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Studies on various culture systems for chondrocytes and osteoblasts
Umeå universitet, Medicinska fakulteten, Institutionen för integrativ medicinsk biologi (IMB). (Chondrogenic and Osteogenic Differentiation)
2017 (Engelska)Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
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.

Ort, förlag, år, upplaga, sidor
Umeå: Umeå University , 2017. , s. 76
Serie
Umeå University medical dissertations, ISSN 0346-6612 ; 1911
Nyckelord [en]
Cell biology, chondrocyte, osteoarthritis, surface materials, topography, tissue engineering, mechanical stimulation
Nationell ämneskategori
Cell- och molekylärbiologi
Forskningsämne
medicinsk cellbiologi
Identifikatorer
URN: urn:nbn:se:umu:diva-138954ISBN: 978-91-7601-757-9 (tryckt)OAI: oai:DiVA.org:umu-138954DiVA, id: diva2:1138324
Disputation
2017-09-27, Hörsal E, Humanisthuset, Umeå, 09:00 (Engelska)
Opponent
Handledare
Tillgänglig från: 2017-09-06 Skapad: 2017-09-04 Senast uppdaterad: 2018-06-09Bibliografiskt granskad
Delarbeten
1. Osteoblast behavior on various ultra short pulsed laser deposited surface coatings
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2013 (Engelska)Ingår i: Materials Science and Engineering: C. Materials for Biological Applications, ISSN 0928-4931, Vol. 33, nr 3, s. 1676-1682Artikel i tidskrift (Refereegranskat) Published
Abstract [en]

Ultra short pulsed laser deposition technique was utilized to create amorphous diamond, alumina and carbon nitride, and two different titania coatings on silicon wafers, thus producing five different surface deposited films with variable physico-chemical properties. The surface characterizations, including the roughness, the contact angle and the zeta potential measurements were performed before we tested the growth properties of human osteoblast-like Saos-2 cells on these surfaces (three separate experiments). The average roughness and hydrophobicity were the highest on titania-deposited surfaces, while carbon nitride was the most hydrophilic one. Osteoblasts on all surfaces showed a flattened, spread-out morphology, although on amorphous diamond the cell shape appeared more elongated than on the other surfaces. On rough titania, the area covered by the osteoblasts was smaller than on the other ones. Cell proliferation assay did not show any statistically significant differences.

Ort, förlag, år, upplaga, sidor
Elsevier, 2013
Nyckelord
Surface coating, adhesion, Cell culture, Osteoblast, Ultra short laser deposition
Nationell ämneskategori
Materialkemi Cell- och molekylärbiologi Ortopedi
Forskningsämne
materialvetenskap; ortopedi; fysikalisk biologi
Identifikatorer
urn:nbn:se:umu:diva-104355 (URN)10.1016/j.msec.2012.12.078 (DOI)23827623 (PubMedID)
Tillgänglig från: 2015-06-10 Skapad: 2015-06-10 Senast uppdaterad: 2018-06-07Bibliografiskt granskad
2. Chondrocyte behavior on nanostructured micropillar polypropylene and polystyrene surfaces
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2014 (Engelska)Ingår i: Materials Science and Engineering. C, Materials for Biological Applications, ISSN 0928-4931, Vol. 43, s. 424-431Artikel i tidskrift (Refereegranskat) Published
Abstract [en]

This study was aimed to investigate whether patterned polypropylene (PP) or polystyrene (PS) could enhance the chondrocytes' extracellular matrix (ECM) production and phenotype maintenance. Bovine primary chondrocytes were cultured on smooth PP and PS, as well as on nanostructured micropillar PP (patterned PP) and PS (patterned PS) for 2 weeks. Subsequently, the samples were collected for fluorescein diacetate-based cell viability tests, for immunocytochemical assays of types I and II collagen, actin and vinculin, for scanning electronic microscopic analysis of cell morphology and distribution, and for gene expression assays of Sox9, aggrecan, procollagen α1(II), procollagen α1(X), and procollagen α2(I) using quantitative RT-PCR assays. After two weeks of culture, the bovine primary chondrocytes had attached on both patterned PP and PS, while practically no adhesion was observed on smooth PP. However, the best adhesion of the cells was on smooth PS. The cells, which attached on patterned PP and PS surfaces synthesized types I and II collagen. The chondrocytes' morphology was extended, and an abundant ECM network formed around the attached chondrocytes on both patterned PP and PS. Upon passaging, no significant differences on the chondrocyte-specific gene expression were observed, although the highest expression level of aggrecan was observed on the patterned PS in passage 1 chondrocytes, and the expression level of procollagen α1(II) appeared to decrease in passaged chondrocytes. However, the expressions of procollagen α2(I) were increased in all passaged cell cultures. In conclusion, the bovine primary chondrocytes could be grown on patterned PS and PP surfaces, and they produced extracellular matrix network around the adhered cells. However, neither the patterned PS nor PP could prevent the dedifferentiation of chondrocytes.

Ort, förlag, år, upplaga, sidor
Elsevier, 2014
Nyckelord
Chondrocyte, polypropylene, polystyrene, micropillar, nanostructure
Nationell ämneskategori
Materialkemi Cell- och molekylärbiologi
Forskningsämne
materialvetenskap; cellforskning
Identifikatorer
urn:nbn:se:umu:diva-103870 (URN)10.1016/j.msec.2014.07.045 (DOI)000342529000052 ()25175232 (PubMedID)
Tillgänglig från: 2015-06-02 Skapad: 2015-06-02 Senast uppdaterad: 2018-06-07Bibliografiskt granskad
3. Effect of gravitational force on the development of articular neocartilage with bovine primary chondrocytes
Öppna denna publikation i ny flik eller fönster >>Effect of gravitational force on the development of articular neocartilage with bovine primary chondrocytes
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(Engelska)Manuskript (preprint) (Övrigt vetenskapligt)
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.

Nyckelord
Centrifugation, cell biology, tissue engineering, cartilage, chondrocyte
Nationell ämneskategori
Cell- och molekylärbiologi
Forskningsämne
medicinsk cellbiologi
Identifikatorer
urn:nbn:se:umu:diva-138953 (URN)
Tillgänglig från: 2017-09-04 Skapad: 2017-09-04 Senast uppdaterad: 2018-06-09

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