Xianliu Wang

Induced pluripotent stem cell-derived mesenchymal stem cells (iPSC-MSCs) are a new type of MSCs that come with attractive merits over the iPSCs per se. Aimed for regenerating bone tissues, this study was designed to investigate osteogenic differentiation and bone regeneration capacities of iPSC-MSCs by using biomimetic nanofibers of hydroxyapatite/collagen/chitosan (HAp/Col/CTS). Murine iPSCs were firstly induced to differentiate into iPSC-MSCs and thoroughly characterized. Effects of HAp/Col/CTS nanofibers prepared from electrospinning of Col-doped HAp/CTS nanocomposite, on osteogenic differentiation of the generated iPSC-MSCs were then evaluated in detail, including cell morphology, proliferation, migration, quantified specific osteogenic gene and protein expressions. Compared with different controls (TCP, CTS, and HAp/CTS), the HAp/Col/CTS scaffold was found to have more favorable effects on attachment and proliferation of iPSC-MSCs than others (P<0.01). Expressions of osteogenic genes, Runx2, Ocn, Alp, and Col, were significantly upregulated in iPSC-MSCs cultured on HAp/Col/CTS than CTS (P<0.01). Similarly, there appeared considerably higher secreting activities of osteogenesis protein markers, ALP and Col. Furthermore, mouse cranial defects were created to investigate efficacy of using iPSC-MSCs in combination with HAp/Col/CTS scaffold for regenerative bone repair in vivo. Examinations by computed tomography (CT) imaging, bone mineral density and hematoxylin eosin (HE) staining corroborated that cell-scaffold construct of iPSC-MSCs+HAp/Col/CTS could effectively promote bone regeneration. After 6 weeks of implantation, bone mineral density of the iPSC-MSCs+HAp/Col/CTS group was found to be nearly 2-fold higher than others. Our results demonstrated that biomimetic nanofibers of HAp/Col/CTS promoted the osteogenic differentiation and bone regeneration of iPSC-MSCs. The iPSC-MSCs+HAp/Col/CTS complex could be used as a new 'stem cell-scaffold' system for realizing personalized and efficacious bone regeneration in future.In bone tissue engineering, stem cells have become the most important source of seed cells. iPSC-MSCs are a new type of MSCs that come with attractive merits over the iPSCs per se. However, how to obtain befitting iPSC-MSCs and regulate their osteogenic differentiation are the key issues to be addressed. Given the great biomimicking capacity to extracellular matrix, electrospun nanofibers may be explored to modulate osteogenic differentiation of the iPSC-MSCs. This study successfully demonstrated that biomimetic nanofibers of HAp/Col/CTS significantly promoted the osteogenic differentiation and bone regeneration of iPSC-MSCs, which thereby suggests that nanofibrous scaffold supported iPSC-MSCs complex may be a new 'stem cell-scaffold' system for regulating the fate of osteogenic differentiation of iPSC-MSCs towards patient-specific bone regeneration in future.

Poor tendon repair is often a clinical challenge due to the lack of ideal biomaterials. Electrospun aligned fibers, resembling the ultrastructure of tendon, have been previously reported to promote tenogenesis. However, the underlying mechanism is unclear and the aligned fibers alone are not capable enough to commit teno-differentiation of stem cells. Here, based on our observation of reduced expression of histone deacetylases (HDACs) in tendon stem/progenitor cells (TSPCs) cultured on aligned fibers, we proposed a strategy to enhance the tenogenesis effect of aligned fibers by using a small molecule Trichostatin A (TSA), an HDAC inhibitor. Such a TSA-laden poly (l-lactic acid) (PLLA) aligned fiber (A-TSA) scaffold was successfully fabricated by a stable jet electrospinning method, and demonstrated its sustained capability in releasing TSA. We found that TSA incorporated aligned fibers of PLLA had an additive effect in directing tenogenic differentiation. Moreover, the in situ implantation study in rat model further confirmed that A-TSA scaffold promoted the structural and mechanical properties of the regenerated Achilles tendon. This study demonstrated that HDAC was involved in the teno-differentiation with aligned fiber topography, and the combination of HDAC with aligned topography might be a more efficient strategy to promote tenogenesis of stem cells. Electrospun aligned fibers, resembling the ultrastructure of tendon, have been previously reported to promote tenogenesis. However, the underlying mechanism is unclear and the aligned fibers alone are not capable enough to commit teno-differentiation of stem cells. The uniqueness of our studies are as follows, based on our observation of reduced expression of histone deacetylases (HDACs) in tendon stem/progenitor cells (TSPCs) cultured on aligned fibers, we proposed a strategy to enhance the tenogenesis effect of aligned fibers by using a small molecule Trichostatin A (TSA), a HDAC inhibitor. Such a TSA-laden poly (l-lactic acid) (PLLA) aligned fiber (A-TSA) scaffold was successfully fabricated by a stable jet electrospinning method, and demonstrated its sustained capability in releasing TSA. The incorporation and subsequent release of bioactive small molecule TSA into electrospun aligned fibers allows a controllable manner for both biochemical and physical regulation of tenogenesis of stem cells both in vitro and in vivo. Collectively, the present study provides a model of “translating the biological knowledge learned from cell-material interaction into optimizing biomaterials (from Biomat-to-Biomat)”.

