Shahmoradi Saeid Reza, Kabir Salmani Maryam, Soleimanpour Hamid Reza, Tavakoli Amir Hossein, Hosaini Kazem, Haghighipour Nooshin, Bonakdar Shahin
1Biomaterials and Tissue Engineering Department, Stem Cell Division, National Institute of Genetic Engineering and Biotechnology, Shahrak-e Pajoohesh, km 15, Tehran - Karaj Highway, Tehran, 1497716316 Iran.
2Iranian Tissue Bank, Imam khomani Hospital, University of Medical Sciences, Keshavarz Blvd, Tehran, 1419733141 Iran.
Tissue Eng Regen Med. 2018 Nov 17;16(1):69-80. doi: 10.1007/s13770-018-0164-4. eCollection 2019 Feb.
Articular cartilage damage is still a troublesome problem. Hence, several researches have been performed for cartilage repair. The aim of this study was to evaluate the chondrogenicity of demineralized bone matrix (DBM) scaffolds under cyclic hydrostatic pressure (CHP) .
In this study, CHP was applied to human bone marrow mesenchymal stem cells (hBMSCs) seeded on DBM scaffolds at a pressure of 5 MPa with a frequency of 0.5 Hz and 4 h per day for 1 week. Changes in chondrogenic and osteogenic gene expressions were analyzed by quantifying mRNA signal level of Sox9, collagen type I, collagen type II, aggrecan (ACAN), Osteocalcin, and Runx2. Histological analysis was carried out by hematoxylin and eosin, and Alcian blue staining. Moreover, DMMB and immunofluorescence staining were used for glycosaminoglycan (GAG) and collagen type II detection, respectively.
Real-time PCR demonstrated that applying CHP to hBMSCs in DBM scaffolds increased mRNA levels by 1.3-fold, 1.2-fold, and 1.7-fold ( < 0.005) for Sox9, Col2, and ACAN, respectively by day 21, whereas it decreased mRNA levels by 0.7-fold and 0.8-fold ( < 0.05) for Runx2 and osteocalcin, respectively. Additionally, in the presence of TGF-β1 growth factor (10 ng/ml), CHP further increased mRNA levels for the mentioned genes (Sox9, Col2, and ACAN) by 1.4-fold, 1.3-fold and 2.5-fold ( < 0.005), respectively. Furthermore, in histological assessment, it was observed that the extracellular matrix contained GAG and type II collagen in scaffolds under CHP and CHP with TGF-β1, respectively.
The osteo-inductive DBM scaffolds showed chondrogenic characteristics under hydrostatic pressure. Our study can be a fundamental study for the use of DBM in articular cartilage defects and lead to production of novel scaffolds with two different characteristics to regenerate both bone and cartilage simultaneously.
关节软骨损伤仍是一个棘手的问题。因此，已经开展了多项软骨修复研究。本研究的目的是评估脱矿骨基质（DBM）支架在循环静水压力（CHP）作用下的软骨形成能力。
在本研究中，将CHP以5MPa的压力、0.5Hz的频率、每天4小时施加于接种在DBM支架上的人骨髓间充质干细胞（hBMSC），持续1周。通过定量Sox9、I型胶原、II型胶原、聚集蛋白聚糖（ACAN）、骨钙素和Runx2的mRNA信号水平，分析软骨形成和成骨基因表达的变化。采用苏木精-伊红染色和阿尔辛蓝染色进行组织学分析。此外，分别使用二甲基亚甲蓝（DMMB）和免疫荧光染色检测糖胺聚糖（GAG）和II型胶原。
实时聚合酶链反应（PCR）表明，在第21天时，对接种在DBM支架上的hBMSC施加CHP分别使Sox9、Col2和ACAN的mRNA水平提高了1.3倍、1.2倍和1.7倍（<0.005），而Runx2和骨钙素的mRNA水平分别降低了0.7倍和0.8倍（<0.05）。此外，在存在转化生长因子-β1（TGF-β1，10ng/ml）的情况下，CHP使上述基因（Sox9、Col2和ACAN）的mRNA水平分别进一步提高了1.4倍、1.3倍和2.5倍（<0.005）。此外，在组织学评估中，观察到在CHP以及CHP联合TGF-β1作用下的支架中，细胞外基质分别含有GAG和II型胶原。
具有骨诱导性的DBM支架在静水压力下表现出软骨形成特性。我们的研究可为DBM在关节软骨缺损中的应用提供基础研究，并有助于生产出具有两种不同特性的新型支架，以同时再生骨和软骨。
