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Biomedical subjects

Kristi S Anseth

Publications and source records attributed to Kristi S Anseth.

At least 19 recordsLinked to original sources
PubMed2007

Contrasting effects of collagen and bFGF-2 on neural cell function in degradable synthetic PEG hydrogels.

Injectable biodegradable cell carriers provide a potential means to improve transplanted cell viability in the nervous system by providing physical protection from compaction, shear forces, and the acute inflammatory response that occurs following transplantation into the host brain environment. Synthetic polyethylene glycol (PEG) hydrogels are ideal candidates for this purpose, as the degradation profile and mechanical properties of the gel can be controlled. Here we introduce biological components into the synthetic gel with the goal of improving neural cell function in the inert PEG environment. In this study, it was found that (1) bFGF-2 is a survival/mitogenic factor for neural precursor cells in degradable hydrogel cultures, (2) collagen has no measurable effect on cell survival, metabolic activity, or proliferation, and (3) co-application of collagen and bFGF-2 to hydrogel cultures targets cell survival and metabolic activity, an effect that is different than either applied individually. Because collagen and bFGF-2 support the survival and growth of neural cells and other cell types, the co-encapsulation approach and functional characterization described in this study can be extended to the development of an array of tissue engineering applications. These findings suggest the importance of understanding and developing strategies to control the chemical microenvironment surrounding cells in three-dimensional biomaterials.

Animals↗
PubMed2007

Nanobiomaterial applications in orthopedics.

Advancements in nanobiotechnology are revolutionizing our capability to understand biological intricacies and resolve biological and medical problems by developing subtle biomimetic techniques. Nanocomposites and nanostructured materials are believed to play a pivotal role in orthopedic research since bone itself is a typical example of a nanocomposite. This article reviews current strategies using nanobiomaterials to improve current orthopedic materials and examines their applications in bone tissue engineering. Preliminary investigations support the potential of nanobiomaterials in orthopedic applications; however, significant advancements are necessary to achieve clinical use. Overall, current trends in nanobiotechnology foreshadow a bright future through the use of nanobiomaterials in the orthopedic domain.

Biocompatible Materials↗
PubMed2006

Encapsulated chondrocyte response in a pulsatile flow bioreactor.

We have developed a bioreactor-based millifluidic technique that allows for dynamic culture conditions and measurement of the fluid flow impinging upon a three-dimensional tissue engineering scaffold. Chondrocytes in scaffolds have been shown to require mechanical stimulation to produce an extracellular matrix that resembles native cartilage. This study investigates the effect of pulsatile flow on chondrocyte response in a model poly(ethylene glycol) dimethacrylate hydrogel. Bovine chondrocytes were encapsulated in the hydrogel and cultured for 7, 14 and 21 days at pulsatile flow frequencies of 0.5 Hz (15ml/min) and 1.5Hz (17ml/min). The scaffolds cultured under dynamic conditions were compared to those cultured under static (non-flow) conditions. Quantitative real-time reverse transcription polymerase chain reaction was used to quantify collagen type I, collagen type II and aggrecan gene copy numbers as markers for chondrocyte phenotypic expression. Histological sections stained with hematoxylin & eosin, and Alcian blue confirmed chondrocyte morphology and matrix formation. Interestingly, regulation of the collagen type II gene was particularly sensitive to the flow conditions. The understanding of the cell response to encapsulation and flow could be used to identify the appropriate culture conditions necessary to design and develop hydrogel carriers to promote the formation of extracellular matrix as well as to further our knowledge of chondrocyte mechanobiology.

Animals↗
PubMed2006

The effect of heparin-functionalized PEG hydrogels on three-dimensional human mesenchymal stem cell osteogenic differentiation.

Poly(ethylene glycol) (PEG) hydrogels functionalized with heparin were utilized as a three-dimensional culture system for human mesenchymal stem cells (hMSCs). Heparin-functionalized hydrogels supported hMSC viability, as quantified through live/dead imaging, and induced osteogenic differentiation, as measured by increased alkaline phosphatase (ALP) production and osteopontin (OPN) and collagen I (COL I) gene expression over the 5-week study. Further exploration of the potential mechanism of heparin-induced osteogenic differentiation was performed. Specifically, the availability of bone morphogenetic protein 2 (BMP2) and fibronectin (FN) in the culture system was controlled and hMSC osteogenic differentiation was evaluated as a function of the microenvironment. BMP2 availability increased both ALP production and OPN gene expression, while FN increased ALP production, but not OPN gene expression. Furthermore, immunostaining of integrin expression revealed that viability and differentiation were differentially affected by integrin production, where both alpha5beta1 and alphavbeta3 integrin-ligand interactions supported viability, while only the alpha5beta1 integrin played a role in hMSC osteogenic differentiation.

