| 1. |
- Amoroso, Matteo, 1984, et al.
(author)
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Functional and morphological studies of in vivo vascularization of 3D-bioprinted human fat grafts
- 2021
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In: Bioprinting. - : Elsevier BV. - 2405-8866. ; 23
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Journal article (peer-reviewed)abstract
- Three-dimensional (3D) bioprinting offers the ability to design and biofabricate 3D structures based on autologous fat; however, the lack of vascularization in larger 3D-bioprinted constructs represents a limiting factor that hampers translation of this technology from bench to bedside. 3D bioprinting using microfractured fat mixed with nanocellulose–alginate hydrogel can promote vascularization through connections of fragments of vessels included in the fat. In this study, we determined the perfusion and diffusion characteristics of 3D-bioprinted fat constructs using magnetic resonance imaging (MRI) and assessed correlations between perfusion and angiogenesis within the printed constructs. Microfractured human fat from liposuction was printed with tunicate nanocellulose–alginate hydrogel, followed by transplantation of the constructs (10 × 10 × 3 mm) into nude mice that underwent longitudinal MRI for up to 99 days. Confirmation of vascularization was undertaken using immunohistochemical and histologic analyses. Before implantation, the constructs contained abundant fat tissue and fragments of human blood vessels (CD31+ and Ku80+), with subsequent in vivo MRI analysis following transplantation indicating low perfusion and suggesting their continued survival mainly by diffusion. Additionally, we observed a high diffusion coefficient (~2 × 10−3 mm2/s) that was preserved throughout the observation period. Following explantation, evaluation revealed that the constructs displayed preserved histology along with a mixture of human (Ku80+) and murine (Ku80−) erythrocyte-containing vessels. These results demonstrated successful interconnection of blood-vessel fragments from microfractured human fat via angiogenesis to form a vascular network with the host circulation, thereby confirming vascularization of the 3D-bioprinted fat constructs.
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| 2. |
- Amoroso, Matteo, 1984, et al.
(author)
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The effect of hemodilution on free flap survival: A systematic review of clinical andexperimental studies.
- 2020
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In: Clinical hemorheology and microcirculation. - 1875-8622. ; 75:4, s. 457-466
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Research review (peer-reviewed)abstract
- Acute normovolemic hemodilution (ANH) has been proposed as a microsurgical technique to improve blood flow in free flaps.Here, we present the first systematic review of clinical and experimental studies on the effect of ANH.We performed a systematic literature search of PubMed, Medline, the Cochrane Library, Google Scholar, and ClinicalTrials.gov using search strategies and a review process in agreement with the PRISMA statement and the Cochrane Handbook for systematic reviews of interventions. PICO criteria were defined before bibliometric processing of the retrieved articles, which were analyzed with the SYRCLE RoB tool for risk of bias and the GRADE scale for level of evidence.We retrieved 74 articles from the literature search, and after processing according to PICO criteria, only four articles remained, all of which were experimental. The rating for risk of bias was uncertain according to SYRCLE RoB results, and the level of evidence was low according to GRADE evaluation.There is no clinical evidence for the effect of ANH on microcirculation in free flaps, and experimental studies provide weak evidence supporting the use of hemodilution in reconstructive microsurgery.
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| 3. |
- Apelgren, Peter, et al.
(author)
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Biomaterial and biocompatibility evaluation of tunicate nanocellulose for tissue engineering.
- 2022
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In: Biomaterials advances. - : Elsevier BV. - 2772-9508. ; 137
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Journal article (peer-reviewed)abstract
- Extracellular matrix fibril components, such as collagen, are crucial for the structural properties of several tissues and organs. Tunicate-derived cellulose nanofibrils (TNC) combined with living cells could become the next gold standard for cartilage and soft-tissue repair, as TNC fibrils present similar dimensions to collagen, feasible industrial production, and chemically straightforward and cost-efficient extraction procedures. In this study, we characterized the physical properties of TNC derived from aquaculture production in Norwegian fjords and evaluated its biocompatibility regarding induction of an inflammatory response and foreign-body reactions in a Wistar rat model. Additionally, histologic and immunohistochemical analyses were performed for comparison with expanded polytetrafluoroethylene (ePTFE) as a control. The average length of the TNC as determined by atomic force microscopy was tunable from 3μm to 2.4μm via selection of a various number of passages through a microfluidizer, and rheologic analysis showed that the TNC hydrogels were highly shear-thinning and with a viscosity dependent on fibril length and concentration. As a bioink, TNC exhibited excellent rheological and printability properties, with constructs capable of being printed with high resolution and fidelity. We found that post-print cross-linking with alginate stabilized the construct shape and texture, which increased its ease of handling during surgery. Moreover, after 30days in vivo, the constructs showed a highly-preserved shape and fidelity of the grid holes, with these characteristics preserved after 90days and with no signs of necrosis, infection, acute inflammation, invasion of neutrophil granulocytes, or extensive fibrosis. Furthermore, we observed a moderate foreign-body reaction involving macrophages, lymphocytes, and giant cells in both the TNC constructs and PTFE controls, although TNC was considered a non-irritant biomaterial according to ISO 10993-6 as compared with ePTFE. These findings represent a milestone for future clinical application of TNC scaffolds for tissue repair. One sentence summary: In this study, the mechanical properties of tunicate nanocellulose are superior to nanocellulose extracted from other sources, and the biocompatibility is comparable to that of ePTFE.
