Cryogenic 3D Research Articles

Cryogenic 3D Printing of GelMA/Graphene Bioinks: Improved Mechanical Strength and Structural Properties for Tissue Engineering.

Tissue engineering aims to recreate natural cellular environments to facilitate tissue regeneration. Gelatin methacrylate (GelMA) is widely utilized for its biocompatibility, ability to support cell adhesion and proliferation, and adjustable mechanical characteristics. This study developed a GelMA and graphene bioink platform at concentrations of 1, 1.5, and 2 mg/mL to enhance scaffold properties for tissue engineering applications. Graphene was incorporated into GelMA matrices to improve mechanical strength and electrical conductivity of the bioinks. The compressive strength and thermal stability of the resulting GelMA/graphene scaffolds were assessed through DSC analysis and mechanical testing. Cytotoxicity assays were conducted to determine cell survival rates. Cryoprinting at -30°C was employed to preserve scaffold structure and function. The chorioallantoic membrane (CAM) assay was used to evaluate biocompatibility and angiogenic potential. The integration of graphene significantly amplified the compressive strength and thermal stability of GelMA scaffolds. Cytotoxicity assays indicated robust cell survival rates of 90%, confirming the biocompatibility of the developed materials. Cryoprinting effectively preserved scaffold integrity and functionality. The CAM assay validated the biocompatibility and angiogenic potential, demonstrating substantial vascularization upon scaffold implantation onto chick embryo CAM. Integrating graphene into GelMA hydrogels, coupled with low-temperature 3D printing, represents a potent strategy for enhancing scaffold fabrication. The resultant GelMA/graphene scaffolds exhibit superior mechanical properties, biocompatibility, and pro-vascularization capabilities, making them highly suitable for diverse tissue engineering and regenerative medicine applications.

Spatiotemporal delivery of BMP-2 and FGF-18 in 3D-bioprinted tri-phasic osteochondral scaffolds enhanced compartmentalized osteogenic and chondrogenic differentiation of mesenchymal stem cells isolated from rats with varied organizational morphologies

Replicating the heterogeneous structure and promoting compartmentalized osteogenesis/chondrogenesis are critical considerations in designing scaffolds for osteochondral tissue regeneration. However, desirable osteochondral regeneration cannot be achieved mainly due to the absence of effective delivery strategies for growth factors (GFs) and the insufficiency of desirable organizational morphologies for seed cells. Herein, we developed a tri-phasic osteochondral scaffold consisting of bone morphogenetic protein-2 (BMP-2)-loaded subchondral layer, fibroblast growth factor-18 (FGF-18)-loaded cartilage layer, and an interface layer that acted as a barrier to reduce the mutual interference of GFs, via cryogenic 3D bioprinting. BMP-2 could exert osteogenic effects for 14 days, and FGF-18 could exert chondrogenic effects for 21 days, demonstrating the time-controlled release function of BMP-2 and FGF-18. By further seeding discrete rat bone marrow mesenchymal stem cells (rBMSCs) and rBMSC microspheres, respectively, onto the subchondral layer and cartilage layer, the engineered cell-laden osteochondral tissue was constructed. The spatiotemporal release of BMP-2 and FGF-18 in the subchondral layer and cartilage layer promoted the osteogenic differentiation of discrete rBMSCs and chondrogenic differentiation of rBMSC microspheres in the subchondral layer and cartilage layer, respectively. In summary, by seeding rBMSCs with varied organizational morphologies in 3D-printed osteochondral scaffolds with a spatiotemporally controlled strategy, engineered osteochondral tissue with compartmentalized osteogenic/chondrogenic differentiation potent can be formed, displaying a facile and promising way to achieve desirable osteochondral tissue regeneration.

Cryogenic temperature 3D mapping via a distributed temperature sensor with centimeter resolution

We conduct 3D mapping of cryogenic temperatures via a Raman-based distributed temperature sensor, employing standard telecom single-mode fibers and polarization-independent superconducting nanowire single photon detectors (SNSPDs). By coiling a test fiber around various stages of a liquid helium cooled cryostat, our device demonstrates a lower temperature sensing limit of (48 ± 2) K, below the nitrogen boiling point. This achievement is made possible by the low dark count rates of SNSPDs, as validated by theoretical simulations. Furthermore, we utilize our device to map cryogenic temperatures on the 350 cm2 surface of a specially designed hollow cylindrical aluminum sample, accommodating approximately 2 m of standard single-mode optical fiber. During nitrogen cooling, we monitor the temporal evolution of the spatially dependent temperature gradient on the metallic sample with a temporal sampling down to one minute. Fiber-based distributed temperature sensing with centimetric spatial resolution can be effectively applied for 3D mapping at cryogenic temperatures of superconducting, quantum computing and aerospace instrumentation.

