Brent L. Vernon

Thermoreversible copolymer gels for extracellular matrix4

To improve the properties of a reversible syn- thetic extracellular matrix based on a thermally reversible polymer, copolymers of N-isopropylacrylamide and acrylic acid were prepared in benzene with varying contents of acrylic acid (0 to 3%) and the thermal properties were evalu- ated. The poly(N-isopropylacrylamide) and copolymers made with acrylic acid had molecular weights from 0.8 to 1.7 ◊10 6 D. Differential scanning calorimetry (DSC) showed the high-molecular-weight acrylic acid copolymers had similar onset temperatures to the homopolymers, but the peak width was considerably increased with increasing acrylic acid content. DSC and cloud point measurements showed that polymers with 0 to 3% acrylic acid exhibit a lower criti- cal solution temperature (LCST) transition between 30° and 37°C. In swelling studies, the homopolymer showed signifi- cant syneresis at temperatures above 31°C. Copolymers with 1 and 1.5% showed syneresis beginning at 32° and 37°C, respectively. At 37°C the copolymers with 1.5-3% acrylic acid showed little or no syneresis. Due to the high water content and a transition near physiologic conditions (below 37°C), the polymers with 1.5-2.0% acrylic acid exhibited properties that would be useful in the development of a refillable synthetic extracellular matrix. Such a matrix could be applied to several cell types, including islets of Langer- hans, for a biohybrid artificial pancreas.

Corticosterone modulation of reproductive and immune systems trade-offs in female tree lizards: long-term corticosterone manipulations via injectable gelling material.

SUMMARY Physiological trade-offs arise because multiple processes compete for the same limiting resources. While competition for resources has been demonstrated between reproduction and immune function, the regulation of this competition remains unclear. Corticosterone (CORT) is a likely mediator due to its dual role in mobilizing energy stores throughout the body and regulating physiological responses to stressors. We manipulated CORT concentrations and resources in pre-reproductive and reproductive female tree lizards (Urosaurus ornatus) to test the hypothesis that CORT regulates the distribution of limiting resources between the reproductive and immune systems. To manipulate circulating concentrations of CORT we utilized a novel method of hormone implantation, in which a polymeric compound is mixed with hormone and injected in liquid form into the animal. After injection, the liquid quickly gels in situ forming a slow release hormone implant. This method of hormone delivery eliminated the need for substantial wounds to the animal or repeated handling required by other methods. In this study, the hormone-treated animals had plasma CORT concentrations comparable to high physiological concentrations. We found that CORT treatment suppressed immune function, but only when animals were energetically compromised. We assessed immune function by measuring the healing rate of a cutaneous biopsy. Healing was suppressed in all CORT-treated reproductive animals and in all CORT-treated animals (pre-reproductive and reproductive) undergoing food restriction, but CORT had no effect in ad libitum non-reproductive females. The context-dependent action of CORT renders its response adjustable to changing environmental conditions and may allow for the suppression of specific functions depending on resource availability.

Extracellular matrix for a rechargeable cell delivery system

Above a critical concentration, aqueous polymer solutions of N-isopropylacrylamide copolymers with small amounts of acrylic acid, synthesized in benzene by radical polymerization, exhibited four distinct phases as the temperature increased; clear solution, opaque solution, gel and shrunken gel. The transition between the opaque solution phase and the gel phase was in the range of 30-34 degrees C and was reversible without syneresis and noticeable hysteresis under the experimental conditions used in this study. Islets of Langerhans, isolated from Sprague-Dawley rat pancreata and entrapped in the gel matrix, remained viable, with no significant decrease in insulin secretion function in vitro for one month. When islets were encapsulated with the gel matrix in hollow fibers [molecular weight cut-off (MWCO)= approximately 400000] and were exposed to dynamic changes in glucose and theophylline concentrations, their insulin secretion patterns demonstrated a smaller lag time and higher amplitude in insulin release than islets entrapped in a conventional alginate matrix under the same experimental conditions. From these two observations, i.e. gel reversibility and islet functionality in the matrix observed in in vitro experiments, the N-isopropylacrylamide copolymers with acrylic acid synthesized in this study are optimum candidates for the extracellular matrix in a diffusion chamber-type cell delivery system in order to recharge the entrapped cells when cell functionality in the system decreases.

