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Acute myeloid leukemia (AML) is a deadly form of leukemia with ineffective traditional treatment and frequent chemoresistance-associated relapse. Personalized drug screening holds promise in identifying optimal regimen, nevertheless, primary AML cells undergo spontaneous apoptosis during cultures, invalidating the drug screening results. Here, we reconstitute a 3D osteogenic niche (3DON) mimicking that in bone marrow to support primary AML cell survival and phenotype maintenance in cultures. Specifically, 3DON derived from osteogenically differentiated mesenchymal stem cells (MSC) from healthy and AML donors are co-cultured with primary AML cells. The AML cells under the AML_3DON niche showed enhanced viability, reduced apoptosis and maintained CD33+ CD34-phenotype, associating with elevated secretion of anti-apoptotic cytokines in the AML_3DON niche. Moreover, AML cells under the AML_3DON niche exhibited low sensitivity to two FDA-approved chemotherapeutic drugs, further suggesting the physiological resemblance of the AML_3DON niche. Most interestingly, AML cells co-cultured with the healthy_3DON niche are highly sensitive to the same sample drugs. This study demonstrates the differential responses of AML cells towards leukemic and healthy bone marrow niches, suggesting the impact of native cancer cell niche in drug screening, and the potential of re-engineering healthy bone marrow niche in AML patients as chemotherapeutic adjuvants overcoming chemoresistance, respectively.

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Intricate microenvironment signals orchestrate to affect cell behavior and fate during tissue morphogenesis. However, the underlying mechanisms on how specific local niche signals influence cell behavior and fate are not fully understood, owing to the lack of in vitro platform able to precisely, quantitatively, spatially, and independently manipulate individual niche signals. Here, microarrays of protein-based 3D single cell micro-niche (3D-SCµN), with precisely engineered biophysical and biochemical niche signals, are micro-printed by a multiphoton microfabrication and micropatterning technology. Mouse embryonic stem cell (mESC) is used as the model cell to study how local niche signals affect stem cell behavior and fate. By precisely engineering the internal microstructures of the 3D SCµNs, we demonstrate that the cell division direction can be controlled by the biophysical niche signals, in a cell shape-independent manner. After confining the cell division direction to a dominating axis, single mESCs are exposed to asymmetric biochemical niche signals, specifically, cell-cell adhesion molecule on one side and extracellular matrix on the other side. We demonstrate that, symmetry-breaking (asymmetric) niche signals successfully trigger cell polarity formation and bias the orientation of asymmetric cell division, the mitosis process resulting in two daughter cells with differential fates, in mESCs.

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OBJECTIVE: To report on preoperative outcomes that guide the choice of surgical techniques to correct equinovarus foot in adults with brain injury. METHODS: Four databases (PubMed, MEDLINE, Cochrane, PEDro) were searched according to the PRISMA guidelines. Studies were included regardless of their level of proof, with no limitation on date of publication, and their quality was assessed with the Methodological Index for Non-Randomized Studies score. RESULTS: We analysed 61 studies (n = 2,293 participants); 523 participants underwent neurotomy, 437 calf musculotendinous lengthening, and 888 tibialis anterior transfer or alternative anterior transfers with the flexor digitorum/hallucis longus (n = 249), the extensor hallucis longus (n = 102), the tibialis posterior (n = 41) and the peroneus longus (n = 41). Two studies were dedicated to osteoarticular surgeries (n = 12 participants). Ankle dorsiflexors motricity was assessed before 70% of neurotomies as compared with 29% before isolated calf lengthening studies, their strength being at least 3/5 in 33% and 50% of the studies concerned, respectively. Passive ankle dorsiflexion was assessed before surgery in 87% of neurotomy studies, with 62% of studies investigating non-retracted spastic equinovarus foot. Before anterior tendon transfer with the tibialis anterior or another muscle, passive ankle dorsiflexion was reported in only 20% and 46% of studies, respectively, and dynamic tibialis anterior activation during gait in 46% and 56%. Although voluntary recruitment of the tibialis anterior produced a better functional result, the presence/correction of varus justified its transfer in 60% of studies as compared with 30% in other transfers, which were justified by hyperactivity or voluntary recruitment of transferred muscle. CONCLUSIONS: This review highlights the poor level of preoperative assessment and the absence of formal criteria to indicate the different surgical approaches in the management of equinovarus foot. It reinforces the interest of a systematic standardized preoperative assessment such as selective motor block and dynamic electromyography to choose the most suitable surgical procedure.

