Injectable In Situ Crosslinking Hydrogel for Autologous Fat Grafting.

Autologous fat grafting is hampered by unpredictable outcomes due to high tissue resorption. Hydrogels based on enzymatically pretreated tunicate nanocellulose (ETC) and alginate (ALG) are biocompatible, safe, and present physiochemical properties capable of promoting cell survival. Here, we compared in situ and ex situ crosslinking of ETC/ALG hydrogels combined with lipoaspirate human adipose tissue (LAT) to generate an injectable formulation capable of retaining dimensional stability in vivo. We performed in situ crosslinking using two different approaches; inducing Ca2+ release from CaCO3 microparticles (CMPs) and physiologically available Ca2+ in vivo. Additionally, we generated ex situ-crosslinked, 3D-bioprinted hydrogel-fat grafts. We found that in vitro optimization generated a CMP-crosslinking system with comparable stiffness to ex situ-crosslinked gels. Comparison of outcomes following in vivo injection of each respective crosslinked hydrogel revealed that after 30 days, in situ crosslinking generated fat grafts with less shape retention than 3D-bioprinted constructs that had undergone ex situ crosslinking. However, CMP addition improved fat-cell distribution and cell survival relative to grafts dependent on physiological Ca2+ alone. These findings suggested that in situ crosslinking using CMP might promote the dimensional stability of injectable fat-hydrogel grafts, although 3D bioprinting with ex situ crosslinking more effectively ensured proper shape stability in vivo.

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 bioconjugationin vitrousing 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.

Correlative Cellular Mass Spectrometry Imaging and Amperometry Show Dose Dependent Changes in Lipid Composition and Exocytosis.

Aberrant functioning of the proteasome has been associated with crucial pathologic conditions including neurodegeneration. Yet, the complex underlying causes at the cellular level remain unclear and there are conflicting reports of neuroprotective to neurodegenerative effects of proteasomal inhibitors such as lactacystin that are utilised as models for neurodegenerative diseases. The conflicting results may be associated with different dose regimes of lactacystin and hence we have performed a dose dependent study of the effects of lactacystin to identify concurrent changes in the cell membrane lipid profile and the dynamics of exocytosis using a combination of surface sensitive mass spectrometry and single cell amperometry. Significant changes of negatively charged lipids were associated with different lactacystin doses that showed a weak correlation with exocytosis while changes in PE and PE-O lipids showed dose dependent changes correlated with initial pore formation and total release of vesicle content respectively.

Biomaterial and biocompatibility evaluation of tunicate nanocellulose for tissue engineering.

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 3 µm to 2.4 µm 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 30 days in vivo, the constructs showed a highly-preserved shape and fidelity of the grid holes, with these characteristics preserved after 90 days 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.

Alginate and tunicate nanocellulose composite microbeads - Preparation, characterization and cell encapsulation.

Alginate has been used for decades for cell encapsulation. Cellulose nanofibrils (CNF) from tunicates are desirable in biomedicine due to high molecular weight, purity, crystallinity, and sustainable production. We prepared microbeads of 400-600 µm of alginate and tunicate CNF. Greater size, dispersity and aspect ratio were observed in microbeads with higher fractions of CNF. CNF content in Ca-crosslinked alginate microbeads decreased stability upon saline exposure, whereas crosslinking with calcium (50 mM) and barium (1 mM) yielded stable microbeads. The Young's moduli of gel cylinders decreased when exchanging alginate with CNF, and slightly increased permeability to dextran was observed in microbeads containing CNF. Encapsulation of MC3T3 cells revealed high cell viability after encapsulation (83.6 ± 0.4%) in beads of alginate and CNF. NHDFs showed lower viability but optimizing mixing and production techniques of microbeads increased cell viability (from 66.2 ± 5.3% to 72.7 ± 7.5%).

Correlated fluorescence microscopy and multi-ion beam secondary ion mass spectrometry imaging reveals phosphatidylethanolamine increases in the membrane of cancer cells over-expressing the molecular chaperone subunit CCTδ.

