December 16, 2020 by admin 0 Comments

Creation of bladder assembloids mimicking tissue regeneration and cancer

Authors
Eunjee Kim, Seoyoung Choi, Byunghee Kang, JungHo Kong, Yubin Kim, Woong Hee Yoon, Hwa-Rim Lee, SungEun Kim, Hyo-Min Kim, HyeSun Lee, Chorong Yang, You Jeong Lee, Minyong Kang, Tae-Young Roh, Sungjune Jung, Sanguk Kim, Ja Hyeon Ku & Kunyoo Shin
Abstract
Current organoid models are limited by their inability to mimic mature organ architecture and associated tissue microenvironments. Here we create multilayer bladder ‘assembloids’ by reconstituting tissue stem cells with stromal components to represent an organized architecture with an epithelium surrounding stroma and an outer muscle layer. These assembloids exhibit characteristics of mature adult bladders in cell composition and gene expression at the single-cell transcriptome level, and recapitulate in vivo tissue dynamics of regenerative responses to injury. We also develop malignant counterpart tumour assembloids to recapitulate the in vivo pathophysiological features of urothelial carcinoma. Using the genetically manipulated tumour-assembloid platform, we identify tumoural FOXA1, induced by stromal bone morphogenetic protein (BMP), as a master pioneer factor that drives enhancer reprogramming for the determination of tumour phenotype, suggesting the importance of the FOXA1–BMP–hedgehog signalling feedback axis between tumour and stroma in the control of tumour plasticity.

November 30, 2020 by admin 0 Comments

Biofabrication of 3D printed hydroxyapatite composite scaffolds for bone regeneration

Authors
Yoontae Kim(1), Eun-Jin Lee(1), Albert V. Davydov(2), Stanislav Frukhbeyen(1), Jonathan E. Seppala(3), Shozo Takagi(1), Laurence Chow(1) and Stella Alimperti(1)
Abstract
Biofabrication has been adapted in engineering patient-specific biosynthetic grafts for bone regeneration. Herein, we developed a 3D high-resolution, room-temperature printing approach to fabricate osteoconductive scaffolds using calcium phosphate cement (CPC). The non-aqueous CPC bioinks were composed of tetracalcium phosphate (TTCP), dicalcium phosphate anhydrous (DCPA), and Polyvinyl butyral (PVB) dissolved in either ethanol (EtOH) or Tetrahydrofuran (THF). They were printed in an aqueous sodium phosphate bath, which performs as a hardening accelerator for hydroxyapatite (HA) formation and as a retainer for 3D microstructure. The PVB solvents, EtOH or THF, affected differently the slurry rheological properties, scaffold microstructure, mechanical properties, and osteoconductivity...

October 21, 2020 by admin 0 Comments

Design and manufacturing of bioprinted gellan gum-based constructs representative of the articular cartilage

Authors
Gianluca Ciardelli, Piergiorgio Gentile, Chiara Tonda Turo
Abstract
Articular cartilage (AC) is a highly specialized tissue which exhibit topographical heterogeneity in terms of matrix composition and mechanical properties. Due to its avascular nature AC shows limited regenerative ability, therefore representing an excellent subject for tissue engineering (TE). Particularly, bioprinting is an emerging additive manufacturing technology that has already demonstrated its potential use in regenerative medicine and cartilage TE. It allows to recapitulate the tissues microstructure by a controlled deposition of “bioinks”, suspensions of cells alone or encapsulated in biomaterials. As cells source, mesenchymal stem cells and chondrocytes, both naturally found in AC, are mainly selected. Hydrogels are largely used as biomaterials for their ability to resemble soft tissues extracellular matrix (ECM), providing an ideal micro-environment for the embedded cells survival, proliferation and differentiation. Hydrogels are produced from synthetic and natural polymers, including gellan gum (GG), a biocompatible polysaccharide that has gained interest in cartilage TE because of its structural similarity to cartilage glycosaminoglycans (GAGs) and chondrogenic potential. The aim of this work was the design and manufacturing of 3D constructs mimicking AC by extrusion bioprinting. Particularly, this thesis objectives (OBJ) were: the synthesis and characterization (physico-chemical, morphological, mechanical) of methacrylated GG-based hydrogels subjected to a dual physical and photo-chemical crosslinking (OBJ1); the subsequent biofabrication via Rokit INVIVO bioprinter of in vitro constructs (OBJ2) and biological characterization of cell-laden constructs in terms of cells viability and AC tissue formation (OBJ3). The final stage of this work dealt with the manufacturing of osteoarthritis (OA) in vitro models, via culturing healthy models in cytokine-enriched culture medium, for future analysis on novel OA therapeutic treatments. Firstly, the success of GG methacrylate (GGMA) synthesis was demonstrated through FTIR and XPS analysis. Then, 4 photo-curable hydrogels were prepared: pure GGMA 2% w/v (GG2) and 3% w/v (GG3), and GGMA (respectively 2% w/v and 0.75% w/v) combined with 5% w/v manuka honey (GG/MH) and 10% w/v gelatin (GG/GEL). Gelation analysis at room temperature showed that GG3 and GG/GEL underwent sol-gel transition in ~1 minute, while GG2 and GG/MH in ~3 minute. Water uptake (WU) analysis demonstrated the strong hydrophilic nature of these hydrogels, reaching WU values up to ~1700%. Morphological analysis evidenced that they had an interconnected porous morphology with a mean pore diameter in the range 100-200 μm, suitable for AC applications. Similarly, mechanical analysis showed that hydrogels had a compressive Young’s modulus between ~25 and ~16 kPa, comparable to other natural hydrogels found in literature. GG2 and GG/MH hydrogels were selected as bioinks encapsulating human TERT immortalised stem cells differentiated into chondrocytes (Y201-C; 7x106 cells/ml). The double-crosslinked bioinks were successfully printed into stable constructs. Live/Dead assay demonstrated high cell viability for both bioprinted constructs. The GAGs quantification assay showed that Y201-C GAGs production increased over time in both hydrogels. Finally, scanning electron microscopy analysis showed that cells exhibited a typical chondrocytes rounded-shaped morphology and tended to aggregate in both healthy and OA GG2 constructs .

