Long-term in vivo survival of 3D-bioprinted human lipoaspirate-derived adipose tissue: proteomic signature and cellular content

Three-dimensional (3D)-bioprinted lipoaspirate-derived adipose tissue (LAT) is a potential alternative to lipo-injection for correcting soft-tissue defects. This study investigated the long-term in vivo survival of 3D-bioprinted LAT and its proteomic signature and cellular composition. We performed proteomic and multicolour flow cytometric analyses on the lipoaspirate and 3D-bioprinted LAT constructs were transplanted into nude mice, followed by explantation after up to 150\ua0days. LAT contained adipose-tissue-derived stem cells (ASCs), pericytes, endothelial progenitor cells (EPCs) and endothelial cells. Proteomic analysis identified 6,067 proteins, including pericyte markers, adipokines, ASC secretome proteins, proangiogenic proteins and proteins involved in adipocyte differentiation and developmental morphogenic signalling, as well as proteins not previously described in human subcutaneous fat. 3D-bioprinted LAT survived for 150\ua0days in vivo with preservation of the construct shape and size. Furthermore, we identified human blood vessels after 30 and 150\ua0days in vivo, indicating angiogenesis from capillaries. These results showed that LAT has a favourable proteomic signature, contains ASCs, EPCs and blood vessels that survive 3D bioprinting and can potentially facilitate angiogenesis and successful autologous fat grafting in soft-tissue reconstruction

Vascularization of tissue engineered cartilage - Sequential in vivo MRI display functional blood circulation

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 7 10 7 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

Autologous endothelialisation by the stromal vascular fraction on laminin-bioconjugated nanocellulose-alginate scaffolds

Establishing a vascular network in biofabricated tissue grafts is essential for ensuring graft survival. Such networks are dependent on the ability of the scaffold material to facilitate endothelial cell adhesion; however, the clinical translation potential of tissue-engineered scaffolds is hindered by the lack of available autologous sources of vascular cells. Here, we present a novel approach to achieving autologous endothelialisation in nanocellulose-based scaffolds by using adipose tissue-derived vascular cells on nanocellulose-based scaffolds. We used sodium periodate-mediated bioconjugation to covalently bind laminin to the scaffold surface and isolated the stromal vascular fraction and endothelial progenitor cells (EPCs; CD31+CD45−) from human lipoaspirate. Additionally, we assessed the adhesive capacity of scaffold bioconjugation in vitro using both adipose tissue-derived cell populations and human umbilical vein endothelial cells. The results showed that the bioconjugated scaffold exhibited remarkably higher cell viability and scaffold surface coverage by adhesion regardless of cell type, whereas control groups comprising cells on non-bioconjugated scaffolds exhibited minimal cell adhesion across all cell types. Furthermore, on culture day 3, EPCs seeded on laminin-bioconjugated scaffolds showed positive immunofluorescence staining for the endothelial markers CD31 and CD34, suggesting that the scaffolds promoted progenitor differentiation into mature endothelial cells. These findings present a possible strategy for generating autologous vasculature and thereby increase the clinical relevance of 3D-bioprinted nanocellulose-based constructs

Successful engraftment, vascularization, and In vivo survival of 3D-bioprinted human lipoaspirate-derived adipose tissue

Autologous fat grafting is commonly used for correction of soft-tissue deformities, despite a high rate of graft resorption and nutrition-supply challenges. Three-dimensional (3D)-bioprinting techniques enable tailor-made architecture of grafts and promote vascularization. In recent years, the importance of adipose tissue-derived stromal/stem cells (ASCs) for graft survival has become evident. This study investigated the printability of mechanically processed lipoaspirate containing ASCs, as well as in vivo survival and neovascularisation of the 3D-bioprinted grafts. Human lipoaspirate-derived adipose tissue was 3D bioprinted in alginate/nanocellulose bioink, implanted into nude mice, and harvested at days 3, 7, and 30, respectively. The processed lipoaspirate showed high viability and good printability when combined with alginate/nanocellulose, and the 3D-bioprinted grafts contained intact vascular structures and a high density of mature adipocytes before and after engraftment. After 30 days in vivo, novel blood vessels were present on the graft surface, showing signs of angiogenesis into the graft, as well as vascularization in the centre of the tissue. Moreover, histologic and immunohistochemical characterisation confirmed the presence of potential ASCs during the first week in vivo. These results demonstrated that human lipoaspirate-derived adipose tissue showed high printability, survived 3D bioprinting and engraftment in vivo, and displayed macroscopic and microscopic evidence of vascularization

Functional and morphological studies of in vivo vascularization of 3D-bioprinted human fat grafts

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 7 10 7 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 7 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

In Vivo Human Cartilage Formation in Three-Dimensional Bioprinted Constructs with a Novel Bacterial Nanocellulose Bioink

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 7 5 7 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 \ub1 13.8 cells per mm 2 observed after 30 days and 85.6 \ub1 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