Effect of chemical immobilization of SDF‐1α into muscle‐derived scaffolds on angiogenesis and muscle progenitor recruitment

The availability of three‐dimensional bioactive scaffolds with enhanced angiogenic capacity that have the capability to recruit tissue specific resident progenitors is of great importance for the regeneration of impaired skeletal muscle. Here, we have investigated whether introduction of chemoattractant factors to tissue specific extracellular matrix promotes cellular behaviour in vitro as well as muscle progenitor recruitment and vascularization in vivo. We developed an interconnective macroporous sponge from decellularized skeletal muscle with maintained biochemical traits of the intact muscle. SDF‐1α, a potent cell homing factor involved in muscle repair, was physically adsorbed or chemically immobilized in these muscle‐derived sponges. The immobilized sponges showed significantly higher SDF‐1α conjugation efficiency along with improved metabolism and infiltration of muscle‐derived stem cells in vitro, and thus generated uniform cellular constructs. In vivo, femoral muscle implantation in rats revealed a negligible immune response in all scaffold groups. We observed enhanced engraftment, neovascularization, and infiltration of CXCR4⁺ cells in the immobilized‐SDF‐1α sponge compared with nonimmobilized controls. Although Pax7⁺ cells identified adjacent to the immobilized‐SDF‐1α implantation site, other factors appear to be necessary for efficient penetration of Pax7⁺ cells into the sponge. These findings suggest that immobilization of cell homing factors via chemical mediators can result in recruitment of cells to the microenvironment with subsequent improvement in angiogenesis.     

Minimally Invasive Approach to the Repair of Injured Skeletal Muscle With a Shape-memory Scaffold

Repair of injured skeletal muscle by cell therapies has been limited by poor survival of injected cells. Use of a carrier scaffold delivering cells locally, may enhance in vivo cell survival, and promote skeletal muscle regeneration. Biomaterial scaffolds are often implanted into muscle tissue through invasive surgeries, which can result in trauma that delays healing. Minimally invasive approaches to scaffold implantation are thought to minimize these adverse effects. This hypothesis was addressed in the context of a severe mouse skeletal muscle injury model. A degradable, shape-memory alginate scaffold that was highly porous and compressible was delivered by minimally invasive surgical techniques to injured tibialis anterior muscle. The scaffold controlled was quickly rehydrated in situ with autologous myoblasts and growth factors (either insulin-like growth factor-1 (IGF-1) alone or IGF-1 with vascular endothelial growth factor (VEGF)). The implanted scaffolds delivering myoblasts and IGF-1 significantly reduced scar formation, enhanced cell engraftment, and improved muscle contractile function. The addition of VEGF to the scaffold further improved functional recovery likely through increased angiogenesis. Thus, the delivery of myoblasts and dual local release of VEGF and IGF-1 from degradable scaffolds implanted through a minimally invasive procedure effectively promoted the functional regeneration of injured skeletal muscle.     

Defining conditions for covalent immobilization of angiogenic growth factors onto scaffolds for tissue engineering

Rapid vascularization of engineered tissues in vitro and in vivo remains one of the key limitations in tissue engineering. We propose that angiogenic growth factors covalently immobilized on scaffolds for tissue engineering can be used to accomplish this goal. The main objectives of this work were: (a) to derive desirable experimental conditions for the covalent immobilization of vascular endothelial growth factor (VEGF) and angiopoietin‐1 (Ang1) on porous collagen scaffolds; and (b) to determine whether primary endothelial cells respond to these scaffolds with covalently immobilized angiogenic factors. VEGF and Ang1 were covalently immobilized onto porous collagen scaffolds, using 1‐ethyl‐3‐(3‐dimethylaminopropyl)carbodiimide hydrochloride (EDC) chemistry. To improve covalent immobilization conditions: (a) different reaction buffers [phosphate‐buffered saline (PBS), distilled water, or 2‐(N‐morpholino)ethanesulphonic acid (MES)] were used; and (b) step immobilization was compared to bulk immobilization. In step immobilization, growth factors are applied after EDC activation of the scaffold, while in bulk immobilization, reagents are simultaneously applied to the scaffold. PBS as the reaction buffer resulted in higher amounts of VEGF and Ang1 immobilized (ELISA), higher cell proliferation rates (XTT) and increased lactate metabolism compared to water and MES as the reaction buffers. Step immobilization in PBS buffer was also more effective than bulk immobilization. Immobilized growth factors resulted in higher cell proliferation and lactate metabolism compared to soluble growth factors used at comparable concentrations. Tube formation by CD31‐positive cells was also observed in collagen scaffolds with immobilized VEGF or Ang1 using H5V and primary rat aortic endothelial cells but not on control scaffolds. Copyright © 2010 John Wiley & Sons, Ltd.     

