Animal experimental models of ischemic wounds - A review of literature.

Critical limb ischemia is a serious form of peripheral arterial disease (PAD). The consequences of lower limb ischemia are pain, claudication and chronic non-healing wounds. Patients with diabetes are especially at a high risk for developing non-healing ulcers. The most serious complication is major amputation. For this reason, there is a significant medical requirement to develop new therapies in order to prevent the progression of PAD. For research purposes, it is crucial to find an appropriate model of chronic ischemia to explore the processes of wound healing. According to recently acquired information, rodents are currently the most commonly used animals in these types of studies. The main advantage of using small animals is the low financial cost due to the relatively small demand for food, water and living space. The disadvantage is their anatomy, which is different from that of humans. Larger animals have a more human-like anatomy and physiology, but they require more expense and space for housing. A bipedicle skin flap and its modifications are popular models for ischemic wounds. In order to secure healing through re-epithelisation, as opposed to contraction in rodents, there is a need to remove the panniculus carnosus muscle. Wounds in other experimental animals heal primarily through re-epithelisation. The application of a silicone mesh underneath the flap prevents vascular regrowth in ischemic tissue. There is an ongoing effort to create in vivo diabetic models for chronic ulcer research. This work presents an overview of existing animal models of ischemic wounds.

Vascular Remodeling of Clinically Used Patches and Decellularized Pericardial Matrices Recellularized with Autologous or Allogeneic Cells in a Porcine Carotid Artery Model.

Background: Cardiovascular surgery is confronted by a lack of suitable materials for patch repair. Acellular animal tissues serve as an abundant source of promising biomaterials. The aim of our study was to explore the bio-integration of decellularized or recellularized pericardial matrices in vivo. Methods: Porcine (allograft) and ovine (heterograft, xenograft) pericardia were decellularized using 1% sodium dodecyl sulfate ((1) Allo-decel and (2) Xeno-decel). We used two cell types for pressure-stimulated recellularization in a bioreactor: autologous adipose tissue-derived stromal cells (ASCs) isolated from subcutaneous fat of pigs ((3) Allo-ASC and (4) Xeno-ASC) and allogeneic Wharton's jelly mesenchymal stem cells (WJCs) ((5) Allo-WJC and (6) Xeno-WJC). These six experimental patches were implanted in porcine carotid arteries for one month. For comparison, we also implanted six types of control patches, namely, arterial or venous autografts, expanded polytetrafluoroethylene (ePTFE Propaten® Gore®), polyethylene terephthalate (PET Vascutek®), chemically stabilized bovine pericardium (XenoSure®), and detoxified porcine pericardium (BioIntegral® NoReact®). The grafts were evaluated through the use of flowmetry, angiography, and histological examination. Results: All grafts were well-integrated and patent with no signs of thrombosis, stenosis, or aneurysm. A histological analysis revealed that the arterial autograft resembled a native artery. All other control and experimental patches developed neo-adventitial inflammation (NAI) and neo-intimal hyperplasia (NIH), and the endothelial lining was present. NAI and NIH were most prominent on XenoSure® and Xeno-decel and least prominent on NoReact®. In xenografts, the degree of NIH developed in the following order: Xeno-decel > Xeno-ASC > Xeno-WJC. NAI and patch resorption increased in Allo-ASC and Xeno-ASC and decreased in Allo-WJC and Xeno-WJC. Conclusions: In our setting, pre-implant seeding with ASC or WJC had a modest impact on vascular patch remodeling. However, ASC increased the neo-adventitial inflammatory reaction and patch resorption, suggesting accelerated remodeling. WJC mitigated this response, as well as neo-intimal hyperplasia on xenografts, suggesting immunomodulatory properties.

Comparison of the effect of stem cell therapy and percutaneous transluminal angioplasty on diabetic foot disease in patients with critical limb ischemia.

