During osteogenesis, mesenchymal stem cells are recruited to the osteoblast lineage and progressively differentiate into osteoblasts that produce a mineralised extracellular matrix. Although most of the organic component of this matrix is...
moreDuring osteogenesis, mesenchymal stem cells are recruited to the osteoblast lineage and progressively differentiate into osteoblasts that produce a mineralised extracellular matrix. Although most of the organic component of this matrix is comprised of collagen, growing evidence suggests the most bioactive element of a developing matrix is its heparan sulfate glycosaminoglycan complement. This species of linear, unbranched sugars contain protein-binding domains that regulate the flow of an astonishing number of mitogenic influences that coordinate mesenchymal stem cell commitment and growth, and ultimately, osteoblast phenotype. Among the heparan sulfate-binding factors known to be important to this process are sonic hedgehog, the fibroblast growth factors and their receptors, members of the transforming growth factor superfamily, as well as the collagens, laminins and fibronectins. How these sugars change during development to bring together the right combination of mitogenic/differ...
Fibroblast growth factor receptors (FGFRs) undergo highly regulated spatial and temporal changes of expression during development. This study describes the use of quantitative reverse transcriptase-polymerase chain reaction and...
moreFibroblast growth factor receptors (FGFRs) undergo highly regulated spatial and temporal changes of expression during development. This study describes the use of quantitative reverse transcriptase-polymerase chain reaction and immunochemistry to assess the changes in expression of FGFR4 as compared to its FGFR4-17a and -17b isoforms in mouse tissues, from early embryogenesis through to adulthood. Compared to FGFR4, the expression of the isoforms is more restricted at all developmental stages tested. The reverse transcriptase-polymerase chain reaction demonstrated that FGFR4 is expressed in more tissue types than either of its isoforms: it was found predominantly in lung, liver, brain, skeletal muscle and kidney, whereas the FGFR4-17a form was detected in lung and skeletal muscle, and the FGFR4-17b form only in lung, liver, skeletal muscle and kidney. Immunohistochemistry confirmed strong FGFR4-17b expression in the postnatal lung. When combined, the results suggest that FGFR4 varia...
Human mesenchymal stem cells (hMSCs) possess great therapeutic potential for the treatment of bone disease and fracture non-union. Too often however, in vitro evidence alone of the interaction between hMSCs and the biomaterial of choice...
moreHuman mesenchymal stem cells (hMSCs) possess great therapeutic potential for the treatment of bone disease and fracture non-union. Too often however, in vitro evidence alone of the interaction between hMSCs and the biomaterial of choice is used as justification for continued development of the material into the clinic. Clearly for hMSC-based regenerative medicine to be successful for the treatment of orthopaedic trauma, it is crucial to transplant hMSCs with a suitable carrier that facilitates their survival, optimal proliferation and osteogenic differentiation in vitro and in vivo. This motivated us to evaluate the use of polycaprolactone-20% tricalcium phosphate (PCL-TCP) scaffolds produced by fused deposition modeling for the delivery of hMSCs. When hMSCs were cultured on the PCL-TCP scaffolds and imaged by a combination of phase contrast, scanning electron and confocal laser microscopy, we observed five distinct stages of colonization over a 21-day period that were characterized by cell attachment, spreading, cellular bridging, the formation of a dense cellular mass and the accumulation of a mineralized extracellular matrix when induced with osteogenic stimulants. Having established that PCL-TCP scaffolds are able to support hMSC proliferation and osteogenic differentiation, we next tested the in vivo efficacy of hMSC-loaded PCL-TCP scaffolds in nude rat critical-sized femoral defects. We found that fluorescently labeled hMSCs survived in the defect site for up to 3 weeks post-transplantation. However, only 50% of the femoral defects treated with hMSCs responded favorably as determined by new bone volume. As such, we show that verification of hMSC viability and differentiation in vitro is not sufficient to predict the efficacy of transplanted stem cells to consistently promote bone formation in orthotopic defects in vivo.