October 6, 2020 by admin 0 Comments

Fabrication of strontium-substituted hydroxyapatite scaffolds using 3D printing for enhanced bone regeneration

Authors
Hyun-Woo Kim & Young-Jin Kim
Abstract
The use of porous three-dimensional (3D) bioceramic scaffolds to facilitate the regeneration of bone defects has attracted great attention because their structures closely mimic the natural extracellular matrix. 3D printing is a versatile method for the fabrication of 3D scaffolds. In this study, 3D strontium-substituted hydroxyapatite (Sr-HA) bioceramic scaffolds were prepared by simple precipitation and 3D printing method. The resulting scaffolds exhibited interconnected microporous structures of strands and a single-phase crystal due to HA, meaning that no changes in the phase composition and microstructure of the scaffolds with the Sr content were observed. However, their dissolution rate and biological performance were substantially influenced by changes in the Sr content of the scaffolds. The optimal Sr content in the Sr-HA scaffolds for enhanced proliferation and differentiation of cells were identified by comparing four compositions of the Sr-HA scaffolds. The results of in vitro bioactivity tests demonstrated that the Sr5-HA scaffold with 0.05 of Sr/(Ca + Sr) molar ratio promoted more rapid cell proliferation, osteogenic differentiation, and cellular mineralization compared with the other scaffolds. Therefore, Sr-HA scaffolds have the potential for application in bone regeneration as new bone graft substitutes.

October 1, 2020 by admin 0 Comments

3D printing of self-healing ferrogel prepared from glycol chitosan, oxidized hyaluronate, and iron oxide nanoparticles

Authors
Eun Seok Ko a,1, Choonggu Kim a,1, Youngtae Choi a, Kuen Yong Lee a,b
Abstract
Hydrogel systems that show self-healing ability after mechanical damage are receiving increasing attention. However, self-healing hydrogels suitable for biomedical applications are limited owing to complex preparation methods. Furthermore, few studies have demonstrated the self-healing property of ferrogels. In this study, we demonstrated that glycol chitosan (GC) and oxidized hyaluronate (OHA) can be used to form a self-healing ferrogel in the presence of superparamagnetic iron oxide nanoparticles (SPIONs) without additional chemical cross-linkers. The overall characteristics of GC/OHA/SPION ferrogel varied based on the GC/OHA ratio, SPION content, and total polymer concentration. Interestingly, GC/OHA/SPION ferrogel was used to fabricate 3D-printed constructs of various shapes via an extrusion printing method. These constructs were responsive to the magnetic field, suggesting their potential application in 4D printing. This approach to developing self-healing ferrogels with biocompatible polysaccharides may prove useful in designing and fabricating drug delivery systems and tissue engineering scaffolds, via 3D printing.

