Xiangxin Lou

Electrospun Biomimetic Fibrous Scaffold from Shape Memory Polymer of PDLLA-co-TMC for Bone Tissue Engineering

Multifunctional fibrous scaffolds, which combine the capabilities of biomimicry to the native tissue architecture and shape memory effect (SME), are highly promising for the realization of functional tissue-engineered products with minimally invasive surgical implantation possibility. In this study, fibrous scaffolds of biodegradable poly(d,l-lactide-co-trimethylene carbonate) (denoted as PDLLA-co-TMC, or PLMC) with shape memory properties were fabricated by electrospinning. Morphology, thermal and mechanical properties as well as SME of the resultant fibrous structure were characterized using different techniques. And rat calvarial osteoblasts were cultured on the fibrous PLMC scaffolds to assess their suitability for bone tissue engineering. It is found that by varying the monomer ratio of DLLA:TMC from 5:5 to 9:1, fineness of the resultant PLMC fibers was attenuated from ca. 1500 down to 680 nm. This also allowed for readily modulating the glass transition temperature Tg (i.e., the switching temperature for actuating shape recovery) of the fibrous PLMC to fall between 19.2 and 44.2 °C, a temperature range relevant for biomedical applications in the human body. The PLMC fibers exhibited excellent shape memory properties with shape recovery ratios of Rr > 94% and shape fixity ratios of Rf > 98%, and macroscopically demonstrated a fast shape recovery (∼10 s at 39 °C) in the pre-deformed configurations. Biological assay results corroborated that the fibrous PLMC scaffolds were cytocompatible by supporting osteoblast adhesion and proliferation, and functionally promoted biomineralization-relevant alkaline phosphatase expression and mineral deposition. We envision the wide applicability of using the SME-capable biomimetic scaffolds for achieving enhanced efficacy in repairing various bone defects (e.g., as implants for healing bone screw holes or as barrier membranes for guided bone regeneration).

Engineering aligned electrospun PLLA microfibers with nano-porous surface nanotopography for modulating the responses of vascular smooth muscle cells

In tissue engineering research, aligned electrospun ultrafine fibers have been shown to regulate cellular alignment and relevant functional expression, but the imposed effect of individual fiber surface nanotopography on cell behaviour has not been examined closely. This work investigates the impact of superimposing a nano-pore feature atop individual fiber surfaces on the responsive behaviour of human vascular smooth muscle cells (vSMCs) for blood vessel tissue engineering. Well-aligned ultrafine poly(l-lactic acid) (PLLA) microfibers with an average fiber diameter of ca. 1.6 μm were fabricated by using a novel stable jet electrospinning (SJES) method. Ellipse-shaped nano-pores with varied aspect ratios (defined as long-to-short axis ratio) of 2.7-3.9, corresponding to a surface nano-roughness in the range of 54.8-110.0 nm, were in situ generated onto individual fiber surfaces by varying ambient humidity from 45% to 75% during the SJES process. The presence of elliptical nano-pores on fiber surfaces affected the characteristic anisotropic wettability of the aligned PLLA fibers and contributed to greater protein adsorption (up to 17.59 μg mg-1). A 7 day in vitro assessment of human umbilical arterial SMCs cultured on these aligned nano-porous fiber substrates indicated that cellular responses were in close correlation with the elliptical nano-pore feature. A pronounced fiber surface nanotopography was superior in soliciting favorable cellular responses, leading to enhanced cell attachment, proliferation, alignment, expression of the vascular matrix proteins and maintenance of a contractile phenotype. This study thus suggests that introduction of an elliptical nano-pore feature to the aligned microfiber surfaces could provide additional dimensionality of topographical cues to modulate the vSMC responses when using the aligned electrospun ultrafine fibers for engineering vascular constructs.

Ultrasound-Modulated Shape Memory and Payload Release Effects in a Biodegradable Cylindrical Rod Made of Chitosan-Functionalized PLGA Microspheres

Minimally invasive implants and/or scaffolds integrated with multiple functionalities are of interest in the clinical settings. In this paper, chitosan (CTS) functionalized poly(lactic-co-glycolic acid) (PLGA) microspheres containing a model payload, lysozyme (Lyz), were prepared by a water-in-oil-in-water emulsion method, from which cylindrical shaped rod (5 mm in diameter) was fabricated by sintering the composite microspheres in a mold. High-intensity focused ultrasound (HIFU) was then employed as a unique technique to enable shape memory and payload release effects of the three-dimensional (3-D) structure. It was found that incorporation of CTS into PLGA microspheres could regulate the transition temperature Ttrans of the microsphere from 45 to 50 °C and affect shape memory ratio of the fabricated cylindrical rod to some extent. Shape memory test and drug release assay proved that HIFU could modulate the shape recovery process and synchronize the release kinetics of the encapsulated Lyz in the rod in a switchable manner. Moreover, the two processes could be manipulated by varying the acoustic power and insonation duration. Mechanical tests of the microspheres-based rod before and after ultrasound irradiation revealed its compressive properties in the range of trabecular bone. Examination of the degradation behavior indicated that the introduction of CTS into the PLGA microspheres also alleviated acidic degradation characteristic of the PLGA-dominant cylindrical rod. With HIFU, this study thus demonstrated the desired capabilities of shape recovery and payload release effects integrated in one microspheres-based biodegradable cylindrical structure.

Genipin-crosslinked electrospun chitosan nanofibers: Determination of crosslinking conditions and evaluation of cytocompatibility

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.