Functionalized BP@(Zn+Ag)/EPLA Nanofibrous Scaffolds Fabricated by Cryogenic 3D Printing for Bone Tissue Engineering.

This study fabricates a functionalized scaffold by cryogenic three-dimensional (3D) printing using an aminated poly-L-lactic acid (EPLA) solution containing nanosilver/zinc-coated black phosphorus (BP@(Zn+Ag)) nanocomposites. The nanocomposites are prepared by a green method of in situ photodeposition of silver and zinc nanoparticles (AgNPs and ZnNPs) on BP nanosheets (BPNs) under visible light irradiation without any chemical reductant. Scanning electron microscope (SEM) and X-ray energy dispersive spectrometer (EDS) confirm the uniform distribution of BP@(Zn+Ag) nanoparticles in the EPLA nanofibrous matrix. The in vitro tests show that the fabricated BP@(Zn+Ag)/EPLA nanofibrous scaffold exhibits excellent antibacterial activity (over 96%) against E. coli and S. aureus, as well as enhanced cell viability and osteogenic activity to facilitate the growth and differentiation of osteoblasts. The in vivo rat calvarial defect model also demonstrates that the BP@(Zn+Ag)/EPLA nanofibrous scaffold promotes new bone tissue formation around the implant site. Therefore, the prepared multifunctional 3D printed BP@(Zn+Ag)/EPLA nanofibrous scaffold has great potential for bone tissue engineering (BTE) applications.

Study on mechanical properties of dual-channel cryogenic 3D printing scaffold for mandibular defect repair.

Mandibular defect repair has always been a clinical challenge, facing technical bottleneck. The new materials directly affect technological breakthroughs in mandibular defect repair field. Our aim is to fabricate a scaffold of advanced biomaterials for repairing of small mandibular defect. Therefore, a novel dual-channel scaffold consisting of silk fibroin/collagen type-I/hydroxyapatite (SCH) and polycaprolactone/hydroxyapatite (PCL/HA) was fabricated by cryogenic 3D printing technology with double nozzles. The mechanical properties and behaviors of the dual-channel scaffold were investigated by performing uniaxial compression, creep, stress relaxation, and ratcheting experiments respectively. The experiments indicated that the dual-channel scaffold was typical non-linear viscoelastic consistent with cancellous tissue; the Young's modulus of this scaffold was 60.1kPa. Finite element analysis (FEA) was employed performing a numerical simulation to evaluate the implantation effect in mandible. The stress distribution of the contact area between scaffold and defect was uniform, the maximum Mises stress of cortical bone and cancellous bone in defect area were 54.520MPa and 3.196MPa, and the maximum displacement of cortical bone and cancellous bone in defect area were 0.1575mm and 0.1555mm respectively, which distributed in the incisor region. The peak maximum Mises stress experienced by the implanted scaffold was 3.128 × 10-3MPa, and the maximum displacement was 6.453 × 10-2mm distributed near incisor area. The displacement distribution of the scaffold was consistent with that of cortical and cancellous bone. The scaffold recovered well when the force applied on it disappeared. Above all, the dual-channel scaffold had excellent bio-mechanical properties in implanting mandible, which provides a new idea for the reconstruction of irregular bone defects in the mandible and has good clinical development prospects.

Three dimensional printed wound dressings: recent progresses.

Three dimensional printed wound dressings: recent progresses.

Vibrationally- and rotationally-resolved photoelectron imaging of cryogenically-cooled SbO2 –

We report a temperature-controlled photoelectron imaging study of SbO2 –, produced from a laser vaporisation source and cooled in a cryogenic 3D Paul trap. Vibrationally resolved photoelectron spectra are obtained for the ground state detachment transition, yielding the bending frequencies for both SbO2 and SbO2 –. Franck-Condon simulations also allow the estimate of the vibrational temperature of the trapped SbO2 – anion. A near-threshold spectrum of SbO2 – at a photon energy of 3.4958 eV reveals partially resolved rotational structure for the 0–0 transition, which yields an accurate electron affinity of 3.4945 ± 0.0004 eV for SbO2. The rotational simulation also yields an estimated rotational temperature of the trapped ions.

