van den Bogaerdt Antoon J, Ulrich Magda M W, van Galen Mieke J M, Reijnen Linda, Verkerk Michelle, Pieper Jeroen, Lamme Evert N, Middelkoop Esther
Research Department, Dutch Burns Foundation, Beverwijk, The Netherlands.
Wound Repair Regen. 2004 Mar-Apr;12(2):225-34. doi: 10.1111/j.1067-1927.2004.012115.x.
Currently, the use of cultured epithelial autografts as an alternative to split-thickness skin autografts for coverage of full-thickness wounds is limited due to fragility of the sheet and variability in the outcome of healing. This could be circumvented by the transfer of proliferating keratinocytes, instead of differentiated sheets, to the wound bed and the "in vivo" regeneration of epidermis. The aim of this study was to achieve re-epithelialization on experimental full-thickness wounds in the pig using a porous, synthetic carrier seeded with proliferating keratinocytes. Porcine keratinocytes were isolated by enzymatic digestion and cultured in Optimem basal medium with mitogens. In a full-thickness wound model, carriers with different seeding densities were transplanted upside down onto the wound bed. Keratinocytes were labeled using a fluorescent red membrane marker, PKH-26 GL. Transfer of keratinocytes and re-epithelialization were recorded macroscopically and histologically. On day 4 after transplantation, transfer of fluorescently labeled keratinocytes was shown by their presence in the granulation tissue. An immature epidermis, as well as epithelial cords and islands, formed as early as day 8. At day 12 a stratified epidermis and wound closure were established and epithelial cysts were formed by differentiation of epithelial islands. Wounds treated with seeding densities as low as 50,000 cells/cm(2) showed wound closure within 12 days, whereas wounds treated with 10,000 cells/cm(2) or the nonseeded (acellular) carriers did not show complete re-epithelialization before day 17 after treatment. This study showed that porcine keratinocytes, transplanted "upside down" in experimental full-thickness wounds using a synthetic carrier, continued to proliferate and started to differentiate, enabling the formation of a new epidermis in a time frame of 12 days.
目前，由于培养的上皮自体移植片的脆弱性以及愈合结果的变异性，其作为全层伤口覆盖的替代物用于替代断层皮片自体移植的应用受到限制。通过将增殖的角质形成细胞而非分化的移植片转移至伤口床并在“体内”再生表皮，可以规避这一问题。本研究的目的是使用接种了增殖角质形成细胞的多孔合成载体，在猪的实验性全层伤口上实现再上皮化。通过酶消化分离猪角质形成细胞，并在含有促细胞分裂剂的Opti-MEM基础培养基中培养。在全层伤口模型中，将具有不同接种密度的载体倒置移植到伤口床上。使用荧光红色膜标记物PKH-26 GL标记角质形成细胞。通过宏观和组织学方法记录角质形成细胞的转移和再上皮化情况。移植后第4天，在肉芽组织中发现荧光标记的角质形成细胞，表明角质形成细胞已发生转移。早在第8天就形成了未成熟的表皮以及上皮索和上皮岛。在第12天，建立了分层表皮并实现伤口闭合，上皮岛分化形成上皮囊肿。接种密度低至50,000个细胞/cm²的伤口在12天内实现伤口闭合，而接种密度为10,000个细胞/cm²的伤口或未接种（无细胞）载体处理的伤口在处理后第17天之前未显示完全再上皮化。本研究表明，使用合成载体在实验性全层伤口中“倒置”移植的猪角质形成细胞能够持续增殖并开始分化，从而在12天的时间内形成新的表皮。
