A Novel Treatment for Connective Tissue in Ehlers-Danlos Patients and Strained and Sprained Ligaments: Investigating Carbon Nanostructure Enhanced Prolotherapy

PI：Freeman，J.，Brolinson，G.和Rylander，N.提案编号：1034026在美国，每年有570万人因扭伤和拉伤而到急诊室就诊。拉伤是由于过度使用或创伤造成的肌肉或肌腱损伤。据估计，2002年有20万美国人需要进行韧带重建手术，价格超过50亿美元。在美国，大约有61，000人受到Ehlers-Danlos综合征（EDS）的影响，这是一种结缔组织疾病。EDS，导致身体产生缺陷和弱胶原蛋白。虽然有几种类型的EDS，但一个共同的特点是关节活动过度和不稳定。患有EDS的人经常经历关节脱位和半脱位，这是痛苦和虚弱的。有效地，这些是持续的扭伤和应变，患者将大大受益于永久形式的内部支撑和关节的稳定。增生疗法是一种可用的，但有点争议，治疗损伤和疼痛的韧带和肌腱。它包括将增殖剂溶液注射到肌腱或骨韧带连接处，刺激身体的伤口愈合级联。受损的组织得到修复，新的胶原蛋白逐渐收缩，形成一个更密集，更强大，更紧密的韧带或肌腱。在具有正常胶原蛋白产生的人中，修复是长期的。EDS患者通常需要持续治疗。为了提高有效性和延长增殖疗法的结果（可能使其成为永久性解决方案），我们提出使用具有碳纳米结构、碳纳米管（CNT）或碳纳米角（CNH）的增殖疗法。这种变革性的治疗将保留正常增生疗法的所有益处，同时提供立即的机械加固（由于nanostructureress）和通过纳米结构长期搅动成纤维细胞以产生胶原蛋白，如果关节在稍后的日期松动（由于损伤或EDS）。我们建议通过以下目标来研究这种疗法：1）测试增生疗法溶液、纳米结构和成纤维细胞增生疗法中常用的麻醉剂。这将确保我们找到这些试剂的组合，其引起最少的细胞损伤，产生最多的胶原蛋白，并且不会导致细胞摄取纳米结构。2)将不同浓度的纳米结构注射到切除的韧带中，以观察改善机械性能的最佳浓度。3)在兔应变MCL模型中使用最佳增殖疗法溶液以及纳米结构的类型和浓度。该模型将测试新疗法的有效性智力优点：这种增强的增殖疗法治疗有可能快速有效地治疗菌株，扭伤，和遗传性疾病削弱的组织。使用碳纳米结构立即增强组织硬度并长期促进新胶原蛋白的生长，使得这种治疗在恢复组织机械性能方面立即有效，并大大降低了损伤复发的机会。后一点对EDS患者来说非常重要，因为他们一生中可能会接受数百到数千次治疗。更广泛的影响：这项拟议的研究将加强治疗扭伤和紧张的组织沿着受伤，由于结缔组织疾病，如EDS。它有可能扩大新的治疗剂在运动医学中的使用，使运动员能够更快地从伤病中恢复过来，更不容易重复受伤。这项研究将为学生提供一个机会，在研究生，本科和高中阶段获得实验设计，工程和细胞生物学的经验。Joseph Freeman博士是一名非裔美国人，Nichole Rylander博士是一名女性，代表了生物医学工程领域中代表性不足的群体，并致力于建立外展计划，以招募代表性不足的学生（少数民族和妇女）进入该领域。他们参与了几个促进组织，包括AdvanceVT（女性招聘计划）和弗吉尼亚理工大学的工程多样性增强中心（CEED）。本科生和研究生将从这些计划中招募，在学年和夏季进行与本项目相关的研究。两名来自代表性不足群体的学术实力雄厚的本科生将被招募，通过弗吉尼亚理工大学的生物工程和生物信息学暑期研究所（BBSI）项目进行为期10周的暑期研究。此外，EDS的一名研究生将执行这里描述的研究，作为她的硕士论文的一部分。

PI: Freeman, J., Brolinson, G. and Rylander, N.Proposal Number: 1034026Sprains and strains account for 5.7 million visits to emergency rooms in the United States each year. A strain is an injury to the muscle or tendon due to overuse or trauma. An estimated 200,000 Americans required reconstructive surgery of ligaments alone in 2002 with a price tag exceeding five billion dollars. There are approximately 61,000 people in the United States affected by Ehlers-Danlos Syndrome (EDS), a connective tissue disorder. EDS, causes the body to produce faulty and weak collagen. While there are several types of EDS, a common trait is hypermobile and unstable joints. People with EDS experience frequent joint dislocations and subluxations which are painful and debilitating. Effectively, these are continual sprains and strains, patients would greatly benefit from a permanent form of internal bracing and stabilization of the joints. Prolotherapy is an available, but somewhat controversial, treatment for damaged and painful ligaments and tendons. It involves the injection of a proliferant solution into the tendon or bone-ligament junction, stimulating the body's wound healing cascade. The damaged tissue is repaired, and new collagen is laid down which gradually shrinks, forming a denser, stronger, and tighter ligament or tendon. In persons with normal collagen production the repair is long term. Those with EDS usually need ongoing treatment. In order to enhance the effectiveness and prolong the results of prolotherapy (possibly making it a permanent solution) we propose the use of prolotherapy