Changes in the membrane composition of sub-populations of cells can influence different properties with importance to tumour growth, metastasis and treatment efficacy. In this study, we use correlated fluorescence microscopy and ToF-SIMS with C60+ and (CO2)6k+ ion beams to identify and characterise sub-populations of cells based on successful transfection leading to over-expression of CCTδ, a component of the multi-subunit molecular chaperone named chaperonin-containing tailless complex polypeptide 1 (CCT). CCT has been linked to increased cell growth and proliferation and is known to affect cell morphology but corresponding changes in lipid composition of the membrane have not been measured until now. Multivariate analysis of the surface mass spectra from single cells, focused on the intact lipid ions, indicates an enrichment of phosphatidylethanolamine species in the transfected cells. While the lipid changes in this case are driven by the structural changes in the protein cytoskeleton, the consequence of phosphatidylethanolamine enrichment may have additional implications in cancer such as increased membrane fluidity, increased motility and an ability to adapt to a depletion of unsaturated lipids during cancer cell proliferation. This study demonstrates a successful fluorescence microscopy-guided cell by cell membrane lipid analysis with broad application to biological investigation.Graphical abstract.

Injectable conductive hydrogel restores conduction through ablated myocardium.

INTRODUCTION: Therapies for substrate-related arrhythmias include ablation or drugs targeted at altering conductive properties or disruption of slow zones in heterogeneous myocardium. Conductive compounds such as carbon nanotubes may provide a novel personalizable therapy for arrhythmia treatment by allowing tissue homogenization. METHODS: A nanocellulose carbon nanotube-conductive hydrogel was developed to have conduction properties similar to normal myocardium. Ex vivo perfused canine hearts were studied. Electroanatomic activation mapping of the epicardial surface was performed at baseline, after radiofrequency ablation, and after uniform needle injections of the conductive hydrogel through the injured tissue. Gross histology was used to assess distribution of conductive hydrogel in the tissue. RESULTS: The conductive hydrogel viscosity was optimized to decrease with increasing shear rate to allow expression through a syringe. The direct current conductivity under aqueous conduction was 4.3 × 10-1 S/cm. In four canine hearts, when compared with the homogeneous baseline conduction, isochronal maps demonstrated sequential myocardial activation with a shift in direction of activation to surround the edges of the ablated region. After injection of the conductive hydrogel, isochrones demonstrated conduction through the ablated tissue with activation restored through the ablated tissue. Gross specimen examination demonstrated retention of the hydrogel within the tissue. CONCLUSIONS: This proof-of-concept study demonstrates that conductive hydrogel can be injected into acutely disrupted myocardium to restore conduction. Future experiments should focus on evaluating long-term retention and biocompatibility of the hydrogel through in vivo experimentation.

Multimodal MALDI Imaging Mass Spectrometry Reveals Spatially Correlated Lipid and Protein Changes in Mouse Heart with Acute Myocardial Infarction.

Acute myocardial infarction (MI) is a cardiovascular disease that remains a major cause of morbidity and mortality worldwide despite advances in its prevention and treatment. During acute myocardial ischemia, the lack of oxygen switches the cell metabolism to anaerobic respiration, with lactate accumulation, ATP depletion, Na+ and Ca2+ overload, and inhibition of myocardial contractile function, which drastically modifies the lipid, protein, and small metabolite profile in the myocardium. Imaging mass spectrometry (IMS) is a powerful technique to comprehensively elucidate the spatial distribution patterns of lipids, peptides, and proteins in biological tissue sections. In this work, we demonstrate an application of multimodal chemical imaging using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), which provided comprehensive molecular information in situ within the same mouse heart tissue sections with myocardial infarction. MALDI-IMS (at 30 µm per pixel) revealed infarct-associated spatial alterations of several lipid species of sphingolipids, glycerophospholipids, lysophospholipids, and cardiolipins along with the acyl carnitines. Further, we performed multimodal MALDI-IMS (IMS3) where dual polarity lipid imaging was combined with subsequent protein MALDI-IMS analysis (at 30 µm per pixel) within the same tissue sections, which revealed accumulations of core histone proteins H4, H2A, and H2B along with post-translational modification products, acetylated H4 and H2A, on the borders of the infarcted region. This methodology allowed us to interpret the lipid and protein molecular pathology of the very same infarcted region in a mouse model of myocardial infarction. Therefore, the presented data highlight the potential of multimodal MALDI imaging mass spectrometry of the same tissue sections as a powerful approach for simultaneous investigation of spatial infarct-associated lipid and protein changes of myocardial infarction.

Lipid Diversity in Cells and Tissue Using Imaging SIMS.

