Evaluation of the materials properties, stability and cell response of a range of PEGDMA hydrogels for tissue engineering applications.

The main aim of this study was to examine the stability of a range of polyethyleneglycol dimethacrylate (PEGDMA) hydrogels over a 28-day period in simulated physiological solution. Upon optimisation of the ultraviolet (UV) curing conditions, the PEGDMA hydrogels were prepared using four different monomer concentrations (25, 50, 75 and 100 wt% PEGDMA) in water and cross-linked by photopolymerisation. Initial results revealed a correlation between monomer concentration and swelling behaviour, where a decrease in swelling was observed with increase in monomer content. On storage in physiological solutions at 37 °C, a decrease in the weight remaining of the hydrogels and the pH of the solutions was observed over a 28-day period. Using scanning electron microscopy, the surface topography of the hydrogels appeared to get smoother and in parallel changes in hydrophilicty were observed, with the biggest changes observed for the higher monomer concentrations where water contact angle values were seen to increase toward 90°. However, the mechanical properties remained relatively unaffected and there was no adverse effect on cell metabolic activity observed for cells grown in the presence of PEGDMA samples or using elution methods. Looking at the combination of mechanical chemical and thermal properties shown these results are an important finding for scaffolds intended for tissue engineering applications, where provision of mechanical support without the elicitation of an inflammatory response due to polymer degradation products is crucial for successful integration and neotissue formation during the first 28 days post implantation.

Evaluation of the Early In Vivo Response of a Functionally Graded Macroporous Scaffold in an Osteochondral Defect in a Rabbit Model.

Cartilage tissue engineering is a multifactorial problem requiring a wide range of material property requirements from provision of biological cues to facilitation of mechanical support in load-bearing diarthrodial joints. The study aim was to design, fabricate and characterize a template to promote endogenous cell recruitment for enhanced cartilage repair. A polylactic acid poly-ε-caprolactone (PLCL) support structure was fabricated using laser micromachining technology and thermal crimping to create a functionally-graded open pore network scaffold with a compressive modulus of 9.98 ± 1.41 MPa and a compressive stress at 50% strain of 8.59 ± 1.35 MPa. In parallel, rabbit mesenchymal stem cells were isolated and their growth characteristics, morphology and multipotency confirmed. Sterilization had no effect on construct chemical structure and cellular compatibility was confirmed. After four weeks implantation in an osteochondral defect in a rabbit model to assess biocompatibility, there was no evidence of inflammation or giant cells. Moreover, acellular constructs performed better than cell-seeded constructs with endogenous progenitor cells homing through microtunnels, differentiating to form neo-cartilage and strengthening integration with native tissue. These results suggest, albeit at an early stage of repair, that by modulating the architecture of a macroporous scaffold, pre-seeding with MSCs is not necessary for hyaline cartilage repair.

A chondromimetic microsphere for in situ spatially controlled chondrogenic differentiation of human mesenchymal stem cells.

Human mesenchymal stem cells (hMSCs) have been identified as a viable cell source for cartilage tissue engineering. However, to undergo chondrogenic differentiation hMSCs require growth factors, in particular members of the transforming growth factor beta (TGF-ß) family. While in vitro differentiation is feasible through continuous supplementation of TGF-ß3, mechanisms to control and drive hMSCs down the chondrogenic lineage in their native microenvironment remain a significant challenge. The release of TGF-ß3 from an injectable microsphere composed of the cartilage-associated extracellular matrix molecule hyaluronan represents a readily translatable approach for in situ differentiation of hMSCs for cartilage repair. In this study, chondromimetic hyaluronan microspheres were used as a growth factor delivery source for hMSC chondrogenesis. Cellular compatibility of the microspheres (1.2 and 14.1 µm) with hMSCs was shown and release of TGF-ß3 from the most promising 14.1 µm microspheres to control differentiation of hMSCs was evaluated. Enhanced accumulation of cartilage-associated glycosaminoglycans by hMSCs incubated with TGF-ß3-loaded microspheres was seen and positive staining for collagen type II and proteoglycan confirmed successful in vitro chondrogenesis. Gene expression analysis showed significantly increased expression of the chondrocyte-associated genes, collagen type II and aggrecan. This delivery platform resulted in significantly less collagen type X expression, suggesting the generation of a more stable cartilage phenotype. When evaluated in an ex vivo osteoarthritic cartilage model, implanted hMSCs with TGF-ß3-loaded HA microspheres were detected within cartilage fibrillations and increased proteoglycan staining was seen in the tissue. In summary, data presented here demonstrate that TGF-ß3-bound hyaluronan microspheres provide a suitable delivery system for induction of hMSC chondrogenesis and their use may represent a clinically feasible tissue engineering approach for the treatment of articular cartilage defects.

