Biology subjects

Moroni, L.

Publications and source records attributed to Moroni, L..

32 records · Page 2Linked to original sources

bioRxiv2021

Effect of reduced graphene oxide (rGO) compaction degree and concentration on rGO-polymer composites printability and cell interactions

Graphene derivatives combined with polymers have attracted enormous attention for bone tissue engineering applications. Among others, reduced graphene oxide (rGO) is one of the preferred graphene-based fillers for the preparation of composites via melt compounding, and their further processing into 3D scaffolds, due to its established large-scale production method, thermal stability, and electrical conductivity. In this study, rGO (low bulk density 10g/L) was compacted by densification using a solvent (either acetone or water) prior to melt compounding, to simplify its handling and dosing into a twin-screw extrusion system. The effects of rGO bulk density (medium and high), densification solvent, and rGO concentration (3, 10 and 15% in weight) on rGO dispersion within the composite, electrical conductivity, printability and cell-material interactions were studied. High bulk density rGO (90 g/L) occupied a low volume fraction within polymer composites, offering poor electrical properties but a reproducible printability up to 15 wt% rGO. On the other hand, the volume fraction within the composites of medium bulk density rGO (50 g/L) was higher for a given concentration, enhancing rGO particle interactions and leading to enhanced electrical conductivity, but compromising the printability window. For a given bulk density (50 g/L), rGO densified in water was more compacted and offered poorer dispersability within the polymer than rGO densified in acetone, and resulted in scaffolds with poor layer bonding or even lack of printability at high rGO percentages. A balance in printability and electrical properties was obtained for composites with medium bulk density rGO densified in acetone. Here, increasing rGO concentration led to more hydrophilic composites with a noticeable increase in protein adsorption. Moreover, scaffolds prepared with such composites presented antimicrobial properties even at low rGO contents (3 wt%). In addition, the viability and proliferation of human mesenchymal stromal cells (hMSCs) was maintained on scaffolds with up to 15% rGO and with enhanced osteogenic differentiation on 3% rGO scaffolds.

bioengineering↗

bioRxiv2021

Additive manufactured scaffolds for bone tissue engineering: physical characterization of thermoplastic composites with functional fillers

Thermoplastic polymer - filler composites are excellent materials for bone tissue engineering (TE) scaffolds, combining the functionality of fillers with suitable load bearing ability, biodegradability, and additive manufacturing (AM) compatibility of the polymer. Two key determinants of their utility are their rheological behavior in the molten state, determining AM processability, and their mechanical load-bearing properties. We report here the characterization of both these physical properties for four bone TE relevant composite formulations with poly(ethylene oxide terephthalate) / poly(butylene terephthalate (PEOT/PBT) as a base polymer, which is often used to fabricate TE scaffolds. The fillers used were reduced graphene oxide (rGO), hydroxyapatite (HA), gentamycin intercalated in zirconium phosphate (ZrP-GTM) and ciprofloxacin intercalated in MgAl layered double hydroxide (MgAl-CFX). The rheological assessment showed that generally the viscous behavior dominated the elastic behavior (G > G) for the studied composites, at empirically determined extrusion temperatures. Coupled rheological-thermal characterization of ZrP-GTM and HA composites showed that the fillers increased the solidification temperatures of the polymer melts during cooling. Both these findings have implications for the required extrusion temperatures and bonding between layers. Mechanical tests showed that the fillers generally made the polymer stiffer but more brittle in proportion to the filler fractions. Furthermore, the elastic moduli of scaffolds did not directly correlate with the corresponding bulk material properties, implying composite-specific AM processing effects on the mechanical properties. Lastly, we show computational models to predict multi-material scaffold elastic moduli using measured single material scaffold and bulk moduli. The reported characterizations are essential for assessing the AM processability and ultimately the suitability of the manufactured scaffolds for the envisioned bone regeneration application.

