Chondrocytes Differentiation- Biological and Biomaterial Perspectives

Articular cartilage is an avascular tissue and once injured it does not heal spontaneously. A successful tissue engineering approach for cartilage reconstruction is autolougus chondrocyte implantation (ACI). In ACI a biopsy is harvested from the knee joint and the chondrocytes are isolated, expanded in vitro and subsequently implanted into the defect to regenerate new hyaline tissue. During the ACI procedure the chondrocytes undergo a process of de- and re-differentiation. To improve cartilage cell therapy, knowledge of how we can control and modulate the chondrocytes behavior is essential. The general aim of this thesis was to give insight in how we can influence the differentiation of chondrocytes. To accomplish this we examined whether and to what extent the redifferentiation capacity of articular chondrocytes was affected by the cell source, in vitro cultivation, and the scaffolds used.We found that human chondrocytes behaviour in vitro was depending on the harvesting site in the knee joint. The chondrocytes from femur had a better attachment, proliferation and post-expansion redifferentiation capacity than chondrocytes from tibia. Focusing on the cell source we further found that some degree of dedifferentiation is needed to obtain high quality redifferentiation in vitro. To further study how we could affect the behavior of in vitro expanded chondrocytes the cells were cultured in scaffolds with defined architecture during the redifferentiation. The effect of scaffolds with varied pore size as well as fiber size was examined. The results revealed that the pore size of the scaffold influenced the redifferentiation capacity whereas the size of the fibers did not. The size of the fiber showed to affect the proliferation of the cells and thus different biological processes can be manipulated by the design of the scaffolds. Furthermore, we found that scaffold mediated chondrogenesis in vitro is a process mimicking the fetal cartilage development. Moreover, we found that that redifferentiation of in vitro expanded articular chondrocytes is needed at the time of implantation for neocartilage formation to take place in vivo.The result presented in this thesis thus show that the outcome of articular cartilage tissue engineering in cartilage reconstruction will depend on harvesting site, degree of differentiation of the chondrocytes as well as the scaffold architecture. This information adds additional knowledge into the field of cartilage tissue engineering to be used for further improvement of the treatment of cartilage repair

Chondrocytes Differentiation- Biological and Biomaterial Perspectives

Articular cartilage is an avascular tissue and once injured it does not heal spontaneously. A successful tissue engineering approach for cartilage reconstruction is autolougus chondrocyte implantation (ACI). In ACI a biopsy is harvested from the knee joint and the chondrocytes are isolated, expanded in vitro and subsequently implanted into the defect to regenerate new hyaline tissue. During the ACI procedure the chondrocytes undergo a process of de- and re-differentiation. To improve cartilage cell therapy, knowledge of how we can control and modulate the chondrocytes behavior is essential. The general aim of this thesis was to give insight in how we can influence the differentiation of chondrocytes. To accomplish this we examined whether and to what extent the redifferentiation capacity of articular chondrocytes was affected by the cell source, in vitro cultivation, and the scaffolds used.We found that human chondrocytes behaviour in vitro was depending on the harvesting site in the knee joint. The chondrocytes from femur had a better attachment, proliferation and post-expansion redifferentiation capacity than chondrocytes from tibia. Focusing on the cell source we further found that some degree of dedifferentiation is needed to obtain high quality redifferentiation in vitro. To further study how we could affect the behavior of in vitro expanded chondrocytes the cells were cultured in scaffolds with defined architecture during the redifferentiation. The effect of scaffolds with varied pore size as well as fiber size was examined. The results revealed that the pore size of the scaffold influenced the redifferentiation capacity whereas the size of the fibers did not. The size of the fiber showed to affect the proliferation of the cells and thus different biological processes can be manipulated by the design of the scaffolds. Furthermore, we found that scaffold mediated chondrogenesis in vitro is a process mimicking the fetal cartilage development. Moreover, we found that that redifferentiation of in vitro expanded articular chondrocytes is needed at the time of implantation for neocartilage formation to take place in vivo.The result presented in this thesis thus show that the outcome of articular cartilage tissue engineering in cartilage reconstruction will depend on harvesting site, degree of differentiation of the chondrocytes as well as the scaffold architecture. This information adds additional knowledge into the field of cartilage tissue engineering to be used for further improvement of the treatment of cartilage repair

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 mu m, 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. \ua9 2008 American Chemical Society

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

Electrospinning of nanofibers for biomedical applications

[No abstract available

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

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

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

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