| 1. |
- Honkanen, Juuso, et al.
(författare)
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Cationic contrast agent diffusion differs between cartilage and meniscus
- 2016
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Ingår i: Annals of Biomedical Engineering. - : Springer. - 0090-6964 .- 1573-9686. ; 44:10, s. 2913-2921
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Tidskriftsartikel (refereegranskat)abstract
- Contrast enhanced computed tomography (CECT) is a non-destructive imaging technique used for the assessment of composition and structure of articular cartilage and meniscus. Due to structural and compositional differences between these tissues, diffusion and distribution of contrast agents may differ in cartilage and meniscus. The aim of this study is to determine the diffusion kinematics of a novel iodine based cationic contrast agent (CA(2+)) in cartilage and meniscus. Cylindrical cartilage and meniscus samples (d = 6 mm, h ≈ 2 mm) were harvested from healthy bovine knee joints (n = 10), immersed in isotonic cationic contrast agent (20 mgI/mL), and imaged using a micro-CT scanner at 26 time points up to 48 h. Subsequently, normalized X-ray attenuation and contrast agent diffusion flux, as well as water, collagen and proteoglycan (PG) contents in the tissues were determined. The contrast agent distributions within cartilage and meniscus were different. In addition, the normalized attenuation and diffusion flux were higher (p < 0.05) in cartilage. Based on these results, diffusion kinematics vary between cartilage and meniscus. These tissue specific variations can affect the interpretation of CECT images and should be considered when cartilage and meniscus are assessed simultaneously.
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| 2. |
- Inkinen, Satu, et al.
(författare)
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Collagen and chondrocyte concentrations control ultrasound scattering in agarose scaffolds
- 2014
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Ingår i: Ultrasound in Medicine and Biology. - : Elsevier. - 0301-5629 .- 1879-291X. ; 40:9, s. 2162-2171
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Tidskriftsartikel (refereegranskat)abstract
- Ultrasound imaging has been proposed for diagnostics of osteoarthritis and cartilage injuries in vivo. However, the specific contribution of chondrocytes and collagen to ultrasound scattering in articular cartilage has not been systematically studied. We investigated the role of these tissue structures by measuring ultrasound scattering in agarose scaffolds with varying collagen and chondrocyte concentrations. Ultrasound catheters with center frequencies of 9 MHz (7.1–11.0 MHz, −6 dB) and 40 MHz (30.1–45.3 MHz, −6 dB) were applied using an intravascular ultrasound device. Ultrasound backscattering quantified in a region of interest starting right below sample surface differed significantly (p < 0.05) with the concentrations of collagen and chondrocytes. An ultrasound frequency of 40 MHz, as compared with 9 MHz, was more sensitive to variations in collagen and chondrocyte concentrations. The present findings may improve diagnostic interpretation of arthroscopic ultrasound imaging and provide information necessary for development of models describing ultrasound propagation within cartilage.
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| 3. |
- Karhula, Sakari, et al.
(författare)
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Effects of articular cartilage constituents on phosphotungstic acid enhanced micro-computed tomography
- 2017
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Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 12:1
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Tidskriftsartikel (refereegranskat)abstract
- Contrast-enhanced micro-computed tomography (CEμCT) with phosphotungstic acid (PTA) has shown potential for detecting collagen distribution of articular cartilage. However, the selectivity of the PTA staining to articular cartilage constituents remains to be elucidated. The aim of this study was to investigate the dependence of PTA for the collagen content in bovine articular cartilage. Adjacent bovine articular cartilage samples were treated with chondroitinase ABC and collagenase to degrade the proteoglycan and the collagen constituents in articular cartilage, respectively. Enzymatically degraded samples were compared to the untreated samples using CEμCT and reference methods, such as Fourier-transform infrared imaging. Decrease in the X-ray attenuation of PTA in articular cartilage and collagen content was observed in cartilage depth of 0-13% and deeper in tissue after collagen degradation. Increase in the X-ray attenuation of PTA was observed in the cartilage depth of 13-39% after proteoglycan degradation. The X-ray attenuation of PTA-labelled articular cartilage in CEμCT is associated mainly with collagen content but the proteoglycans have a minor effect on the X-ray attenuation of the PTA-labelled articular cartilage. In conclusion, the PTA labeling provides a feasible CEμCT method for 3D characterization of articular cartilage.
