Articular cartilage injuries occur frequently in the knee joint. Several methods have been implemented clinically, to treat osteochondral defects but none have been able to produce a long term, durable solution. Photopolymerizable cartilage tissue engineering approaches appear promising; however, fundamentally, forming a stable interface between the tissue engineered cartilage and native tissue, mainly subchondral bone and native cartilage, remains a major challenge. The overall objective of this research is to find a solution for the current problem of dislodgment of tissue engineered cartilage at the defect site for the treatment of degraded cartilage that has been caused due to knee injuries or because of mild to moderate level of osteoarthritis. For this, an in-vitro model was created to analyze the integration of tissue engineered cartilage with the bone, healthy and diseased cartilage over time. We investigated the utility of hydroxyapatite (HA) nanoparticles to promote controlled bone-growth across the bone-cartilage interface in an in vitro engineered tissue model system using bone marrow derived stem cells. We also investigated the application of HA nanoparticles to promote enhance integration between tissue engineered cartilage and native cartilage both in healthy and diseased states. Samples incorporated with HA demonstrated significantly higher interfacial shear strength (at the junction between engineered cartilage and engineered bone and also with diseased cartilage) compared to the constructs without HA (p < 0.05), after 28 days of culture. These findings indicate that the incorporation of HA nanoparticles permits more stable anchorage of the injectable hydrogel-based engineered cartilage construct via augmented integration between bone and cartilage.

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Osteochondral lesions of the talus (OLTs) are the third most common type of osteochondral lesion and can cause pain and instability of the ankle joint. Episurf Medical AB is a medical technology company that develops individualized implants for patients who are suffering from focal cartilage lesions. Episurf have recently started a project that aims to implement their implantation technique in the treatment of OLTs. This master thesis was a part of Episurf’s talus project and the main goal of the thesis was to find the optimal implantation angle of the Episurf implant when treating OLTs. The optimal implantation angle was defined as the angle that minimized the maximum equivalent (von Mises) strain acting on the implant shaft during the stance phase of a normal gait cycle. It is desirable to minimize the strain acting on the implant shaft, since a reduction of the strain can improve the longevity of the implant. To find the optimal implantation angle a finite element model of an ankle joint treated with the Episurf implant was developed. In the model an implant with a diameter of 12 millimeters was placed in the middle part of the medial side of the talar dome. An optimization algorithm was designed to find the implantation angle, which minimized the maximum equivalent strain acting on the implant shaft. The optimal implantation angle was found to be a sagittal angle of 12.5 degrees and a coronal angle of 0 degrees. Both the magnitude and the direction of the force applied to the ankle joint in the simulated stance phase seemed to influence the maximum equivalent strain acting on the implant shaft. A number of simplifications have been done in the simulation of this project, which might affect the accuracy of the results. Therefore it is recommended that further, more detailed, simulations based on this project are performed in order to improve the result accuracy. / Fokala broskskador på talusbenet är den tredje vanligaste typen av fokala broskskador och kan ge upphov till smärta och instabilitet av fotleden. Episurf Medical AB är ett medicintekniskt företag som utvecklar individanpassade implantat för patienter med fokala broskskador. Episurf har nyligen påbörjat ett projekt där deras teknik ska användas i behandlingen av fokala broskskador på talusbenet. Den här masteruppsatsen var en del i Episurfs talusprojekt och dess huvudmål var att finna den optimala implantationsvinkeln av Episurfs implantat i behandlingen av fokala broskskador på talusbenet. Den optimala implanteringsvinkeln definierades som den vinkel som minimerade den effektiva von Mises-töjningen som verkade på implantatskaftet under stance-fasen i en normal gångcykel. Det är eftersträvansvärt att minimera belastningen på implantatskaftet eftersom en reducering av belastningen kan förbättra implantatets livslängd. En finita element-modell av en fotled behandlad med Episurfs implantat utvecklades för att för att finna den optimala implantationsvinkeln. I modellen placerades ett implantat med en diameter på 12 millimeter på mittendelen av talus mediala sida. En optimeringsalgoritm utformades för att finna implantationsvinkeln som minimerade den effektiva von Mises-töjningen på implantatskaftet. Den funna optimala implantationsvinkeln bestod av en vinkel på 12.5 grader i sagittalplan och en vinkel på 0 grader i koronalplan. Både storleken och riktningen på kraften som applicerats på fotleden under den simulerade stance-fasen av gångcykeln verkade påverka belastningen på implantatskaftet. Ett antal förenklingar har gjorts i projektets simuleringar, vilket kan påverka noggrannheten i resultatet. Därför rekommenderas att ytterligare, mer detaljerade simuleringar baserade på det här projektet görs för att förbättra resultatets noggrannhet.

