Beyond osteogenesis: an in vitro comparison of the potentials of six bone morphogenetic proteins

Bone morphogenetic proteins (BMPs) other than the clinically available BMP-2 and BMP-7 may be useful for improving fracture healing through both increasing osteogenesis and creating a favorable healing environment by altering cytokine release by endogenous cells. Given the spectrum of potential applications for BMPs, the objective of this study was to evaluate various BMPs under a variety of conditions to provide further insight into their therapeutic capabilities. The alkaline phosphatase (ALP) activity of both C(2)C(12) and human adipose-derived stem cells (hASCs) was measured after exposure of increasing doses of recombinant human BMP-2, -4, -5, -6, -7, or -9 for 3 and 7 days. BMPs-2, -4, -5, -6, -7, and -9 were compared in terms of their ability to affect the release of stromal derived factor-1 (SDF-1), vascular endothelial growth factor (VEGF), and basic fibroblast growth factor (b-FGF) from human bone marrow stromal cells (hBMSCs). Gene expression of ALP, osteocalcin, SDF-1, VEGF, and b-FGF following shRNA-mediated knockdown of BMP-2 and BMP-6 in hBMSCs or human osteoblasts under osteogenic differentiation conditions was also evaluated. Collectively, BMPs-6 and -9 produced the greatest osteogenic differentiation of C(2)C(12) and hASCs as determined by ALP. The hBMSC secretion of SDF-1 was most affected by BMP-5, VEGF by BMP-4, and b-FGF by BMP-2. The knockdown of BMP-2 in BMSCs had no effect on any of the genes measured whereas BMP-6 knockdown in hBMSCs caused a significant increase in VEGF gene expression. BMP-2 and BMP-6 knockdown in human osteoblasts caused significant increases in VEGF gene expression and trends toward decreases in osteocalcin expression. These findings support efforts to study other BMPs as potential bone graft supplements, and to consider combined BMP delivery for promotion of multiple aspects of fracture healing

The family-specific α4-helix of the kinesin-13, MCAK, is critical to microtubule end recognition

Kinesins that influence the dynamics of microtubule growth and shrinkage require the ability to distinguish between the microtubule end and the microtubule lattice. The microtubule depolymerizing kinesin MCAK has been shown to specifically recognize the microtubule end. This ability is key to the action of MCAK in regulating microtubule dynamics. We show that the a4-helix of the motor domain is crucial to microtubule end recognition. Mutation of the residues K524, E525 and R528, which are located in the C-terminal half of the a4-helix, specifically disrupts the ability of MCAK to recognize the microtubule end. Mutation of these residues, which are conserved in the kinesin-13 family and discriminate members of this family from translocating kinesins, impairs the ability of MCAK to discriminate between the microtubule lattice and the microtubule end

Soluble factors from biofilms of wound pathogens modulate human bone marrow-derived stromal cell differentiation, migration, angiogenesis, and cytokine secretion

BACKGROUND: Chronic, non-healing wounds are often characterized by the persistence of bacteria within biofilms - aggregations of cells encased within a self-produced polysaccharide matrix. Biofilm bacteria exhibit unique characteristics from planktonic, or culture-grown, bacterial phenotype, including diminished responses to antimicrobial therapy and persistence against host immune responses. Mesenchymal stromal cells (MSCs) are host cells characterized by their multifunctional ability to undergo differentiation into multiple cell types and modulation of host-immune responses by secreting factors that promote wound healing. While these characteristics make MSCs an attractive therapeutic for wounds, these pro-healing activities may be differentially influenced in the context of an infection (i.e., biofilm related infections) within chronic wounds. Herein, we evaluated the effect of soluble factors derived from biofilms of clinical isolates of Staphylococcus aureus and Pseudomonas aeruginosa on the viability, differentiation, and paracrine activity of human MSCs to evaluate the influence of biofilms on MSC activity in vitro. RESULTS: Exposure of MSCs to biofilm-conditioned medias of S. aureus and P. aeruginosa resulted in reductions in cell viability, in part due to activation of apoptosis. Similarly, exposure to soluble factors from biofilms was also observed to diminish the migration ability of cells and to hinder multi-lineage differentiation of MSCs. In contrast to these findings, exposure of MSCs to soluble factors from biofilms resulted in significant increases in the release of paracrine factors involved in inflammation and wound healing. CONCLUSIONS: Collectively, these findings demonstrate that factors produced by biofilms can negatively impact the intrinsic properties of MSCs, in particular limiting the migratory and differentiation capacity of MSCs. Consequently, these studies suggest use/application of stem-cell therapies in the context of infection may have a limited therapeutic effect. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12866-015-0412-x) contains supplementary material, which is available to authorized users

The role of resveratrol on skeletal muscle cell differentiation and myotube hypertrophy during glucose restriction