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| 4. |
- Apelgren, Peter, et al.
(author)
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Chondrocytes and stem cells in 3D-bioprinted structures create human cartilage in vivo.
- 2017
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In: PloS one. - : Public Library of Science (PLoS). - 1932-6203. ; 12:12
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Journal article (peer-reviewed)abstract
- Cartilage repair and replacement is a major challenge in plastic reconstructive surgery. The development of a process capable of creating a patient-specific cartilage framework would be a major breakthrough. Here, we described methods for creating human cartilage in vivo and quantitatively assessing the proliferative capacity and cartilage-formation ability in mono- and co-cultures of human chondrocytes and human mesenchymal stem cells in a three-dimensional (3D)-bioprinted hydrogel scaffold. The 3D-bioprinted constructs (5 × 5 × 1.2 mm) were produced using nanofibrillated cellulose and alginate in combination with human chondrocytes and human mesenchymal stem cells using a 3D-extrusion bioprinter. Immediately following bioprinting, the constructs were implanted subcutaneously on the back of 48 nude mice and explanted after 30 and 60 days, respectively, for morphological and immunohistochemical examination. During explantation, the constructs were easy to handle, and the majority had retained their macroscopic grid appearance. Constructs consisting of human nasal chondrocytes showed good proliferation ability, with 17.2% of the surface areas covered with proliferating chondrocytes after 60 days. In constructs comprising a mixture of chondrocytes and stem cells, an additional proliferative effect was observed involving chondrocyte production of glycosaminoglycans and type 2 collagen. This clinically highly relevant study revealed 3D bioprinting as a promising technology for the creation of human cartilage.
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| 5. |
- Apelgren, Peter, et al.
(author)
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In Vivo Human Cartilage Formation in Three-Dimensional Bioprinted Constructs with a Novel Bacterial Nanocellulose Bioink
- 2019
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In: Acs Biomaterials Science & Engineering. - : American Chemical Society (ACS). - 2373-9878. ; 5:5, s. 2482-2490
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Journal article (peer-reviewed)abstract
- Bacterial nanocellulose (BNC) is a 3D network of nanofibrils exhibiting excellent biocompatibility. Here, we present the aqueous counter collision (ACC) method of BNC disassembly to create bioink with suitable properties for cartilage-specific 3D-bioprinting. BNC was disentangled by ACC, and fibril characteristics were analyzed. Bioink printing fidelity and shear-thinning properties were evaluated. Cell-laden bioprinted grid constructs (5 X 5 X 1 mm(3)) containing human nasal chondrocytes (10 M mL(-1)) were implanted in nude mice and explanted after 30 and 60 days. Both ACC and hydrolysis resulted in significantly reduced fiber lengths, with ACC resulting in longer fibrils and fewer negative charges relative to hydrolysis. Moreover, ACC-BNC bioink showed outstanding printability, postprinting mechanical stability, and structural integrity. In vivo, cell-laden structures were rapidly integrated, maintained structural integrity, and showed chondrocyte proliferation, with 32.8 +/- 13.8 cells per mm(2) observed after 30 days and 85.6 +/- 30.0 cells per mm(2) at day 60 (p = 0.002). Furthermore, a full-thickness skin graft was attached and integrated completely on top of the 3D-bioprinted construct. The novel ACC disentanglement technique makes BNC biomaterial highly suitable for 3D-bioprinting and clinical translation, suggesting cell-laden 3D-bioprinted ACC-BNC as a promising solution for cartilage repair.
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| 6. |
- Apelgren, Peter, et al.
