Diarthrodial joint resurfacing: Multi-scale biofabrication of mechanically stable implants. Osteochondral tissue regeneration in the knee joint

In the quest for new treatments for (osteo)chondral defects and to eliminate or postpone total knee replacement surgery, the potential of 3D bioprinting of (osteo)chondral implants as a surgical procedure has been explored. The overarching aim of this thesis was to fabricate a functional (osteo)chondral implant, inspired by native tissue architecture, by combining advanced 3D (bio)fabrication technologies. Part I demonstrated how melt electrowriting (MEW) was used to improve the mechanical properties of (osteo)chondral implants. Here, it was shown that microfibres, deposited in an out-of-plane fashion, improve the shear-properties of hydrogel scaffolds while using only a limited volume-fraction of biodegradable polymer fibres (Chapter 2). Additionally, microfibre meshes were used to improve the interconnection between the cartilage-to-bone interface by interlocking them within a printed calcium phosphate-based (pCaP) bone cement structure (Chapter 3). To further address the zonal structure and composition of the native cartilage tissue through the layered deposition of cells and matrix components, the incorporation of the MEW-based production of microfibres within the bioprinting process was explored in Part II. First, the state-of-the-art concept of combining different manufacturing processes into a single biofabrication platform was reviewed and future perspectives of such approaches were discussed (Chapter 4). Further, it demonstrated, for the first time, the successful convergence of MEW and extrusion-based bioprinting into a single printing platform enabling control over the fibrous and non-fibrous components of chondral grafts (Chapter 5). Moreover, the potential of this converged approach for resurfacing anatomically relevant structures and clinically relevant materials was demonstrated. The importance of ensuring a constant electrical field strength and directing the electrical force normal to the collecting structure for accurate microfibre patterning on non-planar surfaces was shown (Chapter 6). Part III subsequently addressed the pre-clinical application of the developed multi-scale (bio)fabrication approaches. First, it was shown that bone morphogenic protein 9 (BMP-9) can be used to stimulate articular cartilage resident chondroprogenitor cells (ACPCs) to produce large quantities of reinforced cartilage-like matrix in a time-efficient manner which holds promise for the clinical translation of large biofabricated implants (Chapter 7). Next, long-term in vivo evaluation showed that pre-cultured osteochondral plugs with hierarchy in both cell density and microfibre organization were stable enough to withstand the mechanically challenging environment of the stifle joint in an equine model. This study highlights the importance of structural reinforcement and suggests that the use of transplanted cells is, in fact, secondary to the presence of the mechanical structure (Chapter 8). Upscaling from relatively small osteochondral plugs to larger patient-specific implants demonstrated that the size of the implants significantly affected load distribution and that the design of the implant should take into account the position of implantation to effectively restore mechanical functioning of the joint. Additionally, this study introduced a new computer aided design (CAD) to computer aided manufacturing (CAM) software tool to more easily generate the MEW printing trajectory for the resurfacing of patient-specific geometries (Chapter 9)

Diarthrodial joint resurfacing: Multi-scale biofabrication of mechanically stable implants. Osteochondral tissue regeneration in the knee joint

