Biomedical Engineering Reference
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Fig. 3 Collagen scaffold (1 mg/ml) structures and seeded cells were imaged using a Bio-Rad
Radiance Model 2000 (femtosecond pulses at 80 MHz, 0.8 mW) laser scanning multiphoton micro-
scope. Second harmonic generation signals from collagen fi bers (no fl uorophore required) were
excited by a wavelength of 840 nm beam, and emitted signals were acquired with a band-pass fi lter
of 390 ± 70 nm. Cells were stained using the CellTracker orange CMTMR (Molecular Probe, OR,
USA) and visualized with a band-pass fi lter of 560±70 nm. Spectrally separated but combined
images of rat mesenchymal stem cells ( left panel ) and human fi broblasts ( right panel ) are shown
with and remodel the ECM. Finally, collagen-based hydrogels are relatively simple
to construct and does not require expensive equipment. One major disadvantage of
collagen-based scaffold could be the lack of strong mechanical strength essential
for engineering, for example, bone tissue. Although type-I collagen constitutes
~90% of ECM in the bone tissue, collagen polymers alone are insuffi cient to pro-
vide the necessary mechanical strength of the bone tissue. Other materials, such as
hydroxyapatite and calcium phosphate, have been added to the collagen scaffold to
improve mechanical strength (McCarthy et al. 1996 ; Takoaka et al. 1998 ; LeGeros
2002 ; Alhadlaq et al. 2004 ). Using multiphoton microscopy, we were able to obtain
simultaneously the images of collagen fi bers and cells seeded in the 3D collagen
scaffold, allowing us to determine collagen-cell binding. Collagen fi bers were
imaged without fl uorescent probes, but instead relied on Second Harmonic
Generation imaging technique (Zoumi et al. 2002 ; Zipfel et al. 2003 ) , while cells
were loaded with CellTrackers. Seeding rat mesenchymal stem cells (rMSCs) and
human fi broblasts in the collagen scaffold, differential cell adhesion behaviors were
investigated. As shown in Fig. 3 , rMSCs appear to concentrate and reorient collagen
fi bers (left panel) that are consistent with the postulate of strong adhesion, whereas
fi broblasts embedded the same collagen scaffold demonstrated less localized, but
randomized collagen fi bers (right panel). Moreover, the extent of collagen reorgani-
zation around the fi broblast appears not as dramatic as that found around the MSC.
While natural polymers, such as collagen and chitosan, closely mimic the
native extracellular environment, synthetic polymers have been developed for
tissue-engineering applications. Substantial advances have been achieved recently
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