Biomedical subjects

Kristi S Anseth

Publications and source records attributed to Kristi S Anseth.

At least 37 records · Page 2Linked to original sources

PubMed2005

Degradable thiol-acrylate photopolymers: polymerization and degradation behavior of an in situ forming biomaterial.

Degradable thiol-acrylate photopolymers are a new class of biomaterials capable of rapidly polymerizing under physiological conditions upon exposure to UV light, with or without added photoinitiators, and to depths exceeding 10 cm. These materials are formed in situ, and the versatility of their chemistry affords a high degree of control over the final material properties. For example, variations in monomer mole fractions directly affect the final network molecular structure, varying the time required to achieve complete mass loss from 25 to 100 days, the molecular weight distributions of the degradation products, and the swelling ratios and compressive moduli throughout degradation. Additionally, varying the mole fraction of multifunctional thiol monomer in the initial reaction mixture controls the concentration of reactive sites in the network available for post-polymerization modification of the polymer.

Biocompatible Materials↗

PubMed2005

Supercritical fluid crystallization of griseofulvin: crystal habit modification with a selective growth inhibitor.

Poly (sebacic anhydride) (PSA) was used as a growth inhibitor to selectively modify habit of griseofulvin crystals formed via the Precipitation with a compressed-fluid antisolvent (PCA) process. PSA and griseofulvin were coprecipitated within a PCA injector, which provided efficient mixing between the solution and compressed antisolvent process streams. Griseofulvin crystal habit was modified from acicular to bipyramidal when the mass ratio of PSA/griseofulvin in the solution feed stream was <or=1:1. The habit modification was attributed to the preferential adsorption of PSA to the fastest growing crystal face of the acicular crystal form, which inhibited growth. Scanning electron microscopy (SEM) was used to characterize the griseofulvin and PSA particles, and gave results consistent with a selective growth inhibition mechanism. SEM micrographs showed regions on griseofulvin crystals where PSA microparticles had preferentially adsorbed. X-ray powder diffraction (XRPD) and differential scanning calorimetry (DSC) analysis of the griseofulvin crystals indicated no changes in the crystalline form after the habit modification. Powder compressibility decreased from 49 +/- 3% to 28 +/- 7% with the modification in crystal habit. No change in the physical stability of the processed powder was observed after being stored at 25 degrees C/60% RH and 40 degrees C/70% RH for 23 days. Despite the change in crystal habit, griseofulvin crystals achieved 100% dissolution within 60 min in a simulated gastric fluid.

Anhydrides↗

PubMed2005

Crosslinked hyaluronan scaffolds as a biologically active carrier for valvular interstitial cells.

Hyaluronic acid (HA), a major component of the cardiac jelly during heart morphogenesis, is a polysaccharide that upon modification can be photopolymerized into hydrogels. Previous work in our lab has found that photopolymerizable HA hydrogels are suitable scaffolds for the culture and proliferation of valvular interstitial cells (VICs), the most prevalent cell type in native heart valves. The physical properties of HA gels are easily modified through alteration in material crosslink density or by copolymerizing with other reactive macromolecules. Degradation products of HA gels and the starting macromers significantly increased VIC proliferation when added to cell cultures. With low molecular weight HA (<6700 Da) exhibiting greatest stimulation of VIC proliferation. Low molecular weight HA degradation products added to VIC cultures also resulted in a four-fold increase in total matrix production and a two-fold increase in elastin production over untreated controls. VIC internalization of HA, as shown by cellular uptake of fluorescently labeled HA, likely activates signaling cascades resulting in the biological responses seen here. Lastly, VICs encapsulated within HA hydrogels remained viable, and significant elastin production was observed after 6 weeks of culture. This work shows promise for the creation of a tissue-engineered heart valve utilizing the synergistic relationship between hyaluronic acid and VICs.

Animals↗

PubMed2005

The effect on osteoblast function of colocalized RGD and PHSRN epitopes on PEG surfaces.

