Preparation of HD collagen gels resembling desmoplastic matrices.A, High-density mammographic tissue (HMT) and low-density mammographic tissue (LMT) denoted by light and dark radiographic areas respectively, were macrodissected from DCIS mammary tissue and processed. B, Masson’s Trichrome staining showed abundant collagen fibrils in HMT tissue (blue stain) and mostly adipose cells in LMT tissue. C, In vitro HD matrix was prepared by centrifugation to concentrate solubilised collagen followed by polymerisation using vaporised NH4OH. HD matrix along with HMT and LMT samples were stained with picrosirius red and assayed for dye binding at 531 nm wavelength of light. The collagen concentration of HMT (■), LMT tissues (▴) and HD collagen matrix (●) were extrapolated from standards of known collagen concentrations (♦). After 60 min of centrifugation, HD collagen measured 19.16 ± 0.74 mg/cm3, which closely correlated with HMT samples of 19.59 ± 2.91 mg/cm3 collagen. D, SEM of HMT tissues and HD matrix shows that collagen is organized as networks of dense fibrils. Field emission SEM demonstrates that HD collagen consists of small ~30 nm collagen fibrils that form helical coils of larger 200 nm fibrils (arrows). Collagen D-spacing of ~50–60 nm is visible along the lengths of individual collagen fibrils and are aligned relative to adjacent coiled fibrils (arrowheads). E, the fibril density and pore sizes of HD (60 min centrifugation) and LD matrices closely mimicked HMT and LMT counterparts of tumour tissue (n = no. areas sampled). Experiments were repeated three times. Bars indicate standard deviation from triplicate samples. Scale bars represent 200 μm in B and 1 μm in D.
Raviraj et al. BMC Cell Biology 2012 13:12 doi:10.1186/1471-2121-13-12