Cellular Response of Limbal Stem Cells on PHBV/Gelatin Nanofibrous Scaffold for Ocular Epithelial Regeneration

The aim of this study was to develop a blend of nanofibrous poly (3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV)/gelatin substrate for limbal stem cell (LSC) expansion that can serve as a potential alternative substrate to replace human amniotic membrane. The human Limbus stem cell was used to evaluate the biocompatibility of substrates (nanofibrous scaffold, and human amniotic membrane) based on their phenotypic profile, viability, proliferation, and attachment ability. Biocompatibility results indicated that the all substrates were highly biocompatible, as LSCs could favorably attach and proliferate on the nanofibrous surface. Microscopic figures showed that the human LSCs were firmly anchored to the substrates and were able to retain a normal corneal stem cell phenotype. Microscopic analyses illustrated that cells infiltrated the nanofibers and successfully formed a three-dimensional corneal epithelium, which was viable for two weeks. Immunocytochemistry (ICC) and real time–PCR results revealed no change in the expression profile of LSCs grown on nanofibrous substrate when compared to those grown on human amniotic membrane. In addition, electrospun nanofibrous PHBV substrate provides not only a milieu supporting LSCs expansion, but also serves as a useful alternative carrier for ocular surface tissue engineering and could be used as an alternative substrate to amniotic membrane.     

A Decellularized Human Limbal Scaffold for Limbal Stem Cell Niche Reconstruction

The transplantation of ex vivo expanded limbal epithelial progenitor cells (LEPCs) on amniotic membrane or fibrin gel is an established therapeutic strategy to regenerate the damaged corneal surface in patients with limbal stem cell deficiency (LSCD), but the long-term success rate is restricted. A scaffold with niche-specific structure and extracellular matrix (ECM) composition might have the advantage to improve long-term clinical outcomes, in particular for patients with severe damage or complete loss of the limbal niche tissue structure. Therefore, we evaluated the decellularized human limbus (DHL) as a biomimetic scaffold for the transplantation of LEPCs. Corneoscleral tissue was decellularized by sodium deoxycholate and deoxyribonuclease I in the presence or absence of dextran. We evaluated the efficiency of decellularization and its effects on the ultrastructure and ECM composition of the human corneal limbus. The recellularization of these scaffolds was studied by plating cultured LEPCs and limbal melanocytes (LMs) or by allowing cells to migrate from the host tissue following a lamellar transplantation ex vivo. Our decellularization protocol rapidly and effectively removed cellular and nuclear material while preserving the native ECM composition. In vitro recellularization by LEPCs and LMs demonstrated the good biocompatibility of the DHL and intrastromal invasion of LEPCs. Ex vivo transplantation of DHL revealed complete epithelialization as well as melanocytic and stromal repopulation from the host tissue. Thus, the generated DHL scaffold could be a promising biological material as a carrier for the transplantation of LEPCs to treat LSCD.     

Can Human Oral Mucosa Stem Cells Differentiate to Corneal Epithelia?

Human oral mucosa stem cells (hOMSCs) arise from the neural crest, they can self-renew, proliferate, and differentiate to several cell lines and could represent a good source for application in tissue engineering. Because of their anatomical location, hOMSCs are easy to isolate, have multilineage differentiation capacity and express embryonic stem cells markers such as—Sox2, Oct3/4 and Nanog. We have used SHEM (supplemented hormonal epithelial medium) media and cultured hOMSCs over human amniotic membrane and determined the cell’s capacity to differentiate to an epithelial-like phenotype and to express corneal specific epithelial markers—CK3, CK12, CK19, Pan-cadherin and E-cadherin. Our results showed that hOMSCs possess the capacity to attach to the amniotic membrane and express CK3, CK19, Pan-Cadherin and E-Cadherin without induction with SHEM media and expressed CK12 or changed the expression pattern of E-Cadherin to a punctual-like feature when treated with SHEM media. The results observed in this study show that hOMSCs possess the potential to differentiate toward epithelial cells. In conclusion, our results revealed that hOMSCs readily express markers for corneal determination and could provide the ophthalmology field with a therapeutic alternative for tissue engineering to achieve corneal replacement when compared with other techniques. Nevertheless, further studies are needed to develop a predictable therapeutic alternative for cornea replacement.     