Degradation, cytotoxicity, and biocompatibility of NIPAAm-based thermosensitive, injectable, and bioresorbable polymer hydrogels

A thermosensitive, injectable, and bioresorbable polymer hydrogel, poly(N-isopropylacrylamide-co-dimethyl-γ-butyrolactone acrylate-co-acrylic acid) [poly(NDBA)], was synthesized by radical copolymerization with 7.00 mol % dimethyl-γ-butyrolactone acrylate in tetrahydrofuran. The chemical composition was determined by acid titration in conjunction with (1) H NMR quantification. The molecular weight and polydispersity were determined by gel permeation chromatography in conjunction with static light scattering. The degradation properties of the polymer hydrogel were characterized using differential scanning calorimetry, percentage mass loss, cloud point test, and swelling ratio over time. It was found that the initial lower critical solution temperature (LCST) of the polymer is between room temperature and body temperature and that it takes about 2 weeks for the LCST to surpass body temperature under physiological conditions. An indirect cytotoxicity test indicated that this copolymer has relatively low cytotoxicity as seen with 3T3 fibroblast cells. The in vivo-gelation and degradation study showed good agreement with in vitro-degradation findings, and no detrimental effects to adjacent tissues were observed after the complete dissolution of the polymer.

Particle size of fillers affects permeability of polymethylmethacrylate.

Particulate soluble filler added to polymethylmethacrylate increases its permeability, leading to increased elution. We asked whether particle size affects permeability and elution rate associated with a given volume fraction of filler. Permeability of filler-loaded PMMA was measured in 9 mm rods with a 32% volume fraction of four particle sizes (106 μm, 212 μm, 425 μm, 850 μm) and two filler materials (sucrose and xylitol) using a modified phenolphthalein-sodium hydroxide technique, which allowed quantitative serial observation on the same specimens. Fluid penetration was faster for larger particle sizes. The elution rate was greater for smaller particle sizes on qualitative visual assessment. Sucrose fillers were not different from xylitol fillers independent of particle size. For the volume fraction of 32%, larger particles lead to larger caliber porosity, less pore intercon nectivity, and faster fluid penetration. Smaller size particles lead to smaller caliber porosity, greater pore interconnectivity, smaller areas between the pores with no fluid penetration and greater increase in the effective surface area causing a greater elution rate.

PNIPAAm-based biohybrid injectable hydrogel for cardiac tissue engineering

UNLABELLED Injectable biomaterials offer a non-invasive approach to deliver cells into the myocardial infarct region to maintain a high level of cell retention and viability and initiate the regeneration process. However, previously developed injectable matrices often suffer from low bioactivity or poor mechanical properties. To address this need, we introduced a biohybrid temperature-responsive poly(N-isopropylacrylamide) PNIPAAm-Gelatin-based injectable hydrogel with excellent bioactivity as well as mechanical robustness for cardiac tissue engineering. A unique feature of our work was that we performed extensive in vitro biological analyses to assess the functionalities of cardiomyocytes (CMs) alone and in co-culture with cardiac fibroblasts (CFs) (2:1 ratio) within the hydrogel matrix. The synthesized hydrogel exhibited viscoelastic behavior (storage modulus: 1260 Pa) and necessary water content (75%) to properly accommodate the cardiac cells. The encapsulated cells demonstrated a high level of cell survival (90% for co-culture condition, day 7) and spreading throughout the hydrogel matrix in both culture conditions. A dense network of stained F-actin fibers (∼ 6 × 10(4) μm(2) area coverage, co-culture condition) illustrated the formation of an intact and three dimensional (3D) cell-embedded matrix. Furthermore, immunostaining and gene expression analyses revealed mature phenotypic characteristics of cardiac cells. Notably, the co-culture group exhibited superior structural organization and cell-cell coupling, as well as beating behavior (average ∼ 45 beats per min, co-culture condition, day 7). The outcome of this study is envisioned to open a new avenue for extensive in vitro characterization of injectable matrices embedded with 3D mono- and co-culture of cardiac cells prior to in vivo experiments. STATEMENT OF SIGNIFICANCE In this work, we synthesized a new class of biohybrid temperature-responsive poly(N-isopropylacrylamide) PNIPAAm-Gelatin-based injectable hydrogel with suitable bioactivity and mechanical properties for cardiac tissue engineering. A significant aspect of our work was that we performed extensive in vitro biological analyses to assess the functionality of cardiomyocytes alone and in co-culture with cardiac fibroblasts encapsulated within the 3D hydrogel matrix.

