发表
联系我们

登录

需要订阅 JoVE 才能查看此. 登录或开始免费试用。

本文内容

  • 摘要
  • 摘要
  • 引言
  • 研究方案
  • 结果
  • 讨论
  • 披露声明
  • 致谢
  • 材料
  • 参考文献
  • 转载和许可

摘要

脂肪干细胞 （陶瓷） 容易孤立和收获从正常大鼠脂肪。ASC 床单可以使用单元格表工程创建，可以移植到 Zucker 糖尿病脂肪肝大鼠表现出全层皮肤缺损伴骨外露，然后用双层的人工皮肤覆盖。

摘要

人工皮肤取得了相当大的治疗效果，在临床实践中。然而，可能会延长人工皮肤治疗糖尿病患者与阻碍的血流或大的伤口的伤口。作为一种新技术治疗糖尿病溃疡，出现了基于细胞疗法及细胞板工程已改善细胞移植的疗效。大量的报告建议脂肪源性干细胞 （陶瓷），一种间充质基质细胞 (MSC)，表现出由于他们在脂肪组织和其可访问性集合时相比骨髓间充质干细胞从其他组织中的相对丰度的治疗潜力。因此，陶瓷似乎是一个良好的来源的干细胞用于治疗目的。在这项研究，ASC 床单从正常 Lewis 大鼠附睾脂肪已成功创建使用温度反应性培养皿和正常培养基含有抗坏血酸。ASC 床单被移植进 Zucker 糖尿病脂肪酸 (ZDF) 大鼠，大鼠模型的 2 型糖尿病、 肥胖等，表现出减少创面愈合。伤口被创建后颅骨表面、 ASC 床单被移植到伤口，和双层人工皮肤被用来支付床单。收到 ASC 床单的 ZDF 大鼠有更好地愈合比没有移植的 ASC 床单 ZDF 老鼠。这种方法是有限的因为 ASC 床单是敏感的干燥条件，需要一个潮湿的伤口环境的维护。因此，人工皮肤用于封面 ASC，防止干燥。同种异体移植的 ASC 床单结合人工皮肤也可能适用于其他难治性溃疡或烧伤，例如那些观察外周动脉疾病与胶原病和可能给予营养不良或使用类固醇的患者。因此，这种治疗可能改善糖尿病创面愈合的治疗选项的第一步。

引言

糖尿病患者的人口世界范围内增加，达到 4 亿 2015年1;估计有 15-25%的糖尿病患者处于危险从下肢糖尿病溃疡2的进展。糖尿病下肢溃疡是棘手的可能需要长期治疗与康复训练后完全恢复。长治疗期间经常导致显著降低患者的生活质量。因此，治疗糖尿病创面必须开发新的治疗方法，减少或防止恶化。评价糖尿病创面愈合，我们优化糖尿病溃疡创面愈合大鼠模型，模仿实际临床情况，并评估是否移植脂肪源性干细胞 (ASC) 床单使用单元格表工程加速伤口愈合。

骨髓间质细胞 (MSCs) 表现出极大的潜力，加速创面愈合其自我更新的能力，他们的免疫调节作用，它们能够分化成多种细胞谱系3。陶瓷是一种类型的 MSC 来源于脂肪组织，和他们表现出骨髓间充质干细胞来自其他组织，包括其潜在的血管生成和旁分泌活动4，5的几个优势。脂肪组织是较为丰富的人体，并其辅助功能允许收集使用微创手术。因此，陶瓷已用于实验伤口愈合应用6，7。

以前的报道表明，单个单元格 MSC 悬浮液直接注入伤口周围地区可以加快伤口愈合8，9。然而，尽管报告的加速伤口愈合的糖尿病溃疡模型的单细胞悬液注射后，在伤口部位移植细胞的存活时间尚不清楚。

在此研究中，我们应用使用温度反应性培养皿的细胞板工程。这些菜有温度响应聚合物N-异丙基丙烯共价键到10他们表面上。接枝的聚合物层允许温度控制的细胞粘附或脱离表面的培养皿。这道菜的表面变得疏水在 37 ° C，让细胞粘附和增殖，而细胞自发地从表面分离的时候就亲水在温度低于 32 ° c。体外培养的细胞可以收获作为完整细胞到细胞连接与细胞外基质 (ECMs) 的连续单元格表只是通过降低的温度;因此，蛋白水解酶，破坏 ECM，如胰蛋白酶，并非需要的11。因此，细胞板工程可以保护细胞间连接和改善细胞移植的疗效。