(author)
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Long-term in vivo integrity and safety of3D-bioprinted cartilaginous constructs
- 2021
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In: Journal of Biomedical Materials Research Part B-Applied Biomaterials. - : Wiley. - 1552-4973 .- 1552-4981. ; 109:1, s. 126-136
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Journal article (peer-reviewed)abstract
- Long-term stability and biological safety are crucial for translation of 3D-bioprinting technology into clinical applications. Here, we addressed the long-term safety and stability issues associated with 3D-bioprinted constructs comprising a cellulose scaffold and human cells (chondrocytes and stem cells) over a period of 10 months in nude mice. Our findings showed that increasing unconfined compression strength over time significantly improved the mechanical stability of the cell-containing constructs relative to cell-free scaffolds. Additionally, the cell-free constructs exhibited a mean compressive stress and stiffness (compressive modulus) of 0.04 +/- 0.05 MPa and 0.14 +/- 0.18 MPa, respectively, whereas these values for the cell-containing constructs were 0.11 +/- 0.08 MPa (p= .019) and 0.53 +/- 0.59 MPa (p= .012), respectively. Moreover, histomorphologic analysis revealed that cartilage formed from the cell-containing constructs harbored an abundance of proliferating chondrocytes in clusters, and after 10 months, resembled native cartilage. Furthermore, extension of the experiment over the complete lifecycle of the animal model revealed no signs of ossification, fibrosis, necrosis, or implant-related tumor development in the 3D-bioprinted constructs. These findings confirm the in vivo biological safety and mechanical stability of 3D-bioprinted cartilaginous tissues and support their potential translation into clinical applications.
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| 7. |
- Apelgren, Peter
(author)
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Promoting the Clinical Relevance of 3D Bioprinting
- 2022
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Doctoral thesis (other academic/artistic)abstract
- This thesis focuses on the development of methodologies enabling the reconstruction of autologous, functional, and long-term-stable cartilage-like tissue using 3D bioprinting technology and animal experiments. The stability, resilience, and in vivo viability of the printed cells and tissue vascularization, as well as the observed immunogenicity and safety, represent the main issues evaluated and discussed in this thesis. Furthermore, the mechanical properties of the applied biomaterials are evaluated in detail. Study I Background: This study quantitatively assessed the proliferative capacity of chondrocytes in the presence and absence of stem cells in the 3D bioprinting setting. Results: We observed significant increases in the number of chondrocytes and cluster formations during the study period. Compared with pure human nasal chondrocyte (hNC) group, we identified a significant additional proliferative effect in the group containing both hNCs and stem cells, and histologic analysis confirmed the expected production of collagen type II in the extracellular matrix, as well as the distribution of glycosaminoglycans in the cartilage-like tissue. Additionally, fluorescence in situ hybridization analysis confirmed that the chondrocytes were of human origin, and their male phenotype verified the male chon-drocyte-donor source. Study II Background: In this study, we evaluated the results of subcutaneous implantation of 3D-bioprinted constructs mixed with human chondrocytes and stem cells over the course of 10 months. Results: We observed no signs of necrosis, tumors, ossification, or other adverse effects. Moreover, the constructs remained well-preserved, and histologic analyses showed thriving, proliferating chondrocytes in cartilage-like formations. Study III Background: This study mapped the vascularization of gridded 3D-bioprinted constructs. Results: Perfusion data from magnetic resonance imaging revealed progressive vascularization inside of grid holes that were confirmed as being filled with blood vessels connected to host circulation according to histologic analysis. Additionally, immunohistochemical analysis of endothelial cells confirmed the vascular arrangement, with collagen II production further indi-cating chondrocyte proliferation and cartilage formation. Study IV Background: In this study, we evaluated the biocompatibility (according to ISO standards) and mechanical properties of tunicate-derived nanocellulose (TNC) as a novel biomaterial. Results: We determined TNC biocompatibility as equivalent to that of expanded polytetrafluoroethylene while also exhibiting excellent mechanical properties. Keywords 3D bioprinting, cartilage, chondrocytes, stem cells, tissue engineering, nanocellulose, hydrogel, bioink, vascularization, bio-compatibility
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| 8. |
- Apelgren, Peter, et al.
