Ewing's Sarcoma of the Talus in an Adolescent Female: An Unusual Case Presentation With Review of Literature.

Ewing's sarcoma is an aggressive primary malignant bone tumor that affects long and flat bones and is commonly seen in children and adolescents. The involvement of the foot especially the talus is an extremely rare entity with less than 15 cases reported in the literature. The rarity and atypical symptoms often lead to delays in diagnosis affecting the prognosis and survival. We present a 14-year-old female with pain and swelling of the left ankle for 18 months. She was being treated previously for an ankle sprain and was later suspected of avascular necrosis of the talus, before presenting to us. Clinicoradiology-pathological workup confirmed the diagnosis of Ewing sarcoma of the talus. Further, the metastatic workup revealed multiple skeletal metastases at the time of diagnosis. The metastatic lesion of the right femur required prophylactic fixation, otherwise, the patient was treated with palliative chemotherapy and radiotherapy. Ewing's sarcoma of the foot involving the talus is extremely rare and is a commonly misdiagnosed entity, affecting the overall prognosis of the patient. A high index of suspicion and a multidisciplinary approach is imperative for its early diagnosis and definitive management.

Retrospective comparison of outcomes of laparoscopic pyeloplasty using barbed suture versus nonbarbed suture: A single-center experience.

INTRODUCTION: laparoscopic pyeloplasty is an important tool in urology armamentarium. The most important & also the difficult part of this surgery is intracorporial suturing and knotting. There are only a few reports of knotless Barbed sutures for upper tract reconstruction. We report the comparative outcomes of Laparoscopic Pyeloplasty with barbed suture vs non barbed sutures used for uretero-pelvic anastomosis. MATERIALS AND METHODS: We retrospectively reviewed patients' records that underwent Laparoscopic pyeloplasty at our Institution from January 2013 to May 2014. Total 37 patients were underwent LP in this period. Whole of the procedure was same as conventional LP except suture material. 3-0 barbed suture was used in 21 patients and 3-0 vicryl used in 16 patients for uretero-pelvic anastomosis and continuous suturing technique was employed. Patients' demographics, total operative time, intracorporial suturing time, post operative complications, symptoms & renal isotope scan were recorded. RESULTS: Average total operative time was significantly less in barbed suture group vs vicryl group (162 vs 208 minutes) (p=0.0811). Average time taken for intracorporial suturing was 31.2 minutes vs 70 minutes (p=0.0576). 1 patient developed post operative urine leak which persisted for 5 days in barbed group (4.76 %) vs no leak in vicryl group. Most common complication was UTI presented in 2 patients (9.5 %) vs 2 in vicryl (12.5%). JJ stent was removed at 4 weeks. Median follow up was 3 months with 7 patients lost to follow up. None of the patients found to have obstructive drainage or deterioration of split function on follow up isotope renogram at 3 months. CONCLUSIONS: In this study, Laparoscopic pyeloplasty with barbed suture has acceptable outcome when compared to conventional non barbed suture on short term basis. Laparoscopic Pyeloplasty with barbed suture can potentially become the standard approach in near future.

Development of Mithramycin Analogues with Increased Selectivity toward ETS Transcription Factor Expressing Cancers.

Mithramycin A (1) was identified as the top potential inhibitor of the aberrant ETS transcription factor EWS-FLI1, which causes Ewing sarcoma. Unfortunately, 1 has a narrow therapeutic window, compelling us to seek less toxic and more selective analogues. Here, we used MTMSA (2) to generate analogues via peptide coupling and fragment-based drug development strategies. Cytotoxicity assays in ETS and non-ETS dependent cell lines identified two dipeptide analogues, 60 and 61, with 19.1- and 15.6-fold selectivity, respectively, compared to 1.5-fold for 1. Importantly, the cytotoxicity of 60 and 61 is <100 nM in ETS cells. Molecular assays demonstrated the inhibitory capacity of these analogues against EWS-FLI1 mediated transcription in Ewing sarcoma. Structural analysis shows that positioning the tryptophan residue in a distal position improves selectivity, presumably via interaction with the ETS transcription factor. Thus, these analogues may present new ways to target transcription factors for clinical use.

TGF-ß1/CD105 signaling controls vascular network formation within growth factor sequestering hyaluronic acid hydrogels.

