Roles of Collagen and Water in the Fracture Resistance of Bone

开发预防代价高昂的髋部、脊柱和脊柱骨折的新策略的关键障碍 肱骨近端是一个关键的年龄和疾病相关的变化发生在不完全的理解， 骨组织特别是，很少有人知道的致病机制，其中恶化的， 细胞外基质（ECM）降低骨的抗骨折性或增加骨折风险。非酶 翻译后修饰（NE-PTM）是不良ECM的潜在贡献者，因为它们在细胞内积累， 随着骨折风险的增加，基质蛋白。因此，本项目的总体目标是：i）确定 胶原蛋白I的NE-PTM，骨的主要ECM蛋白，预测骨折抵抗力，并且在临床上是有效的。 ii）确定非交联或交联NE-PTM是否有助于骨质疏松症的发生。 骨的抗骨折性降低，以及它们是否通过改变骨的结构和水合作用而降低骨的抗骨折性。 胶原蛋白I为了实现我们的目标，我们将首先生成皮质骨（致密）和松质骨的标本 （海绵状）使用从50岁至100岁的女性和男性捐赠者收集的尸体股骨 年龄（目标1a）。将对这些标本进行全面分析，以量化：骨矿物质密度、骨 体积分数，ECM结合水，胶原蛋白I的二级结构，成熟酶和非酶 胶原蛋白交联、胶原蛋白I原纤维的完整性、抗屈服性（强度）、变形后的能力 屈服（韧性）、抗裂纹扩展性（断裂韧性）和抗损伤性 疲劳（fatigue）。从邻近的骨样本中，我们还将提取ECM蛋白，包括胶原蛋白I和胶原蛋白B。 使用质谱法在形成其三螺旋的特定氨基酸残基处定量NE-PTM。通过拟合 通过将数据与统计模型相结合，我们将确定某些NE-PTM的水平是否有助于解释差异 在抗断裂性、胶原纤维完整性、ECM结合水和光谱标记物方面， 螺旋结构。我们还将从尸体上获得的股骨近端生成骨标本， 骨关节炎（OA）和两种类型的骨科手术病例：OA和半髋关节置换术（THA）。 关节成形术（HA）以固定脆性骨折（目标1b）。将按照目标1a对样本进行分析，以确定 NE-PTM水平是否显著更高，而ECM结合水和抗断裂性是否显著更高， HA（骨质疏松）低于THA（OA）或尸体对照。为了确定NE-PTM 降低骨的抗断裂性，我们将离体处理骨以积累特定类型的NE-PTM（目的 2a）。治疗后，我们将评估骨骼，以确定哪些特定的NE-PTM显著影响骨骼。 以与目标1中确定的老化相似的方式，骨的抗断裂性。最后，我们将表演 胶原蛋白I的分子动力学模拟和分子建模，以确定相关NE-PTM如何影响 三螺旋结构和水合作用（目标2b）。项目的成功完成将改变 从关注骨量到纳入骨ECM的致病贡献，

A critical barrier to the development of new strategies for preventing costly fractures of the hip, spine, and proximal humerus is an incomplete understanding of the key age- and disease-related changes occurring in bone tissue. In particular, little is known about the pathogenic mechanisms by which the deterioration in the extracellular matrix (ECM) reduces the fracture resistance of bone or increases fracture risk. Non-enzymatic post-translational modifications (NE-PTMs) are potential contributors to poor ECM because they accumulate in matrix proteins as fracture risk increases. Therefore, the overall goals of this project are i) to determine which NE-PTMs of collagen I, the predominant ECM protein of bone, predict bone fracture resistance and are clinically relevant in osteoporosis and ii) to establish whether non-crosslinking or crosslinking NE-PTMs contribute to a decrease in fracture resistance of bone and whether they do so via alterations in the structure and hydration of collagen I. To achieve our goals, we will first generate specimens of cortical bone (dense) and trabecular bone (spongy) using cadaveric femurs collected from both female and male donors between 50 years and 100 years of age (Aim 1a). These specimens will be comprehensively analyzed to quantify: bone mineral density, bone volume fraction, ECM-bound water, secondary structure of collagen I, mature enzymatic & non-enzymatic collagen crosslinks, integrity of collagen I fibrils, the resistance to yielding (strength), the ability to deform after yielding (toughness), the resistance to crack growth (fracture toughness), and the resistance to damage accumulation (fatigue). From adjacent bone samples, we will also extract ECM proteins including collagen I and quantify NE-PTMs at specific amino acid residues that form its triple helix using mass spectrometry. By fitting the data to statistical models, we will determine whether the levels of certain NE-PTMs help explain differences among the donors in fracture resistance, collagen fibril integrity, ECM-bound water, and spectroscopic markers of helical structure. We will also generate bone specimens from proximal femurs acquired from cadavers without osteoarthritis (OA) and two types of orthopaedic surgical cases: total hip arthroplasty (THA) for OA and hemi- arthroplasty (HA) to fix a fragility fracture (Aim 1b). The specimens will be analyzed as in Aim 1a to determine whether NE-PTM levels are significantly higher while ECM-bound water and fracture resistance are significantly lower in HA (osteoporosis) vs. THA (OA) or the cadaveric controls. To identify a mechanism whereby NE-PTMs lowers fracture resistance of bone, we will treat bones ex vivo to accumulate specific types of NE-PTMs (Aim 2a). Following treatment, we will assess the bones to determine which specific NE-PTMs significantly affect the fracture resistance of bone in a manner similar to that of aging as determined in Aim 1. Lastly, we will perform molecular dynamics simulations and molecular modeling of collagen I to determine how relevant NE-PTMs affect the triple helix structure and hydration (Aim 2b). Successful completion of the project would shift the paradigm of bone health from a focus on bone mass to the inclusion of pathogenic contributions from bone ECM.