作者要感谢的启动资金，并从南伊利诺伊大学医学院外科研究资助部门;和纪念医学基金会资助。

1，手术取出注意:一个IRB的批准，获得在ACL重建手术来收集的ACL残端。 IRB豁免给出所使用的ACL残端一般都弃之如手术废弃物。 20例患者用于收集的ACL残端。 <ol><li>管理全身麻醉和手术前抗生素作为每机构的协议给患者。 </li><li>应用止血带充气100毫克以上的收缩压。 </li><li>不要将病人仰卧位。做一个横向切口前外侧膝在30-45°屈曲位为5毫米的门户网站，并插入关节镜套管和相机进入髌上囊，在30℃。 </li><li>执行常规诊断关节镜检查。 </li><li>输入一个缺口，并清创膜状滑膜过十字韧带撕裂。 </li><li>使用4.5毫米剃须刀和消融仪来确定ACL残端。 </li><li>使用鸭嘴直苦收集多个SPEcimens从剩余的ACL残端。 </li><li>保持在生理盐水中的样品在无菌容器中。 </li><li>送样病理解码和最终交付给实验室设施。 </li><li>恢复病人和执行术后护理按机构的协议。 </li></ol> 2，组织消化和HACL隔离<ol><li>传输从病理接收人的ACL组织至培养皿用无菌生理盐水/ PBS（磷酸盐缓冲盐水）。 </li><li>剁碎，用无菌剪刀组织成1-2毫米3条并用盐水冲洗组织糜的至少3倍。 </li><li>消化组织糜在DMEM/F-12 0.4％胶原酶在37℃下在补充有10％胎牛血清（FBS）和1％青霉素/链霉素为4-6小时（50/50，1）培养基</li><li>离心将细胞在1000×g离心5分钟，然后重悬细胞于培养基中。 </li><li>与介质洗2〜3次的细胞，则sEED /文化T-25瓶2-3天。 </li></ol> 3，细胞膨胀，冻融<ol><li>使用光学显微镜来可视化培养细胞2-3天以T-25烧瓶中。 </li><li>保持在DMEM/F-12的细胞在37℃下在补充有10％胎牛血清（FBS）和1％青霉素/链霉素（P / S）（50/50，1×）培养基中。 注:更改介质时，细胞显示出坚持和标准形态。用光学显微镜观察细胞。 </li><li>用PBS在第7天（〜1.3×10 6个细胞/瓶）洗涤汇合细胞。添加胰蛋白酶（0.5克/升），并于室温孵育在37℃下5分钟。添加媒体到悬浮细胞中和胰蛋白酶。分三路T-25瓶之间的介质的细胞混合物中。 </li><li>用PBS洗涤汇合细胞，添加胰蛋白酶（0.5克/升），并悬浮在其中含有DMSO，FBS和完全培养基冻存培养基（DMEM/F-12培养基+10％FBS +1％P / S）中的比1:2:7。 </li><li>店该低温小瓶在-80℃下，如果细胞将在一个月内使用，并在液氮中，如果细胞将被存储为更长的持续时间。 </li><li>解冻小瓶在37℃水浴中再利用。 注:通常90-95％的细胞存活。 </li></ol> 4，组织工程化的2-D聚乳酸 - 乙醇酸共聚物（PLAGA）支架的制备<ol><li>溶解1g PLAGA的在12毫升二氯甲烷中，在20ml的闪烁瓶中，并旋涡振荡8小时的溶液以恒定的速度（每分钟800转）。 注:详细描述，Gupta 等人15。 </li><li>转移的溶解液，以玻璃培养皿内衬含氟保护纸。 </li><li>将培养皿真空罩30分钟以下。留在培养皿在-20℃下过夜，然后在室温下进行。将培养皿在冻干机，以确保溶剂完全蒸发，得到的薄膜的聚合物。 </li><li>将聚合物网络连接LMS在干燥器中放置24小时。 </li><li>切聚合物薄膜成12毫米直径的圆盘，并直到需要它们存储在干燥器中。 </li></ol> 5，扫描电子显微镜（SEM）观察<ol><li>洗涤细胞（50,000个细胞/盘）接种在控制TCPS和PLAGA支架用PBS7天之后播种。 </li><li>用在0.1M二甲胂酸盐缓冲液的1.5％戊二醛固定细胞和2.5％的OsO 4在0.1M二甲胂酸盐缓冲液后固定。 </li><li>用0.1M二甲胂酸盐缓冲液洗涤固定的细胞。采用串行乙醇脱水（50％，70％，80％，90％和100％），每次15分钟，并进一步干燥以六甲基二硅氮烷（HMDS）中过夜干燥固定的细胞。 </li><li>发泄SEM溅射涂覆系统的腔与按钮阀，提高顶板。 </li><li>将干燥的样品在室中。降低顶板。 </li><li>切换控制旋钮，水泵开始抽真空后。 </li><li>打开氩气泄漏阀。等待用于真空下降至0.05毫巴。 </li><li>大衣干燥的样品用薄金/钯层（0.13纳米）。 </li><li>返回氩气泄漏阀到它的关闭位置。 </li><li>控制旋钮切换到关闭。 </li><li>发泄室与按钮阀，提高顶板。 </li><li>取出样品，并将其储存在干燥器中放置24小时。 </li><li>观察样品的扫描电子显微镜下。 </li></ol> 6，免疫荧光染色<ol><li>洗涤细胞（50,000个细胞/盘）接种在控制TCPS和PLAGA支架用PBS7天之后播种。 </li><li>用70％乙醇（冷的）10分钟，固定细胞。 </li><li>孵育固定的细胞在室温下用1％牛血清白蛋白（BSA）的PBS中具有0.05％的Triton X-100的20分钟。 </li><li>浸没在样品中1％吐温在室温下20分钟。 </li><li>添加单克隆小鼠抗β-肌动蛋白抗体（1:400）孵育细胞持有者172吨，在4°C。 </li><li>用0.05％吐温洗细胞。二抗（山羊抗小鼠的F（ab&#39;）的IgG 2片段结合有荧光探针，1:400）添加到细胞中，并孵育1小时，在室温下进行。 </li><li>用PBS洗涤细胞。染色的细胞核染色，用80％甘油安装。 </li><li>观察共聚焦显微镜下的细胞。 </li></ol>