Hypoxic preconditioning of myoblasts implanted in a tissue engineering chamber significantly increases local angiogenesis via upregulation of myoblast vascular endothelial growth factor‐A expression and downregulation of miRNA‐1, miRNA‐206 and angiopoietin‐1

Vascularization is a major hurdle for growing three‐dimensional tissue engineered constructs. This study investigated the mechanisms involved in hypoxic preconditioning of primary rat myoblasts in vitro and their influence on local angiogenesis postimplantation. Primary rat myoblast cultures were exposed to 90 min hypoxia at <1% oxygen followed by normoxia for 24 h. Real time (RT) polymerase chain reaction evaluation indicated that 90 min hypoxia resulted in significant downregulation of miR‐1 and miR‐206 (p < 0.05) and angiopoietin‐1 (p < 0.05) with upregulation of vascular endothelial growth factor‐A (VEGF‐A; p < 0.05). The miR‐1 and angiopoietin‐1 responses remained significantly downregulated after a 24 h rest phase. In addition, direct inhibition of miR‐206 in L6 myoblasts caused a significant increase in VEGF‐A expression (p < 0.05), further establishing that changes in VEGF‐A expression are influenced by miR‐206. Of the myogenic genes examined, MyoD was significantly upregulated, only after 24 h rest (p < 0.05). Preconditioned or control myoblasts were implanted with Matrigel? into isolated bilateral tissue engineering chambers incorporating a flow‐through epigastric vascular pedicle in severe combined immunodeficiency mice and the chamber tissue harvested 14 days later. Chambers implanted with preconditioned myoblasts had a significantly increased percentage volume of blood vessels (p = 0.0325) compared with chambers implanted with control myoblasts. Hypoxic preconditioned myoblasts promote vascularization of constructs via VEGF upregulation and downregulation of angiopoietin‐1, miR‐1 and miR‐206. The relatively simple strategy of hypoxic preconditioning of implanted cells ‐ including non‐stem cell types – has broad, future applications in tissue engineering of skeletal muscle and other tissues, as a technique to significantly increase implant site angiogenesis.     

In vitro cellular response to oxidized collagen–PLLA hybrid scaffolds designed for the repair of muscular tissue defects and complex incisional hernias

Unique poly(l ‐lactic acid) (PLLA)‐based scaffolds were constructed by embedding knitted PLLA yarns within a bioresorbable and differentially crosslinked three‐dimensional (3D) oxidized collagen scaffold. The scaffolds were designed specifically for the repair of complex incisional abdominal wall hernias and the repair of defects within planar muscular tissues, such as the bladder. The chemical composition of the collagen matrix and the percentage of scaffold infiltration were compared for the different scaffold compositions. The results demonstrate that the incorporation of the collagen sponge within the PLLA scaffold facilitated bladder smooth muscle cell (bSMC) adhesion and proliferation. The highest dose of oxidized collagen (Oxicol) demonstrated better cell adhesion, resulting in the largest cell densities and most uniform distribution throughout the 3D collagen sponge. This formulation promoted the greatest α‐smooth muscle actin (αSMA) expression detected through immunohistochemical staining and western blotting. For abdominal wall repair applications, the proliferation and differentiation of C2C12 myoblasts and myotube formation were studied. Following 7 days of myogenic induction, the greatest expression of mRNA of the myogenic markers myogenin and MRF4 was observed within the scaffolds with the highest dose of oxidized collagen, 1.5‐ and 3.85‐fold greater expressions, respectively, compared to PLLA with unmodified collagen. Furthermore, in vitro myotube formation and MyMC expression were enhanced in the Oxicol scaffolds. We conclude that the Oxicol scaffold formulation with a high‐dose oxidized collagen ratio provides enhanced myogenesis and αSMA, and the biological induction cues necessary to achieve better tissue integration, than standard PLLA scaffolds in the treatment of complex abdominal wall hernias. Copyright © 2013 John Wiley & Sons, Ltd.     

