預先皮膚擴張於豬模式中用以準備自體脂肪移植之潛能

Abstract

自體脂肪移植 (autologous fat grafting, AFG) 為採集自身的脂肪組織進行注射，目前已經廣泛用於整形外科中之乳房重建；然而，AFG之手術成果通常被視為不可預測，因長期存留容積通常在20%和90%之間變化，其原因一般認為是移植後的早期階段缺乏血液供應所導致的結果。外部體積擴張 (external volume expansion, EVE) 的負壓處理，長期以來被認為具有預先處理受體部位之潛能。此外，機械力已被證實對於間葉幹細胞增殖和分化具有關鍵的作用。因此，為了探討EVE之體內機械力作用對於皮下脂肪中的細胞增殖，血管生長和成熟以及脂肪幹細胞 (adipose stem cells, ASCs) 的細胞標記，增殖能力和分化潛能的影響，利用豬的皮膚和皮下組織與人類之間在解剖和生理上相似性設計為動物模式。 本研究使用負壓50毫米汞柱，在每天相同8小時 (上午9點到下午5點) 的時間間隔連續作用於豬隻側腹表皮直到10或21天。在第11或22天進行採樣之前，使用超音波測量軟組織厚度，結果顯示EVE誘導軟組織擴增是屬於一種暫時性效應。接著將試驗組和對照組所取得之樣本進行各項分析。H&E染色 (hematoxylin and eosin staining) 結果顯示，EVE可以促進血管重塑的過程，但是對脂肪細胞的大小和數量並沒有顯著影響。Ki67免疫組織化學染色的結果顯示，基底角質細胞和脂肪細胞之細胞增殖相較於對照組沒有出現顯著差異；相反地，通過α-SMA染色得到證實，具有平滑肌細胞的血管網絡在EVE處理組中有明顯增加。另外，使用image J測量表皮厚度的結果顯示各組之間並沒有觀察到顯著的差異。CD31免疫螢光染色結果顯示，血管密度會隨著EVE的作用時間而逐漸增加。 基質血管細胞 (stromal vascular fraction, SVF) 和ASCs分別從脂肪組織中分離和純化。ASCs的增殖能力透過倍增時間和集落形成分析進行檢定，結果卻顯示對照組和EVE處理組之間沒有發現統計上的差異。ASCs進行脂肪分化誘導21天，隨後進行油紅染色 (Oil-Red O staining)，結果ASCs的脂肪分化潛能在各組之中也沒有顯著差異。流式細胞術分析結果顯示，無論處理時間長短，ASCs皆會表現諸如CD29, CD44, CD90與CD105等多種間葉細胞標記，惟缺乏表現造血細胞標記如CD34之能力。多色流式細胞儀分析SVF細胞組成，結果顯示各組之間ASCs的比例沒有顯著差異；相較之下，在EVE處理組中的內皮細胞，其百分比，顯著高於對照組者，且亦會隨著其作用時間之延長而顯著增加。 綜合上述，EVE之主要作用機制，在於調節新血管網絡之形成、生長和功能化血管的成熟。EVE的預先處理效果已經在李宋豬模式中得到證實，此結果可以輕易地轉化為臨床實踐，以提高細胞和組織移植存活率。期望本研究成果可以幫助臨床醫生在手術前優化受體組織的血管分布，以進一步改善脂肪移植的存留容積。

Autologous fat grafting (AFG) has been widely used as an injectable substance for breast reconstruction in cosmetic surgery; however, the clinical outcome of AFG is generally considered an unpredictable procedure, with long-term retention commonly varying between 20 and 90%, which is believed to be caused by poor blood supply in the early after grafting. Negative pressure using external volume expansion (EVE) has long been theorized as a potential means to precondition the recipient bed. In addition, the mechanical force has been confirmed to play a pivotal role in mesenchymal stem cells proliferation and differentiation. Accordingly, to investigate the effects of in vivo mechanical loading of EVE on cell proliferation, vascular growth and subsequent maturation as well as cellular markers, proliferation capacity and differentiation potential of adipose stem cells (ASCs) in subcutaneous fat, a swine model was devised to take advantage of anatomical and physiological similarities in skin and subcutaneous tissue between pigs and human. In this study, pigs were treated with continuous suction at -50 mmHg during the same eight-hour (9:00-17:00) interval each day until 10 or 21 days. Before sampling on day-11 or 22, an ultrasonography was performed to study the soft tissue thickness and results revealed that EVE-induced soft tissue enlargement is a transient effect. Specimens from control and treated groups conducted a various analysis. The result of H&E staining showed that EVE can enhance the process of vascular remodeling but has no significant effect on adipocytes size and numbers. IHC stain with Ki67 showed cell proliferation in basal keratinocytes and adipocytes did not appear significant difference as compared with the non-treated group; in contrast, vascular networks layered with smooth muscle cells increased in EVE treated groups as evident by the α-SMA staining. On the other hand, the epidermal thickness was measured by image J but no significant difference was observed across the groups. Immunofluorescence stain with CD31 suggested that blood vessel density would gradually increase with the loading time of EVE. Stromal vascular fraction (SVF) cells and ASCs were isolated and purified from fat tissue, respectively. Proliferation capacity of ASCs was measured by doubling time and colony-forming assay but no statistical difference was found between the control and EVE treated groups. ASCs were subjected to adipogenic induction for 21 days followed by Oil-Red O staining and adipogenic differentiation potential of ASCs had no significant difference across the groups. Flow cytometry analysis showed regardless of treatment interval, ASCs expressed mesenchymal markers such as CD29, CD44, CD90, CD105 while lacking expression of hematopoietic marker such as CD34. Multicolor flow cytometric analysis of SVF cells revealed no significant difference in the ratio of ASCs across the groups; in contrast, the percentage of endothelial cells of EVE treated groups significantly increased as treatment lengthened when compared with the control group. In conclusion, the predominant mechanism of action of EVE, which would modulate neovascular network formation, growth and maturation of functional blood vessels. The preconditioning effect of EVE has been demonstrated in the swine model, which may be easily translated into clinical practices to enhance cell and tissue engraftment. It is expected that this understanding may help clinicians to optimize the vascularity of the recipient bed to further improve fat volume retention before the operation.