Abstract Osteoarthritis (OA) is the commonest joint disease in the world, and it has a major socioeconomic impact. OA causes progressive degenerative changes in the composition and structure of articular cartilage and subchondral bone. Clinical diagnosis of OA is based on physical examination and qualitative evaluation of changes on plain radiographs. Current clinical imaging methods are subjective or insensitive to early OA changes. Therefore, new methods are needed so as to quantify composition of the cartilage and characteristics of the subchondral bone. The aim of this thesis was to evaluate the potential of clinically applicable X-ray-based methods for the assessment of the cartilage proteoglycan content as well as the structure and density of subchondral bone in a knee joint. Subchondral bone density and structure (local binary patterns, Laplacian, and fractal-based algorithms) analysis methods for two-dimensional (2-D) plain radiographs were validated against three-dimensional (3-D) bone microarchitecture obtained from micro-computed tomography ex vivo and applied to plain radiographs in vivo. Furthermore, a method developed for the evaluation of articular cartilage proteoglycan content from computed tomography (CT) was validated against a delayed gadolinium-enhanced magnetic resonance imaging of cartilage (dGEMRIC), which is widely used as a proteoglycan sensitive method, in subjects referred for an arthroscopy of the knee joint. Subchondral bone density and structure evaluated from 2-D radiographs were significantly related to the bone volume fraction and true 3-D microarchitecture of bone, respectively. In addition, bone density- and structure-related parameters from radiographs were significantly different among subjects with different stages of OA. Cartilage proteoglycan content evaluated from CT was significantly related to dGEMRIC method. Furthermore, dGEMRIC was associated with bone structure from a 2-D radiograph. In conclusion, analysis of bone structure and density is feasible from clinically available 2-D radiographs. A novel CT method sensitive to proteoglycan content should be considered when a 3-D view of cartilage quality is needed. / Tiivistelmä Nivelrikko on maailman yleisin nivelsairaus. Se aiheuttaa merkittävää kärsimystä potilaille, ja sillä on suuri taloudellinen vaikutus yhteiskuntaan. Nivelrikko aiheuttaa palautumattomia muutoksia nivelrustokudoksen ja rustonalaisen luun koostumukseen ja rakenteeseen. Nivelrikon diagnoosi perustuu kliiniseen tutkimukseen ja röntgenkuvien silmämääräiseen arviointiin. Nykyiset nivelrikon kliiniset kuvantamismenetelmät ovat subjektiivisia eivätkä riittävän tarkkoja nivelrikon varhaisten muutosten osoittamiseen, minkä vuoksi rustokudoksen koostumuksen ja rustonalaisen luun muutosten arviointiin tarvitaan uusia menetelmiä. Tämän väitöskirjantyön tarkoituksena oli tutkia uusien röntgensäteilyyn perustuvien menetelmien soveltuvuutta polvinivelen rustokudoksen proteoglykaanipitoisuuden sekä luun tiheyden ja rakenteen arviointiin. Rustonalaisen luun tiheyttä ja rakennetta arvioitiin digitaalisesta röntgenkuvasta tietokonepohjaisilla menetelmillä ja tuloksia verrattiin mikrotietokonetomografiassa nähtävään luun kolmiulotteiseen rakenteeseen. Röntgenkuvasta laskettavia muuttujia verrattiin myös eriasteisesta nivelrikosta kärsivien henkilöiden välillä. Rustokudoksen proteoglykaanipitoisuutta epäsuorasti mittaavaa tietokonetomografiamenetelmää verrattiin vastaavaan magneettikuvausmenetelmään henkilöillä, jotka olivat menossa polven niveltähystykseen. Röntgenkuvasta laskettu rustonalaisen luun tiheys ja rakenne olivat tilastollisesti selkeästi yhteydessä luun tilavuusmäärään ja mikrorakenteeseen, ja ne erosivat eriasteisesta nivelrikosta kärsivien henkilöiden välillä. Proteoglykaanipitoisuutta arvioivien tietokonetomografia- ja magneettikuvausmenetelmien välillä oli tilastollisesti merkitsevä korrelaatio. Ruston proteoglykaanipitoisuutta arvioivan magneettikuvausmenetelmän ja röntgenkuvasta laskettavan luun rakenteen välillä oli myös tilastollinen yhteys. Loppupäätelmänä voidaan todeta, että luun tiheyttä ja rakennetta on mahdollista arvioida kliinisesti saatavilla olevista röntgenkuvista. Tietokonetomografiamenetelmän käyttöä tulee harkita tutkimuksissa silloin, kun rustokudoksen tilasta halutaan kolmiulotteista tietoa.