Cell-based strategies for the treatment of ischemic diseases are at the forefront of tissue engineering and regenerative medicine. Cell therapies purportedly can play a key role in the neovascularization of ischemic tissue; however, low survival and poor cell engraftment with the host vasculature following implantation limits their potential to treat ischemic diseases. To overcome these limitations, we previously developed a growth factor sequestering hyaluronic acid (HyA)-based hydrogel that enhanced transplanted mouse cardiosphere-derived cell survival and formation of vasculature that anastomosed with host vessels. In this work, we examined the mechanism by which HyA hydrogels presenting transforming growth factor beta-1 (TGF-ß1) promoted proliferation of more clinically relevant human cardiosphere-derived cells (hCDC), and their formation of vascular-like networks in vitro. We observed hCDC proliferation and enhanced formation of vascular-like networks occurred in the presence of TGF-ß1. Furthermore, production of nitric oxide (NO), VEGF, and a host of angiogenic factors were increased in the presence of TGF-ß1. This response was dependent on the co-activity of CD105 (Endoglin) with the TGF-ßR2 receptor, demonstrating its role in the process of angiogenic differentiation and vascular organization of hCDC. These results demonstrated that hCDC form vascular-like networks in vitro, and that the induction of vascular networks by hCDC within growth factor sequestering HyA hydrogels was mediated by TGF-ß1/CD105 signaling.

A Bioengineering Approach to Myopia Control Tested in a Guinea Pig Model.

Purpose: To investigate the biocompatibility of an injectable hydrogel and its ability to control myopia progression in guinea pigs. Methods: The study used a hydrogel synthesized from acrylated hyaluronic acid with a conjugated cell-binding peptide and enzymatically degradable crosslinker. Seven-day-old guinea pigs were first form deprived (FD) with diffusers for 1 week. One group was kept as an FD-only control; two groups received a sub-Tenon's capsule injection of either hydrogel or buffer (sham surgery) at the posterior pole of the eye. Form deprivation treatments were then continued for 3 additional weeks. Treatment effects were evaluated in terms of ocular axial length and refractive error. Safety was evaluated via intraocular pressure (IOP), visual acuity, flash electroretinograms (ERG), and histology. Results: Both hydrogel and sham surgery groups showed significantly reduced axial elongation and myopia progression compared to the FD-only group. For axial lengths, net changes in interocular difference (treated minus control) were 0.04 ± 0.06, 0.02 ± 0.09, and 0.24 ± 0.08 mm for hydrogel, sham, and FD-only groups, respectively (P = 0.0006). Intraocular pressures, visual acuities, and ERGs of treated eyes were not significantly different from contralateral controls. Extensive cell migration into the implants was evident. Both surgery groups showed noticeable Tenon's capsule thickening. Conclusions: Sub-Tenon's capsule injections of both hydrogel and buffer inhibited myopia progression, with no adverse effects on ocular health. The latter unexpected effect warrants further investigation as a potential novel myopia control therapy. That the hydrogel implant supported significant cell infiltration offers further proof of its biocompatibility, with potential application as a tool for drug and cell delivery.

Rapid generation of hydrogen peroxide contributes to the complex cell death induction by the angucycline antibiotic landomycin E.

Landomycin E (LE) is an angucycline antibiotic produced by Streptomyces globisporus. Previously, we have shown a broad anticancer activity of LE which is, in contrast to the structurally related and clinically used anthracycline doxorubicin (Dx), only mildly affected by multidrug resistance-mediated drug efflux. In the present study, cellular and molecular mechanisms underlying the anticancer activity of landomycin E towards Jurkat T-cell leukemia cells were dissected focusing on the involvement of radical oxygen species (ROS). LE-induced apoptosis distinctly differed in several aspects from the one induced by Dx. Rapid generation of both extracellular and cell-derived hydrogen peroxide already at one hour drug exposure was observed in case of LE but not found before 24h for Dx. In contrast, Dx but not LE induced production of superoxide radicals. Mitochondrial damage, as revealed by JC-1 staining, was weakly enhanced already at 3h LE treatment and increased significantly with time. Accordingly, activation of the intrinsic apoptosis pathway initiator caspase-9 was not detectable before 12h exposure. In contrast, cleavage of the down-stream caspase substrate PARP-1 was clearly induced already at the three hour time point. Out of all caspases tested, only activation of effector caspase-7 was induced at this early time points paralleling the LE-induced oxidative burst. Accordingly, this massive cleavage of caspase-7 at early time points was inhibitable by the radical scavenger N-acetylcysteine (NAC). Additionally, only simultaneous inhibition of multiple caspases reduced LE-induced apoptosis. Specific scavengers of both H2O2 and OH• effectively decreased LE-induced ROS production, but only partially inhibited LE-induced apoptosis. In contrast, NAC efficiently blocked both parameters. Summarizing, rapid H2O2 generation and a complex caspase activation pattern contribute to the antileukemic effects of LE. As superoxide generation is considered as the main cardiotoxic mechanism of Dx, LE might represent a better tolerable drug candidate for further (pre)clinical development.

