Cell Instructive Behavior of Composite Scaffolds in a Co-Culture of Human Mesenchymal Stem Cells and Peripheral Blood Mononuclear Cells.

The in vitro evaluation of 3D scaffolds for bone tissue engineering in mono-cultures is a common practice; however, it does not represent the native complex nature of bone tissue. Co-cultures of osteoblasts and osteoclasts, without the addition of stimulating agents for monitoring cellular cross-talk, remains a challenge. In this study, a growth factor-free co-culture of human bone marrow-derived mesenchymal stem cells (hBM-MSCs) and human peripheral blood mononuclear cells (hPBMCs) has been established and used for the evaluation of 3D-printed scaffolds for bone tissue engineering. The scaffolds were produced from PLLA/PCL/PHBV polymeric blends, with two composite materials produced through the addition of 2.5% w/v nanohydroxyapatite (nHA) or strontium-substituted nanohydroxyapatite (Sr-nHA). Cell morphology data showed that hPBMCs remained undifferentiated in co-culture, while no obvious differences were observed in the mono- and co-cultures of hBM-MSCs. A significantly increased alkaline phosphatase (ALP) activity and osteogenic gene expression was observed in co-culture on Sr-nHA-containing scaffolds. Tartrate-resistant acid phosphatase (TRAP) activity and osteoclastogenic gene expression displayed significantly suppressed levels in co-culture on Sr-nHA-containing scaffolds. Interestingly, mono-cultures of hPBMCs on Sr-nHA-containing scaffolds indicated a delay in osteoclasts formation, as evidenced from TRAP activity and gene expression, demonstrating that strontium acts as an osteoclastogenesis inhibitor. This co-culture study presents an effective 3D model to evaluate the regenerative capacity of scaffolds for bone tissue engineering, thus minimizing time-consuming and costly in vivo experiments.

Mechanical Transfer of Black Phosphorus on a Silk Fibroin Substrate: A Viable Method for Photoresponsive and Printable Biomaterials.

Despite the technological importance of semiconductor black phosphorus (BP) in materials science, maintaining the stability of BP crystals in organic media and protecting them from environmental oxidation remains challenging. In this study, we present the synthesis of bulk BP and the exploitation of the viscoelastic properties of a regenerated silk fibroin (SF) film as a biocompatible substrate to transfer BP flakes, thereby preventing oxidation. A model based on the flow of polymers revealed that the applied flow-induced stresses exceed the yield stress of the BP aggregate. Raman spectroscopy was used to investigate the exfoliation efficiency as well as the environmental stability of BP transferred on the SF substrate. Notably, BP flakes transferred to the SF substrate demonstrated improved stability when SF was dissolved in a phosphate-buffered saline medium, and in vitro cancer cell viability experiments demonstrate the tumor ablation efficiency under visible to near-infrared (Vis-nIR) radiation. Moreover, the SF and BP-enriched SF (SF/BP) solution was shown to be processable via extrusion-based three-dimensional (3D) printing. Therefore, this work paves the way for a general method for the transferring of BP on natural biodegradable polymers and processing them via 3D printing toward novel functionalities and complex shapes for biomedical purposes.

Correction to "Electrospun Nanofibrous UV Filters with Bidirectional Actuation Properties Based on Salmon Sperm DNA/Silk Fibroin for Biomedical Applications".

[This corrects the article DOI: 10.1021/acsomega.3c04563.].

Enhancing quality control in bioprinting through machine learning.

Bioprinting technologies have been extensively studied in literature to fabricate three-dimensional constructs for tissue engineering applications. However, very few examples are currently available on clinical trials using bioprinted products, due to a combination of technological challenges (i.e. difficulties in replicating the native tissue complexity, long printing times, limited choice of printable biomaterials) and regulatory barriers (i.e. no clear indication on the product classification in the current regulatory framework). In particular, quality control (QC) solutions are needed at different stages of the bioprinting workflow (including pre-process optimization, in-process monitoring, and post-process assessment) to guarantee a repeatable product which is functional and safe for the patient. In this context, machine learning (ML) algorithms can be envisioned as a promising solution for the automatization of the quality assessment, reducing the inter-batch variability and thus potentially accelerating the product clinical translation and commercialization. In this review, we comprehensively analyse the main solutions that are being developed in the bioprinting literature on QC enabled by ML, evaluating different models from a technical perspective, including the amount and type of data used, the algorithms, and performance measures. Finally, we give a perspective view on current challenges and future research directions on using these technologies to enhance the quality assessment in bioprinting.

