Shwu Jen Chang

The discrimination of type I and type II collagen and the label-free imaging of engineered cartilage tissue

Using excitation polarization-resolved second harmonic generation (SHG) microscopy, we measured SHG intensity as a function of the excitation polarization angle for type I and type II collagens. We determined the second order susceptibility (χ((2))) tensor ratios of type I and II collagens at each pixel, and displayed the results as images. We found that the χ((2)) tensor ratios can be used to distinguish the two types of collagen. In particular, we obtained χ(zzz)/χ(zxx) = 1.40 ± 0.04 and χ(xzx)/χ(zxx) = 0.53 ± 0.10 for type I collagen from rat tail tendon, and χ(zzz)/χ(zxx) = 1.14 ± 0.09 and χ(xzx)/χ(zxx) = 0.29 ± 0.11 for type II collagen from rat trachea cartilage. We also applied this methodology on the label-free imaging of engineered cartilage tissue which produces type I and II collagen simultaneously. By displaying the χ((2)) tensor ratios in the image format, the variation in the χ((2)) tensor ratios can be used as a contrast mechanism for distinguishing type I and II collagens.

Surface plasmon resonance biosensor with high anti-fouling ability for the detection of cardiac marker troponin T

Designing a surface recognition layer with high anti-fouling ability, high affinity, and high specificity is an important issue to produce high sensitivity biosensing transducers. In this study, a self-assembled monolayer (SAM) consisting of a homogeneous mixture of oligo(ethylene glycol) (OEG)-terminated alkanethiolate and mercaptohexadecanoic acid (MHDA) on Au was employed for immobilizing troponin T antibody and applied in detecting cardiac troponin T by using surface plasmon resonance (SPR). The mixed SAM showed no phase segregation and exhibited human serum albumin resistance, particularly with an antibody-immobilized surface. X-ray photoemission spectra revealed that the chemical composition ratio of OEG to the mixed SAM was 69% and the OEG packing density was 82%. The specific binding of troponin T on the designed surface indicated a good linear correlation (R=0.991, P<0.0009) at concentrations lower than 50 μgmL(-1) with the limit of detection of 100 ngmL(-1) using a SPR measuring instrument. It is concluded that the mixed SAM functions as designed since it has high detection capability, high accuracy and reproducibility, as well as shows strong potential to be applied in rapid clinical diagnosis for label-free detection within 2 min.

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.

Microcapsules prepared from alginate and a photosensitive poly(L-lysine).

A photosensitive polymer, alpha-phenylcinnamylideneacetylated poly(L-lysine), was synthesized and characterized. This photosensitive poly(L-lysine) had 10% of its lysine residues reacted with alpha-phenylcinnamylidene acetyl group and displayed an absorption maximum at 329 nm. The photosensitive poly(L-lysine) was used for the preparation of microcapsules. The capsules formed from this photosensitive poly(L-lysine) and alginate were strengthened significantly by light irradiation. The photo cross-linked capsular membrane was permeable to proteins with mass transfer rate in the descending order: cytochrome C, myoglobin. and serum albumin. GH3 (a rat pituitary tumor cell line) cells were encapsulated and cultured with this microencapsulation system. The cells proliferated to a density of about 4 x 10(5) cells ml(-1) in the capsules after 6 days cultivation.

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.

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.

Amination of polycarbonate surface and its application for cell attachment

The polycarbonate sheet was modified with ammonia gaseous plasma and characterized by the contact angle measurement and ESCA analysis. The contact angles decreased significantly from 77 degrees to about 20 degrees-40 degrees, indicate that the polycarbonate sheet become more hydrophilic after plasma treatment. The ESCA analysis results showed that the hydrophilicity was mainly derived from the amino groups on the modified surface. In this study, a flow-chamber system was also constructed to evaluate the 3T3 fibroblast cells attachment phenomena on these modified sheets. Before the experimental run, the parameters of inoculated cell number and cell passage were examined previously. The results revealed that these two parameters are independent in shear experiment. And besides, 3-hours plating time has the better adhering fraction. The experimental results showed that the 3T3 fibroblast cells adhesion strength increased significantly on the plasma modified sheet.