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Five percent oxygen tension is not beneficial for neocartilage formation in scaffold-free cell cultures
Department of Biosciences, Biocenter Kuopio, University of Eastern Finland, Kuopio, Finland. (Chondrogenic and Osteogenic Differentiation Group)ORCID iD: 0000-0002-1710-7715
Department of Biosciences, University of Eastern Finland, Kuopio, Finland.
Department of Biosciences, University of Eastern Finland, Kuopio, Finland. (Chondrogenic and Osteogenic Differentiation Group)ORCID iD: 0000-0002-9294-7431
Department of Biosciences, Biocenter Kuopio, University of Eastern Finland, Kuopio, Finland. (Chondrogenic and Osteogenic Differentiation Group)ORCID iD: 0000-0002-6181-9904
2012 (English)In: Cell and Tissue Research, ISSN 0302-766X, E-ISSN 1432-0878, Vol. 348, no 1, 109-117 p., 22392735Article in journal (Refereed) Published
Abstract [en]

We have investigated whether 5% oxygen tension (O(2)) is beneficial for neocartilage formation when chondrocytes are cultured in transwell-COL inserts. Six million bovine primary chondrocytes were cultured in an insert with DMEM supplemented with 10% fetal bovine serum and antibiotics, with or without glucosamine sulphate (GS) in a 5% or 20% O(2) environment for 2, 4, or 6 weeks. The samples were collected for the histological staining of proteoglycans (PGs) and type II collagen, quantitative reverse transcription with the polymerase chain reaction (RT-PCR) analyses of the mRNA expression of aggrecan and procollagen α(1)(II), procollagen α(2)(I) and hyaluronan synthase 2, quantitation of PGs, and agarose gel electrophoresis. Neocartilage produced at 20% O(2) appeared larger than that at 5% O(2). Histological staining showed that more PGs and type II collagen and better native cartilage structure were produced at 20% than at 5% O(2). The thickness of neocartilage increased during the culture period. Quantitative RT-PCR showed that the procollagen α(1)(II) mRNA expression level was significantly higher at 20% than at 5% O(2). However, no significant difference in gene expression and PG content was found between control and GS-treated cultures at either 20% or 5% O(2). Thus, in contrast to monolayer cultures, engineered cartilage from scaffold-free cultured chondrocytes at 20% O(2) produced better extracellular matrix (ECM) than that at 5% O(2). PGs were mainly large. Exogenous GS was not beneficial for the ECM in scaffold-free chondrocyte cultures.

Place, publisher, year, edition, pages
Springer Publishing Company, 2012. Vol. 348, no 1, 109-117 p., 22392735
Keyword [en]
Primary chondrocyte, Tissue engineering, Oxygen tension, Glucosamine sulphate, Cell culture, Neocartilage
National Category
Cell and Molecular Biology Orthopedics
Research subject
Biochemistry; cellforskning; Orthopaedics
Identifiers
URN: urn:nbn:se:umu:diva-104719DOI: 10.1007/s00441-012-1366-zISI: 000302324800008PubMedID: 22392735OAI: oai:DiVA.org:umu-104719DiVA: diva2:820853
Available from: 2015-06-12 Created: 2015-06-12 Last updated: 2017-12-04Bibliographically approved
In thesis
1. Production of neocartilage tissues using primary chondrocytes
Open this publication in new window or tab >>Production of neocartilage tissues using primary chondrocytes
2016 (English)Doctoral thesis, comprehensive summary (Other academic)
Alternative title[sv]
Fabrikation av konstgjord brosk med primära broskceller
Abstract [en]

Hyaline cartilage is a highly specialized tissue, which plays an important role in the articulating joints of an individual. It provides the joints with a nearly frictionless, impact resisting surface to protect the ends of the articulating bones. Articular cartilage has a poor self-repair capacity and, therefore, it rarely heals back to normal after an injury. Overweight, injuries, overloading and genetic factors may initiate a degenerative disease of the joint called osteoarthritis.

Osteoarthiritis is a major global public health issue. Currently, the most used treatment for large articular cartilage defects is joint replacement surgery. However, possibilities to replace this highly invasive operation with strategies based on tissue engineering are currently investigated. The idea of the tissue engineering is to optimize the use of the cells, biomaterials and culture conditions to regenerate a new functional tissue for the defect site.

The goal of this thesis was to manufacture cartilage tissue in cell culture conditions in vitro. Bovine primary chondrocytes isolated from the femoral condyles were used in all the experiments for neocartilage production. The samples were collected for histology, gene expression level quantifications, and analyses of proteoglycan (PG) content and quality. The histological sections were stained for type II collagen and PGs, the quantitative RT-PCR was used to observe the relative expressions of aggrecan, Sox9, procollagen α2(I) and procollagen α1(II) genes. The PGs were quantified using a spectrophotometric method, and agarose gel electrophoresis was used to separate the PGs according to their size.

In the two first studies, we optimized the culture conditions of in vitro scaffold-free culture technique to produce the native-type hyaline cartilage of a good quality. We found out that high glucose concentration and hypertonic medium at 20% oxygen tension promoted the best hyaline-like neocartilage tissue production. Glucosamine sulfate supplementation, low oxygen tension, 5 mM glucose concentration and a transient TGF-β3 supplementation were not beneficial for the neocartilage formation in the scaffold-free cell culture system.

