Poly(sophorolipid) structural variation: effects on biomaterial physical and biological properties.

Diacetylated lactonic sophorolipids (polyLSL[6'Ac,6″Ac]), a biosurfactant, can be efficiently polymerized by ring-opening metathesis polymerization (ROMP). In this paper, enzyme-mediated chemical transformations are developed to regioselectively modify LSL[6'Ac,6″Ac] at sophorose primary hydroxyl positions (6' and 6″). The resulting modified LSLs were polymerized to expand polyLSL structural diversity, that is, polyLSL[6'OH,6″Ac], polyLSL[6'OH,6″OH], polyLSL[6'Bu,6″Ac], polyLSL[6'N3,6″Ac], and polyLSL[6'MA,6″Ac]. Controlled placement of azide and methacrylate at sophorolipid moieties enables the use of "click" reactions to introduce bioactive groups. Thermal analyses of polyLSLs showed that the acylation pattern at sugar moieties has a remarkable effect on chain stiffness and crystallinity. Films of polyLSL[6'Ac,6″Ac], polyLSL[6'OH,6″Ac], and polyLSL[6'Bu,6″Ac] exhibited nonbrittle behaviors with compressive elastic moduli ranging from ∼1.5 to ∼4.9 MPa. PolyLSLs were cytocompatible with human mesenchymal stem cells (h-MSCs), and examination of material-induced osteogenic cell lineage progression uncovered a dependence on polyLSL substitution at sophorose 6'-sites. This research reveals opportunities to regulate polyLSL physical properties and cell response behaviors by variation in substituents at polyLSL sophorolipid moieties.

Photo-cross-linked PDMSstar-PEG hydrogels: synthesis, characterization, and potential application for tissue engineering scaffolds.

Inorganic-organic hydrogels with tunable chemical and physical properties were prepared from methacrylated star polydimethylsiloxane (PDMS(star)-MA) and diacrylated poly(ethylene glycol) (PEG-DA) for use as tissue engineering scaffolds. A total of 18 compositionally unique hydrogels were prepared by photo-cross-linking, varying weight ratios of PEG-DA and PDMS(star)-MA of different molecular weights (M(n)): PEG-DA (M(n) = 3.4k and 6k g/mol) and PDMS(star)-MA (M(n) = 1.8k, 5k, and 7k g/mol). Introduction of PDMS(star)-MA caused formation of discrete PDMS-enriched microparticles dispersed within the PEG matrix. The swelling ratio, mechanical properties in tension and compression, nonspecific protein adhesion, controlled introduction of bioactivity, and cytotoxicity of hydrogels were studied. This library of inorganic-organic hydrogels with tunable properties provides a useful platform to study the effect of scaffold properties on cell behavior.

Modeling the effects of hyaluronic acid degradation on the regulation of human astrocyte phenotype using multicomponent interpenetrating polymer networks (mIPNs).

Hyaluronic acid (HA) is a highly abundant component in the extracellular matrix (ECM) and a fundamental element to the architecture and the physiology of the central nervous system (CNS). Often, HA degradation occurs when an overreactive inflammatory response, derived from tissue trauma or neurodegenerative diseases such as Alzheimer's, causes the ECM in the CNS to be remodeled. Herein, we studied the effects of HA content as a key regulator of human astrocyte (HAf) reactivity using multicomponent interpenetrating polymer networks (mIPNs) comprised of Collagen I, HA and poly(ethylene glycol) diacrylate. The selected platform facilities the modulation of HA levels independently of matrix rigidity. Total astrocytic processes length, number of endpoints, the expression of the quiescent markers: Aldehyde Dehydrogenase 1 Family Member L1 (ALDH1L1) and Glutamate Aspartate Transporter (GLAST); the reactive markers: Glial Fibrillary Acidic Protein (GFAP) and S100 Calcium-Binding Protein ß (S100ß); and the inflammatory markers: Inducible Nitric Oxide Synthase (iNOS), Interleukin 1ß (IL-1ß) and Tumor Necrosis Factor Alpha (TNFα), were assessed. Cumulatively, our results demonstrated that the decrease in HA concentration elicited a reduction in the total length of astrocytic processes and an increase in the expression of HAf reactive and inflammatory markers.