In the quest for new treatments for (osteo)chondral defects and to eliminate or postpone total knee replacement surgery, the potential of 3D bioprinting of (osteo)chondral implants as a surgical procedure has been explored. The overarching aim of this thesis was to fabricate a functional (osteo)chondral implant, inspired by native tissue architecture, by combining advanced 3D (bio)fabrication technologies. Part I demonstrated how melt electrowriting (MEW) was used to improve the mechanical properties of (osteo)chondral implants. Here, it was shown that microfibres, deposited in an out-of-plane fashion, improve the shear-properties of hydrogel scaffolds while using only a limited volume-fraction of biodegradable polymer fibres (Chapter 2). Additionally, microfibre meshes were used to improve the interconnection between the cartilage-to-bone interface by interlocking them within a printed calcium phosphate-based (pCaP) bone cement structure (Chapter 3). To further address the zonal structure and composition of the native cartilage tissue through the layered deposition of cells and matrix components, the incorporation of the MEW-based production of microfibres within the bioprinting process was explored in Part II. First, the state-of-the-art concept of combining different manufacturing processes into a single biofabrication platform was reviewed and future perspectives of such approaches were discussed (Chapter 4). Further, it demonstrated, for the first time, the successful convergence of MEW and extrusion-based bioprinting into a single printing platform enabling control over the fibrous and non-fibrous components of chondral grafts (Chapter 5). Moreover, the potential of this converged approach for resurfacing anatomically relevant structures and clinically relevant materials was demonstrated. The importance of ensuring a constant electrical field strength and directing the electrical force normal to the collecting structure for accurate microfibre patterning on non-planar surfaces was shown (Chapter 6). Part III subsequently addressed the pre-clinical application of the developed multi-scale (bio)fabrication approaches. First, it was shown that bone morphogenic protein 9 (BMP-9) can be used to stimulate articular cartilage resident chondroprogenitor cells (ACPCs) to produce large quantities of reinforced cartilage-like matrix in a time-efficient manner which holds promise for the clinical translation of large biofabricated implants (Chapter 7). Next, long-term in vivo evaluation showed that pre-cultured osteochondral plugs with hierarchy in both cell density and microfibre organization were stable enough to withstand the mechanically challenging environment of the stifle joint in an equine model. This study highlights the importance of structural reinforcement and suggests that the use of transplanted cells is, in fact, secondary to the presence of the mechanical structure (Chapter 8). Upscaling from relatively small osteochondral plugs to larger patient-specific implants demonstrated that the size of the implants significantly affected load distribution and that the design of the implant should take into account the position of implantation to effectively restore mechanical functioning of the joint. Additionally, this study introduced a new computer aided design (CAD) to computer aided manufacturing (CAM) software tool to more easily generate the MEW printing trajectory for the resurfacing of patient-specific geometries (Chapter 9)

Combining multi-scale 3D printing technologies to engineer reinforced hydrogel-ceramic interfaces

Multi-material 3D printing technologies that resolve features at different lengths down to the microscale open new avenues for regenerative medicine, particularly in the engineering of tissue interfaces. Herein, extrusion printing of a bone-biomimetic ceramic ink and melt electrowriting (MEW) of spatially organized polymeric microfibres are integrated for the biofabrication of an osteochondral plug, with a mechanically reinforced bone-to-cartilage interface. A printable physiological temperature-setting bioceramic, based on α-tricalciumphosphate, nanohydroxyapatite and a custom-synthesized biodegradable and crosslinkable poloxamer, was developed as bone support. The mild setting reaction of the bone ink enabled to print directly within melt electrowritten polycaprolactone meshes, preserving their micro-architecture. Ceramic-integrated MEW meshes protruded into the cartilage region of the composite plug, and were embedded with mechanically soft gelatin-based hydrogels, laden with articular cartilage chondroprogenitor cells. Such interlocking design enhanced the hydrogel-to-ceramic adhesion strength >6.5-fold, compared with non-interlocking fibre architectures, enabling structural stability during handling and surgical implantation in osteochondral defects ex vivo. Furthermore, the MEW meshes endowed the chondral compartment with compressive properties approaching those of native cartilage (20-fold reinforcement vs. pristine hydrogel). The osteal- and chondral compartment supported osteogenesis and cartilage matrix deposition in vitro, and the neo-synthesized cartilage matrix further contributed to the mechanical reinforcement at the ceramic-hydrogel interface. This multi-material, multi-scale 3D printing approach provides a promising strategy for engineering advanced composite constructs for the regeneration of musculoskeletal and connective tissue interfaces

3D bioprinting of methacrylated hyaluronic acid (MeHA) hydrogel with intrinsic osteogenicity

