During the last decades the number of biologics increased dramatically and several biopharmaceutical drugs such as peptides, therapeutic proteins, hormones, enzymes, vaccines, monoclonal antibodies and antibody-drug conjugates conquered the market. Moreover, administration and local delivery of growth factors has gained substantial importance in the field of tissue engineering. Despite progress that has been made over the last decades formulation and delivery of therapeutic proteins is still a challenge. Thus, we worked on formulation and delivery strategies of therapeutic proteins to improve their biological performance. Phase I of this work deals with protein stability with the main focus on a liquid protein formulation of the dimeric fusion protein PR-15, a lesion specific platelet adhesion inhibitor. In order to develop an adequate formulation ensuring the stability and bioactivity of PR-15 during storage at 4 °C, a pH screening, a forced degradation and a Design of Experiments (DoE) was performed. First the stability and bioactivity of PR-15 in 50 mM histidine buffer in relation to pH was evaluated in a short-term storage stability study at 25 °C and 40 °C for 4 and 8 weeks using different analytical methods. Additionally, potential degradation pathways of PR-15 were investigated under stressed conditions such as heat treatment, acidic or basic pH, freeze-thaw cycles, light exposure, induced oxidation and induced deamidation during the forced degradation study. Moreover, we were able to identify the main degradation product of PR-15 by performing LC/ESI-MS analysis. Further optimization of the injectable PR 15 formulation concerning pH, the choice of buffer and the addition of excipients was studied in the following DoE and finally an optimal PR-15 formulation was found. The growth factors BMP-2, IGF-I and TGF-β3 were selected for the differentiation of stem cells for tissue engineering of cartilage and bone in order to prepare multifunctionalized osteochondral implants for the regeneration of cartilage defects. Silk fibroin (SF) was chosen as biomaterial because of its biocompatibility, mechanical properties and its opportunity for biofunctionalization. Ideal geometry of SF scaffolds with optimal porosity was found in order to generate both tissues on one scaffold. The growth factors BMP-2 and IGF-I were modified to allow spatially restricted covalent immobilization on the generated porous SF scaffolds. In order to perform site-directed covalent coupling by the usage of click chemistry on two opposite sides of the scaffold, we genetically engineered BMP-2 (not shown in this work; performed by Barbara Tabisz) and IGF-I for the introduction of alkyne or azide bearing artificial amino acids. TGF β3 was immobilized to beads through common EDC/NHS chemistry requiring no modification and distributed in the pores of the entire scaffold. For this reason protein modification, protein engineering, protein immobilization and bioconjugation are investigated in phase II. Beside the synthesis the focus was on the characterization of such modified proteins and its conjugates. The field of protein engineering offers a wide range of possibilities to modify existing proteins or to design new proteins with prolonged serum half-life, increased conformational stability or improved release rates according to their clinical use. Site-directed click chemistry and non-site-directed EDC/NHS chemistry were used for bioconjugation and protein immobilization with the aim to underline the preferences of site-directed coupling. We chose three strategies for the incorporation of alkyne or azide functionality for the performance of click reaction into the protein of interest: diazonium coupling reaction, PEGylation and genetic engineering. Azido groups were successfully introduced into SF by implementation of diazonium coupling and alkyne, amino or acid functionality was incorporated into FGF-2 as model protein by means of thiol PEGylation. The proper folding of FGF-2 after PEGylation was assessed by fluorescence spectroscopy, WST-1 proliferation assay ensured moderate bioactivity and the purity of PEGylated FGF-2 samples was monitored with RP-HPLC. Moreover, the modification of