Human three-dimensional fibroblast cultures express angiogenic activity

Abstract

Human neonatal fibroblasts were cultured on a lactate-glycollate copolymer scaffold for 12-16 days to form a three-dimensional dermal equivalent tissue. The cellular content of vascular endothelial growth factor (VEGF) mRNA in these three-dimensional cultures was 22-fold greater than that observed in the same fibroblasts grown as monolayers. No induction was shown by hepatocyte growth factor (HGF) or angiopoietin 1 indicating that the effect was specific to VEGF. The predominant VEGF splice variant, detected by RT-PCR corresponded to the 121 amino acid form, with less of the 165 amino acid form. The cell-associated forms (189 and 206 amino acids) comprised less than 1% of the total VEGF mRNA. VEGF and HGF proteins, determined by ELISA, were secreted in physiologically significant amounts, 0.5-4 ng per 24 h/10(6) cells. Conditioned medium from the three-dimensional cultures stimulated proliferation of endothelial cells in a dose-dependent manner and induced cellular expression of integrin alpha(v)beta(3). Conditioned medium from the same dermal fibroblasts cultured in monolayer showed little angiogenic activity in any of these assays. Using the chorioallantoic membrane (CAM) angiogenesis assay, the cultures stimulated blood vessel production 2.8-fold over scaffold alone. VEGF-neutralizing antibody reduced the vessel development in the CAM to the level in the scaffold control. Anti-HGF antibody had no significant effect. In conclusion, three-dimensional cultures of dermal equivalent tissue express angiogenic activity to a greater extent than monolayer cultures, some of which can be assigned to VEGF.

Scaffold-Based Three-Dimensional Human Fibroblast Culture Provides a Structural Matrix That Supports Angiogenesis in Infarcted Heart Tissue

“…This conclusion is supported by previous work in which the angiogenic capacity of 3DFC material has been demonstrated by in vitro tests of endothelial cell proliferation and ␣ v ␤ 3 integrin induction, 27 new vessel outgrowth and human vascular endothelial cell motility in rat aortic ring assays, 28 and use of in vivo tests of the chick chorioallantoic membrane. 27 Additional clinical studies to assess the healing of diabetic foot ulcers 29 after treatment with allogeneic, viable 3DFC material have further demonstrated the angiogenic ability of this tissue-engineered material in subjects with functional immune systems and suggest that viable 3DFC maintains its angiogenic effect with a negligible host immune response. Therefore, in light of the previously reported angiogenesis data in both in vitro and in vivo models, we anticipated that an angiogenic effect in an immune-incompetent animal similar to that seen in studies in immune-competent, allogeneic humans would take place and that angiogenic effects of viable 3DFC would be functional in other tissues, including damaged myocardium of the heart.…”

“…8,9,12 Data from the present study suggest that the viable 3DFC material is responsible for the observed and quantified increase in new microvessels within the damaged epicardium and myocardium. This conclusion is supported by previous work in which the angiogenic capacity of 3DFC material has been demonstrated by in vitro tests of endothelial cell proliferation and ␣ v ␤ 3 integrin induction, 27 new vessel outgrowth and human vascular endothelial cell motility in rat aortic ring assays, 28 and use of in vivo tests of the chick chorioallantoic membrane. 27 Additional clinical studies to assess the healing of diabetic foot ulcers 29 after treatment with allogeneic, viable 3DFC material have further demonstrated the angiogenic ability of this tissue-engineered material in subjects with functional immune systems and suggest that viable 3DFC maintains its angiogenic effect with a negligible host immune response.…”

“…Viable 3DFC material has been characterized to produce a heterogeneous population of cytokines, growth factors, and extracellular matrix proteins including, but not limited to, VEGF, platelet-derived growth factor A chain, hepatocyte growth factor, keratinocyte growth factor, interleukin 6, interleukin 8, transforming growth factor-␤ 1 , collagen protein, 18 and angiopoietin-1. 18,27 In contrast, a number of investigators have focused on the therapeutic use of individual growth factors, such as either VEGF or bFGF, to stimulate the revascularization of ischemic cardiac tissues. 7,6 These growth factors have been targeted for preclinical and clinical studies because of their well-described angiogenic potential.…”

Vascularization of the Dermal Support Enhances Wound Re-Epithelialization by In Situ Delivery of Epidermal Keratinocytes

“…Tissue-engineered skin substitutes with epidermal and dermal components have been designed to provide the lost cellular functions of the epidermis and dermis, respectively. 7,14,15 Although dermal substitutes may provide wound coverage and reduce pain, they still require an epidermal component, which is often provided through one or more applications of split-thickness autografts, necessitating multiple surgical procedures that injure the donor site and increase scar morbidity. 16 On the other hand, cultured epithelial autografts without dermal component have shown unreliable take rate possibly owing to lack of adequate vascularization and produce an unstable epidermis that is prone to blistering or minor trauma.…”

“…Several polymeric or natural biomaterials can be employed as dermal substrates, including fibroblast-containing nylon, 7 fibroblast-containing biodegradable polyglactin matrix, 15,18 collagen/chondroitin-6-sulfate composite (Integra), [19][20][21] and decellularized dermis (Alloderm/Xenoderm). 22,23 However, vascularization of the dermal support remains a major challenge limiting the survival and take rate of the multilayered epithelial component.…”