Human Wound Contraction: Collagen Organization, Fibroblasts, and Myofibroblasts

Berry, David P.; Harding, Keith; Stanton, Michael R.; Jasani, Bharat; Ehrlich, Paul H.

doi:10.1097/00006534-199807000-00019

Cited by 156 publications

(84 citation statements)

References 11 publications

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“…For example, fibroblast-populated collagen gels demonstrate that fibroblast morphology in 3D is quite distinct from that observed in 2D 10,11 . Similarly, 3D culture can induce tissue-specific differentiation of mammary epithelial cells.…”

Section: Discussionmentioning

confidence: 99%

A Simple Hanging Drop Cell Culture Protocol for Generation of 3D Spheroids

Foty

2011

JoVE

419

374

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Studies of cell-cell cohesion and cell-substratum adhesion have historically been performed on monolayer cultures adherent to rigid substrates. Cells within a tissue, however, are typically encased within a closely packed tissue mass in which cells establish intimate connections with many near-neighbors and with extracellular matrix components. Accordingly, the chemical milieu and physical forces experienced by cells within a 3D tissue are fundamentally different than those experienced by cells grown in monolayer culture. This has been shown to markedly impact cellular morphology and signaling. Several methods have been devised to generate 3D cell cultures including encapsulation of cells in collagen gels 1 or in biomaterial scaffolds 2 . Such methods, while useful, do not recapitulate the intimate direct cell-cell adhesion architecture found in normal tissues. Rather, they more closely approximate culture systems in which single cells are loosely dispersed within a 3D meshwork of ECM products. Here, we describe a simple method in which cells are placed in hanging drop culture and incubated under physiological conditions until they form true 3D spheroids in which cells are in direct contact with each other and with extracellular matrix components. The method requires no specialized equipment and can be adapted to include addition of any biological agent in very small quantities that may be of interest in elucidating effects on cell-cell or cell-ECM interaction. The method can also be used to co-culture two (or more) different cell populations so as to elucidate the role of cell-cell or cell-ECM interactions in specifying spatial relationships between cells. Cell-cell cohesion and cell-ECM adhesion are the cornerstones of studies of embryonic development, tumor-stromal cell interaction in malignant invasion, wound healing, and for applications to tissue engineering. This simple method will provide a means of generating tissue-like cellular aggregates for measurement of biomechanical properties or for molecular and biochemical analysis in a physiologically relevant model. Protocol

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Section: Discussionmentioning

confidence: 99%

A Simple Hanging Drop Cell Culture Protocol for Generation of 3D Spheroids

Foty

2011

JoVE

419

374

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“…Myofibroblasts synthesize and deposit type I collagen into the wound provisional matrix (2). They subsequently contract the type I collagen matrix, facilitating wound closure (3,4).…”