<ol>
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作者宣称，他们有没有竞争的财务权益。

这hACL/2D支架研究的主要目的是利用所获得的细胞修补，以增加局部ACL撕裂初级修复。部分ACL撕裂非手术治疗可能包括固定，支撑，循序渐进的康复计划，并定期随访评估13,16,17的一个短周期。然而，许多研究表明，在运动员的保守治疗已经效果差和故障有关。巴克利等人对25例局部ACL的眼泪在中间随访发现，只有44％的患者在受伤前的水平恢复运动，72％的活性相关的症状8。</sup...

前交叉韧带（ACL）是膝关节的常见的受伤关节内韧带。约200,000（ACL）损伤是在美国每年报告。超过75％的患者经历ACL损伤齐齐整形重建手术1,2,3,4。手术治疗往往是由于固有的愈合不良潜力3所示。 ACL重建通常是通过自体移植或异体移植腱的方法。自体移植和异体移植代表的金标准重建，因为他们拥有较高的成功率，并缝合修复，其他的治疗方案，已经显示出高达94％的5,6,7故障率。 该ACL部分撕裂占10％的所有ACL的眼泪8 28％。在一项前瞻性研究，诺伊斯等人估计，患者的影响超过了ACL半部分撕裂50％进展到完成的ACL功能不全后非手术治疗9。其他的研究报告持续不稳定，并与患者能够恢复到受伤前的活动水平9,10,11,12,13少于30％下降功能。治疗选择很有限，其中包括保守的方式，其余的ACL的热收缩，或ACL重建。最近，出现了增加在增强初级修复的兴趣。这些技术使用生物制剂，以加强基层缝合修复14。最近的研究已尝试收获的间充质样HACL来源的干细胞，以规避接枝限制，31,32，但这些细胞的效力和疗效尚属未知。理想的细胞来源，组织工程应用似乎非mesechymal HACL衍生的成纤维细胞。 目前的研究重点是确定一个合适的基质材料和细胞源工程支架。这是标准程序十字韧带撕裂被丢弃苏在重建手术rgical浪费，但是，这受损的韧带可能是一个质量的来源，收购，开发及提升工程的ACL替换一个理想的组织所需要的细胞。我们实验室已经开发出一种协议，用于体外扩增这些收获HACL来源的细胞。使用一个精心设计的二维矩阵注入HACL来源的细胞，我们设计了一个修补程序，可能会增加部分ACL修复和加强韧带撕裂。

