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Endovascular deployment of drug-coated balloons (DCB) is an emerging strategy for the revascularization of arterial disease. Randomized clinical trials have demonstrated DCB effectiveness, but a recent meta-analysis reported increased mortality risk in humans with use of DCBs containing the common antiproliferative drug paclitaxel. While many factors could have contributed to adverse outcomes, current DCB designs have poor drug delivery efficiency, risk of systemic toxicity, and limited potential to retain therapeutic drug concentrations within the arterial wall following the procedure. Our study focuses on developing a strategy to enhance acute drug transfer from the balloon to the arterial wall over the short procedural window (∼30-120 s). We employed ultraviolet-ozone plasma (UVO) treatment to increase the hydrophilicity of a prototypical balloon material (Nylon-12) and subsequently applied a urea-paclitaxel coating previously shown to undergo favorable adhesive interactions with the arterial wall under simulated ex-vivo deployment. A series of assays were performed to characterize our experimental DCBs in terms of UVO-induced alterations in balloon surface hydrophobicity, formed coating microstructure, coating stability, and acute drug transfer to the arterial wall. Obtained results suggest that the UVO-based surface modification of angioplasty balloons is a promising design strategy and highlights the critical role of coating microstructure in determining the drug transfer efficiency in DCB therapy.

Targeted therapies have changed the treatment of cancer, giving new hope to many patients in recent years. The shortcomings of targeted therapies including acquired resistance, limited susceptible patients, high cost, and high toxicities, have led to the necessity of combining these therapies with other targeted or chemotherapeutic treatments. Natural products are uniquely capable of synergizing with targeted and non-targeted anticancer regimens due to their ability to affect multiple cellular pathways simultaneously. Compounds which provide an additive effect to the often combined immune therapies and cytotoxic chemotherapies, are exceedingly rare. These compounds would however provide a strengthening bridge between the two treatment modalities, increasing their effectiveness and improving patient prognoses. In this study, 7- epi -clusianone was investigated for its anticancer properties. While previous studies have suggested clusianone and its conformational isomers, including 7- epi -clusianone, are chemotherapeutic, few cancer types have been demonstrated to exhibit sensitivity to these compounds and little is known about the mechanism. In this study, 7- epi -clusianone was shown to inhibit the growth of 60 cancer cell types and induce significant cell death in 25 cancer cell lines, while simultaneously modulating the immune system, inhibiting angiogenesis, and inhibiting cancer cell invasion, making it a promising lead compound for cancer drug discovery.

Resveratrol (RSV) and nicotinamide (NAM) have garnered considerable attention due to their anti-inflammatory and anti-aging properties. NAM is a transient inhibitor of class III histone deacetylase SIRTs (silent mating type information regulation 2 homologs) and SIRT1 is an inhibitor of poly-ADP-ribose polymerase-1 (PARP1). The debate on the relationship between RSV and SIRT1 has precluded the use of RSV as a therapeutic drug. Recent work demonstrated that RSV facilitates tyrosyl-tRNA synthetase (TyrRS)-dependent activation of PARP1. Moreover, treatment with NAM is sufficient to facilitate the nuclear localization of TyrRS that activates PARP1. RSV and NAM have emerged as potent agonists of PARP1 through inhibition of SIRT1. In this study, we evaluated the effects of RSV and NAM on pro-inflammatory macrophages. Our results demonstrate that treatment with either RSV or NAM attenuates the expression of pro-inflammatory markers. Strikingly, the combination of RSV with NAM, exerts additive effects on PARP1 activation. Consistently, treatment with PARP1 inhibitor antagonized the anti-inflammatory effect of both RSV and NAM. For the first time, we report the ability of NAM to augment PARP1 activation, induced by RSV, and its associated anti-inflammatory effects mediated through the induction of BCL6 with the concomitant down regulation of COX-2.

Targeted therapies have become the focus of much of the cancer therapy research conducted in the United States. While these therapies have made vast improvements in the treatment of cancer, their results have been somewhat disappointing due to acquired resistances, high cost, and limited populations of susceptible patients. As a result, targeted therapeutics are often combined with other targeted therapeutics or chemotherapies. Compounds which target more than one cancer related pathway are rare, but have the potential to synergize multiple components of therapeutic cocktails. Natural products, as opposed to targeted therapies, typically interact with multiple cellular targets simultaneously, making them a potential source of synergistic cancer treatments. In this study, a rare natural product, deacetylnemorone, was shown to inhibit cell growth in a broad spectrum of cancer cell lines, selectively induce cell death in melanoma cells, and inhibit angiogenesis and invasion. Combined, these results demonstrate that deacetylnemorone affects multiple cancer-related targets associated with tumor growth, drug resistance, and metastasis. Thus, the multi-targeting natural product, deacetylnemorone, has the potential to enhance the efficacy of current cancer treatments as well as reduce commonly acquired treatment resistance., Competing Interests: The authors have declared that no competing interests exist.

