High-efficient serum-free differentiation of trabecular meshwork mesenchymal stem cells into Schwann-like cells on polylactide electrospun nanofibrous scaffolds.

Cell-based therapies of the peripheral nerve injury (PNI) have provided satisfactory outcomes among which Schwann cells (SCs) are the most reliable candidate to improve repair of the damaged nerve, however, it is difficult to obtain sufficient amount of SCs for clinical applications. Trabecular meshwork-derived mesenchymal stem cells (TM-MSCs) are newly introduced neural crest originated MSCs, which may have a desirable potential for Schwann-like differentiation due to their common lineage. On the other hand, one of the challenges of cell-based therapies is usage of serum containing media which is inappropriate for clinical applications. In the present study, we investigated the differentiation potential of TM-MSCs into Schwann-like cells on polylactide (PLA) nanofibrous scaffolds in the presence or absence of serum. Our results revealed that PLA nanofibers had no negative effects on the cell growth and proliferation of TM-MSCs, and improved Schwann-like differentiation compared with tissue culture plates (TCPs). More importantly, when the cells cultured on the scaffold in the presence of serum-free media (SFM), expression mRNA levels of SC markers (S100B, GAP43, GFAP and SOX10) were significantly increased compared with those of serum-rich groups. Immunostaining of TM-MSCs cultured on serum-free PLA nanofibrous scaffolds also showed significant expression of GAP43, GFAP and SOX10 compared to those of control, indicating the efficient role of SFM in the differentiation of TM-MSCs into SCs lineage. Overall, the findings of this study revealed the differentiation potential of TM-MSCs to SC fate for the first time, and also showed the beneficial effects of SFM and PLA nanofibrous scaffolds as a promising approach for peripheral nerve regeneration.

Biomimetic nanoengineered scaffold for enhanced full-thickness cutaneous wound healing.

Wound healing is a complex process based on the coordinated signaling molecules and dynamic interactions between the engineered scaffold and newly formed tissue. So far, most of the engineered scaffolds used for the healing of full-thickness skin wounds do not mimic the natural extracellular matrix (ECM) complexity and therefore are not able to provide an appropriate niche for endogenous tissue regeneration [1]. To address this gap and to accelerate the wound healing process, we present biomimetic bilayer scaffolds compositing of gelatin nanofibers (GFS) and photocrosslinkable composite hydrogels loaded with epidermal growth factors (EGF). The nanofibers operate as the dermis layer, and EGF-loaded composite hydrogels acted as the epidermis matrix for the full-thickness wound healing application. The hydrogels are composed of gelatin metacryloyl (GelMA) modified with silicate nanoplatelets (Laponite). To overcome the challenges of transdermal delivery of EGF, including short half-life and lack of efficient formulation precise, controlled delivery was attained by immobilization of EGF on Laponite. It is shown that the addition of 1wt% silicate nanoplatelet increases the compressive modulus of the hydrogels by 170%. In vitro wound closure analysis also demonstrated improved adhesion of the scaffolds to the native tissue by 3.5 folds. Moreover, the tunable hemostatic ability of the scaffolds due to the negatively charged nanoplatelets is shown. In an established excisional full-thickness wound model, an enhanced wound closure (up to 93.1 ± 1.5%) after 14 days relative to controls (GFS and saline-treated groups) is demonstrated. The engineered adhesive and hemostatic scaffolds with sustained release of the growth factors have the potential to stimulate complete skin regeneration for full-thickness wound healing.

In vitro study of alginate-gelatin scaffolds incorporated with silica NPs as injectable, biodegradable hydrogels.

Porous substrates composed of biodegradable polymers and nanoparticles have found extensive use as three-dimensional (3D) scaffolds to regenerate damaged tissues through the incorporation of cells or growth factors. Here, injectable thermally responsive hydrogels based on SiO2 nanoparticles (NPs), alginate, and gelatin biopolymers, with possible utilization for cartilage tissue engineering, are introduced. The nanocomposites contain different amounts of SiO2 NPs for reinforcement and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC)/N-hydroxysuccinimide (NHS) for chemical crosslinking of polymer chains in the 3D hydrogel network. The cross-sectional structure of the hydrogels containing 0.25, 1.5, and 3.0% SiO2 NPs was observed by FE-SEM, confirming porous morphology with interconnected pores. Based on the rheometer analyses, by increasing the amount of SiO2 NPs, the mechanical strength of the gels can be found. In addition, in vitro biodegradation studies show that the hydrogels without SiO2 are more unstable than the hydrogels containing SiO2 NPs. In vitro biocompatibility of the products tested by MTT assay indicates that cell viability and attachment depend on the presence of SiO2 NPs.

Nanofibrous Scaffolds Containing Hydroxyapatite and Microfluidic-Prepared Polyamidoamin/BMP-2 Plasmid Dendriplexes for Bone Tissue Engineering Applications.

