Roles of Collagen and Water in the Fracture Resistance of Bone

开发新策略以预防代价高昂的髋部、脊柱和 肱骨近端是对发生在年龄和疾病相关的关键改变的不完全理解 骨组织。特别是，人们对糖尿病的致病机制知之甚少。 细胞外基质(ECM)会降低骨骼的抗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 基质蛋白随着骨折风险的增加而增加。因此，这个项目的总体目标是：i)确定 骨中主要的细胞外基质蛋白I型胶原的NE-PTM预测骨折阻力，并在临床上应用 与骨质疏松症相关，以及ii)确定非交联型或交联型NE-PTMS是否有助于 骨骼的抗折性降低，以及它们是否通过改变骨的结构和水化作用而降低 为了达到我们的目标，我们将首先制作皮质骨(致密的)和松质骨的标本。 (海绵状)使用从50岁到100岁之间的男性和女性捐赠者身上收集的身体股骨 年龄(目标1a)。这些标本将被综合分析以量化：骨密度，骨 体积分数、细胞外基质结合水、I型胶原二级结构、成熟酶和非酶 胶原交联物，I型胶原纤维的完整性，抗屈服性(强度)，变形能力 屈服(韧性)、抗裂纹扩展(断裂韧性)和抗损伤 积累(疲劳)。我们还将从邻近的骨骼样本中提取细胞外基质蛋白，包括I型胶原和 利用质谱仪在形成其三螺旋的特定氨基酸残基上对NE-PTMS进行定量。通过拟合 将数据转化为统计模型，我们将确定某些NE-PTM水平是否有助于解释差异 在抗折性、胶原纤维完整性、细胞外基质结合水和光谱标志物方面的供体 螺旋结构的。我们还将从身体上获取股骨近端的骨样本，而不是 骨关节炎(OA)和两种类型的骨科手术病例：全髋关节置换术(THA)治疗骨关节炎和半髋关节置换。 关节成形术(HA)以固定脆性骨折(目标1b)。样本将按照目标1a进行分析，以确定 NE-PTM水平是否显著高于ECM结合水和抗折性显著 HA(骨质疏松)低于THA(骨质疏松症)或身体对照组。确定NE-PTMS的机制 降低骨骼的抗折性，我们将在体外治疗骨骼积累特定类型的NE-PTMS(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 骨的抗折性与目标1中确定的老化方式类似。最后，我们将执行 I型胶原的分子动力学模拟和分子模拟以确定相关的NE-PTM如何影响 三螺旋结构和水合作用(目标2b)。该项目的成功完成将改变这一模式 从关注骨量到包含骨细胞外基质的致病因素，这是骨健康的一个重要方面。

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.