此外，细胞板移植增加细胞存活率时相比，细胞注射12。在此协议中，作为延迟的伤口愈合 2 型糖尿病与肥胖模型选取 Zucker 糖尿病脂肪肝 (ZDF) 鼠。在大约 4 个星期中 ZDF 大鼠自发形成肥胖。他们然后患 2 型糖尿病与肥胖之间 8 和 12 周龄，此时他们表现出高血糖与胰岛素抵抗、 血脂异常和甘油13相关联。延迟的伤口愈合，血流量减少外围及血管中，糖尿病肾病也观察到14，，1516。此外，ZDF 大鼠可能适当模型研究顽固性皮肤溃疡，例如糖尿病溃疡的愈合。

人类和啮齿类动物在创面愈合的机制上的差异是与皮肤的解剖学差异相关联。正常大鼠愈合的伤口基于伤口收缩而创面愈合的人类基于重新上皮和肉芽组织的形成。通常情况下，伤口小夹板固定在啮齿动物模型中使用有助于尽量减少伤口收缩并允许逐步形成的肉芽组织17，虽然非糖尿病大鼠伤口已经几乎完全闭合的收缩。然而，糖尿病创面收缩 ZDF 在大鼠是受损，并且伤口愈合主要发生通过重新上皮和肉芽组织的形成;因此，这一进程是更类似于人类的伤口愈合14。

糖尿病伴骨外露创面后清创术临床上经常遇到。先前的研究审查了直径为 12 毫米全层皮肤伤口的裸鼠裸鼠18，19背上和背上的正常小鼠20直径为 10 毫米全层皮肤伤口。发展为严重的糖尿病创面的临床模型，较大的 (15 x 10 mm2) 全层皮肤缺损暴露骨并没有骨膜被创造了，作为先前描述21，大鼠 2 型糖尿病和肥胖。

通过同种异体移植的 ASC 床单创造了大鼠 ASC （区域审计事务中心） 床单从正常 Lewis 大鼠的陶瓷。在临床实践中，自体移植是行不通的因为溃疡的糖尿病患者常表现出严重的糖尿病并发症，如不加控制高血糖和高身体质量指数，这些并发症原因伤口愈合障碍，增加了从这些患者获得脂肪组织的难度。此外，从动物与糖尿病展览体干改变属性和受损功能22。因此，议定书 》 在这里提出了一种描述区域审计事务中心床单从正常大鼠同种异体移植和人工皮肤对糖尿病大鼠的应用。

在本协议中使用的双层人工皮肤防止伤口的自发性收缩、 促进合成的一种新的结缔组织矩阵，和类似于真正的真皮23。在此协议中，人工皮肤是放置区域审计事务中心工作表上，用尼龙线，防止伤口收缩或扩大造成的松散大鼠皮肤固定。此外，人造皮肤提供一种三维框架为 ASC 床单、 保持潮湿的环境下的移植的 ASC 床单和伤口，和保护伤口免受感染和外部力量。最后，非胶粘剂敷料被放置在伤口保护它免受外部冲击、 保持潮湿的伤口环境，和吸收渗出物。

一个区域审计事务中心表是薄薄的、 灵活的和可变形和可以坚持移动收件人的网站，例如24跳动的心。单元格表工程已被用于各种组织重建和可以生成疗效25，26.表现出临床治疗潜力的 ASC 床单可能加速许多类型的伤口的愈合。此外，ASC 床单，结合人工皮肤，使用同种异体移植可能适用于治疗难治性溃疡或烧伤，例如那些观察外周动脉疾病或胶原病，或者他们可能注射到患者营养不良或使用类固醇。这种方法提高移栽体干的效率。伤口愈合 ZDF 大鼠模型产生严重的伤口情况，类似于人类的伤口愈合过程，模仿在小型实验动物临床条件。

研究方案

All experimental protocols presented below were approved by the Animal Welfare Committee of Tokyo Women's Medical University School of Medicine and abided by all requirements of the Guidelines for Proper Conduct of Animal Experiments.

