Pathological calcium signaling arises from dysregulated intracellular Ca²⁺ microdomains, but current interventions based on channel blockade or systemic chelation cannot selectively correct spatial calcium imbalance without perturbing physiological signaling. Here we report an intracellular Ca²⁺ capacitor (iCaCap), a synthetic organelle-like platform for spatial calcium reprogramming. iCaCap is built from a mesoporous bioactive glass framework whose metastable glassy network enables rapid and reversible ion exchange: boron accelerates Ca²⁺ capture by loosening network connectivity, whereas magnesium prevents crystallization of sequestered calcium and preserves buffering reversibility. As a result, iCaCap clips pathological cytosolic Ca²⁺ spikes, limits mitochondrial Ca²⁺ overload, and restores mitochondrial bioenergetics and suppresses TRPC6–NFAT2-associated profibrotic signalling. In diabetic stress models, iCaCap restores mitochondrial homeostasis, reduces oxidative stress and limits fibrotic remodeling in vivo. In vivo, targeted delivery of iCaCap alleviates renal fibrosis and improves tissue architecture. These findings identify bioactive glass as an active intracellular ionic material, and suggest a nanomaterial-based strategy for spatial regulation of pathological calcium signalling.

Bone tissue engineering materials are crucial for bone repair, but existing repair materials still face many challenges, including poor biocompatibility and bioactivity, slow self-repair processes, limited adaptability, inability to promote angiogenesis and so on. To address these issues, the development of third-generation bone repair materials, which are being designed to stimulate specific cellular responses at the molecular level, such as borate and borosilicate bioactive glasses (BBGs/BSBGs) that activate cells and genes, offers new potential for promoting bone tissue self-renewing. Their unique characteristic lies in a flow of life-giving energy, releasing beneficial ions such as boron, calcium and silicon to stimulate cell proliferation and differentiation, accelerating the regeneration of bones. Through this dynamic repair mechanism, these VitaFlux glasses operate like a "living system" within the body, not only speeding up the healing of damaged tissues but also interacting seamlessly with surrounding tissues during the repair process. In this review, we provide a comprehensive analysis of the current understanding of the osteogenesis mechanisms of BBGs/BSBGs, emphasizing their interactions with cells, including ion release and exchange, protein adsorption, and cell adhesion. We also examine key osteogenic signaling pathways related to the alkaline and ionic microenvironments of BBGs/BSBGs, such as the cell cycle, Wnt, MAPK, and BMP signaling pathways, along with macrophage polarization and angiogenesis. Additionally, strategies and future prospects for advancing BBGs/BSBGs research are discussed. Special attention is given to the NaBC1 and GPCR-mediated signaling pathways, which require further investigation.

Osteosarcoma (OS), with a high tendency for recurrence and metastasis, is associated with severe impairment of bone regeneration. The inherent temperature-sensitive property of tumors positions magnetic hyperthermia (MH) as an increasingly significant area in non-pharmacological cancer treatments. However, the temperature threshold for tumor ablation often causes tissue damage and bone homeostasis imbalance. Therefore, development of moderate MH for OS, capable of achieving tumor ablation while concurrently restoring bone homeostasis, offers significant potential for addressing this challenge. This study integrates magnetothermal nanoparticles with defined temperature thresholds and borosilicate bioactive glass (BSG) to create an injectable magnetothermal bioactive system that allows for regulation of MH temperature. The ionic and alkaline microenvironment from BSG degradation primarily impairs the malignant behavior of OS cells by activating the TNF signaling pathway. This sickening effect diminishes the hyperthermia tolerance of OS cells, thereby boosting apoptosis of OS cells, even in the presence of the limited anti-tumor effects of moderate MH. Furthermore, the combination of moderate MH and BSG also promotes optimal bone formation by stimulating human bone marrow mesenchymal stem cells (hBMSCs) via calcium and JAK-STAT3 signaling pathways. Collectively, this flourishes the therapeutic approaches and theories for the prevention and management of clinically refractory bone tumors.

