Biodegradable and plasma-treated electrospun scaffolds coated with recombinant Olfactomedin-like 3 for accelerating wound healing and tissue regeneration.

Three-dimensional biomimetic scaffolds resembling the native extracellular matrix (ECM) are widely used in tissue engineering, however they often lack optimal bioactive cues needed for acceleration of cell proliferation, neovascularization, and tissue regeneration. In this study, the use of the ECM-related protein Olfactomedin-like 3 (Olfml3) demonstrates the importance and feasibility of fabricating efficient bioactive scaffolds without in vitro cell seeding prior to in vivo implantation. First, in vivo proangiogenic properties of Olfml3 were shown in a murine wound healing model by accelerated wound closure and a 1.4-fold increase in wound vascularity. Second, subcutaneous implantation of tubular scaffolds coated with recombinant Olfml3 resulted in enhanced cell in-growth and neovascularization compared with control scaffolds. Together, our data indicates the potential of Olfml3 to accelerate neovascularization during tissue regeneration by promoting endothelial cell proliferation and migration. This study provides a promising concept for the reconstruction of damaged tissue using affordable and effective bioactive scaffolds.

Porcine carotid artery replacement with biodegradable electrospun poly-e-caprolactone vascular prosthesis.

OBJECTIVE: There is a continuous search for shelf-ready small-caliber vascular prostheses with satisfactory early and late results. Biodegradable scaffolds, repopulated by recipient's cells regenerating a neovessel, can be a suitable option for adult and pediatric, urgent and elective cardiovascular procedures. METHODS: This was a short-term experimental assessment of a new biodegradable vascular prosthesis for arterial replacement in the pig. Eleven pigs underwent bilateral carotid artery replacement with biodegradable electrospun poly-ε-caprolactone (PCL) nanofiber prostheses (internal diameter, 4 mm; length, 5 cm); or expanded polytetrafluoroethylene (ePTFE) prostheses as control. Perioperative anticoagulation was achieved with intravenous heparin (double baseline activated clotting time). Postoperatively, until conclusion of the study at 1 month, animals received aspirin and clopidogrel daily. Transit time flow was measured intraoperatively and at sacrifice. Doppler ultrasound (1 and 4 weeks) and a selective carotid angiography (4 weeks) were performed to assess patency. All explanted grafts were analyzed by histology, morphometry, and scanning electron microscopy in order to study graft-host interaction. RESULTS: Surgical handling and hemostasis of the new prostheses were excellent. Patency rate was 78% (7/9) for PCL grafts, compared with 67% (4/6) for ePTFE grafts. Transit time flow and Doppler ultrasound showed no significant changes in flow and velocity or diameter over time in both groups. Both prostheses showed no detectable in vivo compliance as compared with native carotid artery. Percent neoendothelialization was 86% for PCL and 58% for ePTFE grafts (P = .008). Neointima formation was equal in both grafts. More adventitial infiltration of macrophages, myofibroblasts, and capillaries was seen in PCL grafts with a milder foreign-body reaction when compared with ePTFE implants. Both grafts showed similar endoluminal thrombus formation. CONCLUSIONS: Biodegradable, electrospun PCL grafts showed good surgical and mechanical properties, no aneurysm formation, and similar short-term patency compared with ePTFE grafts. Rapid endothelialization and cell ingrowth confirms favorable PCL graft-recipient biological interaction. Despite good early results, long-term follow-up is required before clinical application.

Paclitaxel-eluting biodegradable synthetic vascular prostheses: a step towards reduction of neointima formation?

BACKGROUND: Clinical small-caliber vascular prostheses are unsatisfactory. Reasons for failure are early thrombosis and late intimal hyperplasia. We thus prepared biodegradable small-caliber vascular prostheses using electrospun polycaprolactone (PCL) with slow-releasing paclitaxel (PTX), an antiproliferative drug. METHODS AND RESULTS: PCL solutions containing PTX were used to prepare nonwoven nanofibre-based 2-mm ID prostheses. Mechanical morphological properties and drug loading, distribution, and release were studied in vitro. Infrarenal abdominal aortic replacement was carried out with nondrug-loaded and drug-loaded prostheses in 18 rats and followed for 6 months. Patency, stenosis, tissue reaction, and drug effect on endothelialization, vascular remodeling, and neointima formation were studied in vivo. In vitro prostheses showed controlled morphology mimicking extracellular matrix with mechanical properties similar to those of native vessels. PTX-loaded grafts with suitable mechanical properties and controlled drug-release were obtained by factorial design. In vivo, both groups showed 100% patency, no stenosis, and no aneurysmal dilatation. Endothelial coverage and cell ingrowth were significantly reduced at 3 weeks and delayed at 12 and 24 weeks in PTX grafts, but as envisioned, neointima formation was significantly reduced in these grafts at 12 weeks and delayed at 6 months. CONCLUSIONS: Biodegradable, electrospun, nanofibre, polycaprolactone prostheses are promising because in vitro they maintain their mechanical properties (regardless of PTX loading), and in vivo show good patency, reendothelialize, and remodel with autologous cells. PTX loading delays endothelialization and cellular ingrowth. Conversely, it reduces neointima formation until the end point of our study and thus may be an interesting option for small caliber vascular grafts.