Toward zonally tailored scaffolds for osteochondral differentiation of synovial mesenchymal stem cells.

Synovium-derived mesenchymal stem cells (SMSCs) are an emerging cell source for regenerative medicine applications, including osteochondral defect (OCD) repair. However, in contrast to bone marrow MSCs, scaffold compositions which promote SMSC chondrogenesis/osteogenesis are still being identified. In the present manuscript, we examine poly(ethylene) glycol (PEG)-based scaffolds containing zonally-specific biochemical cues to guide SMSC osteochondral differentiation. Specifically, SMSCs were encapsulated in PEG-based scaffolds incorporating glycosaminoglycans (hyaluronan or chondroitin-6-sulfate [CSC]), low-dose of chondrogenic and osteogenic growth factors (TGFß1 and BMP2, respectively), or osteoinductive poly(dimethylsiloxane) (PDMS). Initial studies suggested that PEG-CSC-TGFß1 scaffolds promoted enhanced SMSC chondrogenic differentiation, as assessed by significant increases in Sox9 and aggrecan. Conversely, PEG-PDMS-BMP2 scaffolds stimulated increased levels of osteoblastic markers with significant mineral deposition. A "Transition" zone formulation was then developed containing a graded mixture of the chondrogenic and osteogenic signals present in the PEG-CSC-TGFß1 and PEG-PDMS-BMP2 constructs. SMSCs within the "Transition" formulation displayed a phenotypic profile similar to hypertrophic chondrocytes, with the highest expression of collagen X, intermediate levels of osteopontin, and mineralization levels equivalent to "bone" formulations. Overall, these results suggest that a graded transition from PEG-CSC-TGFß1 to PEG-PDMS-BMP2 scaffolds elicits a gradual SMSC phenotypic shift from chondrocyte to hypertrophic chondrocyte to osteoblast-like. As such, further development of these scaffold formulations for use in SMSC-based OCD repair is warranted. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 2019-2029, 2019.

Influence of hydrogel mechanical properties and mesh size on vocal fold fibroblast extracellular matrix production and phenotype.

Current clinical management of vocal fold (VF) scarring produces inconsistent and often suboptimal results. Researchers are investigating a number of alternative treatments for VF lamina propria (LP) scarring, including designer implant materials for functional LP regeneration. In the present study, we investigate the effects of the initial scaffold elastic modulus and mesh size on encapsulated VF fibroblast (VFF) extracellular matrix (ECM) production toward rational scaffold design. Poly(ethylene glycol) diacrylate (PEGDA) hydrogels were selected for this study since their material properties, including mechanical properties, mesh size, degradation rate and bioactivity, can be tightly controlled and systematically modified. Porcine VFF were encapsulated in four PEGDA hydrogels with degradation half lives of approximately 25 days, but with initial elastic compressive moduli and mesh sizes ranging from approximately 30 to 100kPa and from approximately 9 to 27nm, respectively. After 30 days of static culture, VFF ECM production and phenotype in each formulation was assessed biochemically and histologically. Sulfated glycosaminoglycan synthesis increased in similar degree with both increasing initial modulus and decreasing initial mesh size. In contrast, elastin production decreased with increasing initial modulus but increased with decreasing initial mesh size. Both collagen deposition and the induction of a myofibroblastic phenotype depended strongly on initial mesh size but appeared largely unaffected by variations in initial modulus. The present results indicate that scaffold mesh size warrants further investigation as a critical regulator of VFF ECM synthesis. Furthermore, this study validates a systematic and controlled approach for analyzing VFF response to scaffold properties, which should aid in rational scaffold selection/design.

