Institute of Orthopedics, Fourth Medical Center of the General Hospital of CPLA, 100048, Beijing, PR China.
Beijing Engineering Research Center of Orthopaedic Implants, 100048, Beijing, PR China.
J Mater Sci Mater Med. 2022 Jul 15;33(7):58. doi: 10.1007/s10856-022-06678-z.
We mainly proceed from the view of biological effect to study the acellular bovine bone matrix (ABBM) by the low concentration of hydrogen oxidation. After cleaning the bovine bone routinely, it was cleaned with different concentrations of NaOH and stained with hematoxylin-eosin (HE) to observe the effect of decellulization. The effect of bovine bone matrix treated with NaOH were observed by optical microscopy and scanning electron microscopy (SEM), and compared by DNA residue detection. Cell toxicity was also evaluated in MC3T3-E1 cells by CCK-8. For the in vitro osteogenesis detection, alkaline phosphatase (ALP) staining and alizarin red (AR) staining were performed in MC3T3-E1 cells. And the in vivo experiment, Micro CT, HE and Masson staining were used to observe whether the osteogenic effect of the materials treated with 1% NaOH solution was affected at 6 and 12 weeks. After the bovine bone was decellularized with different concentrations of NaOH solution, HE staining showed that ultrasonic cleaning with 1% NaOH solution for 30 min had the best effect of decellularization. The SEM showed that ABBM treated with 1% NaOH solution had few residual cells on the surface of the three-dimensional porous compared to ABBM treated with conventional chemical reagents. DNA residues and cytotoxicity of ABBM treated with 1% NaOH were both reduced. The results of ALP staining and AR staining showed that ABBM treated with 1% NaOH solution had no effect on the osteogenesis effect. The results of micro-CT, HE staining and Masson staining in animal experiments also showed that ABBM treated with 1% NaOH solution had no effect on the osteogenesis ability. The decellularization treatment of ABBM with the low concentration of NaOH can be more cost-effective, effectively remove the residual cellular components, without affecting the osteogenic ability. Our work may provide a novelty thought and a modified method to applicate the acellular bovine bone matrix clinically better. Graphical abstract.
我们主要从生物效应的角度出发，通过低浓度氧化氢来研究脱细胞牛骨基质（ABBM）。常规清洗牛骨后，用不同浓度的 NaOH 清洗并进行苏木精-伊红（HE）染色，以观察脱细胞效果。用光镜和扫描电子显微镜（SEM）观察经 NaOH 处理的牛骨基质的效果，并通过 DNA 残留检测进行比较。通过 CCK-8 法在 MC3T3-E1 细胞中评估细胞毒性。为了进行体外成骨检测，在 MC3T3-E1 细胞中进行碱性磷酸酶（ALP）染色和茜素红（AR）染色。在体内实验中，使用 Micro CT、HE 和 Masson 染色观察经 1%NaOH 溶液处理的材料在 6 和 12 周时的成骨效果是否受到影响。用不同浓度的 NaOH 溶液脱细胞化牛骨后，HE 染色显示，用 1%NaOH 溶液超声清洗 30min 具有最佳的脱细胞效果。SEM 显示，与用常规化学试剂处理的 ABBM 相比，用 1%NaOH 溶液处理的 ABBM 表面三维多孔结构上残留的细胞较少。用 1%NaOH 溶液处理的 ABBM 的 DNA 残留和细胞毒性均降低。ALP 染色和 AR 染色结果表明，用 1%NaOH 溶液处理的 ABBM 对成骨效果没有影响。动物实验的 Micro CT、HE 染色和 Masson 染色结果也表明，用 1%NaOH 溶液处理的 ABBM 对成骨能力没有影响。用低浓度 NaOH 对 ABBM 进行脱细胞化处理可以更具成本效益，有效去除残留的细胞成分，而不影响成骨能力。我们的工作可能为临床更好地应用脱细胞牛骨基质提供了新的思路和改进方法。
