Long-Term Controlled Growth Factor Release Using Layer-by-Layer Assembly for the Development of In Vivo Tissue-Engineered Blood Vessels.

The development of a well-designed tissue-engineered blood vessel (TEBV) still remains a challenge. In recent years, approaches in which the host response to implanted biomaterials is used to generate vascular constructs within the patient's body have gained increasing interest. The delivery of growth factors to these in situ-engineered vascular grafts might enhance myofibroblast recruitment and the secretion of essential extracellular matrix proteins, thereby optimizing their functional properties. Layer-by-layer (LbL) coating has emerged as an innovative technology for the controlled delivery of growth factors in tissue engineering applications. In this study, we combined the use of surface-etched polymeric rods with LbL coatings to control the delivery of TGF-ß1, PDGF-BB, and IGF-1 and steer the foreign body response toward the formation of a functional vascular graft. Results showed that the regenerated tissue is composed of elastin, glycosaminoglycans, and circumferentially oriented collagen fibers, without calcification or systemic spill of the released growth factors. Functional controlled delivery was observed, whereas myofibroblast-rich tissue capsules were formed with enhanced collagen and elastin syntheses using TGF-ß1 and TGF-ß1/PDGF-BB releasing rods, when compared to control rods that were solely surface-engineered by chloroform etching. By combining our optimized LbL method and surface-engineered rods in an in vivo bioreactor approach, we could regulate the fate and ECM composition of in situ-engineered vascular grafts to create a successful in vivo vascular tissue-engineered replacement.

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-ß1, PDGF-ßß, 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-ßß and IGF-1 release, and dual release with TGF-ß1 from COX rods, showed higher cell proliferation when compared to COX rods alone. A substantial increase in α-smooth muscle actin (α-SMA) is also observed in GF releasing COX rods, with TGF-ß1 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-ß1 and IGF-1 providing the highest secretion. TGF-ß1 and IGF-1 releasing COX rods induced higher Glycosaminoglycan (GAG)/DNA amounts than the other GF releasing COX rods. As PDGF-ßß and TGF-ß1/PDGF-ßß 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.

Biological activity of human mesenchymal stromal cells on polymeric electrospun scaffolds.

Electrospinning provides a simple robust method to manufacture scaffolds for tissue engineering applications. Though varieties of materials can be used, optimization and biocompatibility tests are required to provide functional tissue regeneration. Moreover, many studies are limited to 2D electrospun constructs rather than 3D templates due to the production of high density packed fibres, which result in poor cell infiltration. Here, we optimised electrospinning parameters for three different polymers: poly(ε-caprolactone) (PCL), polylactic acid (PLA) and poly(ethylene oxide terephthalate)/poly(butylene terephthalate) (PA) copolymers. Human mesenchymal stromal cells (hMSCs) were cultured on scaffolds for 14 days to study the scaffolds' biocompatibility and their multi-lineage differentiation potential or maintenance of stemness in the absence of chemical stimuli. For all scaffolds, a high and stable metabolic activity was measured throughout the culture time with a high proliferation rate compared to day 1 (PCL 5.8-, PLA 4-, PA 4.9-fold). The metabolism of hMSCs was also measured through glucose and lactate concentrations, showing no cytotoxic levels up to 14 days. Total glycosaminoglycan (GAG) production was the highest in PA electrospun scaffolds. When normalized to DNA, GAG production was the highest in PLA and PA scaffolds. All scaffolds were prone to differentiate to an osteogenic lineage, with PCL providing the highest alkaline phosphatase and collagen type Ia gene upregulation. As PA had the most stable fibre formation, it was chosen as a template to further incorporate stromal cell-derived factor-1 (SDF-1) and granulocyte colony-stimulating factor (G-CSF), and stimulate higher hMSC infiltration. These scaffolds provided significantly higher hMSC infiltration than normal PA scaffolds. In conclusion, our optimized biocompatible electrospun scaffolds have shown promising regulation of hMSC fate. When combined with migratory stimulating cytokines, these scaffolds may overcome the known challenges of poor cellular infiltration typical of micro- and nano-fibrillary random meshes.

