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Keratin is known to be a major protein in hair, but the biological function of keratin in hair growth is unknown, which led us to conduct a pilot study to elucidate biological function of keratin in hair growth via cellular interactions with hair forming cells. Here, we show hair growth is stimulated by intradermal injection of keratin into mice, and show that outer root sheath cells undergo transforming growth factor-β2-induced apoptosis, resulting in keratin exposure. Keratin exposure appears to be critical for dermal papilla cell condensation and hair germ formation as immunodepletion and silencing keratin prevent dermal papilla cell condensation and hair germ formation. Furthermore, silencing keratin in mice resulted in a marked suppression of anagen follicle formation and hair growth. Our study imply a new finding of how to initiate hair regeneration and suggests the potent application of keratin biomaterial for the treatment of hair loss.

Here we show that intradermal injection of keratin promotes hair growth in mice, which results from extracellular interaction of keratin with hair forming cells. Extracellular application of keratin induces condensation of dermal papilla cells and the generation of a P-cadherin-expressing cell population (hair germ) from outer root sheath cells via keratin-mediated microenvironmental changes. Exogenous keratin-mediated hair growth is reflected by the finding that keratin exposure from transforming growth factor beta 2 (TGFβ2)-induced apoptotic outer root sheath cells appears to be critical for dermal papilla cell condensation and P-cadherin-expressing hair germ formation. Immunodepletion or downregulation of keratin released from or expressed in TGFβ2-induced apoptotic outer root sheath cells negatively influences dermal papilla cell condensation and hair germ formation. Our pilot study provides an evidence on initiating hair regeneration and insight into the biological function of keratin exposed from apoptotic epithelial cells in tissue regeneration and development.

Dual stimuli-responsive degradable carbon-based nanoparticles (DS-CNPs) conjugated with Herceptin (HER) and polyethylene glycol (PEG) have been designed for the treatment of HER2-positive breast cancer. Each component has been linked through disulfide linkages that are sensitive to glutathione in a cancer microenvironment. β-cyclodextrin (β-CD) on the surface of DS-CNPs formed an inclusion complex (DL-CNPs) with doxorubicin (DOX) at a high loading capacity of 5.3 ± 0.4%. In response to a high level of glutathione (GSH) and low pH in a tumor environment, DL-CNPs were rapidly degraded and released DOX in a controlled manner via disruption of host–guest inclusion. These novel DL-CNPs exhibited high cellular uptake with low toxicity, which induced the efficient inhibition of antitumor activity both in vitro and in vivo. Cell viability, confocal laser scanning microscopy, and animal studies indicate that DL-CNPs are a great platform with a synergistically enhanced antitumor effect from the dual delivery of HER and DOX in DL-CNPs.

Biophysical properties of the microenvironment, including matrix elasticity and topography, are known to affect various cell behaviors; however, the specific role of each factor is unclear. In this study, fibroblast-derived matrix (FDM) was used as cell culture substrate and physically modified to investigate the influence of its biophysical property changes on human umbilical vein endothelial cells (HUVECs) and human mesenchymal stem cells (hMSCs) behavior in vitro. These FDMs were physically modified by simply storing them at different temperatures: the one stored at 4°C, maintained its original properties, was considered natural FDM, whereas the ones stored at -20°C or -80°C, exhibited a distinct surface morphology, were considered physically modified FDM. Physical modification induced matrix fiber rearrangement in FDM, forming different microstructures on the surface as characterized by focused ion beam (FIB)-cryoSEM. A significant increase of matrix elasticity was found with physically modified FDMs as determined by atomic force microscopy. HUVEC and hMSC behaviors on these natural and physically modified FDMs were observed and compared with each other and with gelatin-coated coverslips. HUVECs showed a similar adhesion level on these substrates at 3 h, but exhibited different proliferation rates and morphologies at 24 h; HUVECs on natural FDM proliferated relatively slower and assembled to capillary-like structures (CLSs). It is observed that HUVECs assembled to CLSs on natural FDMs are independent on the exogenous growth factors and yet dependent on nonmuscle myosin II activity. This result indicates the important role of matrix mechanical properties in regulating HUVECs vascular morphogenesis. As for hMSCs multilineage differentiation, adipogenesis is improved on natural FDM that with lower matrix elasticity, while osteogenesis is accelerated on physically modified FDMs that with higher matrix elasticity, these results further confirm the crucial role of matrix elasticity on cell fate determination.