Poly(ethylene glycol) hydrogels were synthesized with pendant peptide functionalities to examine the influence of synergistic peptide sequences on osteoblast adhesion, spreading, and function. Specifically, acrylated monomers were prepared that contained the peptide sequence, Arg-Gly Asp (RGD), as well as monomers with RGD plus its synergy site, Pro-His-Ser-Arg-Asn (PHSRN), linked via a polyglycine sequence to recapitulate the native spacing of fibronectin. The colocalized RGD-PHSRN sequence improved osteoblast adhesion, spreading, and focal contact formation when compared to RGD alone. In addition, proliferation, metabolic activity, and levels of alkaline phosphatase production, a common marker for osteoblast function, were statistically higher for the colocalized peptide sequences at 1 day, 1 week, and 2 weeks, when compared to control surfaces. Interestingly, increases were not observed in all areas of cell function, as extracellular matrix (ECM) production was the lowest on gels functionalized with the colocalized peptide sequence. This result was attributed to strong receptor-ligand interactions initiating signal transduction cascades that down-regulate ECM production.

Alkaline Phosphatase↗

PubMed2005

Synthetic hydrogel niches that promote hMSC viability.

Photopolymerized poly(ethylene glycol) (PEG) hydrogels were used as a base platform for the encapsulation and culture of human mesenchymal stem cells (hMSCs). The base PEG formulation presents an environment completely devoid of cell-matrix interactions. As such, viability of hMSCs in unmodified PEG hydrogels is very low. This formulation was modified to contain pendant phosphate groups to facilitate the sequestering of osteopontin within the gel, as well as pendant cell-adhesive RGD peptide sequences, which are found in osteopontin and other cell adhesion proteins. The survivability of hMSCs was examined with culture time and as a function of the gel chemistry to examine the role of cell-matrix interactions in promoting long-term viability. In the absence of any adhesive ligands, hMSC viability drops to 15% after 1 week in culture. However, by incorporating the RGD sequence or pendant phosphate groups this low viability was rescued to 75% and 97%, respectively. It is believed that the phosphate groups promote mineralization of the hydrogel network, and this mineral phase sequesters cell-secreted osteopontin, resulting in enhanced cell-matrix interactions and improved cell viability.

Absorption↗

PubMed2005

Activation of valvular interstitial cells is mediated by transforming growth factor-beta1 interactions with matrix molecules.

Strategies for the tissue-engineering of living cardiac valve replacements are limited by a lack of appropriate scaffold materials that both permit cell viability and actively contribute to the growth of functional tissues. Components of the extracellular matrix can localize and modify growth factor signals, and by doing so impart instructional stimuli for direction of cell phenotype. Fibronectin, collagen I, and heparin were explored as affinity matrices for sequestering and presenting soluble signaling molecules to control differentiation of valvular interstitial cells (VICs) to myofibroblasts. VIC differentiation is commonly characterized by expression of stress fibers containing alpha smooth muscle actin (alpha-SMA), and transforming growth factor-beta1 (TGF-beta1) is a central mediator of this transition. Both fibronectin and heparin, which are known to possess TGF-beta1 binding interactions, were found to increase VIC alpha-SMA expression (120% and 258% of expression in controls), while VICs cultured on collagen I-modified substrates had diminished alpha-SMA expression (66% of control). Heparin treatment significantly stimulated VIC production of TGF-beta1 at all concentrations tested (50 to 400 mug/ml). Heparin-modified substrates were found to alter cell morphology through increased adsorption of serum proteins, specifically TGF-beta1. In sum, heparin produced alpha-SMA-positive myofibroblasts through both the de novo production of TGF-beta1, and its localization in the pericellular environment. The addition of heparin to fibronectin-modified substrates led to a synergistic increase in VIC alpha-SMA expression, produced by the reciprocal binding of fibronectin, heparin, cell-produced TGF-beta1. The characterization of molecules, both soluble and insoluble, that control VIC activation will be important for the development of tailored 3D culture environments for tissue-engineering applications.

Actins↗

PubMed2005

Cell-based therapies and tissue engineering.

