The influence of tethered epidermal growth factor on connective tissue progenitor colony formation

Strategies to combine aspirated marrow cells with scaffolds to treat connective tissue defects are gaining increasing clinical attention and use. In situations such as large defects where initial survival and proliferation of transplanted connective tissue progenitors (CTPs) are limiting, therapeutic outcomes might be improved by using the scaffold to deliver growth factors that promote the early stages of cell function in the graft. Signaling by the epidermal growth factor receptor (EGFR) plays a role in cell survival and has been implicated in bone development and homeostasis. Providing epidermal growth factor (EGF) in a scaffold-tethered format may sustain local delivery and shift EGFR signaling to pro-survival modes compared to soluble ligand. We therefore examined the effect of tethered EGF on osteogenic colony formation from human bone marrow aspirates in the context of three different adhesion environments using a total of 39 donors. We found that tethered EGF, but not soluble EGF, increased the numbers of colonies formed regardless of adhesion background, and that tethered EGF did not impair early stages of osteogenic differentiation.National Institute of General Medical Sciences (U.S.) (Grant NIH RO1 AR42997)National Institute of General Medical Sciences (U.S.) (Grant NIH RO1 AG024980)National Institute of General Medical Sciences (U.S.) (Grant NIH RO1 GM59870)National Institute of General Medical Sciences (U.S.) (Grant NIH DE019523

Evaluation of Osteoconductive Scaffolds in the Canine Femoral Multi-Defect Model

Treatment of large segmental bone defects remains an unsolved clinical challenge, despite a wide array of existing bone graft materials. This project was designed to rapidly assess and compare promising biodegradable osteoconductive scaffolds for use in the systematic development of new bone regeneration methodologies that combine scaffolds, sources of osteogenic cells, and bioactive scaffold modifications. Promising biomaterials and scaffold fabrication methods were identified in laboratories at Rutgers, MIT, Integra Life Sciences, and Mayo Clinic. Scaffolds were fabricated from various materials, including poly(L-lactide-co-glycolide) (PLGA), poly(L-lactide-co-ɛ-caprolactone) (PLCL), tyrosine-derived polycarbonate (TyrPC), and poly(propylene fumarate) (PPF). Highly porous three-dimensional (3D) scaffolds were fabricated by 3D printing, laser stereolithography, or solvent casting followed by porogen leaching. The canine femoral multi-defect model was used to systematically compare scaffold performance and enable selection of the most promising substrate(s) on which to add cell sourcing options and bioactive surface modifications. Mineralized cancellous allograft (MCA) was used to provide a comparative reference to the current clinical standard for osteoconductive scaffolds. Percent bone volume within the defect was assessed 4 weeks after implantation using both MicroCT and limited histomorphometry. Bone formed at the periphery of all scaffolds with varying levels of radial ingrowth. MCA produced a rapid and advanced stage of bone formation and remodeling throughout the defect in 4 weeks, greatly exceeding the performance of all polymer scaffolds. Two scaffold constructs, TyrPC[subscript PL]/TCP and PPF4[subscript SLA]/HA[subscript PLGA Dip], proved to be significantly better than alternative PLGA and PLCL scaffolds, justifying further development. MCA remains the current standard for osteoconductive scaffolds.United States. Army Medical Research and Materiel Command (Armed Forces Institute of Regenerative Medicine)United States. Office of Naval ResearchUnited States. Air Force. Office of the Surgeon GeneralUnited States. NavyNational Institutes of Health (U.S.)United States. Veterans AdministrationCleveland Clinic Foundatio

Anisotropic Structure of the Order Parameter in FeSe0.45Te0.55 Revealed by Angle Resolved Specific Heat

The symmetry and structure of the superconducting gap in the Fe-based superconductors are the central issue for understanding these novel materials. So far the experimental data and theoretical models have been highly controversial. Some experiments favor two or more constant or nearly-constant gaps, others indicate strong anisotropy and yet others suggest gap zeros ("nodes"). Theoretical models also vary, suggesting that the absence or presence of the nodes depends quantitatively on the model parameters. An opinion that has gained substantial currency is that the gap structure, unlike all other known superconductors, including cuprates, may be different in different compounds within the same family. A unique method for addressing this issue, one of the very few methods that are bulk and angle-resolved, calls for measuring the electronic specific heat in a rotating magnetic field, as a function of field orientation with respect to the crystallographic axes. In this Communication we present the first such measurement for an Fe-based high-Tc superconductor (FeBSC). We observed a fourfold oscillation of the specific heat as a function of the in-plane magnetic field direction, which allowed us to identify the locations of the gap minima (or nodes) on the Fermi surface. Our results are consistent with the expectations of an extended s-wave model with a significant gap anisotropy on the electron pockets and the gap minima along the \Gamma M (or Fe-Fe bond) direction.Comment: 32 pages, 7 figure

