Garrett B. McGuinness

Physically crosslinked composite hydrogels of PVA with natural macromolecules: structure, mechanical properties, and endothelial cell compatibility.

Polyvinyl alcohol (PVA) hydrogels have been considered potentially suitable for applications as engineered blood vessels because of their structure and mechanical properties. However, PVAs hydrophilicity hinders its capacity to act as a substrate for cell attachment. As a remedy, PVA was blended with chitosan, gelatin, or starch, and hydrogels were formed by subjecting the solutions to freeze-thaw cycles followed by coagulation bath immersion. The structure-property relationships for these hydrogels were examined by measurement of their swelling, rehydration, degradation, and mechanical properties. For the case of pure PVA hydrogels, the equilibrium swelling ratio was used to predict the effect of freeze thaw cycles and coagulation bath on average molecular weights between crosslinks and on mesh size. For all hydrogels, trends for the reswelling ratio, which is indicative of the crosslinked polymer fraction, were consistent with relative tensile properties. The coagulation bath treatment increased the degradation resistance of the hydrogels significantly. The suitability of each hydrogel for cell attachment and proliferation was examined by protein adsorption and bovine vascular endothelial cell culture experiments. Protein adsorption and cell proliferation was highest on the PVA/gelatin hydrogels. This study demonstrates that the potential of PVA hydrogels for artificial blood vessel applications can be improved by the addition of natural polymers, and that freeze-thawing and coagulation bath treatment can be utilized for fine adjustment of the physical characteristics.

Electrospun composites of PHBV, silk fibroin and nano-hydroxyapatite for bone tissue engineering.

Electrospinning of fibrous scaffolds containing nano-hydroxyapatite (nHAp) embedded in a matrix of functional biomacromolecules offers an attractive route to mimicking the natural bone tissue architecture. Functional fibrous substrates will support cell attachment, proliferation and differentiation, while the role of HAp is to induce cells to secrete extracellular matrix (ECM) for mineralization to form bone. Electrospinning of biomaterials composed of polyhydroxybutyrate-co-(3-hydroxyvalerate) with 2% valerate fraction (PHBV), nano-hydroxyapatite (nHAp), and Bombyx mori silk fibroin essence (SF), Mw=90KDa, has been achieved for nHAp and SF solution concentrations of 2 (w/vol) % each and 5 (w/vol) % each. The structure and properties of the nanocomposite fibrous membranes were investigated by means of Scanning Electron Microscopy in combination with Energy Dispersive X-Ray Analysis (SEM/EDX), Fourier Transformed Infrared Spectroscopy (FT-IR), uniaxial tensile and compressive mechanical testing, degradation tests and in vitro bioactivity tests. SEM images showed smooth, uniform and continuous fibre deposition with no bead formation, and fibre diameters of between 10 and 15 μm. EDX and FT-IR confirmed the presence of nHAp and SF. After one month in deionised water, tests showed less than 2% weight loss with the samples retaining their fibrous morphology, confirming that this material biodegrades slowly. After 28 days of immersion in Simulated Body Fluid (SBF) an apatite layer was visible on the surface of the fibres, proving their bioactivity. Preliminary in vitro biological assessment showed that after 1 and 3 days in culture, cells were attached to the fibres, retaining their morphology while presenting a flattened appearance and elongated shape on the surface of fibres. Youngs modulus was found to increase from 0.7 kPa (±0.33 kPa) for electrospun samples of PHBV only to 1.4 kPa (±0.54 kPa) for samples with 2 (w/vol) % each of nHAp and SF. Samples prepared with 5 (w/vol) % each of nHAp and SF did not show a similar improvement.

Cell encapsulation within PVA-based hydrogels via freeze-thawing: a one-step scaffold formation and cell storage technique.

Cryogelation is a physical hydrogel formation method for certain polymers, notably polyvinyl alcohol (PVA). The hypothesis of this study is that a PVA‐based solution with the necessary intracellular cryoprotectant and nutrient supply can be used, first for storage of vascular smooth muscle cells, and subsequently to form a suitable tissue‐engineering scaffold during the thawing process. Bovine arterial smooth muscle cells were encapsulated within PVA–gelatin hydrogels over a wide range of serum, DMSO and cell culture medium concentrations. Several parameters expected to affect gelation and cell viability (PVA viscosity, DMSO concentration, serum presence) were assessed with experimental designs and the optimal conditions for cell survival were determined. Cell viability can be improved by increasing concentration of DMSO and serum without compromising the gelation process. An additional crosslinking step using a coagulation bath was beneficial for hydrogel stability but caused peripheral accumulation of cells. In conclusion, a freeze–thaw process can be utilized to prepare and store cell‐laden hydrogels with adjustable mechanical properties. Copyright

Mechanical properties and in vitro characterization of polyvinyl alcohol- nano-silver hydrogel wound dressings

