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Quantification of the temporal evolution of collagen orientation in mechanically conditioned engineered cardiovascular tissues.

Rubbens MP, Driessen-Mol A, Boerboom RA, Koppert MM, van Assen HC, TerHaar Romeny BM, Baaijens FP, Bouten CV - Ann Biomed Eng (2009)

Bottom Line: Engineered tissues often lack properly organized collagen and consequently do not meet in vivo mechanical demands.Most importantly, intermittent straining improved and accelerated the alignment of the collagen fibers, as compared to constraining the constructs.Both the method and the results are relevant to create and monitor load-bearing tissues with an organized anisotropic collagen network.

View Article: PubMed Central - PubMed

Affiliation: Soft Tissue Biomechanics & Engineering, Department of Biomedical Engineering, Eindhoven University of Technology, WH 4.107, Den Dolech 2, P.O. Box 513, 5600 MB, Eindhoven, The Netherlands. m.p.rubbens@tue.nl

ABSTRACT
Load-bearing soft tissues predominantly consist of collagen and exhibit anisotropic, non-linear visco-elastic behavior, coupled to the organization of the collagen fibers. Mimicking native mechanical behavior forms a major goal in cardiovascular tissue engineering. Engineered tissues often lack properly organized collagen and consequently do not meet in vivo mechanical demands. To improve collagen architecture and mechanical properties, mechanical stimulation of the tissue during in vitro tissue growth is crucial. This study describes the evolution of collagen fiber orientation with culture time in engineered tissue constructs in response to mechanical loading. To achieve this, a novel technique for the quantification of collagen fiber orientation is used, based on 3D vital imaging using multiphoton microscopy combined with image analysis. The engineered tissue constructs consisted of cell-seeded biodegradable rectangular scaffolds, which were either constrained or intermittently strained in longitudinal direction. Collagen fiber orientation analyses revealed that mechanical loading induced collagen alignment. The alignment shifted from oblique at the surface of the construct towards parallel to the straining direction in deeper tissue layers. Most importantly, intermittent straining improved and accelerated the alignment of the collagen fibers, as compared to constraining the constructs. Both the method and the results are relevant to create and monitor load-bearing tissues with an organized anisotropic collagen network.

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Representative multiphoton images of cells (blue) and collagen (green) organization in constrained (a, b) and intermittently strained (c, d) samples after 2 weeks at 15 (a, c) and 50 μm (b, d) imaging depths with the corresponding histograms of collagen orientations at 15 and 50 μm depth. Straining direction was from left to right. Scale bars represent 50 μm
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Fig4: Representative multiphoton images of cells (blue) and collagen (green) organization in constrained (a, b) and intermittently strained (c, d) samples after 2 weeks at 15 (a, c) and 50 μm (b, d) imaging depths with the corresponding histograms of collagen orientations at 15 and 50 μm depth. Straining direction was from left to right. Scale bars represent 50 μm

Mentions: Representative pictures of constrained and intermittently strained samples at 15 and 50 μm depth after 2 weeks of culture are depicted in Fig. 4. Collagen fibers were more randomly distributed in the constrained samples (Figs. 4a and 4b), as compared to the intermittently strained samples (Figs. 4c and 4d), as is depicted in the corresponding histograms. No preferred orientations were found in the constrained samples (r = 0.13 at 15 μm and r = 0.03 at 50 μm), whereas intermittent loading resulted in fiber distributions with more distinct fiber orientations (r = 0.42 at 15 μm and r = 0.37 at 50 μm). The courses of the angle and vector length as a function of depth of the same samples are shown in Fig. 5. No alignment was observed in the constrained samples (Fig. 5a), except for the first few slices. On the contrary, higher values of the vector length were found throughout the imaging stack of the intermittently strained samples (Fig. 5b), indicating a higher degree of alignment. Interestingly, the orientation of the collagen fibers in the intermittently strained samples shifted from an almost perpendicular orientation at the surface to an orientation into the direction of straining deeper into the tissues.Figure 4


Quantification of the temporal evolution of collagen orientation in mechanically conditioned engineered cardiovascular tissues.

