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You are researching: Rizzoli Orthopaedic Institute
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AUTHOR
Year
2024
Journal/Proceedings
Advanced Materials Technologies
Reftype
DOI/URL
DOI
Groups
AbstractAbstract Low back pain is among the main issues in vertebral orthopaedics. Intervertebral disk degeneration can be severe, up to requiring the replacement of the damaged disk by substitutes to achieve spine fusion. Disk removal results in critical size defects, so fusion does not occur naturally, but synthetic bone grafts are needed. Since the surgical procedure is time-consuming, high infection rates occur. Hence, in spine fusion, bone regeneration enhancement and infection prevention are needed. Here, a new dual-component system is proposed, to tackle both issues at one time. To enable spine fusion, 3D extrusion-based printing is employed to develop coherent custom magnesium phosphate (CaMgP)-based cages. The 3D-printed scaffolds are hardened, and the structural properties are evaluated to be within the ranges of physiological bone. To prevent infection, an in-house ice-templating device is employed in combination with a 3D-printed ceramic scaffold, to develop tailored porous alginate structures loaded with vancomycin. Results show that CaMgP can be printed into complex geometries and that the geometry influences the pore orientation during ice-templating. These structures loaded with vancomycin have antibacterial properties against Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) strains.
AUTHOR
Year
2023
Journal/Proceedings
Applied Sciences
Reftype
Groups
Abstract(1) Background: Synovial tissue plays a fundamental role in inflammatory processes. Therefore, understanding the mechanisms regulating healthy and diseased synovium functions, as in rheumatic diseases, is crucial to discovering more effective therapies to minimize or prevent pathological progress. The present study aimed at developing a bioartificial synovial tissue as an in vitro model for drug screening or personalized medicine applications using 3D bioprinting technology. (2) Methods: The volumetric extrusion technique has been used to fabricate cell-laden scaffolds. Gelatin Methacryloyl (GelMA), widely applied in regenerative medicine and tissue engineering, was selected as a bioink and combined with an immortalized cell line of fibroblast-like synoviocytes (K4IM). (3) Results: Three different GelMA formulations, 7.5–10–12.5% w/v, were tested for the fabrication of the scaffold with the desired morphology and internal architecture. GelMA 10% w/v was chosen and combined with K4IM cells to fabricate scaffolds that showed high cell viability and negligible cytotoxicity for up to 14 days tested by Live & Dead and lactate dehydrogenase assays. (4) Conclusions: We successfully 3D bioprinted synoviocytes-laden scaffolds as a proof-of-concept (PoC) towards the fabrication of a 3D synovial membrane model suitable for in vitro studies. However, further research is needed to reproduce the complexity of the synovial microenvironment to better mimic the physiological condition.
AUTHOR
Title
Nanomechanical Mapping of Three Dimensionally Printed Poly-ε-Caprolactone Single Microfibers at the Cell Scale for Bone Tissue Engineering Applications
[Abstract]
Year
2023
Journal/Proceedings
Biomimetics
Reftype
Groups
AbstractPoly-ε-caprolactone (PCL) has been widely used in additive manufacturing for the construction of scaffolds for bone tissue engineering. However, its use is limited by its lack of bioactivity and inability to induce cell adhesion, hence limiting bone tissue regeneration. Biomimicry is strongly influenced by the dynamics of cell–substrate interaction. Thus, characterizing scaffolds at the cell scale could help to better understand the relationship between surface mechanics and biological response. We conducted atomic force microscopy-based nanoindentation on 3D-printed PCL fibers of ~300 µm thickness and mapped the near-surface Young’s modulus at loading forces below 50 nN. In this non-disruptive regime, force mapping did not show clear patterns in the spatial distribution of moduli or a relationship with the topographic asperities within a given region. Remarkably, we found that the average modulus increased linearly with the logarithm of the strain rate. Finally, a dependence of the moduli on the history of nanoindentation was demonstrated on locations of repeated nanoindentations, likely due to creep phenomena capable of hindering viscoelasticity. Our findings can contribute to the rational design of scaffolds for bone regeneration that are capable of inducing cell adhesion and proliferation. The methodologies described are potentially applicable to various tissue-engineered biopolymers.