In bone regenerative medicine there is a need for suitable bone substitutes. Hydrogels have excellent biocompatible and biodegradable characteristics, but their visco-elastic properties limit their applicability, especially with respect to 3D bioprinting. In this study, we modified the naturally occurring extracellular matrix glycosaminoglycan hyaluronic acid (HA), in order to yield photo-crosslinkable hydrogels with increased mechanical stiffness and long-term stability, and with minimal decrease in cytocompatibility. Application of these tailor-made methacrylated hyaluronic acid (MeHA) gels for bone tissue engineering and 3D bioprinting was the subject of investigation. Visco-elastic properties of MeHA gels, measured by rheology and dynamic mechanical analysis, showed that irradiation of the hydrogels with UV light led to increased storage moduli and elastic moduli, indicating increasing gel rigidity. Subsequently, human bone marrow derived mesenchymal stromal cells (MSCs) were incorporated into MeHA hydrogels, and cell viability remained 64.4% after 21 days of culture. Osteogenic differentiation of MSCs occurred spontaneously in hydrogels with high concentrations of MeHA polymer, in absence of additional osteogenic stimuli. Addition of bone morphogenetic protein-2 (BMP-2) to the culture medium further increased osteogenic differentiation, as evidenced by increased matrix mineralisation. MeHA hydrogels demonstrated to be suitable for 3D bioprinting, and were printed into porous and anatomically shaped scaffolds. Taken together, photosensitive MeHA-based hydrogels fulfilled our criteria for cellular bioprinted bone constructs within a narrow window of concentration

3D bioprinting of methacrylated hyaluronic acid (MeHA) hydrogel with intrinsic osteogenicity

In bone regenerative medicine there is a need for suitable bone substitutes. Hydrogels have excellent biocompatible and biodegradable characteristics, but their visco-elastic properties limit their applicability, especially with respect to 3D bioprinting. In this study, we modified the naturally occurring extracellular matrix glycosaminoglycan hyaluronic acid (HA), in order to yield photo-crosslinkable hydrogels with increased mechanical stiffness and long-term stability, and with minimal decrease in cytocompatibility. Application of these tailor-made methacrylated hyaluronic acid (MeHA) gels for bone tissue engineering and 3D bioprinting was the subject of investigation. Visco-elastic properties of MeHA gels, measured by rheology and dynamic mechanical analysis, showed that irradiation of the hydrogels with UV light led to increased storage moduli and elastic moduli, indicating increasing gel rigidity. Subsequently, human bone marrow derived mesenchymal stromal cells (MSCs) were incorporated into MeHA hydrogels, and cell viability remained 64.4% after 21 days of culture. Osteogenic differentiation of MSCs occurred spontaneously in hydrogels with high concentrations of MeHA polymer, in absence of additional osteogenic stimuli. Addition of bone morphogenetic protein-2 (BMP-2) to the culture medium further increased osteogenic differentiation, as evidenced by increased matrix mineralisation. MeHA hydrogels demonstrated to be suitable for 3D bioprinting, and were printed into porous and anatomically shaped scaffolds. Taken together, photosensitive MeHA-based hydrogels fulfilled our criteria for cellular bioprinted bone constructs within a narrow window of concentration

Composite Graded Melt Electrowritten Scaffolds for Regeneration of the Periodontal Ligament-to-Bone Interface

Periodontitis is a ubiquitous chronic inflammatory, bacteria-triggered oral disease affecting the adult population. If left untreated, periodontitis can lead to severe tissue destruction, eventually resulting in tooth loss. Despite previous efforts in clinically managing the disease, therapeutic strategies are still lacking. Herein, melt electrowriting (MEW) is utilized to develop a compositionally and structurally tailored graded scaffold for regeneration of the periodontal ligament-to-bone interface. The composite scaffolds, consisting of fibers of polycaprolactone (PCL) and fibers of PCL-containing magnesium phosphate (MgP) were fabricated using MEW. To maximize the bond between bone (MgP) and ligament (PCL) regions, we evaluated two different fiber architectures in the interface area. These were a crosshatch pattern at a 0/90° angle and a random pattern. MgP fibrous scaffolds were able to promote in vitro bone formation even in culture media devoid of osteogenic supplements. Mechanical properties after MgP incorporation resulted in an increase of the elastic modulus and yield stress of the scaffolds, and fiber orientation in the interfacial zone affected the interfacial toughness. Composite graded MEW scaffolds enhanced bone fill when they were implanted in an in vivo periodontal fenestration defect model in rats. The presence of an interfacial zone allows coordinated regeneration of multitissues, as indicated by higher expression of bone, ligament, and cementoblastic markers compared to empty defects. Collectively, MEW-fabricated scaffolds having compositionally and structurally tailored zones exhibit a good mimicry of the periodontal complex, with excellent regenerative capacity and great potential as a defect-specific treatment strategy