1. Preparation of Animals, Instruments, Culture Media, and Dishes

  1. Prepare complete culture medium using minimum essential medium alpha containing 20% fetal bovine serum (FBS) and 1% penicillin/streptomycin. Store this for several months at 4 °C until use.
  2. Use adult male ZDF rats as a type 2 diabetic obesity model. Use the fat pad of male Lewis rats to isolate adipose tissue to prepare the cell sheets.

2. Isolation and Culture of rASCs

  1. Obtain adipose tissue from Lewis rats (8 - 33 weeks old, male) under general anesthesia using isoflurane.
    1. Prepare a rodent mechanical ventilator and 4% isoflurane. Prepare several sterile cotton tips, clean gauzes, a scalpel, a needle holder, operating scissors, a pair of forceps, and a 5-0 nylon suture. Sterilize the surgical instruments and supplies.
    2. Prepare a 100-mm Petri dish with 10 mL of sterile phosphate-buffered saline (PBS) to temporarily preserve the obtained adipose tissue. Weigh the Petri dish with PBS using a balance before starting surgery to measure the collected adipose tissue. Lay out the surgical instruments and supplies on a sterile drape.
    3. Anesthetize the rats by using isoflurane at 3 - 4% via a rodent mechanical ventilator and maintain the rats at 2 - 3% isoflurane during surgery. Monitor the depth of anesthesia by observing the depth and rate of respiration of each rat.
    4. While wearing sterile gloves, position the rat in a supine position on a sterile drape. Shave the operative area with an electric razor and clean the abdominal section of the rat using sterile gauze soaked in 70% ethanol.
    5. Create an approximately 5 cm-long skin incision with a scalpel in the lateral lower abdomen of the rat. Expose and dissect the external oblique muscle. Then, expose the epididymal adipose tissue surrounding the epididymis. Gently pull the epididymal adipose tissue out.
      NOTE: The adipose tissue can be obtained on either side of the rat's abdomen.
    6. Excise only the epididymal adipose tissue, avoiding damage to the epididymis, testis, and epididymal blood vessels (Figure S1). Soak the excised adipose tissue in the PBS in the Petri dish to prevent dryness and weigh the tissue.
    7. Close the abdominal muscle with 5-0 VICRYL and the skin with a 5-0 nylon suture. Then, return each rat that has undergone surgery to a separate cage (one rat per cage).
      NOTE: Monitor the rats until they fully recover.
  2. Isolate rASCs from 2 - 3 g of adipose tissue (excised from a Lewis rat and processed according to a previously-reported method)27. Briefly, enzymatically digest the excised adipose tissue with 0.1% type A collagenase at 37 °C for 1 h to obtain the stromal-vascular fraction (SVF; Figure S2).
    1. Prepare several 100-µm cell strainers, several 50-mL tubes, several 15 mL tubes, and two scalpels on a biological clean bench. Turn on a centrifuge and warm up a water bath to 37 °C. In addition, prepare approximately 50 mL of sterile PBS in a 50 mL tube with 0.1% (final concentration) of type A collagenase.
    2. Mince the adipose tissue into small pieces using two scalpels.
    3. Move all minced adipose tissue to a 50-mL tube and add PBS to bring the total volume to 15 mL.
    4. Rest the 50-mL tube upright for 5 min. Discard the upper layer and collect the lower layer except for the debris.
    5. Add 0.1% of type A collagenase solution to the 50-mL tube with PBS containing the upper layer in order to enzymatically digest the adipose tissue.
      NOTE: Warm approximately 20 mL of PBS per 2 - 3 g of adipose tissue in a 37 °C water bath.
    6. Tilt and shake the 50 mL tube gently at 120 - 130 rpm for 1 h in a 37 °C water bath.
    7. Rest the 50 mL tube upright for 5 min after shaking.
    8. Filter the solution using a 100 µm cell strainer and pour the solution into a new 50 mL tube. Dispense the filtered solution into two 15 mL tubes.
    9. Centrifuge the solution in the 15 mL tubes for 5 min at 700 x g. Carefully remove the upper layer of supernatant and leave 5 mL of the lower layer of the solution. Do not aspirate the pellet.
    10. Add approximately 5 mL of complete culture medium to each 15 mL tube, up to 10 mL per 15 mL tube, and carefully re-suspend the pellet.
    11. Repeat steps 2.2.8 - 2.2.9. Then, obtain the SVF.
  3. Prepare two 60 cm2 culture dishes. Collect the SVF after two centrifugations at 700 × g for 5 min. Suspend the SVF and plate 10 mL of SVF solution on each 60-cm2 culture dish.
  4. Culture the dishes for 24 h at 37 °C in a 5% CO2 incubator.
  5. Prepare PBS and 0.25% trypsin-ethylenediamine tetraacetic acid (EDTA), storable for several months at 4 °C until use.
  6. After initial plating (24 h), wash the cells with PBS 3 times to remove unattached cells and debris. Add fresh complete medium.
  7. Passage the cells that are nearly sub-confluent on day 3. Add 1 mL of 0.25% trypsin-EDTA and incubate the cells at 37 °C in a 5% CO2 incubator for 3 - 5 min.
    NOTE: Complete the trypsinization step within 10 min to prevent cell damage.
  8. Observe the cells under a light microscope after trypsinization to confirm that all the cells have thoroughly detached from the dishes. Then, add 9 mL of complete culture medium to the dishes.
  9. Count the number of cells using a hemocytometer.
  10. Subculture the cells at a density of 1.7 x 103 cells/cm2 every 3 days until passage 3. Culture the subcultured cells at 37 °C in the humidified atmosphere of a 5% CO2 incubator.

