Cell Spreading Area Research Articles

Effect of chiral polymers on Muse cell proliferation and differentiation

Cell therapy based on Muse cells has shown promising results in clinical trials and various applications in regenerative medicine. However, Muse cells face difficulties maintaining stemness and slow proliferation when cultured in vitro for long periods. In this study, the chiral monomers N-acryloyl-L/d-phenylalanine (NALPHE and NADPHE) were first synthesized and then chiral polymers (Poly(NALPHE) and Poly(NADPHE)) were synthesized by free radical polymerization. The circular dichroism (CD) signals of two chiral monomers (NALPHE and NADPHE) were equal in intensity and opposite in sign. This difference was further amplified owing to the high density of the phenylalanine-based pendants along the side chain formed by free radical polymerization. The influence of chiral polymers on Muse cell adhesion, proliferation, and osteogenic differentiation were then investigated. It was found that Muse cells grown in Poly(NALPHE) medium exhibited higher initial adhesion and higher proliferation rates, as well as larger cell spreading areas, compared to growth in Poly(NADPHE) medium. The higher cellular tension resulting from the good spreading ability of the cells allowed Muse cells to show a higher level of osteogenic differentiation in the Poly(NALPHE) medium. These results provide new insights for designing novel biomaterials more suitable for Muse cells proliferation and differentiation.

Carbon nanotubes perpendicularly grown on graphene oxide nanosheets derived from metal-organic frameworks: Synergistic reinforcement of poly(l-lactic acid) scaffold

The construction of the nanohybrid composed of two carbonaceous nanomaterials is a promising strategy to inhibit their aggregation, and effectively exert the advantages of their excellent mechanical properties as reinforcement for polymers. Here, carbon nanotubes (CNTs) were in situ perpendicularly growing on the surface of graphene oxide (GO) through metal-organic frameworks (MOF)-derived strategy by chemical vapor deposition (CVD), aiming to facilitate their dispersion on poly(l-lactic acid) (PLLA) bone scaffold fabricated by selective laser sintering (SLS). Specifically, GO provided nucleation sites for the growth of MOF, and worked as the templates when CNTs grew, in which MOF provided catalysts and carbon sources simultaneously. The precursor was heated to 550 °C and kept for 2 h, then heated to 900 °C and kept for 2 h before cooling. The obtained nanohybrid (GO@CNT) exhibited a superior dispersion state than the pure GO in the scaffold at the same loading, especially when the loading was 0.75 wt%. Adding 0.75 wt% GO@CNT endowed the scaffold with a remarkable increment in tensile strength and compressive strength of 13.36 MPa and 24.72 MPa with enhancement of 55.35% and 18.85%, respectively, compared to the scaffold containing 0.75 wt% GO. A crack extension model combined with experiment results was proposed to better understand reinforcement mechanisms. Additionally, the scaffold containing GO@CNT exhibited benign cytocompatibility with a cell-spreading area of 86.31% and cell density of 564 cells/mm2 after culturing for 5 d according to the results of cell adhesion and immunofluorescence tests, making it a promising candidate for bone defect repair.

Effect of micropillar density on morphology and migration of low and high metastatic potential breast cancer cells

Study of cell migration in cancer is crucial to the comprehension of the processes and factors that govern tumor spread. Cancer cells migrate invading tissues, causing alterations in cell adhesion, cytoskeleton, and signaling pathways. Little is known about the physical attributes of cancer cells that change when interacting with microenvironments. In this work, the local topography of the ECM has been mimicked through micropillar array substrates. MDA-MB-231 and MCF-7 breast cancer cells, exhibiting high and low metastatic potential, respectively, were analyzed. Differences in morphology and migration of the cells were investigated by examining the cell spreading area, circularity, aspect ratio, migration speed, and migration path. This work encountered that none of the studied cell lines have preferential orientation migrating on uniform patterns. In contrast, cell migration on graded patterns shows preferential orientation along the longitudinal direction from sparser to denser zones which is significantly influenced by substrate stiffness and indicates that both cell lines can sense the spacing gradient and respond to this topographical cue. The migration speed of the breast cancer cell lines significantly decreases from the sparse to medium to dense zones, registering higher values for the MDA-MB-231.

