An Innovative Two-Step Therapeutic Strategy to Maximize the Effect of Stem Cell Therapy for Post-Traumatic Osteoarthritis

项目摘要/摘要 骨关节炎(OA)的病因是多因素的。异常和过度累积的关节应力结果 创伤后骨性关节炎(PTOA)。退伍军人中约30%的膝盖骨关节炎可归因于 职业活动，尤指需要下跪或蹲下并举起重物的工作，如 作为运动损伤。目前，还没有针对骨性关节炎患者的有效治疗方法。最近，干细胞疗法 已经显示出再生受损关节组织的希望。在我们之前的研究中，脂肪来源的干细胞 (ASCs)由于其多谱系，在再生医学中显示出巨大的治疗潜力。 潜在的，免疫抑制活性，有限的免疫原性，以及在培养中相对容易生长。 然而，有几个问题阻碍了干细胞疗法在炎性关节的临床应用。 环境，如ASCs的凋亡、剂量、干预时机、归巢效果和传递途径。 我们还发现，核因子-κB抑制剂，如TPCA-1，可以减轻膝关节机械性损伤的炎症 关节采用我们建立的PTOA小鼠模型和体外模型。 在本项目中，我们的研究基于两点：1)外源性ASCs的预处理 抗氧化剂(如维生素E)在注射到关节前会导致细胞保护作用和抵抗力 移植后对细胞凋亡和中毒性炎症因子的影响。2)TPCA1-纳米体能改善HASH 外源性异体骨移植前抗炎机制对膝关节关节炎的影响 干细胞。因此，我们将展示这种两步治疗应用的协同效应(反 关节炎关节的炎性纳米体治疗，然后移植预适应的ASCs)。 我们有两个目的：(1)研究预适应的细胞保护作用和治疗潜力 PTOA模型中的ASCs。为此，我们将研究抗氧化剂(维生素E-NS)的治疗效果。 在体内使用我们的PTOA小鼠膝关节过载模型对ASCs进行预处理。为了将此方法调整为 适当的退伍军人群体(现役和退役)我们将使用老龄小鼠来调查它们是否表现出 与幼龄小鼠不同的愈合反应和机制。我们将确认 关节中的ASCs，植入水平与受损软骨的减少和修复以及 优化细胞数量和处理间隔。我们将检查关节内ASCs的本地化和 评估关节炎症和软骨完整性。我们将调查与治疗相关的变化 软骨下骨和软骨的生物力学特性及其对疼痛相关行为的影响 功能分析。我们还将使用ASC-软骨细胞共培养来研究修复机制 系统。(2)证明两步应用：TPCA1-纳米体的协同治疗效果 在PTOA小鼠模型中，移植预适应的ASCs之前注射(TPCA1-NS)。在这 目的：我们将使用TPCA1-NS治疗炎症，然后使用维生素E纳米小体 经ASCs处理后移植到本实验室建立的PTOA小鼠模型中。在这个目标3中，我们将 检查两步治疗后软骨、骨和滑膜组织的功能结果 包括联合注射TPCA1-NS和移植预适应的ASCs。这 两步抗炎和抗氧化-纳米体治疗策略可以改善有毒的微 通过注射TPCA1-NS改善关节炎膝关节的环境，并延长ASCs的存活时间以获得 它们的免疫抑制活动有足够的生存时间。 我们相信，从这一项目中获得的数据将作为理解该机制的基础 PTOA与移植ASCs的治疗效果此外，这些数据将有助于开发 未来临床应用的策略。

PROJECT SUMMARY/ABSTRACT The etiology of osteoarthritis (OA) is multi-factorial. Abnormal and excessive cumulative joint stress results in post-traumatic osteoarthritis (PTOA). Approximately 30% of knee OA in Veterans is attributable to occupational activities, particularly jobs requiring kneeling or squatting in combination with heavy lifting, such as sport activity injuries. Currently, there is no effective therapy for OA patients. Recently, the stem cell therapy has shown promise to regenerate the damaged joint tissue. In our previous study, adipose-derived stem cells (ASCs) show great promise as therapeutic agents in regenerative medicine because of their multi-lineage potential, immunosuppressive activities, limited immunogenicity, and relative ease of growth in culture. However, there are several concerns that impede the clinical use of stem cell therapy in the inflammatory joint environment such as apoptosis, dosing, timing of intervention, homing efficacy, and route of delivery of ASCs. We have also found that NF-κB inhibitors such as TPCA-1 decrease inflammation in mechanically injured knee joints using our established PTOA mouse model and in vitro model. In this project, we aim our studies based on two points: 1) Pre-treatment of exogenously derived ASCs with antioxidant (such as Vitamin-E) before injection into the joint can lead to cyto-protective effects and resistance to apoptosis and toxic inflammatory factors after transplantation. 2) TPCA1-nanosome can improve the harsh condition in the arthritic knee joint by anti-inflammatory mechanism before transplantation of an exogenous stem cell. Therefore, we will show the synergistic effect of this two-step therapeutic application (anti- inflammatory nanosome treatment of the arthritic joint followed by transplantation of the Preconditioned ASCs). We have two aims: (1) Investigate the cytoprotective effects and therapeutic potential of preconditioned ASCs in a model of PTOA. In this aim, we will investigate the therapeutic effect of the antioxidant (Vit-E-Ns) pre-treated ASCs in vivo using our PTOA mouse model of knee overloading. In order to tailor this approach to the appropriate veteran population (active vs retired) we will use aged mice to investigate whether they exhibit different healing responses and mechanisms relative to younger mice. We will confirm the persistence of the ASCs in the joint, correlating implantation levels with the reduction and repair of damaged cartilage as well as optimizing the cell number and treatment interval. We will examine localization of the ASCs in the joint and assess joint inflammation and cartilage integrity. We will investigate treatment-associated changes in the biomechanical properties of subchondral bone and cartilage and its effects on pain-related behavior through functional analyses. We will also investigate the recovery mechanism using an ASC-chondrocyte co-culture system. (2) Demonstrate the synergistic therapeutic efficacy of the two-step application: TPCA1-Nanosome (TPCA1-Ns) injection prior to transplantation of the preconditioned ASCs in a mouse model of PTOA. In this aim, we will neutralize inflammation using TPCA1-Ns treatment followed by the utilization of Vit-E nanosome pretreated ASCs for transplantation into the PTOA mouse model established in our lab. In this Aim 3, we will examine the functional outcomes of the cartilage, bone, and synovial tissues after the two-step treatment consisting of the combined injection of the TPCA1-Ns and transplantation of the preconditioned ASCs. This two-step anti-inflammatory and antioxidant-nanosome treatment strategy can improve the toxic micro- environment of the arthritic knee joint via delivery of TPCA1-Ns, and also prolong the ASCs viability to obtain enough time to survive for their immune-suppressive activity. We believe that the data obtained from this project will serve as a basis for understanding the mechanism of PTOA and the therapeutic efficacy of transplanted ASCs. Also, this data will help contribute to developing strategies for clinical applications in the future.