Producing 3D Biomimetic Nanomaterials for Musculoskeletal System Regeneration

The human musculoskeletal system is comprised mainly of connective tissues such as cartilage, tendon, ligaments, skeletal muscle, and skeletal bone. These tissues support the structure of the body, hold and protect the organs, and are responsible of movement. Since it is subjected to continuous strain, the musculoskeletal system is prone to injury by excessive loading forces or aging, whereas currently available treatments are usually invasive and not always effective. Most of the musculoskeletal injuries require surgical intervention facing a limited post-surgery tissue regeneration, especially for widespread lesions. Therefore, many tissue engineering approaches have been developed tackling musculoskeletal tissue regeneration. Materials are designed to meet the chemical and mechanical requirements of the native tissue three-dimensional (3D) environment, thus facilitating implant integration while providing a good reabsorption rate. With biological systems operating at the nanoscale, nanoengineered materials have been developed to support and promote regeneration at the interprotein communication level. Such materials call for a great precision and architectural control in the production process fostering the development of new fabrication techniques. In this mini review, we would like to summarize the most recent advances in 3D nanoengineered biomaterials for musculoskeletal tissue regeneration, with especial emphasis on the different techniques used to produce them

Producing 3D biomimetic nanomaterials for musculoskeletal system regeneration

The human musculoskeletal system is comprised mainly of connective tissues such as cartilage, tendon, ligaments, skeletal muscle, and skeletal bone. These tissues support the structure of the body, hold and protect the organs, and are responsible of movement. Since it is subjected to continuous strain, the musculoskeletal system is prone to injury by excessive loading forces or aging, whereas currently available treatments are usually invasive and not always effective. Most of the musculoskeletal injuries require surgical intervention facing a limited post-surgery tissue regeneration, especially for widespread lesions. Therefore, many tissue engineering approaches have been developed tackling musculoskeletal tissue regeneration. Materials are designed to meet the chemical and mechanical requirements of the native tissue three-dimensional (3D) environment, thus facilitating implant integration while providing a good reabsorption rate. With biological systems operating at the nanoscale, nanoengineered materials have been developed to support and promote regeneration at the interprotein communication level. Such materials call for a great precision and architectural control in the production process fostering the development of new fabrication techniques. In this mini review, we would like to summarize the most recent advances in 3D nanoengineered biomaterials for musculoskeletal tissue regeneration, with especial emphasis on the different techniques used to produce them

Composite biomaterials as long-lasting scaffolds for 3D bioprinting of highly aligned muscle tissue

New biocompatible materials have enabled the direct 3D printing of complex functional living tissues, such as skeletal and cardiac muscle. Gelatinmethacryloyl (GelMA) is a photopolymerizable hydrogel composed of natural gelatin functionalized with methacrylic anhydride. However, it is difficult to obtain a single hydrogel that meets all the desirable properties for tissue engineering. In particular, GelMA hydrogels lack versatility in their mechanical properties and lasting 3D structures. In this work, a library of composite biomaterials to obtain versatile, lasting, and mechanically tunable scaffolds are presented. Two polysaccharides, alginate and carboxymethyl cellulose chemically functionalized with methacrylic anhydride, and a synthetic material, such as poly(ethylene glycol) diacrylate are combined with GelMA to obtain photopolymerizable hydrogel blends. Physical properties of the obtained composite hydrogels are screened and optimized for the growth and development of skeletal muscle fibers from C2C12 murine cells, and compared with pristine GelMA. All these composites show high resistance to degradation maintaining the 3D structure with high fidelity over several weeks. Altogether, in this study a library of biocompatible novel and totally versatile composite biomaterials are developed and characterized, with tunable mechanical properties that give structure and support myotube formation and alignment

Preservation of critical quality attributes of mesenchymal stromal cells in 3D bioprinted structures by using natural hydrogel scaffolds