Tissue engineering is a rapidly evolving discipline that may some-day afford surgeons a limitless supply of autologous tissue for transplantation or allow in situ tissue regeneration. A number of biologic, engineering, and clinical challenges continue to face tissue engineers and surgeons alike. One important example is the choice of an appropriate cell scaffold that promotes growth and is eventually resorbed by the body. Although the application of bioengineered tissue is specific to the anatomic areas of interest,continued advances bring tissue engineering closer to reality in all areas of otolaryngology.

Biocompatible Materials↗

PubMed2004

Incorporation of tissue-specific molecules alters chondrocyte metabolism and gene expression in photocrosslinked hydrogels.

Hydrogels are highly swollen, insoluble networks which can entrap chondrocytes and provide a 3-D environment necessary for the re-growth of cartilaginous tissue. In this study, hydrogels were formulated with a synthetic poly(ethylene glycol) (PEG) component to provide control over the macroscopic gel properties and from a cartilage specific compound, chondroitin sulfate (ChSA), to capture features of the chondrocytes' native environment. PEG was chosen as the base hydrogel chemistry, because it forms a 3-D environment that maintains chondrocyte function. ChSA, a highly negatively charged main component of proteoglycans, was then selectively incorporated into the PEG gel. Macroscopic gel properties were manipulated to obtain high compressive moduli coupled with a high degree of swelling by formulating copolymer gels with these chemistries. The gel compressive modulus of cell-free PEG gels increased from 34 to 140 kPa with the incorporation of ChSA for similar degrees of swelling. When chondrocytes were encapsulated in pure ChSA gels, synthesis of collagen and glycosaminoglycans was inhibited. However, when PEG was introduced into the copolymer gels, both extracellular matrix components were stimulated. Total collagen content increased from non-detectable in the pure ChSA gels to 0.48+/-0.05 mg/g wet weight in the copolymer gels (40/60 ChSA/PEG). Gene expression for collagen type II was also enhanced by the incorporation of PEG into the gel, illustrating an important influence of gel chemistry on chondrocyte function; however, aggrecan gene expression was unaffected. This study demonstrates that the macroscopic properties of chondrocyte gel carriers can be controlled through the incorporation of charge into networks by ChSA, but the neutral, non-interactive base PEG chemistry facilitates extracellular matrix deposition.

Base Sequence↗

PubMed2004

In situ fabrication of macroporous polymer networks within microfluidic devices by living radical photopolymerization and leaching.

Novel fabrication techniques and polymer systems are being explored to enable mass production of low cost microfluidic devices. In this contribution we discuss a new fabrication scheme for making microfluidic devices containing porous polymer components in situ. Contact lithography, a living radical photopolymer (LRPP) system and salt leaching were used to fabricate multilayer microfluidic devices rapidly with various channel geometries and covalently attached porous polymer plugs made of various photopolymerizable substrates. LRPP systems offer the advantages of covalent attachment of microfluidic device layers and facile surface modification via grafting. Several applications of the porous plugs are also explored, including a static mixer, a high surface area-to-volume reactor and a rapidly responding hydrogel valve. Quantitative and qualitative data show an increase in mixing of a fluorescein and a water stream for channels containing porous plugs relative to channels with no porous plugs. Confocal laser scanning microscopy images demonstrate the ability to graft a functional material onto porous plug surfaces. A reaction was carried out on the grafted pore surfaces, which resulted in fluorescent labelling of the grafted material throughout the pores of the plug. Homogenous fluorescence throughout the depth of the porous plug and along pore surfaces indicated that the porous plugs were surface modified by grafting and that reactions can be carried out on the pore surfaces. Finally, porous hydrogel valves were fabricated which swelled in response to contact with various pH solutions. Results indicate that a porous hydrogel valve will swell and close more rapidly than other valve geometries made with the same polymer formulation. The LRPP-salt leaching method provides a means for rapidly incorporating porous polymer components into microfluidic devices, which can be utilized for a variety of pertinent applications upon appropriate selection of porous plug materials and surface treatments.