Polyvinyl alcohol (PVA) hydrogels are materials for potential use in burn healing. Silver nanoparticles can be synthesized within PVA hydrogels giving antimicrobial hydrogels. Hydrogels have to be swollen prior to their application, and the common medium available for that in hospitals is saline solution, but the hydrogel could also take up some of the wounds fluid. This work developed gamma-irradiated PVA/nano-Ag hydrogels for potential use in burn dressing applications. Silver nitrate (AgNO3) was used as nano-Ag precursor agent. Saline solution, phosphate-buffered solution (PBS) pH 7.4 and solution pH 4.0 were used as swelling media. Microstructural evaluation revealed an effect of the nanoparticles on PVA crystallization. The swelling of the PVA-Ag samples in solution pH 4.0 was low, as was their silver delivery, compared with the equivalent samples swollen in the other media. The highest swelling and silver delivery were related to samples prepared with 0.50% AgNO3, and they also presented lower strength in PBS pH 7.4 and solution pH 4.0. Both PVA-Ag samples were also non-toxic and presented antimicrobial activity, confirming that 0.25% AgNO3 concentration is sufficient to establish an antimicrobial effect. Both PVA-Ag samples presented suitable mechanical and swelling properties in all media, representative of potential burn site conditions.

Endothelialization of PVA/gelatin cryogels for vascular tissue engineering: effect of disturbed shear stress conditions.

Mechanically, poly(vinyl alcohol) (PVA)-based cryogels are extremely well suited for vascular tissue engineering applications. However, their surface properties lead to a slow rate of endothelialization, and the mode of cell attachment leaves the endothelium susceptible to removal under physiological shear stress conditions. In this study, abrupt and ramped disturbed shear stress conditions created by a turbulent orbital flow were used to examine endothelialization on PVA/gelatin cryogels. Cell proliferation rate and apoptosis were evaluated by fluorescent activated cell sorter (FACS) analysis, and the expression of cell-adhesion molecules was used to evaluate the response of cells on cryogels to static and shear conditions by real-time polymerase chain reaction (RT-PCR). Application of a ramped shear stress had a profound effect on endothelial cell proliferation (22.30 +/- 0.20-fold increase), necrosis (eliminated), apoptosis (1.04 +/- 0.18 increase), and overall facilitation of endothelialization while concomitantly increasing nitric oxide (NO) synthesis levels. Ramped shear stress was also effective in helping the retention of the endothelial cells on the cryogel surface, whereas abrupt application caused widespread removal. Under static conditions, Selectin-P expression decreased, whereas both inter-cellular adhesion molecule (ICAM) and platelet endothelial cell adhesion molecule (PECAM)-I expression increased on cryogels over a 10-day culture period. Under both shear stress conditions, Selectin-P expression was decreased both on cryogels and tissue culture polystyrene (TCPS). Controlled application of disturbed shear stress shortens endothelialization times on cryogel surfaces, in contrast to the established antiproliferative effect of shear stress caused by laminar flow, without compromising their functionality. This demonstrates how such mechanical stimuli can be exploited to alter cellular behavior and facilitate the required outcomes for tissue engineering applications.

Cell encapsulation and cryostorage in PVA–gelatin cryogels: incorporation of carboxylated ε‐poly‐L‐lysine as cryoprotectant

It is desirable to produce cryopreservable cell‐laden tissue‐engineering scaffolds whose final properties can be adjusted during the thawing process immediately prior to use. Polyvinyl alcohol (PVA)‐based solutions provide platforms in which cryoprotected cell suspensions can be turned into a ready‐to‐use, cell‐laden scaffold by a process of cryogelation. In this study, such a PVA system, with DMSO as the cryoprotectant, was successfully developed. Vascular smooth muscle cell (vSMC)‐encapsulated cryogels were investigated under conditions of cyclic strain and in co‐culture with vascular endothelial cells to mimic the environment these cells experience in vivo in a vascular tissue‐engineering setting. In view of the cytotoxicity DMSO imposes with respect to the production procedure, carboxylated poly‐L‐lysine (COOH–PLL) was substituted as a non‐cytotoxic cryoprotectant to allow longer, slower thawing periods to generate more stable cryogels. Encapsulated vSMC with DMSO as a cryoprotectant responded to 10% cyclic strain with increased alignment and proliferation. Cells were stored frozen for 1 month without loss of viability compared to immediate thawing. SMC‐encapsulated cryogels also successfully supported functional endothelial cell co‐culture. Substitution of COOH–PLL in place of DMSO resulted in a significant increase in cell viability in encapsulated cryogels for a range of thawing periods. We conclude that incorporation of COOH–PLL during cryogelation preserved cell functionality while retaining fundamental cryogel physical properties, thereby making it a promising platform for tissue‐engineering scaffolds, particularly for vascular tissue engineering, or cell preservation within microgels. Copyright

FTIR analysis and quantification of phenols and flavonoids of five commercially available plants extracts used in wound healing

Natural products are used in wound healing in order to prevent infection. Propolis is a well known antimicrobial with phenolic compounds and flavonoid content which vary according to the propolis origin. Besides propolis (from both Brazilian and UK sources), pomegranate, dragons blood and sage are possible antimicrobials to be used in biomaterials. The goal of this work was to analyze the amount of phenols and flavonoid compounds in these natural products, their antioxidant activities and the bonds present by FTIR. The FTIR analysis revealed the presence of active compounds in all drug samples. The phenols quantification showed that Brazilian propolis was rich in phenols compared to the other drugs, followed by pomegranate and UK propolis. UK propolis was the most rich in flavonoids, which is expected on account of its origin. Pomegranate, UK propolis and Dragons blood presented the highest antioxidant activity. All samples presented antioxidant activity > 82%.