An improved correlation to predict molecular weight between crosslinks based on equilibrium degree of swelling of hydrogel networks.

Accurate characterization of hydrogel diffusional properties is of substantial importance for a range of biotechnological applications. The diffusional capacity of hydrogels has commonly been estimated using the average molecular weight between crosslinks (Mc ), which is calculated based on the equilibrium degree of swelling. However, the existing correlation linking Mc and equilibrium swelling fails to accurately reflect the diffusional properties of highly crosslinked hydrogel networks. Also, as demonstrated herein, the current model fails to accurately predict the diffusional properties of hydrogels when polymer concentration and molecular weight are varied simultaneously. To address these limitations, we evaluated the diffusional properties of 48 distinct hydrogel formulations using two different photoinitiator systems, employing molecular size exclusion as an alternative methodology to calculate average hydrogel mesh size. The resulting data were then utilized to develop a revised correlation between Mc and hydrogel equilibrium swelling that substantially reduces the limitations associated with the current correlation. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 1339-1348, 2018.

A canine in vitro model for evaluation of marrow-derived mesenchymal stromal cell-based bone scaffolds.

Tissue engineered bone grafts based on bone marrow mesenchymal stromal cells (MSCs) are being actively developed for craniomaxillofacial (CMF) applications. As for all tissue engineered implants, the bone-regenerating capacity of these MSC-based grafts must first be evaluated in animal models prior to human trials. Canine models have traditionally resulted in improved clinical translation of CMF grafts relative to other animal models. However, the utility of canine CMF models for evaluating MSC-based bone grafts rests on canine MSCs (cMSCs) responding in a similar manner to scaffold-based stimuli as human MSCs (hMSCs). Herein, cMSC and hMSC responses to polyethylene glycol (PEG)-based scaffolds were therefore compared in the presence or absence of osteoinductive polydimethylsiloxane (PDMS). Notably, the conjugation of PDMS to PEG-based constructs resulted in increases in both cMSC and hMSC osteopontin and calcium deposition. Based on these results, cMSCs were further used to assess the efficacy of tethered bone morphogenic protein 2 (BMP2) in enhancing PEG-PDMS scaffold osteoinductivity. Addition of low doses of tethered BMP2 (100 ng/mL) to PEG-PDMS systems increased cMSC expression of osterix and osteopontin compared to both PEG-PDMS and PEG-BMP2 controls. Furthermore, these increases were comparable to effects seen with up to five-times higher BMP2 doses noted in literature. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A:2382-2393, 2018.

Evaluation of late outgrowth endothelial progenitor cell and umbilical vein endothelial cell responses to thromboresistant collagen-mimetic hydrogels.

Bioactive coatings which support the adhesion of late-outgrowth peripheral blood endothelial progenitor cells (EOCs) are actively being investigated as a means to promote rapid endothelialization of "off-the-shelf," small-caliber arterial graft prostheses following implantation. In the present work, we evaluated the behavior of EOCs on thromboresistant graft coatings based on the collagen-mimetic protein Scl2-2 and poly(ethylene glycol) (PEG) diacrylate. Specifically, the attachment, proliferation, migration, and phenotype of EOCs on PEG-Scl2-2 hydrogels were evaluated as a function of Scl2-2 concentration (4, 8, and 12 mg/mL) relative to human umbilical vein endothelial cells (HUVECs). Results demonstrate the ability of each PEG-Scl2-2 hydrogel formulation to support EOC and HUVEC adhesion, proliferation, and spreading. However, only the 8 and 12 mg/mL PEG-Scl2-2 hydrogels were able to support stable EOC and HUVEC confluence. These PEG-Scl2-2 formulations were, therefore, selected for evaluation of their impact on EOC and HUVEC phenotype relative to PEG-collagen hydrogels. Cumulatively, both gene and protein level data indicated that 8 mg/mL PEG-Scl2-2 hydrogels supported similar or improved levels of EOC maturation relative to PEG-collagen controls based on evaluation of CD34, VEGFR2, PECAM-1, and VE-Cadherin. The 8 mg/mL PEG-Scl2-2 hydrogels also appeared to support similar or improved levels of EOC homeostatic marker expression relative to PEG-collagen hydrogels based on von Willebrand factor, collagen IV, NOS3, thrombomodulin, and E-selectin assessment. Combined, the present results indicate that PEG-Scl2-2 hydrogels warrant further investigation as "off-the-shelf" graft coatings. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 1712-1724, 2017.

