Shyh Ming Kuo

Asymmetric Chitosan Membrane Containing Collagen I Nanospheres for Skin Tissue Engineering

A biodegradable chitosan membrane with an asymmetric structure, seeded with fibroblasts, was prepared as a novel skin substitute. Chitosan was cross-linked with genipin and then frozen and lyophilized to yield a porous asymmetric membrane (CG membrane). Nanoscale collagen I particles were injected into the CG membrane to form an asymmetric CGC membrane. The results reveal that the CG membrane treated with 0.125 wt % of genipin had a higher swelling ratio, porosity, and pore size. After 7 d of dynamic culture, many of the adhered cells exhibited a flat morphology and well spread on the surface of CGC membrane treated with 0.125 wt % of genipin. In animal studies, the CGC membrane seeded with fibroblasts and grown in vitro for 7 d was more effective than both gauze and commercial wound dressing, Suile, in healing wounds. An in vivo histological assessment indicated that covering the wound with the asymmetric CGC membrane resulted in its epithelialization and reconstruction. CGC membrane, thus, has great potential in skin tissue engineering.

Three Dimensional Collagen Scaffold Promotes Intrinsic Vascularisation for Tissue Engineering Applications

Here, we describe a porous 3-dimensional collagen scaffold material that supports capillary formation in vitro, and promotes vascularization when implanted in vivo. Collagen scaffolds were synthesized from type I bovine collagen and have a uniform pore size of 80 μm. In vitro, scaffolds seeded with primary human microvascular endothelial cells suspended in human fibrin gel formed CD31 positive capillary-like structures with clear lumens. In vivo, after subcutaneous implantation in mice, cell-free collagen scaffolds were vascularized by host neovessels, whilst a gradual degradation of the scaffold material occurred over 8 weeks. Collagen scaffolds, impregnated with human fibrinogen gel, were implanted subcutaneously inside a chamber enclosing the femoral vessels in rats. Angiogenic sprouts from the femoral vessels invaded throughout the scaffolds and these degraded completely after 4 weeks. Vascular volume of the resulting constructs was greater than the vascular volume of constructs from chambers implanted with fibrinogen gel alone (42.7±5.0 μL in collagen scaffold vs 22.5±2.3 μL in fibrinogen gel alone; p<0.05, n = 7). In the same model, collagen scaffolds seeded with human adipose-derived stem cells (ASCs) produced greater increases in vascular volume than did cell-free collagen scaffolds (42.9±4.0 μL in collagen scaffold with human ASCs vs 25.7±1.9 μL in collagen scaffold alone; p<0.05, n = 4). In summary, these collagen scaffolds are biocompatible and could be used to grow more robust vascularized tissue engineering grafts with improved the survival of implanted cells. Such scaffolds could also be used as an assay model for studies on angiogenesis, 3-dimensional cell culture, and delivery of growth factors and cells in vivo.

Treatment of Acute Unstable Distal Clavicle Fractures with Single Coracoclavicular Suture Fixation

Distal clavicular fractures are less common than fractures involving the middle third of the clavicle. For Neer type IIb distal clavicular unstable fractures associated with disruption of the coracoclavicular ligament, surgical treatment is indicated because of the high risk of nonunion. Various surgical methods can be found in the literature, but no gold standard has been established. We treated 29 consecutive adult patients with unstable distal clavicular fracture with single coracoclavicular suture fixation by using single Mersilene tape (Ethicon, Somerville, New Jersey) and without repair of the torn coracoclavicular ligament or hardware implantation.Twenty-eight patients were followed for at least 46 months (mean, 57.3 months). All fractures healed without further treatment, with a mean time to union of 14.3 weeks. There was no major morbidity, but 2 minor complications occurred. One patient experienced a frozen shoulder on the treated side postoperatively. After adequate rehabilitation, the symptom resolved without any complications at final follow-up. Another patient reported uncomfortable skin tenting due to subcutaneous protrusion of the suture node of the Mersilene tape. After simple subcutaneous surgical removal of the node under local anesthesia, the discomfort resolved. Mean University of California Los Angeles shoulder rating score was 34 (range, 29-35). Twenty patients had excellent results and 8 had good results. All patients resumed their previous levels of activity.

Plasma-modified Nylon Meshes as Supports for Cell Culturing

The polymeric surfaces of three commercially available nylon films with mesh openings of 5 microns, 10 microns and 20 microns were treated with anhydrous ammonia gaseous plasma. Cells cultured on the plasma-treated nylon films have higher proliferation rate and assume morphology distinct from those cultured on the unmodified films. Of the three plasma-modified membranes, the one with 5 microns mesh openings supported a largest population of cell growth. The plasma-treated nylon meshes provided a stronger anchorage for the collagen matrices formed within the mesh openings. Application of this collagen/nylon meshes for cell culturing is demonstrated.

