在压缩的胶原组织糜：含细胞Biotransplant单上演重建修复
Summary
组织工程经常包括在体外扩增 ，以创建用于组织再生自体移植。在这项研究中用于组织扩张，再生， 和体内重建的方法，以便最小化在体外的细胞和生物材料的处理被开发。
Abstract
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.
Introduction
上移植到皮肤和泌尿生殖道最组织工程研究包括来自于专门配备细胞培养设施1,2-健康组织和细胞扩张的自体细胞的收成。
细胞扩增后，将细胞通常存储供以后使用时制备的患者以接收自体移植。氮冷冻允许在-150℃或更低的低温长期贮存。冷冻的过程中一定要小心，为了不失去控制的细胞。细胞死亡的一个危险是在解冻过程中，这可导致细胞膜的破裂的细胞内的水的结晶。细胞冷冻通常通过缓慢和受控冷却（-1℃，每分钟）进行的，使用的细胞，胎牛血清和二甲亚砜的浓度高。解冻后，将细胞需要通过除去冷冻培养基并培养细胞培养塑料或再次处理移植前生物材料回给患者。
所有上述步骤是费时，费力，且昂贵3。此外，针对患者的移植细胞全部排出体外处理是高度管制的，需要训练有素和认可的人员和实验室4。总而言之，促使安全和可靠的制造工艺，这项技术只有在极少数技术先进中心成立，并在常见的外科疾病更广泛的使用是值得怀疑的。
为了克服在实验室环境的细胞培养的局限性，移栽组织糜用于体内细胞扩增的概念是通过使用体本身作为生物反应器引入的。为了这些目的，在自体移植将优先被上一个3D模具按照所需要对i的器官的最终重建的形状移植nterest 5-7。
原来，移栽剁碎上皮细胞的想法，在1958年提出由温顺，当他描述的上皮细胞从伤口的边缘如何生长。他表明，一小片皮肤的将增加其利润率并且由此其为细胞扩增潜力100％通过切割片两次在垂直的方向（图1）8。理论已被使用啮合部分厚度皮肤移植的皮肤移植9支撑 ，并在皮肤伤口愈合模型10。
图1：米克理论根据温顺的理论，上皮生长从伤口的边缘。通过增加切碎技术的曝光区域，组织糜处上皮许多景点的伤口。
本研究是基于低论文，相同的原理可以通过将剁碎上皮围绕模具施加到皮下组织。上皮细胞会从剁碎移植（重组），以便形成连续neoepithelium从内部主体覆盖伤口区域，并分离异物（模具）动员，覆盖伤口区域（迁移）和除（展开）（ 图2）。
图2：根据米克理论三维模具剁碎上皮用于体内intracorporal组织扩张的卡通通过使用放置在模具中，然后移植到皮下组织组织糜，所述假设是，上皮细胞从迁移组织糜的边缘，重组，和扩展，以便形成一个连续的neoepithelium覆盖伤口区域和异物（模具）从内体中分离。
尽管以前的体内研究表明可喜的成果，进一步改善可以通过加强自体移植来实现，使再生上皮细胞可以抵御机械损伤更好7。为了这些目的，一个成功的生物材料的重要先决条件被确定，如：容易扩散营养素和废物的，可能性模具在三维方式和手术操作容易。结论所做的这些需要可以通过添加的复合生物材料的组织糜得到满足。
目前的研究旨在开发一种在组织糜构成的支架塑性压缩胶原含有可生物降解的织物的增强芯。通过这些手段，活细胞能够从组织糜粒子迁移，并与原来的上皮细胞（皮肤或上皮）的形态特征特性增殖。使用塑料压缩，脚手架是减少d在1厘米大小至约420微米的切碎的颗粒在上层胶原进行了包裹。该芯部织物可以是任何聚合物，但需要用亲水表面修饰，以便与覆盖胶原层 11相互连接起来。
该方法通过使用其作为支架用于培养切碎膀胱粘膜或猪碎肉皮肤掺入两个塑料压缩胶原凝胶内的编织网由聚（ε – 己内酯）（PCL）提供了一种增强的支架的完整性。该构建物维持在细胞培养条件为长达6周的体外 ，证明成功地形成一个良好的固结杂交构建体的顶部分层，多层上皮或鳞状皮肤上皮。该构建是易于处理和来代替膀胱增加目的或皮肤缺陷的覆盖物被缝合。组织支架的所有部件都FDA批准和技术可以通过组织收获，切碎，塑性压缩，并移栽回病人作为一个单一阶干预可用于单级的程序。该程序可以在任何普通外科单位在无菌条件下组织扩张和重建进行。
Protocol
Representative Results
Discussion
这项研究提出了一种易于使用的方法，以产生具有用于移植自体组织膀胱壁修补在手术台。贴片通过在中间和胶原有和没有外表面与塑性压缩组合组织糜可生物降解的聚合物针织的组合形成。塑性压缩是以前由其他作者描述的方法，并且可以被定义为从胶原凝胶12,13流体的迅速排出。膀胱粘膜或皮肤的组织糜接种到该支架和膀胱或皮肤上皮的形成可能在6周被遵循。免疫组织化学分析表明?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
The authors thank the Swedish Society for Medical Research, the Promobilia Foundation, the Crown Princess Lovisa Foundation, the Freemason Foundation for Children’s Welfare, the Swedish Society of Medicine, the Solstickan Foundation, Karolinska Institutet, and the Stockholm City Council for financial support.