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Treatments of vision-threatening retinal diseases are often hampered by drug delivery difficulties. Polyelectrolytically-coated alginate encapsulated-cell therapy (ECT) systems have shown therapeutic efficacy through prolonged in vivo drug delivery but still face various biocompatibility, viability, drug delivery and mechanical stability issues in clinical trials. Here, novel, injectable alginate-poly-l-lysine (AP)-coated composite alginate-collagen (CAC) ECT gels were developed for sustained ocular drug delivery, and their long-term performance was compared with non-coated CAC ECT gels. All optimised AP-coated gels (AP1- and AP5.5-CAC ECT: 2 mg/ml collagen, 1.5% high molecular weight alginate, 50,000 cells/gel, with 0.01% or 0.05% poly-l-lysine coating for 5 min, followed by 0.15% alginate coating) and non-coated gels showed effective cell proliferation control, cell viability support and continuous delivery of bioactive glial cell-derived neurotrophic factor (GDNF) with no significant gel degradation in vitro and in rat vitreous. Most importantly, intravitreally injected gels demonstrated therapeutic efficacy in Royal College of Surgeons rats with retinal degeneration, resulting in reduced photoreceptor apoptosis and retinal function loss. At 6 months post-implantation, no host-tissue attachment or ingrowth was detected on the retrieved gels. Non-coated gels were mechanically more stable than AP5.5-coated ones under the current cell loading. This study demonstrated that both coated and non-coated ECT gels can serve as well-controlled, sustained drug delivery platforms for treating posterior eye diseases without immunosuppression.

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The nucleus pulposus (NP) of intervertebral disc represents a soft gel consisting of glycosaminoglycans (GAGs)-rich extracellular matrix (ECM). Significant loss of GAGs and normal functions are the most prevalent changes in degenerated disc. Attempts targeted to incorporate GAGs into collagen fibrous matrices have been made but the efficiency is very low, and the resulting structures showed no similarity with native NP. Inspired by the characteristic composition and structures of the ECM of native NP, here, we hypothesize that by chemically modifying the collagen (Col) and hyaluronic acid (HA) and co-precipitating with GAGs, a bio-inspired nano-material recapitulating the composition, ultra-structure and function of the GAG-rich ECM will be fabricated. Compositionally, the bio-inspired nano-material namely Aminated Collagen-Aminated Hyaluronic Acid-GAG (aCol-aHA-GAG) shows a record high GAG/hydroxyproline ratio up to 39.1:1 in a controllable manner, out-performing that of the native NP. Ultra-structurally, the nano-material recapitulates the characteristic 'nano-beads' (25 nm) and 'bottle-brushes' (133 nm) features as those found in native NP. Functionally, the nano-material supports the viability and maintains the morphological and phenotypic markers of bovine NP cells, and shows comparable mechanical properties of native NP. This work contributes to the development of a compositionally, structurally, and functionally biomimetic nano-material for NP tissue engineering.

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Damage to the hyaline cartilage of the joint surface and osteochondral fractures are key factors leading to the development of osteoarthritis in racehorses, representing a significant cause of racehorse retirement. To tissue-engineer an osteochondral unit that is suitable for joint repair, incorporation of a zone of calcified cartilage should be considered so as to mimic itsin vivocounterpart. To date, equine mesenchymal stem cells (eMSCs) have been reported to have multilineage differentiation potential. Yet the generation of a zone of calcified cartilage using eMSCs has not been reported. This work is an initial attempt to generate a zone of calcified cartilage using eMSCs as the single source of cells and collagen as the scaffolding material. Main advantages of using eMSCs over equine deep zone chondrocytes for the generation of a zone of calcified cartilage include no donor site morbidity and their ease of expansion in culture. Initially, we fabricated cartilage-like tissues and bone-like tissuesin vitroby differentiating eMSCs toward chondrogenic and osteogenic lineages for 21 d, respectively. We then aggregated the cartilage-like and bone-like tissues together with a layer of undifferentiated eMSCs-collagen gel in between to generate a 3-layer osteochondral unit. A zone of calcified cartilage was found between the cartilage-like and bone-like layers after a 14-day culture in chondrogenic differentiation medium. These results provide a solution toward tissue engineering of equine osteochondral units with interfacial zone without using chondrocytes harvested from the deep zone of healthy articular cartilage, and contribute to the future development of osteochondral tissue engineering strategies for human cartilage injuries in the long run.

