In-Bath 3D Printing of Anisotropic Shape-Memory Cryogels Functionalized with Bone-Bioactive Nanoparticles.

Cryogels exhibit unique shape memory with full recovery and structural stability features after multiple injections. These constructs also possess enhanced cell permeability and nutrient diffusion when compared to typical bulk hydrogels. Volumetric processing of cryogels functionalized with nanosized units has potential to widen their biomedical applications, however this has remained challenging and relatively underexplored. In this study, we report a novel methodology that combines suspension 3D printing with directional freezing for the fabrication of nanocomposite cryogels with configurable anisotropy. When compared to conventional bulk or freeze-dried hydrogels, nanocomposite cryogel formulations exhibit excellent shape recovery (>95%) and higher pore connectivity. Suspension printing, assisted with a prechilled metal grid, was optimized to induce anisotropy. The addition of calcium- and phosphate-doped mesoporous silica nanoparticles into the cryogel matrix enhanced bioactivity toward orthopedic applications without hindering the printing process. Notably, the nanocomposite 3D printed cryogels exhibit injectable shape memory while also featuring a lamellar topography. The fabrication of these constructs was highly reproducible and exhibited potential for a cell-delivery injectable cryogel with no cytotoxicity to human-derived adipose stem cells. Hence, in this work, it was possible to combine a gravity defying 3D printed methodology with injectable and controlled anisotropic macroporous structures containing bioactive nanoparticles. This methodology ameliorates highly tunable injectable 3D printed anisotropic nanocomposite cryogels with a user-programmable degree of structural complexity.

Silicone cryogel skeletons enhance the survival and mechanical integrity of hydrogel-encapsulated cell therapies.

The transplantation of engineered cells that secrete therapeutic proteins presents a promising method for addressing a range of chronic diseases. However, hydrogels used to encase and protect non-autologous cells from immune rejection often suffer from poor mechanical properties, insufficient oxygenation, and fibrotic encapsulation. Here, we introduce a composite encapsulation system comprising an oxygen-permeable silicone cryogel skeleton, a hydrogel matrix, and a fibrosis-resistant polymer coating. Cryogel skeletons enhance the fracture toughness of conventional alginate hydrogels by 23-fold and oxygen diffusion by 2.8-fold, effectively mitigating both implant fracture and hypoxia of encapsulated cells. Composite implants containing xenogeneic cells engineered to secrete erythropoietin significantly outperform unsupported alginate implants in therapeutic delivery over 8 weeks in immunocompetent mice. By improving mechanical resiliency and sustaining denser cell populations, silicone cryogel skeletons enable more durable and miniaturized therapeutic implants.

Nitric oxide-releasing photocrosslinked chitosan cryogels.

The highly porous morphology of chitosan cryogels, with submicrometric-sized pore cell walls, provides a large surface area which leads to fast water absorption and elevated swelling degrees. These characteristics are crucial for the applications of nitric oxide (NO) releasing biomaterials, in which the release of NO is triggered by the hydration of the material. In the present study, we report the development of chitosan cryogels (CS) with a porous structure of interconnected cells, with wall thicknesses in the range of 340-881 nm, capable of releasing NO triggered by the rapid hydration process. This property was obtained using an innovative strategy based on the functionalization of CS with two previously synthesized S-nitrosothiols: S-nitrosothioglycolic acid (TGA(SNO)) and S-nitrosomercaptosuccinic acid (MSA(SNO)). For this purpose, CS was previously methacrylated with glycidyl methacrylate and subsequently submitted to photocrosslinking and freeze-drying processes. The photocrosslinked hydrogels thus obtained were then functionalized with TGA(SNO) and MSA(SNO) in reactions mediated by carbodiimide. After functionalization, the hydrogels were frozen and freeze-dried to obtain porous S-nitrosated chitosan cryogels with high swelling capacities. Through chemiluminescence measurements, we demonstrated that CS-TGA(SNO) and CS-MSA(SNO) cryogels spontaneously release NO upon water absorption at rates of 3.34 × 10-2 nmol mg-1 min-1 and 1.27 × 10-1 nmol mg-1 min-1, respectively, opening new perspectives for the use of CS as a platform for localized NO delivery in biomedical applications.