4D bioprinted self-folding scaffolds enhance cartilage formation in the engineering of trachea.

Trachea defects that required surgical interventions are increasing in number in the recent years, especially for pediatric patients. However, current gold standards, such as biological grafts and synthetic prothesis, do not represent an effective solution, due to the lack of mimicry and regeneration capability. Bioprinting is a cutting-edge approach for the fabrication of biomimetic scaffold to empower tissue engineering toward trachea replacement. In this study, we developed a self-folding gelatin-based bilayer scaffold for trachea engineering, exploiting the 4D bioprinting approach, namely the fabrication of dynamic scaffolds, able to shape morph in a predefined way after the application of an environmental stimulus. Indeed, starting form a 2D flat position, upon hydration, this scaffold forms a closed tubular structure. An analytical model, based on Timoshenko's beam thermostats, was developed, and validated to predict the radius of curvature of the scaffold according to the material properties and the scaffold geometry. The 4D bioprinted structure was tested with airway fibroblast, lung endothelial cells and ear chondral progenitor cells (eCPCs) toward the development of a tissue engineered trachea. Cells were seeded on the scaffold in its initial flat position, maintained their position after the scaffold actuation and proliferated over or inside it. The ability of eCPCs to differentiate towards mature cartialge was evaluated. Interestingly, real-time PCR revealed that differentiating eCPCs on the 4D bioprinted scaffold promote healthy cartilage formation, if compared with eCPCs cultured on 2D static scaffold. Thus, eCPCs can perceive scaffold folding and its final curvature and to react to it, towards the formation of mature cartilage for the airway.

Electrospun Nanofibrous UV Filters with Bidirectional Actuation Properties Based on Salmon Sperm DNA/Silk Fibroin for Biomedical Applications.

In this study, we dissolved Bombyx mori degummed silk [i.e., silk fibroin (SF)] and salmon sperm deoxyribonucleic acid (DNA) in water and used a bioinspired spinning process to obtain an electrospun nanofibrous SF-based patch (ESF). We investigated the bidirectional macroscale actuation behavior of ESF in response to water vapor and its UV-blocking properties as well as those of ESF/DNA films. Fourier transform infrared (FTIR) results suggest that the formation of ß-sheet-rich structures promotes the actuation effect. ESF/DNA film with high-ordered and ß-sheet-rich structures exhibits higher electrical conductivity and is water-insoluble. Given the intrinsic ability of both SF and DNA to absorb UV radiation, we performed biological experiments on the viability of keratinocyte HaCaT cells after exposure to solar spectrum components. Our findings indicate that the ESF/DNA patch is photoprotective and can increase the cellular viability of keratinocytes after UV exposure. Furthermore, we demonstrated that ESF/DNA patches treated with water vapor can serve as suitable scaffolds for tissue engineering and can improve tissue regeneration when cellularized with HaCaT cells. The 3D shape morphing capability of these patches, along with their potential as UV filters, could offer significant practical advantages in tissue engineering.

Automatic Photo-Cross-Linking System for Robotic-Based In Situ Bioprinting.