Bone cell viability on methacrylic acid grafted and collagen immobilized porous poly(3‐hydroxybutrate‐co‐3‐hydroxyvalerate)

Porous poly(3‐hydroxybutrate‐co‐3‐hydroxyvalerate) (PHBV) film was prepared by solute leaching of salt/PHBV cast film. The surface chemistry of the PHBV membrane was modified by performing graft polymerization of methacrylic acid (MAA) on ozone treated porous PHBV film, followed by immobilization of type I collagen. The surface characteristics of the modified and nonmodified porous films were measured by water contact angle. The rat osteosarcoma cell line UMR‐106 osteoblast like cells were used as model cells to evaluate the cell viability on surfaces. The initial cell attachment, growth pattern, and proliferation as measured by MTT assay were used to evaluate the bone cell viability on the modified and nonmodified films. Among the PHBV films studied, the nonmodified porous PHBV and the porous PHBV film type I collagen dip coated showed no significant difference in cell attachment and proliferation, while the porous PHBV membrane that was collagen immobilized after MAA grafting showed considerable activity of osteoblast like cells. © 2005 Wiley Periodicals, Inc. J Appl Polym Sci 98: 1916–1921, 2005     

Three-Dimensional Culture of Cartilage Tissue on Nanogel-Cross-Linked Porous Freeze-Dried Gel Scaffold for Regenerative Cartilage Therapy: A Vibrational Spectroscopy Evaluation

This study presents a set of vibrational characterizations on a nanogel-cross-linked porous freeze-dried gel (NanoCliP-FD gel) scaffold for tissue engineering and regenerative therapy. This scaffold is designed for the in vitro culture of high-quality cartilage tissue to be then transplanted in vivo to enable recovery from congenital malformations in the maxillofacial area or crippling jaw disease. The three-dimensional scaffold for in-plate culture is designed with interface chemistry capable of stimulating cartilage formation and maintaining its structure through counteracting the dedifferentiation of mesenchymal stem cells (MSCs) during the formation of cartilage tissue. The developed interface chemistry enabled high efficiency in both growth rate and tissue quality, thus satisfying the requirements of large volumes, high matrix quality, and superior mechanical properties needed in cartilage transplants. We characterized the cartilage tissue in vitro grown on a NanoCliP-FD gel scaffold by human periodontal ligament-derived stem cells (a type of MSC) with cartilage grown by the same cells and under the same conditions on a conventional (porous) atelocollagen scaffold. The cartilage tissues produced by the MSCs on different scaffolds were comparatively evaluated by immunohistochemical and spectroscopic analyses. Cartilage differentiation occurred at a higher rate when MSCs were cultured on the NanoCliP-FD gel scaffold compared to the atelocollagen scaffold, and produced a tissue richer in cartilage matrix. In situ spectroscopic analyses revealed the cell/scaffold interactive mechanisms by which the NanoCliP-FD gel scaffold stimulated such increased efficiency in cartilage matrix formation. In addition to demonstrating the high potential of human periodontal ligament-derived stem cell cultures on NanoCliP-FD gel scaffolds in regenerative cartilage therapy, the present study also highlights the novelty of Raman spectroscopy as a non-destructive method for the concurrent evaluation of matrix quality and cell metabolic response. In situ Raman analyses on living cells unveiled for the first time the underlying physiological mechanisms behind such improved chondrocyte performance.     

Identification,Isolation, and Characterization of Melanocyte Precursor Cells in the Human Limbal Stroma

Given their vital role in the homeostasis of the limbal stem cell niche, limbal melanocytes have emerged as promising candidates for tissue engineering applications. This study aimed to isolate and characterize a population of melanocyte precursors in the limbal stroma, compared with melanocytes originating from the limbal epithelium, using magnetic-activated cell sorting (MACS) with positive (CD117/c-Kit microbeads) or negative (CD326/EpCAM or anti-fibroblast microbeads) selection approaches. Both approaches enabled fast and easy isolation and cultivation of pure limbal epithelial and stromal melanocyte populations, which differed in phenotype and gene expression, but exhibited similar functional properties regarding proliferative potential, pigmentation, and support of clonal growth of limbal epithelial stem/progenitor cells (LEPCs). In both melanocyte populations, limbus-specific matrix (laminin 511-E8) and soluble factors (LEPC-derived conditioned medium) stimulated melanocyte adhesion, dendrite formation, melanogenesis, and expression of genes involved in UV protection and immune regulation. The findings provided not only a novel protocol for the enrichment of pure melanocyte populations from limbal tissue applying easy-to-use MACS technology, but also identified a population of stromal melanocyte precursors, which may serve as a reservoir for the replacement of damaged epithelial melanocytes and an alternative resource for tissue engineering applications.     