Lipids are an important class of biomolecules with many roles within cells and tissue. As targets for study, they present several challenges. They are difficult to label, as many labels lack the specificity to the many different lipid species or the labels maybe larger than the lipids themselves, thus severely perturbing the natural chemical environment. Mass spectrometry provides exceptional specificity and is often used to examine lipid extracts from different samples. However, spatial information is lost during extraction. Of the different imaging mass spectrometry methods available, secondary ion mass spectrometry (SIMS) is unique in its ability to analyze very small features, with probe sizes <50 nm available. It also offers high surface sensitivity and 3D imaging capability on a subcellular scale. This article reviews the current capabilities and some remaining challenges associated with imaging the diverse lipids present in cell and tissue samples. We show how the technique has moved beyond show-and-tell, proof-of-principle analysis and is now being used to address real biological challenges. These include imaging the microenvironment of cancer tumors, probing the pathophysiology of traumatic brain injury, or tracking the lipid composition through bacterial membranes.

Three-Dimensional Bioprinting Using a Coaxial Needle with Viscous Inks in Bone Tissue Engineering - An In vitro Study.

INTRODUCTION: Vascularized autologous tissue grafts are considered "gold standard" for the management of larger bony defects in the craniomaxillofacial area. This modality does however carry limitations, such as the absolute requirement for healthy donor tissues and recipient vessels. In addition, the significant morbidity of large bone graft is deterrent to fibula bone flap use. Therefore, less morbid strategies would be beneficial. The purpose of this study was to develop a printing method to manufacture scaffold structure with viable stem cells. MATERIALS AND METHODS: In total, three different combinations of ground beta tri-calcium phosphate and CELLINK (bioinks) were printed with a nozzle to identify a suitable bioink for three-dimensional printing. Subsequently, a coaxial needle, with three different nozzle gauge combinations, was evaluated for printing of the bioinks. Scaffold structures (grids) were then printed alone and with additional adipose stem cells before being transferred into an active medium and incubated overnight. Following incubation, grid stability was evaluated by assessing the degree of maintained grid outline, and cell viability was determined using the live/dead cell assay. RESULTS: Among the three evaluated combinations of bioinks, two resulted in good printability for bioprinting. Adequate printing was obtained with two out of the three nozzle gauge combinations tested. However, due to the smaller total opening, one combination revealed a better stability. Intact grids with maintained stability were obtained using Ink B23 and Ink B42, and approximately 80% of the printed stem cells were viable following 24 hours. DISCUSSION: Using a coaxial needle enables printing of a stable scaffold with viable stem cells. Furthermore, cell viability is maintained after the bioprinting process.

Biofabrication of bacterial nanocellulose scaffolds with complex vascular structure.

Bacterial nanocellulose (BNC) has proven to be an effective hydrogel-like material for different tissue engineering applications due to its biocompatibility and good mechanical properties. However, as for all biomaterials, in vitro biosynthesis of large tissue constructs remains challenging due to insufficient oxygen and nutrient transport in engineered scaffold-cell matrices. In this study we designed, biofabricated and evaluated bacterial nanocellulose scaffolds with a complex vascular mimetic lumen structure. As a first step a method for creating straight channeled structures within a bacterial nanocellulose scaffold was developed and evaluated by culturing of Human Umbilical Vein Endothelial Cells (HUVECs). In a second step, more complex structures within the scaffolds were produced utilizing a 3D printer. A print mimicking a vascular tree acted as a sacrificial template to produce a network within the nanoporous bacterial nanocellulose scaffolds that could be lined with endothelial cells. In a last step, a method to produce large constructs with interconnected macro porosity and vascular like lumen structure was developed. In this process patient data from x-ray computed tomography scans was used to create a mold for casting a full-sized kidney construct. By showing that the 3D printing technology can be combined with BNC biosynthesis we hope to widen the opportunities of 3D printing, while also enabling the production of BNC scaffolds constructs with tailored vascular architectures and properties.

Relative quantification of deuterated omega-3 and -6 fatty acids and their lipid turnover in PC12 cell membranes using TOF-SIMS.

Understanding FA metabolism and lipid synthesis requires a lot of information about which FAs and lipids are formed within the cells. We focused on the use of deuterated substrates of 100 µM α-linolenic acid and linoleic acid to determine the relative amounts of their converted PUFAs and specific phospholipids that are incorporated into cell plasma membranes. Time-of-flight secondary ion mass spectrometry (TOF-SIMS) was used to image and analyze lipids in model cell membranes with and without FA treatment. Because of its high spatial resolution, TOF-SIMS can be used to simultaneously provide both chemical information and distribution of various molecules in the sample surface down to the subcellular scale. Data obtained from this analysis of isotopes in the cell samples were used to calculate the relative amounts of long-chain PUFAs and phospholipids from their precursors, α-linolenic acid and linoleic acid. Our results show that the FA treatments induced an increase in the amounts of α-linolenic acid and linoleic acid and their long-chain conversion products. Moreover, an enhanced level of phospholipid turnover of these FAs in lipids such as phosphatidylcholines, phosphatidylethanolamines, and phosphatidylinositols was also observed in the cell plasma membrane.

