Ex vivo proof-of-concept of end-to-end scaffold-enhanced laser-assisted vascular anastomosis of porcine arteries.

OBJECTIVE: The low welding strength of laser-assisted vascular anastomosis (LAVA) has hampered the clinical application of LAVA as an alternative to suture anastomosis. To improve welding strength, LAVA in combination with solder and polymeric scaffolds (ssLAVA) has been optimized in vitro. Currently, ssLAVA requires proof-of-concept in a physiologically representative ex vivo model before advancing to in vivo studies. This study therefore investigated the feasibility of ex vivo ssLAVA in medium-sized porcine arteries. METHODS: Scaffolds composed of poly(ε-caprolactone) (PCL) or poly(lactic-co-glycolic acid) (PLGA) were impregnated with semisolid solder and placed over coapted aortic segments. ssLAVA was performed with a 670-nm diode laser. In the first substudy, the optimum number of laser spots was determined by bursting pressure analysis. The second substudy investigated the resilience of the welds in a Langendorf-type pulsatile pressure setup, monitoring the number of failed vessels. The type of failure (cohesive vs adhesive) was confirmed by electron microscopy, and thermal damage was assessed histologically. The third substudy compared breaking strength of aortic repairs made with PLGA and semisolid genipin solder (ssLAVR) to repairs made with BioGlue. RESULTS: ssLAVA with 11 lasing spots and PLGA scaffold yielded the highest bursting pressure (923 ± 56 mm Hg vs 703 ± 96 mm Hg with PCL ssLAVA; P = .0002) and exhibited the fewest failures (20% vs 70% for PCL ssLAVA; P = .0218). The two failed PLGA ssLAVA arteries leaked at 19 and 22 hours, whereas the seven failed PCL ssLAVA arteries burst between 12 and 23 hours. PLGA anastomoses broke adhesively, whereas PCL welds failed cohesively. Both modalities exhibited full-thickness thermal damage. Repairs with PLGA scaffold yielded higher breaking strength than BioGlue repairs (323 ± 28 N/cm(2) vs 25 ± 4 N/cm(2), respectively; P = .0003). CONCLUSIONS: PLGA ssLAVA yields greater anastomotic strength and fewer anastomotic failures than PCL ssLAVA. Aortic repairs with BioGlue were inferior to those produced with PLGA ssLAVR. The results demonstrate the feasibility of ssLAVA/R as an alternative method to suture anastomosis or tissue sealant. Further studies should focus on reducing thermal damage.

Differential response of endothelial and endothelial colony forming cells on electrospun scaffolds with distinct microfiber diameters.

Electrospun scaffolds for in situ tissue engineering can be prepared with different fiber diameters to influence cell recruitment, adhesion, and differentiation. For cardiovascular applications, we investigated the impact of different fiber diameters (2, 5, 8, and 11 µm) in electrospun poly(ε-caprolactone) scaffolds on endothelial colony forming cells (ECFCs) in comparison to mature endothelial cells (HUVECs). In 2D cultures and on 2 µm fiber scaffolds, ECFC morphology and phenotype resemble those of HUVECs. When cultured on scaffolds with 5-11 µm fibers, a different behavior was detected. HUVECs developed a cytoskeleton organized circumferentially around the fibers, with collagen alignment in the same direction. ECFCs, instead, aligned the cytoskeleton along the scaffold fiber axis and deposited a homogeneous layer of collagen over the fibers; moreover, a subpopulation of ECFCs gained the αSMA marker. These results showed that ECFCs do not behave like mature endothelial cells in a 3D fibrous environment.

Characterization of a slowly degrading biodegradable polyester-urethane for tissue engineering scaffolds.