AUTHOR
Title
A 3D Collagen-Based Bioprinted Model to Study Osteosarcoma Invasiveness and Drug Response
[Abstract]
Year
2022
Journal/Proceedings
Polymers
Reftype
Groups
AbstractThe biological and therapeutic limits of traditional 2D culture models, which only partially mimic the complexity of cancer, have recently emerged. In this study, we used a 3D bioprinting platform to process a collagen-based hydrogel with embedded osteosarcoma (OS) cells. The human OS U-2 OS cell line and its resistant variant (U-2OS/CDDP 1 μg) were considered. The fabrication parameters were optimized to obtain 3D printed constructs with overall morphology and internal microarchitecture that accurately match the theoretical design, in a reproducible and stable process. The biocompatibility of the 3D bioprinting process and the chosen collagen bioink in supporting OS cell viability and metabolism was confirmed through multiple assays at short- (day 3) and long- (day 10) term follow-ups. In addition, we tested how the 3D collagen-based bioink affects the tumor cell invasive capabilities and chemosensitivity to cisplatin (CDDP). Overall, we developed a new 3D culture model of OS cells that is easy to set up, allows reproducible results, and better mirrors malignant features of OS than flat conditions, thus representing a promising tool for drug screening and OS cell biology research.
AUTHOR
Title
Methacrylated Silk Fibroin Additive Manufacturing of Shape Memory Constructs with Possible Application in Bone Regeneration
[Abstract]
Year
2022
Journal/Proceedings
Gels
Reftype
Groups
AbstractMethacrylated silk (Sil-MA) is a chemically modified silk fibroin specifically designed to be crosslinkable under UV light, which makes this material applicable in additive manufacturing techniques and allows the prototyping and development of patient-specific 2D or 3D constructs. In this study, we produced a thin grid structure based on crosslinked Sil-MA that can be withdrawn and ejected and that can recover its shape after rehydration. A complete chemical and physical characterization of Sil-MA was first conducted. Additionally, we tested Sil-MA biocompatibility according to the International Standard Organization protocols (ISO 10993) ensuring the possibility of using it in future trials. Sil-MA was also tested to verify its ability to support osteogenesis. Overall, Sil-MA was shown to be biocompatible and osteoconductive. Finally, two different additive manufacturing technologies, a Digital Light Processing (DLP) UV projector and a pneumatic extrusion technique, were used to develop a Sil-MA grid construct. A proof-of-concept of its shape-memory property was provided. Together, our data support the hypothesis that Sil-MA grid constructs can be injectable and applicable in bone regeneration applications.
AUTHOR
Title
3D printing of PCL/nano-hydroxyapatite scaffolds derived from biogenic sources for bone tissue engineering
[Abstract]
Year
2021
Journal/Proceedings
Sustainable Materials and Technologies
Reftype
Groups
AbstractBioactive composites made of ∽85 wt% poly(ε-caprolactone) (PCL) and ∽15 wt% nanometric hydroxyapatite (HA) produced from biogenic sources were 3D printed by an extrusion-based process to obtain porous scaffolds suitable for bone regeneration. Three different composite formulations were considered by using HA synthesized from three distinct natural sources, which were collected as food wastes: cuttlefish bones, mussel shells and chicken eggshells. Composition and thermal properties of the materials were analysed by thermogravimetric analysis (TGA), differential scanning calorimetry (DSC) and x-ray spectroscopy (XRD), while the morphological and mechanical properties of the 3D scaffolds were studied by means of electron microscopy (SEM) and compression tests. Bioactivity was tested by seeding human osteoblast cell line (MG63) onto the scaffolds which were analysed by confocal microscopy and Alamar Blue and PicoGreen® tests after 1 to 7 culture days. The elastic modulus (177–316 MPa) is found to be within the range reported for typical trabecular bones being increased by the presence of the bio-HA particles. Moreover, cells adhesion, viability and proliferation are largely promoted in the scaffolds containing nanometric HA with respect to pure PCL, the best results being revealed when mussel shell-derived HA is used. Indeed, different biological sources result in different cell proliferation rates, pointing that the biological origin has an impact on the cells-scaffold interaction. In general, the results show that PCL/bio-HA scaffolds possess improved mechanical properties and enhanced bioactivity when compared with pure PCL ones.
