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.Proposal编号：1034026在美国，每年有570万人次去急诊室就诊。拉伤是由于过度使用或创伤而导致的肌肉或肌腱损伤。据估计，2002年仅韧带重建手术就需要20万美国人，费用超过50亿美元。在美国，大约有61,000人受到埃勒斯-丹洛斯综合征(EDS)的影响，EDS是一种结缔组织疾病。EDS，导致身体产生有缺陷和脆弱的胶原蛋白。虽然EDS有几种类型，但一个共同的特征是过度活动和关节不稳定。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。实际上，这些都是持续性的扭伤和劳损，患者将极大地受益于永久性的内部支撑和关节稳定。对于受损和疼痛的韧带和肌腱，前路疗法是一种有效的、但有一定争议的治疗方法。它包括将增殖剂溶液注入肌腱或骨韧带交界处，刺激身体伤口愈合的级联反应。受损的组织被修复，新的胶原蛋白逐渐收缩，形成更密集、更坚固和更紧的韧带或肌腱。对于胶原蛋白产生正常的人，修复是长期的。那些患有EDS的人通常需要持续的治疗。为了提高前列腺素治疗的有效性和延长其疗效(可能使其成为一种永久性的解决方案)，我们建议使用碳纳米结构、碳纳米管(CNTs)或碳纳米角(CNHs)的前列腺素治疗。这种变革性的治疗将保留正常治疗的所有好处，同时提供立即的机械强化(由于纳米结构)和纳米结构对成纤维细胞的长期搅拌，以便在以后关节松动(由于受伤或EDS)时产生胶原蛋白。我们建议通过以下目标来研究这种疗法：1)测试用于成纤维细胞的治疗前处理的溶液、纳米结构和麻醉。这将确保我们找到这些药物的组合，对细胞造成最小的损害，产生最多的胶原蛋白，并且不会导致纳米结构被细胞摄取。2)将不同浓度的纳米结构注射到离断韧带中，观察改善力学性能的最佳浓度。3)在兔MCL模型中使用最佳的前处理液和纳米结构的类型和浓度。这一模型将检验新疗法的有效性和智力价值：这种增强的前疗法有可能快速有效地治疗因遗传疾病而变得虚弱的劳损、扭伤和组织。使用碳纳米结构可以立即增强组织硬度，并在长期内促进新胶原的生长，使这种治疗方法在恢复组织机械性能方面立即有效，并显著降低损伤复发的机会。后一点对EDS患者极其重要，因为他们可能在一生中接受数百至数千次治疗。更广泛的影响：这项拟议的研究将加强对扭伤和拉伤组织以及由于结缔组织疾病(如EDS)造成的损伤的治疗。它有可能扩大新的治疗药物在运动医学中的使用，使运动员更快地从受伤中恢复，并减少重复受伤的可能性。这项研究将为学生提供一个在研究生、本科生和高中阶段获得实验设计、工程和细胞生物学经验的机会。作为非裔美国人的约瑟夫·弗里曼博士和作为女性的尼科尔·里兰德博士代表了生物医学工程领域中代表性不足的群体，并致力于建立外联计划，以招募代表性不足的学生(少数族裔和女性)进入该领域。他们参与了几个高级组织，包括AdvanceVT(女性招聘计划)和弗吉尼亚理工大学工程多样性增强中心(CEED)。本科生和研究生将从这些项目中招募，在学年和暑期进行与该项目相关的研究。弗吉尼亚理工大学生物工程和生物信息学暑期研究所(BBSI)项目将招募来自代表性不足群体的两名学业优秀的本科生进行为期10周的暑期研究。此外，一名患有EDS的研究生将进行这里描述的研究，作为她硕士论文的一部分。

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.