Evaluation of articular cartilage repair using biodegradable nanofibrous scaffolds in a swine model: a pilot study

The aim of this study was to evaluate a cell‐seeded nanofibrous scaffold for cartilage repair in vivo. We used a biodegradable poly(ε‐caprolactone) (PCL) nanofibrous scaffold seeded with allogeneic chondrocytes or xenogeneic human mesenchymal stem cells (MSCs), or acellular PCL scaffolds, with no implant as a control to repair iatrogenic, 7 mm full‐thickness cartilage defects in a swine model. Six months after implantation, MSC‐seeded constructs showed the most complete repair in the defects compared to other groups. Macroscopically, the MSC‐seeded constructs regenerated hyaline cartilage‐like tissue and restored a smooth cartilage surface, while the chondrocyte‐seeded constructs produced mostly fibrocartilage‐like tissue with a discontinuous superficial cartilage contour. Incomplete repair containing fibrocartilage or fibrous tissue was found in the acellular constructs and the no‐implant control group. Quantitative histological evaluation showed overall higher scores for the chondrocyte‐ and MSC‐seeded constructs than the acellular construct and the no‐implant groups. Mechanical testing showed the highest equilibrium compressive stress of 1.5 MPa in the regenerated cartilage produced by the MSC‐seeded constructs, compared to 1.2 MPa in the chondrocyte‐seeded constructs, 1.0 MPa in the acellular constructs and 0.2 MPa in the no‐implant group. No evidence of immune reaction to the allogeneically‐ and xenogeneically‐derived regenerated cartilage was observed, possibly related to the immunosuppressive activities of MSCs, suggesting the feasibility of allogeneic or xenogeneic transplantation of MSCs for cell‐based therapy. Taken together, our results showed that biodegradable nanofibrous scaffolds seeded with MSCs effectively repair cartilage defects in vivo, and that the current approach is promising for cartilage repair. Copyright © 2008 John Wiley & Sons, Ltd.     

Bi‐layer silk fibroin grafts support functional tissue regeneration in a porcine model of onlay esophagoplasty

Partial circumferential, full thickness defects of the esophagus can occur as a result of organ perforation or tumour resection, or during surgical reconstruction of strictured segments. Complications associated with autologous tissue flaps conventionally utilized for defect repair necessitate the development of new graft options. In this study, bi‐layer silk fibroin (BLSF) scaffolds were investigated for their potential to support functional restoration of partial circumferential defects in a porcine model of esophageal repair. Onlay thoracic esophagoplasty with BLSF matrices (~3 x 1.5 cm) was performed in adult swine (N = 6) for 3 months of implantation. All animals receiving BLSF grafts survived with no complications and were capable of solid food consumption. Radiographic esophagrams revealed preservation of organ continuity with no evidence of contrast extravasation or strictures. Fluoroscopic analysis demonstrated peristaltic contractions. Ex vivo tissue bath studies displayed contractile responses to carbachol, electric field stimulation, and KCl while isoproterenol produced tissue relaxation. Histological and immunohistochemical evaluations of neotissues showed a stratified, squamous epithelium, a muscularis mucosa composed of smooth muscle bundles, and a muscularis externa organized into circular and longitudinal layers, with a mix of striated skeletal muscle fascicles interspersed with smooth muscle. De novo innervation and vascularization were observed throughout the graft sites and consisted of synaptophysin‐positive neuronal boutons and vessels lined with CD31‐positive endothelial cells. The results of this study demonstrate that BLSF scaffolds can facilitate constructive remodeling of partial circumferential, full thickness esophageal defects in a large animal model. Copyright © 2017 John Wiley & Sons, Ltd.     