Direct printing of patterned three-dimensional ultrafine fibrous scaffolds by stable jet electrospinning for cellular ingrowth

Electrospinning has been widely used to produce ultrafine fibers in microscale and nanoscale; however, traditional electrospinning processes are currently beset by troublesome limitations in fabrication of 3D periodic porous structures because of the chaotic nature of the electrospinning jet. Here we report a novel strategy to print 3D poly(L-lactic acid) (PLLA) ultrafine fibrous scaffolds with the fiber diameter of approximately 2 μm by combining a stable jet electrospinning method and an X-Y stage technique. Our approach allows linearly deposited electrospun ultrafine fibers to assemble into 3D structures with tunable pore sizes and desired patterns. Process conditions (e.g., plotting speed, feeding rate, and collecting distance) were investigated in order to achieve stable jet printing of ultrafine PLLA fibers. The proposed 3D scaffold was successfully used for cell penetration and growth, demonstrating great potential for tissue engineering applications.

HAp incorporated ultrafine polymeric fibers with shape memory effect for potential use in bone screw hole healing

In the clinical setting of bone fracture healing, hardware removal often causes localized microtrauma and residual screw holes may act as stress risers to place the patient at a risk of refracture. To address this noted issue, this study proposed to develop a biologically mimicking and mechanically self-actuated nanofibrous screw-like scaffold/implant for potential in situ bone regeneration. By incorporating nano-hydroxyapatite (HAp) into a shape memory copolymer poly(d,l-lactide-co-trimethylene carbonate) (PLMC) via co-electrospinning, composite nanofibers of HAp/PLMC with various HAp proportions (1, 2 and 3 wt%) were successfully generated. Morphological, thermal and mechanical properties as well as the shape memory effect of the resultant HAp/PLMC nanofibers were characterized using a variety of techniques. Thereafter, osteoblasts isolated from rat calvarial were cultured on the fibrous HAp/PLMC scaffold to assess its suitability for bone regeneration in vitro. We found that agglomerates gradually appeared on the fiber surface with increasing HAp loading fraction. The switching temperature for actuating shape recovery Ts (i.e., glass transition temperature Tg) of the fibrous HAp/PLMC was readily modulated to fall between 43.5 and 51.3 °C by varying the HAp loadings. Excellent shape memory properties were achieved for the HAp/PLMC composite nanofibers with a shape recovery ratio of Rr > 99% and shape fixity ratio of Rf > 99%, and the shape recovery force of the HAp/PLMC nanofibers was also strengthened compared to that of the HAp-free PLMC nanofibers. Moreover, we demonstrated that the engineered screw-like HAp/PLMC scaffold/implant (ϕ = 5 mm) was able to return from a slender bar to its original stumpy shape in a time frame of merely 8 s at 48 °C. Biological assay results corroborated that the incorporation of HAp to PLMC nanofibers significantly enhanced the alkaline phosphatase secretion as well as mineral deposition in bone formation. These attractive results warrant further investigation in vivo on the feasibility of applying the biomimicking nanofibrous HAp/PLMC scaffold with shape memory effect for bone screw hole healing.

Highly aligned core–shell structured nanofibers for promoting phenotypic expression of vSMCs for vascular regeneration

This study was designed to assess the efficacy of hyaluronan (HA) functionalized well-aligned nanofibers of poly-l-lactic acid (PLLA) in modulating the phenotypic expression of vascular smooth muscle cells (vSMCs) for blood vessel regeneration. Highly aligned HA/PLLA nanofibers in core-shell structure were prepared using a novel stable jet electrospinning approach. Formation of a thin HA-coating layer atop each PLLA nanofiber surface endowed the uni-directionally oriented fibrous mats with increased anisotropic wettability and mechanical compliance. The HA/PLLA nanofibers significantly promoted vSMC to elongation, orientation, and proliferation, and also up-regulated the expression of contractile genes/proteins (e.g., α-SMA, SM-MHC) as well as the synthesis of elastin. Six weeks of in vivo scaffold replacement of rabbit carotid arteries showed that vascular conduits made of circumferentially aligned HA/PLLA nanofibers could maintain patency and promoted oriented vSMC regeneration, lumen endothelialization, and capillary formation. This study demonstrated the synergistic effects of nanotopographical and biochemical cues in one biomimetic scaffold design for efficacious vascular regeneration.

Growth factors have a protective effect on neomycin-induced hair cell loss.

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.

Comparing the cultivated cochlear cells derived from neonatal and adult mouse

BackgroundPrevious 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.MethodsOrgans 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.ResultsThe 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.ConclusionsOur 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.

Electrospun nanofibers of hydroxyapatite/collagen/chitosan promote osteogenic differentiation of the induced pluripotent stem cell-derived mesenchymal stem cells.

external conditions, the presence of protease in vivo, and inefficient transmembrane drug delivery [1]. Developing an effective approach to deliver and stabilize targeted functional proteins is therefore necessary and crucial. Herein, we report a novel protein delivery agent synthesis method, based on two-phase microfluidic droplet flow of selfcrosslinking polymer (SCP). SCP was designed with positively charged polymer chains, self-crosslinking thiol group side chains and poly (ethylene glycol) (PEG) grafting. The whole process of protein delivery agent synthesis can be done within 5 min. As shown in Fig. 1a, protein solution, 100 mM pH 7.4 HEPES buffer and SCP solution were injected into three different channels of MFC. After mixing together in the chip, the mixed aqueous solution was carried to the exit of the chip as droplets in the flow of carrying oil. Then the oil/aqueous mixture was centrifuged, and protein delivery agent (hereinafter, protein delivery agent is denoted as nProtein) was separated and collected in the upper aqueous layer. The molar ratio between protein and SCP was adjusted by switching the flow rate of the syringe pump connected to the injection channel. TEM images showed that fabricated nBSA spheres had an average size of ca. 14 nm. nBSA synthesized with this system was fluorescently labeled using Rhodamine B, and then incubated with Hela cells. Fig. 1b shows great transduction efficiency of nBSA.