An Innovative Collagen-Based Cell-Printing Method for Obtaining Human Adipose Stem Cell-Laden Structures Consisting of Core-Sheath Structures for Tissue Engineering.

Three-dimensional (3D) cell printing processes have been used widely in various tissue engineering applications due to the efficient embedding of living cells in appropriately designed micro- or macro-structures. However, there are several issues to overcome, such as the limited choice of bioinks and tailor-made fabricating strategies. Here, we suggest a new, innovative cell-printing process, supplemented with a core-sheath nozzle and an aerosol cross-linking method, to obtain multilayered cell-laden mesh structure and a newly considered collagen-based cell-laden bioink. To obtain a mechanically and biologically enhanced cell-laden structure, we used collagen-bioink in the core region, and also used pure alginate in the sheath region to protect the cells in the collagen during the printing and cross-linking process and support the 3D cell-laden mesh structure. To achieve the most appropriate conditions for fabricating cell-embedded cylindrical core-sheath struts, various processing conditions, including weight fractions of the cross-linking agent and pneumatic pressure in the core region, were tested. The fabricated 3D MG63-laden mesh structure showed significantly higher cell viability (92 ± 3%) compared with that (83 ± 4%) of the control, obtained using a general alginate-based cell-printing process. To expand the feasibility to stem cell-embedded structures, we fabricated a cell-laden mesh structure consisting of core (cell-laden collagen)/sheath (pure alginate) using human adipose stem cells (hASCs). Using the selected processing conditions, we could achieve a stable 3D hASC-laden mesh structure. The fabricated cell-laden 3D core-sheath structure exhibited outstanding cell viability (91%) compared to that (83%) of an alginate-based hASC-laden mesh structure (control), and more efficient hepatogenic differentiations (albumin: ∼ 1.7-fold, TDO-2: ∼ 7.6-fold) were observed versus the control. The selection of collagen-bioink and the new printing strategy could lead to an efficient way to achieve 3D cell-laden mesh structures that mimic the anatomical architecture of a patient's defective region.

Extraction and Characterization of Human Adipose Tissue-Derived Collagen: Toward Xeno-Free Tissue Engineering.

BACKGROUND: Collagen is a key component of connective tissue and has been frequently used in the fabrication of medical devices for tissue regeneration. Human-originated collagen is particularly appealing due to its low immune response as an allograft biomaterial compared to xenografts and its ability to accelerate the regeneration process. Ethically and economically, adipose tissues available from liposuction clinics are a good resource to obtain human collagen. However, studies are still scarce on the extraction and characterization of human collagen, which originates from adipose tissue. The aim of this study is to establish a novel and simple method to extract collagen from human adipose tissue, characterize the collagen, and compare it with commercial-grade porcine collagen for tissue engineering applications. METHODS: We developed a method to extract the collagen from human adipose tissue under quasi-Good Manufacturing Practice (GMP) conditions, including freezing the tissue, blood removal, and ethanol-based purification. Various techniques, including protein quantification, decellularization assessment, SDS-PAGE, FTIR, and CD spectroscopy analysis, were used for characterization. Amino acid composition was compared with commercial collagen. Biocompatibility and cell proliferation tests were performed, and in vitro tests using collagen sponge scaffolds were conducted with statistical analysis. RESULTS: Our results showed that this human adipose-derived collagen was equivalent in quality to commercially available porcine collagen. In vitro testing demonstrated high cell attachment and the promotion of cell proliferation. CONCLUSION: In conclusion, we developed a simple and novel method to extract and characterize collagen and extracellular matrix from human adipose tissue, offering a potential alternative to animal-derived collagen for xeno-free tissue engineering applications.

FeS2-incorporated 3D PCL scaffold improves new bone formation and neovascularization in a rat calvarial defect model.