<table><tbody><tr><td>Petri dish</td><td>Fisher Scientific</td><td>08-757-103C</td><td></td></tr><tr><td>Phosphate Buffered Saline (PBS)</td><td>Fisher Scientific</td><td>BP3994</td><td></td></tr><tr><td>Collagenase</td><td>Gibco</td><td>17018-029</td><td>Store at 4 &amp;#176;C</td></tr><tr><td>DMEM/F-12</td><td>Cellgro</td><td>10-092-CV</td><td>Store at 4 &amp;#176;C. Warm in 37 &amp;#176;C water bath before use.</td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Gibco</td><td>10082</td><td>Store at -80 &amp;#176;C</td></tr><tr><td>Penicillin/Streptomycin</td><td>Lonza</td><td>17-602E</td><td>Store at 4 &amp;#176;C</td></tr><tr><td>Centrifuge Tubes- 15 ml</td><td>Corning</td><td>430790</td><td></td></tr><tr><td>T-25 flasks</td><td>BD Falcon</td><td>3013</td><td></td></tr><tr><td>Trypsin-Versene mixture</td><td>Lonza</td><td>17-161E</td><td>Store at 4 &amp;#176;C. Warm in 37 &amp;#176;C water bath before use.</td></tr><tr><td>DMSO</td><td>Fisher Scientific</td><td>BP231-100</td><td>Combustible liquid. Can cause skin, eye and respiratory tract irritation.</td></tr><tr><td>Cryogenic vials</td><td>Corning</td><td>430489</td><td></td></tr><tr><td>PLAGA</td><td>Purac Biomaterials</td><td>Purasorb PLG8523</td><td>Store at -80 &amp;#176;C</td></tr><tr><td>TCPS disks</td><td>Fisher Scientific</td><td>12-545-82</td><td></td></tr><tr><td>Dichloromethane</td><td>Fisher Scientific</td><td>AC36423-0010</td><td>Possible cancer hazard. Store in a dry, cool place.</td></tr><tr><td>Bytac paper</td><td>Saint gobin performance plastics</td><td>1420652</td><td></td></tr><tr><td>Scintillation vial</td><td>Fisher Scientific</td><td>03-339-21G</td><td></td></tr><tr><td>Bovine Serum Albumin (BSA)</td><td>Sigma Aldrich</td><td>A7906</td><td>Store at 4 &amp;#176;C</td></tr><tr><td>Tween</td><td>Fisher Scientific</td><td>BP337-500</td><td></td></tr><tr><td>mouse Anti-&amp;beta;-actin antibody (1&amp;#176; Ab)</td><td>Sigma Aldrich</td><td>A5441</td><td>Store at -20 &amp;#176;C</td></tr><tr><td>goat-anti-mouse antibody (2&amp;#176; Ab)</td><td>Cell Signaling</td><td>4408</td><td>Store at -20 &amp;#176;C</td></tr><tr><td>Hoechst dye</td><td>Sigma Aldrich</td><td>14530</td><td>Store at -20 &amp;#176;C</td></tr><tr><td>Glycerol</td><td>Fisher Scientific</td><td>BP229-1</td><td></td></tr><tr><td>Glutaraldehyde</td><td>Fisher Scientific</td><td>BP2547-1</td><td>Toxic by inhalation and if swallowed. Causes burns by all exposure routes.</td></tr><tr><td>Hexamethyldisilazane</td><td>Fisher Scientific</td><td>AC43085-1000</td><td>Flammable liquid and vapor. Causes burns by all exposure routes.</td></tr><tr><td>Sterile Scissors</td><td>McKesson</td><td>25-716</td><td></td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td>5804R</td><td></td></tr><tr><td>Light Microscope</td><td>Olympus</td><td>CK40</td><td></td></tr><tr><td>Water Bath</td><td>Thermo scientific</td><td>2845</td><td></td></tr><tr><td>Vortex</td><td>Labnet</td><td>VX-200</td><td></td></tr><tr><td>Glass Petri plates</td><td>fisher Scientific</td><td>S31473</td><td></td></tr><tr><td>Acu-Punch</td><td>Acuderm Inc.</td><td>P1225</td><td>Acu-Punch was used to cut 12 mm disks</td></tr><tr><td>Cacodylate buffer</td><td>Sigma Aldrich</td><td>97068</td><td>Flammable liquid, carcinogen and irritant.</td></tr><tr><td>Osmium tetraoxide</td><td>Sigma Aldrich</td><td>201030</td><td>Highly toxic</td></tr><tr><td>Triton X-100</td><td>Sigma Aldrich</td><td>T8787</td><td>Harmful if swallowed</td></tr><tr><td>Ethanol</td><td>Decon Labs</td><td>2705</td><td>Keep away from heat, sparks, flame and other form of ignition.</td></tr><tr><td>General/regional Anesthesia</td><td>Amphastar pharmaceuticals</td><td></td><td>1% Lidocaine, 0.25% Bupivacaine,Anesthetic agents for induction and maintenance</td></tr><tr><td>Antibiotics</td><td>Hospira</td><td>0409-0805-01</td><td>Ancef 1 g i.v.</td></tr><tr><td>Arthroscopy trocar</td><td>Smith and Nephew</td><td></td><td></td></tr><tr><td>Arthroscopy Camera</td><td>Smith and Nephew</td><td></td><td></td></tr><tr><td>Arthroscopic grasper and bitter</td><td>Arthrex</td><td></td><td></td></tr><tr><td>4.5 mm shaver</td><td>Arthrex</td><td></td><td></td></tr><tr><td>Interference Screws</td><td>Arthrex</td><td></td><td>stainless steel screws</td></tr><tr><td>Sputter Coater</td><td>Polaron</td><td>E5400</td><td></td></tr></tbody></table>

surgical retrieval, isolation and in vitro expansion of human anterior cruciate ligament-derived cells for tissue engineering applications

