Calcium Phosphate Biomaterials for 3D Bioprinting in Bone Tissue Engineering.

Three-dimensional bioprinting is a promising technology for bone tissue engineering. However, most hydrogel bioinks lack the mechanical and post-printing fidelity properties suitable for such hard tissue regeneration. To overcome these weak properties, calcium phosphates can be employed in a bioink to compensate for the lack of certain characteristics. Further, the extracellular matrix of natural bone contains this mineral, resulting in its structural robustness. Thus, calcium phosphates are necessary components of bioink for bone tissue engineering. This review paper examines different recently explored calcium phosphates, as a component of potential bioinks, for the biological, mechanical and structural properties required of 3D bioprinted scaffolds, exploring their distinctive properties that render them favorable biomaterials for bone tissue engineering. The discussion encompasses recent applications and adaptations of 3D-printed scaffolds built with calcium phosphates, delving into the scientific reasons behind the prevalence of certain types of calcium phosphates over others. Additionally, this paper elucidates their interactions with polymer hydrogels for 3D bioprinting applications. Overall, the current status of calcium phosphate/hydrogel bioinks for 3D bioprinting in bone tissue engineering has been investigated.

Modulation of 3D Bioprintability in Polysaccharide Bioink by Bioglass Nanoparticles and Multiple Metal Ions for Tissue Engineering.

BACKGROUND: Bioglasses are used in applications related to bone rehabilitation and repair. The mechanical and bioactive properties of polysaccharides like alginate and agarose can be modulated or improved using bioglass nanoparticles. Further essential metal ions used as crosslinker have the potential to supplement cultured cells for better growth and proliferation. METHOD: In this study, the alginate bioink is modulated for fabrication of tissue engineering scaffolds by extrusion-based 3D bioprinting using agarose, bioglass nanoparticles and combination of essential trace elements such as iron, zinc, and copper. Homogeneous bioink was obtained by in situ mixing and bioprinting of its components with twin screw extruder (TSE) based 3D bioprinting, and then distribution of metal ions was induced through post-printing diffusion of metal ions in the printed scaffolds. The mechanical and 3d bioprinting properties, microscopic structure, biocompatibility of the crosslinked alginate/agarose hydrogels were analyzed for different concentrations of bioglass. The adipose derived mesenchymal stem cells (ADMSC) and osteoblast cells (MC3T3) were used to evaluate this hydrogel's biological performances. RESULTS: The porosity of hydrogels significantly improves with the incorporation of the bioglass. More bioglass concentration results in improved mechanical (compressive, dynamic, and cyclic) and 3D bioprinting properties. Cell growth and extracellular matrix are also enhanced with bioglass concentration. CONCLUSION: For bioprinting of the bioinks, the advanced TSE head was attached to 3D bioprinter and in situ fabrication of cell encapsulated scaffold was obtained with optimized composition considering minimal effects on cell damage. Fabricated bioinks demonstrate a biocompatible and noncytotoxic scaffold for culturing MC3T3 and ADMSC, while bioglass controls the cellular behaviors such as cell growth and extracellular matrix formation.

Incorporation of Cell-Adhesive Proteins in 3D-Printed Lipoic Acid-Maleic Acid-Poly(Propylene Glycol)-Based Tough Gel Ink for Cell-Supportive Microenvironment.

In extrusion-based 3D printing, the use of synthetic polymeric hydrogels can facilitate fabrication of cellularized and implanted scaffolds with sufficient mechanical properties to maintain the structural integrity and physical stress within the in vivo conditions. However, synthetic hydrogels face challenges due to their poor properties of cellular adhesion, bioactivity, and biofunctionality. New compositions of hydrogel inks have been designed to address this limitation. A viscous poly(maleate-propylene oxide)-lipoate-poly(ethylene oxide) (MPLE) hydrogel is recently developed that shows high-resolution printability, drug-controlled release, excellent mechanical properties with adhesiveness, and biocompatibility. In this study, the authors demonstrate that the incorporation of cell-adhesive proteins like gelatin and albumin within the MPLE gel allows printing of biologically functional 3D scaffolds with rapid cell spreading (within 7 days) and high cell proliferation (twofold increase) as compared with MPLE gel only. Addition of proteins (10% w/v) supports the formation of interconnected cell clusters (≈1.6-fold increase in cell areas after 7-day) and spreading of cells in the printed scaffolds without additional growth factors. In in vivo studies, the protein-loaded scaffolds showed excellent biocompatibility and increased angiogenesis without inflammatory response after 4-week implantation in mice, thus demonstrating the promise to contribute to the printable tough hydrogel inks for tissue engineering.

