Bone grafting is the second most common tissue transplantation procedure worldwide. One of the alternative methods for bone repair under investigation is a tissue-engineered bone substitute. An ideal property of tissue-engineered bone substitutes is osteoinductivity, defined as the ability to stimulate primitive cells to differentiate into a bone-forming lineage. In the current study, we use a decellularization and oxidation protocol to produce a porcine bone scaffold and examine whether it possesses osteoinductive potential and can be used to create a tissue-engineered bone microenvironment. The decellularization protocol was patented by our lab and consists of chemical decellularization and oxidation steps using combinations of deionized water, trypsin, antimicrobials, peracetic acid, and triton-X100. To test if the bone scaffold was a viable host, preosteoblasts were seeded and analyzed for markers of osteogenic differentiation. The osteoinductive potential was observed in vitro with similar osteogenic markers being expressed in preosteoblasts seeded on the scaffolds and demineralized bone matrix. To assess these properties in vivo, scaffolds with and without preosteoblasts preseeded were subcutaneously implanted in mice for 4 weeks. MicroCT scanning revealed 1.6-fold increased bone volume to total volume ratio and 1.4-fold increase in trabecular thickness in scaffolds after implantation. The histological analysis demonstrates new bone formation and blood vessel formation with pentachrome staining demonstrating osteogenesis and angiogenesis, respectively, within the scaffold. Furthermore, CD31+ staining confirmed the endothelial lining of the blood vessels. These results demonstrate that porcine bone maintains its osteoinductive properties after the application of a patented decellularization and oxidation protocol developed in our laboratory. Future work must be performed to definitively prove osteogenesis of human mesenchymal stem cells, biocompatibility in large animal models, and osteoinduction/osseointegration in a relevant clinical model in vivo. The ability to create a functional bone microenvironment using decellularized xenografts will impact regenerative medicine, orthopedic reconstruction, and could be used in the research of multiple diseases.

中文翻译：

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研究脱细胞异种移植骨替代物的骨诱导潜力

骨移植是世界上第二常见的组织移植手术。正在研究的骨修复替代方法之一是组织工程骨替代物。组织工程骨替代物的一个理想特性是骨诱导性，定义为刺激原始细胞分化成骨形成谱系的能力。在当前的研究中，我们使用脱细胞和氧化方案来生产猪骨支架，并检查其是否具有骨诱导潜力并可用于创建组织工程骨微环境。去细胞化方案已获得我们实验室的专利，包括使用去离子水、胰蛋白酶、抗菌剂、过氧乙酸和 triton-X100 组合的化学去细胞化和氧化步骤。为了测试骨支架是否是可行的宿主，接种前成骨细胞并分析成骨分化的标记。在体外观察到骨诱导潜力，在支架和脱矿骨基质上接种的前成骨细胞中表达相似的成骨标志物。为了评估体内的这些特性，将预先植入和未植入前成骨细胞的支架皮下植入小鼠体内 4 周。MicroCT 扫描显示，植入后支架的骨体积与总体积之比增加了 1.6 倍，小梁厚度增加了 1.4 倍。组织学分析表明新骨形成和血管形成，五色染色分别显示支架内的骨生成和血管生成。此外，CD31+染色证实了血管内皮层。这些结果表明，在应用我们实验室开发的专利脱细胞和氧化方案后，猪骨仍保持其骨诱导特性。未来的工作必须明确证明人类间充质干细胞的成骨作用、大型动物模型中的生物相容性以及相关体内临床模型中的骨诱导/骨整合。使用脱细胞异种移植物创建功能性骨微环境的能力将影响再生医学、骨科重建，并可用于多种疾病的研究。