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Effect of gravitational force on the development of articular neocartilage with bovine primary chondrocytes
Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB). (Chondrogenic and Osteogenic Differentiation)
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

A lot of effort has been invested into understanding how to assemble cartilage tissue in vitro. Various scaffold types have been used in order to manufacture cartilage tissue with native-like biological properties, while cell-based self-assembly of cartilage without a scaffold material is another strategy utilized. Mechanical forces have also been exploited in the manufacturing process. In this study, we used bovine primary chondrocytes to self-assemble a scaffold-free constructs to investigate whether mechanical loading by gravitational force would be useful in manufacturing cartilage tissue in vitro. Six million chondrocytes were laid on top of defatted bone disks placed inside agarose well in 50 ml culture tubes. The constructs were centrifuged once or three times a day for 15 min at gravitational force of 770 x g for one, two and four weeks. Control samples were cultured under the same conditions without exposure to centrifugation. The samples were analysed by (immuno)histochemistry, Fourier transform infrared imaging, micro-computed tomography, biochemical and gene expression analyses. Biomechanical testing was also performed. Macroscopically, the centrifuged tissues had a more even surface covering a larger area of the bone disk. Fourier transform infrared imaging analysis indicated higher concentration of collagen in the top and bottom edges of the centrifuged samples. Glycosaminoglycan contents increased along the culture, while collagen content appeared to remain at a rather constant level. Aggrecan and procollagen α1(II) gene expression levels had no significant differences, while procollagen α2(I) levels were increased significantly. Biomechanical analyses did not reveal remarkable changes. In conclusion, both of the centrifugation regimes lead to a more uniform tissue constructs, while the biological properties of the native tissue could not be obtained.

Keyword [en]
Centrifugation, cell biology, tissue engineering, cartilage, chondrocyte
National Category
Cell and Molecular Biology
Research subject
Medical Cell Biology
Identifiers
URN: urn:nbn:se:umu:diva-138953OAI: oai:DiVA.org:umu-138953DiVA: diva2:1138317
Available from: 2017-09-04 Created: 2017-09-04 Last updated: 2017-09-05
In thesis
1. Studies on various culture systems for chondrocytes and osteoblasts
Open this publication in new window or tab >>Studies on various culture systems for chondrocytes and osteoblasts
2017 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Osteoarthritis and osteochondral defects are ailments that are increasing in frequency as the lifespan of the population increases and sedentary lifestyle becomes more common. Osteoarthritis is an inflammatory disease that causes the progressive degeneration of articular surfaces and the underlying bone. Accidents and injuries can cause osteochondral defects similar to osteoarthritis. In both cases the structure of the articular cartilage fails, leading to pain and disability. Articular cartilage has a naturally poor ability to regenerate since there is no vasculature and it is aneural. The sparse chondrocytes mainly act to maintain the healthy extracellular matrix. Once the defect is severe enough, a surgical intervention becomes necessary. For small defects and young patients, a cell-based treatment can be used, whereas for larger defects and severe osteoarthritis a partial or whole joint arthroplasty is performed. Methods to repair osteochondral defects have been improving over the years as the inter-disciplinary understanding of joints, and what is required to repair them, has increased. However, there are still issues to solve in order to achieve consistently good results in both joint replacement and repair of cartilage. The main issue faced with current techniques used for joint replacement is poor integration of the artificial joint, leading to loosening at the bone interface over time, while cartilage repair techniques face the problem of generating mechanically inferior fibrocartilage. It is known that surface chemistry and structures at micro- and nanoscale influence cell behaviour, which can be utilised to guide their attachment, proliferation and phenotype. Scaffold-free approaches and mechanical stimulation have previously given promising results in generating articular neocartilage.

This thesis aims at exploring tools and solutions to the problems involved in implant integration, chondrocyte expansion and neocartilage tissue engineering. We hypothesised that 1) ultra-short pulsed laser deposition can be used to create biocompatible coatings; 2) micropillars with nanoscale features can improve the maintenance of the chondrocyte phenotype in culture and 3) hypergravity can aid in the production of more native-like neocartilage constructs.

Our studies showed that ultra-short pulsed laser ablation can be used to create various surfaces for studying cell behaviour. Cell viability was slightly higher on a rough titanium oxide, whereas the cell area was significantly smaller on rough titanium oxide, indicating a lower amount of focal adhesions. Nanopatterned microstructures were not capable of maintaining the chondrocyte phenotype in culture, but they were not disadvantageous either. Hypergravity might help in creating a native-like distribution of collagen and proteoglycans. The constructs were more uniform in shape, but biomechanically the constructs were not different from non-centrifuged controls.

Place, publisher, year, edition, pages
Umeå: Umeå University, 2017. 76 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1911
Keyword
Cell biology, chondrocyte, osteoarthritis, surface materials, topography, tissue engineering, mechanical stimulation
National Category
Cell and Molecular Biology
Research subject
Medical Cell Biology
Identifiers
urn:nbn:se:umu:diva-138954 (URN)978-91-7601-757-9 (ISBN)
Public defence
2017-09-27, Hörsal E, Humanisthuset, Umeå, 09:00 (English)
Opponent
Supervisors
Available from: 2017-09-06 Created: 2017-09-04 Last updated: 2017-09-26Bibliographically approved

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