199Three-dimensional (3D) scaffolds composed of various biomaterials, including metals, ceramics, and synthetic polymers, have been widely used to regenerate bone defects. However, these materials possess clear downsides, which prevent bone regeneration. Therefore, composite scaffolds have been developed to compensate these disadvantages and achieve synergetic effects. In this study, a naturally occurring biomineral, FeS2, was incorporated in PCL scaffolds to enhance the mechanical properties, which would in turn influence the biological characteristics. The composite scaffolds consisting of different weight fractions of FeS2 were 3D printed and compared to pure PCL scaffold. The surface roughness (5.77-fold) and the compressive strength (3.38-fold) of the PCL scaffold was remarkably enhanced in a dose-dependent manner. The in vivo results showed that the group with PCL/ FeS2 scaffold implanted had increased neovascularization and bone formation (2.9-fold). These results demonstrated that the FeS2 incorporated PCL scaffold might be an effective bioimplant for bone tissue regeneration.

Three-dimensional hierarchical composite scaffolds consisting of polycaprolactone, ß-tricalcium phosphate, and collagen nanofibers: fabrication, physical properties, and in vitro cell activity for bone tissue regeneration.

ß-Tricalcium phosphate (ß-TCP) and collagen have been widely used to regenerate various hard tissues, but although Bioceramics and collagen have various biological advantages with respect to cellular activity, their usage has been limited due to ß-TCP's inherent brittleness and low mechanical properties, along with the low shape-ability of the three-dimensional collagen. To overcome these material deficiencies, we fabricated a new hierarchical scaffold that consisted of a melt-plotted polycaprolactone (PCL)/ß-TCP composite and embedded collagen nanofibers. The fabrication process was combined with general melt-plotting methods and electrospinning. To evaluate the capability of this hierarchical scaffold to act as a biomaterial for bone tissue regeneration, physical and biological assessments were performed. Scanning electron microscope (SEM) micrographs of the fabricated scaffolds indicated that the ß-TCP particles were uniformly embedded in PCL struts and that electrospun collagen nanofibers (diameter = 160 nm) were well layered between the composite struts. By accommodating the ß-TCP and collagen nanofibers, the hierarchical composite scaffolds showed dramatic water-absorption ability (100% increase), increased hydrophilic properties (20%), and good mechanical properties similar to PCL/ß-TCP composite. MTT assay and SEM images of cell-seeded scaffolds showed that the initial attachment of osteoblast-like cells (MG63) in the hierarchical scaffold was 2.2 times higher than that on the PCL/ß-TCP composite scaffold. Additionally, the proliferation rate of the cells was about two times higher than that of the composite scaffold after 7 days of cell culture. Based on these results, we conclude that the collagen nanofibers and ß-TCP particles in the scaffold provide good synergistic effects for cell activity.

3D-printed gelatin methacrylate (GelMA)/silanated silica scaffold assisted by two-stage cooling system for hard tissue regeneration.

Among many biomaterials, gelatin methacrylate (GelMA), a photocurable protein, has been widely used in 3D bioprinting process owing to its excellent cellular responses, biocompatibility and biodegradability. However, GelMA still shows a low processability due to the severe temperature dependence of viscosity. To overcome this obstacle, we propose a two-stage temperature control system to effectively control the viscosity of GelMA. To optimize the process conditions, we evaluated the temperature of the cooling system (jacket and stage). Using the established system, three GelMA scaffolds were fabricated in which different concentrations (0, 3 and 10 wt%) of silanated silica particles were embedded. To evaluate the performances of the prepared scaffolds suitable for hard tissue regeneration, we analyzed the physical (viscoelasticity, surface roughness, compressive modulus and wettability) and biological (human mesenchymal stem cells growth, western blotting and osteogenic differentiation) properties. Consequently, the composite scaffold with greater silica contents (10 wt%) showed enhanced physical and biological performances including mechanical strength, cell initial attachment, cell proliferation and osteogenic differentiation compared with those of the controls. Our results indicate that the GelMA/silanated silica composite scaffold can be potentially used for hard tissue regeneration.

Preparation and Characterization of Human Adipose Tissue-Derived Extracellular Matrix, Growth Factors, and Stem Cells: A Concise Review.