The study of double-network carboxymethyl chitosan/sodium alginate based cryogels for rapid hemostasis in noncompressible hemorrhage.

Developing an injectable hemostatic dressing with shape recovery and high blood absorption ratio for rapid hemostasis in noncompressible hemorrhage maintains a critical clinical challenge. Here, double-network cryogels based on carboxymethyl chitosan, sodium alginate, and methacrylated sodium alginate were prepared by covalent crosslinking and physical crosslinking, and named carboxymethyl chitosan/methacrylated sodium alginate (CM) cryogels. Covalent crosslinking was achieved by methacrylated sodium alginate in the freeze casting process, while physical crosslinking was realized by electrostatic interaction between the amino group of carboxymethyl chitosan and the carboxyl group of sodium alginate. CM cryogels exhibited large water swelling ratios (8167 ± 1062 %), fast blood absorption speed (2974 ± 669 % in 15 s), excellent compressive strength (over 160 kPa for CM100) and shape recovery performance. Compared with gauze and commercial gelatin sponge, better hemostatic capacities were demonstrated for CM cryogel with the minimum blood loss of 40.0 ± 8.9 mg and the lowest hemostasis time of 5.0 ± 2.0 s at hemostasis of rat liver. Made of natural polysaccharides with biocompatibility, hemocompatibility, and cytocompatibility, the CM cryogels exhibit shape recovery and high blood absorption rate, making them promising to be used as an injectable hemostatic dressing for rapid hemostasis in noncompressible hemorrhage.

Preparation and assessment of agar/TEMPO-oxidized bacterial cellulose cryogels for hemostatic applications.

In this work, we have demonstrated agar and oxidized bacterial cellulose cryogels as a potential hemostatic dressing material. TEMPO-oxidized bacterial cellulose (OBC) was incorporated into the agar matrix, improving its mechanical and hemostatic properties. The oxidation of bacterial cellulose (BC) was evidenced by chemical characterization studies, confirming the presence of carboxyl groups. The in vitro blood clotting test conducted on agar/OBC composite cryogels demonstrated complete blood clotting within 90 seconds, indicating their excellent hemostatic efficacy. The cryogels exhibited superabsorbent properties with a swelling degree of 4200%, enabling them to absorb large amounts of blood. Moreover, the compressive strength of the composite cryogels was appreciably improved compared to pure agar, resulting in a more stable physical structure. The platelet adhesion test proved the significant ability of the composite cryogels to adhere to and aggregate platelets. Hemocompatibility and cytocompatibility tests have verified the safety of these cryogels for hemostatic applications. Finally, the material exhibited remarkable in vivo hemostatic performance, achieving clotting times of 64 seconds and 35 seconds when tested in the rat tail amputation model and the liver puncture model, respectively. The experiment results were compared with those of commercial hemostat, Axiostat, and Surgispon, affirming the potential of agar/OBC composite cryogel as a hemostatic dressing material.

Killing Two Birds with One Stone: Shape-Memory Cryogels for Hemostasis and Wound Repair.

The development of shape-memory hemostatic agents is crucial for the treatment of deep incompressible bleeding tissue. However, there are few reports on biomaterials that can monitor bacterial infection at the wound site in real time following hemostasis and effectively promote repair. In this study, we propose a multifunctional QCSG/FLZ cryogel composed of glycidyl methacrylate-functionalized quaternary chitosan (QCSG), fluorescein isothiocyanate (FITC), and a lysozyme (LYZ)-modified zeolitic imidazolate framework (ZIF-8) for incompressible bleeding tissue hemostasis and wound repair. QCSG/FLZ cryogels possess interconnected microporous structure and enhanced mechanical properties, allowing them to be molded into different shapes for effective hemostasis in deep incompressible wounds. Furthermore, the fluorescence quench signal of QCSG/FLZ cryogels enables timely monitoring of bacterial infection when wound triggers infection. Meanwhile, the acidic microenvironment of bacterial infection induces structural lysis of ZIF-8, releasing LYZ and Zn2+, which effectively kill bacteria and accelerate wound repair. In conclusion, our study not only provides potential application of QCSG/FLZ cryogels for hemostasis in deep incompressible wounds but promisingly promotes the development of a tissue repair technique.