Surface coating technology is broadly demanded across various fields, including marine and biomedical materials; therefore, a facile and versatile approach is desired. This study proposed an attractive surface coating strategy using photo-crosslinkable benzophenone (BP) moiety for biomaterials application. BP-containing "bioglue" polymer can effectively crosslink with all kinds of surfaces and biomolecules. Upon exposure to ultraviolet (UV) light, free radical reaction from the BP glue facilitates the immobilization of diverse molecules onto different substrates in a straightforward and user-friendly manner. Through either one-step, mixing the bioglue with targeted biomolecules, or two-step methods, pre-coating the bioglue and then adding targeted biomolecules, polyacrylic acid (PAA), cyclic RGD-containing peptides, and proteins (gelatin, collagen, and fibronectin) were successfully immobilized on substrates. After drying the bioglue, targeted biomolecules can still be immobilized on the surfaces preserving their bioactivity. Cell culture on biomolecule-immobilized surfaces using NIH 3T3 fibroblasts and human bone marrow stem cells (hBMSCs) showed significant improvement of cell adhesion and activity compared to the unmodified control in serum-free media after 24 hours. Furthermore, hBMSCs on the fibronectin-immobilized surface showed an increased calcium deposition after 21 days of osteogenic differentiation, suggesting that the immobilized fibronectin is highly bioactive. Given the straightforward protocol and substrate-independent bioglue, the proposed coating strategy is promising in broad-range fields.

Magnesium (Mg) alloys are popular biodegradable metals studied for orthopedic and cardiovascular applications, mainly because Mg ions are essential trace elements known to promote angiogenesis and osteogenesis. However, Mg corrosion consists of oxidation and reduction reactions that produce by-products, such as hydrogen gas, reactive oxygen species, and hydroxides. It is still unclear how all these by-products and Mg ions concomitantly alter the microenvironment and cell behaviors spatially and temporally. This study shows that Mg corrosion can enhance cell proliferation by reducing intracellular ROS. However, Mg cannot decrease ROS and promote cell proliferation in simulated inflammatory conditions, meaning the microenvironment is critical. Furthermore, cells may respond to Mg ions differently in chronic or acute alkaline pH or oxidative stress. Depending on the corrosion rate, Mg modulates HIF1α and many signaling pathways like PI3K/AKT/mTOR, mitophagy, cell cycle, and oxidative phosphorylation. Therefore, this study provides a fundamental insight into the importance of reduction reactions in Mg alloys.

The extracellular matrix (ECM) shapes the stem cell fate during differentiation by exerting relevant biophysical cues. However, the mechanism of stem cell fate decisions in response to ECM-backed complex biophysical cues has not been fully understood due to the lack of versatile ECMs. Here, we designed two versatile ECMs using colloidal self-assembly technology to probe the mechanisms of their effects on mechanotransduction and stem cell fate regulation. Binary colloidal crystals (BCC) with a hexagonally close-packed structure, composed of silica (5 μm) and polystyrene (0.4 μm) particles as well as a polydimethylsiloxane-embedded BCC (BCCP), were fabricated. They have defined surface chemistry, roughness, stiffness, ion release, and protein adsorption properties, which can modulate the cell adhesion, proliferation, and differentiation of human adipose-derived stem cells (hASCs). On the BCC, hASCs preferred osteogenesis at an early stage but showed a higher tendency toward adipogenesis at later stages. In contrast, the results of BCCP diverged from those of BCC, suggesting a unique regulation of ECM-dependent mechanotransduction. The BCC-mediated cell adhesion reduced the size of the focal adhesion complex, accompanying an ordered spatial organization and cytoskeletal rearrangement. This morphological restriction led to the modulation of mechanosensitive transcription factors, such as c-FOS, the enrichment of transcripts in specific signaling pathways such as PI3K/AKT, and the activation of the Hippo signaling pathway. Epigenetic analyses showed changes in histone modifications across different substrates, suggesting that chromatin remodeling participated in BCC-mediated mechanotransduction. This study demonstrates that BCCs are versatile artificial ECMs that can regulate human stem cells' fate through unique biological signaling, which is beneficial in biomaterial design and stem cell engineering.