Large expansion of human mesenchymal stem cells (MSCs) is of great interest for clinical applications. In this study, we examine the feasibility of human fibroblast-derived extracellular matrix (hFDM) as an alternative cell expansion setting. hFDM is obtained from decellularized extracellular matrix (ECM) derived from in vitro cultured human lung fibroblasts. Our study directly compares conventional platforms (tissue culture plastic (TCP), fibronectin (FN)-coated TCP) with hFDM using umbilical cord blood-derived MSCs (UCB-MSCs). Early cell morphology shows a rather rounded shape on TCP but highly elongated morphology on hFDM. Cell proliferation demonstrates that MSCs on hFDM were significantly better compared to the others in both 10 and 2% serum condition. Cell migration assay suggests that cell motility was improved and a cell migration marker CXCR4 was notably up-regulated on hFDM. MSCs differentiation into osteogenic lineage on hFDM was also very effective as examined via gene expression, von Kossa staining and alkaline phosphatase activity. In addition, as the MSCs were expanded on each substrate, transferred to 3D polymer mesh scaffolds and then cultivated for a while, the data found better cell proliferation and more CXCR4 expression with MSCs pre-conditioned on hFDM. Moreover, higher gene expression of stemness and engraftment-related markers was noticed with the hFDM group. Furthermore when UCB-MSCs expanded on TCP or hFDM were injected into emphysema (a lung disease) animal model, the results indicate that MSCs pre-conditioned on hFDM (with 2% serum) retain more advanced therapeutic efficacy on the improvement of emphysema than those on TCP. Current works demonstrate that compared to the conventional platforms, hFDM can be a promising source of cell expansion with a naturally derived biomimetic ECM microenvironment and may find some practical applications in regenerative medicine.

Growth factors (GFs) are major biochemical cues for tissue regeneration. Herein, a novel dual GF delivery system is designed composed of poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) and alginate microcapsules (MCs) via an electrodropping method. While bone morphogenetic protein (BMP)-2 is encapsulated in the PLGA NPs, vascular endothelial growth factor (VEGF) is included in the alginate MCs, where BMP-2-loaded PLGA NPs are entrapped together in the fabrication process. The initial loading efficiencies of BMP-2 and VEGF are 78% ± 3.6% and 43% ± 1.7%, respectively. When our dual GF-loaded MCs are assessed for in vitro osteogenesis of umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs) on 2D and 3D environment, MCs contribute to much better UCB-MSCs osteogenesis as confirmed by von Kossa staining, immunofluorescence (osteocalcin, collagen 1), calcium content measurement, and osteogenic markers expression. In addition, when dual GF-encapsulated MCs are combined with collagen and then applied to 8 mm diameter rat calvarial defect model, the positive effects on vascularized bone regeneration are much more pronounced; micro computed tomography (CT) and histology analyses exhibit 82.3% bone healing coupled with 12.6% vessel occupied area. Put together, current study indicates a synergistic effect of BMP-2/VEGF and highlights the great potential of dual GF delivery modality (PLGA NPs-in-MC) for regeneration of vascularized bone.

Extracellular matrices (ECM) obtained from in vitro-cultured cells have been given much attention, but its application in cardiac tissue engineering is still limited. This study investigates cardiomyogenic potential of fibroblast-derived matrix (FDM) as a novel ECM platform over gelatin or fibronectin, in generating cardiac cell lineages derived from H9c2 cardiomyoblasts. As characterized through SEM and AFM, FDM exhibits unique surface texture and biomechanical property. Immunofluorescence also found fibronectin, collagen, and laminin in the FDM. Cells on FDM showed a more circular shape and slightly less proliferation in a growth medium. After being cultured in a differentiation medium for 7 days, H9c2 cells on FDM differentiated into cardiomyocytes, as identified by stronger positive markers, such as α-actinin and cTnT, along with more elevated gene expression of Myl2 and Tnnt compared to the cells on gelatin and fibronectin. The gap junction protein connexin 43 was also significantly upregulated for the cells differentiated on FDM. A successive work enabled matrix stiffness tunable; FDM crosslinked by 2wt% genipin increased the stiffness up to 8.5 kPa, 100 times harder than that of natural FDM. The gene expression of integrin subunit α5 was significantly more upregulated on FDM than on crosslinked FDM (X-FDM), whereas no difference was observed for β1 expression. Interestingly, X-FDM showed a much greater effect on the cardiomyoblast differentiation into cardiomyocytes over natural one. This study strongly indicates that FDM can be a favorable ECM microenvironment for cardiomyogenesis of H9c2 and that tunable mechanical compliance induced by crosslinking further provides a valuable insight into the role of matrix stiffness on cardiomyogenesis.