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| 4. |
- Prittinen, Juha, 1989-, et al.
(författare)
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Chondrocyte behavior on nanostructured micropillar polypropylene and polystyrene surfaces
- 2014
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Ingår i: Materials Science and Engineering. C, Materials for Biological Applications. - : Elsevier. - 0928-4931. ; 43, s. 424-431
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 5. |
- Prittinen, Juha, 1989-, et al.
(författare)
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Effect of centrifugal force on the development of articular neocartilage with bovine primary chondrocytes
- 2019
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Ingår i: Cell and Tissue Research. - New York : Springer. - 0302-766X .- 1432-0878. ; 375:3, s. 629-639
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Tidskriftsartikel (refereegranskat)abstract
- A lot has been invested into understanding how to assemble cartilage tissue in vitro and various designs have been developed to manufacture cartilage tissue with native-like biological properties. So far, no satisfactory design has been presented. Bovine primary chondrocytes are used to self-assemble scaffold-free constructs to investigate whether mechanical loading by centrifugal force would be useful in manufacturing cartilage tissue in vitro. Six million chondrocytes were laid on top of defatted bone disks placed inside an agarose well in 50-ml culture tubes. The constructs were centrifuged once or three times per day for 15 min at a centrifugal force of 771×g for up to 4 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. The centrifuged tissues had a more even surface covering a larger area of the bone disk. Fourier transform infrared imaging analysis indicated a higher concentration of collagen in the top and bottom edges in some of the centrifuged samples. Glycosaminoglycan contents increased along the culture, while collagen content remained 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. The centrifugation regimes lead to more uniform tissue constructs, whereas improved biological properties of the native tissue could not be obtained by centrifugation.
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| 6. |
- Prittinen, Juha, 1989-, et al.
(författare)
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Effect of gravitational force on the development of articular neocartilage with bovine primary chondrocytes
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- 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.
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| 7. |
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| 8. |
- Puhakka, Pia, et al.
(författare)
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Dependence of light attenuation and backscattering on collagen concentration and chondrocyte density in agarose scaffolds
- 2014
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Ingår i: Physics in Medicine and Biology. - : Institute of Physics Publishing (IOPP). - 0031-9155 .- 1361-6560. ; 59:21, s. 6537-6548
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Tidskriftsartikel (refereegranskat)abstract
- Optical coherence tomography (OCT) has been applied for high resolution imaging of articular cartilage. However, the contribution of individual structural elements of cartilage on OCT signal has not been thoroughly studied. We hypothesize that both collagen and chondrocytes, essential structural components of cartilage, act as important light scatterers and that variation in their concentrations can be detected by OCT through changes in backscattering and attenuation. To evaluate this hypothesis, we established a controlled model system using agarose scaffolds embedded with variable collagen concentrations and chondrocyte densities. Using OCT, we measured the backscattering coefficient (µb) and total attenuation coefficient (µt) in these scaffolds. Along our hypothesis, light backscattering and attenuation in agarose were dependent on collagen concentration and chondrocyte density. Significant correlations were found between µt and chondrocyte density (ρ = 0.853, p < 0.001) and between µt and collagen concentration (ρ = 0.694, p < 0.001). µb correlated significantly with chondrocyte density (ρ = 0.504, p < 0.001) but not with collagen concentration (ρ = 0.103, p = 0.422) of the scaffold. Thus, quantitation of light backscattering and, especially, attenuation could be valuable when evaluating the integrity of soft tissues, such as articular cartilage with OCT.
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| 9. |
- Qu, Chengjuan, 1967-, et al.
(författare)
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Five percent oxygen tension is not beneficial for neocartilage formation in scaffold-free cell cultures
- 2012
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Ingår i: Cell and Tissue Research. - : Springer Publishing Company. - 0302-766X .- 1432-0878. ; 348:1, s. 109-117
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 10. |
- Ylärinne, Janne, 1989-, et al.