Alkaline Phosphatase↗
PubMed2006

Synthesis and characterization of a fluvastatin-releasing hydrogel delivery system to modulate hMSC differentiation and function for bone regeneration.

Increases in bone formation have been demonstrated in mice and rats treated with statins, a group of molecules that increase the production of bone morphogenetic proteins-2 (BMP2) by stimulating its promoter. However, clinical use of statins (e.g., fluvastatin) is limited by the lack of a suitable delivery system to localize and sustain release. To harness the therapeutic effect of statins in orthopedic applications, a fluvastatin-releasing macromer was synthesized. When copolymerized with a dimethacrylated poly(ethylene glycol) solution, this fluvastatin-containing molecule was covalently incorporated into hydrogel networks, and hydrolysis of lactic acid ester bonds resulted in the release of the pendantly tethered fluvastatin from the hydrogel into the surrounding solution. The rate of fluvastatin release was controlled by the length of lactic acid spacer (2-6 repeats), and the dose was controlled by the initial comonomer composition (5-500 microg fluvastatin/gel). Released fluvastatin increased human mesenchymal stem cell (hMSC) gene expression of CBFA1, ALP, and COL I by 34-fold, 2.6-fold, and 1.8-fold, respectively, after 14 days of in vitro culture. In addition, treating hMSCs with the released fluvastatin resulted in an average of 2.0- and 1.5-fold greater BMP2 production whereas mineralization increased an average of 3.0-fold and 2.5-fold for 0.01 and 0.1 microM fluvastatin, respectively, over the 2 week culture period. Therefore, fluvastatin-releasing hydrogels may be useful in bone tissue engineering applications, not only for triggering osteogenic differentiation of hMSCs, but also by modulating their function.

Biocompatible Materials↗
PubMed2006

Detection of antigens in biologically complex fluids with photografted whole antibodies.

A highly sensitive (pM), efficient (t < 20 min) detection assay was developed by designing surfaces with grafted antibodies. Through this approach, a short half-life antigen, glucagon, was rapidly detected in a biologically complex plasma/blood environment. Tailoring of graft composition eliminates the need for time-consuming blocking steps, significantly reducing antigen-antibody incubation times, while maintaining antibody specificity and activity toward target antigen. Grafted antibodies were bound through solvated, mobile polymer chains, thereby circumventing problems associated with antibody accessibility, analyte diffusion, and steric limitations. The efficiency of this assay is provided through grafting synthesized, acrylated antibodies in the presence of PEG monoacrylate. This procedure eliminates the need for blocking steps, due to a decrease in nonspecific protein interactions. These polymerizable antibodies were tethered with a range of densities while retaining biological activity. Moreover, biological activity of acrylated antibodies was compared to that of unmodified antibodies and remained comparable. The acrylated antibodies were grafted from substrate surfaces using controlled radical photopolymerization, maintaining the advantages of classical antibody immobilization techniques while providing improved detection. Through integrating this antibody conjugation chemistry and immunoassay approach with photolithographic techniques, construction of spatial patterns on a microfluidic device was demonstrated for efficient, parallel screening of multiple antigens.

Antibodies↗
PubMed2006

Dexamethasone-functionalized gels induce osteogenic differentiation of encapsulated hMSCs.

Synthetic hydrogels represent highly controlled environments for three-dimensional culture of human mesenchymal stem cells (hMSCs). Encapsulated hMSCs are presented with a "blank" environment, and this environment can be closely controlled in order to elicit an osteogenic response. In vitro, dexamethasone is an efficient and reliable factor that leads to the osteogenic differentiation of human mesenchymal stem cells (hMSCs). The aim of this work was to develop a dexamethasone-releasing poly(ethylene glycol) (PEG)-based hydrogel scaffold to deliver dexamethasone to encapsulated cells in a sustained manner. To accomplish this goal, dexamethasone was covalently linked to a photoreactive mono-acrylated PEG molecule through a degradable lactide bond, and this molecule was covalently incorporated into the PEG hydrogel during photopolymerization. Over time, hydrolysis of the ester bonds resulted in dexamethasone release from the gel. The biological activity of the released dexamethasone was verified in monolayer cell culture and in three-dimensional culture (i.e., in the gel) by the ability of hMSCs to express osteogenic genes, including alkaline phosphatase, osteopontin, and core binding factor alpha 1, as measured using real-time reverse transcription polymerase chain reaction (RT-PCR). These studies indicate that encapsulated hMSCs are capable of osteogenic differentiation in response to released dexamethasone.

