A hydrolytically-tunable photocrosslinked PLA-PEG-PLA/PCL-PEG-PCL dual-component hydrogel that enhances matrix deposition of encapsulated chondrocytes.

In this study, a series of photocrosslinked hydrogels were designed composed of both poly(lactide)-poly(ethylene glycol)-poly(lactide) (PEL) and poly(ε-caprolactone)-poly(ethylene glycol)-poly(ε-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.

The effects of different molecular weight chondroitin-4-sulfates in chondrocyte pellet culture.

For this study, we cultured chondrocyte pellets in Dulbecco's modified Eagle's medium plus a 2 % fetal bovine serum medium, and treated them with 2- to 8-mer oligosaccharides of chondroitin sulfate A to examine the effects of these oligosaccharides on the differentiation and protection of chondrocytes. We found low-molecular-weight CSAs to increase the ratio of the gene expression levels of collagen II/collagen I of chondrocytes from the first day up to 14 days after culture compared with those under a CSA-free medium. Moreover, low-molecular-weight CSAs inhibited the expression of matrix metalloproteinases and peptidases, and stimulated an endogenous tissue inhibitor of metalloproteinases. The dp-8 (8-mer) CSA yielded the most effective response among promoting collagen type II protein secretions compared with other groups.

In vitro and in vivo co-culture of chondrocytes and bone marrow stem cells in photocrosslinked PCL-PEG-PCL hydrogels enhances cartilage formation.

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.

An ectopic approach for engineering a vascularized tracheal substitute.

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.

Evaluation of a mPEG-polyester-based hydrogel as cell carrier for chondrocytes.

Temperature-sensitive hydrogels are attractive alternatives to porous cell-seeded scaffolds and is minimally invasive through simple injection and in situ gelling. In this study, we compared the performance of two types of temperature-sensitive hydrogels on chondrocytes encapsulation for the use of tissue engineering of cartilage. The two hydrogels are composed of methoxy poly(ethylene glycol)- poly(lactic-co-valerolactone) (mPEG-PVLA), and methoxy poly(ethylene glycol)-poly(lactic- co-glycolide) (mPEG-PLGA). Osmolarity and pH were optimized through the manipulation of polymer concentration and dispersion medium. Chondrocytes proliferation in mPEG-PVLA hydrogels was observed as well as accumulation of GAGs and collagen. On the other hand, chondrocytes encapsulated in mPEG-PLGA hydrogels showed low viability and chondrogenesis. Also, mPEG-PVLA hydrogel, which is more hydrophobic, retained physical integrity after 14 days while mPEG-PLGA hydrogel underwent full degradation due to faster hydrolysis rate and more pronounced acidic self-catalyzed degradation. The mPEG-PVLA hydrogel can be furthered tuned by manipulation of molecular weights to obtain hydrogels with different swelling and degradation characteristics, which may be useful as producing a selection of hydrogels compatible with different cell types. Taken together, these results demonstrate that mPEG-PVLA hydrogels are promising to serve as three-dimensional cell carriers for chondrocytes and potentially applicable in cartilage tissue engineering.

Hemocompatibility of poly(vinylidene fluoride) membrane grafted with network-like and brush-like antifouling layer controlled via plasma-induced surface PEGylation.

In this work, the hemocompatibility of PEGylated poly(vinylidene fluoride) (PVDF) microporous membranes with varying grafting coverage and structures via plasma-induced surface PEGylation was studied. Network-like and brush-like PEGylated layers on PVDF membrane surfaces were achieved by low-pressure and atmospheric plasma treatment. The chemical composition, physical morphology, grafting structure, surface hydrophilicity, and hydration capability of prepared membranes were determined to illustrate the correlations between grafting qualities and hemocompatibility of PEGylated PVDF membranes in contact with human blood. Plasma protein adsorption onto different PEGylated PVDF membranes from single-protein solutions and the complex medium of 100% human plasma were measured by enzyme-linked immunosorbent assay (ELISA) with monoclonal antibodies. Hemocompatibility of the PEGylated membranes was evaluated by the antifouling property of platelet adhesion observed by scanning electron microscopy (SEM) and the anticoagulant activity of the blood coagulant determined by testing plasma-clotting time. The control of grafting structures of PEGylated layers highly regulates the PVDF membrane to resist the adsorption of plasma proteins, the adhesion of platelets, and the coagulation of human plasma. It was found that PVDF membranes grafted with brush-like PEGylated layers presented higher hydration capability with binding water molecules than with network-like PEGylated layers to improve the hemocompatible character of plasma protein and blood platelet resistance in human blood. This work suggests that the hemocompatible nature of grafted PEGylated polymers by controlling grafting structures gives them great potential in the molecular design of antithrombogenic membranes for use in human blood.