CMA: Cartilage Repair Strategies to Alleviate Arthritic Pain (CaRe AP): Novel cell-based therapies to increase functional outcomes and alleviate pain in preclinical models of osteoarthritis

总体研究战略：CARA-AP合作项目的总体目标是开发一种治疗方法 对于创伤后骨关节炎(PTOA)，这将缓解疼痛和改善功能。我们假设PTOA 是由适应不良的修复反应引起的，包括激活先天的促炎通路 免疫力进而导致疼痛、功能丧失和结构性衰退。这一计划解决了假设 通过两个高度整合的目标：(1)使用小分子、生物 用于减轻关节疼痛和炎症的抑制剂和免疫调节细胞以及(2)组织工程 使用基于干细胞的疗法重建受损的关节基础设施。调查人员 在这个项目中的合作将协调在四个动物模型中模拟不同的治疗方法的测试 引发患者PTOA的损伤机制。实验将考虑相关因素 影响关节生理和治疗反应。将评估不同治疗方式的效果。 使用对疼痛、功能、炎症和结构的团体标准化测量，以便结果可以 在实验室之间进行比较，并将最有希望的治疗策略优先用于临床试验。 我们提供了令人信服的证据表明，BMP2和非BMP2对间充质祖细胞的序贯治疗 规范的WNT5a在高密度颗粒中培养，在体外可产生关节样细胞。这些效果会被重现 当靶向RUNX1的软骨形成小分子Kartogenin(Kgn)和Wnt5a的Fox5 模拟多肽，顺序使用。此外，植入颗粒，先用BMP2处理，然后再用Wnt5a处理， 形成大鼠软骨缺损区，再生关节样软骨。我们的初步数据也显示了 运动对PTOA模型大鼠软骨保存和关节功能的影响。因此，我们问是否 干细胞的来源可以不同地促进透明软骨的再生和物理康复 可以改善颗粒的整合和治疗结果。具体地说，我们的目标是比较躯体疗法的疗效 细胞(人关节软骨细胞和骨髓间充质干细胞)与患者的细胞- 特定的、容易获得的多能干细胞。我们假设细胞的可塑性将决定 成人干细胞与多能干细胞治疗软骨退行性变的潜力，并取得成功 通过细胞和物理治疗恢复软骨完整性将增强功能 体内结果和减少伤害性感受。 我们将首先建立IPSC来源的间充质干细胞与脂肪来源和人类关节来源的能力。 KGN和Fox5序贯治疗前后软骨间充质干细胞再生关节软骨的研究 将对照或经处理的高密度颗粒植入PTOA大鼠软骨缺损性模型。 将进行组织学评估、基质合成、机械测试和分子成像分析 (目标1)。然后我们将评估动物的细胞和物理治疗对长期 功能性、伤害性和集中性疼痛结果。我们将评估控制组和对照组的功能结果 接受IPSC来源的MSCs或已确定的最有效成体干细胞来源的实验大鼠 目标1.具体来说，步态、自愿跑步、肌肉力量、机械测试和痛觉异常分析将 也会被执行。我们还将评估经常导致慢性关节疼痛的神经变化(目标2)。

Overall Research Strategy: The overall goal of the CaRa-AP Collaborative Program is to develop a treatment for post-traumatic osteoarthritis (PTOA) that will relieve pain and improve function. We hypothesize that PTOA is caused by maladaptive repair responses including activation of the pro-inflammatory pathways of innate immunity that in turn result in pain, loss of function and structural decline. This Program addresses the hypothesis through two highly-integrated aims: (1) innovative intra-articular treatments using small molecules, biologic inhibitors and immunomodulatory cells to reduce pain and inflammation in the joint and (2) tissue engineering using stem cell-based therapies for reconstruction of the damaged joint infrastructure. The investigators collaborating in this program will coordinate testing of therapies in four animal models that mimic different mechanisms of injury that initiate PTOA in patients. The experiments will take into consideration relevant factors affecting joint physiology and treatment response. The effects of different therapeutic modalities will be evaluated using group-standardized measurements of pain, function, inflammation and structure, so that results can be compared across laboratories and the most promising therapeutic strategies prioritized for clinical trials. We provide compelling evidence that sequential treatment of mesenchymal progenitors with BMP2 and non- canonical Wnt5a, cultured in high density pellets, yield articular-like cells in vitro. These effects are reproduced when small molecules Kartogenin (KGN), a chondrogenic small molecule targeting Runx1, and Foxy5, a Wnt5a mimetic peptide, are sequentially used. Moreover, implantation of pellets, treated with BMP2 followed by Wnt5a, into a rat chondral defect regenerate articular-like cartilage. Our preliminary data also demonstrate an influence of physical exercise on cartilage preservation and joint function in a rat model of PTOA. Thus, we ask whether the source of stem cells can differentially promote hyaline cartilage regeneration and if physical rehabilitation can improve pellet integration and treatment outcome. Specifically, we aim to compare the efficacy of somatic cells (human articular chondrocytes and bone marrow derived mesenchymal stem cells) to that of patient- specific, and readily available, pluripotent stem cells. We hypothesize that cell plasticity will determine the potential of adult versus pluripotent stem cells to treat cartilage degeneration, and that successful restoration of cartilage integrity via cellular and physical therapy will lead to enhanced functional outcomes and reduced nociception in vivo. We will first establish the capacity of iPSC-derived MSCs to that of adipose-derived and human articular cartilage MSCs with and without sequential treatment with KGN and Foxy5 to regenerate articular cartilage following implantation of control or treated high-density pellets into a rat chondral defect model of PTOA. Histological evaluations, matrix synthesis, mechanical testing and molecular imaging analyses will be performed (Aim 1). We will then assess the effect of cellular and physical treatment of animals on long-term functional, nociceptive, and centralized pain outcomes. We will evaluate functional outcomes in control and experimental rats that receive either iPSC-derived MSCs or the most effective adult stem cell source identified in Aim 1. Specifically, gait, voluntary running, muscle strength, mechanical testing, and allodynia analyses will also be performed. We will also assess the neurological changes that often lead to chronic joint pain (Aim 2).