Electrospun freestanding hydrophobic fabric as a potential polymer semi-permeable membrane for islet encapsulation.

One of the significant problems associated with islet encapsulation for type 1 diabetes treatment is the loss of islet functionality or cell death after transplantation because of the unfavorable environment for the cells. In this work, we propose a simple strategy to fabricate electrospun membranes that will provide a favorable environment for proper islet function and also a desirable pore size to cease cellular infiltration, protecting the encapsulated islet from immune cells. By electrospinning the wettability of three different biocompatible polymers: cellulose acetate (CA), polyethersulfone (PES), and polytetrafluoroethylene (PTFE) was greatly modified. The contact angle of electrospun CA, PES, and PTFE increased to 136°, 126°, and 155° as compared to 55°, 71°, and 128° respectively as a thin film, making the electrospun membranes hydrophobic. Commercial porous membranes of PES and PTFE show a contact angle of 30° and 118°, respectively, confirming the hydrophobicity of electrospun membranes is due to the surface morphology induced by electrospinning. In- vivo results confirm that the induced hydrophobicity and surface morphology of electrospun membranes impede cell attachment, which would help in maintaining the 3D circular morphology of islet cell. More importantly, the pore size of 0.3-0.6 µm obtained due to the densely packed structure of nanofibers, will be able to restrict immune cells but would allow free movement of molecules like insulin and glucose. Therefore, electrospun polymer fibrous membranes as fabricated in this work, with hydrophobic and porous properties, make a strong case for successful islet encapsulation.

Isogenic-induced endothelial cells enhance osteogenic differentiation of mesenchymal stem cells on silk fibroin scaffold.

Aim: We investigated the role of induced endothelial cells (iECs) in mesenchymal stem cells (MSCs)/iECs co-culture and assessed their osteogenic ability on silk fibroin nanofiber scaffolds. Methods: The osteogenic differentiation was assessed by the ALP assay, calcium assay and gene expression studies. Results: The osteogenic differentiation of the iECs co-cultures was found to be higher than the MSCs group and proximal to endothelial cells (ECs) co-cultures. Furthermore, the usage of isogenic iECs for co-culture increased the osteogenic and endothelial gene expression. Conclusion: These findings suggest that iECs mimic endothelial cells when co-cultured with MSCs and that one MSCs source can be used to give rise to both MSCs and iECs. The isogenic MSCs/iECs co-culture provides a new option for bone tissue engineering applications.

Recent advances in three-dimensional bioprinting of stem cells.

In spite of being a new field, three-dimensional (3D) bioprinting has undergone rapid growth in the recent years. Bioprinting methods offer a unique opportunity for stem cell distribution, positioning, and differentiation at the microscale to make the differentiated architecture of any tissue while maintaining precision and control over the cellular microenvironment. Bioprinting introduces a wide array of approaches to modify stem cell fate. This review discusses these methodologies of 3D bioprinting stem cells. Fabricating a fully operational tissue or organ construct with a long life will be the most significant challenge of 3D bioprinting. Once this is achieved, a whole human organ can be fabricated for the defect place at the site of surgery.

Enhanced osteodifferentiation of MSC spheroids on patterned electrospun fiber mats - An advanced 3D double strategy for bone tissue regeneration.

2D cell culture has been widely developed with various micropatterning and microfabrication techniques over the past few decades for creating and controlling cellular microenvironments including cell-matrix interactions, cell-cell interactions, and bio-mimicking the in-vivo tissue hierarchy and functions. However, the drawbacks of 2D culture have currently paved the way to 3D cell culture which is considered clinically and biologically more relevant. Here we report a 3D double strategy for osteodifferentiation of MSC spheroids on nano- and micro-patterned PLGA/Collagen/nHAp electrospun fiber mats. A comparison of cell alignment, proliferation and differentiation of 2D and 3D MSCs on patterned and non-patterned substrate was done. The study demonstrates the synergistic effect of geometric cues and 3D culture on differentiation of MSC spheroids into osteogenic lineage even in absence of osteoinduction medium.

Effect of patterned electrospun hierarchical structures on alignment and differentiation of mesenchymal stem cells: Biomimicking bone.

