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1.  Biodegradable poly(terephthalate-co-phosphate)s: synthesis, characterization and drug-release properties 
To develop biodegradable polymers with favorable physicochemical and biological properties, we have synthesized a series of poly(terephthalate-co-phosphate)s using a two-step polycondensation. The diol 1,4-bis(2-hydroxyethyl) terephthalate was first reacted with ethylphosphorodichloridate (EOP), and then chain-extended with terephthaloyl chloride (TC). Incorporation of phosphate into the poly(ethylene terephthalate) backbone rendered the co-polymers soluble in chloroform and biodegradable, lowered the Tg, decreased the crystallinity and increased the hydrophilicity. With an EOP/TC molar feed ratio of 80 : 20, the polymer exhibited good film-forming property, yielding at 86.6 ± 1.6% elongation with an elastic modulus of 13.76 ± 2.66 MPa. This polymer showed a favorable toxicity profile in vitro and good tissue biocompatibility in the muscular tissue of mice. In vitro the polymer lost 21% of mass in 21 days, but only 20% for up to 4 months in vivo. It showed no deterioration of properties after sterilization by γ -irradiation at 2.5 Mrad on solid CO2. Release of FITC-BSA from the microspheres was diffusion-controlled and exceeded 80% completion in two days. Release of the hydrophobic cyclosporine-A from microspheres was however much more sustained and near zero-ordered, discharging 60% in 70 days. A limited structure–property relationship has been established for this co-polymer series. The co-polymers became more hydrolytically labile as the phosphate component (EOP) was increased and the side chains were switched from the ethoxy to the methoxy structure. Converting the methoxy group to a sodium salt further increased the degradation rate significantly. The chain rigidity as reflected in the Tg values of the co-polymers decreased according to the following diol structure in the backbone: ethylene glycol > 2-methylpropylene diol > 2,2-dimethylpropylene diol. The wide range of physicochemical properties obtainable from this co-polymer series should help the design of degradable biomaterials for specific biomedical applications.
PMCID: PMC2376812  PMID: 15794482
Poly(terephthalate-co-phosphate); biodegradability; polycondensation; chain extension
2.  Combinatorial Design of Hydrolytically Degradable, Bone–like Biocomposites Based on PHEMA and Hydroxyapatite 
Polymer  2012;54(2):909-919.
With advantages such as design flexibility in modifying degradation, surface chemistry, and topography, synthetic bone–graft substitutes are increasingly demanded in orthopedic tissue engineering to meet various requirements in the growing numbers of cases of skeletal impairment worldwide. Using a combinatorial approach, we developed a series of biocompatible, hydrolytically degradable, elastomeric, bone–like biocomposites, comprising 60 wt% poly(2–hydroxyethyl methacrylate–co–methacrylic acid), poly(HEMA–co–MA), and 40 wt% bioceramic hydroxyapatite (HA). Hydrolytic degradation of the biocomposites is rendered by a degradable macromer/crosslinker, dimethacrylated poly(lactide–b–ethylene glycol–b–lactide), which first degrades to break up 3–D hydrogel networks, followed by dissolution of linear pHEMA macromolecules and bioceramic particles. Swelling and degradation were examined at Hank’s balanced salt solution at 37 °C in a 12–week period of time. The degradation is strongly modulated by altering the concentration of the co–monomer of methacrylic acid and of the macromer, and chain length/molecular weight of the macromer. 95% weight loss in mass is achieved after degradation for 12 weeks in a composition consisting of HEMA/MA/Macromer = 0/60/40, while 90% weight loss is seen after degradation only for 4 weeks in a composition composed of HEMA/MA/Macromer = 27/13/60 using a longer chain macromer. For compositions without a co–monomer, only about 14% is achieved in weight loss after 12–week degradation. These novel biomaterials offer numerous possibilities as drug delivery carriers and bone grafts particularly for low and medium load–bearing applications.
doi:10.1016/j.polymer.2012.12.017
PMCID: PMC3601847  PMID: 23525786
pHEMA; hydrolytic degradation; macromer
3.  Biodegradable Fibrous Scaffolds with Tunable Properties Formed from Photocrosslinkable Poly(glycerol sebacate) 
ACS applied materials & interfaces  2009;1(9):1878-1892.
It is becoming increasingly apparent that the architecture and mechanical properties of scaffolds, particularly with respect to mimicking features of natural tissues, are important for tissue engineering applications. Acrylated poly(glycerol sebacate) (Acr-PGS) is a material that can be crosslinked upon exposure to ultraviolet light, leading to networks with tunable mechanical and degradation properties through simple changes during Acr-PGS synthesis. For example, the number of acrylate functional groups on the macromer dictates the concentration of crosslinks formed in the resulting network. Three macromers were synthesized that form networks that vary dramatically with respect to their tensile modulus (~30 kPa to 6.6 MPa) and degradation behavior (~20 to 100% mass loss at 12 weeks) based on the extent of acrylation (~1 to 24%). These macromers were processed into biodegradable fibrous scaffolds using electrospinning, with gelatin as a carrier polymer to facilitate fiber formation and cell adhesion. The resulting scaffolds were also diverse with respect to their mechanics (tensile modulus ranging from ~60 kPa to 1 MPa) and degradation (~45 to 70% mass loss by 12 weeks). Mesenchymal stem cell adhesion and proliferation on all fibrous scaffolds was indistinguishable from controls. The scaffolds showed similar diversity when implanted on the surface of hearts in a rat model of acute myocardial infarction and demonstrated a dependence on scaffold thickness and chemistry in the host response. In summary, these diverse scaffolds with tailorable chemical, structural, mechanical and degradation properties are potentially useful for the engineering of a wide range of soft tissues.
doi:10.1021/am900403k
PMCID: PMC2765054  PMID: 20160937
electrospinning; elastomer; tissue engineering; scaffold; stem cells
4.  Degradable poly(2-hydroxyethyl methacrylate)-co-polycaprolactone Hydrogels for Tissue Engineering Scaffolds 
Biomacromolecules  2008;9(12):3370-3377.