To improve durability in wet conditions, electrospun chitosan (CTS) nanofibers were submersed into PBS (pH 7.4) solutions containing varied amounts of genipin (GP 0.1, 0.5, and 1% w/v) for crosslinking treatment. GP-crosslinking allowed the electrospun CTS nanofibers to maintain their fibrous morphology in wet state. Maximum tensile strength, 84.2% of the dry state strength, was attained when crosslinking was performed in GP 0.5% solution. GP-crosslinking also endowed the CTS nanofibers with enhanced resistances to swelling and enzymatic degradation. GP-crosslinked CTS nanofibers were found to significantly promote the adhesion and growth of the L929 fibroblasts, with the most suitable sample was the one crosslinked in the GP 0.5% solution as well. Our results suggest that crosslinking with the 0.5% GP in PBS could yield CTS nanofibers with improved wet stability in nanofiber structure and optimized mechanical and biological performances.

Electrospun uniaxially aligned ultrafine fibers show great promise in constructing vascular grafts mimicking the anisotropic architecture of native blood vessels. However, understanding how the stiffness of aligned fibers would impose influences on the functionality of vascular cells has yet to be explored. The present study aimed to explore the stiffness effects of electrospun aligned fibrous substrates (AFSs) on phenotypic modulation in vascular smooth muscle cells (SMCs). A stable jet coaxial electrospinning (SJCES) method was employed to generate highly aligned ultrafine fibers of poly(l-lactide-co-caprolactone)/poly(l-lactic acid) (PLCL/PLLA) in shell–core configuration with a remarkably varying stiffness region from 0.09 to 13.18 N/mm. We found that increasing AFS stiffness had no significant influence on the cellular shape and orientation along the fiber direction with the cultured human umbilical artery SMCs (huaSMCs) but inhibited the cell adhesion rate, promoted cell proliferation and migration, and especially enhanced the F-actin fiber assembly in the huaSMCs. Notably, higher fiber stiffness resulted in significant downregulation of contractile markers like alpha-smooth muscle actin (α-SMA), smooth muscle myosin heavy chain, calponin, and desmin, whereas upregulated the gene expression of pathosis-associated osteopontin (OPN) in the huaSMCs. These results allude to the phenotype of huaSMCs on stiffer AFSs being miserably modulated into a proliferative and pathological state. Consequently, it adversely affected the proliferation and migration behavior of human umbilical vein endothelial cells as well. Moreover, stiffer AFSs also revealed to incur significant upregulation of inflammatory gene expression, such as interleukin-6 (IL-6), monocyte chemoattractant protein-1 (MCP-1), and intercellular adhesion molecule-1 (ICAM-1), in the huaSMCs. This study stresses that although electrospun aligned fibers are capable of modulating native-like oriented cell morphology and even desired phenotype realization or transition, they might not always direct cells into correct functionality. The integrated fiber stiffness underlying is thereby a critical parameter to consider in engineering structurally anisotropic tissue-engineered vascular grafts to ultimately achieve long-term patency.