bioengineering↗

bioRxiv2021

3D soft hydrogels induce human mesenchymal stem cells "deep" quiescence

Human mesenchymal stem/stromal cells (hMSCs) present a great opportunity for tissue regeneration due to their multipotent capacity. However, when cultured on 2D tissue culture polystyrene (TCPS) plates, hMSCs lose their differentiation capacity and clinical potential. It has been reported that cells need a more physiologically relevant micro-environment that allows them to maintain their phenotype. Here, we have developed a 3D alginate hydrogel functionalized with the Arg-Gly-Asp (RGD) sequence and having low mechanical stiffness that mimics the mechanical properties (>5 KPa) of bone marrow. hMSCs cultured in these hydrogels appeared to be halted in G1 phase of the cell cycle and to be non-proliferative, as shown by flow cytometry and 5-Ethynyl-2-deoxyuridine (EdU) staining, respectively. Their quiescent state was characterized by an upregulation of enhancer of zeste homolog 1 (EZH1) at the gene level, forkhead box O3 (FoxO3) and cyclin-dependent kinase inhibitor 1B (p27) at the gene and protein levels compared to hMSCs grown in 2D TCPS. Comparative studies in 3D hydrogels of alginate-RGD presenting higher concentration of the peptide or in collagen hydrogels revealed that independently of the concentration of RGD or the chemistry of the adhesion motives, hMSCs cultured in 3D presented a similar phenotype. This quiescent phenotype was exclusive of 3D cultures. In 2D, even when cells were starved of fetal bovine serum (FBS) and became also non-proliferative, the expression of these markers was not observed. We propose that this difference may be the result of mammalian target of rapamycin complex 1 (mTORC1) being downregulated in hMSCs cultured in 3D hydrogels, which induces cells to be in "deep" quiescence and be kept alive ex vivo for a long period of time. Our results represent a step forward towards understanding hMSCs quiescence and its molecular pathways, providing more insight for hMSCs cell therapies.

bioengineering↗

bioRxiv2021

Effect of Highly Loaded Nanohydroxyapatite Composite Scaffolds Prepared via Melt Extrusion Additive Manufacturing on the Osteogenic Differentiation of Human Mesenchymal Stromal Cells

The field of bone tissue engineering seeks to mimic the bone extracellular matrix composition, balancing the organic and inorganic components. In this regard, additive manufacturing (AM) of highly loaded polymer-calcium phosphate (CaP) composites holds great promise towards the design of bioactive scaffolds. Yet, the biological performance of such scaffolds is still poorly characterized. In this study, melt extrusion AM (ME-AM) was used to fabricate poly(ethylene oxide terephthalate)/poly(butylene terephthalate) (PEOT/PBT)-nanohydroxyapatite (nHA) scaffolds with up to 45 wt% nHA, which presented significantly enhanced compressive mechanical properties, to evaluate their in vitro osteogenic potential as a function of nHA content. While osteogenic gene upregulation and matrix mineralization were observed on all scaffold types when cultured in osteogenic media, human mesenchymal stromal cells did not present an explicitly clear osteogenic phenotype, within the evaluated timeframe, in basic media cultures (i.e. without osteogenic factors). Yet, due to the adsorption of calcium and inorganic phosphate ions from cell culture media and simulated body fluid, the formation of a CaP layer was observed on PEOT/PBT-nHA 45 wt% scaffolds, which is hypothesized to account for their osteoinductivity in the long term in vitro, and osteoconductivity in vivo.

bioengineering↗

bioRxiv2020

Mimicking the Human Tympanic Membrane: the Significance of Geometry

The human tympanic membrane (TM) captures sound waves reaching the outer ear from the environment and transforms them into mechanical motion. The successful transmission of these acoustic vibrations in varying frequency ranges is attributed to the structural architecture of the TM. However, a limited knowledge is available on the contribution of its discrete anatomical features, which is important to fabricate functional biomimetic TM replacements. This work synergizes theoretical and experimental approaches toward understanding the significance of geometry in tissue engineered TM scaffolds. Three test designs along with a plain control were chosen to decouple some of the dominant structural attributes, such as, the radial and circumferential alignment of the collagen fibrils. In silico models suggested a geometrical dependency of their mechanical and acoustical responses, where the presence of radially aligned fibers was observed to have a more prominent effect compared to their circumferential counterparts. Following which, a hybrid fabrication strategy combining electrospinning and additive manufacturing was optimized to manufacture hierarchical scaffolds within the dimensions of the native TM. The experimental characterizations conducted using macro-indentation and laser Doppler vibrometry were in line with the computational models. Finally, biological studies performed with human dermal fibroblasts and human mesenchymal stromal cells, revealed a favorable influence of scaffold hierarchy on cellular alignment and subsequent collagen deposition. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=119 SRC="FIGDIR/small/383299v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@16b3653org.highwire.dtl.DTLVardef@1bbc47forg.highwire.dtl.DTLVardef@619ad8org.highwire.dtl.DTLVardef@1fd1071_HPS_FORMAT_FIGEXP M_FIG Graphical abstract. Schematic diagram illustrating the overall flowchart of the work. 3D: three-dimensional; ES: electrospinning; FDM: fused deposition modeling; TM: tympanic membrane. C_FIG

