Evaluation of Recombinant Human Lactoferrin as an Osteogenic Biomaterial

描述（由申请人提供）：迫切需要更好的颅面骨组织缺损临床治疗。乳铁蛋白（LF）是一种多功能的“兼职”糖蛋白，也是一种有效的骨骼合成代谢效应物。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的强剂量依赖性增殖和抗凋亡作用以及破骨细胞生成的抑制。我们假设，可注射凝胶可以从重组人LF（rhLF）生产，这种生物材料将具有成骨活性。我们建议首先调查的物理性质和表征的rhLF凝胶进一步采用作为再生骨骼生物材料。我们将根据溶胀、机械强度、形态学和体内降解来表征可注射rhLF凝胶的物理性质，以进一步用作可注射生物材料。我们假设，不同浓度的三种凝胶组分，辣根过氧化物酶（HRP），改性rhLF和H2O2，将直接影响水的吸收，机械强度，形态和在体内降解率的rhLF凝胶。将对rhLF凝胶进行流变学试验，以评估三种凝胶组分在不同浓度下的相对机械强度。将通过扫描电子显微镜观察凝胶的形态。将使用皮下小鼠模型评价rhLF凝胶的体内降解。因此，拟议的研究旨在增加我们对rhLF凝胶物理性质的理解。接下来，我们计划探索rhLF凝胶作为一种新型成骨生物材料的生物学效应。第二个具体的目标将是研究rhLF凝胶促进小鼠前成骨细胞的细胞存活、增殖和分化的能力。我们建议使用MC3T3前成骨细胞系来评估rhLF凝胶在体外促进细胞存活、增殖和分化的作用。将细胞加入HRP和修饰的rhLF溶液中，随后加入H2O2将产生载有MC3T3细胞的rhLF凝胶。明胶将类似于rhLF进行修饰以形成交联凝胶，其将用作对照 生物材料在这些研究中。将通过定量活细胞与死细胞来评估细胞存活 比例将通过胸苷掺入测定rhLF凝胶中包封的细胞增殖。培养21天后，将使用茜素红研究矿化程度。此外，还将评估骨钙蛋白、骨桥蛋白和Runx 2、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理解，并进一步确立rhLF作为骨骼再生生物材料的良好候选者。

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.