Abstract Osteoarthritis (OA) is a common degenerative disease of synovial joints becoming more frequent with age. Pain, stiffness and functional disability caused by OA negatively affect the quality of individuals’ lives. In order to prevent the manifestation of symptoms and further OA progress, early diagnosis is essential. Ultrasonography has the potential to detect various abnormalities in the knee joint, however its place in clinical practice remains uncertain. The present study aimed to determine the diagnostic performance of the semi-quantitative wide-area ultrasound (US) scanning of knee femoral cartilage degeneration, osteophytes and meniscal extrusion using magnetic resonance imaging as the reference tool. Diagnostic ability of conventional radiography (CR) was also determined and the performances of both modalities compared. Subsequently, the association of structural US findings with knee pain and function was investigated. Finally, quantitative US image analysis focusing on detection and evaluation of subchondral bone integrity in early OA was developed. The US quantitative outcomes were compared with CR and arthroscopy. Tibio-femoral osteophytes, medial meniscal extrusion and medial femoral cartilage morphological degeneration were identified by US with better or at least comparable accuracy than by CR, in which joint space narrowing was used as a composite measure of cartilage damage and meniscal extrusion. The global femoral cartilage grade associated strongly with increased pain and disability. Site-specifically, especially medial cartilage degeneration and femoral lateral osteophytes were associated with increased pain and disability. Regarding the quantitative outcomes, significant increase in US intensity in the femoral subchondral bone depth 0.35–0.7 mm and decrease in intensity slope up to 0.7 mm depth were observed during radiographic or arthroscopic OA progression. Novel wide-area US scanning provides relevant additional diagnostic information on tissue-specific OA pathology not depicted by CR. US-detected changes of femoral cartilage and osteophytes are also associated with clinical symptoms. Consequently, the use of US as a complementary imaging tool along with CR may enable more accurate diagnostics of knee OA. Furthermore, developed quantitative US analysis is a promising tool for detection of femoral subchondral bone changes in knee OA. / Tiivistelmä Nivelrikko on erittäin yleinen nivelten rappeumasairaus, joka aiheuttaa kipua, jäykkyyttä sekä liikkumisvaikeutta. Nivelrikon nykyistä varhaisempi diagnosointi olisi äärimmäisen tärkeää, jotta voitaisiin vähentää oireiden esiintymistä ja hidastaa sairauden etenemistä. Ultraäänikuvaus on lupaava menetelmä nivelrikon varhaisdiagnostiikkaan, mutta sitä ei kuitenkaan ole vielä yleisesti hyväksytty rutiininomaiseen kliiniseen käyttöön. Tämän tutkimuksen päätavoitteena oli selvittää polven semi-kvantitatiivisen ultraäänikuvauksen diagnostista kykyä verrattuna perinteiseen röntgenkuvaukseen, kun arvioidaan rustokudoksen kulumista, luupiikkejä sekä nivelkierukan siirtymää. Magneettikuvausta käytettiin vertailumenetelmänä. Toisena tavoitteena oli selvittää yhteyttä polven ultraäänilöydösten ja polven kivun sekä liikkuvuuden välillä. Lopuksi selvitettiin, voidaanko kvantitatiivisella analyysilla parantaa ultraäänikuvauksen tarkkuutta varhaisvaiheen nivelrikkopotilaiden rustonalaisen luun kunnon määrittämiseen. Röntgenkuvaukseen verrattuna ultraäänikuvaus osoittautui vähintään yhtä hyväksi tai paremmaksi kuvantamismenetelmäksi, kun arvioitiin reisi- ja sääriluun luupiikkejä, sisemmän nivelkierukan siirtymää tai rustokudoksen kulumista. Reisiluun nivelruston yleinen kulumisen aste oli suoraan verrannollinen potilaiden polvinivelen liikerajoituksiin ja kipuun. Erityisesti reisiluun sisemmän puolen nivelruston kuluminen sekä ulomman puolen luupiikit liittyivät potilaiden oireisiin. Kvantitatiiviset ultraäänitulokset osoittivat, että ultraäänen rusto-luurajapinnalta tulevan heijastuksen maksimi-intensiteetti lisääntyi sekä intensiteetin tason laskunopeus vähentyi nivelrikon vaikeusasteen kasvaessa. Tutkimus osoitti, että ultraäänikuvauksella voidaan saada tärkeää diagnostista lisätietoa polven nivelrikon aiheuttamista kudosmuutoksista, joita ei pystytä havaitsemaan perinteisellä röntgenkuvauksella. Ultraäänikuvauksessa näkyvät kudosmuutokset liittyvät myös potilaan kliinisiin oireisiin. Lisäksi rustonalaista luuta voidaan analysoida kvantitatiivisesti ultraäänikuvien perusteella, mikä edelleen helpottaa kuvien tulkintaa. Tutkimuksen perusteella voidaankin suositella ultraäänikuvauksen nykyistä laajempaa kliinistä käyttöä röntgenkuvausta täydentävänä tutkimusmenetelmänä nivelrikon varhaisdiagnostiikassa.

