皮膚是對抗外來病原的第一道防線，若損傷則非常容易受到感染，因此促進傷口癒合的研究受到高度重視，也有多種促進傷口癒合材料在市場販售。利用組織工程及細胞治療已被用於促進傷口癒合，並且顯示較傳統敷料更佳的效果。本研究建立含有脂肪幹細胞類球體的膠原蛋白凝膠敷料，並測試是否能幫助傷口更快速的癒合。首先觀察脂肪幹細胞與小腸上皮細胞株C2BBe1共培養的結果，發現脂肪幹細胞能形成似微血管的管狀網絡結構。而後我們利用C2BBe1細胞層刮除一道無細胞區域，並使用不同處理模擬傷口癒合，結果顯示加入培養過脂肪幹細胞的條件培液能促使傷口癒合。接著以懸滴培養法製作脂肪細胞類球體，發現經三維培養細胞生長速率提升，並且其條件培養液能降低巨噬細胞發炎因子IL-1β的分泌。後續透過裸鼠皮膚層切除之傷口癒合模式，製造直徑為8 mm的圓形傷口，再以吻合傷口大小的含脂肪幹細胞類球體的膠原蛋白凝膠敷料覆蓋於傷口處，並與其他組比對，發現處理組的傷口具有較佳的皮膚癒合速率。組織切片結果顯示發炎性細胞的浸潤降低，傷口癒合過程中發炎時期也縮短。綜合本研究結果，此脂肪幹細胞類球體的膠原蛋白凝膠敷料對於皮膚傷口是有助益的。製備該敷料簡易，使用也便利，是促進傷口癒合的另一替代方案。

Skin is the first line of barrier to protect the body from pathogens. Patients suffering from burns, diabetes wounds and chronic skin wounds are susceptible to infections. Tissue engineering- based cell therapies have been used to promote wound healing and demonstrated to be more effective than conventional wound dressing. In this study, we examined whether adipose-derived stem cell (ADSC) spheroids, a simple three-dimensional (3D) substitute, can accelerate wound healing. First, we found ADSC co-cultured with the human intestinal epithelial C2BBe1 cells formed tube-like structure. We then investigated whether ADSC could promote cell migration using the scratch wound healing assay on monolayers of C2BBe1 cells. The result showed that wound healing was significantly enhanced after treatment with conditioned medium from ADSC. In addition, we found that ADSC-spheroids formation by hanging drop had higher cells growth rate, and ADSC conditioned medium reduced IL-1β level during inflammation. This study was followed by using the cutaneous excisional nude mice model. We covered the 8 mm-diameter wound on mice with a dressing consisting of ADSC-spheroids embedded in a collagen gel. The ADSC- spheroids collagen dressing treated-group displayed a significant faster healing rate after one week than its untreated counterpart. Data from histology sections of the wounds suggest that the quicker healing rates by ADSC-spheroids embedded collagen dressing treatment may be due to the reduction of inflammation in the wound healing process. The establishment of the ADSC-spheroids collagen dressing is beneficial to patients with skin wounds. The dressing is also simple to prepare and is convenient to be used in patients. Therefore, ADSC-spheroids collagen dressing may provide a clinical alternative to promote wound healing.

1. Clark, R.A., K. Ghosh, and M.G. Tonnesen, Tissue engineering for cutaneous wounds. J Invest Dermatol, 2007. 127(5): p. 1018-29.
2. Madison, K.C., Barrier function of the skin: "la raison d'etre" of the epidermis. J Invest Dermatol, 2003. 121(2): p. 231-41.
3. Sharma, R., et al., Epidermal-like architecture obtained from equine keratinocytes in three-dimensional cultures. J Tissue Eng Regen Med, 2013.
4. Brohem, C.A., et al., Artificial skin in perspective: concepts and applications. Pigment Cell Melanoma Res, 2011. 24(1): p. 35-50.
5. Briggaman, R.A. and C.E. Wheeler, Jr., The epidermal-dermal junction. J Invest Dermatol, 1975. 65(1): p. 71-84.
6. Presland, R.B. and B.A. Dale, Epithelial structural proteins of the skin and oral cavity: function in health and disease. Crit Rev Oral Biol Med, 2000. 11(4): p. 383-408.