September 16, 2020 by admin 0 Comments

Augmented peripheral nerve regeneration through elastic nerve guidance conduits prepared using a porous PLCL membrane with a 3D printed collagen hydrogel

Authors
Jin Yoo ‡a, Ji Hun Park ‡b, Young Woo Kwon ‡c, Justin J. Chung a, In Cheul Choi d, Jae Joon Nam d, Hyun Su Lee e, Eun Young Jeon a, Kangwon Lee e, Soo Hyun Kim af, Youngmee Jung *ag, and Jong Woong Park *d
Abstract
Peripheral nerve injury results in significant sensory and motor functional deficits. Although direct neurorrhaphy in the early phase may reduce its devastating effects, direct end-to-end neurorrhaphy is sometimes impossible owing to a defect at the injured site of the nerve. Autogenous nerve graft is a primary consideration for peripheral nerve defects; however, significant morbidity of the donor site is inevitable. Recently, the treatment using engineered synthetic nerve conduits has been regarded as a promising strategy to promote the regeneration of peripheral nerve defects. In this study, we developed longitudinally oriented collagen hydrogel-grafted elastic nerve guidance conduits (NGC) to reconstruct sciatic nerve defects. An elastic NGC was prepared by using poly(lactide-co-caprolactone) (PLCL), and electrospun PLCL was adopted to fabricate nanoporous structures with appropriate permeability for nerve regeneration. Oriented collagen hydrogels were prepared by the 3D printing method to achieve a microscale hydrogel pattern. Based on sciatic nerve injury models in rats, we confirmed the beneficial effects of the NGC with 3D printed collagen hydrogel on axonal regeneration and remyelination along with superior functional recovery in comparison with the NGC filled with the bulk collagen hydrogel. It is believed that the aligned collagen hydrogels provide a preferable environment for nerve regeneration, functioning as an oriented guidance path. In conclusion, the PLCL nerve guide conduit containing a 3D printed aligned collagen hydrogel can be useful for peripheral nerve regeneration.

July 22, 2020 by admin 0 Comments

Orodispersible Polymer Films with the Poorly Water-Soluble Drug, Olanzapine: Hot-Melt Pneumatic Extrusion for Single-Process 3D Printing

Authors
Hui-Won Cho 1, Seung-Hoon Baek 1, Beom-Jin Lee 1 and Hyo-Eon Jin 1,2,*
Abstract
Amorphous solid dispersions (ASDs) improve the oral delivery of poorly water-soluble drugs. ASDs of olanzapine (OLZ), which have a high melting point and low solubility, are performed using a complicated process. Three-dimensional (3D) printing based on hot-melt pneumatic extrusion (HMPE) is a simplified method for producing ASDs. Unlike general 3D printing, printlet extrusion is possible without the preparation of drug-loaded filaments. By heating powder blends, direct fused deposition modeling (FDM) printing through a nozzle is possible, and this step produces ASDs of drugs. In this study, we developed orodispersible films (ODFs) loaded with OLZ as a poorly water-soluble drug. Various ratios of film-forming polymers and plasticizers were investigated to enhance the printability and optimize the printing temperature. Scanning electron microscopy (SEM) showed the surface morphology of the film for the optimization of the polymer carrier ratios. Differential scanning calorimetry (DSC) was used to evaluate thermal properties. Powder X-ray diffraction (PXRD) confirmed the physical form of the drug during printing. The 3D printed ODF formulations successfully loaded ASDs of OLZ using HMPE. Our ODFs showed fast disintegration patterns within 22 s, and rapidly dissolved and reached up to 88% dissolution within 5 min in the dissolution test. ODFs fabricated using HMPE in a single process of 3D printing increased the dissolution rates of the poorly water-soluble drug, which could be a suitable formulation for fast drug absorption. Moreover, this new technology showed prompt fabrication feasibility of various formulations and ASD formation of poorly water-soluble drugs as a single process. The immediate dissolution within a few minutes of ODFs with OLZ, an atypical antipsychotic, is preferred for drug compliance and administration convenience.