Liposomal surface coatings of metal stents for efficient non-viral gene delivery to the injured vasculature.

Despite the widespread use of drug eluting stents (DES), in-stent restenosis (ISR), delayed arterial healing and thrombosis remain important clinical complications. Gene-eluting stents (GES) represent a potential strategy for the prevention of ISR by delivering a therapeutic gene via a vector from the stent surface to the vessel wall. To this end, a model in vitro system was established to examine whether cationic liposomes could be used for gene delivery to human artery cells. Three different formulations were compared (DOTMA/DOPE, DDAB/DOPE or DDAB/POPC/Chol) to examine the effects of different cationic and neutral lipids on the transfection efficiency of lipoplex-coatings of metal surfaces. Upon completion of the characterization and optimization of the materials for gene delivery in vitro, these coatings were examined on a range of stents and deployed in a rabbit iliac artery injury model in vivo. Maximal transfection efficiencies for all coatings were observed on day 28, followed by declining, but persisting gene expression 42 days after stent placement, thereby, presenting liposomal coatings for gene eluting stents as treatment options for clinical complications associated with stenting procedures.

Enhancing the mesenchymal stem cell therapeutic response: cell localization and support for cartilage repair.

Articular cartilage is a complex, multilayered biological composite material, comprised of chondrocytes encapsulated in a water-based glycosaminoglycan matrix reinforced with collagen fibers. Once damaged by osteoarthritis or traumatic injury, this aneural, avascular tissue has little self-repair capacity. Over the last 20 years, cell therapies and tissue-engineering strategies have shown significant promise for the repair or regeneration of damaged cartilage. In particular, mesenchymal stem cells (MSCs) have great potential owing to their ability to create a reparative environment. Despite the fact that there have been great strides in the design and development of three-dimensional scaffolds, there is an upper limit to the number of viable cells that can be delivered using current approaches. To this end, this review examines current strategies for optimizing MSC localization, evaluates their limitations, and looks to other technologies to devise a combinatorial strategy for the creation of an MSC-seeded composite structure that addresses both the mechanical and biological property requirements for enhanced cartilage repair.

The electrical stimulation of carbon nanotubes to provide a cardiomimetic cue to MSCs.

Once damaged, cardiac muscle has little intrinsic repair capability due to the poor regeneration potential of remaining cardiomyocytes. One method of overcoming this issue is to deliver functional cells to the injured myocardium to promote repair. To address this limitation we sought to test the hypothesis that electroactive carbon nanotubes (CNT) could be employed to direct mesenchymal stem cell (MSC) differentiation towards a cardiomyocyte lineage. Using a two-pronged approach, MSCs exposed to medium containing CNT and MSCs seeded on CNT based polylactic acid scaffolds were electrically stimulated in an electrophysiological bioreactor. After electrical stimulation the cells reoriented perpendicular to the direction of the current and adopted an elongated morphology. Using qPCR, an upregulation in a range of cardiac markers was detected, the greatest of which was observed for cardiac myosin heavy chain (CMHC), where a 40-fold increase was observed for the electrically stimulated cells after 14 days, and a 12-fold increase was observed for the electrically stimulated cells seeded on the PLA scaffolds after 10 days. Differentiation towards a cardioprogenitor cell was more evident from the western blot analysis, where upregulation of Nkx2.5, GATA-4, cardiac troponin t (CTT) and connexin43 (C43) was seen to occur. This was echoed in immunofluorescent staining, where increased levels of CTT, CMHC and C43 protein expression were observed after electrical stimulation for both cells and cell-seeded scaffolds. More interestingly, there was evidence of increased cross talk between the cells as shown by the pattern of C43 staining after electrical stimulation. These results establish a paradigm for nanoscale biomimetic cues that can be readily translated to other electroactive tissue repair applications.

Fabrication, mechanical and in vivo performance of polycaprolactone/tricalcium phosphate composite scaffolds.

