Cytotoxicity and immunological response of gold and silver nanoparticles of different sizes.

The immunological response of macrophages to physically produced pure Au and Ag nanoparticles (NPs) (in three different sizes) is investigated in vitro. The treatment of either type of NP at > or =10 ppm dramatically decreases the population and increases the size of the macrophages. Both NPs enter the cells but only AuNPs (especially those with smaller diamter) up-regulate the expressions of proinflammatory genes interlukin-1 (IL-1), interlukin-6 (IL-6), and tumor necrosis factor (TNF-alpha). Transmission electron microscopy images show that AuNPs and AgNPs are both trapped in vesicles in the cytoplasma, but only AuNPs are organized into a circular pattern. It is speculated that part of the negatively charged AuNPs might adsorb serum protein and enter cells via the more complicated endocytotic pathway, which results in higher cytotoxicity and immunological response of AuNPs as compared to AgNPS.

Evaluation of chondrocyte growth in the highly porous scaffolds made by fused deposition manufacturing (FDM) filled with type II collagen.

Highly porous poly(D,L-lactide-co-glycolide) (PLGA) scaffolds for cartilage tissue engineering were fabricated in this study using the fused deposition manufacturing (FDM) process and were further modified by type II collagen. The average molecular weight of PLGA decreased to about 60% of the original value after the melt-extrusion process. Type II collagen exhibited sponge-like structure and filled the macroporous FDM scaffolds. An increase of the fiber spacing resulted in an increase of the porosity. The storage modulus of FDM scaffolds with a large fiber spacing was comparable to that of the native porcine articular cartilage. Although the FDM hybrid scaffolds were swollen in various extents after 28 days of in vitro culture, the seeded chondrocytes were well distributed in the interior of the scaffolds with a large fiber spacing and neocartilage was formed around the scaffolds. The study also suggested that a low processing temperature may be required to produce PLGA precision scaffolds using FDM.

Fabrication of precision scaffolds using liquid-frozen deposition manufacturing for cartilage tissue engineering.

The fused deposition manufacturing (FDM) system has been used to fabricate tissue-engineered scaffolds with highly interconnecting and controllable pore structure, although the system is limited to a few materials. For this reason, the liquid-frozen deposition manufacturing (LFDM) system based on an improvement of the FDM process was developed. Poly(D,L-lactide-co-glycolide) (PLGA) precision scaffolds were fabricated using LFDM from PLGA solutions of different concentrations. A greater concentration of PLGA solution resulted in greater mechanical strength but also resulted in less water content and smaller pore size on the surface of the scaffolds. LFDM scaffolds in general had mechanical strength closer to that of native articular cartilage than did FDM scaffolds. Neocartilage formation was observed in LFDM scaffolds seeded with porcine articular chondrocytes after 28 days of culture. Chondrocytes in LFDM scaffolds made from low concentrations (15-20%) of PLGA solution maintained a round shape, proliferated well, and secreted abundant extracellular matrix. In contrast, the FDM PLGA scaffolds had low cell numbers and poor matrix production because of heavy swelling. The LFDM system offered a useful way to fabricate scaffolds for cartilage tissue-engineering applications.

The response of articular chondrocytes to type II collagen-Au nanocomposites.

The nanocomposites (denoted "CII-Au") of porcine type II collagen (CII) with 0.05, 0.1, 0.5, 1, or 2.5% (wt/wt) Au nanoparticles ( approximately 5 nm) were fabricated for potential use in cartilage tissue engineering. Au formed clusters on the surface of all nanocomposites and appeared to distribute along the collagen fibrils inside the matrix. The addition of Au at low concentrations (< or =0.5%) increased the modulus and viscosity, as well as the free radical-scavenging ability. These effects decreased at higher concentrations of Au. The chondrocytes on CII-Au became spindle-like with lamellipodia formation. Cell proliferation on CII-Au 0.1% was promoted. Nitric oxide (NO) in the culture medium was reduced by CII-Au 0.05% and CII-Au 0.1%. Type I collagen, aggrecan, and Sox 9 gene expressions increased with an increased Au content, but slightly decreased at 2.5% Au. There was no significant difference in the CII gene expression. The cellular uptake of Au was observed but less than that which occurred when 10 ppm of Au was added in culture medium. Chondrocytes cultured with < or =10 ppm of Au nanoparticles showed neither cytotoxicity nor change in gene expression. Au at an appropriate amount could be well dispersed in CII, and enhanced the material modulus, antioxidant effect, as well as the chondrocyte growth and matrix production.

Effects of unidirectional permeability in asymmetric poly(DL-lactic acid-co-glycolic acid) conduits on peripheral nerve regeneration: an in vitro and in vivo study.

