Poly(trimethylene carbonate) as an elastic biodegradable film for human embryonic stem cell‐derived retinal pigment epithelial cells

Human embryonic stem cell‐derived retinal pigment epithelial (hESC‐RPE) cell therapies show tremendous potential for the treatment of retinal degenerative diseases. A tissue engineering approach, where cells are delivered to the subretinal space on a biodegradable carrier as a sheet, shows great promise for these RPE cell therapies. The aim of the present study was to assess whether a flexible, elastic and biodegradable poly(trimethylene carbonate) (PTMC) film promotes the formation of functional hESC‐RPE and performs better than often used biodegradable poly(d,l ‐lactide) (PDLLA) film. Human ESC‐RPE maturation and functionality on PTMC films was assessed by cell proliferation assays, RPE‐specific gene and protein expression, phagocytic activity and growth factor secretion. It is demonstrated that the mechanical properties of PTMC films have close resemblance to those of the native Bruch's membrane and support the formation hESC‐RPE monolayer in serum‐free culture conditions with high degree of functionality. In contrast, use of PDLLA films did not lead to the formation of confluent monolayers of hESC‐RPE cells and had unsuitable mechanical properties for retinal application. In conclusion, the present study indicates that flexible and elastic biodegradable PTMC films show potential for retinal tissue engineering applications. Copyright © 2017 John Wiley & Sons, Ltd.     

Development of poly(lactide‐co‐glycolide) scaffold‐impregnated small intestinal submucosa with pores that stimulate extracellular matrix production in disc regeneration

The pore size and microstructure of scaffolds influences cell attachment, migration, proliferation and ingrowth, but the optimal pore size of scaffolds for disc tissue formation is not clearly understood. We developed porous poly(lactide‐co‐glycolide) (PLGA) scaffolds with various pore sizes for nucleus pulposus (NP) cell cultures and examined the effects of pore size on cell ingrowth and extracellular matrix (ECM) synthesis. High cell density in the small pores of scaffolds promotes collagen synthesis and cell migration through interconnected pores. Scaffolds with large pores exhibited slower cell proliferation and collagen synthesis. Guided by these results, we investigated a novel, biodegradable, synthetic/natural hybrid scaffold composed of PLGA and small intestinal submucosa (SIS) (PLGA–SIS) with the proper pore size for NP regeneration. We tested the morphological and physical properties of PLGA–SIS scaffolds and initial cell attachment and ECM production of NP in scaffolds. The mechanical and degradable properties of the PLGA–SIS scaffold were superior to those of SIS sponge and were similar to the properties of PLGA scaffold. NP cells grown on PLGA–SIS scaffold exhibited higher initial cell adhesion and ECM production than those grown on pure PLGA scaffold in a biological assay. In conclusion, this study suggests that a proper pore size of scaffolds is critical in NP regeneration, and that PLGA–SIS scaffolds with suitable pores might be useful as substrates for tissue‐engineered biodiscs. Copyright © 2012 John Wiley & Sons, Ltd.     

Differentiation of human pluripotent stem cells to retinal pigmented epithelium in defined conditions using purified extracellular matrix proteins

A potential application of human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) is the generation of retinal pigmented epithelium (RPE) to treat age‐related macular degeneration (AMD), a common but incurable retinal disease. RPE cells derived from hESCs (hESC‐RPEs) and iPSCs (iPSC‐RPEs) express essential RPE markers and can rescue visual function in animal models. However, standard differentiation protocols yield RPE cells at low frequency, especially from iPSC lines, and the common use of Matrigel and xenogeneic feeder cells is not compatible with clinical applications. The extracellular matrix (ECM) can affect differentiation, and therefore changes in ECM composition may improve the frequency of stem cell‐RPE differentiation. We selected several purified ECM proteins and substrates, based on the in vivo RPE ECM environment, and tested their ability to support iPSC‐RPE differentiation and maintenance. iPSCs differentiated on nearly all tested substrates developed pigmented regions, with Matrigel and mouse laminin‐111 supporting the highest pigmentation frequencies. Although iPSC‐RPEs cultured on the majority of the tested substrates expressed key RPE genes, only six substrates supported development of confluent monolayers with normal RPE morphology, including Matrigel and mouse laminin‐111. iPSCs differentiated on mouse laminin‐111 produced iPSC‐RPEs expressing RPE proteins, and hESCs differentiated on mouse laminin‐111 resulted in high yields of functional hESC‐RPEs. This identification of key ECM proteins may assist with future scaffold designs and provide peptide sequences for use in synthetic, xeno‐free, GMP‐compliant generation of RPE from human pluripotent stem cells relevant to clinical translation. Copyright © 2012 John Wiley & Sons, Ltd.     