Alkaline Phosphatase↗
PubMed2006

Exogenously triggered, enzymatic degradation of photopolymerized hydrogels with polycaprolactone subunits: experimental observation and modeling of mass loss behavior.

Degradation plays an important role in the evolution of the extracellular matrix secreted by chondrocytes encapsulated in PEG-based hydrogels. For this study, macromonomers were synthesized by methacrylating both ends of polycaprolactone-b-poly(ethylene glycol)-b-polycaprolactone (PEG-CAP) tri-block copolymers. These divinyl molecules were photopolymerized to form hydrogels with PEG-CAP crosslinks that were subsequently degraded upon exogenous addition of a lipase enzyme. The rate of degradation and subsequent mass loss depends on both the length of the polycaprolactone units and the concentration of enzyme. Control gels that did not receive lipase did not significantly degrade on the time scale of these experiments. A model was developed to predict mass loss using enzyme kinetics and a previously described statistical treatment of bulk network degradation. The model was used to predict mass loss profiles at the specific conditions used, and also to demonstrate the importance of potential changes in reaction rate and enzyme stability on temporal mass loss.

Hydrogels↗
PubMed2006

Controlling network structure in degradable thiol-acrylate biomaterials to tune mass loss behavior.

Degradable thiol-acrylate materials were synthesized from the mixed-mode polymerization of a diacrylate poly(ethylene glycol) (PEG) monomer with thiol monomers of varying functionalities to control the final network structure, ultimately influencing the material's degradation behavior and properties. The influence of the concentration of thiol groups and monomer functionality on the mass loss profiles were examined experimentally and theoretically. Mass loss behavior was also predicted for networks with varying extents of cyclization, PEG molecular weight, and backbone chain length distributions. Experimental results indicate that increasing the thiol concentration from 10 to 50 mol % shifted the reverse gelation time from 35 to 8 days and the extent of mass loss at reverse gelation from 75 to 40%. Similarly, decreasing the thiol functionality from 4 to 1 shifted the reverse gelation time from 18 to 8 days and the mass loss extent at reverse gelation from 70 to 45%.

Acrylates↗
PubMed2006

Manipulations in hydrogel degradation behavior enhance osteoblast function and mineralized tissue formation.

Hydrogels were prepared by copolymerizing a degradable macromer, poly(lactic acid)-b-poly(ethylene glycol)-b-poly(lactic acid) endcapped with methacrylate groups (PEG-LA-DM), with a nondegradable macromer, poly(ethylene glycol) dimethacrylate (PEGDM). Copolymer networks consisted of 100:0, 83:17, 67:33, and 50:50 PEGDM:PEG-LA-DM mass%, essentially creating scaffolds that exhibit 0, 17, 33, and 50% degradation over the time course of the experiment. Osteoblasts were photoencapsulated in these copolymer hydrogels and cultured for 3 weeks in vitro. Metabolic activity, proliferation, and alkaline phosphatase production were enhanced by an increase PEG-LADM content and corresponding degradation. Gene expression of the cultured osteoblasts, normalized to beta-actin, was analyzed, and osteopontin and collagen type I gene expression increased with degradation. Finally, as a measure of mineralized tissue formation, calcium and phosphate deposition was analyzed biochemically and histologically. Mineralization increased with increasing concentration of PEG-LA-DM and biochemically resembled that of hydroxyapatite.

Animals↗
PubMed2005

PEG-based hydrogels as an in vitro encapsulation platform for testing controlled beta-cell microenvironments.