Three dimensional (3D) bioprinting is an emerging technology that enables complex spatial modeling of cell-based tissue engineering products, whose therapeutic potential in regenerative medicine is enormous. However, its success largely depends on the definition of a bioprintable zone, which is specific for each combination of cell-loaded hydrogels (or bioinks) and scaffolds, matching the mechanical and biological characteristics of the target tissue to be repaired. Therefore proper adjustment of the bioink formulation requires a compromise between: (i) the maintenance of cellular critical quality attributes (CQA) within a defined range of specifications to cell component, and (ii) the mechanical characteristics of the printed tissue to biofabricate. Herein, we investigated the advantages of using natural hydrogel-based bioinks to preserve the most relevant CQA in bone tissue regeneration applications, particularly focusing on cell viability and osteogenic potential of multipotent mesenchymal stromal cells (MSCs) displaying tripotency in vitro, and a phenotypic profile of 99.9% CD105(+)/CD45,(-) 10.3% HLA-DR,(+) 100.0% CD90,(+) and 99.2% CD73(+)/CD31(-) expression. Remarkably, hyaluronic acid, fibrin, and gelatin allowed for optimal recovery of viable cells, while preserving MSC's proliferation capacity and osteogenic potency in vitro. This was achieved by providing a 3D structure with a compression module below 8.8 +/- 0.5 kPa, given that higher values resulted in cell loss by mechanical stress. Beyond the biocompatibility of naturally occurring polymers, our results highlight the enhanced protection on CQA exerted by bioinks of natural origin (preferably HA, gelatin, and fibrin) on MSC, bone marrow during the 3D bioprinting process, reducing shear stress and offering structural support for proliferation and osteogenic differentiation

A three-dimensional bioprinted model to evaluate the effect of stiffness on neuroblastoma cell cluster dynamics and behavior

Three-dimensional (3D) bioprinted culture systems allow to accurately control microenvironment components and analyze their effects at cellular and tissue levels. The main objective of this study was to identify, quantify and localize the effects of physical-chemical communication signals between tumor cells and the surrounding biomaterial stiffness over time, defining how aggressiveness increases in SK-N-BE(2) neuroblastoma (NB) cell line. Biomimetic hydrogels with SK-N-BE(2) cells, methacrylated gelatin and increasing concentrations of methacrylated alginate (AlgMA 0%, 1% and 2%) were used. Young's modulus was used to define the stiffness of bioprinted hydrogels and NB tumors. Stained sections of paraffin-embedded hydrogels were digitally quantified. Human NB and 1% AlgMA hydrogels presented similar Young´s modulus mean, and orthotopic NB mice tumors were equally similar to 0% and 1% AlgMA hydrogels. Porosity increased over time; cell cluster density decreased over time and with stiffness, and cell cluster occupancy generally increased with time and decreased with stiffness. In addition, cell proliferation, mRNA metabolism and antiapoptotic activity advanced over time and with stiffness. Together, this rheological, optical and digital data show the potential of the 3D in vitro cell model described herein to infer how intercellular space stiffness patterns drive the clinical behavior associated with NB patients

3D bioprinted functional skeletal muscle models have potential applications for studies of muscle wasting in cancer cachexia

Acquired muscle diseases such as cancer cachexia are responsible for the poor prognosis of many patients suffering from cancer. In vitro models are needed to study the underlying mechanisms of those pathologies. Extrusion bioprinting is an emerging tool to emulate the aligned architecture of fibers while implementing ad- ditive manufacturing techniques in tissue engineering. However, designing bioinks that reconcile the rheological needs of bioprinting and the biological requirements of muscle tissue is a challenging matter. Here we formulate a biomaterial with dual crosslinking to modulate the physical properties of bioprinted models. We design 3D bioprinted muscle models that resemble the mechanical properties of native tissue and show improved prolif- eration and high maturation of differentiated myotubes suggesting that the GelMA-AlgMA-Fibrin biomaterial possesses myogenic properties. The electrical stimulation of the 3D model confirmed the contractile capability of the tissue and enhanced the formation of sarcomeres. Regarding the functionality of the models, they served as platforms to recapitulate skeletal muscle diseases such as muscle wasting produced by cancer cachexia. The genetic expression of 3D models demonstrated a better resemblance to the muscular biopsies of cachectic mouse models. Altogether, this biomaterial is aimed to fabricate manipulable skeletal muscle in vitro models in a non- costly, fast and feasible manne

Development of tunable bioinks to fabricate 3D-printed in vitro models: a special focus on skeletal muscle models with potential applications in metabolic alteration studies