Materials
| Silicone catheter 10-French | Preparing the animal for surgery , Section 1 | ||
| DMEM 10X | Gibco | 31885-023 | Plastic compression section 4 |
| 24 well plates | Falcon | 08-772-1 | Plastic compression section 4 |
| 3'3,'5-Triiodothyronine | Sigma-Aldrich | IRMM469 | In vitro culture; Section 5 |
| 4% PFA | Labmed Solutions | 200-001-8 | Immunocytochemistry; Section 6 |
| 70% ethanol | Histolab | Immunocytochemistry; Section 6 | |
| ABC Elite kit: Biotin -Streptavidin detection kit | Vector | PK6102 | Immunocytochemistry; Section 6 |
| Absolute ethanol | Histolab | 1399.01 | Immunocytochemistry; Section 6 |
| Adenine | Sigma-Aldrich | A8626 | In vitro culture; Section 5 |
| Atropine 25 μg/kg | Temgesic, RB Pharmaceuticals, Great Britain | Preparing the animal for surgery , Section 1 | |
| Azaperone 2 mg/kg | Stresnil, Janssen-Cilag, Pharma, Austria | Preparing the animal for surgery , Section 1 | |
| Biosafety Level 2 hood | Plastic compression; Section 4 | ||
| Blocking solution: Normal serum from the same species as the secondary secondary antibody was generated in. | Vector | The blocking solution depends of the origin of first antibody | Immunocytochemistry; Section 6 |
| Buprenorphine 45 μg/kg | Atropin, Mylan Inc, Canonsburg, PA | Preparing the animal for surgery , Section 1 | |
| Carprofen 3 mg/kg | Rimadyl, Orion Pharma, Sweden | Preparing the animal for surgery , Section 1 | |
| Chlorhexidine gluconate | Hibiscrub 40 mg/mL, Regent Medical, England | Preparing the animal for surgery , Section 1 | |
| Cholera toxin | Sigma-Aldrich | C8052 | In vitro culture; Section 5 |
| Coplin jar: staining jar for boiling | Histolab | 6150 | Immunocytochemistry; Section 6 |
| Stainless mold (33x22x10 mm) custom made | Plastic compression; Section 4 | ||
| DMEM | Gibco | 3188-5023 | Plastic compression section 4. Keep on ice when using it in plastic compression |
| Epidermal growth factor | Sigma-Aldrich | E9644 | In vitro culture; Section 5 |
| Ethilon (non-absorbable monofilament for skin sutures) | Ethicon | Surgery, Section 1 | |
| Fetal bovine serum (FBS) | Gibco | 10437-036 | Plastic compression section 4 |
| Forceps (Adison with tooth) | Preparing the animal for surgery , Section 1 | ||
| Gauze (Gazin Mullkompresse) | Preparing the animal for surgery , Section 1 | ||
| Ham´s F12 | Gibco | 31765-027 | Plastic compression section 4 |
| Hematoxylin | Histolab | 1820 | Immunocytochemistry; Section 6 |
| Humidity chamber | DALAB | Immunocytochemistry; Section 6 | |
| Hydrocortisone | Sigma-Aldrich | H0888 | In vitro culture; Section 5 |
| Hydrogen peroxide Solution 30% | Sigma-Aldrich | H1009 | Immunocytochemistry; Section 6 |
| Insulin | Sigma-Aldrich | I3536 | In vitro culture; Section 5 |
| Isoflurane | Isoflurane, Baxter, Deerfield, IL | Preparing the animal for surgery , Section 1 | |
| Lidocaine 5 mg/ml | Xylocaine, AstraZeneca, Sweden | Preparing the animal for surgery , Section 1 | |