Background: Human adipose tissue is routinely discarded as medical waste. However, this tissue may have valuable clinical applications since methods have been devised to effectively isolate adipose-derived extracellular matrix (ECM), growth factors (GFs), and stem cells. In this review, we analyze the literature that devised these methods and then suggest an optimal method based on their characterization results. Methods: Methods that we analyze in this article include: extraction of adipose tissue, decellularization, confirmation of decellularization, identification of residual active ingredients (ECM, GFs, and cells), removal of immunogens, and comparing structural/physiological/biochemical characteristics of active ingredients. Results: Human adipose ECMs are composed of collagen type I-VII, laminin, fibronectin, elastin, and glycosaminoglycan (GAG). GFs immobilized in GAG include basic fibroblast growth factor (bFGF), transforming growth factor beta 1(TGF-b1), insulin like growth factor 1 (IGF-1), vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), BMP4 (bone morphogenetic protein 4), nerve growth factor (NGF), hepatocyte growth factor (HGF), and epithermal growth factor (EGF). Stem cells in the stromal-vascular fraction display mesenchymal markers, self-renewal gene expression, and multi-differentiation potential. Conclusion: Depending on the preparation method, the volume, biological activity, and physical properties of ECM, GFs, and adipose tissue-derived cells can vary. Thus, the optimal preparation method is dependent on the intended application of the adipose tissue-derived products.

Fabrication of hASCs-laden structures using extrusion-based cell printing supplemented with an electric field.

UNLABELLED: In this study, we proposed a hybrid cell-printing technique that combines a conventional extrusion-based cell-printing process with an electrohydrodynamic jet. The electric field stabilized the extruded struts of cell-embedding-hydrogel and reduced the damage to dispensed cells caused by the high wall shear stress in the dispensing nozzle. The new cell-printing process was optimized in terms of various processing parameters, applied electric field strength, nozzle movement speed, and distance between the nozzle tip and working stage. Using the optimal cell-embedding hydrogel composition (1×10(6)cellsmL(-1) in 4wt% alginate) and cell-printing process parameters (applied voltage, 1kV; nozzle movement speed, 12mms(-1); distance, 0.7mm; current, 10.67±1.1nA), we achieved rapid and stable fabrication of a cell-laden structure without loss of cell viability or proliferation, the values of which were similar to those of the process without an electric field. Furthermore, by applying the same pneumatic pressure to fabricate cell-laden structures, considerably higher volume flow rate and cell viability at the same volume flow rate were achieved by the modified process compared with conventional extrusion-based cell-printing processes. To assess the feasibility of the method, the hydrogel containing human adipose stem cells (hASCs) and alginate (4wt%) was fabricated into a cell-laden porous structure in a layer-by-layer manner. The cell-laden structure exhibited reasonable initial hASC viability (87%), which was similar to that prior to processing of the cell-embedding-hydrogel. STATEMENT OF SIGNIFICANCE: The extrusion-based cell-printing process has shortcomings, such as unstable flow and potential loss of cell viability. The unsteady flow can occur due to the high cell concentration, viscosity, and surface tension of bioinks. Also, cell viability post extrusion can be significantly reduced by damage of the cells due to the high wall shear stress in the extrusion nozzle. To overcome these limitations, we suggested an innovative cell-printing process that combines a conventional extrusion-based cellprinting process with an electric field. The electric field in the cell-printing process stabilized the extruded struts of bioink and dramatically reduced the damage to dispensed cells caused by the high wall shear stress in the dispensing nozzle.

Cell-printed hierarchical scaffolds consisting of micro-sized polycaprolactone (PCL) and electrospun PCL nanofibers/cell-laden alginate struts for tissue regeneration.

Hierarchical scaffolds consisting of micro-sized struts with inter-layered nanofibers between the struts are mechanically stable and biologically superior to conventionally fabricated rapid-prototyped scaffolds and electrospun nanofibers. However, although the hierarchical scaffolds overcome various disadvantages of conventional scaffolds, there are still some limitations, such as low cell migration in the thickness direction and non-homogeneous cell proliferation. To overcome these deficiencies, a new hierarchical scaffold supplemented with osteoblast-like cell (MG63)-laden alginate struts is proposed. To control cell proliferation in the thickness direction of the scaffold, the density of interlayered nanofibers was manipulated using various electrospinning deposition times (2, 5, 10, and 20 s). Using the appropriate interlayered fiber density (electrospin deposition time = 10 s) and cell-laden alginate struts, we can obtain significantly homogeneous cell distribution in the hierarchical scaffold.

Optimal size of cell-laden hydrogel cylindrical struts for enhancing the cellular activities and their application to hybrid scaffolds.