Study on Bioresponsive Gelatin-Hyaluronic Acid-Genipin Hydrogel for High Cell-Density 3D Bioprinting.

The Development of bioresponsive extrudable hydrogels for 3D bioprinting is imperative to address the growing demand for scaffold design as well as efficient and reliable methods of tissue engineering and regenerative medicine. This study proposed genipin (5 mg) cross-linked gelatin (1 to 1.5 g)-hyaluronic acid (0.3 g) hydrogel bioink (20 mL) tailored for 3D bioprinting. The focus is on high cell loading and a less artificial extra-cellular matrix (ECM) effect, as well as exploring their potential applications in tissue engineering. The bioresponsiveness of these hydrogel scaffolds was successfully evaluated at 37 °C and room temperature (at pH 2.5, 7.4, and 9). The rheological and mechanical properties (more than three times) increased with the increase in gelatin content in the hydrogel; however, the hydrogel with the least amount of gelatin showed the best extrusion capability. This optimized hydrogel's high extrusion ability and post-printing shape fidelity were evident from 3D and four-axis printing of complex structures such as hollow tubes, stars, pyramids, and zigzag porous tubular (four-axis) scaffolds (printed at 90 kPa pressure, 70 mm/s speed, 22G needle, fourth axis rotation of 4 rpm). 3 million/mL MC3T3-E1 mouse osteoblast cells were used in preparing 3D bioprinted samples. The in vitro cell culture studies have been carried out in a CO2 incubator (at 37 °C, 5% CO2). In the cytocompatibility study, almost three times more cell viability was observed in 3 days compared to day 1 control, proving the non-toxicity and cell-supportiveness of these hydrogels. High cell viability and cell-to-cell interactions observed at the end of day 3 using this moderately stable hydrogel in 3D bioprinting exhibit high potential for precise cell delivery modes in tissue engineering as well as regenerative medicine.

3D bioprinting of complex tissue scaffolds with in situ homogeneously mixed alginate-chitosan-kaolin bioink using advanced portable biopen.

Control of in situ 3D bioprinting of hydrogel without toxic crosslinker is ideal for tissue regeneration by reinforcing and homogeneously distributing biocompatible reinforcing agent during fabrication of large area and complex tissue engineering scaffolds. In this study, homogeneous mixing, and simultaneous 3D bioprinting of a multicomponent bioink based on alginate (AL)-chitosan (CH), and kaolin was obtained by an advanced pen-type extruder to ensure structural and biological homogeneity during the large area tissue reconstruction. The static, dynamic and cyclic mechanical properties as well as in situ self-standing printability significantly improved with the kaolin concentration for AL-CH bioink-printed samples due to polymer-kaolin nanoclay hydrogen bonding and cross-linking with less amount of calcium ions. The Biowork pen ensures better mixing effectiveness for the kaolin-dispersed AL-CH hydrogels (evident from computational fluid dynamics study, aluminosilicate nanoclay mapping and 3D printing of complex multilayered structures) than the conventional mixing process. Two different cell lines (osteoblast and fibroblast) introduced during large area multilayered 3D bioprinting have confirmed the suitability of such multicomponent bioinks for in vitro even tissue regeneration. The effect of kaolin to promote uniform growth and proliferation of the cells throughout the bioprinted gel matrix is more significant for this advanced pen-type extruder processed samples.

Progress in biomechanical stimuli on the cell-encapsulated hydrogels for cartilage tissue regeneration.

BACKGROUND: Worldwide, many people suffer from knee injuries and articular cartilage damage every year, which causes pain and reduces productivity, life quality, and daily routines. Medication is currently primarily used to relieve symptoms and not to ameliorate cartilage degeneration. As the natural healing capacity of cartilage damage is limited due to a lack of vascularization, common surgical methods are used to repair cartilage tissue, but they cannot prevent massive damage followed by injury. MAIN BODY: Functional tissue engineering has recently attracted attention for the repair of cartilage damage using a combination of cells, scaffolds (constructs), biochemical factors, and biomechanical stimuli. As cyclic biomechanical loading is the key factor in maintaining the chondrocyte phenotype, many studies have evaluated the effect of biomechanical stimulation on chondrogenesis. The characteristics of hydrogels, such as their mechanical properties, water content, and cell encapsulation, make them ideal for tissue-engineered scaffolds. Induced cell signaling (biochemical and biomechanical factors) and encapsulation of cells in hydrogels as a construct are discussed for biomechanical stimulation-based tissue regeneration, and several notable studies on the effect of biomechanical stimulation on encapsulated cells within hydrogels are discussed for cartilage regeneration. CONCLUSION: Induction of biochemical and biomechanical signaling on the encapsulated cells in hydrogels are important factors for biomechanical stimulation-based cartilage regeneration.

