬骨分化效果。脂肪分化方面，油紅-O (Oil red-O) 染色檢測結果顯示 NO > HA2.5 > HA5，應力值較弱的 NO 組具有較好的分化效果。將水膠移植入骨質疏鬆鼠的骨髓腔中進行治療，骨組織切片及電腦斷層掃描 (Micro computed tomography, μCT) 檢測結果顯示，水膠治療可使骨小樑的厚度增加、分散度下降及數量增加。綜合上述結果，本研究製備之水膠具有生物相容性、生物降解性且可改善骨質疏鬆症的骨質情況。

關鍵字

自癒性水膠 ； N-琥珀酰幾丁聚醣 ； 氧化海藻酸鈉 ； 骨髓間葉幹細胞 ； 硬骨分化

並列摘要

Bone is a tissue with self-healing ability. In the case of large bone defects and osteoporosis, biomaterials are required to help bone repair. The aim of this study was to prepare injectable self-healing hydrogels, and to encapsulate bone marrow mesenchymal stem cells (MSCs) to fill bone defects and increase the effect of bone repair. First, Succinic anhydride was used to modify chitosan to prepare N-succinyl chitosan (NSC). Sodium alginate (SA) was oxidized with sodium periodate to multi-aldehyde alginate (Oxidative aliginate, OAlg) as a cross-linker. NSC was mixed with OAlg and 0, 2.5 or 5 wt% hydroxyapatite (HAp) to yield double-network hydrogels of NO, HA2.5 and HA5. Both HA2.5 and HA5 had self-healing property and could be injected into the specific shape by syringe needle. The results of rheology analysis proved both hydrogel had self-healing behavior. The scanning electron microscopy (SEM) analysis showed that the pore size was NO > HA2.5 > HA5. The porosity was NO < HA2.5 < HA5. The results of degradation analysis showed that degradation rate was NO > HA2.5 > HA5. In the biocompatibility test, the MSCs cultured on hydrogels had good cell viability. In addition, the hydrogels were biocompatible and biodegradable when injected subcutaneously in a rat model. In the osteogenic differentiation experiment, alkaline phosphatase (ALP) assay showed that HA5 > HA2.5 > NO. The addition of HAp promoted the osteogenesis. In the adipogenesis differentiation experiment , oil red-O stain showed that NO > HA2.5 > HA5. The hydrogel with lower stiffness had good adipogenesis differentiation. The result of bone histochemistry analysis and micro computed tomography (μCT) showed the trabecular thickness, trabecular number increased and trabecular separation decreased when the hydrogels were transplanted into the bone marrow cavity of osteoporosis rats. In summary, the hydrogels were biocompatible, biodegradable and could improve the bone condition of osteoporosis.

並列關鍵字

self-healing hydrogel ； N-succinyl chitosan ； oxidative alginate ； bone marrow mesenchymal stem cell ； osteogenic differentiation

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