Homogeneous Cell Distribution Research Articles

Impact of topography and added TiN-coating on adult human dermal fibroblasts after seeding on titanium surface in-vitro.

Complications of transcutaneous osseointegrated prosthetic systems (TOPS) focus on the metal-cutaneous interface at the stoma. Besides pain due to scare tissue as well as undefined neuropathic disorders, there is high evidence that the stoma presents the main risk causing hypergranulation and ascending infection. To restore the cutaneous barrier function in this functional area, soft-tissue on- or in-growth providing a vital and mechanically stable bio-artificial conjunction is considered a promising approach. In this study we assessed viability and proliferation of adult human dermal fibroblasts (HDFa) on modifications of a standard prosthetic titanium surface. Un-coated (TiAl6V4) as well as a titanium-nitrite (TiN) coated additive manufactured porous three-dimensional surface structures (EPORE®) were seeded with HDFa and compared to plain TiAl6V4 and polystyrene surfaces as control. Cell viability and proliferation were assessed at 24h and 7days after seeding with a fluorescence-based live-dead assay. Adhesion and cell morphology were analyzed by scanning electron microscopy at the respective measurements. Both EPORE® surface specifications revealed a homogenous cell distribution with flat and spread cell morphology forming filopodia at both measurements. Proliferation and trend to confluence was seen on un-coated EPORE® surfaces with ongoing incubation but appeared substantially lower on the TiN-coated EPORE® specification. While cell viability on both EPORE® specifications was comparable to plain TiAL6V4 and polystyrene controls, cell proliferation and confluence were less pronounced when compared to controls. The EPORE® topography allows for fibroblast adhesion and viability in both standard TiAl6V4 and - to a minor degree - TiN-coated specifications as a proof of principle.

Development and Evaluation of Biodegradable Core-Shell Microfibrous and Nanofibrous Scaffolds for Tissue Engineering Applications

Tissue engineering scaffolds as three-dimensional substrates may serve as ideal templates for tissue regeneration by simulating the structure of the extracellular matrix (ECM). Many biodegradable synthetic polymers, either hydrophobic, like Poly-ε-caprolactone (PCL), or hydrophilic, like Poly(Vinyl Alcohol) (PVA), are widely used as candidate bioactive materials for fabricating tissue engineering scaffolds. However, a combination of good cytocompatibility of hydrophilic polymers with good biomechanical performance of hydrophobic polymers could be beneficial for the in vivo performance of the scaffolds. In this study, we aimed to fabricate biodegradable fibrous scaffolds by combining the properties of hydrophobic PCL with those of hydrophilic PVA and evaluate their properties in comparison with pristine PCL scaffolds. Therefore, single-layered PCL scaffolds, sequential tri-layered (PVA/PCL/PVA), and core-shell (PVA as shell and PCL as core) composite scaffolds were developed utilizing the electrospinning technique. The material structural and biomechanical properties of the electrospun scaffolds, before and after their hydrolytic degradation over a seven-month period following storage in phosphate-buffered saline (PBS) at 37 °C, were comprehensively compared. In addition, human embryonic kidney cells (HEK-293) were cultured on the scaffolds to investigate potential cell attachment, infiltration, and proliferation. The results demonstrated the long-term efficacy of core-shell biodegradable fibrous scaffolds in comparison to single-layers PCL and tri-layers PVA/PCL/PVA, not only due to its superior morphological characteristics and mechanical properties, but also due to its ability to promote homogeneous cell distribution and proliferation, without any external chemical or physical stimuli.Graphical

Cellular Architects at Work: Cells Building their Own Microgel Houses.