Photo-crosslinked gelatin methacryloyl hydrogel strengthened with calcium phosphate-based nanoparticles for early healing of rabbit calvarial defects.

PURPOSE: The aim of this study was to investigate the efficacy of photo-crosslinked gelatin methacryloyl (GelMa) hydrogel containing calcium phosphate nanoparticles (CNp) when applying different fabrication methods for bone regeneration. METHODS: Four circular defects were created in the calvaria of 10 rabbits. Each defect was randomly allocated to the following study groups: 1) the sham control group, 2) the GelMa group (defect filled with crosslinked GelMa hydrogel), 3) the CNp-GelMa group (GelMa hydrogel crosslinked with nanoparticles), and 4) the CNp+GelMa group (crosslinked GelMa loaded with nanoparticles). At 2, 4, and 8 weeks, samples were harvested, and histological and micro-computed tomography analyses were performed. RESULTS: Histomorphometric analysis showed that the CNp-GelMa and CNp+GelMa groups at 2 weeks had significantly greater total augmented areas than the control group (P<0.05). The greatest new bone area was observed in the CNp-GelMa group, but without statistical significance (P>0.05). Crosslinked GelMa hydrogel with nanoparticles exhibited good biocompatibility with a minimal inflammatory reaction. CONCLUSIONS: There was no difference in the efficacy of bone regeneration according to the synthesized method of photo-crosslinked GelMa hydrogel with nanoparticles. However, these materials could remain within a bone defect up to 2 weeks and showed good biocompatibility with little inflammatory response. Further improvement in mechanical properties and resistance to enzymatic degradation would be needed for the clinical application.

Utility and Limitations of Fine-Needle Aspiration Cytology in the Diagnosis of Lymphadenopathy.

BACKGROUND: Fine needle aspiration cytology (FNAC) is a valuable tool for evaluating lymphadenopathy. The purpose of this study was to assess the reliability and effectiveness of FNAC in the diagnosis of lymphadenopathy. METHODS: Cytological characteristics were evaluated in 432 patients who underwent lymph node FNAC and follow-up biopsy at the Korea Cancer Center Hospital from January 2015 to December 2019. RESULTS: Fifteen (3.5%) of the four hundred and thirty-two patients were diagnosed as inadequate by FNAC, with five (33.3%) of these diagnosed as metastatic carcinoma on histological examination. Of the 432 patients, 155 (35.9%) were diagnosed as benign by FNAC, with seven (4.5%) of these diagnosed histologically as metastatic carcinoma. A review of the FNAC slides, however, showed no evidence of cancer cells, suggesting that the negative results may have been due to FNAC sampling errors. An additional five samples regarded as benign on FNAC were diagnosed as non-Hodgkin lymphoma (NHL) by histological examination. Of the 432 patients, 223 (51.6%) were cytologically diagnosed as malignant, with 20 (9.0%) of these diagnosed as tissue insufficient for diagnosis (TIFD) or benign on histological examination. A review of the FNAC slides of these 20 patients, however, showed that 17 (85.0%) were positive for malignant cells. The sensitivity, specificity, positive predictive value (PPV), negative predictive values (NPV), and accuracy of FNAC were 97.8%, 97.5%, 98.7%, 96.0%, and 97.7%, respectively. CONCLUSIONS: Preoperative FNAC was safe, practical, and effective in the early diagnosis of lymphadenopathy. This method, however, had limitations in some diagnoses, suggesting that additional attempts may be required according to the clinical situation.

Control of maleic acid-propylene diepoxide hydrogel for 3D printing application for flexible tissue engineering scaffold with high resolution by end capping and graft polymerization.

