Second Harmonic GenerationSecond harmonic generation (SHG) imaging microscopy was used to investigate the collagen matrix organization within bone tissue. A SHG and two-photon excitation fluorescence (2PEF) microscope was used for this study. The SHG/2PEF microscope was based on a Leica TCS STED CW, Resonant Scanner Spectral confocal scanning head (Leica Microsystems, Mannheim, Germany). The SHG is collected both in back-scattering (BSHG) and in a transmitted light configuration (forward second harmonic generation; FSHG). Since the excitation wavelength is set at 860 nm, the BSHG spectral component of interest, 415–445 nm, is selected by means of the Leica built-in prism based spectrophotometer, whereas the FSHG is selected by two filters, an IR blocking filter and a 430/30 nm band-pass color filter (HQ430/30 m, Chroma Technology Corp., Bellows Falls, VT, USA). All panels have been acquired using a 76 nm pixel size and a 100x 1.4 NA oil-immersion objective (HCX PL APO 100x/1.4 Oil, Leica Microsystems, Mannheim, Germany). SHG imaging was used to gather specific organizational motifs, such as the structure of endogenous collagen proteins26. Moreover, due to the coherent nature of SHG signal, the intensity of the output is proportional to the square of the local concentration of the proteins observed and is intrinsically 3D.
Order versus Disorder
In vivo Bone Formation within Osteoconductive Scaffolds
In modern biomaterial design the generation of an environment mimicking some of the extracellular matrix features is envisaged to support molecular cross-talk between cells and scaffolds during tissue formation/remodeling. In bone substitutes chemical biomimesis has been particularly exploited; conversely, the relevance of pre-determined scaffold architecture for regenerated bone outputs is still unclear. Thus we aimed to demonstrate that a different organization of collagen fibers within newly formed bone under unloading conditions can be generated by differently architectured scaffolds. An ordered and confined geometry of hydroxyapatite foams concentrated collagen fibers within the pores, and triggered their self-assembly in a cholesteric-banded pattern, resulting in compact lamellar bone. Conversely, when progenitor cells were loaded onto nanofibrous collagen-based sponges, new collagen fibers were distributed in a nematic phase, resulting mostly in woven isotropic bone. Thus specific biomaterial design relevantly contributes to properly drive collagen fibers assembly to target bone regeneration.
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S. Scaglione et al.:
Order versus Disorder: in vivo bone formation within osteoconductive scaffolds
Scientific Reports 2 (February 2012) 274, doi:10.1038/srep00274
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