Human platelet lysate (hPL) has emerged as a promising serum substitute to enhance the self-renewal and multipotency of human mesenchymal stem cells (MSCs). Despite its potential, the specific biological mechanisms by which hPL influences MSC phenotypes remain inadequately understood. We investigated the biological signaling activated by hPL in two common types of human MSCs: bone marrow-derived MSCs (BMSCs) and adipose-derived MSCs (ASCs). Cell adhesion and cell-matrix interaction were assessed through immunofluorescence staining and western blotting. The impact of hPL on lipid droplet formation in MSCs was thoroughly examined using oil red O/BODIPY staining, semi-quantitative analysis, and qRT-PCR. RNA sequencing and intracellular inhibition assays were also performed to elucidate the mechanisms by which hPL modulates MSC behavior. MSCs cultured in hPL medium demonstrated a reduction in cell size, spreading area, and vinculin puncta, while enhancing cell proliferation and lipid droplet accumulation compared to those cultured in control media. Notably, the lipid droplets in hPL-treated MSCs were significantly smaller than those in adipocyte-like cells differentiated from MSCs, highlighting hPL’s distinctive role in lipid production. Gene and protein expression profiles of hPL-treated MSCs differed from those in adipocyte-like cells. An angiogenic factor array revealed that hPL-MSCs had a distinct angiogenic factor profile compared to FBS-MSCs, with VEGF expression closely linked to HIF-1α expression. RNA-seq data identified approximately 1,900 differentially expressed genes (DEGs) between hPL-MSCs and FBS-MSCs, with enrichment in focal adhesion, ECM-receptor interaction, and PI3K-Akt/MAPK signaling pathways. Inhibition of MAPK phosphorylation significantly hampered lipid formation in hPL-MSCs, underscoring the pivotal role of MAPK signaling in hPL-driven adipogenesis. This study reveals the biological mechanisms by which hPL infleunces MSC behavior and differentiation, offering new insights into its potential application in regenerative medicine and tissue engineering.

Biophysical and biochemical cues modulate mammalian cell behavior and phenotype simultaneously. Macrophages, indispensable cells in the innate immune system, respond to external threats such as bacterial infections and implanted devices, undergoing the classical M1 polarization to become a pro-inflammatory phenotype. In the study, lipopolysaccharide (LPS)-induced M1 polarization was examined using RAW264.7, THP-1, and primary human PBMCs on a family of artificial extracellular matrix (ECM), named colloidal self-assembled patterns (cSAPs). The results showed that cSAPs were biocompatible, which cannot induce M1 or M2 polarization. Interestingly, specific cSAPs (e.g., cSAP3) suppress the level of M1 polarization (i.e., reduced nitric oxide production, down-regulated gene expression of iNOS, IL-6, TNF-α, IL-1β, and TLR4, and reduced proportion of CD11b+CD86+ cells). Transcriptome analysis showed that cell adhesion and cell-ECM interaction participated in the M1 polarization, and the mechano-sensitive genes such as PIEZO1 were down-regulated on the cSAP3. More interestingly, these genes were also down-regulated under LPS stimulation, indicating that cells became insensitive to the LPS. The abovementioned results indicate that the defined physicochemical cues can govern macrophage polarization. This study illustrates a potential surface design at biointerface, which is critical in tissue engineering and materiobiology. The outcome is also inspiring in ECM-mediated immune responses.

Cell cultures of dispersed cells within hydrogels depict the interaction of the cell–extracellular matrix (ECM) in 3D, while the coculture of different cells within spheroids combines both the effects of cell–cell and cell–ECM interactions. In this study, the cell co-spheroids of human bone mesenchymal stem cells/human umbilical vein endothelial cells (HBMSC/HUVECs) are prepared with the assistance of a nanopattern, named colloidal self-assembled patterns (cSAPs), which is superior to low-adhesion surfaces. A phenol-modified gelatin/hyaluronan (Gel-Ph/HA-Ph) hydrogel is used to encapsulate the multicellular spheroids and the constructs are photo-crosslinked using blue light. The results show that Gel-Ph/HA-Ph hydrogels with a 5%-to-0.3% ratio have the best properties. Cells in HBMSC/HUVEC co-spheroids are more favorable for osteogenic differentiation (Runx2, ALP, Col1a1 and OPN) and vascular network formation (CD31+ cells) compared to HBMSC spheroids. In a subcutaneous nude mouse model, the HBMSC/HUVEC co-spheroids showed better performance than HBMSC spheroids in angiogenesis and the development of blood vessels. Overall, this study paves a new way for using nanopatterns, cell coculturing and hydrogel technology for the generation and application of multicellular spheroids.