This work reports the design and validation of an innovative automatic photo-cross-linking device for robotic-based in situ bioprinting. Photo-cross-linking is the most promising polymerization technique when considering biomaterial deposition directly inside a physiological environment, typical of the in situ bioprinting approach. The photo-cross-linking device was designed for the IMAGObot platform, a 5-degree-of-freedom robot re-engineered for in situ bioprinting applications. The system consists of a syringe pump extrusion module equipped with eight light-emitting diodes (LEDs) with a 405 nm wavelength. The hardware and software of the robot were purposely designed to manage the LEDs switching on and off during printing. To minimize the light exposure of the needle, thus avoiding its clogging, only the LEDs opposite the printing direction are switched on to irradiate the newly deposited filament. Moreover, the LED system can be adjusted in height to modulate substrate exposure. Different scaffolds were bioprinted using a GelMA-based hydrogel, varying the printing speed and light distance from the bed, and were characterized in terms of swelling and mechanical properties, proving the robustness of the photo-cross-linking system in various configurations. The system was finally validated onto anthropomorphic phantoms (i.e., a human humerus head and a human hand with defects) featuring complex nonplanar surfaces. The designed system was successfully used to fill these anatomical defects, thus resulting in a promising solution for in situ bioprinting applications.

4D Printing Shape-Morphing Hybrid Biomaterials for Advanced Bioengineering Applications.

Four-dimensional (4D) printing is an innovative additive manufacturing technology used to fabricate structures that can evolve over time when exposed to a predefined environmental stimulus. 4D printed objects are no longer static objects but programmable active structures that accomplish their functions thanks to a change over time in their physical/chemical properties that usually displays macroscopically as a shapeshifting in response to an external stimulus. 4D printing is characterized by several entangled features (e.g., involved material(s), structure geometry, and applied stimulus entities) that need to be carefully coupled to obtain a favorable fabrication and a functioning structure. Overall, the integration of micro-/nanofabrication methods of biomaterials with nanomaterials represents a promising approach for the development of advanced materials. The ability to construct complex and multifunctional triggerable structures capable of being activated allows for the control of biomedical device activity, reducing the need for invasive interventions. Such advancements provide new tools to biomedical engineers and clinicians to design dynamically actuated implantable devices. In this context, the aim of this review is to demonstrate the potential of 4D printing as an enabling manufacturing technology to code the environmentally triggered physical evolution of structures and devices of biomedical interest.

A Millifluidic Chamber for Controlled Shear Stress Testing: Application to Microbial Cultures.

In vitro platforms such as bioreactors and microfluidic devices are commonly designed to engineer tissue models as well as to replicate the crosstalk between cells and microorganisms hosted in the human body. These systems promote nutrient supply and waste removal through culture medium recirculation; consequently, they intrinsically expose cellular structures to shear stress, be it a desired mechanical stimulus to drive the cell fate or a potential inhibitor for the model maturation. Assessing the impact of shear stress on cellular or microbial cultures thus represents a crucial step to define proper environmental conditions for in vitro models. In this light, the aim of this study was to develop a millifluidic device enabling to generate fully controlled shear stress profiles for quantitatively probing its influence on tissue or bacterial models, overcoming the limitations of previous reports proposing similar devices. Relying on this millifluidic tool, we present a systematic methodology to test how adherent cellular structures react to shear forces, which was applied to the case of microbial biofilms as a proof of concept. The results obtained suggest our approach as a suitable testbench to evaluate culture conditions in terms of shear stress faced by cells or microorganisms.

Chitosan and its derivatives in 3D/4D (bio) printing for tissue engineering and drug delivery applications.

Tissue engineering research has undergone to a revolutionary improvement, thanks to technological advancements, such as the introduction of bioprinting technologies. The ability to develop suitable customized biomaterial inks/bioinks, with excellent printability and ability to promote cell proliferation and function, has a deep impact on such improvements. In this context, printing inks based on chitosan and its derivatives have been instrumental. Thus, the current review aims at providing a comprehensive overview on chitosan-based materials as suitable inks for 3D/4D (bio)printing and their applicability in creating advanced drug delivery platforms and tissue engineered constructs. Furthermore, relevant strategies to improve the mechanical and biological performances of this biomaterial are also highlighted.

Multimaterial and multiscale scaffold for engineering enthesis organ.