MXene-modified 3D printed scaffold for photothermal therapy and facilitation of oral mucosal wound reconstruction

The most prevalent oral and maxillofacial cancer is oral squamous cell carcinoma (OSCC). Patient survival is compromised by relapse due to post-operative tumor remnants and significant mucosal defects. Photothermal therapy (PTT) is used to ablate local malignancies, but the capacity of tumor cells to resist it is correlated with the overexpression of heat shock protein 70 (HSP70). In this work, PTT and tissue engineering scaffold were rationally integrated to construct a Ti3C2 MXene, collagen, silk fibroin and quercetin composite scaffold (M-CSQ scaffold), a 3D printed biomaterial that simultaneously kill OSCC cells and promote the regeneration of the mucosal defects. The M-CSQ scaffolds were prepared by cryogenic 3D printing and freeze-drying techniques with sufficient pores that allow an abundant cell migration and provide a proliferation space. Ti3C2 MXene has excellent photothermal conversion ability and stability. Quercetin targeted HSP70 to decrease its expression in OSCC cells, consequently weakening their resistance to high temperature and enhancing the effect of PTT. The M-CSQ scaffold effectively killed OSCC cells in vitro and inhibited tumor growth in vivo. In addition, the M-CSQ scaffold provided adhesion sites for Sprague-Dawley rat (SD rat) buccal mucosal fibroblasts and promoted the repair of buccal mucosal wounds.

Hierarchical porous ECM scaffolds incorporating GDF-5 fabricated by cryogenic 3D printing to promote articular cartilage regeneration

BackgroundIn recent years, there has been significant research progress on in situ articular cartilage (AC) tissue engineering with endogenous stem cells, which uses biological materials or bioactive factors to improve the regeneration microenvironment and recruit more endogenous stem cells from the joint cavity to the defect area to promote cartilage regeneration.MethodIn this study, we used ECM alone as a bioink in low-temperature deposition manufacturing (LDM) 3D printing and then successfully fabricated a hierarchical porous ECM scaffold incorporating GDF-5.ResultsComparative in vitro experiments showed that the 7% ECM scaffolds had the best biocompatibility. After the addition of GDF-5 protein, the ECM scaffolds significantly improved bone marrow mesenchymal stem cell (BMSC) migration and chondrogenic differentiation. Most importantly, the in vivo results showed that the ECM/GDF-5 scaffold significantly enhanced in situ cartilage repair.ConclusionIn conclusion, this study reports the construction of a new scaffold based on the concept of in situ regeneration, and we believe that our findings will provide a new treatment strategy for AC defect repair.

A Novel Cryogenic Approach to 3D Printing Cytocompatible, Conductive, Hydrogel-Based Inks.

In the field of tissue engineering and regenerative medicine, developing cytocompatible 3D conductive scaffolds that mimic the native extracellular matrix is crucial for the engineering of excitable cells and tissues. In this study, a custom cryogenic extrusion 3D printer was developed, which afforded control over both the ink and printing surface temperatures. Using this approach, aqueous inks were printed into well-defined layers with high precision. A conductive hydrogel ink was developed from chitosan (CS) and edge-functionalised expanded graphene (EFXG). Different EFXG:CS ratios (between 60:40 and 80:20) were evaluated to determine both conductivity and printability. Using the novel customized cryogenic 3D printer, conductive structures of between 2 and 20 layers were produced, with feature sizes as small as 200 μm. The printed structures are mechanically robust and are electrically conducting. The highest Young's modulus and conductivity in a hydrated state were 2.6 MPa and ∼45 S/m, respectively. Cytocompatibility experiments reveal that the developed material supports NSC-34 mouse motor neuron-like cells in terms of viability, attachment, and proliferation. The distinctive mechanical and electrical properties of the 3D-printed structures would make them good candidates for the engineering of 3D-structured excitable cells. Moreover, this novel printing setup can be used to print other hydrogel-based inks with high precision and resolution.

