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To reduce phenolic pollutants in the environment, many countries have imposed firm restrictions on industrial wastewater discharge. In addition, the current industrial process of phenolic resin production uses phenol and formaldehyde as the reactants to perform a polycondensation reaction. Due to the toxicity of formaldehyde and phenolic pollutants, the main purpose of this research was to design a green process using horseradish peroxidase (HRP) enzymatic polymerization to remove phenols and to produce formaldehyde-free phenolic polymers. In this study, the optimal reaction conditions, such as reaction temperature, pH, initial phenol concentration and initial ratio of phenol, and H2O2, were examined. Then, the parameters of the enzyme kinetics were determined. To solve the restriction of enzyme inactivation, several nonionic surfactants were selected to improve the phenol removal efficiency, and the optimal operation conditions in a surfactant-containing system were also confirmed. Importantly, the molecular weight of the synthetic phenolic polymers could be controlled by adjusting the ratio of phenol and H2O2. The content of biphenols in the products was almost undetectable. Collectively, a green chemistry process was proposed in this study and would benefit the treatment of phenol-containing wastewater and the production of formaldehyde-free phenolic resin in the future.

Sobrerol, an oral mucolytic agent, in a recent study showed promise for treating multiple sclerosis. A human equivalent dose of 486 mg of sobrerol administered thrice daily (i.e., 1459 mg of daily dose) demonstrated the highest therapeutic efficacy for repurposing use, which also points out the poor compliance of administration. In this study, oral sustained-release pellets of sobrerol were successfully developed with evaluated manufacturing conditions and drug release kinetics. For design of the target drug product, we used a modeling and simulation approach to establish a predictive model of oral pharmacokinetic profile, by exploring the characteristics and correlations corresponding to the pharmacokinetics and pharmacodynamics of sobrerol, such as absorption lag time (0.18 h), time-scaling in vitro–in vivo correlation (tin-vitro = 0.494 tin-vivo − 0.0904), gastrointestinal transit time (8 h), minimum effective concentration (1.61 μg/mL), and duration of action (12.8 h). Results showed that the frequency of administration and the daily dose remarkably reduced by 33.3% (i.e., from thrice to twice daily) and 22.8%, respectively, which indicates that this prototype approach can be adopted for rapidly developing a modified-release dosage form of sobrerol, with improvement of compliance of administration and therapeutic efficacy.

Tacrolimus (FK506) is a common immunosuppressive drug that is capable of suppressing acute rejection reactions, and is used to treat patients after allotransplantation. A stable and suitable serum concentration of tacrolimus is desirable for better therapeutic effects. However, daily drug administration via oral or injection routes is quite inconvenient and may encounter drug overdose or low patient compliance problems. In this research, our objective was to develop an extended delivery system using a thermosensitive hydrogel of poly ethylene glycol, D,L-lactide (L), and ϵ-caprolactone (CL) block copolymer, mPEG-PLCL, as a drug depot. The formulation of mPEG-PLCL and 0.5% PVP-dissolved tacrolimus was studied and the optimal formulation was obtained. The in vivo data showed that in situ gelling is achieved, a stable and sustained release of the drug within 30 days can be maintained, and the hydrogel was majorly degraded in that period. Moreover, improved allograft survival was achieved. Together, these data imply the potential of the current formulation for immunosuppressive treatments.

We developed an injectable hydrogel system with a sustained release of TGF-β3 through growth factor-loaded microsphere to mimic the cartilage-like microenvironment. Poly(lactic-co-glycolic acid) (PLGA) microspheres incorporated in three dimensional (3D) scaffolds were chosen because of its regulatory approval, good biodegradability, and acting as carriers with sustained release behavior. We evaluated sustained release of TGF-β3 by PLGA microspheres encapsulated in methoxy poly(ethylene glycol)-poly(alanine) (mPA) hydrogels and the resulting enhanced chondrogenic effects. We reported here the effect of the proposed system for sustained release of growth factors on chondrogenesis in cartilage regeneration. PLGA microspheres were used in our thermosensitive mPA hydrogel system with bovine serum albumin as a stabilizing and protecting agent for the emulsion and TGF-β3 enabling sustained release. Gelation, structural properties, and in-vitro release of this composite, that is, microspheres in hydrogel, system were investigated. Using PLGA microspheres to carry growth factors could complement the mPA hydrogel's ability to provide an excellent 3D microenvironment for the promotion of chondrogenic phenotype as compared the systems using mPA hydrogel or microspheres alone. Our study demonstrated that this synthesized composite hydrogel system is capable of modulating the biosynthetic and differentiation activities of chondrocytes. The sustained release of TGF-β3 in this novel hydrogel system could improve biomedical applicability of mPEG-polypeptide scaffolds. The distinctive local growth factor delivery system successfully combined the use of both polymers to be a suitable candidate for prolonged articular cartilage regeneration.

In this study, Saccharomyces cerevisiae was engineered to degrade and utilize xylan, one of the major polysaccharide chains present in hemicellulose. Different hemicellulases from Trichoderma reesei, namely: endoxylanase, β-xylosidase, acetylxylan esterase, α- d -glucuronidase and α- l -arabinofuranosidase, were heterologously secreted by S. cerevisiae. A mixture experimental design was adapted to statistically describe the synergistic interactions between the hemicellulases and to determine the optimum formulations for the hydrolysis of xylan substrates. The hydrolytic activities of the hemicellulase mixtures were then improved by displaying the hemicellulases on the yeast surface as whole-cell biocatalysts. The engineered yeast strains displaying hemicellulases were further engineered to express xylose-utilization genes xylose isomerase (XI) and xylulokinase (XK) which enable its utilization of xylose as a sole carbon source. The resulting consortium was then able to grow and produce ethanol from different xylan substrates.