Glucose restriction (GR) impairs muscle cell differentiation and evokes myotube atrophy. Resveratrol treatment in skeletal muscle cells improves inflammatory-induced reductions in skeletal muscle cell differentiation. We therefore hypothesised that resveratrol treatment would improve muscle cell differentiation and myotube hypertrophy in differentiating C2C12 myoblasts and mature myotubes during GR. Glucose restriction at 0.6 g/L (3.3 mM) blocked differentiation and myotube hypertrophy versus high-glucose (4.5 g/L or 25 mM) differentiation media (DM) conditions universally used for myoblast culture. Resveratrol (10 μM) treatment increased SIRT1 phosphorylation in DM conditions, yet did not improve differentiation when administered to differentiating myoblasts in GR conditions. Resveratrol did evoke increases in hypertrophy of mature myotubes under DM conditions with corresponding elevated Igf-I and Myhc7 gene expression, coding for the ‘slow’ type I MYHC protein isoform. Inhibition of SIRT1 via EX-527 administration (100 nM) also reduced myotube diameter and area in DM conditions and resulted in lower gene expression of Myhc 1, 2 and 4 coding for ‘intermediate’ and ‘faster’ IIx, IIa and IIb protein isoforms, respectively. Resveratrol treatment did not appear to modulate phosphorylation of energy-sensing protein AMPK or protein translation initiator P70S6K. Importantly, in mature myotubes, resveratrol treatment was able to ameliorate reduced myotube growth in GR conditions over an acute 24-h period, but not over 48–72 h. Overall, resveratrol evoked myotube hypertrophy in DM conditions while favouring ‘slower’ Myhc gene expression and acutely ameliorated impaired myotube growth observed during glucose restriction

Current status of molecularly imprinted polymers as alternatives to antibodies in sorbent assays

The literature on the use of molecularly imprinted polymers (MIPs) in antibody-like sorbent assay using radio, fluoro and enzyme-linked approaches is comprehensively reviewed, and their current status discussed. Although immunoassays are still commonly carried out using antibodies, recent developments have demonstrated that molecularly imprinted polymers can be viable alternatives. It is predicted that both traditional antibody-based and MIP sorbent assays will continue to develop in parallel, with each having superiority in certain areas

Mechanisms regulating skeletal muscle satellite cell cycle progression

The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file.Vita."December 2006"Thesis (Ph. D.) University of Missouri-Columbia 2006.[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI AT REQUEST OF AUTHOR.] Skeletal muscle fibers are multinucleated with each myonucleus capable of governing a finite cytoplasmic volume. In healthy skeletal muscle, myonuclear number decreases with muscle atrophy and fiber damage and increases with hypertrophy, to maintain a constant myonuclear to cytoplasmic ratio. Myonuclei are post-mitotic, therefore, the repair, regrowth, and hypertrophy of skeletal muscle relies on satellite cells, muscle precursor cells located between the basal lamina and plasmalemma of mature muscle fibers. Since decreased satellite cell proliferation may limit the regrowth of old skeletal muscle following atrophy or damage, it is important to understand the mechanisms that control satellite cell proliferation to enable the development of countermeasures to treat muscle atrophy that occurs with age. Adenoviral infection of primary satellite cells with the forkhead transcription factor FoxO3a decreased satellite cell cycle progression; this process occurred, in part, through increases in the promoter activity and protein levels of the cyclin dependent kinase (cdk) inhibitor p27superscript Kip1], without altering p21[superscript Waf/Cip1] or cyclin D1 -CDK 4/6 activity. Conversely, increases in satellite cell cycle progression following adenoviral infection of Sirt1 were associated with a decrease in the cyclin dependent kinase inhibitor p21[superscript Waf/Cip1], increased p27[superscript Kip1], and increased cyclin D1 -CDK4/6 activity. In summary, it is speculated that these studies suggest that increasing FoxO3a expression alters factors that would decrease cell cycle progression of primary satellite cells, and this inhibition is likely through the inhibition of cyclin E -CDK2 and/or cyclin A -CDK2 activities via increases in p27[superscript Kip1]. Also, increasing the expression of Sirt1 increases satellite cell cell cycle progression by decreasing the expression levels of p21[superscript Waf/Cip1]. These studies are important for the understanding of the mechanisms that regulate satellite cell proliferation, more specifically, these studies have contributed to the understanding of two molecules that control satellite cell proliferation, FoxO3a and Sirt1.Includes bibliographical reference

Microvascular Fragment Transplantation Improves Rat Dorsal Skin Flap Survival

BACKGROUND: The development of flap necrosis distally remains a concern during microsurgical flap transfers because, at least in part, of decreased perfusion. Microvascular fragments (MVFs) are microvessels isolated from adipose tissue that are capable of improving tissue perfusion in a variety of tissue defects. The aim of this study was to determine whether the transplantation of MVFs in a dorsal rat skin flap model can improve flap survival. METHODS: A 10 × 3 cm flap was raised in a cranial to caudal fashion on the dorsal side of 16 Lewis rats, with the caudal side remaining intact. The rats were equally divided into a treatment group (MVFs) and a control group (sterile saline). At the time of surgery, sterile saline with or without MVFs was injected directly into the flap. Microvessel density was determined after harvesting flap tissue by counting vessels that positively stained for Griffonia simplicifolia lectin I-isolectin B(4). Laser Doppler was used to measure blood flow before and after surgery and 7 and 14 days later. Flap survival was evaluated 7 and 14 days after surgery by evaluating the percentage of viable tissue of the flap with photodigital planimetry. RESULTS: Despite the lack of a significant difference in microvessel density and tissue perfusion, flap survival increased 6.4% (P < 0.05) in MVF-treated animals compared with controls. CONCLUSIONS: The use of MVFs may be a means to improve flap survival. Future studies are required to delineate mechanisms whereby this occurs and to further optimize their application