Hwal Suh

Characterization of porous collagen/hyaluronic acid scaffold modified by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide cross-linking

In order to develop a scaffolding material for tissue regeneration, porous matrices containing collagen and hyaluronic acid were fabricated by freeze drying at -20 degrees C, -70 degrees C or -196 degrees C. The fabricated porous membranes were cross-linked using 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC) in a range of 1-100 mM concentrations for enhancing mechanical stability of the composite matrix. Scanning electron microscope (SEM) views of the matrices demonstrated that the matrices obtained before cross-linking process had interconnected pores with mean diameters of 40, 90 or 230 microm and porosity of 58-66% according to the freezing temperature, and also the porous structures after cross-linking process were retained. The swelling test and IR spectroscopic measurement of different cross-linked membranes were carried out as a measure of the extent of cross-linking. The swelling behavior of cross-linked membranes showed no significant differences as cross-linking degree increased. FT-IR spectra showed the increase of the intensity of the absorbencies at amide bonds (1655, 1546, 1458 cm(-1)) compared to that of CH bond (2930 cm(-1)). In enzymatic degradation test, EDC treated membranes showed significant enhancement of the resistance to collagenase activity in comparison with 0.625% glutaraldehyde treated membranes. In cytotoxicity test using L929 fibroblastic cells, the EDC-cross-linked membranes demonstrated no significant toxicity.

Bacterial adhesion on PEG modified polyurethane surfaces

Polyurethane surface was modified with poly(ethylene glycol) (mol. wt. 1000, PEG1k) carrying terminal hydroxyl, amino and sulfonate groups, poly(ethylene glucol) (mol. wt. 3350, PEG3.4k) and PEG3.4k-Heparin, respectively. These surfaces were investigated for bacterial adhesion using S. epidermidis and E. coli in tryptic soya broth (TSB), brain heart infusion (BHI), and human plasma. All PEG modified surfaces reduced bacterial adhesion significantly and the adhesion level differs depending on surfaces as well as media. In the case of PEG1k surfaces, no reduction of S. epidermidis adhesion was demonstrated in TSB media, regardless of terminal functional groups of PEG1k. However, adhesion in plasma was reduced to the different degree, depending on terminal groups of PEG1k (least adhesion on sulfonated PEG surface). Relatively longer PEG surface (PEG3.4k) and PEG3.4k-heparin surface minimized bacterial adhesion in both media. In the case of E. coli adhesion, significant reduction in adherent bacteria was observed on all PEG1k, PEG3.4k, and PEG-heparin surfaces in both media compared to controls. In contrast, no reduction in bacterial adhesion was demonstrated on poly(propylene glycol) (PPG1k) grafted PU surface as compared to control PU. These results suggest that surface modification with PEG1k-SO3, PEG3.4k and PEG3.4k-heparin seems to be effective for prevention of bacterial adhesion and subsequent infection.

Biological characterization of EDC-crosslinked collagen-hyaluronic acid matrix in dermal tissue restoration.

Porous collagen matrices crosslinked with various amounts of hyaluronic acid (HA) by 1-ethyl-3-(3-dimethyl aminopropyl)carbodiimide (EDC) were developed as scaffolds for dermal tissue regeneration. The effect of HA on cells in accordance with HA concentrations in the collagenous matrices was investigated using cultures of fetal human dermal fibroblasts, and the effect of EDC-crosslinked collagen-HA matrix on wound size reduction was also evaluated in vivo. Scanning electron microscopic views of the matrices demonstrated that all of the collagen-HA matrices had interconnected pores with mean diameters of 150-250 microm. An HA matrix retention test showed that the concentration of HA decreased slowly after an initial rapid decrease over 24h. Fetal human dermal fibroblasts adhered well to all of the collagen-based matrices as compared with the Porous polyurethane matrix used as a control. An 3-(4,5-dimethylthiazolyl)-2,5-diphenyltetrazolium bromide based proliferation test and the hematoxylin and eosin staining of a 2 week cultured matrix showed that the proliferation of fibroblasts was enhanced on a 9.6% HA contained collagen matrix. No significant difference was in terms of fibroblast migration into the various types of scaffolds as HA content was increased. In vivo testing showed that dermis treated with collagen or collagen-HA matrix was thicker than the control, and epithelial regeneration was accelerated, and collagen synthesis increased. However, no significant effect of HA on wound size reduction was found.