The aim of this study was to utilize a new and highly effective bioreactor system, i.e., simultaneous saccharification and fermentation (SSCF), for bioethanol production by the cocultivation of Trichoderma reesei, Aspergillus niger, and Zymomonas mobilis by using a direct conversion process of pretreated-wood dust medium. Wood dust has been effectively used to obtain reducing sugars (glucose, xylose, and other byproducts) through the use of either a supercritical fluid extraction (SFE) or steam explosion (SE) pretreatment step. In addition, experimental results showed that polyurethane as a porous carrier could enhance total saccharification enzyme activity at an inoculum proportion of 1/1 of T. reesei, and A. niger and at a total inoculum concentration of 6.5 × 106 spores/ml. Furthermore, the concentration of alginate beads (3%) and immobilized proportion of Z. mobilis to alginate beads (1:4) were also examined. In accordance with previous reports, bioethanol production was carried out in a SSCF bioreactor by the cocultivation of T. reesei and A. niger in the polyurethane carrier and Z. mobilis immobilized in alginate beads using pretreated-wood dust medium. Experimental results revealed that, after 24 h of cultivation, the yield of bioethanol produced using pretreated-wood dust medium (1%) were 0.069 g/g and 0.049 g/g, for SFE and SE, respectively. Meanwhile, the sugar conversion rate reached 20.72% and 24.39% for SFE and SE, respectively. Thus, the results of this study show that pretreated-wood dust medium has significant potential for use in bioethanol production.

Poly (1,6-bis- (p-carboxyphenoxy hexane)-co- (sebacic anhydride)) (PANH), is a polyanhydride copolymer which has good biocompatibility, and degrades to non-toxic products with a predictable rate of degradation. Nano-sized hydroxyapatite (HAP) is a well-known biomaterial which has been applied in bone regeneration due to its osteoconductivity. However, nano-sized HAP has poor colloidal stability which leads to agglomeration when incorporated into polymeric composites. In this work we describe the surface grafting of poly(ɛ-caprolactone) to HAP (PCL-gHAP) to improve the interfacial adhesion and dispersion of HAP particles in a PANH matrix to form a composite material. The use of scanning electron microscopy-backscattered electron (SEM-BSE) detector and the combination of focused ion beam (FIB)/ transmission electron microscopy (TEM) provided a powerful approach for observing the dispersion of HAP particles in the polymer matrix. We show that surface modification of HAP with PCL improved the homogeneity of the dispersion of HAP particles in the composites and affected the composite morphology during hydrolytic degradation. Composites with high HAP content displayed high compressive strength and a fast rate of degradation. The PCL-gHAP/PANH composites showed superior maintenance of mechanical properties compared with HAP/PANH composites during degradation. A preliminary in vivo study on rat calvaria repair, demonstrated the superior performance of PCL-gHAP/PANH composites. The results suggest that the newly developed PCL-gHAP/PANH composite materials have great potential of serving as a new substrate for bone tissue engineering.

Background The aim of this study was to develop a minimally invasive hydrogel system that can release strontium ions, an element that has been shown to increase osteoblast proliferation and prohibit bone resorption, in a controlled manner. Methods SrCO3 was selected as the salt of choice due to potential acid neutralization reaction between SrCO3 and degradation by-products of methoxy(polyethylene glycol)- co-poly(lactic- co-glycolic acid) (mPEG-PLGA): namely, lactic acid and glycolic acid. SrCO3 was incorporated into mPEG-PLGA hydrogel, and the system was assessed for gelation properties, drug release and biocompatibility. Results SrCO3 incorporation at hydrogel to SrCO3 ratios of 5:1, 3:1 and 1:1 (wt%) did not compromise the thermosensitivity of mPEG-PLGA hydrogels. Furthermore, incorporation of SrCO3 at 1:1 ratio prevented copolymer self-catalysis and decreased hydrogel weight loss from 85% to 61% in vitro after 30 days. During the 30-day time frame, zero-order strontium release was observed and was correlated to hydrogel degradation and acidity. The addition of SrCO3 also improved in vivo hydrogel biocompatibility, due to moderation of acidic microenvironment and amelioration of inflammatory response. Conclusions These results showed that the described system is suitable for the extended release of strontium and exhibits potential for localized treatment for osteoporosis or as a bone void filler.

Poly(methyl methacrylate) (PMMA) is the most frequently used bone void filler in orthopedic surgery. However, the interface between the PMMA-based cement and adjacent bone tissue is typically weak as PMMA bone cement is inherently bioinert and not ideal for bone ingrowth. The present study aims to improve the affinity between the polymer and ceramic interphases. By surface modifying nano-sized hydroxyapatite (nHAP) with ethylene glycol and poly(ɛ-caprolactone) (PCL) sequentially via a two-step ring opening reaction, affinity was improved between the polymer and ceramic interphases of PCL-grafted ethylene glycol-HAP (gHAP) in PMMA. Due to better affinity, the compressive strength of gHAP/PMMA was significantly enhanced compared with nHAP/PMMA. Furthermore, PMMA with 20 wt.% gHAP promoted pre-osteoblast cell proliferation in vitro and showed the best osteogenic activity between the composites tested in vivo . Taken together, gHAP/PMMA not only improves the interfacial adhesion between the nanoparticles and cement, but also increases the biological activity and affinity between the osteoblast cells and PMMA composite cement. These results show that gHAP and its use in polymer/bioceramic composite has great potential to improve the functionality of PMMA cement.