| Lucose 25 mg/mL | Baxter, Deerfield, IL | Preparing the animal for surgery , Section 1 | |
| Marker pen pap pen | Sigma-Aldrich | Z377821-1EA | Immunocytochemistry; Section 6 |
| Medetomidine 25 μg/kg | Domitor, Orion Pharma, Sweden | Preparing the animal for surgery , Section 1 | |
| Mincing device | Applied Tissue Technologies LLC | Minced tissue preparation, section 2 | |
| Monocryl (absorbable monofilament) | Ethicon | Surgery, Section 1 | |
| NaCl | Sigma-Aldrich | S7653 | Immunocytochemistry; Section 6 |
| NaOH 1N | Merck Millipore | 106462 | Plastic compression section 4 and cell culture |
| Nylon mesh, 110 uM thick pore size 0.04 sqmm | Plastic compression; Section 4 | ||
| Oculentum simplex APL: ointment for eye protection | APL | Vnr 336164 | Surgery, Section 1 |
| PBS | Gibco | 14190-094 | Plastic compression section 4 |
| Penicillin-Streptomycin | Gibco | 15140-122 | Plastic compression section 4 |
| Phenobarbiturate 15 mg/kg | Pentobarbital, APL, Sweden | Preparing the animal for surgery , Section 1 | |
| PLGA Knitted fabric | Plastic compression; Section 4 | ||
| Rat-tail collagen | First LINK, Ltd, UK | 60-30-810 | Plastic compression section 4, keep on ice |
| Scalpel blade – 15 | Preparing the animal for surgery , Section 1 | ||
| Shaving shears | Preparing the animal for surgery , Section 1 | ||
| Stainless stell mesh, 400 uM thick pore size | Plastic compression; Section 4 | ||
| Steril gloves | Preparing the animal for surgery , Section 1 | ||
| Sterile gowns | Preparing the animal for surgery , Section 1 | ||
| Sterile drapes | |||
| Sterilium | Bode Chemie HAMBURG | Preparing the animal for surgery , Section 1 | |
| Suture Thread Ethilon | Preparing the animal for surgery , Section 1 | ||
| TE-solution (antigen unmasking solution) consist of 10 mM Tris and 1 mM EDTA, pH 9.0 | 10 mM Tris/1 mM EDTA, adjust pH to 9.0 | ||
| Tiletamine hypochloride 2,5 mg/kg | Preparing the animal for surgery , Section 1 | ||
| Transferrin | Sigma-Aldrich | T8158 | In vitro culture; Section 5 |
| Trizma Base, H2NC | Sigma-Aldrich | T6066 | Immunocytochemistry; Section 6 |
| Vector VIP kit: Enzyme peroxidase substrate kit | Vector | SK4600 | Immunocytochemistry; Section 6 |
| Vicryl (absorbable braded) | Ethicon | Surgery, Section 1 | |
| Tris buffer pH 7.6 (washing buffer) | TE solution: Make 10X (0,5M Tris, 1,5M NaCl) by mixing: 60,6 g Tris (Trizma Base, H2NC(CH2OH)3, M=121.14 g/mol), add 800 ml distilled water adjust the pH till 7.6, add 87,7 g NaCl and fill to 1000 ml with distilled water. Dilute to 1X with distilled water. | ||
| X-tra solv (solvent) | DALAB | 41-5213-810 | Immunocytochemistry; Section 6. Use under fume hood |
| Zolazepam hypochloride | Zoletil, Virbac, France | Preparing the animal for surgery , Section 1 | |
| Depilatory wax strips | Veet | Preparing the animal for surgery , Section 1 | |
| Pentobarbital sodium | Lundbeck | Termination, Section 3 |
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