Injectable Highly Tunable Oligomeric Collagen Matrices for Dental Tissue Regeneration.

Current stem cell transplantation approaches lack efficacy, because they limit cell survival and retention and, more importantly, lack a suitable cellular niche to modulate lineage-specific differentiation. Here, we evaluate the intrinsic ability of type I oligomeric collagen matrices to modulate dental pulp stem cells (DPSCs) endothelial and odontogenic differentiation as a potential stem cell-based therapy for regenerative endodontics. DPSCs were encapsulated in low-stiffness (235 Pa) and high-stiffness (800 Pa) oligomeric collagen matrices and then evaluated for long-term cell survival, as well as endothelial and odontogenic differentiation following in vitro cell culture. Moreover, the effect of growth factor incorporation, i.e., vascular endothelial growth factor (VEGF) into 235 Pa oligomeric collagen or bone morphogenetic protein (BMP2) into the 800 Pa oligomeric collagen counterpart on endothelial or odontogenic differentiation of encapsulated DPSCs was investigated. DPSCs-laden oligomeric collagen matrices allowed long-term cell survival. Real time polymerase chain reaction (RT-PCR) data showed that the DPSCs cultured in 235 Pa matrices demonstrated an increased expression of endothelial markers after 28 days, and the effect was enhanced upon VEGF incorporation. There was a significant increase in alkaline phosphatase (ALP) activity at Day 14 in the 800 Pa DPSCs-laden oligomeric collagen matrices, regardless of BMP2 incorporation. However, Alizarin S data demonstrated higher mineralization by Day 21 and the effect was amplified in BMP2-modified matrices. Herein, we present key data that strongly support future research aimed at clinical translation of an injectable oligomeric collagen system for delivery and fate regulation of DPSCs to enable pulp and dentin regeneration at specific locations of the root canal system.

Curcumin-A Natural Medicament for Root Canal Disinfection: Effects of Irrigation, Drug Release, and Photoactivation.

INTRODUCTION: Curcumin incorporation into polymeric fibers was tested for its antimicrobial properties and potential use in root canal disinfection. METHODS: Curcumin-modified fibers were processed via electrospinning and tested against a 7-day old established Actinomyces naeslundii biofilm. The medicaments tested were as follows: curcumin-modified fibers at 2.5 and 5.0 mg/mL, curcumin-based irrigant at 2.5 and 5.0 mg/mL, saline solution (negative control), and the following positive controls: 2% chlorhexidine, 1% sodium hypochlorite, and triple antibiotic paste (TAP, 1 mg/mL). All medicaments, except for the positive controls, were allocated according to the light exposure protocol (ie, photoactivation with a light-emitting diode every 30 seconds for 4 minutes or without photoactivation). After treatment, the medicaments were removed, and 1 mL saline solution was added; the biofilm was scraped from the well and used to prepare a 1:2000 dilution. Spiral plating was performed using anaerobic blood agar plates. After 24 hours, colony-forming units (colony-forming units/mL, n = 11/group) were counted to determine the antimicrobial effects. RESULTS: Data exhibited significant antimicrobial effects on the positive control groups followed by the curcumin irrigants and, lastly, the photoactivated curcumin-modified fibers. There was a significant reduction of viable bacteria in curcumin-based irrigants, which was greater than the TAP-treated group. Curcumin-free fibers, saline, and the nonphotoactivated curcumin-modified fibers did not display antimicrobial activity. CONCLUSIONS: Curcumin seems to be a potential alternative to TAP when controlling infection, but it requires a minimal concentration (2.5 mg/mL) to be effective. Photoactivation of curcumin-based medicaments seems to be essential to obtain greater antibiofilm activity.

Clindamycin-modified Triple Antibiotic Nanofibers: A Stain-free Antimicrobial Intracanal Drug Delivery System.