BACKGROUND: Control of 3D printing of highly tough hydrogel inks with adequate printability, scaffold fidelity and mechanical properties are highly desirable for biomedical and tissue engineering applications. However, developing a biocompatible tough ink with high-resolution printability, biodegradability, self-healing, adhesion, and integration with surrounding tissues is a big challenge in 3D printing. The aim of this study was to develop extrusion-based 3D printing of viscous hydrogel composing of maleic acid and propylene diepoxide by controlling continuous mechanisms of condensation and radical polymerization. METHODS: The molecular weight of highly adhesive propagating poly(malate-co-propylene oxide) copolymer was controlled by capping its growing chain with mono-functional lipoic acid with different compositions during condensation reaction to form lipoic acid capped gel (LP-capped gel). Poly(ethylene oxide)-diacrylate, PEGDA, is graft-polymerized to the LP-capped backbone polymer (MPLE gel) by UV irradiation during 3D printing process to control the properties of gel printability, mechanical properties, and cell adhesiveness and post-printing fidelity of the printed scaffolds with high resolution and mechanical properties (MPLE scaffold). The scaffolds in complex geometries have been printed out in diverse forms with addition of model drugs with different molecular weights and chemical structures. Both the highly adhesive LP-capped gel and printing-controlled MPLE gel/scaffolds are diversely characterized and compared with for their applications to the extrusion-based printability, including biocompatibility, self-healing, drug releasing, adhesiveness, multi-layered high-resolution printing. Further in vitro/in vivo tests were done to observe cytotoxicity, immune response and tissue formation by using different cells in mice model. RESULTS: LP-capped hydrogel from maleic acid and propylene diepoxide gel showed control of gel properties with lipoic acid with one function group of thiol during condensation reaction, and the ratio at 1:0.3 (w/v) between LP-capped gel and PEGDA was chosen for the optimal results during radical polymerization process for 3D printing at high resolution (90-140 µm in strut thickness) with various complex geometries (lattice, rhombus, and honeycomb). The hydrogel showed excellent properties of self-healing, mechanical strength, biocompatibility, etc. In addition, the long-term release profiles of bioactive molecules were well-controlled by incorporating drugs of high molecular bovine serum albumin (BSA, 21 days, 98.4 ± 0.69%), or small molecule ornidazole (ORN, 14 days, 97.1 ± 1.98%) into the MPLE gel scaffolds for the tests of potential therapeutic applications. More importantly, the MPLE gels represents excellent in vitro cyto-compatibility against osteoblast-like cells (MC3T3) with viability value at 96.43% ± 7.48% over 7 culturing days. For in-vivo studies, the flexible MPLE scaffolds showed significant improvement on angiogenesis with minor inflammatory response after 4-week implantation in mice. CONCLUSION: The MPLE gel inks was well-controlled for the fabrication of flexible complex tissue engineering scaffold with high resolutions, shear-thinning, 3D printability and post-printing fidelity, by modulating the composition of the highly adhesive LP-capped gel and inert PEGDA as well as end capping of lipoic acid to the propagating poly(malate-co-propylene oxide) copolymer. The gel ink demonstrated its excellent printability, in vitro/in vivo biocompatibility and mechanical properties as well as sustained drug release from the gel.

Modulation of bioactive calcium phosphate micro/nanoparticle size and shape during in situ synthesis of photo-crosslinkable gelatin methacryloyl based nanocomposite hydrogels for 3D bioprinting and tissue engineering.

BACKGROUND: The gelatin-methacryloyl (GelMA) polymer suffers shape fidelity and structural stability issues during 3D bioprinting for bone tissue engineering while homogeneous mixing of reinforcing nanoparticles is always under debate. METHOD: In this study, amorphous calcium phosphates micro/nanoparticles (CNP) incorporated GelMA is synthesized by developing specific sites for gelatin structure-based nucleation and stabilization in a one-pot processing. The process ensures homogenous distribution of CNPs while different concentrations of gelatin control their growth and morphologies. After micro/nanoparticles synthesis in the gelatin matrix, methacrylation is carried out to prepare homogeneously distributed CNP-reinforced gelatin methacryloyl (CNP GelMA) polymer. After synthesis of CNP and CNP GelMA gel, the properties of photo-crosslinked 3D bioprinting scaffolds were compared with those of the conventionally fabricated ones. RESULTS: The shape (spindle to spherical) and size (1.753 µm to 296 nm) of the micro/nanoparticles in the GelMA matrix are modulated by adjusting the gelatin concentrations during the synthesis. UV cross-linked CNP GelMA (using Irgacure 2955) has significantly improved mechanical (three times compressive strength), 3D printability (160 layers, 2 cm self-standing 3D printed height) and biological properties (cell supportiveness with osteogenic differentiation). The photo-crosslinking becomes faster due to better methacrylation, facilitating continuous 3D bioprinting or printing. CONCLUSION: For 3D bioprinting using GelMA like photo cross-linkable polymers, where structural stability and homogeneous control of nanoparticles are major concerns, CNP GelMA is beneficial for even bone tissue regeneration within short period.