BACKGROUND AIMS: The aim of our study was to compare the effect of autologous stem cell therapy (SCT) and percutaneous transluminal angioplasty (PTA) on diabetic foot disease (DFD) in patients with critical limb ischemia (CLI). METHODS: Thirty-one patients with DFD and CLI treated by autologous stem cells and 30 patients treated by PTA were included in the study; 23 patients with the same inclusion criteria who could not undergo PTA or SCT formed the control group. Amputation-free survival, transcutaneous oxygen pressure (TcPO2) and wound healing were assessed over 12 months. RESULTS: Amputation-free survival after 6 and 12 months was significantly greater in the SCT and PTA groups compared with controls (P = 0.001 and P = 0.0029, respectively) without significant differences between the active treatment groups. Increase in TcPO2 did not differ between SCT and PTA groups until 12 months (both Ps < 0.05 compared with baseline), whereas TcPO2 in the control group did not change over the follow-up period. More healed ulcers were observed up to 12 months in the SCT group compared with the PTA and control groups (84 versus 57.7 versus 44.4 %; P = 0.042). CONCLUSIONS: Our study showed comparable effects of SCT and PTA on CLI, a major amputation rate that was superior to conservative therapy in patients with diabetic foot and an observable effect of SCT on wound healing. Our results support SCT as a potential promising treatment in patients with CLI and diabetic foot.

External Support of Autologous Internal Jugular Vein Grafts with FRAME Mesh in a Porcine Carotid Artery Model.

BACKGROUND: Autologous vein grafts are widely used for bypass procedures in cardiovascular surgery. However, these grafts are susceptible to failure due to vein graft disease. Our study aimed to evaluate the impact of the latest-generation FRAME external support on vein graft remodeling in a preclinical model. METHODS: We performed autologous internal jugular vein interposition grafting in porcine carotid arteries for one month. Four grafts were supported with a FRAME mesh, while seven unsupported grafts served as controls. The conduits were examined through flowmetry, angiography, macroscopy, and microscopy. RESULTS: The one-month patency rate of FRAME-supported grafts was 100% (4/4), whereas that of unsupported controls was 43% (3/7, Log-rank p = 0.071). On explant angiography, FRAME grafts exhibited significantly more areas with no or mild stenosis (9/12) compared to control grafts (3/21, p = 0.0009). Blood flow at explantation was higher in the FRAME grafts (145 ± 51 mL/min) than in the controls (46 ± 85 mL/min, p = 0.066). Area and thickness of neo-intimal hyperplasia (NIH) at proximal anastomoses were similar for the FRAME and the control groups: 5.79 ± 1.38 versus 6.94 ± 1.10 mm2, respectively (p = 0.558) and 480 ± 95 vs. 587 ± 52 µm2/µm, respectively (p = 0.401). However, in the midgraft portions, the NIH area and thickness were significantly lower in the FRAME group than in the control group: 3.73 ± 0.64 vs. 6.27 ± 0.64 mm2, respectively (p = 0.022) and 258 ± 49 vs. 518 ± 36 µm2/µm, respectively (p = 0.0002). CONCLUSIONS: In our porcine model, the external mesh FRAME improved the patency of vein-to-carotid artery grafts and protected them from stenosis, particularly in the mid regions. The midgraft neo-intimal hyperplasia was two-fold thinner in the meshed grafts than in the controls.

Animal experimental models of ischemic limbs - A systematic review.

BACKGROUND: The objective of this systematic review is to summarize the available animal models of ischemic limbs, and to provide an overview of the advantages and disadvantages of each animal model and individual method of limb ischemia creation. METHODS: A review of literature was conducted using the PubMed and Web of Science pages. Various types of experimental animals and surgical approaches used in creating ischemic limbs were evaluated. Other outcomes of interest were the specific characteristics of the individual experimental animals, and duration of tissue ischemia. RESULTS: The most commonly used experimental animals were mice, followed by rabbits, rats, pigs, miniature pigs, and sheep. Single or double arterial ligation and excision of the entire femoral artery was the most often used method of ischemic limb creation. Other methods comprised single or double arterial electrocoagulation, use of ameroid constrictors, photochemically induced thrombosis, and different types of endovascular methods. The shortest duration of tissue ischemia was 7 days, the longest 90 days. CONCLUSIONS: This review shows that mice are among the most commonly used animals in limb ischemia research. Simple ligation and excision of the femoral artery is the most common method of creating an ischemic limb; nevertheless, it can result in acute rather than chronic ischemia. A two-stage sequential approach and methods using ameroid constrictors or endovascular blinded stent grafts are more suitable for creating a gradual arterial occlusion typically seen in humans. Selecting the right mouse strain or animal with artificially produced diabetes or hyperlipidaemia is crucial in chronic ischemic limb research. Moreover, the observation period following the onset of ischemia should last at least 14 days, preferably 4 weeks.

