Center for the Study of Venoms and Venomous Animals (CEVAP), UNESP - Universidade Estadual Paulista, Botucatu, SP, Brazil.
Botucatu Medical School, UNESP - Universidade Estadual Paulista, Botucatu, SP, Brazil.
Stem Cell Res Ther. 2017 Sep 29;8(1):205. doi: 10.1186/s13287-017-0654-7.
The injection of mesenchymal stem cells (MSCs) directly into the bone of osteoporotic (OP) patients for rapid recovery has been studied worldwide. Scaffolds associated with MSCs are used to maintain and avoid cell loss after application. A unique heterologous fibrin sealant (HFS) derived from snake venom was evaluated for the cytotoxicity of its main components and as a three-dimensional biological scaffold for MSCs to repair a critical femur defect in osteoporotic rats.
The cytotoxicity of HFS was assessed using a 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazoliumbromide (MTT) assay and transmission electron microscopy. The cells were cultured, characterized by flow cytometry and differentiated into the osteogenic lineage. Two-month-old rats underwent ovariectomy to induce OP. After 3 months, a 5 mm critical bone defect was made in the distal end of the rat femurs and filled with HFS; HFS + MSCs; and HFS + MSCs D (differentiated into the osteogenic lineage) to evaluate the effects. An injury control group (injury and no treatment) and blank control group (no injury and no treatment) were also included. The animals were observed at days 14 and 28 by microtomographic (micro-CT) analyses, histologic and biochemical analysis, as well as scanning electron microscopy.
The results revealed that one of the compounds of HFS, the thrombin-like enzyme extracted from snake venom, had no cytotoxic effects on the MSCs. OP was successfully induced, as demonstrated by the significant differences in the levels of 17β-estradiol, Micro-CT analyses and alkaline phosphatase between the ovariectomized (OVX) and non-ovariectomized (NOVX) groups. The histological data revealed that at 14 days after surgery in both the OVX and NOVX animals, the HFS + CTMs and HFS + CTMsD showed a higher formation of bone cells at the site in relation to the control group (without treatment). Collagen formation was evidenced through bone neoformation in all treated and control groups. No morphological differences in the femurs of the NOVX and OVX animals were observed after the surgical procedure. Scanning electron microscopy (SEM) confirmed the histological analysis.
The new HFS composed of two non-toxic components for MSCs showed capacity to promote the recovery of the bone lesions in OVX and NOVX animals at 14 days after surgery. In addition, the HFS enabled the differentiation of MSCs into MSCs D in the group treated with HFS + MSCs. Using the MSCs and/or MSCs D together with this biopharmaceutical could potentially enable significant advances in the treatment of osteoporotic fractures. Future clinical trials will be necessary to confirm these results.
将间充质干细胞（MSCs）直接注射到骨质疏松症（OP）患者的骨骼中以实现快速恢复，这一方法已在全球范围内得到研究。与 MSCs 相关的支架用于在应用后维持和避免细胞丢失。一种源自蛇毒的独特异源纤维蛋白密封剂（HFS），因其主要成分的细胞毒性以及作为修复骨质疏松症大鼠股骨临界缺损的三维生物支架而受到评估。
使用 3-(4,5-二甲基-2-噻唑基)-2,5-二苯基-2H-四唑溴化物（MTT）测定法和透射电子显微镜评估 HFS 的细胞毒性。培养细胞，通过流式细胞术进行表征，并分化为成骨谱系。两个月大的大鼠接受卵巢切除术以诱导 OP。3 个月后，在大鼠股骨远端制造 5 毫米临界骨缺损，并填充 HFS；HFS+MSCs；和 HFS+MSCs D（分化为成骨谱系）以评估效果。还包括损伤对照组（损伤和未治疗）和空白对照组（无损伤和未治疗）。通过微断层扫描（micro-CT）分析、组织学和生化分析以及扫描电子显微镜在第 14 天和第 28 天观察动物。
结果表明，HFS 的一种化合物，即从蛇毒中提取的类凝血酶酶，对 MSCs 没有细胞毒性作用。成功诱导了 OP，卵巢切除术（OVX）和非卵巢切除术（NOVX）组之间 17β-雌二醇、Micro-CT 分析和碱性磷酸酶水平的显著差异证明了这一点。组织学数据显示，在手术 14 天后，OVX 和 NOVX 动物的 HFS+CTMs 和 HFS+CTMsD 显示在与对照组（无治疗）相关的部位形成更高的骨细胞。所有治疗组和对照组均有新骨形成，表明有胶原形成。OVX 和 NOVX 动物的股骨在手术后没有观察到形态学差异。扫描电子显微镜（SEM）证实了组织学分析。
由两种对 MSCs 无毒的成分组成的新型 HFS 显示出在手术后 14 天促进 OVX 和 NOVX 动物骨损伤恢复的能力。此外，HFS 使与 HFS+MSCs 一起治疗的组中的 MSCs 分化为 MSCs D。使用这些 MSC 和/或 MSC D 与这种生物制药一起，可能会在治疗骨质疏松性骨折方面取得重大进展。未来的临床试验将有必要确认这些结果。
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