Evaluation of Recombinant Human Lactoferrin as an Osteogenic Biomaterial

描述（由申请人提供）：颅面骨组织缺损的临床治疗迫切需要。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器。LF在骨形态发生中的合成代谢作用是由于其在成骨细胞发生和破骨细胞发生中具有很强的剂量依赖性增殖和抗凋亡作用。我们假设重组人LF （rhLF）可制成可注射凝胶，该生物材料将具有成骨活性。我们建议首先研究rhLF凝胶的物理性质和特征，以进一步作为再生骨骼生物材料。我们将根据溶胀、机械强度、形态和体内降解来表征可注射rhLF凝胶的物理特性，以进一步作为可注射生物材料应用。我们假设不同浓度的三种凝胶成分，辣根过氧化物酶（HRP）、改性rhLF和H2O2，将直接影响rhLF凝胶的吸水率、机械强度、形态和体内降解率。rhLF凝胶将进行流变学测试，以评估三种凝胶组分在不同浓度下的相对机械强度。用扫描电子显微镜观察凝胶的形貌。rhLF凝胶的体内降解将通过皮下小鼠模型进行评估。因此，所提出的研究旨在增加我们对rhLF凝胶物理性质的理解。下一步，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。第二个具体目标将是研究rhLF凝胶促进小鼠成骨前细胞存活、增殖和分化的能力。我们拟利用MC3T3成骨前细胞系，在体外评价rhLF凝胶对细胞存活、增殖和分化的促进作用。将细胞加入到HRP和修饰的rhLF溶液中，随后加入H2O2将产生MC3T3细胞负载的rhLF凝胶。明胶将被修改类似于rhLF形成交联凝胶，这将作为对照

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. PUBLIC HEALTH RELEVANCE: Lactoferrin (LF), a protein native to the human body, has been demonstrated to possess multifold functions and is well recognized for its influential role in bone growth. The proposed study aims to develop a LF-based biomaterial gel and increase our understanding of the effect of LF gel on bone cells. The development of this novel, bioactive biomaterial for bone repair will address the need for better clinical treatments of craniofacial bone tissue defects.