Nanodiamond enhanced mechanical and biological properties of extrudable gelatin hydrogel cross-linked with tannic acid and ferrous sulphate.

BACKGROUND: The requirements for cell-encapsulated injectable and bioprintable hydrogels are extrusion ability, cell supportive micro-environment and reasonable post-printing stability for the acclimatization of the cells in the target site. Detonation nanodiamond (ND) has shown its potential to improve the mechanical and biological properties of such hydrogels. Enhancing the performance properties of natural biopolymer gelatin-based hydrogels can widen their biomedical application possibilities to various areas including drug delivery, tissue engineering and 3D bioprinting. METHOD: In this study, natural cross-linker tannic acid (TA) is used along with ferrous sulphate (FS) to optimize the swelling and disintegration of extrudable and 3D printable gelatin hydrogels. The amounts of TA and FS are restricted to improve the extrusion ability of the gels in 3D printing. Further, ND particles (detonation type) are dispersed using twin screw extrusion technology to study their effect on mechanical and biological properties of the 3D printing hydrogel. RESULTS: The improved dispersion of ND particles helps to improve compressive strength almost ten times and dynamic modulus three times using 40 mg ND (2% w/w of gelatin). The surface-functional groups of detonation ND also contributed for such improvement in mechanical properties due to higher interaction with the hydrogel matrix. The stability of the hydrogels in water was also improved to 7 days. Four times improvement of the cell growth and proliferation was observed in ND based hydrogel. CONCLUSION: The cell-supportive nature of these moderately stable and extrudable ND dispersed gelatin hydrogels makes them a good candidate for short term regenerative applications of cell-encapsulated injectable hydrogels with better mechanical properties.

Manufacturing of self-standing multi-layered 3D-bioprinted alginate-hyaluronate constructs by controlling the cross-linking mechanisms for tissue engineering applications.

Three-dimensional (3D) bioprinting of self-supporting stable tissue and organ structure is critically important in extrusion-based bioprinting system, especially for tissue engineering and regenerative medicine applications. However, the development of self-standing bioinks with desired crosslinking density, biocompatibility, tunable mechanical strength and other properties like self-healing,in situgelation, drug or protein incorporation is still a challenge. In this study, we report a hydrogel bioink prepared from alginate (Alg) and hyaluronic acid (HA) crosslinked through multiple crosslinking mechanisms, i.e. acyl-hydrazone, hydrazide interactions and calcium ions. These Alg-HA gels were highly dynamic and shear-thinning with exceptional biocompatibility and tunable mechanical properties. The increased dynamic nature of the gels is mainly chemically attributed to the presence of acyl-hydrazone bonds formed between the amine groups of the acyl-hydrazide of alginate and the monoaldehyde of the HA. Among the different combinations of Alg-HA gel compositions prepared, the A5H5 (Alginate-acyl-hydrazide:HA-monoaldehyde, ratio 50:50) gel showed a gelation time of ∼60 s, viscosity of ∼400 Pa s (at zero shear rate), high stability in various pH solutions and increased degradation time (>50 days) than the other samples. The A5H5 gels showed high printability with increased post-printing stability as observed from the 3D printed structures (e.g. hollow tube (∼100 layers), porous cube (∼50 layers), star, heart-in, meniscus and lattice). The scanning electron microscopy analysis of the 3D constructs and hydrogels showed the interconnected pores (∼181µm) and crosslinked networks. Further, the gels showed sustained release of 5-amino salicylic acid and bovine serum albumin. Also, the mechanical properties were tuned by secondary crosslinking via different calcium concentrations.In vitroassays confirmed the cytocompatibility of these gels, where the 3D bioprinted lattice and tubular (∼70 layers) constructs demonstrated high cell viability under fluorescence analysis. Inin vivostudies, Alg-HA gel showed high biocompatibility (>90%) and increased angiogenesis (threefolds) and reduced macrophage infiltration (twofold decrease), demonstrating the promising potential of these hydrogels in 3D bioprinting applications for tissue engineering and regenerative medicine with tunable properties.

The effects of the molecular weights of hyaluronic acid on the immune responses.