Phase behavior of hydrogel bioinks for cryogenic 3D printing

Phase behavior of hydrogel bioinks for cryogenic 3D printing

Photoelectron imaging of cryogenically cooled BiO- and BiO2 - anions.

The advent of ion traps as cooling devices has revolutionized ion spectroscopy as it is now possible to efficiently cool ions vibrationally and rotationally to levels where truly high-resolution experiments are now feasible. Here, we report the first results of a new experimental apparatus that couples a cryogenic 3D Paul trap with a laser vaporization cluster source for high-resolution photoelectron imaging of cold cluster anions. We have demonstrated the ability of the new apparatus to efficiently cool BiO- and BiO2 - to minimize vibrational hot bands and allow high-resolution photoelectron images to be obtained. The electron affinities of BiO and BiO2 are measured accurately for the first time to be 1.492(1) and 3.281(1) eV, respectively. Vibrational frequencies for the ground states of BiO and BiO2, as well as those for the anions determined from temperature-dependent studies, are reported.

Cryogenic 3D printing of bifunctional silicate nanoclay incorporated scaffolds for promoted angiogenesis and bone regeneration

It remains challenging to manage critical-sized bone defects owing to insufficient vascularization. Tissue engineering scaffolds with favorable mechanical strength and excellent bone regenerative ability/angiogenic properties are recognized as promising platforms for bone defects. Various osteoinductive/angiogenic growth factors have been incorporated into scaffolds to enhance bone formation with the required vascularization. However, the instability and ease of inactivation of growth factors under certain physiological conditions limits their effectiveness. In the present study, a bifunctional laponite (LAP) with potent ability to induce both osteogenesis and angiogenesis was incorporated into poly(lactide-coglycolide)/β-tricalcium phosphate (PLGA/β-TCP) composite to form a porous scaffold through micro extrusion-based cryogenic three-dimensional printing. The hierarchically porous PLGA/β-TCP/LAP composite scaffold exhibited favorable initial mechanical strength and displayed a promoted effect towards cell adhesion of rat bone marrow derived mesenchymal stem cells and endothelial progenitor cells. Enhanced in vitro angiogenesis and osteogenesis were simultaneously achieved due to the proangiogenic and osteoinductive ions released from LAP. Furthermore, the PLGA/β-TCP/LAP scaffold promoted the generation of type H vessel and bony regeneration in vivo. Overall, the impartment of inorganic LAP into 3D printed PLGA/β-TCP scaffold provides a simple and efficient way to realize the treatment of critical-sized bone defects via improved angiogenesis and osteogenesis.

Cryogenic 3D Printing of w/o Pickering Emulsions Containing Bifunctional Drugs for Producing Hierarchically Porous Bone Tissue Engineering Scaffolds with Antibacterial Capability

How to fabricate bone tissue engineering scaffolds with excellent antibacterial and bone regeneration ability has attracted increasing attention. Herein, we produced a hierarchical porous β-tricalcium phosphate (β-TCP)/poly(lactic-co-glycolic acid)-polycaprolactone composite bone tissue engineering scaffold containing tetracycline hydrochloride (TCH) through a micro-extrusion-based cryogenic 3D printing of Pickering emulsion inks, in which the hydrophobic silica (h-SiO2) nanoparticles were used as emulsifiers to stabilize composite Pickering emulsion inks. Hierarchically porous scaffolds with desirable antibacterial properties and bone-forming ability were obtained. Grid scaffolds with a macroscopic pore size of 250.03 ± 75.88 μm and a large number of secondary micropores with a diameter of 24.70 ± 15.56 μm can be fabricated through cryogenic 3D printing, followed by freeze-drying treatment, whereas the grid structure of scaffolds printed or dried at room temperature was discontinuous, and fewer micropores could be observed on the strut surface. Moreover, the impartment of β-TCP in scaffolds changed the shape and density of the micropores but endowed the scaffold with better osteoconductivity. Scaffolds loaded with TCH had excellent antibacterial properties and could effectively promote the adhesion, expansion, proliferation, and osteogenic differentiation of rat bone marrow-derived mesenchymal stem cells afterward. The scaffolds loaded with TCH could realize the strategy to “kill bacteria first, then induce osteogenesis”. Such hierarchically porous scaffolds with abundant micropores, excellent antibacterial property, and improved bone-forming ability display great prospects in treating bone defects with infection.