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Background: Collagens of marine origin are applied increasingly as alternatives to mammalian collagens in tissue engineering. The aim of the present study was to develop a biphasic scaffold from exclusively marine collagens supporting both osteogenic and chondrogenic differentiation and to find a suitable setup for in vitro chondrogenic and osteogenic differentiation of human mesenchymal stroma cells (hMSC). Methods: Biphasic scaffolds from biomimetically mineralized salmon collagen and fibrillized jellyfish collagen were fabricated by joint freeze-drying and crosslinking. Different experiments were performed to analyze the influence of cell density and TGF-β on osteogenic differentiation of the cells in the scaffolds. Gene expression analysis and analysis of cartilage extracellular matrix components were performed and activity of alkaline phosphatase was determined. Furthermore, histological sections of differentiated cells in the biphasic scaffolds were analyzed. Results: Stable biphasic scaffolds from two different marine collagens were prepared. An in vitro setup for osteochondral differentiation was developed involving (1) different seeding densities in the phases; (2) additional application of alginate hydrogel in the chondral part; (3) pre-differentiation and sequential seeding of the scaffolds and (4) osteochondral medium. Spatially separated osteogenic and chondrogenic differentiation of hMSC was achieved in this setup, while osteochondral medium in combination with the biphasic scaffolds alone was not sufficient to reach this ambition. Conclusions: Biphasic, but monolithic scaffolds from exclusively marine collagens are suitable for the development of osteochondral constructs.

Le cartilage est un tissu clé des articulations synoviales. Suite à un problème mécanique, traumatique ou inflammatoire, le cartilage est dégradé entrainant des douleurs articulaires et une perte de mobilité. Le cartilage étant un tissu non innervé et non vascularisé, son auto-réparation est très faible. De plus en plus de techniques sont développées pour la réparation des défauts cartilagineux mais aucune n’a encore permis d’obtenir un nouveau cartilage pleinement fonctionnel. En particulier, l’ingénierie tissulaire (IT) est une technique très prometteuse qui consiste à obtenir un greffon de cartilage dont les propriétés mécaniques et structurales soient satisfaisantes une fois implantée dans l’articulation. L’IT est basée sur l’association de cellules, d’un biomatériau et de facteurs de croissance. Le but de cette thèse est d’étudier l’effet de l’âge du donneur des cellules sur la synthèse du greffon par l’IT in vitro et sur la qualité du cartilage obtenu lors de l’implantation dans un modèle de rat NUDE. Puis dans une dernière partie, l’impact de l’environnement chimique et mécanique est étudié sur la qualité du greffon. Nos études montrent ainsi que l’âge du donneur aussi bien dans un contexte in vitro ou in vivo impacte la qualité du greffon et la réparation une fois implanté dans l’animal. En effet, les greffons issus des donneurs âgés ont des propriétés mécaniques légèrement plus élevées et une synthèse des protéines de la matrice extracellulaire (MEC) du cartilage significativement plus élevée que les greffons issus de donneurs jeunes. De plus, la réponse inflammatoire des greffons implantés dans un défaut cartilagineux chez le rat NUDE est plus faible pour les donneurs âgés. Enfin, nous montrons que le microenvironnement mécanique (compression ou pression hydrostatique) et chimique (liquide synovial (LS) ou TGF-β pur) joue un rôle important sur la réponse cellulaire. Par ailleurs, en fonction de l’âge, l’association de ces différents facteurs donnent des résultats différents. Par exemple, pour une sollicitation de type compression, c’est le LS qui est à favoriser pour obtenir les greffons de meilleure qualité dans le cas des donneurs âgés. Au contraire, pour la même sollicitation de type compression, c’est la présence de TGF-β1 qui conduit au greffon de meilleure qualité pour les donneurs jeunes. Ces études mettent en évidence l’importance de l’âge du donneur et montrent de plus qu’un protocole IT patient spécifique est la meilleure solution. / Cartilage is an important tissue of synovial joints. Following a mechanical problem, traumatic or inflammatory, the cartilage is degraded causing joint pain and loss of mobility. Because cartilage is a non-innervated and non-vascularized tissue, its self-repair is very weak. More and more techniques are being developed for the cartilage but none has resulted in a new fully functional cartilage. In particular, tissue engineering (TE) is a very promising technique that consists in obtaining a cartilage graft whose mechanical and structural properties are satisfactory once implanted