伤到ACL是在活跃的人经常遇到的问题。即使是部分的这个关节膝关节韧带导致的损害的功能和生物力学稳定性不足的眼泪。当前选择的部分ACL撕裂的治疗范围从非手术治疗，保守管理多个手术方案，如:热改性，单束修，彻底重建，重建原生韧带的受损部分。一些研究，如果有的话，已经证明管理是一贯出众任何单一的方法，而且在许多情况下，患者继续表现出持续的不稳定等合并症。 本研究的目的是确定可能在部分十字韧带撕裂的修复来实现一个组织工程补丁的发展潜能细胞源的利用率。一种新的协议是从不稳定型心绞痛患者的细胞膨胀发展NTS进行ACL重建。为了分离细胞，ACL重建过程中获得剁碎HACL组织被消化的胶原酶溶液。用补充有10％胎牛血清（FBS）和1％青霉素/链霉素（P / S）的DMEM/F12培养基中进行扩展。然后将细胞保存在-80°C或在液氮中包括DMSO，FBS和膨胀介质的冷冻介质。融化后，将HACL衍生细胞，然后接种到组织工程支架，PLAGA（聚乳酸 - 共 - 乙醇酸）和控制组织培养聚苯乙烯（TCPS）。 7天后，扫描电镜进行到细胞粘附比拟的PLAGA与控制TCPS。采用免疫荧光染色细胞形态进行了评估。 SEM（扫描电子显微镜）显微照片表明，细胞生长和粘附这两种PLAGA和TCPS表面上，并分别由7天汇合，在整个表面上。免疫荧光染色显示正常，非胁迫形态学人图案两面上。这种技术是有前途的ACL中再生和重建应用。

的工作模式为手术取出，组织消化和人类前十字韧带（HACL）衍生细胞的分离示于图1，从外植体迁移和粘附到T-25培养瓶中的细胞。这些细胞培养3天，然后用光学显微镜（ 图2）下进行了可视化。通过7天而得到的汇合单层。健康，活细胞的存在表明HACL来源的细胞的成功的检索和文化。 细胞附着到PLAGA和TCPS的表面通过扫描电子显微镜定性测定图</s...

Watch this Scientific Journal Video about Surgical Retrieval, Isolation and In vitro Expansion of Human Anterior Cruciate Ligament-derived Cells for Tissue Engineering Applications at JoVE.com

Surgical Retrieval, Isolation and In vitro Expansion of Human Anterior Cruciate Ligament-derived Cells for Tissue Engineering Applications

对于未来的应用作为一个补丁来修复前交叉韧带（ACL）的部分撕裂，人类的ACL的细胞从内重建手术， 体外培养扩增和生长在组织工程支架材料获得的组织分离。然后进行细胞粘附和形态学进行确认的生物相容性支架上表面。

手术取出，分离和<em&gt;在体外</em&gt;扩展人类前十字韧带衍生细胞组织工程的应用

Injury to the ACL is a commonly encountered problem in active individuals. Even partial tears of this intra-articular knee ligament lead to biomechanical ...

surgical-retrieval-isolation-vitro-expansion-human-anterior-cruciate

Research

JoVE Journal

Bioengineering

Surgical Retrieval, Isolation and In vitro Expansion of Human Anterior Cruciate Ligament-derived Cells for Tissue Engineering Applications

11.4K 次观看.  Southern Illinois University School of Medicine. 对于未来的应用作为一个补丁来修复前交叉韧带（ACL）的部分撕裂，人类的ACL的细胞从内重建手术， 体外培养扩增和生长在组织工程支架材料获得的组织分离。然后进行细胞粘附和形态学进行确认的生物相容性支架上表面。 

视频: Surgical Retrieval, Isolation and In vitro Expansion of Human Anterior Cruciate Ligament-derived Cells for Tissue Engineering Applications

Use of Human Perivascular Stem Cells for Bone Regeneration

Human perivascular stem cells (PSCs) can be isolated in sufficient numbers from multiple tissues for purposes of skeletal tissue engineering1-3. PSCs are a FACS-sorted population of 'pericytes' (CD146+CD34-CD45-) and 'adventitial cells' (CD146-CD34+CD45-), each of which we have previously reported to have properties of mesenchymal stem cells. PSCs, like MSCs, are able to undergo osteogenic differentiation, as well as secrete pro-osteogenic cytokines1,2. In the present protocol, we demonstrate the osteogenicity of PSCs in several animal models including a muscle pouch implantation in SCID (severe combined immunodeficient) mice, a SCID mouse calvarial defect and a femoral segmental defect (FSD) in athymic rats. The thigh muscle pouch model is used to assess ectopic bone formation. Calvarial defects are centered on the parietal bone and are standardly 4 mm in diameter (critically sized)8. FSDs are bicortical and are stabilized with a polyethylene bar and K-wires4. The FSD described is also a critical size defect, which does not significantly heal on its own4. In contrast, if stem cells or growth factors are added to the defect site, significant bone regeneration can be appreciated. The overall goal of PSC xenografting is to demonstrate the osteogenic capability of this cell type in both ectopic and orthotopic bone regeneration models.