Topographical cues on material surfaces are crucial for guiding the behavior of nerve cells and facilitating the repair of peripheral nerve defects. Previously, micron-grooved surfaces have shown great potential in controlling nerve cell alignment for studying the behavior and functions of those cells and peripheral nerve regeneration. However, the effects of smaller-sized topographical cues, such as those in the submicron- and nano-scales, on Schwann cell behavior remain poorly understood. In this study, four different submicron-grooved polystyrene films (800/400, 800/100, 400/400, and 400/100) were fabricated to study the behavior, gene expression, and membrane potential of Schwann cells. The results showed that all submicron-grooved films could guide the cell alignment and cytoskeleton in a groove depth-dependent manner. Cell proliferation and cell cycle assays revealed that there was no significant difference between the submicron groove samples and the flat control. However, the submicron grooves can direct the migration of cells and upregulate the expression of critical genes in axon regeneration and myelination (e.g., MBP and Smad6). Finally, the membrane potential of the Schwann cells was significantly altered on the grooved sample. In conclusion, this study sheds light on the role of submicron-grooved patterns in regulating the behavior and function of Schwann cells, which provides unique insights for the development of implants for peripheral nerve regeneration.

Direct neuronal reprogramming of somatic cells into induced neurons (iNs) has been recently established as a promising approach to generating neuron cells. Previous studies have reported that the biophysical cues of the in vitro microenvironment are potent modulators in the cell fate decision; thus, the present study explores the effects of a customized pattern (named colloidal self-assembled patterns, cSAPs) on iN generation from human fibroblasts using small molecules. The result revealed that the cSAP, composed of binary particles in a hexagonal-close-packed (hcp) geometry, is capable of improving neuronal reprogramming efficiency and steering the ratio of the iN subtypes. Cells exhibited distinct cell morphology, upregulated cell adhesion markers (i.e., SDC1 and ITGAV), enriched signaling pathways (i.e., Hippo and Wnt), and chromatin remodeling on the cSAP compared to those on the control substrates. The result also showed that the iN subtype specification on cSAP was surface-dependent; therefore, the defined physicochemical cue from each cSAP is exclusive. Our findings show that direct cell reprogramming can be manipulated through specific biophysical cues on the artificial matrix, which is significant in cell transdifferentiation and lineage conversion.

Polydopamine (PDA) is a biocompatible cell-adhesive polymer with versatile applications in biomedical devices. Previous studies have shown that PDA coating could improve cell adhesion and differentiation of human mesenchymal stem cells (hMSCs). However, there is still a knowledge gap in the effect of PDA-mediated protein adsorption on the epigenetic status of MSCs. This work used gelatin-coated cell culture surfaces with and without PDA underlayer (Gel and PDA-Gel) to culture and differentiate primary human adipose-derived stem cells (hASCs). The properties of these two substrates were significantly different, which, in combination with a variation in extracellular matrix (ECM) protein bioactivity, regulated cell adhesion and migration. hASCs reduced focal adhesions by downregulating the expression of integrins such as αV, α1, α2, and β1 on the PDA-Gel compared to the Gel substrate. Interestingly, the ratio of H3K27me3 to H3K27me3+H3K4me3 was decreased, but this only occurred for upregulation of AGG and BMP4 genes during chondrogenic differentiation. This result implies that the PDA-Gel surface positively affects the chondrogenic, but not adipogenic and osteogenic, differentiation. In conclusion, for the first time, this study demonstrates the sequential effects of PDA coating on the biophysical property of adsorbed protein and then focal adhesions and differentiation of hMSCs through epigenetic regulation. This study sheds light on PDA-mediated mechanotransduction.