Collagen type I–PLGA film as an efficient substratum for corneal endothelial cells regeneration

Surface modulations of desired biological construct design for regenerative medical therapy is considered to be highly crucial for cell growth and the subsequent regeneration of biologically competent tissues. In this study, we fabricated stable, transparent, collagen type‐I‐coated PLGA films (Col I–PLGA) as a potential substratum for the regeneration of corneal endothelial cells. Morphological and structural properties were analysed by FE–SEM, AFM, FTIR, contact angle, etc., and in vitro biocompatibility of the Col I–PLGA films was further tested in primary rabbit corneal endothelial cells (rCEnCs) as models. Compared with bare PLGA films, the Col I–PLGA films displayed the requisite surface roughness, with higher Ra (nm) values, transparency, good hydrophilicity, stability and water uptake. Next, cultured rCEnCs on Col I–PLGA films showed the characteristic polygonal shape of rCEnCs with enhanced initial attachment, proliferation and expression of mRNAs. Collectively, these results indicate that Col I–PLGA can be employed as a suitable alternative for high‐quality corneal tissue expansion and transplantation. Copyright © 2016 John Wiley & Sons, Ltd.     

Hybrid scaffolds based on PLGA and silk for bone tissue engineering

Porous silk scaffolds, which are considered to be natural polymers, cannot be used alone because they have a long degradation rate, which makes it difficult for them to be replaced by the surrounding tissue. Scaffolds composed of synthetic polymers, such as PLGA, have a short degradation rate, lack hydrophilicity and their release of toxic by‐products makes them difficult to use. The present investigations aimed to study hybrid scaffolds fabricated from PLGA, silk and hydroxyapatite nanoparticles (Hap NPs) for optimized bone tissue engineering. The results from variable‐pressure field emission scanning electron microscopy (VP–FE–SEM), equipped with EDS, confirmed that the fabricated scaffolds had a porous architecture, and the location of each component present in the scaffolds was examined. Contact angle measurements confirmed that the introduction of silk and HAp NPs helped to change the hydrophobic nature of PLGA to hydrophilic, which is the main constraint for PLGA used as a biomaterial. Thermo‐gravimetric analysis (TGA) and FT–IR spectroscopy confirmed thermal decomposition and different vibrations caused in functional groups of compounds used to fabricate the scaffolds, which reflected improvement in their mechanical properties. After culturing osteoblasts for 1, 7 and 14 days in the presence of scaffolds, their viability was checked by MTT assay. The fluorescent microscopy results revealed that the introduction of silk and HAp NPs had a favourable impact on the infiltration of osteoblasts. In vivo experiments were conducted by implanting scaffolds in rat calvariae for 4 weeks. Histological examinations and micro‐CT scans from these experiments revealed beneficial attributes offered by silk fibroin and HAp NPs to PLGA‐based scaffolds for bone induction. Copyright © 2015 John Wiley & Sons, Ltd.     

S100a7蛋白在体外培养人视网膜色素上皮细胞中的表达及功能

背景：S100a7蛋白在细胞增殖、血管形成等病理生理过程中发挥重要作用,但在视网膜色素上皮细胞中的作用少有报道。目的：探讨S100a7蛋白在体外培养人视网膜色素上皮细胞中的表达及作用。方法：体外培养ARPE-19细胞株,通过免疫荧光及Western blot技术,检测S100a7在ARPE-19细胞中的表达;不同稀释度（1∶1000,1∶10000,1∶100000）S100a7蛋白抗体与ARPE-19细胞共培养,通过MTT技术检测S100a7蛋白在人视网膜色素上皮细胞增殖过程中的作用。结果与结论：免疫荧光及Westernblot技术证实该蛋白在ARPE-19细胞中表达;MTT实验结果显示,人视网膜色素上皮细胞加入S100a7抗体72h后,各浓度处理组的吸光度值较对照组显著均降低（P〈0.05）。结果证实S100a7蛋白在人视网膜色素上皮细胞中的表达,并明显促进人视网膜色素上皮细胞增殖。     

A Bruch's membrane substitute fabricated from silk fibroin supports the function of retinal pigment epithelial cells in vitro

