Controlled Delivery of Sdf-1 Alpha and Igf-1: Cxcr4(+) Cell Recruitment and Functional Skeletal Muscle Recovery

Therapeutic delivery of regeneration-promoting biological factors directly to the site of injury has demonstrated its efficacy in various injury models. Several reports describe improved tissue regeneration following local injection of tissue specific growth factors, cytokines and chemokines. Evidence exists that combined cytokine/growth factor treatment is superior for optimizing tissue repair by targeting different aspects of the regeneration response. The purpose of this study was to evaluate the therapeutic potential of the controlled delivery of stromal cell-derived factor-1alpha (SDF-1 alpha) alone or in combination with insulin-like growth factor-I (SDF-1 alpha/IGF-I) for the treatment of tourniquet-induced ischemia/reperfusion injury (TK-I/R) of skeletal muscle. We hypothesized that SDF-1 alpha will promote sustained stem cell recruitment to the site of muscle injury, while IGF-I will induce progenitor cell differentiation to effectively restore muscle contractile function after TK-I/R injury while concurrently reducing apoptosis. Utilizing a novel poly-ethylene glycol PEGylated fibrin gel matrix (PEG-Fib), we incorporated SDF-1 alpha alone (PEG-Fib/SDF-1 alpha) or in combination with IGF-I (PEG-Fib/SDF-1 alpha/IGF-I) for controlled release at the site of acute muscle injury. Despite enhanced cell recruitment and revascularization of the regenerating muscle after SDF-1 alpha treatment, functional analysis showed no benefit from PEG-Fib/SDF-1 alpha therapy, while dual delivery of PEG-Fib/SDF-1 alpha/IGF-I resulted in IGF-I-mediated improvement of maximal force recovery and SDF-1 alpha-driven in vivo neovasculogenesis. Histological data supported functional data, as well as highlighted the important differences in the regeneration process among treatment groups. This study provides evidence that while revascularization may be necessary for maximizing muscle force recovery, without modulation of other effects of inflammation it is insufficient.Kinesiology and Health Educatio

Phenotypic Basis for Matrix Stiffness-Dependent Chemoresistance of Breast Cancer Cells to Doxorubicin

The persistence of drug resistant cell populations following chemotherapeutic treatment is a significant challenge in the clinical management of cancer. Resistant subpopulations arise via both cell intrinsic and extrinsic mechanisms. Extrinsic factors in the microenvironment, including neighboring cells, glycosaminoglycans, and fibrous proteins impact therapy response. Elevated levels of extracellular fibrous proteins are associated with tumor progression and cause the surrounding tissue to stiffen through changes in structure and composition of the extracellular matrix (ECM). We sought to determine how this progressively stiffening microenvironment affects the sensitivity of breast cancer cells to chemotherapeutic treatment. MDA-MB-231 triple negative breast carcinoma cells cultured in a 3D alginate-based hydrogel system displayed a stiffness-dependent response to the chemotherapeutic doxorubicin. MCF7 breast carcinoma cells cultured in the same conditions did not exhibit this stiffness-dependent resistance to the drug. This differential therapeutic response was coordinated with nuclear translocation of YAP, a marker of mesenchymal differentiation. The stiffness-dependent response was lost when cells were transferred from 3D to monolayer cultures, suggesting that endpoint ECM conditions largely govern the response to doxorubicin. To further examine this response, we utilized a platform capable of dynamic ECM stiffness modulation to allow for a change in matrix stiffness over time. We found that MDA-MB-231 cells have a stiffness-dependent resistance to doxorubicin and that duration of exposure to ECM stiffness is sufficient to modulate this response. These results indicate the need for additional tools to integrate mechanical stiffness with therapeutic response and inform decisions for more effective use of chemotherapeutics in the clinic

Mechanical properties of α-tricalcium phosphate-based bone cements incorporating regenerative biomaterials for filling bone defects exposed to low mechanical loads

Calcium phosphate-based cements with enhanced regenerative potential are promising biomaterials for the healing of bone defects. With a view to the use of such cements for low load bearing applications such as sinus augmentation or filling extraction sites, we have prepared α-tricalcium phosphate (α-TCP)-based bone cements including materials that we would expect to improve their regenerative potential, and describe the mechanical properities of the resulting formulations herein. Formulations incorporated α-TCP, hydroxyapatite, biopolymer-thickened wetting agents, sutures, and platelet poor plasma. The mechanical properties of the composites were composition dependent, and optimized formulations had clinically relevant mechanical properties. Such calcium phosphate-based cements have potential as replacements for cements such as those based on polymethylmethacrylate (PMMA)

Development of poly(propylene fumarate-co-ethylene glycol): An injectable, biodegradable implant for cardiovascular applications

A novel block copolymer consisting of poly(propylene fumarate), PPF, and poly(ethylene glycol), PEG, was fabricated and evaluated for use as a cardiovascular stent to prevent reclosure of the vessel lumen following balloon angioplasty. This copolymer has been fabricated in a block configuration with two to three homopolymer units in series through a transesterification reaction between the linear polyester and the terminal hydroxyl functionalities of the PEG. This material has been characterized in terms of structure and composition as well as thermal properties and solubility behavior. We described the preparation and bulk characterization of crosslinked P(PF-co-EG) hydrogels. The extent of the crosslinking reaction and the degree of swelling in aqueous solution were determined on several different copolymer formulations. Mechanical properties were evaluated and were shown to increase with increasing PPF molecular weight and decrease with increasing PEG content. The degradation behavior was examined in vitro at pH 7.4 and in vivo in a subcutaneous rat model, in terms of mass loss, dimensional changes, mechanical properties, morphology, and biocompatibility over a twelve week time course. The P(PF-co-EG) hydrogels demonstrated a pattern typical of bulk degradation. They retained at least a 20% of their initial ultimate tensile stress after three weeks with no apparent changes in morphology. Platelet adhesion and aggregation on P(PF-co-EG) hydrogels was examined under both static and flow conditions. We demonstrated a significant decrease in platelet attachment on the copolymer hydrogel films relative to PPF. In addition, there were also reductions in attachment resulting from an increase in PEG weight percent or molecular weight. The copolymer surfaces showed no thrombus formation or platelet spreading