The purpose of this research was to develop and characterize a novel, slowly degrading polyester-urethane. In this study, a polyester-urethane with a crystalline segment of poly((R)-3-hydroxybutyric acid)-diol linked by a diisocyanate to an amorphous segment of poly(epsilon-caprolactone-co-glycolide)-diol was synthesized. Porous and nonporous scaffolds were processed using electrospinning and solvent casting respectively. The morphology, pore size, and filament diameter of the mesh and film were characterized using scanning electron microscopy (SEM). The thermal properties were examined using differential scanning calorimetry (DSC). A degradation study was initiated to characterize the change in mechanical properties, molecular weight, and surface morphology over 12 months using tensile testing, gel permeation chromatography (GPC), and SEM respectively. Concomitantly, cell morphology and viability on these variants were investigated using fibroblasts. The mechanical test data indicated a gradual decrease in the ultimate tensile strength and strain to break while the modulus of elasticity remained stable. GPC data suggested a slow decrease in the molecular weight while SEM examination revealed changed surface morphologies. The in vitro studies implied that the novel polyester-urethane was not cytotoxic and that the mesh was a more favorable scaffold towards cell viability. The summation of these results suggests that this polyester-urethane has the potential for tissue engineering applications.

Polyesterurethane foam scaffold for smooth muscle cell tissue engineering.

Reconstruction of the genitourinary tract, using engineered urological tissues, requires a mechanically stable biodegradable and biocompatible scaffold and cultured cells. Such engineered autologous tissue would have many clinical implications. In this study a highly porous biodegradable polyesterurethane-foam, DegraPol was evaluated with tissue engineered human primary bladder smooth muscle cells. The cell-polymer constructs were characterized by histology, scanning electron microscopy, immunohistochemistry and proliferation assays. Smooth muscle cells grown on DegraPol showed the same morphology as when grown on control polystyrene surface. Positive immunostaining with alpha smooth muscle actin indicated the preservation of the specific cell phenotype. Micrographs from scanning electron microscopy showed that the cells grew on the foam surface as well as inside the pores. In addition they grew as cell aggregates within the foam. The smooth muscle cells proliferated on the Degrapol; however, proliferation rate decreased due to apoptosis with time in culture. This study showed that Degrapol has the potential to be used as a scaffold.

Development of Non-Cell Adhesive Vascular Grafts Using Supramolecular Building Blocks.

Cell-free approaches to in situ tissue engineering require materials that are mechanically stable and are able to control cell-adhesive behavior upon implantation. Here, the development of mechanically stable grafts with non-cell adhesive properties via a mix-and-match approach using ureido-pyrimidinone (UPy)-modified supramolecular polymers is reported. Cell adhesion is prevented in vitro through mixing of end-functionalized or chain-extended UPy-polycaprolactone (UPy-PCL or CE-UPy-PCL, respectively) with end-functionalized UPy-poly(ethylene glycol) (UPy-PEG) at a ratio of 90:10. Further characterization reveals intimate mixing behavior of UPy-PCL with UPy-PEG, but poor mechanical properties, whereas CE-UPy-PCL scaffolds are mechanically stable. As a proof-of-concept for the use of non-cell adhesive supramolecular materials in vivo, electrospun vascular scaffolds are applied in an aortic interposition rat model, showing reduced cell infiltration in the presence of only 10% of UPy-PEG. Together, these results provide the first steps toward advanced supramolecular biomaterials for in situ vascular tissue engineering with control over selective cell capturing.

Laser-assisted vascular welding: optimization of acute and post-hydration welding strength.

