Extracellular vesicles as therapeutic vehicles for chondroprotection

单一关节损伤可导致创伤后骨关节炎（PTOA）的发展。关节功能丧失的核心是关节软骨的进行性退化。虽然外科手术可以纠正结构缺陷，但没有治疗方法可以在受伤后的几周内保护软骨细胞的代谢和活力。临床前研究表明，体外生长的间充质干细胞/基质细胞（MSC）在PTOA动物模型中具有软骨保护作用;这些作用现在被认为是由关节内递送后的MSC旁分泌信号介导的。最近，MSC衍生的细胞外囊泡（MSC-EV）已被证明在几种非骨骼损伤模型中刺激与其供体细胞相当的促再生作用，表明它们是MSC旁分泌作用的重要组成部分。MSC-EV预防或延迟PTOA的潜力可能取决于它们直接向软骨细胞传递信号的能力，这需要通过致密的带负电荷的软骨基质进行运输。虽然EV大小和净电荷表明在健康软骨内的渗透有限，但其精确的表面组成可能会影响软骨吸收和扩散。此外，在关节损伤后的急性炎症期发生的软骨基质变化可以改变较大溶质的运输，EV可能会做出类似的反应。如果MSC-EV可以在软骨基质发生不可逆变化之前传递改善软骨细胞功能的信号，则它们可以用于在关节创伤后的关键时期恢复组织稳态。该项目将通过更好地了解调节其货物输送到软骨细胞的因素，更好地确定MSC-EV对PTOA的治疗潜力。我们将首先测试软骨糖胺聚糖消耗（比胶原损失更可逆的软骨基质变化）是否影响MSC-EV货物递送（目标1）。我们还将确定MSC-EV表面电荷和与软骨基质的静电相互作用如何影响递送（目的2）。最后，我们将通过全基因组功能获得性筛选（Aim 3）鉴定控制MSC-EV货物递送的软骨细胞基因。在短期内，该项目将为研究预防PTOA的潜在EV作用模式奠定基础;从长远来看，它将为合成囊泡的设计提供信息，用于将治疗分子递送到滑膜关节细胞。

A single joint injury can result in the development of post-traumatic osteoarthritis (PTOA). Central to loss of joint function is the progressive degeneration of articular cartilage. While surgical procedures can correct structural defects, there are no treatments that can protect chondrocyte metabolism and viability in the weeks after an injury. Preclinical studies have shown that mesenchymal stem/stromal cells (MSCs) grown in vitro can have chondroprotective effects in animal models of PTOA; these effects are now thought to be mediated by MSC paracrine signaling following intra-articular delivery. Recently, MSC-derived extracellular vesicles (MSC-EVs) have been shown to stimulate pro-regenerative effects equivalent to their donor cells in several non-skeletal injury models, suggesting they are an important component of MSC paracrine actions. The potential of MSC-EVs for preventing or delaying PTOA may depend on their ability to deliver signals directly to chondrocytes, which requires transport through the dense, negatively-charged matrix of cartilage. While EV size and net charge suggest limited penetration within healthy cartilage, their precise surface composition may influence cartilage uptake and diffusion. Moreover, cartilage matrix changes that occur during the acute inflammatory period after a joint injury can alter the transport of larger solutes, and EVs may respond similarly. If MSC-EVs can deliver signals that improve chondrocyte function before irreversible changes occur to the cartilage matrix, they can be used to restore tissue homeostasis during the critical period following joint trauma. This project will better define the therapeutic potential of MSC-EVs for PTOA through an improved understanding of the factors regulating their cargo delivery to chondrocytes. We will first test whether cartilage glycosaminoglycan depletion, a more reversible change to the cartilage matrix than collagen loss, impacts MSC-EV cargo delivery (Aim 1). We will also determine how delivery is influenced by MSC-EV surface charge and altered electrostatic interactions with the cartilage matrix (Aim 2). Finally, we will identify chondrocyte genes that control MSC-EV cargo delivery through a genome-wide gain-of-function screen (Aim 3). In the short term, this project will lay the groundwork for studying potential EV modes of action for the prevention of PTOA; in the long term, it will inform the design of synthetic vesicles for delivering therapeutic molecules to synovial joint cells.