Fracture healing is a complex process regulated by numerous growth and adhesive factors expressed at specific stages during healing. The naturally occurring, cell surface-expressed sugar, heparan sulfate (HS), is known to bind to and...
moreFracture healing is a complex process regulated by numerous growth and adhesive factors expressed at specific stages during healing. The naturally occurring, cell surface-expressed sugar, heparan sulfate (HS), is known to bind to and potentiate the effects of many classes of growth factors, and as such, may be a potential candidate therapy for enhancing bone repair. This study investigated the local application of bone-derived HS in the repair of rat femoral fractures. After 2 weeks, there was a significant increase in the callus size of rats administered with 5 microg HS compared to the control and 50 microg HS groups, presumably due to increased trabecular bone volume rather than increased cartilage production. In addition, 5 microg HS increased the expression of ALP, Runx2, FGF-1, IGF-II, TGF-beta1, and VEGF. It is hypothesized that these increases resulted from changes in HS-mediated receptor/ligand interactions that increase local growth factor production to augment bone formation. The findings of this study demonstrate the anabolic potential of HS in bone repair by recruiting and enhancing the production of endogenous growth factors at the site of injury.
Most research strategies for cartilage tissue engineering use extended culture with complex media loaded with costly GFs (growth factors) to drive tissue assembly and yet they result in the production of cartilage with inferior mechanical...
moreMost research strategies for cartilage tissue engineering use extended culture with complex media loaded with costly GFs (growth factors) to drive tissue assembly and yet they result in the production of cartilage with inferior mechanical and structural properties compared with the natural tissue. Recent evidence suggests that GAGs (glycosaminoglycans) incorporated into tissue engineering scaffolds can sequester and/or activate GFs and thereby more effectively mimic the natural ECM (extracellular matrix). Such approaches may have potential for the improvement of cartilage engineering. However, natural GAGs are structurally complex and heterogeneous, making structure-function relationships hard to determine and clinical translation difficult. Importantly, subfractions of GAGs with specific chain lengths and sulfation patterns have been shown to activate key signalling processes during stem cell differentiation. In addition, recently, GAGs have been bound to synthetic biomaterials, su...
Human mesenchymal stem cells (hMSCs) are an attractive tissue engineering avenue for the repair and regeneration of bone. In this study we detail the in vivo performance of a novel electrospun polycaprolactone scaffold incorporating the...
moreHuman mesenchymal stem cells (hMSCs) are an attractive tissue engineering avenue for the repair and regeneration of bone. In this study we detail the in vivo performance of a novel electrospun polycaprolactone scaffold incorporating the glycosaminoglycan heparan sulfate (HS) as a carrier for hMSC. HS is a multifunctional regulator of many key growth factors expressed endogenously during bone wound repair, and we have found it to be a potent stimulator of proliferation in hMSCs. To assess the potential of the scaffolds to support hMSC function in vivo, hMSCs pre-committed to the osteogenic lineage (human osteoprogenitor cells) were seeded onto the scaffolds and implanted subcutaneously into the dorsum of nude rats. After 6 weeks the scaffolds were retrieved and examined by histological methods. Implanted human cells were identified using a human nuclei-specific antibody. The host response to the implants was characterized by ED1 and ED2 antibody staining for monocytes/macrophages and...
The therapeutic use of VEGF165 to stimulate blood vessel formation for the treatment of peripheral arterial disease or cardiovascular-related disease has met with limited success. Here we describe an affinity-isolated heparan sulfate...
moreThe therapeutic use of VEGF165 to stimulate blood vessel formation for the treatment of peripheral arterial disease or cardiovascular-related disease has met with limited success. Here we describe an affinity-isolated heparan sulfate glycotherapeutic (HS7(+ve)) that binds to, and enhances the bioactivity of, VEGF165. Application of HS7(+ve) complexed with VEGF165 results in enhanced VEGF165-VEGFR2 interaction, prolonged downstream pErk1/2 signalling, and increased cell proliferation and tube formation in HUVECs, compared with VEGF165 alone. The pro-angiogenic potential of HS7(+ve) was further assessed in vivo using the chick embryo chorioallantoic membrane (CAM) assay. Exogenous dosing with HS7(+ve) alone significantly enhanced the formation of new blood vessels with potencies comparable to VEGF165. These results demonstrate the potential for vascular therapy of glycotherapeutic agents targeted at augmenting the bioactivity of VEGF165.