Biodegradable poly(2-hydroxyethyl methacrylate) hydrogels for engineered tissue constructs were developed using atom transfer radical polymerization (ATRP), a degradable crosslinker and a macroinitiator. Hydrogels are appropriate materials for tissue engineering scaffolds due to their tissue-like mechanical compliance and mass transfer properties. However, many hydrogels that have seen wide application in medicine are not biodegradable or cannot be easily cleared from the body. Poly(2-hydroxyethyl methacrylate) (pHEMA) was selected for the scaffold material due to its reasonable mechanical strength, elasticity, long history of successful use in medicine and because it can be easily fabricated into numerous configurations. pHEMA was studied at various molecular weights between 2 kDa and 50 kDa. The molecular weight range suitable for renal clearance was an important factor in the experimental design. The fabricated hydrogels contain oligomeric blocks of polycaprolactone (PCL), a hydrolytically and enzymatically degradable polymer, as a crosslinking agent. In addition a degradable macroinitiator also containing oligomeric PCL was used to initiate the ATRP. The chain length, crosslink density, and polymerization solvent were found to greatly affect the mechanical properties of the pHEMA hydrogels. Degradation of the pHEMA hydrogels was characterized using 0.007 M NaOH, lipase solutions and phosphate buffered saline. Mass loss, swelling ratio and tensile modulus were evaluated. Degradation products from the sodium hydroxide were measured using gel permeation chromatography (GPC) to verify the polymer lengths and polydispersity. Erosion was only observed in the sodium hydroxide and lipase solutions. However, swelling ratio and tensile modulus indicate bulk degradation in all PCL containing samples. Degradable hydrogels in enzymatic solutions showed 30% mass loss in 16 weeks. Initial cell toxicity studies indicate no adverse cellular response to the hydrogels or their degradation products. These hydrogels have appropriate mechanical properties, a tunable degradation rate, and are composed of materials currently in FDA approved devices. Thus the degradable pHEMA developed in this study has considerable potential as a scaffold for tissue engineering in cardiac and other applications.
doi:10.1021/bm800686h
PMCID: PMC2650022  PMID: 19061434
PolyHEMA; Polycaprolactone; ATRP; Cardiac Tissue Engineering; Degradation
5.  Controlled Proteolytic Cleavage Site Presentation in Biomimetic PEGDA Hydrogels Enhances Neovascularization In Vitro 
Tissue Engineering. Part A  2012;18(23-24):2477-2486.
The volume of tissue that can be engineered is limited by the extent to which vascularization can be stimulated within the scaffold. The ability of a scaffold to induce vascularization is highly dependent on its rate of degradation. We present a novel approach for engineering poly (ethylene glycol) diacrylate (PEGDA) hydrogels with controlled protease-mediated degradation independent of alterations in hydrogel mechanical and physical properties. Matrix metalloproteinase (MMP)-sensitive peptides containing one (SSite) or three (TriSite) proteolytic cleavage sites were engineered and conjugated to PEGDA macromers followed by photopolymerization to form PEGDA hydrogels with tethered cell adhesion ligands of YRGDS and with either single or multiple MMP-sensitive peptide domains between cross links. These hydrogels were investigated as provisional matrices for inducing neovascularization, while maintaining the structural integrity of the hydrogel network. We show that hydrogels made from SSite and TriSite peptide-containing PEGDA macromers polymerized under the same conditions do not result in alterations in hydrogel swelling, mesh size, or compressive modulus, but result in statistically different hydrogel degradation times with TriSite gels degrading in 1–3 h compared to 2–4 days in SSite gels. In both polymer types, increases in the PEGDA concentration result in decreases in hydrogel swelling and mesh size, and increases in the compressive modulus and degradation time. Furthermore, TriSite gels support vessel invasion over a 0.3–3.6 kPa range of compressive modulus, while SSite gels do not support invasion in hydrogels above compressive modulus values of 0.4 kPa. In vitro data demonstrate that TriSite gels result in enhanced vessel invasion areas by sevenfold and depth of invasion by twofold compared to SSite gels by 3 weeks. This approach allows for controlled, localized, and cell-mediated matrix remodeling and can be tailored to tissues that may require more rapid regeneration and neovascularization.
doi:10.1089/ten.tea.2012.0173
PMCID: PMC3501121  PMID: 22725267
6.  Controlling poly(β-amino ester) network properties through macromer branching 
Acta biomaterialia  2007;4(2):207-217.
Photopolymerizable and degradable biomaterials are becoming important in the development of advanced materials in the fields of tissue engineering, drug delivery, and microdevices. We have recently developed a library of poly(β-amino ester)s (PBAEs) that form networks with a wide range of mechanical properties and degradation rates that are controlled by simple alterations in the macromer molecular weight or chemical structure. In this study, the influence of macromer branching on network properties was assessed by adding the trifunctional monomer pentaerythritol triacrylate (PETA) during synthesis. This led to a dose-dependent increase in the network compressive modulus, tensile modulus, and glass transition temperature, and a decrease in the network soluble fraction, yet led to only minor variations in degradation profiles and reaction behavior. For instance, the tensile modulus increased from 1.98 ± 0.09 MPa to 3.88 ± 0.20 MPa when the macromer went from a linear structure to a more branched structure with the addition of PETA. When osteoblast-like cells were grown on thin films, there was an increase in cell adhesion and spreading as the amount of PETA incorporated during synthesis increased. Towards tissue engineering applications, porous scaffolds were fabricated by photopolymerizing around a poragen and then subsequently leaching the poragen. Interconnected pores were observed in the scaffolds and observed trends translated to the porous scaffold (i.e., increasing mechanics with increasing branching). These findings demonstrate a simple variation during macromer synthesis that can be used to further tune the physical properties of scaffolds for given applications, particularly for candidates from the PBAE library.
doi:10.1016/j.actbio.2007.10.002
PMCID: PMC2350244  PMID: 18033746
Macromer; Biodegradable; Tissue Engineering; Branching; Photopolymerization
7.  Visible light cured thiol-vinyl hydrogels with tunable degradation for 3D cell culture 
Acta biomaterialia  2013;10(1):10.1016/j.actbio.2013.08.044.
