Vascularization is an important strategy to overcome diffusion limits and enable the formation of... more Vascularization is an important strategy to overcome diffusion limits and enable the formation of complex, physiologically relevant engineered tissues and organoids. Self‐assembly is a technique to generate in vitro vascular networks, but engineering the necessary network morphology and function remains challenging. Here, autofluorescence multiphoton microscopy (aMPM), a label‐free imaging technique, is used to quantitatively evaluate in vitro vascular network morphology. Vascular networks are generated using human embryonic stem cell–derived endothelial cells and primary human pericytes encapsulated in synthetic poly(ethylene glycol)‐based hydrogels. Two custom‐built bioreactors are used to generate distinct fluid flow patterns during vascular network formation: recirculating flow or continuous flow. aMPM is used to image these 3D vascular networks without the need for fixation, labels, or dyes. Image processing and analysis algorithms are developed to extract quantitative morpholo...
The commercial success of tissue engineering products requires efficacy, cost effectiveness, and ... more The commercial success of tissue engineering products requires efficacy, cost effectiveness, and the possibility of scaleup. Advances in tissue engineering require increased sophistication in the design of biomaterials, often challenging the current manufacturing techniques. Interestingly, several of the properties that are desirable for biomaterial design are embodied in the structure and function of plants. This study demonstrates that decellularized plant tissues can be used as adaptable scaffolds for culture of human cells. With simple biofunctionalization technique, it is possible to enable adhesion of human cells on a diverse set of plant tissues. The elevated hydrophilicity and excellent water transport abilities of plant tissues allow cell expansion over prolonged periods of culture. Moreover, cells are able to conform to the microstructure of the plant frameworks, resulting in cell alignment and pattern registration. In conclusion, the current study shows that it is feasibl...
Despite significant advances in the fabrication of bioengineered scaffolds for tissue engineering... more Despite significant advances in the fabrication of bioengineered scaffolds for tissue engineering, delivery of nutrients in complex engineered human tissues remains a challenge. By taking advantage of the similarities in the vascular structure of plant and animal tissues, we developed decellularized plant tissue as a prevascularized scaffold for tissue engineering applications. Perfusion-based decellularization was modified for different plant species, providing different geometries of scaffolding. After decellularization, plant scaffolds remained patent and able to transport microparticles. Plant scaffolds were recellularized with human endothelial cells that colonized the inner surfaces of plant vasculature. Human mesenchymal stem cells and human pluripotent stem cell derived cardiomyocytes adhered to the outer surfaces of plant scaffolds. Cardiomyocytes demonstrated contractile function and calcium handling capabilities over the course of 21 days. These data demonstrate the poten...
Extracellular Matrix for Tissue Engineering and Biomaterials, 2018
The extracellular matrix (ECM) has unique biochemical, mechanical and organisational properties t... more The extracellular matrix (ECM) has unique biochemical, mechanical and organisational properties through which it provides a physical scaffolding for cells; a barrier that protects tissues; several signals that affect cell behaviour; and a reservoir for biologically active molecules. Considering the importance of ECM in regulating many fundamental cell processes, a myriad of strategies and materials has been developed to reproduce its properties. The first part of the chapter covers various approaches aiming to generate scaffolds whose fibre size, orientation and stiffness could mimic the ECM nanofibrous structure. In particular, the use of natural fibrous proteins, the application of electrospinning and freeze-drying and examples of tissue engineering applications are presented. The second part discusses strategies aiming to address the ECM ligand-binding function and to reproduce the dynamic, reciprocal, dialogue between cells and their microenvironment; examples of 3D scaffolds for controlled release of growth factors, drugs and genetic material are reported. Researchers have also used native ECM components to recapitulate the biochemical and biophysical properties of ECM. In the third part of the chapter, the use of fibrinogen and fibrin is presented as an example of natural scaffolds recapitulating ECM functions. Fibrinogen and fibrin can be used as provisional matrix in regenerating tissues; moreover, by varying the fabrication method and by blending them with other materials, it is possible to produce biodegradable scaffolds with reasonable control of degradation rate and drug release.