INTRODUCTION: A biocompatible strategy to promote bacterial eradication within the root canal system after pulpal necrosis of immature permanent teeth is critical to the success of regenerative endodontic procedures. This study sought to synthesize clindamycin-modified triple antibiotic (metronidazole, ciprofloxacin, and clindamycin [CLIN]) polymer (polydioxanone [PDS]) nanofibers and determine in vitro their antimicrobial properties, cell compatibility, and dentin discoloration. METHODS: CLIN-only and triple antibiotic CLIN-modified (CLIN-m, minocycline-free) nanofibers were processed via electrospinning. Scanning electron microscopy, Fourier-transform infrared spectroscopy (FTIR), and tensile testing were performed to investigate fiber morphology, antibiotic incorporation, and mechanical strength, respectively. Antimicrobial properties of CLIN-only and CLIN-m nanofibers were assessed against several bacterial species by direct nanofiber/bacteria contact and over time based on aliquot collection up to 21 days. Cytocompatibility was measured against human dental pulp stem cells. Dentin discoloration upon nanofiber exposure was qualitatively recorded over time. The data were statistically analyzed (P < .05). RESULTS: The mean fiber diameter of CLIN-containing nanofibers ranged between 352 ± 128 nm and 349 ± 128 nm and was significantly smaller than PDS fibers. FTIR analysis confirmed the presence of antibiotics in the nanofibers. Hydrated CLIN-m nanofibers showed similar tensile strength to antibiotic-free (PDS) nanofibers. All CLIN-containing nanofibers and aliquots demonstrated pronounced antimicrobial activity against all bacteria. Antibiotic-containing aliquots led to a slight reduction in dental pulp stem cell viability but were not considered toxic. No visible dentin discoloration upon CLIN-containing nanofiber exposure was observed. CONCLUSIONS: Collectively, based on the remarkable antimicrobial effects, cell-friendly, and stain-free properties, our data suggest that CLIN-m triple antibiotic nanofibers might be a viable alternative to minocycline-based antibiotic pastes.

Advanced Scaffolds for Dental Pulp and Periodontal Regeneration.

No current therapy promotes root canal disinfection and regeneration of the pulp-dentin complex in cases of pulp necrosis. Antibiotic pastes used to eradicate canal infection negatively affect stem cell survival. Three-dimensional easy-to-fit antibiotic-eluting nanofibers, combined with injectable scaffolds, enriched or not with stem cells and/or growth factors, may increase the likelihood of achieving predictable dental pulp regeneration. Periodontitis is an aggressive disease that impairs the integrity of tooth-supporting structures and may lead to tooth loss. The latest advances in membrane biomodification to endow needed functionalities and technologies to engineer patient-specific membranes/constructs to amplify periodontal regeneration are presented.

Tetracycline-incorporated polymer nanofibers as a potential dental implant surface modifier.

This study investigated the antimicrobial and osteogenic properties of titanium (Ti) disks superficially modified with tetracycline (TCH)-incorporated polymer nanofibers. The experiments were carried out in two phases. The first phase dealt with the synthesis and characterization (i.e., morphology, mechanical strength, drug release, antimicrobial activity, and cytocompatibility) of TCH-incorporated fibers. The second phase was dedicated to evaluating both the antimicrobial and murine-derived osteoprecursor cell (MC3T3-E1) response of Ti-modified with TCH-incorporated fibers. TCH was successfully incorporated into the submicron-sized and cytocompatible fibers. All TCH-incorporated mats presented significant antimicrobial activity against periodontal pathogens. The antimicrobial potential of the TCH-incorporated fibers-modified Ti was influenced by both the TCH concentration and bacteria tested. At days 5 and 7, a significant increase in MC3T3-E1 cell number was observed for TCH-incorporated nanofibers-modified Ti disks when compared to that of TCH-free nanofibers-modified Ti-disks and bare Ti. A significant increase in alkaline phosphatase (ALP) levels on the Ti disks modified with TCH-incorporated nanofiber on days 7 and 14 was seen, suggesting that the proposed surface promotes early osteogenic differentiation. Collectively, the data suggest that TCH-incorporated nanofibers could function as an antimicrobial surface modifier and osteogenic inducer for Ti dental implants. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 2085-2092, 2017.

