Efficient Wound Healing Using a Synthetic Nanofibrous Bilayer Skin Substitute in Murine Model.

Treatment of full-thickness skin wounds with minimal scarring and complete restoration of native tissue properties still exists as a clinical challenge. A bilayer skin substitute was fabricated by coating human amniotic membrane (AM) with electrospun silk fibroin nanofibers, and its in vivo biological behavior was studied using murine full-thickness skin wound model. Donut-shaped silicon splints were utilized to prevent wound contraction in mouse skin and simulate re-epithelialization, which is the normal path of human wound healing. Skin regeneration using the bilayer scaffold was compared with AM and untreated defect after 30 d. Tissue samples were taken from healed wound areas and investigated through histopathological and immunohistochemical staining to visualize involucrin (IVL), P63, collagen I, CD31, and vascular endothelial growth factor. In addition, mRNA expression of IVL, P63, interleukin-6, and cyclooxygenase-2 was studied. The application of bilayer scaffold resulted in the best epidermal and dermal regeneration, demonstrated by histopathological examination and molecular analysis. In regenerated wounds of the bilayer scaffold group, the mRNA expression levels of inflammatory markers (interleukin-6 and cyclooxygenase-2) were downregulated, and the expression pattern of keratinocyte markers (IVL and P63) at both mRNA and protein levels was more similar to native tissue in comparison with AM and no-treatment groups. There was no significant difference in the expression level of collagen I, CD31, and vascular endothelial growth factor among different groups. Conclusively, these promising results serve as a supporting evidence for proceeding to clinical phase to examine the capacity of this bilayer scaffold for human skin regeneration.

Differentiation of Menstrual Blood Stem Cells into Keratinocyte-Like Cells on Bilayer Nanofibrous Scaffold.

Skin tissue engineering is a high-throughput technology to heal the wounds. Already, considerable advances have been achieved using stem cells for wound healing applications. Menstrual blood stem cell (MenSC) is an available and accessible source of stem cells that have differentiation potential into a wide range of lineages like keratinocytes. Extracellular matrix like substratum plays an impressive role in skin regeneration as an attachment site for stem cells by transmitting the bioactive signals and provoking stem cells to differentiate into keratinocyte lineage. The biomimetic nanofibrous scaffold especially in bilayer format has been extensively utilized to develop skin equivalents. This chapter explains detailed protocols of keratinocyte differentiation of MenSCs on bilayer scaffold comprising amniotic membrane and fibroin nanofibers. The isolated MenSCs are seeded on the nanofibers and subsequently differentiated into keratinocyte lineage in co-culture with foreskin-derived keratinocytes. Immunofluorescence staining is used to evaluate the development of seeded MenSCs in bilayer scaffold into keratinocyte-like cells.

Correction to: "Comparative repair capacity of knee osteochondral defects using regenerated silk fiber scaffolds and fibrin glue with/without autologous chondrocyes during 36 weeks in rabbit model.

The published online of the original version contains mistakes.

Bilayer Amniotic Membrane/Nano-fibrous Fibroin Scaffold Promotes Differentiation Capability of Menstrual Blood Stem Cells into Keratinocyte-Like Cells.

The skin provides a dynamic barrier separating and protecting human body from the exterior world, and then immediate repair and rebuilding of the epidermal barrier is crucial after wound and injury. Wound healing without scars and complete regeneration of skin tissue still remain as a clinical challenge. The demand to engineer scaffolds that actively promote regeneration of damaged areas of the skin has been increased. In this study, menstrual blood-derived stem cells (MenSCs) have been induced to differentiate into keratinocytes-like cells in the presence of human foreskin-derived keratinocytes on a bilayer scaffold based on amniotic membrane and silk fibroin. Based on the findings, newly differentiated keratinocytes from MenSCs successfully expressed the keratinocytes specific markers at both mRNA and protein levels judged by real-time PCR and immunostaining techniques, respectively. We could show that the differentiated cells over bilayer composite scaffolds express the keratinocytes specific markers at higher levels when compared with those cultured in conventional 2D culture system. Based on these findings, bilayer amniotic membrane/nano-fibrous fibroin scaffold represents an efficient natural construct with broad applicability to generate keratinocytes from MenSCs for stem cell-based skin wounds healing and regeneration.

Hardystonite-Coated Poly(l-lactide) Nanofibrous Scaffold and Efficient Osteogenic Differentiation of Adipose-Derived Mesenchymal Stem Cells.