In vivo evaluation of injectable thermosensitive polymer with time-dependent LCST

The focus of this study was to examine the biocompatibility, time-dependent LCST, and bioerodable properties of a copolymer system composed of NIPAAm, dimethyl-gamma-butyrolactone (DMBL), and acrylic acid (AAc). Sprague Dawley rats were subcutaneously injected with 25 wt % solutions of poly(NIPAAm-co-DMBL-co-AAc). At predetermined times, animals were sacrificed and polymer implants were recovered for characterization via 1H-NMR. In addition, polymer-contacting tissue sections were harvested and processed for histology. The biocompatibility of the implants was assessed by counting the number of fibroblasts and leukocytes present at the tissue-implant interface. The LCST data obtained from the in vivo implants was shown to agree with that of in vitro findings. Implant mass was shown to decrease after 4 days, indicating accelerated diffusion rates with increased implant swelling, hydrolytic degradation was confirmed with 1H-NMR measurements. The cellular presence at the copolymer implant-tissue interface was shown to return to that of normal tissue 30 days postimplantation, which suggests a normal wound healing response.

Bioresponsive copolymers of poly(N-isopropylacrylamide) with enzyme-dependent lower critical solution temperatures.

Novel thermoreversible copolymers of N-isopropylacrylamide (NIPAAm) with collagenase-sensitive solubility behavior were synthesized by radical polymerization of poly(NIPAAm-co-NASI) and nucleophilic substitution of custom peptides GAPGL-NH(2) and GAPGLF-NH(2). The materials were characterized by nuclear magnetic resonance spectroscopy (NMR), gel permeation chromatography in conjunction with static light scattering, differential scanning calorimetry (DSC), and cloud point determination. Successful synthesis and specific degradation by collagenase above and below the material LCST was confirmed by NMR. The LCST behavior of the polymers was affected by collagenase. The LCST of the copolymers, as measured by cloud point determination, increased by 1 and 9 degrees C, respectively, after enzymatic degradation. DSC thermographs indicated increased polymer solubility after enzymatic degradation because of a reduced energy of gelation. These results demonstrate the significant impact of a single amino acid on the LCST behavior of thermosensitive copolymers. Furthermore, the results suggest that comonomers in similar systems could be designed to elicit phase transitions or conformation changes in response to a variety of enzymes for which the substrate structure is known.

In situ forming, resorbable graft copolymer hydrogels providing controlled drug release.

In situ forming hydrogels are promising drug delivery vehicles due to their ease of delivery as liquids and their ability to be used in sites with irregular geometries. In this work, we report on in situ forming, resorbable hydrogels based on N-isopropylacrylamide (NIPAAm) as a fluid-like controlled release gel. These gels are the first resorbable NIPAAm-based gels providing controlled release without relying on affinity between the drug and device. Therefore, these gels provide a more flexible delivery system which can be used to deliver any drug at a controlled rate. The polymers contain repeat units of NIPAAm with (R)-α-Acryloyloxy-β,β-dimethyl-γ-butyrolactone (DBLA) and varying amounts of hydrophilic Jeffamine® M-1000 acrylamide (JAAm) grafts. The graft copolymer architecture allows the water content of the hydrogels to be tuned over a wide range while keeping the initial gelation temperature below body temperature. Incorporation of JAAm in the polymers led to greater water content, faster gel degradation, and reduced burst release. Sustained release of the antimicrobial drugs cefazolin and vancomycin (over about 5 and 7 days, respectively) was observed from gels containing an intermediate amount of grafts which combined reduced phase separation with a degradation time of 40 days. The degradation byproducts of one hydrogel formulation were cytocompatible to NIH 3T3 fibroblasts at concentrations up to 2.5 wt %. This class of terpolymer hydrogels is a promising local delivery system for a wide variety of drugs, particularly for applications involving irregular geometries such as implant interfaces.

Temperature-Responsive Graft Copolymer Hydrogels for Controlled Swelling and Drug Delivery

Temperature-responsive graft copolymers of N-isopropylacrylamide and Jeffamine® M-1000 acrylamide were synthesized to provide controlled swelling without introducing degradable moieties or increasing the LCST above body temperature. Jeffamine® M-1000 caused a small LCST increase (0.24–0.27°C/wt%) and a broader sol-gel transition. Twenty wt% copolymer gels (Mw> 225 kDa) retained their initial volume after 42 days, while homopolymer gels shrank by more than 50%. Copolymer gels eluted <20% of ovalbumin over 6 days whereas homopolymer gels released >90% within 3 h. These results suggest that Jeffamine® M-1000 acrylamide is suitable for inclusion in N-isopropylacrylamide-based biomaterials to control swelling and drug release nearly independently of LCST.