Evaluation of the ability of xanthan gum/gellan gum/hyaluronan hydrogel membranes to prevent the adhesion of postrepaired tendons

After tendon-repair surgery, adhesion between the surgical tendon and the synovial sheath is often presented resulting in poor functional repair of the tendon. This may be prevented using a commercially available mechanical barrier implant, Seprafilm, which is composed of hyaluronan (HA) and carboxymethyl cellulose hydrogels. In a rat model, prepared membranes of various compositions of gellan gum (GG), xanthan gum (XG) and HA as well as Seprafilm were wrapped around repaired tendons and the adhesion of the tendons was examined grossly and histologically after 3 weeks of healing. Certain formulations of the XG/GG/HA hydrogel membranes reduced tendon adhesion with equal efficacy but without reducing the tendon strength compared to Seprafilm. The designed membranes swelled rapidly and blanketed onto the tendon tissue more readily and closely than Seprafilm. Also they degraded slowly, which allowed the membranes to function as barriers for extended periods.

Nano-sized collagen I molecules enhanced the differentiation of rat mesenchymal stem cells into cardiomyocytes

The aim of this study was to investigate the ability of nano-sized collagen I molecules (nanoparticles or nanofibrils) and a 5-azacytidine (5-aza) treatment to enhance the differentiation of rat mesenchymal stem cells (MSCs) toward a cardiomyogenic phenotype in vitro. Second passaged MSCs were cocultured with nano-sized collagen I molecules for 24 h and then treated with 10 μM 5-aza for 24 h. The results demonstrated that the size of the cells increased significantly and acquired a flattened, triangular-shaped morphology after treatment with nano-sized collagen I molecules and 5-aza. The cells are connecting with adjoining cells by forming myotube-like structures. Additional treatment of the MSCs with nano-sized collagen I fibrils significantly increased two transcription factors GATA-4 (12.6-fold increase) and Nkx2.5 (4.8-fold increase) expressions compared with MSC groups treated only with 5-aza at 3-day culturing. Furthermore, MSCs pretreated with nano-sized collagen fibrils significantly increased the expressions of cardiac genes of troponin I, β-myosin heavy chain, and cardiac α-actin compared with MSC groups treated only with 5-aza (all, p < 0.01 or better). These results indicate that culturing MSCs with nano-sized collagen I molecules, which may act as scaffolds or soluble protein ingredients, leads to alterations in gene expression and affects the differentiation fate induced with 5-aza.

Preparation and characterization of hyaluronan/collagen II microspheres under an electrostatic field system with disc electrodes.

Collagen II and hyaluronan are the two major components of the native extracellular matrix (ECM). Both biopolymers are responsible for providing the associated tissues with tensile strength, and also serve as a structural scaffold for cell adhesion and growth. Over the years, many researchers have focused on the preparation and evaluation of man-made ECM comprising the two polymers in the form of a membrane for chondrocyte culture applications. Here, a simple and in situ method, involving the injection of the hyaluronan/collagen II (HA/Col II) mixture solution through a pair of hollow-centered parallel disc electrodes (HCPDEs) of a high-voltage electrostatic field system, was developed and employed to prepare HA/Col II microspheres in watery phase. The HA/Col II microspheres were firmed up by a two-step cross-linking treatment (first by FeCl(3) and then by 1-ethyl-3-(3-dimethyl aminopropyl) carbodimide, EDC) to secure the spherical structure shape. Then, at 37 degrees C, reconstitution treatment of the Col II molecules was conducted to further strengthen the microspheres. Depending on treatment conditions, the resulting series of HA/Col II microspheres all exhibited good sphericity in the range of 486+/-43 to 679+/-24microm in diameter. Furthermore, the ratio and amount of HA/Col II in the mixture solutions would affect the morphological structure and basic characteristics, including mechanical strength, thermal properties and water content. In the preliminary study, the HA/Col II microspheres have shown to provide favorable ECM characteristics, with appropriate mechanical strength, and exhibited a 3D inclination.

