Hsu-Wei Fang

Distinct gene expression of receptor activator of nuclear factor-κB and rank ligand in the inflammatory response to variant morphologies of UHMWPE particles

Recent studies have examined the role of wear debris-induced bone resorption in the aseptic loosening of orthopedic prostheses. Research has shown that inflammation depends not only on the amount of particulate debris, but also the shape and size of the accumulated wear particles. Our previous studies have demonstrated that variant shapes of ultra-high molecular weight polyethylene (UHMWPE) particles induce diverse cellular and apoptotic responses in a murine inflammation model. Since enhanced osteoclastogenesis is recognized as a hallmark of bone loss in prosthetic loosening, we have now investigated the gene expression of receptor activator of nuclear factor-kappaB (RANK) and receptor activator of nuclear factor-kappaB ligand (RANKL) during the inflammatory response to different shapes of UHMWPE particles. Two shapes of UHMWPE particles (globular or elongated) were implanted in established air pouches on BALB/c mice, and pouches harvested 7 days after stimulation with UHMWPE particles. Gene levels of RANK, RANKL, TNFalpha, IL-1beta, and cathepsin K (CK) were quantified by real time RT-PCR, and TRAP staining of pouch membrane was used to evaluate osteoclastogenesis. We found that (i) elongated particles generated significantly higher RANK and RANKL gene expression than globular particles in pouch tissue; (ii) elongated particles provoked significantly higher IL-1beta and TNFalpha gene expression; (iii) a positive association was found between tissue inflammation status and the gene level of RANK/RANKL; and (iv) elongated particles stimulated significantly higher CK gene expression in comparison with globular particles. Histology revealed that clusters of TRAP+ cells were located in regions in contact with elongated particles. Overall, these data suggest that the morphology of wear debris may be a critical factor in the pathogenesis of prosthetic loosening.

Dose-dependent effect of Lactobacillus rhamnosus on quantitative reduction of faecal rotavirus shedding in children

Beneficial effects of probiotics in acute infectious diarrhoea in children are mainly seen in watery diarrhoea and viral gastroenteritis. Lactobacillus rhamnosus, one the most extensively studied probiotic strains, is effective in shortening courses of acute diarrhoea in children. However, the dose-dependent effect of Lactobacillus upon quantification of faecal rotavirus shedding in humans remains little known. Thus, an open-label randomized trial in 23 children with acute rotaviral gastroenteritis was undertaken by randomly allocating patients to receive one of the three regimens for 3 days: daily Lactobacillus rhamnosus 35 (Lcr35) with 0 CFU/day to six patients in the control group, 2 x 10(8) CFU/day to nine patients in the low-dose group, and 6 x 10(8) CFU/day to eight patients in the high-dose group. Faecal samples were collected before and after the 3-day regimen for measurements of rotavirus concentrations by ELISA. There was no statistically significant change in faecal rotavirus concentrations in either the control group (119.2 x 10(5) particles/ml vs. 23.7 x 10(5) particles/ml, p = 0.075) or the low-dose group (36.1 x 10(5) particles/ml vs. 73.5 x 10(5) particles/ml, p = 0.859). However, the high-dose group had a significant reduction of faecal rotavirus concentration (64.2 x 10(5) particles/ml vs. 9.0 x 10(5) particles/ml, p = 0.012). Without any exception, the faecal rotavirus concentrations of all eight patients in the high-dose Lcr35 group declined by 86% after 3 days when compared with those before Lcr35 administration. In conclusion, this is the first report to provide quantitative evidence of the dose-dependent effect of Lactobacillus rhamnosus, a minimal effective dose of 6 x 10(8) CFU for 3 days, upon the faecal rotavirus shedding in paediatric patients.

Quantitative analysis of osteoblast‐like cells (MG63) morphology on nanogrooved substrata with various groove and ridge dimensions

Nanotextured silicon substrata with parallel ridges separated by grooves with equal width from 90 to 500 nm, were fabricated by electron beam lithography and dry etching techniques. Osteoblast-like cells, MG-63, were cultured on the sterilized nanopatterned substrata for 4 or 24 h, and then imaged by scanning electron microscopy. The influence of substrate topography on cell morphology was analyzed by image software. We found the initially cells spread faster on the nanopatterned surfaces than on the flat surface, suggesting that surface anisotropic feature facilitates initial cell extension along its direction. However, because of inhibition of cell lateral expansion across nanogrooved surfaces, the cells on the nanogrooved surface did not further expand laterally, and cell spreading area was less than that on the flat surface after 24 h of incubation. Cells elongated and aligned along the direction of grooves on all the nanopatterned substrata. Furthermore, fluorescence staining of cell nuclei indicated that the nuclei of the cells cultured on the nanopatterned surfaces also displayed a more elongated and aligned morphology along the direction of the grooves. Since cell shape and orientation influence cell functions and alignment of extracellular matrix secreted by cells, our results may provide the information regarding responses of osteoblasts to the nanostructure of collagen fibrils, and benefit bone tissue engineering and surface design of orthopedic implants.