OBJECTIVE: The aim of this study is to fabricate functional scaffolds to gene delivery bone morphogenetic protein-2 (BMP-2) plasmid for bone formation in bone tissue engineering. METHODS: Dendriplexes (DPs) of generation 4 polyamidoamin (G4-PAMAM)/BMP-2 plasmid were prepared through microfluidic (MF) platform. The physiochemical properties and toxicity of DPs were evaluated by DLS, AFM, FESEM and MTT assay. In order to create a suitable environment for stem cell growth and differentiation, poly-l-lactic acid (PLLA) and poly-l-lactic acid/poly (ethylene oxide) (PLLA/PEO) scaffolds containing hydroxyapatite nanoparticles (HA) and DPs were fabricated by the electrospinning method. The osteogenic potency of the scaffolds on human adipose tissue-derived mesenchymal stem cells (hASCs) was investigated. RESULTS: The results revealed that tuning the physical properties of DPs by adjusting flow parameters in microfluidic platform can easily improve the cell viability compared to conventional bulk mixing method. Also, the result showed that the presence of HA and DPs in PLLA/PEO scaffold enhanced alkaline phosphatase (ALP) activity and increased the amount of deposited Ca, as well as, related to osteogenesis gen markers. CONCLUSION: This study indicated that on using the MF platform in preparation of DPs and loading them along with HA in PLLA/PEO scaffold, the osteogenic differentiation of hASCs could be tuned.

Microfluidic fabrication of alendronate-loaded chitosan nanoparticles for enhanced osteogenic differentiation of stem cells.

AIMS: In this study, we used a cross-junction microfluidic device for preparation of alendronate-loaded chitosan nanoparticles with desired characteristics to introduce a suitable element for bone tissue engineering scaffolds. MAIN METHODS: By controlling the reaction condition in microfluidic device, six types of alendronate-loaded chitosan nanoparticles were fabricated which had different physical properties. Hydrodynamic diameter of synthetized particles was evaluated by dynamic light scattering (102 to 215 nm). Nanoparticle morphology was determined by SEM and AFM images. The osteogenic effects of prepared selected nanoparticles on human adipose stem cells (hA-MSCs) were evaluated by assessment of alkaline phosphatase (ALP) activity, calcium deposition, ALP and osteopontin gene expression. KEY FINDINGS: The highest loading efficiency percentage (%LE) was %32.42 ± 2.02. Based on MTT assessment, two samples which had no significant cytotoxicity were chosen for further studies (particle sizes and %LE were 142 ± 6.1 nm, 198 ± 16.56 nm, %16.76 ± 3.91 and %32.42 ± 2.02, respectively). In vitro release behavior of nanoparticles displayed pH responsive characteristics. Significant faster release was seen in acidic pH = 5.8 than neutral pH = 7.4. The selected nanoparticles demonstrated higher ALP activity at 14 days in comparison to selected blank sample and osteogenic differentiation media (ODM) and a downregulation at 21 days in comparison to 14 days. Calcium content assay at 21 days displayed significant differences between alendronate-loaded nanoparticles and ODM. ALP and osteopontin mRNA expression was significantly higher than the cells cultured in ODM at 14 and 21 days. SIGNIFICANCE: We concluded that our prepared nanoparticles significantly enhanced osteogenic differentiation of hA-MSCs and can be a suitable compartment of bone tissue engineering scaffolds.

Extraction of Hydroxyapatite Nanostructures from Marine Wastes for the Fabrication of Biopolymer-Based Porous Scaffolds.

Three-dimensional porous nanocomposites consisting of gelatin-carboxymethylcellulose (CMC) cross-linked by carboxylic acids biopolymers and monophasic hydroxyapatite (HA) nanostructures were fabricated by lyophilization, for soft-bone-tissue engineering. The bioactive ceramic nanostructures were prepared by a novel wet-chemical and low-temperature procedure from marine wastes containing calcium carbonates. The effect of surface-active molecules, including sodium dodecyl sulfate (SDS) and hexadecyltrimethylammonium bromide (CTAB), on the morphology of HA nanostructures is shown. It is demonstrated that highly bioactive and monophasic HA nanorods with an aspect ratio > 10 can be synthesized in the presence of SDS. In vitro studies on the bioactive biopolymer composite scaffolds with varying pore sizes, from 100 to 300 µm, determine the capacity of the developed procedure to convert marine wastes to profitable composites for tissue engineering.

Discovery of Novel Cell Surface Markers for Purification of Embryonic Dopamine Progenitors for Transplantation in Parkinson's Disease Animal Models.

Despite the progress in safety and efficacy of cell replacement therapy with pluripotent stem cells (PSCs), the presence of residual undifferentiated stem cells or proliferating neural progenitor cells with rostral identity remains a major challenge. Here we report the generation of a LIM homeobox transcription factor 1 alpha (LMX1A) knock-in GFP reporter human embryonic stem cell (hESC) line that marks the early dopaminergic progenitors during neural differentiation to find reliable membrane protein markers for isolation of midbrain dopaminergic neurons. Purified GFP positive cells in vitro exhibited expression of mRNA and proteins that characterized and matched the midbrain dopaminergic identity. Further quantitative proteomics analysis of enriched LMX1A+ cells identified several membrane-associated proteins including a polysialylated embryonic form of neural cell adhesion molecule (PSA-NCAM) and contactin 2 (CNTN2), enabling prospective isolation of LMX1A+ progenitor cells. Transplantation of human-PSC-derived purified CNTN2+ progenitors enhanced dopamine release from transplanted cells in the host brain and alleviated Parkinson's disease-related phenotypes in animal models. This study establishes an efficient approach for purification of large numbers of human-PSC-derived dopaminergic progenitors for therapeutic applications.