bioengineering↗

bioRxiv2020

A hybrid additive manufacturing platform to create bulk and surface composition gradients on scaffolds for tissue regeneration

Scaffolds with gradients of physico-chemical properties and controlled 3D architectures are crucial for engineering complex tissues. These can be produced using multi-material additive manufacturing (AM) techniques. However, they typically only achieve discrete gradients using separate printheads to vary compositions. Achieving continuous composition gradients, to better mimic tissues, requires material dosing and mixing controls. No such AM solution exists for most biomaterials. Existing AM techniques also cannot selectively modify scaffold surfaces to locally stimulate cell adhesion. We report a hybrid AM solution to cover these needs. On one platform, we combine a novel dosing- and mixing-enabled, dual-material printhead with an atmospheric pressure plasma jet to selectively activate/coat scaffold filaments during manufacturing. We fabricated continuous composition gradients in both 2D hydrogels and 3D thermoplastic scaffolds. We demonstrated an improvement in mechanical properties of continuous gradients compared to discrete gradients in the 3D scaffolds, and the ability to selectively enhance cell adhesion.Competing Interest StatementP.S. and A.P. own part of the plasma jet manufacturer Nadir s.r.l.. E.V.F. is also affiliated with Nadir. R.S., L.M. and C.M. have applied for a patent (EP18200211.3, submitted: 12 Oct. 2018, PCT/EP2019/077519, submitted: 10 Oct. 2019) on the developed printhead and aim to license it to interested printer companies. M.C.-T. has no financial interests to declare.View Full Text

bioengineering↗

bioRxiv2020

3D additive manufactured composite scaffolds with antibiotic-loaded lamellar fillers for bone infection prevention and tissue regeneration

Bone infections following open bone fracture or implant surgery remain a challenge in the orthopedics field. In order to avoid high doses of systemic drug administration, optimized local antibiotic release from scaffolds is required. 3D additive manufactured (AM) scaffolds made with biodegradable polymers are ideal to support bone healing in non-union scenarios and can be given antimicrobial properties by the incorporation of antibiotics. In this study, ciprofloxacin and gentamicin intercalated in the interlamellar spaces of magnesium aluminum layered double hydroxides (MgAl) and -zirconium phosphates (ZrP), respectively, are dispersed within a thermoplastic polymer by melt compounding and subsequently processed via high temperature melt extrusion AM ([~]190 {degrees}C) into 3D scaffolds. The inorganic fillers enable a sustained antibiotics release through the polymer matrix, controlled by antibiotics counterions exchange or pH conditions. Importantly, both antibiotics retain their functionality after the manufacturing process at high temperatures, as verified by their activity against both Gram + and Gram - bacterial strains. Moreover, scaffolds loaded with filler-antibiotic do not impair human mesenchymal stromal cells osteogenic differentiation, allowing matrix mineralization and the expression of relevant osteogenic markers. Overall, these results suggest the possibility of fabricating dual functionality 3D scaffolds via high temperature melt extrusion for bone regeneration and infection prevention.

bioengineering↗

bioRxiv2020

Full cell infiltration and thick tissue formation in vivo in tailored electrospun scaffolds