Biodegradable aliphatic polyesters, especially polylactide (PLA), polyglycolide (PGA), and their copolymer poly(lactide-co-glycolide) (PLGA), are the most representative and widely used synthetic polymers in the field of tissue engineering and regenerative medicine. However, these polyesters often give rise to aseptic inflammation because of their acidic degradation products after implantation. Here, unidirectional shell-core structured fibers of chitosan/poly(lactide-co-glycolide) (i.e., CTS/PLGA) with acid-neutralizing capability were developed for addressing the noted issue by coating the PLGA fiber surfaces with a layer of the alkaline chitosan by coaxial electrospinning. Our results showed that during a period of 8-week degradation, the shell-layer of chitosan with its unique alkaline nature for acid-neutralization obviously hindered the pH decrease as a result of the degradation of PLGA-core. In a mocked acidic environment testing of the human dermal fibroblasts, chitosan-enabled acidity neutralization could significantly reduce in vitro the secretion of inflammatory factors and downregulate the expression of related inflammatory genes. Thereafter, biocompatibility assessment in vitro showed that the CTS/PLGA fibers had poorer cell adhesion capacity than the PLGA fibers but were cytocompatible and promoted cell migration and secretion of collagen. Moreover, subcutaneous embedding for two and four weeks in vivo revealed that the CTS/PLGA fibers significantly reduced the recruitment of inflammatory cells and the formation of foreign body giant cells (FBGCs). This study thereby demonstrated the evident acid-neutralizing effect of the chitosan-coating layer on alleviating the inflammatory responses caused by the acidic degradation products of the PLGA-core. Our highly aligned CTS/PLGA fibers, as a kind of quasi "pH-neutral fibers" with the acid-neutralizing capability, could be potentially applied for engineering those architecturally anisotropic tissues (e.g., tendon/ligament) toward improved efficacy of regeneration. STATEMENT OF SIGNIFICANCE: It is well known that acidic degradation products from representative aliphatic polyesters (e.g., PLA, PGA, and PLGA) give rise to the problem of aseptic inflammation. Various alkaline components acting as neutralizing agents have been used to address the noted issue. However, rather less attention has been paid to engineer these polyesters into a fibrous form with acid-neutralizing functionality. The present study proposes the concept of "pH-neutral fibers" and develops shell-core structured unidirectional fibers of chitosan/poly(lactide-co-glycolide) with acid-neutralizing capability for ameliorating inflammatory responses caused by the acidic degradation products of PLGA. It provides a comprehensive study encompassing fiber characterization and in vitro and in vivo evaluation, which would pave the way for developing sophisticated pH-neutral fibers for functional tissue regeneration.

The high performance of SF fibers is attributed to the high fiber alignment, molecular orientation and contents of the β-pleated sheet.

HAp nanoparticle incorporated PLMC nanofibers with enhanced shape memory effect can potentially be used for bone screw hole healing.

Promoting healthy endothelialization of the tissue-engineered vascular grafts is of great importance in preventing the occurrence of undesired post-implantation complications including neointimal hyperplasia, late thrombosis, and neoatherosclerosis. Previous researches have demonstrated the crucial role of scaffold topography or stiffness in modulating the behavior of the monolayer endothelial cells (ECs). However, effects of the stiffness of scaffolds with anisotropic topography on ECs within vivo like oriented morphology has received little attention. In this study, aligned fibrous substrates (AFSs) with tunable stiffness (14.68–2141.72 MPa), similar to the range of stiffness of the healthy and diseased subendothelial matrix, were used to investigate the effects of fiber stiffness on ECs' attachment, orientation, proliferation, function, remodeling and dysfunction. The results demonstrate that stiffness of the AFSs, capable of providing topographical cues, is a crucial endothelium-protective microenvironmental factor by maintaining stable and quiescent endothelium with in vivo like orientation and strong cell–cell junctions. Stiffer AFSs exacerbated the disruption of endothelium integrity, the occurrence of endothelial-to-mesenchymal transition (EndMT), and the inflammation-induced activation in the endothelial monolayer. This study provides new insights into the understanding on how the stiffness of biomimicking anisotropic substrate regulates the structural and functional integrity of the in vivo like endothelial monolayer, and offers essential designing parameters in engineering biomimicking small-diameter vascular grafts for the regeneration of viable blood vessels. In vascular tissue engineering, promoting endothelialization on scaffold surface has been considered as a paramount strategy to reduce post-implantation complications. Electrospun aligned fibers have been known to provide contact guidance effect in directing endothelial cells' oriented growth, however, whether the formed EC monolayer in 'correct' orientation shape is of 'correct' function hasn't been explored yet. Given the recognized important role of substrate stiffness in endothelial function, AFSs across physiologically relevant range of moduli (14.68–2141.72 MPa) while maintaining consistent surface chemistry and topographical features were employed to investigate the fiber stiffness effects on ECs function in anisotropic morphology. This study will provide more insightful perspectives in the physiologically remodeling progression of vascular endothelium and design of vascular scaffolds.

