design-validation-volumetric-extrusion-bioprinter-for-bioprinting

Design and Validation of a Volumetric-extrusion Bioprinter for Bioprinting of Soluble Basement Membrane Extract for Translational Research

In translational research, 3D in vitro models are essential for advancing scientific understanding and therapeutic development. To this aim, Soluble ...

Politecnico di Milano

The in vitro replication of physiological mechanical conditioning through bioreactors plays a crucial role in the development of functional Small-Caliber Tissue-Engineered Blood Vessels. An in silico scaffold-specific model under pulsatile perfusion provided by a bioreactor was implemented using a fluid-structure interaction (FSI) approach for viscoelastic tubular scaffolds (e.g. decellularized swine arteries, DSA). Results of working pressures, circumferential deformations, and wall shear stress on DSA fell within the desired physiological range and indicated the ability of this model to correctly predict the mechanical conditioning acting on the cells-scaffold system. Consequently, the FSI model allowed us to a priori define the stimulation pattern, driving in vitro physiological maturation of scaffolds, especially with viscoelastic properties.

Estimation of the physiological mechanical conditioning in vascular tissue engineering by a predictive fluid-structure interaction approach.

A tissue engineered oesophagus could overcome limitations associated with oesophageal substitution. Combining decellularized scaffolds with patient-derived cells shows promise for regeneration of tissue defects. In this proof-of-principle study, a two-stage approach for generation of a bio-artificial oesophageal graft addresses some major challenges in organ engineering, namely: (i) development of multi-strata tubular structures, (ii) appropriate re-population/maturation of constructs before transplantation, (iii) cryopreservation of bio-engineered organs and (iv) in vivo pre-vascularization. The graft comprises decellularized rat oesophagus homogeneously re-populated with mesoangioblasts and fibroblasts for the muscle layer. The oesophageal muscle reaches organised maturation after dynamic culture in a bioreactor and functional integration with neural crest stem cells. Grafts are pre-vascularised in vivo in the omentum prior to mucosa reconstitution with expanded epithelial progenitors. Overall, our optimised two-stage approach produces a fully re-populated, structurally organized and pre-vascularized oesophageal substitute, which could become an alternative to current oesophageal substitutes.

Multi-stage bioengineering of a layered oesophagus with in vitro expanded muscle and epithelial adult progenitors.

Aiming to perfuse porous tubular scaffolds for vascular tissue engineering (VTE) with controlled flow rate, prevention of leakage through the scaffold lumen is required. A gel coating made of 8% w/v alginate and 6% w/v gelatin functionalized with fibronectin was produced using a custom-made bioreactor-based method. Different volumetric proportions of alginate and gelatin were tested (50/50, 70/30, and 90/10). Gel swelling and stability, and rheological, and uniaxial tensile tests reveal superior resistance to the aggressive biochemical microenvironment, and their ability to withstand physiological deformations (~10%) and wall shear stresses (5-20 dyne/cm). These are prerequisites to maintain the physiologic phenotypes of vascular smooth muscle cells and endothelial cells (ECs), mimicking blood vessels microenvironment. Gels can induce ECs proliferation and colonization, especially in the presence of fibronectin and higher percentages of gelatin. The custom-designed bioreactor enables the development of reproducible and homogeneous tubular gel coating. The permeability tests show the effectiveness of tubular scaffolds coated with 70/30 alginate/gelatin gel to occlude wadding pores, and therefore prevent leakages. The synthesized double-layered tubular scaffolds coated with alginate/gelatin gel and fibronectin represent both promising substrate for ECs and effective leakproof scaffolds, when subjected to pulsatile perfusion, for VTE applications.

Shear-resistant hydrogels to control permeability of porous tubular scaffolds in vascular tissue engineering.

Explosion of gene therapy approaches for treating rare monogenic and common liver disorders created an urgent need for disease models able to replicate human liver cellular environment. Available models lack 3D liver structure or are unable to survive in long-term culture. We aimed to generate and test a 3D culture system that allows long-term maintenance of human liver cell characteristics. The  whole-organ "Bioreactor grown Artificial Liver Model" (BALM) employs a custom-designed bioreactor for long-term 3D culture of human induced pluripotent stem cells-derived hepatocyte-like cells (hiHEPs) in a mouse decellularized liver scaffold. Adeno-associated viral (AAV) and lentiviral (LV) vectors were introduced by intravascular injection. Substantial AAV and LV transgene expression in the BALM-grown hiHEPs was detected. Measurement of secreted proteins in the media allowed non-invasive monitoring of the system. We demonstrated that humanized whole-organ BALM is a valuable tool to generate pre-clinical data for investigational medicinal products.

An  Whole-Organ Liver Engineering for Testing of Genetic Therapies.

Cancer is one of the most life-threatening diseases worldwide. Despite the huge efforts, the failure rate of therapies remains high due to cells heterogeneity, so physiologically relevant models are strictly necessary. Bioprinting is a technology able to form highly complex 3D tissue models and enables the creation of large-scale constructs. In cancer research, Matrigel is the most widely used matrix, but it is hardly bioprinted pure, without the use of any other bioink as reinforcement. Its complex rheological behavior makes the control with a standard bioprinting process nearly impossible. In this work, we present a customized bioprinting strategy to produce pure Matrigel scaffolds with good shape fidelity. To this aim, we realized a custom-made volumetric dispensing system and performed printability evaluations. To determine optimal printing parameters, we analyzed fibers spreading ratio on simple serpentines. After identifying an optimal flow rate of 86.68 ± 5.77 µL/min and a printing speed of 10 mm/min, we moved forward to evaluate printing accuracy, structural integrity and other key parameters on single and multi-layer grids. Results demonstrated that Matrigel was able to maintain its structure in both simple and complex designs, as well as in single and multilayer structures, even if it does not possess high mechanical strength. In conclusion, the use of volumetric dispensing allowed printing pure Matrigel constructs with a certain degree of shape fidelity on both single and multiple layers.

Bioprinting of Matrigel Scaffolds for Cancer Research.

Patients affected by microgastria, severe gastroesophageal reflux, or those who have undergone subtotal gastrectomy, have commonly described reporting dumping syndromes or other symptoms that seriously impair the quality of their life. Gastric tissue engineering may offer an alternative approach to treating these pathologies. Decellularization protocols have great potential to generate novel biomaterials for large gastric defect repair. There is an urgency to define more reliable protocols to foster clinical applications of tissue-engineered decellularized gastric grafts.

Article	Year
Estimation of the physiological mechanical conditioning in vascular tissue engineering by a predictive fluid-structure interaction approach. Computer methods in biomechanics and biomedical engineering\| PubMed ID: 28569086	2017
Multi-stage bioengineering of a layered oesophagus with in vitro expanded muscle and epithelial adult progenitors. Nature communications\| PubMed ID: 30327457	2018
Shear-resistant hydrogels to control permeability of porous tubular scaffolds in vascular tissue engineering. Materials science & engineering. C, Materials for biological applications\| PubMed ID: 31546369	2019
An Whole-Organ Liver Engineering for Testing of Genetic Therapies. iScience\| PubMed ID: 33305175	2020
Bioprinting of Matrigel Scaffolds for Cancer Research. Polymers\| PubMed ID: 34205767	2021
Stomach engineering: region-specific characterization of the decellularized porcine stomach. Pediatric surgery international\| PubMed ID: 38032517	2023

Alessandro Filippo Pellegata

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