7. Markeson, D., et al., Scarring, stem cells, scaffolds and skin repair. J Tissue Eng Regen Med, 2015. 9(6): p. 649-68.
8. You, H.J. and S.K. Han, Cell therapy for wound healing. 2014. 29(3): p. 311-9.
9. Zahorec, P., et al., Mesenchymal stem cells for chronic wounds therapy. Cell Tissue Bank, 2015. 16(1): p. 19-26.
10. YU-WEN, Y., Epidemiological Characteristics and Medical Utilization Profiles of Patients Hospitalzed of Burn injury in Taiwan,2000-2010. 2014, National Defense Medical Institute of Public Health
11. Lee, S.H., J.H. Lee, and K.H. Cho, Effects of Human Adipose-derived Stem Cells on Cutaneous Wound Healing in Nude Mice. Ann Dermatol, 2011. 23(2): p. 150-5.
12. Jones, J.E., E.A. Nelson, and A. Al-Hity, Skin grafting for venous leg ulcers. Cochrane Database Syst Rev, 2013. 1: p. Cd001737.
13. Enshaei, A. and N. Masoudi, Survey of early complications of primary skin graft and secondary skin graft (delayed) surgery after resection of burn waste in hospitalized burn patients. Glob J Health Sci, 2014. 6(7 Spec No): p. 98-102.
14. Cordeiro, J.V. and A. Jacinto, The role of transcription-independent damage signals in the initiation of epithelial wound healing. Nat Rev Mol Cell Biol, 2013. 14(4): p. 249-62.
15. O’Loughlin, A. and T. O’Brien, Topical Stem and Progenitor Cell Therapy for Diabetic Foot Ulcers. Stem Cells in Clinic and Research. 2011.
16. Arnoux, V., et al., Cutaneous Wound Reepithelialization, in Rise and Fall of Epithelial Phenotype. 2005, Springer US. p. 111-134.
17. Hong, S.J., et al., Topically delivered adipose derived stem cells show an activated-fibroblast phenotype and enhance granulation tissue formation in skin wounds. PLoS One, 2013. 8(1): p. e55640.
18. Lee, J.C., S. Kandula, and N.S. Sherber, Beyond wet-to-dry: a rational approach to treating chronic wounds. Eplasty, 2009. 9: p. e14.
19. Halim, A.S., T.L. Khoo, and S.J. Mohd Yussof, Biologic and synthetic skin substitutes: An overview. Indian J Plast Surg, 2010. 43(Suppl): p. S23-8.
20. Sarabahi, S., Recent advances in topical wound care. Indian J Plast Surg, 2012. 45(2): p. 379-87.
21. Vindenes, H., [Skin transplantation]. Tidsskr Nor Laegeforen, 1999. 119(27): p. 4050-3.
22. Lin, S.D., et al., Microskin autograft with pigskin xenograft overlay: a preliminary report of studies on patients. Burns, 1992. 18(4): p. 321-5.
23. Hasegawa, T., et al., Keratinocyte progenitor cells reside in human subcutaneous adipose tissue. PLoS One, 2015. 10(2): p. e0118402.
24. Potten, C.S. and R.J. Morris, Epithelial stem cells in vivo. J Cell Sci Suppl, 1988. 10: p. 45-62.
25. Grossman, Z., The stem cell concept revisited: self-renewal capacity is a dynamic property of hemopoietic cells. Leuk Res, 1986. 10(8): p. 937-50.
26. Rodrigues, M., et al., Progenitor Cell Dysfunctions Underlie Some Diabetic Complications. Am J Pathol, 2015.
27. Yu, J., et al., Stemness and transdifferentiation of adipose-derived stem cells using L-ascorbic acid 2-phosphate-induced cell sheet formation. Biomaterials, 2014. 35(11): p. 3516-26.
28. Hassan, W.U., U. Greiser, and W. Wang, Role of adipose-derived stem cells in wound healing. Wound Repair Regen, 2014. 22(3): p. 313-25.
