Molecular Engineering of Cartilage PCM Mechanotransduction in Osteoarthritis Using Biomimetic Proteoglycans

摘要 在过去的50年里，骨关节炎软骨的再生在很大程度上是一个生物医学上没有解决的挑战。 许多策略被用来利用细胞的合成能力来产生新的细胞外 Matrix，希望逆转与骨关节炎(OA)相关的疼痛和功能障碍，以符合 美国国立卫生研究院的使命是寻求有关生命系统的基本知识和这些知识的应用 增进健康，延年益寿，减少疾病和残疾。尤其令人感兴趣的是， 细胞周围基质(PCM)，紧邻软骨细胞周围的区域，由于其显示的 在健康和骨关节炎软骨中介导软骨细胞机械转导的重要性。在办公自动化中， PCM的退变是疾病发生的主要事件之一，导致软骨细胞破坏 机械转导和不可逆的软骨降解。因此，如果我们能够设计出PCM的特性， 我们有可能调节软骨细胞的机械敏感活动，进而促进软骨形成。 再生和/或减轻骨关节炎软骨退变。我们的仿生蛋白多糖(BPG) 具有工程软骨PCM的利基效应。我们用化学方法末端连接7-8硫酸软骨素 糖胺多聚糖(CS-GAG)到聚丙烯酸(PAA)核(MW~10 kDa)上，形成仿生的 Proteoglycan，BPG10，具有模仿本地aggrecan的瓶刷纳米结构。当渗透到 BPG10在体外或关节腔内注射入兔膝关节内优先 已在PCM中本地化。这种定位导致了PCM在体外的微模数显著增加，并且 反过来，显著增强了软骨细胞内钙信号的活性。BPG10的作用还包括 与办公自动化相关。当BPG10渗透到人骨关节炎软骨中时，BPG10也定位于PCM中，并增强 局部PCM模数，表明有可能恢复退化的PCM并挽救受损的PCM 软骨细胞的机械敏感性活动。考虑到合成的PAA核心不受生理因素的影响 与天然蛋白多糖一样，BPG10也可以抵抗体内软骨细胞的分解代谢。我们的 中心假设是仿生蛋白多糖将分子工程PCM，增加 通过与天然PCM分子相互作用使PCM微弹性，从而促进软骨细胞 机械转导和减轻骨性关节炎诱导的软骨退变。为了验证这一假设，我们将：(1) 研究BPG10与软骨基质生物分子的物理相互作用；(2)确定BPG10是否 增强3D培养中软骨细胞的新的PCM和降解软骨移植的PCM，从而， 调节软骨细胞的机械转导和代谢活动以及(3)关节内给药的检测 BPG10可延缓兔在体骨关节炎的进展。在这些研究中，将测试单个CS-GAG 作为对照，考察BPG10‘S独特结构的作用。

Abstract Regeneration of osteoarthritic cartilage has been a largely unmet biomedical challenge for the past fifty years. Numerous strategies are being employed to harness the synthetic power of cells to generate new extracellular matrix in the hope of reversing the pain and dysfunction associated with osteoarthritis (OA), in keeping with the mission of the NIH to seek fundamental knowledge about of living systems and the application of that knowledge to enhance health, lengthen life, and reduce illness and disability. Of particular interest is the emerging role of the pericellular matrix (PCM), the region immediately surrounding the chondrocyte, due to its demonstrated importance in mediating chondrocyte mechanotransduction in both healthy and OA cartilage. In OA, degeneration of the PCM is one leading event of disease initiation, contributing to disrupted chondrocyte mechanotransduction and irreversible cartilage degradation. Thus, if we can engineer the properties of the PCM, there is a potential for us to modulate chondrocyte mechanosensitive activities, and in turn, to promote cartilage regeneration and/or to attenuate osteoarthritic cartilage degeneration. Our biomimetic proteoglycans (BPGs) have the niche effect of engineering cartilage PCM. We chemically end-attached 7-8 chondroitin sulfate glycosaminoglycans (CS-GAGs) to a poly(acrylic acid) (PAA) core (Mw ~10 kDa), resulting in a biomimetic proteoglycan, BPG10, with a bottle-brush nanostructure mimicking the native aggrecan. When infiltrated into bovine cartilage explants in vitro or intra-articularly injected into rabbit knees in vivo, BPG10 was preferentially localized in the PCM. This localization led to a significant increase in the micromodulus of the PCM in vitro, and in turn, significantly enhanced chondrocyte intracellular calcium signaling activities. The role of BPG10 is also relevant to OA. When infiltrated into human OA cartilage, BPG10 was also localized in the PCM, and enhanced the local PCM modulus, indicating a potential for restoring degenerative PCM and rescuing disrupted chondrocyte mechanosensitive activities. Given that the synthetic PAA core is not susceptible to physiologic enzymes, as are natural proteoglycans, BPG10 could also be resistant to chondrocyte catabolism in vivo. Our central hypothesis is that biomimetic proteoglycans will molecularly engineer the PCM, increasing the micromodulus of the PCM through interactions with native PCM molecules, thus promoting chondrocyte mechanotransduction and attenuating OA-induced cartilage degeneration. To test this hypothesis, we will: (1) study the physical interactions between BPG10 and cartilage matrix biomolecules; (2) determine if BPG10 augments the neo-PCM of chondrocytes in 3D culture and the PCM of degrading cartilage explants, and thus, modulates chondrocyte mechanotransduction and metabolic activities and (3) test if intra-articular administration of BPG10 attenuates the progression of OA in rabbits in vivo. In these studies, individual CS-GAGs will be tested as a control to examine the role of BPG10's unique structure.