native FGF-2 with 10 kDa PEG chains resulted in enhanced thermal stability. Additionally, we genetically engineered one IGF-I mutant by incorporating the unnatural amino acid propargyl-L-lysine (plk) at position 65 into the IGF-I amino acid sequence and were able to express hardly verifiable amounts of plk-IGF-I. Consequently, plk-IGF-I expression has to be further optimized in future studies in order to generate plk-IGF-I with higher yields. Bioconjugation of PEGylated FGF-2 with functionalized silk was performed in solution and was successful for click as well as EDC/NHS chemistry. However, substantial amounts of unreacted PEG-FGF-2 were adsorbed to SF and could not be removed from the reaction mixture making it impossible to expose the advantages of click chemistry in relation to EDC/NHS chemistry. The immobilization of PEG-FGF-2 to microspheres was a trial to increase product yield and to remove unreacted PEG-FGF-2 from reaction mixture. Bound PEG-FGF-2 was visualized by fluorescence imaging or flow cytometry and bioactivity was assessed by analysis of the proliferation of NIH 3T3 cells. However, immobilization on beads raised the same issue as in solution: adsorption caused by electrostatic interactions of positively charged FGF-2 and negatively charged SF or beads. Finally, we were not able to prove superiority of site-directed click chemistry over non-site-directed EDC/NHS. The skills and knowledge in protein immobilization as well as protein characterization acquired during phase II helped us in phase III to engineer cartilage tissue in biofunctionalized SF scaffolds. The approach of covalent immobilization of the required growth factors is relevant because of their short in vivo half-lives and aimed at controlling their bioavailability. So TGF-β3 was covalently coupled by means of EDC/NHS chemistry to biocompatible and biostable PMMA beads. Herein, we directly compared bioactivity of covalently coupled and adsorbed TGF-β3. During the so-called luciferase assay bioactivity of covalent coupled as well as adsorbed TGF-β3 on PMMA beads was ensured. In order to investigate the real influence of EDC/NHS chemistry on TGF-β3’s bioactivity, the amount of immobilized TGF-β3 on PMMA beads was determined. Therefore, an ELISA method was established. The assessment of total amount of TGF-β3 immobilized on the PMMA beads allowed as to calculate coupling efficiency. A significantly higher coupling efficiency was determined for the coupling of TGF-β3 via EDC/NHS chemistry compared to the reaction without coupling reagents indicating a small amount of adsorbed TGF-β3. These results provide opportunity to determine the consequence of coupling by means of EDC/NHS chemistry for TGF β3 bioactivity. At first sight, no statistically significant difference between covalent immobilized and adsorbed TGF-β3 was observed regarding relative luciferase activities. But during comparison of total and active amount of TGF-β3 on PMMA beads detected by ELISA or luciferase assay, respectively, a decrease of TGF-β3’s bioactivity became apparent. Nevertheless, immobilized TGF β3 was further investigated in combination with SF scaffolds in order to drive BMSCs to the chondrogenic lineage. According to the results obtained through histological and immunohistochemical studies, biochemical assays as well as qRT-PCR of gene expression from BMSCs after 21 days in culture immobilized TGF-β3 was able to engineer cartilage tissue. These findings support the thesis that local presentation of TGF β3 is superior towards exogenous TGF β3 for the development of hyaline cartilage. Furthermore, we conclude that covalent immobilized TGF β3 is not only superior towards exogenously supplemented TGF-β3 but also superior towards adsorbed TGF-β3 for articular hyaline cartilage tissue engineering. Diffusion processes were inhibited through covalent immobilization of TGF-β3 to PMMA beads and thereby a stable and consistent TGF-β3 concentration was maintained in the target area. With the knowledge acquired during phase II and III as well as during the studies of Barbara Tabisz concerning the expression and purification of plk-BMP-2 we made considerable progress towards the formation of multifunctionalized