Multimodal freezing system for cryogenic 3D printing

Cryogenic 3D printing (Cryo-3DP) technique is used to realize the frozen 3D objects by layered deposition of the liquids inside an insulated chamber. The operating temperature of the process ranges from − 20 to − 25 °C. Cryo-3DP demands high cooling rates initially to reach the process temperature followed by lower cooling rates to sustain it. Multimodal freezing system proposed in this study uses more than one cooling mode that helps in achieving the variable cooling rates. The present system uses two modes of cooling, viz., vapor compression refrigeration (VCR) and CO2 injection. A detailed design of the multimodal freezing system is carried out for the chamber size of 200 mm × 200 mm × 195 mm. The performance of the system is analysed numerically. The results were experimentally validated using the prototype of the multimodal system developed as a part of the present study. The results show that the multimodal system reduces the initial cooling time substantially by providing a high cooling rate that rapidly cools the chamber to initiate the cryo-3DP. VCR system provides a low but sustained cooling rate that maintains the desired temperature. The present study proves the significance of multimodality in cooling system that is fit to deploy in a commercial cryogenic 3D printer.

Recent Advances in Cryogenic 3D Printing Technologies

Cryogenic 3D printing, or freeze 3D printing, is an additive manufacturing process that prints an object at “low” temperature atmosphere. It uses a temperature lower than the melting point of the printing material (commonly a water‐based suspension or slurry) to solidify the layered part. Cryogenic 3D printing compasses several different processes including ice 3D printing, freeze from extrusion, freeze nano‐printing (FNP), etc. However, no systematic survey of these technologies is present. In the current review, various 3D printing technologies that use low temperature to solidify the layer are reviewed. The technical aspects such as path planning, heat transfer, and diffusion in multi‐material are investigated. The applications of cryogenic 3D printing are presented including the investment casting, ice sculpture, and microfluidic channel from original ice 3D printing, and functional porous materials from the emerging FNP and low‐temperature deposition. Overall, this article gives a summary of cryogenic 3D printing technologies, including a survey on its technical aspects and potential applications for future research and development.

The fabrication of multifunctional sodium alginate scaffold incorporating ibuprofen-loaded modified PLLA microspheres based on cryogenic 3D printing

A strategy to develop a multifunctional sodium alginate personalized scaffold with enhanced mechanical stability, osteogenesis activity and excellent anti-inflammatory activity by cryogenic 3 D printing combined with subsequent crosslinking with Sr2+ is proposed in this study. The ink for 3 D printing was prepared by dispersing modified PLLA droplets containing ibuprofen into sodium alginate aqueous solution using lecithin as stabilizer. The results showed that the drug-loaded microspheres formed from the low-temperature solidifying of the modified PLLA droplets were homogeneously dispersed in sodium alginate substrate, and the scaffold displayed a sustained drug release performance toward ibuprofen which endowed the scaffold with persistent anti-inflammatory effects. In vitro cell culture indicated that the lecithin not only acted as the stabilizer, but also stimulated the proliferation and mineralization of osteoblastic cells on the scaffold. Sr2+-crosslinking improved the mechanical properties and osteogenic activity of the scaffold.

Cryogenically 3D printed biomimetic scaffolds containing decellularized small intestinal submucosa and Sr2+/Fe3+ co-substituted hydroxyapatite for bone tissue engineering

