206 Nanomechanical properties of collagen VII anchoring fibrils in recessive dystrophic epidermolysis bullosa patients

Our aim was to apply novel nanomechanical tests to investigate the biophysical properties of collagen VII anchoring fibrils in skin sections in a healthy control and in two recessive dystrophic epidermolysis bullosa (RDEB) patients. One patient with localized RDEB was a compound heterozygote with a consequent R1957W change within the hinge region and a premature termination (Y2819*) within the acidic region of collagen VII a1 chain. The other was diagnosed with generalized severe RDEB, was homozygous and had truncated collagen VII a1 chains in the NC-1 domain (K142R). Sections of skin biopsies from patient and control were investigated with atomic force microscopy (AFM) combined with fluorescence imaging. In the localized RDEB patient and healthy control, the anchoring fibrils were first immunostained, identified and scanned using AFM. In the generalized severe RDEB patient (no collagen VII staining), the dermal surface of the basement membrane zone (BMZ) adjacent to bulla formation was scanned. Nanoindentation was used to obtain force-volume maps and to characterize local viscoelasticity. Force-displacement curves were fitted with the Hertz model to calculate the local Young modulus. The control skin specimen could be partitioned into high-, intermediate- and low-Young-modulus areas that correspond to the dermis, BMZ and the epidermis, respectively. Remarkably, the dermis was stiffer than the epidermis by an order of magnitude. The region with intermediate Young modulus could not be clearly identified in the RDEB samples. The healthy skin may thus be seen as a highly compliant epidermis woven onto a stiff dermis. Hence, collagen VII plays an important role in coupling the mechanically distinct epidermis and dermis. The applied biophysical measurements enabled us to obtain clinically relevant data and may be used in the future to evaluate protein or gene therapies in RDEB patients.