29. Goodell, M.A., H. Nguyen, and N. Shroyer, Somatic stem cell heterogeneity: diversity in the blood, skin and intestinal stem cell compartments. Nat Rev Mol Cell Biol, 2015. 16(5): p. 299-309.
30. van der Veen, V.C., et al., Stem cells in burn eschar. Cell Transplant, 2012. 21(5): p. 933-42.
31. Chavez-Munoz, C., et al., Transdifferentiation of adipose-derived stem cells into keratinocyte-like cells: engineering a stratified epidermis. PLoS One, 2013. 8(12): p. e80587.
32. Gimble, J. and F. Guilak, Adipose-derived adult stem cells: isolation, characterization, and differentiation potential. Cytotherapy, 2003. 5(5): p. 362-9.
33. Ong, W.K. and S. Sugii, Adipose-derived stem cells: fatty potentials for therapy. Int J Biochem Cell Biol, 2013. 45(6): p. 1083-6.
34. Uysal, C.A., et al., The Effect of Bone-Marrow-Derived Stem Cells and Adipose-Derived Stem Cells on Wound Contraction and Epithelization. Adv Wound Care (New Rochelle), 2014. 3(6): p. 405-413.
35. Brett, D., A Review of Collagen and Collagen-based Wound Dressings. Wounds, 2008. 20(12): p. 347-56.
36. Li, H., et al., Effects of multiple agents on epithelial differentiation of rabbit adipose-derived stem cells in 3D culture. Tissue Eng Part A, 2012. 18(17-18): p. 1760-70.
37. Janeczek Portalska, K., et al., Endothelial differentiation of mesenchymal stromal cells. PLoS One, 2012. 7(10): p. e46842.
38. Deveza, L., et al., Paracrine release from nonviral engineered adipose-derived stem cells promotes endothelial cell survival and migration in vitro. Stem Cells Dev, 2013. 22(3): p. 483-91.
39. Du, Y., et al., Adipose-derived stem cells differentiate to keratocytes in vitro. Mol Vis, 2010. 16: p. 2680-9.
40. Wang, C.C., et al., Spherically symmetric mesenchymal stromal cell bodies inherent with endogenous extracellular matrices for cellular cardiomyoplasty. Stem Cells, 2009. 27(3): p. 724-32.
41. Abbott, R.D. and D.L. Kaplan, Strategies for improving the physiological relevance of human engineered tissues. Trends Biotechnol, 2015. 33(7): p. 401-7.
42. Lin, R.Z. and H.Y. Chang, Recent advances in three-dimensional multicellular spheroid culture for biomedical research. Biotechnol J, 2008. 3(9-10): p. 1172-84.
43. Hsu, S.H., et al., Substrate-dependent modulation of 3D spheroid morphology self-assembled in mesenchymal stem cell-endothelial progenitor cell coculture. Biomaterials, 2014. 35(26): p. 7295-307.
44. Zhang, S., et al., The effects of spheroid formation of adipose-derived stem cells in a microgravity bioreactor on stemness properties and therapeutic potential. Biomaterials, 2015. 41: p. 15-25.
45. Liu, B.H., et al., Spheroid formation and enhanced cardiomyogenic potential of adipose-derived stem cells grown on chitosan. Biores Open Access, 2013. 2(1): p. 28-39.
46. Bartosh, T.J., et al., Aggregation of human mesenchymal stromal cells (MSCs) into 3D spheroids enhances their antiinflammatory properties. Proc Natl Acad Sci U S A, 2010. 107(31): p. 13724-9.
47. Chung, H.M., C.H. Won, and J.H. Sung, Responses of adipose-derived stem cells during hypoxia: enhanced skin-regenerative potential. Expert Opin Biol Ther, 2009. 9(12): p. 1499-508.
48. Lee, E.Y., et al., Hypoxia-enhanced wound-healing function of adipose-derived stem cells: increase in stem cell proliferation and up-regulation of VEGF and bFGF. Wound Repair Regen, 2009. 17(4): p. 540-7.