Effect of implantation site on outcome of tissue-engineered vascular grafts.

OBJECTIVE: Vascular prostheses for small caliber bypass grafts in cardiac and vascular diseases or for access surgery are still missing. Poly (Ɛ-caprolactone) (PCL) has been previously investigated by our group and showed good biocompatibility and mechanical properties in vitro and rapid endothelialisation, cellular infiltration and vascularisation in vivo yielding optimal patency in the abdominal aortic position. The aim of the present study is to evaluate our PCL graft in the carotid position and to compare its outcome to the grafts implanted in the abdominal aortic position. METHODS: PCL grafts (1 mm ID/10 mm long) were implanted into the left common carotid artery in 20 Sprague-Dawley rats and compared to our previously published series of abdominal aortic implants. The animals were followed up to 3, 6, 12 and 24 weeks. At each time point, in vivo compliance, angiography and histological examination with morphology were performed. RESULTS: PCL grafts showed good mechanical properties and ease of handling. The average graft compliance was 14.5 ±â€¯1.7%/ mmHg compared to 7.8 ±â€¯0.9% for the abdominal position and 45.1 ±â€¯3.2%/ mmHg for the native carotid artery. The overall patency for the carotid position was 65% as compared to 100% in the abdominal position. Complete endothelialisation was achieved at 3 weeks and cell invasion was more rapid than in the aortic position. In contrast, intimal hyperplasia (IH) and vascular density were less pronounced than in the aortic position. CONCLUSION: Our PCL grafts in the carotid position were well endothelialised with early cellular infiltration, higher compliance, lower IH and calcification compared to the similar grafts implanted in the aortic position. However, there was a higher occlusion rate compared to our abdominal aorta series. Anatomical position, compliance mismatch, flow conditions may answer the difference in patency seen.

Histologic Assessment of Drug-Eluting Grafts Related to Implantation Site.

Drug-eluting vascular prostheses represent a new direction in vascular surgery to reduce early thrombosis and late intimal hyperplasia for small calibre grafts. Subcutaneous implantation in rats is a rapid and cost-effective screening model to assess the drug-elution effect and could, to some extent, be useful to forecast results for vascular prostheses. We compared biological and histological responses to scaffolds in different implantation sites. Polycaprolactone (PCL), paclitaxel-loaded PCL (PCL-PTX) and dexamethasone-loaded PCL (PCL-DXM) electrospun scaffolds were implanted subcutaneously and in an infrarenal abdominal aortic model in rats for up to 12 weeks. At the conclusion of the study, a histological analysis was performed. Cellular graft invasion revealed differences in the progression of cellular infiltration between PCL-PTX and PCL/PCL-DXM groups in both models. Cell infiltration increased over time in the aortic model compared to the subcutaneous model for all groups. Cell counting revealed major differences in fibroblast, macrophage and giant cell graft colonisation in all groups and models over time. Macrophages and giant cells increased in the PCL aortic model; whereas in the subcutaneous model these cell types increased only after three weeks or even decreased in the drug-eluting PCL groups. Other major findings were observed only in the aortic replacement such as extracellular matrix deposition and neo-angiogenesis. The subcutaneous implant model can be used for screening, especially when drug-eluting effects are studied. However, major histological differences were observed in cell type reaction and depth of cell penetration compared to the aortic model. Our results demonstrate that the implantation site is a critical determinant of the biological response.

Experimental noninferiority trial of synthetic small-caliber biodegradable versus stable vascular grafts.