We report here a synthetically simple yet highly tunable and diverse visible light mediated thiol-vinyl gelation system for fabricating cell-instructive hydrogels. Gelation was achieved via a mixed-mode step-and-chain-growth photopolymerization using functionalized 4-arm poly(ethylene glycol) as backbone macromer, eosin-Y as photosensitizer, and di-thiol containing molecule as dual purpose co-initiator/cross-linker. N-vinylpyrrolidone (NVP) was used to accelerate gelation kinetics and to adjust the stiffness of the hydrogels. Visible light (wavelength: 400–700nm) was used to initiate rapid gelation (gel points: ~20 seconds) that reached completion within a few minutes. The major differences between current thiol-vinyl gelation and prior visible light mediated photopolymerization are that: (1) the co-initiator triethanolamine (TEOA) used in the previous systems was replaced with multifunctional thiols and (2) mixed-mode polymerized gels contain less network heterogeneity. The gelation kinetics and gel properties at the same PEG macromer concentration could be tuned by changing the identity of vinyl groups and di-thiol cross-linkers, as well as concentration of cross-linker and NVP. Specifically, acrylate-modified PEG afforded the fastest gelation rate, followed by acrylamide and methacrylate-functionalized PEG. Increasing NVP concentration also accelerated gelation and led to a higher network cross-linking density. Further, increasing di-thiol peptide concentration in the gel formulation increased hydrogel swelling and decreased gel stiffness. Due to the formation of thiol-ether-ester bonds following thiol-acrylate reaction, the gels degraded hydrolytically following a pseudo first order degradation kinetics. Degradation rate was controlled by adjusting thiol or NVP content in the polymer precursor solution. The cytocompatibility and utility of this hydrogel system were evaluated using in situ encapsulation of human mesenchymal stem cells (hMSC). Encapsulated hMSCs remained alive (>90%) throughout the duration of the study and the cells were differentiated down osteogenic lineage with varying degrees by controlling the rate and mode of gel degradation.
doi:10.1016/j.actbio.2013.08.044
PMCID: PMC3840055  PMID: 24021231
Visible light; photopolymerization; thiol-ene; hydrogels; mesenchymal stem cells
8.  Crosslinking and degradation of step-growth hydrogels formed by thiol-ene photo-click chemistry 
Biomacromolecules  2012;13(7):2003-2012.
Thiol-ene photo-click hydrogels have been used for a variety of tissue engineering and controlled release applications. In this step-growth photopolymerization scheme, multi-arm poly(ethylene glycol) norbornene (PEG4NB) was crosslinked with di-thiol containing crosslinkers to form chemically crosslinked hydrogels. While the mechanism of thiol-ene gelation was well described in the literature, its network ideality and degradation behaviors are not well-characterized. Here, we compared the network crosslinking of thiol-ene hydrogels to Michael-type addition hydrogels and found thiol-ene hydrogels formed with faster gel points and higher degree of crosslinking. However, thiol-ene hydrogels still contained significant network non-ideality, demonstrated by a high dependency of hydrogel swelling on macromer contents. In addition, the presence of ester bonds within the PEG-norbornene macromer rendered thiol-ene hydrogels hydrolytically degradable. Through validating model predictions with experimental results, we found that the hydrolytic degradation of thiol-ene hydrogels was not only governed by ester bond hydrolysis, but also affected by the degree of network crosslinking. In an attempt to manipulate network crosslinking and degradation of thiol-ene hydrogels, we incorporated peptide crosslinkers with different sequences and characterized the hydrolytic degradation of these PEG-peptide hydrogels. In addition, we incorporated a chymotrypsin-sensitive peptide as part of the crosslinkers to tune the mode of gel degradation from bulk degradation to surface erosion.
doi:10.1021/bm300752j
PMCID: PMC4016800  PMID: 22708824
Thiol-ene chemistry; hydrogel; photopolymerization; degradation
9.  Mechanical and Cell Viability Properties of Crosslinked Low and High Molecular Weight Poly(ethylene glycol) Diacrylate Blends 
There is a strong need for tissue engineering scaffolds that are mechanically robust, exhibit good biocompatibility, and can be made from readily available materials. To this end, blends of commercially available poly(ethylene glycol) diacrylate (PEGDA) with molecular weights of 400 and 3400 were UV-crosslinked at total polymer concentrations that varied systematically from 20 to 40 wt %. The compressive strength and cell viability were determined for each PEGDA mixture. The compressive modulus of the blends was maximized when the wt % ratio PEGDA3400/400 was about 40/60, with the compressive strength reaching 1.7 MPa. Cell viability results with a LIVE/DEAD fluorescence assay show an average viability of ~ 80% at a total PEGDA concentration of 20 wt %, for all blends. Increasing the total polymer concentration increased the compressive modulus of a polymer, but adversely affected cell viability for all the PEGDA blend compositions. The blend composition affected the mechanical behavior of the discs, where a higher degree of crosslinking was achieved by increasing the concentration of shorter chained PEGDA400, whereas elasticity was gained by incorporating longer chained PEGDA3400 into the blends. These results can be exploited for use in tissue engineering applications, where a mechanically robust scaffold is advantageous.
doi:10.1002/jbm.a.32563
PMCID: PMC2845736  PMID: 19585581
Cell encapsulation; Hydrogel; Mechanical properties; Photopolymerization; Tissue Engineering
10.  Evaluation of Physical and Mechanical Properties of Porous Poly (Ethylene Glycol)-co-(L-Lactic Acid) Hydrogels during Degradation 
PLoS ONE  2013;8(4):e60728.
