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Exposure of aged mice to a young systemic milieu revealed remarkable rejuvenation effects on aged tissues, including skeletal muscle. Although some candidate factors have been identified, the exact identity and the underlying mechanisms of putative rejuvenating factors remain elusive, mainly due to the complexity of in vivo parabiosis. Here, we present an in vitro muscle parabiosis system that integrates young- and old-muscle stem cell vascular niche on a three-dimensional microfluidic platform designed to recapitulate key features of native muscle stem cell microenvironment. This innovative system enables mechanistic studies of cellular dynamics and molecular interactions within the muscle stem cell niche, especially in response to conditional extrinsic stimuli of local and systemic factors. We demonstrate that vascular endothelial growth factor (VEGF) signaling from endothelial cells and myotubes synergistically contribute to the rejuvenation of the aged muscle stem cell function. Moreover, with the adjustable on-chip system, we can mimic both blood transfusion and parabiosis and detect the time-varying effects of anti-geronic and pro-geronic factors in a single organ or multi-organ systems. Our unique approach presents a complementary in vitro model to supplement in vivo parabiosis for identifying potential anti-geronic factors responsible for revitalizing aging organs.

Synthetic substrates with defined chemical and structural characteristics may potentially be prepared to mimic the living ECM to regulate cell adhesion and growth. Hydrogels with cell-adhesive peptides (0.28 ± 0.03 nmol peptide cm(-2) , TTA-R-0.5; and 0.91 ± 0.12 nmol peptide cm(-2) , TTA-R-2.0) and/or micro-scaled topographical patterns (10, 25, and 80 µm grooves) are prepared using enzymatic polymerization. The adherent morphology and proliferation of C2C12 skeletal myoblasts and human aortic smooth muscle cells (hAoSM) on the hydrogels are studied. The newly developed hydrogels may be useful in investigating the roles of cell adhesion and substrate surface properties in the communication of adherent cells with the ECM.

The efficacy of three-dimensional (3D) culture on the proliferation and maturation of chondrocytes seeded into a hydrogel scaffold was assessed. Three types of hydrogel were prepared for the 3D culture of primary isolated chondrocytes. Chondrocyte proliferation was assessed using a live/dead viability/cytotoxicity assay and semiquantitative RT-PCR after 3D culture in hydrogel. Cylindrical defects in the center of rat xyphoids were used for the implantation of platelet-rich plasma (PRP)/hydrogel composites. Rats were killed at day 7 postoperatively and evaluated histochemically and immunohistologically. Xyphoid chondrocytes proliferated well with time in hydrogels. In the PRP-containing hydrogels, xyphoid defects displayed early formation of chondroid matrix with massive peripheral infiltration of spindle cells. These results were consistent with Safranin-O staining for proteoglycans and immunohistochemistry for type II collagen. Gene expression analyses in vitro revealed aggrecan, type II collagen, and ChM-1 and CB1 upregulation by PRP/hydrogel. PRP/hydrogel provided a suitable environment for hyaline cartilaginous regeneration, leading to anti-inflammation by significant increase of CB1 and inhibiting vascular ingrowth via considerable upregulation of ChM-1. The results provide a valuable reference for the clinical application of hydrogel scaffolds for hyaline cartilage regeneration, as well as the use of autologous PRP to improve cellular proliferation and maturation of xyphoid repair. © 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 100A:3099–3107, 2012.

The structure of tissue plays a critical role in its function and therefore a great deal of attention has been focused on engineering native tissue-like constructs for tissue engineering applications. Transfer printing of cell layers is a new technology that allows controlled transfer of cell layers cultured on smart substrates with defined shape and size onto tissue-specific defect sites. Here, the temperature-responsive swelling-deswelling of the hydrogels with groove patterns and their versatile and simple use as a template to harvest cell layers with anisotropic extracellular matrix assembly is reported. The hydrogels with a cell-interactive peptide and anisotropic groove patterns are obtained via enzymatic polymerization. The results show that the cell layer with patterns can be easily transferred to new substrates by lowering the temperature. In addition, multiple cell layers are stacked on the new substrate in a hierarchical manner and the cell layer is easily transplanted onto a subcutaneous region. These results indicate that the evaluated hydrogel can be used as a novel substrate for transfer printing of artificial tissue constructs with controlled structural integrity, which may hold potential to engineer tissue that can closely mimic native tissue architecture.