osteochondral implants for the regeneration of cartilage defects. However, further studies are required for the translation of these insights into the development of multifunctionalized osteochondral SF scaffolds. / In den letzten Jahrzehnten stieg die Zahl der Biologika dramatisch an und mehrere biopharmazeutische Arzneimittel wie Peptide, therapeutische Proteine, Hormone, Enzyme, Impfstoffe, monoklonale Antikörper und Antikörper-Wirkstoff-Konjugate eroberten den Markt. Darüber hinaus hat die Applikation und lokale Verabreichung von Wachstumsfaktoren im Bereich des Tissue Engineerings eine wesentliche Bedeutung erlangt. Trotz der in den letzten Jahrzehnten erzielten Fortschritte ist die Formulierung und Verabreichung therapeutischer Proteine noch immer eine Herausforderung. Daher haben wir uns in dieser Arbeit mit der Formulierung und Verabreichung therapeutischer Proteine beschäftigt und Strategien entwickelt, um deren biologische Wirkung zu verbessern. In Phase I dieser Arbeit konzentrieren wir uns auf die Stabilität des dimeren Fusionsproteins PR 15, einem Inhibitor der Adhäsion von Plättchen an arterielle Gefäßläsionen. Um eine geeignete flüssige Formulierung zu entwickeln, welche die Stabilität und Bioaktivität von PR-15 während der Lagerung bei 4 °C sicherstellt, wurde ein pH Screening, eine Forced Degradation Studie und ein Design of Experiments (DoE) durchgeführt. Zuerst wurde die Stabilität und Bioaktivität von PR-15 bei verschiedenen pH Werten in 50 mM Histidinpuffer in einer Kurzzeitstabilitätsstudie bei 25 °C und 40 °C nach 4 und 8 Wochen mit Hilfe verschiedener analytischer Methoden beobachtet. Des Weiteren wurden mögliche Abbauwege von PR-15 unter Stressbedingungen wie erhöhter Temperatur, saurem oder basischem pH-Wert, Einfrier-Auftau-Zyklen, Lichteinwirkung, induzierter Oxidation sowie induzierter Deamidierung während der Forced Degradation Studie untersucht. Darüber hinaus konnten wir das Hauptabbauprodukt von PR-15 durch LC/ESI-MS Analysen identifizieren. Im folgenden DoE wurde die injizierbare PR-15 Formulierung weiter optimiert und bezüglich pH, der Wahl des Puffers sowie der Zugabe von Hilfsstoffen analysiert, bis letztendlich eine optimale PR 15-Formulierung gefunden wurde. Die Wachstumsfaktoren BMP-2, IGF-I und TGF-β3 wurden zur Differenzierung von Stammzellen für das Tissue Engineering von Knochen und Knorpel ausgewählt, um multifunktionalisierte osteochondrale Implantate zur Regeneration von Knorpeldefekten herzustellen. Seidenfibroin (SF) wurde aufgrund seiner Biokompatibilität, seiner mechanischen Eigenschaften und seiner Möglichkeiten zur Biofunktionalisierung als Biomaterial gewählt. Zuerst wurden SF-Scaffolds mit idealer Geometrie und optimaler Porosität erzeugt, um sowohl Knochen also auch Knorpel auf einem Scaffold herzustellen. Um eine räumlich begrenzte kovalente Immobilisierung der Wachstumsfaktoren BMP-2 und IGF-I auf den porösen SF-Scaffolds zu ermöglichen, wurden diese mit unnatürlichen Aminosäuren genetisch modifiziert. Das Einführen von Alkin- bzw. Azidresten in die Aminosäuresequenz von BMP-2 (in dieser Arbeit nicht gezeigt; von Barbara Tabisz durchgeführt) und IGF-I erlaubt unter Verwendung der Click-Chemie eine ortsgerichtete kovalente Kopplung der Wachstumsfaktoren an zwei gegenüberliegenden Seiten der Scaffolds. TGF-β3 wurde durch gewöhnliche EDC/NHS-Chemie, welche keine Modifikation erforderte, kovalent an Mikrosphären immobilisiert und in den Poren des gesamten SF-Scaffolds verteilt. Daher beschäftigen wir uns in Phase II mit der Modifikation von Proteinen, dem Protein Engineering, der Immobilisation von Proteinen und mit Biokonjugation. Neben der Synthese lag der Fokus auf der Charakterisierung modifizierter Proteine und deren Konjugaten. Das Gebiet des Protein Engineerings bietet eine Vielzahl von Möglichkeiten, bestehende Proteine zu modifizieren oder neue Proteine mit verlängerter Serumhalbwertszeit, erhöhter konformativer Stabilität oder verbesserten Freisetzungsraten entsprechend der klinischen Anwendung zu entwickeln. Die ortsspezifische Click-Chemie und die nicht-ortsspezifische EDC/NHS-Chemie wurden für die Biokonjugation und die Immobilisierung von Proteinen verwendet mit dem Ziel, die Vorzüge der