Articular cartilage provides an almost frictionless surface for the articulating ends of the bone. Cartilage functions to lubricate and transmit compressive forces resulting from joint loading and impact. If the cartilage is damaged, through traumatic injury or disease, it lacks the ability of self-repairing as the tissue lacks vascular system. If the injuries to articular cartilage are left untreated, they may progress to Osteoarthritis. Osteoarthritis, a degenerative disease, is one of the leading disabilities in the United States. Tissue engineering has the potential to regenerate healthy hyaline cartilage, which can alleviate pain and restore the functions of normal tissue. This study explores the production of engineered cartilage on top of composite calcium phosphate scaffold. The current research is related to a biphasic approach to cartilage tissue engineering — in which one layer supports to form subchondral bone (osteogenesis) and another supports cartilage formation (chondrogenesis). Chondrocyte and bone marrow-derived stem cell attachment to chitosan will be investigated for producing a bilayered construct for osteochondral repair. The main objectives of my research include the following: attachment and proliferation of human mesenchymal stem cells on chitosan calcium phosphate scaffolds, techniques to create a biphasic construct, the effect of coating chitosan calcium phosphate scaffolds with type I collagen and determining the ideal bead size for making chitosan calcium phosphate scaffolds.

The purpose of the present study is to develop techniques for manufacturing anatomically-shaped substrates of implants made from calcium polyphosphate (CPP) ceramic. These substrates have tissue-engineered cartilage growing on their top surfaces and can be used as implants for osteochondral focal defect repair. While many research groups have been fabricating such substrates using standard material shapes, e.g., rectangles and circular discs, it is considered beneficial to develop methods that can be integrated in the substrate fabrication process to produce an implant that is specific to a patient’s own anatomy (as obtained from computer tomography data) to avoid uneven and/or elevated stress distribution that can affect the survival of cartilage. The custom-made, porous CPP substrates were fabricated with three-dimensional printing (3DP) and computer numerically controlled (CNC) machining for the first time to the best of the author’s knowledge. The 3DP technique was employed in two routines: indirect- and direct-3DP. In the former, 3DP was used to fabricate molds for pre-shaping of the CPP substrates from two different powder size ranges (<75 μm and 106-150 μm). In the latter, CPP substrates were produced directly from the retrofitted 3DP apparatus in a layer-by-layer fashion from 45-75 μm CPP powder with a polymeric binder. The prototyped samples were then sintered to obtain the required porosity and mechanical properties. These substrates were characterized in terms of their dimensional shrinkage and density. Also, SEM images were used to assess the particle distribution and neck and bond formations. The substrates produced using the indirect-3DP method yielded densities (<75 μm: 66.28 ± 11.62% and 106-150 μm: 65.87 ± 6.12%), which were comparable to the substrates used currently and with some success in animal studies. Geometric adjustment factors were devised to compensate for the slight expansion inherent in the 3DP mold fabricating process. These equations were used to bring the plaster molds into true dimension. The direct-3DP method has proven to be the ultimate choice due to its ability to produce complex anatomically-shaped substrates without the use of a chemical solvent. In addition, it allows for precise control of both pore size and internal architectures of the substrates. Thus, the direct-3DP was considered to be superior than the indirect-3DP as a fabrication method. In the alternative CNC machining approach to fabrication, the ability to machine the CPP ceramic was feasible and by careful selection of the machining conditions, anatomically-shaped CPP substrates were produced. To develop strategies for optimizing the machining process, a mechanistic model was developed based on curve fitting the average cutting forces to determine the cutting coefficients for CPP. These cutting coefficients were functions of workpiece material, axial depth of cut, chip width, and cutter geometry. To explore the utility of this modelling approach, cutting forces were predicted for a helical ball-end mill and compared with experimental results. The cutting force simulation exhibits good agreement in predicting the fundamental force magnitude and general shape of the actual forces. However, there were some discrepancies between the predicted and measured forces. These differences were attributed to internal microstructure defects, density gradients, and the use of a shear plane model in force prediction that was not entirely appropriate for brittle materials such as CPP. The present study successfully developed 3DP and CNC fabrication methods for manufacturing anatomically-shaped CPP substrates. Future studies were recommended to explore further optimization of these fabrication methods and to demonstrate the utility of accurate substrates shapes to the clinical application of focal defect repair implants.