This paper explores the use of selective laser sintering (SLS) for the generation of bone tissue engineering scaffolds from polycaprolactone (PCL) and PCL/tricalcium phosphate (TCP). Different scaffold designs are generated, and assessed from the point of view of manufacturability, porosity and mechanical performance. Large scaffold specimens are produced, with a preferred design, and are assessed through an in vivo study of the critical size bone defect in sheep tibia with subsequent microscopic, histological and mechanical evaluation. Further explorations are performed to generate scaffolds with increasing TCP content. Scaffold fabrication from PCL and PCL/TCP mixtures with up to 50 mass% TCP is shown to be possible. With increasing macroporosity the stiffness of the scaffolds is seen to drop; however, the stiffness can be increased by minor geometrical changes, such as the addition of a cage around the scaffold. In the animal study the selected scaffold for implantation did not perform as well as the TCP control in terms of new bone formation and the resulting mechanical performance of the defect area. A possible cause for this is presented.

In vitro characterization of an electroactive carbon-nanotube-based nanofiber scaffold for tissue engineering.

In an effort to reduce organ replacement and enhance tissue repair, there has been a tremendous effort to create biomechanically optimized scaffolds for tissue engineering applications. In contrast, the development and characterization of electroactive scaffolds has attracted little attention. Consequently, the creation and characterization of a carbon nanotube based poly(lactic acid) nanofiber scaffold is described herein. After 28 d in physiological solution at 37 °C, a change in the mass, chemical properties and polymer morphology is seen, while the mechanical properties and physical integrity are unaltered. No adverse cytotoxic affects are seen when mesenchymal stem cells are cultured in the presence of the scaffold. Taken together, these data auger well for electroactive tissue engineering.

Behavior of human mesenchymal stem cells in fibrin-based vascular tissue engineering constructs.

A limitation of current tissue engineering vascular graft technology is the provision of an expandable, autologous cell source. By harnessing the multipotency of mesenchymal stem cells (MSC), it is hoped that functional vascular cells can be produced. To date, a range of 2D and 3D environments have been investigated for the manipulation of MSC differentiation pathways. To this end, this study aims to test the hypothesis that MSC seeded in various fibrin gel environments will exhibit evidence of a smooth muscle cell (SMC) phenotype. Initially, a range of cell-seeding densities were screened for 2D and 3D fibrin constructs, where it was observed that a seeding densities of 500,000 cells/mL facilitated gel compaction without degradation or loss in cell viability. Additionally, positive expression of CD49, CD73, CD105 markers and negative expression of hemopoietic stem cell-associated CD34 and CD45 indicated that MSC phenotype was retained within the fibrin gel. Nonetheless, a decrease in the gene expression of alpha-smooth cell actin and calponin was observed for MSC cultured in static 3D fibrin gels. Although a slight recovery was observed after 24 h mechanical stimulation, the fold-change remained significantly lower than that observed for cells cultured on 2D tissue culture plastic. While MSC differentiation toward a SMC appears possible in both 2D and 3D environments, scaffold architecture and mechanical stimulation undoubtedly play an important role in the creation of a functional SMC phenotype.

Evaluation of human endothelial cells post stent deployment in a cardiovascular simulator in vitro.

Percutaneous stent implantation has revolutionized the clinical treatment of occluded arteries. Nevertheless, there is still a large unmet need to prevent re-occlusion after implantation. Consequently, a niche exists for a cost-effective pre-clinical method of evaluating novel interventional devices in human models. Therefore, the development of a coronary model artery offers tremendous potential for the treatment of endothelial cell dysfunction and restenosis. As a first step, we employ tissue-engineering principles to examine the effect of stent deployment upon endothelial cells in a tubular in vitro system capable of replicating the coronary artery biomechanical environment. In particular, the cellular and molecular changes pertaining to inflammation, proliferation, and death were assessed after stent deployment. Real-time quantitative PCR demonstrated increased expression of genes encoding for E-Selectin, ICAM-1, and VCAM-1; markers associated with an inflammatory response in vivo. Further, an increase in the pro-apoptotic protein Bax was paralleled with a decrease in the anti-apoptotic protein Bcl-2; however, apoptotic morphology was not observed. Interestingly, transcription of c-fos increased, whereas Ki67 levels fell over the same period. One hypothesis is that these results are in response to the altered local hemodynamic environment induced by stent deployment. Most significantly, this study highlights the potential of a biomimetic hemodynamic bioreactor combined with a gene expression analysis to evaluate, with greater specificity, the performance and interaction of stents with the endothelial layer in a controlled environment.

Human coronary artery smooth muscle cell response to a novel PLA textile/fibrin gel composite scaffold.

