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Hydrogel complex scaffolds (hydrogel scaffolds) are prepared by coating precursor solutions onto heparin-modified poly(ε-caprolactone) (PCLH) scaffolds followed by subsequent in situ gelation. Here, we show that hydrogel complexation can significantly strengthen the scaffold and slow its degradation. The hydrogel scaffold was implanted into the abdominal aorta of a rat model, and the aneurysm incidence rate of the hydrogel scaffolds sharply decreased compared with that of the hydrogel-free scaffolds. Histological and immunohistological analyses showed that the implanted grafts had good vascular regeneration. The absence of calcification and occurrence of contractile smooth muscle cells (SMCs) at the first month was found in the hydrogel-free PCLH scaffold due to the presence of surface-modified heparin, whereas the hydrogel scaffold exhibited mild calcification and later occurrence of contractile SMCs as the complexed hydrogel covered the fibers and blocked the interaction between heparin and cells. Heparin was further physically encapsulated into the hydrogel before gelation, and its sustainable release was demonstrated by an in vitro release test. A pilot implantation in a rabbit carotid model showed that the encapsulated heparin modulated the scaffold characteristics including anticoagulation, anticalcification, and the early occurrence of contractile SMCs in vivo. Consequently, hydrogel complexation can significantly improve the in vivo regeneration property of the scaffold due to its multiple beneficial characteristics.

A continuous electrospinning technique was applied to fabricate double layer tubular tissue engineering vascular graft (TEVG) scaffold. The luminal layer was made from poly(ɛ-caprolac-tone)(PCL) ultrafine fibers via common single axial electrospinning followed by the outer layer of core-shell structured nanofibers via coaxial electrospinning. For preparing the outer layernano-fibers, the PCL was electrospun into the shell and both bovine serum albumin (BSA) and tetrapeptide val-gal-pro-gly (VAPG) were encapsulated into the core. The core-shell structure in the outer layer fibers was observed by transmission electron microscope (TEM). The in vitro release tests exhibited the sustainable release behavior of BSA and VAPG so that they provided a better cell growth environment in the interior of tubular scaffold wall. The in vitro culture of smooth muscle cells (SMCs) demonstrated their potential to penetrate into the scaffold wall for the 3D cell culture. Subsequently, 3D cell coculture was conducted. First, SMCs were seeded on the luminal surface of the scaffold and cultured for 5 days, and then endothelial cells (ECs) were also seeded on the luminal surface and cocultured with SMCs for another 2 days. After stained with antibodies, 3D cell distribution on the scaffold was revealed by confocal laser scanning microscopy (CLSM) where ECs were mainly located on the luminal surface whereas SMCs penetrated into the surface and distributed inside the scaffold wall. This double layer tubular scaffold with 3D cell distribution showed the promise to develop it into a novel TEVG for clinical trials in the near future., (© 2015 Wiley Periodicals, Inc.)

Aim: Aim of the study was to construct and evaluate of autologous cell derived vein grafts based on tissue engineering concept., Methods: In this study, we constructed venous grafts (VGs) in 12 days based on tissue engineering concept. We draw out 8-12 mL of bone marrow from the intended recipient canines (N.=8) to culture and expand endothelial progenitor cells (EPCs). After having been labeled with PKH26-GL, the cells were seeded onto the luminal surface of decellularized scaffolds (DSs) with single, rotative method for 4 hours. Following static culture for 24-72 hours, the hybrids were implanted to recipient canine inferior vena cava. Non-seeded DSs (N.=4) were performed as control., Results: Angiography disclosed that patent number of test (control) group were 7/7 (2/4), 6/6 (2/2) and 4/4 (1/2) at postoperative 10 days, 4 weeks and 12 weeks, respectively. At 12 weeks, confluenced endothelial cells which covered the whole inner luminal surface of the explants were detected. Meanwhile, fibroblasts and α-actin positive cells in the matrices were found. PKH26-GL labeled EPCs sustained on the luminal surface accompanied by newly formed endothelial cells. However, the explants in both groups showed partial stenosis., Conclusion: These results indicate that such constructed VGs based on autologous bone marrow-derived EPCs and porcine DSs are promising and deserve to further improvement and testing.

