Biomedical subjects

Steven T Boyce

Publications and source records attributed to Steven T Boyce.

At least 19 recordsLinked to original sources

PubMed2006

Wound closure with EDC cross-linked cultured skin substitutes grafted to athymic mice.

Collagen-glycosaminoglycan (C-GAG) sponges are commonly utilized as a substitute for the extracellular matrix of dermal tissue. Cultured skin substitutes (CSS) were assessed, after fabrication using sponges cross-linked with 1-ethyl-3-3-dimethylaminopropylcarbodiimide hydrochloride (EDC) at 0, 1, 5, or 50 mm, for development of viable, stratified skin tissue anatomy in vitro, and for wound contraction and cell viability in vivo. Cross-linking the C-GAG sponges with EDC reduced in vitro contraction of the CSS from a 39% reduction in area in the 0 mm CSS to 0% in the 50 mm group. Conversely, the wounds closed with 0, 1 and 5 mm EDC groups exhibited significantly less wound contraction than the 50 mm group. Engraftment of human cells occurred in 86%, 83%, and 83% of the wounds treated with CSS fabricated using 0, 1, and 5 mm EDC cross-linked sponges, respectively, which were significantly higher engraftment rates than the 50 mm group (17%). These data suggest that low concentrations of EDC can be used to improve the biochemical stability of the C-GAG component of CSS in vitro, and promote stable wound closure.

Animals↗

PubMed2006

Improvement of epidermal barrier properties in cultured skin substitutes after grafting onto athymic mice.

Barrier function in cultured skin substitutes (CSS) prepared from human cell sources was measured by noninvasive (surface hydration, transepidermal water loss) and invasive methods (water permeation, niacinamide flux) before and after grafting onto athymic mice. In vitro measurements were made on days 7 and 14. Although three of the four measures of barrier function improved markedly from day 7 to 14, the values obtained were still far from those obtained with native human skin controls. Additional CSS were grafted onto athymic mice on day 14, and skin was harvested 2 and 6 weeks after grafting. Grafting brought about a substantial decrease in all measurements by 2 weeks and almost complete normalization of barrier function after 6 weeks. The most sensitive measure of this recovery was niacinamide permeability, which decreased from (280 +/- 40) x 10(-4) cm/h in vitro to (17 +/- 30) x 10(-4) cm/h 2 weeks after grafting and (5 +/- 2) x 10(-4) cm/h 6 weeks after grafting, versus control values of (2 +/- 2) x 10(-4) cm/h in human cadaver skin and (0.6 +/- 0.4) x 10(-4) cm/h in human epidermal membrane prepared from freshly excised breast skin. These results demonstrate the reformation of epidermal barrier function after transplantation and provide insights for the development of a functional epidermal barrier in CSS in vitro.

Animals↗

PubMed2006

EDC cross-linking improves skin substitute strength and stability.

Collagen-based scaffolds are extensively utilized as an analog for the extracellular matrix in cultured skin substitutes (CSS). To improve the mechanical properties and degradation rates of collagen scaffolds, chemical cross-linking is commonly employed. In this study, freeze-dried collagen-GAG sponges were crosslinked with increasing concentrations of 1-ethyl-3-3-dimethylaminopropylcarbodiimide hydrochloride (EDC; 0, 1, 5, 10, 50mm). Cross-linking with EDC at concentrations >1mm was shown to greatly decrease degradation by collagenase up to 21 days. Ultimate tensile strength (UTS) of acellular collagen sponges scaled positively with EDC concentration up to 10mm. At 50mm EDC, the UTS decreased dramatically likely due to the brittle nature of the highly crosslinked material. Co-culture of human fibroblasts (HF) and keratinocytes (HK) on these substrates reveals an apparent cytotoxicty of the EDC at high concentrations with reduced cell viability and poor cellular organization in CSS fabricated with scaffolds crosslinked with 10 or 50mm EDC. From the data gathered in this study, intermediate concentrations of EDC, specifically 5mm, increase collagen sponge stability and strength while providing an environment in which HF and HK can attach, proliferate and organize in a manner conducive to dermal and epidermal regeneration.

Coculture Techniques↗

PubMed2006

Keratin expression in cultured skin substitutes suggests that the hyperproliferative phenotype observed in vitro is normalized after grafting.