Introduktion: Hyalint brosk består av kondrocyter inbäddade i ett extracellulärt matrix som täcker alla kroppens synoviala leder. I dag är skador på hyalint brosk ett folkhälsoproblem med begränsade behandlingsmöjligheter. Mesenkymala stamceller har i flertalet studier visat sig vara en lovande kandidat för framtida behandling av broskskador, eftersom dessa kan differentiera till broskproducerande kondrocyter. Modern behandling av broskskador innefattar odling av patientens egna kondrocyter eller stamceller för att sedan återföra dem till broskskadan. För att göra detta effektivt behöver optimering av odlingsförhållanden för aktuella celler utföras. Syfte: Att undersöka tillväxt hos kondrocyter samt mesenkymala stromaceller från benmärg i varierande odlingsförhållanden, detta med avseende att utvärdera vilka tillsatser som mest gynnar cellernas viabilitet och proliferation. Material & Metod: Framtagning av kondrocyter och mesenkymala stromaceller från benmärg i denna studie utfördes på överblivet ortopediskt material från en individ. Cellerna kollagenasbehandlades innan de odlades ut i ett standardmedium under tre veckor. Därefter odlades de i närvaro av olika tillsatser under sex dagar. Cellernas viabilitet mättes genom relativ fluorescens med CellTiter-Blue® reagens medan proliferationen mättes genom cellräkning med flödescytometri. Resultat: Det fanns en signifikant skillnad (ANOVA, p<0,05) mellan minst två tillsatser inom grupperna för viabilitetsanalys samt cellräkning för både kondrocyter och benmärgderiverade mesenkymala stromaceller (BMMSC). Kondrocyterna som odlades i närvaro av fetalt bovint serum (FBS) hade högre proliferation och viabilitet än de celler som odlades i frånvaro av FBS. FBS i kombination med insulin-transferrin-selen (ITS) och kondrocytfaktorerna (KF) dexametason, prolin, insulin samt transforming growth factor beta hade störst positiv effekt på proliferationen. Vidare visade Minimum Essential Medium α med tillsats av FBS 10 % ha störst effekt, av den panel som undersöktes, på både viabilitet och proliferation hos BMMSC. Slutsats: FBS i kombination med ITS och KF hade störst stimulerande effekt på proliferation hos kondrocyter. Val av standardmedium hade större betydelse än tillsats av faktorer vid odling av BMMSC in vitro. / Introduction: Articular cartilage consists of chondrocytes and covers all the synovial joints in the body. Articular cartilage injuries are today a public health issue with limited orthopedic treatments. Mesenchymal stem cells have shown to be a promising candidate for future treatment of articular cartilage injuries due to their many abilities. Modern treatment of cartilage damage involves cultivation of the patient´s own chondrocytes and stem cells. To make this treatment as effective as possible the culture conditions for chondrocytes and stem cells must be optimized. Aim: To study cell growth in chondrocytes and mesenchymal stromal cells from bone marrow in varying culture conditions, this regarding to evaluate the most optimal culture conditions for cell viability and proliferation. Method: Cell extraction in this study was performed on leftover orthopedic material. The cells were treated with collagenase and then cultured in a standard medium for three weeks before they were cultured in presence of various factors for six days. Cell viability was measured by relative fluorescence while proliferation was measured by cell counting. Result: There was a statistically significant difference (ANOVA, p<0,05) between at least two additives within the groups for viability analysis and cell count for both chondrocytes and bone marrow-derived mesenchymal stromal cells (BMMSC). The chondrocytes cultured in presence of fetal bovine serum (FBS) had a higher proliferation and viability compared to the cells cultured in the absence of FBS. FBS in combination with insulin-transferrin-selenium (ITS) and the chondrocyte factors (CF) dexamethasone, proline, insulin and transforming growth factor beta had the biggest impact on proliferation. Minimum Essential Medium α with FBS 10% additive showed positive effects on both viability and proliferation of BMMSCs. Conclusion: FBS in combination with ITS and CF had a good effect on the proliferation of chondrocytes. The choice of standard medium seems to have a more important role than growth factor additive when studying BMMSCs expansion in vitro.

Articular chondral lesions in the knee joint can be diagnosed at an early stage using MRI. Segmenting and visualizing lesions and the overall joint structure allows improved communication between the radiologist and referring physician. It can also be of help when determining diagnosis or conducting surgical planning. Although there are a variety of studies proving good results of segmentation of larger structures such as bone and cartilage in the knee, there are no studies available researching segmentation of articular cartilage lesions. Automating the segmentation will save time and money since manual segmentation is very time-consuming. In this thesis, a U-Net based convolutional neural network is used to perform automatic segmentation of chondral lesions located on the distal part of the femur, in the knee joint. Using two different techniques, batch normalization and dropout, a network was trained and tested using MRI sequences collected from Episurf Medical's database. The network was then evaluated using a segmentation approach and a detection approach. For the segmentation approach, the highest achieved dice coefficient and sensitivity of 0.4059 ± 0.1833 and 0.4591 ± 0.2387, was obtained using batch normalization and 260 training subjects, consisting of MRI sequence and corresponding masks. Using a detection approach, the predicted output could correctly identify 81.8% of the chondral lesions in the MRI sequences. Although there is a need for improvement of technique and datasets used in this thesis, the achieved results show prerequisites for future improvement and possible implementation. / Skador i knäledens brosk kan diagnostiseras i ett tidigt stadie med hjälp av MR. Segmentering och visualisering av skadorna, samt ledens struktur i helhet, bidrar till en förbättrad kommunikation mellan radiolog och remitterande läkare. Det kan också underlätta för att ställa diagnos eller utföra operationsplanering. I dagsläget finns flertalet studier som påvisar goda resultat för segmentering av större strukturer, t.ex. ben och brosk. Det finns dock få studier som studerar segmentering av skador i ledbrosk. Genom att automatisera segmenteringsprocessen kan både tid och pengar sparas. Detta eftersom att manuell segmentering är mycket tidskrävande. I detta arbete kommer ett U-Net baserat convolutional neural network att användas för att utföra automatisk segmentering av skador på distala femur i knäleden. Nätverket kommer att tränas med två olika tekniker, batch normalization och dropout. Nätverket kommer att tränas med data som är hämtad från Episurf Medicals databas och består av MR sekvenser. Nätverket kommer att tränas och utvärderas med hjälp av två metoder, en segmenteringsmetod och detekteringsmetod. Den högsta uppnådda dice koefficienten och sensitiviteten vid utvärderingen av segmenteringsmetoden uppmätte 0,4059 ± 0,1833 och 0,4591 ± 0,2387. Den upnåddes med hjälp av batch normalization och 260 MR sekvenser för träning och testning. För detektionsmetoden kunde programmet identifiera 81,8% av skadorna synliga på MR sekvenserna. Även om tekniken och datan som används behöver optimeras, så visar det uppnådda resultatet på bra förutsättningar för fortsatta studier och i framtiden möjligen även implementering av tekniken.