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Upon monolayer cultures on flat and rigid plastic dishes, many cells de-differentiate and lose their native phenotype. Technologies able to identify and reconstitute the cell niche factors that best maintain the physiological cellular phenotype in cultures are critical. We have developed a multiphoton microfabrication and micropatterning (MMM) technology, a robust 3D micro-printing platform capable to fabricate protein microstructures and micropatterns with quantitative, spatial and independent control of the mechanical, topological and extracellular matrix properties. Here, using bovine nucleus pulposus cells (bNPCs) as an example, we aim to reconstitute a spectrum of individual cell niche factors (2 mechanical, 9 topological and 4 matrices) in vitro for multiplex cell niche factor screening, and fabricate the optimal combinations of a series of shortlisted cell niche factors that best maintain the bNPC phenotype. Among all factors screened, two topological (micropillar array; fiber-bead structure) and two matrix (laminin; vitronectin) factors were shortlisted and the combinatory cell niche factors reconstituted from the shortlisted factors were found to synergistically augmented the expression of selected bNPC phenotype markers (Col II, SNAP25 and Keratin 8) and maintained their morphology and phenotype. These optimal cell niches can be micro-printed on culture dishes for physiologically relevant cultures and contribute to biomimetic scaffold design for intervertebral disc tissue engineering.

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Hybrid perovskites are emerging as a promising, high-performance luminescent material; however, the technological challenges associated with generating high-resolution, free-form perovskite structures remain unresolved, limiting innovation in optoelectronic devices. Here, we report nanoscale three-dimensional (3D) printing of colored perovskite pixels with programmed dimensions, placements, and emission characteristics. Notably, a meniscus comprising femtoliters of ink is used to guide a highly confined, out-of-plane crystallization process, which generates 3D red, green, and blue (RGB) perovskite nanopixels with ultrahigh integration density. We show that the 3D form of these nanopixels enhances their emission brightness without sacrificing their lateral resolution, thereby enabling the fabrication of high-resolution displays with improved brightness. Furthermore, 3D pixels can store and encode additional information into their vertical heights, providing multilevel security against counterfeiting. The proof-of-concept experiments demonstrate the potential of 3D printing to become a platform for the manufacture of smart, high-performance photonic devices without design restrictions.

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Cells can sense mechanical signals through cytoskeleton reorganization. We previously discovered the formation of omni-directional actin protrusions upon compression loading, namely compression-induced actin protrusions (CAPs), in human mesenchymal stem cells (MSCs) in 3D micro-tissues. Here, the regulatory roles of three RhoGTPases (CDC42, Rac1 and RhoA) in the formation of CAPs were investigated. Upon compression loading, extensive formation of CAPs was found, significantly associated with an upregulated mRNA expression of Rac1 only, but not CDC42, nor RhoA. Upon chemical inhibition of these RhoGTPase activity during compression, only Rac1 activity was significantly suppressed, associating with the reduced CAP formation. Silencing the upstream regulators of these RhoGTPase pathways including Rac1 by specific siRNA dramatically disrupted actin cytoskeleton, distorted cell morphology and aborted CAP formation. Silencing cortactin (CTTN), a downstream effector of the Rac1 pathway, induced a compensatory upregulation of Rac1, enabling the MSCs to respond to the compression loading stimulus in terms of CAP formation, although at a reduced number. The importance of Rac1 signalling in CAP formation and the corresponding upregulation of lamellipodial markers also suggest that these CAPs are lamellipodia in nature. This study delineates the mechanism of compression-induced cytoskeleton reorganization, contributing to rationalizing mechanical loading regimes for functional tissue engineering.

