3D Printing Elastic Biomimetic Scaffolds For Tissue Engineering Applications

Tissue engineering aims to use the body's regenerative ability to repair damaged tissues via artificial implants.Bioelastomers can mimic the mechanical properties of soft tissues,and sustain and recover from various deformations without mechanical irritations to the surrounding tissues when implanted in a mechanically dynamic environment in the body.Thus,bioelastomers have attracted great attentions in tissue engineering.Recently,three-dimensional(3D)printing technology possesses a strong ability to customize macro morphology and precisely control microstructure,thus has become widely applied in regenerative medicine and promoted significant innovations.However,mimicking native vascular networks to facilitate mass exchange and allow long-term survival of engineered tissues in vitro and in vivo remains a key challenge in tissue engineering.Moreover,the available biomaterials for 3D printing are very limited,especially thermosets.As such,thermosetting bioelastomer require a long term curing at harsh conditions,which are not compatible with rapid 3D printing processing.Furthermore,the design of tubular scaffold with customized structure remains a great challenge in clinical tubular tissue reconstruction.It's difficult to fabricate porous and thin-walled tubular scaffold due to the printed fibers lack the buttressing effect.To address the aforementioned challenges,we established new and general strategies to efficiently fabricate elastic biomimetic scaffold with hierarchical and porous structure via widely available extrusion-based 3D printing.The main contents are as follows:(1)We established a 3D printed sacrificial caramel-based template strategy to create Perfusable and Permeable Hierarchical Microchannel-networks(PHMs)scaffolds.Inspired by the Chinese traditional caramel painting art,we extended the heat treatment of sucrose to produce caramel inks.The resultant caramel inks could be printable by conventional thermoplastic processing techniques like injection molding extrusion.A series of conditions of the precaramelization(temperature and duration)and following printing procedure(nozzle size,extrusion rate,and nozzle moving rate)were tried to enable a stable,continuous,smooth printing of caramel with appropriate viscoelastic properties.Based on these 3D printed caramel templates,we used polycaprolactone(PCL)as the representative biomaterial to create PHMs via solution immersion and template leaching.Sacrificial caramel-based filaments were readily dissolved out by distilled water to form an interconnected vascular scaffold.Furthermore,we fabricated a human-scale ear-shape PCL PHMs based on the digital model of a human ear.This approach was designed to impart the polymer scaffolds with well-organized hierarchical structures resembling the native vascular networks at multiple levels including a 3D framework,perfusable microchannels and permeable porous walls.Abundant micropores were distributed throughout the external surface,inside of the channel walls,and lumen surface with relatively uniform pore size in normal distributions.Next,we investigated the coating process via the phase separation mechanism in detail including different molecular weight,concentrations and solvents of PCL.These thin channel walls with controllable micropores is seldom reported previously.Importantly,the PHMs manufacturing strategy is highly versatile and can be readily applied to diverse materials to vary the properties of resultant PHMs.As a proof of principle,the representative thermoplastic PCL-polyurethane(PCL-PU)and thermoset poly(glycerol sebacate urethane)(PGSU)bioelastomers were used.We fabricated elastic PHMs to both mimic the mechanical property and structure of native vasculatures.The PHMs can be readily integrated with various tissue engineering scaffolds(such as spongy scaffold,hydrogel scaffold,electrospun scaffold and nanofibrillar bacterial cellulose scaffold)to produce sophisticated composite scaffolds with built-in PHMs.The hydrogel encapsulated PHMs were used as an example to evaluate the function of build-in PHMs to support the mass transportation of whole scaffolds.Sodium alginate matrices were mixed with living cardiomyocytes homogeneously and then encapsulated PHMs to form 3D tissue constructs.The microchannels of built-in PHMs provided a favorable microenvironment for cell proliferation and maintained cell viability in the 3D tissue constructs with cell-laden gels during the process of in vitro culture.In addition,the unique hierarchical structure of PHMs promoted tissue integration and revascularization compared to typical 3D printed scaffold when implanted subcutaneously and epicardially.When implanted on infarcted myocardium,cardiomyocyte/ fibroblasts ratio of the PHM group was significantly higher than other groups,indicating more viable myocardium and less fibration in the infarct region.Accordingly,this PHMs may serve as a new type of cardiac patch to treat myocardial infarction.(2)To address the aforementioned challenges of 3D printing thermosets,we report a new strategy for direct 3D printing of various thermosets via readily available extrusion-based technology,exemplified by crosslinked polyester,polyurethane and epoxy resins.The key was the design of composite inks,which containing both the thermoplastic precursors of thermosets and sacrificial carrier materials.As a proof of principle,we first investigated the 3D printing of a widely used thermoset biomaterial poly(glycerol sebacate)(PGS).The performance of various formulations of composite inks was investigated to optimize the parameters.Increasing the salt content in inks resulted in better shape retention,but reduced their rheological property and extrusion ability.The PGS/salt weight ratio of 1:2 was the optimized formulation,which enabled good rheological property,stable printability and better shape retention.As designed,the printed PGS scaffolds showed well-organized hierarchical structures,which were difficult to fabricate by previous methods.Controlling the digital models and printing parameters readily customized the primary multi-layered 3D framework and the secondary patterned