Triple Antibiotic Polymer Nanofibers for Intracanal Drug Delivery: Effects on Dual Species Biofilm and Cell Function.

INTRODUCTION: Root canal disinfection and the establishment of an intracanal microenvironment conducive to the proliferation/differentiation of stem cells play a significant role in regenerative endodontics. This study was designed to (1) investigate the antimicrobial efficacy of triple antibiotic-containing nanofibers against a dual-species biofilm and (2) evaluate the ability of dental pulp stem cells (DPSCs) to adhere to and proliferate on dentin upon nanofiber exposure. METHODS: Seven-day-old dual-species biofilm established on dentin specimens was exposed for 3 days to the following: saline (control), antibiotic-free nanofibers (control), and triple antibiotic-containing nanofibers or a saturated triple antibiotic paste (TAP) solution (50 mg/mL in phosphate buffer solution). Bacterial viability was assessed using the LIVE/DEAD assay (Molecular Probes, Inc, Eugene, OR) and confocal laser scanning microscopy. For cytocompatibility studies, dentin specimens after nanofiber or TAP (1 g/mL in phosphate buffer solution) exposure were evaluated for cell adhesion and spreading by actin-phalloidin staining. DPSC proliferation was assessed on days 1, 3, and 7. Statistics were performed, and significance was set at the 5% level. RESULTS: Confocal laser scanning microscopy showed significant bacterial death upon antibiotic-containing nanofiber exposure, differing significantly (P < .05) from antibiotic-free fibers and the control (saline). DPSCs showed enhanced adhesion/spreading on dentin specimens treated with antibiotic-containing nanofibers when compared with its TAP counterparts. The DPSC proliferation rate was similar on days 1 and 3 in antibiotic-free nanofibers, triple antibiotic-containing nanofibers, and TAP-treated dentin. Proliferation was higher (9-fold) on dentin treated with antibiotic-containing nanofibers on day 7 compared with TAP. CONCLUSIONS: Triple antibiotic-containing polymer nanofibers led to significant bacterial death, whereas they did not affect DPSC attachment and proliferation on dentin.

Synthesis and characterization of CaO-loaded electrospun matrices for bone tissue engineering.

OBJECTIVES: This study aims to synthesize and characterize biodegradable polymer-based matrices loaded with CaO nanoparticles for osteomyelitis treatment and bone tissue engineering. MATERIALS AND METHODS: Poly(ε-caprolactone) (PCL) and PCL/gelatin (1:1, w/w) solutions containing CaO nanoparticles were electrospun into fibrous matrices. Scanning (SEM) and transmission (TEM) electron microscopy, Fourier transformed infrared (FTIR), energy dispersive X-ray spectroscopy (EDS), contact angle (CA), tensile testing, and antibacterial activity (agar diffusion assay) against Staphylococcus aureus were performed. Osteoprecursor cell (MC3T3-E1) response (i.e., viability and alkaline phosphatase expression/ALP) and infiltration into the matrices were evaluated. RESULTS: CaO nanoparticles were successfully incorporated into the fibers, with the median fiber diameter decreasing after CaO incorporation. The CA decreased with the addition of CaO, and the presence of gelatin made the matrix very hydrophilic (CA = 0°). Increasing CaO concentrations progressively reduced the mechanical properties (p ≤ 0.030). CaO-loaded matrices did not display consistent antibacterial activity. MC3T3-E1 cell viability demonstrated the highest levels for CaO-loaded matrices containing gelatin after 7 days in culture. An increased ALP expression was consistently seen for PCL/CaO matrices when compared to PCL and gelatin-containing counterparts. CONCLUSIONS: Despite inconsistent antibacterial activity, CaO nanoparticles can be effectively loaded into PCL or PCL/gelatin fibers without negatively affecting the overall performance of the matrices. More importantly, CaO incorporation enhanced cell viability as well as differentiation capacity, as demonstrated by an increased ALP expression. CLINICAL SIGNIFICANCE: CaO-loaded electrospun matrices show potential for applications in bone tissue engineering.