An in vitro encapsulation platform for systematically testing the effects of microenvironmental parameters on encapsulated islets was developed. The base encapsulation matrix was a biocompatible hydrogel formed via the photoinitiated polymerization of dimethacrylated poly(ethylene glycol) (PEGDM). The resulting inert encapsulation matrix affords control over the biochemical and biophysical cellular microenvironment and the introduction of systematic changes to this environment. The compatibility of the PEG-based encapsulation platform with pancreatic beta-cells was first established using a murine beta-cell line, MIN6. When cell-cell contacts were introduced via aggregation of MIN6 beta-cells prior to encapsulation, MIN6 beta-cells remained viable within the PEG hydrogel platform throughout 3weeks of in vitro culture. Proliferating cells were observed within encapsulated MIN6 aggregates qualitatively with bromodeoxyuridine staining and quantitatively by measuring the DNA content of encapsulation samples with time. MIN6 beta-cells were encapsulated in hydrogels formed from three PEGDM macromers of varying molecular weights (M (n)=4,000, 8,000, 10,000g/mol), and the resulting differences in hydrogel crosslinking density, which influences transport properties, did not affect encapsulated beta-cell survival. Encapsulated MIN6 beta-cells transplanted into diabetic mice returned blood glucose levels to normal levels, indicating in vivo function. Finally, the compatibility of the PEG encapsulation system with freshly isolated islets was confirmed.

Animals↗
PubMed2005

Three-dimensional growth and function of neural tissue in degradable polyethylene glycol hydrogels.

Graft survival and integration are major factors that limit the efficacy of cell therapies for the treatment of disease and injury in the central nervous system. Efforts to improve cell survival and integration have focused in part on the development of biocompatible scaffolds that support neural cell growth and function. Here we photoencapsulate neural cells within degradable hydrogels and use confocal microscopy to non-invasively monitor these key cell functions over time. By directly imaging fluorescently labeled cells we show that neural cells cultured within three-dimensional polymer networks create their own cellular microenvironment to survive, proliferate and differentiate and form neurons and glia that are electrophysiologically responsive to neurotransmitter. By changing the degradation rate of the polymer network, the time-scale over which neural cells extend processes throughout the hydrogel could be tuned on a time-scale that ranged from 1-3 weeks. These studies were carried out in the absence of serum and extracellular matrix molecules that can be immunogenic and identify degradable PEG hydrogels as suitable synthetic cell carriers for neural transplantation.

Animals↗
PubMed2005

Surface grafted antibodies: controlled architecture permits enhanced antigen detection.

The attachment of antibodies to substrate surfaces is useful for achieving specific detection of antigens and toxins associated with clinical and field diagnostics. Here, acrylated whole antibodies were produced through conjugation chemistry, with the goal of covalently photografting these proteins from surfaces in a controlled fashion, to facilitate rapid and sensitive antigenic detection. A living radical photopolymerization chemistry was used to graft the acrylated whole antibodies on polymer surfaces at controlled densities and spatial locations by controlling the exposure time and area, respectively. Copolymer grafts containing these antibodies were synthesized to demonstrate two principles. First, PEG functionalities were introduced to prevent nonspecific protein interactions and improve the reaction kinetics by increasing solvation and mobility of the antibody-containing chains. Both of these properties lead to sensitive (pM) and rapid (<20 min) detection of antigens with this surface modification technique. Second, graft composition was tailored to include multiple antibodies on the same grafted chains, establishing a means for simultaneously detecting multiple antigens on one grafted surface area. Finally, the addition of PEG spacers between the acrylate functionality and the pendant detection antibodies was tuned to enhance the detection of a short-half-life molecule, glucagon, in a complex biological environment, plasma.

Acrylates↗
PubMed2005

Serum deprivation improves seeding and repopulation of acellular matrices with valvular interstitial cells.

Cell-extracted valvular tissues (acellular scaffolds, or aScaffolds) offer unique advantages over synthetic polymers for cardiac valve engineering applications in that they retain extracellular matrix molecules to support cellular ingrowth. The extracellular matrix is important in directing many cellular pathways, such as adhesion, proliferation, migration, differentiation, and survival. However, repopulating this type of scaffold often requires high seeding densities or recurrent cell delivery. The optimization of valvular interstitial cell (VIC) seeding onto aScaffolds is reported herein. VICs (the most prevalent cell type in valve leaflets) have maximal growth in 15-20% serum concentrations on tissue-culture polystyrene. Interestingly, after VIC seeding onto aScaffolds, a reduction of serum content, from 15% serum to 5% or less, was found to increase significantly the number of adherent cells, as well as induce transfer of VICs from a tissue-culture polystyrene surface to the aScaffold. aScaffolds seeded and cultured with periods of reduced serum levels were shown to support and enhance VIC viability and attachment, as well as accelerate VIC migration into the aScaffold, leading to a uniformly repopulated valve leaflet construct after 4 weeks of static culture.