Interaction of human smooth muscle cells on random and aligned nanofibrous scaffolds of PHBV and PHBV-gelatin

Tissue-engineered scaffolds with nanofibrous morphology have been shown to be effective in regeneration of tissues because nanofibers mimic the native architecture of the extracellular matrix. The unique alignment in the native tissue motivated the authors to fabricate aligned nanofibers of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) and PHBV-gelatin. The in vitro potential of the scaffolds was evaluated using human smooth muscle cells. MTS study confirmed that PHBV aligned nanofibrous scaffold promotes better cell proliferation as well as gene expression of key contractile and extracellular matrix markers than their PHBV-gelatin counterparts. Hence, the PHBV aligned nanofibers can be used as a biomimetic scaffold for the regeneration of esophagus. Electrospinning system for aligned nanofibers fabrication (A) and interaction of human smooth muscle cells on aligned nanofibers (B).     

Synthetic substrates for long-term stem cell culture

Stem cells have a host of applications in regenerative medicine and basic research. However, clinical translation hinges on the availability of effective stem cell expansion. Stem cell expansion has been limited due to the use of xenogenic factors in the culture system, batch-to-batch variation, and processes that do not readily lend themselves to scale-up. Synthetic substrates represent attractive alternatives to standard feeder layer culture, as they address many of these pressing limitations. Specifically, we use a grafting-to approach to create a zwitterionic hydrogel capable of maintaining human pluripotent stem cells in long-term culture. This approach enables the control of substrate physiochemical properties, is relatively inexpensive, and results in a substrate with good storage and sterilization stability. In this feature, we focus on the contributions of our culture system to prolonged stem cell culture and compare it to other culture systems currently available.     

Signaling Pathways Impact on Induction of Corneal Epithelial-like Cells Derived from Human Wharton’s Jelly Mesenchymal Stem Cells

Corneal epithelium, the outmost layer of the cornea, comprises corneal epithelial cells (CECs) that are continuously renewed by limbal epithelial stem cells (LESCs). Loss or dysfunction of LESCs causes limbal stem cell deficiency (LSCD) which results in corneal epithelial integrity loss and visual impairment. To regenerate the ocular surface, transplantation of stem cell-derived CECs is necessary. Human Wharton’s jelly derived mesenchymal stem cells (WJ-MSCs) are a good candidate for cellular therapies in allogeneic transplantation. This study aimed to test the effects of treatments on three signaling pathways involved in CEC differentiation as well as examine the optimal protocol for inducing corneal epithelial differentiation of human WJ-MSCs. All-trans retinoic acid (RA, 5 or 10 µM) inhibited the Wnt signaling pathway via suppressing the translocation of β-catenin from the cytoplasm into the nucleus. SB505124 downregulated the TGF-β signaling pathway via reducing phosphorylation of Smad2. BMP4 did not increase phosphorylation of Smad1/5/8 that is involved in BMP signaling. The combination of RA, SB505124, BMP4, and EGF for the first 3 days of differentiation followed by supplementing hormonal epidermal medium for an additional 6 days could generate corneal epithelial-like cells that expressed a CEC specific marker CK12. This study reveals that WJ-MSCs have the potential to transdifferentiate into CECs which would be beneficial for further applications in LSCD treatment therapy.     

Preparation and characterization of polyhydroxybutyrate‐co‐hydroxyvalerate/silk fibroin nanofibrous scaffolds for skin tissue engineering

Nanofibrous scaffolds were obtained by co‐electrospinning poly (3‐hydroxybuty‐rate‐co‐3‐hydroxyvalerate) (PHBV) and fibroin regenerated from silk in different proportions using 1,1,1,3,3,3‐hexafluoro‐2‐isopropanol (HFIP) as solvent. Field emission scanning electron microscope (FESEM) investigation showed that the fiber diameters of the nanofibrous scaffolds ranged from 190 to 460 nm. X‐ray diffraction (XRD) and Fourier transform infrared spectroscopy analysis (FT‐IR) showed that the main structure of silk fibroin (SF) in the nanofibrous scaffold was β‐sheet. Compared to the PHBV nanofibrous scaffold, the surface hydrophilicity and water‐uptake capability of the PHBV/SF nanofibrous scaffold with 50/50 were improved. The results of cell adhesion experiment showed that the fibroblasts adhered more to the PHBV/SF nanofibrous scaffold with 50/50 than the pure PHBV nanofibrous scaffold. The proliferation of fibroblast on the PHBV/SF nanofibrous scaffold with 50/50 was higher than that on the pure PHBV nanofibrous scaffold. Our results indicated that the PHBV/SF nanofibrous scaffold with 50/50 may be a better candidate for biomedical applications such as skin tissue engineering and wound dressing. POLYM. ENG. SCI., 55:907–916, 2015. © 2014 Society of Plastics Engineers     