3. Creation of rASC Sheets

  1. Prepare several 35-mm diameter temperature-responsive culture dishes. Pre-coat the dishes with FBS (or complete medium containing FBS) at 37 °C in a 5% CO2 incubator for more than 1 h.
    NOTE: A 35 mm diameter temperature-responsive culture dish is a commercially available product.
  2. Prepare a thermo-plate and incubate at 37 °C for the following experimental procedures.
    NOTE: Perform every procedure using temperature-responsive culture dishes on a 37 °C thermo-plate to prevent the cells from spontaneously detaching from the dish.
    1. Warm the complete culture medium used in the procedures to 37 °C prior to experimentation.
    2. Seed passage 3 rASCs derived from Lewis rats onto a 35 mm diameter temperature-responsive culture dish at a density of 1.5 x 105 cells/dish for 3 days using a 2 mL total volume of complete culture medium.
      NOTE: When rASCs are plated onto a new culture dish, the dish should be slowly rocked back and forth and left and right in an incubator to achieve uniform rASC seeding and a uniformly thick rASC sheet.
    3. Change 2 mL of the medium to 2 mL of complete culture medium containing 16.4 µg/mL L-ascorbic acid phosphate magnesium salt n-hydrate (AA) on day 3 after seeding to the temperature-responsive culture dishes incubated on a 37 °C thermo-plate.
      NOTE: Dissolved AA can be stored at -30 °C for months.
    4. Culture the cells for an additional 4 - 5 days and replace the medium every 2 days with fresh medium containing AA.
    5. Confirm the proliferation and generation of ASCs under a light microscope to determine whether gaps occurred between the cells in a contiguous cell sheet.
  3. Transfer the cell sheets from the incubator to the benchtop for 15 - 20 min to cool them to room temperature.; observe the cells spontaneously detach as a contiguous cell sheet. Harvest the rASC sheet from the dish surface with a pair of forceps.
    NOTE: Usually, rASC sheets can be handled with a pair of forceps. If necessary, a transfer membrane can be used to transfer a cell sheet from the culture dish to the wound site if the cell sheet is brittle and fragile.