Gap structure in the electron-doped Iron-Arsenide Superconductor Ba(Fe0.92Co0.08)2As2: low-temperature specific heat study

We report the field and temperature dependence of the low-temperature specific heat down to 400 mK and in magnetic fields up to 9 T of the electron-doped Ba(Fe0.92Co0.08)2As2 superconductor. Using the phonon specific heat obtained from pure BaFe2As2 we find the normal state Sommerfeld coefficient to be 18 mJ/mol.K^2 and a condensation energy of 1.27 J/mol. The temperature dependence of the electronic specific heat clearly indicate the presence of the low-energy excitations in the system. The magnetic field variation of field-induced specific heat cannot be described by single clean s- or d-wave models. Rather, the data require an anisotropic gap scenario which may or may not have nodes. We discuss the implications of these results.Comment: New Journal of Physics in press, 10 pages, 5 figure

Regulation of mammary gland branching morphogenesis by the extracellular matrix and its remodeling enzymes.

A considerable body of research indicates that mammary gland branching morphogenesis is dependent, in part, on the extracellular matrix (ECM), ECM-receptors, such as integrins and other ECM receptors, and ECM-degrading enzymes, including matrix metalloproteinases (MMPs) and their inhibitors, tissue inhibitors of metalloproteinases (TIMPs). There is some evidence that these ECM cues affect one or more of the following processes: cell survival, polarity, proliferation, differentiation, adhesion, and migration. Both three-dimensional culture models and genetic manipulations of the mouse mammary gland have been used to study the signaling pathways that affect these processes. However, the precise mechanisms of ECM-directed mammary morphogenesis are not well understood. Mammary morphogenesis involves epithelial 'invasion' of adipose tissue, a process akin to invasion by breast cancer cells, although the former is a highly regulated developmental process. How these morphogenic pathways are integrated in the normal gland and how they become dysregulated and subverted in the progression of breast cancer also remain largely unanswered questions

Heart Valve Tissue Engineering: Concepts, Approaches, Progress, and Challenges

Potential applications of tissue engineering in regenerative medicine range from structural tissues to organs with complex function. This review focuses on the engineering of heart valve tissue, a goal which involves a unique combination of biological, engineering, and technological hurdles. We emphasize basic concepts, approaches and methods, progress made, and remaining challenges. To provide a framework for understanding the enabling scientific principles, we first examine the elements and features of normal heart valve functional structure, biomechanics, development, maturation, remodeling, and response to injury. Following a discussion of the fundamental principles of tissue engineering applicable to heart valves, we examine three approaches to achieving the goal of an engineered tissue heart valve: (1) cell seeding of biodegradable synthetic scaffolds, (2) cell seeding of processed tissue scaffolds, and (3) in-vivo repopulation by circulating endogenous cells of implanted substrates without prior in-vitro cell seeding. Lastly, we analyze challenges to the field and suggest future directions for both preclinical and translational (clinical) studies that will be needed to address key regulatory issues for safety and efficacy of the application of tissue engineering and regenerative approaches to heart valves. Although modest progress has been made toward the goal of a clinically useful tissue engineered heart valve, further success and ultimate human benefit will be dependent upon advances in biodegradable polymers and other scaffolds, cellular manipulation, strategies for rebuilding the extracellular matrix, and techniques to characterize and potentially non-invasively assess the speed and quality of tissue healing and remodeling

Autologous bone marrow stem cell intralesional transplantation repairing bisphosphonate related osteonecrosis of the jaw