BACKGROUND: Liquid solder laser-assisted vascular welding using biocompatible polymeric scaffolds (ssLAVW) is a novel technique for vascular anastomoses. Although ssLAVW has pronounced advantages over conventional suturing, drawbacks include low welding strength and extensive thermal damage. AIM: To determine optimal ssLAVW parameters for maximum welding strength and minimal thermal damage. METHODS: Substudy 1 compared breaking strength (BS) of aortic strips welded with electrospun poly(ε-caprolactone) (PCL) or poly(lactic-co-glycolic acid) (PLGA) scaffold, 670-nm laser, 50-s single-spot continuous lasing (SSCL), and semi-solid solder (48% bovine serum albumin (BSA)/0.5% methylene blue (MB)/3% hydroxypropylmethylcellulose (HPMC)). Substudy 2 compared the semi-solid solder to 48% BSA/0.5% MB/0.38% genipin and 48% BSA/0.5% MB/3% HPMC/0.38% genipin solder. Substudy 3 compared SSCL to single-spot pulsed lasing (SSPL). RESULTS: PCL-ssLAVW yielded an acute BS of 248.0 ± 54.0 N/cm2 and remained stable up to 7d of hydration. PLGA-ssLAVW obtained higher acute BS (408.6 ± 78.8 N/cm2) but revealed structural defects and a BS of 109.4 ± 42.6 N/cm2 after 14 d of hydration. The addition of HPMC and genipin improved the 14-d BS of PLGA-sLAVW (223.9 ± 19.1 N/cm2). Thermal damage was reduced with SSPL compared with SSCL. CONCLUSIONS: PCL-ssLAVW yielded lower but more stable welds than PLGA-ssLAVW. The addition of HPMC and genipin to the solder increased the post-hydration BS of PLGA-ssLAVW. SSPL regimen reduced thermal damage. PLGA-ssLAVW using 48% BSA/0.5% MB/3% HPMC/0.38% genipin solder and SSPL constitutes the most optimal welding modality. RELEVANCE FOR PATIENTS: Surgical patients requiring vascular anastomoses may benefit from the advantages that ssLAVW potentially offers over conventional sutures (gold standard). These include no needle trauma and remnant suture materials in the patient, reduction of foreign body reaction, immediate liquid-tight sealing, and the possibility of a faster and easier procedure for minimally invasive and endoscopic anastomotic techniques.

Tailoring the void space and mechanical properties in electrospun scaffolds towards physiological ranges.

Electrospinning has proven to be a promising method to produce scaffolds for tissue engineering despite the frequently encountered limitations in 3-dimensional tissue formation due to a lack of cell infiltration. To fully unlock the potential of electrospun scaffolds for tissue engineering, the void space within the fibrous network needs to be increased substantially and in a controlled manner. Low-temperature electrospinning (LTE) increases the fiber to fiber distance by embedding ice particles as void spacers during fiber deposition. Scaffold porosities up to 99.5% can be reached and in line with the increase in void space, the mechanical properties of the scaffolds shift towards the range for native biological tissue. While both the physiological mechanical properties and high porosity were promising for tissue engineering applications, control of the porosity in three dimensions was still limited when using LTE methods. Based on a range of LTE spun scaffolds made of poly(lactic acid) and poly(ε-caprolactone), we found that changing the ratio between the rate of ice crystal formation and polymer fiber deposition only had a small effect on the 3D-porosity of the final scaffold architecture. Varying the fiber stiffness, however, offers considerable control over the scaffold void space.

Biodegradable polymer scaffold, semi-solid solder, and single-spot lasing for increasing solder-tissue bonding in suture-free laser-assisted vascular repair.

We recently showed the fortifying effect of poly-caprolactone (PCL) scaffold in liquid solder-mediated laser-assisted vascular repair (ssLAVR) of porcine carotid arteries, yielding a mean ± SD leaking point pressure of 488 ± 111 mmHg. Despite supraphysiological pressures, the frequency of adhesive failures was indicative of weak bonding at the solder-tissue interface. As a result, this study aimed to improve adhesive bonding by using a semi-solid solder and single-spot vs. scanning irradiation. In the first experiment, in vitro ssLAVR (n=30) was performed on porcine abdominal aorta strips using a PCL scaffold with a liquid or semi-solid solder and a 670-nm diode laser for dual-pass scanning. In the second experiment, the scanning method was compared to single-spot lasing. The third experiment investigated the stability of the welds following hydration under quasi-physiological conditions. The welding strength was defined by acute breaking strength (BS). Solder-tissue bonding was examined by scanning electron microscopy and histological analysis was performed for thermal damage analysis. Altering solder viscosity from liquid to semi-solid solder increased the BS from 78 ± 22 N/cm(2) to 131 ± 38 N/cm(2) . Compared to scanning ssLAVR, single-spot lasing improved adhesive bonding to a BS of 257 ± 62 N/cm(2) and showed fewer structural defects at the solder-tissue interface but more pronounced thermal damage. The improvement in adhesive bonding was associated with constantly stronger welds during two weeks of hydration. Semi-solid solder and single-spot lasing increased welding strength by reducing solder leakage and improving adhesive bonding, respectively. The improvement in adhesive bonding was associated with enhanced weld stability during hydration.

Optimization of suture-free laser-assisted vessel repair by solder-doped electrospun poly(ε-caprolactone) scaffold.