Journal Article↗

PubMed2004

Designing scaffolds for valvular interstitial cells: cell adhesion and function on naturally derived materials.

Valvular interstitial cells (VICs) possess many properties that make them attractive for use in the construction of a tissue-engineered valve; however, we have found that the surfaces to which VICs will adhere and spread are limited. For example, VICs adhere and spread on collagen and laminin-coated surfaces, but display altered morphology and do not proliferate. Interestingly, fibronectin (FN) was one adhesion protein that facilitated VIC adhesion and proliferation. Yet VICs did not spread on surfaces modified with RGD, a ubiquitous cell-adhesive peptide, nor with other FN-specific peptide sequences such as EILDV and PHSRN. Hyaluronic acid (HA) is a highly elastic polysaccharide that is involved in natural valve morphogenesis and possesses binding interactions with FN. Hyaluronic acid was modified to form photopolymerizable hydrogels, and VICs were found to spread and proliferate on HA-based gels, forming a confluent monolayer on the gels within 4 days. Modified HA retained its ability to specifically bind FN, allowing for the formation of gels containing both HA and FN. Valvular interstital cells cultured on HA surfaces displayed significantly increased production of extracellular matrix proteins, indicating that HA-based scaffolds may provide useful biological cues to stimulate heart valve tissue formation.

Animals↗

PubMed2004

Robust polymer microfluidic device fabrication via contact liquid photolithographic polymerization (CLiPP).

Microfluidic devices are commonly fabricated in silicon or glass using micromachining technology or elastomers using soft lithography methods; however, invariable bulk material properties, limited surface modification methods and difficulty in fabricating high aspect ratio devices prevent these materials from being utilized in numerous applications and/or lead to high fabrication costs. Contact Liquid Photolithographic Polymerization (CLiPP) was developed as an alternative microfabrication approach that uniquely exploits living radical photopolymerization chemistry to facilitate surface modification of device components, fabrication of high aspect ratio structures from many different materials with numerous covalently-adhered layers and facile construction of three-dimensional devices. This contribution describes CLiPP and demonstrates unique advantages of this new technology for microfabrication of polymeric microdevices. Specifically, the procedure for fabricating devices with CLiPP is presented, the living radical photopolymerization chemistry which enables this technology is described, and examples of devices made using CLiPP are shown.

Equipment Design↗

PubMed2004

Encapsulating chondrocytes in copolymer gels: bimodal degradation kinetics influence cell phenotype and extracellular matrix development.

Hydrogels provide an ideal environment for encapsulating chondrocytes and facilitating the production of cartilaginous tissue. However, the deposition of extracellular matrix (ECM) and ultimate tissue function are significantly affected by degradation of gel scaffolds. It was hypothesized that a bimodal degradation process would capture the critical features necessary for neotissue development. Specifically, most of the initial crosslinks would degrade quickly and enable ECM deposition, whereas a critical amount would remain or degrade much more slowly to provide structural integrity over a longer time period. In this study, chondrocytes were encapsulated in copolymer gels of nondegradable [poly(ethylene glycol) dimethacrylate] and degradable [poly(lactic acid)-b-poly(ethylene glycol)-b-poly(lactic acid) dimethacrylate] macromers to investigate the effects of gel degradation on ECM evolution. All gels were synthesized from 10 wt % total macromer solutions consisting of 0, 19, 21, 23, 25, or 100 mol % nondegradable units. The copolymer constructs were found to have lower DNA content than completely degradable constructs after 8 weeks. However, total biochemical content was very similar among the various copolymer constructs. Histological analysis gave more interesting insight, showing a more uniform spatial distribution of ECM components in copolymer samples than in constructs with 100 mol % nondegradable units. In addition, a number of major structural defects were present in constructs with 0 mol % nondegradable units that became less apparent as the amount of nondegradable units was increased. Overall, the copolymer gels had a higher compressive modulus during neotissue development and also showed no evidence of chondrocyte dedifferentiation. With their bimodal degradation profile, copolymer gels with carefully selected ratios of degrading to slow or nondegrading crosslinks provide distinct advantages for ECM development in tissue-engineered cartilage.