The growth and differentiation of mesenchymal stem cells (MSCs) is controlled by various growth factors, the activities of which can be modulated by heparan sulfates (HSs). We have previously noted the necessity of sulfated...
moreThe growth and differentiation of mesenchymal stem cells (MSCs) is controlled by various growth factors, the activities of which can be modulated by heparan sulfates (HSs). We have previously noted the necessity of sulfated glycosaminoglycans for the fibroblast growth factor type 2 (FGF-2)-stimulated differentiation of osteoprogenitor cells. Here we show that exogenous application of HS to cultures of primary rat MSCs stimulates their proliferation, leading to increased expression of osteogenic markers and enhanced bone nodule formation. FGF-2 can also increase the proliferation, and osteogenic differentiation of rat bone marrow stem cells (rMSCs) when applied exogenously during their linear growth. However, as opposed to exogenous HS, the continuous use of FGF-2 during in vitro differentiation completely blocked rMSC mineralization. We show that the effects of both FGF-2 and HS are mediated through FGF receptor 1 (FGFR1) and that inhibition of signaling through this receptor arrests cell growth, resulting in the cells being unable to reach the critical density necessary to induce differentiation. Blocking FGFR1 signaling in postconfluent osteogenic cultures significantly increased calcium deposition. Taken together our data suggest that FGFR1 signaling plays an important role during osteogenic differentiation, first by stimulating cell growth that is closely followed by an inhibitory effect once the cells have reached confluence. It also confirms the importance of HS as a coreceptor for the signaling of endogenous FGF-2 and suggests that purified glycosaminoglycans may be attractive alternatives to growth factors for improved ex vivo growth and differentiation of MSCs.
While defining the environment for human embryonic stem cell (hESC) culture on 2-dimensional (2D) surfaces has made rapid progress, the industrial-scale implementation of this technology will benefit from translating this knowledge into a...
moreWhile defining the environment for human embryonic stem cell (hESC) culture on 2-dimensional (2D) surfaces has made rapid progress, the industrial-scale implementation of this technology will benefit from translating this knowledge into a 3-dimensional (3D) system, thus enabling better control, automation, and volumetric scale-up in bioreactors. The current study describes a system with defined conditions that are capable of supporting the long-term 2D culture of hESCs and the transposing of these conditions to 3D microcarrier (MC) cultures. Vitronectin (VN) and laminin (LN) were chosen as matrices for the long-term propagation of hESCs in a defined culture medium (STEMPRO(®)) for conventional 2D culture. Adsorption of these proteins onto 2D tissue culture polystyrene (TCPS) indicated that surface density saturation of 510 and 850 ng/cm(2) for VN and LN, respectively, was attained above 20 μg/mL deposition solution concentration. Adsorption of these proteins onto spherical (97±10 μm), polystyrene MC followed a similar trend and coating surface densities of 450 and 650 ng/cm(2) for VN and LN, respectively, were used to support hESC propagation. The long-term expansion of hESCs was equally successful on TCPS and MC, with consistently high expression (>90%) of pluripotent markers (OCT-4, MAB-84, and TRA-1-60) over 20 passages and maintenance of karyotypic normality. The average fold increase in cell numbers on VN-coated MC per serial passage was 8.5±1.0, which was similar to LN-coated MC (8.5±0.9). Embryoid body differentiation assays and teratoma formation confirmed that hESCs retained the ability to differentiate into lineages of all 3 germ layers, thus demonstrating the first translation to a fully defined MC-based environment for the expansion of hESCs.