Synergic effects of VEGF‐A and SDF‐1 on the angiogenic properties of endothelial progenitor cells

Here we investigated the impact of hypoxic environment on the angiogenic properties of early‐outgrowth endothelial progenitor cells (EPCs), with particular focus on the role of secreted vascular endothelial growth factor‐A (VEGF‐A) and stromal derived factor‐1 (SDF‐1) in mediating these effects. We found that cultured EPCs secreted factors with paracrine effects on chemotaxis, migration, proliferation and tube formation of mature endothelial cells (ECs), and these properties were not affected by hypoxia. Depletion of VEGF‐A did not change the ability of EPC‐conditioned medium (CM) to promote EC migration and tube formation in vitro, suggesting that the pro‐angiogenic paracrine effects of EPCs did not totally rely on the presence of VEGF‐A. These findings were confirmed by in vivo experiments, on a mouse model of hind limb ischaemia, which showed that VEGF‐depleted EPC‐CM sustained tissue perfusion at the same level as complete EPC‐CM. However, concomitant deletion of VEGF‐A and SDF‐1 in EPC‐CM impaired the pro‐angiogenic properties of EPC‐CM, by inhibition of EC spreading in culture, tube‐like structure formation on Matrigel support, in vivo neovessels formation and ischaemic hind limb regeneration. Taken together, our data demonstrate that: (i) hypoxia does not affect the capacity of EPCs to support the angiogenic process; (ii) the absence of either VEGF‐A or SDF‐1 from EPC‐CM can be rescued by the presence of the other one, so that the overall angiogenic effects remain unchanged; and (iii) and the concomitant deletion of VEGF‐A and SDF‐1 from EPC‐CM impairs its pro‐angiogenic effect, both in vitro and in vivo. Copyright © 2016 John Wiley & Sons, Ltd.     

Treatment of growth plate injury using IGF‐I‐loaded PLGA scaffolds

Growth plate fracture can lead to retarded growth and unequal limb length, which may have a lifelong effect on a person's physical stature. The goal of this research was to develop an in vivo tissue‐engineering approach for the treatment of growth plate injury via localized delivery of insulin‐like growth factor I (IGF‐I) from cell‐free poly(lactic‐co‐glycolic acid) (PLGA) scaffolds. Mass loss and drug release studies were conducted to study the scaffold degradation and IGF‐I release patterns. In vitro cell studies showed that rat bone marrow stromal cells seeded on the porous scaffolds colonized the pores and deposited matrix within the scaffolds. These in vitro evaluations were followed by a proof‐of‐concept animal study involving implantation of scaffolds in proximal tibial growth plate defects in New Zealand white rabbits. Histological analysis of tissue sections from the in vivo studies showed regeneration of cartilage, albeit with disorganized structure, at the site of implantation of IGF‐I‐releasing scaffolds; in contrast, only bone was formed in empty defects and those treated with IGF‐free scaffolds. The present findings show the potential for treating growth plate injury using in vivo tissue engineering techniques. Copyright © 2012 John Wiley & Sons, Ltd.     

Controlled release of vascular endothelial growth factor from spray‐dried alginate microparticles in collagen–hydroxyapatite scaffolds for promoting vascularization and bone repair

A major limitation with current tissue‐engineering approaches is creating functionally vascularized constructs that can successfully integrate with the host; this often leads to implant failure, due to avascular necrosis. In order to overcome this, the objective of the present work was to develop a method to incorporate growth factor‐eluting alginate microparticles (MPs) into freeze‐dried, collagen‐based scaffolds. A collagen–hydroxyapatite (CHA) scaffold, previously optimized for bone regeneration, was functionalized for the sustained delivery of an angiogenic growth factor, vascular endothelial growth factor (VEGF), with the aim of facilitating angiogenesis and enhancing bone regeneration. VEGF was initially encapsulated in alginate MPs by spray‐drying, producing particles of < 10 µm in diameter. This process was found to effectively encapsulate and control VEGF release while maintaining its stability and bioactivity post‐processing. These VEGF‐MPs were then incorporated into CHA scaffolds, leading to homogeneous distribution throughout the interconnected scaffold pore structure. The scaffolds were capable of sustained release of bioactive VEGF for up to 35 days, which was proficient at increasing tubule formation by endothelial cells in vitro. When implanted in vivo in a rat calvarial defect model, this scaffold enhanced vessel formation, resulting in increased bone regeneration compared to empty‐defect and VEGF‐free scaffolds. This biologically functionalized scaffold, composed entirely of natural‐based materials, may offer an ideal platform to promote angiogenesis and tissue regeneration. Copyright © 2015 John Wiley & Sons, Ltd.     