AUTHOR
Title
Composite Scaffolds for Bone Tissue Regeneration Based on PCL and Mg-Containing Bioactive Glasses
[Abstract]
Year
2021
Journal/Proceedings
Biology
Reftype
DOI/URL
DOI
Groups
AbstractPolycaprolactone (PCL) is widely used in additive manufacturing for the construction of scaffolds for tissue engineering because of its good bioresorbability, biocompatibility, and processability. Nevertheless, its use is limited by its inadequate mechanical support, slow degradation rate and the lack of bioactivity and ability to induce cell adhesion and, thus, bone tissue regeneration. In this study, we fabricated 3D PCL scaffolds reinforced with a novel Mg-doped bioactive glass (Mg-BG) characterized by good mechanical properties and biological reactivity. An optimization of the printing parameters and scaffold fabrication was performed; furthermore, an extensive microtopography characterization by scanning electron microscopy and atomic force microscopy was carried out. Nano-indentation tests accounted for the mechanical properties of the scaffolds, whereas SBF tests and cytotoxicity tests using human bone-marrow-derived mesenchymal stem cells (BM-MSCs) were performed to evaluate the bioactivity and in vitro viability. Our results showed that a 50/50 wt% of the polymer-to-glass ratio provides scaffolds with a dense and homogeneous distribution of Mg-BG particles at the surface and roughness twice that of pure PCL scaffolds. Compared to pure PCL (hardness H = 35 ± 2 MPa and Young’s elastic modulus E = 0.80 ± 0.05 GPa), the 50/50 wt% formulation showed H = 52 ± 11 MPa and E = 2.0 ± 0.2 GPa, hence, it was close to those of trabecular bone. The high level of biocompatibility, bioactivity, and cell adhesion encourages the use of the composite PCL/Mg-BG scaffolds in promoting cell viability and supporting mechanical loading in the host trabecular bone.
AUTHOR
Title
Modeling and Fabrication of Silk Fibroin-Gelatin-Based Constructs Using Extrusion-Based Three-Dimensional Bioprinting
[Abstract]
Year
2021
Journal/Proceedings
ACS Biomater. Sci. Eng.
Reftype
DOI/URL
DOI
Groups
AbstractRobotic dispensing-based 3D bioprinting represents one of the most powerful technologies to develop hydrogel-based 3D constructs with enormous potential in the field of regenerative medicine. The optimization of hydrogel printing parameters, proper geometry and internal architecture of the constructs, and good cell viability during the bioprinting process are the essential requirements. In this paper, an analytical model based on the hydrogel rheological properties was developed to predict the extruded filament width in order to maximize the printed structure’s fidelity to the design. Viscosity data of two natural hydrogels were imputed to a power-law model to extrapolate the filament width. Further, the model data were validated by monitoring the obtained filament width as the output. Shear stress values occurring during the bioprinting process were also estimated. Human mesenchymal stromal cells (hMSCs) were encapsulated in the silk fibroin-gelatin (G)-based hydrogel, and a 3D bioprinting process was performed to produce cell-laden constructs. Live and dead assay allowed estimating the impact of needle shear stress on cell viability after the bioprinting process. Finally, we tested the potential of hMSCs to undergo chondrogenic differentiation by evaluating the cartilaginous extracellular matrix production through immunohistochemical analyses. Overall, the use of the proposed analytical model enables defining the optimal printing parameters to maximize the fabricated constructs’ fidelity to design parameters before the process execution, enabling to achieve more controlled and standardized products than classical trial-and-error approaches in the biofabrication of engineered constructs. Employing modeling systems exploiting the rheological properties of the hydrogels might be a valid tool in the future for guaranteeing high cell viability and for optimizing tissue engineering approaches in regenerative medicine applications. Robotic dispensing-based 3D bioprinting represents one of the most powerful technologies to develop hydrogel-based 3D constructs with enormous potential in the field of regenerative medicine. The optimization of hydrogel printing parameters, proper geometry and internal architecture of the constructs, and good cell viability during the bioprinting process are the essential requirements. In