Ion-induced electrospinning of hierarchical spiderweb-like bioscaffolds

Tissue engineered scaffolds need to possess various functionalities, including biocompatibility, mechanical support, bioactivity, and vascularization. The design and fabrication of bioscaffolds to attain mutual coordination among these functionalities with minimal processing complexity are a highly challenging but rewarding task. In this study, a simple, effective one-step electrospinning method was developed to fabricate hierarchical spiderweb-like bioscaffolds that achieve both superior biological and mechanical functionalities. By incorporating ionic drugs of deferoxamine mesylate and lithium chloride, the spiderweb-inspired structures with adjustable coverages (up to 100 %) were successfully created, imparting the fibrous bioscaffolds with remarkable tensile strengths (∼88.28 MPa). The strengthening mechanisms endowed by the spiderweb structure in optimizing stress distribution to delay damage and enhance load-bearing ability were elucidated through finite element simulations. Furthermore, this hierarchical spiderweb-like bioscaffold demonstrated favorable biological characteristics, including biocompatibility, osteogenesis, angiogenesis, and hemostasis. The presence of the nano-spiderweb structures significantly improved cell adhesion and differentiation on the scaffold and increased the spreading area of cells by 2–3 times. The dual-drug loaded bioscaffolds with full coverages of the spiderweb structure exhibited the least amount of bleeding (45.33 ± 27.47 mg) and the fastest hemostasis speed (82 ± 8.19 s) in the hemostasis test, compared to the control group. Overall, the outstanding performance makes the developed bioscaffolds a promising alternative for tissue repair and regeneration in the field of tissue engineering.

Early Stages of Ex Vivo Collagen Glycation Disrupt the Cellular Interaction and Its Remodeling by Mesenchymal Stem Cells-Morphological and Biochemical Evidence.

Mesenchymal stem cells (MSCs), pivotal for tissue repair, utilize collagen to restore structural integrity in damaged tissue, preserving its organization through concomitant remodeling. The non-enzymatic glycation of collagen potentially compromises MSC communication, particularly upon advancing the process, underlying various pathologies such as late-stage diabetic complications and aging. However, an understanding of the impact of early-stage collagen glycation on MSC interaction is lacking. This study examines the fate of in vitro glycated rat tail collagen (RTC) upon exposure to glucose for 1 or 5 days in contact with MSCs. Utilizing human adipose tissue-derived MSCs (ADMSCs), we demonstrate their significantly altered interaction with glycated collagen, characterized morphologically by reduced cell spreading, diminished focal adhesions formation, and attenuated development of the actin cytoskeleton. The morphological findings were confirmed by ImageJ 1.54g morphometric analysis with the most significant drop in the cell spreading area (CSA), from 246.8 μm2 for the native collagen to 216.8 μm2 and 163.7 μm2 for glycated ones, for 1 day and 5 days, respectively, and a similar trend was observed for cell perimeter 112.9 μm vs. 95.1 μm and 86.2 μm, respectively. These data suggest impaired recognition of early glycated collagen by integrin receptors. Moreover, they coincide with the reduced fibril-like reorganization of adsorbed FITC-collagen (indicating impaired remodeling) and a presumed decreased sensitivity to proteases. Indeed, confirmatory assays reveal diminished FITC-collagen degradation for glycated samples at 1 day and 5 days by attached cells (22.8 and 30.4%) and reduced proteolysis upon exogenous collagenase addition (24.5 and 40.4%) in a cell-free system, respectively. The mechanisms behind these effects remain uncertain, although differential scanning calorimetry confirms subtle structural/thermodynamic changes in glycated collagen.