Cell factory‐derived bioactive molecules with polymeric cryogel scaffold enhance the repair of subchondral cartilage defect in rabbits

We have explored the potential of cell factory‐derived bioactive molecules, isolated from conditioned media of primary goat chondrocytes, for the repair of subchondral cartilage defects. Enzyme‐linked immunosorbent assay (ELISA) confirms the presence of transforming growth factor‐β1 in an isolated protein fraction (12.56 ± 1.15 ng/mg protein fraction). These bioactive molecules were used alone or with chitosan–agarose–gelatin cryogel scaffolds, with and without chondrocytes, to check whether combined approaches further enhance cartilage repair. To evaluate this, an in vivo study was conducted on New Zealand rabbits in which a subchondral defect (4.5 mm wide × 4.5 mm deep) was surgically created. Starting after the operation, bioactive molecules were injected at the defect site at regular intervals of 14 days. Histopathological analysis showed that rabbits treated with bioactive molecules alone had cartilage regeneration after 4 weeks. However, rabbits treated with bioactive molecules along with scaffolds, with or without cells, showed cartilage formation after 3 weeks; 6 weeks after surgery, the cartilage regenerated in rabbits treated with either bioactive molecules alone or in combinations showed morphological similarities to native cartilage. No systemic cytotoxicity or inflammatory response was induced by any of the treatments. Further, ELISA was done to determine systemic toxicity, which showed no difference in concentration of tumour necrosis factor‐α in blood serum, before or after surgery. In conclusion, intra‐articular injection with bioactive molecules alone may be used for the repair of subchondral cartilage defects, and bioactive molecules along with chondrocyte‐seeded scaffolds further enhance the repair. Copyright © 2015 John Wiley & Sons, Ltd.     

Sequential identification of a degradable phosphate glass scaffold for skeletal muscle regeneration

Tissue engineering has the potential to overcome limitations associated with current management of skeletal muscle defects. This study aimed to sequentially identify a degradable phosphate glass scaffold for the restoration of muscle defects. A series of glass compositions were investigated for the potential to promote bacterial growth. Thereafter, the response of human craniofacial muscle‐derived cells was determined. Glass compositions containing Fe4‐ and 5 mol% did not promote greater Staphylococcus aureus and Staphylococcus epidermidis growth compared to the control (p > 0.05). Following confirmation of myogenicity, further studies assessed the biocompatibility of glasses containing Fe5 mol%. Cells seeded on collagen‐coated disks demonstrated comparable cellular metabolic activity to control. Upregulation of genes encoding for myogenic regulatory factors (MRFs) confirmed myofibre formation and there was expression of developmental MYH genes. The use of 3‐D aligned fibre scaffolds supported unidirectional cell alignment and upregulation of MRF and developmental MYH genes. Compared to the 2‐D disks, there was also expression of MYH2 and MYH7 genes, indicating further myofibre maturation on the 3‐D scaffolds and confirming the importance of key biophysical cues. Copyright © 2012 John Wiley & Sons, Ltd.     