Porous hydrogels of poly(ethylene glycol) (PEG) have been shown to facilitate vascularized tissue formation. However, PEG hydrogels exhibit limited degradation under physiological conditions which hinders their ultimate applicability for tissue engineering therapies. Introduction of poly(L-lactic acid) (PLLA) chains into the PEG backbone results in copolymers that exhibit degradation via hydrolysis that can be controlled, in part, by the copolymer conditions. In this study, porous, PEG-PLLA hydrogels were generated by solvent casting/particulate leaching and photopolymerization. The influence of polymer conditions on hydrogel architecture, degradation and mechanical properties was investigated. Autofluorescence exhibited by the hydrogels allowed for three-dimensional, non-destructive monitoring of hydrogel structure under fully swelled conditions. The initial pore size depended on particulate size but not polymer concentration, while degradation time was dependent on polymer concentration. Compressive modulus was a function of polymer concentration and decreased as the hydrogels degraded. Interestingly, pore size did not vary during degradation contrary to what has been observed in other polymer systems. These results provide a technique for generating porous, degradable PEG-PLLA hydrogels and insight into how the degradation, structure, and mechanical properties depend on synthesis conditions.
doi:10.1371/journal.pone.0060728
PMCID: PMC3621899  PMID: 23593296
11.  Injectable PolyHIPEs as High Porosity Bone Grafts 
Biomacromolecules  2011;12(10):3621-3628.
Polymerization of high internal phase emulsions (polyHIPEs) is a relatively new method for the production of high porosity scaffolds. The tunable architecture of these polyHIPE foams make them attractive candidates for tissue engineered bone grafts. Previously studied polyHIPE systems require either toxic diluents or high cure temperatures which prohibit their use as an injectable bone graft. In contrast, we have developed an injectable polyHIPE that cures at physiological temperatures to a rigid, high-porosity foam. First, a biodegradable macromer, propylene fumarate dimethacrylate (PFDMA), was synthesized that has appropriate viscosity and hydrophobicity for emulsification. The process of surfactant selection is detailed with particular focus on the key structural features of both polymer (log P values, hydrogen bond acceptor sites) and surfactant (HLB values, hydrogen bond donor sites) that enable stable HIPE formation. Incubation of HIPEs at 37°C was used to initiate radical crosslinking of the unsaturated double bond of the methacrylate groups to polymerize the continuous phase and lock in the emulsion geometry. The resulting polyHIPEs exhibited ~75% porosity, pore sizes ranging from 4 to 29 μm, and an average compressive modulus and strength of 33 and 5 MPa, respectively. These findings highlight the great potential of these scaffolds as injectable, tissue engineered bone grafts.
doi:10.1021/bm2008839
PMCID: PMC3190649  PMID: 21861465
12.  Bioactive Hydrogels Made from Step-Growth Derived PEG-Peptide Macromers 
Biomaterials  2010;31(13):3736-3743.
Synthetic hydrogels based on poly(ethylene glycol) (PEG) have been used as biomaterials for cell biology and tissue engineering investigations. Bioactive PEG-based gels have largely relied on heterobifunctional or multi-arm PEG precursors that can be difficult to synthesize and characterize or expensive to obtain. Here, we report an alternative strategy, which instead uses inexpensive and readily available PEG precursors to simplify reactant sourcing. This new approach provides a robust system in which to probe cellular interactions with the microenvironment. We used the step-growth polymerization of PEG diacrylate (PEGDA, 3400 Da) with bis-cysteine matrix metalloproteinase (MMP)-sensitive peptides via Michael-type addition to form biodegradable photoactive macromers of the form acrylate-PEG-(peptide-PEG)m-acrylate. The molecular weight (MW) of these macromers is controlled by the stoichiometry of the reaction, with a high proportion of resultant macromer species greater than 500 kDa. In addition, the polydispersity of these materials was nearly identical for three different MMP-sensitive peptide sequences subjected to the same reaction conditions. When photopolymerized into hydrogels, these high MW materials exhibit increased swelling and sensitivity to collagenase-mediated degradation as compared to previously published PEG hydrogel systems. Cell-adhesive acrylate-PEG-CGRGDS was synthesized similarly and its immobilization and stability in solid hydrogels was characterized with a modified Lowry assay. To illustrate the functional utility of this approach in a biological setting, we applied this system to develop materials that promote angiogenesis in an ex vivo aortic arch explant assay. We demonstrate the formation and invasion of new sprouts mediated by endothelial cells into the hydrogels from embedded embryonic chick aortic arches. Furthermore, we show that this capillary sprouting and three-dimensional migration of endothelial cells can be tuned by engineering the MMP-susceptibility of the hydrogels and the presence of functional immobilized adhesive ligands (CGRGDS vs. CGRGES peptide). The facile chemistry described and significant cellular responses observed suggest the usefulness of these materials in a variety of in vitro and ex vivo biologic investigations, and may aid in the design or refinement of material systems for a range of tissue engineering approaches.
doi:10.1016/j.biomaterials.2010.01.058
PMCID: PMC2837100  PMID: 20138664
13.  Morphology of Photopolymerized End-linked Poly(ethylene glycol) Hydrogels by Small Angle X-ray Scattering 
Macromolecules  2010;43(16):6861-6870.
Due to the biocompatibility of poly(ethylene glycol) (PEG), PEG-based hydrogels have attracted considerable interest for use as biomaterials in tissue engineering applications. In this work, we show that PEG-based hydrogels prepared by photopolymerization of PEG macromonomers functionalized with either acrylate or acrylamide end-groups generate networks with crosslink junctions of high functionality. Although the crosslink functionality is not well controlled, the resultant networks are sufficiently well ordered to generate a distinct correlation peak in the small angle x-ray scattering (SAXS) related to the distance between crosslink junctions within the PEG network. The crosslink spacing is a useful probe of the PEG chain conformation within the hydrogel and ranges from approximately 6 to 16 nm, dependent upon both the volume fraction of polymer and the molecular weight of the PEG macromonomers. The presence of a peak in the scattering of photopolymerized PEG networks is also correlated with an enhanced compressive modulus in comparison to PEG networks reported in the literature with much lower crosslink functionality that exhibit no scattering peak. This comparison demonstrates that the method used to link together PEG macromonomers has a critical impact on both the nanoscale structure and the macroscopic properties of the resultant hydrogel network.
doi:10.1021/ma101070s
PMCID: PMC3055273  PMID: 21403767
Poly(ethylene glycol); hydrogel; small angle x-ray scattering
14.  Poly(ε-Caprolactone)-Based Copolymers Bearing Pendant Cyclic Ketals and Reactive Acrylates for the Fabrication of Photocrosslinked Elastomers 
Acta biomaterialia  2013;9(9):8232-8244.