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Engineered biomimetic cell niches represent a valuable in vitro tool for investigating physiological and pathological cellular activities, while developing an all-in-one technology to engineer cell niches, particularly soluble cell niche factors, with retained bioactivities, remains challenging. Here, we report a mask-free, non-contact and biocompatible multiphoton microfabrication and micropatterning (MMM) technology in engineering a spatially and quantitatively controllable bone morphogenetic protein-2 (BMP-2) soluble niche, by immobilizing optimally biotinylated BMP-2 (bBMP-2) on micro-printed neutravidin (NA) micropatterns. Notably, the micropatterned NA bound-bBMP-2 niche elicited a more sustained and a higher level of the downstream Smad signaling than that by free BMP-2, in C2C12 cells, suggesting the advantages of immobilizing soluble niche factors on engineered micropatterns or scaffold materials. This work reports a universal all-in-one cell niche engineering platform and contributes to reconstituting heterogeneous native soluble cell niches for signal transduction modeling and drug screening studies.

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Mechanical signal is important for regulating stem cell fate, but the molecular mechanisms involved are unclear. Cell-matrix adhesions are important molecular mechanosensors that their formation and maturation are force-dependent processes. However, most studies focused on the role of cell contractility or substrate stiffness in these processes. How external mechanical force stimulates the formation and maturation of cell-matrix adhesions is largely unknown. Here, by using human mesenchymal stem cells (hMSCs)-collagen microtissues as a 3D model, we found that upon short-term dynamic compression, integrin αV binding, focal adhesion formation, and subsequent FAK activation, are stimulated. This compression-stimulated FAK signaling also leads to YAP activation, suggesting crosstalk between integrin-based signaling and mechanosensing. More importantly, long-term compression induces maturation of α5-integrin based adhesions to form long, slender 3D-matrix adhesions (3DMAs), which are distinct from 2D focal adhesions in composition and morphology and previously found only in cell-derived matrices and native tissues. Mechanical preconditioning hMSCs with long-term compression loading induces the formation of mature integrin α5-dependent 3DMAs and potentiates their osteogenesis. Collectively, this work shows that active mechanical stimulation can modulate cell-matrix interactions significantly at the cell-material interfaces in a dynamic manner, and affects cell fate decisions, demonstrating the significance of loading-based functional tissue engineering.

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Direct mass-transfer via liquid nanodroplets is one of the most powerful approaches for additive micro/nanofabrication. Electrohydrodynamic (EHD) dispensing has made the delivery of nanosized droplets containing diverse materials a practical reality; however, in its serial form it has insufficient throughput for large-area processing. Here, a parallel, nanoscale EHD method is developed that offers both improved productivity and material diversity in 3D nanoprinting. The method exploits a double-barreled glass nanopipette filled with material inks to parallelize nanodripping ejections, enabling a dual 3D nanoprinting process. It is discovered that an unusual electric field distribution created by cross talk of neighboring pipette apertures can be used to steer the microscopic ejection paths of the ink at will, enabling on-demand control over shape, placement, and material mixing in 3D printed nanostructures. After thorough characterizations of the printing conditions, the parallel fabrication of nanomeshes and nanowalls of silver, CdSe/ZnS quantum dots, and their composites, with programmed designs is demonstrated. This method is expected to advance productivity in the heterogeneous integration of functional 3D nanodevices in a facile manner.

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Mesenchymal condensation is a critical transitional stage that precedes cartilage or bone formation. A microencapsulation technique was previously established to entrap mesenchymal stem cells (MSC) in nanofibrous collagen meshwork. We hypothesize that collagen microencapsulation of MSCs mimics the mesenchymal cell condensation process. Specifically, human MSCs at different concentrations were microencapsulated in collagen for different time points before evaluation for early skeletogenesis markers. A transient upregulation of mesenchymal condensation markers including peanut agglutinin, fibronectin, integrins α5 and αv, an enhanced nuclear localization of SOX9 and binding interactions with COL2A1, and other changes in chondrogenic, hypertropic and osteogenic marker were demonstrated. Collagen microencapsulation upregulated both the chondrogenic and the osteogenic transcription factors and the encapsulated hMSCs hold the potential to differentiate towards both chondrogenic and osteogenic lineages. We also hypothesize that collagen microencapsulation potentiates MSC chondrogenesis. Particularly, chondrogenic differentiation of hMSCs were induced at different time post-encapsulation before evaluation for chondrogenesis outcomes. Sustained SOX9, ACAN and COL2A1 expression were noted and the timing to induce supplement chondro-inductive factors matters. This study reports an extracellular matrix-based in vitro model of mesenchymal condensation, an early stage in skeletogenesis, contributing to rationalizing development-inspired tissue engineering.