Effect of VEGF on the Regenerative Capacity of Muscle Stem Cells in Dystrophic Skeletal Muscle

We have isolated a population of muscle-derived stem cells (MDSCs) that, when compared with myoblasts, display an improved regeneration capacity, exhibit better cell survival, and improve myogenesis and angiogenesis. In addition, we and others have observed that the origin of the MDSCs may reside within the blood vessel walls (endothelial cells and pericytes). Here, we investigated the role of vascular endothelial growth factor (VEGF)–mediated angiogenesis in MDSC transplantation–based skeletal muscle regeneration in mdx mice (an animal model of muscular dystrophy). We studied MDSC and MDSC transduced to overexpress VEGF; no differences were observed in vitro in terms of phenotype or myogenic differentiation. However, after in vivo transplantation, we observe an increase in angiogenesis and endogenous muscle regeneration as well as a reduction in muscle fibrosis in muscles transplanted with VEGF-expressing cells when compared to control cells. In contrast, we observe a significant decrease in vascularization and an increase in fibrosis in the muscles transplanted with MDSCs expressing soluble forms-like tyrosine kinase 1 (sFlt1) (VEGF-specific antagonist) when compared to control MDSCs. Our results indicate that VEGF-expressing cells do not increase the number of dystrophin-positive fibers in the injected mdx muscle, when compared to the control MDSCs. Together the results suggest that the transplantation of VEGF-expressing MDSCs improved skeletal muscle repair through modulation of angiogenesis, regeneration and fibrosis in the injected mdx skeletal muscle.     

In vivo vascular tissue engineering: influence of cytokine and implant location on tissue specific cellular recruitment

In vivo tissue engineering has been explored as a means to create autologous vascular replacements. Elastin is necessary to sustain continual pulsatile flow and to prevent the dilatation of vascular tissues. Unfortunately, elastogenesis in tissue‐engineered constructs has been very limited. To overcome this limitation, we have created tubular elastin scaffolds from porcine carotid arteries. Elastin would provide the necessary elasticity to the graft on implanting these scaffolds as vascular grafts. In this study, elastin tubes with agarose gel containing either stromal‐derived factor‐1 α [SDF; for homing of endothelial cells (ECs)] or basic fibroblast growth factor (bFGF; for homing of myofibroblasts) were implanted into adipose tissue, as it is a known source of stem/progenitor cells. We also implanted these tubes into subdermal pouches (as a control location). We observed a difference in the types of cells recruited—ECs were recruited in large numbers by SDF in the adipose tissue, whereas the adipose‐FGF group had a vascularized (smooth muscle and EC‐positive), collagenous capsule (adventitia) with many smooth muscle α‐actin (SMA)‐positive cells in the elastin scaffold layer (media). These results were in contrast to the subdermal group, which only recruited fibroblasts and some SMA‐positive cells. Also, more cell infiltration and neo‐collagen formation was seen in adipose implants. This study provides novel results by the use of specific cytokines and implant locations to recruit tissue‐specific cells to create autologous vascular grafts. Copyright © 2009 John Wiley & Sons, Ltd.     

Silk fibroin scaffolds loaded with angiogenic genes in adenovirus vectors for tissue regeneration

Vascularization remains a critical challenge in dermal tissue regeneration. In this study, a vascular endothelial growth factor (VEGF165) and angiopoietin‐1 (Ang‐1) dual gene coexpression vector that encoded green fluorescent protein (GFP) was constructed from an arginine–glycine–aspartic acid‐modified adenovirus. Silk fibroin (SF) scaffolds loaded with adenovirus vectors were fabricated by freeze‐drying method. In vitro, the human endothelial‐derived cell line EA.hy926 was infected with adenovirus vectors and then expressed GFP, secreted VEGF165 and Ang‐1, and promoted cell proliferation effectively. The VEGF165 and Ang‐1 genes loaded in the SF scaffolds significantly promoted the formation of abundant microvascular networks in the chick embryo chorioallantoic membrane. In vivo, angiogenic genes loaded in the scaffolds promoted vascularization and collagen deposition in scaffolds, thus effectively accelerating dermal tissue regeneration in a dorsal full‐thickness skin defect wound model in Sprague–Dawley rats. In conclusion, SF scaffolds loaded with arginine–glycine–aspartic acid‐modified adenovirus vectors encoding VEGF165 and Ang‐1 could stimulate the formation of vascular networks through the effective expression of target genes in vascular endothelial cells, thereby accelerating the regeneration of dermal tissue.     