Osteoinductive PolyHIPE Foams as Injectable Bone Grafts.

We have recently fabricated biodegradable polyHIPEs as injectable bone grafts and characterized the mechanical properties, pore architecture, and cure rates. In this study, calcium phosphate nanoparticles and demineralized bone matrix (DBM) particles were incorporated into injectable polyHIPE foams to promote osteoblastic differentiation of mesenchymal stem cells (MSCs). Upon incorporation of each type of particle, stable monoliths were formed with compressive properties comparable to control polyHIPEs. Pore size quantification indicated a negligible effect of all particles on emulsion stability and resulting pore architecture. Alizarin red calcium staining illustrated the incorporation of calcium phosphate particles at the pore surface, while picrosirius red collagen staining illustrated collagen-rich DBM particles within the monoliths. Osteoinductive particles had a negligible effect on the compressive modulus (∼30 MPa), which remained comparable to human cancellous bone values. All polyHIPE compositions promoted human MSC viability (∼90%) through 2 weeks. Furthermore, gene expression analysis indicated the ability of all polyHIPE compositions to promote osteogenic differentiation through the upregulation of bone-specific markers compared to a time zero control. These findings illustrate the potential for these osteoinductive polyHIPEs to promote osteogenesis and validate future in vivo evaluation. Overall, this work demonstrates the ability to incorporate a range of bioactive components into propylene fumarate dimethacrylate-based injectable polyHIPEs to increase cellular interactions and direct specific behavior without compromising scaffold architecture and resulting properties for various tissue engineering applications.

Characterization of sequential collagen-poly(ethylene glycol) diacrylate interpenetrating networks and initial assessment of their potential for vascular tissue engineering.

Collagen hydrogels have been widely investigated as scaffolds for vascular tissue engineering due in part to the capacity of collagen to promote robust cell adhesion and elongation. However, collagen hydrogels display relatively low stiffness and strength, are thrombogenic, and are highly susceptible to cell-mediated contraction. In the current work, we develop and characterize a sequentially-formed interpenetrating network (IPN) that retains the benefits of collagen, but which displays enhanced mechanical stiffness and strength, improved thromboresistance, high physical stability and resistance to contraction. In this strategy, we first form a collagen hydrogel, infuse this hydrogel with poly(ethylene glycol) diacrylate (PEGDA), and subsequently crosslink the PEGDA by exposure to longwave UV light. These collagen-PEGDA IPNs allow for cell encapsulation during the fabrication process with greater than 90% cell viability via inclusion of cells within the collagen hydrogel precursor solution. Furthermore, the degree of cell spreading within the IPNs can be tuned from rounded to fully elongated by varying the time delay between the formation of the cell-laden collagen hydrogel and the formation of the PEGDA network. We also demonstrate that these collagen-PEGDA IPNs are able to support the initial stages of smooth muscle cell lineage progression by elongated human mesenchymal stems cells.

Thermoresponsive double network micropillared hydrogels for controlled cell release.

Thermoresponsive poly(N-isopropylacrylamide) hydrogels (PNIPAAm) have been widely used for controlled cell detachment. In this study, cell release is enhanced via deswelling with a two-pronged approach combining a double network (DN) design and micropatterning. PNIPAAm hydrogels are prepared as DNs comprised of a tightly crosslinked 1st network and a loosely crosslinked 2nd network. Moreover, the PNIPAAm DN hydrogels are prepared as both planar 1.5 mm-thick slabs as well as micropillar arrays (≈200 µm pillar diameter). Compared to the corresponding conventional single network (SN) hydrogels, DN hydrogels exhibit enhanced thermosensitivity and cell release efficiency, particularly for the micropillar arrays.