Radiologic Evaluation of Uterine Vasculature of Uterus Transplant Living Donor Candidates: DUETS Classification.

Uterus transplantation is a treatment solution for women suffering from absolute uterine factor infertility. As much as 19.5% of uterus-transplanted patients underwent urgent graft hysterectomy due to thrombosis/hypoperfusion. The necessity to identify candidates with high-quality uterine vasculature is paramount. We retrospectively evaluated and compared the imaging results with actual vascular findings from the back table. In this article, we present a novel radiologic grading scale (DUETS classification) for evaluating both uterine arteries and veins concerning their suitability for uterus procurement and transplantation. This classification defines several criteria for arteries (caliber, tapering, atherosclerosis, tortuosity, segment, take-off, and course) and veins (caliber, tapering, plethora, fenestrations, duplication/multiplicity, dominant route of venous drainage, radiologist's confidence with imaging and assessment). In conclusion, magnetic resonance angiography can provide reliable information on uterine venous characteristics if performed consistently according to a well-established protocol and assessed by a dedicated radiologist. The caliber of uterine arteries seems to be inversely related to the time passed since the last delivery. We recommend that the radiologist comments on the reliability and confidence of the imaging study. It cannot be over-emphasized that the most crucial aspect of surgical imaging is the necessity of high-quality communication between a surgeon and a radiologist.

[Eversion carotid endarterectomy: evaluation of results after changing the operation technique]. / Everzní endarterektomie arterie carotis interna: hodnocení výsledku po zmene operacní techniky.

BACKGROUND: Stroke is the third most common cause of mortality, and carotid artery stenosis causes up to 30% of all ischemic strokes. Eversion CEA (ECEA) has been proposed as an acceptable alternative to the standard bifurcation endarterectomy in many patients. This study was designed to analyze the long-term durability of ECEA in symptomatic and asymptomatic patients. Data were retrospectively reviewed to determine the incidence of major adverse cardiovascular events (MACEs) within 30 days of surgery, late survival, and the incidence of restenosis. METHODS AND RESULTS: From January 1999 to June 2010, 344 ECEAs were performed on 324 patients (34% female, 38% symptomatic). The mean follow-up period was 30 months. CONCLUSIONS: MACEs occurred in 28 patients (8.6%). The overall incidence of stroke or death after ECEA was 1.7% and 0.9% at discharge. The overall incidence of stoke or death after CEA was 4.3% and 21% (14 strokes, 69 deaths, 8 of which were stroke-related). The overall occurrence of any restenosis (> 50%) after CEA was 4.3% (14 of 324 procedures).

Human Uterus Transplantation from Living and Deceased Donors: The Interim Results of the First 10 Cases of the Czech Trial.

INTRODUCTION: Uterus transplantation (UTx) is a rapidly evolving treatment of uterine-factor infertility. We report the results of the first 10 UTx procedures performed at our institution. METHODS: The program started in April 2016 as a two-arm study comparing the efficacy of UTx from live donors (LD) and deceased donors (DD). RESULTS: Between April 2016 and April 2018, we performed five DD UTx and five LD UTx. Two grafts had to be removed early due to thrombosis. One graft was removed due to chronic rejection and previous herpes simplex infection at month 7. Graft survival is 70% at one year. Recipient survival is 100% at two years. Live donor survival is 100% at three years. Three live-births have been achieved, two from a LD and one from a graft from a nulliparous DD. Vaginal anastomotic stenosis occurred in 63% (5/8) of grafts. Self-expanding stents have shown preliminary suitability for the treatment of vaginal stenosis. Three recipients developed severe acute rejection. CONCLUSION: The interim results of our study demonstrate mid-term viability in 70% of grafts. The LD UTx produced two live births and the DD UTx produced one live birth. Nulliparous donors should be considered for donation.

Live Birth Following Uterine Transplantation From a Nulliparous Deceased Donor.