In the third study, we used these newly defined, optimized culture conditions to produce the neocartilage tissues in the HyStem™ and the HydroMatrix™ scaffold materials and we compared these tissues to the ones grown as scaffold-free control cultures. We noticed that there was no difference between the controls and the scaffolds, and occasionally the scaffold-free controls had produced better quality cartilage than the ones with the scaffolds. Overall, the neocartilage tissues were of good hyaline-like quality in the third study. Their extracellular matrix contents were close to the native cartilage, although the neotissues lacked the zonal organization typical to the normal articular cartilage. The tissues had the right components, but their ultrastructure differed from the native cartilage.

In conclusion, we were able to optimize our in vitro neocartilage culture method further, and discovered a good combination of the culture conditions to produce hyaline-like cartilage of good quality. Surprisingly, the scaffold materials were not beneficial for the cartilage formation.

 

Abstract [fi]

Lasi- eli hyaliinirusto on pitkälle erikoistunutta kudosta, jolla on erittäin tärkeä rooli yksilön nivelten toiminnassa. Kudos suojaa ruston alapuolista luuta muodostamalla lähes kitkattoman ja joustavan liikkumista helpottavan pinnan. Lasiruston oma uusiutumiskyky on hyvin heikko, ja näin ollen kudos vain harvoin paranee alkuperäisen kaltaiseksi vaurion jälkeen. Ylipaino, vammat, liiallinen kuormitus tai geneettiset tekijät voivat käynnistää rustokudoksen rappeutumisen. Tätä tilaa kutsutaan nivelrikoksi.

Nivelrikko on valtava kansanterveydellinen ongelma. Keinonivelleikkaus on nykyisellään ainoa hoitokeino pinta-alaltaan laajojen nivelruston vaurioiden hoitoon. Vaihtoehtoja tämän suuren ja invasiivisen kirurgisen operaation korvaamiseksi tutkitaan kuitenkin koko ajan ympäri maailmaa. Kudosteknologian ajatuksena on optimoida solujen, biomateriaalien ja erilaisten kasvatusolosuhteiden käyttö uuden, alkuperäisen kaltaisen toiminnallisen kudoksen luomiseksi vauriokohtaan.

Väitöskirjan kaikissa kolmessa osatutkimuksessa uudisrustokudoksia tuotettiin käyttäen naudan polven rustosta eristettyjä primäärisiä rustosoluja. Näytteet kerättiin histologisia analyysejä, geenin ilmentymistutkimuksia ja proteoglykaanisisällön ja -jakauman (PG) analyyseja varten. Histologisista leikkeistä värjättiin tyypin II kollageeni ja PG:t, ja kvantitatiivista RT-PCR -menetelmää käytettiin aggrekaani-, Sox9-, prokollageeni α2(I)- ja prokollageeni α1(II)-geenien suhteellisten ilmentymistasojen määrittämiseen. Proteoglykaanisisältö analysoitiin käyttäen spektrofotometristä menetelmää, ja PG:t eroteltiin kokonsa perusteella agaroosigeelielektroforeesia käyttäen.

Kahdessa ensimmäisessä osatutkimuksessa optimoitiin tukirakenteetta kasvattujen uudisrustojen kasvatusolosuhteita natiivin kaltaisen lasiruston tuottamiseksi. Havaitsimme, että korkea glukoosipitoisuus ja hypertoninen elatusaine yhdistettynä 20 % happiosapaineeseen tuotti parhaimman laatuista uudisrustokudosta tutkituista yhdistelmistä. Glukosamiinisulfaatin lisäys, matala happiosapaine, 5 mM glukoosi konsentraatio tai TGF-β3:n lisääminen alkuvaiheessa eivät edesauttaneet uudisrustokudosten muodostumisessa.

Kolmannessa osatutkimuksessa otettiin käyttöön uudet, hyväksi havaitut kasvatusolosuhteet yhdistettynä HyStem™ and HydroMatrix™ -tukimateriaaleihin, ja niitä verrattiin tukirakenteettomaan kasvatusmenetelmään. Tutkimuksessa havaittiin, ettei tukirakenteettoman kontrollin tai tukimateriaalien välillä ollut mitään eroa, ja että kontrollikasvatukset tuottivat ajoittain jopa parempaa rustoa kuin tukimateriaalein kasvatetut. Kaiken kaikkiaan kaikki tuotetut uudiskudokset muistuttivat laadullisesti lasiruston kaltaista kudosta. Molekyylisisältö lähenteli natiivia rustoa, vaikkakin uudiskudoksista puuttui normaalille nivelrustolle tyypillinen vyöhykkeinen järjestäytyminen. Kudoksissa oli parhaimmillaan oikea määrä oikeita komponentteja, mutta ne eivät vain olleet järjestäytyneet oikealla tavalla.

Onnistuimme optimoimaan uudisrustokudosten kasvatusmenetelmäämme. Löysimme hyvän kasvatusolosuhteiden yhdistelmän, jonka avulla kykenimme tuottamaan lasiruston kaltaista uudisrustokudosta. Hivenen yllättäenkin, tukimateriaalit eivät olleet avuksi tutkimuksessamme uudisrustokudoksia muodostettaessa.

Place, publisher, year, edition, pages
Umeå: Umeå University, 2016. 70 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1769
Keyword
primary chondrocytes, articular cartilage, tissue engineering, neocartilage, scaffold, scaffold-free, hyaluronan, self-assembling peptide, TGF-β3, glucosamine sulfate, hypoxia, hypertonic medium, glucose
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:umu:diva-113929 (URN)978-91-7601-391-5 (ISBN)
Public defence
2016-01-26, N430, Naturvetarhuset, Umeå, 09:00 (English)
Opponent
Supervisors
Available from: 2016-01-08 Created: 2016-01-06 Last updated: 2016-01-07Bibliographically approved

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