 |
|
Modeling Aberrant Wound Healing Using Tissue-Engineered Skin Constructs and Multiphoton Microscopy
Behrooz A. Torkian, MD;
Alvin T. Yeh, PhD;
Rodney Engel, BA;
Chung-Ho Sun, PhD;
Bruce J. Tromberg, PhD;
Brian J. F. Wong, MD, PhD
Arch Facial Plast Surg. 2004;6:180-187.
Background Keloids and hypertrophic scars result from aberrant wound healing and remain a potential complication of any surgical procedure or trauma. Investigation of aberrant wound healing has been limited to the study of growth factors, collagen precursors, and DNA synthesis in simple in vitro systems, which necessitate removal or destruction of cells or factors in the growth environment of cell cultures. Multiphoton microscopy (MPM) can use endogenous chromophores such as collagen and nicotinamide adenine dinucleotide hydrogenase to produce thin optical sections of thick living tissues without the use of dyes or stains. Endogenous second-harmonic-generation (SHG) signals in collagen can be collected to form an MPM image.
Objective To present a novel wound-healing model used to investigate keloid-derived fibroblast activity and collagen production in the same intact tissue-engineered construct over time.
Methods Artificial tissue constructs called RAFTs (produced by suspension of keloid or normal dermal fibroblasts in type I collagen gel with an overlying keratinocyte layer) were cultured at air-fluid interface. Multiphoton microscopy SHG images of collagen in the intact tissue constructs consisting of normal or keloid-derived fibroblasts were obtained. The constructs were then incised with a scalpel. Serial MPM and phase-contrast microscopy images were obtained to monitor changes in the extracellular matrix in response to wounding of the artificial skin construct over 8 days.
Results The tissue-engineered constructs formed a bilayer resembling the dermis and epidermis of human skin. Phase-contrast microscopy revealed migration of keratinocytes into the defect created by scalpel wounding. The constructs were found to contract with time after wounding. The MPM SHG images showed collagen deposition in the tissue constructs after wounding. Tissue constructs with keloid-derived fibroblasts were found to deposit collagen at a higher rate than those with normal fibroblasts.
Conclusions The MPM model described herein permits serial observation of the same intact specimens without the need for fixation or cytotoxic stains. Furthermore, it demonstrates the biologic activity of RAFT artificial tissue constructs.
From Departments of Otolaryngology–Head and Neck Surgery (Drs Torkian and Wong) and Biomedical Engineering (Drs Tromberg and Wong), University of California, Irvine Medical Center, Orange; Beckman Laser Institute, University of California, Irvine (Drs Yeh, Sun, Tromberg, and Wong and Mr Engel). Dr Yeh is currently at the Department of Biomedical Engineering, Texas A&M University, College Station. Mr Engel is currently a medical student at Albert Einstein School of Medicine, Yeshiva University, Bronx, NY.
THIS ARTICLE HAS BEEN CITED BY OTHER ARTICLES
Noninvasive Assessment of Collagen Gel Microstructure and Mechanics Using Multiphoton Microscopy
Raub et al.
Biophys. J 2007;92:2212-2222.
ABSTRACT
| FULL TEXT
|
