Hanna Stenhamre

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.

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.

Influence of pore size on the redifferentiation potential of human articular chondrocytes in poly(urethane urea) scaffolds.

The chemical and physical properties of scaffolds affect cellular behaviour, which ultimately determines the performance and outcome of tissue‐engineered cartilage constructs. The objective of this study was to assess whether a degradable porous poly(urethane urea) scaffold could be a suitable material for cartilage tissue engineering. We also investigated whether the post‐expansion redifferentiation and cartilage tissue formation of in vitro expanded adult human chondrocytes could be regulated by controlled modifications of the scaffold architecture. Scaffolds with different pore sizes, < 150 µm, 150–300 µm and 300–500 µm, were seeded with chondrocytes and subjected to chondrogenic and osteogenic induction in vitro. The poly(urethane urea) scaffold with the smaller pore size enhanced the hyaline‐like extracellular matrix and thus neocartilage formation. Conversely, the chondrocytes differentiated to a greater extent into the osteogenic pathway in the scaffold with the larger pore size. In conclusion, our results demonstrate that poly(urethane urea) may be useful as a scaffold material in cartilage tissue engineering. Furthermore, the chondrogenic and the osteogenic differentiation capacity of in vitro expanded human articular chondrocytes can be influenced by the scaffold architecture. By tailoring the pore sizes, the performance of the tissue‐engineered cartilage constructs might be influenced and thus also the clinical outcome in the long run. Copyright

Topographic variation in redifferentiation capacity of chondrocytes in the adult human knee joint

OBJECTIVES The aim of this study was to investigate the topographic variation in matrix production and cell density in the adult human knee joint. Additionally, we have examined the redifferentiation potential of chondrocytes expanded in vitro from the different locations. METHOD Full thickness cartilage-bone biopsies were harvested from seven separate anatomical locations of healthy knee joints from deceased adult human donors. Chondrocytes were isolated, expanded in vitro and redifferentiated in a pellet mass culture. Biochemical analysis of total collagen, proteoglycans and cellular content as well as histology and immunohistochemistry were performed on biopsies and pellets. RESULTS In the biochemical analysis of the biopsies, we found lower proteoglycan to collagen (GAG/HP) ratio in the non-weight bearing (NWB) areas compared to the weight bearing (WB) areas. The chondrocytes harvested from different locations in femur showed a significantly better attachment and proliferation ability as well as good post-expansion chondrogenic capacity in pellet mass culture compared with the cells harvested from tibia. CONCLUSION These results demonstrate that there are differences in extra cellular content within the adult human knee in respect to GAG/HP ratio. Additionally, the data show that clear differences between chondrocytes harvested from femur and tibia from healthy human knee joints exist and that the differences are not completely abolished during the process of de- and redifferentiation. These findings emphasize the importance of the understanding of topographic variation in articular cartilage biology when approaching new cartilage repair strategies.

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.

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.

Contents Vol. 187, 2008

Stem Cells and Tissue Engineering A. Bader, Leipzig E-Mail: augustinus.bader@bbz.uni-leipzig.de S.F. Badylak, Pittsburgh, Pa. E-Mail: badylaks@msx.upmc.edu A. Müller, Würzburg E-Mail: albrecht.müller@mail.uni-wuerzburg.de A. Ratcliffe, San Diego, Calif. E-Mail: anthonyratcliffe@synthasome.com A.M. Wobus, Gatersleben E-Mail: wobusam@ipk-gatersleben.de Neurosciences M. Frotscher, Freiburg i.Br. E-Mail: frotsch@sun2.ruf.uni-freiburg.de W.L. Neuhuber, Erlangen E-Mail: winfried.neuhuber@rzmail.uni-erlangen.de