PDMS(star)-PEG hydrogels prepared via solvent-induced phase separation (SIPS) and their potential utility as tissue engineering scaffolds.

Inorganic-organic hydrogels based on methacrylated star polydimethylsiloxane (PDMS(star)-MA) and diacrylated poly(ethylene glycol) (PEG-DA) macromers were prepared via solvent-induced phase separation (SIPS). The macromers were combined in a dichloromethane precursor solution and sequentially photopolymerized, dried and hydrated. The chemical and physical properties of the hydrogels were further tailored by varying the number average molecular weight (M(n)) of PEG-DA (M(n)=3.4k and 6k gmol(-1)) as well as the weight percent ratio of PDMS(star)-MA (M(n)=7k gmol(-1)) to PEG-DA from 0:100 to 20:80. Compared to analogous hydrogels fabricated from aqueous precursor solutions, SIPS produced hydrogels with a macroporous morphology, a more even distribution of PDMS(star)-MA, increased modulus and enhanced degradation rates. The morphology, swelling ratio, mechanical properties, bioactivity, non-specific protein adhesion, controlled introduction of cell adhesion, and cytocompatibility of the hydrogels were characterized. As a result of their tunable properties, this library of hydrogels is useful to study material-guided cell behavior and ultimate tissue regeneration.

An approach for assessing hydrogel hydrophobicity.

Hydrogel hydrophobicity is an important modulator of mammalian cell behavior, drug payload release, and medical device fouling. Contact angle and protein adsorption measures are two common methods for evaluating hydrogel hydrophobicity. However, protein adsorption is a complex phenomenon which is challenging to interpret in terms of gel hydrophobicity alone. In addition, the permeability of hydrogels can be problematic for contact angle assessment, as this method can only be strictly applied to smooth, solid, and nonpermeable surfaces. Therefore, the development of a technique for measuring hydrogel hydrophobicity which is simple, sensitive, and independent of variations in gel permeability would significantly advance the ability to finely tune this variable. The present technical note develops a method for quantifying the hydrophobicity of hydrogels by exploiting their capacity to swell differentially in solvents of distinct polarities. To validate this technique, hydrogels of varying hydrophobicities were prepared by combining hydrophilic poly(ethylene glycol) diacrylate (PEGDA) with either hydrophobic 3-(trimethoxysilyl) propyl methacrylate (TMSPM) or hydrophilic 2-hydroxyethyl methacrylate (HEMA). The ratio of hydrogel swelling in 70% isopropanol to that in water was termed the hydrophobicity index (H-index) and was determined for each gel type. The measured H-indices reflected known differences in the hydrophobicities of HEMA, TMSPM, and PEGDA and, in contrast to contact angle assessments, appeared to be independent of variations in hydrogel permeability. In addition, the trend in H-indices agreed well with the trend in protein adsorption across hydrogel formulations, although the H-indices appeared to be able to resolve more subtle differences in gel hydrophobicity than protein adsorption measures.

Influence of select extracellular matrix proteins on mesenchymal stem cell osteogenic commitment in three-dimensional contexts.