Laser-assisted synthesis of nano-hydroxyapatite and functionalization with bone active molecules for bone regeneration.

The main goal of bone tissue engineering research is to replace the allogenic and autologous bone graft substitutes that can promote bone repair. Owing to excellent biocompatibility and osteoconductivity, hydroxyapatite is in extensive research and high demand for both medical and non-medical applications. Although various methods have been developed for the synthesis of hydroxyapatite, in the present study we have shown the use of nanosecond laser energy in the wet precipitation method of nano-hydroxyapatite (nHAP) synthesis without using ammonium solution or any other chemicals for pH maintenance. Here, the present study aimed to fabricate the nanohydroxyapatite using a nanosecond laser. The X-ray diffraction and Fourier transform infrared spectroscopy have confirmed the hydroxyapatite formation under laser irradiation in less time without aging. A transmission electron microscopy confirmed the nano size of synthesized nHAP, which is comparable to conventional nHAP. The length and width of the laser-assisted nHAP were found to be in the range of 50-200 nm and 15-20 nm, respectively, at various laser parameters. The crystallite size obtained by Debye Scherrer formulae was found to be in the range of ∼ 16-36 nm. In addition, laser-assisted nHAP based composite cryogel (nanohydroxyapatite/gelatin/collagen I) was synthesized and impregnated with bioactive molecules (bone morphogenic protein and zoledronic acid) that demonstrated significant osteogenic potential both in vitro in cell experiment and in vivo rat muscle pouch model (abdomen and tibia muscles). Dual-energy X-ray analysis, micro-CT, and histological analysis confirmed ectopic bone regeneration. Micro-CT based histomorphometry showed a higher amount (more than 10-fold) of mineralization for animal groups implanted with composite cryogels loaded with bioactive molecules compared to only composite cryogels groups. Our findings thus demonstrate a controlled and rapid synthetic method for the synthesis of nHAP with various physical, chemical, and biological properties exhibited as comparable to conventionally synthesized nHAP.

Enzymatic crosslinking of lignin nanoparticles and nanocellulose in cryogels improves adsorption of pharmaceutical pollutants.

Pharmaceuticals, designed for treating diseases, ironically endanger humans and aquatic ecosystems as pollutants. Adsorption-based wastewater treatment could address this problem, however, creating efficient adsorbents remains a challenge. Recent efforts have shifted towards sustainable bio-based adsorbents. Here, cryogels from lignin-containing cellulose nanofibrils (LCNF) and lignin nanoparticles (LNPs) were explored as pharmaceuticals adsorbents. An enzyme-based approach using laccase was used for crosslinking instead of fossil-based chemical modification. The impact of laccase treatment on LNPs alone produced surface-crosslinked water-insoluble LNPs with preserved morphology and a hemicellulose-rich, water-soluble LNP fraction. The water-insoluble LNPs displayed a significant increase in adsorption capacity, up to 140 % and 400 % for neutral and cationic drugs, respectively. The crosslinked cryogel prepared by one-pot incubation of LNPs, LCNF and laccase showed significantly higher adsorption capacities for various pharmaceuticals in a multi-component system than pure LCNF or unmodified cryogels. The crosslinking minimized the leaching of LNPs in water, signifying enhanced binding between LNPs and LCNF. In real wastewater, the laccase-modified cryogel displayed 8-44 % removal for cationic pharmaceuticals. Overall, laccase treatment facilitated the production of bio-based adsorbents by improving the deposition of LNPs to LCNF. Finally, this work introduces a sustainable approach for engineering adsorbents, while aligning with global sustainability goals.

Facile preparation of fatigue-resistant Mxene-reinforced chitosan cryogel for accelerated hemostasis and wound healing.

