Biomedical Engineering: Applications, Basis and Communications

Maintenance of differentiated functional phenotype within in vitro chondrocyte culture requires seeding at high densities with large numbers of cells. However, optimal cell seeding numbers and densities remain elusive due to multiple varying parameters and different methodologies utilized in previous studies. In the current study, we tried to investigate the relationship between cell seeding number and differentiated functional phenotype of in vitro cultured chondrocytes. Varying numbers of primary porcine chondrocytes (0.25, 2.5, 25 and 250 K) were seeded in 96 well-plates and cultured for 4 weeks. Cell proliferation, glycosaminoglycan (GAG) production and gene expression levels of Sox9, aggrecan, COL II and COL I were evaluated. The results showed that GAG content was high in the 0.25 and 25 K groups, gene expression of Sox9 was high in the 2.5, 25 and 250 K groups and expression of COL II was high in the 25 K group, whereas expression of COL I was low in the 0.25, 25 and 250 K groups. It is concluded that the seeding number and density of the 25 K (78 K cells/cm²) group achieved the optimal balance between functional phenotype of individual cells and the total ECM production for in vitro cultured chondrocytes.

Recent development of tissue engineering scaffolds that mimic anatomical structures exhibits a tendency to use rapid prototyping technology, because it can be applied to precisely manufacture the designed objects from the computer-generated model. Among all of rapid prototyping approaches, combining with lithography is characterized with a high throughput of fabrication, especially for the fabrication of polymeric scaffolds. In this study, the aims were to: (1) synthesize the 2-hydroxyethyl methacrylate (HEMA)-capped poly(ethylene glycol) (PEG), which served as the cross-linker of the continuous phase of a poly(lactide-co-glycolide) (PLGA) scaffold and (2) fabricate the composite scaffolds through stereolithography. The synthetic process of the cross-linker was traced, and the end-point of the process was found to lie in 3 to 4 h depending on the molecular weight of the PEG used. The chemical structure of the cross-linker was found to be linear and symmetric to PEG and with a 1:2 molar ratio of PEG and HEMA. It was anticipated to form an interpenetrating network upon irradiating under UV light with PLGA serving as the main body of the scaffold. PEG1000–HEMA had better biocompatibility than those with shorter PEG chains. Scaffolds with two structural variants, square and hexagonal pores, designed by computer were demonstrated. It may further combine medical images to reconstruct tissues and organs for regenerative medicine.

Introduction: The formation of a stereocomplex between PLLA and PDLA has been studied intensively because it increases the mechanical performance and thermal/hydrolytic resistance of polylactide-based materials; however, few studies have investigated the stereocomplex formation between PLLA and the (D-lactide)-containing PLLA copolymer. To investigate the effect of the D-lactide content of PLLA on the thermal behaviors and mechanical properties, (5D/95L) polylactide [(5D/95L)PLA], which contains a molar ratio of 5% of the D-form and 95% of the L-form of the monomer, and (15D/85L) polylactide [(15D/85L)PLA], which contains a molar ratio of 15% of the D-form and 85% of the L-form of the monomer, were used in a series of specimens. For the hydrolytic degradation test, the specimens were placed in 20-mL vials, which were filled with phosphate-buffered solution; the vials were allowed to stand at 57°C for 91 days in accordance with the ASTM F1635-95 (2000) standard test method for in vitro studies. The mechanical properties, thermal properties and crystallization behaviors were investigated using DSC and MTS, respectively. Results: The initial bending strength of the (5D/95L)PLA and (15D/85L)PLA were 35.4 and 31.1 N, respectively. After 1 week, the binding strength of the (5D/95L)PLA increased by 9.8%, and the binding strength of the (15D/85L)PLA decreased by 26%. In addition, the DSC curve of the (5D/95L)PLA demonstrated a higher melting temperature in the 1st week, and this Tc was observed in the DSC curve of the (5D/95L)PLA only during this time. The DSC curve of the (15D/85L)PLA was irregular. Discussion & Conclusions: In the (5D/95L)PLA, the recrystallization that occurred during the hydrolysis process was confirmed by the Tc and the increase in the bending strength. The stereocomplex crystallites may be formed in the (15D/85L)PLA during the degradation process. Because of the increase in the D-form monomer, the stereocomplexes were generated more easily and acted as nucleation sites. The PLLA crystal near the stereocomplex crystallites exhibited an incomplete structure, which led to a faster decrease in the bending strength. The low D-lactide content in the matrix of the PLLA did not form a stereocomplex crystallite because the surface area was not large enough to act as a nucleation site. However, the higher D-lactide-containing fraction formed a large stereocomplex crystallite. The (5D/95L)PLA demonstrated better thermal/hydrolytic resistance and mechanical stability than the (15D/85L)PLA.