mentioning

confidence: 99%

PTEN Regulates Fibroblast Elimination during Collagen Matrix Contraction

Nho

Xia

Diebold

et al. 2006

Journal of Biological Chemistry

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During tissue repair, excess fibroblasts are eliminated by apoptosis. This physiologic process limits fibrosis and restores normal anatomic patterns. Replicating physiologic apoptosis associated with tissue repair, fibroblasts incorporated into type I collagen matrices undergo apoptosis in response to collagen matrix contraction. In this in vitro model of wound repair, fibroblasts first attach to collagen via ␣2␤1 integrin. This provides a survival signal via activation of the phosphatidylinositol 3-kinase/Akt signal pathway. However, during subsequent collagen matrix contraction, the level of phosphorylated Akt progressively declines, triggering apoptosis. The mechanism underlying the fall in phosphorylated Akt is incompletely understood. Here we show that PTEN phosphatase becomes activated during collagen matrix contraction and is responsible for antagonizing phosphatidylinositol 3-kinase activity and promoting a decline in phosphorylated Akt and fibroblast apoptosis in response to collagen contraction. PTEN null fibroblasts displayed enhanced levels of phosphorylated Akt and were resistant to collagen matrix contraction-induced apoptosis. Reconstitution of PTEN in PTEN null cells conferred susceptibility to apoptosis in response to contraction of collagen matrices. Consistent with this, knockdown of PTEN in PTEN ؉/؉ embryonic fibroblasts by small interfering RNA augmented Akt activity and suppressed apoptosis in contractile collagen matrices. Furthermore, inhibition of Akt activity restored the sensitivity of PTEN null cells to collagen contraction-induced apoptosis, indicating that the mechanism by which PTEN alters fibroblast viability is through modulation of phosphorylated Akt levels. Our work suggests that collagen matrix contraction activates PTEN by a mechanism involving cytoskeletal disassembly. Our studies indicate a key role for PTEN in regulating fibroblast viability during tissue repair.Fibroblasts play an essential role in tissue repair. In response to injury, interstitial fibroblasts differentiate into myofibroblasts (1). Myofibroblasts synthesize and deposit type I collagen into the wound provisional matrix (2). They subsequently contract the type I collagen matrix, facilitating wound closure (3, 4).Following contraction of the type I collagen matrix, myofibroblasts are eliminated by apoptosis, reducing wound cellularity (3). The precise molecular mechanisms governing removal of fibroblasts during tissue repair are unclear.Using fibroblasts cultured in three-dimensional contractile type I collagen matrices as a model of wound/tissue repair, the molecular signals regulating fibroblast viability during collagen contraction have begun to be elucidated. When fibroblasts are incorporated into the type I collagen matrix, they utilize ␣2␤1 integrin to attach and spread (5). This results in phosphorylation of serine 473 of Akt. Activation of Akt provides a survival signal that is mediated through phosphatidylinositol 3-kinase (PI3K) 3 (5-12). However, upon collagen matrix contraction, this integrin sur...

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“…Microarray analysis has also shown the importance of ECM glypicans which are proteins that can bind a multitude of GF and ECM molecules which are implicated in signal transduction cascaded that most likely regulate cell proliferation. Glypicans activate or determine activities of morphogens and GF such as FGF, BMPs and IGFs (Berry et al, 1998;De Cat and David, 2001;Hwang et al, 2001). We have seen that glycipans are up-regulated in fetal dermal skin cells (microarray data, GPC4 and GPC3 with 2.02 and 2.03 up-regulated, respectively) and in another recent study by Lener et al (2006) they had seen the same effect with down-regulation of glycipans in aged skin.…”

Section: Discussionmentioning

confidence: 56%

Chronic wound healing by fetal cell therapy may be explained by differential gene profiling observed in fetal versus old skin cells

Ramelet¹,

Hirt‐Burri

Raffoul³

et al. 2009

Experimental Gerontology

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a b s t r a c tEngineering of fetal tissue has a high potential for the treatment of acute and chronic wounds of the skin in humans as these cells have high expansion capacity under simple culture conditions and one organ donation can produce Master Cell Banks which can fabricate over 900 million biological bandages (9 Â 12 cm). In a Phase 1 clinical safety study, cases are presented for the treatment of therapy resistant leg ulcers. All eight patients, representing 13 ulcers, tolerated multiple treatments with fetal biological bandages showing no negative secondary effects and repair processes similar to that seen in 3rd degree burns. Differential gene profiling using Affymetrix gene chips (analyzing 12,500 genes) were accomplished on these banked fetal dermal skin cells compared to banked dermal skin cells of an aged donor in order to point to potential indicators of wound healing. Families of genes involved in cell adhesion and extracellular matrix, cell cycle, cellular signaling, development and immune response show significant differences in regulation between banked fetal and those from banked old skin cells: with approximately 47.0% of genes over-expressed in fetal fibroblasts. It is perhaps these differences which contribute to efficient tissue repair seen in the clinic with fetal cell therapy.

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Human Wound Contraction: Collagen Organization, Fibroblasts, and Myofibroblasts

Cited by 156 publications

References 11 publications

A Simple Hanging Drop Cell Culture Protocol for Generation of 3D Spheroids

A Simple Hanging Drop Cell Culture Protocol for Generation of 3D Spheroids

PTEN Regulates Fibroblast Elimination during Collagen Matrix Contraction

Chronic wound healing by fetal cell therapy may be explained by differential gene profiling observed in fetal versus old skin cells

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