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.

Objective

To characterize tissue destruction after CO(2) laser-ablation of the vocal cords with the use of optical coherence tomography (OCT).

Study design and setting

OCT was used to image fresh porcine vocal cords after laser ablation. OCT and histology estimates of the ablation crater dimensions and the depth of thermal injury were obtained.

Results

The vocal cord substructures up to 2.29 mm in depth at 10 microm resolution, and the thermal disruption after laser ablation were identified by OCT. OCT and histology estimates of the lesion dimensions showed no significant differences. Crater depth is directly proportional to laser power, whereas crater width and the zone of thermal injury appear to be unrelated to laser power.

Conclusions

OCT may be used to accurately characterize the native states and the laser-induced thermal injury of laryngeal mucosa, within the inherent limitation in its depth of penetration. OCT may be a useful diagnostic and monitoring tool in an otolaryngology practice.

Wound healing is a physiologic process that acts to repair disruptions in the continuity of tissue caused by injury or surgical incision. Keloids and hypertrophic scars are forms of aberrant wound healing, which are characterized by the overproduction of collagen, resulting in an excessive amount of scar tissue. Keloid tumors, by definition, grow outside the boundary of the original tissue damage. Multiphoton microscopy (MPM) is an imaging technique which allows imaging of living specimens, without the use of fixation or stains. Images of collagen fibers are produced by the second harmonic signal intensity generated by endogenous fluorescence through excitation by infrared laser light. A postauricular keloid tumor was excised from a patient. The tissue was dissected, and a portion was imaged using MPM. Normal skin tissue was isolated from a patient undergoing a facelift. A portion of this tissue was also dissected and imaged using MPM. MPM images were taken using a 63X water immersion objective lens on a two-photon microscope and a titanium-sapphire laser. Images were taken beginning at the surface of the tissue and moving in at intervals of 200 nm to a final depth of 30 μm. The two-photon images were used to reconstruct three-dimensional representations of the collagen matrix within the tissues, which are readily contrasted. Density of the collagen within each tissue was also ascertained using depth dependant decay of the image intensity. Multiphoton imaging was successfully used to image the collagen matrix of normal skin and a keloid scar, demonstrating differences in their microstructures.