Sustained release of ascorbate-2-phosphate and dexamethasone from porous PLGA scaffolds for bone tissue engineering using mesenchymal stem cells

The purpose of this research was to develop porous poly(D,L-lactide-co-glycolide) (PLGA) scaffolds from which ascorbate-2-phosphate (AsAP) and dexamethasone (Dex) are continuously released for a month for osteogenesis of mesenchymal stem cells for bone tissue engineering. Porous PLGA matrices containing AsAP and Dex were prepared by solvent casting/particulate leaching method. In vitro release and water uptake studies were performed in Dulbeccos phosphate buffered saline at 37 degrees C and 15 rpm. Drug loading and release rates were determined by high performance liquid chromatography. Release studies of Dex and AsAP showed that, after an initial burst release lasting 4 and 9 days, respectively, release rates followed zero order kinetics with high correlation coefficients at least until 35 days. Incorporation of AsAP into the scaffolds increased the release rates of Dex and AsAP, and the scaffold water uptake. When mesenchymal stem cells (MSCs) were cultured in the AsAP and Dex containing scaffolds in vitro, the amount of mineralization was significantly higher than in control scaffolds. In conclusion, AsAP and Dex were incorporated into porous PLGA scaffolds and continuously released over a month and osteogenesis of MSCs was increased by culture in these scaffolds.

Development of collagenase-resistant collagen and its interaction with adult human dermal fibroblasts

Collagen is regarded as one of the most useful biomaterials. The excellent biocompatibility and safety due to its biological characteristics, such as biodegradability and weak antigenecity, made collagen the primary source in biomedical application. Collagen has been widely used in the crosslinked form to extend the durability of collagen. The chemical treatment influences the structural integrity of collagen molecule resulting in the loss of triple helical characteristic. The structural characteristic of collagen is importantly related to its biological function for the interaction with cell. In this study, structural stability of collagen was enhanced thought EGCG treatment, resulting in high resistance against degradation by bacterial collagenase and MMP-1, which is confirmed by collagen zymography. The triple helical structure of EGCG-treated collagen could be maintained at 37 degrees C in comparison with collagen, which confirmed by CD spectra analysis, and EGCG-treated collagen showed high free-radical scavenging activity. Also, with fibroblasts culture on EGCG-treated collagen, the structural stability of EGCG-treated collagen provided a favorable support for cell function in cell adhesion and actin filament expression. These observations underscore the need for native, triple helical collagen conformation as a prerequisite for integrin-mediated cell adhesion and functions. According to this experiment, EGCG-treated collagen assumes to provide a practical benefit to resist the degradation by collagenase retaining its structural characteristic, and can be a suitable biomaterial for biomedical application.

Protective effects of green tea polyphenol against reactive oxygen species-induced oxidative stress in cultured rat calvarial osteoblast.

The injurious effects of reactive oxygen species on osteoblasts and the potential protective role played by green tea polyphenols (GtPP) were investigated using primarily cultured rat calvarial osteoblasts. Oxidative stress was induced in cultured osteoblasts, either by adding 100 mmol/L H2O2 or by the action of 40 U/L xanthine oxidase (XO) in the presence of xanthine (250 μmol/L). After incubation, the cellular viability, function and morphology were evaluated. Both treatments produced a significant reduction in osteoblast viability, as assessed by a two-colored fluorescence staining method combined with flow cytometric analysis and MTT assay. A significant reduction in the alkaline phosphatase activity was observed after H2O2 addition, whereas XO did not have the same effect. On the microscopic observations, the morphological changes and intracellular ultrastructural damages were remarkably induced by both treatments. The H2O2-induced alterations were prevented by pre-incubating the osteoblasts with 200 μg/ml GtPP for 1 h. When the oxidative stress was induced by XO, the cellular viability and morphology was also maintained at the same polyphenol concentration. These results demonstrate that GtPP can act as a biological antioxidant in a cell culture experimental model and protect cells from oxidative stress-induced toxicity.

Corneal Dystrophy-associated R124H Mutation Disrupts TGFBI Interaction with Periostin and Causes Mislocalization to the Lysosome

The 5q31-linked corneal dystrophies are heterogeneous autosomal-dominant eye disorders pathologically characterized by the progressive accumulation of aggregated proteinaceous deposits in the cornea, which manifests clinically as severe vision impairment. The 5q31-linked corneal dystrophies are commonly caused by mutations in the TGFBI (transforming growth factor-β-induced) gene. However, despite the identification of the culprit gene, the cellular roles of TGFBI and the molecular mechanisms underlying the pathogenesis of corneal dystrophy remain poorly understood. Here we report the identification of periostin, a molecule that is highly related to TGFBI, as a specific TGFBI-binding partner. The association of TGFBI and periostin is mediated by the amino-terminal cysteine-rich EMI domains of TGFBI and periostin. Our results indicate that the endogenous TGFBI and periostin colocalize within the trans-Golgi network and associate prior to secretion. The corneal dystrophy-associated R124H mutation in TGFBI severely impairs interaction with periostin in vivo. In addition, the R124H mutation causes aberrant redistribution of the mutant TGFBI into lysosomes. We also find that the periostin-TGFBI interaction is disrupted in corneal fibroblasts cultured from granular corneal dystrophy type II patients and that periostin accumulates in TGFBI-positive corneal deposits in granular corneal dystrophy type II (also known as Avellino corneal dystrophy). Together, our findings suggest that TGFBI and periostin may play cooperative cellular roles and that periostin may be involved in the pathogenesis of 5q31-linked corneal dystrophies.