High-viscosity driven modulation of biomechanical properties of human mesenchymal stem cells promotes osteogenic lineage

Biomechanical cues could effectively govern cell gene expression to direct the differentiation of specific stem cell lineage. Recently, the medium viscosity has emerged as a significant mechanical stimulator that regulates the cellular mechanical properties and various physiological functions. However, whether the medium viscosity can regulate the mechanical properties of human mesenchymal stem cells (hMSCs) to effectively trigger osteogenic differentiation remains uncertain. The mechanism by which cells sense and respond to changes in medium viscosity, and regulate cell mechanical properties to promote osteogenic lineage, remains elusive. In this study, we demonstrated that hMSCs, cultured in a high-viscosity medium, exhibited larger cell spreading area and higher intracellular tension, correlated with elevated formation of actin stress fibers and focal adhesion maturation. Furthermore, these changes observed in hMSCs were associated with activation of TRPV4 (transient receptor potential vanilloid sub-type 4) channels on the cell membrane. This feedback loop among TRPV4 activation, cell spreading and intracellular tension results in calcium influx, which subsequently promotes the nuclear localization of NFATc1 (nuclear factor of activated T cells 1). Concomitantly, the elevated intracellular tension induced nuclear deformation and promoted the nuclear localization of YAP (YES‐associated protein). The concurrent activation of NFATc1 and YAP significantly enhanced alkaline phosphatase (ALP) for pre-osteogenic activity. Taken together, these findings provide a more comprehensive view of how viscosity-induced alterations in biomechanical properties of MSCs impact the expression of osteogenesis-related genes, and ultimately promote osteogenic lineage.

Dynamic RGD ligands derived from highly mobile cyclodextrins regulate spreading and proliferation of endothelial cells to promote vasculogenesis

A thiolated RGD was incorporated into the threaded allyl-β-cyclodextrins (Allyl-β-CDs) of the polyrotaxane (PR) through a thiol-ene click reaction, resulting in the formation of dynamic RGD ligands on the PR surface (dRGD-PR). When maintaining consistent RGD density and other physical properties, endothelial cells (ECs) cultured on dRGD-PR exhibited significantly increased cell proliferation and a larger cell spreading area compared to those on the non-dynamic RGD (nRGD-PCL). Furthermore, ECs on dRGD-PR demonstrated elevated expression levels of FAK, p-FAK, and p-AKT, along with a larger population of cells in the G2/M stage during cell cycle analysis, in contrast to cells on nRGD-PCL. These findings suggest that the movement of the RGD ligands may exert additional beneficial effects in promoting EC spreading and proliferation, beyond their essential adhesion and proliferation-promoting capabilities, possibly mediated by the RGD-integrin-FAK-AKT pathway. Moreover, in vitro vasculogenesis tests were conducted using two methods, revealing that ECs cultured on dRGD-PR exhibited much better vasculogenesis than nRGD-PCL in vitro. In vivo testing further demonstrated an increased presence of CD31-positive tissues on dRGD-PR. In conclusion, the enhanced EC spreading and proliferation resulting from the dynamic RGD ligands may contribute to improved in vitro vasculogenesis and in vivo vascularization.

Synergistic effects of graphene microgrooves and electrical stimulation on M2 macrophage polarization

Macrophages play a crucial role in host response and wound healing, with M2 polarization contributing to the reduction of foreign-body reactions induced by the implantation of biomaterials and promoting tissue regeneration. Electrical stimulation (ES) and micropatterned substrates have a significant impact on the macrophage polarization. However, there is currently a lack of well-established cell culture platforms for studying the synergistic effects of these two factors. In this study, we prepared a graphene free-standing substrate with 20 μm microgrooves using capillary forces induced by water evaporation. Subsequently, we established an ES cell culture platform for macrophage cultivation by integrating a self-designed multi-well chamber cell culture device. We observed that graphene microgrooves, in combination with ES, significantly reduce cell spreading area and circularity. Results from immunofluorescence, ELISA, and flow cytometry demonstrate that the synergistic effect of graphene microgrooves and ES effectively promotes macrophage M2 phenotypic polarization. Finally, RNA sequencing results reveal that the synergistic effects of ES and graphene microgrooves inhibit the macrophage actin polymerization and the downstream PI3K signaling pathway, thereby influencing the phenotypic transition. Our results demonstrate the potential of graphene-based microgrooves and ES to synergistically modulate macrophage polarization, offering promising applications in regenerative medicine.