Block copolymers of poly(ethylene glycol) (PEG) and poly(ε-caprolactone) (PCL) with chemically addressable functional groups were synthesized and characterized. Ring opening polymerization of ε-caprolactone (CL) and 1,4,8-trioxaspiro-[4,6]-9-undecanone (TSU) using α-methoxy, ω-hydroxyl poly(ethylene glycol) (mPEG) as the initiator afforded a copolymer with cyclic ketals being randomly distributed in the hydrophobic PCL block. At an initiator/catalyst molar ratio of 10/1 and a TSU/CL weight ratio of 1/4, a ketal-carrying copolymer (ECT2-CK) with Mn of 52 kDa and a ketal content of 15 mol% was obtained. Quantitative side chain deacetalization revealed the reactive ketones without noticeable polymer degradation. In our study, 10 mol% of cyclic ketals were deprotected and the ketone-containing copolymer was designated as ECT2-CO. Reaction of ECT2-CO with 2-(2-(aminooxy)acetoxy)-ethyl acrylate gave rise to an acrylated product (ECT2-AC) containing an estimated 3–5 acrylate groups per chain. UV-initiated radical polymerization of ECT2-AC in dichloromethane resulted in a crosslinked network (xECT2-AC). Thermal and morphological analyses employing Differential Scanning Calorimetry (DSC) and Atomic Force Microscopy (AFM) operated in PeakForce Tapping mode revealed the semicrystalline nature of the network, containing stiff crystalline lamellae dispersed in a softer amorphous interstitial. Macroscopic and nanoscale mechanical characterizations showed that ECT2-CK exhibited a significantly lower modulus than PCL of a similar molecular weight. While ECT2-CK undergoes a plastic deformation with a distinct yield point and a cold drawing region, xECT2-AC exhibited a compliant, elastomeric deformation with a Young’s modulus of 0.5 ± 0.1 MPa at 37 °C. When properly processed, the crosslinked network exhibited shape memory behaviors, with shape fixity and shape recovery values close to 1 and a shape recovery time of less than 4 s at 37 °C. In vitro studies showed that xECT2-AC films did not induce any cytotoxic effects to the cultured mesenchymal stem cells. The crosslinkable polyester copolymers can be potentially used as tissue engineering scaffolds and minimally invasive medical devices.
doi:10.1016/j.actbio.2013.06.005
PMCID: PMC3732508  PMID: 23770222
Poly(ε-caprolactone); Poly(ethylene glycol); Copolymers; Functional Groups; Photocrosslinking; Elastomeric; Shape Memory; Tissue Engineering
15.  Helix versus coil polypeptide macromers: gel networks with decoupled stiffness and permeability 
Soft matter  2012;42(8):10887-10895.
As a platform for investigating the individual effects of substrate stiffness, permeability, and ligand density on cellular behavior, we developed a set of hydrogels with stiffness tuned by polymer backbone rigidity, independent of cross-link density and concentration. Previous studies report that poly(propargyl-L-glutamate) (PPLG), synthesized by ring-opening polymerization of the N-carboxy anhydride of γ-propargyl-L-glutamate (γpLglu), adopts a rigid a-helix conformation: we hypothesized that a random copolymer (PPDLG) with equal amounts of γpLglu and γ-propargyl-D-glutamate (γpDglu) monomers would exhibit a more flexible random coil conformation. The resulting macromers exhibited narrow molecular weight distributions (PDI = 1.15) and were grafted with ethylene glycol groups using a highly efficient “click” azide/alkyne cycloaddition reaction with average grafting efficiency of 97% for PPLG and 85% for PPDLG. The polypeptide secondary structure, characterized via circular dichroism spectroscopy, FTIR spectroscopy, and dynamic light scattering, is indeed dependent upon monomer chirality: PPLG exhibits an α-helix conformation while PPDLG adopts a random coil conformation. Hydrogel networks produced by cross-linking either helical or random coil polypeptides with poly(ethylene glycol) (PEG) were analyzed for amount of swelling, gelation efficiency, and permeability to a model protein. In addition, the elastic modulus of helical and coil polypeptide gels was determined by AFM indentation in fluid. Importantly, we found that helical and coil polypeptide gels exhibited similar swelling and permeability but different stiffnesses, which correspond to predictions from the theory of semi-flexible chains.
doi:10.1039/C2SM26487K
PMCID: PMC3932710  PMID: 24575148
16.  Impact of Degradable Macromer Content in a Poly(Ethylene Glycol) Hydrogel on Neural Cell Metabolic Activity, Redox State, Proliferation, and Differentiation 
Tissue Engineering. Part A  2010;16(6):1857-1866.
Hydrogels that degrade at different rates were prepared by copolymerizing slowly degrading macromer poly(ethylene glycol) (PEG) dimethacrylate with a faster degrading macromer poly(lactic acid)-b-PEG-b-poly(lactic acid) dimethacrylate. A clinically relevant population of neural cells composed of differentiated neurons and multipotent precursor cells was cultured within hydrogels. Within 2 h after encapsulation, metabolic activity was higher in hydrogels prepared with increasing levels of degradable content. This improvement was accompanied by a reduction in intracellular redox state and an increase in the fraction of glutathione in the reduced state, both of which persisted throughout 7 days of culture and which may be the result of radical scavenging by lactic acid. Importantly, an increase in cellular proliferation was observed in gels prepared with increasing degradable macromer content after 7 days of growth without a shift in the cellular composition of the culture toward the glial cell phenotype. The findings of this study provide additional insight into the growth of neural cells in PEG-based hydrogels. Results suggest that lactic acid released during gel degradation may impact the function of encapsulated cells, a finding of general interest to biomaterials scientists who focus on the development of degradable polymers for cell culture and drug delivery devices.
doi:10.1089/ten.tea.2009.0509
PMCID: PMC2949233  PMID: 20067398
17.  Continuous gradient scaffolds for rapid screening of cell-material interactions and interfacial tissue regeneration 
Acta biomaterialia  2013;9(9):8254-8261.