Recently, we demonstrated the feasibility of subcutaneous transplantation of MIN6 cells embedded in a scaffold with poly(ethylene glycol) methyl ether (mPEG)-poly(Ala) hydrogels. In this study, we further tracked these grafts using magnetic resonance (MR) and bioluminescence imaging. After being incubated overnight with chitosan-coated superparamagnetic iron oxide (CSPIO) nanoparticles and then mixed with mPEG-poly(Ala) hydrogels, MIN6 cells appeared as dark spots on MR scans. For in vivo experiments, we transfected MIN6 cells with luciferase and/or incubated them overnight with CSPIO overnight, 5 × 106 MIN6 cells embedded in mPEG-poly(Ala) hydrogels were transplanted into the subcutaneous space of each nude mouse. The graft of CSPIO-labeled MIN6 cells was visualized as a distinct hypointense area on MR images located at the implantation site before day 21. However, this area became hyperintense on MR scans for up to 64 days. In addition, positive bioluminescence images were also observed for up to 64 days after transplantation. The histology of removed grafts showed positive insulin and iron staining. These results indicate mPEG-poly(Ala) is a suitable scaffold for β-cell encapsulation and transplantation. Moreover, MR and bioluminescence imaging are useful noninvasive tools for detecting and monitoring mPEG-poly(Ala) hydrogel-embedded MIN6 cells at a subcutaneous site.

Herein, we report the synthesis and characterization of a novel positively charged thermosensitive hydrogel prepared from poloxamer (PLX)-poly(L-alanine-lysine) (Lys-Ala-PLX-Ala-Lys) that demonstrates potential in biomedical applications including tissue engineering and drug delivery. At a dilute concentration, the copolymer self-assembled into micelles that were sensitive to temperature elevation. In the concentration range of 3–7 wt%, the aqueous copolymer solution underwent sol–gel transition near the physiological temperature and exhibited changes in its secondary structure content, as evidenced by FT-IR. Excellent viability of cells cultured within the scaffold was observed after 72 h of incubation. On the other hand, negatively charged bovine serum albumin was incorporated into the hydrogel without diminishing material integrity. Over the course of seven days, the protein underwent extended zero-order release. These findings highlight the potential of this system as a cell scaffold as well as a depot for the extended delivery of drugs and proteins, specifically those which bear a charge opposite to that of the hydrogel.

5-hydroxymethylfurfural (HMF) is a versatile biomass-derived chemical for synthesizing useful compounds. In this work an attempt is made to produce HMF by developing a low-cost process for doing that using cellulosic waste and deep eutectic solvents (DESs). The most effective DES was choline chloride/citric acid (ChCl/citric acid, 2/1), which provided a greater glucose conversion percentage (around 100 mol%), HMF yield (8 mol%), and HMF selectivity (8 %) than other DESs with 0.1 g of pure glucose. To increase the HMF yield further, the species of catalyst, reaction time and reaction temperature were varied. The highest glucose conversion percentage (91.76 mol%), HMF yield (15.02 g/g) and HMF selectivity (8%) were obtained with a reaction time of five minutes, a reaction temperature of 130 ℃, sulfuric acid as the catalyst, and a ChCl/citric acid ratio of the DES of 2/1. These optimal conditions were used with the enzymatic hydrolysate of pretreated wood dusts as a substrate. The experimental results thus obtained revealed a glucose conversion percentage, HMF yield and HMF selectivity of 34.86 mol%, 16.46 g/g and 67.43 %, respectively, under these conditions. These results reveal that using enzymatic hydrolysate from pretreated wood in the production of HMF can yield approximately as much (16.46 g/g) as the use of pure glucose, indicating that this low-cost process may be commercially feasible.

A system for localized drug delivery composed of polyanhydride polymers was studied. The polymers were made via melt-condensation of sebacic acid (SA) with different amount of 1, 3-bis (p-carboxyphenoxy) propane (CPP) as copolymers. By double emulsion technique, microspheres with core/shell-like structure were fabricated. Hydrophilic compound Brilliant Blue G (BBG) was encapsulated in the inner core(s) region with loading efficiencies of 50% ~ 60% for poly (SA) and 40% ~ 45% for poly(SA-CPP) copolymer. Hydrophobic compound, curcumin, was encapsulated in the outer shell region at efficiencies about 90%. Drug release profiles show no initial burst and stable release rates. The studied delivery system was showed to provide stable release for both hydrophilic and hydrophobic compounds simultaneously.

Objective Quercetin (Que), a bioflavonoid, is both anti-inflammatory and antioxidative. Que has been used as an oral supplement for osteoarthritis (OA) with inconsistent findings because of its low bioavailability. We encapsulated Que in a mPEG-polypeptide thermogel to prolong its bioactivity. The efficacy of this formulation was evaluated in a posttraumatic OA rat model. Design Methoxy-poly(ethylene glycol)-l-poly(alanine) (mPEG-PA) polymer was synthesized and characterized in terms of cytotoxicity and release kinetics in vitro. At 12 weeks old, Sprague-Dawley rats underwent anterior cruciate ligament transection (ACLT). At 24 weeks post-operation, rats received either an intra-articular (IA) injection of saline, hydrogel, or hydrogel with Que (50 or 500 μg). Gait analysis was performed at pre-ACLT, pre-treatment, and at 4, 8, and 12 weeks post-treatment. At 12 weeks post-treatment, knee joints were collected for histopathological evaluation. Results In vitro studies showed that chondrocytes were viable after 72 hours of incubation with mPEG-PA, and the release of Que could be sustained for >28 days. Among all OA rats, the limb idleness index (LII) were significantly increased at 24 weeks post-ACLT. Rats that received hydrogel with Que (50 μg) showed the most reduction in LII at both 4 and 8 weeks post-treatment. The Osteoarthritis Research Society International score of rats received hydrogel with Que (50 μg) was significantly lower than the control group. All rats suffered from low-grade synovitis (Krenn score: 2-4). Conclusion This study suggests that a sustained delivery of Que (50 μg) could provide symptom relief and also delay the progression of OA in the knee.

