Contribution of bone marrow-derived cells to in situ engineered tissue capsules in a rat model of chronic kidney disease

Nephrolog

Enhancement of synthesis of extracellular matrix proteins on retinoic acid loaded electrospun scaffolds

Electrospinning is a renowned technique for the generation of ultrafine, micro- and nanoscale fibres due to its simplicity, versatility and tunability. Owing to its adaptability to a wide selection of materials and scaffold architectures, electrospun meshes have been developed as biocompatible scaffolds and drug delivery systems for tissue engineering. Here, we developed a drug delivery scaffold by electrospinning poly(ε-caprolactone) (PCL) directly blended with a therapeutic agent, retinoic acid (RA), at different concentrations. The release profile, DNA, and elastin analysis of direct and transwell seeded RA-loaded PCL electrospun scaffolds showed desirable controlled release at 15 kV fabrication, with 0.01% RA as the optimum concentration. The selected 0.01% (w/v) RA-loaded PCL meshes were further analysed using five different seeding cultures to investigate and extensively distinguish the effects of RA release with or without cell contact to the PCL electrospun meshes for cell morphology, proliferation and extracellular matrix (ECM) protein secretion of collagen and elastin. Upon exposure to RA-loaded PCL scaffolds, an increase of human dermal fibroblast (HDF) proliferation was observed. In contrast, human mesenchymal stromal cell (hMSC) cultures showed a decrease in cell proliferation. For both hMSC and HDF cultures, exposure to RA-loaded PCL scaffolds provided a significant increase in elastin production per cell. For collagen expression, a slight increase was measured and was outperformed by the 3D geometry stimulation from PCL scaffolds. In contrast to hMSCs, HDFs showed enhanced stress actin fibres in cultures with RA-loaded PCL scaffolds. Both cell types exhibited more vinculin expression when seeded to RA-loaded PCL scaffolds. Hence, electrospun scaffolds releasing RA in a controlled manner were able to regulate cell proliferation, morphology and ECM secretion, and present an attractive approach for optimizing tissue regeneration

Control Delivery of Multiple Growth Factors to Actively Steer Differentiation and Extracellular Matrix Protein Production

In tissue engineering, biomaterials have been used to steer the host response. This determines the outcome of tissue regeneration, which is modulated by multiple growth factors (GFs). Hence, a sustainable delivery system for GFs is necessary to control tissue regeneration actively. A delivery technique of single and multiple GF combinations, using a layer-by-layer (LBL) procedure to improve tissue remodeling, is developed. TGF-beta 1, PDGF-beta beta, and IGF-1 are incorporated on tailor-made polymeric rods, which could be used as a tool for potential tissue engineering applications, such as templates to induce the formation of in situ tissue engineered blood vessels (TEBVs). Cell response is analyzed in vitro using rat and human dermal fibroblasts for cellular proliferation, fibroblast differentiation, and extracellular matrix (ECM) protein synthesis. Results revealed a higher loading efficiency and control release of GFs incorporated on chloroform and oxygen plasma-activated (COX) rods. Single PDGF-beta beta and IGF-1 release, and dual release with TGF-beta 1 from COX rods, showed higher cell proliferation when compared to COX rods alone. A substantial increase in alpha -smooth muscle actin (alpha -SMA) is also observed in GF releasing COX rods, with TGF-beta 1 COX rods providing the most pronounced differentiation. A significant increase in collagen and elastin synthesis is observed on all GF releasing COX rods compared to control, with COX rods releasing TGF-beta 1 and IGF-1 providing the highest secretion. TGF-beta 1 and IGF-1 releasing COX rods induced higher Glycosaminoglycan (GAG)/DNA amounts than the other GF releasing COX rods. As PDGF-beta beta and TGF-beta 1/PDGF-beta beta COX rods displayed the highest fibroblast attachment, these rods provided the highest total collagen and elastin production. The attractive results from efficiently incorporating single and multiple GFs on COX rods and their sustainable release to steer cellular behavior suggest a promising route to enrich the formation of in situ engineered tissues

Control Delivery of Multiple Growth Factors to Actively Steer Differentiation and Extracellular Matrix Protein Production

In tissue engineering, biomaterials have been used to steer the host response. This determines the outcome of tissue regeneration, which is modulated by multiple growth factors (GFs). Hence, a sustainable delivery system for GFs is necessary to control tissue regeneration actively. A delivery technique of single and multiple GF combinations, using a layer-by-layer (LBL) procedure to improve tissue remodeling, is developed. TGF-beta 1, PDGF-beta beta, and IGF-1 are incorporated on tailor-made polymeric rods, which could be used as a tool for potential tissue engineering applications, such as templates to induce the formation of in situ tissue engineered blood vessels (TEBVs). Cell response is analyzed in vitro using rat and human dermal fibroblasts for cellular proliferation, fibroblast differentiation, and extracellular matrix (ECM) protein synthesis. Results revealed a higher loading efficiency and control release of GFs incorporated on chloroform and oxygen plasma-activated (COX) rods. Single PDGF-beta beta and IGF-1 release, and dual release with TGF-beta 1 from COX rods, showed higher cell proliferation when compared to COX rods alone. A substantial increase in alpha -smooth muscle actin (alpha -SMA) is also observed in GF releasing COX rods, with TGF-beta 1 COX rods providing the most pronounced differentiation. A significant increase in collagen and elastin synthesis is observed on all GF releasing COX rods compared to control, with COX rods releasing TGF-beta 1 and IGF-1 providing the highest secretion. TGF-beta 1 and IGF-1 releasing COX rods induced higher Glycosaminoglycan (GAG)/DNA amounts than the other GF releasing COX rods. As PDGF-beta beta and TGF-beta 1/PDGF-beta beta COX rods displayed the highest fibroblast attachment, these rods provided the highest total collagen and elastin production. The attractive results from efficiently incorporating single and multiple GFs on COX rods and their sustainable release to steer cellular behavior suggest a promising route to enrich the formation of in situ engineered tissues

