Electrospun silk biomaterial scaffolds for regenerative medicine

Electrospinning is a versatile technique that enables the development of nanofiber-based biomaterial scaffolds. Scaffolds can be generated that are useful for tissue engineering and regenerative medicine since they mimic the nanoscale properties of certain fibrous components of the native extracellular matrix in tissues. Silk is a natural protein with excellent biocompatibility, remarkable mechanical properties as well as tailorable degradability. Integrating these protein polymer advantages with electrospinning results in scaffolds with combined biochemical, topographical and mechanical cues with versatility for a range of biomaterial, cell and tissue studies and applications. This review covers research related to electrospinning of silk, including process parameters, post treatment of the spun fibers, functionalization of nanofibers, and the potential applications for these material systems in regenerative medicine. Research challenges and future trends are also discussed.     

Electrospinning of polysaccharides for regenerative medicine

Electrospinning techniques enable the production of continuous fibers with dimensions on the scale of nanometers from a wide range of natural and synthetic polymers. The number of recent studies regarding electrospun polysaccharides and their derivatives, which are potentially useful for regenerative medicine, is increasing dramatically. However, difficulties regarding the processibility of the polysaccharides (e.g., poor solubility and high surface tension) have limited their application. In this review, we summarize the characteristics of various polysaccharides such as alginate, cellulose, chitin, chitosan, hyaluronic acid, starch, dextran, and heparin, which are either currently being used or have potential to be used for electrospinning. The recent progress of nanofiber matrices electrospun from polysaccharides and their biomedical applications in tissue engineering, wound dressings, drug delivery, and enzyme immobilization are discussed.     

Defined culture medium for stem cell differentiation: Applicability of serum-free conditions in the mouse embryonic stem cell test

The embryonic stem cell test (EST) is a validated method to assess the developmental toxicity potency of chemicals. It was developed to reduce animal use and allow faster testing for hazard assessment. The cells used in this method are maintained and differentiated in media containing foetal calf serum. This animal product is of considerable variation in quality, and individual batches require extensive testing for their applicability in the EST. Moreover, its production involves a large number of foetuses and possible animal suffering. We demonstrate the serum-free medium and feeder cell-free maintenance of the mouse embryonic stem cell line D3 and investigate the use of specific growth factors for induction of cardiac differentiation. Using a combination of bone morphogenetic protein-2, bone morphogenetic protein-4, activin A and ascorbic acid, embryoid bodies efficiently differentiated into contracting myocardium. Additionally, examining levels of intracellular marker proteins by flow cytometry not only confirmed differentiation into cardiomyocytes, but demonstrated significant differentiation into neuronal cells in the same time frame. Thus, this approach might allow for simultaneous detection of developmental effects on both early mesodermal and neuroectodermal differentiation. The serum-free conditions for maintenance and differentiation of D3 cells described here enhance the transferability and standardisation and hence the performance of the EST.     

The stem cell and tissue engineering research in Chinese ophthalmology

Much has been considerably developed recently in the ophthalmic research of stem cell (SC) and tissue engineering (TE). They have become closer to the clinical practice, standardized and observable. Leading edge research of SC and TE on the ocular surface reconstruction, neuroregeneration and protection, and natural animal model has become increasingly available. However, challenges remain on the way, especially on the aspects of function reconstruction and specific differentiation. This paper reviews the new developments in this area with an intention of identifying research priorities for the future.     

Methods to assess stem cell lineage, fate and function

Stem cell therapy has the potential to regenerate injured tissue. For stem cells to achieve their full therapeutic potential, stem cells must differentiate into the target cell, reach the site of injury, survive, and engraft. To fully characterize these cells, evaluation of cell morphology, lineage specific markers, cell specific function, and gene expression must be performed. To monitor survival and engraftment, cell fate imaging is vital. Only then can organ specific function be evaluated to determine the effectiveness of therapy. In this review, we will discuss methods for evaluating the function of transplanted cells for restoring the heart, nervous system, and pancreas. We will also highlight the specific challenges facing these potential therapeutic areas.     