Osteoarthritis is one of the most common causes of disability in adults in America. It is a progressive and degenerative disease where the articular cartilage is broken down and lost from the surfaces of bones causing chronic pain and swelling in the joints, and currently has no cure. The most commonly osteoarthritis starts from a focal lesion on the cartilage surface, which will expand on the surface and downwards through the thickness of the tissue. The current gold standard for correcting cartilage focal lesions is the osteochondral autograft/allograft transplantation (OAT), which replaces the defect with a fresh osteochondral graft. The main limiting factor for using the OAT comes from the limited number of autograft and allografts that are available for implantation. To address the concern of graft availability, this study will look at the development of a porcine osteochondral xenograft (OCXG). The first aim of this research is to establish a decellularization protocol that will remove the antigens and cellular debris, which are the leading causes of graft rejection when implanting animal tissue in humans. The second aim of this study is restoring the mechanical strength of the OCXG that was lost during the decellularization process through crosslinking the tissue using genipin and epigallocatechin gallate (EGCG). The third aim is comparing the performance of the complete crosslinked OCXG at different degrees of crosslinking in a long-term goat animal model. The final aim is an alternative way to correct focal lesions through the development of an injectable collagen stabilizing treatment with genipin and punicalagin that will slow or stop the growth of a lesion and prevent osteoarthritis.

Objective: Anterior cruciate ligament (ACL) injuries are increasing in adolescents and increase the risk for early-onset knee osteoarthritis (OA). Biomarkers can be a non-invasive measure to assess physiological properties following knee injury or trauma. The objective of this thesis was to i) perform a systematic review to determine the most studied biomarkers of knee healing following ACL reconstruction (ACLR), and age of these patients, and ii) explore the feasibility of measuring these biomarkers in adolescents with ACL injuries. Design: Studies were included if i) participants underwent ACLR, and ii) at least one biomarker of healing was measured. Participant age, sample(s) collected, and biomarker(s) studied were recorded. Interleukin-6 (IL-6), c-terminal crosslinking telopeptide of type II collagen (CTX-II) and procollagen type II collagen propeptide (PIICP) were then measured using ELISA in adolescents prior to ACLR in urine (u) and synovial fluid (sf). Spearman’s Rho (rs) coefficients were calculated to determine the association between uCTX-II/sfCTX-II, and uIL-6/sfIL-6. A ratio of PIICP: CTX-II was calculated to represent the ratio of cartilage synthesis to degradation. Results: The review produced six studies evaluating healing following ACLR. IL-6 and CTX-II were the most studied (3/6 studies), and only one study included adolescents (age 19.6±4.5). Due to multiple undetectable biomarker levels, we could only report rs for uCTX-II/sfCTX-II (rs = -.200, p-value = .800, n=4). We also reported a ratio for sfPIICP: sfCTX-II (23.06 ±19.23). Conclusion: Exploring biomarkers in adolescents was motivated by their unique physiology due to puberty, and this was the first study to do so. The findings from this pilot study indicate that further analysis is required to determine optimal sample preparation. This will allow for reliable results while studying the feasibility of these biomarkers during ACLR recovery. This insight can ensure more informed decision making by clinicians clearing patients for return-to-activity.