BACKGROUND: Nulliparous uterine grafts have never been used in uterus transplantation (UTx), possibly due to presumed infertility. Our objective was to verify the feasibility of nulliparous uterine graft transplantation. METHODS: The Czech Uterus Transplant Trial (registered under ClinicalTrials.gov, identifier NCT03277430) is a 2-arm trial comparing the efficacy of deceased donor (DD) versus live-donor uterus transplant (10 patients in both arms). A 25-year-old patient suffering from inborn absolute uterine factor infertility underwent a DD uterus transplant. The donor was a 20-year-old nulliparous brain-dead donor. RESULTS: The transplant procedure was uneventful. The posttransplant period was complicated by (1) recurrent episodes of acute cellular rejection, (2) neutropenia necessitating the administration of granulocyte colony-stimulating factor, (3) vaginal anastomotic stenosis treated with the insertion of a self-expanding stent, (4) the concurrence of Clostridium difficile colitis and acute appendicitis, and (5) temporary renal function impairment of a combined cause. Two years after the UTx, after the fourth embryo transfer, the patient became pregnant. Apart from gestational diabetes mellitus, the pregnancy was uneventful. Due to preterm contractions, delivery was achieved via caesarean section at gestational age 34 + 6 years. The postoperative course was uneventful for both the mother and the newborn. CONCLUSIONS: Herein, we report the first live birth after a DD UTx in Europe. This report provides a proof of concept that nulliparous uteri may present a suitable source of uterine grafts for UTx. Stenting may serve as a feasible treatment method for vaginal anastomotic stenosis.

Hybrid Repair Combined with Fresh Arterial Allograft Extra-Anatomical Reconstruction: The Treatment of Infrarenal Abdominal Aneurysm above an Aortobifemoral Bypass Complicated by an Infected Pseudoaneurysm in the Left Groin.

INTRODUCTION: A 72-year-old male patient was admitted into our centre with large infected pseudoaneurysm (PSA) in the left groin. The patient underwent a CT angiography (CTA) that confirmed a large partly thrombosed 6.5 × 5.5 cm PSA in the left groin arising from the distal anastomosis of the aortobifemoral bypass (ABF). Furthermore, the CTA revealed 11 cm juxtarenal abdominal aortic aneurysm (JAAA) from which the proximal anastomosis of the ABF was arising. METHOD: Aorto-uni-iliac stent graft Cook was placed from the right groin trough native severely stenotic right iliac arteries with proximal landing zone below the renal arteries, excluding the JAAA and the ABF. The distal landing zone was in the common iliac artery maintaining patent right internal iliac artery. Afterwards, a femoro-femoral crossover bypass from right to left was performed using a fresh arterial allograft. Postprocedurally, the hospital stay was uneventful. The left groin PSA cultures came positive for Staphylococcus epidermidis and Corynebacterium tuberculostearicum, both sensitive to vancomycin and rifampicin. RESULT: The patient underwent intravenous ATB treatment with vancomycin for two weeks, followed by four weeks of oral rifampicin. The patient was discharged on the 20th postoperative days. CONCLUSION: Hybrid repair combining aortic stent graft and extra-anatomical bypass in the treatment of infected distal parts of an aortofemoral bypass is an acceptable treatment modality.

Uterus transplant graft's arterial atherosclerotic remodeling veracity.

BACKGROUND: Uterus transplantation is a complex, multi-step experimental procedure used for the treatment of uterus absence or uterus anomaly that prevents embryo implantation or pregnancy completion. METHOD: To date, only 51 uterus transplants worldwide had been performed. When simplified, it is vascularized composite allograft transplantation. While it is still an experimental procedure with encouraging results for the future, there are still many issues that have to be clarified. The most serious complications of uterus transplantation are graft rejection or grafts vascular failure. RESULTS: So far, no reference to the atherosclerotic arterial infiltration of the uterus arteries was suggested and studied as one of the main causes of graft's failure. CONCLUSION: In this review we summarized current knowledge and possible role of uterus arterial damage, including atherosclerotic changes on the graft's survival.

Toll-like receptor dual-acting agonists are potent inducers of PBMC-produced cytokines that inhibit hepatitis B virus production in primary human hepatocytes.