Microporous annealed particle (MAP) scaffolds are investigated for their application as injectable 3D constructs in the field of regenerative medicine and tissue repair. While available MAP scaffolds provide a stable interlinked matrix of microgels for cell culture, the infiltration depth and space for cells to grow inside the scaffolds is pre-determined by the void fraction during the assembly. In the case of MAP scaffolds fabricated from interlinked spherical microgels, a cellularity gradient can be observed with the highest cell density on the scaffold surface. Additionally, the interlinked microgel network limits the ability of cells to remodel their environment, which contradicts native tissue dynamics. In this work, a cell-induced interlinking method for MAP scaffold formation is established, which avoids the necessity of chemical crosslinkers and pre-engineered pores to achieve micro- or macropores in these 3D frameworks. This method enables cells to self-organize with microgels into dynamic tissue constructs, which can be further controlled by altering the microgel properties, the cell/microgel ratio, and well shape. To form a cell-induced interlinked scaffold, the cells are mixed with dextran-based microgels and function as a glue between the microgels, resulting in a more homogenous cell distribution throughout the scaffold with efficient cell-cell interactions.

Neuro-regenerative behavior of adipose-derived stem cells in aligned collagen I hydrogels.

Peripheral nerve injuries persist as a major clinical issue facing the US population and can be caused by stretch, laceration, or crush injuries. Small nerve gaps are simple to treat, and the nerve stumps can be reattached with sutures. In longer nerve gaps, traditional treatment options consist of autografts, hollow nerve guidance conduits, and, more recently, manufactured fibrous scaffolds. These manufactured scaffolds often incorporate stem cells, growth factors, and/or extracellular matrix (ECM) proteins to better mimic the native environment but can have issues with homogenous cell distribution or uniformly oriented neurite outgrowth in scaffolds without fibrous alignment. Here, we utilize a custom device to fabricate collagen I hydrogels with aligned fibers and encapsulated adipose-derived mesenchymal stem cells (ASCs) for potential use as a peripheral nerve repair graft. Initial results of our scaffold system revealed significantly less cell viability in higher collagen gel concentrations; 3mg/mL gels showed 84.8±7.3% viable cells, compared to 6mg/mL gels viability of 76.7±9.5%. Mechanical testing of the 3mg/mL gels showed a Young's modulus of 6.5±0.8kPa nearly matching 7.45kPa known to support Schwann cell migration. Further analysis of scaffolds coupled with stretching in vitro revealed heightened angiogenic and factor secretion, ECM deposition, fiber alignment, and dorsal root ganglia (DRG) neurite outgrowth along the axis of fiber alignment. Our platform serves as an in vitro testbed to assess neuro-regenerative potential of ASCs in aligned collagen fiber scaffolds and may provide guidance on next-generation nerve repair scaffold design.

A Fibrin-Based Human Multicellular Gingival 3D Model Provides Biomimicry and Enables Long-Term In Vitro Studies.

Collagen-type I gels are widely used for the fabrication of 3D in vitro gingival models. Unfortunately, their long-term stability is low, which limits the variety of in vitro applications. To overcome this problem and achieve better hydrolytic stability of 3D gingival models, fibrin-based hydrogel blends with increased long-term stability in vitro are investigated. Two different fibrin-based hydrogels are tested: fibrin 2.5% (w/v) and fibrin 1% (w/v)/gelatin 5% (w/v). Appropriate numbers of primary human gingival fibroblasts (HGFs) and OKG4/bmi1/TERT (OKG) keratinocytes are optimized to achieve a homogeneous distribution of cells under the assumed 3D conditions. Both hydrogels support the viability of HGFs and the stability of the hydrogel over 28 days. In vitro cultivation at the air-liquid interface triggers keratinization of the epithelium and increases its thickness, allowing the formation of multiple tissue-like layers. The presence of HGFs in the hydrogel further enhances epithelial differentiation. In conclusion, a fibrin-based 3D gingival model mimics the histology of native gingiva in vitro and ensures its long-term stability in comparison with the previously reported collagen paralogs. These results open new perspectives for extending the period within which specific biological or pathological conditions of artificial gingival tissue can be evaluated.