[eng] In vitro engineered three-dimensional tissue models are attracting an increasing interest due to their potential applications in preclinical assays. On the one hand, they are an alternative to the high costs, ethical issues and time-consuming experiments associated with animal models. On the other hand, unlike traditional monolayer cultures, 3D models are fabricated with polymer matrices that can mimic the spatial organization and physiological environment of native tissue. The scalability of these models to the market is currently limited by the fabrication methods. Additive manufacturing techniques, as extrusion bioprinting, provide the automated and controlled deposition of biomaterials with encapsulated cells to fabricate 3D models with unlimited shapes. However, few biomaterials fulfill the rheological, mechanical and biological needs for tissue engineering approaches. Printable biomaterials are commonly highly concentrated viscous fluids that could resemble the mechanical properties of stiff tissues, as skeletal muscle. However, they result in restrictive matrices with closed pores that can hamper the migration and proliferation of cells. As a solution, biomaterials have been chemically modified to obtain photocrosslinkable hydrogels, which provide 3D cultures with flexible physical properties. Nevertheless, current bioprinted muscle tissue models show poorly differentiated fibers and lack of functionality. Based on these precedents, this thesis is focused on the development of photocrosslinkable bioinks with tunable physical properties to fabricate customized in vitro 3D models of skeletal muscle tissue and neuroblastoma. To that end, gelatin, alginate and cellulose natural polymers are chemically modified to obtain UV- crosslinkable hydrogels with disparate physical properties. It is found that composite biomaterials of gelatin methacryloyl and alginate methacrylate present the best mechanical properties for stiff tissues as skeletal muscle and tumors. On this basis, the physical properties and composition of GelMA-AlgMA are modified to obtain matrices that resemble the physiological conditions of each tissue. It is found that neuroblastoma models require dense polymer networks to mimic the restrictive matrices found in solid tumors of high-risk patients. Neuroblastoma cell clusters in bioprinted cultures with a high concentration of AlgMA display the characteristic phenotype of aggressive solid tumors. Therefore, this bioink is proposed for the fabrication of stiff neuroblastoma tumor models. In contrast, this formulation was found unsuitable for skeletal muscle models, which present low proliferation and differentiation. Instead, the physical properties of GelMA-AlgMA bioink are tuned by changing the fabrication parameters, and fibrin is added to the composition to obtain a highly porous bioprinted model that resembles the mechanical properties of muscle tissue. As a result, muscle precursor cells are spontaneously differentiated into highly aligned mature fibers that, in combination with an electric pulse stimulation system, develop into mature muscle fibers with contraction capability and pronounced sarcomere units. The functionality of the bioprinted tissue agrees with the metabolic activity analysis, which corresponds to the behavior of native tissue. Hence, this model could be used to monitor the effects of drugs in the metabolic respiration of muscle mitochondria, simplifying the traditional protocols based on the isolation of single fibers. As an approach to obtain faithful platforms for the study of muscle pathologies as cancer cachexia, bioprinted muscle rings are treated with medium conditioned with colorectal cancer cells. This study shows that soluble factors secreted by cancer cells induce the upregulation of protein degradation pathways and degeneration of muscle fibers. In particular, high levels of soluble TNFRI are associated with severe cachexia, which correlates with the plasma level of tumor-bearing mice. The results indicate a close resemblance between the gene expression pattern of the bioprinted model and muscle tissue of cachectic mice, whereas monolayer cultures present several disparities. Together, GelMA-AlgMA-Fibrin is presented as a promising biomaterial for the fabrication of bioprinted models of healthy skeletal muscle tissue and muscle wasting on cancer cachexia disease.[spa] La bioimpresión por extrusión es una técnica prometedora para el escalado y la fabricación automatizada de modelos tisulares in vitro. Sin embargo, las propiedades fisicoquímicas de las biotintas actuales pocas veces cumplen los requisitos de los tejidos naturales. Esta tesis describe la modificación química de polímeros naturales como gelatina y alginato para obtener biotintas fotopolimerizables con propiedades físicas maleables. La composición de las biotintas y los parámetros de fabricación de los diseños bioimpresos son modulados para conseguir dos tipos de formulaciones adecuadas para el desarrollo de modelos de músculo esquelético y neuroblastoma. En el estudio, ambos modelos muestran mayor semblanza a los tejidos originales que los tradicionales cultivos en monocapa. Por un lado, los modelos de neuroblastoma recapitulan el comportamiento de los tumores sólidos en pacientes de alto riesgo. Por otro lado, la composición de la biotinta para modelos musculares induce la diferenciación de células precursoras a fibras musculares maduras y alineadas que, en combinación con un sistema de estimulación por pulsos eléctricos, dan como resultado fibras musculares funcionales con capacidad contráctil. Este modelo imita el comportamiento del músculo esquelético y se puede utilizar para monitorizar los efectos de compuestos químicos sobre el tejido. Al final del estudio, se trabaja en la generación de un modelo de músculo atrofiado por caquexia derivada del cáncer. El modelo bioimpreso reproduce la característica sobreexpresión de genes relacionados con la degradación proteica hallada en ratones. En conjunto, se adaptan la composición y propiedades físicas de biotintas basadas en gelatina y alginato para la fabricación de modelos de tejido muscular y neuroblastoma por medio de fabricación aditiva. Además, el modelo muscular se presenta como un posible candidato para realizar estudios in vitro del tejido sano y atrofiado por caquexia derivada de cáncer