Engineering scaffolds with structurally and biochemically biomimicking cues is essential for the success of tissue-engineered cartilage. Chitosan (CS)-based scaffolds have been widely used for cartilage regeneration due to its chemostructural similarity to the glycosaminoglycans (GAGs) found in the extracellular matrix of cartilage. However, the weak mechanical properties and inadequate chondroinduction capacity of CS give rise to compromised efficacy of cartilage regeneration. In this study, we incorporated short fiber segments, processed from electrospun aligned poly(lactic-co-glycolic acid) (PLGA) fiber arrays, into a citric acid-modified chitosan (CC) hydrogel scaffold for mechanical strengthening and structural biomimicking and meanwhile introduced cartilage-decellularized matrix (CDM) for biochemical signaling to promote the chondroinduction activity. We found that the incorporation of PLGA short fibers and CDM remarkably strengthened the mechanical properties of the CC hydrogel (+349% in compressive strength and +153% in Young’s modulus), which also exhibited a large pore size, appropriate porosity, and fast water absorption ability. Biologically, the engineered CDM-Fib/CC scaffold significantly promoted the adhesion and proliferation of chondrocytes and supported the formation of matured cartilage tissue with a cartilagelike structure and deposition of abundant cartilage ECM-specific GAGs and type II collagen (+42% in GAGs content and +295% in type II collagen content). The enhanced mechanical competency and chondroinduction capacity with the engineered CDM-Fib/CC scaffold eventually fulfilled successful in situ osteochondral regeneration in a rabbit model. This study thereby demonstrated a great potential of the engineered highly biomimetic chitosan-based scaffold in cartilage tissue repair and regeneration.

Poly(l-lactide) (PLLA) as one of the most well-known biodegradable polyesters has been studied extensively for bone tissue engineering. If being properly programmed, scaffolds from PLLA can also be endowed with the capability of shape memory. However, several noted issues, for example, mechanical brittleness, high glass transition temperature Tg, and relatively poor shape retention and recovery properties, necessitate modification of the PLLA to improve its application efficacy in physiological conditions. This study is proposed to modify PLLA by having the biodegradable poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) incorporated to form ultrafine composite fibers (i.e., PLLA-PHBV) through electrospinning. Different pairs of PLLA-PHBV at the varying mass ratios of 10:0, 9:1, 8:2, 7:3, 6:4, and 0:10 can be successfully electrospun into fibrous form with the fineness of 2-3 μm. Incorporation of PHBV enables to give rise to desired Tg decreases and also, interestingly, increases in the Young's modulus of the PLLA-PHBV blends, while gradually increasing the PHBV mass ratios up to 30%. The PLLA-PHBV (7:3) formulation is identified to present excellent shape memory properties with high shape fixing ratio (>98%) and shape recovery ratio (>96%) compared to the unmodified PLLA fiber counterpart. Moreover, the PLLA-PHBV (7:3) fibers also show enhanced osteogenesis-inducing ability in the mouse bone mesenchymal stem cells, even under nonosteoinductive conditions. Collectively, for the first time this study demonstrates the enhanced shape memory and osteogenesis capabilities of the electrospun PLLA-PHBV composite fibers, and the researched PLLA-PHBV (7:3) fiber system could be potentially applied as a multifunctional scaffolding material for applications in bone tissue repair and regeneration. Impact statement By first converting the poly(l-lactide) (PLLA)-poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) hybrids into fibrous form at varied mass ratios followed by a thorough characterization, we reasonably demonstrated that incorporation of an appropriate amount of PHBV (i.e., 30%) into the PLLA fibers could give rise to significant improvement on the shape memory capability of the PLLA, along with the desired decreases in the transition temperature (Tg). Moreover, the fibrous PLLA-PHBV (7:3) scaffold was also found to significantly promote the osteogenic commitment in bone mesenchymal stem cells with osteoinductive factors in a synergistic manner. Our biomimicking and shape memory enabled fibrous scaffold of PLLA-PHBV could be used to construct multifunctional three-dimensional scaffold with shape memory effect for bone regeneration.