The development of highly effective chitosan-based hemostatic materials that can be utilized for deep wound hemostasis remains a considerable challenge. In this study, a hemostatic antibacterial chitosan/N-hydroxyethyl acrylamide (NHEMAA)/Ti3C2Tx (CSNT) composite cryogel was facilely prepared through the physical interactions between the three components and the spontaneous condensation of NHEMAA. Because of the formation of strong crosslinked network, the CSNT cryogel showed a developed pore structure (~ 99.07 %) and superfast water/blood-triggered shape recovery, enabling it to fill the wound after contacting the blood. Its capillary effect, amino groups, negative charges, and affinity with lipid collectively induced rapid hemostasis, which was confirmed by in vitro and in vivo analysis. In addition, CSNT cryogel showed excellent photothermal antibacterial activities, high biosafety, and in vivo wound healing ability. Furthermore, the presence of chitosan effectively prevented the oxidation of MXene, thus enabling the long-term storage of the MXene-reinforced cryogel. Thus, our hemostatic cryogel demonstrates promising potential for clinical application and commercialization, as it combines high resilience, rapid hemostasis, efficient sterilization, long-term storage, and easy mass production.

Creating and exploring carboxymethyl cellulose aerogels as drug delivery devices.

Carboxymethyl cellulose (CMC) is a well-known cellulose derivative used in biomedical applications due to its biocompatibility and biodegradability. In this work, novel porous CMC materials, aerogels, were prepared and tested as a drug delivery device. CMC aerogels were made from CMC solutions, followed by non-solvent induced phase separation and drying with supercritical CO2. The influence of CMC characteristics and of processing conditions on aerogels' density, specific surface area, morphology and drug release properties were investigated. Freeze-drying of CMC solutions was also used as an alternative process to compare the properties of the as-obtained "cryogels" with those of aerogels. Aerogels were nanostructured materials with bulk density below 0.25 g/cm3 and high specific surface area up to 143 m2/g. Freeze drying yields highly macroporous materials with low specific surface areas (around 5-18 m2/g) and very low density, 0.01 - 0.07g/cm3. Swelling and dissolution of aerogels and cryogels in water and in a simulated wound exudate (SWE) were evaluated. The drug was loaded in aerogels and cryogels, and release kinetics in SWE was investigated. Drug diffusion coefficients were correlated with material solubility, morphology, density, degree of substitution and drying methods, demonstrating tuneability of new materials' properties in view of their use as delivery matrices.

BM-MSC-Loaded Graphene-Collagen Cryogels Ameliorate Neuroinflammation in a Rat Spinal Cord Injury Model.

A major obstacle to axonal regeneration following spinal cord injury (SCI) is neuroinflammation mediated by astrocytes and microglial cells. We previously demonstrated that graphene-based collagen hydrogels alone can decrease neuroinflammation in SCI. Their regenerative potential, however, is poorly understood and incomplete. Furthermore, stem cells have demonstrated both neuroprotective and regenerative properties in spinal cord regeneration, although there are constraints connected with the application of stem cell-based therapy. In this study, we have analyzed the regeneration capability of human bone marrow mesenchymal stem cell (BM-MSC)-loaded graphene-cross-linked collagen cryogels (Gr-Col) in a thoracic (T10-T11) hemisection model of SCI. Our study found that BM-MSC-loaded Gr-Col improves axonal regeneration, reduces neuroinflammation by decreasing astrocyte reactivity, and promotes M2 macrophage polarization. BM-MSC-loaded-Gr-Col demonstrated enhanced regenerative potential compared to Gr-Col and the injury group control. Next-generation sequencing (NGS) analysis revealed that BM-MSC-loaded-Gr-Col modulates the JAK2-STAT3 pathway, thus decreasing the reactive and scar-forming astrocyte phenotype. The decrease in neuroinflammation in the BM-MSC-loaded-Gr-Col group is attributed to the modulation of Notch/Rock and STAT5a/b and STAT6 signaling. Overall, Gene Set Enrichment Analysis suggests the promising role of BM-MSC-loaded-Gr-Col in promoting axonal regeneration after SCI by modulating molecular pathways such as the PI3/Akt pathway, focal adhesion kinase, and various inflammatory pathways.

Tailoring microstructure and mechanical properties of pectin cryogels by modulate intensity of ionic interconnection.