The liver is the current site of choice for clinical islet transplantation, but many intraportal grafts were lost in the early stage of transplantation. Other disadvantages include the difficulty of monitoring the islets and procedure-associated complications. In contrast, the subcutaneous space offers the advantages of a large space, a less invasive procedure and easy graft monitoring and removal. However, islet transplantation into an unmodified subcutaneous site has poor efficacy; possibly due to poor oxygenation and inadequate vascularization of the transplanted tissue. In this study, we tested the feasibility of subcutaneous transplantation of MIN6 beta cells embedded in temperature-sensitive poly(ethylene glycol) methyl ether (mPEG)-Ala hydrogels as scaffolds. After culturing for 14 days, the viability of MIN6 beta cells in mPEG-Ala hydrogel was comparable with those in medium, either assayed by MTT or LIVE/DEAD. Static incubation showed that both had comparable insulin secretion. For in vivo experiments, we infected MIN6 beta cells with luciferase by AAV and then transplanted 5*106 cells embedded in mPEG-Ala hydrogel into the subcutaneous space of each nude mouse’s upper back. Positive In Vivo Imaging System (IVIS) images and positive insulin staining of tissue histology were observed up to 41 days after transplantation. Meanwhile, we incubated MIN6 beta cells with chitosan-coated superparamagnetic iron oxide (CSPIO) overnight and then transplanted 5*106 MIN6 beta cells into the subcutaneous tissue of each nude mouse’s left flank. The graft of CSPIO-labeled MIN6 beta cells was visualized on Magnetic Resonance (MR) scans as distinct hypointense spots on T2-weighted images located at the transplantation site at day 6 and 20. These results indicate that subcutaneous transplantation of MIN6 beta cells embedded in temperature-sensitive mPEG-Ala hydrogels is feasible. Moreover, the IVIS and MR imaging are useful tools for detecting and monitoring beta cells at the subcutaneous site. Disclosure J. Juang: None.H. Lin: None.C. Chen: None.C. Kao: None.S. Wu: None.S. Lin: None.C. Shen: None.J. Wang: None.I. Chu: None.

Cupriavidus taiwanensis 187 is reportedly efficient in achieving the degradation of phenol and accumulation of polyhydroxybutyrate (PHB). This study attempted to optimize the cultivation conditions and fermentation strategies for phenol degradation and PHA accumulation by C. taiwanensis 187. After the cultivation conditions were optimized, the conditions required for achieving phenol degradation (100%) and PHB accumulation (51 mg/L) by C. taiwanensis 187 were identified as 30 °C and 200 rpm, when the cultivation time was around 7 h. The accumulation of PHB was further increased from 72 to 213 mg/L by feeding phenol in three rounds into the fermenter along with the exhaustion of dissolved oxygen, which could totally degrade the phenol at around 1500 mg/L. Production of PHB by C. taiwanensis 187 was confirmed by GC, 1H-NMR, and 13C-NMR analyses. Each analytical result proved that C. taiwanensis 187 was able to use phenol as the sole carbon source for producing PHB. Finally, these results revealed that the phenol degraded by C. taiwanensis 187 mainly contributed to cell growth rather than PHB accumulation. These results indicated that the strain C. taiwanensis 187 could be used to degrade phenol to obtain usable biological polyesters.

Climate change is directly linked to the rapid depletion of our non-renewable fossil resources and has posed concerns on sustainability. Thus, imploring the need for us to shift from our fossil based economy to a sustainable bioeconomy centered on biomass utilization. The efficient bioconversion of lignocellulosic biomass (an ideal feedstock) to a platform chemical, such as bioethanol, can be achieved via the consolidated bioprocessing technology, termed yeast surface engineering, to produce yeasts that are capable of this feat. This approach has various strategies that involve the display of enzymes on the surface of yeast to degrade the lignocellulosic biomass, then metabolically convert the degraded sugars directly into ethanol, thus elevating the status of yeast from an immobilization material to a whole-cell biocatalyst. The performance of the engineered strains developed from these strategies are presented, visualized, and compared in this article to highlight the role of this technology in moving forward to our quest against climate change. Furthermore, the qualitative assessment synthesized in this work can serve as a reference material on addressing the areas of improvement of the field and on assessing the capability and potential of the different yeast surface display strategies on the efficient degradation, utilization, and ethanol production from lignocellulosic biomass.