Poor welding strength constitutes an obstacle in the clinical employment of laser-assisted vascular repair (LAVR) and anastomosis. We therefore investigated the feasibility of using electrospun poly(ε-caprolactone) (PCL) scaffold as reinforcement material in LAVR of medium-sized vessels. In vitro solder-doped scaffold LAVR (ssLAVR) was performed on porcine carotid arteries or abdominal aortas using a 670-nm diode laser, a solder composed of 50% bovine serum albumin and 0.5% methylene blue, and electrospun PCL scaffolds. The correlation between leaking point pressures (LPPs) and arterial diameter, the extent of thermal damage, structural and mechanical alterations of the scaffold following ssLAVR, and the weak point were investigated. A strong negative correlation existed between LPP and vessel diameter, albeit LPP (484±111 mmHg) remained well above pathophysiological pressures. Histological analysis revealed that thermal damage extended into the medial layer with a well-preserved internal elastic lamina and endothelial cells. Laser irradiation of PCL fibers and coagulation of solder material resulted in a strong and stiff scaffold. The weak point of the ssLAVR modality was predominantly characterized by cohesive failure. In conclusion, ssLAVR produced supraphysiological LPPs and limited tissue damage. Despite heat-induced structural/mechanical alterations of the scaffold, PCL is a suitable polymer for weld reinforcement in medium-sized vessel ssLAVR.

Cyto- and hemocompatibility of a biodegradable 3D-scaffold material designed for medical applications.

In this study, the polyester urethane Degrapol (DP) was explored for medical applications. Electrospun DP-fiber fleeces were characterized with regard to fiber morphology, swelling, and interconnectivity of interfiber spaces. Moreover, DP was assayed for cell proliferation and hemocompatibility being a prerequisite to any further in vivo application. It was shown that DP-fiber fleeces produced at different humidity while spinning affects interconnectivity of interfiber spaces, such that the higher the humidity the looser the resulting fiber fleeces. When the spinning target was cooled with dry ice, the resulting DP-fibers remained less fused to each other. However, permeability for fluorescent beads was not significantly increased. Fibroblast adhesion and proliferation occurred in a comparable manner on native as well as on fibronectin or collagen I adsorbed DP-fiber fleeces. On DP-surfaces fibroblasts proliferated equally well as compared with glass or PLGA surfaces or DP-surfaces adsorbed with fibronectin or collagen I. In contrast, human umbilical vein endothelial cells proliferated only after adsorption of DP-surfaces with fibronectin or collagen I, indicating that different cell types respond differently to DP-surfaces. Furthermore, hemocompatibility of DP-surfaces was found to be similar or better to PLGA or stainless steel, both medically used materials. These experiments indicate that DP-fiber fleeces or surfaces might be useful for tissue engineering.

Cotton wool-like nanocomposite biomaterials prepared by electrospinning: in vitro bioactivity and osteogenic differentiation of human mesenchymal stem cells.

The present study evaluates the in vitro biomedical performance of an electrospun, flexible, and cotton wool-like poly(lactide-co-glycolide) (PLGA)/amorphous tricalcium phosphate (ATCP) nanocomposite. Experiments on in vitro biomineralization, applicability in model defects and a cell culture study with human mesenchymal stem cells (hMSC) allowed assessing the application of the material for potential use as a bone graft. Scaffolds with different flame made ATCP nanoparticle loadings were prepared by electrospinning of a PLGA-based composite. Immersion in simulated body fluid showed significant deposition of a hydroxyapatite layer only on the surface of ATCP doped PLGA (up to 175% mass gain within 15 days for PLGA/ATCP 60:40). Proliferation and osteogenic differentiation of hMSC on different nanocomposites were assessed by incubating cells in osteogenic medium for 4 weeks. Proper adhesion and an unaffected morphology of the cells were observed by confocal laser scanning microscopy for all samples. Fluorometric quantification of dsDNA and analysis of ALP activity revealed no significant difference between the tested scaffolds and excluded any acute cytotoxic effects of the nanoparticles. The osteocalcin content for all scaffolds was 0.12-0.19 ng/ng DNA confirming osteogenic differentiation of human mesenchymal stem cells on these flexible bone implants.