Assessing the Effects of VEGF Releasing Microspheres on the Angiogenic and Foreign Body Response to a 3D Printed Silicone-Based Macroencapsulation Device.

Macroencapsulation systems have been developed to improve islet cell transplantation but can induce a foreign body response (FBR). The development of neovascularization adjacent to the device is vital for the survival of encapsulated islets and is a limitation for long-term device success. Previously we developed additive manufactured multi-scale porosity implants, which demonstrated a 2.5-fold increase in tissue vascularity and integration surrounding the implant when compared to a non-textured implant. In parallel to this, we have developed poly(ε-caprolactone-PEG-ε-caprolactone)-b-poly(L-lactide) multiblock copolymer microspheres containing VEGF, which exhibited continued release of bioactive VEGF for 4-weeks in vitro. In the present study, we describe the next step towards clinical implementation of an islet macroencapsulation device by combining a multi-scale porosity device with VEGF releasing microspheres in a rodent model to assess prevascularization over a 4-week period. An in vivo estimation of vascular volume showed a significant increase in vascularity (* p = 0.0132) surrounding the +VEGF vs. -VEGF devices, however, histological assessment of blood vessels per area revealed no significant difference. Further histological analysis revealed significant increases in blood vessel stability and maturity (** p = 0.0040) and vessel diameter size (*** p = 0.0002) surrounding the +VEGF devices. We also demonstrate that the addition of VEGF microspheres did not cause a heightened FBR. In conclusion, we demonstrate that the combination of VEGF microspheres with our multi-scale porous macroencapsulation device, can encourage the formation of significantly larger, stable, and mature blood vessels without exacerbating the FBR.

Post-loading of proangiogenic growth factors in PLGA microspheres.

Active self-encapsulation (ASE) is a recently developed post-loading method based on absorption of (positively charged) proteins in microporous PLGA microspheres loaded with negatively charged polysaccharides (trapping agents). The aim of this study was to investigate ASE for simultaneous loading and controlled release of multiple growth factors. For this purpose, vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF) and insulin-like growth factor (IGF) were loaded in microspheres containing high molecular weight dextran sulfate (HDS) as trapping agent; loading was performed in a concentrated growth factor solution of low ionic strength and of pH 5 under conditions at which the proteins are positively charged. Subsequent pore closure was induced by incubation of the growth factor-loaded microspheres at 42.5 °C, i.e. above the Tg of (hydrated) PLGA (~30 °C). A 1:1:1 combination of VEGF, FGF and IGF was loaded with high loading (4.3%) and loading efficiency (91%). The in vitro release kinetics and bioactivity of loaded growth factors were studied for 4 weeks using ELISA and an endothelial cell proliferation assay, respectively. While IGF was released quickly, VEGF and FGF were continuously released for 4 weeks in their bioactive form, whereby a growth factor combination had a synergistic angiogenic effect. Therefore, ASE is a suitable method for co-loading growth factors which can provide sustained release profiles of bioactive growth factors, which is attractive for vascularization of biomaterial implants.

Phenylglyoxaldehyde-Functionalized Polymeric Sorbents for Urea Removal from Aqueous Solutions.

For realization of a wearable artificial kidney based on regeneration of a small volume of dialysate, efficient urea removal from dialysate is a major challenge. Here a potentially suitable polymeric sorbent based on phenylglyoxaldehyde (PGA), able to covalently bind urea under physiological conditions, is described. Sorbent beads containing PGA groups were obtained by suspension polymerization of either styrene or vinylphenylethan-1-one (VPE), followed by modification of the aromatic groups of poly(styrene) and poly(VPE) into PGA. It was found that PGA-functionalized sorbent beads had maximum urea binding capacities of 1.4-2.2 mmol/g and removed ∼0.6 mmol urea/g in 8 h at 37 °C under static conditions from urea-enriched phosphate-buffered saline, conditions representative of dialysate regeneration. This means that the daily urea production of a dialysis patient can be removed with a few hundred grams of this sorbent which, is an important step forward in the development of a wearable artificial kidney.

A Ninhydrin-Type Urea Sorbent for the Development of a Wearable Artificial Kidney.

