Biomedical Engineering Reference
In-Depth Information
sulting collagen layers were characterized with respect to dry layer thickness
utilizing ellipsometry, immobilized protein quantities using acidic hydrolysis
of collagen and subsequent amino acid-based HPLC-analysis [108], and as-
sessment of surface topography by scanning force microscopy and scanning
electron microscopy. Furthermore, confocal laser scanning microscopy was
used for the detection of surface-bound collagen-glycosaminoglycan assem-
blies. The data obtained revealed differences in the deposited collagen layers
including thickness, protein amount, and surface character.
While pure collagen provided a dry layer thickness of about 80 nm, signifi-
cantly thinner layers in the range between 14 and 32 nm (for fixed, pre-set
refractive index values) were measured if heparin or hyaluronic acid were
added to fibril-forming solutions (Table 3). The quantification of surface-
bound proteins confirmed the results of the ellipsometric measurements:
noticeable lower collagen amounts were quantified if fibril formation and
immobilization was performed in the presence of glycosaminoglycans. The
quantification was supported by surface topographic analysis of the attached
fibrillar components (Fig. 7).
In comparison to the meshwork formed by pure collagen different surface
patterns concerning shape and dimensions of the deposited fibrils depend-
ing on the glycosaminoglycans could be demonstrated. Collagen fibrils which
had been reconstituted in the presence of polymer-coated substrates provided
well-established networks without gaps between the separate fibres whereas
fibril formation by addition of heparin or hyaluronic acid gave rise to modi-
fied morphologies of the deposited layers. In the case of heparin cofibrils
Table 3
Deposition of collagen-glycosaminoglycan conjugates on poly(octadecen-
alt
-
maleic anhydride)-coated substrates. Fibrillogenesis and immobilization of fibrillar colla-
gen (1.2 mg
/
ml) was performed for 2 hat37
◦
C in the presence of heparin and hyaluronic
acid (0.4, 1.2 and 5.0 mg
ml, respectively). Thickness of the layers was determined ellip-
sometrically using the refractive index of 1.6035 for the dried collagen layer. The collagen
amount was determined by amino acid-based HPLC analysis after acidic hydrolysis of
surface-bound collagen
/
Immobilized assemblies
Layer thickness
Collagen amount
cm
2
]
[nm]
[
µ
g
/
Collagen
81.3
±
5.8
10.4
±
0.4
Collagen/Heparin 0.4 mg
/
ml
n.d.
2.7
±
1.2
Collagen/Heparin 1.2 mg
/
ml
29.3
±
8.5
2.3
±
0.3
Collagen/Heparin 5.0 mg
/
ml
32.3
±
1.6
2.3
±
0.9
Collagen/Hyaluronic acid 0.4 mg
/
ml
n.d.
3.4
±
1.3
Collagen/Hyaluronic acid 1.2 mg
/
ml
14.8
±
2.0
1.8
±
0.5
Collagen/Hyaluronic acid 5.0 mg
/
ml
30.9
±
8.7
3.0
±
0.8