Shhedding New Light on the Role of Hedgehog Signaling in Corneal Wound Healing

The cornea, an anterior ocular tissue that notably serves to protect the eye from external insults and refract light, requires constant epithelium renewal and efficient healing following injury to maintain ocular homeostasis. Although several key cell populations and molecular pathways implicated in corneal wound healing have already been thoroughly investigated, insufficient/impaired or excessive corneal wound healing remains a major clinical issue in ophthalmology, and new avenues of research are still needed to further improve corneal wound healing. Because of its implication in numerous cellular/tissular homeostatic processes and oxidative stress, there is growing evidence of the role of Hedgehog signaling pathway in physiological and pathological corneal wound healing. Reviewing current scientific evidence, Hedgehog signaling and its effectors participate in corneal wound healing mainly at the level of the corneal and limbal epithelium, where Sonic Hedgehog-mediated signaling promotes limbal stem cell proliferation and corneal epithelial cell proliferation and migration following corneal injury. Hedgehog signaling could also participate in corneal epithelial barrier homeostasis and in pathological corneal healing such as corneal injury-related neovascularization. By gaining a better understanding of the role of this double-edged sword in physiological and pathological corneal wound healing, fascinating new research avenues and therapeutic strategies will undoubtedly emerge.     

PHBV扩链改性及其发泡行为研究

以乙烯⁃甲基丙烯酸缩水甘油酯共聚物（CE）为扩链剂与聚（3?羟基丁酸酯?co?3?羟基戊酸酯）（PHBV）进行熔融共混,制备了扩链PHBV样品,然后采用超临界CO₂（scCO₂）釜压发泡法将其在160 ℃、20 MPa下进行物理发泡,成功制备了不同PHBV泡沫。结果表明,与常压差示扫描量热法相比,采用高压差示扫描量热法测量的各组PHBV的结晶温度和熔融温度略微下降;CE的加入提高了PHBV样品的复数黏度（G'）和储能模量（η^* ）;随着CE含量的增加,PHBV发泡样品的泡孔密度和发泡倍率逐渐增加,泡孔尺寸逐渐减小。     

Corneal Epithelial Regeneration: Old and New Perspectives

Corneal blindness is the fifth leading cause of blindness worldwide, and therapeutic options are still often limited to corneal transplantation. The corneal epithelium has a strong barrier function, and regeneration is highly dependent on limbal stem cell proliferation and basement membrane remodeling. As a result of the lack of corneal donor tissues, regenerative medicine for corneal diseases affecting the epithelium is an area with quite advanced basic and clinical research. Surgery still plays a prominent role in the treatment of epithelial diseases; indeed, innovative surgical techniques have been developed to transplant corneal and non-corneal stem cells onto diseased corneas for epithelial regeneration applications. The main goal of applying regenerative medicine to clinical practice is to restore function by providing viable cells based on the use of a novel therapeutic approach to generate biological substitutes and improve tissue functions. Interest in corneal epithelium rehabilitation medicine is rapidly growing, given the exposure of the corneal outer layers to external insults. Here, we performed a review of basic, clinical and surgical research reports on regenerative medicine for corneal epithelial disorders, classifying therapeutic approaches according to their macro- or microscopic target, i.e., into cellular or subcellular therapies, respectively.     

Characterization of Mesenchymal Stem Cell Differentiation within Miniaturized 3D Scaffolds through Advanced Microscopy Techniques

