AFM is a non-destructive alternative for imaging biological tissues under aqueous conditions; however, imaging bulk skin tissue using AFM can be challenging because collagen fibril bundles are surrounded by a sol-gel of hydrophilic GAGs and subcutaneous adipose fat. Recently Graham and coworkers reported a combined tissue cryo-sectioning and AFM imaging method that provides excellent resolution of the ECM components in skin, cartilage, aorta, and lung.(Graham et al., 2010
) The sample preparation greatly facilitates AFM imaging and characterization of biological tissues while in the meantime avoids fixation, chemical staining, and high vacuum.
In order to evaluate the nanomorphology of collagen fibrils present in dermis, we selected the D-spacing as a reliable quantitative marker. We have previously demonstrated that the application of 2-dimensional Fast Fourier Transforms (2D-FFT) allows an accurate evaluation of this prominent fibril feature. The D-spacing arises from a parallel staggered packing of collagen monomers which lead to alternating gap and overlap zones along the longitudinal axis of a fibril, as illustrated by the two-dimensional Hodge-Petruska model.(Hodge and Petruska, 1963
) Recent X-ray crystallographic work by Orgel et al.
provides additional three-dimensional insight which supports a supertwist microfibril model.(Orgel et al., 2001
) These structural models indicate that quantitative analysis of the D-spacing should be sensitive to changes in the collagen molecule triple helix, the molecular packing, and intermolecular cross-linking effects. For example, the single amino acid substitution of a cysteine residue for glycine-349 results in nanoscale morphology changes observed in the collagen fibril D-spacing distribution. Moreover, the free energy changes induced by amino substitution correlate with clinical severity of Osteogenis Imperfecta
.(Lee et al., 2011
Quantitative analysis of ovine dermis collagen D-spacings indicates a distribution of values is present ranging from 56 to 67
nm with a mean value of 62
nm. Although AFM has an excellent ability to differentiate differences in the D-spacing within tissue, the absolute value is limited by the calibration process. The average value of the distribution of 62 nm is close to
previous literature values obtained by X-ray scattering. Purslow reported 67 nm D-spacing in rat skin;(Purslow et al., 1998
) others reported lower values of about 65 nm for skin.(Brodsky et al., 1980
; Gathercole et al., 1987
; Stinson and Sweeny, 1980
) These techniques have spot sizes of microns and thus average over too large an area of the skin structure for observation of a D-spacing distribution. The observation of this distribution in dermal collagen provides further evidence that a distribution of values is an intrinsic aspect of collagen fibrillar structure. A similar distribution has previously been observed for another non-mineralized Type 1 collagen tissue, murine tail tendon, as well as for the mineralized collagen tissues murine dentin and bone and ovine bone.(Wallace et al., 2010a
; Wallace et al., 2010b
; Wallace et al., 2011
) The observation of the distribution is possible because of the fibril by fibril analysis using the AFM data.
The influence of bulk tissue stress on collagen fibril D-spacings has been subject of numerous studies. Gupta et al. (2008)
demonstrated a connection between fibril stain and D-spacing. They noted a 0.3 nm increase in D-spacing in bone as measured by small angle X-ray scattering (SAXS) under mechanical stretching. For bone, fibril strain accounts for only a fraction of the total tissue strain, suggesting that interfibrillar sliding and shear of the proteoglycan-rich matrix takes up the remainder of the tissue strain. With regards to tendon, Puxkandl et al.
(2002) demonstrated up to a 1 nm change when a 3% macroscopic strain was employed and a 0.2 nm change at a 1% strain. D-spacing changes varied between 0.2 and 2 nm at tendon fracture. The most general conclusion from the comparison of this data to the distribution of D-spacings that we report, which has a width of 12 nm, is that materials strain effects on D-spacing are not large enough to explain the D-spacing distribution observed in either mineralized or non-mineralized biological tissues. The strain effects tend to be about an order of magnitude too small.
One limitation of the current study is that we used dorsal skin exposed to ultraviolet (UV) radiation as opposed to skin protected from extrinsic UV radiation. Ovine dermis is considerably thicker than human dermis;(Dellmann and Eurell, 1998
) in addition a layer of wool equivalent of SPF 30 protection also makes it difficult to assess how much photoageing is induced in these dermal tissue samples as compared with human samples.(Fleet, 2006
; Forrest and Fleet, 1986
) However given that the Sham and OVX ovine were provided with the same sheltering condition, the effects observed in this study signify change in the hormonal level rather than differential UV radiation exposure.
Estrogen is known to play important roles in mediating connective tissue physiology and function. Estrogen depletion associated with menopause causes detrimental effects on connective tissues. In skin, estrogen depletion is associated with declining dermal collagen content, skin thickness, water-holding capacity, and skin elasticity. In terms of mechanical properties, a steep increase in skin extensibility was noted in women during perimenopause (Pierard et al., 1995
) and ovariectomized rats exhibit an increased Young's Modulus in the skin.(Ozyazgan et al., 2002
) Reduced estrogen level also impairs the rate and quality of wound healing: in postmenopausal women and in ovariectomized female rodents, a marked delay in wound healing was reported.(Ashcroft et al., 1997
; Calvin et al., 1998
) Hormone replacement therapy was found to partially reverse these effects and topical application of estrogen on wounded skin accelerated wound healing.(Ashcroft et al., 1999
) In addition, Pierard and coworkers noted a positive correlation between bone mineral density and skin viscoelasticity in women.(Pierard et al., 2001
Collagen ultrastructure in ovine bone demonstrated significant change with estrogen depletion, 28 % of fibrils in OVX ovine have D-spacings lower than 64 nm, while sham-operated ovine contained 7% of such fibrils with low D-spacings.(Wallace et al., 2010b
) The results presented here show that similar changes occur in dermal collagen nanomorphology of upon estrogen depletion. Although the percentage of low D-spacing fibrils (less than 59 nm
) is lower in dermis, 14.6%
in OVX group and 1.6%
in Sham group, the result is persistent in all five OVX animals we examined. Bone is a mineralized connective tissue while dermis is only constituted of macromolecular proteins. Thus, the results indicate the changes in collagen nanomorphology results from changes in the protein structure, most likely post-translational modifications, and/or the structural interactions with other tissue proteins such as decorin,(Danielson et al., 1997
) and is not a mineralization related structural change.
Fibril diameter has been employed previously as a key measure of ultrastructural change. A number of diseases and tissue malfunctions are associated with changes in collagen fibril diameter. Decorin and lumican knockout rats and type V collagen deficient mice showed one-fold increase in fibril diameters.(Wenstrup et al., 2004
; Yeh et al., 2010
) Ovarectomy has been shown to decrease expression level of proteoglycans including decorin(Danielson et al., 1997
) and lumican.(Markiewicz et al., 2007
) In this study, average collagen fibril diameter in sham is about 130 ± 30 nm, and 120 ± 20 nm in OVX, the difference is less than 10 % and considered negligible given the limited accuracy in the analysis. Thus, estrogen depletion exerts an anisotropic effect on skin collagen's ultrastructure. It is unclear whether decorin and lumican deficiency are associated to collagen fibril D-spacing changes, this will be the subject of future studies.
In conclusion, estrogen depletion causes a change in the nanoscale morphology of dermal collagen, quantitatively demonstrated by change in the D-spacing metric. The morphology changes are similar to those previously observed for the changes in bone collagen suggesting that estrogen depletion acts upon a structural aspect of the collagen molecule and/or associated proteins and is intrinsic to the fibril formation process.