In this study, a ceramic-coated nanofibrous scaffold has been fabricated to biomimic the microstructure of natural extracellular matrix and the stiffening inorganic compartment of bone. Poly-l-lactic acid (PLLA) nanofibers were electrospun and exposed to oxygen plasma to induce hydrophilicity and promote ceramic adsorption. Hardystonite (HS), which possesses superior osteoinduction potential over hydroxyapatite, was coated on plasma-treated PLLA nanofibers by drenching the nanofibers in HS suspension. Pure and composite PLLA-based scaffolds were characterized in terms of physical and biological properties. In vitro cultivation of adipose-derived mesenchymal stem cells (AMSCs) on the scaffolds displayed that the composite scaffold is able to further support cell attachment and proliferation. In case of osteogenic differentiation of AMSCs, HS coating significantly increased the synthesis and activity of alkaline phosphate over 21 days period. In addition, the composite scaffold showed improved mineralization. The expression level of osteonectin and osteocalcin genes was significantly enhanced by HS coating of nanofibers. The biological improvement of PLLA nanofibrous matrix in the presence of HS nanoparticles could either be attributed to the release and stimulatory effect of constituent ions of HS or to the modification of chemico-physical properties of the resultant ceramic by silicon and zinc present in HS.

Current State of Cartilage Tissue Engineering using Nanofibrous Scaffolds and Stem Cells.

Cartilage is an avascular, aneural, and alymphatic connective tissue with a limited capacity caused by low mitotic activity of its resident cells, chondrocytes. Natural repair of full thickness cartilage defects usually leads to the formation of fibrocartilage with lower function and mechanical force compared with the original hyaline cartilage and further deterioration can occur. Tissue engineering and regenerative medicine is a promising strategy to repair bone and articular cartilage defects and rehabilitate joint functions by focusing on the optimal combination of cells, material scaffolds, and signaling molecules. The unique physical and topographical properties of nanofibrous structures allow them to mimic the extracellular matrix of native cartilage, making an appropriate resemblance to induce cartilage tissue regeneration and reconstruction. To improve simulation of native cartilage, the incorporation of nanofibrous scaffolds with suitable corresponsive cells could be effective. In this review article, an attempt was made to present the current state of cartilage tissue engineering using nanofibrous scaffolds and stem cells as high proliferative immune privilege cells with chondrogenic differentiation ability. The comprehensive information was retrieved by search of relevant subject headings in Medline/Pubmed and Elsevier databases.

Comparative capability of menstrual blood versus bone marrow derived stem cells in neural differentiation.

In order to characterize the potency of menstrual blood stem cells (MenSCs) for future cell therapy of neurological disorders instead of bone marrow stem cells (BMSCs) as a well-known and conventional source of adult stem cells, we examined the in vitro differentiation potential of these stem cells into neural-like cells. The differentiation potential of MenSCs to neural cells in comparison with BMSCs was assessed under two step neural differentiation including conversion to neurosphere-like cells and final differentiation. The expression levels of Nestin, Microtubule-associated protein 2, gamma-aminobutyric acid type B receptor subunit 1 and 2, and Tubulin, beta 3 class III mRNA and/or protein were up-regulated during development of MenSCs into neurosphere-like cells (NSCs) and neural-like cells. The up-regulation level of these markers in differentiated neural-like cells from MenSCs was comparable with differentiated cells from BMSCs. Moreover, both differentiated MenSCs and BMSCs expressed high levels of potassium, calcium and sodium channel genes developing functional channels with electrophysiological recording. For the first time, we demonstrated that MenSCs are a unique cell population with differentiation ability into neural-like cells comparable to BMSCs. In addition, we have introduced an approach to generate NSCs from MenSCs and BMSCs and their further differentiation into neural-like cells in vitro. Our results hold a promise to future stem cell therapy of neurological disorders using NSCs derived from menstrual blood, an accessible source in every woman.

Extended Culture of Encapsulated Human Blastocysts in Alginate Hydrogel Containing Decidualized Endometrial Stromal Cells in the Presence of Melatonin.