(author)
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Skin Grafting on 3D Bioprinted Cartilage Constructs In Vivo
- 2018
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In: Plastic and Reconstructive Surgery-Global Open. - : Ovid Technologies (Wolters Kluwer Health). - 2169-7574. ; 6:9
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Journal article (peer-reviewed)abstract
- Background: Three-dimensional (3D) bioprinting of cartilage is a promising new technique. To produce, for example, an auricle with good shape, the printed cartilage needs to be covered with skin that can grow on the surface of the construct. Our primary question was to analyze if an integrated 3D bioprinted cartilage structure is a tissue that can serve as a bed for a full-thickness skin graft. Methods: 3D bioprinted constructs (10x10x1.2mm) were printed using nanofibrillated cellulose/alginate bioink mixed with mesenchymal stem cells and adult chondrocytes and implanted subcutaneously in 21 nude mice. Results: After 45 days, a full-thickness skin allograft was transplanted onto the constructs and the grafted construct again enclosed subcutaneously. Group 1 was sacrificed on day 60, whereas group 2, instead, had their skin-bearing construct uncovered on day 60 and were sacrificed on day 75 and the explants were analyzed morphologically. The skin transplants integrated well with the 3D bioprinted constructs. A tight connection between the fibrous, vascularized capsule surrounding the 3D bioprinted constructs and the skin graft were observed. The skin grafts survived the uncovering and exposure to the environment. Conclusions: A 3D bioprinted cartilage that has been allowed to integrate in vivo is a sufficient base for a full-thickness skin graft. This finding accentuates the clinical potential of 3D bioprinting for reconstructive purposes.
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| 9. |
- Apelgren, Peter, et al.
(author)
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Skin Grafting on 3D Bioprinted Cartilage Constructs In Vivo
- 2018
-
In: Plastic and Reconstructive Surgery - Global Open. - 2169-7574. ; 6:9
-
Journal article (peer-reviewed)abstract
- Background: Three-dimensional (3D) bioprinting of cartilage is a promising new technique. To produce, for example, an auricle with good shape, the printed cartilage needs to be covered with skin that can grow on the surface of the construct. Our primary question was to analyze if an integrated 3D bioprinted cartilage structure is a tissue that can serve as a bed for a full-thickness skin graft. Methods: 3D bioprinted constructs (10x10x1.2mm) were printed using nanofibrillated cellulose/alginate bioink mixed with mesenchymal stem cells and adult chondrocytes and implanted subcutaneously in 21 nude mice. Results: After 45 days, a full-thickness skin allograft was transplanted onto the constructs and the grafted construct again enclosed subcutaneously. Group 1 was sacrificed on day 60, whereas group 2, instead, had their skin-bearing construct uncovered on day 60 and were sacrificed on day 75 and the explants were analyzed morphologically. The skin transplants integrated well with the 3D bioprinted constructs. A tight connection between the fibrous, vascularized capsule surrounding the 3D bioprinted constructs and the skin graft were observed. The skin grafts survived the uncovering and exposure to the environment. Conclusions: A 3D bioprinted cartilage that has been allowed to integrate in vivo is a sufficient base for a full-thickness skin graft. This finding accentuates the clinical potential of 3D bioprinting for reconstructive purposes.
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| 10. |
- Apelgren, Peter, et al.
(author)
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Vascularization of tissue engineered cartilage-Sequential in vivo MRI display functional blood circulation
- 2021
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In: Biomaterials. - : Elsevier BV. - 0142-9612 .- 1878-5905. ; 276
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Journal article (peer-reviewed)abstract
- Establishing functional circulation in bioengineered tissue after implantation is vital for the delivery of oxygen and nutrients to the cells. Native cartilage is avascular and thrives on diffusion, which in turn depends on proximity to circulation. Here, we investigate whether a gridded three-dimensional (3D) bioprinted construct would allow ingrowth of blood vessels and thus prove a functional concept for vascularization of bioengineered tissue. Twenty 10 x 10 x 3-mm 3Dbioprinted nanocellulose constructs containing human nasal chondrocytes or cell-free controls were subcutaneously implanted in 20 nude mice. Over the next 3 months, the mice were sequentially imaged with a 7 T small-animal MRI system, and the diffusion and perfusion parameters were analyzed. The chondrocytes survived and proliferated, and the shape of the constructs was well preserved. The diffusion coefficient was high and well preserved over time. The perfusion and diffusion patterns shown by MRI suggested that blood vessels develop over time in the 3D bioprinted constructs; the vessels were confirmed by histology and immunohistochemistry. We conclude that 3D bioprinted tissue with a gridded structure allows ingrowth of blood vessels and has the potential to be vascularized from the host. This is an essential step to take bioengineered tissue from the bench to clinical practice.
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