Cell surface heparan sulfate (HS) and chondroitin sulfate (CS) proteoglycans have been implicated in a multitude of biological processes, including embryonic implantation, tissue morphogenesis, wound repair, and neovascularization through...
moreCell surface heparan sulfate (HS) and chondroitin sulfate (CS) proteoglycans have been implicated in a multitude of biological processes, including embryonic implantation, tissue morphogenesis, wound repair, and neovascularization through their ability to regulate growth factor activity and morphogenic gradients. However, the direct role of the glycosaminoglycan (GAG) sugar-side chains in the control of human mesenchymal stem cell (hMSC) differentiation into the osteoblast lineage is poorly understood. Here, we show that the abundant cell surface GAGs, HS and CS, are secreted in proteoglycan complexes that directly regulate the bone morphogenetic protein (BMP)-mediated differentiation of hMSCs into osteoblasts. Enzymatic depletion of the HS and CS chains by heparinase and chondroitinase treatment decreased HS and CS expression but did not alter the expression of the HS core proteins perlecan and syndecan. When digested separately, depletion of HS and CS chains did not effect hMSC proliferation but rather increased BMP bioactivity through SMAD1/5/8 intracellular signaling at the same time as increasing canonical Wnt signaling through LEF1 activation. Long-term culturing of cells in HS- and CS-degrading enzymes also increased bone nodule formation, calcium accumulation, and the expression of such osteoblast markers as alkaline phosphatase, RUNX2, and osteocalcin. Thus, the enzymatic disruption of HS and CS chains on cell surface proteoglycans alters BMP and Wnt activity so as to enhance the lineage commitment and osteogenic differentiation of hMSCs.
Bone morphogenetic protein-2 (BMP-2) is considered a promising adjuvant for the treatment of skeletal non-union and spinal fusion. However, BMP-2 delivery in a conventional collagen scaffold necessitates a high dose to achieve an...
moreBone morphogenetic protein-2 (BMP-2) is considered a promising adjuvant for the treatment of skeletal non-union and spinal fusion. However, BMP-2 delivery in a conventional collagen scaffold necessitates a high dose to achieve an efficacious outcome. To lower its effective dose, we precomplexed BMP-2 with the glycosaminoglycans (GAGs) dermatan sulfate (DS) or heparin (HP), prior to loading it into a hyaluronic acid (HA) hydrogel. In vitro release studies showed that BMP-2 precomplexed with DS or HP had a prolonged delivery compared to without GAG. BMP-2-DS complexes achieved a slightly faster release in the first 24 h than HP; however, both delivered BMP-2 for an equal duration. Analysis of the kinetic interaction between BMP-2 and DS or HP showed that HP had approximately 10 times higher affinity for BMP-2 than DS, yet it equally stabilized the protein, as determined by alkaline phosphatase activity. Ectopic bone formation assays at subcutaneous sites in rats demonstrated that HA hydrogel-delivered BMP-2 precomplexed with GAG induced twice the volume of bone compared with BMP-2 delivered uncomplexed to GAG.
This article reports the method of preparation of gadolinium oxide-doped silica nanoparticles (NPs) whose surface has been functionalized to anchor DNA onto it. The silica...