Evaluation of a synthetic peptide-based bioink (PeptiInk Alpha 1) for in vitro 3D bioprinting of cartilage tissue models

Cartilage pathology in human disease is poorly understood and requires further research. Various attempts have been made to study cartilage pathologies using in vitro human cartilage models as an alternative for preclinical research. Three-dimensional (3D) bioprinting is a technique that has been used to 3D-bioprint cartilage tissue models in vitro using animal-derived materials such as gelatine or hyaluronan, which present challenges in terms of scalability, reproducibility, and ethical concerns. We present an assessment of synthetic self-assembling peptides as bioinks for bioprinted human in vitro cartilage models. Primary human chondrocytes were mixed with PeptiInk Alpha 1, 3D-bioprinted and cultured for 14 days, and compared with 3D chondrocyte pellet controls. Cell viability was assessed through LIVE/DEAD assays and DNA quantification. High cell viability was observed in the PeptiInk culture, while a fast decrease in DNA levels was observed in the 3D pellet control. Histological evaluation using hematoxylin and eosin staining and immunofluorescence labeling for SOX-9, collagen type II, and aggrecan showed a homogeneous cell distribution in the 3D-bioprinted PeptiInks as well as high expression of chondrogenic markers in both control and PeptiInk cultures. mRNA expression levels assessed by - qRT-PCR (quantitative real time-polymerase chain reaction) confirmed chondrogenic cell behavior. These data showed promise in the potential use of PeptiInk Alpha 1 as a bioprintable manufacturing material for human cartilage in vitro models.

Impact of 3D collagen-based model and hydrostatic pressure on periodontal ligament fibroblast: A morpho-biochemical analysis

This study developed a customized hydrostatic pressure-based loading environment to investigate the effect of static hydrostatic pressure on the periodontal ligament fibroblasts (PDLf) in a three-dimensional (3D) collagen-based model. The cylindrical tissue constructs were comprised of PDL fibroblast cells seeded in type I collagen matrices and divided into three experimental groups: Control (no load), low-load (∼0.07 kPa), and high-load (∼60 kPa), all subjected to 24 h of experimental duration. Cells in the 3D construct were stained with fluorophore-conjugated antibodies for cytoskeletal protein F-actin and matricellular protein periostin. Cell culture supernatant was assessed for receptor activator of nuclear factor kappaB ligand (RANKL) and osteoprotegerin (OPG) expression. Transmission electron microscopy examined the contact between the cells and the collagen matrix. Ultrastructural changes in the 3D collagen matrix were also analyzed using scanning electron microscopy. Experiments were performed in triplicates, and data was analyzed using one-way ANOVA (p < 0.05). The 3D PDLf constructs from the low-load group demonstrated the highest levels of homogeneous cell distribution and higher expression of F-actin and periostin with enhanced interaction with the matrix. The collagen matrix in this group showed more closely packed fibers forming thicker bundles when compared to the control and the high-load 3D PDLf constructs. Nonuniform cell distribution with decreased expression of F-actin and periostin was observed in the control and high-load PDLf constructs. The high-load group showed the highest RANKL/OPG expression. This study demonstrated low-level hydrostatic pressure's role in regulating PDLf functions and extracellular matrix response, while excessive hydrostatic pressure may be detrimental to PDL fibroblast cell function.

The potential role of hydrophilic and hydrophobic liquid emulsifier-tailored sunflower wax/sunflower oil oleogels on the properties of whole wheat batter and sponge cakes

Abstract Sunflower oil (SO) oleogel was initially prepared using 5 % (w/w) sunflower wax, hydrophobic (Span 80), or hydrophilic (Tween 80) emulsifiers. This study involved the physicochemical characterization of the batter and cake prepared through partial and complete replacement of butter with the oleogels. Batter and cake properties were improvised in T80, prepared with oleogel containing 0.015 % (w/w) of Tween 80. The polarized micrograph of the T80 batter displayed a large number of air bubbles stabilized by the wax crystals. Starch gelatinization was found highest in T80 batter and could be related to the ability of hydrophilic emulsifiers to form a complex with starch. The FTIR spectra in the T80 batter and cake displayed a reduced peak for gluten content. T80 cake crumb showed a homogenous distribution of smaller air cells supporting its softness. A reduction in the firmness and hardness of T80 was obtained from the texture studies.