Molecular magnetic resonance probe targeting VEGF165: preparation and in vitro and in vivo evaluation

A new method for imaging the tumor human vascular endothelial growth factor 165 (VEGF 165) is presented. A magnetic resonance imaging (MRI) probe was prepared by crosslinking ultrasmall superparamagnetic iron oxide (USPIO) nanoparticles to the aptamer for tumor vascular endothelial growth factor 165 (VEGF165‐aptamer). The molecular probe was evaluated for its in vitro and in vivo activities toward VEGF165. Enzyme‐linked immunosorbent assay showed that the VEGF165‐aptamer–USPIO nanoparticles conjugate specifically binds to VEGF165 in vitro. A cell proliferation test showed that VEGF165‐aptamer–USPIO seems to block the proliferation of human umbilical vein endothelial cells induced by free VEGF165, suggesting that VEGF165 is an effective target of this molecular probe. In xenograft mice carrying liver cancer that expresses VEGF165, T₂‐weighted imaging of the tumor displayed marked negative enhancement 3 h after the intravenous administration of VEGF165‐aptamer–USPIO. The enhancement disappeared 6 h after administration of the probe. These results suggest the targeted imaging effect of VEGF165‐aptamer–USPIO probe in vivo for VEGF165‐expressing tumors. This is the first report of a targeted MRI molecular probe based on USPIO and VEGF165‐aptamer. Copyright © 2014 John Wiley & Sons, Ltd.     

Silk fibroin scaffolds with muscle‐like elasticity support in vitro differentiation of human skeletal muscle cells

Human adult skeletal muscle has a limited ability to regenerate after injury and therapeutic options for volumetric muscle loss are few. Technologies to enhance regeneration of tissues generally rely upon bioscaffolds to mimic aspects of the tissue extracellular matrix (ECM). In the present study, silk fibroins from four Lepidoptera (silkworm) species engineered into three‐dimensional scaffolds were examined for their ability to support the differentiation of primary human skeletal muscle myoblasts. Human skeletal muscle myoblasts (HSMMs) adhered, spread and deposited extensive ECM on all the scaffolds, but immunofluorescence and quantitative polymerase chain reaction analysis of gene expression revealed that myotube formation occurred differently on the various scaffolds. Bombyx mori fibroin scaffolds supported formation of long, well‐aligned myotubes, whereas on Antheraea mylitta fibroin scaffolds the myotubes were thicker and shorter. Myotubes were oriented in two perpendicular layers on Antheraea assamensis scaffolds, and scaffolds of Philosamia/Samia ricini (S. ricini) fibroin poorly supported myotube formation. These differences were not caused by fibroin composition per se, as HSMMs adhered to, proliferated on and formed striated myotubes on all four fibroins presented as two‐dimensional fibroin films. The Young's modulus of A. mylitta and B. mori scaffolds mimicked that of normal skeletal muscle, but A. assamensis and S. ricini scaffolds were more flexible. The present study demonstrates that although myoblasts deposit matrix onto fibroin scaffolds and create a permissive environment for cell proliferation, a scaffold elasticity resembling that of normal muscle is required for optimal myotube length, alignment, and maturation. © 2016 The Authors Journal of Tissue Engineering and Regenerative Medicine Published by John Wiley & Sons Ltd. StartCopTextStartCopText© 2016 The Authors Journal of Tissue Engineering and Regenerative Medicine Published by John Wiley & Sons Ltd.     

Promotion of diabetic wound healing by collagen scaffold with collagen‐binding vascular endothelial growth factor in a diabetic rat model