Considering the complex hierarchical structure of bone, biomimicking the micro and nano level features should be an integral part of scaffold fabrication for successful bone regeneration. We aim to biomimic the microstructure and nanostructure of bone and study the effect of physical cues on cell alignment, proliferation, and differentiation. To achieve this, we have divided the scaffolds into groups: electrospun SU-8 nanofibers, electrospun SU-8 nanofibers with UV treatment, and micropatterned (20 µm sized ridges and grooves) SU-8 nanofibers by photolithography with UV treatment. Two types of culture conditions were applied: with and without osteoinduction medium. In vitro cell proliferation assays, protein estimation, alkaline phosphatase osteodifferentiation assay, live dead assay, and cell alignment studies were performed on these micropatterned nanofiber domains. Our findings show that patterned surface induced an early osteodifferentiation of mesenchymal stem cells even in absence of osteoinduction medium. An interesting similarity with the helicoidal plywood model of the bone was observed. The cells showed layering and rotation along the patterns with time. This resembles the in vivo anisotropic multilamellar bone tissue architecture thus, closely mimicking the subcellular features of bone. This might serve as a smart biomaterial surface for mesenchymal stem cell differentiation in therapeutics where the addition of external chemical factors is a challenge.

Electrospun nanofibres to mimic natural hierarchical structure of tissues: application in musculoskeletal regeneration.

Biomimetic scaffolds mimicking the natural hierarchical structure of tissues have recently attracted the interest of researchers and provide a promising strategy to resemble the nonhomogeneous property of tissues. This review provides an overview of the various hierarchical length scales in the native tissues of the musculoskeletal system. It further focuses on electrospinning as a technique to mimic the tissue structures with specific emphasis on bone. The effect of cellular alignment, infiltration, vascularisation, and differentiation in these nanostructures has also been discussed. An outline of the various additive manufacturing techniques in combination with electrospinning has been elaborated. The review concludes with the challenges and future directions to understand the intricacies of bottom-up approach to engineer the systems at a macroscale. Copyright © 2016 John Wiley & Sons, Ltd.

Electrospun Fibers for Recruitment and Differentiation of Stem Cells in Regenerative Medicine.

Electrospinning is a popular technique used to mimic the natural sub-micron features of the native tissue. The ultra-fine fibers provide a favorable extracellular matrix-like environment for regulation of cellular functions. This article summarizes and reviews the current advances in electrospun fiber application and focuses on the novel strategies applied for tissue regeneration and repair. It explores the different factors affecting the attachment and proliferation of mesenchymal stem cells (MSCs) on the electrospun substrates. The influence of different features of electrospun fibers in the differentiation of MSCs into specific lineages (bone, cartilage, tendon/ligament, and nerves) has been elaborated. In addition, the different techniques to mimic the hierarchical features of tissues and its effect on cellular functions are reviewed. Additionally, the new developments like three-dimensional (3D) electrospinning, 3D spheroid double strategy and the comparative analysis of dynamic and static culture on electrospun scaffolds are discussed. With the intricate understanding of the interaction between the cells and the electrospun fiber matrix we can aim to combine the newer strategies to overcome the existing challenges and improve the potential application of electrospun fibers in the field of tissue regeneration and repair.

Oxidized Alginate-Gelatin Hydrogel: A Favorable Matrix for Growth and Osteogenic Differentiation of Adipose-Derived Stem Cells in 3D.

Alginate-based hydrogels are extensively used matrices for cell encapsulation, but they need to be modified to recapitulate chemical, microstructural, and mechanical properties of the native extracellular matrix. Like other cell types, mesenchymal stem cells exhibit rounded and clustered morphologies when they are embedded in alginate hydrogels. In this study, we use covalently cross-linked oxidized alginate-gelatin hydrogels to encapsulate human adipose-derived stem cells in order to investigate cell growth, viability, and morphology during osteogenic differentiation taking advantage of the different physicochemical properties of this modified alginate-based hydrogel in comparison to those of the pristine alginate hydrogel. We investigate the effect of hydrogel compositions on stem cell behavior in 3D. Higher viability and the spreading morphology of encapsulated cells with interconnected networks were observed in high gelatin containing compositions. More filopodial protrusions from multicellular nodules were noticed during osteogenic differentiation in the hydrogels having a high amount of gelatin, confirming their suitability for cell encapsulation and bone tissue engineering applications.

Adipose- and bone marrow-derived mesenchymal stem cells display different osteogenic differentiation patterns in 3D bioactive glass-based scaffolds.