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Effective retinal drug delivery remains a challenge for treating vision-threatening diseases. Encapsulated-cell therapy (ECT) can provide local drug delivery without repeated invasive injections but is plagued by unsteady performance and biosafety issues. Here, an injectable composite alginate-collagen (CAC) ECT gel with a Tet-on inducible pro-caspase 8 mechanism that acted as an orally-inducible biosafety switch was developed for safer drug delivery. The optimised gels (2 mg/ml collagen, 1.5% high molecular weight alginate and 50,000 cells/gel) could be effectively terminated in vitro (≥20 pg/ml Doxycycline) and in vivo (1 mg/ml oral Doxycycline after 48 h). Also, they displayed effective proliferation control and continuous delivery of bioactive glial-cell derived neurotrophic factor (GDNF) with no significant gel degradation in vitro and in rat vitreous. Most importantly, intravitreally injected gels demonstrated therapeutic efficacy in Royal College of Surgeons rats with degenerating retina in reducing photoreceptor apoptosis and retina function loss. Furthermore, double gel injections into the same eye yielded better outcomes without compromising gel viability. Retrieved gels showed no host-tissue attachment or cell-protrusion 6 months post-implantation. The CAC ECT system exhibited mechanical stability, good encapsulation power, cell viability support, multiplexed GDNF dosage, and compatibility with different cell types (HEK293 and ARPE-19) without immunosuppressant, making it an attractive, safe and well-controlled platform for treating various eye diseases.

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Immunomodulatory activity of mesenchymal stem cells (MSCs) is largely mediated by paracrine factors. Our previous studies showed that activation of nuclear factor-kappa B (NF-κB) regulates cytokine/growth factor secretion by MSCs. This study aimed to elucidate the role of Rap1 (repressor/activator protein), a novel modulator involved in the NF-κB pathway, in regulating the immunomodulatory potency of MSCs in acute allograft rejection of heart transplantation. The immunosuppressive potency of wild-type MSCs (WT-MSCs) or Rap1-deficient MSCs (Rap1-/--MSCs) was examined in mice with acute allograft rejection following heart transplantation. With a combination of immunosuppressant rapamycin at a dose of 1 mg/kg/d, WT-MSCs notably prolonged the survival of the transplanted heart compared with Rap1-/--MSCs. Rap1-/--MSCs displayed a marked insensitivity to inhibit the mixed lymphocyte reaction (MLR) due to impaired cytokine production and a significantly reduced activity of NF-κB signaling in vitro. Finally, transplantation of encapsulated WT-MSCs greatly prolonged the survival of the heart allograft compared with encapsulated Rap1-/--MSCs. Our results indicate that Rap1 is essential to maintain the immunomodulatory function of MSCs. Deletion of Rap1 results in impaired immunomodulatory function of MSCs.

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Porous bioscaffolds are applied to facilitate skin repair since the early 1990s, but a perfect regeneration outcome has yet to be achieved. Until now, most efforts have focused on modulating the chemical properties of bioscaffolds, while physical properties are traditionally overlooked. Recent advances in mechanobiology and mechanotherapy have highlighted the importance of biomaterials' physical properties in the regulation of cellular behaviors and regenerative processes. In skin repair, the mechanical and structural features of porous bioscaffolds are two major physical properties that determine therapeutic efficacy. Here, first an overview of natural skin repair with an emphasis on the major biophysically sensitive cell types involved in this multistage process is provided, followed by an introduction of the four roles of bioscaffolds as skin implants. Then, how the mechanical and structural features of bioscaffolds influence these four roles is discussed. The mechanical and structural features of porous bioscaffolds should be tailored to balance the acceleration of wound closure and functional improvements of the repaired skin. This study emphasizes that decoupling and precise control of the mechanical and structural features of bioscaffolds are significant aspects that should be considered in future biomaterial optimization, which can build a foundation to ultimately achieve perfect skin regeneration outcomes.