OBJECTIVE: Long-term evolution of polycaprolactone vascular prostheses has been investigated recently. The goal of this study was to evidence a noninferiority of such grafts compared with expanded polytetrafluoroethylene (ePTFE) implants in an aortic replacement model in the rat. METHODS: Fourteen anesthetized Sprague-Dawley rats received an infrarenal aortic graft (biodegradable, n = 8; expanded polytetrafluoroethylene, n = 6) replacement (end to end; inner diameter, 2 mm). Biodegradable grafts (polycaprolactone) were produced by random micro-/nanofiber electrospinning. After a median survival of 16.5 months, in vivo ultrasonography and angiography as well as postexplantation microcomputed tomography, histomorphometry, immunohistochemistry, and scanning electron microscopy were performed. RESULTS: Patency was 100% for polycaprolactone and 67% for ePTFE. No aneurysmal dilatation or stenoses were found in either group. Compliance was significantly higher for polycaprolactone compared with ePTFE (8.2 ± 1.0%/100 mm Hg vs 5.7 ± 0.7%/100 mm Hg; P < .01), but markedly reduced compared with adjacent native aortas and the control group. Histologically, low cellular in-growth was found in ePTFE whereas polycaprolactone showed significantly greater homogenous cellularity, producing an autologous extracellular matrix (10.8% ± 4.0% vs 32.1% ± 9.2%, P < .0001). Morphometry showed 100% neo-endothelialization for both grafts with a totally confluent endothelial coverage for polycaprolactone grafts by scanning electron microscope. More intimal hyperplasia was found in ePTFE compared with polycaprolactone grafts. Calcification was higher in ePTFE than in polycaprolactone grafts (15.8% vs 7.0%, P = .04) and was absent in controls. CONCLUSIONS: Outcomes of synthetic biodegradable nanofiber polycaprolactone grafts are not inferior compared with the clinically used expanded polytetrafluoroethylene grafts after long-term implantation in the rat aorta. Moreover, these implants show better patency, compliance, endothelialization, and cell in-growth, and less intimal hyperplasia and calcification than their counterparts.

Long term performance of polycaprolactone vascular grafts in a rat abdominal aorta replacement model.

In the active field of vascular graft research, polycaprolactone is often used because of its good mechanical strength and its biocompatibility. It is easily processed into micro and nano-fibers by electrospinning to form a porous, cell-friendly scaffold. However, long term in vivo performance of polycaprolactone vascular grafts had yet to be investigated. In this study, polycaprolactone micro and nano-fiber based vascular grafts were evaluated in the rat abdominal aorta replacement model for 1.5, 3, 6, 12, and 18 months (n = 3 for each time point). The grafts were evaluated for patency, thrombosis, compliance, tissue regeneration, and material degradation. Results show excellent structural integrity throughout the study, with no aneurysmal dilation, and perfect patency with no thrombosis and limited intimal hyperplasia. Endothelialization, cell invasion, and neovascularization of the graft wall rapidly increased until 6 months, but at 12 and 18 months, a cellular regression is observed. On the medium term, chondroid metaplasia takes place in the intimal hyperplasia layers, which contributes to calcification of the grafts. This study presents issues with degradable vascular grafts that cannot be identified with short implantation times or in vitro studies. Such findings should allow for better design of next generation vascular grafts.

A high-throughput microfluidic assay to study neurite response to growth factor gradients.

Studying neurite guidance by diffusible or substrate bound gradients is challenging with current techniques. In this study, we present the design, fabrication and utility of a microfluidic device to study neurite guidance under chemogradients. Experimental and computational studies demonstrated the establishment of a steep gradient of guidance cue within 30 min and stable for up to 48 h. The gradient was found to be insensitive to external perturbations such as media change and movement of device. The effects of netrin-1 (0.1-10 µg mL(-1)) and brain pulp (0.1 µL mL(-1)) were evaluated for their chemoattractive potential on neurite turning, while slit-2 (62.5 or 250 ng mL(-1)) was studied for its chemorepellant properties. Hippocampal or dorsal root ganglion (DRG) neurons were seeded into a micro-channel and packed onto the surface of a 3D collagen gel. Neurites grew into the matrix in three dimensions, and a gradient of guidance cue was created orthogonal to the direction of neurite growth to impact guidance. The average turning angle of each neurite was measured and averaged across multiple devices cultured under similar conditions to quantify the effect of guidance cue gradient. Significant positive turning towards gradient was measured in the presence of brain pulp and netrin-1 (1 µg mL(-1)), relative to control cultures which received no external guidance cue (p < 0.001). Netrin-1 released from transfected fibroblasts had the most positive turning effect of all the chemoattractive cues tested (p < 0.001). Slit-2 exhibited strong chemorepellant characteristics on both hippocampal and DRG neurite guidance at 250 ng mL(-1) concentration. Slit-2 also showed similar behavior on DRG neuron invasion into 3D collagen gel (p < 0.01 relative to control cultures). Taken together, the results suggest the utility of this microfluidic device to generate stable chemogradients for studying neurobiology, cell migration and proliferation, matrix remodeling and co-cultures with other cell lines, with potential applications in cancer biology, tissue engineering and regenerative medicine.