in the joint. TE is based on the association of cells, biomaterial and growth factors. The aim of this thesis is to study the effect of cell donor’s age on graft synthesis by TE in vitro and on the quality of the cartilage obtained during implantation in a NUDE rat model. Then in a last part, the impact of the chemical and mechanical environment is studied on the quality of the graft. Our studies show that the age of the donor both in vitro and in vivo has an impact on graft quality and repair once implanted in the animal. In fact, grafts from older donors have slightly higher mechanical properties and significantly higher synthesis of extracellular matrix proteins (ECM) than grafts from younger donors. In addition, the inflammatory response of grafts implanted in a cartilage defect in the NUDE rat is lower for older donors. Finally, we show that the mechanical microenvironment (compression or hydrostatic pressure) and chemical microenvironment (synovial fluid (SF) or TGF-β) play an important role in the cellular response. Moreover, depending on age, the combination of these different factors gives different results. For example, for a compression solicitation, it is the SF that is to be favored to obtain better quality grafts in the case of elderly donors. On the contrary, for the same compression stress, it is the presence of TGF-β1 that leads to the best quality graft for young donors. These studies highlight the importance of donor age and further show that a specific patient protocol of TE is the best solution.

The focus of this dissertation is on the development of a solid freeform fabrication (SFF) process for the design and manufacture of porous biodegradable orthopaedic implants from calcium polyphosphate (CPP). Porous CPP structures are used as bone substitutes for regenerating bone defects and/or as substrates in formation of so-called “biphasic” implants for repair of damaged osteochondral tissues. The CPP implants can be utilized in the treatment of many musculoskeletal diseases, osteochondral defects, and bone tumours while replacement of the defect site is required. In this study, the fabrication of CPP structures was developed through a powder-based SFF technique known as adhesive bonding 3D-printing. SFF is an advanced alternative to the “conventional” fabrication method consisting of gravity sintering of CPP pre-forms followed by machining to final form, as SFF enables rapid manufacturing of complex-shaped bio-structures with controlled internal architecture. To address the physical and structural properties of the porous SFF-made components, they were characterized using scanning electron microscopy, micro-CT scanning and mercury intrusion porosimetry. Specific surface area and permeability of the porous structures were also determined. Additionally, the chemical properties (crystallinity) of the specimens were identified by X-ray diffraction. The mechanical properties of the crystalline CPP material were also measured by micro- and nano-indentation. Moreover, the porous structures were tested by uniaxial and diametral mechanical compression to determine the compressive and tensile strengths, respectively. Furthermore, the effect of the stacked-layer orientation on the mechanical properties of the SFF-made constructs was investigated through the production of samples with horizontal or vertical stacked-layers. The properties of the SFF-made samples were compared with those of the conventionally-made CPP constructs. The SFF-made implants showed drastically higher compressive mechanical strength compared to the conventionally-formed samples with identical porosity. It was also shown that the orientation of the stacked-layer has substantial influence on the mechanical strengths. Moreover, this thesis examined the ability of in vitro forming of cartilaginous tissue on the SFF-made substrates where the chondrocytes cellular response to the CPP implants was evaluated histologically and biochemically. In addition, an initial in vivo assessment of the CPP structures as bone substitutes was conducted using a rabbit medial femoral site model. Significant amount of new-bone was formed within the CPP porous constructs during the 6-week implantation period demonstrating appropriate biological response of SFF-made CPP structures for bone substitute applications. Another accomplishment of this thesis was the development of a mathematical model which predicts the compact density of powder layers spread by a counter-rotating roller in the SFF technique. The results may be used in the control of the apparent density of the final implant. The potential of the developed SFF method as an efficient and reproducible technique for the production of porous CPP structures for use in orthopaedics and musculoskeletal tissue regenerative applications was concluded.

