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Background: Decellularized xenogenic scaffolds represent a promising substrate for tissue-engineered vascular prostheses, particularly those with smaller diameters (<6â mm). Despite their benefits, a notable limitation presents itself during decellularization, namely, the diminished mechanical strength that introduces the risk of aneurysmal dilations in the early post-implantation period. This study introduces a strategy for modification the mechanical properties of these biological scaffolds through the forming of an external polymeric reinforcement via thermal extrusion. Methods: The study utilized scaffolds fabricated from bovine internal mammary arteries through decellularization and preservation. The scaffolds were divided into subgroups and reinforced with polymeric helices made of Polyvinylidene fluoride (PVDF) and Polycaprolactone (PCL), n = 5 for each. An experimental setup for external reinforcement coating was designed. Computed microtomography was employed to obtain accurate 3D models of the scaffolds. Mechanical properties were evaluated through in vitro uniaxial tension tests (Z50, Zwick/Roell, Germany), compliance evaluation and numerical simulations (Abaqus/CAE, Dassault Systemes, France) to investigate the effect of external reinforcement on aneurysm growth. Results: Using a double-layer helix for the reinforcement significantly enhanced the radial tensile strength of the scaffolds, increasing it up to 2.26 times. Yet, the comparison of vessel's compliance between two reinforced and the Control scaffolds within the physiological pressures range did not reveal any significant differences. Numerical simulation of aneurysm growth showed that thin-walled regions of the Control scaffold developed aneurysmal-type protrusions, bulging up to 0.7â mm, with a substantial degradation of mechanical properties. In contrast, both PVDF and PCL reinforced scaffolds did not exhibit significant property degradation, with deformations ranging 0.1-0.13â mm depending on the model, and a maximum decrease in the modulus of elasticity of 23%. Conclusion: The results of the study demonstrated that the external polymer helical reinforcement of decellularized scaffolds via thermal extrusion enables a controlled modification of mechanical properties, notably enhancing radial strength while maintaining sufficient compliance within the physiological pressure range. A series of in vitro tests demonstrated the consistency and potential of this approach for decellularized xenogenic scaffolds, a concept that had not been explored before.
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The lack of suitable autologous grafts and the impossibility of using synthetic prostheses for small artery reconstruction make it necessary to develop alternative efficient vascular grafts. In this study, we fabricated an electrospun biodegradable poly(ε-caprolactone) (PCL) prosthesis and poly(3-hydroxybutyrate-co-3-hydroxyvalerate)/poly(ε-caprolactone) (PHBV/PCL) prosthesis loaded with iloprost (a prostacyclin analog) as an antithrombotic drug and cationic amphiphile with antibacterial activity. The prostheses were characterized in terms of their drug release, mechanical properties, and hemocompatibility. We then compared the long-term patency and remodeling features of PCL and PHBV/PCL prostheses in a sheep carotid artery interposition model. The research findings verified that the drug coating of both types of prostheses improved their hemocompatibility and tensile strength. The 6-month primary patency of the PCL/Ilo/A prostheses was 50%, while all PHBV/PCL/Ilo/A implants were occluded at the same time point. The PCL/Ilo/A prostheses were completely endothelialized, in contrast to the PHBV/PCL/Ilo/A conduits, which had no endothelial cells on the inner layer. The polymeric material of both prostheses degraded and was replaced with neotissue containing smooth-muscle cells; macrophages; proteins of the extracellular matrix such as type I, III, and IV collagens; and vasa vasorum. Thus, the biodegradable PCL/Ilo/A prostheses demonstrate better regenerative potential than PHBV/PCL-based implants and are more suitable for clinical use.
