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Effects of Intermittent and Incremental Cyclic Stretch on ERK Signaling and Collagen Production in Engineered Tissue.

ABSTRACT: Intermittent cyclic stretching and incrementally increasing strain amplitude cyclic stretching were explored to overcome the reported adaptation of fibroblasts in response to constant amplitude cyclic stretching, with the goals of accelerating collagen production and understanding the underlying cell signaling. The effects of constant amplitude, intermittent, and incremental cyclic stretching regimens were investigated for dermal fibroblasts entrapped in a fibrin gel by monitoring the extracellular signal-regulated kinase (ERK1/2) and p38 pathways, collagen transcription, and finally the deposited collagen protein. Activation of ERK1/2, which has been shown to be necessary for stretch-induced collagen transcription, was maximal at 15 min and decayed by 1 h. ERK1/2 was reactivated by an additional onset of stretching or by an increment in the strain amplitude 6 h after the initial stimulus, which was approximately the lifetime of activated p38, a known ERK1/2 inhibitor. While both intermittent and incremental regimens reactivated ERK1/2, only incremental stretching increased collagen production compared to samples stretched with constant amplitude, resulting in a 37% increase in collagen per cell after 2 weeks. This suggests that a regimen with small, frequent increments in strain amplitude is optimal for this system and should be used in bioreactors for engineered tissues requiring high collagen content.

SUBMITTER: Schmidt JB

PROVIDER: S-EPMC4840278 | biostudies-literature | 2016 Mar

REPOSITORIES: biostudies-literature

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Effects of Intermittent and Incremental Cyclic Stretch on ERK Signaling and Collagen Production in Engineered Tissue.

Schmidt Jillian B JB Chen Kelley K Tranquillo Robert T RT

Cellular and molecular bioengineering 20150811 1

Intermittent cyclic stretching and incrementally increasing strain amplitude cyclic stretching were explored to overcome the reported adaptation of fibroblasts in response to constant amplitude cyclic stretching, with the goals of accelerating collagen production and understanding the underlying cell signaling. The effects of constant amplitude, intermittent, and incremental cyclic stretching regimens were investigated for dermal fibroblasts entrapped in a fibrin gel by monitoring the extracellu ...[more]

PMID: 27114743

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Project description:Cyclic stretching and growth factors like TGF-? have been used to enhance extracellular matrix (ECM) production by cells in engineered tissue to achieve requisite mechanical properties. In this study, the effects of TGF-?1 were evaluated during long-term cyclic stretching of fibrin-based tubular constructs seeded with neonatal human dermal fibroblasts. Samples were evaluated at 2, 5, and 7 weeks for tensile mechanical properties and ECM deposition. At 2 weeks, +TGF-?1 samples had 101% higher collagen concentration but no difference in ultimate tensile strength (UTS) or modulus compared to -TGF-?1 samples. However, at weeks 5 and 7, -TGF-?1 samples had higher UTS/modulus and collagen concentration, but lower elastin concentration compared to +TGF-?1 samples. The collagen was better organized in -TGF-?1 samples based on picrosirius red staining. Western blot analysis at weeks 5 and 7 showed increased phosphorylation of ERK in -TGF-?1 samples, which correlated with higher collagen deposition. The TGF-?1 effects were further evaluated by western blot for ?SMA and SMAD2/3 expression, which were 16-fold and 10-fold higher in +TGF-?1 samples, respectively. The role of TGF-?1 activated p38 in inhibiting phosphorylation of ERK was evaluated by treating samples with SB203580, an inhibitor of p38 activation. SB203580-treated cells showed increased phosphorylation of ERK after 1 hour of stretching and increased collagen production after 1 week of stretching, demonstrating an inhibitory role of activated p38 via TGF-?1 signaling during cyclic stretching. One advantage of TGF-?1 treatment was the 4-fold higher elastin deposition in samples at 7 weeks. Further cyclic stretching experiments were thus conducted with constructs cultured for 5 weeks without TGF-?1 to obtain improved tensile properties followed by TGF-?1 supplementation for 2 weeks to obtain increased elastin content, which correlated with a reduction in loss of pre-stress during preconditioning for tensile testing, indicating functional elastin. This study shows that a sequential stimulus approach - cyclic stretching with delayed TGF-?1 supplementation - can be used to engineer tissue with desirable tensile and elastic properties.

Project description:Tissue-engineered blood vessels (TEVs) are typically produced using the pulsatile, uniaxial circumferential stretch to mechanically condition and strengthen the arterial grafts. Despite improvements in the mechanical integrity of TEVs after uniaxial conditioning, these tissues fail to achieve critical properties of native arteries such as matrix content, collagen fiber orientation, and mechanical strength. As a result, uniaxially loaded TEVs can result in mechanical failure, thrombus, or stenosis on implantation. In planar tissue equivalents such as artificial skin, biaxial loading has been shown to improve matrix production and mechanical properties. To date however, multiaxial loading has not been examined as a means to improve mechanical and biochemical properties of TEVs during culture. Therefore, we developed a novel bioreactor that utilizes both circumferential and axial stretch that more closely simulates loading conditions in native arteries, and we examined the suture strength, matrix production, fiber orientation, and cell proliferation. After 3 months of biaxial loading, TEVs developed a formation of mature elastic fibers that consisted of elastin cores and microfibril sheaths. Furthermore, the distinctive features of collagen undulation and crimp in the biaxial TEVs were absent in both uniaxial and static TEVs. Relative to the uniaxially loaded TEVs, tissues that underwent biaxial loading remodeled and realigned collagen fibers toward a more physiologic, native-like organization. The biaxial TEVs also showed increased mechanical strength (suture retention load of 303?±?14.53?g, with a wall thickness of 0.76?±?0.028?mm) and increased compliance. The increase in compliance was due to combinatorial effects of mature elastic fibers, undulated collagen fibers, and collagen matrix orientation. In conclusion, biaxial stretching is a potential means to regenerate TEVs with improved matrix production, collagen organization, and mechanical properties.

Dataset Information

Effects of Intermittent and Incremental Cyclic Stretch on ERK Signaling and Collagen Production in Engineered Tissue.

Publications

Effects of Intermittent and Incremental Cyclic Stretch on ERK Signaling and Collagen Production in Engineered Tissue.

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