The physiology of wound healing is dependent on the crosstalk between inflammatory mediators and cellular components of skin regeneration including fibroblasts and endothelial cells. Therefore, strategies to promote healing must regulate this crosstalk to achieve maximum efficacy. In light of the remarkable potential of natural compounds to target multiple signaling mechanisms, this study aims to demonstrate the potential of hypermongone C, a polycyclic polyprenylated acylphloroglucinol (PPAP), to accelerate wound closure by concurrently enhancing fibroblast proliferation and migration, promoting angiogenesis, and suppressing pro-inflammatory cytokines. This compound belongs to a family of plants ( Hypericum ) that traditionally have been used to treat injuries. Nevertheless, the exact biological evidence to support the claims is still missing. The results were obtained using a traditional model of cell scratch assay and endothelial cell tube formation, combined with the analysis of protein and gene expression by macrophages. In summary, the data suggest that hypermongone C is a multi-targeting therapeutic natural compound for the promotion of tissue repair and the regulation of inflammation.

Cell polarity, the asymmetric distribution of proteins, organelles, and cytoskeleton, plays an important role in development, homeostasis, and disease. Understanding the mechanisms that govern cell polarity is critical for creating strategies to treat developmental defects, accelerate tissue regeneration, and hinder cancer progression. This review focuses on the role of cell polarity in a number of physiological processes, including asymmetric division, cell migration, immune response mediated by T lymphocytes, and cancer progression and metastasis, and highlights microfabrication techniques to systematically parse the role of microenvironmental cues in the regulation of cell polarity.

Human stem cells hold significant potential for the treatment of various diseases. However, their use as a therapy is hampered because of limited understanding of the mechanisms by which they respond to environmental stimuli. Efforts to understand extracellular biophysical cues have demonstrated the critical roles of geometrical and mechanical signals in determining the fate of stem cells. The goal of this study was to explore the interplay between cell polarity and matrix stiffness in stem cell lineage specification. We hypothesize that confining cells to asymmetric extracellular matrix islands will impart polarity at a single-cell level and will interact with mechanical signals to define the lineage of stem cells. To test these hypotheses, we employed microcontact printing to create patterned symmetric and asymmetric hydrogel islands of soft and hard surface stiffness. Human mesenchymal stem cells (hMSCs) were confined to these islands at the single-cell level and given the ability to differentiate along adipogenic or osteogenic routes. Our results demonstrated that cell polarity defines the lineage specification of hMSCs only on islands with low stiffness. Insight gained from this study provides a rational basis for designing stem cell cultures to enhance tissue engineering and regenerative medicine strategies.

Wound healing is a complex physiological process that is controlled by a well-orchestrated cascade of interdependent biochemical and cellular events, which has spurred the development of therapeutics that simultaneously target these active cellular constituents. We assessed the potential of Parrotia persica (Hamamelidaceae) in wound repair by analyzing the regenerative effects of its two main phenolic compounds, myricetin-3- O -β-rhamnoside and chlorogenic acid. To accomplish this, we performed phytochemical profiling and characterized the chemical structure of pure compounds isolated from P. persica , followed by an analysis of the biological effects of myricetin-3- O -β-rhamnoside and chlorogenic acid on three cell types, including keratinocytes, fibroblasts, and endothelial cells. Myricetin-3- O -β-rhamnoside and chlorogenic acid exhibited complementary pro-healing properties. The percentage of keratinocyte wound closure as measured by a scratch assay was four fold faster in the presence of 10 µg/mL chlorogenic acid, as compared to the negative control. On the other hand, myricetin-3- O -β-rhamnoside at 10 µg/mL was more effective in promoting fibroblast migration, demonstrating a two-fold higher rate of closure compared to the negative control group. Both compounds enhanced the capillary-like tube formation of endothelial cells in an in vitro angiogenesis assay. Our results altogether delineate the potential to synergistically accelerate the fibroblastic and remodelling phases of wound repair by administering appropriate amounts of myricetin-3- O -β-rhamnoside and chlorogenic acid., Competing Interests: The authors declare no conflicts of interest.

The cancer stem cell hypothesis has been used to explain many cancer complications resulting in poor patient outcomes including induced drug resistance, metastases to distant organs, and tumor recurrence. While the validity of the cancer stem cell model continues to be the cause of much scientific debate, a number of putative cancer stem cell markers have been identified making studies concerning the targeting of cancer stem cells possible. In this review, a number of identifying properties of cancer stem cells have been outlined including properties contributing to the drug resistance and metastatic potential commonly observed in supposed cancer stem cells. Due to cancer stem cells' numerous survival mechanisms, the diversity of cancer stem cell markers between cancer types and tissues, and the prevalence of cancer stem cell markers among healthy stem and somatic cells, it is likely that currently utilized treatments will continue to fail to eradicate cancer stem cells. The successful treatment of cancer stem cells will rely upon the development of anti-neoplastic drugs capable of influencing many cellular mechanisms simultaneously in order to prevent the survival of this evasive subpopulation. Natural compounds represent a historically rich source of novel, biologically active compounds which are able to interact with a large number of cellular targets while limiting the painful side-effects commonly associated with cancer treatment. A brief review of select natural products that have been demonstrated to diminish the clinically devastating properties of cancer stem cells or to induce cancer stem cell death is also presented., Competing Interests: None.