Dimensionally stable and bioactive membrane for guided bone regeneration: An in vitro study.

Composite fibrous electrospun membranes based on poly(dl-lactide) (PLA) and poly(ε-caprolactone) (PCL) were engineered to include borate bioactive glass (BBG) for the potential purposes of guided bone regeneration (GBR). The fibers were characterized using scanning and transmission electron microscopies, which respectively confirmed the submicron fibrous arrangement of the membranes and the successful incorporation of BBG particles. Selected mechanical properties of the membranes were evaluated using the suture pullout test. The addition of BBG at 10 wt % led to similar stiffness, but more importantly, it led to a significantly stronger (2.37 ± 0.51 N mm) membrane when compared with the commercially available Epiguide® (1.06 ± 0.24 N mm) under hydrated conditions. Stability (shrinkage) was determined after incubation in a phosphate buffer solution from 24 h up to 9 days. The dimensional stability of the PLA:PCL-based membranes with or without BBG incorporation (10.07-16.08%) was similar to that of Epiguide (14.28%). Cell proliferation assays demonstrated a higher rate of preosteoblasts proliferation on BBG-containing membranes (6.4-fold) over BBG-free membranes (4- to 5.8-fold) and EpiGuide (4.5-fold), following 7 days of in vitro culture. Collectively, our results demonstrated the ability to synthesize, via electrospinning, stable, polymer-based submicron fibrous BBG-containing membranes capable of sustaining osteoblastic attachment and proliferation-a promising attribute in GBR.

Differentiation of porcine mesenchymal stem cells into epithelial cells as a potential therapeutic application to facilitate epithelial regeneration.

Epithelial denudation is one of the characteristics of chronic asthma. To restore its functions, the airway epithelium has to rapidly repair the injuries and regenerate its structure and integrity. Mesenchymal stem cells (MSCs) have the ability to differentiate into many cell lineages. However, the differentiation of MSCs into epithelial cells has not been fully studied. Here, we examined the differentiation of MSCs into epithelial cells using three different media compositions with various growth supplementations. The MSCs were isolated from porcine bone marrow by density gradient centrifugation. The isolated MSCs were CD11(-) CD34(-) CD45(-) CD44(+) CD90(+) and CD105(+) by immunostaining and flow cytometry. MSCs were stimulated with EpiGRO (Millipore), BEpiCM (ScienCell) and AECGM (PromoCell) media for 5 and 10 days, and epithelial differentiation was assessed by qPCR (keratin 14, 18 and EpCAM), fluorometry (cytokeratin 7-8, cytokeratin 14-15-16-19 and EpCAM), western blot analysis (pancytokeratin, EpCAM) and flow cytometry (cytokeratin 7-8, cytokeratin 14-15-16-19 and EpCAM). The functional marker MUC1 was also assessed after 10 days of air-liquid interface (ALI) culture in optimized media. Cells cultured in BEpiCM containing fibroblast growth factor and prostaglandin E2 showed the highest expression of the epithelial markers: CK7-8 (85.90%); CK-14-15-16-19 (10.14%); and EpCAM (64.61%). The cells also expressed functional marker MUC1 after ALI culture. The differentiated MSCs when cultured in BEpiCM medium ex vivo in a bioreactor on a decellularized trachea for 10 days retained the epithelial-like phenotype. In conclusion, porcine bone marrow-derived MSCs demonstrate commitment to the epithelial lineage and might be a potential therapy for facilitating the repair of denuded airway epithelium.