Recombinant interferon-α (IFN-α) treatment functionally cures chronic hepatitis B virus (HBV) infection in some individuals and suppresses virus replication in hepatocytes infected in vitro. We studied the antiviral effect of conditioned media (CM) from peripheral blood mononuclear cells (PBMCs) stimulated with agonists of Toll-like receptors (TLRs) 2, 7, 8 and 9. We found that CM from PBMCs stimulated with dual-acting TLR7/8 (R848) and TLR2/7 (CL413) agonists were more potent drivers of inhibition of HBe and HBs antigen secretion from HBV-infected primary human hepatocytes (PHH) than CM from PBMCs stimulated with single-acting TLR7 (CL264) or TLR9 (CpG-B) agonists. Inhibition of HBV in PHH did not correlate with the quantity of PBMC-produced IFN-α, but it was a complex function of multiple secreted cytokines. More importantly, we found that the CM that efficiently inhibited HBV production in freshly isolated PHH via various cytokine repertoires and mechanisms did not reduce covalently closed circular (ccc)DNA levels. We confirmed our data with a cell culture model based on HepG2-NTCP cells and the plasmacytoid dendritic cell line GEN2.2. Collectively, our data show the importance of dual-acting TLR agonists inducing broad cytokine repertoires. The development of poly-specific TLR agonists provides novel opportunities towards functional HBV cure.

The Effect of Different Thawing Rates on Cryopreserved Human Iliac Arteries Allograft's Structural Damage and Mechanical Properties.

INTRODUCTION: The rate of thawing of cryopreserved human iliac arteries allografts (CHIAA) directly affects the severeness of structural changes that occur during this process. METHOD: The experiment was performed on ten CHIAA. The 10% dimethylsulphoxide in 6% hydroxyethyl starch solution was used as the cryoprotectant; all CHIAA were cooled at a controlled rate and stored in the vapor phase of liquid nitrogen (-194°C). Two thawing protocols were tested: (1) placing the CHIAA in a water bath at 37°C, and (2) the CHIAA were thawed in a controlled environment at 5°C. All samples underwent analysis under a scanning electron microscope. Testing of the mechanical properties of the CHIAA was evaluated on a custom-built single axis strain testing machine. Longitudinal and circumferential samples were prepared from each tested CHIAA. RESULTS: Ultrastructural analysis revealed that all five CHIAA thawed during the thawing protocol 1 which showed significantly more damage to the subendothelial structures when compared to the samples thawed in protocol 2. Mechanical properties: Thawing protocol 1-longitudinal UTS 2, 53 ± 0, 47 MPa at relative strain 1, 27 ± 0, 12 and circumferential UTS 1, 94 ± 0, 27 MPa at relative strain 1, 33 ± 0, 09. Thawing protocol 2-longitudinal ultimate tensile strain (UTS) 2, 42 ± 0, 34 MPa at relative strain 1, 32 ± 0, 09 and circumferential UTS 1, 98 ± 0, 26 MPa at relative strain 1, 29 ± 0, 07. Comparing UTS showed no statistical difference between thawing methods. CONCLUSION: Despite the significant differences in structural changes of presented thawing protocols, the ultimate tensile strain showed no statistical difference between thawing methods.

Large Hepatic Artery Pseudoaneurysm Resection After Orthotopic Liver Transplantation.

BACKGROUND Hepatic artery (HA) pseudoaneurysm (PSA) after liver transplantation (OLTx) is rare but often fatal complication requiring quick repair. Its prevalence in patients after OLTx is around 0.94%. CASE REPORT A 41-year-old female patient underwent a full-graft orthotopic liver transplantation (OLTx) for alcoholic liver cirrhosis in 2017. During regular postoperative Doppler ultrasonography (DU) check-ups, a large 3-cm pseudoaneurysm (PSA) was detected on the hepatic artery. The patient underwent a computed angiography (CTA) to verify the PSA anatomical localization and relationship with the transplanted liver graft. Selective celiac arteriography showed HA PSA and 90% stenosis of the hepatic artery after PSA. The stent graft placement was unsuccessful as the guiding wire was unable to pass through the post-PSA HA stenosis. The patient was scheduled for an open repair under general anesthesia. Through a right subcostal incision, the HA PSA was resected and the HA was mobilized and re-anastomosed using an end-to-end technique. Three months after the procedure, the patient has a good liver graft perfusion through the HA with no sign of PSA reoccurrence or stenosis. CONCLUSIONS Early detection of the HA PSA after OLTx is a life-threatening complication requiring prompt treatment. If endovascular treatment options fail, open surgical repair, despite its challenges, is the only possible treatment option.