Cultured skin substitutes, consisting of fibroblasts and keratinocytes in a biopolymer matrix, are an adjunctive treatment for full thickness burn wounds. Previous studies revealed that cultured skin substitutes in vitro exhibit a gene expression profile similar to hyperproliferative skin or wounded normal skin. In the present study, we sought to determine whether this hyperproliferative phenotype is maintained after healing of grafted cultured skin in vivo. Immunohistochemistry was used to localize multiple keratin proteins in native human skin, and in cultured skin substitutes in vitro and after grafting to athymic mice. Keratin 6, keratin 16, and keratin 17, which are known to be upregulated during keratinocyte activation and in hyperproliferative epidermis, were highly expressed in cultured skin substitutes in vitro. These proteins were low or undetectable in native human skin, and were reduced in cultured skin after grafting. Conversely, keratin 15, which is downregulated in activated keratinocytes, was not detected in cultured skin substitutes in vitro but was upregulated after grafting to mice. The results confirm previous observations suggesting a hyperproliferative or activated phenotype in cultured skin substitutes in vitro, similar to wounded native skin. After grafting to athymic mice, the expression patterns suggest a normalization of cultured skin substitutes to a phenotype more closely resembling uninjured human skin.

Adult↗

PubMed2006

Norepinephrine modulates the inflammatory and proliferative phases of wound healing.

BACKGROUND: Injury results in the massive release of norepinephrine (NE) into the peripheral circulation. Recent investigations have demonstrated functional adrenoreceptors on the cellular mediators of cutaneous wound healing and NE-induced phenotypic alterations in immune cells have been demonstrated in vitro. Despite this, there is little description of how NE might alter the phases of wound healing in vivo. The purpose of this study was to compare cutaneous wound healing in norepinephrine-intact and norepinephrine-depleted mice. METHODS: Norepinephrine-depleted (NED) mice were generated by chemical axotomy with 6-hydroxydopamine and compared with norepinephrine-intact (NEI) animals (n = 6-12 per group, per time point). Using an excisional wound model, neutrophil recruitment was measured by myeloperoxidase assay. Macrophage recruitment and angiogenesis were measured by immunohistochemistry and re-epithelialization was determined histologically. The development of incisional wound disruption strength was determined over time. Finally, macrophage scavenger function was assessed by an in vitro latex bead phagocytosis assay. RESULTS: Wounds from NEI mice demonstrated greater neutrophil infiltration than NED wounds (24, 72 hours; p < 0.05). Wound macrophage recruitment was initially higher in NEI animals (24 hours, p < 0.05), but was eventually surpassed by that of NED animals (120 hours, p < 0.05). Angiogenesis was decreased while re-epithelialization was accelerated in NEI animals (p < 0.05). Wound disruption strength and macrophage scavenger function were unaltered between NED and NEI mice. CONCLUSIONS: Norepinephrine modulates the inflammatory and proliferative phases of wound healing in a temporally defined, cell-specific manner. By increasing recruitment of innate immune cells and expediting wound closure, norepinephrine appears to play a protective role in defense against infection.

Animals↗

PubMed2006

Quantitative assay for quality assurance of human cells for clinical transplantation.

Transplantation of human cells after isolation and culture has become an important alternative for treatment of acute or chronic skin wounds. To increase the efficacy and reduce cost for transplantation of skin cells, more efficient and accurate techniques for evaluation of cell proliferation are needed. Hemocytometer counts provide a valid assessment of cell proliferation and viability, but they are very labor intensive and require removal of the cells from their substrate. In this study, hemocytometer counts were compared with a fluorometric assay (n = 21 per condition) that uses the commercially available reagent alamarBlue, which is reduced to a fluorescent substrate by cellular dehydrogenases. Human epidermal keratinocytes were inoculated at 200, 600, 2000, and 6000 cells/cm2 incubated for 6 days in modified MCDB 153 medium. Alamar Blue was incubated with cells for 2 h at 37 degrees C, and fluorescence was measured with a microplate reader at 590 nm. Hemocytometer counts (x10(-4)) from the respective cell inoculation densities were 0.30 +/- 0.04, 1.07 +/- 0.10, 6.37 +/- 0.62, and 16.99 +/- 0.96. Fluorescence values (x10(-3)) for the respective inoculation densities were 0.14 +/- 0.01, 0.34 +/- 0.02, 1.20 +/- 0.09, and 1.79 +/- 0.12. Regression analysis showed a statistical significant (p < 0.0001) correlation (r2 = 0.87) between cell counts and optical density from the alamarBlue assay. These data demonstrate that alamarBlue provides a valid substitute for cell counts to assess cell proliferation before clinical transplantation of engineered skin. AlamarBlue also allows repeated, nondamaging assessment of living cells over time. These advantages are expected to increase the validity and reliability of quality assurance standards for transplanted skin cells, and to increase the efficacy of healing of cutaneous wounds.

