3D Culture Facilitates VEGF-Stimulated Endothelial Differentiation of Adipose-Derived Stem Cells.

De novo vascularization of implantable tissue and whole organ constructs has been a significant challenge in the field of tissue engineering; the use of endothelial cell populations for this task is constrained by the cell population's limited regeneration capacity and potential for loss of function. Thus, there is a need for a stem-cell population that may be induced into an endothelial cell phenotype reliably. Adipose derived stem cells (ADSCs) are multipotent cells that can be readily isolated from donor fat and may have the potential to be readily induced into endothelial cells. The ability to stimulate endothelial differentiation of these cells has been limited in standard 2D culture. We hypothesized that 3D culture would yield better differentiation. To study the influence of cell density and culture conditions on the potential of ADSCs to differentiate into an endothelial-like state, we seeded these cells types within a 3D cell-adhesive, proteolytically degradable, peptide-modified poly(ethylene-glycol) (PEG) hydrogel. ADSCs were either cultured in basal media or pro-angiogenic media supplemented with 20 ng/mL of VEGF in 2D and then encapsulated at low or high densities within the PEG-based hydrogel. These encapsulated cells were maintained in either basal media or pro-angiogenic media. Cells were then isolated from the hydrogels and cultured in Matrigel to assess the potential for tubule formation. Our work shows that maintenance of ADSCs in a pro-angiogenic medium in 2D monoculture alone does not result in any CD31 expression. Furthermore, the level of CD31 expression was affected by the density of the cells encapsulated within the PEG-based hydrogel. Upon isolation of these cells, we found that these induced ADSCs were able to form tubules within Matrigel, indicative of endothelial function, while ADSCs cultured in basal medium could not. This finding points to the potential for this stem-cell population to serve as a safe and reliable source of endothelial cells for tissue engineering and regenerative medicine purposes.

Visible light photoinitiation of mesenchymal stem cell-laden bioresponsive hydrogels.

Biological activity can be added to synthetic scaffolds by incorporating functional peptide sequences that provide enzyme-mediated degradation sites, facilitate cellular adhesion or stimulate signaling pathways. Poly(ethylene glycol) diacrylate is a popular synthetic base for tissue engineering scaffolds because it creates a hydrophilic environment that can be chemically manipulated to add this biological functionality. Furthermore, the acrylate groups allow for encapsulation of cells using photopolymerization under physiological conditions. One complication with the addition of these peptides is that aromatic amino acids absorb light at 285 nm and compete with the ultraviolet (UV)-sensitive photoinitiators such as IrgacureTM 2959 (I2959), the most commonly used initiator for cytocompatible photoencapsulation of cells into synthetic scaffolds. In this study we define non-toxic conditions for photoencapsulation of human mesenchymal stem cells (hMSC) in PEGDA scaffolds using a visible light photoinitiator system composed of eosin Y, triethanolamine and 1-vinyl-2-pyrrolidinone. This visible light photoinitiator produced hydrogel scaffolds with an increased viability of encapsulated hMSCs and a more tightly crosslinked network in one-third the time of UV polymerization with I2959.

Development and optimization of a dual-photoinitiator, emulsion-based technique for rapid generation of cell-laden hydrogel microspheres.

A growing number of clinical trials explore the use of cell-based therapies for the treatment of disease and restoration of damaged tissue; however, limited cell survival and engraftment remains a significant challenge. As the field continues to progress, microencapsulation strategies are proving to be a valuable tool for protecting and supporting these cell therapies while preserving minimally invasive delivery. This work presents a novel, dual-photoinitiator technique for encapsulation of cells within hydrogel microspheres. A desktop vortexer was used to generate an emulsion of poly(ethylene glycol) diacrylate (PEGDA) or PEGDA-based precursor solution in mineral oil. Through an optimized combination of photoinitiators added to both the aqueous and the oil phase, rapid gelation of the suspended polymer droplets was achieved. The photoinitiator combination provided superior cross-linking consistency and greater particle yield, and required lower overall initiator concentrations compared with a single initiator system. When cells were combined with the precursor solution, these benefits translated to excellent microencapsulation yield with 60-80% viability for the tested cell types. It was further shown that the scaffold material could be modified with cell-adhesive peptides to be used as surface-seeded microcarriers, or additionally with enzymatically degradable sequences to support three-dimensional spreading, migration and long-term culture of encapsulated cells. Three cell lines relevant to neural stem cell therapies are demonstrated here, but this technology is adaptable, scalable and easy to implement with standard laboratory equipment, making it a useful tool for advancing the next generation of cell-based therapeutics.