The aim of this study is to develop polymeric chemisorbents with a high density of ninhydrin groups, able to covalently bind urea under physiological conditions and thus potentially suitable for use in a wearable artificial kidney. Macroporous beads are prepared by suspension polymerization of 5-vinyl-1-indanone (vinylindanone) using a 90:10 (v/v) mixture of toluene and nitrobenzene as a porogen. The indanone groups are subsequently oxidized in a one-step procedure into ninhydrin groups. Their urea absorption kinetics are evaluated under both static and dynamic conditions at 37 °C in simulated dialysate (urea in phosphate buffered saline). Under static conditions and at a 1:1 molar ratio of ninhydrin: urea the sorbent beads remove ≈0.6-0.7 mmol g-1 and under dynamic conditions and at a 2:1 molar excess of ninhydrin ≈0.6 mmol urea g-1 sorbent in 8 h at 37 °C, which is a step toward a wearable artificial kidney.

Vascular Endothelial Growth Factor-Releasing Microspheres Based on Poly(ε-Caprolactone-PEG-ε-Caprolactone)-b-Poly(L-Lactide) Multiblock Copolymers Incorporated in a Three-Dimensional Printed Poly(Dimethylsiloxane) Cell Macroencapsulation Device.

Pancreatic islet transplantation is a promising advanced therapy that has been used to treat patients suffering from diabetes type 1. Traditionally, pancreatic islets are infused via the portal vein, which is subsequently intended to engraft in the liver. Severe immunosuppressive treatments are necessary, however, to prevent rejection of the transplanted islets. Novel approaches therefore have focused on encapsulation of the islets in biomaterial implants which can protect the islets and offer an organ-like environment. Vascularization of the device's surface is a prerequisite for the survival and proper functioning of transplanted pancreatic islets. We are pursuing a prevascularization strategy by incorporation of vascular endothelial growth factor (VEGF)-loaded microspheres in 3-dimensional printed poly(dimethylsiloxane)-based devices prior to their prospective loading with transplanted cells. Microspheres (~50 µm) were based on poly(ε-caprolactone-PEG-ε-caprolactone)-b-poly(L-lactide) multiblock copolymers and were loaded with 10 µg VEGF/mg microspheres, and subsequently dispersed in a hyaluronic acid carrier liquid. In vitro release studies at 37°C demonstrated continuous release of fully bioactive VEGF for 4 weeks. In conclusion, our results demonstrate that incorporation of VEGF-releasing microspheres ensures adequate release of VEGF for a time window of 4 weeks, which is attractive in view of the vascularization of artificial pancreas implants.

Sustained Release of Vascular Endothelial Growth Factor from Poly(ε-caprolactone-PEG-ε-caprolactone)-b-Poly(l-lactide) Multiblock Copolymer Microspheres.

Vascular endothelial growth factor (VEGF) is the major regulating factor for the formation of new blood vessels, also known as angiogenesis. VEGF is often incorporated in synthetic scaffolds to promote vascularization and to enhance the survival of cells that have been seeded in these devices. Such applications require sustained local delivery of VEGF of around 4 weeks for stable blood vessel formation. Most delivery systems for VEGF only provide short-term release for a couple of days, followed by a release phase with very low VEGF release. We now have developed VEGF-loaded polymeric microspheres that provide sustained release of bioactive VEGF for 4 weeks. Blends of two swellable poly(ε-caprolactone)-poly(ethylene glycol)-poly(ε-caprolactone)-b-poly(l-lactide) ([PCL-PEG-PCL]-b-[PLLA])-based multiblock copolymers with different PEG content and PEG molecular weight were used to prepare the microspheres. Loading of the microspheres was established by a solvent evaporation-based membrane emulsification method. The resulting VEGF-loaded microspheres had average sizes of 40-50 µm and a narrow size distribution. Optimized formulations of a 50:50 blend of the two multiblock copolymers had an average VEGF loading of 0.79 ± 0.09%, representing a high average VEGF loading efficiency of 78 ± 16%. These microspheres released VEGF continuously over 4 weeks in phosphate-buffered saline pH 7.4 at 37 °C. This release profile was preserved after repeated and long-term storage at -20 °C for up to 9 months, thereby demonstrating excellent storage stability. VEGF release was governed by diffusion through the water-filled polymer matrix, depending on PEG molecular weight and PEG content of the polymers. The bioactivity of the released VEGF was retained within the experimental error in the 4-week release window, as demonstrated using a human umbilical vein endothelial cells proliferation assay. Thus, the microspheres prepared in this study are suitable for embedment in polymeric scaffolds with the aim of promoting their functional vascularization.