Mesenchymal Stem Cell Paracrine Factors in Vascular Repair and Regeneration.

Mesenchymal stem cell therapy show great optimism in the treatment of several diseases. MSCs are attractive candidates for cell therapy because of easy isolation, high expansion potential giving unlimited pool of transplantable cells, low immunogenicity, amenability to ex vivo genetic modification, and multipotency. The stem cells orchestrate the repair process by various mechanisms such as transdifferentiation, cell fusion, microvesicles or exosomes and most importantly by secreting paracrine factors. The MSCs release several angiogenic, mitogenic, anti-apoptotic, anti-inflammatory and anti-oxidative factors that play fundamental role in regulating tissue repair in various vascular and cardiac diseases. The therapeutic release of these factors by the cells can be enhanced by several strategies like genetic modification, physiological and pharmacological preconditioning, improved cell culture and selection methods, and biomaterial based approaches. The current review describes the impact of paracrine factors released by MSCs on vascular repair and regeneration in myocardial infarction, restenosis and peripheral artery disease, and the various strategies adopted to enhance the release of these paracrine factors to enhance organ function.

SOCS3 promotor hypermethylation and STAT3-NF-κB interaction downregulate SOCS3 expression in human coronary artery smooth muscle cells.

Suppressor of cytokine signaling-3 (SOCS3) is an intracellular negative regulator of cytokine signaling pathway. We recently found significant reduction in SOCS3 expression in coronary artery smooth muscle cells (CASMCs) of atherosclerotic swine and also in vitro cultured cells. Here, we investigated the underlying mechanisms of SOCS3 downregulation by IGF-1 and TNF-α in human CASMCs(hCASMCs). We propose that hypermethylation of CpG islands in the SOCS3 promoter is responsible for decrease in SOCS3 expression involving STAT3 and NFkB-p65 interaction. Western blot and qPCR data revealed significant upregulation of SOCS3 (6- to 10-fold) in hCASMC when treated individually with TNF-α (100 ng/ml) or IGF-1 (100 ng/ml). However, a significant decrease (5-fold) was observed by the combined treatment with TNF-α and IGF-1 compared with individual stimulation. IGF-1 phosphorylated STAT3 and TNF-α-activated NF-κB in hCASMCs. In the nuclear extract of hCASMCs stimulated with both TNF-α and IGF-1, there was an interaction between NF-κB-p65 and pSTAT3, as determined by co-immunoprecipitation. Knockdown of STAT3 by small interfering RNA abolished SOCS3 expression in response to IGF-1. Methylation-specific PCR confirmed hypermethylation of SOCS3 promoter in hCASMCs stimulated with both TNF-α and IGF-1, and this was positively associated with elevated levels of DNA methyltransferase-I (9- to 10-fold). Knockdown of DNMT1 increased SOCS3 expression in IGF-1+TNF-α-stimulated cells. Downregulation of SOCS3 in the presence of both TNF-α and IGF-1 in hCASMCs is due to SOCS3 promoter hypermethylation involving STAT3-NFkBp65 interaction. Because TNF-α and IGF-1 are released due to mechanical injury during coronary intervention, hypermethylation of SOCS3 gene could be an underlying mechanism of intimal hyperplasia and restenosis.

In vitro differentiation of bone marrow derived porcine mesenchymal stem cells to endothelial cells.