The high outflow permeability of the nerve conduit used to emit the drained waste generated from the traumatized host nerve stump is critical in peripheral nerve regeneration. Our earlier studies have established that asymmetric conduits fulfill the basic requirements for use as nerve guide conduits. In this study, the inflow characteristics of optimal nerve conduits were further examined using in vivo and in vitro trials. Various asymmetric poly(DL-lactic acid-co-glycolic acid) (PLGA) conduits were controlled by modifying precipitation baths using 0, 20, and 95% isopropyl alcohol, with high-porosity (permeability), medium-porosity (high outflow and low inflow), and low-porosity (permeability), respectively. In the in vitro trial, the Schwann cells and fibroblasts were seeded on either side of the asymmetric PLGA films in a newly designed coculture system that simulated the repaired nerve conduit environment. The results of the directional permeable films indicated the statistically significant proliferation of Schwann cells and the inhibition of the division of fibroblasts in lactate dehydrogenase release and inhibition of 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl-tetrazolium bromide (MTT) reduction, compared with the other films. In the in vivo trial, the PLGA conduits seeded with Schwann cells were implanted into 10 mm right sciatic nerve defects in rats. After 6 weeks, implanted conduits were harvested. Histological examination verified that directional permeable conduits had markedly more A-type and B-type myelin fibers in the midconduit and distal nerve. In this work, the directional transport characteristics were established as an extremely important factor to the design and development of optimal nerve guide conduits in peripheral nerve regeneration.

Evaluation of the growth of chondrocytes and osteoblasts seeded into precision scaffolds fabricated by fused deposition manufacturing.

In this study, fused deposition manufacturing (FDM) was utilized to fabricate the precision scaffolds for cartilage and bone regeneration. Cell seeding into such scaffolds was evaluated. For poly(D,l-lactide) (PLA) scaffolds used for cartilage regeneration, the structure with larger inner space, four direction stacking (4D) and small interval of fibers were better. Chondrocyte proliferated well with matrix accumulation in precision scaffolds coated with type II collagen at 4 weeks of in vitro culture. The seeding efficiency of osteoblasts in most polycaprolactone (PCL) scaffolds used for bone regeneration could arrive 50% of original cell seeding density, and the amount of cells in scaffolds increased to double fold after 2 weeks of in vitro culture. The histological cross-section also revealed proliferation and mineralization of osteoblasts among the PCL fibers. The results indicated that the highly porous and interconnected structure of precision scaffolds could benefit cell ingrowth.

Evaluation of biodegradable polyesters modified by type II collagen and Arg-Gly-Asp as tissue engineering scaffolding materials for cartilage regeneration.

Synthetic biodegradable polyesters poly(L-lactide) (PLLA) and poly(D,L-lactide-coglycolide) (PLGA) (50:50) modified by porcine type II collagen and an Arg-Gly-Asp (RGD)-containing protein were evaluated as scaffolds for cartilage regeneration in this study. Cytocompatibility of the polymer films was tested using immortalized chondrocytes. Neocartilage formation in vitro on cell-seeded scaffolds was further examined using primary porcine chondrocytes. The inflammatory response of the scaffolds was evaluated subcutaneously in rats. A pilot animal study was conducted, in which rabbit allogeneic chondrocyte-seeded scaffolds were implanted to repair the defected rabbit knee cartilage. The results demonstrated that PLGA as well as its blends with PLLA had better cell growth than pure PLLA, and that type II collagen enhanced, but RGD inhibited cell proliferation. Scaffolds made of blended PLLA/PLGA had larger dynamic compressive modulus compared to scaffolds made of PLLA or PLGA single polymer. Chondrocyte-seeded scaffolds modified by type II collagen without RGD had the greater number of cells as well as higher glycosaminoglycan (GAG) and collagen contents compared to scaffolds without type II collagen modification or scaffolds further modified with RDG. Type II collagen modification prevented infiltration by host tissue and capsule formation. Unmodified PLLA and PLLA/PLGA constructs demonstrated persisting inflammatory response after 6 months, while all type II collagen-modified PLLA/PLGA constructs showed complete repair and no inflammation. Partial or full repair was observed after 2 months of postimplantation in type II collagen-modified PLLA/PLGA constructs, with equal cellularity and 75-80% matrix contents of a normal rabbit articular cartilage. It was concluded that PLLA/PLGA blended scaffolds modified by type II collagen were a potential tissue engineering scaffold for cartilage regeneration.

The effect of two different bioreactors on the neocartilage formation in type II collagen modified polyester scaffolds seeded with chondrocytes.

The effect of dynamic culture conditions on neocartilage formation in type II collagen modified polyester scaffolds was studied. Porcine or human articular chondrocytes were seeded in the scaffolds. The cell-scaffold constructs were cultivated statically, in a rotating-type bioreactor or in a shaker for up to 4 weeks. The cell proliferation, morphology, NO production, synthesis of proteoglycans and collagen, and mechanical properties were evaluated. The results demonstrated that the rotating-type bioreactor promoted the growth of primary porcine chondrocytes, helped to maintain their phenotype, and increased the production of extracellular matrix. The constructs also had the largest dynamic compressive modulus. In the static condition, chondrocytes occupied only the outer margin of the cell-polymer constructs. The poor mass transfer in static condition may have caused a lower pH value in the middle of the constructs and lead further to faster scaffold degradation as well as the weakest neocartilage. Constructs in the shaker produced the highest amount of NO as well as the lowest amount of cells and matrix production. Human or porcine chondrocytes of the second passage seeded in scaffolds were much less viable, with the largest amount of cells and matrix when cultured in rotating-type bioreactors. A larger seeding density was required to form neocartilage from passaged adult chondrocytes.