Fibers produced from electrospinning are well-known to be extremely fine with diameters ranging from tens of nanometers to a few microns. Such ultrafine fibers not only allow for engineering scaffolds resembling the ultrastructure of the native extracellular matrix, but also offer possibility to explore the remodeling behavior of cells in vitro , due to their mechanically ‘adequate’ softness endowed by their ultrafine fineness. However, the remodeling effect of cells on the biomimicking fibrous substrates remains to be understood, because the crisscrossing and entangling among nanofibers in those tightly packed fibrous mats ultimately lead to merely a topological phenomenon, similar to that of the nanofiber-like topography embossed on the surface of a solid matter. In this study, the effect of nanofiber density on cellular response behavior was investigated by reducing the density of electrospun fiber networks. Using polycaprolactone (PCL) as a model polymer, randomly oriented fiber networks with various densities, namely, 37.7 ± 16.3 μg/cm 2 (D1), 103.8 ± 16.3 μg/cm 2 (D2), 198.2 ± 40.0 μg/cm 2 (D3), and 471.8 ± 32.7 μg/cm 2 (D4), were prepared by electrospinning for varied collection durations (10 s, 50 s, 100 s, and 10 min, respectively). By examining the responsive behavior of the human induced pluripotent stem cell-derived mesenchymal stem cells (hiPS-MSCs) cultured on these nanofibrous networks, we showed that the fiber network with a moderate density (D2) is beneficial to the cell attachment, spreading, actin polymerization, contractility and migration. There also showed an increased tendency in nuclear localization of the Yes-associated protein (YAP) and subsequent activation of YAP responsive gene transcription, and cell proliferation and collagen synthesis were also enhanced on the D2. However, further increasing the fiber density (D3, D4) gave rise to weakened induction effect of fibers on the cellular responses. These results enrich our understanding on the effect of fiber density on cell behavior, and disclose the dependence of cellular responses on fiber density. This study paves the way to precisely design biomimetic fibrous scaffolds for achieving enhanced cell-scaffold interactions and tissue regeneration. • A better understanding on cell-fiber interactions can be achieved by reducing the density of electrospun fiber networks. • Electrospun fiber network with a moderate density is optimal in supporting the cell function of hiPS-MSCs. • Topological and mechanical cues of the moderate-density fiber network could stimulate the cellular mechanotransduction.

Electrospun-aligned fibers in ultrathin fineness have previously demonstrated a limited capacity in driving stem cells to differentiate into tendon-like cells. In view of the tendon's mechanoactive nature, endowing such aligned fibrous structure with mechanoactivity to exert in situ mechanical stimulus by itself, namely, without any forces externally applied, is likely to potentiate its efficiency of tenogenic induction. To test this hypothesis, in this study, a shape-memory-capable poly(l-lactide-co-caprolactone) (PLCL) copolymer was electrospun into aligned fibrous form followed by a "stretching-recovery" shape-programming procedure to impart shape memory capability. Thereafter, in the absence of tenogenic supplements, human adipose-derived stem cells (ADSCs) were cultured on the programmed fibrous substrates for a duration of 7 days, and the effects of constrained recovery resultant stress-stiffening on cell morphology, proliferation, and tenogenic differentiation were examined. The results indicate that the in situ enacted mechanical stimulus due to shape memory effect (SME) did not have adverse influence on cell viability and proliferation, but significantly promoted cellular elongation along the direction of fiber alignment. Moreover, it revealed that tendon-specific protein markers such as tenomodulin (TNMD) and tenascin-C (TNC) and gene expression of scleraxis (SCX), TNMD, TNC, and collagen I (COL I) were significantly upregulated on the mechanoactive fibrous substrate with higher recovery stress compared to the counterparts. Mechanistically, the Rho/ROCK signaling pathway was identified to be involved in the substrate self-actuation-induced enhancement in tenodifferentiation. Together, these results suggest that constrained shape recovery stress may be employed as an innovative loading modality to regulate the stem cell tenodifferentiation by presenting the fibrous substrate with an aligned tendon-like topographical cue and an additional mechanoactivity. This newly demonstrated paradigm in modulating stem cell tenodifferentiation may improve the efficacy of tendon tissue engineering strategy for tendon healing and regeneration.