Growth factors (GFs) are very critical in stem cell differentiation and tissue regeneration. Therefore GF delivery carriers have been a major subject in tissue engineering research. In this study, we prepare and optimize core-shell microcapsules (C-S MCs) for dual GF delivery. The C-S MCs, composed of an alginate shell and poly(lactic-co-glycolic) acid (PLGA) core, are fabricated using an electrodropping method via custom-made coaxial needles. They are 198±38 µm in diameter with an average core size of 90±13 µm, and they are fabricated using an alginate concentration of 1% (w/v), an electrical voltage of 11 kV, and an inner syringe flow rate of 50 µL/min. Using this platform, dual GFs, bone morphogenetic protein (BMP-2) and vascular endothelial growth factor (VEGF) are encapsulated in the alginate shell and PLGA core, respectively. In vitro release tests of dual GF-loaded C-S MCs reveal early release of BMP-2, followed by VEGF on a temporal release profile of 28 days. In vitro study of the dual GF-loaded MCs demonstrates their osteogenic activity with preosteoblasts; osteogenic markers (osteocalcin and type I collagen) are upregulated and both calcium content and alkaline phosphatase (ALP) activity also increased. In addition, C-S MCs combined with collagen and preosteoblasts were subcutaneously transplanted to the dorsal region of nude mice for 3 weeks. Analysis of the retrieved constructs exhibits that both osteogenesis and angiogenesis were more active in the group containing dual GF-loaded MCs, along with deep penetration of blood vessels inside the construct, compared to blank MCs or single GF (BMP-2)-loaded MCs. This study proposes a dual GF delivery carrier using C-S MCs and demonstrates the feasibility of C-S MCs in the induction of osteogenesis and angiogenesis.

An artificial matrix (Fn-Tigra), consisting of graphene oxide (GO) and fibronectin (Fn), is developed on pure titanium (Ti) substrates via an electrodropping technique assisted with a custom-made coaxial needle. The morphology and topography of the resulting artificial matrix is orderly aligned and composed of porous microcavities. In addition, Fn is homogenously distributed and firmly bound onto GO as determined via immunofluorescence and elemental mapping, respectively. The artificial matrix is moderately hydrophobic (63.7°), and exhibits an average roughness of 546 nm and a Young's modulus (E) of approximately 4.8 GPa. The biocompatibility, cellular behavior, and osteogenic potential of preosteoblasts on Fn-Tigra are compared to those of cells cultured on Ti and Ti-GO (Tigra). Cell proliferation and viability are significantly higher on Fn-Tigra and Tigra than that of cells grown on Ti. Focal adhesion molecule (vinculin) expression is highly activated at the central and peripheral area of preosteoblasts when cultured on Fn-Tigra. Furthermore, we demonstrate enhanced in vitro osteogenic differentiation of preosteoblasts cultured on Fn-Tigra over those cultured on bare Ti, as determined via Alizarin red and von Kossa staining, and the analysis of osteocalcin, type I collagen, alkaline phosphatase activity, and calcium contents. Finally, we investigate the biophysical and biomechanical properties of the cells using AFM. While the height and roughness of preosteoblasts increased with time, cell surface area decreased during in vitro osteogenesis over 2 weeks. In addition, the E of cells cultured on Tigra and Fn-Tigra increase in a statistically significant and time-dependent manner by 30%, while those cultured on bare Ti retain a relatively consistent E. In summary, we engineer a biocompatible artificial matrix (Fn-Tigra) capable of osteogenic induction and consequently demonstrate its potential in bone tissue engineering applications.