Producing 3D biomimetic nanomaterials for musculoskeletal system regeneration

The human musculoskeletal system is comprised mainly of connective tissues such as cartilage, tendon, ligaments, skeletal muscle, and skeletal bone. These tissues support the structure of the body, hold and protect the organs, and are responsible of movement. Since it is subjected to continuous strain, the musculoskeletal system is prone to injury by excessive loading forces or aging, whereas currently available treatments are usually invasive and not always effective. Most of the musculoskeletal injuries require surgical intervention facing a limited post-surgery tissue regeneration, especially for widespread lesions. Therefore, many tissue engineering approaches have been developed tackling musculoskeletal tissue regeneration. Materials are designed to meet the chemical and mechanical requirements of the native tissue three-dimensional (3D) environment, thus facilitating implant integration while providing a good reabsorption rate. With biological systems operating at the nanoscale, nanoengineered materials have been developed to support and promote regeneration at the interprotein communication level. Such materials call for a great precision and architectural control in the production process fostering the development of new fabrication techniques. In this mini review, we would like to summarize the most recent advances in 3D nanoengineered biomaterials for musculoskeletal tissue regeneration, with especial emphasis on the different techniques used to produce them

Composite biomaterials as long-lasting scaffolds for 3D bioprinting of highly aligned muscle tissue

New biocompatible materials have enabled the direct 3D printing of complex functional living tissues, such as skeletal and cardiac muscle. Gelatinmethacryloyl (GelMA) is a photopolymerizable hydrogel composed of natural gelatin functionalized with methacrylic anhydride. However, it is difficult to obtain a single hydrogel that meets all the desirable properties for tissue engineering. In particular, GelMA hydrogels lack versatility in their mechanical properties and lasting 3D structures. In this work, a library of composite biomaterials to obtain versatile, lasting, and mechanically tunable scaffolds are presented. Two polysaccharides, alginate and carboxymethyl cellulose chemically functionalized with methacrylic anhydride, and a synthetic material, such as poly(ethylene glycol) diacrylate are combined with GelMA to obtain photopolymerizable hydrogel blends. Physical properties of the obtained composite hydrogels are screened and optimized for the growth and development of skeletal muscle fibers from C2C12 murine cells, and compared with pristine GelMA. All these composites show high resistance to degradation maintaining the 3D structure with high fidelity over several weeks. Altogether, in this study a library of biocompatible novel and totally versatile composite biomaterials are developed and characterized, with tunable mechanical properties that give structure and support myotube formation and alignment

Composite biomaterials as long-lasting scaffolds for 3D bioprinting of highly aligned muscle tissue

New biocompatible materials have enabled the direct 3D printing of complex functional living tissues, such as skeletal and cardiac muscle. Gelatinmethacryloyl (GelMA) is a photopolymerizable hydrogel composed of natural gelatin functionalized with methacrylic anhydride. However, it is difficult to obtain a single hydrogel that meets all the desirable properties for tissue engineering. In particular, GelMA hydrogels lack versatility in their mechanical properties and lasting 3D structures. In this work, a library of composite biomaterials to obtain versatile, lasting, and mechanically tunable scaffolds are presented. Two polysaccharides, alginate and carboxymethyl cellulose chemically functionalized with methacrylic anhydride, and a synthetic material, such as poly(ethylene glycol) diacrylate are combined with GelMA to obtain photopolymerizable hydrogel blends. Physical properties of the obtained composite hydrogels are screened and optimized for the growth and development of skeletal muscle fibers from C2C12 murine cells, and compared with pristine GelMA. All these composites show high resistance to degradation maintaining the 3D structure with high fidelity over several weeks. Altogether, in this study a library of biocompatible novel and totally versatile composite biomaterials are developed and characterized, with tunable mechanical properties that give structure and support myotube formation and alignment