Tissue engineering-based cartilage repair with mesenchymal stem cells in a porcine model†‡

This in vivo pilot study explored the use of mesenchymal stem cell (MSC) containing tissue engineering constructs in repair of osteochondral defects. Osteochondral defects were created in the medial condyles of both knees of 16 miniature pigs. One joint received a cell/collagen tissue engineering construct with or without pretreatment with transforming growth factor β (TGF‐β) and the other joint from the same pig received no treatment or the gel scaffold only. Six months after surgery, in knees with no treatment, all defects showed contracted craters; in those treated with the gel scaffold alone, six showed a smooth gross surface, one a hypertrophic surface, and one a contracted crater; in those with undifferentiated MSCs, five defects had smooth, fully repaired surfaces or partially repaired surfaces, and one defect poor repair; in those with TGF‐β‐induced differentiated MSCs, seven defects had smooth, fully repaired surfaces or partially repaired surfaces, and three defects showed poor repair. In Pineda score grading, the group with undifferentiated MSC, but not the group with TGF‐β‐induced differentiated MSCs, had significantly lower subchondral, cell morphology, and total scores than the groups with no or gel‐only treatment. The compressive stiffness was larger in cartilage without surgical treatment than the treated area within each group. In conclusion, this preliminary pilot study suggests that using undifferentiated MSCs might be a better approach than using TGF‐β‐induced differentiated MSCs for in vivo tissue engineered treatment of osteochondral defects.

Molecular Dynamics Simulations to Investigate the Aggregation Behaviors of the Aß(17–42) Oligomers

Abstract The amyloid β-peptides (Aßs) are the main protein components of amyloid deposits in Alzheimers disease (AD). Detailed knowledge of the structure and assembly dynamics of Aß is important for the development of properly targeted AD therapeutics. So far, the process of the monomeric Aß assembling into oligomeric fibrils and the mechanism underlying the aggregation process remain unclear. In this study, several molecular dynamics simulations were conducted to investigate the aggregation behaviors of the Aß(17–42) oligomers associated with various numbers of monomers (dimer, trimer, tetramer, and pentamer). Our results showed that the structural stability of the Aß(17–42) oligomers increases with increasing the number of monomer. We further demonstrated that the native hydrophobic contacts are positive correlated with the ß-sheet contents, indicating that hydrophobic interaction plays an important role in maintaining the structural stability of the Aß(17–42) oligomers, particularly for those associated with more monomers. Our results also showed that the stability of the C-terminal hydrophobic segment 2 (residues 30–42) is higher than that of the N-terminal hydrophobic segment 1 (residues 17–21), suggesting that hydrophobic segment 2 may act as the nucleation site for aggregation. We further identified that Met35 residue initiates the hydrophobic interactions and that the intermolecular contact pairs, Gly33-Gly33 and Gly37-Gly37, form a stable “molecular notch”, which may mediate the packing of the ß-sheet involving many other hydrophobic residues during the early stage of amyloid-like fibril formation.

Cartilage fragments from osteoarthritic knee promote chondrogenesis of mesenchymal stem cells without exogenous growth factor induction.

Extracellular matrix (ECM) is thought to participate significantly in guiding the differentiation process of mesenchymal stem cells (MSCs). In this study, we hypothesized that cartilage fragments from osteoarthritic knee could promote chondrogenesis of MSCs. Nonworn parts of cartilage tissues were obtained during total knee arthroplasty (TKA) surgery. Cartilage fragments and MSCs were wrapped into fibrin glue; and the constructs were implanted subcutaneously into nude mice. Histological analysis showed neocartilage‐like structure with positive Alcian blue staining in the cartilage fragment–fibrin–MSC constructs. However, constructs with only MSCs in fibrin showed condensed appearance like MSCs in the pellet culture. Gene expression of type II collagen in the constructs with 60 mg cartilage fragments were significantly elevated after 4 weeks of implantation. Conversely, the constructs without cartilage fragments failed to express type II collagen, which indicated MSCs did not differentiate into a chondrogenic lineage. In conclusion, we demonstrated the effect of cartilage fragments from osteoarthritic knee in promoting chondrogenic differentiation of MSCs. This may be a favorable strategy for MSC chondrogenesis without exogenous growth factor induction.