Animals↗

PubMed2004

Encapsulating chondrocytes in degrading PEG hydrogels with high modulus: engineering gel structural changes to facilitate cartilaginous tissue production.

A major challenge when designing cell scaffolds for chondrocyte delivery in vivo is creating scaffolds with sufficient mechanical properties to restore initial function while simultaneously controlling temporal changes in the gel structure to facilitate tissue formation. To address this design challenge, degradable photocrosslinked hydrogels based on poly(ethylene glycol) were investigated. To alter the gel's initial mechanical properties, hydrogels were fabricated by varying the initial macromer concentration from 10% to 15% to 20%. A twofold increase in macromer concentration resulted in an eightfold increase in the initial compressive modulus from 60 to 500 kPa. Gel degradation was tailored by incorporating fast-degrading crosslinks that enable maximal extracellular matrix (ECM) diffusion with time and a minimal number of nondegrading (or slowly degrading) crosslinks to maintain scaffold integrity and prevent complete gel erosion during tissue formation. Chondrocytes encapsulated in these gels produced cartilaginous tissue rich in glycosaminoglycans and collagen as seen biochemically and histologically. Interestingly, mass loss appeared to more closely match tissue secretion in gels fabricated from a 15% macromer concentration. However, the spatial ECM distribution was grossly similar in all three gels. By tailoring gel degradation and controlling network evolution during degradation, gels with optimal properties can be fabricated to support initially physiologic compressive loads while simultaneously supporting the formation of a neotissue.

Animals↗

PubMed2004

Valvular myofibroblast activation by transforming growth factor-beta: implications for pathological extracellular matrix remodeling in heart valve disease.

The pathogenesis of cardiac valve disease correlates with the emergence of muscle-like fibroblasts (myofibroblasts). These cells display prominent stress fibers containing alpha-smooth muscle actin (alpha-SMA) and are believed to differentiate from valvular interstitial cells (VICs). However, the biological factors that initiate myofibroblast differentiation and activation in valves remain unidentified. We show that transforming growth factor-beta1 (TGF-beta1) mediates differentiation of VICs into active myofibroblasts in vitro in a dose-dependent manner, as determined by a significant increase in alpha-SMA and the dramatic augmentation of stress fiber formation and alignment. Additionally, TGF-beta1 and increased mechanical stress function synergistically to enhance contractility. In turn, contractile valve myofibroblasts exert tension on the extracellular matrix, resulting in a dramatic realignment of extracellular fibronectin fibrils. TGF-beta1 also inhibits valve myofibroblast proliferation without enhancing apoptosis. Our results are consistent with activation of a highly contractile myofibroblast phenotype by TGF-beta1 and are the first to connect valve myofibroblast contractility with pathological valve matrix remodeling. We suggest that the activation of contractile myofibroblasts by TGF-beta1 may be a significant first step in promoting alterations to the valve matrix architecture that are evident in valvular heart disease.

Actins↗

PubMed2004

Delivering DNA from photocrosslinked, surface eroding polyanhydrides.

Sustained delivery of DNA has the potential to enhance long-term gene therapy; however, precise control of a wide range of DNA release profiles may be needed. In this work, multifunctional anhydride monomers were photocrosslinked to produce hydrophobic, highly crosslinked polymer networks that degrade by surface erosion. Surface-eroding polymers can deliver molecules of a wide range of sizes at sustained, steady rates, which is advantageous for DNA delivery, where the high molecular weight may complicate control of the release profiles. When plasmid DNA was released from photocrosslinked polyanhydride matrices, DNA recovery was low (approximately 25%). Electrophoresis indicated that the plasmid DNA was released primarily in the relaxed and supercoiled forms, yet the relative fraction of released DNA in the supercoiled form decreased over time. To improve DNA recovery and reduce the damaging effects of polymer degradation, DNA was pre-encapsulated in alginate microparticles, which served as a temporary coating that quickly dissolved upon microparticle release from the polyanhydride matrix. As photocrosslinked polyanhydrides have highly predictable drug release profiles that depend on the polymer erosion rate and implant geometry and not on the entrapped molecule size, they can serve dual purposes in many biomaterial applications where structural support and drug release would be beneficial.