The purpose of the present study is to develop techniques for manufacturing anatomically-shaped substrates of implants made from calcium polyphosphate (CPP) ceramic. These substrates have tissue-engineered cartilage growing on their top surfaces and can be used as implants for osteochondral focal defect repair. While many research groups have been fabricating such substrates using standard material shapes, e.g., rectangles and circular discs, it is considered beneficial to develop methods that can be integrated in the substrate fabrication process to produce an implant that is specific to a patient’s own anatomy (as obtained from computer tomography data) to avoid uneven and/or elevated stress distribution that can affect the survival of cartilage. The custom-made, porous CPP substrates were fabricated with three-dimensional printing (3DP) and computer numerically controlled (CNC) machining for the first time to the best of the author’s knowledge. The 3DP technique was employed in two routines: indirect- and direct-3DP. In the former, 3DP was used to fabricate molds for pre-shaping of the CPP substrates from two different powder size ranges (<75 μm and 106-150 μm). In the latter, CPP substrates were produced directly from the retrofitted 3DP apparatus in a layer-by-layer fashion from 45-75 μm CPP powder with a polymeric binder. The prototyped samples were then sintered to obtain the required porosity and mechanical properties. These substrates were characterized in terms of their dimensional shrinkage and density. Also, SEM images were used to assess the particle distribution and neck and bond formations. The substrates produced using the indirect-3DP method yielded densities (<75 μm: 66.28 ± 11.62% and 106-150 μm: 65.87 ± 6.12%), which were comparable to the substrates used currently and with some success in animal studies. Geometric adjustment factors were devised to compensate for the slight expansion inherent in the 3DP mold fabricating process. These equations were used to bring the plaster molds into true dimension. The direct-3DP method has proven to be the ultimate choice due to its ability to produce complex anatomically-shaped substrates without the use of a chemical solvent. In addition, it allows for precise control of both pore size and internal architectures of the substrates. Thus, the direct-3DP was considered to be superior than the indirect-3DP as a fabrication method. In the alternative CNC machining approach to fabrication, the ability to machine the CPP ceramic was feasible and by careful selection of the machining conditions, anatomically-shaped CPP substrates were produced. To develop strategies for optimizing the machining process, a mechanistic model was developed based on curve fitting the average cutting forces to determine the cutting coefficients for CPP. These cutting coefficients were functions of workpiece material, axial depth of cut, chip width, and cutter geometry. To explore the utility of this modelling approach, cutting forces were predicted for a helical ball-end mill and compared with experimental results. The cutting force simulation exhibits good agreement in predicting the fundamental force magnitude and general shape of the actual forces. However, there were some discrepancies between the predicted and measured forces. These differences were attributed to internal microstructure defects, density gradients, and the use of a shear plane model in force prediction that was not entirely appropriate for brittle materials such as CPP. The present study successfully developed 3DP and CNC fabrication methods for manufacturing anatomically-shaped CPP substrates. Future studies were recommended to explore further optimization of these fabrication methods and to demonstrate the utility of accurate substrates shapes to the clinical application of focal defect repair implants.

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.

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.

Knorpeldefekte des Kniegelenks zeichnen sich durch eine sehr begrenzte spontane Heilungstendenz aus und führen im Verlauf häufig zur Arthrose. Trotz intensiver Forschungsbemühungen konnte bisher keine neue Therapieoption eine zufrieden-stellende Alternative zu den bisherigen Therapien hervorbringen. Eine ACI in Kombination mit einem künstlich hergestellten Knorpel-Knochen-Ersatzmaterial scheint jedoch großes Potential für die Therapie von Knorpel-Knochen-Schäden zu besitzen. Im vorliegenden Langzeittierversuch mit Kaninchen wurde eine einzeitige ACI mit einem biphasischen Ersatzmaterial (TruFit®) und platelet-rich-plasma (PRP) kombiniert. Zu diesem Zweck wurde in der medialen Femurkondyle ein critical-size-Defekt mit einem Durchmesser von 4,5 mm gesetzt. In der ersten Versuchsgruppe blieb der Defekt unbehandelt (Leer). Bei der zweiten Gruppe wurde die Defekthöhle mit einem TruFit®-Zylinder aufgefüllt (TFP). Gruppe drei erhielt zusätzlich PRP (TFP+PRP) und Gruppe vier wurde darüber hinaus mit einer einzeitigen ACI kombiniert (TFP+PRP+C), bei der Chondrozyten mit Hilfe eines speziellen Kollagenase-Schnellverdaus isoliert werden konnten. Die Auswertung der Knorpel-Knochen-Regeneration erfolgte nach 12 Monaten durch eine Mikroradiographie, eine intravitale Fluoreszenzmarkierung des Knochens und durch Toluidinblau-O- und Safranin-O-Färbungen. Verwendet wurden die Scores nach Wakitani und O’Driscoll. Dabei konnte gezeigt werden, dass eine TruFit®-Therapie die Knochenregeneration positiv beeinflussen kann. Die Zugabe von PRP bewirkte die Bildung von zahlreichen dünnen Trabekeln mit einer erhöhten Anzahl trabekulärer Verbindungen, allerdings auch eine schlechtere Rekonvaleszenz der subchondralen Knochenschicht. Bezüglich der Knorpelheilung schnitt die Gruppe TFP+PRP+C am besten ab, wobei die Unterschiede nicht signifikant waren. Insgesamt zeigten alle Versuchsgruppen eine unzureichende osteochondrale Regeneration, so dass für die Therapie am Menschen zunächst weitere Studien nötig sind, die sowohl ossär als auch chondral eine verbesserte Heilungspotenz demonstrieren können. Bisher fehlen groß angelegte Studien um Therapieempfehlungen bezüglich des Ersatzmaterials, der genauen Durchführung der einzeitigen ACI und Zusätzen wie Wachstumsfaktoren zu machen.