ortsgerichteten Kopplung hervorzuheben. Für den Einbau der für die Durchführung der Click-Reaktion erforderlichen Alkin- bzw. Azidfunktionalität in das betreffende Protein wurden drei Strategien ausgewählt: die Azokupplung, die PEGylierung und die gentechnische Modifizierung. Azidgruppen wurden mittels Azokupplung erfolgreich in SF eingebaut und die Alkin-, Amino- oder Säurefunktionalität wurde mittels PEGylierung der Cysteine in das Modellprotein FGF-2 integriert. Die korrekte Faltung von FGF-2 nach erfolgreicher PEGylierung wurde durch Fluoreszenzspektroskopie bestätigt, im WST-1 Proliferationsassay wurde eine angemessene Bioaktivität festgestellt und die Reinheit von PEGylierten FGF-2 wurde mittels RP-HPLC analysiert. Darüber hinaus führte die Modifikation von nativem FGF-2 mit 10 kDa PEG-Ketten zu einer erhöhten thermischen Stabilität. Des Weiteren wurde ein IGF-I-Mutant gentechnisch hergestellt, indem die unnatürliche Aminosäure Propargyl-L-Lysin (Plk) an Position 65 in die IGF-I-Sequenz eingebaut wurde. Da letztendlich lediglich kaum nachweisbare Mengen an Plk-IGF-I exprimiert werden konnten, muss die Plk-IGF-I-Expression in anschließenden Studien weiter optimiert werden, um Plk-IGF-I mit höheren Ausbeuten erzeugen zu können. Die Biokonjugation von PEGyliertem FGF-2 und funktionalisierter Seide wurde sowohl mittels Click- als auch mittels EDC/NHS-Chemie erfolgreich durchgeführt. Allerdings wurden erhebliche Mengen PEG-FGF-2 lediglich an SF adsorbiert und nicht kovalent gekoppelt und konnten schlussendlich nicht aus dem Reaktionsgemisch entfernt werden. Die anschließende Immobilisierung von PEG-FGF-2 an Mikrosphären, war ein Versuch die Ausbeute der Reaktion zu erhöhen und adsorbiertes PEG-FGF-2 leichter zu entfernen. Immobilisiertes PEG-FGF-2 wurde mittels Fluoreszenzmikroskopie und/oder Durchflusszytometrie nachgewiesen und die Bioaktivität wurde durch die Analyse der Proliferation von NIH-3T3-Zellen ermittelt. Die Immobilisierung auf Mikrosphären führte jedoch zu demselben Problem wie in Lösung: Adsorption von positiv geladenem FGF-2 an negativ geladenes SF bzw. negativ geladenen Mikrosphären durch elektrostatische Wechselwirkungen. Schließlich waren wir nicht in der Lage, die Überlegenheit der ortsgerichteten Click-Chemie gegenüber der nicht-ortsgerichteten EDC/ NHS-Chemie zu beweisen. Die während Phase II erworbenen Fähigkeiten und Kenntnisse im Bereich der Immobilisierung und Charakterisierung von Proteinen halfen uns in Phase III Knorpelgewebe in biofunktionalisierten SF-Scaffolds zu erzeugen. Der Ansatz der kovalenten Immobilisierung, der für das Tissue Engineering von Knorpel erforderlichen Wachstumsfaktoren, ist aufgrund ihrer kurzen in vivo Halbwertszeiten von Bedeutung und zielt darauf ab, ihre Bioverfügbarkeit zu kontrollieren. So wurde TGF-β3 mittels EDC/NHS-Chemie kovalent an biokompatible und biostabile PMMA-Mikrosphären gekoppelt. Mit Hilfe des sogenannten Luciferase-Assays wurden die Bioaktivitäten von kovalent gekoppeltem sowie von adsorbiertem TGF-β3 auf PMMA-Mikrosphären ermittelt. Um die Kopplungseffizienz zu berechnen und den tatsächlichen Einfluss der EDC/NHS-Chemie auf die Bioaktivität von TGF-β3 zu untersuchen, wurde die Menge an immobilisiertem TGF-β3 auf PMMA-Mikrosphären mittels ELISA bestimmt. Für die Kopplung von TGF-β3 mittels EDC/NHS-Chemie wurde eine signifikant höhere Kopplungseffizienz im Vergleich zu der Reaktion ohne Kopplungsreagenzien, welche eine geringe Menge an adsorbiertem TGF-β3 zeigte, bestimmt. Bei alleiniger Betrachtung der Ergebnisse des Luciferase-Assays, bei welchem kein statistisch signifikanter Unterschied zwischen kovalent immobilisiertem und adsorbiertem TGF-β3 bezüglich der relativen Luciferase-Aktivität beobachtet wurde, scheint es als hätte die EDC/NHS-Kopplung keinen Einfluss auf die Bioaktivität von TGF β3. Beim Vergleich der mittels ELISA bestimmten TGF β3 Gesamtmenge und der mittels Luciferase-Assay bestimmten Menge an aktivem TGF-β3 auf den PMMA-Mikrosphären, wurde jedoch ein Verlust der Bioaktivität von TGF-β3 durch die EDC/NHS-Kopplung deutlich. Ungeachtet dessen, wurde immobilisiertes TGF-β3 genutzt, um Knorpelgewebe in SF-Scaffolds aus BMSCs zu generieren. Nach den Ergebnissen der histologischen