Supplemental material, Supplementary for Intra-Articular Delivery of Quercetin Using Thermosensitive Hydrogel Attenuate Cartilage Degradation in an Osteoarthritis Rat Model by Sze-Wing Mok, Sai-Chuen Fu, Yau-Chuk Cheuk, I-Ming Chu, Kai-Ming Chan, Ling Qin, Shu-Hang Yung and Ki-Wai Kevin Ho in CARTILAGE

Thermosensitive hydrogels are attractive alternative scaffolding materials for minimally invasive surgery through a simple injection and in situ gelling. In this study, a novel poly(ester-amide) polymer, methoxy poly(ethylene glycol)-poly(pyrrolidone-co-lactide) (mPDLA, P3L7) diblock copolymer, was synthesized and characterized for cartilage tissue engineering. A series of amphiphilic diblock copolymers was synthesized by ring-opening polymerization of mPEG 550, D,L-lactide, and 2-pyrrolidone. By dynamic light scattering analysis and tube-flipped-upside-down method, viscoelastic properties of the mPDLA diblock copolymer solution exhibited sol-gel transition behavior as a function of temperature. An in vitro degradation assay showed that degradation acidity was effectively reduced by introducing the 2-pyrrolidone monomer into the polyester hydrogel. Besides, mPDLA exhibited great biocompatibility in vitro for cell encapsulation due to a high swelling ratio. Moreover, cell viability and biochemical analysis proved that the mPDLA hydrogel presented a great chondrogenic response. Taken together, these results demonstrate that mPDLA hydrogels are promising injectable scaffolds potentially applicable to cartilage tissue engineering.

Cyclodextrins (CDs) coupled with oils forms an insoluble inclusion complex that is able to adsorb to the interface between oils and aqueous phases; it thereby stabilizes Pickering emulsions. Three types of oils (triglyceride, linear chain oil, and ring-structured oil) were chosen to work with CDs to prepare bupivacaine (BPC)-encapsulated Pickering emulsions. We also investigated the relationship between oils and CDs; as well as their influences on stability, drug-releasing capability and skin permeability. Particle sizes and microstructures were determined by dynamic light scattering and scanning electron microscopy, respectively. In vitro drug release studies and in vitro skin permeation studies were evaluated by using Franz diffusion model. Particle sizes of all Pickering emulsions were larger than 1 μm, and the morphology was spherical and covered with rough surfaces. BPC was released over an extended period, and the releasing ratios from Pickering emulsions were only 12.2%–23.1% after 48 h. In skin permeation studies, compared with other formulations, a formula involved with ring-structured oil allowed the highest permeation amount through skin. However, after 24 h of exposure, formulation operated with linear chain oil showed the highest skin-retaining amount. These results suggest that Pickering emulsions could regulate the target site of skin depending on various types of oil used.

The aim of this work was to study the feasibility of surface displaying synthetic phytochelatin (EC) on Saccharomyces cerevisiae to overcome the inhibitory effect of heavy metals on ethanol production. Via the fusion of a gene encoding EC to an α-agglutinin gene, the engineered S. cerevisiae was able to successfully display EC on its surface. This surface engineered yeast strain exhibited an efficient cadmium adsorption capability and a remarkably enhanced cadmium tolerance. Moreover, its ethanol production efficiency was significantly improved as compared to a control strain in the presence of cadmium. Similar results could also be observed in the presence of other metals, such as nickel, lead and copper. Overall, this method allows simultaneous biorefinery and heavy metal removal when using heavy metal-contaminated biomass as raw materials.

In this study, a series of photocrosslinked hydrogels were designed composed of both poly(lactide)-poly(ethylene glycol)-poly(lactide) (PEL) and poly(e-caprolactone)-poly(ethylene glycol)-poly(e-caprolactone) (PEC) macromers. The PEL/PEC hydrogels at ratios of 100:0, 75:25, 50;50, 25:75 and 0:100 were studied for their degradation characteristics and their ability to support chondrogenesis of encapsulated chondrocytes. Difference in hydrolytic susceptibility between copolymers led to different degradation patterns where higher PEC content correlated with slower degradation. Increased chondrogenic gene expression was observed in chondrocyte-laden hydrogels within a 4-week culture period. Biochemical and histological evaluations revealed significant accumulation of extracellular matrix proteins such as glycosaminoglycans and collagen in the 50/50 hydrogel owing to appropriate tuning of hydrogel degradation. These results demonstrate that the dual-component photocrosslinked hydrogel system is suitable for use as scaffold to support chondrogenesis and, moreover, the tunability of these systems opens up possibilities for use in different cell culturing applications. Copyright © 2014 John Wiley & Sons, Ltd.

Curcumin is a naturally occurring compound that has been shown to have anti-oxidant, anti-inflammatory, and anti-carcinogenic activities. However, its pharmaceutical potential has been limited due to its low solubility in water. The use of amphiphilic nanocarriers is an attractive and simple method to solubilize curcumin. In this study, we modified Pluronic F-127 [poly(ethylene glycol)100-block-poly(propylene glycol)65-block-poly(ethylene glycol)100] (PF-127) with oligomers of alanine, an amino acid, to increase the drug entrapment efficiency of curcumin through core stabilization. Alanine-modified PF-127 exhibited lower critical micelle concentration and decreased molecular motion in both the hydrophilic and hydrophobic segments ((1)H NMR). Nanocarriers in the size range of 54.2-68.4 nm were observed. Entrapment efficiency of curcumin increased by at most 66% (from 25.3 to 91.3%) and the difference in solubility was clearly visualized by increased transparency of the nanocarrier solutions. Curcumin was released continuously up to 120 h from modified carriers, while drug release from unmodified carriers plateaued within 24 h. These modified nanocarriers exhibited no cytotoxicity and more efficiently delivered drugs to HeLa cells as confirmed by fluorescent microscopy. This study demonstrated that alanine modification of FDA-approved PF-127 affects copolymer nanoassembly and has a profound impact on curcumin loading and possibly on other hydrophobic drugs as well.