Tissue engineering offers a promising strategy to restore injuries resulting from trauma, infection, tumor resection, or other diseases. In spite of significant progress, the field faces a significant bottleneck; the critical need to understand and exploit the interdependencies of tissue healing, angiogenesis, and inflammation. Inherently, the balance of these interacting processes is affected by a number of injury site conditions that represent a departure from physiological environment, including reduced pH, increased concentration of free radicals, hypoglycemia, and hypoxia. Efforts to harness the potential of immune response as a therapeutic strategy to promote tissue repair have led to identification of natural compounds with significant anti-inflammatory properties. This article provides a concise review of the body's inflammatory response to biomaterials and describes the role of oxygen as a physiological cue in this process. We proceed to highlight the potential of natural compounds to mediate inflammatory response and improve host-graft integration. Herein, we discuss the use of natural compounds to map signaling molecules and checkpoints that regulate the cross-linkage of immune response and skeletal repair., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

The success of bone tissue engineering strategies critically depends on the rapid formation of a mature vascular network in the scaffolds after implantation. Conventional methods to accelerate the infiltration of host vasculature into the scaffolds need to consider the role of host response in regulation of bone tissue ingrowth and extent of vascularization. The long term goal of this study was to harness the potential of inflammatory response to enhance angiogenesis and bone formation in three dimensional (3D) scaffolds. Towards this goal, we explored the use of resveratrol, a natural compound commonly used in complementary medicine, to enable the concurrently (i) mediate M1 to M2 macrophage plasticity, (ii) impart natural release of angiogenic factors by macrophages and (iii) enhance osteogenic differentiation of human mesenchymal stem cells (hMSCs). We mapped the time-dependent response of macrophage gene expression as well as hMSC osteogenic differentiation to varying doses of resveratrol. The utility of this approach was evaluated in 3D poly (lactide-co-glycolide) (PLGA) sintered microsphere scaffolds for bone tissue engineering applications. Our results altogether delineate the potential to synergistically accelerate angiogenic factor release and upregulate osteogenic signaling pathways by "dialing" the appropriate degree of resveratrol release.

Cell-based angiogenic therapies offer potential for the repair of ischemic injuries, while avoiding several of the limitations associated with material-based growth factor delivery strategies. Evidence supports that applying MSCs as spheroids rather than dispersed cells can improve retention and enhance therapeutic effect through increased secretion of angiogenic factors due to hypoxia. However, while spheroid culture appears to modulate MSC behavior, there has been little investigation of how major culture parameters that affect cellular oxygen tension, such as external oxygenation and culture size, impact the angiogenic potential of spheroids. We cultured equal numbers of adipose-derived stem cells (ASCs) as spheroids containing 10,000 (10k) or 60,000 (60k) cells each, in 20% and 2% oxygen. VEGF secretion varied among the sample groups, with 10k, 2% O2 spheroids exhibiting the highest production. Spheroid-conditioned media was applied to HUVEC monolayers, and proliferation was assessed. Spheroids of either size in 2% oxygen induced comparable proliferation compared to a 2 ng/ml VEGF control sample, while spheroids in 20% oxygen induced less proliferation. Spheroids were also applied in coculture with HUVEC monolayers, and induction of migration through a Transwell membrane was evaluated. Sixty thousand, 2% O2 spheroids induced similar levels of migration as VEGF controls, while 10k, 2% O2 spheroids induced significantly more. Ten thousand, 20% spheroids performed no better than VEGF-free controls. We conclude that the therapeutic ability of ASC spheroids to stimulate angiogenesis in endothelial cells is affected by both culture size and oxygenation parameters, suggesting that, while ASC spheroids offer potential in the treatment of injured and ischemic tissues, careful consideration of culture size in respect to in vivo local oxygen tension will be necessary for optimal results.

