WO2014117597A2 - 快速组织工程皮肤构建修复皮肤缺损的方法 - Google Patents
快速组织工程皮肤构建修复皮肤缺损的方法 Download PDFInfo
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- WO2014117597A2 WO2014117597A2 PCT/CN2013/088833 CN2013088833W WO2014117597A2 WO 2014117597 A2 WO2014117597 A2 WO 2014117597A2 CN 2013088833 W CN2013088833 W CN 2013088833W WO 2014117597 A2 WO2014117597 A2 WO 2014117597A2
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/36—Skin; Hair; Nails; Sebaceous glands; Cerumen; Epidermis; Epithelial cells; Keratinocytes; Langerhans cells; Ectodermal cells
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
- A61L27/60—Materials for use in artificial skin
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- the present invention relates to the field of medical methods, and more particularly to a method for rapid tissue engineering skin construction to repair skin defects. Background technique
- the best alternative is still autologous epidermal dermal substitute because most of the remaining skin substitutes are only effective in patients with chronic ulcers and burns, and they also have different degrees of difficulty in cutting, easy infection, immune rejection. And allergies and other issues. Due to the lack of support of the dermis component, the cultured epidermis is highly sensitive to mechanical damage, tends to form wound contraction and scar hyperplasia after transplantation, and does not achieve true skin reconstruction due to lack of dermis. However, the autologous epidermis dermal substitute is lack of epidermis, has no living cells, is susceptible to infection, and is easily degraded and contracted after transplantation, and is likely to form scars at the edge of the wound.
- a method for rapidly repairing a skin defect to repair a skin defect comprising the following steps: (1) taking a skin piece, the area of the skin piece being larger than about 10% of the wound area to be repaired; (2) The acellular allogeneic dermal matrix is covered on the wound surface to be covered, and the surface is covered with the previously taken skin piece, and the excess skin piece is trimmed; (3) the cell suspension is injected or sprayed to the front composite skin.
- the above-mentioned leather sheet may be a leather sheet obtained by cutting a self-curtain blade with a dermatome.
- the present invention may further comprise the following steps: (4) injecting a cell suspension between the skin graft and the acellular allogeneic dermal matrix through the edge of the percutaneous sheet, and then performing the method of composite skin grafting. Post processing.
- Normal skin consists of epidermis and dermis, and all current methods of repairing skin defects have failed to achieve the desired purpose of restoring the skin.
- the key to fully recovering skin function is the structural structure of the epidermis and dermis, which functionally restores the functions of normal skin, such as Pain, warmth, touch, perspiration function, and appearance are consistent with normal skin.
- the invention comprises acellular allogeneic dermis on the skin structure, and the seed cell is applied with an autoactive cell suspension, which not only has the structure including the epidermis and the dermis, but also functions to achieve the purpose of repair to the greatest extent, the autologous activity. Cell suspensions will maximize the function of repairing skin defects.
- the invention can effectively promote wound healing, improve the survival rate of the skin, and improve the texture of the skin after the healing.
- the cell suspension can be prepared by placing the skin in a 0.2% trypsin solution heated to 37 degrees for 15-20 minutes to separate the epidermis from the dermis.
- the bacteria scalpel scrapes the cells from the surface of the dermis and epidermis, and the basal cells are scraped off by a compound Ringer's solution to prepare a cell suspension.
- the cell suspension can also be prepared by placing the skin in a 0.2% trypsin solution heated to 37 degrees for 15-20 minutes, using sterile mash from trypsin. Remove the skin from the solution, rinse the skin with the compound forest format solution, neutralize the trypsin digestion, separate the epidermis and the dermis with two forceps, and scrape the cells from the surface of the dermis and epidermal junction with a sterile scalpel. The Grignard solution is rinsed to collect the scraped basal cells to make a cell suspension.