BACKGROUND: The molecular weight of hyaluronic acid (HyA) depends on the type of organ in the body. When HyA of the desired molecular weight is implanted into the human body for regeneration of damaged tissue, it is degraded by hyaluronidase in associated with an inflammatory response. This study sought to evaluate the effects of HyA molecular weight and concentration on pro- and anti-inflammatory responses in murine macrophages. METHODS: The structures and molecular weights of HyAs (LMW-10, MMW-100, MMW-500, and HMW-1,500) were confirmed by 1 H NMR and gel permeation chromatography (GPC), respectively. After treatment of murine macrophages with a low (10 µg/mL) or high (100 µg/mL) concentration of each molecular weight HyA, cells were stimulated with lipopolysaccharide (LPS) and changes in immune response in both LPS-stimulated and untreated macrophages were evaluated by assessing nitric oxide (NO) production, and analyzing expression of pro- and anti-inflammatory genes including by RT-PCR. RESULTS: Molecular weights of LMW-10, MMW-100, MMW-500, and HMW-1,500 were 13,241 ± 161, 96,531 ± 1,167, 512,657 ± 8,545, and 1,249,500 ± 37,477 Da, respectively. NO production by LPS-stimulated macrophages was decreased by increasing concentrations and molecular weights of HyA. At a high concentration of 100 µg/mL, HMW-1,500 reduced NO production in LPS-stimulated macrophages to about 45 %. Using NanoString technology, we also found that the immune-related genes TNF-α, IL-6, IL-1ß, TGF-ß1, IL-10, IL-11, CCL2, and Arg1 were specifically over-expressed in LPS-stimulated macrophages treated with various molecular weights of HyA. An RT-PCR analysis of gene expression showed that HMW-1,500 decreased expression of classically activated (M1) macrophage genes, such as TNF-α, IL-6, CCL2, and IL-1ß, in LPS-stimulated macrophages, whereas medium molecular-weight HyA (MMW-100 and MMW-500) instead increased expression levels of these genes. HMW-1,500 at a high concentration (100 µg/mL) significantly decreased expression of pro-inflammatory genes in LPS-stimulated macrophages. Expression of genes associated with anti-inflammatory responses (M2 phenotype), such as TGF-ß1, IL-10, IL-11, and Arg1, were increased by high concentrations of MMW-500 and HMW-1,500 in LPS-stimulated macrophages. CONCLUSIONS: High molecular-weight HyA (i.e., > 1,250 kDa) inhibits pro-inflammatory responses in LPS-stimulated macrophages and induces anti-inflammatory responses in a concentration dependent manner.

Three-Dimensional Printed Design of Antibiotic-Releasing Esophageal Patches for Antimicrobial Activity Prevention.

Pharyngoesophageal defects can cause exposure to various bacterial flora and severe inflammation. We fabricated a biodegradable polycaprolactone (PCL) patch composed of both thin film and three-dimensional (3D) printed lattice, and then investigated the efficacy of pharyngoesophageal reconstruction by using 3D printed antibiotic-releasing PCL patches that inhibited early inflammation by sustained tetracycline (TCN) release from both thin PCL films and printed rods implanted in esophageal partial defects. PCL was 3D printed in lattice form on a presolution casted PCL thin film at ∼100 µm resolution. TCN was loaded onto the PCL-printed patches by 3D printing a mixture of TCN and PCL particles melted at 100°C. TCN exhibited sustained release in vitro for over 1 month. After loading TCN, the patches showed decreased tensile strength and Young's modulus, and less than 20% TCN was slowly released from the 2.5% TCN-loaded PCL patches over 150 days. Cytotoxicity tests of extract solutions from patch samples demonstrated excellent in vitro cell compatibility. Antibiotic-releasing PCL patches were then transplanted into partial esophageal defects in rats. Microcomputed tomography analysis revealed no leak of orally injected contrast agent in the entire esophagus. Tissue remodeling was examined through histological responses of M1 and M2 macrophages. In particular, the 1% and 3% TCN patch groups exhibited significant muscle layer regeneration by desmin immunostaining. Further histological and immunofluorescence analyses revealed that the 1% and 3% TCN patch groups exhibited the best esophageal regeneration according to reepithelialization, neovascularization, and elastin texture around the implanted sites. Our antibiotic-releasing patch successfully consolidates the regenerative potential of esophageal muscle and mucosa and the antibacterial activity of TCN for 3D esophageal reconstruction. Impact statement Anastomosis site leakage and necrosis after pharyngoesophageal transplantation inevitably causes mortality because the mediastinum and neck compartments become contaminated. Herein, we present antibiotic-releasing pharyngoesophageal patch that prevents saliva leakage and has an antimicrobial effect. We have demonstrated antibiotic release profile and mechanical properties for esophageal transplantation. Upon esophageal transplantation of antibiotic-releasing polycaprolactone patches, antimicrobial effects and muscle regeneration around the graft sites were clearly identified in the group containing 1% and 3% of tetracycline. The esophageal graft led to the remarkable recovery throughout reepithelialization, neovascularization, and elastin texture of around the implanted sites. We believe that current system is capable of various applications that require antibacterial in vivo.