this paper, an analytical model based on the hydrogel rheological properties was developed to predict the extruded filament width in order to maximize the printed structure’s fidelity to the design. Viscosity data of two natural hydrogels were imputed to a power-law model to extrapolate the filament width. Further, the model data were validated by monitoring the obtained filament width as the output. Shear stress values occurring during the bioprinting process were also estimated. Human mesenchymal stromal cells (hMSCs) were encapsulated in the silk fibroin-gelatin (G)-based hydrogel, and a 3D bioprinting process was performed to produce cell-laden constructs. Live and dead assay allowed estimating the impact of needle shear stress on cell viability after the bioprinting process. Finally, we tested the potential of hMSCs to undergo chondrogenic differentiation by evaluating the cartilaginous extracellular matrix production through immunohistochemical analyses. Overall, the use of the proposed analytical model enables defining the optimal printing parameters to maximize the fabricated constructs’ fidelity to design parameters before the process execution, enabling to achieve more controlled and standardized products than classical trial-and-error approaches in the biofabrication of engineered constructs. Employing modeling systems exploiting the rheological properties of the hydrogels might be a valid tool in the future for guaranteeing high cell viability and for optimizing tissue engineering approaches in regenerative medicine applications.
AUTHOR
Title
Multifunctional 3D-Printed Magnetic Polycaprolactone/Hydroxyapatite Scaffolds for Bone Tissue Engineering
[Abstract]
Year
2021
Journal/Proceedings
Polymers
Reftype
Groups
AbstractMultifunctional and resistant 3D structures represent a great promise and a great challenge in bone tissue engineering. This study addresses this problem by employing polycaprolactone (PCL)-based scaffolds added with hydroxyapatite (HAp) and superparamagnetic iron oxide nanoparticles (SPION), able to drive on demand the necessary cells and other bioagents for a high healing efficiency. PCL-HAp-SPION scaffolds with different concentrations of the superparamagnetic component were developed through the 3D-printing technology and the specific topographical features were detected by Atomic Force and Magnetic Force Microscopy (AFM-MFM). AFM-MFM measurements confirmed a homogenous distribution of HAp and SPION throughout the surface. The magnetically assisted seeding of cells in the scaffold resulted most efficient for the 1% SPION concentration, providing good cell entrapment and adhesion rates. Mesenchymal Stromal Cells (MSCs) seeded onto PCL-HAp-1% SPION showed a good cell proliferation and intrinsic osteogenic potential, indicating no toxic effects of the employed scaffold materials. The performed characterizations and the collected set of data point on the inherent osteogenic potential of the newly developed PCL-HAp-1% SPION scaffolds, endorsing them towards next steps of in vitro and in vivo studies and validations.
AUTHOR
Title
The effect of silk-gelatin bioink and TGF-β3 on mesenchymal stromal cells in 3D bioprinted chondrogenic constructs: A proteomic study
[Abstract]
Year
2021
Journal/Proceedings
Journal of Materials Research
Reftype
Chawla2021
DOI/URL
DOI
Groups
AbstractMajor limitation of 3D bioprinting is the poor understanding of the role of bioink in modulating molecular signaling pathways. Phenotypically stable engineered articular cartilage was fabricated using silk fibroin-gelatin (SF-G) bioink and progenitor cells or mature articular chondrocytes. In the current study, role of SF-G bioink in modulating in vitro chondrogenic signaling pathways in human bone marrow-derived stromal cells (hMSCs) is elucidated. The interaction between SF-G bioink and hMSCs augmented several chondrogenic pathways, including Wnt, HIF-1, and Notch. We explored the debatable role of TGF-β signaling, by assessing the differential protein expression by hMSCs-laden bioprinted constructs in the presence and absence of TGF-β3. hMSCs-laden bioprinted constructs contained a large percentage of collagen type II and Filamin-B, typical to the native articular cartilage. Hypertrophy markers were not identified following TGF-β3 addition. This is first detailed proteomics analysis to identify articular cartilage-specific pathways in SF-G-based 3D bioprinted construct.
