Polyester elastomers for soft tissue engineering.

Polyester elastomers are soft, biodegradable and biocompatible and are commonly used in various biomedical applications, especially in tissue engineering. These synthetic polyesters can be easily fabricated using various techniques such as solvent casting, particle leaching, molding, electrospinning, 3-dimensional printing, photolithography, microablation etc. A large proportion of tissue engineering research efforts have focused on the use of allografts, decellularized animal scaffolds or other biological materials as scaffolds, but they face the major concern of triggering immunological responses from the host, on top of other issues. This review paper will introduce the recent developments in elastomeric polyesters, their synthesis and fabrication techniques, as well as their application in the biomedical field, focusing primarily on tissue engineering in ophthalmology, cardiac and vascular systems. Some of the commercial and near-commercial polyesters used in these tissue engineering fields will also be described.

New Linear and Star-Shaped Thermogelling Poly([R]-3-hydroxybutyrate) Copolymers.

The synthesis of multi-arm poly([R]-3-hydroxybutyrate) (PHB)-based triblock copolymers (poly([R]-3-hydroxybutyrate)-b-poly(N-isopropylacrylamide)-b-[[poly(methyl ether methacrylate)-g-poly(ethylene glycol)]-co-[poly(methacrylate)-g-poly(propylene glycol)]], PHB-b-PNIPAAM-b-(PPEGMEMA-co-PPPGMA), and their subsequent self-assembly into thermo-responsive hydrogels is described. Atom transfer radical polymerization (ATRP) of N-isopropylacrylamide (NIPAAM) followed by poly(ethylene glycol) methyl ether methacrylate (PEGMEMA) and poly(propylene glycol) methacrylate (PPGMA) was achieved from bromoesterified multi-arm PHB macroinitiators. The composition of the resulting copolymers was investigated by (1) H and (13) C J-MOD NMR spectroscopy as well as size-exclusion chromatography (SEC), thermogravimetric analysis (TGA), and differential scanning calorimetry (DSC). The copolymers featuring different architectures and distinct hydrophilic/hydrophobic contents were found to self-assemble into thermo-responsive gels in aqueous solution. Rheological studies indicated that the linear one-arm PHB-based copolymer tend to form a micellar solution, whereas the two- and four-arm PHB-based copolymers afforded gels with enhanced mechanical properties and solid-like behavior. These investigations are the first to correlate the gelation properties to the arm number of a PHB-based copolymer. All copolymers revealed a double thermo-responsive behavior due to the NIPAAM and PPGMA blocks, thus allowing first the copolymer self-assembly at room temperature, and then the delivery of a drug at body temperature (37 °C). The non-significant toxic response of the gels, as assessed by the cell viability of the CCD-112CoN human fibroblast cell line with different concentrations of the triblock copolymers ranging from 0.03 to 1 mg mL(-1) , suggest that these PHB-based thermo-responsive gels are promising candidate biomaterials for drug-delivery applications.

Experimental analysis of gene assembly with TopDown one-step real-time gene synthesis.

Herein we present a simple, cost-effective TopDown (TD) gene synthesis method that eliminates the interference between the polymerase chain reactions (PCR) assembly and amplification in one-step gene synthesis. The method involves two key steps: (i) design of outer primers and assembly oligonucleotide set with a melting temperature difference of >10 degrees C and (ii) utilization of annealing temperatures to selectively control the efficiencies of oligonucleotide assembly and full-length template amplification. In addition, we have combined the proposed method with real-time PCR to analyze the step-wise efficiency and the kinetics of the gene synthesis process. Gel electrophoresis results are compared with real-time fluorescence signals to investigate the effects of oligonucleotide concentration, outer primer concentration, stringency of annealing temperature, and number of PCR cycles. Analysis of the experimental results has led to insights into the gene synthesis process. We further discuss the conditions for preventing the formation of spurious DNA products. The TD real-time gene synthesis method provides a simple and efficient method for assembling fairly long DNA sequence, and aids in optimizing gene synthesis conditions. To our knowledge, this is the first report that utilizes real-time PCR for gene synthesis.

TmPrime: fast, flexible oligonucleotide design software for gene synthesis.