Emerging evidence indicates that cellular bioenergetics is critical in determining the self-renewal and differentiation of stem cells. Human platelet lysate (hPL) contains abundant proteins, which has been shown to improve self-renewal and osteogenic differentiation of mesenchymal stem cells (MSCs). However, the detailed modulating effect of hPL on MSCs energy metabolism remains unexplored. This study showed that MSCs cultured in hPL displayed a reduced cell size and cell spreading, but an improved proliferation and osteogenic capability compared with cells maintained in fetal bovine serum (FBS). RNA sequencing revealed widespread transcriptome differences between hPL-and FBS-MSCs where the differential expressed genes (DEGs) were enriched mainly in the PI3K-Akt and metabolic signal pathways. We found a significant downregulation of HIF1A (hypoxia-inducible factor 1 alpha) and altered mitochondrial features in hPL-MSCs, indicating a metabolism switch of the hPL-treated cells from glycolysis towards mitochondrial oxidative phosphorylation (OxPhos). It was also demonstrated that hPL-MSCs tend to differentiate towards the aerobic metabolism-demanded osteocytes or adipocytes rather than the anaerobic metabolism-demanded chondrocytes using a differentiation medium. Finally, hPL-MSCs showed an impaired paracrine function where the secreted factors cannot stimulate M2 polarization of THP1 cells and angiogenesis of HUVECs. We concluded that the PI3K-Akt/HIF1A-mediated metabolic state dominated the physiological property and lineage commitment of MSCs in hPL. For the first time, this study demonstrates the molecular mechanism of hPL in the regulation of metabolism and functions of MSCs, which implies the potential of hPL as an efficient biological material for stem cell engineering and regenerative medicine. (c) 2021 Elsevier Ltd. All rights reserved.

Hybrid surfaces with tunable topography, chemistry, and stiffness have potential to rebuild native extracellular matrix (ECM) and manipulate cell behavior in vitro. However, the fabrication of controllable hybrid surfaces is still challenging. In this study, colloidal self-assembly technology was used to program particles into highly ordered structures with hybrid chemistry and stiffness at biointerfaces. These colloidal self-assembled patterns (cSAPs), including unary, binary, and ternary cSAPs, composed of silicon (Si), polystyrene (PS), and/or poly(N-isopropylacrylamide) (pNIPAM) nanogels (PNGs), were fabricated using either coassembly or layer-by-layer (LBL) methods. The selected binary cSAPs (i.e., PS/PNG and PNG/PS) have a tunable surface topography and wettability between 25 and 37 °C; thus, they can be used as dynamic cell culture substrates. Human adipose-derived mesenchymal stem cells (hASCs), bone marrow-derived mesenchymal stem cells (hBMSCs), and macrophages (THP-1) were investigated on these hybrid cSAPs under a static or dynamic system. The results showed that hybrid cSAPs significantly influenced the focal adhesions, cell morphology, cell migration, and gene expressions of stem cells. In general, stem cells had more vinculin puncta, smaller spreading size, and faster migration speed than the TCPS control. Hybrid cSAPs up-regulated gene expressions of focal adhesion kinase (FAK) and chondrocytes (AGG and SOX9) under static culture, while they also up-regulated osteocytes (COL1 and RUNX2) under dynamic culture. THP-1 macrophages were at M0 state on all cSAPs under static culture. However, cells became sensitive under dynamic culture. For example, some M1 genes (i.e., IL6, CD68, and TNFα) and M2 genes (i.e., IL10 and CD206) were down-regulated, while other M1 genes (i.e., IL1β) and M2 genes (i.e., TGF-β and IL1ra) were up-regulated, depending on the particle combinations. In conclusion, new hybrid cSAPs with thermoresponsive surface properties are versatile materials for stem cells and macrophages manipulation.

The generation of complex physicochemical signals on the surface of biomedical materials is still challenging despite the fact that a broad range of surface modification methods have been developed over the last few decades. Colloidal self-assembled patterns (cSAPs) are combinations of unique colloids differing in size and surface chemistry acting as building blocks that can be programmed to generate surface patterns with exquisite control of complexity. This study reports on producing a variety of pre-modified colloids for the fabrication of cSAPs as well as post-assembly modifications to yield complex surfaces. The surface of cSAPs presents hierarchical micro- and nanostructures, localized hydrophilic/hydrophobic characteristics, and tunable surface functionality imparted by the individual colloids. The selected cSAPs can control bacterial adhesion (S. aureus, P. aeruginosa, and E. coli) and affect the cell cycle of human bone marrow stem cells (hBMSCs). Moreover, in a mouse subcutaneous model, cSAPs with selective [2-(methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium (SBMA) modification can reduce the inflammatory response after being challenged with bacteria. This study reveals that functionalized cSAPs are versatile tools for controlling cellular responses at biointerfaces, which is instructive for biomaterials or biodevices.