Previous studies have demonstrated the potential of fibrin as a cell carrier for cardiovascular tissue engineering applications. Unfortunately, fibrin exhibits poor mechanical properties. One method of addressing this issue is to incorporate a textile in fibrin to provide structural support. However, it is first necessary to develop a deeper understanding of the effect of the textile on cell response. In this study, the cytotoxicity of a polylactic acid (PLA) warp-knit textile was assessed with human coronary artery smooth muscle cells (HCASMC). Subsequently, quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) was employed to examine the gene expression of HCASMC embedded in fibrin with and without the textile. Five genes were examined over a 3-week period: smooth muscle alpha-actin (SMalphaA), myosin heavy chain 11 smooth muscle (SM1/SM2), calponin, myosin heavy chain 10 non-muscle (SMemb) and collagen. Additionally, a microarray analysis was performed to examine a wider range of genes. The knitting process did not adversely affect the cell response; there was no dramatic change in cell number or metabolic rate compared to the negative control. After 3 weeks, there was no significant difference in gene expression, except for a slight decrease of 10% in SMemb in the fibrin with textile. After 3 weeks, there were no obvious cytotoxic effects observed as a result of the knitting process and the gene expression profile did not appear to be altered in the presence of the mesh in the fibrin gel.

Carbon nanotubes and mesenchymal stem cells: biocompatibility, proliferation and differentiation.

The synergy of the unique properties of carbon nanotubes (CNT) with the remarkable potential of human mesenchymal stem cells (hMSC) provides an exciting opportunity for novel therapeutic modalities. However, little is known about the impact of CNT on hMSC behavior. We report the effect of CNT on hMSC renewal, metabolic activity, and differentiation. Furthermore, we tracked the intracellular movement of CNT through the cytoplasm to a nuclear location and assessed effects on cellular ultra structure.

Response of mesenchymal stem cells to the biomechanical environment of the endothelium on a flexible tubular silicone substrate.

Understanding the response of mesenchymal stem cells (MSCs) to forces in the vasculature is very important in the field of cardiovascular intervention for a number of reasons. These include the development of MSC seeded tissue engineered vascular grafts, targeted or systemic delivery of MSCs in the dynamic environment of the coronary artery and understanding the potential pathological calcifying role of mechanically conditioned multipotent cells already present in the vessel wall. In vivo, cells present in the coronary artery are exposed to the primary biomechanical forces of shear stress, radial stress and hoop stress. To date, many studies have examined the effect of these stresses in isolation, thereby not presenting the complete picture. Therefore, the main aim of this study is to examine the combined role of these stresses on MSC behaviour. To this end, a bioreactor was configured to expose MSCs seeded on flexible silicone substrates to physiological forces - namely, a pulsatile pressure between 40 and 120mmHg (5.33-1.6x10(4)Pa), radial distention of 5% and a shear stress of 10dyn/cm(2) (1Pa) at frequency of 1Hz for up to 24h. Thereafter, the 'pseudovessel' was assessed for changes in morphology, orientation and expression of endothelial and smooth muscle cell (SMC) specific markers. Hematoxylin and eosin (H&E) staining revealed that MSCs exhibit a similar mechanosensitive response to that of endothelial cells (ECs); they reorientate parallel with direction of flow and have adapted their morphology to be similar to that of ECs. However, gene expression results show the cells exhibit greater levels of SMC-associated markers alpha-smooth muscle actin and calponin (p<0.05).

The effect of physiological cyclic stretch on the cell morphology, cell orientation and protein expression of endothelial cells.

In vivo, endothelial cells are constantly exposed to pulsatile shear and tensile stresses. The main aim of this study was to design and build a physiological simulator, which reproduced homogenous strain profiles of the tensile strain experienced in vivo, and to investigate the effect of this cyclic tensile strain on the cell morphology, cell orientation and protein expression of endothelial cells. The biological response of human umbilical vein endothelial cells to a uniaxial cyclic stretch, in this newly developed simulator, was examined experimentally using immunohistostaining and confocal imaging and it was found that the cells elongated and oriented at 58.9 degrees +/- 4.5 degrees . This value was compared to a mathematical model where it was revealed that endothelial cells would orient at an angle of 60 degrees . This model also revealed that endothelial cells have an axial strain threshold value of 1.8% when exposed to a 10% cyclic strain at 1 Hz for 3 h. Cells cultured under conditions of cyclic strain showed increased ICAM-1 immunostaining when compared to static cells whereas, a marked decrease in the levels of VCAM-1 receptor staining was also observed. Haemodynamic stresses can modulate the endothelial cell adhesion response in vivo thus, taken together; this data validates the bioreactor as replicating the physiological environment.