49. Kang, S., S.M. Kim, and J.H. Sung, Cellular and molecular stimulation of adipose-derived stem cells under hypoxia. Cell Biol Int, 2014. 38(5): p. 553-62.
50. Rehman, J., et al., Secretion of angiogenic and antiapoptotic factors by human adipose stromal cells. Circulation, 2004. 109(10): p. 1292-8.
51. Grayson, W.L., et al., Hypoxia enhances proliferation and tissue formation of human mesenchymal stem cells. Biochem Biophys Res Commun, 2007. 358(3): p. 948-53.
52. Kim, W.S., B.S. Park, and J.H. Sung, The wound-healing and antioxidant effects of adipose-derived stem cells. Expert Opin Biol Ther, 2009. 9(7): p. 879-87.
53. Buravkova, L.B., et al., [Characteristics of human lipoaspirate-isolated mesenchymal stromal cells cultivated under a lower oxygen tension]. Tsitologiia, 2009. 51(1): p. 5-11.
54. Rubina, K., et al., Adipose stromal cells stimulate angiogenesis via promoting progenitor cell differentiation, secretion of angiogenic factors, and enhancing vessel maturation. Tissue Eng Part A, 2009. 15(8): p. 2039-50.
55. Gehmert, S., et al., Angiogenesis: the role of PDGF-BB on adipose-tissue derived stem cells (ASCs). Clin Hemorheol Microcirc, 2011. 48(1): p. 5-13.
56. Yang, E.K., et al., Tissue engineered artificial skin composed of dermis and epidermis. Artif Organs, 2000. 24(1): p. 7-17.
57. Hsu, S.H. and P.S. Hsieh, Self-assembled adult adipose-derived stem cell spheroids combined with biomaterials promote wound healing in a rat skin repair model. Wound Repair Regen, 2015. 23(1): p. 57-64.
58. Dyson, M., et al., Comparison of the effects of moist and dry conditions on dermal repair. J Invest Dermatol, 1988. 91(5): p. 434-9.
59. Hinman, C.D. and H. Maibach, EFFECT OF AIR EXPOSURE AND OCCLUSION ON EXPERIMENTAL HUMAN SKIN WOUNDS. Nature, 1963. 200: p. 377-8.
60. Junker, J.P., et al., Clinical Impact Upon Wound Healing and Inflammation in Moist, Wet, and Dry Environments. Adv Wound Care (New Rochelle), 2013. 2(7): p. 348-356.
61. Liang, C.C., A.Y. Park, and J.L. Guan, In vitro scratch assay: a convenient and inexpensive method for analysis of cell migration in vitro. Nat Protoc, 2007. 2(2): p. 329-33.
62. Gao, S., et al., Mouse bone marrow-derived mesenchymal stem cells induce macrophage M2 polarization through the nuclear factor-kappaB and signal transducer and activator of transcription 3 pathways. Exp Biol Med (Maywood), 2014. 239(3): p. 366-75.
63. Xue, J., et al., Gene-modified mesenchymal stem cells protect against radiation-induced lung injury. Mol Ther, 2013. 21(2): p. 456-65.
64. Wang, X., et al., The mouse excisional wound splinting model, including applications for stem cell transplantation. Nat Protoc, 2013. 8(2): p. 302-9.
65. Cheng, N.C., et al., Short-term spheroid formation enhances the regenerative capacity of adipose-derived stem cells by promoting stemness, angiogenesis, and chemotaxis. Stem Cells Transl Med, 2013. 2(8): p. 584-94.
66. Jung, H., et al., Transforming growth factor-beta 1 in adipose derived stem cells conditioned medium is a dominant paracrine mediator determines hyaluronic acid and collagen expression profile. Cytotechnology, 2011. 63(1): p. 57-66.
67. Shang, Q., et al., Delivery of Adipose-derived Stem Cells Attenuates Adipose Tissue Inflammation and Insulin Resistance in Obese Mice through Remodeling Macrophage Phenotypes. Stem Cells Dev, 2015.
68. Carvalho, J.L., et al., Innovative Strategies for Tissue Engineering. Advances in Biomaterials Science and Biomedical Applications. 2013.