Sinus augmentation is a common approach for patients with severe alveolar ridge atrophy but an ideal material to increase the bone volume for dental implantation is still needed. The present study evaluated the effect of a newly developed polymeric bone-filling powder (formed from acrylic acid and N-isopropylacrylamide, ANa) mixed with platelet-rich plasma (PRP) for bone generation in a rabbit model of sinus augmentation. A total of 12 New Zealand White rabbits were randomly divided into three groups based on filling material. All animals underwent a bilateral maxillary sinus augmentation. PRP was prepared using an automatic separation system to obtain a high platelet concentration. ANa powder was individually mixed with phosphate-buffered saline (PBS) or PRP for sinus floor elevation. The left maxillary sinus received the ANa/PBS filling, whereas the right maxillary sinus received a mixture of ANa/PRP gel. Equal volumes of filling material were inserted in each maxillary sinus. Thus, defects with no implantation served as controls. Animals were sacrificed at 4 and 12 weeks, and then all specimens were harvested for micro-computed tomography (micro-CT) and histological analysis. On micro-CT evaluation, ANa/PRP significantly increased the bone volume in maxillary sinus augmentation relative to the negative control and ANa/PBS after 12 weeks. New bone areas with osteocytes and osteon formation were found in all three groups at 12 weeks post-implantation. This study confirms that ANa mixed with PRP can effectively increase the bone volume in the sinus cavity. This benefit for sinus augmentation may increase the success of dental implantation because of the formation of a thin layer of alveolar bone.

Protein kinase (PK)-responsive gene carriers modified with polyethylene glycol (PEG) chains using an acid-labile linker were developed. These carriers were obtained by modifying the PEG chains and substrate peptides for the PKs (PKA or PKCα) on the branched polyethyleneimine main chain. Polyplexes formed from these carriers and plasmid DNA (pDNA) were stably dispersed under neutral pH medium. The polyplexes were also taken up by cells on the release of the PEG chains under the slightly acidic extracellular pH associated with cancer cells. The polyplexes taken up by cells resulted in gene expression when the substrate peptides were phosphorylated by the intracellular PKs to release pDNA from the polyplexes. These novel gene carriers are expected to be promising for cancer-specific gene therapy via intravenous administration.

It has been reported in many literatures that different kinds of bone graft augmentations have been applied in the treatment of distal radial fractures with favorable results. The authors applied calcium sulfate cement in the treatment of distal radial fractures and evaluated the radiological outcomes of 33 patients. The radiological changes of cement statuses, implants, and radiological parameter of distal radius were analyzed. After closed reduction of the displacement, fractures were fixed by Kirschner pins and stabilized by percutaneous injection of premixed calcium sulfate bone cement. Periodic X-ray evaluations were obtained at immediate post-operation and at every month. A total of 28 patients completed the follow-up and the cement extravasations into wrist joint or into soft tissues had been seen in almost every patient. The gradual settling and losses of radial height, radial inclination angle, and volar tilting angles were significant even after removal of Kirschner pins after 8 weeks. Gradual absorptions of cured cement block inside and outside distal radial cortices were observed in every case and complete absorptions of intraosseous cements after 8 weeks happened in 53.6% (15/28) of patients. The extravasated cements took longer time to fade away. The radiological results of current study revealed negative results of calcium sulfate applied in the treatment of distal radial fracture in terms of reduction parameters and cement block longevity.

Boosting angiogenesis is a crucial process to enhance tissue growth in tissue engineering (TE). Hepatoma-derived growth factor (HDGF) has been identified as an angiogenic factor, but its involvement in angiogenesis in an arteriovenous loop-based TE chamber developed by the laboratory is unclear. In this study, the authors first examined the effects of HDGF on angiogenic responses in endothelial cells and in a corneal model of neovascularization, and then characterized the expression of HDGF in the TE chamber. HDGF (1–500 ng/mL) induced concentration-dependent angiogenic responses in human endothelial cells in vitro (proliferation, migration, and tube formation). Local application of HDGF stimulated neovascularization in a rat model of corneal angiogenesis. In the TE chamber, there was an increase in blood vessel volume from day 3 to day 14. Immunofluorescence microscopy revealed that HDGF is highly expressed in the neovessels in the chamber. Peak expression of HDGF (day 3) coincided with the infiltration of inflammatory cells, and the mRNA level of endogenous HDGF correlated with that of tumor necrosis factor α (TNFα). In vitro, TNFα stimulated HDGF expression in endothelial cells. The data suggest that HDGF may be involved in angiogenic responses in the TE chamber and the proinflammatory cytokine TNFα may have a pivotal role in stimulating HDGF expression. Enhancing HDGF signaling may be a new approach to extend vascularization for TE.

