Optimization of Human Tendon Tissue Engineering Using Bioreactors

使用生物反应器优化人体肌腱组织工程

• 批准号: 8838111
• 负责人: James Chang
• 金额: --
• 依托单位: VETERANS ADMIN PALO ALTO HEALTH CARE SYS
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-06-01 至 2014-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8838111
• 关键词: Aging; Allografting; Articular Range of Motion; Autologous; Biocompatible; Biocompatible Materials; Biomedical Engineering; Biopsy; Bioreactors; Blast Injuries; Cadaver; Cartilage; Cells; Cicatrix; Clinical; Collagen; Connective Tissue; Custom; Defect; Degenerative polyarthritis; Dermal; Digit structure; Disease; Effectiveness; Evolution; Fibroblasts; Fingers; Flexor; Freezing; Goals; Gunshot wound; Hand; Hand Injuries; Hand functions; Hematoxylin and Eosin Staining Method; Histocompatibility Testing; Histology; Hour; Human; In Vitro; Incidence; Injury; Lead; Limb structure; Lower Extremity; Mechanics; Methods; Microinjections; Microscopy; Military Personnel; Modeling; Motor; Muscle; Operative Surgical Procedures; Oryctolagus cuniculus; Outcome; Patients; Ploidies; Postoperative Period; Production; Proliferating; Protocols documentation; Reconstructive Surgical Procedures; Research; Rest; Rheumatoid Arthritis; Risk; Rupture; Seeds; Silicones; Skin; Soldier; Source; Specimen; Staging; Stem cells; Stress; Stretching; Structure; Surgeon; Suspension substance; Suspensions; System; Techniques; Tendon Injuries; Tendon structure; Tensile Strength; Testing; Time; Tissue Banking; Tissue Banks; Tissue Engineering; Tissues; Translating; Translational Research; Triton; Tritons; Upper Extremity; Veterans; Work; Wrist joint; abstracting; base; bone; clinical application; density; disability; efficacy testing; functional restoration; human tissue; improved functioning; injured; novel; reconstruction; repaired; retinal rods; scaffold; tissue culture; vehicular accident

DESCRIPTION (provided by applicant): Project Summary/Abstract The objective of this research is to translate previous work on flexor tendon tissue engineering in the rabbit model to human clinical cases. Specifically, the goals & objectives are to: 1) Optimize techniques for acellularization of human flexor tendons based on work in the rabbit; 2) Seed acellularized human tendons with candidate cells to create tissue-engineered tendon constructs; 3) Maximize tissue-engineered tendon construct strength and viability in vitro by applying cyclic shear and strain forces using a novel large-scale tissue bioreactor; and 4) Translate to select human clinical cases by using these tissue-engineered tendon constructs for tendon reconstruction in severe cases of mutilating hand injuries. Human flexor tendons will be dissected and preserved in culture. Conditions using SDS, Triton x-100, and freeze-thaw cycles will be varied until the optimal protocol to minimize antigenicity while maintaining structural integrity is established. Effectiveness will be determined by histology, fluorescent cytostaining, and DNA content. Structural strength will be determined by tensiometry for ultimate tensile strength and elastic modulus. Primary cultures of tenocytes, dermal fibroblasts, and adipoderived stem cells will be expanded in culture and seeded at a density of 2x106 cells/cc. Seeding will consist of combinations of cell suspension, microinjection, and ultrasonication. The tendon constructs will be kept in culture for 7 days. Cell seeding efficacy will be determined by H&E microscopy, cytostaining, and quantitative analysis of collagen I & III. A custom tissue bioreactor providing uniaxial tendon strain will be used. The tendon constructs will be subjected to a uniaxial stretch force 1.25N each over a 5 day course. The initial cycle parameters will be 1cycle/min in alternating 1 hour periods of mechanical loading and rest. After bioreactor treatment or stationary incubation, the specimens will undergo tensile testing to compare ultimate tensile stress and elastic modulus. In Veteran patients with severe upper extremity injuries, both sets of flexor tendons are missing [flexor digitorum profundus (FDP) and flexor digitorum superficialis (FDS)]. The FDS tendons are a redundant system that is usually not reconstructed. This provides a unique opportunity to test efficacy of the tissue-engineered tendon grafts with minimal additional operative time and risk to the patient. The FDPs will be reconstructed using normal tendon grafts and the FDSs will be reconstructed using the new tissue-engineered tendon grafts. Outcomes will consist of postoperative range of motion, histology on biopsies, and need for revision surgery. After these techniques are developed, VA surgeons could remove a small portion of tendon (or other cell source) and then allow cells to proliferate in culture while the patient is stabilized. Then, cadaver allograft tendons from a tissue bank can be acellularized and seeded with the patient's own cells. When reconstruction of the extremity is undertaken, large amounts of biocompatible tendon would be available. This bioengineering research may be translated to direct clinical applications for a significant need in injured soldiers.

描述（由申请人提供）： 项目摘要/摘要这项研究的目的是将兔模型中屈肌肌腱组织工程的先前工作转化为人类临床病例。具体而言，目标和目标是：1）根据兔子的工作优化人屈肌肌腱的技术； 2）带有候选细胞的种子细胞化的人肌腱，形成组织工程肌腱构建体； 3）使用新型的大型组织生物反应器施加环状剪切和应变力，从而在体外最大化组织肌腱构建强度和生存力； 4）通过在严重的肢体损伤病例中使用这些组织工程肌腱构建体进行肌腱重建，从而选择人类临床病例。人体屈肌将在文化中进行解剖和保存。使用SDS，Triton X-100和Freeze-Thaw周期的条件将变化，直到建立最小化抗原性的最佳方案，同时确定结构完整性。有效性将由组织学，荧光细胞抑制和DNA含量确定。结构强度将通过张力计测定，以实现最终的拉伸强度和弹性模量。培养基，皮肤成纤维细胞和脂肪构成干细胞的原发性培养物将在培养中扩展，并以2x106细胞/CC的密度播种。播种将包括细胞悬浮液，显微注射和超声化的组合。肌腱构建体将在文化中保存7天。细胞播种功效将通过H＆E显微镜，细胞固化和胶原蛋白I和III的定量分析确定。将使用提供单轴肌腱应变的定制组织生物反应器。在5天的课程中，肌腱构建体将每轴伸展力1.25N。在1小时的机械加载和休息时间交替的时间内，初始循环参数将为1周/分钟。生物反应器处理或固定孵育后，样品将进行拉伸测试，以比较最终的拉伸应力和弹性模量。在严重上肢损伤的退伍军人患者中，两组屈肌肌腱都缺失[屈肌profundus（FDP）和屈肌表面（FDS）]。 FDS肌腱是通常不重建的冗余系统。这提供了一个独特的机会，可以测试组织工程肌腱移植物的功效，其额外的手术时间很少，并且对患者的风险很小。 FDP将使用正常肌腱移植物重建，FDS将使用新的组织工程肌腱移植物重建。结果将包括术后运动范围，组织学的组织学以及对修改手术的需求。开发这些技术后，VA外科医生可以去除一小部分肌腱（或其他细胞来源），然后在患者稳定时使细胞在培养中增殖。然后，来自组织库的尸体同种异体肌腱可以被化为细胞并与患者自身的细胞接种。当进行四肢重建时，将提供大量的生物相容性肌腱。这项生物工程研究可能会转化为直接临床应用，以便受伤的士兵的重大需求。