Current methods of treating critical size bone defects include autografts and allografts, however, both present major limitations including donor-site morbidity, risk of disease transmission, and immune rejection. Tissue engineering provides a promising alternative to circumvent these shortcomings through the use of autologous cells, three-dimensional scaffolds, and growth factors. We investigated the development of a scaffold with native bone extracellular matrix (ECM) components for directing the osteogenic differentiation of human embryonic stem cells (hESCs). Toward this goal, a microsphere-sintering technique was used to fabricate poly(lactic-co-glycolic acid) (PLGA) scaffolds with optimum mechanical and structural properties. Human osteoblasts (hOBs) were seeded on these scaffolds to deposit bone ECM for 14 days. This was followed by a decellularization step leaving the mineralized matrix intact. Characterization of the decellularized PLGA scaffolds confirmed the deposition of calcium, collagen II, and alkaline phosphatase by osteoblasts. hESCs were seeded on the osteomimetic substrates in the presence of osteogenic growth medium, and osteogenicity was determined according to calcium content, osteocalcin expression, and bone marker gene regulation. Cell proliferation studies showed a constant increase in number for hESCs seeded on both PLGA and ECM-coated PLGA scaffolds. Calcium deposition by hESCs was significantly higher on the osteomimetic scaffolds compared with the control groups. Consistently, immunofluorescence staining demonstrated an increased expression of osteocalcin in hESCs seeded on ECM-coated osteomimetic PLGA scaffolds. Gene expression analysis of RUNX2 and osteocalcin further confirmed osteogenic differentiation of hESCs at the highest expression level on osteomimetic PLGA. These results together demonstrate the potential of PLGA scaffolds with native bone ECM components to direct osteogenic differentiation of hESCs and induce bone formation.

Tissue engineering utilizes cells, signaling molecules, and scaffolds towards creating functional tissue to repair damaged organs. Pluripotent stem cells (PSCs) are a promising cell source due to their ability to self-renewal indefinitely and their potential to differentiate into almost any cell type. Great strides have been taken to parse the physiological mechanisms by which PSCs respond to their microenvironment and commit to a specific lineage. The combination of physical cues and chemical factors is thought to have the most profound influence on stem cell behavior, therefore a major focus of tissue engineering strategies is scaffold design to incorporate these signals. One overlooked component of the in vivo microenvironment researchers attempt to recapitulate with three dimensional (3D) substrates is the nanoarchitecture formed by the fibrillar network of extracellular matrix (ECM) proteins. These nanoscale features have the ability to impact cell adhesion, migration, proliferation, and lineage commitment. Significant advances have been made in deciphering how these nanoscale cues interact with stem cells to determine phenotype, but much is still unknown as to how the interplay between physical and chemical signals regulate in vitro and in vivo cellular fate. This review dives deeper to investigate nanoscale platforms for engineering tissue, as well use the use of these nanotechnologies to drive pluripotent stem cell lineage determination.

We investigated the biological response of human pluripotent stem cells (hPSCs) cultured on a carbon nanotube (CNT) array-based substrate with the long term goal to direct hPSC germ layer specification for a wide variety of tissue engineering applications. CNT arrays were fabricated using a chemical vapor deposition system allowing for control over surface roughness and mechanical stiffness. Our results demonstrated that hPSCs readily attach to hydrophilized and extracellular matrix coated CNT arrays. hPSCs cultured as colonies in conditions supporting self-renewal demonstrated the morphology and marker expression of undifferentiated hPSCs. Conditions inducing spontaneous differentiation lead to hPSC commitment to all three embryonic germ layers as assessed by immunostaining and RT-PCR analysis. Strikingly, the physical characteristics of CNT arrays favored mesodermal specification of hPSCs. This is contradictory to the behavior of hPSCs on traditional tissue culture plastic which promotes the development of ectoderm. Altogether, these results demonstrate the potential of CNT arrays to be used in the generation of new platforms that allow for precise control of hPSC differentiation by tuning the characteristics of their physical microenvironment., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

A wide variety of environmental factors including physical and biochemical signals are responsible for stem cell behavior and function. In particular, matrix elasticity and cell shape have been shown to determine stem cell function, yet little is known about the interplay between how these physical cues control cell differentiation. For the first time, by using ultraviolet (UV) lithography to pattern poly(ethylene) glycol (PEG) hydrogels we are able to manufacture microenvironments capable of parsing the effects of matrix elasticity, cell shape, and cell size in order to explore the relationship between matrix elasticity and cell shape in mesenchymal stem cell (MSC) lineage commitment. Our data shows that cells cultured on 1,000 μm 2 circles, squares, and rectangles were primarily adipogenic lineage regardless of matrix elasticity, while cells cultured on 2,500 and 5,000 μm 2 shapes more heavily depended on shape and elasticity for lineage specification. We further went on to characterize how modifying the cell cytoskeleton through pharmacological inhibitors can modify cell behavior. By showing MSC lineage commitment relationships due to physical signals, this study highlights the importance of cell shape and matrix elasticity in further understanding stem cell behavior for future tissue engineering strategies.

Stem cells offer a promising tool in tissue engineering strategies, as their differentiated derivatives can be used to reconstruct most biological tissues. These approaches rely on controlling the biophysical cues that tune the ultimate fate of cells. In this context, significant effort has gone to parse out the role of conflicting matrix-elicited signals (e.g., topography and elasticity) in regulation of macroscopic characteristics of cells (e.g., shape and polarity). A critical hurdle, however, lies in our inability to recapitulate the nanoscale spatiotemporal pattern of these signals. The study presented in this manuscript took an initial step to overcome this challenge by developing a carbon nanotube (CNT)-based substrate for nanoresolution control of focal adhesion formation and cell alignment. The utility of this system was studied using human umbilical vascular endothelial cells (HUVECs) and human embryonic stem cells (hESCs) at a single cell level. Our results demonstrated the ability to control cell orientation by merely controlling the alignment of focal adhesions at a nanoscale size. Our long-term vision is to use these nanoengineered substrates to mimic cell orientation in earlier development and explore the role of polarity in asymmetric division and lineage specification of dividing cells.