Human perivascular stem cells (PSCs) are a novel stem cell class for skeletal tissue regeneration similar to mesenchymal stem cells (MSCs). PSCs can be isolated by FACS (fluorescence activated cell sorting) from adipose tissue procured during standard liposuction procedures, then combined with an osteoinductive scaffold to achieve bone formation in vivo.

人类血管周围干细胞用于骨再生

Human perivascular stem cells (PSCs) can be isolated in sufficient numbers from multiple tissues for purposes of skeletal tissue engineering1-3. PSCs are ...

科研

生物工程

Matrix-assisted Autologous Chondrocyte Transplantation for Remodeling and Repair of Chondral Defects in a Rabbit Model

Articular cartilage defects are considered a major health problem because articular cartilage has a limited capacity for self-regeneration 1. Untreated cartilage lesions lead to ongoing pain, negatively affect the quality of life and predispose for osteoarthritis. During the last decades, several surgical techniques have been developed to treat such lesions. However, until now it was not possible to achieve a full repair in terms of covering the defect with hyaline articular cartilage or of providing satisfactory long-term recovery 2-4. Therefore, articular cartilage injuries remain a prime target for regenerative techniques such as Tissue Engineering. In contrast to other surgical techniques, which often lead to the formation of fibrous or fibrocartilaginous tissue, Tissue Engineering aims at fully restoring the complex structure and properties of the original articular cartilage by using the chondrogenic potential of transplanted cells. Recent developments opened up promising possibilities for regenerative cartilage therapies.
The first cell based approach for the treatment of full-thickness cartilage or osteochondral lesions was performed in 1994 by Lars Peterson and Mats Brittberg who pioneered clinical autologous chondrocyte implantation (ACI) 5. Today, the technique is clinically well-established for the treatment of large hyaline cartilage defects of the knee, maintaining good clinical results even 10 to 20 years after implantation 6. In recent years, the implantation of autologous chondrocytes underwent a rapid progression. The use of an artificial three-dimensional collagen-matrix on which cells are subsequently replanted became more and more popular 7-9.
MACT comprises of two surgical procedures: First, in order to collect chondrocytes, a cartilage biopsy needs to be performed from a non weight-bearing cartilage area of the knee joint. Then, chondrocytes are being extracted, purified and expanded to a sufficient cell number in vitro. Chondrocytes are then seeded onto a three-dimensional matrix and can subsequently be re-implanted. When preparing a tissue-engineered implant, proliferation rate and differentiation capacity are crucial for a successful tissue regeneration 10. The use of a three-dimensional matrix as a cell carrier is thought to support these cellular characteristics 11.
The following protocol will summarize and demonstrate a technique for the isolation of chondrocytes from cartilage biopsies, their proliferation in vitro and their seeding onto a 3D-matrix (Chondro-Gide, Geistlich Biomaterials, Wollhusen, Switzerland). Finally, the implantation of the cell-matrix-constructs into artificially created chondral defects of a rabbit's knee joint will be described. This technique can be used as an experimental setting for further experiments of cartilage repair.

An experimental technique for the treatment of chondral defects in the rabbit's knee joint is described. The implantation of autologous chondrocytes seeded on a matrix is a well-accepted method for the remodeling and repair of articular cartilage lesions providing satisfying long-term results. Matrix-assisted autologous chondrocyte transplantation (MACT) offers a standardized and clinically established implantation method.

基质辅助重塑和修复软骨缺损的自体软骨细胞移植在兔模型

Articular cartilage defects are considered a major health problem because articular cartilage has a limited capacity for self-regeneration 1. Untreated ...

医学

Treatment of Osteochondral Defects in the Rabbit's Knee Joint by Implantation of Allogeneic Mesenchymal Stem Cells in Fibrin Clots