June 30, 2020 by admin 0 Comments

Preparation and evaluation of identifiable quick response (QR)-coded orodispersible films using 3D printer with directly feeding nozzle

Authors
Byung-Cheol Oh (a), Gang Jin (a), Chulhun Park (b), Jun-Bom Park (c), Beom-Jin Lee (a),(d)
Abstract
3D-printing technology is growing in importance due to increased availability and a wider range of applications. Here, we prepared and evaluated a hot melt pneumatic (HMP) 3D-printed QR (Quick Response)-coded orodispersible film (QRODF) containing a poorly water-soluble aripiprazole (ARP). Moreover, QRODF was formulated to evaluate the extrusion process and characterize physicochemical properties of drug-loaded films. QRODF was designed with a 30-mm length/width and 0.3-mm thickness by varying QRODF formulations with different polyethylene oxide 100,000(PEO)/poloxamer 188(POX188) ratios and then optimized for extrusion accessibility and film-forming capability. The optimal QRODF formulation was further controlled by ARP and citric acid addition (pH control) for salivary applicability and dissolution rate. Physicochemical evaluation of QRODF was performed by scanning electron microscopy, differential scanning calorimetry, and powder X-ray diffraction. Dissolution studies were performed in buffer media (pH 1.2) following USP Apparatus type II method. Drug-loaded QRODF was scannable using a smartphone. Drug release from QRODF rapidly reached over 95% and was dependent on polymer/poloxamer ratios. By optimizing PEO/POX/drug ratio, the morphology and physical properties of the oral film were changed. Furthermore, disintegration and dissolution rates of ARP-loaded QRODF were successfully established in a controlled manner.

June 15, 2020 by admin 0 Comments

Dual-crosslinked methylcellulose hydrogels for 3D bioprinting applications

Authors
Ji Youn Shin (a), Yong Ho Yeo (a), Jae Eun Jeong (b), Su A. Park (b), Won Ho Park (a)
Abstract
Thermo-sensitive methylcellulose (MC) hydrogel has been widely used as a scaffold material for biomedical applications. However, due to its poor mechanical properties, the MC-based hydrogel has rarely been employed in 3D bioprinting for tissue engineering scaffolds. In this study, the dual crosslinkable tyramine-modified MC (MC-Tyr) conjugate was prepared via a two-step synthesis, and its hydrogel showed excellent mechanical properties and printability for 3D bioprinting applications. The MC-Tyr conjugate formed a dual-crosslinked hydrogel by modulating the temperature and/or visible light. A combination of reversible physical crosslinking (thermal crosslinking) and irreversible chemical crosslinking (photocrosslinking) was used in this dual crosslinked hydrogel. Also, the photocrosslinking of MC-Tyr solution was facilitated by visible light exposure in the presence of biocompatible photoinitiators (riboflavin, RF and riboflavin 5’-monophophate, RFp). The RF and RFp were used to compare the cytotoxicity and salting-out effect of MC-Tyr hydrogel, as well as the initiation ability, based on the difference in chemical structure. Also, the influence of the printing parameters on the printed MC hydrogel was investigated. Finally, the cell-laden MC-Tyr bioink was successfully extruded into stable 3D hydrogel constructs with high resolution via a dual crosslinking strategy. Furthermore, the MC-Tyr scaffolds showed excellent cell viability and proliferation.

June 10, 2020 by admin 0 Comments

pH-dependent nanodiamonds enhance the mechanical properties of 3D-printed hyaluronic acid nanocomposite hydrogels

Authors
Dae Gon Lim, Eunah Kang & Seong Hoon Jeong
Abstract
Nanocomposite hydrogels capable of undergoing manufacturing process have recently attracted attention in biomedical applications due to their desired mechanical properties and high functionality. 3D printing nanocomposite hydrogels of hyaluronic acid (HA)/nanodiamond (ND) revealed that the addition of ND with the low weight ratio of 0.02 wt% resulted in higher compressive force and gel breaking point, compared with HA only nanocomposites. These HA nanocomposite hydrogels loaded with surface functionalized ND allowed for the enforced compressive stress to be tuned in a pH-dependent manner. HA nanocomposite hydrogels with ND-OH at pH 8 showed an increase of 1.40-fold (0.02%: 236.18 kPa) and 1.37-fold (0.04%: 616.72 kPa) the compressive stress at the composition of 0.02 wt% and 0.04 wt, respectively, compared to those of ND-COOH (0.02%: 168.31 kPa, 0.04%: 449.59 kPa) at the same pH. Moreover, the compressive stress of HA/ND-OH (0.04 wt%) at pH 8 was mechanically enhanced 1.29-fold, compared to that of HA/ND-OH (0.04 wt%) at pH 7. These results indicate that the tunable buffering environment and interaction with the long chains of HA at the molecular level have a critical role in the dependency of the mechanical properties on pH. Due to the pH stability of the ND-OH nanophase, filament-based processing and layer-based deposition at microscale attained enforced mechanical properties of hydrogel. Fine surface tuning of the inorganic ND nanophase and controlled 3D printing leads to improved control over the pH-dependent mechanical properties of the nanocomposite hydrogels reported herein.