Towards functional 3D-stacked electrospun composite scaffolds of PHBV, silk fibroin and nanohydroxyapatite: Mechanical properties and surface osteogenic differentiation.

Bone tissue engineering scaffolds have two challenging functional tasks to fulfil: to encourage cell proliferation, differentiation and matrix synthesis and to provide suitable mechanical stability upon implantation. Composites of biopolymers and bioceramics combine the advantages of both types of materials, resulting in better processability and enhanced mechanical and biological properties through matrix reinforcement. In the present study, novel thick bone composite scaffolds were successfully fabricated using electrospun flat sheets of polyhydroxybutyrate–polyhydroxyvalerate/nanohydroxyapatite/silk fibroin essence (2% nanohydroxyapatite – 2% silk fibroin essence and 5% nanohydroxyapatite – 5% silk fibroin essence, respectively). Their potential as in vitro bone regeneration scaffolds was evaluated using mouse calvarian osteoblast cells (MC3T3-E1), in terms of morphology (scanning electron microscope), cell attachment, cell proliferation, Col type I, osteopontin and bone alkaline phosphatase activity (Quantitative Real Time Polymerase Chain Reaction [qRT-PCR], enzyme-linked immunosorbent assay, immunocytochemistry). Electrospun polyhydroxybutyrate–polyhydroxyvalerate scaffolds were used as reference constructs. The results showed that the compressive and tensile mechanical properties of the scaffolds are dependent on the change in their composition, and the treatment these underwent. Furthermore, methanol-treated and autoclaved (MA) P2 (2% nanohydroxyapatite, 2% silk fibroin essence) samples appeared to exhibit more promising tensile properties. Additionally, the compressive tests results confirmed that the methanol pre-treatment and the autoclaving step lead to an increase in the P2 secant modulus when compared to the non-methanol-treated ones, P2 and P5 (5% nanohydroxyapatite, 5% silk fibroin essence), respectively. Both formulations of polyhydroxybutyrate–polyhydroxyvalerate/nanohydroxyapatite/silk fibroin essence composite promoted greater cell adhesion and proliferation than the corresponding polyhydroxybutyrate–polyhydroxyvalerate control ones. Cells seeded on the composite fibrous scaffolds were extensively expanded and elongated on the fibre surface after one day in culture, whereas those seeded on the polyhydroxybutyrate–polyhydroxyvalerate scaffolds were not completely elongated. In addition, cells grown on P2 and P5 scaffolds had higher alkaline phosphatase activity when compared to those containing no nanohydroxyapatite/silk fibroin essence.

Electrospun nanofibre bundles and yarns for tissue engineering applications: A review

Nanofibre membranes produced through the electrospinning process have been studied extensively over the past decade for a number of high demand applications including use as tissue engineered scaffolds. Despite possessing desirable properties including high surface area to volume ratios and enhanced mechanical properties, they ultimately suffer from a lack of cellular infiltration. Variations on the process include the production of highly aligned filaments of electrospun fibres referred to as bundles and yarns. Nanofibre bundle and yarn-based scaffolds have been shown to demonstrate superior cell infiltration rates compared to traditional electrospun nonwovens while also offering the capability to be incorporated into a wider array of post-processing technologies. In this review, fibre collection techniques currently employed within the literature for the fabrication of electrospun bundles and yarns along with their applications in the field of tissue engineering will be discussed.

High Power, Low Frequency Ultrasound: Meniscal Tissue Interaction and Ablation Characteristics

This study evaluates high power low frequency ultrasound transmitted via a flat vibrating probe tip as an alternative technology for meniscal debridement in the bovine knee. An experimental force controlled testing rig was constructed using a 20 kHz ultrasonic probe suspended vertically from a load cell. Effect of variation in amplitude of distal tip displacement (242-494 μm peak-peak) settings and force (2.5-4.5 N) on tissue removal rate (TRR) and penetration rate (PR) for 52 bovine meniscus samples was analyzed. Temperature elevation in residual meniscus was measured by embedded thermocouples and histologic analysis. As amplitude or force increases, there is a linear increase in TRR (Mean: 0.9 to 11.2 mg/s) and PR (Mean: 0.08 to 0.73 mm/s). Maximum mean temperatures of 84.6°C and 52.3°C were recorded in residual tissue at 2 mm and 4 mm from the ultrasound probe-tissue interface. There is an inverse relationship between both amplitude and force, and temperature elevation, with higher settings resulting in less thermal damage.