Osteoarthritis (OA) is a degenerative condition of articular cartilage that affects more than 25 million people in the US. Joint injuries, like anterior cruciate ligament (ACL) tears, can lead to OA due to a change in articular cartilage loading. Gait analysis combined with knee joint finite element modeling (FEM) has been used to predict the articular cartilage loading. To predict the change of articular cartilage loading during gait due to various ACL injuries, a tibiofemoral FEM was developed from magnetic resonance images (MRIs) of a 33 year male, with no prior history of knee injuries. The FEM was validated for maximum contact pressure and anterior tibial translation using cadaver knee studies. The FEM was used to model gait of knees with an intact ACL, anteromedial (AM) bundle injury, posterolateral (PL) bundle injury, complete ACL injury, AM deficiency, PL deficiency, complete ACL rupture, as well as a bone-patellar tendon-bone (BPTB) graft. Generally, the predicted maximum contact pressure and contact area increased for all the ACL injuries when compared to intact ACLs. While an increase in maximum contact pressure and contact area is an indication of an increased risk of the development of OA, the percent of increase was typically small suggesting that walking is a safe activity for individuals with ACL injuries.

[EN] Articular cartilage is a tissue with low capacity for self-restoration due to its avascularity and low cell population. It is located on the surface of the subchondral bone covering the diarthrodial joints. Degeneration of articular cartilage can appear in athletes, in people with genetic degenerative processes (osteoarthritis or rheumatoid arthritis) or due to a trauma; what produces pain, difficulties in mobility and progressive degeneration that finally leads to joint failure. Self-restoration is only produced when the defect reaches the subchondral bone and bone marrow mesenchymal stem cells (MSCs) invade the defect. However, this new formed tissue is a fibrocartilaginous type cartilage and no a hyaline cartilage, which finally leads to degeneration. Transplantation of autologous chondrocytes has been proposed to regenerate articular cartilage but this therapy fails mainly to the absence of a material support (scaffold) for the adequate stimulation of cells. Matrix-induced autologous chondrocyte implantation uses a collagen hydrogel as scaffold for chondrocytes; however, it does not have the adequate mechanical properties, does not provide the biological cues for cells and regenerated tissue is not articular cartilage but fibrocartilage. Different approaches have been done until now in order to obtain a scaffold that mimics better articular cartilage properties and composition. Hydrogels are a good option as they retain high amounts of water, in a similar way to the natural tissue, and can closely mimic the composition of natural tissue by the combination of natural derived hydrogels. Their three-dimensionality plays a critical role in articular cartilage tissue engineering to maintain chondrocyte function, since monolayer culture of chondrocytes makes them dedifferentiate towards a fibroblast-like phenotype secreting fibrocartilage. Recently, injectable hydrogels have attracted attention for the tissue engineering of articular cartilage due to their ability to encapsulate cells, injectability in the injury with minimal invasive surgeries and adaptability to the shape of the defect. Following this new approach we aimed at synthesizing two new families of injectable hydrogels based on the natural protein gelatin for the tissue engineering of articular cartilage. The first series of materials consisted on the combination of injectable gelatin with loose reinforcing polymeric microfibers to obtain injectable composites with improved mechanical properties. Our results demonstrate that there is an influence of the shape and distribution of the fibers in the mechanical properties of the composite. More importantly bad fiber-matrix interaction is not able to reinforce the hydrogel. Due to this, our composites were optimized by improving matrix-fiber interaction through a hydrophilic grafting onto the microfibers, with very successful results. The second series of materials were inspired in the extracellular matrix of articular cartilage and consisted of injectable mixtures of gelatin and hyaluronic acid. Gelatin molecules in the mixtures provided integrin adhesion sites to cells, and hyaluronic acid increased the mechanical properties of gelatin. This combination demonstrated ability for the differentiation of MSCs towards the chondrocytic lineage and makes these materials very good candidates for the regeneration of articular cartilage. The last part of this thesis is dedicated to the synthesis of a non-biodegradable material with mechanical properties, swelling and permeability similar to cartilage. This material intends to be used as a platform in a bioreactor in which the typical loads of the joint are simulated, so that the hydrogels or scaffolds would fit in the recesses in the platform. The function of the platform is to simulate the effect of the surrounding tissue on the scaffold after implantation and could reduce animal experimentation by simulating in vivo conditions. / [ES] El cartílago articular es un tejido con baja capacidad de auto-reparación debida a su avascularidad y baja población celular. Se encuentra en la superficie del hueso subcondral cubriendo las articulaciones. La degeneración del cartílago articular puede aparecer en atletas, en personas con procesos genéticos degenerativos o debido a un trauma; lo que produce dolor, dificultades en la movilidad y degeneración progresiva que lleva al fallo de la articulación. La auto-reparación sólo se produce cuando