Combining multi-scale 3D printing technologies to engineer reinforced hydrogel-ceramic interfaces

Multi-material 3D printing technologies that resolve features at different lengths down to the microscale open new avenues for regenerative medicine, particularly in the engineering of tissue interfaces. Herein, extrusion printing of a bone-biomimetic ceramic ink and melt electrowriting (MEW) of spatially organized polymeric microfibres are integrated for the biofabrication of an osteochondral plug, with a mechanically reinforced bone-to-cartilage interface. A printable physiological temperature-setting bioceramic, based on α-tricalciumphosphate, nanohydroxyapatite and a custom-synthesized biodegradable and crosslinkable poloxamer, was developed as bone support. The mild setting reaction of the bone ink enabled to print directly within melt electrowritten polycaprolactone meshes, preserving their micro-architecture. Ceramic-integrated MEW meshes protruded into the cartilage region of the composite plug, and were embedded with mechanically soft gelatin-based hydrogels, laden with articular cartilage chondroprogenitor cells. Such interlocking design enhanced the hydrogel-to-ceramic adhesion strength >6.5-fold, compared with non-interlocking fibre architectures, enabling structural stability during handling and surgical implantation in osteochondral defects ex vivo. Furthermore, the MEW meshes endowed the chondral compartment with compressive properties approaching those of native cartilage (20-fold reinforcement vs. pristine hydrogel). The osteal- and chondral compartment supported osteogenesis and cartilage matrix deposition in vitro, and the neo-synthesized cartilage matrix further contributed to the mechanical reinforcement at the ceramic-hydrogel interface. This multi-material, multi-scale 3D printing approach provides a promising strategy for engineering advanced composite constructs for the regeneration of musculoskeletal and connective tissue interfaces

Osteogenic differentiation of MSCs in MeHA hydrogels.

<p>A. Calcium mineralization per mg of hydrogel scaffold after 21 days of culturing in αMEM (control, white bars) or αMEM supplemented with BMP-2 (black bars). Experiments performed in duplicate and replicated with 3 MSC donors. Data shown as mean ± SD. N/A: The 1% (w/v) hydrogels without BMP-2 disintegrated before the day 21 time point. * Represents <i>p</i><0.05. B. Whole-mount Alizarin red staining of the MeHA hydrogels after 21 days of incubation in the presence of BMP-2. At lower MeHA concentration (1–1.5% (w/v)) gels, only some cells stained red. When the gel’s MeHA polymer concentration was increased, larger areas around the cells stained red, indicating calcium deposition into the surrounding matrix. Most intense staining is seen in the 2.5% (w/v) gels. Representative pictures are shown. Scale bars = 100 μm.</p

MeHA hydrogel swelling and degradation as a function of gel concentration.

<p>A. Increase of hydrogel wet weight (swelling) during 3 weeks incubation at 37°C, which was most pronounced at lower MeHA concentrations. B. Decrease of hydrogel dry weight during incubation. C. Decrease of hydrogel dry weight followed in time in the presence of hyaluronidase. Gel degradation was slower with increasing MeHA concentration. Data presented as mean ± SD, n = 5 for all measurements.</p

MeHA printability.

<p>Porous cubes (upper rows) and non-porous human L3 vertebrae shapes (lower rows) were 3D bioprinted, using the designs shown in the left column, and subsequently UV irradiated. After crosslinking, scaffolds were lifted using a spatula (horizontal bar) to test handling. Scale bars = 500 μm.</p