In tissue engineering, the physical and chemical properties of the scaffold mediates cell behavior including regeneration. Thus, a strategy that permits rapid screening of cell-scaffold interactions is critical. Herein, we have prepared eight “hybrid” hydrogel scaffolds in the form of continuous gradients such that a single scaffold contains spatially varied properties. These scaffolds are based on combining an inorganic macromer [methacrylated star polydimethylsiloxane, PDMSstar-MA] and organic macromer [poly(ethylene glycol)diacrylate, PEG-DA] as well both aqueous and organic fabrication solvents. Having previously demonstrated its bioactivity and osteoinductivity, PDMSstar-MA is a particularly powerful component to incorporate into instructive gradient scaffolds based on PEG-DA. The following parameters were varied to produce the different gradients or gradual transitions in: (1) the wt% ratio of PDMSstar-MA to PEG-DA macromers, (2) the total wt% macromer concentration, (3) the number average molecular weight (Mn) of PEG-DA and (4) the Mn of PDMSstar-MA. Upon dividing each scaffold into four “zones” perpendicular to the gradient, we were able to demonstrate the spatial variation in morphology, bioactivity, swelling and modulus. Among these gradient scaffolds are those in which swelling and modulus are conveniently decoupled. In addition to rapid screening of cell-material interactions, these scaffolds are well-suited for regeneration of interfacial tissues (e.g. osteochondral tissues) that transition from one tissue type to another.
doi:10.1016/j.actbio.2013.05.012
PMCID: PMC3732554  PMID: 23707502
poly(ethylene glycol); polydimethylsiloxane; hydrogel; scaffold; gradient
18.  Cartilage-like mechanical properties of poly (ethylene glycol)-diacrylate hydrogels 
Biomaterials  2012;33(28):6682-6690.
Hydrogels prepared from poly-(ethylene glycol) (PEG) have been used in a variety of studies of cartilage tissue engineering. Such hydrogels may also be useful as a tunable mechanical material for cartilage repair. Previous studies have characterized the chemical and mechanical properties of PEG-based hydrogels, as modulated by precursor molecular weight and concentration. Cartilage mechanical properties vary substantially, with maturation, with depth from the articular surface, in health and disease, and in compression and tension. We hypothesized that PEG hydrogels could mimic a broad range of the compressive and tensile mechanical properties of articular cartilage. The objective of this study was to characterize the mechanical properties of PEG hydrogels over a broad range and with reference to articular cartilage. In particular, we assessed the effects of PEG precursor molecular weight (508 Da, 3.4 kDa, 6 kDa, and 10 kDa) and concentration (10–40%) on swelling property, equilibrium confined compressive modulus (HA0), compressive dynamic stiffness, and hydraulic permeability (kp0) of PEG hydrogels in static/dynamic confined compression tests, and equilibrium tensile modulus (Eten) in tension tests. As molecular weight of PEG decreased and concentration increased, hydrogels exhibited a decrease in swelling ratio (31.5–2.2), an increase in HA0 (0.01–2.46 MPa) and Eten (0.02–3.5 MPa), an increase in dynamic compressive stiffness (0.055–42.9 MPa), and a decrease in kp0 (1.2 × 10−15 to 8.5 × 10−15 m2/(Pa s)). The frequency-dependence of dynamic compressive stiffness amplitude and phase, as well as the strain-dependence of permeability, were typical of the time- and strain-dependent mechanical behavior of articular cartilage. HA0 and Eten were positively correlated with the final PEG concentration, accounting for swelling. These results indicate that PEG hydrogels can be prepared to mimic many of the static and dynamic mechanical properties of articular cartilage.
doi:10.1016/j.biomaterials.2012.06.005
PMCID: PMC3572364  PMID: 22749448
PEG; Biomechanics; Crosslink; Compression; Tension; Modulus
19.  Synthesis of photodegradable hydrogels as dynamically tunable cell culture platforms 
Nature protocols  2010;5(12):1867-1887.
We describe a detailed procedure to create photolabile, poly(ethylene glycol)-based (PEG) hydrogels and manipulate material properties in situ. The cytocompatible chemistry and degradation process enable dynamic, tunable changes for applications in 2D or 3D cell culture. The materials are created by synthesizing an o-nitrobenzylether-based photodegradable monomer that can be coupled to primary amines. Here, we provide coupling procedures to PEG-bis-amine to form a photodegradable crosslinker or to the fibronectin-derived peptide RGDS to form a photoreleasable tether. Hydrogels are synthesized with the photodegradable crosslinker in the presence or absence of cells, allowing direct encapsulation or seeding on surfaces. Cell-material interactions can be probed in 2D or 3D by spatiotemporally controlling the gel microenvironment, which allows unique experiments to be performed to monitor cell response to changes in their niche. Degradation is readily achieved with cytocompatible wavelengths of low intensity flood irradiation (365 to 420 nm) in minutes or with highintensity laser irradiation (405 nm) in seconds. In this protocol, synthesis and purification of the photodegradable monomers take approximately 2 weeks, but can be substantially shortened by purchasing the o-nitrobenzylether precursor. Preparation of the sterile solutions for hydrogel fabrication takes hours, while the reaction to form the final hydrogel is complete in minutes. Hydrogel degradation occurs on-demand, in seconds to minutes, with user-directed light exposure. This comprehensive protocol is useful for controlling peptide presentation and substrate modulus during cell culture on or within an elastic matrix. These PEG-based materials are useful for probing the dynamic influence of cell-cell and cell-material interactions on cell function in 2D or 3D. While other protocols are available for controlling peptide presentation or modulus, few allow manipulation of material properties in situ and in the presence of cells down to the micrometer scale.
doi:10.1038/nprot.2010.139
PMCID: PMC3897936  PMID: 21127482
cell culture; tissue engineering; responsive materials; hydrogels; biomaterials; photolabile; poly(ethylene glycol); RGDS; mesenchymal stem cells
20.  Synthesis and characterization of a biodegradable elastomer featuring a dual crosslinking mechanism 
Soft matter  2010;6(11):2449-2461.