Type I atelocollagen grafting onto ozone‐treated polyurethane films: Cell attachment, proliferation, and collagen synthesis

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 x 10(-8)mol/cm(2) 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.

Tissue Engineering of the Intervertebral Disc With Cultured Nucleus Pulposus Cells Using Atelocollagen Scaffold and Growth Factors

Study Design. In vitro experiment using rabbit nucleus pulposus (NP) cells seeded in atelocollagen scaffolds under the stimulation of growth factors. Objective. To demonstrate the effect of anabolic growth factors in rabbit NP cells cultured in atelocollagen type I and type II. Summary of Background Data. Atelocollagen provides intervertebral disc (IVD) cells for a biocompatible environment to produce extracellular matrix. IVD cells with exogenous transforming growth factor-beta 1 (TGF-&bgr;1) and bone morphogenetic protein-2 (BMP-2) also render an increase in matrix synthesis. However, the effect of anabolic growth factors in NP cells cultured in atelocollagens was not elucidated before. Methods. Rabbit NP cell was harvested, enzymatically digested, and cultured. The NP cells were seeded to atelocollagen type I and type II scaffolds, and then cultures were exposed to TGF-&bgr;1 (10 ng/mL) and/or BMP-2 (100 ng/mL). DNA synthesis was measured using [4H]-thymidine incorporation. Newly synthesized proteoglycan was measured using [35S]-sulfate incorporation. Reverse transcription-polymerase chain reactions (RT-PCRs) for mRNA expression of aggrecan, collagen type I, collagen type II, and osteocalcin were performed. Results. Rabbit NP cells cultured in atelocollagen type I scaffold showed an increase (1.7 to 2.4-fold) in DNA synthesis in response to TGF-&bgr;1 and/or BMP-2 (P < 0.05), whereas NP cultures in atelocollagen type II demonstrated a 30% increase in DNA synthesis only with combination of both growth factors compared with control (P < 0.05). Rabbit NP cells in atelocollagen type II scaffold with TGF-&bgr;1 and combination of both growth factors exhibited robust 5.3- and 5.4-fold increases in proteoglycan synthesis (P < 0.05), whereas any cultures in atelocollagen type I failed to show any significant increase compared with control. Rabbit NP cells in atelocollagen type I and type II scaffolds with TGF-&bgr;1 and/or BMP-2 demonstrated the upregulation of aggrecan, collagen type I, and collagen type II mRNA expression compared with saline control (P < 0.05). The response in transcriptional level was more robust in atelocollagen type II than in type I. In any event, there is no recognizable expression of osteocalcin (P < 0.05). Conclusion. NP cells in atelocollagens under the stimulation of TGF-&bgr;1 and BMP-2 exhibited anabolic responses in transcriptional and translational levels. Hence, such an approach can provide a suitable engineered tissue for IVD regeneration with potential for robust refurbishment of matrix.

Heparinized bovine pericardium as a novel cardiovascular bioprosthesis

A novel chemical modification of biological tissues was developed by the direct coupling heparin to bovine pericardium (BP). The heparinization involves pretreatment of BP using GA and followed by grafting heparin to BP by the reaction of residual aldehyde and amine group of heparin. BP was modified by direct coupling of heparin and the effect of heparin coupling on calcification was evaluated in vitro and in vivo. Heparinized BP was characterized by measuring shrinkage temperature, mechanical properties, digestion resistance to collagenase enzyme, in vitro cytotoxicity, and in vivo calcification. Thermal and mechanical properties showed that the durability of heparin-treated tissue increased as compared with fresh tissue and GA-treated tissue. Resistance to collagenase digestion revealed that heparin-treated tissue has greater resistance to enzyme digestion than did fresh tissue and GA-treated tissue. Heparinized tissue had shown to be non-cytotoxic, however, relatively high cytotoxicity was observed in the GA-treated tissues due to the release of GA. In vivo calcification study demonstrated much less calcium deposition on heparin-treated BP than GA-treated one. Obtained results attest to the usefulness of heparinized BP for cardiovascular bioprostheses.