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Prótese Vascular , Enxerto Vascular , Animais , Ovinos , Polímeros , Poliésteres , Implantação de PróteseRESUMO
Background Whereas the risk factors for structural valve degeneration (SVD) of glutaraldehyde-treated bioprosthetic heart valves (BHVs) are well studied, those responsible for the failure of BHVs fixed with alternative next-generation chemicals remain largely unknown. This study aimed to investigate the reasons behind the development of SVD in ethylene glycol diglycidyl ether-treated BHVs. Methods and Results Ten ethylene glycol diglycidyl ether-treated BHVs excised because of SVD, and 5 calcified aortic valves (AVs) replaced with BHVs because of calcific AV disease were collected and their proteomic profile was deciphered. Then, BHVs and AVs were interrogated for immune cell infiltration, microbial contamination, distribution of matrix-degrading enzymes and their tissue inhibitors, lipid deposition, and calcification. In contrast with dysfunctional AVs, failing BHVs suffered from complement-driven neutrophil invasion, excessive proteolysis, unwanted coagulation, and lipid deposition. Neutrophil infiltration was triggered by an asymptomatic bacterial colonization of the prosthetic tissue. Neutrophil elastase, myeloblastin/proteinase 3, cathepsin G, and matrix metalloproteinases (MMPs; neutrophil-derived MMP-8 and plasma-derived MMP-9), were significantly overexpressed, while tissue inhibitors of metalloproteinases 1/2 were downregulated in the BHVs as compared with AVs, together indicative of unbalanced proteolysis in the failing BHVs. As opposed to other proteases, MMP-9 was mostly expressed in the disorganized prosthetic extracellular matrix, suggesting plasma-derived proteases as the primary culprit of SVD in ethylene glycol diglycidyl ether-treated BHVs. Hence, hemodynamic stress and progressive accumulation of proteases led to the extracellular matrix degeneration and dystrophic calcification, ultimately resulting in SVD. Conclusions Neutrophil- and plasma-derived proteases are responsible for the loss of BHV mechanical competence and need to be thwarted to prevent SVD.
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Bioprótese , Insuficiência Cardíaca , Próteses Valvulares Cardíacas , Humanos , Metaloproteinase 9 da Matriz/metabolismo , Próteses Valvulares Cardíacas/efeitos adversos , Proteólise , Proteômica , Valvas Cardíacas/metabolismo , Valva Aórtica/cirurgia , Valva Aórtica/metabolismo , Insuficiência Cardíaca/etiologia , Peptídeo Hidrolases/metabolismo , Lipídeos , Bioprótese/efeitos adversosRESUMO
Fibrin is widely used in vascular tissue engineering. Typically, fibrin polymerization is initiated by adding exogenous thrombin. In this study, we proposed a protocol for the preparation of completely autologous fibrin without the use of endogenous thrombin and compared the properties of the prepared fibrin matrix with that obtained by the traditional method. Fibrinogen was obtained by ethanol precipitation followed by fibrin polymerization by adding either exogenous thrombin and calcium chloride (ExThr), or only calcium chloride (EnThr). We examined the structure, mechanical properties, thrombogenicity, degradation rate and cytocompatibility of fibrin matrices. Factor XIII (FXIII) quantitative assay was performed by ELISA, and FXIII activity was assessed by SDS-PAGE detection of γ-γ cross-links. The results show that network structure of EnThr fibrin was characterized by thinner fibers. The EnThr fibrin matrices had higher strength, stiffness and resistance to proteolytic degradation compared to ExThr fibrin. EnThr fibrin matrices exhibited less thrombogenicity in vitro than ExThr, and retained high cytocompatibility. Thus, the proposed approach has several advantages over the traditional method, namely the fabrication of a completely autologous coating material that has better mechanical properties, higher resistance to proteolysis and lower thrombogenicity.
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Nanocomposites based on poly(styrene-block-isobutylene-block-styrene) (SIBS) and single-walled carbon nanotubes (CNTs) were prepared and characterized in terms of tensile strength as well as bio- and hemocompatibility. It was shown that modification of CNTs using dodecylamine (DDA), featured by a long non-polar alkane chain, provided much better dispersion of nanotubes in SIBS as compared to unmodified CNTs. As a result of such modification, the tensile strength of the nanocomposite based on SIBS with low molecular weight (Mn = 40,000 g mol-1) containing 4% of functionalized CNTs was increased up to 5.51 ± 0.50 MPa in comparison with composites with unmodified CNTs (3.81 ± 0.11 MPa). However, the addition of CNTs had no significant effect on SIBS with high molecular weight (Mn~70,000 g mol-1) with ultimate tensile stress of pure polymer of 11.62 MPa and 14.45 MPa in case of its modification with 1 wt% of CNT-DDA. Enhanced biocompatibility of nanocomposites as compared to neat SIBS has been demonstrated in experiment with EA.hy 926 cells. However, the platelet aggregation observed at high CNT concentrations can cause thrombosis. Therefore, SIBS with higher molecular weight (Mn~70,000 g mol-1) reinforced by 1-2 wt% of CNTs is the most promising material for the development of cardiovascular implants such as heart valve prostheses.