(författare)
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Comparison of the neocartilages generated in scaffolds and scaffold-free agarose gel supported primary chondrocyte culture : Generation of neocartilage in vitro
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Objective: The present study was conducted to compare the neocartilages generated in scaffolds and scaffold-free, agarose gel-supported primary chondrocyte cultures.Design: Six million bovine primary chondrocytes were embedded in HyStem™ or HydroMatrix™ scaffolds, or scaffold-free in chondrocyte culture medium, and then loaded to agarose gel supported culture wells. Neocartilages were grown in the presence of hypertonic high glucose DMEM medium in 37 °C incubator at 20% O2 and 5% CO2 for one, three or six weeks. By the end of culture periods, the formed tissues were analyzed by histological staining for proteoglycans (PGs) and type II collagen, gene expression measurements of chondrocyte-specific genes aggrecan, Sox9 and procollagen α1(II), and procollagen α2(I) were performed using quantitative RT-PCR, and analyses of PG contents and structure were conducted by spectrophotometric and agarose gel electrophoretic methods, respectively.Results: The neocartilage generated in scaffold-free cultures appeared slightly bigger in size than in HyStem™- or HydroMatrix™-containing scaffolds. Histology visualized that the PGs and type II collagen were abundantly present in both in scaffold-free and scaffold-containing tissues. The PG content gradually increased following the culture period. However, the mRNA expression levels of the cartilage-specific genes of aggrecan, procollagen α1(II) and Sox9 gradually decreased following culture period, while procollagen α2(I) levels increased.Conclusions: After six weeks cultivations, the PG concentrations in neocartilage tissues manufactured with HyStem™ or HydroMatrix™ scaffolds, and in scaffold-free agarose gel-supported cell cultures, were similar to native cartilage. No obvious benefits could be seen on the extracellular matrix assembly in HyStem™ or HydroMatrix™ scaffolds cultures.
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| 11. |
- Ylärinne, Janne, 1990-, et al.
(författare)
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Hypertonic conditions enhance cartilage formation in scaffold-free primary chondrocyte cultures
- 2014
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Ingår i: Cell and Tissue Research. - Berlin : Springer Berlin/Heidelberg. - 0302-766X .- 1432-0878. ; 358:2, s. 541-550
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Tidskriftsartikel (refereegranskat)abstract
- The potential of hypertonic conditions at in vivo levels to promote cartilage extracellular matrix accumulation in scaffold-free primary chondrocyte cultures was investigated. Six million bovine primary chondrocytes were cultured in transwell inserts in low glucose (LG), high glucose (HG), or hypertonic high glucose (HHG) DMEM supplemented with fetal bovine serum, antibiotics, and ascorbate under 5 % or 20 % O2 tension with and without transforming growth factor (TGF)-β3 for 6 weeks. Samples were collected for histological staining of proteoglycans (PGs) and type II collagen, analysis by quantitative reverse transcription plus the polymerase chain reaction (RT-PCR) of mRNA expression of aggrecan and procollagen α1 (II) and of Sox9 and procollagen α2 (I), and quantitation of PGs and PG separation in agarose gels. Cartilage tissues produced at 20 % O2 tension were larger than those formed at 5 % O2 tension. Compared with LG, the tissues grew to larger sizes in HG or HHG medium. Histological staining showed the strongest PG and type II collagen staining in cartilage generated in HG or HHG medium at 20 % O2 tension. Quantitative RT-PCR results indicated significantly higher expression of procollagen α1 (II) mRNA in cartilage generated in HHG medium at 20 % O2 tension compared with that in the other samples. TGF-β3 supplements in the culture medium provided no advantage for cartilage formation. Thus, HHG medium used at 20 % O2 tension is the most beneficial combination of the tested culture conditions for scaffold-free cartilage production in vitro and should improve cell culture for research into cartilage repair or tissue engineering.
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| 12. |
- Ylärinne, Janne, et al.