Tendon and ligament injuries are relevant clinical problems in modern society, and the current medical approaches do not guarantee complete recovery of the physiological functionalities. Moreover, they present a non-negligible failure rate after surgery. Failures often occur at the enthesis, which is the area of tendons and ligaments insertion to bones. This area is highly anisotropic and composed of four distinct zones: tendon or ligament, non-mineralized fibrocartilage, mineralized fibrocartilage, and bone. The organization of these regions provides a gradient in mechanical properties, biochemical composition, cellular phenotype, and extracellular matrix organization. Tissue engineering represents an alternative to traditional medical approaches. This work presents a novel biofabrication approach for engineering the enthesis. Gradient-based scaffolds were fabricated by exploiting the combination of electrospinning and three-dimensional (3D) bioprinting technologies. Studies were conducted to evaluate scaffold biocompatibility by seeding bone marrow-derived mesenchymal stem cells (BM-MSCs). Then, the scaffold's ability to promote cellular adhesion, growth, proliferation, and differentiation in both tenogenic and osteogenic phenotypes was evaluated. Fabricated scaffolds were also morphologically and mechanically characterized, showing optimal properties comparable to literature data. The versatility and potentiality of this novel biofabrication approach were demonstrated by fabricating clinical-size 3D enthesis scaffolds. The mechanical characterization highlighted their behavior during a tensile test was comparable to tendons and ligaments in vivo.

3D Bioprinting in Microgravity: Opportunities, Challenges, and Possible Applications in Space.

3D bioprinting has developed tremendously in the last couple of years and enables the fabrication of simple, as well as complex, tissue models. The international space agencies have recognized the unique opportunities of these technologies for manufacturing cell and tissue models for basic research in space, in particular for investigating the effects of microgravity and cosmic radiation on different types of human tissues. In addition, bioprinting is capable of producing clinically applicable tissue grafts, and its implementation in space therefore can support the autonomous medical treatment options for astronauts in future long term and far-distant space missions. The article discusses opportunities but also challenges of operating different types of bioprinters under space conditions, mainly in microgravity. While some process steps, most of which involving the handling of liquids, are challenging under microgravity, this environment can help overcome problems such as cell sedimentation in low viscous bioinks. Hopefully, this publication will motivate more researchers to engage in the topic, with publicly available bioprinting opportunities becoming available at the International Space Station (ISS) in the imminent future.

Effect of Uniaxial Compression Frequency on Osteogenic Cell Responses in Dynamic 3D Cultures.

The application of mechanical stimulation on bone tissue engineering constructs aims to mimic the native dynamic nature of bone. Although many attempts have been made to evaluate the effect of applied mechanical stimuli on osteogenic differentiation, the conditions that govern this process have not yet been fully explored. In this study, pre-osteoblastic cells were seeded on PLLA/PCL/PHBV (90/5/5 wt.%) polymeric blend scaffolds. The constructs were subjected every day to cyclic uniaxial compression for 40 min at a displacement of 400 µm, using three frequency values, 0.5, 1, and 1.5 Hz, for up to 21 days, and their osteogenic response was compared to that of static cultures. Finite element simulation was performed to validate the scaffold design and the loading direction, and to assure that cells inside the scaffolds would be subjected to significant levels of strain during stimulation. None of the applied loading conditions negatively affected the cell viability. The alkaline phosphatase activity data indicated significantly higher values at all dynamic conditions compared to the static ones at day 7, with the highest response being observed at 0.5 Hz. Collagen and calcium production were significantly increased compared to static controls. These results indicate that all of the examined frequencies substantially promoted the osteogenic capacity.

Analysis of the Robotic-Based In Situ Bioprinting Workflow for the Regeneration of Damaged Tissues through a Case Study.