el defecto alcanza el hueso subcondral y las células madre (MSCs) de la médula ósea invaden el defecto. Sin embargo, este nuevo tejido es un cartílago de tipo fibrocartilaginoso y no un cartílago hialino, el cual finalmente lleva a la degeneración. El trasplante de condrocitos autólogos ha sido propuesto para regenerar el cartílago articular pero esta terapia falla principalmente por la ausencia de un material soporte (scaffold) que estimule adecuadamente a las células. El implante de condrocitos autólogos mediante un hidrogel de colágeno no tiene las propiedades mecánicas apropiadas, no proporciona las señales biológicas a las células y el tejido regenerado no es cartílago articular sino fibrocartílago. Se han realizado diferentes enfoques para obtener un scaffold que mimetice mejor las propiedades y la composición del cartílago articular. Los hidrogeles son una buena opción ya que retienen elevadas cantidades de agua, de forma similar al tejido natural, y pueden imitar de cerca la composición del tejido natural mediante la combinación de derivados de hidrogeles naturales. Su tridimensionalidad juega un papel crítico para mantener la función de los condrocitos, ya que el cultivo en monocapa de los condrocitos hace que desdiferencien hacia un fenotipo similar al fibroblasto secretando fibrocartílago. Los hidrogeles inyectables han acaparado la atención en la ingeniería tisular de cartílago articular debido a su capacidad para encapsular células, su inyectabilidad en el daño con cirugías mínimamente invasivas y su adaptabilidad a la forma del defecto. Siguiendo este nuevo enfoque hemos sintetizado dos nuevas familias de hidrogeles inyectables basados en la proteína natural gelatina para la ingeniería tisular del cartílago articular. La primera serie de materiales combina una gelatina inyectable con microfibras poliméricas sueltas de refuerzo para obtener composites inyectables con propiedades mecánicas mejoradas. Nuestros resultados demuestran que hay una influencia de la forma y la distribución de las fibras en las propiedades mecánicas del composite. Además, la mala interacción entre las fibras y la matriz no es capaz de reforzar el hidrogel. Debido a esto, nuestros composites han sido optimizados mediante la mejora de la interacción fibra-matriz a través de un injerto hidrófilo sobre las microfibras, con resultados muy exitosos. La segunda serie de materiales se ha inspirado en la matriz extracelular del cartílago articular y ha consistido en mezclas inyectables de gelatina y ácido hialurónico. Las moléculas de gelatina proporcionan los dominios de adhesión mediante integrinas a las células, y el ácido hialurónico aumenta las propiedades mecánicas de la gelatina. Esta combinación ha demostrado la habilidad para la diferenciación de MSCs hacia el linaje condrocítico y convierte a estos materiales en buenos candidatos para la regeneración del cartílago articular. La última parte de esta tesis se dedica a la síntesis de un material no biodegradable con propiedades mecánicas, hinchado y permeabilidad similar al cartílago. Este material pretende ser empleado como plataforma en un biorreactor en el que se simulan las cargas típicas de las articulaciones, de forma que los scaffolds encajarían en los huecos de la plataforma. Su función es simular el efecto del tejido circundante en el scaffold después de su implantación y podría reducir la experimentación anim / [CA] El cartílag articular es un teixit amb baixa capacitat d'auto-reparació deguda a la seua avascularitat i baixa població cel·lular. Es troba en la superfície de l'ós subcondral cobrint les articulacions. La degeneració del cartílag articular pot aparèixer en atletes, en persones amb processos genètics degeneratius o degut a un trauma; produeix dolor, dificultats a la mobilitat i degeneració progressiva que finalment porta a la fallida de l'articulació. L'auto-reparació es produeix quan el defecte arriba fins a l'ós subcondral i les cèl·lules mare (MSCs) de la medul·la òssia envaeixen el defecte. No obstant això, aquest nou teixit format es un cartílag de tipus fibrocartilaginós i no un cartílag hialí, el qual finalment porta a la degeneració. El transplantament de condròcits autòlegs ha sigut proposat per a regenerar el cartílag articular però aquesta teràpia falla principalment per la absència d'un material de suport (scaffold) que estimuli adequadament a les cèl·lules. L'implant de condròcits autòlegs en un hidrogel de col·lagen per als condròcits no té les propietats mecàniques apropiades, no proporciona les senyals biològiques a les cèl·lules i el teixit regenerat no és cartílag articular sinó fibrocartílag. Diferents enfocs han sigut realitzats fins ara per a obtenir un scaffold que mimetitzi millor les propietats i la composició del cartílag articular. Els hidrogels son una bona opció ja que retenen elevades quantitats d'aigua, de forma similar al teixit natural, i poden imitar acuradament la composició del teixit natural mitjançant la combinació d'hidrogels naturals. La seua tridimensionalitat juga un paper crític per a mantenir la funció dels condròcits, ja que el cultiu en monocapa dels condròcits fa que aquests desdiferencien cap a un fenotip similar al fibroblàstic secretant fibrocartílag. Recentment, els hidrogels injectables han acaparat l'atenció en l' enginyeria tissular de cartílag articular degut a la seua capacitat per a encapsular cèl·lules, la seua injectabilitat en el dany amb cirurgies mínimament invasives i la seua adaptabilitat a la forma del defecte. Seguint aquesta nova aproximació hem sintetitzat dues noves famílies d'hidrogels injectables basats en la proteïna natural gelatina per a l'enginyeria tissular del cartílag articular. La primera sèrie de materials combina una gelatina injectable amb microfibres polimèriques