und immunhistochemischen Untersuchungen, der biochemischen Assays sowie der qRT-PCR der Genexpression von BMSCs nach 21 Tagen in Kultur, gelang es uns unter Verwendung von immobilisiertem TGF-β3 Knorpelgewebe aufzubauen. Diese Ergebnisse unterstützen die These, dass die lokale Präsentation von TGF-β3 gegenüber exogen zugegebenem TGF-β3 für die Entwicklung von hyalinem Knorpel überlegen ist. Außerdem schließen wir daraus, dass kovalent immobilisiertes TGF-β3 nicht nur gegenüber exogen zugegebenem TGF-β3 für die Entwicklung von hyalinem Knorpelgewebe überlegen ist, sondern auch gegenüber adsorbiertem TGF-β3. Diffusionsprozesse konnten durch kovalente Immobilisierung von TGF-β3 an PMMA-Mikrosphären verhindert werden und damit eine stabile und gleichmäßige TGF β3-Konzentration am Wirkort aufrechterhalten werden. Mit den in Phase II und III gewonnenen Erkenntnissen und den Untersuchungen von Barbara Tabisz zur Expression und Aufreinigung von plk-BMP-2 haben wir erhebliche Fortschritte bei der Entwicklung multifunktionaler osteochondraler Implantate zur Regeneration von Knorpeldefekten gemacht. Für die Umsetzung dieser Erkenntnisse zur Herstellung multifunktionaler osteochondraler SF-Scaffolds sind jedoch weitere Studien erforderlich.

Despite the ongoing political debate regarding the legality of medical marijuana, clinical investigations of the therapeutic use of cannabinoids are now more prevalent than at any time in history. Cannabinoids have been shown to have analgesic, anti-spasmodic, anticonvulsant, anti-tremor, anti-psychotic, anti-inflammatory, anti-oxidant, anti-emetic and appetite-stimulant properties. There are mainly two well-known cannabinoid receptors, CB1 and CB2, located in the central (CB1) and peripheral (CB2) nervous systems as well as the immune system. More recently, endocannabinoids (ligands) and their receptors have also been found in the skeleton which appear as the main body system and physiologically regulated by CB2. This study aimed to examine the effect of both CB1 and CB2 receptor stimulation on wound closure response of MG-63 osteoblast bone cell monolayers using different treatments with cannabinoid such as Winn55,212-2, URB602 and HU308. Also, cell adhesion, cell proliferation and cell length was investigated. The study also aimed to examine the effect of HU308 treatments in combination with TGF-β3 (transforming growth factor beta -3) on wound healing, cell adhesion and extracellular matrix up regulation (collagen type I, fibronectin and protien S-100A6) as well as other biological factors such as secretion of matrix metalloproteinase (MMP-2) and nitric oxide (NO). Finally, this study investigated HU308/TGF-β3 combination treatment on the regulation of extracellular matrix (collagen type I, fibronectin and protien S-100A6) in a 3D multilayer system of MG-63 osteoblast bone cells. Wound healing assays of MG-63 monolayers revealed accelerated wound repair as well as increased cell proliferation mainly regulated through CB2 receptors, and that treatments with HU308 and HU308/TGF-β3 achieved minimum closure timings compared with control groups (P<0.05). Our finding suggested that proliferation rate with 500nM HU308 was significantly higher than control and TGF-β3/HU308 combination groups (P<0.05). Interestingly, percentage of wound remained open after 15 hours for combination groups was 17.6%±1.32 whereas treatment with 500nM HU308 had 20%±2.25 indicating that the combination groups took the lead throughout wound healing. It was also observed that bridge formation in all treatment groups was taking place between 15 to 20 hour periods whereas within control treatments bridge formation started to take place after 25 hours. Cell surface attachment was examined via the trypsinization assay in which the time taken to trypsinize cells from the surface provided a means of assessing the strength of attachment. The results indicated that higher concentrations of HU308 (2μM), induced significant force of cell attachment compared with control and concentrations of 500nM and 1μM (P<0.05). However, groups treated with TGF-β3 and combination HU308/TGF-β3 indicated reduced cell surface attachment compared with control groups, indicating enhanced