Animals↗
PubMed2005

The effect of ethylene glycol methacrylate phosphate in PEG hydrogels on mineralization and viability of encapsulated hMSCs.

Owing to their resistance to protein adsorption, poly(ethylene glycol) (PEG) hydrogels are unable to support the adhesion of human mesenchymal stem cells (hMSCs). Human MSCs attach to various substrates through cell adhesion proteins, and osteopontin is an important cell adhesion protein for both MSCs and osteoblasts found in bone. As such, we hypothesized that cell adhesion to a PEG hydrogel could be dramatically improved by actively promoting the formation of a mineral phase throughout the hydrogel. This was accomplished using the photoreactive, phosphate-containing molecule ethylene glycol methacrylate phosphate (EGMP). When EGMP was incorporated into the PEG hydrogel, it resulted in the formation of a bone-like mineral phase, as verified using compositional analysis and X-ray diffraction. Furthermore, cell viability of gel-encapsulated hMSCs was increased in the presence of EGMP from 15% in the absence of EGMP to 97% in the presence of 50 mm EGMP. This improvement in cell viability was thought to be due to the ability of mineralized hydrogels to sequester cell-secreted osteopontin. It was found that EGMP-containing PEGDA hydrogels are able to sequester the important cell adhesion protein osteopontin, and these hydrogels promoted hMSC adhesion and spreading.

Calcium Phosphates↗
PubMed2005

Thermomechanics of the shape memory effect in polymers for biomedical applications.

We examine the shape memory effect in polymer networks intended for biomedical, and specifically cardiovascular, applications. The polymers were synthesized by photopolymerization from a tert-butyl acrylate monomer with a diethyleneglycol diacrylate crosslinker. Three-point flexural tests were used to systematically investigate the thermomechanics of shape storage (predeformation) and shape recovery. The glass transition temperature, T(g), of the polymers was determined to be approximately 65 degrees C. The polymers show 100% strain recovery, at low and high predeformation temperatures, up to maximum strains of approximately 80%. The polymers show a sigmoidal free strain recovery response as a function of increasing temperature at a constant heating rate. Free strain recovery was determined to depend on the temperature during predeformation; lower predeformation temperatures (T < T(g)) decreased the temperature required for free strain recovery. Constrained stress recovery shows a complex evolution as a function of temperature and also depends on the temperature during predeformation. Stress recovery after low-temperature predeformation (T < T(g)) shows a peak in the generated recovery stress, whereas stress recovery after high-temperature predeformation (T > T(g)) is sigmoidal. The isothermal free strain recovery rate was found to increase with increasing temperature or decreasing predeformation temperature. The thermomechanical results are discussed in light of potential biomedical applications, and a prototype device is presented.

Biocompatible Materials↗
PubMed2005

Heparin functionalized PEG gels that modulate protein adsorption for hMSC adhesion and differentiation.

Heparin was modified with methacrylate groups, copolymerized with dimethacrylated poly(ethylene glycol), and analyzed as a localized delivery vehicle for bFGF and synthetic extracellular matrix for the differentiation of hMSCs. By deriving cues from molecules normally present in the extracellular matrix (ECM), a complex network of collagens, laminin, fibronectin, glycosaminoglycans, and growth factors, synthetic cell scaffolds can be designed that actively sequester important bioactive signals. Among the glycosaminoglycans, heparin binds reversibly with many proteins, therefore, poly(ethylene glycol) based biomaterials, normally resistant to cell adhesion, functionalized with heparin in order to sequester important proteins, can actively and selectively stimulate desired cell functions. Results demonstrate that methacrylate-modified heparin retained its ability to bind heparin-binding proteins both in solution and when copolymerized with dimethacrylated PEG in a hydrogel. In addition, the heparin functionalized gels can deliver biologically active bFGF for up to 5 weeks. Finally, the gels were examined as a potential scaffold for hMSC culture and were found to promote adhesion, proliferation, and osteogenic differentiation.

Adsorption↗