Biological Assay↗

PubMed2005

Differential expression of matrix metalloproteinase-1 in vitro corresponds to tissue morphogenesis and quality assurance of cultured skin substitutes.

OBJECTIVE: To determine if matrix metalloproteinase-1 (MMP-1) was involved in the premature degradation of the dermal component in cultured skin substitutes (CSS) prepared with cells from burn patients. METHODS AND RESULTS: CSS 645 and 647 were prepared from clinical human fibroblasts (HF) and keratinocytes (HK) that demonstrated premature degradation of collagen-glycosaminoglycan sponges in vitro. The control CSS were prepared from clinical HF and HK, CSS 648, and a pre-clinical cell strain, CSS 644 that did not degrade the sponges. Surface electrical capacitance measures surface hydration and was significantly higher for CSS 647 from days 9 through 14. MTT (3-[4,5-dimethylthiazol-2-yl]- diphenyltetrazolium bromide) conversion, an indicator of cellular viability was significantly lower for the 6-mm punch biopsies from CSS 645 and 647 at day 15 as compared to control CSS. MMP-1 protein levels measured by ELISA were significantly higher in medium from HF 645 and 647 than controls on the day of CSS inoculation. At day 14 of incubation, the mean MMP-1 concentration was significantly elevated in the medium from CSS 645 and 647 versus the controls, CSS 644 and 648. Western blots, and casein zymography demonstrated the presence of the latent and active forms of MMP-1 in the HF and CSS media, respectively. CONCLUSION: MMP-1 was significantly higher in the media from two of the four HF strains and CSS after a 24 h incubation period. Elevated MMP-1 coincided with premature degradation of the dermal substitute in vitro, and reduced numbers of CSS that met quality assurance standards for clinical transplantation.

Burns↗

PubMed2005

Microarray analysis of gene expression in cultured skin substitutes compared with native human skin.

Cultured skin substitutes (CSS), prepared using keratinocytes, fibroblasts, and biopolymers, can facilitate closure of massive burn wounds by increasing the availability of autologous tissue for grafting. But because they contain only two cell types, skin substitutes cannot replace all of the functions of native human skin. To better understand the physiological and molecular differences between CSS and native skin, we undertook a comprehensive analysis of gene expression in native skin, cultured keratinocytes, cultured fibroblasts, and skin substitutes using Affymetrix gene chip microarrays. Hierarchical tree clustering identified six major clusters of coordinately regulated genes, using a list of 1030 genes that were the most differentially expressed between groups. These clusters correspond to biomarker pools representing expression signatures for native skin, fibroblasts, keratinocytes, and cultured skin. The expression analysis revealed that entire clusters of genes were either up- or downregulated upon combination of fibroblasts and keratinocytes in cultured skin grafts. Further, several categories of genes were overexpressed in CSS compared with native skin, including genes associated with hyperproliferative skin or activated keratinocytes. The observed pattern of expression indicates that CSS in vitro, which display a well-differentiated epidermal layer, exhibit a hyperproliferative phenotype similar to wounded native skin.

Cluster Analysis↗

PubMed2004

Expression of human beta-defensins HBD-1, HBD-2, and HBD-3 in cultured keratinocytes and skin substitutes.

Defensins are effector molecules of the innate host defense system with antimicrobial activity against a variety of pathogens, including microorganisms commonly found in burn units. beta-Defensins are variably expressed in the epithelia of skin and other organs. Cultured skin substitutes (CSS) grafted to burn wounds lack a vascular plexus and are therefore more susceptible to microbial contamination than split thickness skin autograft. To investigate whether beta-defensins can contribute to host defense in CSS, we examined expression of human beta-defensins HBD-1, HBD-2, and HBD-3 in cultured keratinocytes and CSS from uninjured donors and burn patients. HBD-1 was expressed in all keratinocyte strains analyzed. HBD-2 expression in keratinocyte monolayers was highly variable but did not correlate with burn injury. HBD-3 was expressed at variable levels in all but one keratinocyte strain. CSS were prepared from two donors that lacked expression of HBD-2 in keratinocyte monolayers. All three genes were readily detected in CSS from both donors, suggesting up-regulation of HBD-2 and HBD-3. In sections of CSS, HBD-1, HBD-2, and HBD-3 proteins were localized to distinct epidermal regions. We conclude that beta-defensins can potentially contribute to innate immunity in CSS, but their levels may be too low to prevent contamination after grafting.