Mesenchymal stem cells (MSCs) hold potential for the regeneration of damaged tissues in cardiovascular diseases. In this study, we investigated the potential of porcine MSCs to differentiate into endothelial cells (ECs) in vitro. The cultured bone marrow-derived cells were CD11b⁻CD34⁻CD44⁺CD45⁻CD90⁺ and showed mesodermal lineage differentiation, which is characteristic of MSCs. The MSCs were induced to differentiate into ECs using endothelial growth medium (EGM), with and without high concentrations of VEGF (EGM + VEGF; 50 ng/ml). Endothelial basal medium (EBM) without growth factors served as the control. The EC differentiation was assessed by the presence of vWF, ability to take up acetylated LDL, in vitro angiogenesis assay, flow cytometry and qPCR of EC markers vWF, VE-cadherin, PECAM-1, VEGF-R1 and VEGF-R2 after 10 days of stimulation. Cells cultured in EGM + VEGF medium demonstrated higher amounts of DiI-AcLDL-positive cells and enhanced the presence of vWF (90%), VE-Cadherin- (60%) and PECAM-1 (48%)-positive cells, than in EBM. These cells showed profuse sprouting of capillary tubes and closed polygon formation in the angiogenesis assay. There was 1.5-2-fold increase in the mRNA expression of endothelial markers in the cells stimulated with EGM + VEGF medium when compared to control. The results demonstrate the ability of porcine MSCs to differentiate into ECs under in vitro inducing conditions. The differentiated cells would provide new options for re-endothelialization following interventional procedures and tissue engineering.

Successful transfection of genes using AAV-2/9 vector in swine coronary and peripheral arteries.

BACKGROUND: Gene therapy has attracted attention for its potential to treat several cardiovascular diseases. The use of adeno-associated viral (AAV) vectors to facilitate therapeutic gene transfer to suppress intimal hyperplasia is a promising concept. The objective of this study was to analyze the in vivo transduction of a novel recombinant AAV-2/9 vector with SM22α promoter, containing ß-galactosidase gene (LacZ) or green fluorescent protein (GFP) as reporter genes, to the medial layer smooth muscle cells (SMCs) of swine coronary and peripheral arteries. METHODS: The AAV-2/9 vector containing SM22α (1 × 10(13) pfu) were administered into carotid/femoral/coronary arteries of domestic swine using irrigating balloon catheter-based gene delivery. Following gene transfer, cryosections of arteries were processed for X-Gal and GFP analysis. Fluorescence microscopy and Western blotting were done to analyze the GFP expression in the SMCs. RESULTS: LacZ mRNA expression was visualized in the medial layer 7 d after vector administration. The GFP expression was detected at day 7 and lasted for at least 2 mo showing the longer-lasting expression of the AAV-2/9 vector. Control arteries did not show any expression of GFP or LacZ. There was no significant effect of AAV-2/9 viral transduction on serum amylase, fibrinogen, and serum CRP levels. CONCLUSION: These finding support the use of AAV-2/9 as a vector to effectively transduce a gene in SMCs of coronary and peripheral arteries without causing inflammation.

Neuropeptide Y receptors in carotid plaques of symptomatic and asymptomatic patients: effect of inflammatory cytokines.

AIMS: Cytokines released by the immune cells at the site of plaque milieu induce smooth muscle cell apoptosis to promote plaque instability. But, neuropeptide Y (NPY), a pleotropic factor, may modulate the effects of cytokines in atherosclerotic plaques of patients with carotid stenosis. Our aim was to investigate the relative expression of NPY-Y1, NPY-Y2 and NPY-Y5 receptors on carotid plaque vascular smooth muscle cells (pVSMCs) of symptomatic (S) and asymptomatic (AS) patients and examine the effect of inflammatory cytokines on the expression of NPY receptors, that may attenuate plaque rupture. METHODS AND RESULTS: In healthy carotid artery, there were significantly increased immunopositivity and increased mRNA transcripts of NPY-Y1 and NPY-Y5 receptors in thin sections and isolated VSMCs, respectively, compared to S and AS plaques. However, the NPY-Y2 expression was higher in S and AS pVSMCs than controls. Stimulation of the cells with TNF-α, IL-12 or IFN-γ (50 ng/ml) decreased mRNA transcripts of NPY-Y1 and NPY-Y5 and increased NPY-Y2 mRNAs in VSMCs of healthy carotid artery. The effect of the cytokines on mRNA transcripts of NPY-Y5 and NPY-Y2 in pVSMCs of S and AS patients was similar to healthy VSMCs, but with variable effect on NPY-Y1. CONCLUSION: Increased expression of NPY-Y2 receptors in symptomatic pVSMCs than in healthy and asymptomatic subjects suggests a potential role of NPY-Y2 in plaque instability. This is further supported by the pronounced effect of atheroma-associated cytokines to increase NPY-Y2 mRNA transcripts in pVSMCs of patients with carotid stenosis.