Development of bioactive photocrosslinkable fibrous hydrogels.

Three-dimensional (3D) fibrous hydrogels were fabricated by blending two photoactive polymers, poly(ethylene glycol) diacrylate (PEGDA) and poly(vinyl alcohol) (PVA), and the resulting solution was electrospun. PEGDA is a commonly used hydrogel material for tissue engineering applications since its interaction with cells can be tuned by crosslinking in a variety of bioactive molecules including peptides and proteins. The PVA in these materials aids in fiber formation and stabilizes the fibrous network when hydrated. The average dry fiber diameter in the hydrogels was 1.02 µm and upon swelling, the fiber diameter increased approximately six-fold. Fibers were stable under cell culture conditions for up to 5 days. The adhesive ligand, RGDS, was readily incorporated into the fibrous network via the conjugation of RGDS to PEG-monoacrylate which was then crosslinked with the fibers. The bioactivity of the fibrous hydrogels was compared with peptide-modified PEGDA-based hydrogels. The two hydrogel materials had similar cell adhesion and viability. Cell morphology on the fibrous hydrogels was dendritic showing a more in vivo like representation, as compared to spread cell morphology on the PEGDA gels. The ability to generate 3D fibrous architectures in hydrogel systems opens up new areas of investigation in cell-material interactions and tissue formation.

Blood vessel matrix: a new alternative for abdominal wall reconstruction.

BACKGROUND: Biologic matrices offer a new approach to the management of abdominal wall defects when the use of other foreign material is not ideal. A member of our team (GEA) developed a biological decellularized matrix generated from harvested blood vessels of swine blood vessel matrix (BVMx). The aim of our study was to investigate whether this novel collagen-based biological matrix is safe and effective for the repair of abdominal wall hernia defects in a rat model. METHODS: Full thickness abdominal wall defects were created in rats and repaired with our BVMx. After implantation as an underlay for 30 and 90 days, animals were sacrificed and the implanted material evaluated for herniation, adhesions, breaking strength, inflammation, and revascularization. RESULTS: No evidence of herniation was noted at 30 (n = 7) or 90 (n = 7) days after repair. Adhesions, if present, were filmy and easily separated. The mean area of visceral adhesions to the BVMx was 18.9 +/- 11.0% at 30 days and 7.1 +/- 3.1% at 90 days post implantation (P = 0.33). The breaking strength of the BVMx-fascial interface was 4.5 +/- 0.8 N at 30 days and 4.5 +/- 2.4 N at 90 days post implantation (P = 0.98). Histologic analysis demonstrated that the BVMx elicited a mild transient inflammatory response and supported fibroblast migration, deposition of newly formed collagen, and neovascularization. CONCLUSIONS: These data confirm that this BVMx supports vascular ingrowth and provides adequate strength for the repair of abdominal wall defects. Future studies in a large animal model are required to assess its validity for human application.

Colloidal particles at a nematic-isotropic interface: effects of confinement.

When captured by a flat nematic-isotropic interface, colloidal particles can be dragged by it. As a result spatially periodic structures may appear, with the period depending on particle mass, size, and interface velocity (J.L. West, A. Glushchenko, G.X. Liao, Y. Reznikov, D. Andrienko, M.P. Allen, Phys. Rev. E 66, 012702 (2002)). If liquid crystal is sandwiched between two substrates, the interface takes a wedge-like shape, accommodating the interface-substrate contact angle and minimizing the director distortions on its nematic side. Correspondingly, particles move along complex trajectories: they are first captured by the interface and then "glide" towards its vertex point. Our experiments quantify this scenario, and numerical minimization of the Landau-de Gennes free energy allows for a qualitative description of the interfacial structure and the drag force.

Independent Optical Control of Microfluidic Valves Formed from Optomechanically Responsive Nanocomposite Hydrogels.

Independent optical control of microfluidic valves formed from optomechanically responsive nanocomposite hydrogels is achieved using strongly absorbing Au nanoparticles or nanoshells embedded within a thermally responsive polymer. Valves formed from composites with different nanoparticles could be independently controlled by changing the illumination wavelength.

Nanoshell-mediated near-infrared thermal therapy of tumors under magnetic resonance guidance.