4. Preparation of the Full-thickness Skin Defect Wound Model and Transplantation of rASC Sheets

  1. Prepare a rodent mechanical ventilator and 4% isoflurane. Prepare several sterile cotton tips, clean gauze, 5-0 nylon suture, a scalpel, a periosteal raspatory, a needle holder, operating scissors, and a pair of forceps. Sterilize the surgical instruments and supplies. Lay out the surgical instruments and supplies on a sterile drape.
  2. Prepare PBS and maintain it at room temperature, along with some artificial skin and a non-adhesive dressing. Precut the artificial skin (15 x 10 mm) and non-adhesive dressing (20 × 15 mm). Soak the inner collagen sponge layer of the artificial skin in saline before using the artificial skin.
    NOTE: The artificial skin is made of two layers: an outer silicone sheet layer and an inner collagen sponge.
  3. Use ZDF rats (16 - 18 weeks old, male, 500 - 600 g) as a wound-healing model for type 2 diabetes and obesity.
  4. Anesthetize the ZDF rats by inhalation of 4% isoflurane using a rodent mechanical ventilator. Induce anesthesia by using isoflurane at 4 - 5% via a rodent mechanical ventilator and maintain it at 3 - 4% during surgery. Monitor the depth of anesthesia by observing the depth and rate of respiration of each rat via a toe pinch.
  5. While wearing sterile gloves, position the rat in the prone position on a sterile drape. Clean the head of the rat using sterile gauze soaked in 70% ethanol, shave the operative area with an electric razor, and clean the skin after shaving using sterile gauze soaked in 70% ethanol.
  6. Create a squared full-thickness skin defect (15 x 10 mm2) on the head of the anesthetized ZDF rats by removing the cutaneous tissue from the epidermis to the periosteum. Excise the skin and cutaneous tissue with a scalpel and remove the periosteum with a periosteal raspatory.
  7. Move the rASCs on the 35 mm temperature-responsive culture dish from the incubator to a room-temperature environment immediately prior to transplantation.
  8. Observe the rASCs on the 35 mm temperature-responsive culture dish spontaneously detach as a sheet from the dish surface after the formation of a wound. Harvest the rASC sheets after 7 days of culturing on temperature-responsive culture dishes. Remove the cultured medium and wash the rASC sheet with PBS three times. Soak the rASC sheet in PBS until transplantation to prevent desiccation.
  9. Place the rASC sheet immediately over the skull defect using a pair of forceps. If the cell sheet is brittle and fragile, a membrane can be used as a scaffold for transferring the cell sheet from the culture dish to the wound site.
  10. Cover the rASC sheet and defect with the artificial skin (15 x 10 mm2) and close the wound with approximately 10 stiches using the 5-0 nylon suture. To protect the wound, place a non-adhesive dressing (20 x 15 mm2) over the artificial skin, using 5-0 nylon sutures to keep the wound environment moist and to absorb exudates.
    NOTE: The non-adhesive dressings will be removed by the ZDF rats within a few days after application. Therefore, it is necessary to regularly monitor the rats after transplantation. Usually, the non-adhesive dressing is replaced every 2 days under general anesthesia.
  11. Return each rat that has undergone surgery to a separate cage until they have fully recovered (one rat per cage). After an observation period, euthanize the rats using the approved method of isoflurane overdose.

结果

本议定书试图建立一种新的基于单元格的难治性糖尿病伤口疗法。简要 （如图 1所示），同种异体区域审计事务中心床单从正常大鼠使用单元格表工程被创造了，然后移植对糖尿病大鼠使用双层人工皮肤全层皮肤缺损。光镜图像的区域审计事务中心表 (图 2A) 的一个好例子和一个坏的榜样的区域审计事务中心表 (图 2B) 如图 2

讨论

成功地培养区域审计事务中心表最关键的步骤如下: 1) 温度必须在温度反应性培养皿培养过程维持在大约 37 ° C。在区域审计事务中心表的创建，过程中每道工序进行 37 ° C 的热板，和每个试剂被加热到 37 ° C，以防止细胞自发地分离从菜31。2) 收件人 ZDF 大鼠必须进行监测，以防止被删除的非胶粘剂的酱汁，是区域审计事务中心床单移植成功的关键。如果删除了更衣室，必须应用...

披露声明

以下作者披露与此发布相关的财务关系: 辉冈野是创始人和董事的细胞种子有限公司许可证技术和专利从东京女子医科大学，并照雄冈野和雅之大和利益相关者在细胞种子公司东京女子医科大学接收从细胞种子公司研究基金其他作者宣称，他们并没有到此出版物有关的财务关系。

致谢

作者感谢塑料部和整形外科手术，东京大学医学院博士由纪子古贺提供实用的建议。我们还感谢先生秀和村田的糖尿病中心的东京妇女医疗大学医学院为优异的技术支持。这项研究被支持由创新中心创造先进的跨学科研究领域项目的发展中国家创新系统"单元格表组织工程中心 (CSTEC)"项目从部的教育、 文化、 体育、 科学和技术 （文部科学省） 的日本。