We have demonstrated that selected growth factors are involved in regulating survival and proliferation of progenitor cells derived from the neonatal rat organ of Corti (OC). The protective and regenerative effects of these defined growth factors on the injured organ of Corti were therefore investigated. The organ of Corti dissected from the Wistar rat pups (P3-P5) was split into apical, middle, and basal parts, explanted and cultured with or without neomycin and growth factors. Insulin-like growth factor-1 (IGF-1), fibroblast growth factor-2 (FGF-2), and epidermal growth factor (EGF) protected the inner hair cells (IHCs) and outer hair cells (OHCs) from neomycin ototoxicity. Using EGF, IGF-1, and FGF-2 alone induced no protective effect on the survival of auditory hair cells. Combining 2 growth factors (EGF + IGF-1, EGF + FGF-2, or IGF-1 + FGF-2) gave statistically protective effects. Similarly, combining all three growth factors effectively protected auditory hair cells from the ototoxic insult. None of the growth factors induced regeneration of hair cells in the explants injured with neomycin. Thus various combinations of the three defined factors (IGF-1, FGF-2, and EGF) can protect the auditory hair cells from the neomycin-induced ototoxic damage, but no regeneration was seen. This offers a possible novel approach to the treatment of hearing loss.

Significant progress has been made in the design of smart fibers toward achieving improved efficacy in tissue regeneration. While electrospun fibers can be engineered with shape memory capability, both the fiber structure and applied shape-programming parameters are the determinants of final performance in applications. Herein, we report a comparison study on the shape memory responses compared between electrospun random and aligned fibers by varying the programming temperature T prog and the deforming strain ε deform . A PLLA–PHBV (6:4 mass ratio) polymer blend was first electrospun into random and aligned fibrous mat forms; thereafter, the effects of applying specific T prog (37°C and 46°C) and ε deform (30%, 50%, and 100%) on the morphological change, shape recovery efficiency, and switching temperature T sw of the two types of fibrous structures were examined under stress-free condition, while the maximum recovery stress σ max was determined under constrained recovery condition. It was identified that the applied T prog had less impact on fiber morphology, but increasing ε deform gave rise to attenuation in fiber diameters and bettering in fiber orientation, especially for random fibers. The efficiency of shape recovery was found to correlate with both the applied T prog and ε deform , with the aligned fibers exhibiting relatively higher recovery ability than the random counterpart. Moreover, T sw was found to be close to T prog , thereby revealing a temperature memory effect in the PLLA–PHBV fibers, with the aligned fibers showing more proximity, while the σ max generated was ε deform -dependent and 2.1–3.4 folds stronger for the aligned one in comparison with the random counterpart. Overall, the aligned fibers generally demonstrated better shape memory properties, which can be attributed to the macroscopic structural orderliness and increased molecular orientation and crystallinity imparted during the shape-programming process. Finally, the feasibility of using the shape memory effect to enable a mechanoactive fibrous substrate for regulating osteogenic differentiation of stem cells was demonstrated with the use of aligned fibers.

Previous reports showed the presence of limited numbers of stem cells in neonatal murine cochlear sensory epithelia and these cells are progressively lost during the postnatal development. The goal of this study was to investigate whether stem cells can be derived from mature mouse cochleae under suspension culture conditions, and to analyze the expression of the stem cell and inner ear progenitor cell markers in cells dissociated from neonatal and adult mouse organs of Corti.Organs of Corti were dissected from postnatal day 1 (P1) or postnatal day 60 (P60) mouse. The dissociated cells were cultivated under suspension cultures conditions. Reverse transcription-polymerase chain reaction (RT-PCR) and immunocytochemistry were conducted for phenotype characterization.The number of cochlear stem cells (otospheres) yielded from P1 organ of Corti was significantly higher than that of the P60 organ of Corti. RT-PCR analyses showed that the stem markers, such as nanog, sox2, klf4, and nestin can be found to be distributed similarly in the cells derived from both of organisms, but the inner ear developmental/progenitor cell markers showed lower expression in P60 organ of Corti compared to P1. Immunocytochemistry results also revealed the evidence that P60 otospheres lacking of differentiation potential in vitro, which opposed to the strong differentiation potential of otospheres at P1 stage.Our findings suggest that the loss of numbers and features of stem cells in the adult organ of Corti is associated with the substantial down-regulation of inner ear progenitor key-markers during maturation of the cells in organ of Corti.

The presence of stem cells in the organ of Corti raises the hope of regeneration of mammalian inner ear cells. However, little is known about the distribution of endogenous stem cells in the inner ear as well as their sphere-forming abilities. The aim of this study is to evaluate the stem cells derived from different turns of the organ of Corti and analyze the sphere-forming capabilities of these stem cells. We dissected and isolated cochleae from postnatal day 1, 7 and 14 mice to separate organ of Corti into apical, middle and basal turns for cell isolation. Our results show that different turns of the organ of Corti harbor distinctly different populations of stem cells. Apical turn-derived cells give rise to more sphere-forming cells than middle and basal turn-derived cells, and middle turn-derived cells give rise to sphere cells number significantly higher than basal turn. Our findings indicate that apical turn of young age murine organ of Corti is best suited for isolation of endogenous stem cells for regeneration of hearing loss.