The treatment of osteochondral articular defects has been challenging physicians for many years. The better understanding of interactions of articular cartilage and subchondral bone in recent years led to increased attention to restoration of the entire osteochondral unit. In comparison to chondral lesions the regeneration of osteochondral defects is much more complex and a far greater surgical and therapeutic challenge. The damaged tissue does not only include the superficial cartilage layer but also the subchondral bone. For deep, osteochondral damage, as it occurs for example with osteochondrosis dissecans, the full thickness of the defect needs to be replaced to restore the joint surface 1. Eligible therapeutic procedures have to consider these two different tissues with their different intrinsic healing potential 2. In the last decades, several surgical treatment options have emerged and have already been clinically established 3-6.
Autologous or allogeneic osteochondral transplants consist of articular cartilage and subchondral bone and allow the replacement of the entire osteochondral unit. The defects are filled with cylindrical osteochondral grafts that aim to provide a congruent hyaline cartilage covered surface 3,7,8. Disadvantages are the limited amount of available grafts, donor site morbidity (for autologous transplants) and the incongruence of the surface; thereby the application of this method is especially limited for large defects.
New approaches in the field of tissue engineering opened up promising possibilities for regenerative osteochondral therapy. The implantation of autologous chondrocytes marked the first cell based biological approach for the treatment of full-thickness cartilage lesions and is now worldwide established with good clinical results even 10 to 20 years after implantation 9,10. However, to date, this technique is not suitable for the treatment of all types of lesions such as deep defects involving the subchondral bone 11.
The sandwich-technique combines bone grafting with current approaches in Tissue Engineering 5,6. This combination seems to be able to overcome the limitations seen in osteochondral grafts alone. After autologous bone grafting to the subchondral defect area, a membrane seeded with autologous chondrocytes is sutured above and facilitates to match the topology of the graft with the injured site. Of course, the previous bone reconstruction needs additional surgical time and often even an additional surgery. Moreover, to date, long-term data is missing 12.
Tissue Engineering without additional bone grafting aims to restore the complex structure and properties of native articular cartilage by chondrogenic and osteogenic potential of the transplanted cells. However, again, it is usually only the cartilage tissue that is more or less regenerated. Additional osteochondral damage needs a specific further treatment. In order to achieve a regeneration of the multilayered structure of osteochondral defects, three-dimensional tissue engineered products seeded with autologous/allogeneic cells might provide a good regeneration capacity 11.
Beside autologous chondrocytes, mesenchymal stem cells (MSC) seem to be an attractive alternative for the development of a full-thickness cartilage tissue. In numerous preclinical in vitro and in vivo studies, mesenchymal stem cells have displayed excellent tissue regeneration potential 13,14. The important advantage of mesenchymal stem cells especially for the treatment of osteochondral defects is that they have the capacity to differentiate in osteocytes as well as chondrocytes. Therefore, they potentially allow a multilayered regeneration of the defect.
In recent years, several scaffolds with osteochondral regenerative potential have therefore been developed and evaluated with promising preliminary results 1,15-18. Furthermore, fibrin glue as a cell carrier became one of the preferred techniques in experimental cartilage repair and has already successfully been used in several animal studies 19-21 and even first human trials 22.
The following protocol will demonstrate an experimental technique for isolating mesenchymal stem cells from a rabbit's bone marrow, for subsequent proliferation in cell culture and for preparing a standardized in vitro-model for fibrin-cell-clots. Finally, a technique for the implantation of pre-established fibrin-cell-clots into artificial osteochondral defects of the rabbit's knee joint will be described.

An experimental technique for the treatment of osteochondral defects in the rabbit's knee joint is described. The implantation of allogeneic mesenchymal stem cells into osteochondral defects provides a promising development in the field of tissue engineering. The preparation of fibrin-cell-clots in vitro offers a standardized method for implantation.

异基因骨髓间充质干细胞植入兔子的膝关节软骨缺损治疗纤维蛋白凝块

The treatment of osteochondral articular defects has been challenging physicians for many years. The better understanding of interactions of articular ...

Athymic Rat Model for Evaluation of Engineered Anterior Cruciate Ligament Grafts

Anterior cruciate ligament (ACL) rupture is a common ligamentous injury that often requires surgery because the ACL does not heal well without intervention. Current treatment strategies include ligament reconstruction with either autograft or allograft, which each have their associated limitations. Thus, there is interest in designing a tissue-engineered graft for use in ACL reconstruction. We describe the fabrication of an electrospun polymer graft for use in ACL tissue engineering. This polycaprolactone graft is biocompatible, biodegradable, porous, and is comprised of aligned fibers. Because an animal model is necessary to evaluate such a graft, this paper describes an intra-articular athymic rat model of ACL reconstruction that can be used to evaluate engineered grafts, including those seeded with xenogeneic cells. Representative histology and biomechanical testing results at 16 weeks postoperatively are presented, with grafts tested immediately post-implantation and contralateral native ACLs serving as controls. The present study provides a reproducible animal model with which to evaluate tissue engineered ACL grafts, and demonstrates the potential of a regenerative medicine approach to treatment of ACL rupture.

Animal models are important tools for the evaluation of tissue-engineered grafts. This paper presents the protocol for preparing an electrospun biodegradable polymer graft for use in anterior cruciate ligament tissue engineering, as well as a surgical protocol for implantation in a rat model.

无胸​​腺大鼠模型工程前交叉韧带移植评估

Anterior cruciate ligament (ACL) rupture is a common ligamentous injury that often requires surgery because the ACL does not heal well without ...