Therapeutic factors secreted by mesenchymal stem cells (MSCs) promote angiogenesis in vivo. Howev... more Therapeutic factors secreted by mesenchymal stem cells (MSCs) promote angiogenesis in vivo. However, delivery of MSCs in the absence of a cytoprotective environment offers limited efficacy due to low cell retention, poor graft survival and the non-maintenance of a physiologically relevant dose of growth factors at the injury site. The delivery of stem cells on an extracellular matrix (ECM)-based platform alters cell behaviour including migration, proliferation and paracrine activity, which are essential for angiogenesis. We demonstrate the biophysical and biochemical effects of pre-conditioning human MSCs for 96 hours on a three-dimensional ECM-based microgel platform. By altering the macromolecular concentration surrounding cells in the microgels, the pro-angiogenic phenotype of hMSCs can be tuned in a controlled manner through cell-driven changes in extracellular stiffness and ‘outside-in’ integrin signaling. The microgels tested at a low-cell dose (5×104 cells) in a pre-clinical ...
The ability to locally deliver bioactive molecules to distinct regions of the skeleton may provid... more The ability to locally deliver bioactive molecules to distinct regions of the skeleton may provide a novel means by which to treat osteomyelitis,improve bone healing, or accelerate growth. In this manuscript we demonstrate that biologically active molecules can be released from bioabsorbable membranes and have a local effect on the skeleton. Herein, we introduce a mineral-coated poly-ε-caprolactone film capable of binding and releasing HGH and TGF-β1both <i>in vitro</i> and <i>in vivo</i>. These mineral coated membranes loaded with either HGH or TGF-β1 were surgically placed underneath the periosteum, and multiple effects on neighboring osseous tissue, periosteal tissue, and local bone marrow cellularity were observed. Importantly, the observed effects in the presence of HGH orTGF-β1 were distinct from sham surgeries and implanted mineral-coated poly-ε-caprolactone films without these growth factors. Such results suggest that HGH and TGF-β1 released from the mineral-coated films are indeed bioactive <i>in vivo</i> and are exerting localized effects on the skeleton. We posit that this work represents the first step towards the development of technologies to locally affect the skeleton, and that further studies to develop therapeutic applications and optimize these systems can now be pursued.
Journal of visualized experiments : JoVE, May 31, 2018
The autologous, synthetic, and animal-derived grafts currently used as scaffolds for tissue repla... more The autologous, synthetic, and animal-derived grafts currently used as scaffolds for tissue replacement have limitations due to low availability, poor biocompatibility, and cost. Plant tissues have favorable characteristics that make them uniquely suited for use as scaffolds, such as high surface area, excellent water transport and retention, interconnected porosity, preexisting vascular networks, and a wide range of mechanical properties. Two successful methods of plant decellularization for tissue engineering applications are described here. The first method is based on detergent baths to remove cellular matter, which is similar to previously established methods used to clear mammalian tissues. The second is a detergent-free method adapted from a protocol that isolates leaf vasculature and involves the use of a heated bleach and salt bath to clear the leaves and stems. Both methods yield scaffolds with comparable mechanical properties and low cellular metabolic impact, thus allowi...
Gene delivery to primary human cells is a technology of critical interest to both life science re... more Gene delivery to primary human cells is a technology of critical interest to both life science research and therapeutic applications. However, poor efficiencies in gene transfer and undesirable safety profiles remain key limitations in advancing this technology. Here, we describe a materials-based approach whereby application of a bioresorbable mineral coating improves microparticle-based transfection of plasmid DNA lipoplexes in several primary human cell types. In the presence of these mineral-coated microparticles (MCMs), we observed up to 4-fold increases in transfection efficiency with simultaneous reductions in cytotoxicity. We identified mechanisms by which MCMs improve transfection, as well as coating compositions that improve transfection in three-dimensional cell constructs. The approach afforded efficient transfection in primary human fibroblasts as well as mesenchymal and embryonic stem cells for both two- and three-dimensional transfection strategies. This MCM-based tra...
Cell therapy of the degenerated intervertebral disc is limited by the lack of appropriate cell so... more Cell therapy of the degenerated intervertebral disc is limited by the lack of appropriate cell sources, thus new strategies for the differentiation of stem cells towards a nucleus pulposus (NP)-like phenotype need investigation. In the current study, it is hypothesized that spherical niche-like structures composed of type II collagen and hyaluronan (HA) mimic the NP microenvironment and promote the differentiation of adipose-derived stem cells (ADSCs) towards an NP-like phenotype. ADSCs are embedded in microgels of different concentrations of collagen II/HA. Cells' response to the different environments is studied by characterizing differences in cells' viability, morphology, and gene expression. After 21 days of culture, ADSCs maintain ± 80% viability in all the conditions tested. Moreover, microgels with higher concentration of collagen are stable and maintain cells in a rounder shape. In presence of differentiation media, cells are able to differentiate in all the conditi...