The focus of this dissertation is on the development of a solid freeform fabrication (SFF) process for the design and manufacture of porous biodegradable orthopaedic implants from calcium polyphosphate (CPP). Porous CPP structures are used as bone substitutes for regenerating bone defects and/or as substrates in formation of so-called “biphasic” implants for repair of damaged osteochondral tissues. The CPP implants can be utilized in the treatment of many musculoskeletal diseases, osteochondral defects, and bone tumours while replacement of the defect site is required. In this study, the fabrication of CPP structures was developed through a powder-based SFF technique known as adhesive bonding 3D-printing. SFF is an advanced alternative to the “conventional” fabrication method consisting of gravity sintering of CPP pre-forms followed by machining to final form, as SFF enables rapid manufacturing of complex-shaped bio-structures with controlled internal architecture. To address the physical and structural properties of the porous SFF-made components, they were characterized using scanning electron microscopy, micro-CT scanning and mercury intrusion porosimetry. Specific surface area and permeability of the porous structures were also determined. Additionally, the chemical properties (crystallinity) of the specimens were identified by X-ray diffraction. The mechanical properties of the crystalline CPP material were also measured by micro- and nano-indentation. Moreover, the porous structures were tested by uniaxial and diametral mechanical compression to determine the compressive and tensile strengths, respectively. Furthermore, the effect of the stacked-layer orientation on the mechanical properties of the SFF-made constructs was investigated through the production of samples with horizontal or vertical stacked-layers. The properties of the SFF-made samples were compared with those of the conventionally-made CPP constructs. The SFF-made implants showed drastically higher compressive mechanical strength compared to the conventionally-formed samples with identical porosity. It was also shown that the orientation of the stacked-layer has substantial influence on the mechanical strengths. Moreover, this thesis examined the ability of in vitro forming of cartilaginous tissue on the SFF-made substrates where the chondrocytes cellular response to the CPP implants was evaluated histologically and biochemically. In addition, an initial in vivo assessment of the CPP structures as bone substitutes was conducted using a rabbit medial femoral site model. Significant amount of new-bone was formed within the CPP porous constructs during the 6-week implantation period demonstrating appropriate biological response of SFF-made CPP structures for bone substitute applications. Another accomplishment of this thesis was the development of a mathematical model which predicts the compact density of powder layers spread by a counter-rotating roller in the SFF technique. The results may be used in the control of the apparent density of the final implant. The potential of the developed SFF method as an efficient and reproducible technique for the production of porous CPP structures for use in orthopaedics and musculoskeletal tissue regenerative applications was concluded.

Background: Osteochondral allograft (OCA) transplantation is a useful treatment for posttraumatic ankle arthritis in young patients, but failure rates are high and reoperations are not uncommon. The aim of this study was to evaluate the outcomes of failed ankle OCA transplantation converted to ankle arthrodesis (AA) or total ankle arthroplasty (TAA). Methods: We evaluated 24 patients who underwent salvage procedures (13 AA and 11 TAA) after primary failed ankle OCA transplantation. Reoperations were assessed. Failure of the salvage procedure was defined as an additional surgery that required a revision AA/TAA or amputation. Evaluation among nonfailing ankles included the American Academy of Orthopaedic Surgeons Foot and Ankle Module (AAOS-FAM), pain, and satisfaction. Results: In the salvage AA cohort, 3 patients were classified as failures (2 revision AA and 1 amputation). The 10 nonfailing patients had a mean follow-up of 7.4 years. Eighty-eight percent were satisfied with the procedure, but 63% reported continued problems with their ankle (eg, pain, swelling, stiffness). Mean pain level was 1.9 and AAOS-FAM core score was 83±13. In the salvage TAA cohort, 2 patients were classified as failures (both revision TAA). The 9 nonfailing patients had a mean follow-up of 3.8 years. Fifty percent were satisfied with the procedure, but 40% reported continued problems with their ankle. The mean pain level was 1.3, and the median AAOS-FAM core score was 82±26. Conclusion: Revision and reoperation rates for salvage procedures following failed OCA transplantation of the ankle are higher compared to published data for primary AA and TAA procedures. However, we believe OCA transplantation can serve as an interim procedure for younger patients with advanced ankle joint disease who may not be ideal candidates for primary AA or TAA at the time of initial presentation. Level of Evidence: Level IV, case series.