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Purpose/aim: Corneal injury is a major cause of impaired vision around the globe. The fine structure of the corneal stroma plays a pivotal role in the phenotype and behavior of the embedded cells during homeostasis and healing after trauma or infection. In order to study healing processes in the cornea, it is important to create culture systems that functionally mimic the natural environment.

Materials and methods: Collagen solution was vitrified on top of a grated film to achieve thin collagen films with parallel microgrooves. Keratocytes (corneal stromal cells) were cultured on the films either as a single layer or as stacked layers of films and cells. SEM and F-actin staining were used to analyze the pattern transference onto the collagen and the cell orientation on the films. Cell viability was analyzed with MTS and live/dead staining. Keratocytes, fibroblasts, and myofibroblasts were cultured to study the pattern’s effect on phenotype.

Results: A microstructured collagen film-based culture system that guides keratocytes (stromal cells) to their native, layerwise perpendicular orientation in 3D and that can support fibroblasts and myofibroblasts was created. The films are thin and transparent enough to observe cells at least three layers deep. The cells maintain viability in 2D and 3D cultures and the films can support fibroblast and myofibroblast phenotypes.

Conclusions: The films provide an easily reproducible stroma model that maintains high cell viability and improves the preservation of the keratocyte phenotype in keratocytes that are differentiated to fibroblasts.

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.

A correct articular cartilage ultrastructure regarding its structural components and cellularity is important for appropriate performance of tissue-engineered articular cartilage. Various scaffold-based, as well as scaffold-free, culture models have been under development to manufacture functional cartilage tissue. Even decellularized tissues have been considered as a potential choice for cellular seeding and tissue fabrication. Pore size, interconnectivity, and functionalization of the scaffold architecture can be varied. Increased mechanical function requires a dense scaffold, which also easily restricts cellular access within the scaffold at seeding. High pore size enhances nutrient transport, while small pore size improves cellular interactions and scaffold resorption. In scaffold-free cultures, the cells assemble the tissue completely by themselves; in optimized cultures, they should be able to fabricate native-like tissue. Decellularized cartilage has a native ultrastructure, although it is a challenge to obtain proper cellular colonization during cell seeding. Bioprinting can, in principle, provide the tissue with correct cellularity and extracellular matrix content, although it is still an open question as to how the correct molecular interaction and structure of extracellular matrix could be achieved. These are challenges facing the ongoing efforts to manufacture optimal articular cartilage.

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.

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.

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.

The adhesion and spreading of human bone marrow-derived mesenchymal stem cells (hMSCs), bovine primary chondrocytes and human osteoblastic osteosarcoma cell line (Saos-2) cultured on various coated surfaces were examined to determine whether different materials coated on the silicon wafer could affect the growth of the different cell types. The amorphous diamond (AD), titania (TiO₂), alumina (Al₂O₃) or carbon nitride (C₃N₄) coating on the silicon wafer was obtained by using ultra short pulsed laser deposition. The differences in surface characteristics were characterized with atomic force microscope and contact angles and zeta potential measurements. Human MSCs, bovine primary chondrocytes and Saos-2 osteoblasts were cultured for 48 h in direct contact with AD-, TiO₂-, Al₂O₃- and C₃N₄-coated surfaces. Cell proliferation was assayed with MTT assay. The morphology, adhesion and spreading of the cultured cells were examined with scanning electron microscope. Human MSCs had the highest cell number after 48-h-culture on all of the different coated surfaces, followed by Saos-2 osteoblasts, and then bovine primary chondrocytes. The morphological appearance of MSCs, chondrocytes and Saos-2 osteoblasts remained as original. No statistically significant differences on cell proliferation were found among the different coated surfaces. Ultra short pulsed laser deposited high quality AD-, TiO₂-, Al₂O₃- and C₃N₄-coated surfaces, and provided a good environment for the adhesion and spreading of the hMSCs, primary chondrocytes and Saos-2 osteoblasts

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.