Aims: Studies show that VEGF can promote tissue regeneration in diabetic wounds. The aim of this study was to evaluate the effects of a new composite biomaterial, a collagen scaffold with CBD‐VEGF, for wound healing in a diabetic rat model. Materials and methods: We produced a collagen scaffold loaded with CBD‐VEGF, which allowed VEGF to bind to the collagen scaffold. The diabetic rat model was constructed by injecting streptozocin (STZ) peritoneally and removing a 2 x 2.5 cm thick slice of skin from the back of the animal. Animals were randomly divided into 4 groups: blank control (BC Group, n = 24), collagen scaffold loaded with PBS (PBS Group, n = 24), collagen scaffold loaded with NAT‐VEGF (NAT‐VEGF Group, n = 24), and collagen scaffold loaded with CBD‐VEGF (CBD‐VEGF Group, n = 24). Wounds of the BC Group were covered with gauze and those of the PBS, NAT‐VEGF and CBD‐VEGF Groups were grafted by corresponding collagen scaffolds, respectively. Healing rates were calculated and compared among groups. Wound tissue was evaluated by histologic analysis. Results: The CBD‐VEGF group showed a higher wound healing rate, better vascularization and higher level of VEGF in the granulation tissue wound compared with NAT‐VEGF and PBS groups. Conclusions: The collagen scaffold with CBD‐VEGF promoted wound healing in a diabetic rat model, which could potentially provide better therapeutic options for the treatment of diabetic wounds. Copyright © 2012 John Wiley & Sons, Ltd.     

Osteoprotegerin,a new actor in vasculogenesis,stimulates endothelial colony‐forming cells properties

Summary. Background: Osteoprotegerin (OPG), a soluble receptor of the tumour necrosis factor family, and its ligand, the receptor activator of nuclear factor‐κB ligand (RANKL), are emerging as important regulators of vascular pathophysiology. Objectives: We evaluated their effects on vasculogenesis induced by endothelial colony‐forming cells (ECFC) and on neovessel formation in vivo. Methods: Effects of OPG and RANKL on in vitro angiogenesis were evaluated after ECFC incubation with OPG or RANKL (0–50 ng mL^?1). Effects on microvessel formation were evaluated with an in vivo murin Matrigel plug assay. Vascularization was evaluated by measuring plug hemoglobin and vascular endothelial growth factor (VEGF)‐R2 content 14 days after implantation. Results: We found that ECFC expressed OPG and RANK but not RANKL mRNA. Treatment of ECFC with VEGF or stromal cell‐derived factor‐1 (SDF‐1) upregulated OPG mRNA expression. OPG stimulated ECFC migration (P < 0.05), chemotaxis (P < 0.05) and vascular cord formation on Matrigel^® (P < 0.01). These effects were correlated with SDF‐1 mRNA overexpression, which was 30‐fold higher after 4 h of OPG stimulation (P < 0.01). OPG‐mediated angiogenesis involved the MAPK signaling pathway as well as Akt or mTOR cascades. RANKL also showed pro‐vasculogenic effects in vitro. OPG combined with FGF‐2 promoted neovessel formation in vivo, whereas RANKL had no effect. Conclusions: OPG induces ECFC activation and is a positive regulator of microvessel formation in vivo. Our results suggest that the OPG/RANK/RANKL axis may be involved in vasculogenesis and strongly support a modulatory role in tissue revascularization.     

Asymmetrical seeding of MSCs into fibrin–poly(ester‐urethane) scaffolds and its effect on mechanically induced chondrogenesis

Mesenchymal stem cells (MSCs) are currently being investigated as candidate cells for regenerative medicine approaches for the repair of damaged articular cartilage. For these cells to be used clinically, it is important to understand how they will react to the complex loading environment of a joint in vivo. In addition to investigating alternative cell sources, it is also important for the structure of tissue‐engineered constructs and the organization of cells within them to be developed and, if possible, improved. A custom built bioreactor was used to expose human MSCs to a combination of shear and compression loading. The MSCs were either evenly distributed throughout fibrin‐poly(ester‐urethane) scaffolds or asymmetrically seeded with a small proportion seeded on the surface of the scaffold. The effect of cell distribution on the production and deposition of cartilage‐like matrix in response to mechanical load mimicking in vivo joint loading was then investigated. The results show that asymmetrically seeding the scaffold led to markedly improved tissue development based on histologically detectable matrix deposition. Consideration of cell location, therefore, is an important aspect in the development of regenerative medicine approaches for cartilage repair. This is particularly relevant when considering the natural biomechanical environment of the joint in vivo and patient rehabilitation protocols. © 2016 The Authors Journal of Tissue Engineering and Regenerative Medicine Published by John Wiley & Sons Ltd.