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Currently, an ultrastructural analysis of cardiovascular tissues is significantly complicated. Routine histopathological examinations and immunohistochemical staining suffer from a relatively low resolution of light microscopy, whereas the fluorescence imaging of plaques and bioprosthetic heart valves yields considerable background noise from the convoluted extracellular matrix that often results in a low signal-to-noise ratio. Besides, the sectioning of calcified or stent-expanded blood vessels or mineralised heart valves leads to a critical loss of their integrity, demanding other methods to be developed. Here, we designed a conceptually novel approach that combines conventional formalin fixation, sequential incubation in heavy metal solutions (osmium tetroxide, uranyl acetate or lanthanides, and lead citrate), and the embedding of the whole specimen into epoxy resin to retain its integrity while accessing the region of interest by grinding and polishing. Upon carbon sputtering, the sample is visualised by means of backscattered scanning electron microscopy. The technique fully preserves calcified and stent-expanded tissues, permits a detailed analysis of vascular and valvular composition and architecture, enables discrimination between multiple cell types (including endothelial cells, vascular smooth muscle cells, fibroblasts, adipocytes, mast cells, foam cells, foreign-body giant cells, canonical macrophages, neutrophils, and lymphocytes) and microvascular identities (arterioles, venules, and capillaries), and gives a technical possibility for quantitating the number, area, and density of the blood vessels. Hence, we suggest that our approach is capable of providing a pathophysiological insight into cardiovascular disease development. The protocol does not require specific expertise and can be employed in virtually any laboratory that has a scanning electron microscope.
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An association between high serum calcium/phosphate and cardiovascular events or death is well-established. However, a mechanistic explanation of this correlation is lacking. Here, we examined the role of calciprotein particles (CPPs), nanoscale bodies forming in the human blood upon its supersaturation with calcium and phosphate, in cardiovascular disease. The serum of patients with coronary artery disease or cerebrovascular disease displayed an increased propensity to form CPPs in combination with elevated ionised calcium as well as reduced albumin levels, altogether indicative of reduced Ca2+-binding capacity. Intravenous administration of CPPs to normolipidemic and normotensive Wistar rats provoked intimal hyperplasia and adventitial/perivascular inflammation in both balloon-injured and intact aortas in the absence of other cardiovascular risk factors. Upon the addition to primary human arterial endothelial cells, CPPs induced lysosome-dependent cell death, promoted the release of pro-inflammatory cytokines, stimulated leukocyte adhesion, and triggered endothelial-to-mesenchymal transition. We concluded that CPPs, which are formed in the blood as a result of altered mineral homeostasis, cause endothelial dysfunction and vascular inflammation, thereby contributing to the development of cardiovascular disease.
