In clinical practice, plastic surgeons are often faced with large skin defects that are difficult to close primarily. Management of large skin wounds e.g. burns or traumatic lacerations requires knowledge of skin biomechanic properties. Research into skin microstructural adaptation to mechanical deformation has only been performed using static regimes due to technical limitations. Here, we combine uniaxial stretch tests with fast second harmonic generation imaging and we apply this for the first time to investigate dynamic collagen rearrangement in reticular human dermis. Ex vivo human skin from the abdomen and upper thigh was simultaneously uniaxially stretched while either periodically visualizing 3D reorganization, or visualizing 2D changes in real time. We determined collagen alignment via orientation indices and found pronounced variability across samples. Comparing mean orientation indices at the different stages of the stress strain curves (toe, heel, linear) showed a significant increase in collagen alignment during the linear part of the mechanical response. We conclude that fast SHG imaging during uni-axial extension is a promising research tool for future studies on skin biomechanic properties.

Osteogenesis imperfecta (OI) and Ehlers–Danlos syndrome (EDS) are inherited connective tissue disorders caused by diverse genetic defects, many of which affect collagen biosynthesis. However, the identified genetic variants do not always fully explain the clinical heterogeneity observed in patients, highlighting the need for advanced models and imaging techniques to assess collagen structure and fibroblast behavior at the microscopic level. In this study, we employed 5-week three-dimensional (3D) dermal fibroblast cultures derived from patients with haploinsufficient (HI) and dominant-negative (DN) OI, EDS, and healthy controls. Using label-free higher harmonic generation microscopy (HHGM), we visualized and quantified secreted collagen fibers and fibroblast morphology in situ. We analyzed fibroblast 3D orientation, collagen fiber diameter, collagen amount per cell, and the spatial alignment between fibroblasts and collagen fibers. HI OI fibroblasts secreted significantly less collagen than both control and EDS-derived cells, while EDS samples exhibited thinner collagen fibers compared to controls. Across all groups, collagen fiber orientation was strongly correlated with fibroblast alignment, in line with the role of fibroblasts in matrix organization. In healthy controls and HI OI samples, we observed a depth-dependent, counterclockwise rotation in fibroblast orientation from the culture bottom to the surface—a pattern that was less prominent in DN OI and EDS samples, potentially reflecting altered matrix guidance in diseased tissues. Overall, the quantity and quality of collagen, as well as fibroblast morphology and organization, were markedly altered in the OI and EDS model systems. These alterations may mirror tissue-level manifestations of the diseases, demonstrating the physiological relevance of patient-derived 3D fibroblast models for OI and EDS, as well as the power of harmonic generation microscopy in probing the cellular and extracellular consequences of disease-related gene defects in collagen or its biosynthetic pathways. Extensions of this methodological approach provide a way towards deeper understanding of tissue-level manifestations of collagen dysregulation in connective tissue disorders.

SPIE Digital Library Proceedings

The inherited bone disease Osteogenesis Imperfecta (OI) caused by genetic mutation can lead to brittle bones that fracture easily and serious skeletal deformities due to collagen defects. To understand this disease, in vitro models of 2D monolayer fibroblast cultures obtained from subdermal skin biopsies were developed by worldwide research groups, and nowadays were extended to 3D models to further mimic this disease. The models allow for studying of the collagen formation and possible abnormalities in extracellular matrix (ECM) and will enable studies of the mechanisms underlying defects in the quality and quantity of collagen fibres in OI skin fibroblasts when compared to the healthy cells. The most common methods for measuring the components in fibroblasts cultures are collagen immunostaining, which is time consuming and prohibits time-lapse measurements, or second harmonic generation microscopy (SHG) which only enables visualization of collagen fibres but not fibroblasts. Here by our label-free higher harmonic generation microscopy with 1050nm / 1070nm laser, we have extended SHG to with third harmonic generation microscopy (THG) to visualize fibroblasts cell morphology and their nuclei, also with multiphoton autofluorescence (MAF) signal from the cytoplasm of the fibroblasts. Our techniques allow for the visualisation of the 3D cultures in real-time, resulting in the possibility of making time-lapse videos. For both OI patient and control fibroblast cultures, fibroblasts and their nuclei were clearly visualised by forward THG signal; the collagen fibres by the forward SHG channel, and the cytoplasm signal with the backward autofluorescence channel. First results on the comparison between cultures of OI patients and controls reveal differences in fibroblasts orientation, collagen orientation and density.