Herein we present TmPrime, a computer program to design oligonucleotide sets for gene assembly by both ligase chain reaction (LCR) and polymerase chain reaction (PCR). TmPrime offers much flexibility with no constraints on the gene and oligonucleotide lengths. The program divides the long input DNA sequence based on the input desired melting temperature, and dynamically optimizes the length of oligonucleotides to achieve homologous melting temperatures. The output reports the melting temperatures, oligonucleotide sequences and potential formation of secondary structures. Our program also provides functions on sequence pooling to separate long genes into smaller pieces for multi-pool assembly and codon optimization for expression. The software has been successfully used in the design and synthesis of green fluorescent protein fragment (GFPuv) (760 bp), human protein kinase B-2 (PKB2) (1446 bp) and the promoter of human calcium-binding protein A4 (S100A4) (752 bp) using real-time PCR assembly with LCGreen I, which offers a novel approach to compare the efficiency of gene synthesis. The purity of assembled products is successfully estimated with the use of melting curve analysis, which would potentially eliminate the necessity for agarose gel electrophoresis. This program is freely available at http://prime.ibn.a-star.edu.sg.

Integrated two-step gene synthesis in a microfluidic device.

Herein we present an integrated microfluidic device capable of performing two-step gene synthesis to assemble a pool of oligonucleotides into genes with the desired coding sequence. The device comprised of two polymerase chain reactions (PCRs), temperature-controlled hydrogel valves, electromagnetic micromixer, shuttle micromixer, volume meters, and magnetic beads based solid-phase PCR purification, fabricated using a fast prototyping method without lithography process. The fabricated device is combined with a miniaturized thermal cycler to perform gene synthesis. Oligonucleotides were first assembled into genes by polymerase chain assembly (PCA), and the full-length gene was amplified by a second PCR. The synthesized gene was further separated from the PCR reaction mixture by the solid-phase PCR purification. We have successfully used this device to synthesize a green fluorescent protein fragment (GFPuv) (760 bp), and obtained comparable synthesis yield and error rate with experiments conducted in a PCR tube within a commercial thermal cycler. The resulting error rate determined by DNA sequencing was 1 per 250 bp. To our knowledge, this is the first microfluidic device demonstrating integrated two-step gene synthesis.

Electrospinning of poly(glycerol sebacate)-based nanofibers for nerve tissue engineering.

Nerve tissue engineering (TE) requires biomimetic scaffolds providing essential chemical and topographical cues for nerve regeneration. Poly(glycerol sebacate) (PGS) is a biodegradable and elastic polymer that has gained great interest as a TE scaffolding biomaterial. However, uncured PGS is difficult to be electrospun into nanofibers. PGS would, therefore, require the addition of electrospinning agents. In this study, we modified PGS by using atom transfer radical polymerization (ATRP) to synthesize PGS-based copolymers with methyl methacrylate (MMA). The synthesized PGS-PMMA copolymer showed a molecular weight of 82kDa and a glass transition temperature of 115°C. More importantly, the PGS-PMMA could be easily electrospun into nanofiber with a fiber diameter of 167±33nm. Blending gelatin into PGS-PMMA nanofibers was found to increase its hydrophilicity and biocompatibility. Rat PC12 cells were seeded onto the PGS-PMMA/gelatin nanofibers to investigate their potential for nerve regeneration. It was found that gelatin-containing PGS-based nanofibers promoted cell proliferation. The elongated cell morphology observed on such nanofibers indicated that the scaffolds could induce the neurite outgrowth of the nerve stem cells. Overall, our study suggested that the synthesis of PGS-based copolymers might be a promising approach to enhance their processability, and therefore advancing bioscaffold engineering for various TE applications.

A Thixotropic Polyglycerol Sebacate-Based Supramolecular Hydrogel as an Injectable Drug Delivery Matrix.

We have developed a "self-healing" polyglycerol sebacate-polyethylene glycol methyl ether methacrylate (PGS-PEGMEMA)/α-Cyclodextrin (αCD) hydrogel which could be sheared into a liquid during injection and has the potential to quickly "heal" itself back into gel post-injection. This hydrogel was shown to be biocompatible and biodegradable and therefore appropriate for use in vivo. Furthermore, the storage and loss moduli of the hydrogels could be tuned (by varying the concentration of αCD) between a fraction of a kPa to a few 100 kPa, a range that coincides with the moduli of cells and human soft tissues. This property would allow for this hydrogel to be used in vivo with maximal mechanical compatibility with human soft tissues. In vitro experiments showed that the hydrogel demonstrated a linear mass erosion profile and a biphasic drug (doxorubicin) release profile: Phase I was primarily driven by diffusion and Phase II was driven by hydrogel erosion. The diffusion mechanism was modeled with the First Order equation and the erosion mechanism with the Hopfenberg equation. This established fitting model could be used to predict releases with other drugs and estimate the composition of the hydrogel required to achieve a desired release rate.

