Achieving Sustained Control of Inflammation to Prevent Post-Traumatic Osteoarthritis (PTOA)

膝骨关节炎（KOA）在美国军人和退伍军人中非常普遍， 关节创伤和过度使用损伤。其社会经济影响估计为每年600亿美元， 存在改善疾病的治疗方法。关节炎症与疾病的严重程度和进展有关4，但 目前的抗炎药物不能防止疾病进展，并且具有相当大的副作用。 本项目的目的是开发和采用一种新的方法来调节局部炎症反应， 创伤后关节炎（PTOA）是一种以持续的方式在关节损伤后的反应，以预防创伤后骨关节炎（PTOA）。 具体地说，我们假设通过阻断Toll样受体（TLR）激活， TLR共受体CD 14，将减少PTOA病理的发展和膝关节后功能下降 损伤我们是第一个在OA患者中发现高水平的CD 14，并表明它增强了TLR-1。 介导的炎症反应。我们现在已经证明，CD 14的遗传缺陷减少了 在PTOA小鼠模型中，我们的初步数据显示， 该相同的模型表明用于治疗目的的药理学阻断是可能的。我们建议 建立TLR共受体CD 14阻断作为预防PTOA病理和功能的有效策略 下降，使用由我们的团队开发的膝关节OA的大型动物（小型猪）模型。我们将优化内部 关节（IA）递送临床开发的CD 14阻断抗体，以在治疗期间实现持续释放。 通过将抗体封装在创新的机械活化微胶囊中， （MAMCs）。最后，我们将测试KOA退伍军人的TLR激活标志物是否可以预测临床 对现有抗炎治疗的反应，重点是功能恢复，以优化 患者分层，用于TLR靶向抗炎治疗的未来临床试验。 在目标1中，尤卡坦小型猪将接受内侧半月板（DMM）的双侧关节镜失稳 双后肢手术。从术后一周开始， （Implicit Biosciences）将通过IA输送至一侧膝关节;对侧将接受同种型匹配的 抗体对照功能结局将包括活动和膝关节屈曲角，每两周测量一次 使用可穿戴运动传感器设备。将在DMM前采集滑液（SF）和血清 和治疗效果的分子/细胞分析的终点。动物组将在 术后6个月的终点，以及使用新型MR成像技术评价的PTOA病理学 允许检测临床相关的早期软骨分子变化，并验证成像数据 使用标准化组织病理学。在目标2中，我们将使用以下方法优化MAMC中抗CD 14的包封： 微流控复乳技术，并确认包封后活性药物的持续释放 使用体外和离体分析。然后将包封的抗-CD 14 IA递送至受试者的一个后膝关节。 DMM操作的小型猪，并将包封的对照抗体置于对侧。单剂量将是 DMM后一周注射包封的抗CD 14与未包封的抗CD 14对 将比较病理和功能结果。MAMC在关节中的保留和分布将 通过IVIS成像评估。最后，在目标3中，我们将利用参与者提供的SF样本， 一项VA资助的临床试验（MOVE-OK研究），测试IA皮质类固醇在膝关节OA退伍军人中的疗效。 将在收集并储存的SF中测量与TLR活性相关的生物标志物（包括sCD 14、sTLR 4、LBP） 作为MOVE-OK方案的一部分，使用ELISA/多重测定。生物标志物水平之间的关联，以及 将使用线性回归分析测试治疗后活动水平和残疾评分的变化。 追求这个项目将导致一个创新的方法来控制关节炎后的发展 损伤，并为TLR靶向免疫调节方法的未来临床试验提供患者选择。

Knee Osteoarthritis (KOA) is highly prevalent in U.S. military service members and Veterans due to the impact of joint trauma and overuse injury. Its socioeconomic impact is estimated at $60 billion per year, and no disease-modifying treatments exist. Joint inflammation is linked to severity and progression of disease 4, but current anti-inflammatory medications do not protect against progression, and have substantial side effects. The purpose of this project is to develop and employ a novel method to modulate the local inflammatory response in the joint after an injury in a sustained manner, to prevent post-traumatic osteoarthritis (PTOA). Specifically, we hypothesize that modulation of Toll-like receptor (TLR) activation, via blockade of the TLR co-receptor CD14, will reduce development of PTOA pathology and functional decline after knee injury. We were the first to discover high levels of CD14 in OA patients and show that it augmented TLR- mediated inflammatory responses. We have now demonstrated that genetic deficiency of CD14 reduces signs of disability and prevents progression of cartilage damage in a murine model of PTOA; Our preliminary data in this same model shows that pharmacologic blockade for therapeutic purposes is possible. We propose to establish blockade of TLR co-receptor CD14 as an effective strategy to prevent PTOA pathology and functional decline, using a large-animal (mini-pig) model of knee OA developed by our team. We will optimize intra- articular (IA) delivery of a clinically developed CD14 blocking antibody to achieve sustained release during the post-injury period, by encapsulating the antibody in innovative Mechanically Activated Microcapsules (MAMCs). Finally, we will test whether TLR activation markers in Veterans with KOA can predict clinical response to an existing anti-inflammatory therapeutic, with a focus on functional recovery, in order to optimize patient stratification for future clinical trials of TLR-targeted anti-inflammatory therapies. In Aim 1, Yucatan mini-pigs will undergo bilateral arthroscopic destabilization of the medial meniscus (DMM) surgery of both hind limbs. Starting one week after surgery, a neutralizing anti-CD14 monoclonal antibody (Implicit Biosciences) will be delivered IA to one knee; the contra-lateral side will receive isotype-matched antibody control. Functional outcomes will include activity and knee joint flexion angles measured every two weeks using a wearable motion-sensor device. Synovial fluid (SF) and serum will be collected prior to DMM and at endpoints for molecular/cellular analysis of effects of treatment. Groups of animals will be euthanized at endpoints up to six months after surgery, and PTOA pathology evaluated using novel MR imaging techniques allowing clinically-relevant early cartilage molecular changes to be detected, and imaging data will be validated using standardized histopathology. In Aim 2 we will optimize encapsulation of anti-CD14 in MAMCs using microfluidic double emulsion technology, and confirm sustained delivery of active drug after encapsulation using in vitro and ex vivo analyses. Encapsulated anti-CD14 will then be delivered IA to one hind knee joint of DMM-operated mini-pigs, and encapsulated control antibody to the contra-lateral side. A single dose will be given one week after DMM. The effects of encapsulated anti-CD14 vs. unencapsulated anti-CD14 on pathology and functional outcomes will be compared. Retention and distribution of MAMCs in the joint will be assessed by IVIS imaging. Finally, in Aim 3, we will leverage the availability of SF samples from participants in a VA-funded clinical trial (MOVE-OK study) testing the efficacy of IA corticosteroids in Veterans with knee OA. Biomarkers related to TLR activity (including sCD14, sTLR4, LBP) will be measured in SF collected and stored as part of the MOVE-OK protocol, using ELISA/multiplex assays. Associations between biomarker levels, and change in activity levels and disability scores with therapy will be tested using linear regression analysis. Pursuit of this project will result in the development of an innovative method to control joint inflammation after injury, and inform patient selection for future clinical trials of TLR-targeted immunomodulatory approaches.