Growth factors have been shown to be powerful mediators of mesenchymal stem cell (MSC) osteogenic differentiation. However, their use in tissue engineered scaffolds not only can be costly but also can induce undesired responses in surrounding tissues. Thus, the ability to specifically promote MSC osteogenic differentiation in the absence of exogenous growth factors via the manipulation of scaffold material properties would be beneficial. The current work examines the influence of select extracellular matrix (ECM) proteins on MSC osteogenesis toward the goal of developing scaffolds with intrinsically osteoinductive properties. Fibrinogen (FG), fibronectin (FN) and laminin-1 (LN) were chosen for evaluation due to their known roles in bone morphogenesis or bone fracture healing. These proteins were conjugated into poly(ethylene glycol) diacrylate (PEGDA) hydrogels and their effects on encapsulated 10T½ MSCs were evaluated. Specifically, following 1week of culture, mid-term markers of various MSC lineages were examined in order to assess the strength and specificity of the observed osteogenic responses. PEG-LN gels demonstrated increased levels of the osteogenic transcription factor osterix relative to day 0 levels. In addition, PEG-FG and PEG-LN gels were associated with increased deposition of bone ECM protein osteocalcin relative to PEG-FN gels and day 0. Importantly, the osteogenic response associated with FG and LN appeared to be specific in that markers for chondrocytic, smooth muscle cell and adipocytic lineages were not similarly elevated relative to day 0 in these gels. To gain insight into the integrin dynamics underlying the observed differentiation results, initial integrin adhesion and temporal alterations in cell integrin profiles were evaluated. The associated results suggest that α(2), α(v) and α(6) integrin subunits may play key roles in integrin-mediated osteogenesis.

A comparative study of the cytotoxicity and corrosion resistance of nickel-titanium and titanium-niobium shape memory alloys.

Nickel-titanium (NiTi) shape memory alloys (SMAs) are commonly used in a range of biomedical applications. However, concerns exist regarding their use in certain biomedical scenarios due to the known toxicity of Ni and conflicting reports of NiTi corrosion resistance, particularly under dynamic loading. Titanium-niobium (TiNb) SMAs have recently been proposed as an alternative to NiTi SMAs due to the biocompatibility of both constituents, the ability of both Ti and Nb to form protective surface oxides, and their superior workability. However, several properties critical to the use of TiNb SMAs in biomedical applications have not been systematically explored in comparison with NiTi SMAs. These properties include cytocompatibility, corrosion resistance, and alterations in alloy surface composition in response to prolonged exposure to physiological solutions. Therefore, the goal of the present work was to comparatively investigate these aspects of NiTi (49.2 at.% Ti) and TiNb (26 at.% Nb) SMAs. The results from the current studies indicate that TiNb SMAs are less cytotoxic than NiTi SMAs, at least under static culture conditions. This increased TiNb cytocompatibility was correlated with reduced ion release as well as with increased corrosion resistance according to potentio-dynamic tests. Measurements of the surface composition of samples exposed to cell culture medium further supported the reduced ion release observed from TiNb relative to NiTi SMAs. Alloy composition depth profiles also suggested the formation of calcium phosphate deposits within the surface oxide layers of medium-exposed NiTi but not of TiNb. Collectively, the present results indicate that TiNb SMAs may be promising alternatives to NiTi for certain biomedical applications.

Relative impact of form-induced stress vs. uniaxial alignment on multipotent stem cell myogenesis.

Tissue engineering strategies based on multipotent stem cells (MSCs) hold significant promise for the repair or replacement of damaged smooth muscle tissue. To design scaffolds which specifically induce MSC smooth muscle lineage progression requires a deeper understanding of the relative influence of various microenvironmental signals on myogenesis. For instance, MSC myogenic differentiation has been shown to be promoted by increases in active RhoA and FAK, both of which can be induced via increased cell-substrate stress. Separate studies have demonstrated MSC myogenesis to be enhanced by uniaxial cell alignment. The goal of the present study was to compare the impact of increased peak cell-substrate stresses vs. increased uniaxial cell alignment on MSC myogenic differentiation. To this end, MSC fate decisions were compared within two distinct multicellular "forms". A "stripe" multicellular pattern was designed to induce uniaxial cell alignment. In contrast, a second multicellular pattern was designed with "loops" or curves, which altered cell directionality while simultaneously generating regional peak stresses significantly above that intrinsic to the "stripe" form. As anticipated, the higher peak stress levels of the "loop" pattern were associated with increased fractions of active RhoA and active FAK. In contrast, two markers of early smooth muscle lineage progression, myocardin and SM-α-actin, were significantly elevated in the "stripe" pattern relative to the "loop" pattern. These results indicate that scaffolds which promote uniaxial MSC alignment may be more inductive of myogenic differentiation than those associated with increased peak, cell-substrate stress but in which cell directionality varies.