Symbiotic culture of nanocellulose pellicle: A potential matrix for 3D bioprinting.

Nanocellulose pellicle is produced as a byproduct during the symbiotic culture of bacteria and yeast in kombucha. It shows good mechanical strength, biocompatibility and hydrophilicity. However, it has limited application in tissue engineering due to its low processability. In this work, bacterial cellulose-based sustainable kombucha (KBC) sheet has been produced and it was acid-treated to partially hydrolyse. This controlled process improves its extrusion and shape formation ability. The physical, functional and biological properties were studied to assess its potential as a 3D printed scaffold. Two different cell lines (Human dermal fibroblast cells and mouse osteoblast cells) were used to study the cytocompatibility. Both the cell types showed good attachment, growth and proliferation on the pure and treated KBC. They attained almost full confluence within 3 days. This study indicates that the controlled partial hydrolysis of KBC can make it suitable for 3D printing retaining its mechanical strength and cytocompatibility. This sustainable microbial biopolymer shows the possibility to be used as a bioink for 3D bioprinting.

Major Clues and Pitfalls in the Differential Diagnosis of Parathyroid and Thyroid Lesions Using Fine Needle Aspiration Cytology.

BACKGROUND: It is difficult to distinguish parathyroid lesions (PLs) from thyroid lesions using fine needle aspiration cytology (FNAC) because of their proximity and their similar cytomorphological features. METHODS: FNAC smears of 46 patients with pathologically proven PLs that were histologically diagnosed as parathyroid adenoma (PA, n = 35), parathyroid hyperplasia (PH, n = 3), atypical parathyroid adenoma (APA, n = 1), and parathyroid carcinoma (PC, n = 7) were retrospectively reviewed and analyzed. RESULTS: Our initial cytological diagnoses indicated correct diagnoses in 31 of 46 PL patients (67%). The 15 erroneous diagnoses were 5 patients with non-specific benign disease (11%), 4 with nodular hyperplasia of the thyroid (9%), 5 with atypical cells (11%), and 1 with a metastatic papillary thyroid carcinoma (2%). Follicular pattern, papillary structures, colloid-like material, and macrophages, which often suggest thyroid lesions, were also present in some PLs. We found that branching capillaries along the papillary structures, stippled nuclear chromatin, and frequent occurrence of naked nuclei were useful for determining a parathyroid origin. CONCLUSIONS: It is important to be aware that PLs are frequently mistaken for thyroid lesions based on FNAC. The specific and unique characteristics of PLs identified here may be helpful in diagnosis.

Self-crosslinking hyaluronic acid-carboxymethylcellulose hydrogel enhances multilayered 3D-printed construct shape integrity and mechanical stability for soft tissue engineering.

One of the primary challenges in extrusion-based 3D bioprinting is the ability to print self-supported multilayered constructs with biocompatible hydrogels. The bioinks should have sufficient post-printing mechanical stability for soft tissue and organ regeneration. Here, we report on the synthesis, characterization and 3D printability of hyaluronic acid (HA)-carboxymethylcellulose (CMC) hydrogels cross-linked through N-acyl-hydrazone bonding. The hydrogel's hydrolytic stability was acquired by the effects of both the prevention of the oxidation of the six-membered rings of HA, and the stabilization of acyl-hydrazone bonds. The shear-thinning and self-healing properties of the hydrogel allowed us to print different 3D constructs (lattice, cubic and tube) of up to 50 layers with superior precision and high post-printing stability without support materials or post-processing depending on their compositions (H7:C3, H5:C5 and H3:C7). Morphological analyses of different zones of the 3D-printed constructs were undertaken for verification of the interconnection of pores. Texture profile analysis (TPA) (hardness (strength), elastic recovery, etc) and cyclic compression studies of the 3D-printed constructs demonstrated exceptional elastic properties and fast recovery after 50% strain, respectively, which have been attributed to the addition of CMC into HA. A model drug quercetin was released in a sustained manner from hydrogels and 3D constructs. In vitro cytotoxicity studies confirmed the excellent cyto-compatibility of these gels. In vivo mice studies prove that these biocompatible hydrogels enhance angiogenesis. The results indicate that controlling the key properties (e.g. self-crosslinking capacity, composition) can lead to the generation of multilayered constructs from 3D-bioprintable HA-CMC hydrogels capable of being leveraged for soft tissue engineering applications.