Enhanced neural stem cell functions in conductive annealed carbon nanofibrous scaffolds with electrical stimulation

Carbon-based nanomaterials have shown great promise in regenerative medicine because of their unique electrical, mechanical, and biological properties; however, it is still difficult to engineer 2D pure carbon nanomaterials into a 3D scaffold while maintaining its structural integrity. In the present study, we developed novel carbon nanofibrous scaffolds by annealing electrospun mats at elevated temperature. The resultant scaffold showed a cohesive structure and excellent mechanical flexibility. The graphitic structure generated by annealing renders superior electrical conductivity to the carbon nanofibrous scaffold. By integrating the conductive scaffold with biphasic electrical stimulation, neural stem cell proliferation was promoted associating with upregulated neuronal gene expression level and increased microtubule-associated protein 2 immunofluorescence, demonstrating an improved neuronal differentiation and maturation. The findings suggest that the integration of the conducting carbon nanofibrous scaffold and electrical stimulation may pave a new avenue for neural tissue regeneration.     

异基因造血干细胞移植治疗儿童血液病

目的探讨异基因造血干细胞移植治疗儿童血液病的价值。方法采用异基因造血干细胞移植治疗11例儿童血液病，其中再生障碍性贫血2例、白血病7例、骨髓异常增生症1例、非霍奇金淋巴瘤1例；预处理采用氟达拉滨／环磷酰胺（FLU／CTX）或全身放疗／环磷酰胺（TBI／CTX），观察患儿的临床治疗效果。结果10例患儿均移植成功，9例患儿生存，其中7例无病存活，存活最长时间为5．5年；CMV感染3例，继发出血性膀胱炎1例，继发弥漫性GVHD1例。结论异基因造血干细胞移植是治疗儿童血液病的有效方法，值得临床进一步推广。     

Cisplatin-induced alterations in the functional spermatogonial stem cell pool and niche in C57/BL/6J mice following a clinically relevant multi-cycle exposure

A typical clinical cis-diamminedichloroplatinum(II) (cisplatin) dosing regimen consists of repeated treatment cycles followed by a recovery period. While effective, this dosing structure results in a prolonged, often permanent, infertility in men. Spermatogonial stem cells (SSCs) are theoretically capable of repopulating the seminiferous tubules after exposure has ceased. We propose that an altered spermatogonial environment during recovery from the initial treatment cycle drives an increase in SSC mitotic cell activity, rendering the SSC pool increasingly susceptible to cisplatin-induced injury from subsequent cycles. To test this hypothesis, the undifferentiated spermatogonia population and niche of the adult mouse (C57/BL/6J) were examined during the recovery periods of a clinically-relevant cisplatin exposure paradigm. Histological examination revealed a disorganization of spermatogenesis correlating with the number of exposure cycles. Quantification of terminal deoxynucleotidyl transferase-mediated digoxigenin-dUTP nick end labeling (TUNEL) staining indicated an increase in apoptotic frequency following exposure. Immunohistochemical examination of Foxo1 and incorporated BrdU showed an increase in the undifferentiated spermatogonial population and mitotic activity in the recovery period in mice exposed to one cycle, but not two cycles of cisplatin. Immunohistochemical investigation of glial cell line-derived neurotrophic factor (GDNF) revealed an increase in production along the basal Sertoli cell membrane throughout the recovery period in all treatment groups. Taken together, these data establish that the impact of cisplatin exposure on the functional stem cell pool and niche correlates with: (1) the number of dosing cycles; (2) mitotic activity of early germ cells; and (3) alterations in the basal Sertoli cell GDNF expression levels after cisplatin-induced testicular injury.     

逆向撕除联合角膜缘干细胞移植治疗翼状胬肉效果观察

目的探讨逆向撕除联合角膜缘干细胞移植治疗翼状胬肉的疗效。方法通过对34眼行常规单纯翼状胬肉切除术,42眼行逆向撕除联合角膜缘干细胞移植术。观察术后视力,结膜移植片愈合情况、并发症,并对1年后两种术式复发率进行对比分析。结果1年后随访,34眼行常规单纯翼状胬肉切除术后12眼（35.3％）复发,42眼行逆向撕除联合角膜缘干细胞移植术后有6眼（14．2％）复发,复发率比较差异具有显著性（p〈0．05）。全部患者视力均有不同程度提高,无睑球粘连,无持续性角膜浸润及其他并发症发生。结论逆向撕除联合角膜缘干细胞移植治疗翼状胬肉疗效好,无并发症,复发率低,值得临床推广。     

Molecular multiple endpoint embryonic stem cell test--a possible approach to test for the teratogenic potential of compounds