Metal nanoshells are a class of nanoparticles with tunable optical resonances. In this article, an application of this technology to thermal ablative therapy for cancer is described. By tuning the nanoshells to strongly absorb light in the near infrared, where optical transmission through tissue is optimal, a distribution of nanoshells at depth in tissue can be used to deliver a therapeutic dose of heat by using moderately low exposures of extracorporeally applied near-infrared (NIR) light. Human breast carcinoma cells incubated with nanoshells in vitro were found to have undergone photothermally induced morbidity on exposure to NIR light (820 nm, 35 W/cm2), as determined by using a fluorescent viability stain. Cells without nanoshells displayed no loss in viability after the same periods and conditions of NIR illumination. Likewise, in vivo studies under magnetic resonance guidance revealed that exposure to low doses of NIR light (820 nm, 4 W/cm2) in solid tumors treated with metal nanoshells reached average maximum temperatures capable of inducing irreversible tissue damage (DeltaT = 37.4 +/- 6.6 degrees C) within 4-6 min. Controls treated without nanoshells demonstrated significantly lower average temperatures on exposure to NIR light (DeltaT < 10 degrees C). These findings demonstrated good correlation with histological findings. Tissues heated above the thermal damage threshold displayed coagulation, cell shrinkage, and loss of nuclear staining, which are indicators of irreversible thermal damage. Control tissues appeared undamaged.

A whole blood immunoassay using gold nanoshells.

A rapid immunoassay capable of detecting analyte within complex biological media without any sample preparation is described. This was accomplished using gold nanoshells, layered dielectric-metal nanoparticles whose optical resonance is a function of the relative size of its constituent layers. Aggregation of antibody/nanoshell conjugates with extinction spectra in the near-infrared was monitored spectroscopically in the presence of analyte. Successful detection of immunoglobulins was achieved in saline, serum, and whole blood. This system constitutes a simple immunoassay capable of detecting sub-nanogram-per-milliliter quantities of various analytes in different media within 10-30 min.

The use of school milk as a vehicle for fluoride in Knowsley, UK; an evaluation of effectiveness.

OBJECTIVE: To compare caries in children initially aged 3 to 5 years who had participated for four years in a fluoridated school milk programme with a group of children in a similar community drinking non-fluoridated milk. STUDY DESIGN: A four-year longitudinal study measuring caries experience and caries increment in primary molars and caries experience in permanent molars and incisors. METHOD: 478 children in Knowsley (test group) and 396 in Skelmersdale (comparison group) were examined for caries (dmft/dfs) at baseline in 1997. Of these, 318 in Knowsley and 233 in Skelmersdale were re-examined at follow-up (dmft/dfs and DMFT/DFS) in 2001. RESULTS: The mean ages at baseline of the children from the test and comparison groups were 4.7 and 4.8 years respectively. The baseline dmft/dfs was 1.73/2.51 in the test group and 1.29/2.15 in the comparison group. The 4-year dmft/dfs mean increments were 2.28/4.49 and 1.96/4.12 in test and comparison groups respectively. The DMFT/DFS at age 7-9 years in the test and comparison groups were 0.40/0.45 and 0.40/0.55 respectively. CONCLUSION: The fluoridated school milk scheme, as configured in Knowsley. Merseyside, did not reduce caries within the primary dentition and, at best, had a small clinical impact on the permanent dentition up to 8 years of age.

Photo-orientation of liquid crystals due to light-induced desorption and adsorption of dye molecules on an aligning surface.

We show that adsorption of dye molecules control the light-induced alignment of dye-doped nematic liquid crystal (LC) on a nonphotosensitive polymer surface. The dependencies of light-induced twist structures on exposure, thermal baking, thickness, and aging before irradiation of the LC cells allowed us to propose the following mechanism for the alignment. Before irradiation, the "dark"-adsorbed layer on the tested surface is formed from dye molecules predominantly aligned along the initial direction of the director. Irradiation of the cell with linearly polarized light produces an additional layer with different orientational ordering of dye molecules. The final easy axis is determined by the competition of "dark" and light-induced contributions to anchoring and is aligned between the "dark" easy axes and polarization of the light. For quantitative interpretation, we apply the tensor model of anchoring and assume that the photoalignment in the mesophase is a cumulative effect of the light-induced anchoring on the background of the already existing anisotropic "dark" dye layer.

Smooth muscle cell growth in photopolymerized hydrogels with cell adhesive and proteolytically degradable domains: synthetic ECM analogs for tissue engineering.