Enhanced apoptotic effects of dihydroartemisinin-aggregated gelatin and hyaluronan nanoparticles on human lung cancer cells

Recent studies suggest that dihydroartemisinin (DHA), a derivative of artemisinin isolated from the traditional Chinese herb Artemisia annua L., has anticancer properties. Due to poor water solubility, poor oral activity, and a short plasma half-life, large doses of DHA have to be injected to achieve the necessary bioavailability. This study examined increasing DHA bioavailability by encapsulating DHA within gelatin (GEL) or hyaluronan (HA) nanoparticles via an electrostatic field system. Observations from transmission electron microscopy show that DHA in GEL and HA nanoparticles formed GEL/DHA and HA/DHA aggregates that were approximately 30-40 nm in diameter. The entrapment efficiencies for DHA were approximately 13 and 35% for the GEL/DHA and HA/DHA aggregates, respectively. The proliferation of A549 cells was inhibited by the GEL/DHA and HA/DHA aggregates. Fluorescent annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI) staining displayed low background staining with annexin V-FITC or PI on DHA-untreated cells. In contrast, annexin V-FITC and PI stains dramatically increased when the cells were incubated with GEL/DHA and HA/DHA aggregates. These results suggest that DHA-aggregated GEL and HA nanoparticles exhibit higher anticancer proliferation activities than DHA alone in A549 cells most likely due to the greater aqueous dispersion after hydrophilic GEL or HA nanoparticles aggregation. These results demonstrate that DHA can aggregate with nanoparticles in an electrostatic field environment to form DHA nanosized aggregates.

Evaluation of nanoarchitectured collagen type II molecules on cartilage engineering.

Scaffold architecture, including the geometry and dimension of scaffolds, is an important parameter in cell adhesion, migration, proliferation, and differentiation. Following the characterization of collagen type II nanoarchitectured molecules, collagen fibrils (CNFs) and collagen spheres (CNPs) prepared using a high-voltage electric field in our laboratory, we proposed to use these nanoarchitectured molecules to assess their influence on the culturing of chondrocytes in stirred bioreactors. The results demonstrate that chondrocytes rapidly formed more and larger chondrocyte pellets (spheroids) after the addition of nanoarchitectured molecules into the culture medium. The maintenance of chondrocytes with round morphology and increased glycosaminoglycan secretion indicated that these spheroids contained viable and un-dedifferentiated chondrocytes. No significant increases in DNA content were detected. These results show that the introduction of these molecules did not affect chondrocyte proliferation during a 3-day culture period. After the addition of CNPs and CNFs into the culture medium, the expression levels of collagen type II and aggrecan genes in chondrocytes increased significantly as demonstrated by real-time PCR analysis. Interestingly, chondrocytes exhibited distinct collagen type II and aggrecan gene expression profiles in culture with CNPs and CNFs. The aggrecan gene expression level of the chondrocytes was 2.5-fold greater following CFN addition than following the addition of CNPs. In contrast, the collagen type II expression level of the chondrocytes was 2.2-fold greater following the addition of CNPs than following the addition of CNFs. The chondrocyte pellets rapidly restored defects in articular cartilage during a 1-month implantation period in a rabbit model.

EVALUATION OF CHITOSAN/CaSO4/PLATELET-RICH PLASMA MICROSPHERE COMPOSITES AS ALVEOLUS OSTEOGENESIS MATERIAL

Periodontal disease is the manifestation of serious bacteria infection that may extend to the gingival, periodontal ligaments, and alveolus bone. One commonly administrated treatment is the debridement therapy with the removal of infected area including the soft and hard lesion tissues. In some critical case, osteogenetic materials are being filled into the defective voids to improve the regeneration of slow-growing bony tissues. In attempt to improve bone regeneration, chitosan microsphere composites embedded with two osteogenesis beneficial ingredients, CaSO4 and platelet-rich plasma (PRP), were fabricated by using a high voltage electrostatic field system. Three groups, chitosan/CaSO4 microspheres (Group A), chitosan/CaSO4 microspheres mixed with thrombin (Group B), and chitosan/CaSO4/PRP microspheres mixed with thrombin (Group C) were prepared. And, these chitosan-based composites were evaluated together with a control group in pig oral model for the bone regeneration study. The chitosan/CaSO4/PRP microsphere composites, exhibiting good sphericity, were in the range of 457.5 ± 59.3 μm in diameter. Defects filled with Group B and Group C showed increases in new bone formation along with fibrous tissue regeneration as compared to that filled with Group A. The Massons Trichrome stain observations suggested more abundant presence of fibrous collagen matrices around the defects after implanted with Group B over that of Group C microsphere composites. The preparation of chitosan/CaSO4-based microspheres was straight forward by using high voltage electrostatic field system. Furthermore, Chitosan/CaSO4-based microspheres with thrombin could be used successfully in regenerating new bone around the alveolus bone area.