- the cell suspension can be prepared by the method of placing the skin piece heated to 37 degrees. Soak in 0.2% trypsin solution for 15 ⁇ 20 minutes, then re-seat the skin in a heated (37 °C) trypsin solution for 5-10 minutes (the cell suspension digestion time takes 15 minutes or more, twice The total digestion time is less than 60 minutes), the epidermis and the dermis are separated, and the cells are scraped from the surface of the dermis and epidermis by a sterile scalpel, and the scraped basal cells are collected by a compound Ringer's solution to prepare a fine suspension.
- the cell suspension can also be prepared by the following method.
- the cell suspension can be prepared by placing the skin in a 0.2% trypsin solution that has been heated to 37 degrees. ⁇ 20 minutes, then re-seat the skin in heated trypsin solution for 5-10 minutes, remove the skin from trypsin solution with sterile sputum, rinse the skin with compound lining solution, neutralize trypsin Digestion reaction, the epidermis and dermis were separated by two forceps, and the cells were scraped from the surface of the dermis and epidermis by a sterile scalpel, and the basal cells were scraped off by a compound Ringer's solution to prepare a cell suspension.
- the cell suspension of the present invention can be obtained quickly, the entire step can be completed quickly during the operation, and the entire steps of preparing the cell suspension are generally completed in half an hour. Compared with traditional cell transplantation (removing cells in an in vitro laboratory for primary culture, and passage amplification, it takes a long time), it is efficient and rapid.
- the cell suspension of the present invention is derived from the dermal and epidermal junction cells, ie, basal cells, having an active, multi-directional differentiation potential (multi-directional differentiation potential may also form directional differentiation in a specific microenvironment of the surgical wound, Different directional differentiations meet the different needs of surgical wound healing, respectively, and they contain a variety of active cellular components with different roles. detailed description
- the invention is further illustrated in the following.
- the invention relates to a method for rapidly repairing skin defects and repairing skin defects, which comprises a method of thick skin graft and cell suspension combined with composite skin grafting.
- the skin knife to cut the skin piece with its own blade thickness.
- the area of the skin should be larger than about 10% of the wound area to be repaired.
- the acellular allogeneic dermal matrix is covered on the wound surface to be covered, and the surface is covered with the skin blade of the original blade thickness which is previously taken to form a composite skin grafting area, and the excess skin piece is trimmed.
- the ideal artificial skin consists of a two-layer structure of epidermis and dermis with a tight connection between the two. It has flexibility and a certain mechanical strength. It is attached to the wound quickly after transplanting to the wound surface. It has the advantages of being safe and not carrying viruses. . At this time, the epidermal and dermal components can complete their own proliferation, differentiation and functional maturity as soon as possible, forming a permanent skin substitute that is closer to the physiology.
- the cell suspension is injected or sprayed into the above-mentioned composite skin grafting area, and the cell suspension is injected between the skin piece and the acellular allogeneic dermal matrix through the edge of the skin piece; and then the post-treatment is performed according to the method of composite skin transplantation.
- the above cell suspension is prepared by the following method: The skin is placed in a 0.2% trypsin solution heated to 37 ° C and soaked for 15 to 20 minutes. The skin pieces were removed from the trypsin solution using sterile mash and two tweezers were used to try to peel off the epidermis and dermis. If separation is not easy to achieve, reattach the skin to 0.2% trypsin solution that has been heated to 37 ° C and incubate for another 5 to 10 minutes. The skin pieces were removed from the trypsin solution using sterile mash, and the skin pieces were rinsed with the compound Ringer's solution to neutralize the trypsin digestion reaction.
- the digested skin pieces were completely separated into two layers of dermis and epidermis using two tweezers to reveal the dermis-epidermal junction.
- the skin layer was fixed with tweezers, and the cells were scraped from the surface of the dermis and epidermis by a sterile scalpel, and the basal cells were scraped off by a compound Ringer's solution to prepare a cell suspension.
- the cell suspension is prepared as described above, the total amount is not too large.
- the number of cells is constant, the more the total amount of the suspension, the lower the cell concentration.
- Digestion time may vary depending on factors such as skin thickness and room temperature, but in general, the digestion time should be more than 15 minutes, and the total digestion time should not exceed 60 minutes.