Sustained delivery of growth factors with high loading efficiency in a layer by layer assembly

Layer by layer (LBL) assembly has garnered considerable interest due to its ability to generate multifunctional films with high tunability and versatility in terms of substrates and polyelectrolytes, allowing the option to use complex devices and drugs. Polyelectrolytes, such as growth factors (GFs), are essential, but costly, delicate, biological molecules that have been used in various tissue regeneration applications. For this reason, the controlled drug delivery of efficiently loaded GFs via LBL assembly (GF-LBL) can contribute to the establishment of cost-effective biologically triggered biomedical applications. We have developed an LBL method to load GFs (specifically, transforming growth factor beta 1, platelet-derived growth factor beta beta, and insulin growth factor 1), with up to 90% efficiency approximately, by gas plasma surface activation and tuning the pH to increase the ionic strength of polyelectrolytes. Poly(styrenesulfonate) (PSS) and poly(ethyleneimine) (PEI) have been used to provide the initial necessary charge for multilayer build-up. Heparin and dextran sulphate have been investigated as counter polyelectrolytes to enhance the activity of GFs by protecting their ligands, where heparin resulted in the highest achievable loading efficiency for all GFs. Oxygen gas plasma and acidic pH levels also resulted in a significant increase in GF loading efficiency. The three GFs were released by diffusion and erosion in a controlled manner over lengthy time scales and the bioactivity was maintained for up to 14 days. When tested as implants in vitro, GF-LBL constructs increased fibroblast proliferation, influenced cell morphology and migration, and enhanced myofibroblast differentiation, indicating that the biological functionalities of the GFs were preserved. In conclusion, this developed LBL assembly method can provide a simple drug delivery system, which may yield more effective applications for tissue regeneration as well as biomedical sciences at large

Development and evaluation of in vivo tissue engineered blood vessels in a porcine model

BACKGROUND: There's a large clinical need for novel vascular grafts. Tissue engineered blood vessels (TEBVs) have great potential to improve the outcome of vascular grafting procedures. Here, we present a novel approach to generate autologous TEBV in vivo. Polymer rods were engineered and implanted, evoking an inflammatory response that culminates in encapsulation by a fibrocellular capsule. We hypothesized that, after extrusion of the rod, the fibrocellular capsule differentiates into an adequate vascular conduit once grafted into the vasculature. METHODS AND RESULTS: Rods were implanted subcutaneously in pigs. After 4 weeks, rods with tissue capsules grown around it were harvested. Tissue capsules were grafted bilaterally as carotid artery interposition. One and 4-week patency were evaluated by angiography whereupon pigs were sacrificed. Tissue capsules before and after grafting were evaluated on tissue remodeling using immunohistochemistry, RNA profiling and mechanical testing. Rods were encapsulated by thick, well-vascularized tissue capsules, composed of circumferentially aligned fibroblasts, collagen and few leukocytes, with adequate mechanical strength. Patency was 100% after 1 week and 87.5% after 4 weeks. After grafting, tissue capsules remodeled towards a vascular phenotype. Gene profiles of TEBVs gained more similarity with carotid artery. Wall thickness and αSMA-positive area significantly increased. Interestingly, a substantial portion of (myo)fibroblasts present before grafting expressed smooth muscle cell markers. While leukocytes were hardly present anymore, the lumen was largely covered with endothelial cells. Burst pressure remained stable after grafting. CONCLUSIONS: Autologous TEBVs were created in vivo with sufficient mechanical strength enabling vascular grafting. Grafts differentiated towards a vascular phenotype upon grafting

Development and evaluation of in vivo tissue engineered blood vessels in a porcine model

BACKGROUND: There's a large clinical need for novel vascular grafts. Tissue engineered blood vessels (TEBVs) have great potential to improve the outcome of vascular grafting procedures. Here, we present a novel approach to generate autologous TEBV in vivo. Polymer rods were engineered and implanted, evoking an inflammatory response that culminates in encapsulation by a fibrocellular capsule. We hypothesized that, after extrusion of the rod, the fibrocellular capsule differentiates into an adequate vascular conduit once grafted into the vasculature. METHODS AND RESULTS: Rods were implanted subcutaneously in pigs. After 4 weeks, rods with tissue capsules grown around it were harvested. Tissue capsules were grafted bilaterally as carotid artery interposition. One and 4-week patency were evaluated by angiography whereupon pigs were sacrificed. Tissue capsules before and after grafting were evaluated on tissue remodeling using immunohistochemistry, RNA profiling and mechanical testing. Rods were encapsulated by thick, well-vascularized tissue capsules, composed of circumferentially aligned fibroblasts, collagen and few leukocytes, with adequate mechanical strength. Patency was 100% after 1 week and 87.5% after 4 weeks. After grafting, tissue capsules remodeled towards a vascular phenotype. Gene profiles of TEBVs gained more similarity with carotid artery. Wall thickness and αSMA-positive area significantly increased. Interestingly, a substantial portion of (myo)fibroblasts present before grafting expressed smooth muscle cell markers. While leukocytes were hardly present anymore, the lumen was largely covered with endothelial cells. Burst pressure remained stable after grafting. CONCLUSIONS: Autologous TEBVs were created in vivo with sufficient mechanical strength enabling vascular grafting. Grafts differentiated towards a vascular phenotype upon grafting