Multivalent hyaluronic acid bioconjugates improve sFlt-1 activity in vitro.

Anti-VEGF drugs that are used in conjunction with laser ablation to treat patients with diabetic retinopathy suffer from short half-lives in the vitreous of the eye resulting in the need for frequent intravitreal injections. To improve the intravitreal half-life of anti-VEGF drugs, such as the VEGF decoy receptor sFlt-1, we developed multivalent bioconjugates of sFlt-1 grafted to linear hyaluronic acid (HyA) chains termed mvsFlt. Using size exclusion chromatography with multiangle light scattering (SEC-MALS), SDS-PAGE, and dynamic light scattering (DLS), we characterized the mvsFlt with a focus on the molecular weight contribution of protein and HyA components to the overall bioconjugate size. We found that mvsFlt activity was independent of HyA conjugation using a sandwich ELISA and in vitro angiogenesis assays including cell survival, migration and tube formation. Using an in vitro model of the vitreous with crosslinked HyA gels, we demonstrated that larger mvsFlt bioconjugates showed slowed release and mobility in these hydrogels compared to low molecular weight mvsFlt and unconjugated sFlt-1. Finally, we used an enzyme specific to sFlt-1 to show that conjugation to HyA shields sFlt-1 from protein degradation. Taken together, our findings suggest that mvsFlt bioconjugates retain VEGF binding affinity, shield sFlt-1 from enzymatic degradation, and their movement in hydrogel networks (in vitro model of the vitreous) is controlled by both bioconjugate size and hydrogel network mesh size. These results suggest that a strategy of multivalent conjugation could substantially improve drug residence time in the eye and potentially improve therapeutics for the treatment of diabetic retinopathy.

Matrix metalloproteinase-13 mediated degradation of hyaluronic acid-based matrices orchestrates stem cell engraftment through vascular integration.

A critical design parameter for the function of synthetic extracellular matrices is to synchronize the gradual cell-mediated degradation of the matrix with the endogenous secretion of natural extracellular matrix (ECM) (e.g., creeping substitution). In hyaluronic acid (HyA)-based hydrogel matrices, we have investigated the effects of peptide crosslinkers with different matrix metalloproteinases (MMP) sensitivities on network degradation and neovascularization in vivo. The HyA hydrogel matrices consisted of cell adhesive peptides, heparin for both the presentation of exogenous and sequestration of endogenously synthesized growth factors, and MMP cleavable peptide linkages (i.e., QPQGLAK, GPLGMHGK, and GPLGLSLGK). Sca1(+)/CD45(-)/CD34(+)/CD44(+) cardiac progenitor cells (CPCs) cultured in the matrices with the slowly degradable QPQGLAK hydrogels supported the highest production of MMP-2, MMP-9, MMP-13, VEGF165, and a range of angiogenesis related proteins. Hydrogels with QPQGLAK crosslinks supported prolonged retention of these proteins via heparin within the matrix, stimulating rapid vascular development, and anastomosis with the host vasculature when implanted in the murine hindlimb.

Matrix-Assisted Transplantation of Functional Beige Adipose Tissue.

Novel, clinically relevant, approaches to shift energy balance are urgently needed to combat metabolic disorders such as obesity and diabetes. One promising approach has been the expansion of brown adipose tissues that express uncoupling protein (UCP) 1 and thus can uncouple mitochondrial respiration from ATP synthesis. While expansion of UCP1-expressing adipose depots may be achieved in rodents via genetic and pharmacological manipulations or the transplantation of brown fat depots, these methods are difficult to use for human clinical intervention. We present a novel cell scaffold technology optimized to establish functional brown fat-like depots in vivo. We adapted the biophysical properties of hyaluronic acid-based hydrogels to support the differentiation of white adipose tissue-derived multipotent stem cells (ADMSCs) into lipid-accumulating, UCP1-expressing beige adipose tissue. Subcutaneous implantation of ADMSCs within optimized hydrogels resulted in the establishment of distinct UCP1-expressing implants that successfully attracted host vasculature and persisted for several weeks. Importantly, implant recipients demonstrated elevated core body temperature during cold challenges, enhanced respiration rates, improved glucose homeostasis, and reduced weight gain, demonstrating the therapeutic merit of this highly translatable approach. This novel approach is the first truly clinically translatable system to unlock the therapeutic potential of brown fat-like tissue expansion.