The properties of regenerated cartilage using bone marrow-derived mesenchymal stem cells (MSCs) and poly lactic-co-glycolic acid (PLGA) scaffold composites pretreated with TGF-beta3 were investigated and compared to the non-TGF-beta3 treated MSCs/PLGA composites in a rabbit model. We prepared MSCs/PLGA scaffold composites and pretreated it with TGF-beta3 for 3 weeks prior to transplantation. Then, composites were transplanted to the osteochondral defect in the rabbit knee. After 12 weeks of transplantation, 10 of the 12 rabbits in which TGF-beta3 pretreated MSCs/PLGA scaffold composites were transplanted showed cartilaginous regeneration. In gross morphology, regenerated cartilage showed smooth, flush, and transparent features. In indentation test, this had about 80% of Young's modulus of normal articular cartilage. Histological examination demonstrated hyaline like cartilage structures with glycosaminoglycan and type II collagen expression. Histological scores were not statistically different to the normal articular cartilage. These results showed improvement of cartilage regeneration compared to the non-TGF-beta3 pretreated MSCs/PLGA scaffold composite transplanted group. Thus, we have successfully regenerated improved hyaline-like cartilage and determined the feasibility of treating damaged articular cartilage using MSCs/PLGA scaffold composite pretreated with TGF-beta3. Also, we suggest this treatment modality as another concept of cartilage tissue engineering.

The purpose of this study is to investigate the effects of intermittent hydrostatic pressure with various resting periods on the cell adhesive forces and other parameters related to spreading in early stage of cell adhesion. For this, bovine pulmonary arterial endothelium (CPAE, cell line), porcine articular chondrocytes, and human endothelial cells (HECs) were used. The cells were divided into six different experimental groups. Control group was cultured without stimulation, while the constant pressure was applied to group 1 for 2 h. Groups 2-5 were intermittently pressurized for 2 min at a time over a 2-h period with 5, 10, 15, and 20-min resting periods, respectively. Each group was then split into two subgroups, depending whether it experienced extra 60 min stabilization period after stimulation. The average adhesive force and the number and area of focal contacts were significantly higher in the group 4 subgroup, which received an extra 60 min of culture than in the other groups. Similarly, other parameters in this subgroup were significantly different from those in the other groups. The focal contact area and adhesive force were closely related (r = 0.990). We concluded that the mechanical stimuli affect cell adhesion and that the length of the resting period influences the adhesive forces generated at the early stages of adhesion.

A novel chemical modification of biological tissue was developed by the immobilization of heparin to small intestinal submucosa (SIS) using glutaraldehyde (GA). Heparinized SIS tissue was characterized by measuring in vitro plasma protein adsorption, platelet adhesion, in vitro fibroblast attachment, and in vivo calcification. Lower protein adsorption and platelet adhesion were observed on heparinized SIS than non-modified SIS. The attachment of human dermal fibroblast on the heparinized SIS was significantly increased, and the amount of calcium deposited on the modified SIS was much less than fresh SIS. These results demonstrated that heparin immobilized SIS can be applied as a novel bioprosthesis for a variety of cardiovascular applications.

Biodegradable polymer scaffolds that have dual pores on the matrix were successfully prepared using a combination of gas foaming and phase separation methods. PLLA and PLGA were dissolved in dioxane/water at the ratios of 90/10, 87/13, or 85/15 (v/v), respectively. Sodium bicarbonate was then added to the polymer solution and freezed-dried, then subjected to a gas foaming process. Dual pore scaffolds have two distinct pore sizes: large pores in 200∼300 µm and small ones in 5∼20 µm, with the interconnected structure. Their porosities were ranged from 92 to 98%, significantly higher than those of unipore scaffold (90%). Two variables, water content (10, 13, and 15%) and freeze-drying temperature (FDT; −196, −70, and −20 °C), were introduced during the scaffold preparation. Mechanical compressive strength decreased as the polymer solution was gradually diluted with water (100/0 to 85/15, v/v). Earlier weight loss of the different scaffolds was found with FDT at −20 °C but the final result was the same, regardless of different FDTs used. These dual pore polymer scaffolds may thus be useful in tissue engineering applications.