Study on NaOH improving the surface morphology of three-dimensional printed poly- L- lactic acid mesh scaffolds

To explore the effect of NaOH on the surface morphology of three-dimensional (3D) printed poly- L-lactic acid (PLLA) mesh scaffolds. The 3D printed PLLA mesh scaffolds were prepared by fused deposition molding technology, then the scaffold surfaces were etched with the NaOH solution. The concentrations of NaOH solution were 0.01, 0.1, 0.5, 1.0, and 3.0 mol/L, and the treatment time was 1, 3, 6, 9, and 12 hours, respectively. There were a total of 25 concentration and time combinations. After treatment, the microstructure, energy spectrum, roughness, hydrophilicity, compressive strength, as well as cell adhesion and proliferation of the scaffolds were observed. The untreated scaffolds were used as a normal control. 3D printed PLLA mesh scaffolds were successfully prepared by using fused deposition molding technology. After NaOH etching treatment, a rough or micro porous structure was constructed on the surface of the scaffold, and with the increase of NaOH concentration and treatment time, the size and density of the pores increased. The characterization of the scaffolds by energy dispersive spectroscopy showed that the crystal contains two elements, Na and O. The surface roughness of NaOH treated scaffolds significantly increased ( P<0.05) and the contact angle significantly decreased ( P<0.05) compared to untreated scaffolds. There was no significant difference in compressive strength between the untreated scaffolds and treated scaffolds under conditions of 0.1 mol/L/12 h and 1.0 mol/L/3 h ( P>0.05), while the compression strength of the other treated scaffolds were significantly lower than that of the untreated scaffolds ( P<0.05). After co-culturing the cells with the scaffold, NaOH treatment resulted in an increase in the number of cells on the surface of the scaffold and the spreading area of individual cells, and more synapses extending from adherent cells. NaOH treatment is beneficial for increasing the surface hydrophilicity and cell adhesion of 3D printed PLLA mesh scaffolds.

Argon plasma-modified bacterial nanocellulose: Cell-specific differences in the interaction with fibroblasts and endothelial cells

Unique properties of bacterial nanocelullose (BNC), such as high purity, water retention, nanofibrillar structure and non-cytotoxicity, allow its application in regenerative medicine, particularly for active wound healing and for skin tissue engineering. However, its biological activity is not fully sufficient to be considered a good cell scaffold. This can be enhanced by physicochemical modifications, particularly plasma treatment. In this work, air-dried (AD) or lyophilized (L) BNC samples were modified in a direct current Ar+ plasma discharge (PM). BNC_AD samples showed higher surface roughness, tensile strength and Young's modulus but a lower swelling ratio than BNC_L samples. The swelling ratio of BNC_L samples further increased after PM. The samples were subjected to six-day in vitro cell culture tests with normal human dermal fibroblasts (NHDFs) or human umbilical vein endothelial cells (HUVECs), i.e. cell types important for skin defect healing. NHDFs adhered in higher numbers, by a larger cell spreading area and reached higher cell population densities on PM samples than on unmodified samples. However, in HUVECs, this effect of PM on cell growth was less pronounced or even opposite, especially on mechanically weaker and more swellable BNC_L. At the same time, both cell types preferred mechanically stronger BNC_AD for their adhesion and growth, which was more apparent in HUVECs. The expression of mRNA for markers of cell adhesion and phenotypic maturation in NHDFs (talin, vinculin, CD90) was generally similar on BNC_PM and on tissue culture polystyrene. In HUVECs, the expression of vinculin and PECAM-1 on all BNC samples was lower than on polystyrene, but the expression of KDR (VEGF receptor 2) was higher, especially on BNC_PM. These results indicate cell type-specific differences in the response to various BNC modifications, which must be properly balanced to accommodate various cell types needed for wound healing and skin tissue engineering.