Graphene oxide (GO) and reduced graphene oxide (rGO) are two of the most representative graphene derivatives that can be employed to engineer bioactive and/or electroactive scaffolds for neural tissue engineering (NTE). However, a comparison with regard to their chemical structural impact on cytocompatibility has yet to be performed. In this study, GO was prepared using a modified Hummers' method followed by thermal treatment to obtain the rGO. Thereafter, a stable jet electrospinning approach was used to electrospin a viscous mixed solution of polycaprolactone (PCL)/poly(ethylene oxide) (PEO) (mass ratio 4:1) containing a tiny amount of GO and rGO (1% w/w), for the generation of highly-aligned nanoscale fibers of PCL-PEO-GO and PCL-PEO-rGO, respectively. Structural characteristics and physicochemical properties of the GO and/or rGO incorporated PCL-PEO nanofibers were characterized by various analytical techniques including Raman, FTIR, SEM, water drop contact angle, cyclic voltammetry and tensile testing. Finally, biological study in vitro by culturing the fibrous scaffolds with murine Schwann cells for cytocompatibility evaluation demonstrated that comparatively PCL-PEO-GO scaffold could more favorably promote the proliferation of the Schwann cells. However, considering the noted higher electroconductivity, the fibrous PCL-PEO-rGO may be applied to induce stem cell differentiation towards neural lineage. This research could provide a guidance for judicious selection of the GO and rGO incorporated nanofibers to construct electroactive scaffolds for engineering functional neural tissues.

In this work, the new calcium-magnesium-silicate Ca5MgSi3O12 ceramic was made via traditional solid-state reaction. The bioactivities were investigated by immerging the as-made ceramics in simulated body fluid (SBF) for different time at body temperature (37 °C). Then the samples were taken to measure X-ray powder diffraction (XRD), Scanning electron microscopy (SEM), X-ray energy-dispersive spectra (EDS), and Fourier transform infrared spectroscopy (FT-IR) measurements. The bone-like hydroxyapatite nanoparticles formation was observed on the ceramic surfaces after the immersion in SBF solutions. Ca5MgSi3O12 ceramics possess the Young’s modulus and the bending strength and of 96.3 ± 1.2 GPa and 98.7 ± 2.3 MPa, respectively. The data suggest that Ca5MgSi3O12 ceramics can quickly induce HA new layers after soaking in SBF. Ca5MgSi3O12 ceramics are potential to be used as biomaterials for bone-tissue repair. The cell adherence and proliferation experiments are conducted confirming the reliability of the ceramics as a potential candidate.

A capacitance-type MWCNTs/SiO2 composites humidity sensor based on capillary condensation and percolation phenomenon was developed in this paper. To study the model of this humidity sensor, the porosity structure of the sensing film (MWCNTs/SiO2) was observed by SEM image, and the pore volumes in MWCNTs/SiO2 film at different pore radius were investigated by an Accelerated Surface Area and Porosimetry System. The model of the MWCNTs/SiO2 humidity sensor was built based on the capillary condensation and percolation theory. To verify the sensor model, MWCNTs/SiO2 humidity sensors were presented and the sensors capacitances were tested under different RH. According to the testing results, the sensor with MWCNTs concentration of 1 wt% has the best linearity (about 5.2%), and the sensitivity of the sensors is about 673pF/%RH.