In vivo ultrasound and photoacoustic monitoring of mesenchymal stem cells labeled with gold nanotracers.

Longitudinal monitoring of cells is required in order to understand the role of delivered stem cells in therapeutic neovascularization. However, there is not an imaging technique that is capable of quantitative, longitudinal assessment of stem cell behaviors with high spatial resolution and sufficient penetration depth. In this study, in vivo and in vitro experiments were performed to demonstrate the efficacy of ultrasound-guided photoacoustic (US/PA) imaging to monitor mesenchymal stem cells (MSCs) labeled with gold nanotracers (Au NTs). The Au NT labeled MSCs, injected intramuscularly in the lower limb of the Lewis rat, were detected and spatially resolved. Furthermore, our quantitative in vitro cell studies indicate that US/PA imaging is capable of high detection sensitivity (1×10⁴ cells/mL) of the Au NT labeled MSCs. Finally, Au NT labeled MSCs captured in the PEGylated fibrin gel system were imaged in vivo, as well as in vitro, over a one week time period, suggesting that longitudinal cell tracking using US/PA imaging is possible. Overall, Au NT labeling of MSCs and US/PA imaging can be an alternative approach in stem cell imaging capable of noninvasive, sensitive, quantitative, longitudinal assessment of stem cell behaviors with high spatial and temporal resolutions at sufficient depths

Laminar construct for tissue-engineered dermal equivalent

Compositions and methods for creating a laminar construct for tissue-engineered dermal equivalent are provided. One composition provided herein comprises a hydrogel matrix comprising two or more hydrogels layers and a population of stem cells. Associated methods are also provided.Board of Regents, University of Texas Syste

Design and Evaluation of Short Self-Assembling Depsipeptides as Bioactive and Biodegradable Hydrogels

Described herein is the design of a cell-adherent and degradable hydrogel. Our goal was to create a self-assembling, backbone ester-containing analogue of the cell adhesion motif, arginine–glycine–aspartic acid (RGD). Two depsipeptides containing Fmoc (<i>N</i>-(fluorenyl)-9-methoxycarbonyl), Fmoc-FR-Glc-D, and Fmoc-F-Glc-RGD (where “Glc” is glycolic acid) were designed based on the results of integrin-binding affinity and cell interaction analyses. Two candidate molecules were synthesized, and their gelation characteristics, degradation profiles, and ability to promote cell attachment were analyzed. We found that ester substitution within the RGD sequence significantly decreases the integrin-binding affinity and subsequent cell attachment, but when the ester moiety flanks the bioactive sequence, the molecule can maintain its integrin-binding function while permitting nonenzymatic hydrolytic degradation. A self-assembled Fmoc-F-Glc-RGD hydrogel showed steady, linear degradation over 60 days, and when mixed with Fmoc-diphenylalanine (Fmoc-FF) for improved mechanical stiffness, the depsipeptide gel exhibited improved cell attachment and viability. Though the currently designed depsipeptide has several inherent limitations, our results indicate the potential of depsipeptides as the basis for biologically functional and degradable self-assembling hydrogel materials

Mesenchymal Stem Cell Response to TGF-Beta 1 in Both 2D and 3D Environments

Smooth muscle cells (SMC) are critical in stabilizing developing vascular networks, and transforming growth factor beta 1 (TGF-beta 1) has been shown to promote SMC differentiation from stem cells. Previously, our lab has developed a chemically modified fibrin-based hydrogel that induces endothelial cell (EC) phenotype and network formation from human mesenchymal stem cells (hMSCs) without exogenous cytokines. Additionally, we have shown that this hydrogel system is capable of releasing growth factors in a controlled manner. In the present work, the effects of TGF-beta 1 on hMSCs in both monolayer and fibrin-based gel culture systems were demonstrated. The objective was to enhance SMC properties through TGF-beta 1 signaling for vessel stability while maintaining EC gene expression and morphology. Proliferation was decreased with higher TGF-beta 1 concentration in both monolayer and 3D gel cultures. EC genes were predominantly downregulated in the presence of TGF-beta 1 in monolayer cultures, while SMC genes were generally upregulated. In fibrin-based gels, several SMC genes were significantly upregulated at high concentrations of TGF-beta 1. Even at elevated TGF-beta 1 concentrations, no significant differences were seen in EC genes for hMSCs in gels compared to controls. Network formation and growth occurred in PEGylated fibrin gels loaded with TGF-beta 1 and were not significantly different from gels without loaded growth factor. Additionally, production of smooth muscle a-actin (SMA) was significantly increased in gels loaded with TGF-beta 1. These results demonstrate a simultaneous response of hMSCs to both the 3D biomatrix and cytokine signaling cues.American Heart AssociationNational Science FoundationCBET-0853996 ARRABiomedical Engineerin