Functional stability of endothelial cells on a novel hybrid scaffold for vascular tissue engineering.

Porous and pliable conduits made of biodegradable polymeric scaffolds offer great potential for the development of blood vessel substitutes but they generally lack signals for cell proliferation, survival and maintenance of a normal phenotype. In this study we have prepared and evaluated porous poly(ε-caprolactone) (PCL) integrated with fibrin composite (FC) to get a biomimetic hybrid scaffold (FC PCL) with the biological properties of fibrin, fibronectin (FN), gelatin, growth factors and glycosaminoglycans. Reduced platelet adhesion on a human umbilical vein endothelial cell-seeded hybrid scaffold as compared to bare PCL or FC PCL was observed, which suggests the non-thrombogenic nature of the tissue-engineered scaffold. Analysis of real-time polymerase chain reaction (RT-PCR) after 5 days of endothelial cell (EC) culture on a hybrid scaffold indicated that the prothrombotic von Willebrand factor and plasminogen activator inhibitor (PAI) were quiescent and stable. Meanwhile, dynamic expressions of tissue plasminogen activator (tPA) and endothelial nitric oxide synthase indicated the desired cell phenotype on the scaffold. On the hybrid scaffold, shear stress could induce enhanced nitric oxide release, which implicates vaso-responsiveness of EC grown on the tissue-engineered construct. Significant upregulation of mRNA for extracellular matrix (ECM) proteins, collagen IV and elastin, in EC was detected by RT-PCR after growing them on the hybrid scaffold and FC-coated tissue culture polystyrene (FC TCPS) but not on FN-coated TCPS. The results indicate that the FC PCL hybrid scaffold can accomplish a remodeled ECM and non-thrombogenic EC phenotype, and can be further investigated as a scaffold for cardiovascular tissue engineering.

Scaffolds in tissue engineering of blood vessels.

Tissue engineering of small diameter (<5 mm) blood vessels is a promising approach for developing viable alternatives to autologous vascular grafts. It involves in vitro seeding of cells onto a scaffold on which the cells attach, proliferate, and differentiate while secreting the components of extracellular matrix that are required for creating the tissue. The scaffold should provide the initial requisite mechanical strength to withstand in vivo hemodynamic forces until vascular smooth muscle cells and fibroblasts reinforce the extracellular matrix of the vessel wall. Hence, the choice of scaffold is crucial for providing guidance cues to the cells to behave in the required manner to produce tissues and organs of the desired shape and size. Several types of scaffolds have been used for the reconstruction of blood vessels. They can be broadly classified as biological scaffolds, decellularized matrices, and polymeric biodegradable scaffolds. This review focuses on the different types of scaffolds that have been designed, developed, and tested for tissue engineering of blood vessels, including use of stem cells in vascular tissue engineering.

Design of fibrin matrix composition to enhance endothelial cell growth and extracellular matrix deposition for in vitro tissue engineering.