This study aims to critically analyse the workflow of the in situ bioprinting procedure, presenting a simulated neurosurgical case study, based on a real traumatic event, for collecting quantitative data in support of this innovative approach. After a traumatic event involving the head, bone fragments may have to be removed and a replacement implant placed through a highly demanding surgical procedure in terms of surgeon dexterity. A promising alternative to the current surgical technique is the use of a robotic arm to deposit the biomaterials directly onto the damaged site of the patient following a planned curved surface, which can be designed pre-operatively. Here we achieved an accurate planning-patient registration through pre-operative fiducial markers positioned around the surgical area, reconstructed starting from computed tomography images. Exploiting the availability of multiple degrees of freedom for the regeneration of complex and also overhanging parts typical of anatomical defects, in this work the robotic platform IMAGObot was used to regenerate a cranial defect on a patient-specific phantom. The in situ bioprinting process was then successfully performed showing the great potential of this innovative technology in the field of cranial surgery. In particular, the accuracy of the deposition process was quantified, as well as the duration of the whole procedure was compared to a standard surgical practice. Further investigations include a biological characterisation over time of the printed construct as well as an in vitro and in vivo analysis of the proposed approach, to better analyse the biomaterial performances in terms of osteo-integration with the native tissue.

A computational analysis of a novel therapeutic approach combining an advanced medicinal therapeutic device and a fracture fixation assembly for the treatment of osteoporotic fractures: Effects of physiological loading, interface conditions, and fracture fixation materials.

The occurrence of periprosthetic femoral fractures (PFF) has increased in people with osteoporosis due to decreased bone density, poor bone quality, and stress shielding from prosthetic implants. PFF treatment in the elderly is a genuine concern for orthopaedic surgeons as no effective solution currently exists. Therefore, the goal of this study was to determine whether the design of a novel advanced medicinal therapeutic device (AMTD) manufactured from a polymeric blend in combination with a fracture fixation plate in the femur is capable of withstanding physiological loads without failure during the bone regenerative process. This was achieved by developing a finite element (FE) model of the AMTD together with a fracture fixation assembly, and a femur with an implanted femoral stem. The response of both normal and osteoporotic bone was investigated by implementing their respective material properties in the model. Physiological loading simulating the peak load during standing, walking, and stair climbing was investigated. The results showed that the fixation assembly was the prime load bearing component for this configuration of devices. Within the fixation assembly, the bone screws were found to have the highest stresses in the fixation assembly for all the loading conditions. Whereas the stresses within the AMTD were significantly below the maximum yield strength of the device's polymeric blend material. Furthermore, this study also investigated the performance of different fixation assembly materials and found Ti-6Al-4V to be the optimal material choice from those included in this study.

A Computational Model for the Release of Bioactive Molecules by the Hydrolytic Degradation of a Functionalized Polyester-Based Scaffold.

This work presents a computational model to study the degradation behavior of polyester-based three-dimensional (3D) functionalized scaffolds for bone regeneration. As a case study, we investigated the behavior of a 3D-printed scaffold presenting a functionalized surface with ICOS-Fc, a bioactive protein able to stimulate bone regeneration and healing, inhibiting osteoclast activity. The aim of the model was to optimize the scaffold design to control its degradation and thus the release of grafted protein over time and space. Two different scenarios were considered: (i) a scaffold without macroporosity presenting a functionalized external surface; and (ii) a scaffold presenting an internal functionalized macroporous architecture with open channels to locally deliver the degradation products.

Promotion of In Vitro Osteogenic Activity by Melt Extrusion-Based PLLA/PCL/PHBV Scaffolds Enriched with Nano-Hydroxyapatite and Strontium Substituted Nano-Hydroxyapatite.

