Janne Ylärinne

Production of neocartilage tissues using primary chondrocytes

Opponent

Professor Mats Brittberg, Sahlgrenska Hospital, Göteborg, Sweden

Reviewers

Docent André Struglics, University of Lund, Sweden

Professor Anna Engström-Laurent, University of Umeå, Sweden

Professor Mikael Wiberg, University of Turku

Supervisors

Professor Mikko Lammi, University of Umeå, Sweden

Docent Chengjuan Qu, University of Umeå, Sweden

Articles of the thesis

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.

  1. Qu C, Lindeberg H, Ylärinne JH, Lammi MJ: Five percent oxygen tension is not beneficial for the neocartilage formation in scaffold-free cell culture. Cell Tissue Res 348(1): 109-117, 2012 [Pubmed] [Full text]

  2. Ylärinne JH, Qu C, Lammi MJ: Hypertonic conditions enhance cartilage formation in scaffold-free primary chondrocyte cultures. Cell Tissue Res 358(2): 541-550, 2014 [Pubmed] [Full text]

  3. Ylärinne J, Qu C, Lammi MJ: Scaffold-free approach produces similar quality neo-cartilage tissues as HyStem™ and Hydromatrix™ scaffolds. J Mater Sci Mater Med 28(4): 59, 2017 [Pubmed] [Full text]

Related articles

  1. Inkinen S, Liukkonen J, Ylärinne J, Puhakka PH, Lammi MJ, Viren T, Jurvelin JS, Töyräs J: Collagen and chondrocyte concentrations control ultrasound scattering in agarose scaffolds. Ultrasound Med Biol 40(9): 2162-2171, 2014 [Pubmed] [Full text]

  2. Puhakka P, Ylärinne JH, Lammi MJ, Saarakkala S, Tiitu V, Kröge H, Virén T, Jurvelin JS, Töyräs J: Dependence of light attenuation and backscattering on collagen concentration and chondrocyte density in agarose scaffolds. Phys Med Biol 59(21): 6537-6548, 2014 [Pubmed] [Full text]

  3. Karhula SS, Finnilä MA, Lammi MJ, Ylärinne, JH, Kauppinen A, Pritzker KPH, Nieminen HJ, Saarakkala S: Effects of articular cartilage constituents on phosphotungstic acid enhanced micro-computed tomography imaging. PLoS One 12(1): e017075, 2017 [Pubmed] [Full text]

  4. Prittinen J, Ylärinne J, Piltti J, Karhula S, Rieppo L, Ojanen P, Korhonen RK, Saarakkala S, Lammi MJ, Qu C: Effect of centrifugal force on the development of articular neocartilage with bovine primary chondrocytes. Cell Tissue Res 375(3): 629-639, 2019 [Pubmed] [Full text]

Last updated March 7, 2019