Effect of Hydrogel Matrix and Fluid Shear Stress on the Behavioral Regulation of Mesenchymal Stem Cells

Development of a micromodel that recapitulates multiple mechanical properties to mimic the complex mechanical microenvironment is crucial for cell‐based research. Herein, a microsystem combining structure of hydrogel matrix and acoustic streaming (AS) to mimic the cellular microenvironment is proposed, which can realize multiplex cellular mechanical cues, including matrix stiffness, fluid shear stress (FSS) generated by AS, and matrix roughness. The results demonstrate that the cell spreading area enlarges with the increase of matrix stiffness, and cell spreading is encouraged by integrin β1 cluster to polymerize actin when combines the hydrogel matrix with FSS. In addition, FSS has the influence on the roughness of the hydrogel, which further affects the cell morphology and mechanical properties, inducing mesenchymal stem cells (MSCs) differentiation into astrocytes rapidly. Meanwhile, cell migration is also enhanced by FSS stimulation, particularly, undifferentiated cells at 22 kPa hydrogel have the fastest migration speed, and change the movement model from contact inhibition to contact stimulation migration. Especially, matrix roughness and stiffness dominantly control of cell spreading and differentiation, whereas FSS affects cell migration. In conclusion, the microsystem in this work shows superior performance in regulating the spreading, differentiation, and migration of MSCs, which provides a new tool for cell‐microenvironment study.

Multi-site enhancement of osteogenesis: peptide-functionalized GelMA hydrogels with three-dimensional cultures of human dental pulp stem cells.

Human dental pulp stem cells (hDPSCs) have demonstrated greater proliferation and osteogenic differentiation potential in certain studies compared to other types of mesenchymal stem cells, making them a promising option for treating craniomaxillofacial bone defects. However, due to low extracting concentration and long amplifying cycles, their access is limited and utilization rates are low. To solve these issues, the principle of bone-forming peptide-1 (BFP1) in situ chemotaxis was utilized for the osteogenic differentiation of hDPSCs to achieve simultaneous and synergistic osteogenesis at multiple sites. BFP1-functionalized gelatin methacryloyl hydrogel provided a 3D culture microenvironment for stem cells. The experimental results showed that the 3D composite hydrogel scaffold constructed in this study increased the cell spread area by four times compared with the conventional GelMA scaffold. Furthermore, the problems of high stem cell dosage and low rate of utilization were alleviated by orchestrating the programmed proliferation and osteogenic differentiation of hDPSCs. In vivo, high-quality repair of critical bone defects was achieved using hDPSCs extracted from a single tooth, and multiple 'bone island'-like structures were successfully observed that rapidly induced robust bone regeneration. In conclusion, this study suggests that this kind of convenient, low-cost, island-like osteogenesis strategy involving a low dose of hDPSCs has great potential for repairing craniomaxillofacial critical-sized bone defects.

Combining QCM-D with live-cell imaging reveals the impact of serum proteins on the dynamics of fibroblast adhesion on tannic acid-functionalised surfaces.

Nanocoatings based on plant polyphenols have been recently suggested as a potent strategy for modification of implant surfaces for enhancing host cell attachment and reducing bacterial colonisation. In this study we aimed to investigate how serum proteins impact the early adhesion dynamics of human gingival fibroblasts onto titanium surfaces coated with tannic acid (TA). Silicate-TA nanocoatings were formed on titanium and pre-conditioned in medium supplemented with 0, 0.1, 1 or 10% FBS for 1 hour. Dynamics of fibroblasts adhesion was studied using quartz crystal microbalance with dissipation (QCM-D). Time-lapse imaging was employed to assess cell area and motility, while immunofluorescence microscopy was used to examine cell morphology and focal adhesion formation. Our results showed that in serum-free medium, fibroblasts demonstrated enhanced and faster adhesion to TA coatings compared to uncoated titanium. Increasing the serum concentration reduced cell adhesion to nanocoatings, resulting in nearly complete inhibition at 10% FBS. This inhibition was not observed for uncoated titanium at 10% FBS, although cell adhesion was delayed and progressed slower compared to serum-free conditions. In addition, 1% FBS dramatically reduced cell adhesion on uncoated titanium. We revealed a positive relationship between changes in dissipation and changes in cell spreading area, and a negative relationship between dissipation and cell motility. In conclusion, our study demonstrated that serum decreases fibroblasts interaction with surfaces coated with TA in a concentration dependent manner. This suggests that controlling serum concentration can be used to regulate or potentially prevent fibroblasts adhesion onto TA-coated titanium surfaces.