Human embryonic stem cells (hESCs), due to their self-renewal capacity and pluripotency, have become a potential source of transplantable β-cells for the treatment of diabetes. However, it is imperative that the derived cells fulfill the criteria for clinical treatment. In this study, we replaced common Matrigel with a synthetic peptide-acrylate surface (Synthemax) to expand undifferentiated hESCs and direct their differentiation in a defined and serum-free medium. We confirmed that the cells still expressed pluripotent markers, had the ability to differentiate into three germ layers, and maintained a normal karyotype after 10 passages of subculture. Next, we reported an efficient protocol for deriving nearly 86% definitive endoderm cells from hESCs under serum-free conditions. Moreover, we were able to obtain insulin-producing cells within 21 days following a simple three-step protocol. The results of immunocytochemical and quantitative gene expression analysis showed that the efficiency of induction was not significantly different between the Synthemax surface and the Matrigel-coated surface. Thus, we provided a totally defined condition from hESC culture to insulin-producing cell differentiation, and the derived cells could be a therapeutic resource for diabetic patients in the future.

Tissue engineering can provide alternatives to current methods for tracheal reconstruction. Here we describe an approach for ectopic engineering of vascularized trachea based on the implantation of co-cultured scaffolds surrounded by a muscle flap. Poly(L-lactic-co-glycolic acid) (PLGA) or poly(ε-caprolactone) (PCL) scaffolds were seeded with chondrocytes, bone marrow stem cells and co-cultured both cells respectively (8 groups), wrapped in a pedicled muscle flap, placed as an ectopic culture on the abdominal wall of rabbits (n = 24), and harvested after two and four weeks. Analysis of the biochemical and mechanical properties demonstrated that the PCL scaffold with co-culture cells seeding displayed the optimal chondrogenesis with adequate rigidity to maintain the cylindrical shape and luminal patency. Histological analysis confirmed that cartilage formed in the co-culture groups contained a more homogeneous and higher extracellular matrix content. The luminal surfaces appeared to support adequate epithelialization due to the formation of vascularized capsular tissue. A prefabricated neo-trachea was transferred to the defect as a tracheal replacement and yielded satisfactory results. These encouraging results indicate that our co-culture approach may enable the development of a clinically applicable neo-trachea.

We reveal that a slight change in the functional group of the oligopeptide block incorporated into the poloxamer led to drastically different hierarchical assembly behavior and rheological properties in aqueous media. An oligo(L-Ala-co-L-Phe-co-β-benzyl L-Asp)-poloxamer-oligo(β-benzyl-L-Asp-co-L-Phe-co-L-Ala) block copolymer (OAF-(OAsp(Bzyl))-PLX-(OAsp(Bzyl))-OAF, denoted as polymer 1), which possessed benzyl group on the aspartate moiety of the peptide block, was synthesized through ring-opening polymerization. The benzyl group on aspartate was then converted to carboxylic acid to yield oligo(L-Ala-co-L-Phe-co-L-Asp)-poloxamer-oligo(L-Asp-co-L-Phe-co-L-Ala) (OAF-(OAsp)-PLX-(OAsp)-OAF, denoted as polymer 2). Characterization of the peptide secondary structure in aqueous media by circular dichroism revealed that the oligopeptide block in polymer 1 exhibited mainly an α-helix conformation, whereas that in polymer 2 adopted predominantly a β-sheet conformation at room temperature. The segmental dynamics of the PEG in polymer 1 remained essentially unperturbed upon heating from 10 to 50 °C; by contrast, the PEG segmental motion in polymer 2 became more constrained above ca. 35 °C, indicating an obvious change in the chemical environment of the block chains. Meanwhile, the storage modulus of the polymer 2 solution underwent an abrupt increase across this temperature, and the solution turned into a gel. Wet-cell TEM observation revealed that polymer 1 self-organized to form microgel particles of several hundred nanometers in size. The microgel particle was retained as the characteristic morphological entity such that the PEG chains did not experience a significant change of their chemical environment upon heating. The hydrogel formed by polymer 2 was found to contain networks of nanofibrils, suggesting that the hydrogen bonding between the carboxylic acid groups led to an extensive stacking of the β sheets along the fibril axis at elevated temperature. The in vitro cytotoxicity of the polymer 2 aqueous solution was found to be low in human retinal pigment epithelial cells. The low cytotoxicity coupled with the sol-gel transition makes the corresponding hydrogel a good candidate for biomedical applications.

Chondrocytes (CH) and bone marrow stem cells (BMSCs) are sources that can be used in cartilage tissue engineering. Co-culture of CHs and BMSCs is a promising strategy for promoting chondrogenic differentiation. In this study, articular CHs and BMSCs were encapsulated in PCL-PEG-PCL photocrosslinked hydrogels for 4 weeks. Various ratios of CH:BMSC co-cultures were investigated to identify the optimal ratio for cartilage formation. The results thus obtained revealed that co-culturing CHs and BMSCs in hydrogels provides an appropriate in vitro microenvironment for chondrogenic differentiation and cartilage matrix production. Co-culture with a 1:4 CH:BMSC ratio significantly increased the synthesis of GAGs and collagen. In vivo cartilage regeneration was evaluated using a co-culture system in rabbit models. The co-culture system exhibited a hyaline chondrocyte phenotype with excellent regeneration, resembling the morphology of native cartilage. This finding suggests that the co-culture of these two cell types promotes cartilage regeneration and that the system, including the hydrogel scaffold, has potential in cartilage tissue engineering. Copyright © 2013 John Wiley & Sons, Ltd.