The need for advanced materials in emerging technologies such as tissue engineering has prompted increased research to produce novel biodegradable polymers elastic in nature and mechanically compliant with the host tissue. We have developed a soft biodegradable elastomeric platform biomaterial created from citric acid, maleic anhydride, and 1,8-octanediol, poly(octamethylene maleate (anhydride) citrate) (POMaC), which is able to closely mimic the mechanical properties of a wide range of soft biological tissues. POMaC features a dual crosslinking mechanism, which allows for the option of the crosslinking POMaC using UV irradiation and/or polycondensation to fit the needs of the intended application. The material properties, degradation profiles, and functionalities of POMaC thermoset networks can all be tuned through the monomer ratios and the dual crosslinking mechanism. POMaC polymers displayed an initial modulus between 0.03 and 1.54 MPa, and elongation at break between 48% and 534% strain. In vitro and in vivo evaluation using cell culture and subcutaneous implantation, respectively, confirmed cell and tissue biocompatibility. POMaC biodegradable polymers can also be combined with MEMS technology to fabricate soft and elastic 3D microchanneled scaffolds for tissue engineering applications. The introduction of POMaC will expand the choices of available biodegradable polymeric elastomers. The dual crosslinking mechanism for biodegradable elastomer design should contribute to biomaterials science.
doi:10.1039/C001605E
PMCID: PMC3233194  PMID: 22162975
21.  Tuning the Properties of Elastin Mimetic Hybrid Copolymers via a Modular Polymerization Method 
Biomacromolecules  2012;13(6):1774-1786.
We have synthesized elastin mimetic hybrid polymers (EMHPs) via the step-growth polymerization of azide-functionalized poly(ethylene glycol) (PEG) and alkyne-terminated peptide (AKAAAKA)2 (AK2) that is abundant in the crosslinking domains of the natural elastin. 1 The modular nature of our synthesis allows facile adjustment of the peptide sequence to modulate the structural and biological properties of EMHPs. Thus, EMHPs containing cell-binding domains (CBD) were constructed from α,ω-azido-PEG and two types of alkyne-terminated AK2 peptides with sequences of DGRGX(AKAAAKA)2X (AK2-CBD1) and X(AKAAAKA)2XGGRGDSPG (AK2-CBD2, X = propargylglycine) via a step-growth, click coupling reaction. The resultant hybrid copolymers contain an estimated five to seven repeats of PEG and AK2 peptides. The secondary structure of EMHPs is sensitive to the specific sequence of the peptidic building blocks, with CBD-containing EMHPs exhibiting a significant enhancement in the α-helical content as compared to the peptide alone. Elastomeric hydrogels formed by covalent crosslinking of the EMHPs had a compressive modulus of 1.06 ± 0.1MPa. Neonatal human dermal fibroblasts (NHDFs) were able to adhere to the hydrogels within 1 h, and to spread and develop F-actin filaments 24 h post-seeding. NHDF proliferation was only observed on hydrogels containing RGDSP domains, demonstrating the importance of integrin engagement for cell growth and the potential use of these EMHPs as tissue engineering scaffolds. These cell-instructive, hybrid polymers are promising candidates as elastomeric scaffolds for tissue engineering.
doi:10.1021/bm3002705
PMCID: PMC3372701  PMID: 22533503
22.  pHEMA-nHA Encapsulation and Delivery of Vancomycin and rhBMP-2 Enhances its Role as a Bone Graft Substitute 
Background
Bone grafts are widely used in orthopaedic procedures. Autografts are limited by donor site morbidity while allografts are known for considerable infection and failure rates. A synthetic composite bone graft substitute poly(2-hydroxyethyl methacrylate)-nanocrystalline hydroxyapatite (pHEMA-nHA) was previously developed to stably press-fit in and functionally repair critical-sized rat femoral segmental defects when it was preabsorbed with a single low dose of 300 ng recombinant human bone morphogenetic protein-2/7 (rhBMP-2/7).
Questions/purposes
To facilitate clinical translation of pHEMA-nHA as a synthetic structural bone graft substitute, we examined its ability to encapsulate and release rhBMP-2 and the antibiotic vancomycin.
Methods
We analyzed the compressive behavior and microstructure of pHEMA-nHA as a function of vancomycin incorporation doses using a dynamic mechanical analyzer and a scanning electron microscope. In vitro release of vancomycin was monitored by ultraviolet-visible spectroscopy. Release of rhBMP-2 from pHEMA-nHA-vancomycin was determined by ELISA. Bioactivity of the released vancomycin and rhBMP-2 was examined by bacterial inhibition and osteogenic transdifferentiation capabilities in cell culture, respectively.
Results
Up to 4.8 wt% of vancomycin was incorporated into pHEMA-nHA without compromising its structural integrity and compressive modulus. Encapsulated vancomycin was released in a dose-dependent and sustained manner in phosphate-buffered saline over 2 weeks, and the released vancomycin inhibited Escherichia coli culture. The pHEMA-nHA-vancomycin composite released preabsorbed rhBMP-2 in a sustained manner over 8 days and locally induced osteogenic transdifferentiation of C2C12 cells in culture.
Conclusions
pHEMA-nHA can encapsulate and deliver vancomycin and rhBMP-2 in a sustained and localized manner with reduced loading doses.
Clinical Relevance
The elasticity, osteoconductivity, and rhBMP-2/vancomycin delivery characteristics of pHEMA-nHA may benefit orthopaedic reconstructions or fusions with enhanced safety and efficiency and reduced infection risk.
doi:10.1007/s11999-012-2644-5
PMCID: PMC3705044  PMID: 23070662
23.  Effect of Swelling Ratio of Injectable Hydrogel Composites on Chondrogenic Differentiation of Encapsulated Rabbit Marrow Mesenchymal Stem Cells In Vitro 
Biomacromolecules  2009;10(3):541-546.