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 ßß, 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.

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

Tissue engineered blood vessels (TEBVs) hold great promise for clinical use in patients with end stage renal disease (ESRD) requiring vascular access for hemodialysis. A promising way to make TEBVs is to exploit foreign body response (FBR) of polymeric rods used as templates. However, since the FBR predominantly involves bone-marrow (BM) derived cells and ESRD coincides with impaired function of BM, it is important to assess the generation of TEBVs in conditions of renal failure. To this end, we implanted polymer rods in the subcutis of rats after BM-transplantation with GFP-labeled BM cells in a model of chronic kidney disease (CKD). At 3 weeks after implantation, rods were encapsulated by tissue capsule (TC) composed of collagen, myofibroblasts and macrophages. On average, 13% of CD68+ macrophages were GFP+, indicating BM origin. Macrophage-to-myofibroblasts differentiation appeared to play an important role in TC formation as 26% of SMA+/GFP+ myofibroblasts co-expressed the macrophage marker CD68. Three weeks after rod implantation, the cellular response changed towards tissue repair, characterized by 40% increase in CD68+/CD163+ repair associated macrophages and 95% increase in TGFß and IL10 gene expression as compared to TCs harvested at 1 week. These results show that both BM derived and tissue resident cells, contribute to TC formation, whereas macrophages serve as precursors of myofibroblasts in mature TCs. Finally, the presence of CKD did not significantly alter the process of TC formation, which holds the potential to support our approach for future clinical use in ESRD patients.

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.

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.

Tailoring the foreign body response for in situ vascular tissue engineering.

This study describes a screening platform for a guided in situ vascular tissue engineering approach. Polymer rods were developed that upon 3 weeks of subcutaneous implantation evoke a controlled inflammatory response culminating in encapsulation by a tube-shaped autologous fibrocellular tissue capsule, which can form a basis for a tissue-engineered blood vessel. Rods of co-polymer were produced using different ratios of poly(ethylene oxide terephthalate) and poly(butylene terephthalate) to create a range of physicochemical properties. In addition, a set of different physical, chemical, and biological surface modifications were tested on their ability to actively steer this tissue capsule formation using a rat model as testing platform. Tissue capsules were mainly composed of circumferentially aligned collagen and myofibroblasts. Different implant material resulted in distinct differences in tissue capsule formation. Compared to its unmodified counterparts, all surface modifications resulted in increased wall thickness, collagen, and myofibroblasts. Oxygen plasma-treated rods resulted in loose tissue arrangement, collagen, and collagen/TGF-ß-coated rods yielded thick, collagen-rich, densely packed tissue capsules, though with a random distribution of myofibroblasts. In contrast, chloroform-etched rods provided homogenous densely packed tissue capsules, completely populated by myofibroblasts. In conclusion, by varying the implant's surface characteristics, tissue capsule composition, cell distribution, and tissue arrangement could be tailored, enabling controlled guidance of the tissue response for in vivo vascular tissue engineering.

Towards an in vitro model mimicking the foreign body response: tailoring the surface properties of biomaterials to modulate extracellular matrix.

Despite various studies to minimize host reaction following a biomaterial implantation, an appealing strategy in regenerative medicine is to actively use such an immune response to trigger and control tissue regeneration. We have developed an in vitro model to modulate the host response by tuning biomaterials' surface properties through surface modifications techniques as a new strategy for tissue regeneration applications. Results showed tunable surface topography, roughness, wettability, and chemistry by varying treatment type and exposure, allowing for the first time to correlate the effect of these surface properties on cell attachment, morphology, strength and proliferation, as well as proinflammatory (IL-1ß, IL-6) and antiinflammatory cytokines (TGF-ß1, IL-10) secreted in medium, and protein expression of collagen and elastin. Surface microstructuring, derived from chloroform partial etching, increased surface roughness and oxygen content. This resulted in enhanced cell adhesion, strength and proliferation as well as a balance of soluble factors for optimum collagen and elastin synthesis for tissue regeneration. By linking surface parameters to cell activity, we could determine the fate of the regenerated tissue to create successful soft tissue-engineered replacement.