Enhanced survival and engraftment of transplanted stem cells using growth factor sequestering hydrogels.

We have generated a bioinspired tunable system of hyaluronic acid (HyA)-based hydrogels for Matrix-Assisted Cell Transplantation (MACT). With this material, we have independently evaluated matrix parameters such as adhesion peptide density, mechanical properties, and growth factor sequestering capacity, to engineer an environment that imbues donor cells with a milieu that promotes survival and engraftment with host tissues after transplantation. Using a versatile population of Sca-1(+)/CD45(-) cardiac progenitor cells (CPCs), we demonstrated that the addition of heparin in the HyA hydrogels was necessary to coordinate the presentation of TGFß1 and to support the trophic functions of the CPCs via endothelial cell differentiation and vascular like tubular network formation. Presentation of exogenous TGFß1 by binding with heparin improved differentiated CPC function by sequestering additional endogenously-produced angiogenic factors. Finally, we demonstrated that TGFß1 and heparin-containing HyA hydrogels can promote CPC survival when implanted subcutaneously into murine hind-limbs and encouraged their participation in the ensuing neovascular response, which included blood vessels that had anastomosed with the host's blood vessels.

Controlling osteogenic stem cell differentiation via soft bioinspired hydrogels.

Osteogenic differentiation of human mesenchymal stem cells (hMSCs) is guided by various physical and biochemical factors. Among these factors, modulus (i.e., rigidiy) of the ECM has gained significant attention as a physical osteoinductive signal that can contribute to endochondral ossification of a cartilaginous skeletal template. However, MSCs also participate in intramembranous bone formation, which occurs de novo from within or on a more compliant tissue environment. To further understand the role of the matrix interactions in this process, we evaluated osteogenic differentiation of hMSCs cultured on low moduli (102, 390 or 970 Pa) poly(N-isopropylacrylamide) (p(NIPAAm)) based semi-interpenetrating networks (sIPN) modified with the integrin engaging peptide bsp-RGD(15) (0, 105 or 210 µM). Cell adhesion, proliferation, and osteogenic differentiation of hMSCs, as measured by alkaline phosphatase (ALP), runt-related transcription factor 2 (RUNX2), bone sialoprotein-2 (iBSP), and osteocalcien (OCN) protein expression, was highest on substrates with the highest modulus and peptide concentrations. However, within this range of substrate stiffness, many osteogenic cellular functions were enhanced by increasing either the modulus or the peptide density. These findings suggest that within a compliant and low modulus substrate, a high affinity adhesive ligand serves as a substitute for a rigid matrix to foster osteogenic differentiation.

Integrin-mediated adhesion and proliferation of human MSCs elicited by a hydroxyproline-lacking, collagen-like peptide.

In this study, we evaluated the competence of a rationally designed collagen-like peptide (CLP-Cys) sequence - containing the minimal essential Glycine-Glutamic acid-Arginine (GER) triplet but lacking the hydroxyproline residue - for supporting human mesenchymal stem cell (hMSC) adhesion, spreading and proliferation. Cellular responses to the CLP-Cys sequence were analyzed by conjugating the peptide to two different substrates - a hard, planar glass surface and a soft hyaluronic acid (HA) particle-based hydrogel. Integrin-mediated cell spreading and adhesion were observed for hMSCs cultivated on the CLP-Cys functionalized surfaces, whereas on control surfaces lacking the peptide motif, cells either did not adhere or maintained a round morphology. On the glass surface, CLP-Cys-mediated spreading led to the formation of extended and well developed stress fibers composed of F-actin bundles and focal adhesion complexes while on the soft gel surface, less cytoskeletal reorganization organization was observed. The hMSCs proliferated significantly on the surfaces presenting CLP-Cys, compared to the control surfaces lacking CLP-Cys. Competitive binding assay employing soluble CLP-Cys revealed a dose-dependent inhibition of hMSC adhesion to the CLP-Cys-presenting surfaces. Blocking the α(2)ß(1) receptor on hMSC also resulted in a reduction of cell adhesion on both types of CLP-Cys surfaces, confirming the affinity of CLP-Cys to α(2)ß(1) receptors. These results established the competence of the hydroxyproline-free CLP-Cys for eliciting integrin-mediated cellular responses including adhesion, spreading and proliferation. Thus, CLP-Cys-modified HA hydrogels are attractive candidates as bioactive scaffolds for tissue engineering applications.

Heparin-decorated, hyaluronic acid-based hydrogel particles for the controlled release of bone morphogenetic protein 2.