材料

NameCompanyCatalog NumberComments
α-MEM glutamaxInvitrogen32571-036Carlsbad, CA
Fetal bovine serum (FBS)Japan Bioserum Co Ltd.S1650-500
Penicillin/streptomycinLife Technologies15140-122
Collagenase ARoche Diagnostics10 103 578 001Mannheim, Germany
60-cm2 Primaria tissue culture dishBD Biosciences353803Franklin Lakes, NJ
Dulbecco's Phosphate Buffer Saline (PBS)Life Technologies1490-144
0.25% Trypsin-ethylenediamine tetraacetic acid (EDTA)Life Technologies25200-056
L-ascorbic acid phosphate magnesium salt n-hydrateWako013-19641
35-mm temperature-responsive culture dish (UpcellTM)CellSeedNUNC-174904Tokyo, Japan
Microwarm plate (MP-1000)Kitazato Science Co., Ltd.1111
Rodent mechanical ventilatorStoelting#50206Wood Dale, IL
4% isofluranePfizer Japan114-13340-3Tokyo, Japan
Artificial skin (Pelnac®)Smith & NephewPN-R40060 Tokyo, Japan
Non-adhesive dressing (Hydrosite plus®)Smith & Nephew66800679Known as Allevyn non-adhessing® in the United State
5-0 nylon sutureAlfresaEP1105NB45-KF2
20 CELLSTAR TUBESgreiner bio-one227 261
15mL Centrifuge TubeCorning Incorporated430791
14 GOLDMAN-FOX PERIOSTEALHu-FriedyP14Chicago, IL