cell migration. Immunofluorescence staining as well as Elisa based semi-quantification technique indicated that both collagen type I and fibronectin were unregulated using higher concentrations of HU308 with decreased cell proliferation compared to lower concentrations. Nevertheless, protein S-100A6 was up-regulated in treatments with HU308, TGF-β3 and their combination HU308/TGF-β3 (P<0.05), indicating the positive role of these treatments in promoting cell differentiation. MMP-2 levels in the current study were also shown to be concentration-dependent, i.e. higher concentrations of HU308 significantly reduced MMP-2 secretion leading to decreased cell migration, while HU308/TGF-β3 combination treatment increased MMP-2 levels, indicating an increase in cell migration. The current study also examined levels of nitric oxide synthesis in relation to different treatments with HU308, TGF-β3 and HU308/TGF-β3 combination. It was found that nitric oxide up-regulation influences rate of MG-63 osteoblast wound healing in a concentration dependent manner. Lastly, UpCell culture dishes proved to have efficacy in obtaining a multilayer model of MG-63 osteoblast system in-vitro through changes in cell morphology. It was also found that treatments with HU308, TGF-β3 and HU308/TGF-β3 combination influenced collagen type I, fibronecton and protein S-100A6 secretion. These findings supported the earlier Elisa based semi-quantification results obtained for monolayer cultures.

Yes / Articular cartilage is an avascular and flexible connective tissue found in joints. It produces a cushioning effect at the joints and provides low friction to protect the ends of the bones from wear and tear/damage. It has poor repair capacity and any injury can result pain and loss of mobility. Transforming growth factor-beta (TGF-β), a cytokine superfamily, regulates cell function, including differentiation and proliferation. Although the function of the TGF-βs in various cell types has been investigated, their function in cartilage repair is as yet not fully understood. The effect of TGF-β3 in biological regulation of primary chondrocyte was investigated in this work. TGF-β3 provided fibroblastic morphology to chondrocytes and therefore overall reduction in cell proliferation was observed. The length of the cells supplemented with TGF-β3 were larger than the cells without TGF-β3 treatment. This was caused by the fibroblast like cells (dedifferentiated chondrocytes) which occupied larger areas compared to cells without TGF-β3 addition. The healing process of the model wound closure assay of chondrocyte multilayer was slowed down by TGF-β3, and this cytokine negatively affected the strength of chondrocyte adhesion to the cell culture surface.

no / There is evidence that both the herb Sanicle and the cytokine TGF- β3 can be beneficial in enhancing wound repair. In this study 3T3 fibroblast cells were cultured and the confluent monolayers were wounded (scarred) using a disposable plastic pipette. Various amounts of TGF-β3 (a growth factor) and Sanicle extract were applied to the cell monolayers. TGF-β3 was applied at concentrations of 50ng/ml, 5ng/ml, 500pg/ml, 50pg/ml and 5pg/ml to five different culture flasks with one additional flask acting as control. Sanicle was applied at concentrations of 100μg/ml, 10μg/ml, 1μg/ml, 100ng/ml and 10ng/ml with one additional flask as a control. The cells were imaged over a period of 20 hours with or without presence of TGF-β3 and Sanicle. The results indicated that although there were no significant increases in the rate of wound closure in relation to application of TGF-β3, there is an indication that TGF-β3 may enhance model wound closure at optimum working concentration between 5ng/ml and 50ng/ml. However, the sanicle extract did not stimulate enhanced repair of the model in vitro wound, but instead seemed to promote cell death along the wound margin. These results indicate that sanicle may be used in the care of wounds, but not as a growth promoter, but because it acts as an antibiotic agent, and possibly because it aids wound debridement.

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. / 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.