Silk fibroin provides a promising biomaterial for ocular tissue reconstruction, including the damaged outer blood–retinal barrier of patients afflicted with age‐related macular degeneration (AMD). The aim of the present study was to evaluate the function of retinal pigment epithelial (RPE) cells in vitro, when grown on fibroin membranes manufactured to a thickness similar to that of Bruch's membrane (3 µm). Confluent cultures of RPE cells (ARPE‐19) were established on fibroin membranes and maintained under conditions designed to promote maturation over 4 months. Control cultures were grown on polyester cell culture well inserts (Transwell^®). Cultures established on either material developed a cobblestone morphology, with partial pigmentation, within 12 weeks. Immunocytochemistry at 16 weeks revealed a similar distribution pattern between cultures for F‐actin, ZO‐1, ezrin, cytokeratin pair 8/18, RPE‐65 and Na⁺/K⁺‐ATPase. Electron microscopy revealed that cultures grown on fibroin displayed a rounder apical surface with a more dense distribution of microvilli. Both cultures avidly ingested fluorescent microspheres coated with vitronectin and bovine serum albumin (BSA), but not controls coated with BSA alone. VEGF and PEDF were detected in the conditioned media collected from above and below the two membrane types. Levels of PEDF were significantly higher than for VEGF on both membranes and a trend was observed towards larger amounts of PEDF in apical compartments. These findings demonstrated that RPE cell functions on fibroin membranes are equivalent to those observed for standard test materials (polyester membranes). As such, these studies support advancement to studies of RPE cell implantation on fibroin membranes in a preclinical model. Copyright © 2015 John Wiley & Sons, Ltd.     

Embryonic stem cells as a treatment for macular degeneration

Introduction: Retinal degenerations are typically characterized by loss of highly differentiated cell types within the neurosensory retina, such as photoreceptors, or retinal pigment epithelium (RPE). RPE loss is the final common pathway in a number of degenerations including the leading cause of new blindness in the developed world: age-related macular degeneration (AMD).

Areas covered: This paper presents the pathophysiologic case for RPE transplantation with stem cell (SC)-derived tissue, a review of the preclinical data substantiating the hypothesis and the initial clinical trials safety data from early human trials.

Expert opinion: Targeting the RPE for transplantation with SC-derived tissue presents a reasonable therapeutic opportunity in a variety of important, otherwise untreatable, blinding conditions. Success of cellular replacement strategies is contingent on finding a viable source of replacement cells, establishing a safe technique for delivery and survival of transplanted cells within the host, restoration of normal retinal architecture and stabilization or improvement of vision.     

Effect of pore sizes of PLGA scaffolds on mechanical properties and cell behaviour for nucleus pulposus regeneration in vivo

This study investigated the influence of pore sizes of poly(lactic‐co‐glycolic acid) (PLGA) scaffolds on the compressive strength of tissue‐engineered biodiscs and selection of the best suitable pore size for cells to grow in vivo. PLGA scaffolds were fabricated by solvent casting/salt‐leaching with pore sizes of 90–180, 180–250, 250–355 and 355–425 µm. Nucleus pulposus (NP) cells were seeded on PLGA scaffolds with various pore sizes. Each sample was harvested at each time point, after retrieval of PLGA scaffolds seeded with NP cells, which were implanted into subcutaneous spaces in nude mice at 4 and 6 weeks. MTT assay, glycosaminoglycan (GAG) assay, haematoxylin and eosin (H&E) staining, safranin O staining and immunohistochemistry (for collagen type II) were performed at each time point. As the pores became smaller, the value of the compressive strength of the scaffold was increased. The group of scaffolds with pore sizes of 90–250 µm showed better cell proliferation and ECM production. These results demonstrated that the compressive strength of the scaffold was improved while the scaffold had pore sizes in the range 90–250 µm and good cell interconnectivity. Suitable space in the scaffold for cell viability is a key factor for cell metabolism. Copyright © 2014 John Wiley & Sons, Ltd.     