Icariin (ICA) is a small molecule drug capable of promoting cartilage repair and ameliorating inflammation. Loading ICA into a biomaterial scaffold for cartilage tissue engineering will thus potentially enhance the biological functionality of the engineered scaffold. In this study, short fibers processed from electrospun poly(l-lactide-co-caprolactone) (PLCL) fibers which were prior coated with polydopamine (PDA), were mixed with citric acid doped chitosan solution (CC) for preparing short fibers reinforced chitosan hydrogel (PDA@PLCL/CC) by a freeze-thawing combined freeze-drying method. Thereafter, ICA was loaded into the PDA@PLCL/CC scaffold through physical adsorption to generate a newly engineered biomimetic cartilage scaffold (ICA-PDA@PLCL/CC). Finally, ICA-mediated chondrogenic and ameliorated inflammatory effects of the ICA-PDA@PLCL/CC scaffold were examined in vitro using rabbit chondrocytes. The results showed that the ICA-free PDA@PLCL/CC scaffold possessed appropriate pore size and porosity (> 80%), high water absorbance capacity and improved mechanical performance, and also promoted chondrocyte proliferation and adhesion. The ICA-laden ICA-PDA@PLCL/CC scaffold was evidenced to maintain cytomorphology, upregulate the expression of chondrogenic gene (sox-9), glycosaminoglycan gene (gag), and type Ⅱ collagen gene (col Ⅱ) as well as the synthesis of the cartilage matrix. In the presence of a simulated inflammation, the ICA-PDA@PLCL/CC scaffold was found to reduce chondrocyte fibrosis, effectively downregulate the expression of proinflammatory factors interleukin-6 (il-6), interleukin-1 (il-1), and inducible nitric oxide synthase (inos) in chondrocytes. It can also reduce matrix metalloproteinase-3 (mmp-3) expression and promote the synthesis of the extracellular matrix glycosaminoglycan (GAG) and type II collagen (Col II). The newly developed ICA-PDA@PLCL/CC scaffold may find applications in the regeneration and repair of cartilage defects.

Biodegradable aliphatic polyesters, especially polylactide (PLA), polyglycolide (PGA), and their copolymer poly(lactide-co-glycolide) (PLGA), are the most representative and widely used synthetic polymers in the field of tissue engineering and regenerative medicine. However, these polyesters often give rise to the aseptic inflammation problem triggered by their acidic degradation products after implantation. Here, shell-core structured unidirectional fibers of chitosan/poly(lactide-co-glycolide) (i.e., CTS/PLGA) with acid-neutralizing capability were proposed for addressing the noted issue so as to achieve improved biocompatibility. Our results showed that during a period of 8-week degradation, the shell-layer of chitosan with its unique alkaline nature for acid-neutralization obviously hindered pH decrease as a result of the degradation of PLGA-core. In a mocked acidic environment testing with the human dermal fibroblasts, chitosan-enabled acidity neutralization could significantly reduce in vitro the secretion of inflammatory factors and down-regulate the expression of related inflammatory genes, such as Interleukin-6 (IL-6) and Interleukin-8 (IL-8) in the fibroblasts. Thereafter, biocompatibility assesments in vitro showed that CTS/PLGA nanofibers had poorer cell adhesion capacity than PLAG nanofibers, but were cytocompatible and promoted the cell migration and secretion of collagen. Moreover, two and four weeks of subcutaneous embedding in vivo revealed that the CTS/PLGA nanofibers significantly reduced the recruitment of inflammatory cells and the formation of foreign body giant cells (FBGCs). This study thereby demonstrated the excellent acid-neutralizing effect of the chitosan-coating layer on alleviating the inflammatory response caused by the acidic degradation products of the PLGA-core. Our highly-aligned CTS/PLGA nanofibers, as a kind of quasi ‘pH-neutral fibers’ with acid-neutralizing capability, may be potentially applied for engineering those architecturally anisotropic tissues (e.g., tendon/ligament) toward improved efficacy of regeneration.

Promoting healthy endothelialization of the tissue-engineered vascular grafts is of great importance in preventing the occurrence of undesired post-implantation complications including neointimal hyperplasia, late thrombosis, and neoatherosclerosis. Previous researches have demonstrated the crucial role of scaffold topography or stiffness in modulating the behavior of the monolayer endothelial cells (ECs). However, stiffness influence of the substrate/scaffold with anisotropic topography on ECs with in vivo like oriented morphology has rarely been attempted. In this study, aligned fibrous substrates (AFSs) with tunable stiffness (14.68~2141.72 MPa) resembling that of the healthy and diseased subendothelial matrix were used to investigate the effects of aligned fiber stiffness on EC attachment, orientation, proliferation, function, remodeling and dysfunction. The results demonstrate that stiffness of the AFSs capable of providing topographical cues is a crucial endothelium-protective microenvironmental factor by maintaining the long-standing, stable and quiescent endothelium with in vivo like orientation and strong cell-cell junctions. Stiffer AFSs exacerbated disruption of endothelium integrity, occurrence of endothelial-to-mesenchymal transition (EndMT), and inflammation-induced activation in endothelial monolayer. This study provides new insights into the understanding on how the stiffness of biomimicking anisotropic substrate regulates the structural and functional integrity of the in vivo like endothelial monolayer, and offers essential designing parameters in engineering biomimicking small-diameter vascular grafts for the regeneration of viable blood vessels.