Cancer has arisen to be of the most prominent health care issues across the world in recent years. Doctors have used physiological intervention as well as chemical and radioactive therapeutics to treat cancer thus far. As an alternative to current methods, gene delivery systems with high efficiency, specificity, and safety that can reduce side effects such as necrosis of tissue are under development. Although viral vectors are highly efficient, concerns have arisen from the fact that viral vectors are sourced from lethal diseases. With this in mind, rod shaped nano-materials such as carbon nanotubes (CNTs) have become an attractive option for drug delivery due to the enhanced permeability and retention effect in tumors as well as the ability to penetrate the cell membrane. Here, we successfully engineered poly (lactic-co-glycolic) (PLGA) functionalized CNTs to reduce toxicity concerns, provide attachment sites for pro-apoptotic protein caspase-3 (CP3), and tune the temporal release profile of CP3 within bone cancer cells. Our results showed that CP3 was able to attach to functionalized CNTs, forming CNT-PLGA-CP3 conjugates. We show this conjugate can efficiently transduce cells at dosages as low as 0.05 μg/ml and suppress cell proliferation up to a week with no further treatments. These results are essential to showing the capabilities of PLGA functionalized CNTs as a non-viral vector gene delivery technique to tune cell fate.

Significant effort has gone towards parsing out the effects of surrounding microenvironment on macroscopic behavior of stem cells. Many of the microenvironmental cues, however, are intertwined, and thus, further studies are warranted to identify the intricate interplay among the conflicting downstream signaling pathways that ultimately guide a cell response. In this contribution, by patterning adhesive PEG (polyethylene glycol) hydrogels using Dip Pen Nanolithography (DPN), we demonstrate that substrate elasticity, subcellular elasticity, ligand density, and topography ultimately define mesenchymal stem cells (MSCs) spreading and shape. Physical characteristics are parsed individually with 7 kilopascal (kPa) hydrogel islands leading to smaller, spindle shaped cells and 105 kPa hydrogel islands leading to larger, polygonal cell shapes. In a parallel effort, a finite element model was constructed to characterize and confirm experimental findings and aid as a predictive tool in modeling cell microenvironments. Signaling pathway inhibition studies suggested that RhoA is a key regulator of cell response to the cooperative effect of the tunable substrate variables. These results are significant for the engineering of cell-extra cellular matrix interfaces and ultimately decoupling matrix bound cues presented to cells in a tissue microenvironment for regenerative medicine.

Studies of cell-extracellular matrix (ECM) interactions at a single cell level have drawn interest from scientists around the world. Subcellular ECM micropatterning techniques allow researchers to control cell shape, migration, and spindle orientation during mitosis potentially influencing the stem cell fate. Generally these studies have been limited to somatic cells rather than human pluripotent stem cells (hPSCs) which are capable of enormous differentiation potential. hPSCs require a defined ECM for attachment and express characteristic integrins mediating cell-substrate interactions. hPSCs also rely on cell-cell contacts for survival and to maintain self-renewal properties, but these circumstances also significantly limit hPSC observation at a single cell level. In addition, currently available methods for ECM micropatterning generally require a facility with trained personnel and intricate equipment to produce protein micropatterns. To overcome this problem, we have developed a protocol for vitronectin micropatterning using simple UV/ozone modification of polystyrene. Single hPSCs were able to attach and form characteristic stress fibers and focal adhesions similar to somatic cell types which demonstrate hPSC responsiveness to extracellular adhesive cues. Micropatterned hPSCs were able to be cultured for up to 48 hours while maintaining expression of pluripotency-associated transcription factor OCT4. Although further studies are necessary, the results of our investigation will potentially have a large impact on cell regenerative medicine and tissue engineering.

Despite their indisputable clinical value, current tissue engineering strategies face major challenges in recapitulating the natural nano-structural and morphological features of native bone. The aim of this study is to take a step forward by developing a porous scaffold with appropriate mechanical strength and controllable surface roughness for bone repair. This was accomplished by homogenous dispersion of carbon nanotubes (CNTs) in a poly(lactide-co-glycolide) (PLGA) solution followed by a solvent casting/particulate leaching scaffold fabrication. Our results demonstrated that CNT/PLGA composite scaffolds possessed a significantly higher mechanical strength as compared to PLGA scaffolds. The incorporation of CNTs led to an enhanced surface roughness and resulted in an increase in the attachment and proliferation of MC3T3-E1 osteoblasts. Most interestingly, the in vitro osteogenesis studies demonstrated a significantly higher rate of differentiation on CNT/PLGA scaffolds compared to the control PLGA group. These results all together demonstrate the potential of CNT/PLGA scaffolds for bone tissue engineering as they possess the combined effects of mechanical strength and osteogenicity.