(författare)
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HyStemTM and HydroMatrixTM scaffolds for articular cartilage tissue engineering
- 2016
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Ingår i: Osteoarthritis and Cartilage. - : Elsevier. - 1063-4584 .- 1522-9653. ; 24, s. S466-S466
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- Purpose: The purpose of the articular cartilage is to protect the subchondral bone from both impacts and abrasion caused by normal movement of an individual. In osteoarthritis, the tissue wearing leads in the worst case to almost complete erosion of cartilage tissue in the joint. The goal of cartilage tissue engineering is to develop methods to manufacture implants for cartilage repair. This study was conducted to compare neocartilages generated in scaffolds and scaffold-free, agarose gel-supported primary chondrocyte cultures.Methods: Six million bovine primary chondrocytes were embedded in the HyStem™ (HS) or the HydroMatrix™ (HM)scaffolds, or suspended in chondrocyte culture medium (scaffold-free), and then loaded to the agarose gel supported culture wells. The neocartilages were grown in the presence of hypertonic high glucose DMEM medium in 37° C incubator at 20% O2 and 5% CO2 for one, three or six weeks. By the end of the culture periods, the formed tissues were analyzed by toluidine blue staining of their histological sections for proteoglycans (PGs) and immunostaining for type II collagen. The gene expression measurements of the chondrocyte-specific genes aggrecan, Sox9 and procollagen α1(II), as well as those of procollagen α2(I), were performed using quantitative RT-PCR, and analyses of the PG contents and the structure were conducted by spectrophotometric and agarose gel electrophoretic methods, respectively.Results: The neocartilages generated in scaffold-free cultures appeared slightly bigger in size than those tissues generated in the HyStem™ or the HydroMatrix™ scaffolds (Figure 1). The histological stainings showed that the PGs and type II collagen were abundantly present both in the scaffold-free and the scaffold-containing tissues (Figures 2 and 3). The PG content of the tissues generated in both scaffold-free and scaffold culture gradually increased following the culture period. However, the mRNA expression levels of the cartilage-specific genes of aggrecan, procollagen α1(II) and Sox9 gradually decreased following culture period, while procollagen α2(I) levels increased.Conclusions: The results from our present study showed that there was no obvious difference in quality of the tissues generated in the scaffolds or the scaffold-free cell culture system. We were able to grow regularly-shaped, smooth-surfaced neotissues with all the methods used in the present study. The HyStem™ scaffold appeared to be helpful for the formation of the tissue in the beginning of the cultivation, whereas the HydroMatrix™ proved harder to handle. The culture methods produced good hyaline cartilage-like tissues. The molecular contents were similar to the native cartilage. However, the structural organization did not have the same assembly as the native articular cartilage. Further development of the culture technique needs to be solved in the future research.
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| 13. |
- Ylärinne, Janne, 1989-
(författare)
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Production of neocartilage tissues using primary chondrocytes
- 2016
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- 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.
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| 14. |
- Ylärinne, Janne, 1990-, et al.
(författare)
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Scaffold-free approach produces neocartilage tissue of similar quality as the use of HyStem™ and Hydromatrix™ scaffolds
- 2017
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Ingår i: Journal of materials science. Materials in medicine. - : Springer Publishing Company. - 0957-4530 .- 1573-4838. ; 28:4
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Tidskriftsartikel (refereegranskat)abstract
- Numerous biomaterials are being considered for cartilage tissue engineering, while scaffold-free systems have also been introduced. Thus, it is important to know do the scaffolds improve the formation of manufactured neocartilages. This study compares scaffold-free cultures to two scaffold-containing ones. Six million bovine primary chondrocytes were embedded in HyStem™ or HydroMatrix™ scaffolds, or suspended in scaffold-free chondrocyte culture medium, and then loaded into agarose gel supported culture well pockets. Neocartilages were grown in the presence of hypertonic high glucose DMEM medium for up to 6 weeks. By the end of culture periods, the formed tissues were analyzed by histological staining for proteoglycans (PGs) and type II collagen, gene expression measurements of aggrecan, Sox9, procollagen α1(II), and procollagen α2(I) were performed using quantitative RT-PCR, and analyses of PG contents and structure were conducted by spectrophotometric and agarose gel electrophoretic methods. Histological stainings showed that the PGs and type II collagen were abundantly present in both the scaffold-free and the scaffold-containing tissues. The PG content gradually increased following the culture period. However, the mRNA expression levels of the cartilage-specific genes of aggrecan, procollagen α1(II) and Sox9 gradually decreased following culture period, while procollagen α2(I) levels increased. After 6-week-cultivations, the PG concentrations in neocartilage tissues manufactured with HyStem™ or HydroMatrix™ scaffolds, and in scaffold-free agarose gel-supported cell cultures, were similar to native cartilage. No obvious benefits could be seen on the extracellular matrix assembly in HyStem™ or HydroMatrix™ scaffolds cultures.
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