Minced Tissue in Compressed Collagen: A Cell-containing Biotransplant for Single-staged Reconstructive Repair

Conventional techniques for cell expansion and transplantation of autologous cells for tissue engineering purposes can take place in specially equipped human cell culture facilities. These methods include isolation of cells in single cell suspension and several laborious and time-consuming events before transplantation back to the patient. Previous studies suggest that the body itself could be used as a bioreactor for cell expansion and regeneration of tissue in order to minimize ex vivo manipulations of tissues and cells before transplanting to the patient. The aim of this study was to demonstrate a method for tissue harvesting, isolation of continuous epithelium, mincing of the epithelium into small pieces and incorporating them into a three-layered biomaterial. The three-layered biomaterial then served as a delivery vehicle, to allow surgical handling, exchange of nutrition across the transplant, and a controlled degradation. The biomaterial consisted of two outer layers of collagen and a core of a mechanically stable and slowly degradable polymer. The minced epithelium was incorporated into one of the collagen layers before transplantation. By mincing the epithelial tissue into small pieces, the pieces could be spread and thereby the propagation of cells was stimulated. After the initial take of the transplants, cell expansion and reorganization would take place and extracellular matrix mature to allow ingrowth of capillaries and nerves and further maturation of the extracellular matrix. The technique minimizes ex vivo manipulations and allow cell harvesting, preparation of autograft, and transplantation to the patient as a simple one-stage intervention. In the future, tissue expansion could be initiated around a 3D mold inside the body itself, according to the specific needs of the patient. Additionally, the technique could be performed in an ordinary surgical setting without the need for sophisticated cell culturing facilities.

Tissue engineering often includes in vitro expansion in order to create autografts for tissue regeneration. In this study a method for tissue expansion, regeneration, and reconstruction in vivo was developed in order to minimize the processing of cells and biological materials outside the body.

在压缩的胶原组织糜:含细胞Biotransplant单上演重建修复

Conventional techniques for cell expansion and transplantation of autologous cells for tissue engineering purposes can take place in specially equipped ...

Treatment of Ligament Constructs with Exercise-conditioned Serum: A Translational Tissue Engineering Model

In vitro experiments are essential to understand biological mechanisms; however, the gap between monolayer tissue culture and human physiology is large, and translation of findings is often poor. Thus, there is ample opportunity for alternative experimental approaches. Here we present an approach in which human cells are isolated from human anterior cruciate ligament tissue remnants, expanded in culture, and used to form engineered ligaments. Exercise alters the biochemical milieu in the blood such that the function of many tissues, organs and bodily processes are improved. In this experiment, ligament construct culture media was supplemented with experimental human serum that has been &#39;conditioned&#39; by exercise. Thus the intervention is more biologically relevant since an experimental tissue is exposed to the full endogenous biochemical milieu, including binding proteins and adjunct compounds that may be altered in tandem with the activity of an unknown agent of interest. After treatment, engineered ligaments can be analyzed for mechanical function, collagen content, morphology, and cellular biochemistry. Overall, there are four major advantages versus traditional monolayer culture and animal models, of the physiological model of ligament tissue that is presented here. First, ligament constructs are three-dimensional, allowing for mechanical properties (i.e., function) such as ultimate tensile stress, maximal tensile load, and modulus, to be quantified. Second, the enthesis, the interface between boney and sinew elements, can be examined in detail and within functional context. Third, preparing media with post-exercise serum allows for the effects of the exercise-induced biochemical milieu, which is responsible for the wide range of health benefits of exercise, to be investigated in an unbiased manner. Finally, this experimental model advances scientific research in a humane and ethical manner by replacing the use of animals, a core mandate of the National Institutes of Health, the Center for Disease Control, and the Food and Drug Administration.

We present a model of ligament tissue in which three-dimensional constructs are treated with the human exercise-conditioned serum and analyzed for collagen content, function, and cellular biochemistry.

用运动调节血清治疗韧带结构:转化组织工程模型

In vitro experiments are essential to understand biological mechanisms; however, the gap between monolayer tissue culture and human physiology is large, ...

Evaluation of Stem Cell Therapies in a Bilateral Patellar Tendon Injury Model in Rats