soltes de reforç per a obtenir compòsits injectables amb propietats mecàniques millorades. Els nostres resultats demostren que hi ha una influència de la forma i la distribució de les fibres en les propietats mecàniques del compòsit. Més importantment, la mala interacció entre les fibres i la matriu no és capaç de reforçar l'hidrogel. Degut a això, els nostres compòsits han segut optimitzats mitjançant la millora de la interacció fibra-matriu a traves d'un empelt hidròfil sobre les fibres, amb resultats molt exitosos. La segona sèrie de materials està inspirada en la matriu extracel·lular del cartílag articular i ha consistit en mescles injectables de gelatina i àcid hialurònic. Les molècules de gelatina proporcionen els dominis d'adhesió mitjançant integrines a les cèl·lules, i l'àcid hialurònic augmenta les propietats mecàniques de la gelatina. Esta combinació ha demostrat l'habilitat per a la diferenciació de MSCs cap al llinatge condrocític i converteix a aquests materials en bons candidats per a la regeneració del cartílag articular. L'última part d'aquesta tesi és dedicada a la síntesi d'un material no biodegradable amb propietats mecàniques, inflat i permeabilitat similar al cartílag. Aquest material pretén ser utilitzat com a plataforma a un bioreactor que simula les cargues típiques de les articulacions, de manera que els hidrogels o scaffolds encaixarien als buits de la plataforma. La seua funció es simular l'efecte del teixit circumdant al scaffold després d / Poveda Reyes, S. (2016). Protein-based injectable hydrogels towards the regeneration of articular cartilage [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/61392 / Premios Extraordinarios de tesis doctorales

Tesis por compendio / [ES] El cartílago hialino es un tejido conectivo denso con poca capacidad de auto regeneración cuando es afectado por patologías degenerativas. Por lo tanto, la estimulación eléctrica se ha propuesto como una terapia alternativa no invasiva para mejorar la reparación del cartílago hialino. De acuerdo con esto, este trabajo presenta un enfoque computacional y experimental combinado para entender mejor la biología del cartílago hialino y su respuesta a la estimulación eléctrica usando diferentes modelos in vitro. En primer lugar, se ha desarrollado un modelo mecanobiológico para simular el proceso de osificación endocondral. Por otro lado, se ha evaluado el efecto de la estimulación eléctrica sobre el cartílago hialino en tres escenarios diferentes. Inicialmente se ha analizado la proliferación celular y la síntesis de glicosaminoglicanos de condrocitos cultivados en monocapa y estimulados con campos eléctricos. Luego, se ha realizado un análisis histomorfométrico a explantes de condroepífisis que fueron estimulados eléctricamente. Por último, se ha evaluado el efecto de los campos eléctricos sobre la diferenciación condrogénica de células madre mesenquimales cultivadas en hidrogeles. Los resultados indican que la estimulación eléctrica es un estímulo biofísico prometedor, ya que este tipo de estimulación mejora la viabilidad y la proliferación celular, induce cambios morfológicos en los condrocitos, y estimula la síntesis de las principales moléculas que componen el cartílago hialino, tales como SOX-9, glicosaminoglicanos y agrecan. Además, este proyecto es el primer paso hacia la implementación de un estímulo biofísico alternativo que modifica la dinámica celular de los condrocitos de la placa de crecimiento en condiciones ex vivo. Adicionalmente, este estudio resalta el efecto potencial de los campos eléctricos para inducir el proceso de condrogénesis de células madre mesenquimales cultivadas en condiciones basales. En general, la evaluación de la estimulación eléctrica sobre condrocitos, tejidos y andamios es una herramienta útil que puede contribuir al conocimiento actual de las terapias regenerativas enfocadas en la regeneración del cartílago hialino. / [CA] El cartílag hialí és un teixit connectiu dens amb poca capacitat d'auto regeneració quan es veu afectat per patologies degeneratives. Per tant, l'estimulació elèctrica s'ha proposat com una teràpia alternativa no invasiva per millorar la reparació del cartílag articular. D'acord amb això, aquest treball presenta un enfoc computacional i experimental combinat per entendre millor la biologia del cartílag hialí i la seva resposta a l'estimulació elèctrica usant diferents models in vitro. En primer lloc, s'ha desenvolupat un model mecanobiològic per simular el procés d'ossificació endocondral. D'altra banda, s'ha avaluat l'efecte de l'estimulació elèctrica sobre el cartílag hialí en tres escenaris diferents. Inicialment s'ha analitzat la proliferació cel·lular i la síntesi de glicosaminoglicans de condròcits cultivats en monocapa i estimulats amb camps elèctrics. Després, s'ha realitzat una anàlisi histomorfomètrica a explants de condroepífisis que van ser estimulats elèctricament. Finalment, s'ha avaluat l'efecte dels camps elèctrics sobre la diferenciació condrogénica de cèl·lules mare mesenquimals cultivades en hidrogels. Els resultats indiquen que l'estimulació elèctrica és un estímul biofîsic prometedor, ja que aquest tipus d'estimulació millora la viabilitat i la proliferació cel·lular, indueix canvis morfològics en els condròcits, i estimula la síntesi de les principals molècules que componen el cartílag hialí, com ara SOX-9, glicosaminoglicans i agrecan. A més, aquest projecte és el primer pas cap a la implementació d'un estímul biofísic alternatiu que modifica la dinàmica cel·lular dels condròcits de la placa de creixement en condicions ex vivo. Addicionalment, aquest estudi ressalta l'efecte potencial dels camps elèctrics per induir el procés de condrogènesi de cèl·lules mare mesenquimals cultivades en condicions basals. En