Porous morphology and mechanical properties determine the applications of cryogels. To understand the influence of the ionic network on the microstructure and mechanical properties of pectin cryogels, we prepared low-methoxyl pectin (LMP) cryogels with different Ca2+ concentrations (measured as R-value, ranging from 0 to 2) through freeze-drying (FD). Results showed that the R-values appeared to be crucial parameters that impact the pore morphology and mechanical characteristics of cryogels. It is achieved by altering the network stability and water state properties of the cryogel precursor. Cryogel precursors with a saturated R-value (R = 1) produced a low pore diameter (0.12 mm) microstructure, obtaining the highest crispness (15.00 ± 1.85) and hardness (maximum positive force and area measuring 2.36 ± 0.31 N and 12.30 ± 1.57 N·s respectively). Hardness showed a negative correlation with Ca2+ concentration when R ≤ 1 (-0.89), and a similar correlation with the porosity of the gel network when R ≥ 1 (-0.80). Given the impacts of crosslinking on the pore structure, it is confirmed that the pore diameter can be designed between 56.24 and 153.58 µm by controlling R-value in the range of 0-2.

Decellularized brain extracellular matrix based NGF-releasing cryogel for brain tissue engineering in traumatic brain injury.

Traumatic brain injuries(TBI) pose significant challenges to human health, specifically neurological disorders and related motor activities. After TBI, the injured neuronal tissue is known for hardly regenerated and recovered to their normal neuron physiology and tissue compositions. For this reason, tissue engineering strategies that promote neuronal regeneration have gained increasing attention. This study explored the development of a novel neural tissue regeneration cryogel by combining brain-derived decellularized extracellular matrix (ECM) with heparin sulfate crosslinking that can perform nerve growth factor (NGF) release ability. Morphological and mechanical characterizations of the cryogels were performed to assess their suitability as a neural regeneration platform. After that, the heparin concnentration dependent effects of varying NGF concentrations on cryogel were investigated for their controlled release and impact on neuronal cell differentiation. The results revealed a direct correlation between the concentration of released NGF and the heparin sulfate ratio in cryogel, indicating that the cryogel can be tailored to carry higher loads of NGF with heparin concentration in cryogel that induced higher neuronal cell differentiation ratio. Furthermore, the study evaluated the NGF loaded cryogels on neuronal cell proliferation and brain tissue regeneration in vivo. The in vivo results suggested that the NGF loaded brain ECM derived cryogel significantly affects the regeneration of brain tissue. Overall, this research contributes to the development of advanced neural tissue engineering strategies and provides valuable insights into the design of regenerative cryogels that can be customized for specific therapeutic applications.

Hyaluronidase-Responsive Bactericidal Cryogel for Promoting Healing of Infected Wounds: Inflammatory Attenuation, ROS Scavenging, and Immune Regulation.

Wounds infected with multidrug-resistant (MDR) bacteria are increasingly threatening public health and challenging clinical treatments because of intensive bacterial colonization, excessive inflammatory responses, and superabundant oxidative stress. To overcome this malignant burden and promote wound healing, a multifunctional cryogel (HA/TA2/KR2) composed of hyaluronic acid (HA), tannic acid (TA), and KR-12 peptides is designed. The cryogel exhibited excellent shape-memory properties, strong absorption performance, and hemostatic capacity. In vitro experiments demonstrated that KR-12 in the cryogel can be responsively released by stimulation with hyaluronidase produced by bacteria, reaching robust antibacterial activity against Escherichia coli (E. coli), MDR Pseudomonas aeruginosa (MDR-PA), and methicillin-resistant Staphylococcus aureus (MRSA) by disrupting bacterial cell membranes. Furthermore, the synergetic effect of KR-12 and TA can efficiently scavenge ROS and decrease expression of pro-inflammatory cytokines (tumor necrosis factor (TNF)-α & interleukin (IL)-6), as well as modulate the macrophage phenotype toward the M2 type. In vivo animal tests indicated that the cryogel can effectively destroy bacteria in the wound and promote healing process via accelerating angiogenesis and re-epithelialization. Proteomic analysis revealed the underlying mechanism by which the cryogel mainly reshaped the infected wound microenvironment by inhibiting the Nuclear factor kappa B (NF-κB) signaling pathway and activating the Janus kinase-Signal transducer and activator of transcription (JAK-STAT6) signaling pathway. Therefore, the HA/TA2/KR2 cryogel is a promising dressing candidate for MDR bacteria-infected wound healing.