Osteogenic potential of poly(ethylene glycol)-poly(dimethylsiloxane) hybrid hydrogels.

Growth factors have been shown to be potent mediators of osteogenesis. However, their use in tissue-engineered scaffolds not only can be costly but also can induce undesired responses in surrounding tissues. Thus, the ability to specifically induce osteogenic differentiation in the absence of exogenous growth factors through manipulation of scaffold material properties would be desirable for bone regeneration. Previous research indicates that addition of inorganic or hydrophobic components to organic, hydrophilic scaffolds can enhance multipotent stem cell (MSC) osteogenesis. However, the combined impact of scaffold inorganic content and hydrophobicity on MSC behavior has not been systematically explored, particularly in three-dimensional (3D) culture systems. The aim of the present study was therefore to examine the effects of simultaneous increases in scaffold hydrophobicity and inorganic content on MSC osteogenic fate decisions in a 3D culture environment toward the development of intrinsically osteoinductive scaffolds. Mouse 10T½ MSCs were encapsulated in a series of novel scaffolds composed of varying levels of hydrophobic, inorganic poly(dimethylsiloxane) (PDMS) and hydrophilic, organic poly(ethylene glycol) (PEG). After 21 days of culture, increased levels of osteoblast markers, runx2 and osteocalcin, were observed in scaffolds with increased PDMS content. Bone extracellular matrix (ECM) molecules, collagen I and calcium phosphate, were also elevated in formulations with higher PDMS:PEG ratios. Importantly, this osteogenic response appeared to be specific in that markers for chondrocytic, smooth muscle cell, and adipocytic lineages were not similarly affected by variations in scaffold PDMS content. As anticipated, the increase in scaffold hydrophobicity accompanying increasing PDMS levels was associated with elevated scaffold serum protein adsorption. Thus, scaffold inorganic content combined with alterations in adsorbed serum proteins may underlie the observed cell behavior.

Influence of glycosaminoglycan identity on vocal fold fibroblast behavior.

Poly(ethylene glycol) (PEG) hydrogels have recently begun to be studied for the treatment of scarred vocal fold lamina propria due, in part, to their tunable mechanical properties, resistance to fibroblast-mediated contraction, and ability to be polymerized in situ. However, pure PEG gels lack intrinsic biochemical signals to guide cell behavior and generally fail to mimic the frequency-dependent viscoelastic response critical to normal superficial lamina propria function. Recent results suggest that incorporation of viscoelastic bioactive substances, such as glycosaminoglycans (GAGs), into PEG networks may allow these gels to more closely approach the mechanical responses of normal vocal fold lamina propria while also stimulating desired vocal fold fibroblast behaviors. Although a number of vocal fold studies have examined the influence of hyaluronan (HA) on implant mechanics and vocal fold fibroblast responses, the effects of other GAG types have been relatively unexplored. This is significant, since recent studies have suggested that chondroitin sulfate C (CSC) and heparan sulfate (HS) are substantially altered in scarred lamina propria. The present study was therefore designed to evaluate the effects of CSC and HS incorporation on the mechanical response of PEG gels and vocal fold fibroblast behavior relative to HA. As with PEG-HA, the viscoelasticity of PEG-CSC and PEG-HS gels more closely approached that of the normal vocal fold lamina propria than pure PEG hydrogels. In addition, collagen I deposition and fibronectin production were significantly higher in CSC than in HA gels, and levels of the myofibroblast marker smooth muscle α-actin (SM α-actin) were greater in CSC and HS gels than in HA gels. Since collagen I, fibronectin, and SM α-actin are generally elevated in scarred lamina propria these results suggest that CSC and HS may be undesirable for vocal fold implants relative to HA. Investigation of various signaling intermediates indicated that alterations in NFκB-p50, NFκB-p65, or pERK1/2 levels may underlie the observed differences among the PEG-GAG gels.