- the method of basal cell suspension of skin obtained by the above method is applied to clinical practice, and has good effects in promoting wound healing, improving skin survival rate, and improving skin color and texture after healing.
- the cell suspension contains basal cells, Langerhans cells, melanocytes and fibroblasts, as well as epidermal stem cells.
- the rich active cell population is the cause of its clinical effects.
- Epidermal stem cells are currently one of the hotspots used as seed cells in skin tissue engineering research, and acellular allogeneic dermis as a scaffold has also been proved to be an ideal tissue engineering skin material. Combining these two technologies, a quick Effectively construct a method with a full-featured skin.
- An electric dermatome was used to cut a 0.25 mm thick blade of skin on the thighs of the child.
- About 6 cm 2 of the obtained skin piece was digested with trypsin at 37 ° C for 15 minutes.
- the epidermis layer of the digested skin was separated from the dermis layer, and the cells located in the basal layer were collected to prepare a cell suspension of about 4 ml for use.
- 1 ml of the cell suspension was sprayed on the bilateral thigh donor site, and the wound was covered with the yarn of the Saslin oil and then pressure-wrapped.
- the right upper limb black sputum resection area covered the allogeneic acellular dermal matrix, and then the stromal cell suspension was sprayed on the operation area, and then the autologous blade thick skin was covered, and the skin grafting area was pressurized and bandaged. Postoperative routine anti-inflammatory fluid replacement and other treatments.