Studies on three-dimensional tissue engineered graft (3DTEG) have attracted great interest among researchers as they present a means to meet the pressing clinical demand for tissue engineering scaffolds. To explore the feasibility of 3DTEG, high porosity poly-ε-caprolactone (PCL) was obtained via the co-electrospinning of polyethylene glycol and PCL, and used to construct small-diameter poly-ε-caprolactone-lysine (PCL-LYS-H) scaffolds, whereby heparin was anchored to the scaffold surface by lysine groups. A variety of small-diameter 3DTEG models were constructed with different PCL layers and the mechanical properties of the resulting constructs were evaluated in order to select the best model for 3DTEGs. Bone marrow mononuclear cells were induced and differentiated to endothelial cells (ECs) and smooth muscle cells (SMCs). A 3DTEG (labeled '10-4%') was successfully produced by the dynamic co-culture of ECs on the PCL-LYS-H scaffolds and SMCs on PCL. The fluorescently labeled cells on the 3DTEG were subsequently observed by laser confocal microscopy, which showed that the ECs and SMCs were embedded in the 3DTEG. Nitric oxide and endothelial nitric oxide synthase assays showed that the ECs behaved normally in the 3DTEG. This study consequently provides a new thread to produce small-diameter tissue engineered grafts, with excellent mechanical properties, that are perfusable to vasculature and functional cells.

Poly(ε-caprolactone) (PCL) was conjugated with heparin and fabricated into nonwoven tubular scaffold by electrospinning. The dynamic contact angle analysis revealed the hydrophilicity improvement due to heparin concentrating on the conjugate surface. The microbicinchoninic acid and quartz crystal microbalance measurements implied that the conjugate can significantly reduce the absorption of plasma protein, such as albumin and fibrinogen, indicative of the good blood biocompatibility. As evidenced by Enzyme Linked Immunosorbent Assay, the electrospun conjugate scaffolds possessed a higher loading capability of vascular endothelial growth factor (VEGF) than that of the blank PCL in aqueous solution via static interaction. The viability of loaded VEGF was evaluated by cell culture and adhesion tests. The amount and morphology of cells were substantially improved after VEGF was loaded into scaffolds exhibiting excellent cell biocompatibility. To assess the in vivo biocompatibility, a tubular scaffold (L = 4 cm, D = 2 mm) was transplanted into dog's femoral artery. The scaffold patency was inspected by carotid artery angiography 4 weeks after implantation. The explanted scaffold was also investigated by histological analysis including hematoxyline eosin, Millere Masson (collagen and elastin), and von Kossa (calcium) stain. Furthermore, von Willebrand factor immunohistochemical stain was performed to examine the formation of endothelial layer. The conjugate shows the potential to be used as scaffold materials in vascular tissue engineering., (Copyright © 2012 Wiley Periodicals, Inc.)

We used epsilon-caprolactone/L-lactide (PCLA) as a biodegradable scaffold and bone marrow (BM) mesenchymal stem cells (MSCs) as seeding cells for vascular tissue engineering: we expected MSCs to grow in the scaffolds in a bioreactor. The MSCs we used were from the BM of dogs, and vascular scaffolds were carried out on the electrospinning process of PCLA copolymers. MSCs expressed CD44 and CD105 but did not express CD34 or CD14 at an identical time point. Scaffolds were nontoxic to cells and were favorable for the growth and migration of MSCs. After culture in a bioreactor with mechanical stimulation, cells completely covered the surfaces of PCLA scaffolds and penetrated or infiltrated into the inside of the scaffold structure.