• Novel SIS/SrFeHA biomimetic scaffolds are developed by extrusion cryogenic 3D printing. • The cryogenic printing endows scaffolds with improved porous and bioactive features. • SIS/SrFeHA coordinates favorable immunomodulation with enhanced angio-/osteogenesis and biomineralization. • A new finding of angiogenic mechanism of SIS/SrFeHA is clarified to be Amot + /YAP − /Hippo signaling pathway. • Combination of SIS and SrFeHA provides a new potential for bone tissue engineering. Currently, advanced bone biomaterials are preferred able to mimic the architecture/composition of natural bone and possess sufficient biological multifunctionality to chronologically dictate bone regeneration events. Such biomaterials are increasingly being researched and quite promising in bone tissue engineering. In this view, novel biomimetic scaffolds composed of decellularized small intestinal submucosa matrix (SIS-ECM) and Sr 2+ /Fe 3+ co-substituted hydroxyapatite (SrFeHA) were constructed via extrusion cryogenic 3D printing method. The produced SIS/SrFeHA scaffolds revealed desirable 3D interconnected macro/micro-porous structures, rough microsurfaces and improved mechanical strength, along with appreciable synergic releasing of bioactive ECM components and Sr 2+ /Fe 3+ . These favorable physico-chemical properties rendered composite SIS/SrFeHA inducing a highly biomimetic bony environment which manipulated desired immunoregulation coupled with enhanced angiogenesis, osteogenesis, and biomineralization. Consistently, in vivo cranial defects and subcutaneous implantation outcomes yet affirmed the superior ability of SIS/SrFeHA to well dictate early immune reaction, neovascularization, and in vivo bone regeneration, suggesting the potent combined actions of SIS and SrFeHA to accelerate bone healing. Moreover, as examined by RNA sequencing assay, a new finding of up-regulated Amot + /YAP − /Hippo signaling pathway responsible for the enhanced angiogenesis in SIS/SrFeHA scaffolds was further clarified. Consequently, for the first time, present research highlights the considerable potential of SIS/SrFeHA scaffolds for inducing bone healing and simultaneously afford a new finding for better understanding underlying mechanism of bioactive SrFeHA components, thus may generating a new promising alternative for future bone tissue engineering.

Cryogenic 3D Printing of ß-TCP/PLGA Composite Scaffolds Incorporated With BpV (Pic) for Treating Early Avascular Necrosis of Femoral Head.

Avascular necrosis of femoral head (ANFH) is a disease that is characterized by structural changes and collapse of the femoral head. The exact causes of ANFH are not yet clear, but small advances in etiopathogenesis, diagnosis and treatment are achieved. In this study, ß-tricalcium phosphate/poly lactic-co-glycolic acid composite scaffolds incorporated with bisperoxovanadium [bpV (pic)] (bPTCP) was fabricated through cryogenic 3D printing and were utilized to treat rat models with early ANFH, which were constructed by alcohol gavage for 6 months. The physical properties of bPTCP scaffolds and in vitro bpV (pic) release from the scaffolds were assessed. It was found that the sustained release of bpV (pic) promoted osteogenic differentiation and inhibited adipose differentiation of bone marrow-derived mesenchymal stem cells. Micro-computed tomography scanning and histological analysis confirmed that the progression of ANFH in rats was notably alleviated in bPTCP scaffolds. Moreover, it was noted that the bPTCP scaffolds inhibited phosphatase and tensin homolog and activated the mechanistic target of rapamycin signaling. The autophagy induced by bPTCP scaffolds could partially prevent apoptosis, promote osteogenesis and angiogenesis, and hence eventually prevent the progression of ANFH, suggesting that the bPTCP scaffold are promising candidate to treat ANFH.

Cryogenic 3D printing of modified polylactic acid scaffolds with biomimetic nanofibrous architecture for bone tissue engineering

The individualized polylactic acid (PLA) scaffolds fabricated by 3D printing technique have a good application prospect in the bone tissue engineering field. However, 3D printed PLA scaffold mainly manufactured by using a Fused Deposition Modelling fabrication technique (FDM) has some disadvantages, such as having smooth surface, strong hydrophobicity, poor cell adhesion, undesirable bioactivity, the degradation and deterioration at a high temperature triggering an inflammatory response. In this work, the aminated modified polylactic acid nanofibrous scaffold prepared by cryogenic 3D printing technology is designed to provide a feasible countermeasure to solve the key problems existing at present. The prepared scaffolds were fully characterized in terms of physico-chemical and morphological analyses, and the collected results revealed that the using of the cryogenic 3D printing technology can effectively avoid the degradation and deterioration of PLA at a high temperature required by FDM technique and promote the formation of nanofibrous structures. The in vitro tests with MC3T3-E1 cells confirmed that the cell-responsive biomimetic fibrous architecture and improved hydrophilicity due to the introduction of hydrophilic active amino groups provided a bioactive interface for cell adhesion and growth. Meanwhile, the active amino groups introduced by ammonolysis reaction can act as active sites for biomineralization. Thus, the as-prepared scaffolds may hold great potential for bone tissue engineering applications.