Electrospun (ESP) scaffolds are a promising type of tissue engineering constructs for large defects with limited depth. To form new functional tissue, the scaffolds need to be infiltrated with cells, which will deposit extracellular matrix. However, due to dense fiber packing and small pores, cell and tissue infiltration of ESP scaffolds is limited. Here, we combine two established methods, increasing fiber diameter and co-spinning sacrificial fibers, to create a porous ESP scaffold that allows robust tissue infiltration. Full cell infiltration across 2 mm thick scaffolds is seen 3 weeks after subcutaneous implantation in rats. After 6 weeks, the ESP scaffolds are almost fully filled with de novo tissue. Cell infiltration and tissue formation in vivo in this thickness has not been previously achieved. In addition, we propose a novel method for in vitro cell seeding to improve cell infiltration and a model to study 3D migration through a fibrous mesh. This easy approach to facilitate cell infiltration further improves previous efforts and could greatly aid tissue engineering approaches utilizing ESP scaffolds. Statement of significanceElectrospinning creates highly porous scaffolds with nano- to micrometer sized fibers and are a promising candidate for a variety of tissue engineering applications. However, smaller fibers also create small pores which are difficult for cells to penetrate, restricting cells to the top layers of the scaffolds. Here, we have improved the cell infiltration by optimizing fiber diameter and by co-spinning a sacrificial polymer. We developed novel culture technique that can be used to improve cell seeding and to study cytokine driven 3D migration through fibrous meshes. After subcutaneous implantation, infiltration of tissue and cells was observed up to throughout up to 2 mm thick scaffolds. This depth of infiltration in vivo had not yet been reported for electrospun scaffolds. The scaffolds we present here can be used for in vitro studies of migration, and for tissue engineering in defects with a large surface area and limited depth.

bioengineering↗

bioRxiv2019

A one-step biofunctionalization strategy of electrospun scaffolds enables spatially selective presentation of biological cues

To recapitulate the heterogeneous complexity of tissues in our body with synthetic mimics of the extracellular matrix (ECM), it is important to develop methods that can easily allow the selective functionalization of defined spatial domains. Here, we introduce a facile method to functionalize microfibrillar meshes with different reactive groups able to bind biological moieties in a one-step reaction. The resulting scaffolds proved to selectively support a differential neurite growth after being seeded with dorsal root ganglia. Considering the general principles behind the method developed, this is a promising strategy to realize enhanced biomimicry of native ECM for different regenerative medicine applications.

bioengineering↗

bioRxiv2019

Meso-scale multi-material fabrication of a Synthetic ECM Mimic for In vivo-like Peripheral Nerve Regeneration

A growing focus and continuing challenge for biological sciences is creating representative in vitro environments to study and influence cell behavior. Here, we describe the synthetic recreation of the highly ordered extracellular matrix (ECM) of the peripheral nervous system (PNS) in terms of structure and scale, providing a versatile 3D culturing platform that achieves some of the highest in vitro neurite growth rates so far reported. By combining electrospinning technology with a unique multi-material processing sequence that harnesses intrinsic material properties, a hydrogel construct is realized that incorporates oriented 6 m-diameter microchannels decorated with topographical nanofibers. We show that this mimics the native PNS ECM architecture and promotes extensive growth from primary neurons; through controlled variation in design, we show that the open lumens of the microchannels directing rapid axon invasion of the hydrogel while the nanofibers provide essential cues for cell adhesion and topographical guidance. Furthermore, these microstructural and nanofibrillar elements enabled a typically bioinert hydrogel (PEGDA) to achieve similar neurite extension when compared to a biocompatible collagen hydrogel, with PEGDA-based devices approaching neurite growth rates similar to what is observed in vivo. Through the accessible fabrication approach developed here, multi-material scaffolds were designed with cell-relevant architectures ranging from meso-to nanoscale and shown to support nerve growth to mimic PNS regeneration, with potential for regenerative medicine and neural engineering applications.

bioengineering↗

bioRxiv2019

Tandem electrospinning for heterogeneous nanofiber patterns

Smart nanofibrillar constructs can be a promising technological solution for many emerging and established fields, facilitating the potential impact of nano-scale strategies to address relevant technological challenges. As a fabrication technique, electrospinning (ESP) is relatively well-known, accessible, economic, and fast, though until now has shown limitation over control and design of the fibrillar constructs which can be produced. Here, we introduce "Tandem Electrospinning" (T-ESP), a novel technique able to create increasingly complex patterns of fibrous polymeric constructs on a micro and nano-scale. Modifying a standard ESP configuration results in a focusing electric field that is able to spatially define the deposition pattern of multiple polymer jets simultaneously. Additional spatially defined heterogeneity is achieved by tuning polymer solution properties to obtain a gradient of fiber alignment. Heterogeneous fibrous meshes are created with either random, aligned, or a divergent fiber patterns. This approach holds potential for many fields, with application examples shown for Tissue Engineering and Separation Technologies. The technique outlined here provides a rapid, versatile, and accessible method for polymeric nanofabrication of patterned heterogeneous fibrous constructs. Polymer properties are also shown to dictate fiber alignment, providing further insight into the mechanisms involved in the electrospinning fabrication process.