- the external dressing was removed 7 days after surgery, and most of the skin grafting areas survived well. After three months of follow-up, the skin color of the skin graft area was good, close to normal skin, good elasticity and normal joint function.
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Description
快速组织工程皮肤构建修复皮肤缺损的方法 技术领域
本发明涉及医疗方法领域， 特别是一种快速组织工程皮肤构建修复皮肤缺损 的方法。 背景技术
各种原因引起的大面积皮肤缺损， 如： 烧伤、 创伤， 是烧伤外科、 整形修 复外科面临的主要问题。 为了有效解决皮源和修复后效果的问题， 新的技术与 方法不断出现， 如皮片移植、 皮瓣移植、 邮票皮片移植、 微粒皮移植、 网状皮 移植、 复合皮移植等。 组织工程皮肤的研究也在不断的深入， 目前的组织工程
3种， 而最佳 替代物依然是自体表皮片真皮替代物 因为其余的皮肤替代物大 多数仅显效于慢性溃疡和烧伤的患者， 同时它们也都不同程度的存在切取困难、 易于感染、 免疫排斥和过敏等问题。 培养表皮片由于缺乏真皮成分的支持， 对 机械损伤高度敏感， 移植后易形成创面收缩和瘢痕增生， 而且由于缺乏真皮， 没有实现真正的皮肤重建。 然而， 自体表皮片真皮替代物由于缺乏表皮， 无活 细胞， 易感染， 移植后易降解、 收缩， 在创面边缘易形成瘢痕。
虽然新的方法都在一定程度上改善与推进了创面修复的治疗水平， 但也都 存在着各自的不足之处， 无法克服皮片存活率低、 产品周期长的缺点。 发明内容
本发明的目的是提供一种快速组织工程皮肤构建修复皮肤缺损的方法， 以 解决上述技术中的至少一个。
根据本发明的一个方面， 提供了一种快速组织工程皮肤构建修复皮肤缺损 的方法， 包括以下步骤： （1)取皮片， 皮片面积大于需修复的创面面积约 10%左 右； （2)将脱细胞异体真皮基质覆盖于所需覆盖的创面， 其表面再覆盖上之前所 取的皮片， 修剪出多余的皮片； （3)再将细胞悬液注射或喷洒到前面进行的复合 皮植皮区内。 上述的皮片可以是用取皮刀切取自体刃厚的皮片。
在一些具体的实施方式中， 本发明还可以包括下述步骤： （4)经皮片边缘将 细胞悬液注射到皮片与脱细胞异体真皮基质之间， 再按复合皮移植的方法进行 术后处理。
正常的皮肤由表皮和真皮组成， 而目前所有的手术修复皮肤缺损的方法， 都未能达到理想的修复目的， 即完全恢复皮肤的功能。 而完全恢复皮肤功能的 关键在于结构上包含表皮和真皮的结构， 功能上恢复正常皮肤应有的功能， 如
痛、 温、 触觉、 排汗功能、 外观上与正常皮肤一致。 本发明在皮肤结构上包括 了脱细胞异体真皮， 且种子细胞应用的是自体活性的细胞悬液， 既在结构上达 到包含表皮和真皮， 功能上也最大程度地达到修复的目的， 自体的活性细胞悬 液将最大程度的修复皮肤缺损的功能。 与现有技术相比， 本发明可以有效地促 进创面愈合， 提高皮片成活率， 改善愈后皮肤色泽质地。
在一些具体的实施方式中， 细胞悬液可以由下述方法制成， 将皮片置于已 加热至 37度的 0.2%胰蛋白酶溶液浸泡消化 15~20分钟，使表皮和真皮分离，用 无菌解剖刀从真皮与表皮结合部表面刮削细胞， 应用复方林格氏液沖洗收集刮 下的基底层细胞制成细胞悬液。
在一些具体的实施方式中， 细胞悬液还可以由下述方法制成， 将皮片置于 已加热至 37度的 0.2%胰蛋白酶溶液浸泡消化 15~20分钟，用无菌镊从胰蛋白酶 溶液中取出皮片， 用复方林格式液沖洗皮片， 中和胰酶消化反应， 用两把镊子 使表皮和真皮分离， 用无菌解剖刀从真皮与表皮结合部表面刮削细胞， 应用复 方林格氏液沖洗收集刮下的基底层细胞制成细胞悬液。