Tissue-engineered blood vessel substitutes should closely resemble native vessels in terms of structure, composition, mechanical properties, and function. Successful cardiovascular tissue engineering requires optimization of in vitro culture environment that would produce functional constructs. The extracellular matrix (ECM) protein elastin plays an essential role in the cardiovascular system to render elasticity to blood vessel wall, whereas collagen is responsible for providing mechanical strength. The objective of this study was to understand the significance of various ECM components on endothelial cell (EC) growth and tissue generation. We demonstrate that, even though fibrin is a good matrix for EC growth, fibronectin is the crucial component of the fibrin matrix that enhances EC adhesion, spreading, and proliferation. Vascular EC growth factor is known to influence in vitro growth of EC, but, so far, ECM deposition in in vitro culture has not been reported. In this study, it is shown that incorporation of a mixture of hypothalamus-derived angiogenic growth factors with fibrin matrix enhances synthesis and deposition of insoluble elastin and collagen in the matrix, within 10 days of in vitro culture. The results suggest that a carefully engineered fibrin composite matrix may support EC growth, survival, and remodeling of ECM in vitro and impart optimum properties to the construct for resisting the shear stress at the time of implantation.

Development of a fibrin composite-coated poly(epsilon-caprolactone) scaffold for potential vascular tissue engineering applications.

Poor cell adhesion, cytotoxicity of degradation products and lack of biological signals for cell growth, survival, and tissue generation are the limitations in the use of a biodegradable polymer scaffold for vascular tissue engineering. We have fabricated a hybrid scaffold by integrating physicochemical characteristics of poly(epsilon-caprolactone) (PCL) and biomimetic property of a composite of fibrin, fibronectin, gelatin, growth factors, and proteoglycans to improve EC growth on the scaffold. Solvent cast porous films of poly(epsilon-caprolactone) was prepared using PEG as a porogen. Porosity varied between 5 and 200 microm, and FTIR spectroscopy confirmed structural aspects of PCL. Films kept in PBS for 60 days showed tensile strength and elongation matching native blood vessel. Slow degradation of the scaffold was demonstrated by gravimetric analysis and molecular weight determination. Human umbilical vein endothelial cell (HUVEC) adhesion and proliferation on bare films were minimal. FTIR spectroscopy and environmental scanning electron microscopy (ESEM) of PCL-fibrin hybrid scaffold confirmed the presence of fibrin composite on PCL film. HUVEC was subsequently cultured on hybrid scaffold, and continuous EC lining was observed in 15 and 30 days of culture using ESEM. Results suggest that the new hybrid scaffold can be a suitable candidate for cardiovascular tissue engineering.

Vascular tissue generation in response to signaling molecules integrated with a novel poly(epsilon-caprolactone)-fibrin hybrid scaffold.

A major constraint in the use of biodegradable polymer scaffolds for vascular tissue engineering is poor cell adhesion and lack of signals for new tissue generation. The presence of extracellular matrix (ECM) within the scaffold is desirable for growth of endothelial cells and in vitro formation of remodelled vascular conduit. In this study, we have produced a hybrid scaffold by coating porous poly-caprolactone (PCL) film with biomimetic ECM components consisting of fibrin, gelatin, fibronectin, angiogenic growth factors and proteoglycans. Human umbilical vein endothelial cells (HUVECs) adhered, spread, proliferated and survived for long periods in culture on the hybrid scaffold. As compared to bare PCL, enhanced cell adhesion, spreading and cytoskeletal organization were demonstrated on the hybrid scaffold, using confocal microscopy of EC-actin stained with Texas red-conjugated phalloidin. Population doubling of endothelial cells (ECs) on the hybrid scaffold and bare scaffold was estimated as 42 h and 136 h, respectively, as assessed by a 3H-thymidine uptake method. Analysis of proliferating cell nuclear antigen (PCNA) also indicated low proliferation on bare scaffold. Flow cytometric analysis of annexin V-stained cells showed poor survival of ECs on bare PCL as compared to the hybrid scaffold. Deposition of insoluble collagen and elastin was identified on the hybrid scaffold by cells recovered after 15 days and 30 days of EC culture, using fluorochrome-tagged specific antibodies and confocal microscopy, and the fluorescence intensity corresponding to elastin and collagen after 30 days was similar to that of 15 days. The results indicate that ECM deposition by endothelial cells is a regulated process without excessive accumulation after 30 days.