Rubbens MP, Driessen-Mol A, Boerboom RA, Koppert MM, van Assen HC, TerHaar Romeny BM, Baaijens FP, Bouten CV - Ann Biomed Eng (2009)

Representative multiphoton images of cells (blue) and collagen (green) organization in constrained (a, b) and intermittently strained (c, d) samples after 2 weeks at 15 (a, c) and 50 μm (b, d) imaging depths with the corresponding histograms of collagen orientations at 15 and 50 μm depth. Straining direction was from left to right. Scale bars represent 50 μm
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2690830&req=5

Fig4: Representative multiphoton images of cells (blue) and collagen (green) organization in constrained (a, b) and intermittently strained (c, d) samples after 2 weeks at 15 (a, c) and 50 μm (b, d) imaging depths with the corresponding histograms of collagen orientations at 15 and 50 μm depth. Straining direction was from left to right. Scale bars represent 50 μm
Mentions: Representative pictures of constrained and intermittently strained samples at 15 and 50 μm depth after 2 weeks of culture are depicted in Fig. 4. Collagen fibers were more randomly distributed in the constrained samples (Figs. 4a and 4b), as compared to the intermittently strained samples (Figs. 4c and 4d), as is depicted in the corresponding histograms. No preferred orientations were found in the constrained samples (r = 0.13 at 15 μm and r = 0.03 at 50 μm), whereas intermittent loading resulted in fiber distributions with more distinct fiber orientations (r = 0.42 at 15 μm and r = 0.37 at 50 μm). The courses of the angle and vector length as a function of depth of the same samples are shown in Fig. 5. No alignment was observed in the constrained samples (Fig. 5a), except for the first few slices. On the contrary, higher values of the vector length were found throughout the imaging stack of the intermittently strained samples (Fig. 5b), indicating a higher degree of alignment. Interestingly, the orientation of the collagen fibers in the intermittently strained samples shifted from an almost perpendicular orientation at the surface to an orientation into the direction of straining deeper into the tissues.Figure 4

Bottom Line: Engineered tissues often lack properly organized collagen and consequently do not meet in vivo mechanical demands.Most importantly, intermittent straining improved and accelerated the alignment of the collagen fibers, as compared to constraining the constructs.Both the method and the results are relevant to create and monitor load-bearing tissues with an organized anisotropic collagen network.

View Article: PubMed Central - PubMed

Affiliation: Soft Tissue Biomechanics & Engineering, Department of Biomedical Engineering, Eindhoven University of Technology, WH 4.107, Den Dolech 2, P.O. Box 513, 5600 MB, Eindhoven, The Netherlands. m.p.rubbens@tue.nl

ABSTRACT
Load-bearing soft tissues predominantly consist of collagen and exhibit anisotropic, non-linear visco-elastic behavior, coupled to the organization of the collagen fibers. Mimicking native mechanical behavior forms a major goal in cardiovascular tissue engineering. Engineered tissues often lack properly organized collagen and consequently do not meet in vivo mechanical demands. To improve collagen architecture and mechanical properties, mechanical stimulation of the tissue during in vitro tissue growth is crucial. This study describes the evolution of collagen fiber orientation with culture time in engineered tissue constructs in response to mechanical loading. To achieve this, a novel technique for the quantification of collagen fiber orientation is used, based on 3D vital imaging using multiphoton microscopy combined with image analysis. The engineered tissue constructs consisted of cell-seeded biodegradable rectangular scaffolds, which were either constrained or intermittently strained in longitudinal direction. Collagen fiber orientation analyses revealed that mechanical loading induced collagen alignment. The alignment shifted from oblique at the surface of the construct towards parallel to the straining direction in deeper tissue layers. Most importantly, intermittent straining improved and accelerated the alignment of the collagen fibers, as compared to constraining the constructs. Both the method and the results are relevant to create and monitor load-bearing tissues with an organized anisotropic collagen network.

Show MeSH