在一些具体的实施方式中， 经过胰蛋白酶溶液浸泡消化后， 仍不易实现表 皮和真皮的分离， 此时， 细胞悬液可以由下述方法制成， 将皮片置于已加热至 37度的 0.2%胰蛋白酶溶液浸泡消化 15~20分钟， 再把皮片重新置于已加热（37 度） 的胰蛋白酶溶液中孵育 5~10分钟 (细胞悬液的消化时间需 15分钟以上， 两次的消化总时间不超过 60分钟）， 使表皮和真皮分离， 用无菌解剖刀从真皮 与表皮结合部表面刮削细胞， 应用复方林格氏液沖洗收集刮下的基底层细胞制 成细 悬液。
在一些具体的实施方式中， 细胞悬液还可以由下述方法制成， 细胞悬液可 以由下述方法制成， 将皮片置于已加热至 37度的 0.2%胰蛋白酶溶液浸泡消化 15~20分钟， 再把皮片重新置于已加热的胰蛋白酶溶液中孵育 5~10分钟， 用无 菌镊从胰蛋白酶溶液中取出皮片， 用复方林格式液沖洗皮片， 中和胰酶消化反 应， 用两把镊子使表皮和真皮分离， 用无菌解剖刀从真皮与表皮结合部表面刮 削细胞， 应用复方林格氏液沖洗收集刮下的基底层细胞制成细胞悬液。
本发明的细胞悬液可以快速获得， 整个步骤可以在术中快速完成， 制备细 胞悬液的全部步骤一般只需半小时即可完成。 与传统的细胞移植（需取下细胞 在体外实验室中完成原代培养， 并传代扩增， 耗时长）相比， 具有高效， 快速 的特点。 另外， 本发明的细胞悬液是取自真皮与表皮结合部细胞， 即基底层细 胞， 具有活性， 多向分化潜力 （多向分化潜力也可能会在手术创面特定的微环 境下形成定向分化， 不同的定向分化分别满足手术创面愈合的不同需要）， 同时 其包含多种活性细胞成分， 具有不同的作用。
具体实施方式
下面对本发明作进一步详细地说明。
本发明一种快速组织工程皮肤构建修复皮肤缺损的方法， 由刃厚皮片、 细 胞悬液结合复合皮移植的方法组成。
先应用取皮刀切取自体刃厚的皮片备用， 皮片面积应大于需修复的创面面 积约 10%左右。
将脱细胞异体真皮基质覆盖于所需覆盖的创面， 其表面覆盖上之前所取的 自体刃厚的皮片， 形成复合皮植皮区， 修剪多余的皮片。 理想的人工皮肤包含 表皮和真皮两层结构并且两者之间连接紧密， 其具有柔韧性和一定的机械强度， 移植到创面后很快即与创面良好贴附， 具有安全、 不携带病毒的优点。 此时， 表皮与真皮成分能尽快完成自身增殖、 分化和功能成熟， 形成更接近于生理的 永久性的皮肤替代物等。
再将细胞悬液注射或喷洒到上述复合皮植皮区内， 经皮片边缘将细胞悬液 注射到皮片与脱细胞异体真皮基质之间； 再按复合皮移植的方法进行术后处理。
上述的细胞悬液由下述方法制成： 将皮片置于已加热至 37摄氏度的 0.2% 胰蛋白酶溶液浸泡消化 15~20分钟。 用无菌镊从胰蛋白酶溶液中取出皮片， 使 用两把镊子， 尝试剥离表皮和真皮。 如果分离不易实现， 把皮片重新置于已加 热至 37摄氏度的 0.2%的胰蛋白酶溶液中， 再孵育 5~10分钟。 用无菌镊从胰蛋 白酶溶液中取出皮片， 用复方林格氏液沖洗皮片， 中和胰酶消化反应。 用两把 镊子将消化后的皮片完全分离成真皮和表皮两层， 显露真皮-表皮结合部。 用镊 子固定皮片层， 用无菌解剖刀从真皮与表皮结合部表面刮削细胞， 应用复方林 格氏液沖洗收集刮下的基底层细胞制成细胞悬液。 按上述方法制细胞悬液时总 量不要过大， 当细胞数量一定时， 悬液总量越多细胞浓度越低。 消化时间可能 因皮片厚度、 室温等因素有所不同而定， 但就一般而言， 消化时间需 15分钟以 上， 两次的消化总时间不超过 60分钟为宜。
通过上述方法获得的皮肤基底细胞悬液的方法应用于临床， 在促进创面愈 合、 提高皮片成活率、 改善愈后皮肤色泽质地等方面都有良好的效果。 经过初 步的研究， 该种细胞悬液中含有基底细胞、 朗格罕斯细胞、 黑色素细胞和成纤 维细胞以及表皮干细胞， 丰富的活性细胞群落是其产生临床效果的原因。 表皮 干细胞是目前作为种子细胞应用于皮肤组织工程研究的热点之一， 而脱细胞异 体真皮作为支架也被证实为理想的组织工程皮肤材料， 将这两种技术结合在一 起应用， 得到一种快捷、 有效的构建出有完整功能皮肤的方法。
病例 1 :
吴某， 男， 5岁。 因诊为 "先天性右上肢巨大黑色素细胞痣" 入院求治。 入 院查体见患儿右上肢大部分皮肤呈黑色， 稍增生， 大部分黑色皮肤处有增粗毛