Mesenchymal stem cells can be isolated from a variety of different sources, each having their own peculiar merits and drawbacks. Although a number of studies have been conducted comparing these stem cells for their osteo-differentiation ability, these are mostly done in culture plastics. We have selected stem cells from either adipose tissue (ADSCs) or bone marrow (BMSCs) and studied their differentiation ability in highly porous three-dimensional (3D) 45S5 Bioglass®-based scaffolds. Equal numbers of cells were seeded onto 5 × 5 × 4 mm3 scaffolds and cultured in vitro, with or without osteo-induction medium. After 2 and 4 weeks, the cell-scaffold constructs were analysed for cell number, cell spreading, viability, alkaline phosphatase activity and osteogenic gene expression. The scaffolds with ADSCs displayed osteo-differentiation even without osteo-induction medium; however, with osteo-induction medium osteogenic differentiation was further increased. In contrast, the scaffolds with BMSCs showed no osteo-differentiation without osteo-induction medium; after application of osteo-induction medium, osteo-differentiation was confirmed, although lower than in scaffolds with ADSCs. In general, stem cells in 3D bioactive glass scaffolds differentiated better than cells in culture plastics with respect to their ALP content and osteogenic gene expression. In summary, 45S5 Bioglass-based scaffolds seeded with ADSCs are well-suited for possible bone tissue-engineering applications. Induction of osteogenic differentiation appears unnecessary prior to implantation in this specific setting. Copyright © 2013 John Wiley & Sons, Ltd.

Bioactive copper-doped glass scaffolds can stimulate endothelial cells in co-culture in combination with mesenchymal stem cells.

Bioactive glass (BG) scaffolds are being investigated for bone tissue engineering applications because of their osteoconductive and angiogenic nature. However, to increase the in vivo performance of the scaffold, including enhancing the angiogenetic growth into the scaffolds, some researchers use different modifications of the scaffold including addition of inorganic ionic components to the basic BG composition. In this study, we investigated the in vitro biocompatibility and bioactivity of Cu2+-doped BG derived scaffolds in either BMSC (bone-marrow derived mesenchymal stem cells)-only culture or co-culture of BMSC and human dermal microvascular endothelial cells (HDMEC). In BMSC-only culture, cells were seeded either directly on the scaffolds (3D or direct culture) or were exposed to ionic dissolution products of the BG scaffolds, kept in permeable cell culture inserts (2D or indirect culture). Though we did not observe any direct osteoinduction of BMSCs by alkaline phosphatase (ALP) assay or by PCR, there was increased vascular endothelial growth factor (VEGF) expression, observed by PCR and ELISA assays. Additionally, the scaffolds showed no toxicity to BMSCs and there were healthy live cells found throughout the scaffold. To analyze further the reasons behind the increased VEGF expression and to exploit the benefits of the finding, we used the indirect method with HDMECs in culture plastic and Cu2+-doped BG scaffolds with or without BMSCs in cell culture inserts. There was clear observation of increased endothelial markers by both FACS analysis and acetylated LDL (acLDL) uptake assay. Only in presence of Cu2+-doped BG scaffolds with BMSCs, a high VEGF secretion was demonstrated by ELISA; and typical tubular structures were observed in culture plastics. We conclude that Cu2+-doped BG scaffolds release Cu2+, which in turn act on BMSCs to secrete VEGF. This result is of significance for the application of BG scaffolds in bone tissue engineering approaches.

In vitro and in vivo Biocompatibility of Alginate Dialdehyde/Gelatin Hydrogels with and without Nanoscaled Bioactive Glass for Bone Tissue Engineering Applications.

In addition to good mechanical properties needed for three-dimensional tissue engineering, the combination of alginate dialdehyde, gelatin and nano-scaled bioactive glass (45S5) is supposed to combine excellent cellular adhesion, proliferation and differentiation properties, good biocompatibility and predictable degradation rates. The goal of this study was to evaluate the in vitro and in vivo biocompatibility as a first step on the way to its use as a scaffold in bone tissue engineering. In vitro evaluation showed good cell adherence and proliferation of bone marrow derived mesenchymal stem cells seeded on covalently crosslinked alginate dialdehyde-gelatin (ADA-GEL) hydrogel films with and without 0.1% nano-Bioglass® (nBG). Lactate dehydrogenase (LDH)- and mitochondrial activity significantly increased in both ADA-GEL and ADA-GEL-nBG groups compared to alginate. However, addition of 0.1% nBG seemed to have slight cytotoxic effect compared to ADA-GEL. In vivo implantation did not produce a significant inflammatory reaction, and ongoing degradation could be seen after four weeks. Ongoing vascularization was detected after four weeks. The good biocompatibility encourages future studies using ADA-GEL and nBG for bone tissue engineering application.

Osteoinduction and survival of osteoblasts and bone-marrow stromal cells in 3D biphasic calcium phosphate scaffolds under static and dynamic culture conditions.