Magnetic nanoparticles in 3D-printed scaffolds for biomedical applications

Abstract Magnetic nanoparticles (MNPs) have recently attracted considerable attention, mainly due to their unique magnetic properties and biocompatibility. Although MNPs have been extensively studied for biomedical applications, there are still very few studies on them as part of three-dimensional (3D)-printed scaffolds. Thus, this review aims to show the potential of MNPs to modulate various properties of 3D-printed scaffolds. 3D Printing is for itself a contemporary method in biomedicine, owing to its ability to produce versatile scaffolds with complex shapes enabling a homogeneous distribution of cells or other entrapped compounds, as well as possible precise control of pore size and shape, porosity, and interconnectivity of pores that contribute to structural stability. All mentioned properties can be upgraded or complemented with the specific properties of MNPs (e.g., biocompatibility and positive effect on cell proliferation). Considering the latest related literature and a steadily increasing number of related publications, the fabrication of magnetically responsive scaffolds is among the most interesting strategies in tissue engineering. According to the literature, incorporating MNPs into scaffolds can improve their mechanical properties and significantly affect biological properties, such as cellular responses. Moreover, under the influence of an external magnetic field, MNPs significantly promoted cell adhesion, proliferation, and differentiation.

A perfusion-based three-dimensional cell culture system to model alveolar rhabdomyosarcoma pathological features

Although a rare disease, rhabdomyosarcoma (RMS) is one of the most common cancers in children the more aggressive and metastatic subtype is the alveolar RMS (ARMS). Survival outcomes with metastatic disease remain dismal and the need for new models that recapitulate key pathological features, including cell-extracellular matrix (ECM) interactions, is warranted. Here, we report an organotypic model that captures cellular and molecular determinants of invasive ARMS. We cultured the ARMS cell line RH30 on a collagen sponge in a perfusion-based bioreactor (U-CUP), obtaining after 7 days a 3D construct with homogeneous cell distribution. Compared to static culture, perfusion flow induced higher cell proliferation rates (20% vs. 5%), enhanced secretion of active MMP-2, and upregulation of the Rho pathway, associated with cancer cell dissemination. Consistently, the ECM genes LAMA1 and LAMA2, the antiapoptotic gene HSP90, identified in patient databases as hallmarks of invasive ARMS, were higher under perfusion flow at mRNA and protein level. Our advanced ARMS organotypic model mimics (1) the interactions cells-ECM, (2) the cell growth maintenance, and (3) the expression of proteins that characterize tumor expansion and aggressiveness. In the future, the perfusion-based model could be used with primary patient-derived cell subtypes to create a personalized ARMS chemotherapy screening system.

Skin substitutes based on gellan gum with mechanical and penetration compatibility to native human skin.

The study reports on a simple system to fabricate skin substitutes consisting of a naturally occurring bacterial polysaccharide gellan gum. Gelation was driven by the addition of a culture medium whose cations induced gellan gum crosslinking at physiological temperature, resulting in hydrogels. Human dermal fibroblasts were incorporated in these hydrogels and their mechanical, morphological, and penetration characteristics were studied. The mechanical properties were determined by means of oscillatory shear rheology, and a short linear viscoelastic regime was noted up to less than 1% of strain amplitude. The storage modulus increased with an increasing polymer concentration. The moduli were in the range noted for native human skin. After 2 weeks of fibroblast cultivation, the storage moduli showed signs of deterioration, so that a culture time of 2 weeks was proposed for further studies. Microscopic and fluorescent staining observations were documented. These depicted a crosslinked network structure in the hydrogels with a homogeneous distribution of cells and an assured cell viability of 2 weeks. H&E staining was also performed, which showed some traces of ECM formation in a few sections. Finally, caffeine penetration experiments were carried out with Franz diffusion cells. The hydrogels with a higher concentration of polymer containing cells showed an improved barrier function against caffeine compared to previously studied multicomponent hydrogels as well as commercially available 3D skin models. Therefore, these hydrogels displayed both mechanical and penetration compatibility with the ex vivo native human skin.