There is a profound need for orthopaedic grafting strategies due to various trauma and musculoskeletal diseases. Tissue engineering offers a promising avenue to develop viable grafts for bone repair. The transfer of bone tissue engineering strategies to clinical applications is limited by the failure to adequately vascularize scaffolds after implantation. This review focuses on the natural processes for bone and vessel formation as well as the microenvironmental cues and microscale fabrication techniques to properly coordinate these events towards successful vascularization of tissue engineered scaffolds.

Regenerative engineering approaches utilizing biomimetic synthetic scaffolds provide alternative strategies to repair and restore damaged bone. The efficacy of the scaffolds for functional bone regeneration critically depends on their ability to induce and support vascular infiltration. In the present study, three-dimensional (3D) biomimetic poly(lactide-co-glycolide) (PLAGA) sintered microsphere scaffolds were developed by sintering together PLAGA microspheres followed by nucleation of minerals in a simulated body fluid. Further, the angiogenic potential of vascular endothelial growth factor (VEGF)-incorporated mineralized PLAGA scaffolds were examined by monitoring the growth and phenotypic expression of endothelial cells on scaffolds. Scanning electron microscopy micrographs confirmed the growth of bone-like mineral layers on the surface of microspheres. The mineralized PLAGA scaffolds possessed interconnectivity and a compressive modulus of 402 ± 61 MPa and compressive strength of 14.6 ± 2.9 MPa. Mineralized scaffolds supported the attachment and growth and normal phenotypic expression of endothelial cells. Further, precipitation of apatite layer on PLAGA scaffolds resulted in an enhanced VEGF adsorption and prolonged release compared to nonmineralized PLAGA and, thus, a significant increase in endothelial cell proliferation. Together, these results demonstrated the potential of VEGF-incorporated biomimetic PLAGA sintered microsphere scaffolds for bone tissue engineering as they possess the combined effects of osteointegrativity and angiogenesis., (Copyright © 2012 Wiley Periodicals, Inc.)

Objective: This is the new comparative geogenetic molecular evolution research of M. tuberculosis in Iran and Belarus. Thus, we researched the genetic patterns of samples collected in the first survey of anti-tuberculosis drug-resistance by gene coding of RNA polymerase as part of the international project of on tuberculosis., Method: DNA extraction and amplification of rpoB gene was performed. All PCR products of gene were sequenced using the Amersham auto sequencer. For analysing phenogram has been demonstrated by method UPGMA and Neighbour-Joining. Clinical isolates (70/473) were analyzed by using sequencing gene rpoB and genotyped by program DNAMAN and MEGA., Results: The all data were compared with the international database of national center for biotechnology information website. Multi drug resistant of tuberculosis patient (MDR-TB) was 92% in never treated and 8% in previously treated. Mutations in rpoB gene and katG genes were showed in 95% and 84% of the MDR isolates, respectively. Two clusters were found to be identical by the four different analysis methods, presumably representing cases of recent transmission of MDR tuberculosis. The other isolates are divided in Iran into 2 groups: group A - similar to the Eastern strains (China, Taiwan) and group B - strains of another genotype. And 3 groups in Belarus: group A - Strains of the first group are more similar to the standard European and Eastern ones China and Taiwan) which diverged in the last 10 years (Genetic evolution rate), i.e. they are relatively new ones, and that is confirmed by the mutations, group B - Strains of the second group diverged earlier; they are older than the strains of the first group (16 years old- time and rate of evolution) and group C - Strains of the third group are similar to European strains and only circulate in Brest region. They are grouped separately on the phenogram and became prevalent in Iran (they are called Iranian residential strains and also is genetic analogy between group A from Iran and Belarusian isolates., Conclusion: This research gives a first result on genetic evolution of the M. tuberculosis strains distributing in the Iran and Belarus during the first survey of anti-tuberculosis drug-resistance and is homologies between groups A from Iran with group A from Belarus. It may aid in the creation of a national database that will be a valuable support for further studies.

The aim of this study was to investigate the significance of multiple-mutations in the katG gene, predominant nucleotide changes and its correlation with high level of resistance to isoniazid in Mycobacterium tuberculosis isolates that were randomly collected from sputa of 42 patients with primary and secondary active pulmonary tuberculosis from different geographic regions of Iran. Drug susceptibility testing was determined using the CDC standard conventional proportional method. DNA extraction, katG gene amplification, and DNA sequencing analysis were performed. Thirty four (80%) isolates were found to have multiple-mutations (composed of 2-5 mutations) in the katG gene. Increased number of predominant mutations and nucleotide changes were demonstrated in codons 315 (AGC-->ACC), 316 (GGC-->AGC), 309 (GGT-->GTT) with a higher frequency among patients bearing secondary tuberculosis infection with elevated levels of resistance to isoniazid (MIC ≥ 5-10 µg/mL). Furthermore, it was demonstrated that the combination of mutations with their predominant nucleotide changes were also observed in codons 315, 316, and 309 indicating higher frequencies of mutations among patients with secondary infection respectively. In this study, 62% (n= 21) of multi-mutated isolates found to have combination of mutations with predominant nucleotide changes in codons 315 (AGC-->ACC), 316 (GGC-->GTT), 309 (GGT-->GGT), and also demonstrated to be more frequent in isolates of patients with secondary infections, bearing higher level of resistance to isoniazid (≥ 5-10 µg/mL).