In this study, a biodegradable in situ gel-forming controlled drug delivery system based on a thermosensitive methoxy polyethylene glycol-co-poly (lactic acid-co-aromatic anhydride) (mPEG-PLCPPA) hydrogel was studied. The hydrogels were formed by micelle aggregation with rising temperature. The hydrogels underwent a temperature-dependent sol–gel–sol transition, which was a flowing sol at ambient temperature and a non-flowing gel at the physiological body temperature. The residual weight and pH value changes after degradation and the viscosity properties of the hydrogel were investigated. The in vitro release behavior of vancomycin from the mPEG-PLCPPA hydrogels at different concentrations was also investigated. The results showed that the mPEG-PLCPPA amphiphilic copolymer could self-assemble to form micelles at low concentrations, and that the particle sizes gradually increased with increasing temperature. The hydrogel maintained a stable degradation rate and provided a moderate pH microenvironment after degradation for 30 days. Vancomycin sustained a stable release profile from the hydrogel over a 10-day period. Furthermore, good biocompatibility was proven by MTT assay and live and dead test. Therefore, the mPEG-PLCPPA hydrogel shows promise as an injectable local antibiotic delivery system.

Bioceramic or inorganic nanoparticles made of SiO2, TiO2, SrO, and hydroxyapatite (HAP) have been reported to improve cell adhesion onto polymers. However, direct mixing of these nanoparticles with polymers often leads to their aggregation within the polymer matrix and subsequent deterioration of the material’s mechanical strength. A novel method for modifying the surfaces of the nanoparticles by grafting e-caprolactone using a ring-opening condensation reaction was developed to improve the interconnection of the nanoparticles within the polymer matrix. The mechanical studies showed that adding grafted nanoparticles into the polycaprolactone (PCL) matrix improved the initial mechanical strength. MTT assay and a live/dead stain showed higher cell viability in the tablets with grafted SiO2, TiO2, and HAP nanoparticles, except the SrO-containing tablets. The cell adhesion and alkaline phosphatase activity assay confirmed that the composite tablets with PCL-grafted HAP nanoparticles had better osteoconductivity. HE stains showed that composite tablets with PCL-grafted SiO2, TiO2, and HAP nanoparticles produce less immune response than the pure PCL. We thus conclude that a PCL matrix incorporating PCL-grafted HAP nanoparticles has enhanced mechanical strength, improved osteoconductivity, and a slower degradation rate than pure nanoparticles.

Human embryonic stem cells (hESCs), due to their self-renewal capacity and pluripotency, are an important source of cells for regenerative medicine. The immediate obstacles that need to be addressed are the poor cell survival rate of hESCs and their cell quality after cryopreservation. In this study, we used the Cell Alive System (CAS) which combines a programmed freezer with an oscillating magnetic field to reduce cryo-injury during the freezing process. The hESC clumps suspended in freezing medium were divided into three groups: (i) cells frozen by a conventional freezing container, Mr. Frosty and kept in a −80 °C freezer (MF); (ii) cells frozen to −32 °C by CAS, and then transferred to a −80 °C freezer (CAS); (iii) cells frozen to −32 °C by CAS, and then transferred to a pre-cooled Mr. Frosty and kept in a −80 °C freezer (CAS-MF) for overnight. All cryovials were placed in liquid nitrogen for one week, and hESCs were then thawed and cultured on feeder for 7 days. The results of alkaline phosphatase (AP) staining showed that the attachment efficiency of the cells cryopreserved by CAS and CAS-MF was significantly higher (29.0% and 44.0%) than in the MF method (7.0%). Furthermore, we confirmed the cells cryopreserved using CAS-MF could be subcultured while expressing pluripotent markers, differentiate into three germ layers, and maintain a normal karyotype. These results demonstrate that the use of CAS-MF offers an efficient method of hESC banking.

The acinus-mimicking microfluidic chip, which simulates the in vivo condition of the liver, was developed and reported in this paper. The gradient microenvironment of the liver acinus is replicated within this proposed microfluidic chip. The advantage of this acinus-mimicking chip is capable of adjusting the concentration gradient in a relatively short period of time at around 10 s. At the same instance the non-linear concentration gradient can be presented in the various zones within this microfluidic chip. The other advantage of this proposed design is in the convenience of allowing the direct injection of the cells into the chip. The environment within the chip is multi-welled and gel-free with high cell density. The multi-row pillar microstructure located at the entrance of the top and bottom flow channels is designed to be able to balance the pressure of the perfusion medium. Through this mechanism the shear stress experienced by the cultured cells can be minimized to reduce the potential damage flow from the perfusion process. The fluorescence staining and the observations of the cell morphology verify the life and death of the cells. The shear stress experienced by the cells in the various zones within the chip can be effectively mapped. The serum glutamic oxaloacetic transaminase (SGOT) collected from the supernatants was used to determine the effects of the degassing process and the shear stress of the medium flow on the cultured cells.

The purpose of this study was to design a thermosensitive composite gel to be used as a bone graft substitute. This gel can provide a more suitable microenvironment by using the amphiphilic triblock copolymer (mPEG 550 PLGA 1405 ) consisting of methoxy poly(ethylene glycol) (mPEG), poly(lactic- co -glycolic acid) (PLGA). An aqueous dispersion of mPEG 550 PLGA 1405 mixed with different ratios of HAP/β-TCP (composite gel) underwent a sol–gel–sol transition as the temperature was increased from 4 to 70 °C. The particle size and critical micellization concentration (CMC) were increased by adding ceramics. During the in vitro degradation process, composite gels demonstrated a slight decrease in pH value, a slower degradation rate, less toxicity, and a higher cell survival rate. The biocompatibility of the composite gels was validated by hemolysis test. In vivo animal studies demonstrated both radiographic and gross bone union when the ratio of HAP/β-TCP was 7:3. Based on the results, we have developed novel thermosensitive composite gels as bone substitutes.