Spontaneous Isolated Common Iliac Artery Dissection Treated with Self-Expandable Stent in a 38-year-old Patient: A Case Report.

INTRODUCTION: Isolated iliac artery dissection (ISIAD) without the involvement of the aorta is a rare medical condition. REPORT: A case of a 38-year-old man with sudden onset of rest pain and paraesthesia on the right lower limb (RLL) is presented. Upon admission, the RLL was pulseless, with mild paraesthesia in the foot. The patient underwent computed tomography angiography, which revealed isolated common iliac artery (CIA) dissection followed by endovascular treatment (stenting) of the CIA dissection, with an instant therapeutic effect. Hospital stay was uneventful. The patient was discharged on the third post-procedural day. DISCUSSION: Endovascular treatment of ISIAD is a viable treatment modality, with low periprocedural complications, mortality, and morbidity. Owing to its mini-invasiveness, it is a viable treatment modality.

Acute appendicitis in a patient after a uterus transplant: A case report.

BACKGROUND: Acute appendicitis in a solid organ transplant recipient is a rare occurrence, and experience remains limited. Appendicitis in uterine transplant recipients has never been reported. Immunocompromised patients with acute abdomen often present late and with attenuated symptoms. The differential diagnosis in a transplanted patient is broad and challenging due to possible existing complications associated with the graft, effects of immunosuppression, and altered anatomical relations. CASE SUMMARY: A 26-year-old woman suffering from absolute uterine factor infertility received a uterus transplant. In the post-transplant period, she suffered from leukopenia and recurrent acute cellular rejection. Her compliance was suboptimal. She travelled to an exotic destination despite the physician's recommendation not to do so. Following her vacation, she presented with abdominal discomfort, nausea and diarrhoea. There was no sign of acute abdomen; the abdominal ultrasound was negative on day 0. Clostridium difficile colitis was verified and treated with perorally administered vancomycin. On day 4, the discomfort changed to pain; the ultrasound scan revealed a finding suggestive of appendicitis. Surgical exploration revealed perforated appendicitis, and appendectomy was performed. From a surgical point of view, the patient's follow-up was uneventful. The patient underwent a successful embryo transfer 6 months after the appendectomy. The patient gave birth to a healthy boy at the 35th week of gestation. CONCLUSION: A high index of suspicion of an atypical course and symptomatology of acute abdomen should be maintained in immunosuppressed patients.

Simultaneous living donor orthotopic renal transplantation and bilateral nephrectomy for recurrent renal cell carcinoma and renal failure: case report and review of literature.

BACKGROUND: We report the case of a 43-year-old female patient with systemic lupus erythematosus, class III lupus nephritis, with predialysis creatinine levels around 350 µmol/L (3.95 mg/dL) after partial resection of the left kidney with histologically verified papillary carcinoma in 2010. Preoperative computed tomography of the abdomen revealed a small 8 mm tumor in the left upper kidney pole. The patient was indicated for simultaneous bilateral nephrectomy and orthotopic renal transplantation with the aim to minimize invasiveness of the procedure as well as for curable tumor removal. METHOD: The procedure was performed under the full anesthesia trough upper middle laparotomy. As the first step, bilateral transperitoneal nephrectomy was performed. The live donor surgery started in a parallel theater to shorten the cold ischemic time of the graft. The renal graft had singe vessels and ureter; it was placed into the recipient's right orthotopic position. End-to-end anastomosis of the right renal vein and artery anastomosis were performed; ureter was anastomosed end-to-end using recipient's ureter. RESULTS: The postoperative period was uneventful with repeatedly excellent ultrasonography check-up of the graft's perfusion. The patient was discharged after 13 days with a good renal function of the graft (urea: 15 mmol/L, creatinine 160 µmol/L [1.80 mg/dL]). CONCLUSION: Orthotopic renal transplantation is a technically challenging but valid alternative for patients who are unsuitable candidates for heterotopic renal transplantation or in cases where there is a clear benefit of orthotopic renal transplantation.