Benefits of NaCl addition for time-of-flight secondary ion mass spectrometry analysis including the discrimination of diacylglyceride and triacylglyceride ions.

RATIONALE: Diacylglycerides (DAGs) and triacylglycerides (TAGs) are two important lipid classes present in all mammalian cells that share similar chemical structures but differ in biological function in cells and tissues. Differentiation of these two species during time-of-flight secondary ion mass spectrometry (ToF-SIMS) analysis is therefore important, but has been difficult due to the formation of DAG-like ions during the ionization process of TAGs. METHODS: We investigated the use of salt adduct formation as a quick and simple method to determine the origin of the DAG-like ions in ToF-SIMS spectra. NaCl was added to lipid standards of a DAG and a TAG and differences in fragmentation patterns were identified. The salt was then applied to prepared tissue samples by spraying with a saturated solution of NaCl in methanol and samples were analysed with ToF-SIMS using a 40 keV (CO2 )6k + primary ion beam. RESULTS: A 40 Da peak shift was observed in the DAG spectrum that was not observed in the TAG spectrum ([M + H - H2 O]+ to [M + Na]+ ) while the isobaric [M - RCOO]+ peak did not shift allowing differentiation between the two species. Spraying NaCl on to tissue sections indicated that the DAG-like ions originated from TAGs. CONCLUSIONS: With the method described in this paper, simple addition of salt by spraying on the sample leads to better interpretation of complex mass spectra from biological tissue samples, discriminating DAG and TAG fragment peaks.

Significant enhancement of negative secondary ion yields by cluster ion bombardment combined with cesium flooding.

In secondary ion mass spectrometry (SIMS), the beneficial effect of cesium implantation or flooding on the enhancement of negative secondary ion yields has been investigated in detail for various semiconductor and metal samples. All results have been obtained for monatomic ion bombardment. Recent progress in SIMS is based to a large extent on the development and use of cluster primary ions. In this work we show that the enhancement of negative secondary ions induced by the combination of ion bombardment with simultaneous cesium flooding is valid not only for monatomic ion bombardment but also for cluster primary ions. Experiments carried out using C60+ and Ar4000+ bombardment on silicon show that yields of negative secondary silicon ions can be optimized in the same way as by Ga+ and Cs+ bombardment. Both for monatomic and cluster ion bombardment, the optimization does not depend on the primary ion species. Hence, it can be assumed that the silicon results are also valid for other cluster primary ions and that results obtained for monatomic ion bombardment on other semiconductor and metal samples are also valid for cluster ion bombardment. In SIMS, cluster primary ions are also largely used for the analysis of organic matter. For polycarbonate, our results show that Ar4000+ bombardment combined with cesium flooding enhances secondary ion signals by a factor of 6. This can be attributed to the removal of charging effects and/or reduced fragmentation, but no major influence on ionization processes can be observed. The use of cesium flooding for the imaging of cells was also investigated and a significant enhancement of secondary ion yields was observed. Hence, cesium flooding has also a vast potential for SIMS analyses with cluster ion bombardment.

Universal method for protein bioconjugation with nanocellulose scaffolds for increased cell adhesion.

Bacterial nanocellulose (BNC) is an emerging biomaterial since it is biocompatible, integrates well with host tissue and can be biosynthesized in desired architecture. However, being a hydrogel, it exhibits low affinity for cell attachment, which is crucial for the cellular fate process. To increase cell attachment, the surface of BNC scaffolds was modified with two proteins, fibronectin and collagen type I, using an effective bioconjugation method applying 1-cyano-4-dimethylaminopyridinium (CDAP) tetrafluoroborate as the intermediate catalytic agent. The effect of CDAP treatment on cell adhesion to the BNC surface is shown for human umbilical vein endothelial cells and the mouse mesenchymal stem cell line C3H10T1/2. In both cases, the surface modification increased the number of cells attached to the surfaces. In addition, the morphology of the cells indicated more healthy and viable cells. CDAP activation of bacterial nanocellulose is shown to be a convenient method to conjugate extracellular proteins to the scaffold surfaces. CDAP treatment can be performed in a short period of time in an aqueous environment under heterogeneous and mild conditions preserving the nanofibrillar network of cellulose.