Functionalized gelatin-alginate based bioink with enhanced manufacturability and biomimicry for accelerating wound healing

Three-dimensional (3D) bioprinting is a promising technique to construct heterogeneous architectures that mimic cell microenvironment. However, the current bioinks for 3D bioprinting usually show some limitations, such as low printing accuracy, unsatisfactory mechanical properties and compromised cytocompatibility. Herein, a novel bioink comprising hydroxyphenyl propionic acid-conjugated gelatin and tyramine-modified alginate is developed for printing 3D constructs. The bioink takes advantage of an ionic/covalent intertwined network that combines covalent bonds formed by photo-mediated redox reaction and ionic bonds formed by chelate effect. Benefiting from the thermosensitivity of gelatin and the double-crosslinking mechanism, the developed bioink shows controllable rheological behaviors, enhanced mechanical behavior, improved printing accuracy and structure stability. Moreover, the printed cell-laden hydrogels exhibit a homogeneous cell distribution and considerable cell survival because the pre-crosslinking of the bioink prevents cellular sedimentation and the visible light crosslinking mechanism preserves cell viability. Further in vivo studies demonstrate that resulting cell-laden hydrogels are beneficial for the reduction of inflammation response and the promotion of collagen deposition and angiogenesis, thereby improving the quality of skin wound healing. This convenient and effective strategy is of great significance for accelerating the development of multifunctional bioinks and broadening the biomedical applications of 3D bioprinting.

Preparation of compact alginate films for 2D in vitro studies: Challenges and strategies for improvement

Alginate-based hydrogels show high biocompatibility and therefore are broadly used for cell cultivation and tissue engineering approaches in general. When hydrogel films for cell seeding were prepared by following published protocols, attached but inhomogeneously distributed cells were observed, which was attributed to the fact that casted hydrogel films were uneven and irregularly shaped. The causes of these irregularities resulting in an inhomogeneous cell distribution, e.g., due to accumulation of cells in cracks, were investigated. This study established a universal protocol for preparing planar hydrogel films that allow a homogenous cell distribution by varying several parameters such as alginate type, freezing, and crosslinking conditions. This finding can be applied to investigations of two-dimensional cell-material interactions of advanced alginate-based materials, e.g., when coupled with peptides.

Customizable 3D printed perfusion bioreactor for the engineering of stem cell microenvironments.

Faithful modeling of tissues and organs requires the development of systems reflecting their dynamic 3D cellular architecture and organization. Current technologies suffer from a lack of design flexibility and complex prototyping, preventing their broad adoption by the scientific community. To make 3D cell culture more available and adaptable we here describe the use of the fused deposition modeling (FDM) technology to rapid-prototype 3D printed perfusion bioreactors. Our 3D printed bioreactors are made of polylactic acid resulting in reusable systems customizable in size and shape. Following design confirmation, our bioreactors were biologically validated for the culture of human mesenchymal stromal cells under perfusion for up to 2weeks on collagen scaffolds. Microenvironments of various size/volume (6-12mm in diameter) could be engineered, by modulating the 3D printed bioreactor design. Metabolic assay and confocal microscopy confirmed the homogenous mesenchymal cell distribution throughout the material pores. The resulting human microenvironments were further exploited for the maintenance of human hematopoietic stem cells. Following 1 week of stromal coculture, we report the recapitulation of 3D interactions between the mesenchymal and hematopoietic fractions, associated with a phenotypic expansion of the blood stem cell populations.Our data confirm that perfusion bioreactors fit for cell culture can be generated using a 3D printing technology and exploited for the 3D modeling of complex stem cell systems. Our approach opens the gates for a more faithful investigation of cellular processes in relation to a dynamic 3D microenvironment.

Formation of low-density electrospun fibrous network integrated mesenchymal stem cell sheet.