filaments unit.Tertiary structure was the abundant interconnected micropores distributed throughout the filaments,which were built by particle leaching and resulted in higher porosity and specific surface area.The pore sizes were determined by the salt particulate sizes,which can be tuned from tens to hundreds micrometers by grinding and sieving.Although the highly porous,PGS scaffolds exhibited a good ductility,superior elasticity and anti-fatigue property with negligible hysteresis under dynamic deformation,which enabled the constructs to resist multiple tensile and compression deformations and retain their original hierarchical and porous structure during tissue engineering application in vivo.Furthermore,we screened the in vitro degradation of 3D printed PGS scaffold in a high concentrated lipase solution.The PGS constructs degraded continuously with a total mass loss of 93.5 ± 2.1% within 5 hours confirming its good degradability.The rat subcutaneous implantation results showed that 3D printed PGS scaffolds had partially degraded and integrated with host tissues,with newly formed tissue infiltration and vascular ingrowth without apparent inflammation reaction.Besides the excellent biocompatibility and biodegradability,one of the most important advantages of cured PGS is its robust elasticity due to the stable chemical crosslinking structure.Thus,PGS are very promising for the treatment of elastic tissues such as myocardium.By implanting the 3D printed PGS patches onto the infarcted area,we aimed to attenuate myocardial remodeling post myocardial infarction.As evaluated by the H&E staining and immunohistochemical staining,the gross morphology of hearts explanted 28 days post-implantation indicated that the 3D printed PGS patches thickened the left ventricular wall,attenuated left ventricular expansion and reduce the degree of fibrosis.(3)We present a reliable method to rapidly fabricate tissue-engineered tubular scaffold with hierarchical structure using a 4-axis printing system.3D printer and a rotary receiver were cooperative worked to build 4-axis printing system of X/Y/Z/Rotation.The fabrication process can be adapted to various biomaterials including hydrogels,thermoplastic materials and thermosetting materials.Biomaterials was extruded out and deposited on the surface of receiver with spiral-arranged structure.The periodic fibers were bound solidly together in upper and lower layers to form a stable network by the reciprocating motion of printer.Stable braided structure could maintain the original tubular structure without collapsing.It only takes several minutes to fabricate tubular scaffolds with more than 10 centimeters length.This method has high efficiency compared to the processing techniques of tubular scaffold.Based on this 4-axis printing method,we used PCL as the representative biomaterial to investigate the controllability of morphological structure.This method could be readily constructed hollow scaffolds with diverse geometries by changing the shape of receivers.By varying the rotational rate of receiver,while holding constant other parameters,we fabricated diverse tubular scaffolds with different microstructures.By theoretical calculation and analysis,the thread pitch,fiber diameter,fiber distance and fiber angle of interweaved of fibrous-networks were in inverse ratio to the rotational rate.These experimental data were closely matched with the relationships derive from the model,which allowed the generation of tubular scaffold in predefined and reproducible morphology.Although the PCL is a typical plastic material,the scaffolds exhibited superior elasticity and fatigue resistance under dynamic deformation.Furthermore,their mechanical property could be efficiently tuned by controlling the meshwork architecture.By combining this method with previously described 3D printing method of thermosets,we further fabricated the PGS into the bio-spring.The bio-spring showed good flexibility,elasticity and well-organized hierarchical structures.To demonstrate the potential application of the 4-axis printing technology,we fabricated a PGS/Gelatin hybrid tubular scaffold by electrospinning gelatin nanofibers on the exterior surface of PGS bio-spring,and verify its efficiency on engineering tubular cartilage both in vitro and in vivo.After cell seeding,chondrocytes were able to effectively proliferate on the hybrid scaffold,indicating good biocompatibility and low apoptosis.After cultured in vitro for 8 weeks,the hybrid tubular scaffold formed cartilage-like tissue.Histological examinations showed that chondrocytes proliferated on the scaffold surface and secreted cartilage ECM to form tubular cartilage.After 12 weeks of subcutaneous implantation on nude mice,the hybrid tubular scaffold formed mature neocartilage with enough strength and elasticity.The engineered tubular cartilage had higher wet weight,thickness,and DNA content compared to normal tracheal cartilage.Moreover,Young's modulus,GAG content,and total collagen content of the engineered tubular cartilage achieved a level over 70% of normal tracheal cartilage.In conclusion,this study aims to solve the challenges of artificial vascular networks,3D printing thermosets and customized tubular scaffolds.Based on 3D printing technology,we established general strategies to efficiently fabricate a series of elastic biomimetic tissue engineered scaffolds,such as Perfusable and Permeable Hierarchical Microchannel-network scaffolds,thermosetting PGS porous scaffolds and tubular scaffolds with controllable structures.These scaffolds show great potential applications in the tissue engineering.Scaffolds could be used as heart patches for the treatment of myocardial infarction,and could effectively prevent the left ventricular expansion,thicken the heart muscle,promote angiogenesis and reduce fibrosis.Furthermore,the tubular scaffolds could regenerate tubular mature cartilage that was similar to the native tracheal cartilage.Based on these general strategies,various biomaterials can be fabricated into customized scaffolds with hierarchical structures.It is expected that these methods can be further developed for other biomedical applications such as bone,nerve,vessels and other tissues.