We are interested in developing hydrophilic particulate systems that are capable of sequestering growth factors, regulating their release and potentiating their biological functions. To this end heparin (HP)-decorated, hyaluronic acid (HA)-based hydrogel particles (HGPs) were synthesized using an inverse emulsion polymerization technique employing divinyl sulfone as the crosslinker. By varying the feed composition of the aqueous phase the amount of HP integrated in the particles can be systematically tuned. The resulting microscopic particles are spherical in shape and contain nanosized pores suitable for growth factor encapsulation. The covalently immobilized HP retained its ability to bind bone morphogenetic protein-2 (BMP-2) specifically, and its release kinetics can be adjusted by tuning the particle composition. Compared with pure HA particles the hybrid HA/HP HGPs show a higher BMP-2 loading capacity. While BMP-2 was released from HA HGPs with a significant initial burst, a near zero order release kinetics was observed from HA/HP hybrid particles with an optimized heparin content of 0.55 µg per mg HGPs. The ability of HA/HP hybrid particles to present BMP-2 in a controlled manner, combined with the innate bioactivity of HA, induced robust and consistent chondrogenic differentiation of murine mesenchymal stem cells, as shown by up-regulation of the mRNA levels of chondrogenic markers and the production of cartilage-specific extracellular matrix components. The simplicity of the particle synthesis, combined with the defined biological activities of the constituent building blocks, renders the HP-decorated, HA-based hydrogel particle system an attractive candidate for the sustained release of BMP-2, possibly for cartilage repair and regeneration.

Controlling the adhesion and differentiation of mesenchymal stem cells using hyaluronic acid-based, doubly crosslinked networks.

We have created hyaluronic acid (HA)-based, cell-adhesive hydrogels that direct the initial attachment and the subsequent differentiation of human mesenchymal stem cells (MSCs) into pre-osteoblasts without osteogenic supplements. HA-based hydrogel particles (HGPs) with an average diameter of 5-6 µm containing an estimated 2.2 wt% gelatin (gHGPs) were synthesized by covalent immobilization of gelatin to HA HGPs prepared via an inverse emulsion polymerization technique. Separately, a photocrosslinkable HA macromer (HAGMA) was synthesized by chemical modification of HA with glycidyl methacrylate (GMA). Doubly crosslinked networks (DXNs) were engineered by embedding gHGPs in a secondary network established by HAGMA at a particle concentration of 2.5 wt%. The resultant composite gels, designated as HA-gHGP, have an average compressive modulus of 21 kPa, and are non-toxic to the cultured MSCs. MSCs readily attached to these gels, exhibiting an early stage of stress fiber assembly 3 h post seeding. By day 7, stellate-shaped cells with extended filopodia were found on HA-gHGP gels. Moreover, cells had migrated deep into the matrix, forming a three dimensional, branched and interconnected cell community. Conversely, MSCs on the control gels lacking gelatin moieties formed isolated spheroids with rounded cell morphology. After 28 days of culture on HA-gHGP, Type I collagen production and mineral deposition were detected in the absence of osteogenic supplements, suggesting induction of osteogenic differentiation. In contrast, cells on the control gels expressed markers for adipogenesis. Overall, the HA-gHGP composite matrix has great promise for directing the osteogenic differentiation of MSCs by providing an adaptable environment through the spatial presentation of cell-adhesive modules.

Hyaluronic acid-based hydrogels as 3D matrices for in vitro evaluation of chemotherapeutic drugs using poorly adherent prostate cancer cells.

The current investigation aimed to develop a biomimetic, three-dimensional (3D) culture system for poorly adherent bone metastatic prostate cancer cells (C4-2B) for use as an in vitro platform for anti-cancer drug screening. To this end, hyaluronic acid (HA) derivatives carrying complementary aldehyde (HAALD) and hydrazide (HAADH) groups were synthesized and characterized. In situ encapsulation of C4-2B cells was achieved by simple mixing of HAALD and HAADH in the presence of the cells. Unlike two-dimensional (2D) monolayer culture in which cells adopt an atypical spread morphology, cells residing in the HA matrix formed distinct clustered structures which grew and merged, reminiscent of real tumors. Anti-cancer drugs added to the media surrounding the cell/gel construct diffused into the gel and killed the embedded cells. The HA hydrogel system was used successfully to test the efficacy of anti-cancer drugs including camptothecin, docetaxel, and rapamycin, alone and in combination, including specificity, dose and time responses. Responses of cells to anti-neoplastics differed between the 3D HA hydrogel and 2D monolayer systems. We suggest that the data obtained from 3D HA systems is superior to that from conventional 2D monolayers as the 3D system better reflects the bone metastatic microenvironment of the cancer cells.