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Angina Pectoris/fisiopatologia , Isquemia Encefálica/fisiopatologia , Cloreto de Cálcio/sangue , Doença da Artéria Coronariana/fisiopatologia , Células Endoteliais/patologia , Infarto do Miocárdio/fisiopatologia , Fosfatos/sangue , Angina Pectoris/sangue , Angina Pectoris/genética , Animais , Aorta/metabolismo , Aorta/patologia , Isquemia Encefálica/sangue , Isquemia Encefálica/genética , Cloreto de Cálcio/química , Estudos de Casos e Controles , Morte Celular , Doença da Artéria Coronariana/sangue , Doença da Artéria Coronariana/genética , Células Endoteliais/metabolismo , Transição Epitelial-Mesenquimal , Floculação , Regulação da Expressão Gênica , Humanos , Inflamação , Molécula 1 de Adesão Intercelular/genética , Molécula 1 de Adesão Intercelular/metabolismo , Leucócitos/metabolismo , Leucócitos/patologia , Lisossomos/metabolismo , Lisossomos/patologia , Masculino , Infarto do Miocárdio/sangue , Infarto do Miocárdio/genética , Fosfatos/química , Cultura Primária de Células , Ratos , Ratos Wistar , Fatores de Transcrição da Família Snail/genética , Fatores de Transcrição da Família Snail/metabolismo , Túnica Íntima/metabolismo , Túnica Íntima/patologia , Molécula 1 de Adesão de Célula Vascular/genética , Molécula 1 de Adesão de Célula Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
Tissue-engineered vascular graft for the reconstruction of small arteries is still an unmet clinical need, despite the fact that a number of promising prototypes have entered preclinical development. Here we test Poly(3-hydroxybutyrate-co-3-hydroxyvalerate)Poly(ε-caprolactone) 4-mm-diameter vascular grafts equipped with vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) and stromal cell-derived factor 1α (SDF-1α) and surface coated with heparin and iloprost (PHBV/PCL[VEGF-bFGF-SDF]Hep/Ilo, n = 8) in a sheep carotid artery interposition model, using biostable vascular prostheses of expanded poly(tetrafluoroethylene) (ePTFE, n = 5) as a control. Primary patency of PHBV/PCL[VEGF-bFGF-SDF]Hep/Ilo grafts was 62.5% (5/8) at 24 h postimplantation and 50% (4/8) at 18 months postimplantation, while all (5/5) ePTFE conduits were occluded within the 24 h after the surgery. At 18 months postimplantation, PHBV/PCL[VEGF-bFGF-SDF]Hep/Ilo grafts were completely resorbed and replaced by the vascular tissue. Regenerated arteries displayed a hierarchical three-layer structure similar to the native blood vessels, being fully endothelialised, highly vascularised and populated by vascular smooth muscle cells and macrophages. The most (4/5, 80%) of the regenerated arteries were free of calcifications but suffered from the aneurysmatic dilation. Therefore, biodegradable PHBV/PCL[VEGF-bFGF-SDF]Hep/Ilo grafts showed better short- and long-term results than bio-stable ePTFE analogues, although these scaffolds must be reinforced for the efficient prevention of aneurysms.
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The implantation of bioprosthetic heart valves (BHVs) is increasingly becoming the treatment of choice in patients requiring heart valve replacement surgery. Unlike mechanical heart valves, BHVs are less thrombogenic and exhibit superior hemodynamic properties. However, BHVs are prone to structural valve degeneration (SVD), an unavoidable condition limiting graft durability. Mechanisms underlying SVD are incompletely understood, and early concepts suggesting the purely degenerative nature of this process are now considered oversimplified. Recent studies implicate the host immune response as a major modality of SVD pathogenesis, manifested by a combination of processes phenocopying the long-term transplant rejection, atherosclerosis, and calcification of native aortic valves. In this review, we summarize and critically analyze relevant studies on (1) SVD triggers and pathogenesis, (2) current approaches to protect BHVs from calcification, (3) obtaining low immunogenic BHV tissue from genetically modified animals, and (4) potential strategies for SVD prevention in the clinical setting.