Three-dimensional culture systems and suitable substrates topographies demonstrated to drive stem cell fate in vitro by mechanical conditioning. For example, the Nichoid 3D scaffold remodels stem cells and shapes nuclei, thus promoting stem cell expansion and stemness maintenance. However, the mechanisms involved in force transmission and in biochemical signaling at the basis of fate determination are not yet clear. Among the available investigation systems, confocal fluorescence microscopy using fluorescent dyes enables the observation of cell function and shape at the subcellular scale in vital and fixed conditions. Contrarily, nonlinear optical microscopy techniques, which exploit multi-photon processes, allow to study cell behavior in vital and unlabeled conditions. We apply confocal fluorescence microscopy, coherent anti-Stokes Raman scattering (CARS), and second harmonic generation (SHG) microscopy to characterize the phenotypic expression of mesenchymal stem cells (MSCs) towards adipogenic and chondrogenic differentiation inside Nichoid scaffolds, in terms of nuclear morphology and specific phenotypic products, by comparing these techniques. We demonstrate that the Nichoid maintains a rounded nuclei during expansion and differentiation, promoting MSCs adipogenic differentiation while inhibiting chondrogenesis. We show that CARS and SHG techniques are suitable for specific estimation of the lipid and collagenous content, thus overcoming the limitations of using unspecific fluorescent probes.     

Electrospun Biopolyesters as Drug Screening Platforms for Corneal Keratocytes

In vitro drug screening techniques provide rapid and easy to analyze data, while saving a lot of animals from being sacrificed. An important part of any in vitro drug screening platform is a biomaterial which promotes cell growth and proliferation. The potential of electrospun scaffolds made of polyhydroxybutyrate (PHB), poly (3-hydroxybutyric acid-co-3-hydroxyvaleric acid) (PHBV), and polycaprolactone (PCL) were studied to serve as drug screening platform for corneal keratocyte tissues. The results showed that the proliferation rate was slightly higher for PCL and PHBV on day 7. Gene expression results showed that PCL was the best in maintaining keratocyte genes.     

Bioengineering of Dental Pulp Stem Cells in a Microporous PNIPAAm-PLGA Scaffold

In the present article a novel bio absorbable polymeric scaffold using poly(N-isopropyl acrylamide-block-poly(L-lactide-co-glycolide) (PNIPAAm-b-PLGA) copolymers is developed for in vitro culture of human dental pulp stem cells (DPSCs). The processing of porous scaffolds has been carried out by emulsion freeze-drying and salt leaching out methods. DPSCs were cultured on scaffolds for up to 14 days. The morphology of the scaffolds, cell viability and interaction between DPSCs and scaffold was characterized by using SEM. The results of cells implantation indicated that scaffold has good cell biocompatibility. Therefore PNIPAAm-PLGA scaffolds have great potential to be used as cell carrier in tissue engineering.     

Ocular Stem Cell Research from Basic Science to Clinical Application: A Report from Zhongshan Ophthalmic Center Ocular Stem Cell Symposium

Stem cells hold promise for treating a wide variety of diseases, including degenerative disorders of the eye. The eye is an ideal organ for stem cell therapy because of its relative immunological privilege, surgical accessibility, and its being a self-contained system. The eye also has many potential target diseases amenable to stem cell-based treatment, such as corneal limbal stem cell deficiency, glaucoma, age-related macular degeneration (AMD), and retinitis pigmentosa (RP). Among them, AMD and glaucoma are the two most common diseases, affecting over 200 million people worldwide. Recent results on the clinical trial of retinal pigment epithelial (RPE) cells from human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) in treating dry AMD and Stargardt’s disease in the US, Japan, England, and China have generated great excitement and hope. This marks the beginning of the ocular stem cell therapy era. The recent Zhongshan Ophthalmic Center Ocular Stem Cell Symposium discussed the potential applications of various stem cell types in stem cell-based therapies, drug discoveries and tissue engineering for treating ocular diseases.     

Three-dimensional silk impregnated HAp/PHBV nanofibrous scaffolds for bone regeneration

Three-dimensional silk fibroin impregnated poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) nanofibrous scaffolds with or without hydroxyapatite (HAp) were prepared by wet-electrospinning method followed by freeze-drying. Scaffolds with cotton wool-like structure have the average fiber diameter of 450–850?nm with 80–85% porosity. In-vitro cell culture tests using MG-63 osteosarcoma human cells revealed improved cell viability, alkaline phosphatase (ALP) activity and total cellular protein amount on the silk impregnated scaffolds compared to PHBV and HAp/PHBV scaffolds after 10 days of cell culture. Immunohistochemical analyses on the silk impregnated scaffolds showed that HAp triggered cell penetration and type I collagen production. Besides, HAp mineralization tendency increased with a decrease in percent crystallinity of the scaffolds comprising HAp and silk after 4 weeks of incubation in simulated body fluid. Consequently, cotton wool-like HAp/PHBV-SF scaffold would be a promising candidate as a bone-filling material for tissue regeneration.