Sustained, low-dose intraperitoneal cisplatin improves treatment outcome in ovarian cancer mouse models.

Intraperitoneal (IP) chemotherapy for ovarian cancer treatment prolongs overall survival by 16 months compared to intravenous chemotherapy but is not widely practiced due to catheter-related complications and complexity of administration. An implantable, nonresorbable IP microdevice was used to release chemotherapeutic agent at a constant rate of approximately 1.3 µg/h in vitro and 1.0 µg/h in vivo. Studies conducted in two orthotopic murine models bearing human xenografts (SKOV3 and UCI101) demonstrate that continuous dosing reduces tumor burden to the same extent as weekly IP bolus drug injections. Treatment-induced toxicity was quantified via body weight loss and complete blood count. The microdevice resulted in significantly less toxicity than IP bolus injections, despite administration of higher cumulative doses (total area under the concentration-time curve of 3049 ng day/mL with the microdevice vs. 2118 ng-day/mL with IP bolus injections). This preclinical study supports the concept that reduced toxicity with similar efficacy outcomes can be achieved by continuous dosing in ovarian cancer patients currently treated with IP therapy.

Current treatment options and drug delivery systems as potential therapeutic agents for ovarian cancer: a review.

Ovarian cancer is one of the most common and deadliest gynecologic cancer with about 75% of the patients presenting in advanced stages. The introduction of intraperitoneal chemotherapy in 2006 had led to a 16 month improvement in the overall survival. However, catheter-related complication and the complexity of the procedure had deterred intraperitoneal route as the preferred route of treatment. Other alternative treatments had been developed by incorporating other FDA-approved agents or procedures such as pegylated liposomal doxorubicin (PLD), hyperthermic intraoperative intraperitoneal chemotherapy (HIPEC) and the administration of bevacizumab. Various clinical trials were conducted on these alternatives as both the first-line treatment and second- or third-line therapy for the recurrent disease. The outcome of these studies were summarized and discussed. A prospective improvement in the treatment of ovarian cancer could be done through the use of a drug delivery system. Selected promising recent developments in ovarian cancer drug delivery systems using different delivery vehicles, surface modifications, materials and drugs were also reviewed.

TopDown real-time gene synthesis.

This chapter introduces a simple, cost-effective TopDown one-step gene synthesis method, which is suitable for the sequence assembly of fairly long DNA. This method can be distinguished from conventional gene synthesis methods by two key features: (1) the melting temperature of the outer primers is designed to be ∼8°C lower than that of the assembly oligonucleotides, and (2) different annealing temperatures are utilized to selectively control the efficiencies of oligonucleotide assembly and full-length template amplification. This method eliminates the interference between polymerase chain reactions (PCR) assembly and amplification in one-step gene synthesis. Additionally, the TopDown gene synthesis has been combined with the LCGreen I DNA fluorescence dye in a real-time gene synthesis approach for investigating the stepwise efficiency and kinetics of PCR-based gene synthesis. The obtained real-time fluorescence signals are compared with gel electrophoresis results to optimize gene synthesis conditions.

Hydrodynamic spinning of hydrogel fibers.

Hydrogel scaffolds are highly hydrated polymer networks that allow cells to adhere, proliferate and differentiate in the treatment of diseased or injured tissues and organs. Using hydrodynamic shaping and in situ cross-linking of hydrogel precursors, we have developed a highly efficient "hydrodynamic spinning" approach for synthesizing hydrogel fibers of different diameters in a multiphase coaxial flow. A triple-orifice spinneret has been created, and three different types of hydrogel precursors have been examined. Without changing the spinning head, hollow and solid hydrogel fibers with different diameters have been spun by simply manipulating the ratio of input flow rates. Together with the ability of simultaneous cell-seeding in the hydrogel matrix, hydrodynamic spinning can be broadly applied to many hydrogel materials, providing a powerful technique in the preparation of fiber-like and tubule-like hydrogel constructs for tissue engineering.