Photopolymerizable polyethylene glycol (PEG) derivatives have been investigated as hydrogel tissue engineering scaffolds. These materials have been modified with bioactive peptides in order to create materials that mimic some of the properties of the natural extracellular matrix (ECM). The PEG derivatives with proteolytically degradable peptides in their backbone have been used to form hydrogels that are degraded by enzymes involved in cell migration, such as collagenase and elastase. Cell adhesive peptides, such as the peptide RGD, have been grafted into photopolymerized hydrogels to achieve biospecific cell adhesion. Cells seeded homogeneously in the hydrogels during photopolymerization remain viable, proliferate, and produce ECM proteins. Cells can also migrate through hydrogels that contain both proteolytically degradable and cell adhesive peptides. The biological activities of these materials can be tailored to meet the requirements of a given tissue engineering application by creating a mixture of various bioactive PEG derivatives prior to photopolymerization.

Tissue engineering in the cardiovascular system: progress toward a tissue engineered heart.

Achieving the lofty goal of developing a tissue engineered heart will likely rely on progress in engineering the various components: blood vessels, heart valves, and cardiac muscle. Advances in tissue engineered vascular grafts have shown the most progress to date. Research in tissue-engineered vascular grafts has focused on improving scaffold design, including mechanical properties and bioactivity; genetically engineering cells to improve graft performance; and optimizing tissue formation through in vitro mechanical conditioning. Some of these same approaches have been used in developing tissue engineering heart valves and cardiac muscle as well. Continued advances in scaffold technology and a greater understanding of vascular cell biology along with collaboration among engineers, scientists, and physicians will lead to further progress in the field of cardiovascular tissue engineering and ultimately the development of a tissue-engineered heart.

Hidden photoalignment of liquid crystals in the isotropic phase.

We found the effect of a hidden photoalignment of a dye-doped nematic liquid crystal (LC) on a nonphotosensitive polymer surface after polarized irradiation of the cell in the isotropic phase. We observed that irradiation resulted in a uniform planar orientation of the LC after cooling to the mesophase. The direction of a light-induced easy axis on the polymer can be either parallel or perpendicular to the polarization of the incident light, depending on the light intensity. We attribute this behavior to two mechanisms of photoalignment: light-induced adsorption of dye molecules on the substrate, and anisotropic desorption in a previously adsorbed dye layer. The experimental results on photoalignment of a LC on a thin dye film confirm our model.

Tethered-TGF-beta increases extracellular matrix production of vascular smooth muscle cells.

Biomaterials developed for tissue engineering and wound healing applications need to support robust cell adhesion, yet also need to be replaced by new tissue synthesized by those cells. In order to maintain mechanical integrity of the tissue, the cells must generate sufficient extracellular matrix before the scaffold is degraded. We have previously shown that materials containing cell adhesive ligands to promote or improve cell adhesion can decrease extracellular matrix production (Mann et al., Modification of surfaces with cell adhesion peptides alters extracellular matrix deposition. Biomaterials 1999;20:2281-6). Such decreased matrix production by cells in tissue engineering scaffolds may result in tissue failure. However, we have found that TGF-beta1 can be used in scaffolds to dramatically increase matrix production. Matrix production by vascular smooth muscle cells grown on adhesive ligand-modified glass surfaces and in PEG hydrogels containing covalently bound adhesive ligands was increased in the presence of 0.04 pmol/ml (1 ng/ml) TGF-beta1. TGF-beta1 can counteract the effect of these adhesive ligands on matrix production; matrix production could be increased even above that observed in the absence of adhesive peptides. Further, TGF-beta1 covalently immobilized to PEG retained its ability to increase matrix production. Tethering TGF-beta1 to the polymer scaffold resulted in a significant increase in matrix production over the same amount of soluble TGF-beta1.

Nitric oxide-generating polymers reduce platelet adhesion and smooth muscle cell proliferation.

We have developed polymeric biomaterials capable of providing localized and sustained production of nitric oxide (NO) for the prevention of thrombosis and restenosis. In the current study, we have characterized the kinetics of NO production by these materials and investigated their efficacy in reducing platelet adhesion and smooth muscle cell proliferation in vitro. Three nitric oxide donors with different half-lives were covalently incorporated into photopolymerized polyethylene glycol hydrogels. Under physiological conditions, NO was produced by these hydrogels over periods ranging from hours to months, depending upon the polymer formulation. NO production was inhibited at acidic pH, which may be useful for storage of the materials. The NO-releasing materials successfully inhibited smooth muscle cell growth in culture. Platelet adhesion to collagen-coated surfaces was also inhibited following exposure of whole blood to NO-producing hydrogels. The effects of NO production by these hydrogels on platelet adhesion and the proliferation of smooth muscle cells suggest that these materials could reduce thrombosis and restenosis following procedures such as balloon angioplasty.