This paper reports a gel-free multi-well microfluidic chip for cell cultures. Polydimethylsiloxane (PDMS) material was used because of its hydrophobic and gas-permeable features. Surface tension and fluidic channel resistance simplified the procedure of cell injection into the system. The shear stress and the mass-transfer perfusion medium reached a balance through the multi-row square-pillar microstructure and the driven pressure. The cell seeding was completed in 10-mm scaled culture wells in less than 6 s using the microstructure composed of PDMS. The adjustable initial cell density and length of the culture area facilitate its use in desired biological experiments. To verify whether the operation caused cell damage or not, the biological index of supernatants with glutamic oxaloacetic transaminase (GOT) and blood urea nitrogen (BUN) were monitored for 4 days. The 1–4-mm-long multi-rows square-pillar microstructure can sustain HA22T cells for 5 days and still maintain cell viability for up to 90% of seeded cells.

Background/Purpose Durable mechanical strength and biocompatibility are the two major requirements for osteogenic scaffolds. Polyanhydrides are a class of biodegradable polymers characterized by anhydride bonds that connect repeating units of the polymer backbone chain. Hydroxyapatite (HAP) is the main component of human bone and is a good osteoinductive factor that promotes bone mineralization. This work validates the combination of polyanhydrides and HAP for biomedical application. Methods Polyanhydride copolymers were fabricated from sebacic acid (SA) and 1,6-bis(p-carboxyphenoxy)hexane (CPH). HAP was surface-modified by polycaprolactone (PCL), and testing tablets were made using different ratios of copolymers and surface-grafted HAP (g-HAP). Degradation tests were performed to evaluate mechanical strength, pH, and weight loss. Biocompatibility was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and live/dead stain test. Cell affinity was measured using scanning electronic microscopy (SEM). Results The favorable surface erosion property of polyanhydrides prevented marked changes in the mechanical properties over time. In addition, the degradation byproducts of the copolymer did not cause a serious decline in pH and were less harmful to the cells. g-HAP increased cell affinity for the polymer surface. Conclusion The research team synthesized polyanhydride/g-HAP composites with high mechanical strength, slow degradation, and excellent biocompatibility. The result showed that a CPH/SA ratio of 7:3 in combination with 10 wt% g-HAP was optimal as bone substitute.

The aim of this study was to use a modified bioreactor system for simultaneous saccharification of cellulose and bioethanol production. We tested Aspergillus niger and Trichoderma reesei for cellulose saccharification and Zymomonas mobilis for bioethanol production simultaneously in this modified bioreactor. The results showed that various carboxymethylcellulose (CMC) concentrations (10, 15, or 20 g/L) as a substrate for A. niger and T. reesei yielded bioethanol production of 0.51, 0.78, and 0.89 g/L and a CMC conversion rate of 10.2%, 10.7%, and 8.89%, respectively. These data suggested that at 10 g/L CMC as a substrate, the CMC conversion rate was higher than that at the other concentrations. When CMC concentration exceeded 15 g/L, bioethanol production was prolonged to 40 h. These results were attributed to the viscosity of CMC. This study also tested Napier grass (an agricultural byproduct) for bioethanol production. The results revealed bioethanol production and the theoretical Napier grass conversion rate were 0.38 g/L and 12.6%, respectively, for 13-h cultivation when the feeding concentration of Napier grass was 10 g/L. When Napier grass concentration was increased to 15 g/L, bioethanol production and the Napier grass conversion rate reached 0.51 g/L and 23%, respectively, after 14-h cultivation. Eventually, the experimental results indicated using agricultural waste for bioethanol production has been become a potential strategy.

Hydrogels are considered to be attractive cell-matrix for chondrocytes due to their similarity in properties to the natural cartilage. However, the formation of chondrocyte cell clusters in hydrogels has been mostly limited to naturally-derived or relatively fast degrading materials. In this study, a series of diblock copolymer poly(ethylene glycol)-poly(alanine) (mPEG-PA) was synthesized and investigated as injectable biomimic hydrogels for the culturing of chondrocytes. Depending on the poly(alanine) chain length, afforded hydrogels exhibited variable mechanical property and microarchitecture due to difference in secondary structure arrangement. After 21days of culture, cell clusters were observed in all hydrogels with longer PA chains and these hydrogels supported more homogenous and established clustering as well as significantly higher glycosaminoglycan and collagen deposition. Interestingly, scanning electron microscopy revealed a distinct micron range fibrillar-like microarchitecture that may be responsible for maintaining chondrocyte phenotype and matrix production. In addition, micrographs revealed the presence of collagen fibrils and an extensive extracellular matrix network. Therefore, it is reasonable to conclude that mPEG-PA hydrogels possess the desirable properties for chondrocyte cluster formation and serve as potential candidate in cartilage tissue engineering.

The composite of methoxy polyethylene glycol (mPEG) and poly(lactic-co- glycolic acid) (PLGA) thermosensitive hydrogel mixed with different mass raio of hydroxyapatite and β-tricalcium phosphate (β-TCP) were used as bone graft substitutes. The physical properties of a series of composite gels, including the critical micelle concentration (CMC), particle sizes, zeta potential, rheological behavior, morphology of composite gels, and sol–gel transition, were characterized in vitro. These composite gels could form a gel at body temperature and could be controlled easily at room temperature. During the in vitro degradation process, composite gels demonstrated a slight decrease in pH value, a slower degradation rate, less toxicity, and a higher cell survival rate. The biocompatibility of the composite gels was validated by hemolysis test. In vivo animal studies demonstrated both radiographic and gross bone union when the ratio of HAP/ β-TCP was 7:3.