Alginates↗

PubMed2004

DNA delivery from photocrosslinked PEG hydrogels: encapsulation efficiency, release profiles, and DNA quality.

Sustained DNA delivery from polymer matrices provides a means for enhanced and prolonged gene therapy; however, limitations exist with respect to tailoring the DNA release profiles and maintaining the quality of the encapsulated DNA over time. To address these issues, PEG-based macromolecular monomers were photopolymerized to produce hydrogels with various degradation rates to control the DNA release profiles. Photocrosslinked PEG-based hydrogels were designed that released DNA for periods of 6-100 days with either nearly linear or delayed burst release profiles. Plasmid DNA was released primarily in the relaxed and supercoiled forms, and the released DNA showed high biological activity in plated cell cultures. The addition of both chemical and physical protective agents helped preserve the supercoiled form of the plasmid DNA during photoencapsulation (up to 75% compared to non-encapsulated plasmid controls), thereby enhancing the biological activity of the released DNA.

Cross-Linking Reagents↗

PubMed2004

In vitro osteogenic differentiation of human mesenchymal stem cells photoencapsulated in PEG hydrogels.

Much research has focused on the differentiation of human mesenchymal stem cells (hMSCs) in monolayer culture; however, little is known about their differentiation potential in three-dimensional culture conditions. In this research, hMSCs were encapsulated in a photocrosslinkable, injectable scaffolding system based on poly(ethylene glycol) (PEG) hydrogels. To demonstrate the ability of hMSCs to differentiate in PEG hydrogels, cell/polymer constructs were cultured in osteogenic differentiation media to elicit an osteoblastic response. First, viability of encapsulated hMSCs up to 4 weeks in culture was investigated using a membrane integrity assay. Second, gene expression of encapsulated cells was determined with reverse transcription polymerase chain reaction (RT-PCR) as a function of media composition. After 1 week in osteogenic differentiation media, encapsulated hMSCs expressed osteonectin, osteopontin, and alkaline phosphatase, which are all characteristic of osteoblasts. Finally, von Kossa staining was used to evaluate mineralization of the PEG gels. Results support the hypothesis that hMSCs photoencapsulated in PEG hydrogels and cultured in the presence of osteogenic differentiation media are able to differentiate to osteoblasts inside the gel and mineralize the matrix. These experiments demonstrate the feasibility of using a PEG-based, photocrosslinkable system to culture and deliver human mesenchymal stem cells for bone tissue regeneration and repair.

Bone Substitutes↗

PubMed2004

Crosslinking density influences the morphology of chondrocytes photoencapsulated in PEG hydrogels during the application of compressive strain.

Chondrocyte deformation, which occurs during mechanical loading, is thought to play an important role in the mechanotransduction pathway. In designing a scaffold that can be gelled in situ for cartilage tissue engineering, an important consideration is the influence of mechanical loading. This study tested the hypothesis that changes in the crosslinking density of a hydrogel scaffold influence the morphology of encapsulated chondrocytes in response to an applied load. Chondrocytes were entrapped in photo-crosslinkable hydrogel scaffolds based on poly(ethylene glycol) (PEG) with two crosslinking densities, 0.119 and 0.376 mol/l, with the higher density having a 11-fold higher compressive modulus. The cell-embedded hydrogels were subjected to static compressive strains between 0% and 20% after 1 and 6 days of culture. Using confocal laser scanning microscopy, chondrocytes in the highly crosslinked gel at day 1 deformed more than gels in the more loosely crosslinked gel. By day 6, this finding was reversed. When single cells within a region were followed, heterogeneities in cell deformation were observed on both a macroscopic and microscopic scale. These heterogeneities were greater in the highly crosslinked gel. These findings demonstrate that different levels of cell deformation and heterogeneity may be obtained by varying the crosslinking density in PEG hydrogels.

Animals↗