参考文献

  1. Boulton, A. J., Vileikyte, L., Ragnarson-Tennvall, G., Apelqvist, J. The global burden of diabetic foot disease. Lancet. 366 (9498), 1719-1724 (2005).
  2. Zannettino, A. C., et al. Multipotential human adipose-derived stromal stem cells exhibit a perivascular phenotype in vitro and in vivo. J Cell Physiol. 214 (2), 413-421 (2008).
  3. Kern, S., Eichler, H., Stoeve, J., Kluter, H., Bieback, K. Comparative analysis of mesenchymal stem cells from bone marrow, umbilical cord blood, or adipose tissue. Stem Cells. 24 (5), 1294-1301 (2006).
  4. Casteilla, L., Planat-Benard, V., Laharrague, P., Cousin, B. Adipose-derived stromal cells: Their identity and uses in clinical trials, an update. World J Stem Cells. 3 (4), 25-33 (2011).
  5. Zuk, P. A., et al. Multilineage cells from human adipose tissue: implications for cell-based therapies. Tissue Eng. 7 (2), 211-228 (2001).
  6. Zuk, P. . The ASC: Critical Participants in Paracrine-Mediated Tissue Health and Function. , (2013).
  7. Nie, C., et al. Locally administered adipose-derived stem cells accelerate wound healing through differentiation and vasculogenesis. Cell Transplant. 20 (2), 205-216 (2011).
  8. Shin, L., Peterson, D. A. Human mesenchymal stem cell grafts enhance normal and impaired wound healing by recruiting existing endogenous tissue stem/progenitor cells. Stem Cells Transl Med. 2 (1), 33-42 (2013).
  9. Okano, T., Yamada, N., Sakai, H., Sakurai, Y. A novel recovery system for cultured cells using plasma-treated polystyrene dishes grafted with poly(N-isopropylacrylamide). J Biomed Mater Res. 27 (10), 1243-1251 (1993).
  10. Yamato, M., et al. Thermo-responsive culture dishes allow the intact harvest of multilayered keratinocyte sheets without dispase by reducing temperature. Tissue Eng. 7 (4), 473-480 (2001).
  11. Sekine, H., et al. Cardiac cell sheet transplantation improves damaged heart function via superior cell survival in comparison with dissociated cell injection. Tissue Engineering Part A. 17 (23-24), 2973-2980 (2011).
  12. Kuhlmann, J., et al. Intramyocellular lipid and insulin resistance: a longitudinal in vivo 1H-spectroscopic study in Zucker diabetic fatty rats. Diabetes. 52 (1), 138-144 (2003).
  13. Slavkovsky, R., et al. Zucker diabetic fatty rat: a new model of impaired cutaneous wound repair with type II diabetes mellitus and obesity. Wound Repair Regen. 19 (4), 515-525 (2011).
  14. Oltman, C. L., et al. Progression of vascular and neural dysfunction in sciatic nerves of Zucker diabetic fatty and Zucker rats. Am J Physiol Endocrinol Metab. 289 (1), E113-E122 (2005).
  15. Coppey, L. J., Gellett, J. S., Davidson, E. P., Dunlap, J. A., Yorek, M. A. Changes in endoneurial blood flow, motor nerve conduction velocity and vascular relaxation of epineurial arterioles of the sciatic nerve in ZDF-obese diabetic rats. Diabetes Metab Res Rev. 18 (1), 49-56 (2002).
  16. Galiano, R. D., Michaels, V., Dobryansky, M., Levine, J. P., Gurtner, G. C. Quantitative and reproducible murine model of excisional wound healing. Wound Repair Regen. 12 (4), 485-492 (2004).
  17. Lin, Y. C., et al. Evaluation of a multi-layer adipose-derived stem cell sheet in a full-thickness wound healing model. Acta Biomater. 9 (2), 5243-5250 (2013).
  18. McLaughlin, M. M., Marra, K. G. The use of adipose-derived stem cells as sheets for wound healing. Organogenesis. 9 (2), 79-81 (2013).
  19. Cerqueira, M. T., et al. Human adipose stem cells cell sheet constructs impact epidermal morphogenesis in full-thickness excisional wounds. Biomacromolecules. 14 (11), 3997-4008 (2013).
  20. Koga, Y., et al. Recovery course of full-thickness skin defects with exposed bone: an evaluation by a quantitative examination of new blood vessels. J Surg Res. 137 (1), 30-37 (2007).
  21. Cianfarani, F., et al. Diabetes impairs adipose tissue-derived stem cell function and efficiency in promoting wound healing. Wound Repair Regen. 21 (4), 545-553 (2013).
  22. Matsuda, K., Suzuki, S., Isshiki, N., Ikada, Y. Re-freeze dried bilayer artificial skin. Biomaterials. 14 (13), 1030-1035 (1993).
  23. Miyahara, Y., et al. Monolayered mesenchymal stem cells repair scarred myocardium after myocardial infarction. Nat Med. 12 (4), 459-465 (2006).
  24. Iwata, T., et al. Cell sheet engineering and its application for periodontal regeneration. J Tissue Eng Regen Med. , (2013).
  25. Elloumi-Hannachi, I., Yamato, M., Okano, T. Cell sheet engineering: a unique nanotechnology for scaffold-free tissue reconstruction with clinical applications in regenerative medicine. J Intern Med. 267 (1), 54-70 (2010).
  26. Watanabe, N., et al. Genetically modified adipose tissue-derived stem/stromal cells, using simian immunodeficiency virus-based lentiviral vectors, in the treatment of hemophilia. B. Hum Gene Ther. 24 (3), 283-294 (2013).
  27. Kim, W. S., et al. Wound healing effect of adipose-derived stem cells: a critical role of secretory factors on human dermal fibroblasts. J Dermatol Sci. 48 (1), 15-24 (2007).
  28. Nakagami, H., et al. Novel autologous cell therapy in ischemic limb disease through growth factor secretion by cultured adipose tissue-derived stromal cells. Arterioscler Thromb Vasc Biol. 25 (12), 2542-2547 (2005).
  29. Asahara, T., et al. VEGF contributes to postnatal neovascularization by mobilizing bone marrow-derived endothelial progenitor cells. EMBO J. 18 (14), 3964-3972 (1999).
  30. Kato, Y., et al. Allogeneic transplantation of an adipose-derived stem cell (ASC) sheet combined with artificial skin accelerates wound healing in a rat wound model of type 2 diabetes and obesity. Diabetes. , db141133 (2015).

转载和许可

请求许可使用此 JoVE 文章的文本或图形

请求许可

探索更多文章

126

This article has been published

Video Coming Soon

JoVE Logo

政策

使用条款

隐私

联系我们

向图书馆推荐

JoVE 新闻简报

科研

教育

发表

图书馆员

关于 JoVE

版权所属 © 2025 MyJoVE 公司版权所有，本公司不涉及任何医疗业务和医疗服务。