<p>Abstract</p> <p>Purpose</p> <p>Bisphosphonate - related osteonecrosis of the JAW (BRONJ) is a well known side effect of bisphosphonate therapies in oncologic and non oncologic patients. Since to date no definitive consensus has been reached on the treatment of BRONJ, novel strategies for the prevention, risk reduction and treatment need to be developed. We report a 75 year old woman with stage 3 BRONJ secondary to alendronate and pamidronate treatment of osteoporosis. The patient was unresponsive to recommended treatment of the disease, and her BRONJ was worsening. Since bone marrow stem cells are know as being multipotent and exhibit the potential for differentiation into different cells/tissue lineages, including cartilage, bone and other tissue, we performed autologous bone marrow stem cell transplantation into the BRONJ lesion of the patient.</p> <p>Methods</p> <p>Under local anesthesia a volume of 75 ml of bone marrow were harvested from the posterior superior iliac crest by aspiration into heparinized siringes. The cell suspension was concentrated, using Ficoll - Hypaque^®centrifugation procedures, in a final volume of 6 ml. Before the injection of stem cells into the osteonecrosis, the patient underwent surgical toilet, local anesthesia was done and spongostan was applied as a carrier of stem cells suspension in the bone cavity, then 4 ml of stem cells suspension and 1 ml of patient's activated platelet-rich plasma were injected in the lesion of BRONJ.</p> <p>Results</p> <p>A week later the residual spongostan was removed and two weeks later resolution of symptoms was obtained. Then the lesion improved with progressive superficialization of the mucosal layer and CT scan, performed 15 months later, shows improvement also of bone via concentric ossification: so complete healing of BRONJ (stage 0) was obtained in our patient, and 30 months later the patient is well and without signs of BRONJ.</p> <p>Conclusion</p> <p>To our knowledge this is the first case of BRONJ successfully treated with autologous stem cells transplantation with a complete response.</p

DNA Damage, Somatic Aneuploidy, and Malignant Sarcoma Susceptibility in Muscular Dystrophies

Albeit genetically highly heterogeneous, muscular dystrophies (MDs) share a convergent pathology leading to muscle wasting accompanied by proliferation of fibrous and fatty tissue, suggesting a common MD–pathomechanism. Here we show that mutations in muscular dystrophy genes (Dmd, Dysf, Capn3, Large) lead to the spontaneous formation of skeletal muscle-derived malignant tumors in mice, presenting as mixed rhabdomyo-, fibro-, and liposarcomas. Primary MD–gene defects and strain background strongly influence sarcoma incidence, latency, localization, and gender prevalence. Combined loss of dystrophin and dysferlin, as well as dystrophin and calpain-3, leads to accelerated tumor formation. Irrespective of the primary gene defects, all MD sarcomas share non-random genomic alterations including frequent losses of tumor suppressors (Cdkn2a, Nf1), amplification of oncogenes (Met, Jun), recurrent duplications of whole chromosomes 8 and 15, and DNA damage. Remarkably, these sarcoma-specific genetic lesions are already regularly present in skeletal muscles in aged MD mice even prior to sarcoma development. Accordingly, we show also that skeletal muscle from human muscular dystrophy patients is affected by gross genomic instability, represented by DNA double-strand breaks and age-related accumulation of aneusomies. These novel aspects of molecular pathologies common to muscular dystrophies and tumor biology will potentially influence the strategies to combat these diseases

Two-Photon Microscopy for Non-Invasive, Quantitative Monitoring of Stem Cell Differentiation

BACKGROUND: The engineering of functional tissues is a complex multi-stage process, the success of which depends on the careful control of culture conditions and ultimately tissue maturation. To enable the efficient optimization of tissue development protocols, techniques suitable for monitoring the effects of added stimuli and induced tissue changes are needed. METHODOLOGY/PRINCIPAL FINDINGS: Here, we present the quantitative use of two-photon excited fluorescence (TPEF) and second harmonic generation (SHG) as a noninvasive means to monitor the differentiation of human mesenchymal stem cells (hMSCs) using entirely endogenous sources of contrast. We demonstrate that the individual fluorescence contribution from the intrinsic cellular fluorophores NAD(P)H, flavoproteins and lipofuscin can be extracted from TPEF images and monitored dynamically from the same cell population over time. Using the redox ratio, calculated from the contributions of NAD(P)H and flavoproteins, we identify distinct patterns in the evolution of the metabolic activity of hMSCs maintained in either propagation, osteogenic or adipogenic differentiation media. The differentiation of these cells is mirrored by changes in cell morphology apparent in high resolution TPEF images and by the detection of collagen production via SHG imaging. Finally, we find dramatic increases in lipofuscin levels in hMSCs maintained at 20% oxygen vs. those in 5% oxygen, establishing the use of this chromophore as a potential biomarker for oxidative stress. CONCLUSIONS/SIGNIFICANCE: In this study we demonstrate that it is possible to monitor the metabolic activity, morphology, ECM production and oxidative stress of hMSCs in a non-invasive manner. This is accomplished using generally available multiphoton microscopy equipment and simple data analysis techniques, such that the method can widely adopted by laboratories with a diversity of comparable equipment. This method therefore represents a powerful tool, which enables researchers to monitor engineered tissues and optimize culture conditions in a near real time manner