Vascularization is an important strategy to overcome diffusion limits and enable the formation of... more Vascularization is an important strategy to overcome diffusion limits and enable the formation of complex, physiologically relevant engineered tissues and organoids. Self‐assembly is a technique to generate in vitro vascular networks, but engineering the necessary network morphology and function remains challenging. Here, autofluorescence multiphoton microscopy (aMPM), a label‐free imaging technique, is used to quantitatively evaluate in vitro vascular network morphology. Vascular networks are generated using human embryonic stem cell–derived endothelial cells and primary human pericytes encapsulated in synthetic poly(ethylene glycol)‐based hydrogels. Two custom‐built bioreactors are used to generate distinct fluid flow patterns during vascular network formation: recirculating flow or continuous flow. aMPM is used to image these 3D vascular networks without the need for fixation, labels, or dyes. Image processing and analysis algorithms are developed to extract quantitative morpholo...
The commercial success of tissue engineering products requires efficacy, cost effectiveness, and ... more The commercial success of tissue engineering products requires efficacy, cost effectiveness, and the possibility of scaleup. Advances in tissue engineering require increased sophistication in the design of biomaterials, often challenging the current manufacturing techniques. Interestingly, several of the properties that are desirable for biomaterial design are embodied in the structure and function of plants. This study demonstrates that decellularized plant tissues can be used as adaptable scaffolds for culture of human cells. With simple biofunctionalization technique, it is possible to enable adhesion of human cells on a diverse set of plant tissues. The elevated hydrophilicity and excellent water transport abilities of plant tissues allow cell expansion over prolonged periods of culture. Moreover, cells are able to conform to the microstructure of the plant frameworks, resulting in cell alignment and pattern registration. In conclusion, the current study shows that it is feasibl...
Despite significant advances in the fabrication of bioengineered scaffolds for tissue engineering... more Despite significant advances in the fabrication of bioengineered scaffolds for tissue engineering, delivery of nutrients in complex engineered human tissues remains a challenge. By taking advantage of the similarities in the vascular structure of plant and animal tissues, we developed decellularized plant tissue as a prevascularized scaffold for tissue engineering applications. Perfusion-based decellularization was modified for different plant species, providing different geometries of scaffolding. After decellularization, plant scaffolds remained patent and able to transport microparticles. Plant scaffolds were recellularized with human endothelial cells that colonized the inner surfaces of plant vasculature. Human mesenchymal stem cells and human pluripotent stem cell derived cardiomyocytes adhered to the outer surfaces of plant scaffolds. Cardiomyocytes demonstrated contractile function and calcium handling capabilities over the course of 21 days. These data demonstrate the poten...
Extracellular Matrix for Tissue Engineering and Biomaterials, 2018
The extracellular matrix (ECM) has unique biochemical, mechanical and organisational properties t... more The extracellular matrix (ECM) has unique biochemical, mechanical and organisational properties through which it provides a physical scaffolding for cells; a barrier that protects tissues; several signals that affect cell behaviour; and a reservoir for biologically active molecules. Considering the importance of ECM in regulating many fundamental cell processes, a myriad of strategies and materials has been developed to reproduce its properties. The first part of the chapter covers various approaches aiming to generate scaffolds whose fibre size, orientation and stiffness could mimic the ECM nanofibrous structure. In particular, the use of natural fibrous proteins, the application of electrospinning and freeze-drying and examples of tissue engineering applications are presented. The second part discusses strategies aiming to address the ECM ligand-binding function and to reproduce the dynamic, reciprocal, dialogue between cells and their microenvironment; examples of 3D scaffolds for controlled release of growth factors, drugs and genetic material are reported. Researchers have also used native ECM components to recapitulate the biochemical and biophysical properties of ECM. In the third part of the chapter, the use of fibrinogen and fibrin is presented as an example of natural scaffolds recapitulating ECM functions. Fibrinogen and fibrin can be used as provisional matrix in regenerating tissues; moreover, by varying the fabrication method and by blending them with other materials, it is possible to produce biodegradable scaffolds with reasonable control of degradation rate and drug release.