AUTHOR
Title
Design of a novel procedure for the optimization of the mechanical performances of 3D printed scaffolds for bone tissue engineering combining CAD, Taguchi method and FEA
[Abstract]
Year
2019
Journal/Proceedings
Medical Engineering and Physics
Reftype
Groups
AbstractIn order to increase manufacturing and experimental efficiency, a certain degree of control over design performances before realization phase is recommended. In this context, this paper presents an integrated procedure to design 3D scaffolds for bone tissue engineering. The procedure required a combination of Computer Aided Design (CAD), Finite Element Analysis (FEA), and Design methodologies Of Experiments (DOE), firstly to understand the influence of the design parameters, and then to control them. Based on inputs from the literature and limitations imposed by the chosen manufacturing process (Precision Extrusion Deposition), 36 scaffold architectures have been drawn. The porosity of each scaffold has been calculated with CAD. Thereafter, a generic scaffold material was considered and its variable parameters were combined with the geometrical ones according to the Taguchi method, i.e. a DOE method. The compressive response of those principal combinations was simulated by FEA, and the influence of each design parameter on the scaffold compressive behaviour was clarified. Finally, a regression model was obtained correlating the scaffold's mechanical performances to its geometrical and material parameters. This model has been applied to a novel composite material made of polycaprolactone and innovative bioactive glass. By setting specific porosity (50%) and stiffness (0.05 GPa) suitable for trabecular bone substitutes, the model selected 4 of the 36 initial scaffold architectures. Only these 4 more promising geometries will be realized and physically tested for advanced indications on compressive strength and biocompatibility.
AUTHOR
Title
Investigating the Role of Sustained Calcium Release in Silk-Gelatin-Based Three-Dimensional Bioprinted Constructs for Enhancing the Osteogenic Differentiation of Human Bone Marrow Derived Mesenchymal Stromal Cells
Year
2019
Journal/Proceedings
ACS Biomaterials Science & Engineering
Reftype
DOI/URL
DOI
AUTHOR
Title
Patient-specific meniscus prototype based on 3D bioprinting of human cell-laden scaffold
[Abstract]
Year
2019
Journal/Proceedings
Bone and Joint Research
Reftype
DOI/URL
DOI
Groups
AbstractObjectives Meniscal injuries are often associated with an active lifestyle. The damage of meniscal tissue puts young patients at higher risk of undergoing meniscal surgery and, therefore, at higher risk of osteoarthritis. In this study, we undertook proof-of-concept research to develop a cellularized human meniscus by using 3D bioprinting technology. Methods A 3D model of bioengineered medial meniscus tissue was created, based on MRI scans of a human volunteer. The Digital Imaging and Communications in Medicine (DICOM) data from these MRI scans were processed using dedicated software, in order to obtain an STL model of the structure. The chosen 3D Discovery printing tool was a microvalve-based inkjet printhead. Primary mesenchymal stem cells (MSCs) were isolated from bone marrow and embedded in a collagen-based bio-ink before printing. LIVE/DEAD assay was performed on realized cell-laden constructs carrying MSCs in order to evaluate cell distribution and viability. Results This study involved the realization of a human cell-laden collagen meniscus using 3D bioprinting. The meniscus prototype showed the biological potential of this technology to provide an anatomically shaped, patient-specific construct with viable cells on a biocompatible material. Conclusion This paper reports the preliminary findings of the production of a custom-made, cell-laden, collagen-based human meniscus. The prototype described could act as the starting point for future developments of this collagen-based, tissue-engineered structure, which could aid the optimization of implants designed to replace damaged menisci. Cite this article: G. Filardo, M. Petretta, C. Cavallo, L. Roseti, S. Durante, U. Albisinni, B. Grigolo. Patient-specific meniscus prototype based on 3D bioprinting of human cell-laden scaffold. Bone Joint Res 2019;8:101–106. DOI: 10.1302/2046-3758.82.BJR-2018-0134.R1.