Decellularized human lung fibroblast-derived matrix (hFDM) has demonstrated its excellent proangiogenic capability. In this study, we propose a self-assembled, injectable multicellular microspheres containing human umbilical vein endothelial cells (HUVECs) and mesenchymal stem cell (MSCs), collagen hydrogel (Col), and hFDM toward therapeutic angiogenesis. Those multicellular microspheres are spontaneously formed by the mixtures of cell and hydrogel after being dropped on the parafilm for several hours. The size of microspheres can be manipulated via adjusting the initial volume of droplets and the culture period. The cells in the microspheres are highly viable. Multicellular microspheres show good capability of cell migration on 2D culture plate and also exhibit active cell sprouting in 3D environment (Col) forming capillary-like structures. We also find that multiple angiogenic-related factors are significantly upregulated with the multicellular microspheres prepared via Col and hFDM (Col/hFDM) than those prepared using Col alone or single cells (harvested from cocultured HUVECs/MSCs in monolayer). For therapeutic efficacy evaluation, three different groups of single cells, Col and Col/hFDM microspheres are injected to a hindlimb ischemic model, respectively, along with PBS injection as a control group. It is notable that Col/hFDM microspheres significantly improve the blood reperfusion and greatly attenuate the fibrosis level of the ischemic regions. In addition, Col/hFDM microspheres show higher cell engraftment level than that of the other groups. The incorporation of transplanted cells with host vasculature is detectable only with the treatment of Col/hFDM. Current results suggest that hFDM plays an important role in the multicellular microspheres for angiogenic cellular functions in vitro as well as in vivo. Taken together, our injectable multicellular microspheres (Col/hFDM) offer a very promising platform for cell delivery and tissue regenerative applications.

A decellularized extracellular matrix (dECM) is an excellent biomaterial in regenerative medicine, due to its biomimetic nature in targeting tissues and organs. In this study, we prepared cell-derived ECMs (CDM) derived from four different cell sources, characterized them individually, and found that intrinsic properties of each CDM were substantially different in terms of the fibrous matrix, total protein, and biochemical factors. Based on such information, we selected two ECM candidates, the human lung fibroblast derived matrix (hFDM) and the umbilical cord-blood mesenchymal stem cell derived matrix (UMDM) for the study of ECM-macrophage interactions in vitro and in vivo. In fact, UMDM was the richer in both total protein and angiogenic-related cytokines than any other CDM. When THP-1 cell-derived macrophages (M0) were seeded onto the UMDM or the hFDM, it showed a mixed cell morphology of macrophage phenotype and the macrophages (M0) preconditioned on UMDM presented more diverse cytokine release profiles. The treatment of conditioned medium obtained from CDM-seeded macrophages showed that UMDM could yield significantly advanced wound closure in 24 h via the human dermal fibroblast scratch model. To investigate the role of ECM on macrophage polarization in vivo, we prepared an ECM hydrogel, a mixture of each CDM and Pluronic F127/hyaluronan, and applied them onto a full-thickness mouse skin wound model for 2 weeks. The therapeutic efficacy as assessed via histology and immunofluorescence staining (α-SMA and CD206) revealed that the UMDM-treated group showed more effective wound healing compared to the other groups, as proven via the thinner epidermal layer, significant recovery of skin appendage, better neovascularization, and higher recruitment of myofibroblasts and larger number of macrophages (M2) at 7 days. The difference between UMDM and hFDM was marginal. Taken together, among the CDMs, UMDM and hFDM are promising resources of ECM, showing a great potential for wound healing. Although the mechanism is not fully understood, bioactive innate factors in UMDM may contribute individually and/or collectively to advance wound healing.