Extended in vitro culture of human embryos beyond blastocyst stage could serve as a tool to explore the molecular and physiological mechanisms underlying embryo development and to identify factors regulating pregnancy outcomes. This study presents the first report on the maintenance of human embryo in vitro by alginate co-encapsulation of human blastocyst and decidualized endometrial stromal cells (EnSCs) under melatonin-fortified culture conditions. The effectiveness of the 3D culture system was studied through monitoring of embryo development in terms of survival time, viability, morphological changes, and production of the two hormones of 17b-oestradiol and human chorionic gonadotropin. The embryo structural integrity was preserved during alginate encapsulation; however, only 23 % of the encapsulated embryos could retain in the hydrogels over time and survived until day 4 post-encapsulation. The culture medium fortification with melatonin significantly elevated the maintenance rate of expanded embryos in alginate beads by 65 % and prolonged survival time of human embryos to day 5. Furthermore, embryo co-culture with EnSCs using melatonin-fortified medium increased the survival time of encapsulated embryos to 44 %. The levels of two measured hormones significantly rose at day 4 in comparison with day 2 post-encapsulation especially in the group co-encapsulated with EnSCs and cultivated in melatonin-fortified culture medium. These data are the first evidence representing in vitro development of human embryos until day 10 post-fertilization. This achievement can facilitate the investigation of the mechanisms regulating human embryo development.

Comparative repair capacity of knee osteochondral defects using regenerated silk fiber scaffolds and fibrin glue with/without autologous chondrocytes during 36 weeks in rabbit model.

The reconstruction capability of osteochondral (OCD) defects using silk-based scaffolds has been demonstrated in a few studies. However, improvement in the mechanical properties of natural scaffolds is still challengeable. Here, we investigate the in vivo repair capacity of OCD defects using a novel Bombyx mori silk-based composite scaffold with great mechanical properties and porosity during 36 weeks. After evaluation of the in vivo biocompatibility and degradation rate of these scaffolds, we examined the effectiveness of these fabricated scaffolds accompanied with/without autologous chondrocytes in the repair of OCD lesions of rabbit knees after 12 and 36 weeks. Moreover, the efficiency of these scaffolds was compared with fibrin glue (FG) as a natural carrier of chondrocytes using parallel clinical, histopathological and mechanical examinations. The data on subcutaneous implantation in mice showed that the designed scaffolds have a suitable in vivo degradation rate and regenerative capacity. The repair ability of chondrocyte-seeded scaffolds was typically higher than the scaffolds alone. After 36 weeks of implantation, most parts of the defects reconstructed by chondrocytes-seeded silk scaffolds (SFC) were hyaline-like cartilage. However, spontaneous healing and filling with a scaffold alone did not eventuate in typical repair. We could not find significant differences between quantitative histopathological and mechanical data of SFC and FGC. The fabricated constructs consisting of regenerated silk fiber scaffolds and chondrocytes are safe and suitable for in vivo repair of OCD defects and promising for future clinical trial studies.

Fabrication and characterization of nano-fibrous bilayer composite for skin regeneration application.

Full thickness wound healing with minimal scarring and complete restoration of normal skin properties still remains as a clinical challenge. In this study, a bilayer skin substitute has been fabricated to biomimic the microstructure of natural extracellular matrix of the skin. Human amniotic membrane (HAM) and silk fibroin nano-fibers were combined to produce bilayer construct, which was further treated and characterized. HAM was obtained from healthy mothers and de-epithelized by means of fine enzymatic method to preserve the extracellular structure. Fibroin protein was extracted from fresh Bombyx mori cocoons and transformed to uniform nano-fiberous structure, which was used as a coating layer on the de-epithelized membrane. Surface modification through oxygen plasma treatment was attempted to further induce hydrophilicity. Subsequently, scaffolds were fully characterized in terms of morphology, mechanical properties, hydrophilicity and cell culture response. Histological and immunohistological staining demonstrated localization of fibronectin, cell denudation and structural integrity of HAM after de-epithelization. Scanning electron microscopy images showed bead-free silk fibroin nano-fibers with the average diameter of 250nm. Water contact angle of bilayer scaffolds reduced dramatically to 26.34° after oxygen plasma treatment, which is correlated with more hydrophilic surface. Due to fibroin nano-fiber coating, mechanical properties of HAM improved significantly. Tensile Young's modulus and tensile strength increased from 16.14MPa and 68.46MPa to 25.69MPa and 108.03MPa, respectively. 14days in vitro cultivation of mouse embryonic fibroblasts on the scaffolds revealed that bilayer scaffolds are able to support cell attachment and proliferation. Plasma-etched scaffolds provided the best niche for cell-matrix crosstalk by allowing cells to penetrate beneath the pores and to integrate in fibers direction. The obtained results suggest that the presented nano-fibrous bilayer composite based on HAM is a potential substitute for skin regeneration application.