A facile method to fabricate versatile keratin cryogels for tissue engineering applications.

Human hair keratin (HHK) has been extensively explored as a biomaterial for soft tissue regeneration due to their excellent bioactivity and biocompatibility. The possibility to fabricate HHK into three-dimensional (3D) hydrogels with physical properties resembling soft tissues has been well demonstrated. However, conventional keratin hydrogels often exhibit a dense architecture that could hinder cell filtration. In the present study, HHK-based cryogels were fabricated using a freeze-thaw (FT) method, where oxidized dopamine (ODA) was employed to covalently crosslink thiol/amine rich-keratin molecules at sub-zero temperatures. The obtained HHK-ODA cryogels have micron-sized pores ranging between 100 and 200 µm and mechanical properties that can be tuned by varying the crosslinking density between ODA and HHK. Through optimization of the weight content of ODA and the number of FT cycles, the compressive strengths and stiffnesses of these cryogels achieved 15-fold increments from ∼1.5 kPa to ∼22 kPa and ∼300 Pa to ∼5000 Pa, respectively. The HHK-ODA cryogels competently supported human dermal fibroblast spreading and proliferation. Overall, this study exhibited a facile method to fabricate mechanically superior keratin-based cryogels with cell compatible microarchitecture, circumventing the need for complicated chemical modifications and the use of cytotoxic crosslinkers.

The design and characterisation of sinusoidal toolpaths using sub-zero bioprinting of polyvinyl alcohol.

Sub-zero (°C) additive manufacturing (AM) systems present a promising solution for the fabrication of hydrogel structures with complex external geometry or a heterogeneous internal structure. Polyvinyl alcohol cryogels (PVA-C) are promising tissue-mimicking materials, with mechanical properties that can be designed to satisfy a wide variety of soft tissues. However, the design of more complex mechanical properties into additively manufactured PVA-C samples, which can be enabled using the toolpath, is a largely unstudied area. This research project will investigate the effect of toolpath variation on the elastic and viscoelastic properties of PVA-C samples fabricated using a sinusoidal toolpath. Samples were fabricated using parametric variation of a sinusoidal toolpath, whilst retaining the same overall cross-sectional area, using a sub-zero AM system. To mechanically characterise the samples, they were tested under tension in uniaxial ramp tests, and through dynamic mechanical analysis (DMA). The elastic and viscoelastic moduli of the samples are presented. No correlations between the parametric variation of the design and the Young's modulus were observed. Analysis of the data shows high intra-sample repeatability, demonstrated robust testing protocols, and variable inter-sample repeatability, indicating differences in the printability and consistency of fabrication between sample sets. DMA of the wavelength samples, show a frequency-dependent loss moduli. The storage modulus demonstrates frequency independence, and a large increase in magnitude as the sample increases to 3 wavelengths.

DNA Cryogels with Anisotropic Mechanical and Responsive Properties for Specific Cell Capture and Release.

Due to their programmable stimuli-responsiveness, excellent biocompatibility, and water-rich and soft structures similar to biological tissues, smart DNA hydrogels hold great promise for biosensing and biomedical applications. However, most DNA hydrogels developed to date are composed of randomly oriented and isotropic polymer networks, and the resulting slow response to biotargets and lack of anisotropic properties similar to those of biological tissues have limited their extensive applications. Herein, anisotropic DNA hydrogels consisting of unidirectional void channels internally oriented up to macroscopic length scales were constructed by a directional cryopolymerization method, as exemplified by a DNA-incorporated covalently cross-linked DNA cryogel and a DNA duplex structure noncovalently cross-linked DNA cryogel. Results showed that the formation of unidirectional channels significantly improved the responsiveness of the gel matrix to biomacromolecular substances and further endowed the DNA cryogels with anisotropic properties, including anisotropic mechanical properties, anisotropic swelling/shrinking behaviors, and anisotropic responsiveness to specific biotargets. Moreover, the abundant oriented and long macroporous channels in the gel matrix facilitated the migration of cells, and through the introduction of aptamer structures and thermosensitive polymers, an anisotropic DNA cryogel-based platform was further constructed to achieve the highly efficient capture and release of specific cells. These anisotropic DNA hydrogels may provide new opportunities for the development of anisotropic separation and biosensing systems.