Cell sheets combined with electrospun fibrous mats represent an attractive approach for the repair and regeneration of injured tissues. However, the conventional dense electrospun mats as supportive substrates in forming "cell sheet on fiber mat" complexes suffer from problems of limiting the cellular function and eliciting a host response upon implantation. To give full play to the role of electrospun biomimicking fibers in forming quality cell sheets, this study proposed to develop a cell-fiber integrated sheet (CFIS) featuring a spatially homogeneous distribution of cells within the fiber structure by using a low-density fibrous network for cell sheet formation. A low-density electrospun polycaprolactone (PCL) fibrous network at a density of 103.8 ± 16.3 μg cm-2 was produced by controlling the fiber deposition for a short period of 1 min and subsequently transferred onto polydimethylsiloxane rings for facilitating cell sheet formation, in which rat bone marrow-derived mesenchymal cells were used. Using a dense electrospun PCL fibrous mat (481.5 ± 7.5 μg cm-2) as the control, it was found that cells on the low-density fibrous network (L-G) exhibited improved capacities in spreading, proliferation, stemness maintenance and matrix-remodeling during the process of CFIS formation. Structurally, the CFIS constructs revealed strong integration between the cells and the fibrous network, thus providing excellent cohesion and physical integrity to enable strengthening of the formed cell sheet. By contrast, the cell sheet formed on the dense fibrous mat (D-G) showed a two-layer (biphasic) structure due to the limitation of cellular invasion. Moreover, such engineered CFIS was identified with enhanced immunomodulatory effects by promoting LPS-stimulated macrophages towards an M2 phenotype in vitro. Our results suggest that the CFIS may be used as a native tissue equivalent "cell sheet" for improving the efficacy of the tissue engineering approach for the repair and regeneration of impaired tissues.

Homogenic Evaluation for Spatial Distribution in Osteoclast Differentiation.

Cells have heterogeneous cellular diversity in size, morphology, cell cycle, metabolism, differentiation degree, and spatial distribution. The shift of specific cells towards the desired cells is crucial for maintaining uniform cellular function and can be represented by homogeneity and heterogeneity. Here, we developed a simple and direct method for evaluating the homogeneous distribution of desired cells in a constant region. We differentiated osteoclast progenitors into bone-resorbing multinucleated giant osteoclasts in a 2-dimensional culture plate under 2 conditions. Cells were stained with tartrate-resistant acid phosphatase to assess osteoclast differentiation, images were taken using a microscope and divided into sectors, and the number of osteoclasts (≥3 nuclei) in each sector was counted. To assess the homogeneity of the spatial distribution of osteoclasts, the standard deviation (SD) was calculated from the mean number of osteoclasts within each sector. From the 2 groups, a value with a SD close to 0 indicates high spatial homogeneity while a relatively high SD represents low spatial homogeneity. Our findings suggest that spatial homogeneity can be represented as SD.

Biomimetic Keratin-Coated Gold Nanoparticles for Photo-Thermal Therapy in a 3D Bioprinted Glioblastoma Tumor Model.

Before entering human clinical studies to evaluate their safety and effectiveness, new drugs and novel medical treatments are subject to extensive animal testing that are expensive and time-consuming. By contrast, advanced technologies enable the development of animal-free models that allow the efficacy of innovative therapies to be studied without sacrificing animals, while providing helpful information and details. We report on the powerful combination of 3D bioprinting (3DB) and photo-thermal therapy (PTT) applications. To this end, we realize a 3DB construct consisting of glioblastoma U87-MG cells in a 3D geometry, incorporating biomimetic keratin-coated gold nanoparticles (Ker-AuNPs) as a photo-thermal agent. The resulting plasmonic 3DB structures exhibit a homogeneous cell distribution throughout the entire volume while promoting the localization of Ker-AuNPs within the cells. A 3D immunofluorescence assay and transmission electron microscopy (TEM) confirm the uniform distribution of fluorescent-labeled Ker-AuNPs in the volume and their capability to enter the cells. Laser-assisted (λ = 532 nm) PTT experiments demonstrate the extraordinary ability of Ker-AuNPs to generate heating, producing the highest temperature rise of about 16 °C in less than 2 min.