Objective: To investigate the method of constructing small-diameter vascular grafts from xenogenic decellularized arterial matrices and mesenchymal stem cells (MSCs)., Methods: Porcine iliac arteries were decellularized by detergent and trypsin treatment. Histology, mechanical strength and porosity experiments were performed to evaluate the properties of decellularized matrices. MSCs were isolated from bone marrow of dogs and expanded ex vivo. Decellularized matrices were seeded with MSCs and further cultured in a pulsatile bioreactor. Morphological features of the tissue engineered grafts were assayed by HE staining and scanning electron microscopy., Results: After cell extraction, absence of cellular components and preservation of extracellular matrix were verified. Mechanical strength of decellularized matrices was slightly reduced compared with native arteries. Porosity of decellularized matrices was 94.9%. Decellularized matrices were successfully seeded with MSCs, which grew to a near-confluent monolayer under flow conditions and MSCs were highly elongated and oriented to the flow direction., Conclusion: Small-diameter vascular grafts can be constructed by seeding MSCs onto xenogenic decellularized arterial matrices and culturing in a pulsatile bioreactor.

Biodegradable triblock copolymers of epsilon-caprolactone and L-lactide with varying compositions and molecular weights have been synthesized. They were then used to fabricate compliant small-diameter tissue engineered vascular scaffolds by using an electrospinning technique. The in vitro and in vivo degradation of the ultrafine fabrics was monitored to be faster than their counterpart cast films. A favorable interaction between the scaffolds and the mouse fibroblast L929 cells was demonstrated via MTT assay. A confluent, adherent monolayer of canine mesenchymal stem cells was observed in the tubular scaffold lumen after culture in a bioreactor for 3 days. The scaffold mechanical strength was strong enough to be transplanted into the canine carotid artery.

Aim: Recent studies have demonstrated that mesenchymal stem cells (MSCs) can differentiate into endothelial cells. The effect of shear stress on MSC differentiation is incompletely understood, and most studies have been based on two-dimensional systems. We used a model of tissue-engineered vascular grafts (TEVGs) to investigate the effects of shear stress on MSC differentiation., Methods: MSCs were isolated from canine bone marrow. The TEVG was constructed by seeding MSCs onto poly-epsilon-caprolactone and lactic acid (PCLA) scaffolds and subjecting them to shear stress provided by a pulsatile bioreactor for four days (two days at 1 dyne/cm(2) to 15 dyne/cm(2) and two days at 15 dyne/cm(2))., Results: Shear stress significantly increased the expression of endothelial cell markers, such as platelet-endothelial cell adhesion molecule-1 (PECAM-1), VE-cadherin, and CD34, at both the mRNA and protein levels as compared with static control cells. Protein levels of alpha-smooth muscle actin (alpha-SMA) and calponin were substantially reduced in shear stress-cultured cells. There was no significant change in the expression of alpha-SMA, smooth muscle myosin heavy chain (SMMHC) or calponin at the mRNA level., Conclusion: Shear stress upregulated the expression of endothelial cell-related markers and downregulated smooth muscle-related markers in canine MSCs. This study may serve as a basis for further investigation of the effects of shear stress on MSC differentiation in TEVGs.

Purpose: Uniform and tubular surface seeding is a prerequisite for tissue-engineered blood vessels to mature properly in a bioreactor. Our objective was to investigate reendothelialization of tubular scaffolds by the synergistic forces of sedimentation and rotation, so as to fabricate tissue-engineered venous grafts in vitro., Materials and Methods: Canine bone-marrow-derived endothelial progenitor cells were expanded in vitro. By means of a homemade horizontally rotative device, enzymatically decellularized porcine aortic scaffolds were tubularly seeded with the cells by precoating different matrices, under different rotation speeds, culture durations, and seeding techniques. Incorporation of lipoprotein and antiplatelet aggregation functions of seeded cells were evaluated. The seeding efficacies of various methods were compared by histology and scanning electronic microscopy., Results: Uniform distribution and a larger area of cell coverage were demonstrated by precoating with fibronectin (Fn) and a rotation speed of 2.5 revolutions per hour (rph). The efficacy of rotative seeding was comparable to its static counterpart at 4 h, but decreased at 72 h. The result of single-spin seeding was not different from that of three-spin seeding. The seeded cells showed their natural functions of lipoprotein uptake and antiplatelet aggregative properties. Based on these, we constructed 12 tissue-engineered venous grafts with a cell coverage area of 87.4+/-6.2%., Conclusions: Efficient and reproducible endothelialization was demonstrated by precoating scaffolds with Fn and by performing single-spin seeding at a speed of 2.5 rph.