Bone tissue engineering has emerged as a promising strategy to overcome the limitations of current treatments for bone-related disorders, but the trade-off between mechanical properties and bioactivity remains a concern for many polymeric materials. To address this need, novel polymeric blends of poly-L-lactic acid (PLLA), polycaprolactone (PCL) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) have been explored. Blend filaments comprising PLLA/PCL/PHBV at a ratio of 90/5/5 wt% have been prepared using twin-screw extrusion. The PLLA/PCL/PHBV blends were enriched with nano-hydroxyapatite (nano-HA) and strontium-substituted nano-HA (Sr-nano-HA) to produce composite filaments. Three-dimensional scaffolds were printed by fused deposition modelling from PLLA/PCL/PHBV blend and composite filaments and evaluated mechanically and biologically for their capacity to support bone formation in vitro. The composite scaffolds had a mean porosity of 40%, mean pores of 800 µm, and an average compressive modulus of 32 MPa. Polymer blend and enriched scaffolds supported cell attachment and proliferation. The alkaline phosphatase activity and calcium production were significantly higher in composite scaffolds compared to the blends. These findings demonstrate that thermoplastic polyesters (PLLA and PCL) can be combined with polymers produced via a bacterial route (PHBV) to produce polymer blends with excellent biocompatibility, providing additional options for polymer blend optimization. The enrichment of the blend with nano-HA and Sr-nano-HA powders enhanced the osteogenic potential in vitro.

Biomaterial Inks from Peptide-Functionalized Silk Fibers for 3D Printing of Futuristic Wound-Healing and Sensing Materials.

This study illustrates the sensing and wound healing properties of silk fibroin in combination with peptide patterns, with an emphasis on the printability of multilayered grids, and envisions possible applications of these next-generation silk-based materials. Functionalized silk fibers covalently linked to an arginine-glycine-aspartic acid (RGD) peptide create a platform for preparing a biomaterial ink for 3D printing of grid-like piezoresistors with wound-healing and sensing properties. The culture medium obtained from 3D-printed silk fibroin enriched with RGD peptide improves cell adhesion, accelerating skin repair. Specifically, RGD peptide-modified silk fibroin demonstrated biocompatibility, enhanced cell adhesion, and higher wound closure rates at lower concentration than the neat peptide. It was also shown that the printing of peptide-modified silk fibroin produces a piezoresistive transducer that is the active component of a sensor based on a Schottky diode harmonic transponder encoding information about pressure. We discovered that such biomaterial ink printed in a multilayered grid can be used as a humidity sensor. Furthermore, humidity activates a transition between low and high conductivity states in this medium that is retained unless a negative voltage is applied, paving the way for utilization in non-volatile organic memory devices. Globally, these results pave the way for promising applications, such as monitoring parameters such as human wound care and being integrated in bio-implantable processors.

Biocompatible and Printable Ionotronic Sensing Materials Based on Silk Fibroin and Soluble Plant-Derived Polyphenols.

The emergence of ionotronic materials has been recently exploited for interfacing electronics and biological tissues, improving sensing with the surrounding environment. In this paper, we investigated the synergistic effect of regenerated silk fibroin (RS) with a plant-derived polyphenol (i.e., chestnut tannin) on ionic conductivity and how water molecules play critical roles in regulating ion mobility in these materials. In particular, we observed that adding tannin to RS increases the ionic conductivity, and this phenomenon is accentuated by increasing the hydration. We also demonstrated how silk-based hybrids could be used as building materials for scaffolds where human fibroblast and neural progenitor cells can highly proliferate. Finally, after proving their biocompatibility, RS hybrids demonstrate excellent three-dimensional (3D) printability via extrusion-based 3D printing to fabricate a soft sensor that can detect charged objects by sensing the electric fields that originate from them. These findings pave the way for a viable option for cell culture and novel sensors, with the potential base for tissue engineering and health monitoring.

Engineering Auxetic Cylinders and Intestine to Improve Longitudinal Intestinal Lengthening and Tailoring Procedure.

Auxetic materials can be exploited for coupling different types of tissues. Herein, we designed a material where the microorganism metabolic activity yields the formation of buckled/collapsed bubbles within gelling silicone cylinders thus providing auxetic properties. The finite element model of such hollow auxetic cylinders demonstrated the tubular structure to promote worm-like peristalsis. In this scenario, the described hybrid auxetic structures may be applied to the longitudinal intestinal lengthening and tailoring procedure to promote enteral autonomy in short bowel syndrome. The presented material and analytical design synergistic approach offer a pioneering step for the clinical translation of hybrid auxetic materials.