The embryonic stem cell test (EST) examines the cytotoxicity of chemical compounds on embryonic stem (ES) cells and 3T3.A31 fibroblasts. Additionally, the EST measures the ability of ES cells to differentiate into contracting cardiomyocytes following drug exposure. In this study, we introduce new endpoints to obtain a molecular multiple endpoint EST (mme-EST), enabling the identification of potential chemical effects on osteogenic, chondrogenic and neural differentiation in addition to the traditional endpoint of cardiomyocyte differentiation. Six compounds in three classes with known teratogenic in vivo potential were assayed with the mme-EST in a pilot study: penicillin G (non-teratogenic), 5-fluorouracil and retinoic acid (strongly teratogenic), diphenylhydantoin, valproic acid and thalidomide (moderately teratogenic). While the traditional EST measures a morphological endpoint, we included molecular markers of differentiation as endpoints. With the mme-EST, every compound could be classified correctly according to its known teratogenic potential in vivo. Penicillin G, 5-fluorouracil and diphenylhydantoin inhibited differentiation of all endpoints equally. Interestingly, valproic acid showed the strongest inhibition of neural differentiation, while thalidomide specifically inhibited osteogenic development. Retinoic acid, on the other hand, supported neural but inhibited chondrogenic and osteogenic differentiation concentration-dependently. Valproic acid and thalidomide, classified incorrectly with the established EST model, were classified correctly with the mme-EST according to their effects on specific endpoints. This pilot study indicates that the predictive value of the EST may be enhanced by including further differentiation endpoints.     

Developing osteoblasts as an endpoint for the mouse embryonic stem cell test

The mouse Embryonic Stem cell Test (EST) using cardiomyocyte differentiation is a promising in vitro assay for detecting potential embryotoxicity; however, the addition of another differentiation endpoint, such as osteoblasts, may improve the predictive value of the test. A number of variables such as culture conditions and starting cell number were investigated. A 14 day direct plating method of D3 mouse embryonic stem cells (mESCs) was used to test the predictivity of osteoblast differentiation as an endpoint in the EST. Twelve compounds were tested using the prediction model developed in the ECVAM validation study. Eight of the compounds selected from the EST validation study served as model compounds; four additional compounds known to produce skeletal defects were also tested. Our results indicate comparable chemical classification between the validated cardiomyocyte endpoint and the osteoblast endpoint. These results suggest that differentiation to osteoblasts may provide confirmatory information in predicting embryotoxicity.     

In vivo relevant mixed urolithins and ellagic acid inhibit phenotypic and molecular colon cancer stem cell features: A new potentiality for ellagitannin metabolites against cancer

Colon cancer stem cells (CSCs) offer a novel paradigm for colorectal cancer (CRC) treatment and dietary polyphenols may contribute to battle these cells. Specifically, polyphenol-derived colon metabolites have the potential to interact with and affect colon CSCs. We herein report the effects against colon CSCs of two mixtures of ellagitannin (ET) metabolites, ellagic acid (EA) and the gut microbiota-derived urolithins (Uro) at concentrations detected in the human colon tissues following the intake of ET-containing products (pomegranate, walnuts). These mixtures reduce phenotypic and molecular features in two models of colon CSCs: Caco-2 cells and primary tumour cells from a patient with CRC. The mixture containing mostly Uro-A (85% Uro-A, 10% Uro-C, 5% EA) was most effective at inhibiting the number and size of colonospheres and aldehyde dehydrogenase activity (ALDH, a marker of chemoresistance) whereas the mixture containing less Uro-A but IsoUro-A and Uro-B (30% Uro-A, 50% IsoUro-A, 10% Uro-B, 5% Uro-C, 5% EA) had some effects on the number and size of colonospheres but not on ALDH. These data support a role for polyphenols metabolites in the control of colon cancer chemoresistance and relapse and encourage the research on the effects of polyphenols against CSCs.     

Design of electrospun nanofibrous mats for osteogenic differentiation of mesenchymal stem cells

The clinical translation potential of mesenchymal stem cells (MSCs) in regenerative medicine has been greatly exploited. With the merits of high surface area to volume ratio, facile control of components, well retained topography, and the capacity to mimic the native extracellular matrix (ECM), nanofibers have received a great deal of attention as bone tissue engineering scaffolds. Electrospinning has been considered as an efficient approach for scale-up fabrication of nanofibrous materials. Electrospun nanofibers are capable of stimulating cell–matrix interaction to form a cell niche, directing cellular behavior, and promoting the MSCs adhesion and proliferation. In this review, we give a comprehensive literature survey on the mechanisms of electrospun nanofibers in supporting the MSCs differentiation. Specifically, the influences of biological and physical osteogenic inductive cues on the MSCs osteogenic differentiation are reviewed. Along with the significant advances in the field, current research challenges and future perspectives are also discussed.