Enzyme-Mineralized PVASA Hydrogels with Combined Toughness and Strength for Bone Tissue Engineering.

Enzymatic mineralization is an advanced mineralization method that is often used to enhance the stiffness and strength of hydrogels, but often accompanied by brittle behavior. Moreover, the hydrogel systems with dense networks currently used for enzymatic mineralization are not ideal materials for bone repair applications. To address these issues, two usual bone repair hydrogels, poly(vinyl alcohol) (PVA) and sodium alginate (SA), were selected to form a double-network structure through repeated freeze-thawing and ionic cross-linking, followed by enzyme mineralization. The results demonstrated that both enzymatic mineralization and double-network structure improved the mechanical and biological properties and even exhibited synergistic effects. The mineralized PVASA hydrogels exhibited superior comprehensive mechanical properties, with a Young's modulus of 1.03 MPa, a storage modulus of 103 kPa, and an equilibrium swelling ratio of 132%. In particular, the PVASA hydrogel did not suffer toughness loss after mineralization, with a high toughness value of 1.86 MJ/m3. The prepared hydrogels also exhibited superior biocompatibility with a cell spreading area about 13 times that of mineralized PVA. It also effectively promoted cellular osteogenic differentiation in vitro and further promoted the formation of new bone in the femur defect region in vivo. Overall, the enzyme-mineralized PVASA hydrogel demonstrated combined strength and toughness and great potential for bone tissue engineering applications.

A multiscale theory for spreading and migration of adhesion-reinforced mesenchymal cells.

We present a chemomechanical whole-cell theory for the spreading and migration dynamics of mesenchymal cells that can actively reinforce their adhesion to an underlying viscoelastic substrate as a function of its stiffness. Our multiscale model couples the adhesion reinforcement effect at the subcellular scale with the nonlinear mechanics of the nucleus-cytoskeletal network complex at the cellular scale to explain the concurrent monotonic area-stiffness and non-monotonic speed-stiffness relationships observed in experiments: we consider that large cell spreading on stiff substrates flattens the nucleus, increasing the viscous drag force on it. The resulting force balance dictates a reduction in the migration speed on stiff substrates. We also reproduce the experimental influence of the substrate viscosity on the cell spreading area and migration speed by elucidating how the viscosity may either maintain adhesion reinforcement or prevent it depending on the substrate stiffness. Additionally, our model captures the experimental directed migration behaviour of the adhesion-reinforced cells along a stiffness gradient, known as durotaxis, as well as up or down a viscosity gradient (viscotaxis or anti-viscotaxis), the cell moving towards an optimal viscosity in either case. Overall, our theory explains the intertwined mechanics of the cell spreading, migration speed and direction in the presence of the molecular adhesion reinforcement mechanism.

Varying the RGD concentration on a hyaluronic acid hydrogel influences dormancy versus proliferation in brain metastatic breast cancer cells.

