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Comparative Study
. 2010 Aug;93(8):690-707.
doi: 10.1002/bip.21431.

Polymerization and matrix physical properties as important design considerations for soluble collagen formulations

S T Kreger  1 , B J Bell, J Bailey, E Stites, J Kuske, B Waisner, S L Voytik-Harbin
Affiliations
Comparative Study

Polymerization and matrix physical properties as important design considerations for soluble collagen formulations

S T Kreger et al. Biopolymers. 2010 Aug.

Abstract

Despite extensive use of type I collagen for research and medical applications, its fibril-forming or polymerization potential has yet to be fully defined and exploited. Here, we describe a type I collagen formulation that is acid solubilized from porcine skin collagen (PSC), quality controlled based upon polymerization potential, and well suited as a platform polymer for preparing three-dimensional (3D) culture systems and injectable/implantable in vivo cellular microenvironments in which both relevant biochemical and biophysical parameters can be precision-controlled. PSC is compared with three commercial collagens in terms of composition and purity as well as polymerization potential, which is described by kinetic parameters and fibril microstructure and mechanical properties of formed matrices. When subjected to identical polymerization conditions, PSC showed significantly decreased polymerization times compared to the other collagens and yielded matrices with the greatest mechanical integrity and broadest range of mechanical properties as characterized in oscillatory shear, uniaxial extension, and unconfined compression. Compositional and intrinsic viscosity analyses suggest that the enhanced polymerization potential of PSC may be attributed to its unique oligomer composition. Collectively, this work demonstrates the importance of standardizing next generation collagen formulations based upon polymerization potential and provides preliminary insight into the contribution of oligomers to collagen polymerization properties.

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Figures

Fig. 1
Fig. 1
Polypeptide composition of PSC (A; Lane 1), Sigma (Lane 2), BD-RTC (Lane 3), and PureCol (Lane 4) collagen sources as determined using SDS-PAGE (4%) under reducing conditions. PSC contained a prominent protein band intermediate in molecular weight to β and γ bands (arrow). Western blot analysis (B; Lane 2) confirmed that this band contained the type I collagen epitope with corresponding Coomassie Blue-stained bands shown in Lane 1.
Fig. 2
Fig. 2
Polymerization kinetics of PSC and commercial collagen sources measured spectrophotometrically at 405 nm. Sources were polymerized at concentrations of 0.5 mg/ml and 1.0 mg/ml (lower and upper curves for each collagen source, respectively). Curves represent mean ± SD (n = 3) with SD plotted at 15-minute intervals for clarity. Kinetic parameters calculated from curves are summarized in Table 1.
Fig. 3
Fig. 3
Collagen fibril microstructure of matrices prepared with PSC and commercial collagen sources as visualized using CRM. Representative CRM images are shown for each source (PureCol (A,B), Sigma (C,D), BD-RTC (E,F), and PSC (G,H)) polymerized at 0.5 mg/ml (top row) and 2.0 mg/ml (bottom row; scale bar = 10 μm). Subtle differences in fibril network organization and fibril-fibril interactions, such as bundling/coalescence (arrows E,G) were observed between the sources. Increasing collagen concentration appeared to increase fibril density while maintaining similar fibril diameters and network organizations.
Fig. 4
Fig. 4
Fibril volume fraction (fibril density, A) and average fibril diameter (B) measured from CRM images of matrices prepared with different collagen sources and concentrations. Fibril volume fraction increased linearly with collagen concentration and was not significantly dependent on source (with the exception of PureCol at 2 mg/ml (p<0.05), n = 6–10 images per matrix formulation). Average fibril diameter showed a small, but significant dependence on collagen source (p<0.05 at each concentration, n = 48 fibrils per matrix formulation). In general, fibril diameters did not change with collagen concentration.
Fig. 5
Fig. 5
Shear storage modulus (G', A) and phase shift (δ, B) of matrices prepared with PSC or commercial collagen sources at varied concentrations (values reported at 1 % strain, 1 Hz frequency, n = 3). PSC had the largest G' and lowest δ at each concentration (p<0.05). G' increased with increasing collagen concentration, but with significantly different relationships for each source (p<0.05 at each concentration with the exception of PureCol and BD-RTC (p≥0.9 for all comparisons)). δ was significantly different for each source (p<0.05) and did not change with collagen concentration.
Fig. 6
Fig. 6
Unconfined compression stress-strain curves (A) and modulus (EC, B) for matrices prepared with PSC or commercial collagen sources at varied concentrations. Representative stress-strain curves for each source prepared at 2 mg/ml are shown to illustrate source-dependent compressive behavior (curves are mean ± SD (n = 3) with SD plotted at 10% strain intervals for clarity). EC, measured in a linear region of curves from 15 to ~60 % strain as indicated in (A), increased with collagen concentration in a source dependent fashion. PSC exhibited the largest EC at each concentration (p<0.05).
Fig. 7
Fig. 7
Matrices produced with PSC or commercial collagens at various concentrations were subjected to uniaxial tensile testing and ET (A,B), σU (C,D), and εF (E) measured. ET and σU were calculated based upon “engineering” (A,C) “true” (B,D) assumptions. All measured parameters are summarized in Table 2 (n = 3–10, note σU and εF groups not shown when > 50% of samples failed outside of gauge section). PSC exhibited the largest ET and σU at each concentration (p<0.05), despite whether the “engineering” or “true” definition was used. In general, εF showed no dependence on collagen source or concentration.
Fig. 8
Fig. 8
Time-lapse brightfield images showing the failure of PSC (A–C) and BD-RTC (D–F) matrices (4 mg/ml) during uniaxial extension tests. PSC failure was observed as an abrupt, elastic tearing across the gauge section (also representative of Sigma failure). BD-RTC failure was observed as a slow dissociation throughout the gauge section (note fibrous strands of matrix pulled apart between separating ends, also representative of PureCol failure; scale bar = 5 mm).
Fig. 9
Fig. 9
Tuning the physical properties of 3D PSC matrices effectively directed different patterns of lineage specific differentiation by embedded MSC. MSC (5×104 cells/ml) were cultured for up to 14 days within collagen matrices whose physical properties corresponded to G' of 45Pa or 695Pa, as prepared by modulating the collagen content of polymerization reaction. Cultures were stained with oil red O (Upper Panel (A and D)) or alizarin red (Upper Panel (B and C)) for visualization of adipocytes (white arrows) or calcified nodules (black arrow), respectively. The number of adipocytes and calcified nodules within the 3D constructs also was quantified (Lower Panel). Asterix denotes statistically significant relationships (p<0.05). Scale bar = 50 μm.
Fig. 10
Fig. 10
Relative expression levels of LPL and CBFA1 as determined by RT-PCR for MSC cultured for 4 or 14 days on tissue culture plastic (circles) or within 3D PSC matrices. PSC matrices were prepared with physical properties corresponding to G' of 45Pa (squares) or 695Pa (triangles). MSC were cultured in the presence of regular medium (A and B) or adipogenic medium (C and D).Note differences in y-axis scales.
Fig. 11
Fig. 11
Intra- and inter- hide variation in polymerization potential of PSC preparations. Data are provided for 6 different PSC lots prepared from 3 different market-weight pig hides. Highly consistent relationships are observed between matrix mechanical properties (as measured by G') and collagen concentration used in the polymerization reaction. The first digit of the lot number designates the source pig, the second number represents the production lot number, and the last 6 digits indicate the production date.
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