Fabrication of a Coculture Organoid Model in the Biomimetic Matrix of Alginate to Investigate Breast Cancer Progression in a TAMs-Leading Immune Microenvironment.

The current research models of breast cancer are usually limited in their capacity to recapitulate the tumor microenvironment in vitro. The lack of an extracellular matrix (ECM) oversimplifies cell-cell or cell-ECM cross-talks. Moreover, the lack of tumor-associated macrophages (TAMs), that can comprise up to 50% of some solid neoplasms, poses a major problem for recognizing various hallmarks of cancer. To address these concerns, a type of direct breast cancer cells (BCCs)-TAMs coculture organoid model was well developed by a sequential culture method in this study. Alginate cryogels were fabricated with appropriate physical and mechanical properties to serve as an alternative ECM. Then, our previous experience was leveraged to polarize TAMs inside of the cryogels for creating an in vitro immune microenvironment. The direct coculture significantly enhanced BCCs organoid growth and cancer aggressive phenotypes, including the stemness, migration, ECM remodeling, and cytokine secretion. Furthermore, transcriptomic analysis and protein-protein interaction networks implied certain pathways (PI3K-Akt pathway, MAPK signaling pathway, etc.) and targets (TNF, PPARG, TLR2, etc.) during breast cancer progression in a TAM-leading immune microenvironment. Future studies to advance treatment strategies for BCC patients may benefit from using this facile model to reveal and target the interactions between cancer signaling and the immune microenvironment.

Aligned cryogel fibers incorporated 3D printed scaffold effectively facilitates bone regeneration by enhancing cell recruitment and function.

Reconstructing extensive cranial defects represents a persistent clinical challenge. Here, we reported a hybrid three-dimensional (3D) printed scaffold with modification of QK peptide and KP peptide for effectively promoting endogenous cranial bone regeneration. The hybrid 3D printed scaffold consists of vertically aligned cryogel fibers that guide and promote cell penetration into the defect area in the early stages of bone repair. Then, the conjugated QK peptide and KP peptide further regulate the function of the recruited cells to promote vascularization and osteogenic differentiation in the defect area. The regenerated bone volume and surface coverage of the dual peptide-modified hybrid scaffold were significantly higher than the positive control group. In addition, the dual peptide-modified hybrid scaffold demonstrated sustained enhancement of bone regeneration and avoidance of bone resorption compared to the collagen sponge group. We expect that the design of dual peptide-modified hybrid scaffold will provide a promising strategy for bone regeneration.

Cellulose nanocrystals-reinforced dual crosslinked double network GelMA/hyaluronic acid injectable nanocomposite cryogels with improved mechanical properties for cartilage tissue regeneration.

Improvement of mechanical properties of injectable tissue engineering scaffolds is a current challenge. The objective of the current study is to produce a highly porous injectable scaffold with improved mechanical properties. For this aim, cellulose nanocrystals-reinforced dual crosslinked porous nanocomposite cryogels were prepared using chemically crosslinked methacrylated gelatin (GelMA) and ionically crosslinked hyaluronic acid (HA) through the cryogelation process. The resulting nanocomposites showed highly porous structures with interconnected porosity (>90%) and mean pore size in the range of 130-296 µm. The prepared nanocomposite containing 3%w/v of GelMA, 20 w/w% of HA, and 1%w/v of CNC showed the highest Young's modulus (10 kPa) and excellent reversibility after 90% compression and could regain its initial shape after injection by a 16-gauge needle in the aqueous media. The in vitro results demonstrated acceptable viability (>90%) and migration of the human chondrocyte cell line (C28/I2), and chondrogenic differentiation of human adipose stem cells. A two-month in vivo assay on a rabbit's ear model confirmed that the regeneration potential of the prepared cryogel is comparable to the natural autologous cartilage graft, suggesting it is a promising alternative for autografts in the treatment of cartilage defects.