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Bioprótese , Doenças das Valvas Cardíacas/cirurgia , Implante de Prótese de Valva Cardíaca/efeitos adversos , Próteses Valvulares Cardíacas , Falha de Prótese , Implante de Prótese de Valva Cardíaca/instrumentação , Implante de Prótese de Valva Cardíaca/tendências , HumanosRESUMO
In this study, we incorporated carbon nanotubes (CNTs) into poly(styrene-block-isobutylene-block-styrene) (SIBS) to investigate the physical characteristics of the resulting nanocomposite and its cytotoxicity to endothelial cells. CNTs were dispersed in chloroform using sonication following the addition of a SIBS solution at different ratios. The resultant nanocomposite films were analyzed by X-ray microtomography, optical and scanning electron microscopy; tensile strength was examined by uniaxial tension testing; hydrophobicity was evaluated using a sessile drop technique; for cytotoxicity analysis, human umbilical vein endothelial cells were cultured on SIBS-CNTs for 3 days. We observed an uneven distribution of CNTs in the polymer matrix with sporadic bundles of interwoven nanotubes. Increasing the CNT content from 0 wt% to 8 wt% led to an increase in the tensile strength of SIBS films from 4.69 to 16.48 MPa. The engineering normal strain significantly decreased in 1 wt% SIBS-CNT films in comparison with the unmodified samples, whereas a further increase in the CNT content did not significantly affect this parameter. The incorporation of CNT into the SIBS matrix resulted in increased hydrophilicity, whereas no cytotoxicity towards endothelial cells was noted. We suggest that SIBS-CNT may become a promising material for the manufacture of implantable devices, such as cardiovascular patches or cusps of the polymer heart valve.
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Modification by Arg-Gly-Asp (RGD) peptides is a promising approach to improve the biocompatibility of biodegradable vascular patches for arteriotomy. In this study, we evaluated the performance of vascular patches electrospun using a blend of polycaprolactone (PCL) and polyhydroxybutyrate/valerate (PHBV) and additionally modified with RGDK, AhRGD, and c[RGDFK] peptides using 1,6-hexamethylenediamine or 4,7,10-trioxa-1,13-tridecanediamine (TTDDA) linkers. We examined mechanical properties and hemocompatibility of resulting patches before implanting them in rat abdominal aortas to assess their performance in vivo. Patches were explanted 1, 3, 6, and 12 months postoperation followed by histological and immunofluorescence analyses. Patches manufactured from the human internal mammary artery or commercially available KemPeriplas-Neo xenopericardial patches were used as a control. The tensile strength and F max of KemPeriplas-Neo patches were 4- and 16.7-times higher than those made of human internal mammary artery, respectively. Both RGD-modified and unmodified PHBV/PCL patches demonstrated properties similar to a human internal mammary artery patch. Regardless of RGD modification, experimental PHBV/PCL patches displayed fewer lysed red blood cells and resulted in milder platelet aggregation than KemPeriplas-Neo patches. Xenopericardial patches failed to form an endothelial layer in vivo and were prone to calcification. By contrast, TTDDA/RGDK-modified biodegradable patches demonstrated a resistance to calcification. Modification by TTDDA/RGDK and TTDDA/c[RGDFK] facilitated the formation of neovasculature upon the implantation in vivo.
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Polymeric heart valves seem to be an attractive alternative to mechanical and biological prostheses as they are more durable, due to the superior properties of novel polymers, and have the biocompatibility and hemodynamics comparable to tissue substitutes. This study reports a comprehensive assessment of a nanocomposite based on the functionalised graphene oxide and poly(carbonate-urea)urethane with the trade name "Hastalex" in comparison with GORE-TEX, a commercial polymer routinely used for cardiovascular medical devices. Experimental data have proved that GORE-TEX has a 2.5-fold (longitudinal direction) and 3.5-fold (transverse direction) lower ultimate tensile strength in comparison with Hastalex (p < 0.05). The contact angles of Hastalex surfaces (85.2 ± 1.1°) significantly (p < 0.05) are lower than those of GORE-TEX (127.1 ± 6.8°). The highest number of viable cells Ea.hy 926 is on the Hastalex surface exceeding 7.5-fold when compared with the GORE-TEX surface (p < 0.001). The platelet deformation index for GORE-TEX is 2-fold higher than that of Hastalex polymer (p < 0.05). Calcium content is greater for GORE-TEX (8.4 mg/g) in comparison with Hastalex (0.55 mg/g). The results of this study have proven that Hastalex meets the main standards required for manufacturing artificial heart valves and has superior mechanical, hemocompatibility and calcific resistance properties in comparison with GORE-TEX.
