Shalumon K T, Sheu Chialin, Fong Yi Teng, Liao Han-Tsung, Chen Jyh-Ping
Department of Chemical and Materials Engineering, Chang Gung University, Kwei-San, Taoyuan 33302, Taiwan.
Department of Plastic and Reconstructive Surgery and Craniofacial Research Center, Chang Gung Memorial Hospital, Kwei-San, Taoyuan 33305, Taiwan.
Polymers (Basel). 2016 Dec 10;8(12):429. doi: 10.3390/polym8120429.
This study aims to prepare biphasic osteochondral scaffolds based on seamless joining of sintered polymer and polymer/ceramic microspheres for co-culture of chondrocytes and bone marrow stem cells (BMSCs). Poly(lactide--glycolide) (PLGA) microspheres and 10% nanohydroxyapatite (nHAP)-incorporated PLGA (PGA/nHAP) microspheres were prepared through the oil-in-water precipitation method. Virgin (V) and composite (C) scaffolds were prepared from 250⁻500 µm PLGA and PLGA/nHAP microspheres, respectively, while osteochondral (OC) scaffolds were fabricated through the combination of V and C scaffolds. Physico-chemical properties of scaffolds were characterized through microscopic-spectroscopic evaluations. The effect of nHAP in scaffolds was investigated through thermogravimetric analysis and mechanical testing, while surface hydrophobicity was tested through contact angle measurements. Rabbit chondrocytes and BMSCs were used for cell culture, and cell morphology and proliferation were determined from SEM and DNA assays. Alizarin red and Alcian blue stains were used to identify the in vitro bone and cartilage tissue-specific regeneration, while cetylpyridinium chloride was used to quantitatively estimate calcium in mineralized bone. For co-culture in OC scaffolds, BMSCs were first seeded in the bone part of the scaffold and cultured in osteogenic medium, followed by seeding chondrocytes in the cartilage part, and cultured in chondrocyte medium. High cell viability was confirmed from the Live/Dead assays. Actin cytoskeleton organization obtained by DAPI-phalloidin staining revealed proper organization of chondrocytes and BMSCs in OC scaffolds. Immunofluorescent staining of bone (type I collagen and osteocalcin (OCN)) and cartilage marker proteins (type II collagen (COL II)) confirmed cellular behavior of osteoblasts and chondrocytes in vitro. Using an ectopic osteochondral defect model by subcutaneous implantation of co-cultured OC scaffolds in nude mice confirmed cell proliferation and tissue development from gross view and SEM observation. IF staining of OCN and COL II in the bone and cartilage parts of OC scaffolds and tissue-specific histological analysis exhibited a time-dependent tissue re-modeling and confirmed the potential application of the biphasic scaffold in osteochondral tissue engineering.
本研究旨在制备基于烧结聚合物与聚合物/陶瓷微球无缝连接的双相骨软骨支架，用于软骨细胞和骨髓干细胞（BMSCs）的共培养。通过水相沉淀法制备了聚（丙交酯-乙交酯）（PLGA）微球和掺入10%纳米羟基磷灰石（nHAP）的PLGA（PGA/nHAP）微球。分别由250⁻500 µm的PLGA和PLGA/nHAP微球制备原始（V）支架和复合（C）支架，而骨软骨（OC）支架则通过V支架和C支架的组合制成。通过微观光谱评估对支架的物理化学性质进行表征。通过热重分析和力学测试研究了支架中nHAP的作用，同时通过接触角测量测试了表面疏水性。使用兔软骨细胞和BMSCs进行细胞培养，并通过扫描电子显微镜（SEM）和DNA检测确定细胞形态和增殖情况。采用茜素红和阿尔新蓝染色鉴定体外骨和软骨组织特异性再生，而用十六烷基吡啶氯化物定量估计矿化骨中的钙。对于在OC支架中共培养，首先将BMSCs接种在支架的骨部分并在成骨培养基中培养，然后将软骨细胞接种在软骨部分，并在软骨细胞培养基中培养。通过活/死检测证实了高细胞活力。通过DAPI-鬼笔环肽染色获得的肌动蛋白细胞骨架组织显示OC支架中软骨细胞和BMSCs的组织良好。骨（I型胶原蛋白和骨钙素（OCN））和软骨标记蛋白（II型胶原蛋白（COL II））的免疫荧光染色证实了体外成骨细胞和软骨细胞的细胞行为。通过在裸鼠皮下植入共培养的OC支架建立异位骨软骨缺损模型，从大体观察和SEM观察证实了细胞增殖和组织发育。OC支架的骨和软骨部分中OCN和COL II的免疫荧光染色以及组织特异性组织学分析显示了时间依赖性的组织重塑，并证实了双相支架在骨软骨组织工程中的潜在应用。