bioengineering↗

bioRxiv2019

Improving cell distribution on 3D additive manufactured scaffolds through engineered seeding media density and viscosity

In order to ensure the long-term in vitro and in vivo functionality of cell-seeded 3D scaffolds, an effective and reliable method to control cell seeding efficiency and distribution is crucial. Static seeding on 3D additive manufactured scaffolds made of synthetic polymers still remains challenging, as it often results in poor cell attachment, high cell sedimentation and non-uniform cell distribution, due to gravity and to the intrinsic macroporosity and surface chemical properties of the scaffolds. In this study, the bio-inert macromolecules dextran and Ficoll were used for the first time as temporary supplements to alter the viscosity and density of the seeding media, respectively, and improve the static seeding output. The addition of these macromolecules drastically reduced the cell sedimentation velocities, allowing for homogeneous cell attachment to the scaffold filaments. Both dextran- and Ficoll-based seeding methods supported human mesenchymal stromal cells viability and osteogenic differentiation post-seeding. Interestingly, the improved cell distribution led to increased matrix production and mineralization compared to scaffolds seeded by conventional static method. These results suggest a simple and universal method for an efficient seeding of 3D additive manufactured scaffolds, independent of their material and geometrical properties, and applicable for bone and various other tissue regeneration.

bioengineering↗

bioRxiv2019

Mechanosensitive regulation of FGFR1 through the MRTF-SRF pathway

Controlling basic fibroblast growth factor (bFGF) signaling is important for both tissue-engineering purposes, controlling proliferation and differentiation potential, and for cancer biology, influencing tumor progression and metastasis. Here, we observed that human mesenchymal stromal cells (hMSCs) no longer responded to soluble or covalently bound bFGF when cultured on microfibrillar substrates, while fibroblasts did. This correlated with a downregulation of FGF receptor 1 (FGFR1) expression of hMSCs on microfibrillar substrates, compared to hMSCs on conventional tissue culture plastic (TCP). hMSCs also expressed less SRF on ESP scaffolds, compared to TCP, while fibroblasts maintained high FGFR1 and SRF expression. Inhibition of actin-myosin tension or the MRTF/SRF pathway decreased FGFR1 expression in hMSCs, fibroblasts and MG63 osteosarcoma cells. This downregulation was functional, as hMSCs became irresponsive to bFGF in the presence of MRTF/SRF inhibitor. Together, our data show that hMSCs, but not fibroblasts, are irresponsive to bFGF when cultured on microfibrillar susbtrates by downregulation of FGFR1 through the MRTF/SRF pathway. This is the first time FGFR1 expression has been shown to be mechanosensitive and adds to the sparse literature on FGFR1 regulation. These results could open up new targets for cancer treatments and could aid designing tissue engineering constructs that better control cell proliferation.

bioengineering↗

bioRxiv2019

Dimensionality changes actin network through lamin A and C and zyxin

The actin cytoskeleton plays a key role in differentiation of human mesenchymal stromal cells (hMSCs), but its regulation in 3D tissue engineered scaffolds remains poorly studied. hMSCs cultured on 3D electrospun scaffolds made of a stiff material do not form actin stress fibers, contrary to hMSCs on 2D films of the same material. On 3D electrospun- and 3D additive manufactured scaffolds, hMSCs also displayed fewer focal adhesions, lower lamin A and C expression and less YAP1 nuclear localization. Together, this shows that dimensionality prevents the build-up of cellular tension, even on stiff materials. Knock down of either lamin A and C or zyxin resulted in fewer stress fibers in the cell center. Zyxin knock down reduced lamin A and C expression, but not vice versa, showing that this signal chain starts from the outside of the cell. Our study demonstrates that dimensionality changes the actin cytoskeleton through lamin A and C and zyxin, an important insight for future scaffold design, as the actin network, focal adhesions and nuclear stiffness are all critical for hMSC differentiation.

bioengineering↗