A majority of breast cancer deaths occur due to metastasis of cancer cells to distant organs. In particular, brain metastasis is very aggressive with an extremely low survival rate. Breast cancer cells that metastasize to the brain can enter a state of dormancy, which allows them to evade death. The brain microenvironment provides biophysical, biochemical, and cellular cues, and plays an important role in determining the fate of dormant cancer cells. However, how these cues influence dormancy remains poorly understood. Herein, we employed hyaluronic acid (HA) hydrogels with a stiffness of ~0.4 kPa as an in vitro biomimetic platform to investigate the impact of biochemical cues, specifically alterations in RGD concentration, on dormancy versus proliferation in MDA-MB-231Br brain metastatic breast cancer cells. We applied varying concentrations of RGD peptide (0, 1, 2, or 4 mg/mL) to HA hydrogel surfaces and confirmed varying degrees of surface functionalization using a fluorescently labeled RGD peptide. Post functionalization, ~10,000 MDA-MB-231Br cells were seeded on top of the hydrogels and cultured for 5 days. We found that an increase in RGD concentration led to changes in cell morphology, with cells transitioning from a rounded to spindle-like morphology as well as an increase in cell spreading area. Also, an increase in RGD concentration resulted in an increase in cell proliferation. Cellular dormancy was assessed using the ratio of phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK) to phosphorylated p38 (p-p38) positivity, which was significantly lower in hydrogels without RGD and in hydrogels with lowest RGD concentration compared to hydrogels functionalized with higher RGD concentration. We also demonstrated that the HA hydrogel-induced cellular dormancy was reversible. Finally, we demonstrated the involvement of β1 integrin in mediating cell phenotype in our hydrogel platform. Overall, our results provide insight into the role of biochemical cues in regulating dormancy versus proliferation in brain metastatic breast cancer cells.

Preparation of porous GelMA microcarriers by microfluidic technology for Stem-Cell culture

Gelatin methacrylamide porous microcarriers (GelMA PMS), one of the most promising three-dimensional cell culture scaffolds, are limited in large-scale applications due to lack of ideal preparation method, unclear regulation of pore size on cell behavior and a lack of research on the entire cell culture process. Herein, combining microfluidic synchronous photo-crosslinking and ice-templating method, a low-cost, easy-assembly and suitable for scalability method is proposed to prepare GelMA PMS for bone marrow mesenchymal stem cells (BMSCs) expansion. The pore size of GelMA PMS can be controlled by adjusting the freezing temperature (-20 ℃, −60 ℃, −196℃). As a result, PMS 60 (frozen at −60 ℃) with the optimized pore size of 25.3 ± 3.2 μm exhibited the highest cell attachment ratio (90.2%) and the largest cell spreading area (0.516 mm2/MS). More importantly, PMS 60 displayed higher cell attachment efficiency, proliferation rate, adipogenic, osteogenic, and chondrogenic differentiation potential than commercial microcarriers Cytodex-1. Furthermore, BMSCs not only achieved bead-to-bead transfer on PMS 60 but also exhibited potent proliferation rate after transfer to new blank microcarriers. Cultured BMSCs were efficiently harvested from PMS 60 without damaging cell viability. In addition, the cryopreservation and thawing process did not affect the PMS 60 to support cell proliferation, while cells loaded on Cytodex-1 could not continue to proliferate. Therefore, GelMA PMS fabricated in this work shows great potential application and provides insights for MSCs culture.

Effect of microstructure and chemical composition on cell orientation and proliferation viability of titanium alloy processed by pulse lasers

Samples with different surface microstructures and elemental composition were fabricated by pulsed lasers with different pulse duration, including the millisecond laser remelting (RE), the bioceramic coating (BC), and nanosecond laser processing (NL) samples. The morphologies, chemical elements, phases, and valences of these samples were analyzed and characterized by scanning electron microscopy (SEM), energy dispersive spectroscopy (EDS), X-ray diffraction (XRD), and X-ray photoelectron spectroscopy (XPS). The contact angles of the samples were measured in air and under water. Their cell activity and behavior were analyzed by cell proliferation and cytoskeleton staining experiments. The results show that the hydrophilicity and aerophobicity increase after laser treatment, and the wetting behavior of underwater bubbles is strongly related to the wettability of the sample in the air. None of the samples examined in the experiment are cytotoxic. During the initial culture, the greater unevenness of the BC and NL sample surfaces hinders the initial expansion of the cells. In contrast, the cell spreading area of the ground (G) and the RE samples is larger. The inclined sidewall in the V-shaped structure weakens the energy barrier in the groove-ridge combination (NL sample), allowing cells that rarely adhere to the sidewall to adhere successfully. In addition, the orderly distribution of adhesion sites created by laser processing further promotes cell adhesion on the inclined sidewalls. When the cells have been cultured to Day 7, the NL sample forms a clear directional distribution of cells with its unique V-shaped structure mimicking the placoid scale on the shark skin surface.