The aim of this study was to identify the signal transduction pathways and mechano-transducers that play critical roles in the processes induced by changes in cyclic hydrostatic pressure and fluid shear in 3-dimensional (3D) culture systems. Mesenchymal stem cells were loaded into a polymeric scaffold and divided into three groups according to the stress treatment: static, fluid shear, and hydrostatic pressure with fluid shear. Cells were exposed daily to a hydrostatic pressure of 0.2 MPa for 1 min followed by 14 min rest with fluid flow at 30 rpm. Protein extracts were analyzed by Western blot for extracellular signal-regulated kinase 1/2 (ERK1/2). The complexes were cultured under the mechanical stimuli for 21 days with or without phospho-ERK1/2 inhibitor (U0126) and evaluated by RT-PCR, calcium contents, and immunohistochemistry. Under conditions of mechanical stimulation, the activation of ERK1/2 was sustained or increased with time. U0126 suppressed mechanical stimuli-induced expression of osteocalcin. In addition, calcium contents and the degrees of osteocalcin and osteopontin staining were decreased by this inhibitor. These results demonstrate that mechanical stimuli, particularly hydrostatic pressure with fluid shear, enhance osteogenesis in 3D culture systems via ERK1/2 activation.

Two kinds of water soluble thermosensitive chitosan copolymers were prepared by graft polymerization of N-isopropylacrylarnide (NIPAAm) onto chitosan using cerium ammonium nitrate (CAN) as an initiator (chitosan-g-NIPAAm) and by coupling monocarboxy Pluronic® with chitosan using 1 -ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) and N-hydroxysuccinimide (NHS) as coupling agents (chitosan-Pluronic®). The physicochemical properties of the resulting copolymers were characterized and they formed thermally reversible hydrogel, which exhibits a lower critical solution temperature (LCST) at 34°C in aqueous solutions. The human mesenchymal stem cells (hMSCs) were cultured in the chiotsan-g-NIPAAm in vitro. Chondrogenic differentiation was induced in the chitosan-g-NIPAAm gel. Therefore, chondrogenic differentiated cells from MSCs with a thermosensitive chitosan-g-NIPAAm could be used as an injectable cell-polymer complex. In summary, chitosan-g-NIPAAm and chitosan-Pluronic® attest to their usefulness as injectable materials because of their thermally reversible property and relatively good biocompatibility.

A novel chemical modification of biological tissues was developed aimed at improving biocompatibility and calcification resistance. This method involved the additional grafting of sulfonated PEO (PEO-SO(3)) or heparin after conventional glutaraldehyde (GA) fixation of bovine pericardium (BP). The amino groups of PEO-SO(3) or heparin were utilized to react to the GA residues to block them. The PEO-SO(3) or heparin grafted tissues demonstrated a slightly higher shrinkage temperature and tensile strength, but greater resistance to collagenase digestion, than GA treated ones. These results suggest that modified tissues have improved durability due to the grafting and filling effect of PEO-SO(3) or heparin in addition to the GA cross-linking. At the direct contact cytotoxicity test in vitro, PEO-SO(3) or heparin grafted tissue was shown to be nontoxic, while relatively significant cytotoxicity was observed for the GA treated tissues, possibly due to the release of GA. From the in vivo calcification study, calcium contents deposited on the modified tissues were much less than those on GA treated tissues. Such a decreased calcification might be explained by the decrease of residual GA groups during the additional treatment, and the space-filling effect and the nonadhesive property and/or the blood compatibility of PEO-SO(3) or heparin grafted covalently. The newly modified tissue patch was observed to show improved pathological assessibility including less inflammation and tissue reactions. This simple modification method may be useful for calcification-resistant and blood-compatible tissue patches for cardiovascular implants.