S100a7蛋白在体外培养人视网膜色素上皮细胞中的表达及功能

背景:S100a7蛋白在细胞增殖、血管形成等病理生理过程中发挥重要作用,但在视网膜色素上皮细胞中的作用少有报道。目的:探讨S100a7蛋白在体外培养人视网膜色素上皮细胞中的表达及作用。方法:体外培养ARPE-19细胞株,通过免疫荧光及Western blot技术,检测S100a7在ARPE-19细胞中的表达;不同稀释度(1∶1000,1∶10000,1∶100000)S100a7蛋白抗体与ARPE-19细胞共培养,通过MTT技术检测S100a7蛋白在人视网膜色素上皮细胞增殖过程中的作用。结果与结论:免疫荧光及Westernblot技术证实该蛋白在ARPE-19细胞中表达;MTT实验结果显示,人视网膜色素上皮细胞加入S100a7抗体72h后,各浓度处理组的吸光度值较对照组显著均降低(P<0.05)。结果证实S100a7蛋白在人视网膜色素上皮细胞中的表达,并明显促进人视网膜色素上皮细胞增殖。     

Development of a protocol for maintaining viability while shipping organoid‐derived retinal tissue

Retinal organoid technology enables generation of an inexhaustible supply of three‐dimensional retinal tissue from human pluripotent stem cells (hPSCs) for regenerative medicine applications. The high similarity of organoid‐derived retinal tissue and transplantable human fetal retina provides an opportunity for evaluating and modeling retinal tissue replacement strategies in relevant animal models in the effort to develop a functional retinal patch to restore vision in patients with profound blindness caused by retinal degeneration. Because of the complexity of this very promising approach requiring specialized stem cell and grafting techniques, the tasks of retinal tissue derivation and transplantation are frequently split between geographically distant teams. Delivery of delicate and perishable neural tissue such as retina to the surgical sites requires a reliable shipping protocol and also controlled temperature conditions with damage‐reporting mechanisms in place to prevent transplantation of tissue damaged in transit into expensive animal models. We have developed a robust overnight tissue shipping protocol providing reliable temperature control, live monitoring of the shipment conditions and physical location of the package, and damage reporting at the time of delivery. This allows for shipping of viable (transplantation‐competent) hPSC‐derived retinal tissue over large distances, thus enabling stem cell and surgical teams from different parts of the country to work together and maximize successful engraftment of organoid‐derived retinal tissue. Although this protocol was developed for preclinical in vivo studies in animal models, it is potentially translatable for clinical transplantation in the future and will contribute to developing clinical protocols for restoring vision in patients with retinal degeneration.     

Collagen vitrigels with low‐fibril density enhance human embryonic stem cell‐derived retinal pigment epithelial cell maturation

Structural and biochemical cues of extracellular matrix can substantially influence the differentiation and maturation of cultured retinal pigment epithelial (RPE) cells. In this study, thin collagen vitrigels were engineered to create collagen nanofibrillar structures of different fibril densities in an effort to evaluate the maturation of human embryonic stem cell–derived retinal pigment epithelial (hESC‐RPE) cells. The ultrastructure of the different collagen vitrigels was characterized by transmission electron microscopy, and the mechanical properties were evaluated by tensile testing. The pigmentation and polarization of cells, in addition to key RPE marker gene and protein expression levels, were analyzed to determine the differentiation of hESCs on the gels. The hESC‐RPE differentiation was most significant in collagen vitrigels with low fibril density with mature collagen fibrils with diameter of around 60 nm and Young's modulus of 2.41 ± 0.13 MPa. This study provides insight into the influence of collagen nanofibrillar structures on hESC‐RPE maturation and presents a potential bioengineered substratum for hESC‐RPE for future preclinical and clinical applications.     

Design,fabrication and in vitro evaluation of a novel polymer‐hydrogel hybrid scaffold for bone tissue engineering

The development of a bone mechanically‐compatible and osteoinductive scaffold is important for bone tissue engineering applications, particularly for the repair and regeneration of large area critically‐sized bone defects. Although previous studies with weight‐bearing scaffolds have shown promising results, there is a clear need to develop better osteoinductive strategies for effective scaffold‐based bone regeneration. In this study, we designed and fabricated a novel polymer‐hydrogel hybrid scaffold system in which a load‐bearing polymer matrix and a peptide hydrogel allowed for the synergistic combination of mechanical strength and great potential for osteoinductivity in a single scaffold. The hybrid scaffold system promoted increased pre‐osteoblastic cell proliferation. Further, we biotinylated human recombinant bone morphogenetic protein 2 (rhBMP2), and characterized the biotin addition and its effect on rhBMP2 biological activity. The biotinylated rhBMP2 was tethered to the hybrid scaffold using biotin‐streptavidin complexation. Controlled release studies demonstrated increased rhBMP2 retention with the tethered rhBMP2 hybrid scaffold group. In vitro evaluation of the hybrid scaffold was performed with rat bone marrow stromal cells and mouse pre‐osteoblast cell line MC3T3‐E1 cells. Gene expression of alkaline phosphatase (ALP), collagen I (Col I), osteopontin (OPN), bone sialoprotein (BSP), Runx‐2 and osteocalcin (OC) increased in MC3T3‐E1 cells seeded on the rhBMP2 tethered hybrid scaffolds over the untethered counterparts, demonstrating osteoinductive potential of the hybrid graft. These findings suggest the possibility of developing a novel polymer‐hydrogel hybrid system that is weight bearing and osteoinductive for effective bone tissue engineering. Copyright © 2012 John Wiley & Sons, Ltd.     