Inorganic-organic hybrid scaffolds for osteochondral regeneration.

Ligament graft failure frequently results from poor integration of the replacement tissue with associated bone. Thus, the ability to regenerate the bone-ligament osteochondral interface would be advantageous in ligament reconstruction. At the osteochondral interface, the tissue transitions from a bone-like matrix to fibrocartilage. Therefore, a scaffold which promotes a spatially regulated transition in cell behavior from osteoblast-like to chondrocyte-like would be desirable. Previous research indicates that addition of inorganic components to organic scaffolds can enhance the deposition of bone-like matrix by associated osteoblasts. We therefore reasoned that a gradient in the inorganic content of a hybrid inorganic-organic scaffold may induce an osteochondral-like transition in cell phenotype and matrix production. To test this hypothesis, hydrogels were prepared from poly(ethylene glycol) (PEG) and star poly(dimethylsiloxane) (PDMS(star)). As anticipated, both the matrix deposition and phenotype of encapsulated osteoblasts varied with scaffold inorganic content, although the directionality of this modulation was contrary to expectation. Specifically, osteoblasts appeared to transdifferentiate into chondrocyte-like cells with increasing scaffold inorganic content, as indicated by increased chondroitin sulfate and collagen type II production and by upregulation of sox9, a transcription factor associated with chondrocytic differentiation. Furthermore, the deposition of bone-like matrix (collagen type I, calcium phosphate, and osteocalcin) decreased with increasing PDMS(star) content. The resistance of the PDMS(star)-PEG scaffolds to protein adsorption and/or the changes in gel modulus/mesh structure accompanying PDMS(star) incorporation may underlie the unexpected increase in chondrocytic phenotype with increasing inorganic content. Combined, the present results indicate that PDMS(star)-PEG hybrid gels may prove promising for osteochondral regeneration. (c) 2010 Wiley Periodicals, Inc. J Biomed Mater Res, 2010.

Approach for fabricating tissue engineered vascular grafts with stable endothelialization.

A major roadblock in the development of tissue engineered vascular grafts (TEVGs) is achieving construct endothelialization that is stable under physiological stresses. The aim of the current study was to validate an approach for generating a mechanically stable layer of endothelial cells (ECs) in the lumen of TEVGs. To accomplish this goal, a unique method was developed to fabricate a thin EC layer using poly(ethylene glycol) diacrylate (PEGDA) as an intercellular "cementing" agent. This EC layer was subsequently bonded to the lumen of a tubular scaffold to generate a bi-layered construct. The viability of bovine aortic endothelial cells (BAECs) through the "cementing" process was assessed. "Cemented" EC layer expression of desired phenotypic markers (AcLDL uptake, VE-cadherin, eNOS, PECAM-1) as well as of injury-associated markers (E-selectin, SM22alpha) was also examined. These studies indicated that the "cementing" process allowed ECs to maintain high viability and expression of mature EC markers while not significantly stimulating primary injury pathways. Finally, the stability of the "cemented" EC layers under abrupt application of high shear pulsatile flow (approximately 11 dyn/cm(2), P (avg) approximately 95 mmHg, DeltaP approximately 20 mmHg) was evaluated and compared to that of conventionally "seeded" EC layers. Whereas the "cemented" ECs remained fully intact following 48 h of pulsatile flow, the "seeded" EC layers delaminated after less than 1 h of flow. Furthermore, the ability to extend this approach to degradable PEGDA "cements" permissive of cell elongation was demonstrated. Combined, these results validate an approach for fabricating bi-layered TEVGs with stable endothelialization.