Evaluation of Recombinant Human Lactoferrin as an Osteogenic Biomaterial

描述(由申请人提供)：迫切需要更好的临床治疗头面部骨组织缺损。乳铁蛋白(LF)是一种多功能的兼职糖蛋白，也是骨骼的有效合成代谢效应器。Lf在骨形态发生中的合成代谢作用归因于其在成骨细胞生成中具有强烈的剂量依赖性的增殖和抗凋亡作用以及对破骨细胞生成的抑制作用。我们假设可以从重组人乳铁蛋白(Rh Lf)中制备出可注射凝胶，这种生物材料将具有成骨活性。我们建议首先研究rhLF凝胶的物理性质，并对其进行表征，以进一步将其用作再生骨骼生物材料。我们将根据溶胀、机械强度、形态和体内降解来表征可注射的rhLF凝胶的物理性质，以进一步作为可注射的生物材料应用。我们推测，不同浓度的辣根过氧化物酶(HRP)、修饰的重组人乳糖凝胶和过氧化氢将直接影响重组人乳糖凝胶的吸水率、机械强度、形态和体内降解率。将对rhLF凝胶进行流变学测试，以评估三种凝胶成分在不同浓度下的相对机械强度。凝胶的形态将通过扫描电子显微镜可视化。将使用小鼠皮下模型评估rhLF凝胶的体内降解。因此，建议的研究旨在增加我们对rhLF凝胶物理性质的了解。接下来，我们计划探索重组人乳铁蛋白凝胶作为一种新型的成骨生物材料的生物学效应。第二个具体目标将是研究重组人乳铁蛋白凝胶促进小鼠成骨前细胞的存活、增殖和分化的能力。我们建议使用MC3T3前成骨细胞系来评价rhLF凝胶在体外促进细胞存活、增殖和分化的作用。细胞将被加入到HRP和改良的重组人乳铁蛋白溶液中，随后加入过氧化氢将产生MC3T3细胞负载的重组人乳铁蛋白凝胶。明胶将类似于重组人乳铁蛋白进行修饰，形成交联凝胶，作为对照 这些研究中的生物材料。细胞存活率将通过量化活细胞到死亡细胞来评估。 比率包被在rhLF凝胶中的细胞的增殖将通过胸腺嘧啶核苷掺入来确定。培养21d后，用茜素红研究其矿化程度。此外，还将评估骨钙素、骨桥蛋白和Runx2、碱性磷酸酶的表达水平，以研究rhLF凝胶对细胞分化的影响。这项拟议的研究将增加我们对rhLF凝胶在调节骨骼细胞中的生物学效应的了解，并进一步确立rhLF作为骨骼再生生物材料的优秀候选者。

DESCRIPTION (provided by applicant): There is a critical need for better clinical treatments of craniofacial bone tissue defects. Lactoferrin (LF), a multifunctional "moonlighting" glycoprotein, is also a potent anabolic effector of the skeleton. LF's anabolic effect in bone morphongenesis is attributed to its strong dose-dependent proliferative and anti-apoptotic actions in osteoblastogenesis and inhibition of osteoclastogenesis. We hypothesize that an injectable gel can be produced from recombinant human LF (rhLF) and this biomaterial will possess osteogenic activities. We propose to first investigate the physical properties and characterize the rhLF gels to be further employed as a regenerative skeletal biomaterial. We will characterize the physical properties of the injectable rhLF gels based on swelling, mechanical strength, morphology and in vivo degradation to be further applied as an injectable biomaterial. We hypothesize that varying concentrations of the three gel components, horseradish peroxidase (HRP), modified rhLF and H2O2, will directly affect the water uptake, mechanical strength, morphology and in vivo degradation rates of the rhLF gels. rhLF gels will be subjected to rheological testing to assess the relative mechanical strengths at varying concentrations of the three gel components. The morphology of the gels will be visualized by scanning electron microscope. The in vivo degradation of the rhLF gels will be evaluated using a subcutaneous mouse model. Thus, the proposed studies are aimed to increase our understanding of the rhLF gel physical properties. Next, we plan to explore the biological effects of rhLF gel as a novel osteogenic biomaterial. The second specific aim will be to investigate the ability of rhLF gels to promote cell survival, proliferation and differentiation of murine preosteoblast cells. We propose to use the MC3T3 preosteoblastic cell line to evaluate the effects of rhLF gel in promoting cell survival, proliferation and differentiation in vitro. Cells will be added to the HRP and modified rhLF solution and the subsequent addition of H2O2 will produce MC3T3 cell - laden rhLF gels. Gelatin will be modified similar to rhLF to form cross-linked gels, which will serve as the control biomaterial in these studies. Cell survival will be assessed by quantification of live to dead cell ratio. Proliferation of the cells encapsulated in the rhLF gels will be determined through thymidine incorporation. Alizarin Red will be utilized to study the degree of mineralization after 21 days of culture. Additionally, osteocalcin, osteopontin and Runx2, alkaline phosphatase expression levels will be assessed to study the effects of the rhLF gel on cell differentiation. Th proposed study will increase our understanding of the biological effect of rhLF gel in modulating skeletal cells and further establish rhLF as an excellent candidate for a skeletal regenerative biomaterial.