In many tissue engineering approaches, the basic difference between in vitro and in vivo conditions for cells within three-dimensional (3D) constructs is the nutrition flow dynamics. To achieve comparable results in vitro, bioreactors are advised for improved cell survival, as they are able to provide a controlled flow through the scaffold. We hypothesize that a bioreactor would enhance long-term differentiation conditions of osteogenic cells in 3D scaffolds. To achieve this either primary rat osteoblasts or bone marrow stromal cells (BMSC) were implanted on uniform-sized biphasic calcium phosphate (BCP) scaffolds produced by a 3D printing method. Three types of culture conditions were applied: static culture without osteoinduction (Group A); static culture with osteoinduction (Group B); dynamic culture with osteoinduction (Group C). After 3 and 6 weeks, the scaffolds were analysed by alkaline phosphatase (ALP), dsDNA amount, SEM, fluorescent labelled live-dead assay, and real-time RT-PCR in addition to weekly alamarBlue assays. With osteoinduction, increased ALP values and calcium deposition are observed; however, under static conditions, a significant decrease in the cell number on the biomaterial is observed. Interestingly, the bioreactor system not only reversed the decreased cell numbers but also increased their differentiation potential. We conclude from this study that a continuous flow bioreactor not only preserves the number of osteogenic cells but also keeps their differentiation ability in balance providing a suitable cell-seeded scaffold product for applications in regenerative medicine.

Development of a pre-vascularized 3D scaffold-hydrogel composite graft using an arterio-venous loop for tissue engineering applications.

Hyaluronic acid (HA) and fibrin glue (FG) are effective hydrogels for tissue engineering applications as they support tissue in-growth, retain growth factors, and release them slowly with time. The scaffolds, in combination with a hydrogel, effectuate a successful graft. However, the survival of a graft entirely depends upon a functional vascular supply. Therefore, hydrogels must support the in-growing vasculature. To study and compare the vascular patterns, HA and FG hydrogel-containing PLDLLA-TCP-PCL scaffolds were implanted in the groin of male Lewis rats and supplied with a micro-surgically prepared arterio-venous (A-V) loop. The rats were perfused with a vascular contrast media after 4 and 8 weeks and sacrificed for further analysis. The specimens were scanned with micro-CT to find the vascular growth patterns. Corrosion casting of blood vessels followed by SEM demonstrated a high vascular density near the parent blood vessels. Histologically, HA and FG implanted animal groups showed significant angiogenetic activity, especially within the pores of the scaffold. However, formation of new blood vessels was more conspicuously observed at 4 weeks in FG than HA implants. Furthermore, by 8 weeks, the number and pattern of blood vessels were comparable between them. At this time, HA was still present indicating its slow degradation. The finding was confirmed by histomorphometric analysis. This experimental study demonstrates that HA containing composite scaffold systems permit stabile in-growth of blood vessels due to sustained degradation over 8 weeks. HA is a potential matrix for a tissue engineered composite graft.

Hyaluronan-based heparin-incorporated hydrogels for generation of axially vascularized bioartificial bone tissues: in vitro and in vivo evaluation in a PLDLLA-TCP-PCL-composite system.

Smart matrices are required in bone tissue-engineered grafts that provide an optimal environment for cells and retain osteo-inductive factors for sustained biological activity. We hypothesized that a slow-degrading heparin-incorporated hyaluronan (HA) hydrogel can preserve BMP-2; while an arterio-venous (A-V) loop can support axial vascularization to provide nutrition for a bio-artificial bone graft. HA was evaluated for osteoblast growth and BMP-2 release. Porous PLDLLA-TCP-PCL scaffolds were produced by rapid prototyping technology and applied in vivo along with HA-hydrogel, loaded with either primary osteoblasts or BMP-2. A microsurgically created A-V loop was placed around the scaffold, encased in an isolation chamber in Lewis rats. HA-hydrogel supported growth of osteoblasts over 8 weeks and allowed sustained release of BMP-2 over 35 days. The A-V loop provided an angiogenic stimulus with the formation of vascularized tissue in the scaffolds. Bone-specific genes were detected by real time RT-PCR after 8 weeks. However, no significant amount of bone was observed histologically. The heterotopic isolation chamber in combination with absent biomechanical stimulation might explain the insufficient bone formation despite adequate expression of bone-related genes. Optimization of the interplay of osteogenic cells and osteo-inductive factors might eventually generate sufficient amounts of axially vascularized bone grafts for reconstructive surgery.