Conformational and adsorptive characteristics of albumin affect interfacial protein boundary lubrication: from experimental to molecular dynamics simulation approaches.

The lifetime of artificial joints is mainly determined by their biotribological properties. Synovial fluid which consists of various biological molecules acts as the lubricant. Among the compositions of synovial fluid, albumin is the most abundant protein. Under high load and low sliding speed articulation of artificial joint, it is believed the lubricants form protective layers on the sliding surfaces under the boundary lubrication mechanism. The protective molecular layer keeps two surfaces from direct collision and thus decreases the possibility of wear damage. However, the lubricating ability of the molecular layer may vary due to the conformational change of albumin in the process. In this study, we investigated the influence of albumin conformation on the adsorption behaviors on the articulating surfaces and discuss the relationship between adsorbed albumin and its tribological behaviors. We performed the friction tests to study the effects of albumin unfolding on the frictional behaviors. The novelty of this research is to further carry out molecular dynamics simulation, and protein adsorption experiments to investigate the mechanisms of the albumin-mediated boundary lubrication of arthroplastic materials. It was observed that the thermal processes induce the loss of secondary structure of albumin. The compactness of the unfolded structure leads to a higher adsorption rate onto the articulating material surface and results in the increase of friction coefficient.

Molecular Dynamics Simulations to Gain Insights into the Stability and Morphologies of K3 Oligomers from β2-microglobulin

Abstract β2-Microglobulin (β2-m) forms amyloid fibrils in patients undergoing long-term hemodialysis. K3 peptide, a Ser20-Lys41 fragment of β2-m, has been known to form fibrils over a wide range of pH and solvent conditions. Recent solid-state NMR has revealed that K3 oligomer adopts a parallel U-shaped β-strand-turn-β-strand motif. In order to investigate the stability and morphologies of K3 oligomers with different sizes (dimer, trimer, and tetrameri and organizations (single and double layers), several all-atom molecular dynamics simulations were conducted at 310 K and pH 2 in water and 2,2,2-trifluoroethanol (TFE). For single-layered organizations, our results show that TFE destabilizes the stacking of K3 peptides due to the fact that TFE weakens the intermolecular hydrophobic interactions of K3 oligomers. In addition, we also identified that the loop region is stabilized by the hydrophobic cluster involving resides Y7, Fll, and I16. Our results further suggest that K3 tetramer is a potential minimal nucleus seed for the formation of K3 protofibrils. For dou-ble-layered organizations in water, our data demonstrate that K3 peptides can form various stable assemblies through different interfacial arrangements, such as NN, NC, and CC, by different driving forces. We further propose that the stacking of different interfaces between two facing β-sheets of K3 peptides could be related to different fibril morphologies, which is in good agreement with the previous experimental results, showing that K3 protofibrils associated to formed mature fibrils with a wide range of diameters from 4 to 15 nm when they were transferred from 20% (v/v) TFE to aqueous solution.

Dip Coating Assisted Polylactic Acid Deposition on Steel Surface: Film Thickness Affected by Drag Force and Gravity

Article history: Dip coating process has the Received 9 February 2008 Accepted 10 April 2008 Available online 16 April 2008

Chondrogenesis From Immortalized Human Mesenchymal Stem Cells: Comparison Between Collagen Gel and Pellet Culture Methods

Human mesenchymal stem cells (hMSCs) can differentiate into cells of connective tissue lineages, including cartilage. To overcome the limiting autogenous chondrocyte populations available in cartilage repair, various methods have been developed to maximize chondrogenesis of hMSCs in vitro, most of which use cells derived from primary culture. In this study, we compared chondrogenesis of immortalized hMSCs embedded in collagen gel to those grown in pellet culture. The hMSCs in collagen scaffolds expressed more glycosaminoglycan than those in pellet culture. Real-time reverse transcriptase-polymerase chain reaction (RT-PCR) analysis demonstrated that the expression of genes encoding sox-9, aggrecan, and types I and II collagen increased in pellet culture over time. However, in the collagen cultures, only type II collagen and aggrecan expression increased over time, whereas sox-9 expression remained unchanged and type I collagen expression decreased. These results indicate that the immortalized hMSC line is a promising tool for further in vitro chondrogenic studies.