An injectable, biodegradable hydrogel composite of oligo(poly(ethylene glycol) fumarate) (OPF) and gelatin microparticles (MPs) has been investigated as a cell and growth factor carrier for cartilage tissue engineering applications. In this study, hydrogel composites with different swelling ratios were prepared by crosslinking OPF macromers with poly(ethylene glycol) (PEG) repeating units of varying molecular weights from 1,000 ~ 35,000. Rabbit marrow mesenchymal stem cells (MSCs) and MPs loaded with transforming growth factor-β1 (TGF-β1) were encapsulated in the hydrogel composites in order to examine the effect of the swelling ratio of the hydrogel composites on the chondrogenic differentiation of encapsulated rabbit marrow MSCs both in the presence and absence of TGF-β1. The swelling ratio of the hydrogel composites increased as the PEG molecular weight in the OPF macromers increased. Chondrocyte-specific genes were expressed at higher levels in groups containing TGF-β1-loaded MPs and varied with the swelling ratio of the hydrogel composites. OPF hydrogel composites with PEG repeating units of molecular weight 35,000 and 10,000 with TGF-β1-loaded MPs exhibited a 159 ± 95 and a 89 ± 31 fold increase in type II collagen gene expression at day 28, respectively, while OPF hydrogel composites with PEG repeating units of molecular weight 3,000 and 1,000 with TGF-β1-loaded MPs showed a 27 ± 10 and a 17 ± 7 fold increase in type II collagen gene expression, respectively, as compared to the composites with blank MPs at day 0. The results indicate that chondrogenic differentiation of encapsulated rabbit marrow MSCs within OPF hydrogel composites could be affected by their swelling ratio, thus suggesting the potential of OPF composite hydrogels as part of a novel strategy for controlling the differentiation of stem cells.
doi:10.1021/bm801197m
PMCID: PMC2765566  PMID: 19173557
injectable hydrogels; crosslinking; marrow mesenchymal stem cells; gelatin microparticles; TGF-β1; chondrogenic differentiation; cartilage tissue engineering
24.  FGF-1 and proteolytically-mediated cleavage site presentation influence 3D fibroblast invasion in biomimetic PEGDA hydrogels 
Acta Biomaterialia  2012;8(6):2213-2222.
Controlled scaffold degradation is a critical design criterion for the clinical success of tissue engineered constructs. Here, we exploited a biomimetic poly(ethylene glycol) diacrylate (PEGDA) hydrogel system immobilized with tethered YRGDS as the cell adhesion ligand and with either single (SSite) or multiple (MSite) collagenase-sensitive domains between crosslinks, to systematically study the effect of proteolytic cleavage site presentation on hydrogel degradation rate and 3D fibroblast invasion in vitro. Through the incorporation of multiple collagenase-sensitive domains between crosslinks, hydrogel degradation rate was controlled and enhanced independent of alterations in compressive modulus. As compared to SSite hydrogels, MSite hydrogels resulted in increased 3D fibroblast invasion in vitro which occurred over a wider range of compressive modulus. Furthermore, encapsulated soluble acidic fibroblast growth factor (FGF-1), a potent mitogen during processes such as vascularization and wound healing, was incorporated into SSite and MSite PEGDA scaffolds to determine its in vitro potential on fibroblast cell invasion. Hydrogels containing soluble FGF-1 significantly enhanced 3D fibroblast invasion in a dose-dependent manner within the different types of PEG matrices investigated over a period of 15 days. The methodology presented provides flexibility in designing PEG scaffolds with desired mechanical properties, but with increased susceptibility to proteolytically-mediated degradation. These results indicate that effective tuning of initial matrix stiffness and hydrogel degradation kinetics plays a critical role in effectively designing PEG scaffolds that promote controlled 3D cellular behavior and in situ tissue regeneration.
doi:10.1016/j.actbio.2012.03.017
PMCID: PMC3348386  PMID: 22426138
Biodegradation; Biomimetic Material; Free-radical; Cross-linking; Fibroblast growth factor; fibroblasts
25.  Synthesis and Characterization of Elastin-Mimetic Hybrid Polymers with Multiblock, Alternating Molecular Architecture and Elastomeric Properties 
Macromolecules  2009;42(7):2532-2541.
We are interested in developing elastin–mimetic hybrid polymers (EMHPs) that capture the multiblock molecular architecture of tropoelastin as well as the remarkable elasticity of mature elastin. In this study, multiblock EMHPs containing flexible synthetic segments based on poly(ethylene glycol) (PEG) alternating with alanine-rich, lysine-containing peptides were synthesized by step-growth polymerization using α,ω-azido-PEG and alkyne-terminated AKA3KA (K = lysine, A = alanine) peptide, employing orthogonal click chemistry. The resulting EMHPs contain an estimated three to five repeats of PEG and AKA3KA and have an average molecular weight of 34 kDa. While the peptide alone exhibited α-helical structures at high pH, the fractional helicity for EMHPs was reduced. Covalent cross-linking of EMHPs with hexamethylene diisocyanate (HMDI) through the lysine residue in the peptide domain afforded an elastomeric hydrogel (xEMHP) with a compressive modulus of 0.12 MPa when hydrated. The mechanical properties of xEMHP are comparable to a commercial polyurethane elastomer (Tecoflex SG80A) under the same conditions. In vitro toxicity studies showed that while the soluble EMHPs inhibited the growth of primary porcine vocal fold fibroblasts (PVFFs) at concentrations ≥0.2 mg/mL, the cross-linked hybrid elastomers did not leach out any toxic reagents and allowed PVFFs to grow and proliferate normally. The hybrid and modular approach provides a new strategy for developing elastomeric scaffolds for tissue engineering.
doi:10.1021/ma802791z
PMCID: PMC2743465  PMID: 19763157

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