with carbon nanostructures, carbon nanotubes (CNTs) or carbon nanohorns (CNHs). This transformative treatment would retain all of the benefits of normal prolotherapy while providing immediate mechanical reinforcement (due to nanostructuress) and long term agitation of fibroblasts by nanostructures for the production of collagen if the joint is loose at a later date (as a result of injury or EDS). We propose to investigate this therapy through the following objectives: 1) Testing prolotherapy solutions, nanostructures, and anesthesia commonly used in prolotherapy with fibroblasts. This will insure that we find a combination of these agents that causes the least cell damage, produces the most collagen and does not lead to nanostructure ingestion by the cells. 2) Injecting different concentrations of nanostructures into excised ligaments to view the optimal concentration for improved mechanical properties. 3) The use of the best prolotherapy solution and types and concentration of nanostructures in a rabbit strained MCL model. This model will test the effectiveness of the new therapy Intellectual Merit: This enhanced prolotherapy treatment has the potential to quickly and effectively treat strains, sprains, and tissues weakened by genetic disorders. The use of carbon nanostructures to immediately enhance tissue stiffness and promote the growth of new collagen over the long term makes this treatment immediately effective in restoring tissue mechanical properties and drastically reduces the chances of injury recurrence. The later point is of extreme importance to people with EDS because they may undergo hundreds to thousands of treatments over a lifetime. Broader Impact: This proposed research will enhance the treatment of sprained and strained tissues along with injuries due to connective tissue disorders, such as EDS. It has the possibility of expanding the use of new therapeutic agents in sports medicine allowing athletes to return from injury quicker and less prone to repeat the injury. This research will provide an opportunity for students to gain experience in experimental design, engineering, and cell biology at the graduate, undergraduate, and high school levels. Dr. Joseph Freeman as an African American and Dr. Nichole Rylander as a female represent underrepresented groups within the biomedical engineering field and are devoted to establishing outreach programs to recruit underrepresented students (minorities and women) into the field. They are involved with several advancement organizations including AdvanceVT (female recruiting program) and Center for the Enhancement of Engineering Diversity (CEED) at Virginia Tech. Undergraduate and graduate students will be recruited from these programs to conduct research related to this project during the academic year and summer. Two scholastically strong undergraduate students from underrepresented groups will be recruited to perform summer research for 10 weeks through the Bioengineering and Bioinformatics Summer Institute (BBSI) program at Virginia Tech. In addition, a graduate student with EDS will perform the research described here as part of her Master's Thesis.