发生长。 入院后完善术前常规检查未见明显手术禁忌。 于气管内麻下行 "右上 肢巨大黑毛痣切除 +大腿取皮右上肢快速组织工程皮肤植皮术"。 术中完整切除 右上肢黑痣（术后病理确诊为黑色素细胞痣）。 电动取皮刀于患儿双侧大腿处切 取 0.25mm厚刃厚皮片备用。 将所取皮片中约 6cm2应用胰蛋白酶于 37摄氏度消 化 15分钟。 将消化之皮片表皮层与真皮层分离后收集位于基底层的细胞制备成 细胞悬液约 4ml备用。将 1ml细胞悬液喷洒于双侧大腿供皮区后创面覆盖纱士林 油纱后加压包扎。 右上肢黑痣切除区域格覆盖异体脱细胞真皮基质后将基质细 胞悬液喷洒于手术区， 再覆盖自体刃厚皮片， 植皮区加压包扎固定。 术后常规 抗炎补液等治疗。 患儿右上肢植皮区于术后 7 天拆除外敷料， 见绝大部分植皮 区成活良好。 术后三个月复诊见植皮区皮肤色泽质地良好， 接近正常皮肤， 弹 性良好， 关节功能正常。
以上所述的仅是本发明的一种实施方式。 对于本领域的普通技术人员来说， 在不脱离本发明创造构思的前提下， 还可以做出若干变形和改进， 这些都属于 本发明的保护范围。
Claims
1.快速组织工程皮肤构建修复皮肤缺损的方法， 其特征在于， 包括以下步 骤： （1)取皮片， 皮片面积大于需修复的创面面积约 10%左右； （2)将脱细胞异体 真皮基质覆盖于所需覆盖的创面， 其表面再覆盖上述所取的皮片， 形成复合皮 植皮区， 修剪多余的皮片； （3)将细胞悬液注射或喷洒到上述复合皮植皮区内。
2.根据权利要求 1所述的快速组织工程皮肤构建修复皮肤缺损的方法，其特 征在于， 还包括下述步骤： （4)经皮片边缘将细胞悬液注射到皮片与脱细胞异体 真皮基质之间， 再按复合皮移植的方法进行术后处理。
3.根据权利要求 1或 2所述的快速组织工程皮肤构建修复皮肤缺损的方法， 其特征在于， 所述的皮片是用取皮刀切取自体刃厚的皮片。
4.根据权利要求 3所述的快速组织工程皮肤构建修复皮肤缺损的方法，其特 征在于， 所述细胞悬液由下述方法制成，将皮片置于已加热至 37摄氏度的 0.2% 胰蛋白酶溶液浸泡消化 15~20分钟， 使表皮和真皮分离， 用无菌解剖刀从真皮 与表皮结合部表面刮削细胞， 应用复方林格氏液沖洗收集刮下的基底层细胞制 成细 悬液。
5.根据权利要求 3所述的快速组织工程皮肤构建修复皮肤缺损的方法，其特 征在于， 所述细胞悬液由下述方法制成，将皮片置于已加热至 37摄氏度的 0.2% 胰蛋白酶溶液浸泡消化 15~20分钟， 用无菌镊从胰蛋白酶溶液中取出皮片， 用 复方林格氏液沖洗皮片， 中和胰酶消化反应， 用两把镊子使表皮和真皮分离， 用无菌解剖刀从真皮与表皮结合部表面刮削细胞， 应用复方林格氏液沖洗收集 刮下的基底层细胞制成细胞悬液。
6.根据权利要求 3所述的快速组织工程皮肤构建修复皮肤缺损的方法，其特 征在于， 所述细胞悬液由下述方法制成，将皮片置于已加热至 37摄氏度的 0.2% 胰蛋白酶溶液浸泡消化 15~20分钟， 再把皮片重新置于已加热的胰蛋白酶溶液 中孵育 5~10分钟， 使表皮和真皮分离， 用无菌解剖刀从真皮与表皮结合部表面 刮削细胞， 应用复方林格氏液沖洗收集刮下的基底层细胞制成细胞悬液。
7.根据权利要求 3所述的快速组织工程皮肤构建修复皮肤缺损的方法，其特 征在于， 所述细胞悬液由下述方法制成，将皮片置于已加热至 37摄氏度的 0.2%
胰蛋白酶溶液浸泡消化 15~20分钟， 再把皮片重新置于已加热的胰蛋白酶溶液 中孵育 5~10分钟， 用无菌镊从胰蛋白酶溶液中取出皮片， 用复方林格氏液沖洗 皮片， 中和胰酶消化反应， 用两把镊子使表皮和真皮分离， 用无菌解剖刀从真 皮与表皮结合部表面刮削细胞， 应用复方林格氏液沖洗收集刮下的基底层细胞 制成细胞悬液。
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| CN103989531B (zh) * | 2014-06-04 | 2016-05-11 | 朱家源 | 快速构建组织工程皮肤的器械包 |
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