Adolescent↗

PubMed2003

Assessment of a silver-coated barrier dressing for potential use with skin grafts on excised burns.

Acticoat burn dressing is a silver-coated dressing with antimicrobial activity purported to reduce infection from environmental organisms in partial and full-thickness wounds. Acticoat was tested for activity as an antimicrobial treatment and as an antimicrobial barrier dressing in three in vitro assays. It was found that a modified disc assay method gave false negative results but in an assay in which bacteria were inoculated on top of samples of Acticoat, bacterial numbers were reduced, over time, with all microorganisms tested. Acticoat served as a barrier for bacteria, inoculated onto it, from contaminating the surface of an agar plate under the Acticoat. The data show that Acticoat has: antimicrobial capabilities, but to be effective hours of contact between Acticoat and the microorganisms are required; and the capacity to serve as an antimicrobial barrier dressing. These findings support the conclusion that Acticoat has activity to reduce microbial contamination of wounds from environmental sources.

Anti-Bacterial Agents↗

PubMed2002

Principles and practices for treatment of cutaneous wounds with cultured skin substitutes.

BACKGROUND: Skin substitutes prepared from cultured skin cells and biopolymers may reduce requirements for donor skin autograft, and have been shown to be effective in treatment of excised burns, burn scars, and congenital skin lesions. DATA SOURCES: Cultured skin substitutes (CSS) generate skin phenotypes (epidermal barrier, basement membrane) in the laboratory, and restore tissue function and systemic homeostasis. Healed skin is smooth, soft and strong, but develops irregular degrees of pigmentation. Quantitative analysis demonstrates that CSS closes 67 times the area of the donor skin, compared to less than 4 times for split-thickness skin autograft. CONCLUSIONS: CSS reduce requirements for donor skin autograft for closure of excised, full-thickness cutaneous wounds, and demonstrate qualitative outcome that is not different from meshed, split-thickness autograft. These results offer reductions in morbidity and mortality for the treatment of burns and chronic wounds, and for cutaneous reconstruction.

Biocompatible Materials↗

PubMed2002

Vitamin C regulates keratinocyte viability, epidermal barrier, and basement membrane in vitro, and reduces wound contraction after grafting of cultured skin substitutes.

Cultured skin substitutes have become useful as adjunctive treatments for excised, full-thickness burns, but no skin substitutes have the anatomy and physiology of native skin. Hypothetically, deficiencies of structure and function may result, in part, from nutritional deficiencies in culture media. To address this hypothesis, vitamin C was titrated at 0.0, 0.01, 0.1, and 1.0 mM in a cultured skin substitute model on filter inserts. Cultured skin substitute inserts were evaluated at 2 and 5 wk for viability by incorporation of 5-bromo-2'-deoxyuridine (BrdU) and by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) conversion. Subsequently, cultured skin substitute grafts consisting of cultured human keratinocytes and fibroblasts attached to collagen-glycosaminoglycan substrates were incubated for 5 wk in media containing 0.0 mM or 0.1 mM vitamin C, and then grafted to athymic mice. Cultured skin substitutes (n = 3 per group) were evaluated in vitro at 2 wk of incubation for collagen IV, collagen VII, and laminin 5, and through 5 wk for epidermal barrier by surface electrical capacitance. Cultured skin substitutes were grafted to full-thickness wounds in athymic mice (n = 8 per group), evaluated for surface electrical capacitance through 6 wk, and scored for percentage original wound area through 8 wk and for HLA-ABC-positive wounds at 8 wk after grafting. The data show that incubation of cultured skin substitutes in medium containing vitamin C results in greater viability (higher BrdU and MTT), more complete basement membrane development at 2 wk, and better epidermal barrier (lower surface electrical capacitance) at 5 wk in vitro. After grafting, cultured skin substitutes with vitamin C developed functional epidermal barrier earlier, had less wound contraction, and had more HLA-positive wounds at 8 wk than without vitamin C. These results suggest that incubation of cultured skin substitutes in medium containing vitamin C extends cellular viability, promotes formation of epidermal barrier in vitro, and promotes engraftment. Improved anatomy and physiology of cultured skin substitutes that result from nutritional factors in culture media may be expected to improve efficacy in treatment of full-thickness skin wounds.