Natural polymer chitosan and synthetic polymer poly(lactide-co-glycolide) (PLAGA) have been investigated for a variety of tissue engineering applications. We have previously reported the fabrication and in vitro evaluation of a novel chitosan/PLAGA sintered microsphere scaffold for load-bearing bone tissue engineering applications. In this study, the in vitro degradation characteristics of the chitosan/PLAGA scaffold and the in vivo bone formation capacity of the chitosan/PLAGA-based scaffolds in a rabbit ulnar critical-sized-defect model were investigated. The chitosan/PLAGA scaffold showed slower degradation than the PLAGA scaffold in vitro. Although chitosan/PLAGA scaffold showed a gradual decrease in compressive properties during the 12-week degradation period, the compressive strength and compressive modulus remained in the range of human trabecular bone. Chitosan/PLAGA-based scaffolds were able to guide bone formation in a rabbit ulnar critical-sized-defect model. Microcomputed tomography analysis demonstrated that successful bridging of the critical-sized defect on the sides both adjacent to and away from the radius occurred using chitosan/PLAGA-based scaffolds. Immobilization of heparin and recombinant human bone morphogenetic protein-2 on the chitosan/PLAGA scaffold surface promoted early bone formation as evidenced by complete bridging of the defect along the radius and significantly enhanced mechanical properties when compared to the chitosan/PLAGA scaffold. Furthermore, histological analysis suggested that chitosan/PLAGA-based scaffolds supported normal bone formation via intramembranous formation., (2010 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

One of the fundamental principles underlying tissue engineering approaches is that newly formed tissue must maintain sufficient vascularization to support its growth. Efforts to induce vascular growth into tissue-engineered scaffolds have recently been dedicated to developing novel strategies to deliver specific biological factors that direct the recruitment of endothelial cell (EC) progenitors and their differentiation. The challenge, however, lies in orchestration of the cells, appropriate biological factors, and optimal factor doses. This study reports an approach as a step forward to resolving this dilemma by combining an ex vivo gene transfer strategy and EC transplantation. The utility of this approach was evaluated by using 3D poly(lactide-co-glycolide) (PLAGA) sintered microsphere scaffolds for bone tissue engineering applications. Our goal was achieved by isolation and transfection of adipose-derived stromal cells (ADSCs) with adenovirus encoding the cDNA of VEGF. We demonstrated that the combination of VEGF releasing ADSCs and ECs results in marked vascular growth within PLAGA scaffolds. We thereby delineate the potential of ADSCs to promote vascular growth into biomaterials.

Bone tissue engineering offers promising alternatives to repair and restore tissues. Our laboratory has employed poly(lactide-co-glycolide) PLAGA microspheres to develop a three dimensional (3-D) porous bioresorbable scaffold with a biomimetic pore structure. Osseous healing and integration with the surrounding tissue depends in part on new blood vessel formation within the porous structure. Since endothelial cells play a key role in angiogenesis (formation of new blood vessels from pre-existing vasculature), the purpose of this study was to better understand human endothelial cell attachment, viability, growth, and phenotypic expression on sintered PLAGA microsphere scaffold. Scanning electron microscopy (SEM) examination showed cells attaching to the surface of microspheres and bridging the pores between the microspheres. Cell proliferation studies indicated that cell number increased during early stages and reached a plateau between days 10 and 14. Immunofluorescent staining for actin showed that cells were proliferating three dimensionally through the scaffolds while staining for PECAM-1 (platelet endothelial cell adhesion molecule) displayed typical localization at cell-cell contacts. Gene expression analysis showed that endothelial cells grown on PLAGA scaffolds maintained their normal characteristic phenotype. The cell proliferation and phenotypic expression were independent of scaffold pore architecture. These results demonstrate that PLAGA sintered microsphere scaffolds can support the growth and biological functions of human endothelial cells. The insights from this study should aid future studies aimed at enhancing angiogenesis in three dimensional tissue engineered scaffolds.