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Reconstituting biomimetic matrix niche in vitro and culturing cells at the cell niche interface is necessary to understand the effect and function of the specific matrix niche. Here we attempted to reconstitute a biomimetic extracellular matrix (ECM) niche by culturing nucleus pulposus cells (NPCs) in a collagen microsphere system previously established and allowing them to remodel the template matrix. The reconstituted NPC-derived complex ECM was obtained after decellularization and the composition of such niche was evaluated by proteomic analysis. Results showed that a complex acellular matrix niche consisting of ECM proteins and cytoskeletal proteins by comparing with the template collagen matrix starting material. In order to study the significance of the NPC-derived matrix niche, dermal fibroblasts were repopulated in such niche and the phenotypes of these cells were changed, gene expression of collagen type II and CA12 increased significantly. A biomimetic NPC-derived cell niche consisting of complex ECM can be reconstituted in vitro, and repopulating such matrix niche with fibroblasts resulted in changes in phenotypic markers. This work reports a 3D in vitro model to study cell niche factors, contributing to future understanding of cellular interactions at the cell-niche interface and rationalized scaffold design for tissue engineering.

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While cells are known to sense and respond to their niche including the matrix and the mechanical microenvironment, whether they preferentially sense and react to the stiffness of their microenvironment regardless of its intrinsic material properties is unknown. In this work, protein micropillar arrays with independently controllable stiffness via alterations in pillar height and elastic modulus via laser power used during photochemical cross-linking, were fabricated using a recently developed multiphoton-based 3D protein micro-patterning technology. Human dermal fibroblasts were cultured on these micropillar arrays and the specific interactions between cells and the protein micropatterns particularly on the formation and maturation of the cell-matrix adhesions, were investigated via immunofluorescence staining of the major molecular markers of the adhesions and the measurement of their cluster size, respectively. Our results showed that the cluster size of focal adhesions increased as the stiffness of the micropillar arrays increased, but it was insensitive to the elastic modulus of the protein micropillars that is one of the intrinsic material properties. This finding provides evidence to the notion that cells preferentially sense and react to the stiffness, but not the elastic modulus of their microenvironment.

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Cell-matrix adhesions are important structures governing the interactions between cells and their microenvironment at the cell-matrix interface. The focal complex (FC) and focal adhesion (FA) have been substantially investigated in conventional planar culture systems using fibroblasts as an in vitro model. However, the formation of more mature types of cell-matrix adhesion in human mesenchymal stem cells (hMSCs), including fibrillar adhesion (FBA) and 3D matrix adhesion (3DMA), have not been fully elucidated. Here we investigate the niche factor(s) that influence(s) the maturation of FBA and 3DMA by using multiphoton fabrication-based micropatterning. First, the bovine serum albumin (BSA)-made protein micropatterns were functionalized by incorporating various concentrations of fibronectin (FN) in fabrication solution. The amount of cross-linked FN is positively correlated with the initial concentration of FN in the reaction liquid, as verified by immunofluorescence staining. On the other hand, the anisotropic FN-functionalized micropatterns were fabricated by varying the length (i.e., in-plane stiffness) and height (i.e., bending stiffness) of micropatterns, respectively. Finally, hMSCs were cultured on these micropatterns for 2 h and 1 day to determine the formation of FBA and 3DMA, respectively, using immunofluorescence staining. Results demonstrated that FN-functionalized micropatterns with high anisotropy in x-y dimension benefit FBA maturation. Furthermore, niche factors such as higher bending and in-plane stiffness and the presence of abundant fibronectin have a positive effect on the maturation of FN-based cell-matrix adhesion. These findings could provide some new perspectives on designing platforms for further cell niche study and rationalizing scaffold design for tissue engineering.

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