3D printable and injectable lactoferrin-loaded carboxymethyl cellulose-glycol chitosan hydrogels for tissue engineering applications.

In this study, carboxymethyl cellulose (CMC)-glycol chitosan (GC) hydrogel, a potential three-dimensional (3D) printing biomaterial ink for tissue engineering applications was synthesized using simple, biocompatible in situ-gelling Schiff's base reaction and ionic interactions. Different grades of hydrogels (C70G30, C50G50 and C30G70) were synthesized at physiological conditions. The oxidation of CMC and imine bond formation in the hydrogel were confirmed spectroscopically. Scanning electron microscopic images revealed the crosslinked interconnected pores in the cross-sectioned hydrogels (dried). Swelling (equilibrium: 1 h), porosity (~75%), in vitro degradation (>30 days) and thermal gravimetric analyses of the dried gels were studied. Initially, cytotoxicity assay was evaluated using mouse osteoblastic cells (MC3T3). These experiments revealed that CMC-GC gels formed stable hydrogel networks and were biocompatible. Particularly, C50G50 gels showed high printability (continuous extrusion) and post-printing stability (without secondary crosslinking). Gel 3D printing was optimized by varying the air pressure, temperature, needle size and nozzle speed, to obtain stable lattice structures (2 to 16 layers). The printed (2 and 5 layers) hydrogels showed high stability in phosphate buffer saline (PBS) solution (1 h), under UV light (1 h) and after autoclaving. The strut dimensions and porosity of the printed gels before and after the stability tests were analyzed. The hydrogel stability may be attributed to both the imine bond and ionic interaction between the cationic and anionic polymer side chains. Lactoferrin (glycoprotein) incorporated C50G50 gels showed sustained release up to 21 days in PBS (pH 7.4) solution and demonstrated increased biocompatibility (>80%) during in vitro cytotoxicity assays (MC3T3 cells and bone marrow mesenchymal stem cells) and Live/Dead assay (MC3T3 cells). A higher number of live osteoblast cells on the C50G50 hydrogels with increasing lactoferrin concentration was observed. These results show that the CMC-GC gels are promising bio-ink candidates for 3D printing and loading proteins or drugs for tissue engineering applications.

Effects of Molar Ratios of Two Immiscible Monomers toward Development of an Amphiphilic, Highly Stretchable, Bioadhesive, Self-Healing Copolymeric Hydrogel and its Mineral-Active Cellular Behavior.

Here, we report the striking properties such as high stretchability, self-healing, and adhesiveness of an amphiphilic copolymeric hydrogel (poly(acrylic acid)-poly(methyl methacrylate) (PAA-PMMA) gel) synthesized from two immiscible monomers-acrylic acid (AA) and methyl methacrylate (MMA)-through a simple free radical polymerization in an aqueous medium. The developed hydrogel, with a specific molar ratio of MMA and AA, is self-healable, which is attributed to the hydrophobic interaction arising from methyl groups of PMMA, as well as the breakdown and reformation of sacrificial noncovalent cross-linking through the weak hydrogen bonds between the carboxylic acid groups of PAA and methoxy groups of PMMA. The energy dissipation values in the hysteresis test signify the excellent self-recoverability of the hydrogel. The developed hydrogel showed adhesive behavior to the surfaces of polystyrene, glass, wood, metal, stone, ceramics, pork skin, and human skin. The physical and mechanical properties of the PAA-PMMA gel were fine-tuned through changes in the MMA/AA ratio and pH. Moreover, the PAA-PMMA hydrogel can serve as a template for calcium phosphate mineralization to yield a hydrogel composite, which improved MC3T3 cell adhesion and proliferation. Overall, we propose that depending on synthesis parameters and other scenarios, the synthesized PAA-PMMA hydrogel could potentially be employed in varying biomedical and industrial applications.