Control over angiogenesis (formation of new capillaries from preexisting vessels) is often a crucial requirement for implantable porous biomaterials serving as scaffolds for tissue regeneration. Angiogenesis is influenced by the transport of chemoattractants such as vascular endothelial growth factor (VEGF) through the implant. To investigate this influence, we developed a computational model of capillary formation based on endothelial cell migration by modeling the random motion of sprout tips biased along spatially and temporally evolving concentration gradients of VEGF. The model focuses on the effect of diffusive VEGF transport inside a 2D domain on the directed migration of sprouts to test several chemical and physical strategies to stimulate and control angiogenesis. We considered a 2D porous membrane that is located between the primary vessel and a line source of VEGF. We assess the vascular network formed in 2 cases of a high and zero VEGF degradation rates applying 3 strategies of VEGF production: (1) only a line source; (2) a line source plus controlled release from a small number of VEGF sources that are randomly dispersed on the pore boundaries; and (3) a line source plus controlled release of VEGF from the pore boundaries themselves. Results show that in the limiting cases where VEGF degradation rate is relatively high, strategies 2 and 3 lead to a substantial increase in the number of vessels. This increase depends on the relative rates at which the line source and embedded sources or solid boundaries produce VEGF. Using strategy 2 results in a newly formed capillary network that is highly localized around the embedded sources. However, using strategy 3 leads to a more uniformly distributed vessel network and a higher degree of vessel ingrowth inside the porous membrane. In addition, the duration at which we engineer the embedded sources or pore boundaries to release VEGF determines the morphology of the capillary network. Although a higher release duration leads to a dense network of newly formed vessels near the primary vessel, it hinders further vessel penetration inside the porous membrane. Therefore, in applying both strategies 2 and 3, there is an optimum release duration that leads to a deeper penetration of vessels inside the membrane. It is hoped that insights from this study will aid in the design of materials with optimal structural and chemical properties to facilitate controlled angiogenesis.

We discuss a generic computational model of eukariotic chemotaxis in 2D random porous domains. The model couples the fully time-dependent finite-difference solution of a reaction-diffusion equation for the concentration field of a chemoattractant to biased random walks representing individual chemotactic cells. We focus in particular on the influence of consumption of chemoattractant by the boundaries of obstacles with irregular shapes which are distributed randomly in the domain on the chemotactic response of the cells. Cells are stimulated to traverse a field of obstacles by a line source of chemoattractant. We find that the reactivity of the obstacle boundaries with respect to the chemoattractant strongly determines the transit time of cells through two primary mechanisms. The channeling effect arises because cells are effectively repelled from surfaces which consume chemoattractant, and opposing surfaces therefore act to keep cells in the middle of channels. This reduces traversal times relative to the case with unreactive boundaries, provided that the appropriate Péclet number relating the strength of reactivity to diffusion in governing chemoattractant transport is neither too low nor too high. The dead-zone effect arises due to a realistic threshold on the chemotactic response, which at steady state results in portions of the domain having no detectable gradient. Of these two, the channeling effect is responsible for 90% of the sensitivity of transit times to boundary reactivity. Based on these results, we speculate that it may be possible to tune the rates of cellular penetration into porous domains by engineering the reactivity of the internal surfaces to cytokines.

A number of bone tissue engineering approaches are aimed at (i) increasing the osteconductivity and osteoinductivity of matrices, and (ii) incorporating bioactive molecules within the scaffolds. In this study we examined the growth of a nano-crystalline mineral layer on poly(lactide-co-glycolide) (PLAGA) sintered microsphere scaffolds for tissue engineering. In addition, the influence of the mineral precipitate layer on protein adsorption on the scaffolds was studied. Scaffolds were mineralized by incubation in simulated body fluid (SBF). Scanning electron microscopy (SEM) analysis revealed that mineralized scaffolds possess a rough surface with a plate-like nanostructure covering the surface of microspheres. The results of protein adsorption and release studies showed that while the protein release pattern was similar for PLAGA and mineralized PLAGA scaffolds, precipitation of the mineral layer on PLAGA led to enhanced protein adsorption and slower protein release. Mineralization of tissue-engineered surfaces provides a method for both imparting bioactivity and controlling levels of protein adsorption and release.

We discuss a generic computational model which captures the effects of transient chemoattractant concentration on the chemotactic motility of individual cells. The model solves the appropriate unsteady chemoattractant transport equation using finite differences, while simultaneously executing biased random walks representing individual cells. The simulations were implemented for a 2D homogeneous domain, and two case studies were considered. In the first case study, we consider a single-point source at the origin of the domain which produces chemoattractant, while other cells execute biased random walks toward this point source. We observe that for continuous chemoattractant production, chemoattractant diffusivity has no effect on cell motility, as measured by the mean of time to reach the source. However, in the case of pulsed random production with a specific average duty cycle, the mean time-to-contact is generally minimal with respect to chemoattractant diffusivity over a moderate range of diffusivities. In the second case study, two mobile cells which simultaneously secrete chemoattractant are initially placed a certain distance apart and are then allowed to execute biased random walks. Our model shows that a pulsed random protocol for chemoattractant production facilitates the two cells "finding" one another compared to continuous production. From this case study we also learn that there exists a range of moderate chemoattractant diffusivities for which the mean time-to-contact is minimal when cells both produce/detect chemoattractant and chemotactically migrate. Using these case studies, we discuss how transience in chemoattractant concentration becomes important in characterizing the effectiveness of chemotaxis.