Objective: To explore the feasibility of the tissue engineered venous grafts (TEVGs) constructed in vitro based on canine autologous bone marrow-derived endothelial progenitor cells (EPCs) and porcine decellularized aortic scaffolds implanted into the canine inferior vena cava., Methods: To draw out a volume of 8 - 12 ml of bone marrow from the canine (n = 8), to culture and expand EPCs in vitro using conditioned medium. After labeled with a red fluorescent dye PKH26-GL, the cells were seeded onto the luminal surface of decellularized porcine scaffolds with single, rotative method for 4 h. Following static culture for 24 - 72 h, the hybrids were implanted to replace autologous canine inferior vena cava about 4 cm long. Meantime one femoral artery-venous shunt about 1 cm long was performed. The non-seeded decellularized scaffolds (n = 4) were performed the same as control. Angiography was performed and the hybrids were explanted for morphology and labeled cells' immuno-fluorescence examinations at postoperative 10 d, 4 weeks and 12 weeks, respectively., Results: The patent number of experiment (control) group were 7/7 (2/4), 6/6 (2/2) and 4/4 (1/2) at postoperative 10 d, 4 weeks and 12 weeks, respectively. At 12 weeks, tightly confluence endothelial cells which covered the whole inner luminal surface of the explants were detected by immunohistochemistry of factor VIII and scanning electron microscopy, while fibrin-based pseudo-intima was detected on the inner luminal surface of matrix in the solo patent dog from the control group. Meanwhile, fibroblasts and alpha-actin positive cells in the matrices were found by transmission electron microscopy and alpha-actin immunohistochemistry. PKH26-GL labeled EPCs sustained on the luminal surface at a rather proportion accompanied by newly formed endothelial cells. However, the explants in both groups showed partial stenosis., Conclusions: Such constructed tissue engineered venous graft based on canine autologous bone marrow-derived endothelial progenitor cells and porcine decellularized aortic matrices is promising and deserve to further improvement and testing.

Objective: To explore the primary experimental methods to construct tissue engineering blood vessel., Methods: Using the collagen-chitosan to prefabricate the scaffolds with 3-dimensional structure, the proliferated human endothelial cells (ECs), smooth muscle cells (SMCs) and fibroblasts act as the seed cells. The cells were seeded to scaffolds in two-step method, and engineering tissue were matured by static culture or bioreactor culture. Extracellular matrix contents and the platelet aggregation were examined in engineering tissue, tissue engineering blood vessels were taken as patches to repair the man-made defaults on the rats aorta., Results: The proliferated human ECs, SMCs and fibroblast can hold activity and act as seed cells. The prefabricated scaffolds, with excellently cell and tissue biocompatibility, can facilitated cells adherence and upgrowth, the cells quantities and extracellular matrix contents in engineered tissue are time dependent increase (P < 0.05). Platelet aggregation tests confirm the tissue engineering blood vessel have some anti-coagulability. Using the engineering tissue patch to repair the default, 6 aortas in 8 animal were patency till 10 days post-operation., Conclusions: The seeding cells can be seeded on the 3-dimensional collagen-chitosan scaffolds and matured, the tissue engineering blood vessel can be constructed primarily.