Biomedical scaffolds must be mechanically stable and highly porous three-dimensional (3D) structures to allow efficient cell-to-cell and cell-to-substrate interactions, induce blood vessel formation, and transfer oxygen, nutrients, and metabolic waste. A 3D cell-laden hybrid scaffold consisting of a combination of structural synthetic polymers and a cell-laden hydrogel is an outstanding biomedical scaffold due to its controllable mechanical properties, multiple cell loading, and homogeneous cell-distribution within the scaffold. But although this hybrid scaffold is better than conventional scaffolds, some issues must still be overcome. One is the controllability of cell release from the cell-embedded hydrogel. Here, we propose a method to solve this problem using a geometric cell-laden hydrogel. Various cylindrical cell-laden strut sizes (diameter: 100, 200, 400, and 800 µm) using osteoblast-like-cells (MG63) were investigated A diameter of 200 µm was the most attractive to efficiently induce cell release and proliferation based on cell viability and fluorescence analyses. In addition, cell-laden alginate struts (200 and 800 µm) were used to fabricate poly(ε-caprolactone) hybrid scaffolds; the hybrid scaffolds were interlayered with a cell-laden hydrogel (200 µm), demonstrating significantly high osteogenic expression compared to scaffolds laden with 800 µm struts.

Regeneration of chronic tympanic membrane perforation using 3D collagen with topical umbilical cord serum.

Chronic tympanic membrane (TM) perforation is one of the most common otology complications. Current surgical management of TM perforation includes myringoplasty and tympanoplasty. The purpose of this study was to evaluate the efficacy and feasibility of three dimensional (3D) porous collagen scaffolds with topically applied human umbilical cord serum (UCS) for the regeneration of chronic TM perforation in guinea pigs. To achieve this goal, we fabricated porous 3D collagen scaffolds (avg. strut diameter of 236 ± 51 µm, avg. pore size of 382 ± 67 µm, and a porosity of 96%) by using a 3 axis robot dispensing and low temperature plate systems. Guinea pigs were used in a model of chronic TM perforation. In the experimental group (n=10), 3D collagen scaffold was placed on the perforation and topically applied of UCS every other day for a period of 8 days. The control group ears (n=10) were treated with paper discs and phosphate buffered saline (PBS) only using the same regimen. Healing time, acoustic-mechanical properties, and morphological analysis were performed by otoendoscopy, auditory brainstem response (ABR), single-point laser Doppler vibrometer (LDV), optical coherence tomography (OCT), and light microscopic evaluation. The closure of the TM perforation was achieved in 100% of the experimental group vs. 43% of the control group, and this difference was statistically significant (p=0.034). The ABR threshold at all frequencies of the experimental group was significantly recovered to the normal level compared to the control group. TM vibration velocity in the experimental group recovered similar to the normal control level. The difference is very small and they are not statistically significant below 1 kHz (p=0.074). By OCT and light microscopic examination, regenerated TM of the experimental group showed thickened fibrous and mucosal layer. In contrast, the control group showed absence of fibrous layer like a dimeric TM.

Designed hybrid scaffolds consisting of polycaprolactone microstrands and electrospun collagen-nanofibers for bone tissue regeneration.

Biomedical scaffolds used in bone tissue engineering should have various properties including appropriate bioactivity, mechanical strength, and morphologically optimized pore structures. Collagen has been well known as a good biomaterial for various types of tissue regeneration, but its usage has been limited due to its low mechanical property and rapid degradation. In this work, a new hybrid scaffold consisting of polycaprolactone (PCL) and collagen is proposed for bone tissue regeneration. The PCL enhances the mechanical properties of the hybrid scaffold and controls the pore structure. Layered collagen nanofibers were used to enhance the initial cell attachment and proliferation. The results showed that the hybrid scaffold yielded better mechanical properties of pure PCL scaffold as well as enhanced biological activity than the pure PCL scaffold did. The effect of pore size on bone regeneration was investigated using two hybrid scaffolds with pore sizes of 200 ± 20 and 300 ± 27 µm. After post-seeding for 7 days, the cell proliferation with pore size, 200 ± 20 µm, was greater than that with pore size, 300 ± 27 µm, due to the high surface area of the scaffold.