Nanosized fibers' effect on adult human articular chondrocytes behavior.

Tissue engineering with chondrogenic cell based therapies is an expanding field with the intention of treating cartilage defects. It has been suggested that scaffolds used in cartilage tissue engineering influence cellular behavior and thus the long-term clinical outcome. The objective of this study was to assess whether chondrocyte attachment, proliferation and post-expansion re-differentiation could be influenced by the size of the fibers presented to the cells in a scaffold. Polylactic acid (PLA) scaffolds with different fiber morphologies were produced, i.e. microfiber (MS) scaffolds as well as nanofiber-coated microfiber scaffold (NMS). Adult human articular chondrocytes were cultured in the scaffolds in vitro up to 28 days, and the resulting constructs were assessed histologically, immunohistochemically, and biochemically. Attachment of cells and serum proteins to the scaffolds was affected by the architecture. The results point toward nano-patterning onto the microfibers influencing proliferation of the chondrocytes, and the overall 3D environment having a greater influence on the re-differentiation. In the efforts of finding the optimal scaffold for cartilage tissue engineering, studies as the current contribute to the knowledge of how to affect and control chondrocytes behavior.

Behavior of human chondrocytes in engineered porous bacterial cellulose scaffolds.

Regeneration of articular cartilage damage is an area of great interest due to the limited ability of cartilage to self-repair. The latest cartilage repair strategies are dependent on access to biomaterials to which chondrocytes can attach and in which they can migrate and proliferate, producing their own extracellular matrix. In the present study, engineered porous bacterial cellulose (BC) scaffolds were prepared by fermentation of Acetobacter xylinum (A. xylinum) in the presence of slightly fused wax particles with a diameter of 150-300 microm, which were then removed by extrusion. This porous material was evaluated as a scaffold for cartilage regeneration. Articular chondrocytes from young adult patients as well as neonatal articular chondrocytes were seeded with various seeding techniques onto the porous BC scaffolds. Scanning electron microscopy (SEM) analysis and confocal microscopy analysis showed that cells entered the pores of the scaffolds and that they increasingly filled out the pores over time. Furthermore, DNA analysis implied that the chondrocytes proliferated within the porous BC. Alcian blue van Gieson staining revealed glycosaminoglycan (GAG) production by chondrocytes in areas where cells were clustered together. With some further development, this novel biomaterial can be a suitable candidate for cartilage regeneration applications.

Electrospinning of highly porous scaffolds for cartilage regeneration.

This study presents a new innovative method where electrospinning is used to coat single microfibers with nanofibers. The nanofiber-coated microfibers can be formed into scaffolds with the combined benefits of tailored porosity for cellular infiltration and nanostructured surface morphology for cell growth. The nanofiber coating is obtained by using a grounded collector rotating around the microfiber, to establish an electrical field yet allow collection of nanofibers on the microfiber. A Teflon tube surrounding the fibers and collector is used to force the nanofibers to the microfiber. Polycaprolactone nanofibers were electrospun onto polylactic acid microfibers and scaffolds of 95 and 97% porosities were made. Human chondrocytes were seeded on these scaffolds and on reference scaffolds of purely nanofibers and microfibers. Thereafter, cellular infiltration was investigated. The results indicated that scaffold porosity had great effects on cellular infiltration, with higher porosity resulting in increased infiltration, thereby confirming the advantage of the presented method.

Neither Notch1 expression nor cellular size correlate with mesenchymal stem cell properties of adult articular chondrocytes.

BACKGROUND: Tissue repair is thought to be regulated by progenitor cells, which in other tissues are characterized by their Notch1 expression or small cellular size. Here we studied if these traits affect the chondrogenic potential and are markers for multipotent progenitor cell populations in adult articular cartilage. METHODS: Directly isolated articular chondrocytes were sorted with regard to their Notch1 expression or cellular size. Their colony forming efficiency (CFE) and their potential to differentiate towards adipogenic, osteogenic and chondrogenic lineages were investigated. The different sorted populations were also expanded in monolayer and analyzed in the same manner as the directly isolated cells. RESULTS: No differences in CFE or adipogenic, osteogenic and chondrogenic potentials were detected among the sorted populations. Expanded cells displayed a higher osteochondral potential than directly isolated cells. CONCLUSION: Cellular size or Notch1 expression is not per se a specific marker for mesenchymal progenitor cells in adult articular cartilage. Monolayer-expanded adult chondrocytes contain a larger mesenchymal progenitor cell-like population than directly isolated cells, highly likely as a result of dedifferentiation. If there are resident Notch1-positive cells or cells of a specific size in adult articular cartilage with functional features of progenitor cells, the population consists of only a very small number of cells.

Limited mutagenesis increases the stability of human carboxypeptidase U (TAFIa) and demonstrates the importance of CPU stability over proCPU concentration in down-regulating fibrinolysis.

Procarboxypeptidase U [proCPU, thrombin-activatable fibrinolysis inhibitor (TAFI), EC 3.4.17.20] belongs to the metallocarboxypeptidase family and is a zymogen found in human plasma. ProCPU has been proposed to be a molecular link between coagulation and fibrinolysis. Upon activation of proCPU, the active enzyme (CPU) rapidly becomes inactive due to its intrinsic instability. The inherent instability of CPU is likely to be of major importance for the in vivo down-regulation of its activity, but the underlying structural mechanisms of this fast and spontaneous loss of activity of CPU have not yet been explained, and they severely inhibit the structural characterization of CPU. In this study, we screened for more thermostable versions of CPU to increase our understanding of the mechanism underlying the instability of CPU's activity. We have shown that single as well as a few 2-4 mutations in human CPU can prolong the half-life of CPU's activity at 37 degrees C from 0.2 h of wild-type CPU to 0.5-5.5 h for the mutants. We provide evidence that the gain in stable activity is accompanied by a gain in thermostability of the enzyme and increased resistance to proteolytic digest by trypsin. Using one of the stable mutants, we demonstrate the importance of CPU stability over proCPU concentration in down-regulating fibrinolysis.