Animals↗

PubMed2002

Human dermal microvascular endothelial cells form vascular analogs in cultured skin substitutes after grafting to athymic mice.

Cultured skin substitutes (CSS) consisting of autologous fibroblasts and keratinocytes combined with biopolymers are an adjunctive treatment for large excised burns. CSS containing two cell types are limited by anatomical deficiencies, including lack of a vascular plexus, leading to slower vascularization after grafting than split-thickness autograft. To address this limitation, CSS were prepared containing human keratinocytes, fibroblasts, and dermal microvascular endothelial cells (HDMEC) isolated from a single skin sample. After 16 days in culture, control CSS and CSS containing HDMEC (CSS+EC) were grafted to full-thickness wounds in athymic mice. In CSS+EC in vitro, HDMEC persisted in the dermal substitutes and formed multicellular aggregates. One wk after grafting, HDMEC in CSS+EC organized into multicellular structures, some containing lumens. By 4 wk after grafting, HDMEC were found in linear and circular organizations resembling vascular analogs associated with basement membrane deposition. In some cases, colocalization of HDMEC with mouse perivascular cells was observed. The results demonstrate HDMEC transplantation in a clinically relevant cultured skin model, persistence of HDMEC after grafting, and HDMEC organization into vascular analogs in vitro and in vivo. All cells were derived from the same donor tissue, indicating the feasibility of preparing CSS containing autologous HDMEC for grafting to patients.

Adolescent↗

PubMed2002

Cultured skin substitutes reduce donor skin harvesting for closure of excised, full-thickness burns.

OBJECTIVE: Comparison of cultured skin substitutes (CSS) and split-thickness skin autograft (AG) was performed to assess whether donor-site harvesting can be reduced quantitatively and whether functional and cosmetic outcome is similar qualitatively in the treatment of patients with massive cutaneous burns. SUMMARY BACKGROUND DATA: Cultured skin substitutes consisting of collagen-glycosaminoglycan substrates populated with autologous fibroblasts and keratinocytes have been shown to close full-thickness skin wounds in preclinical and clinical studies with acceptable functional and cosmetic results. METHODS: Qualitative outcome was compared between CSS and AG in 45 patients on an ordinal scale (0, worst; 10, best) with primary analyses at postoperative day 28 and after about 1 year for erythema, pigmentation, pliability, raised scar, epithelial blistering, and surface texture. In the latest 12 of the 45 patients, tracings were performed of donor skin biopsies and wounds treated with CSS at postoperative days 14 and 28 to calculate percentage engraftment, the ratio of closed wound:donor skin areas, and the percentage of total body surface area closed with CSS. RESULTS: Measures of qualitative outcome of CSS or AG were not different statistically at 1 year after grafting. Engraftment at postoperative day 14 exceeded 75% in the 12 patients evaluated. The ratio of closed wound:donor skin areas for CSS at postoperative day 28 was significantly greater than for conventional 4:1 meshed autografts. The percentage of total body surface area closed with CSS at postoperative day 28 was significantly less than with AG. CONCLUSIONS: The requirement for harvesting of donor skin for CSS was less than for conventional skin autografts. These results suggest that acute-phase recovery of patients with extensive burns is facilitated and that complications are reduced by the use of CSS together with conventional skin grafting.

Burns↗

PubMedDate not supplied

Engineered skin substitutes: practices and potentials.

Wound healing can be problematic in several clinical settings because of massive tissue injury (burns), wound healing deficiencies (chronic wounds), or congenital conditions and diseases. Engineered skin substitutes have been developed to address the medical need for wound coverage and tissue repair. Currently, no engineered skin substitute can replace all of the functions of intact human skin. A variety of biologic dressings and skin substitutes have however contributed to improved outcomes for patients suffering from acute and chronic wounds. These include acellular biomaterials and composite cultured skin analogs containing allogeneic or autologous cultured skin cells.

Humans↗

PubMedDate not supplied

Regulation of cutaneous pigmentation by titration of human melanocytes in cultured skin substitutes grafted to athymic mice.