69. Imamura, M., [Extracellular matrix components and angiogenesis]. Nihon Yakurigaku Zasshi, 1996. 107(3): p. 153-60.
70. Grant, D.S., et al., Angiogenesis as a component of epithelial-mesenchymal interactions. Exs, 1995. 74: p. 235-48.
71. Rankin, C.R., et al., Annexin A2 regulates beta1 integrin internalization and intestinal epithelial cell migration. J Biol Chem, 2013. 288(21): p. 15229-39.
72. Kim, W.S., B.S. Park, and J.H. Sung, Protective role of adipose-derived stem cells and their soluble factors in photoaging. Arch Dermatol Res, 2009. 301(5): p. 329-36.
73. Bhang, S.H., et al., Efficacious and clinically relevant conditioned medium of human adipose-derived stem cells for therapeutic angiogenesis. Mol Ther, 2014. 22(4): p. 862-72.
74. Skiles, M.L., et al., Use of culture geometry to control hypoxia-induced vascular endothelial growth factor secretion from adipose-derived stem cells: optimizing a cell-based approach to drive vascular growth. Tissue Eng Part A, 2013. 19(21-22): p. 2330-8.
75. Stolarska, M.A., Y. Kim, and H.G. Othmer, Multi-scale models of cell and tissue dynamics. Philos Trans A Math Phys Eng Sci, 2009. 367(1902): p. 3525-53.
76. Freyer, J.P. and R.M. Sutherland, Proliferative and clonogenic heterogeneity of cells from EMT6/Ro multicellular spheroids induced by the glucose and oxygen supply. Cancer Res, 1986. 46(7): p. 3513-20.
77. Pasparakis, M., I. Haase, and F.O. Nestle, Mechanisms regulating skin immunity and inflammation. Nat Rev Immunol, 2014. 14(5): p. 289-301.
78. Thiel, D.J., et al., Observation of an unexpected third receptor molecule in the crystal structure of human interferon-gamma receptor complex. Structure, 2000. 8(9): p. 927-36.
79. Dunn, L., et al., Murine model of wound healing. J Vis Exp, 2013(75): p. e50265.
80. Seo, G., et al., Adipose-derived stem cell conditioned medium accelerates keratinocyte differentiation via the up-regulation of miR-24. Exp Dermatol, 2015.
81. Kuo, Y.R., et al., Adipose-derived Stem Cells Accelerate Diabetic Wound Healing through the Induction of Autocrine and Paracrine Effects. Cell Transplant, 2015.
82. Hu, R., et al., Fibroblast-like cells differentiated from adipose-derived mesenchymal stem cells for vocal fold wound healing. PLoS One, 2014. 9(3): p. e92676.
83. Lin, Y.H., et al., The effects of microenvironment on wound healing by keratinocytes derived from mesenchymal stem cells. Ann Plast Surg, 2013. 71 Suppl 1: p. S67-74.
84. Uysal, A.C. and H. Mizuno, Tendon regeneration and repair with adipose derived stem cells. Curr Stem Cell Res Ther, 2010. 5(2): p. 161-7.
85. Li, H., et al., Epithelial-differentiated adipose-derived stem cells seeded bladder acellular matrix grafts for urethral reconstruction: an animal model. Tissue Eng Part A, 2014. 20(3-4): p. 774-84.
86. Varghese, M.C., et al., Local environment of chronic wounds under synthetic dressings. Arch Dermatol, 1986. 122(1): p. 52-7.
87. Alvarez, O.M., P.M. Mertz, and W.H. Eaglstein, The effect of occlusive dressings on collagen synthesis and re-epithelialization in superficial wounds. J Surg Res, 1983. 35(2): p. 142-8.
88. Rubio, P.A., Use of semiocclusive, transparent film dressings for surgical wound protection: experience in 3637 cases. Int Surg, 1991. 76(4): p. 253-4.
89. Moon CH, C.T., New wound dressing techniques to accelerate healing. Curr Treat Options Infect Dis. 2003;5:251–60., 2003.