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: 7861379
• 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/7861379
• 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. PUBLIC HEALTH RELEVANCE: Project Narrative This translational research will optimize techniques to produce tissue engineered tendons. After these techniques are developed, military and VA surgeons could remove a small portion of remaining tendon (or other cell source) and then allow these cells to proliferate in tissue culture while the patient is stabilized. Then, cadaver allograft tendons from a human tissue bank can be acellularized and seeded with the patient's own cells. When definitive reconstruction of the extremity is undertaken, large amounts of biocompatible tendon material would be available. Therefore, recent advances in bioengineering research may be translated to direct clinical applications for a unique and significant need in injured soldiers. Furthermore, aging veterans have increased incidence of rheumatoid arthritis and osteoarthritis which lead to degeneration of tendons and decreased hand function. Tissue engineered flexor tendon grafts would also be useful for reconstruction in these cases.

描述(由申请人提供)： 项目概述/摘要本研究的目的是将兔模型屈肌腱组织工程的研究成果应用于人类临床。具体地说，目标和目标是：1)基于在兔身上的工作，优化人屈肌肌腱去细胞的技术；2)用候选细胞接种去细胞人肌腱，以创建组织工程肌腱构建；3)通过使用新型大型组织生物反应器施加循环剪切力和应变力，最大限度地提高组织工程肌腱的体外构建强度和生存能力；以及4)通过使用这些组织工程肌腱构建物选择人类临床病例，使用这些组织工程肌腱构建物重建严重手部损伤的肌腱。人体屈肌腱将被解剖并在培养中保存。使用十二烷基硫酸钠、Triton x-100和冻融循环的条件将有所不同，直到建立了在保持结构完整性的同时将抗原性降至最低的最佳方案。有效性将由组织学、荧光细胞染色和DNA含量来确定。结构强度将通过极限抗拉强度和弹性模数的张力法来确定。腱细胞、真皮成纤维细胞和脂肪干细胞的原代培养将在培养中扩增，并以2x106个/毫升的密度播种。播种将包括细胞悬浮液、微量注射和超声波处理的组合。肌腱结构将在培养中保存7天。细胞种植效率将通过H&E显微镜、细胞染色和I型和III型胶原的定量分析来确定。将使用定制的组织生物反应器提供单轴肌腱应变。肌腱结构将在5天的过程中分别承受1.25N的单轴拉力。初始循环参数为1周/分钟，机械加载和休息交替进行1小时。在生物反应器处理或静止孵化后，试件将接受拉伸测试，以比较极限拉应力和弹性模量。在有严重上肢损伤的退伍军人患者中，两套屈肌腱都缺失[指深屈肌(FDP)和指浅屈肌(FDS)]。FDS肌腱是一个冗余的系统，通常不会重建。这提供了一个独特的机会来测试组织工程化肌腱移植物的有效性，并将额外的手术时间和对患者的风险降至最低。FDPs将使用普通肌腱移植物重建，FDS将使用新的组织工程肌腱移植物重建。结果将包括术后活动范围、活检的组织学检查以及是否需要翻修手术。在这些技术开发出来后，退伍军人外科医生可以移除一小部分肌腱(或其他细胞来源)，然后在患者病情稳定的情况下，允许细胞在培养中增殖。然后，来自组织库的身体同种异体肌腱可以去细胞并种植患者自己的细胞。当进行四肢重建时，将有大量的生物相容肌腱可用。这项生物工程研究可能会被转化为直接临床应用，以满足受伤士兵的重大需求。 公共卫生相关性： 这项翻译研究将优化生产组织工程化肌腱的技术。在开发出这些技术后，军事和退伍军人外科医生可以移除剩余肌腱的一小部分(或其他细胞来源)，然后在患者病情稳定的情况下，允许这些细胞在组织培养中增殖。然后，来自人类组织库的身体同种异体肌腱可以去细胞并种植患者自己的细胞。当最终重建四肢时，将有大量的生物相容的肌腱材料可用。因此，生物工程研究的最新进展可能会转化为对受伤士兵独特而重要的需求的直接临床应用。此外，老年退伍军人类风湿性关节炎和骨性关节炎的发病率增加，这会导致肌腱退化和手功能下降。在这些情况下，组织工程化屈肌腱移植也将用于重建。