Engineering of cardiac microtissues by microfluidic cell encapsulation in thermoshrinking non-crosslinked PNIPAAm gels

Multicellular agglomerates in form of irregularly shaped or spherical clusters can recapitulate cell–cell interactions and are referred to as microtissues. Microtissues gain increasing attention in several fields including cardiovascular research. Cardiac microtissues are evolving as excellent model systems for drug testing in vitro (organ-on-a-chip), are used as tissue bricks in 3D printing processes and pave the way for improved cell replacement therapies in vivo. Microtissues are formed for example in hanging drop culture or specialized microwell plates; truly scalable methods are not yet available. In this study, a novel method of encapsulation of cells in poly-N-isopropylacrylamid (PNIPAAm) spheres is introduced. Murine induced pluripotent stem cell-derived cardiomyocytes and bone marrow-derived mesenchymal stem cells were encapsulated in PNIPAAm by raising the temperature of droplets formed in a microfluidics setup above the lower critical solute temperature (LCST) of 32 °C. PNIPAAM precipitates to a water-insoluble physically linked gel above the LCST and shrinks by the expulsion of water, thereby trapping the cells in a collapsing polymer network and increasing the cell density by one order of magnitude. Within 24 h, stable cardiac microtissues were first formed and later released from their polymer shell by washout of PNIPAAm at temperatures below the LCST. Rhythmically contracting microtissues showed homogenous cell distribution, age-dependent sarcomere organizations and action potential generation. The novel approach is applicable for microtissue formation from various cell types and can be implemented into scalable workflows.

Injectable hydrogels for bone and cartilage tissue engineering: a review.

Tissue engineering, using a combination of living cells, bioactive molecules, and three-dimensional porous scaffolds, is a promising alternative to traditional treatments such as the use of autografts and allografts for bone and cartilage tissue regeneration. Scaffolds, in this combination, can be applied either through surgery by implantation of cell-seeded pre-fabricated scaffolds, or through injection of a solidifying precursor and cell mixture, or as an injectable cell-seeded pre-fabricated scaffold. In situ forming and pre-fabricated injectable scaffolds can be injected directly into the defect site with complex shape and critical size in a minimally invasive manner. Proper and homogeneous distribution of cells, biological factors, and molecular signals in these injectable scaffolds is another advantage over pre-fabricated scaffolds. Due to the importance of injectable scaffolds in tissue engineering, here different types of injectable scaffolds, their design challenges, and applications in bone and cartilage tissue regeneration are reviewed.

Three-dimensional-printed collagen/chitosan/secretome derived from HUCMSCs scaffolds for efficient neural network reconstruction in canines with traumatic brain injury.

The secretome secreted by stem cells and bioactive material has emerged as a promising therapeutic choice for traumatic brain injury (TBI). We aimed to determine the effect of 3D-printed collagen/chitosan/secretome derived from human umbilical cord blood mesenchymal stem cells scaffolds (3D-CC-ST) on the injured tissue regeneration process. 3D-CC-ST was performed using 3D printing technology at a low temperature (−20°C), and the physical properties and degeneration rate were measured. The utilization of low temperature contributed to a higher cytocompatibility of fabricating porous 3D architectures that provide a homogeneous distribution of cells. Immediately after the establishment of the canine TBI model, 3D-CC-ST and 3D-CC (3D-printed collagen/chitosan scaffolds) were implanted into the cavity of TBI. Following implantation of scaffolds, neurological examination and motor evoked potential detection were performed to analyze locomotor function recovery. Histological and immunofluorescence staining were performed to evaluate neuro-regeneration. The group treated with 3D-CC-ST had good performance of behavior functions. Implanting 3D-CC-ST significantly reduced the cavity area, facilitated the regeneration of nerve fibers and vessel reconstruction, and promoted endogenous neuronal differentiation and synapse formation after TBI. The implantation of 3D-CC-ST also markedly reduced cell apoptosis and regulated the level of systemic inflammatory factors after TBI.