Polyethylene glycol (PEG) has been grafted onto surface of many medical devices to reduce or eliminate protein adsorption that often leads to infection or inflammatory responses. Besides commonly used surface activation strategies, here in this work, the novel functional brush copolymer poly (vinyl alcohol)-g-PEG-co-polyurethane (PVA-g-(PEG-co-PU)) was first synthesized, characterized, and evaluated. PVA is used here as a versatile backbone platform for grafting functional molecules. On the one hand, the PU molecules can attach to the surface of desired PU-based implants, on the other hand, the presence of densely grafted PEG at the materials–tissue interface effectively impedes nonspecific protein adsorption. These copolymers with various grafting ratio of PEG and PU were analyzed by Fourier transform infrared spectroscopy and gel permeation chromatography. The brush copolymers that were obtained were further coated on the PU surface and contact angle measurements were performed. It was found that the brush copolymers turn the surface more hydrophilic with increasing PEG grafting density. Furthermore, the effects of PEG ratio on protein adsorption-resistant behavior were investigated. Protein adsorption was found to be the lowest on the surfaces with the highest PEG grafting density on PVA backbone. The study demonstrated unique brush copolymer to work as a potentially useful coating material.

Y-shaped amphiphilic copolymers have been synthesized by various strategies. They can self-assemble to form stable micellar drug carriers. Higher ratio of hydrophobic segment in these micelles is known to stabilize encapsulated drugs. This study used methoxy poly(ethylene glycol) (mPEG, A block) and poly(ε-caprolactone) (PCL, B block) to synthesize an AB₂ type amphiphilic block copolymer to encapsulate the anticancer drug, doxorubicin (DOX). The synthesized mPEG-(PCL)₂ self-assembled into nanoparticles and its critical micelle concentration was 43.7 × 10^-3 mg/mL. The particle size of empty micelle and DOX-loaded micelle was 95.1 and 21.4 nm, respectively. DOX-loaded micelles have a drug- loading efficiency of 22.3% and could be released from micelles up to 50% at pH 5 and 40% at pH 7.4 in 48 h in an in vitro release experiment. The nitric oxide (NO) assay indicated that the micelles could avoid the cytotoxic recognition by murine macrophage cells. The half-lethal dose (IC₅₀) of DOX-loaded micelles for two human breast cancer cell lines (MCF-7/WT for wild-type and MCF-7/ADR for adriamycin-resistant) were 0.937 and 7.476 μg/mL, respectively. For MCF-7/ADR cells, the ratio of IC₅₀ of free DOX vs. that of DOX-loaded micelles, which reported as resistance reversion index, was 0.125. Confocal images showed that DOX-loaded micelles accumulated mostly in the cytoplasm instead of nuclei. On the contrary, free DOX diffused throughout the cells. Flow cytometric histograms indicated that the fluorescence intensity in MCF-7/ADR cell line was about 83.2% and 50.9% for DOX-loaded micelles and free DOX, respectively. This result indicates that the DOX-loaded micelles formed by AB₂ copolymer could overcome multidrug resistance of breast cancer cells as it can accumulate more in MCF-7/ADR cells compared with free DOX.

Diabetes mellitus (DM) is associated with bone loss and leads to osteopenia and osteoporosis. This study was undertaken to investigate whether the impaired functions of mesenchymal stem cells (MSCs) derived from bone marrow play a role in pathogenesis of DM-associated bone loss. Bone marrow MSCs were taken from the alloxan-induced diabetic rats and normal rats. Bone mineral densities of tibias and femurs in diabetic rats decreased compared to those of normal rats as shown by dual energy X-ray absorptiometry. MSCs from diabetic rats exhibited reduced colony formation activity. The in vitro effects of high glucose (HG) (20 or 33 mM) on the growth, oxidative stress, apoptosis, and differentiation MSCs were next assessed. The viability and proliferation of MSCs derived from diabetic rats decreased significantly compared with that from normal rats. HG further suppressed the proliferation and viability of MSCs from both diabetic and normal rats. HG was associated with 38–40% increase in reactive oxygen species level and had significantly downregulated the activities of superoxide dismutase (SOD) and catalase (CAT) which could be recovered by the addition of L-ascorbic acid. The phenomena of apoptosis such as chromatin condensation and DNA fragmentation were found in cells cultured under HG conditions. As compared with 5.5 mM glucose, exposure of MSCs to HG enhanced adipogenic induction of triacylglycerol accumulation and inhibited osteogenic induction of alkaline phosphatase activity. HG increased peroxisome proliferator-activated receptor gamma expression during adipogenesis and reduced RUNX2 expression during osteoblastogenesis. These results indicate that MSCs derived from diabetic rats exhibited the inhibitory effects on cell growth and osteogenic ability. The oxidative stress, apoptosis, and adipogenic capability of MSCs were increased by HG. Furthermore, it is suggested that HG induces bone loss via attenuating the proliferation and osteoblastogenesis and enhancing adipogenesis mediated by the oxidative stress in rat bone marrow MSCs.