Effect of double growth factor release on cartilage tissue engineering

The effects of double release of insulin‐like growth factor I (IGF‐I) and growth factor β1 (TGF–β1) from nanoparticles on the growth of bone marrow mesenchymal stem cells and their differentiation into cartilage cells were studied on PLGA scaffolds. The release was achieved by using nanoparticles of poly(lactic acid‐co‐glycolic acid) (PLGA) and poly(N‐isopropylacrylamide) (PNIPAM) carrying IGF‐I and TGF–β1, respectively. On tissue culture polystyrene (TCPS), TGF‐β1 released from PNIPAM nanoparticles was found to have a significant effect on proliferation, while IGF‐I encouraged differentiation, as shown by collagen type II deposition. The study was then conducted on macroporous (pore size 200–400 µm) PLGA scaffolds. It was observed that the combination of IGF‐I and TGF‐β1 yielded better results in terms of collagen type II and aggrecan expression than GF‐free and single GF‐containing applications. It thus appears that gradual release of a combination of growth factors from nanoparticles could make a significant contribution to the quality of the engineered cartilage tissue. Copyright © 2011 John Wiley & Sons, Ltd.     

A simple and static preservation system for shipping retinal pigment epithelium cell sheets

The ability to move cells and tissues from bench to bedside is an essential aspect of regenerative medicine. In this study, we propose a simple and static shipping system to deliver tissue‐engineered cell sheets. Notably, this system is electronic‐device‐free and simplified to minimize the number of packing and opening steps involved. Shipping conditions were optimized, and application and verification of the system were performed using human iPS cell‐derived or fetal retinal pigment epithelium (RPE) cell sheets. The temperature of the compartments within the insulated container was stable at various conditions, and filling up the cell vessel with medium effectively prevented turbulence‐induced mechanical damage to the RPE cell sheets. Furthermore, no abnormal changes were observed in RPE morphology, transepithelial electrical resistance, or mRNA expression after transit by train and car. Taken together, our simple shipping system has the potential to minimize the costs and human error associated with bench to bedside tissue transfer. This specially designed regenerative tissue shipping system, validated for use in this field, can be used without any special training. This study provides a procedure for easily sharing engineered tissues with the goal of promoting collaboration between laboratories and hospitals and enhancing patient care.     

Peptide nanostructures on nanofibers for peripheral nerve regeneration

Self‐assembled peptide nanofibrous scaffolds with designer sequences, similar to neurite growth promoting molecules enhance the differentiation of neural stem cells. However, self‐assembled peptide nanofibrous scaffolds lack the required mechanical strength to suffice to bridge long critical‐sized peripheral nerve defects. Hence, there is a demand for a potential neural substrate, which could be biomimetic coupled with bioactive nanostructures to regrow the denuded axons towards the distal end. In the present study, we developed designer self‐assembling peptide‐based aligned poly(lactic‐co‐glycolic acid) (PLGA) nanofibrous scaffolds by simple surface coating of peptides or coelectrospinning. Retention of secondary structures of peptides in peptide‐coated and cospun fibers was confirmed by circular dichroism spectroscopy. The rod‐like peptide nanostructures enhance the typical bipolar morphology of Schwann cells. Although the peptide‐coated PLGA scaffolds exhibited significant increase in Schwann cell proliferation than pristine PLGA and PLGA‐peptide cospun scaffolds (p < .05), peptide cospun scaffolds demonstrated better cellular infiltration and significantly higher gene expression of neural cell adhesion molecule, glial fibrillary acidic protein, and peripheral myelin protein22 compared to the pristine PLGA and PLGA‐peptide‐coated scaffolds. Our results demonstrate the positive effects of aligned peptide coelectrospun scaffolds with biomimetic cell recognition motifs towards functional proliferation of Schwann cells. These scaffolds could subsequently repair peripheral nerve defects by augmenting axonal regeneration and functional nerve recovery.