general, l'avaluació de l'estimulació elèctrica sobre condròcits, teixits i scaffolds és una eina útil que pot contribuir al coneixement actual de les teràpies regeneratives enfocades a la regeneració del cartílag hialí. / [EN] Hyaline cartilage is a dense connective tissue with low self-healing capacity when is affected by degenerative pathologies. Therefore, electrical stimulation has been proposed as a possible non-invasive alternative therapy to enhance the restoration of the cartilaginous tissue. Accordingly, this work presents a combined computational and experimental approach to understand better the hyaline cartilage biology and its response to electrical stimulation using different in vitro models. On the one hand, a mechanobiological model was developed to simulate the endochondral ossification process. On the other hand, the electrical stimulation on hyaline cartilage was evaluated in three different scenarios. Initially, cell proliferation and glycosaminoglycans synthesis of chondrocytes, cultured in monolayer and stimulated with electric fields, was analyzed. Then, a histomorphometric analysis was performed to chondroepiphysis explants that were electrically stimulated. Finally, the effects of the electric fields on chondrogenic differentiation of mesenchymal stem cells cultured in hydrogels was assessed. The results indicated that electrical stimulation is a promising biophysical stimulus, due to the fact that this type of stimulation enhances the viability and the proliferation of cells, induces morphological changes in the chondrocytes, and stimulates the synthesis of the main molecules that compose the hyaline cartilage, such as SOX-9, glycosaminoglycans and aggrecan. Moreover, this project is the first step towards the implementation of an alternative biophysical stimulus that modifies the cellular dynamics of growth plate chondrocytes in ex vivo conditions. Additionally, this study highlights the potential effect of electric fields to induce the chondrogenesis process of mesenchymal stem cells cultured in basal conditions. Overall, the assessment of electrical stimulation on chondrocytes, tissues and scaffolds is a useful tool that may contribute to the current knowledge of regenerative therapies focused on hyaline cartilage healing. / To the financial support from COLCIENCIAS – COLFUTURO through the fellowship No. 647 for national doctorates. To the financial support from COLCIENCIAS through the research grant 712-2015 No. 50457. To the financial support from the Spanish Ministry of Economy and Competitiveness through the MAT2016-76039-C4-1-R project. / Vaca González, JJ. (2019). The effect of electric fields on hyaline cartilage: an in vitro and in silico study [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/120023 / Compendio

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. One of the common forms of articular cartilage disease which has a huge impact on patient's life is arthritis. Research on cartilage cell/tissue engineering will help patients to improve their physical activity by replacing or treating the diseased/damaged cartilage tissue. Cartilage cell, called chondrocyte is embedded in the matrix (Lacunae) and has round shape in vivo. The in vitro monolayer culture of primary chondrocyte causes morphological change characterized as dedifferentiation. Transforming growth factor-beta (TGF-β), a cytokine superfamily, regulates cell function, including differentiation and proliferation. The effect of TGF-β1, 2, 3, and their manipulated forms in biological regulation of primary chondrocyte was investigated in this work. A novel method was developed to isolate and purify the primary chondrocytes from knee joint of neonate Sprague-Dawley rat, and the effect of some supplementations such as hyaluronic acid and antibiotics were also investigated to provide the most appropriate condition for in vitro culture of chondrocyte cells. Addition of 0.1mg/ml hyaluronic acid in chondrocyte culture media resulted an increase in primary chondrocyte proliferation and helped the cells to maintain chondrocytic morphology. TGF-β1, 2 and 3 caused chondrocytes to obtain fibroblastic phenotype, alongside an increase in apoptosis. The healing process of the wound closure assay of chondrocyte monolayers were slowed down by all three isoforms of TGF-β. All three types of TGF-β negatively affected the strength of chondrocyte adhesion. TGF-β1, 2 and 3 up regulated the expression of collagen type-II, but decreased synthesis of collagen type-I, Chondroitin sulfate glycoprotein, and laminin. They did not show any significant change in production of S-100 protein and fibronectin. TGF-β2, and 3 did not change expression of integrin-β1 (CD29), but TGF-β1 decreased the secretion of this adhesion protein. Manipulated TGF-β showed huge impact on formation of fibroblast like morphology of chondrocytes with chondrocytic phenotype. These isoforms also decreased the expression of laminin, chondroitin sulfate glycoprotein, and collagen type-I, but they increased production of collagen type-II and did not induce synthesis of fibronectin and S-100 protein. In addition, the strength of cell adhesion on solid surface was reduced by manipulated TGF-β. Only manipulated form of TGF-β1 and 2 could increase the proliferation rate. Manipulation of TGF-β did not up regulate the expression of integrin-β1 in planar culture system. The implications of this R&D work are that the manipulation of TGF-β by combination of TGF-β1, 2, and 3 can be utilized in production of superficial zone of cartilage and perichondrium. The collagen, fibronectin and hyaluronic acid could be recruited for the fabrication of a biodegradable scaffold that promotes chondrocyte growth for autologous chondrocyte implantation or for formation of cartilage.