An approach is presented for the graft copolymerization of type I atelocollagen onto the surface of polyurethane (PU) films treated with ozone. Through inducing oxidization to modify PU surface by ozone, peroxide groups are easily generated on the surface. Those peroxides are broken by redox-polymerization, and provide active species which initiate graft polymerization by reacting with amines in the collagen molecules. The ozone oxidation time and voltage could readily control the amount of peroxide production. The surface density of generated peroxides on PU surface was determined by iodide method. The maximum concentration of peroxide was about 10.20 × 10−8mol/cm2 when ozone oxidation was performed at 60 V for 30 min. After the reaction of PU by ozone oxidation, type I atelocollagen was graft-copolymerized onto the PU film. All the physical measurements on the collagen-grafted surface indicated that the PU surface was effectively covered with type I atelocollagen. The interaction of the collagen-grafted PU surface with fibroblasts could be greatly enhanced by the surface graft polymerization with type I atelocollagen. Attachment and proliferation of fibroblasts on the grafted type I atelocollagen were significantly enhanced, and it is assumed that the atelocollagen matrix supported the initial attachment and growth of cells. In the early stage of proliferation, collagen synthesis in fibroblasts was not activated and remained at a relatively low level due to the grafted type I atelocollagen, increasing only with fibroblast differentiation. © 2000 John Wiley & Sons, Inc. J Biomed Mater Res, 52, 669–677, 2000.

In an attempt to produce surfaces that show low levels of adsorption of protein and adhesion of platelets, different molecular weights of polyethylene glycol methacrylate (PEG-MA) were grafted onto polyethylene film by a preirradiation grafting process. The extent of grafting was found to be dependent on the storage condition of the irradiated polyethylene film, the preirradiated dose, reaction time and temperature, molecular weight of PEG-MA, and the type of solvent. The grafting yield was found to decrease rapidly with storage time for irradiated polyethylene film stored at room temperature. On the other hand, the grafting yield in the irradiated polyethylene stored at −130°C remained nearly constant up to 20 days after irradiation. The grafting yield decreased with an increased PEG-MA molecular weight. Human plasma protein was adsorbed onto control and PEG-MA-grafted polyethylene film surfaces, and the relative adsorbed amount of proteins on the surfaces was evaluated by electron spectroscopy for chemical analysis. The adsorbed protein and platelet adhesion on the polyethylene film surface decreased rapidly with the grafting yield. © 1999 John Wiley & Sons, Inc. J Appl Polym Sci 71: 631–641, 1999

Polyethylene oxide (PEO) surfaces were prepared by the addition of PEO- and sulfonated PEO-containing amphiphilic block copolymers as surface-modifying additives in a segmented polyurethane (PU). PEO-PPO-PEO triblock copolymers (Pluronics) with different PEO chain lengths (from 2 to 80) were used as additives. The prepared film surfaces were characterized by the measurement of dynamic water contact angles and electron spectroscopy for chemical analysis. It was observed that the PU films containing 10 wt % of PEO additives were surface-saturated with the additives regardless of their PEO chain length, but the PEO chains were more projected from the film surfaces containing the additives with longer PEO chains. The water absorption of the films increased largely with the increasing PEO chain length of the additives. The addition of PEO additives produced film surfaces that were in a gel-like state. The films demonstrated some extraction of the PEO additives. However, the additives with higher molecular weights were entrapped more stably into the PU matrix. The mechanical properties (tensile strength and elongation) of the films were changed by the addition of PEO additives, but the differences were not significant compared to the control PU. The platelet adhesion on the film surfaces decreased with increasing PEO chain length of the additives. The film surface containing additives with long PEO chains (chain length of 80) was particularly effective in preventing platelet adhesion. The effect of negatively charged sulfonate groups on the prevention of platelet adhesion appeared only on the film surfaces containing additives with short PEO chains. For longer PEO chains, the chain mobility effect was more dominant than the negative charge effect on the prevention of platelet adhesion. © 1998 John Wiley & Sons, Inc. J Biomed Mater Res, 40, 314–323, 1998.

To improve the biocompatibility of polyurethane (PU) and bioprosthetic tissue (BT), they were chemically grafted with a hydrophilic poly(ethylene oxide) (PEO) and further negatively charged sulfonate groups (SO 3 ) to produce PU-PEO-SO 3 and BT-PEO-SO 3 , respectively. PU-PEO-SO 3 was much more blood compatible than untreated PU and PU-PEO, and the degree of surface cracking and calcification on implanted PUs was decreased in the following order: PU > PU-PEO > PU-PEO-SO 3 . Also, less calcium deposition of BT-PEO-SO 3 than that of BT control was observed in in vivo animal tests. Such superior blood compatibility, biostability, and anticalcification of sulfonated PEO-grafted PUs and tissues might be attributed to synergistic effects of nonadhesive and mobile PEO and negative sulfonate acid groups via a negative cilia model.