Pigmentation of healed cultured skin substitutes in burn patients is frequently irregular and unpredictable which compromises solar protection and the patient's self-image. To address these morbidities, human fibroblasts were inoculated on a collagen-glycosaminoglycan substrate followed 1 day later by the addition of keratinocytes at 1.1 x 10(6)/cm2 combined with either 0, 1.1 x 10(2), 1.1 x 10(3), or 1.1 x 10(4) melanocytes/cm2. The skin substitutes were incubated in vitro for 3 weeks and grafted to athymic mice. In vitro, the number of L-Dopa-positive melanocytes in the skin substitutes increased proportionately to the number of melanocytes inoculated. The melanocytes localized to the basal epidermis when labeled for MEL-5. The skin substitutes with 1.1 x 10(4) melanocytes/cm2 were significantly darker than other groups in vitro by chromameter evaluation. By 12 weeks after grafting, the cultured skin ranged from no pigment in the control group, to 75% pigmented area in the 1.1 x 10(3) melanocytes/cm2 group, to complete pigmentation in the 1.1 x 10(4) melanocytes/cm2 group. In vivo, the mean chromameter values were significantly darker for the grafts with 1.1 x 10(3) and 1.1 x 10(4) melanocytes/cm2. These results suggest that complete restoration of cutaneous pigmentation can be accomplished by addition of between 0.1 and 1.0 x 10(4) melanocytes/cm2 to skin substitutes.

Animals↗

PubMedDate not supplied

Overexpression of vascular endothelial growth factor accelerates early vascularization and improves healing of genetically modified cultured skin substitutes.

Cultured skin substitutes (CSS) lack a vascular plexus, leading to slower vascularization after grafting than split-thickness skin autograft. CSS containing keratinocytes genetically modified to overexpress vascular endothelial growth factor (VEGF) were previously shown to exhibit enhanced vascularization up to 2 weeks after grafting to athymic mice. The present study examines whether enhanced vascularization compared with controls persists after stable engraftment is achieved and analyzes VEGF expression, wound contraction, and engraftment. Control and VEGF-modified (VEGF+) CSS were grafted onto full-thickness wounds in athymic mice. VEGF expression was detected in VEGF+ CSS 14 weeks after grafting. Graft contraction was significantly lower in VEGF+ CSS compared with controls, suggesting more stable engraftment and better tissue development. Positive HLA-ABC staining, indicating persistence of human cells, was seen in 86.7% (13/15) of grafted VEGF+ CSS, compared with 58.3% (7/12) of controls. Differences in dermal vascularization between control and VEGF+ grafts were significant 1 week after surgery, but not at later times. However, the distribution of vessels was different, with more vessels in the upper dermis of VEGF+ grafts. These results suggest that VEGF overexpression in genetically modified CSS acts to accelerate early graft vascularization and can contribute to improved healing of full-thickness skin wounds.

Adult↗

PubMedDate not supplied

Vascular endothelial growth factor overexpression increases vascularization by murine but not human endothelial cells in cultured skin substitutes grafted to athymic mice.

Cultured skin substitutes (CSS) consisting of fibroblasts, keratinocytes, and biopolymers are an adjunctive treatment for large burns. Because CSS lack a vascular plexus, they vascularize more slowly than split-thickness autografts. Previously, CSS were prepared with dermal microvascular endothelial cells (ECs), which formed vascular analogs at a low frequency but did not contribute to increased vascularization after grafting. The present study addressed whether keratinocytes genetically modified to overexpress vascular endothelial growth factor (VEGF), an endothelial cell mitogen, could improve the persistence and organization of ECs in CSS. CSS were prepared with control or VEGF-modified keratinocytes, with (CSS + ECs) or without added ECs, and were grafted to full-thickness wounds in athymic mice. Elevated VEGF expression was detected in VEGF-modified CSS and CSS + ECs compared with controls, but no significant difference in EC density in vitro was observed. After grafting, VEGF-modified CSS and CSS + ECs showed enhanced vascularization, and organization of human ECs into multicellular structures in CSS + ECs was observed. However, VEGF overexpression did not significantly enhance the proliferation of human ECs, suggesting that other factors may be required. Improved persistence and organization of human ECs in vitro will likely be required for their participation in vascularization of CSS + ECs after grafting.

Animals↗