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Materiais Biocompatíveis , Grafite , Próteses Valvulares Cardíacas , Nanocompostos , Poliuretanos , Células A549 , Animais , Materiais Biocompatíveis/toxicidade , Calcinose/induzido quimicamente , Bovinos , Módulo de Elasticidade , Grafite/toxicidade , Hemólise/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana , Humanos , Hibridomas/efeitos dos fármacos , Teste de Materiais , Microscopia Eletrônica de Varredura , Nanocompostos/toxicidade , Nanocompostos/ultraestrutura , Pericárdio , Adesividade Plaquetária/efeitos dos fármacos , Polímeros/toxicidade , Politetrafluoretileno/toxicidade , Poliuretanos/toxicidade , Desenho de Prótese , Ratos , Ratos Wistar , Propriedades de Superfície , Resistência à TraçãoRESUMO
Modification with Arg-Gly-Asp (RGD) peptides is a promising approach to improve biocompatibility of small-calibre vascular grafts but it is unknown how different RGD sequence composition impacts graft performance. Here we manufactured 1.5 mm poly(3-hydroxybutyrate-co-3-hydroxyvalerate)/poly(ε-caprolactone) grafts modified by distinct linear or cyclic RGD peptides immobilized by short or long amine linker arms. Modified vascular prostheses were tested in vitro to assess their mechanical properties, hemocompatibility, thrombogenicity and endothelialisation. We also implanted these grafts into rat abdominal aortas with the following histological examination at 1 and 3 months to evaluate their primary patency, cellular composition and detect possible calcification. Our results demonstrated that all modes of RGD modification reduce ultimate tensile strength of the grafts. Modification of prostheses does not cause haemolysis upon the contact with modified grafts, yet all the RGD-treated grafts display a tendency to promote platelet aggregation in comparison with unmodified counterparts. In vivo findings identify that cyclic Arg-Gly-Asp-Phe-Lys peptide in combination with trioxa-1,13-tridecanediamine linker group substantially improve graft biocompatibility. To conclude, here we for the first time compared synthetic small-diameter vascular prostheses with different modes of RGD modification. We suggest our graft modification regimen as enhancing graft performance and thus recommend it for future use in tissue engineering.
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The blend of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) and poly(ε-caprolactone) (PCL) has recently been considered promising for vascular tissue engineering. However, it was shown that PHBV/PCL grafts require biofunctionalization to achieve high primary patency rate. Here we compared immobilization of arginine-glycine-aspartic acid (RGD)-containing peptides and the incorporation of vascular endothelial growth factor (VEGF) as two widely established biofunctionalization approaches. Electrospun PHBV/PCL small-diameter grafts with either RGD peptides or VEGF, as well as unmodified grafts were implanted into rat abdominal aortas for 1, 3, 6, and 12 months following histological and immunofluorescence assessment. We detected CD31âº/CD34âº/vWF⺠cells 1 and 3 months postimplantation at the luminal surface of PHBV/PCL/RGD and PHBV/PCL/VEGF, but not in unmodified grafts, with the further observation of CD31âºCD34-vWF⺠phenotype. These cells were considered as endothelial and produced a collagen-positive layer resembling a basement membrane. Detection of CD31âº/CD34⺠cells at the early stages with subsequent loss of CD34 indicated cell adhesion from the bloodstream. Therefore, either conjugation with RGD peptides or the incorporation of VEGF promoted the formation of a functional endothelial cell layer. Furthermore, both modifications increased primary patency rate three-fold. In conclusion, both of these biofunctionalization approaches can be considered as equally efficient for the modification of tissue-engineered vascular grafts.
