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(e) Inhibition of FGF-2-induced matrix neovascularization in gels containing glass fibers (We further investigated the importance of contraction for neovascularization by using contraction-resistant gels.When biochemically inert borosilicate glass fibers (10–100 m long) are mixed with the rat tail collagen matrix, hydrogen bonds form between surface silanol groups in the glass fibers and amino acids.

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Formation of vessels in wound and tumor granulation tissue has traditionally been explained by angiogenic mechanisms, defined as the formation of new branchpoints in the preexisting capillary network, by outgrowth of endothelial sprouts.

In this report, we show that formation of granulation tissue does not require immediate angiogenesis.

In response to fibroblast growth factor-2 (FGF-2), the gel gradually contracted and was partly digested by invading cells (Fig. In parallel to these physical changes in the gel, we observed ingrowth of vascularized tissue. This double staining showed that all neovessels were functionally connected with the circulation.(a) A fibrin and collagen gel placed on the CAM.

We visualized blood-filled structures by permanent staining of erythrocytes with 3,3'-diaminobenzidine (DAB) and H (Fig. Subsequent clarification with benzyl benzoate–benzyl alcohol (BBBA), which renders opaque tissues transparent, visualized the entire vasculature of ingrown tissue (Fig. Intracardiac ink injection distinguished vessels with functional circulation from those filled with blood but with no or low circulation (Fig. (b) Response to FGF-2; the gel contracted and its opacity increased. (c) Neovessels in the ingrown tissue, stained for hemoglobin. injection of India ink and staining for hemoglobin to discriminate functional vessels (black, sometimes appearing greenish) from blunt-ended or underperfused ones (red-brown). Preexisting vessels hence expanded within the growing tissue and did not enter the provisional matrix as independent vessels.

Protomyofibroblasts are induced by tensile stress and contract the wound matrix through a stress fiber–rich contractile apparatus.

Tissue integrity is then restored by formation of granulation tissue and its growth into the wound void at days 4–5 (refs. Later, in response to tensile stress, ED-A fibronectin and macrophage-derived growth factors such as transforming growth factor-1, protomyofibroblasts differentiate into highly contractile myofibroblasts that express -smooth muscle actin (SMA).

3b) and late stages of ingrown tissue (g,h; horizontal section, plane in Supplementary Fig. In the normal CAM (c) and early tissue buds (e), SMA vascular mural cells (arrows) and elongated interconnected nonvascular cells (arrowheads) that in the outer parts of the implant were aligned parallel to the gel border (b).

Except for perivascular areas, collagen was evenly distributed in the normal CAM (d).

Gel contraction was a prerequisite for neovascularization, as replacement of acid-solubilized rat tail collagen type I with pepsin-solubilized collagen type I (Vitrogen) reduced implant contraction (data not shown) and impaired neovascular ingrowth (Fig. The lower cell-driven contractibility of Vitrogen is due to pepsin-mediated cleavage, which results in removal of both collagen telopeptides and cryptic binding sites for integrins.

Removal of these cellular binding sites inhibits development of cell-mediated tension because cells are unable to translocate matrix molecules.

Using neovascularization models based on the chick chorioallantoic membrane and the healing mouse cornea, we found that tissue tension generated by activated fibroblasts or myofibroblasts during wound contraction mediated and directed translocation of the vasculature.

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