Improved adhesion and differentiation of endothelial cells on surface-attached fibrin structures containing extracellular matrix proteins.

Currently used vascular prostheses are hydrophobic and do not allow endothelial cell (EC) adhesion and growth. The aim of this study was to prepare fibrin (Fb)-based two-dimensional (2D) and three-dimensional (3D) assemblies coated with extracellular matrix (ECM) proteins and to evaluate the EC adhesion, proliferation and differentiation on these assemblies in vitro. Coating of Fb with collagen, laminin (LM), and fibronectin (FN) was proved using the surface plasmon resonance technique. On all Fb assemblies, ECs reached higher cell densities than on polystyrene after 3 and 7 days of culture. Immunoflurescence staining showed better assembly of talin and vinculin into focal adhesion plaques, and also more apparent staining of vascular endothelial cadherin on surface-attached 3D Fb and protein-coated Fb assemblies. On these samples, ECs also contained a lower concentration of intercellular adhesion molecule-1, measured by enzyme-linked immunosorbent assay. Higher concentrations of CD31 (platelet-endothelial cell adhesion molecule-1) were found on 3D Fb coated with LM, and higher concentrations of von Willebrand factor were found on 3D Fb coated with type I collagen or LM in comparison to 2D Fb layers. The results indicate that ECM protein-coated 2D and 3D Fb assemblies can be used for versatile applications in various tissue replacements where endothelialization is desirable, for example, vascular prostheses and heart valves.

The gene expression of human endothelial cells is modulated by subendothelial extracellular matrix proteins: short-term response to laminar shear stress.

Vascular surgery for atherosclerosis is confronted by the lack of a suitable bypass material. Tissue engineering strives to produce bio-artificial conduits to provide resistance to thrombosis. The objectives of our study were to culture endothelial cells (EC) on composite assemblies of extracellular matrix proteins, and to evaluate the cellular phenotype under flow. Cell-adhesive assemblies were fabricated on glass slides as combinations of collagen (Co), laminin (LM), and fibronectin (FN), resulting in three samples: Co, Co/LM, and Co/FN. Surface topography, roughness, and wettability were determined. Human saphenous vein EC were harvested from cardiac patients, cultured on the assemblies and submitted to laminar shear stress (SS) of 12 dyn/cm(2) for 40, 80, and 120 min. Cell retention was assessed and qRT-PCR of adhesion genes (VE-cadherin, vinculin, KDR, CD-31 or PECAM-1, ß1-integrins) and metabolic genes (t-PA, NF-κB, eNOS and MMP-1) was performed. Quantitative immunofluorescence of VE cadherin, vinculin, KDR, and vonWillebrand factor was performed after 2 and 6 h of flow. Static samples were excluded from shearing. The cells reached confluence with similar growth curves. The cells on Co/LM and Co/FN were resistant to flow up to 120 min but minor desquamation occurred on Co corresponding with temporary downregulation of VE cadherin and vinculin-mRNA and decreased fluorescence of vinculin. The cells seeded on Co/LM initially more upregulated vinculin-mRNA and also the inflammatory factor NF-κB, and the cells plated on Co/FN changed the expression profile minimally in comparison with the static control. Fluorescence of VE cadherin and vonWillebrand factor was enhanced on Co/FN. The cells cultured on Co/LM and Co/FN increased the vinculin fluorescence and expressed more VE cadherin and KDR-mRNA than the cells on Co. The cells plated on Co/FN upregulated the mRNA of VE cadherin, CD-31, and MMP 1 to a greater extent than the cells on Co/LM and they enhanced the fluorescence of VE cadherin, KDR, and vonWillebrand factor. Some of these changes sustained up to 6 h of flow, as confirmed by immunofluorescence. Combined matrices Co/LM and Co/FN seem to be more suitable for EC seeding and retention under flow. Moreover, Co/FN matrix promoted slightly more favorable cellular phenotype than Co/LM under SS of 2-6 h.