The fusion of skeletal myoblasts is a critical step in myotube formation and muscle maturation. Previously, we demonstrated that nanogrooves improved myoblast fusion via end-to-end fusion, which in turn generated parallel myotubes. However, the effect of serum components on the end-to-end fusion mechanism is unclear. In the current study, the synergistic effect of nanogrooves and human platelet lysate (hPL) on the growth and fusion of skeletal myoblasts was studied. Four types of nanogrooves (400 and 800 nm width; 100 and 400 nm depth) were used. Cell spreading, growth, and differentiation were screened on these nanogrooves in media formulated with hPL or fetal bovine serum (FBS), along with the flat substrate as a control group. The results showed that the deeper nanogrooves induced better alignment of myoblasts. hPL-adapted skeletal myoblasts (i.e., myoblasts@hPL) showed a smaller cell size with a more elongated morphology than myoblasts@FBS. During cell growth, the expression of myogenic genes (Myf5, MyoD, and MyoG) in myoblasts@hPL was lower than in myoblasts@FBS. During cell differentiation, myoblasts@hPL also expressed a lower level of myogenic and myosin heavy chain (MHC) genes. MHC-positive myoblasts@hPL without myotubes were found on all surfaces. Myomaker, an essential myoblast fusion gene, was upregulated during growth but downregulated during differentiation in myoblasts@hPL. Fibronectin-coated surfaces facilitate cell spreading and growth but still cannot support myoblast@hPL fusion. The results implied that hPL either lacks promotor factors or contains inhibitors on mouse skeletal myoblast fusion. This study reveals the effect of biophysical and biochemical cues on myoblast fusion and their potential for muscle tissue engineering.

Decellularized extracellular matrix (ECM)-based scaffold has been a very useful resource for effective tissue regeneration. In this study, we report a novel ECM patch that physically combines human fibroblast-derived matrix (hFDM) and poly(vinyl alcohol) (PVA) hydrogel. hFDM was obtained after decellularization of in vitro cultured human fibroblasts. We investigated the basic characteristics of hFDM alone using immunofluorescence (fibronectin, collagen type I) and angiogenesis-related factor analysis. Successful incorporation of hFDM with PVA produced an hFDM/PVA patch, which showed excellent cytocompatibility with human mesenchymal stem cells (hMSCs), as assessed via cell adhesion, viability, and proliferation. Moreover, in vitro scratch assay using human dermal fibroblasts showed a significant improvement of cell migration when treated with the paracrine factors originated from the hMSC-incorporated hFDM. To evaluate the therapeutic effect on wound healing, hMSCs were seeded on the hFDM/PVA patch and they were then transplanted into a mouse full-thickness wound model. Among four experimental groups (control, PVA, hFDM/PVA, hMSC/hFDM/PVA), we found that hMSC/hFDM/PVA patch accelerated the wound closure with time. More notably, histology and immunofluorescence demonstrated that compared to the other interventions tested, hMSC/hFDM/PVA patch could lead to significantly advanced tissue regeneration, as confirmed via nearly normal epidermis thickness, skin adnexa regeneration (hair follicle), mature collagen deposition, and neovascularization. Additionally, cell tracking of prelabeled hMSCs suggests the in vivo retention of transplanted cells in the wound region after the transplantation of hMSC/hFDM/PVA patch. Taken together, our engineered ECM patch supports a strong regenerative potential toward advanced wound healing.

Skin wound healing is an intricate orchestration that involves different cell types, an extracellular matrix (ECM), cytokines, and growth factors. On the basis of the great benefits of cell-derived ECM in regenerative applications, here we propose an electrospun fibrous membrane that combines poly(L-lactide-co-caprolactone) (PLCL) and human fibroblast-derived ECM (hFDM). hFDM-deposited PLCL (hFDM-PLCL) was obtained via decellularization of a confluent layer of fibroblasts cultivated on PLCL. An organized assembly of fibrillar structure on hFDM-PLCL was notable via immunostaining. As human umbilical vein endothelial cells (HUVECs) were seeded on the hFDM-PLCL, they proliferated faster and exhibited more elongated, capillary-like morphology than those on PLCL or fibronectin-coated PLCL (Fn-PLCL). HUVECs have a relatively large aspect ratio, spreading area, and vinculin-positive area per cell on the fibrillary structure of hFDM-PLCL. In addition, transwell cell migration assay showed the chemoattractant effect of hFDM for HUVECs and human dermal fibroblasts. Furthermore, HUVECs-loaded hFDM-PLCL membranes showed the most promising therapeutic effects on a mouse skin wound model as proved via the wound closure rate, neovascularization effect, regenerated epidermis, and skin appendage. This study shows that biodegradable PLCL fibers not only support the weak mechanical properties of hFDM but also allow hFDM to reserve ECM macromolecules and to maintain structural integrity. Current results also demonstrate the critical role of hFDM with biochemical and biophysical cues on HUVECs adhesion, proliferation, and vascular morphogenesis in vitro and even on the wound healing process in vivo. Taken together, hFDM-functionalized PLCL patch should be a promising platform for cell delivery and regenerative applications.