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Prótese Vascular , Materiais Revestidos Biocompatíveis/química , Proteínas Imobilizadas/química , Oligopeptídeos/química , Fator A de Crescimento do Endotélio Vascular/química , Animais , Antígenos CD34/análise , Implante de Prótese Vascular , Adesão Celular , Células Endoteliais/citologia , Masculino , Molécula-1 de Adesão Celular Endotelial a Plaquetas/análise , Ratos Wistar , Engenharia TecidualRESUMO
The combination of a natural polymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) and a synthetic hydrophobic polymer poly(ε-caprolactone) (PCL) is promising for the preparation of biodegradable and biocompatible small-diameter vascular grafts for bypass surgery. However, physico-mechanical properties and endothelialization rate of PHBV/PCL grafts are poor. We suggested that incorporation of vascular endothelial growth factor (VEGF) into PHBV/PCL grafts may improve their physico-mechanical properties and enhance endothelialization. Here we compared morphology, physico-mechanical properties, and in vivo performance of electrospun small-diameter vascular grafts prepared from PHBV/PCL with and without VEGF. Structure of the graft surface and physico-mechanical properties were examined by scanning electron microscopy and universal testing machine, respectively. Grafts were implanted into rat abdominal aorta for 1, 3, and 6 months with the further histological, immunohistochemical, and immunofluorescence examination. PHBV/PCL grafts with and without VEGF were highly porous and consisted mostly of nanoscale and microscale fibers, respectively. Mean pore diameter and mean pore area were significantly lower in PHBV/PCL/VEGF compared to PHBV/PCL grafts (1.47 µm and 10.05 µm(2); 2.63 µm and 47.13 µm(2), respectively). Durability, elasticity, and stiffness of PHBV/PCL grafts with VEGF were more similar to internal mammary artery compared to those without, particularly 6 months postimplantation. Both qualitative examination and quantitative image analysis showed that three-fourths of PHBV/PCL grafts with VEGF were patent and had many CD31-, CD34-, and vWF-positive cells at their inner surface. However, all PHBV/PCL grafts without VEGF were occluded and had no or a few CD31-positive cells at the inner surface. Therefore, VEGF enhanced endothelialization and improved graft patency at all the time points in a rat abdominal aorta replacement model. In conclusion, PHBV/PCL grafts with VEGF have better biocompatibility and physico-mechanical properties compared to those without. Incorporation of VEGF improves graft patency and accelerates formation of endothelial cell monolayer.
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Small diameter arterial bypass grafts are considered as unmet clinical need since the current grafts have poor patency of 25% within 5 years. We have developed a 3D scaffold manufactured from natural and synthetic biodegradable polymers, poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) and poly(ð-caprolactone) (PCL), respectively. Further to improve the biophysical properties as well as endothelialisation, the grafts were covalently conjugated with arginine-glycine-aspartic acid (RGD) bioactive peptides. The biophysical properties as well as endothelialisation of PHBV/PCL and PCL 2 mm diameter bypass grafts were assessed with and without biofunctionalisation with RGD peptides in vitro and in vivo. Morphology of the grafts was assessed by scanning electron microscopy, whereas physico-mechanical properties were evaluated using a physiological circulating system equipped with a state of art ultrasound vascular wall tracking system. Endothelialisation of the grafts in vitro and in vivo were assessed using a cell viability assay and rat abdominal aorta replacement model, respectively. The biofunctionalisation with RGD bioactive peptides decreased mean fiber diameter and mean pore area in PHBV/PCL grafts; however, this was not the case for PCL grafts. Both PHBV/PCL and PCL grafts with RGD peptides had lower durability compared to those without; these durability values were similar to those of internal mammary artery. Modification of PHBV/PCL and PCL grafts with RGD peptides increased endothelial cell viability in vitro by a factor of eight and enhanced the formation of an endothelial cell monolayer in vivo 1 month postimplantation. In conclusion, PHBV/PCL small-caliber graft can be a suitable 3D scaffold for the development of a tissue engineering arterial bypass graft.
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Calcium phosphate bions (CPB) are biomimetic mineralo-organic nanoparticles which represent a physiological mechanism regulating the function, transport and disposal of calcium and phosphorus in the human body. We hypothesised that CPB may be pathogenic entities and even a cause of cardiovascular calcification. Here we revealed that CPB isolated from calcified atherosclerotic plaques and artificially synthesised CPB are morphologically and chemically indistinguishable entities. Their formation is accelerated along with the increase in calcium salts-phosphates/serum concentration ratio. Experiments in vitro and in vivo showed that pathogenic effects of CPB are defined by apoptosis-mediated endothelial toxicity but not by direct tissue calcification or functional changes in anti-calcification proteins. Since the factors underlying the formation of CPB and their pathogenic mechanism closely resemble those responsible for atherosclerosis development, further research in this direction may help us to uncover triggers of this disease.