Regenerative medicine provides novel alternatives to conditions that challenge traditional treatments. The prevalence and morbidity of tendinopathy across species, combined with the limited healing properties of this tissue, have prompted the search for cellular therapies and propelled the development of experimental models to study their efficacy. Umbilical cord matrix-derived mesenchymal stem cells (UCM-MSC) are appealing candidates because they are abundant, easy to collect, circumvent the ethical concerns and risk of teratoma formation, yet resemble primitive embryonic stem cells more closely than adult tissue-derived MSCs. Significant interest has focused on chitosan as a strategy to enhance the properties of MSCs through spheroid formation. This paper details techniques to isolate UCM-MSCs, prepare spheroids on chitosan film, and analyze the effect of spheroid formation on surface marker expression. Consequently, creation of a bilateral patellar tendon injury model in rats is described for in vivo implantation of UCM-MSC spheroids formed on chitosan film. No complication was observed in the study with respect to morbidity, stress rising effects, or tissue infection. The total functional score of the operated rats at 7 days was lower than that of normal rats, but returned to normal within 28 days after surgery. Histological scores of tissue-healing confirmed the presence of a clot in treated defects evaluated at 7 days, absence of foreign body reaction, and progressing healing at 28 days. This bilateral patella tendon defect model controls inter-individual variation via creation of an internal control in each rat, was associated with acceptable morbidity, and allowed detection of differences between untreated tendons and treatments.

This paper describes the preparation and evaluation of umbilical cord matrix-derived mesenchymal stem cells spheroids with a bilateral patellar tendon defect model in a rat. This model was associated with an acceptable morbidity and was found to detect differences between untreated and treated tendons, and between the two treatments tested.

大鼠双侧髌腱损伤模型中干细胞治疗的评价

Regenerative medicine provides novel alternatives to conditions that challenge traditional treatments. The prevalence and morbidity of tendinopathy across ...

关节镜双后内侧入路用于 PCL 囊肿切除术

Arthroscopic Excision of Posterior Cruciate Ligament Cysts Using a Double Posteromedial Approach

Cruciate ligament cysts of the knee are a rare condition. Posterior cruciate ligament cysts of the knee are less common than anterior cruciate ligament cysts. In patients with asymptomatic isolated cruciate ligament cysts of the knee, conservative treatment is recommended. Symptomatic cruciate ligament cysts of the knee are mostly manifested as knee hyperflexion pain, straightening pain, knee discomfort after standing for a long time or walking for a long time, etc., which seriously affects the quality of life, surgical treatment can be performed. The surgical treatments can be divided into ultrasound-guided cyst puncture and fluid extraction procedure and arthroscopic cystectomy. Cysts are mostly lobulated with a multi-layer cyst wall, cyst fluid extraction does not remove the cyst wall completely but simply extracts cyst fluid, leading to a high recurrence rate. Arthroscopic surgery can completely remove the cyst wall with little trauma, a low recurrence rate, and fast postoperative recovery, so arthroscopic resection is the most common and preferred method of treatment. Since posterior cruciate ligament cysts mostly occur posterior to the ligament, we remove the cyst wall by adding a double posteromedial approach to the knee joint, and the cyst wall is removed under direct vision, which is simple to operate, the cyst wall is completely cleared, the trauma is small, the postoperative recovery is fast, and there is no recurrence. Here, 8 posterior cruciate ligament cysts were removed with complete postoperative symptom relief, no surgical complications, and no recurrence at 1-year follow-up.

作者聚焦: 双后内侧入路治疗后交叉韧带囊肿

We present a surgical approach to treat posterior cruciate ligament cysts by an arthroscopic double posteromedial approach.

关节镜下使用双后内侧入路切除后交叉韧带囊肿

Cruciate ligament cysts of the knee are a rare condition. Posterior cruciate ligament cysts of the knee are less common than anterior cruciate ligament ...

手术取出，分离和

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人类血管周围干细胞用于骨再生

基质辅助重塑和修复软骨缺损的自体软骨细胞移植在兔模型

异基因骨髓间充质干细胞植入兔子的膝关节软骨缺损治疗纤维蛋白凝块

无胸腺大鼠模型工程前交叉韧带移植评估

在压缩的胶原组织糜:含细胞Biotransplant单上演重建修复

用运动调节血清治疗韧带结构:转化组织工程模型

大鼠双侧髌腱损伤模型中干细胞治疗的评价

关节镜下使用双后内侧入路切除后交叉韧带囊肿

关节镜下使用双后内侧入路切除后交叉韧带囊肿

关节镜下使用双后内侧入路切除后交叉韧带囊肿

手术取出，分离和

摘要

探索更多视频

此视频中的章节

人类血管周围干细胞用于骨再生

基质辅助重塑和修复软骨缺损的自体软骨细胞移植在兔模型

异基因骨髓间充质干细胞植入兔子的膝关节软骨缺损治疗纤维蛋白凝块

无胸​​腺大鼠模型工程前交叉韧带移植评估

在压缩的胶原组织糜:含细胞Biotransplant单上演重建修复

用运动调节血清治疗韧带结构:转化组织工程模型

大鼠双侧髌腱损伤模型中干细胞治疗的评价

关节镜下使用双后内侧入路切除后交叉韧带囊肿

关节镜下使用双后内侧入路切除后交叉韧带囊肿

关节镜下使用双后内侧入路切除后交叉韧带囊肿

无胸腺大鼠模型工程前交叉韧带移植评估