Dual-Crosslinking of Gelatin-Based Hydrogels: Promising Compositions for a 3D Printed Organotypic Bone Model.

Gelatin-based hydrogels have emerged as a popular scaffold material for tissue engineering applications. The introduction of variable crosslinking methods has shown promise for fabricating stable cell-laden scaffolds. In this work, we examine promising composite biopolymer-based inks for extrusion-based 3D bioprinting, using a dual crosslinking approach. A combination of carefully selected printable hydrogel ink compositions and the use of photoinduced covalent and ionic crosslinking mechanisms allows for the fabrication of scaffolds of high accuracy and low cytotoxicity, resulting in unimpeded cell proliferation, extracellular matrix deposition, and mineralization. Three selected bioink compositions were characterized and the respective cell-laden scaffolds were bioprinted. Temporal stability, morphology, swelling, and mechanical properties of the scaffolds were thoroughly studied and the biocompatibility of the constructs was assessed using rat mesenchymal stem cells while focusing on osteogenesis. Experimental results showed that the composition of 1% alginate, 4% gelatin, and 5% (w/v) gelatine methacrylate, was found to be optimal among the examined, with shape fidelity of 88%, large cell spreading area and cell viability at around 100% after 14 days. The large pore diameters that exceed 100 µm, and highly interconnected scaffold morphology, make these hydrogels extremely potent in bone tissue engineering and bone organoid fabrication.

Stiffened fibre-like microenvironment based on patterned equidistant micropillars directs chondrocyte hypertrophy.

Articular cartilage, composed of collagen type II as a major extracellular matrix and chondrocyte as a unique cell type, is a specialized connective tissue without blood vessels, lymphatic vessels and nerves. This distinctive characteristic of articular cartilage determines its very limited ability to repair when damaged. It is well known that physical microenvironmental signals regulate many cell behaviors such as cell morphology, adhesion, proliferation and cell communication even determine chondrocyte fate. Interestingly, with increasing age or progression of joint diseases such as osteoarthritis (OA), the major collagen fibrils in the extracellular matrix of articular cartilage become larger in diameter, leading to stiffening of articular tissue and reducing its resistance to external tension, which in turn aggravates joint damage or progression of joint diseases. Therefore, designing a physical microenvironment closer to the real tissue and thus obtaining data closer to the real cellular behaviour, and then revealing the biological mechanisms of chondrocytes in pathological states is of crucial importance for the treatment of OA disease. Here we fabricated micropillar substrates with the same topology but different stiffnesses to mimic the matrix stiffening that occurs in the transition from normal to diseased cartilage. It was first found that chondrocytes responded to stiffened micropillar substrates by showing a larger cell spreading area, a stronger enhancement of cytoskeleton rearrangement and more stability of focal adhesion plaques. The activation of Erk/MAPK signalling in chondrocytes was detected in response to the stiffened micropillar substrate. Interestingly, a larger nuclear spreading area of chondrocytes at the interface layer between the cells and top surfaces of micropillars was observed in response to the stiffened micropillar substrate. Finally, it was found that the stiffened micropillar substrate promoted chondrocyte hypertrophy. Taken together, these results revealed the cell responses of chondrocytes in terms of cell morphology, cytoskeleton, focal adhesion, nuclei and cell hypertrophy, and may be beneficial for understanding the cellular functional changes affected by the matrix stiffening that occurs during the transition from a normal state to a state of osteoarthritis.