Therapeutic factors secreted by mesenchymal stem cells (MSCs) promote angiogenesis in vivo. Howev... more Therapeutic factors secreted by mesenchymal stem cells (MSCs) promote angiogenesis in vivo. However, delivery of MSCs in the absence of a cytoprotective environment offers limited efficacy due to low cell retention, poor graft survival and the non-maintenance of a physiologically relevant dose of growth factors at the injury site. The delivery of stem cells on an extracellular matrix (ECM)-based platform alters cell behaviour including migration, proliferation and paracrine activity, which are essential for angiogenesis. We demonstrate the biophysical and biochemical effects of pre-conditioning human MSCs for 96 hours on a three-dimensional ECM-based microgel platform. By altering the macromolecular concentration surrounding cells in the microgels, the pro-angiogenic phenotype of hMSCs can be tuned in a controlled manner through cell-driven changes in extracellular stiffness and ‘outside-in’ integrin signaling. The microgels tested at a low-cell dose (5×104 cells) in a pre-clinical ...
The ability to locally deliver bioactive molecules to distinct regions of the skeleton may provid... more The ability to locally deliver bioactive molecules to distinct regions of the skeleton may provide a novel means by which to treat osteomyelitis,improve bone healing, or accelerate growth. In this manuscript we demonstrate that biologically active molecules can be released from bioabsorbable membranes and have a local effect on the skeleton. Herein, we introduce a mineral-coated poly-ε-caprolactone film capable of binding and releasing HGH and TGF-β1both <i>in vitro</i> and <i>in vivo</i>. These mineral coated membranes loaded with either HGH or TGF-β1 were surgically placed underneath the periosteum, and multiple effects on neighboring osseous tissue, periosteal tissue, and local bone marrow cellularity were observed. Importantly, the observed effects in the presence of HGH orTGF-β1 were distinct from sham surgeries and implanted mineral-coated poly-ε-caprolactone films without these growth factors. Such results suggest that HGH and TGF-β1 released from the mineral-coated films are indeed bioactive <i>in vivo</i> and are exerting localized effects on the skeleton. We posit that this work represents the first step towards the development of technologies to locally affect the skeleton, and that further studies to develop therapeutic applications and optimize these systems can now be pursued.
Journal of visualized experiments : JoVE, May 31, 2018
The autologous, synthetic, and animal-derived grafts currently used as scaffolds for tissue repla... more The autologous, synthetic, and animal-derived grafts currently used as scaffolds for tissue replacement have limitations due to low availability, poor biocompatibility, and cost. Plant tissues have favorable characteristics that make them uniquely suited for use as scaffolds, such as high surface area, excellent water transport and retention, interconnected porosity, preexisting vascular networks, and a wide range of mechanical properties. Two successful methods of plant decellularization for tissue engineering applications are described here. The first method is based on detergent baths to remove cellular matter, which is similar to previously established methods used to clear mammalian tissues. The second is a detergent-free method adapted from a protocol that isolates leaf vasculature and involves the use of a heated bleach and salt bath to clear the leaves and stems. Both methods yield scaffolds with comparable mechanical properties and low cellular metabolic impact, thus allowi...
Gene delivery to primary human cells is a technology of critical interest to both life science re... more Gene delivery to primary human cells is a technology of critical interest to both life science research and therapeutic applications. However, poor efficiencies in gene transfer and undesirable safety profiles remain key limitations in advancing this technology. Here, we describe a materials-based approach whereby application of a bioresorbable mineral coating improves microparticle-based transfection of plasmid DNA lipoplexes in several primary human cell types. In the presence of these mineral-coated microparticles (MCMs), we observed up to 4-fold increases in transfection efficiency with simultaneous reductions in cytotoxicity. We identified mechanisms by which MCMs improve transfection, as well as coating compositions that improve transfection in three-dimensional cell constructs. The approach afforded efficient transfection in primary human fibroblasts as well as mesenchymal and embryonic stem cells for both two- and three-dimensional transfection strategies. This MCM-based tra...
Cell therapy of the degenerated intervertebral disc is limited by the lack of appropriate cell so... more Cell therapy of the degenerated intervertebral disc is limited by the lack of appropriate cell sources, thus new strategies for the differentiation of stem cells towards a nucleus pulposus (NP)-like phenotype need investigation. In the current study, it is hypothesized that spherical niche-like structures composed of type II collagen and hyaluronan (HA) mimic the NP microenvironment and promote the differentiation of adipose-derived stem cells (ADSCs) towards an NP-like phenotype. ADSCs are embedded in microgels of different concentrations of collagen II/HA. Cells' response to the different environments is studied by characterizing differences in cells' viability, morphology, and gene expression. After 21 days of culture, ADSCs maintain ± 80% viability in all the conditions tested. Moreover, microgels with higher concentration of collagen are stable and maintain cells in a rounder shape. In presence of differentiation media, cells are able to differentiate in all the conditi...
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Papers by Gianluca Fontana