He Shukun, Hu Ruonan, Yao Xuan, Cui Jing, Liu Huimin, Zhu Min, Ning Liangju
Department of Orthopedic Surgery and Orthopedic Research Institute, Stem Cell and Tissue Engineering Research Center, State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, Chengdu, 610041, China.
Department of Orthopedics, First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou, 310058, China.
Regen Biomater. 2024 Sep 25;11:rbae116. doi: 10.1093/rb/rbae116. eCollection 2024.
Recent studies have indicated that demineralized cortical bone (DCB) may be used to repair tendons and ligaments, such as the patellar tendon and anterior cruciate ligament (ACL). Hydrogen peroxide (HO) has been shown to reduce the osteoinductivity of DCB, and heat treatment may also decrease the osteoinductivity of DCB. The purpose of this study was (i) to determine whether heat treatment reduces the osteoinductivity of DCB and (ii) to compare the effectiveness of heat treatment and HO treatment on BMP-2 inactivation. DCB was prepared by immersion in 0.6 N hydrochloric acid, and DCB-H and DCB-HO were prepared by heat treatment (70°C for 8 h) and HO treatment (3% HO for 8 h), respectively. The surface topographies, elemental distributions and histological structures of the scaffolds were observed by scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FT-IR) and histological staining. The viability and osteogenic differentiation of TDSCs cultured on the scaffolds were evaluated live/dead cell staining and Cell Counting Kit-8 (CCK-8) testing, real-time polymerase chain reaction (RT-PCR) and western bolt (WB) analysis, alkaline phosphatase activity (ALP) and alizarin red S (ARS) staining. The intramuscular implantation of the scaffolds in rats was also used to evaluate the effect of heat treatment and HO treatment on the osteoinductivity of DCB. Our results demonstrated that both treatments removed BMP-2 and osteocalcin (OCN) within the DCB and that DCB-H and DCB-HO had good cytocompatibility and reduced the osteogenic differentiation of TDSCs. Moreover, the results indicated that the DCB-H and DCB-HO groups had smaller areas of osteoid formation than did the DCB group, and the DCB-HO group had the smallest area among the three groups. Our study demonstrated that heat treatment could reduce the osteoinductivity of DCB, and that HO treatment was more effective than heat treatment.
近期研究表明，脱矿皮质骨（DCB）可用于修复肌腱和韧带，如髌腱和前交叉韧带（ACL）。过氧化氢（HO）已被证明可降低DCB的骨诱导活性，热处理也可能降低DCB的骨诱导活性。本研究的目的是：（i）确定热处理是否会降低DCB的骨诱导活性；（ii）比较热处理和HO处理对骨形态发生蛋白-2（BMP-2）失活的效果。通过将其浸入0.6 N盐酸中来制备DCB，分别通过热处理（70°C，8小时）和HO处理（3% HO，8小时）制备DCB-H和DCB-HO。通过扫描电子显微镜（SEM）、傅里叶变换红外光谱（FT-IR）和组织学染色观察支架的表面形貌、元素分布和组织结构。通过活/死细胞染色和细胞计数试剂盒-8（CCK-8）检测、实时聚合酶链反应（RT-PCR）和蛋白质免疫印迹（WB）分析、碱性磷酸酶活性（ALP）和茜素红S（ARS）染色来评估在支架上培养的肌腱衍生干细胞（TDSCs）的活力和成骨分化。还通过将支架肌肉内植入大鼠来评估热处理和HO处理对DCB骨诱导活性的影响。我们的结果表明，两种处理均去除了DCB中的BMP-2和骨钙素（OCN），并且DCB-H和DCB-HO具有良好的细胞相容性，并降低了TDSCs的成骨分化。此外，结果表明，DCB-H组和DCB-HO组的类骨质形成面积比DCB组小，且DCB-HO组在三组中面积最小。我们的研究表明，热处理可降低DCB的骨诱导活性，且HO处理比热处理更有效。