Adsorption of proteins (fibrinogen, albumin, and gamma globulin) from plasma onto surface-modified PUs (PU-PEO, PU-SO3, and PU-PEO-SO3) was evaluated. Adsorbed fibrinogen at steady state decreased in the order PU-SO3 > PU > PU-PEO-SO3 > PU-PEO, suggesting that sulfonate groups have specific high affinity to fibrinogen. The intermediate fibrinogen adsorption on PU-PEO-SO3 can be explained by the compensatory effect between the low protein binding affinity of the PEO chain and the high fibrinogen binding affinity of the sulfonate group. In addition, PU-PEO-SO3 showed a very fast fibrinogen adsorption due to the high accessibility of the sulfonate group to fibrinogen by the poly(ethylene oxide) (PEO) spacer. The kinetic profiles of their surfaces showed that as the adsorption time increases, fibrinogen initially adsorbed was decreased and a plateau reached, demonstrating that all the surfaces exhibited the Vroman effect (the fibrinogen displacement phenomenon). PU-PEO showed the least fibrinogen and albumin adsorption among PUs, confirming the known nonadhesive property of PEO chains. It is very interesting that PU-PEO-SO3 exhibited the highest adsorption of albumin and the lowest adsorption of IgG. Therefore, it may be concluded that such adsorption behaviors of proteins to PU-PEO-SO3 contribute to improved blood compatibility.

To examine the biostability and calcification-resistance of polyurethanes (PUs), the surface of PU was grafted with hydrophobic perfluorodecanoic acid (PFDA) (PU-PFDA), hydrophilic polyethyleneoxide (PEO) (PU-PEO1000), and further negatively charged sulfonate groups (PU-PEO1000-SO3). An in vivo animal test was conducted by subcutaneous implantation in rats during 2, 4 and 6 months. A scanning electron microscope study demonstrated that the degree of surface cracking on explanted PUs was increased in the following order: PU-PFDA > PU > PU-PEO1000 > PU-PEO1000-SO3. In the results of energy dispersive x-ray analysis and inductively coupled plasma atomic emission spectrometry, the deposition of calcium was found abundantly, but that of phosphorus was hardly in existence in all implanted PUs, suggesting that this calcium compound is not a hydroxyapatite. The calcium contents, regardless of implantation time, were also increased in the same order (PU-PFDA > PU > PU-PEO1000 > PU-PEO1000-SO3). After 6 months implantation, no severe tissue reactions were observed and calcification almost occurred on polymer surfaces in all implants. Such superior biostability and anticalcification of PU-PEO1000-SO3 might be attributed to synergistic effects of its excellent surface smoothness, sulfonate acid (SO3−) groups, nonadhesive and mobile PEO, and the high hydrophilicity and enhanced blood compatibility. Therefore, PU-PEO1000-SO3 is promising as biostable and calcification-resistant biomaterial. © 1993 John Wiley & Sons, Inc.

Surfaces of commercial polyurethanes (PUs) were modified by poly(ethylene oxide) (PEO) grafting and/or heparin immobilization for long-term biomedical applications. PU surfaces were treated with diisocyanate and then reacted with PEO or heparin. The heparin immobilized by various methods on the PU surface was very stable, with concentrations of 1.45-1.84 micrograms/cm2. Surface structure and characteristics of each modified PU were examined by performing the following surface analyses: attenuated total reflection infrared (ATR-IR), electron spectroscopy for chemical analysis (ESCA), scanning electron microscopy (SEM), and dynamic contact angle measurements. The reaction scheme and surface chemical structure of modified PUs were confirmed by ATR-IR and ESCA, respectively. SEM results showed that the PU-PEO surface was very smooth and that the smoothness of the heparinized PU surfaces varied, depending upon the solvent and coupling agent used in the process. The hydrophilicity of the surface was significantly increased after PEO grafting or heparin immobilization. Increase in the chain length of the grafted PEO resulted in significant increases in hydrophilicity and surface mobility.