Temperature-sensitive polymer-nanoshell composites for photothermally modulated drug delivery.

Composites of thermally sensitive hydrogels and optically active nanoparticles have been developed for the purpose of photothermally modulated drug delivery. Copolymers of N-isopropylacrylamide (NIPAAm) and acrylamide (AAm) exhibit a lower critical solution temperature (LCST) that is slightly above body temperature. When the temperature of the copolymer exceeds the LCST, the hydrogel collapses, causing a burst release of any soluble material held within the hydrogel matrix. Gold-gold sulfide nanoshells, a new class of nanoparticles designed to strongly absorb near-infrared light, have been incorporated into poly(NIPAAm-co-AAm) hydrogels for the purpose of initiating a temperature change with light; light at wavelengths between 800 and 1200 nm is transmitted through tissue with relatively little attenuation, absorbed by the nanoparticles, and converted to heat. Significantly enhanced drug release from composite hydrogels has been achieved in response to irradiation by light at 1064 nm. We have investigated the release of methylene blue and proteins of varying molecular weight. Additionally, the nanoshell-composite hydrogels can release multiple bursts of protein in response to repeated near-IR irradiation.

Comparison of insufflation vs. retractional technique for laparoscopic-assisted intervertebral fusion of the lumbar spine.

Laparoscopic transperitoneal fusion of the L5-S1 spinal interspace has become a common procedure. Retroperitoneal retraction and laparoscopic instrumentation without insufflation also allows visualization of the upper lumbar spaces, but this procedure is much more difficult to accomplish. We review and compare our results using each of these techniques for the treatment of mechanical instability and chronic back pain. A total of 35 selected patients underwent intervertebral fusion between February 1996 and August 1998. Their mean age was 48 years. There were 22 female and 13 male patients. Standard CO2 insufflation was used in 10 patients with L5-S1 fusions. Retractional gasless technique was used in nine patients with fusions at L5-S1, 16 patients at L4-L5, one patient at L3-L4, three patients at L2-3, and one patient at L1-L2. Thus, we performed a total of 40 lumbar fusions in 35 patients. In the 19 patients with the gasless technique, a balloon dissector and retractor facilitated the retroperitoneal exposure. Seven of these 19 patients were converted to open procedures, most commonly due to lacerations of the peritoneal lining that prohibited visualization. None of the L5-S1 patients with insufflation were converted to open. Mean operative time in the insufflated patients was 152 min vs. 181 min for the retractional technique. There were seven complications in the transperitoneal group: one fusion device migration, one postoperative UTI, one intracerebral hemorrhage, one severe postoperative pancreatitis, and three iliac vein lacerations. There were 16 complications in the retroperitoneal group: one deep vein thromboses, one serosal bowel injury, one small tear in the spleen, one cage migration, one postoperative pulmonary atelectasis, one postoperative hydrocele, four postoperative ileus, and six peritoneal tears. The mean postoperative stay was three days for both groups. There were no deaths. The L5-S1 interspace is best approached transperitoneally for anterior fusion. Although the retroperitoneal retractional technique is much more difficult and has a longer and steeper learning curve, it does allow laparoscopic anterior fusion of the upper lumbar spine.

Applications of nanotechnology to biotechnology commentary.

The ability to systematically modify the properties of nanostructures by controlling their structure and their surface properties at a nanoscale level makes them extremely attractive candidates for use in biological contexts, from fundamental scientific studies to commercially viable technologies.

Modification of surfaces with cell adhesion peptides alters extracellular matrix deposition.

The goal of the current study was to evaluate matrix protein synthesis by cells cultured on materials that had been modified with cell adhesion ligands. We examined the effects of surface peptide density and of peptides with different affinities on the extracellular matrix production of smooth muscle cells, endothelial cells and fibroblasts. While initial adhesion was greatest on the higher density peptide surfaces, all cell types exhibited decreased matrix production on the more highly adhesive surfaces. Similarly, when different peptides were evaluated, matrix production was the lowest on the most adhesive surface and highest on the least adhesive surface. These results suggest that extracellular matrix synthesis may be regulated, to some extent, by signal transduction initiated by adhesion events. This may pose limitations for use of bioactive materials as tissue engineering scaffolds, as matrix production is an important aspect of tissue formation. However, it may be possible to increase matrix production on highly adhesive surfaces using exogenous factors. TGF-beta was shown to increase matrix production by both smooth muscle cells and endothelial cells.