moreThis article reports the method of preparation of gadolinium oxide-doped silica nanoparticles (NPs) whose surface has been functionalized to anchor DNA onto it. The silica NP's surface was modified by 3-aminopropyltrimethoxysilane for DNA to bind electrostatically. Silica NPs with low polydispersity and encapsulating gadolinium oxide were prepared in the aqueous core of the reverse micelles. The average size of these spherical silica NPs doped with gadolinium oxide and dispersed in water is ∼ 50 nm as measured by dynamic light scattering and transmission electron microscopy. The plasmid DNA electrostatically held over NP's surface was firmly immobilized and protected from DNase attack. The gadolinium oxide-doped silica NPs are paramagnetic as observed from the nuclear magnetic resonance (NMR) line-broadening effect on proton spectrum of the surrounding water. In vitro transfection efficiencies of these gadolinium oxide-doped and DNA-conjugated silica NPs in COS-7 and 293T cells were found to be about 75% and 77% respectively of that of 'Polyfect®' as positive control. This article reports the method of preparation of gadolinium oxide-doped silica nanoparticles (NPs) whose surface has been functionalized to anchor DNA. These NPs are paramagnetic with in vitro transfection efficiencies in COS-7 and 293T cells of about 75% and 77% compared to 'Polyfect®' as positive control.
Following injury, it is inherently difficult to completely restore the biomechanical properties of ligaments. Relatively little is known about the cellular mechanisms controlling ligament healing. Numerous studies have implicated...
moreFollowing injury, it is inherently difficult to completely restore the biomechanical properties of ligaments. Relatively little is known about the cellular mechanisms controlling ligament healing. Numerous studies have implicated fibroblast growth factors (FGFs) as key molecules during the initiation of the cellular proliferation, differentiation, migration and matrix deposition that characterise wound healing. While current surgical emphasis concentrates on growth factor intervention, the role of their cognate receptors (FGFRs) has largely been overlooked. Following transection of the medial collateral ligament (MCL) in rabbits, we examined FGFR expression over a 14-day healing period. Using semi-quantitative RT-PCR, we observed a significant upregulation in FGFR2 expression after 3 days. By 7 days post injury, FGFR2 expression fell to basal levels in line with those of FGFR1 and 3, both of which remained unaffected by surgical transection. These results demonstrate a role for FGFR2 in fibroblast and endothelial cell proliferation in damaged ligament, and suggest a window for FGF therapy.
Human mesenchymal stem cells (hMSCs) are an attractive tissue engineering avenue for the repair and regeneration of bone. In this study we detail the in vivo performance of a novel electrospun polycaprolactone scaffold incorporating the...
moreHuman mesenchymal stem cells (hMSCs) are an attractive tissue engineering avenue for the repair and regeneration of bone. In this study we detail the in vivo performance of a novel electrospun polycaprolactone scaffold incorporating the glycosaminoglycan heparan sulfate (HS) as a carrier for hMSC. HS is a multifunctional regulator of many key growth factors expressed endogenously during bone wound repair, and we have found it to be a potent stimulator of proliferation in hMSCs. To assess the potential of the scaffolds to support hMSC function in vivo, hMSCs pre-committed to the osteogenic lineage (human osteoprogenitor cells) were seeded onto the scaffolds and implanted subcutaneously into the dorsum of nude rats. After 6 weeks the scaffolds were retrieved and examined by histological methods. Implanted human cells were identified using a human nuclei-specific antibody. The host response to the implants was characterized by ED1 and ED2 antibody staining for monocytes/macrophages and mature tissue macrophages, respectively. It was found that the survival of the implanted human cells was affected by the host response to the implant regardless of the presence of HS, highlighting the importance of controlling the host response to tissue engineering devices.
Glycosaminoglycan (GAG) sugars are largely responsible for the bioactivity of the proteoglycan proteins they decorate, and are particularly important for mediating the processes of cell attachment and growth factor signaling. Here, we...
moreGlycosaminoglycan (GAG) sugars are largely responsible for the bioactivity of the proteoglycan proteins they decorate, and are particularly important for mediating the processes of cell attachment and growth factor signaling. Here, we show that chlorate-induced de-sulfation of GAGs expressed by MG-63 osteosarcoma cells results in delayed cell proliferation when the cells are exposed to chlorate for short or medium periods, but a disrupted mineralization without altered cell proliferation in response to long-term chlorate exposure. Analysis of GAG-binding growth factor activity indicated that chlorate disrupted BMP2/noggin signaling, but not FGF2 activity. Microarray analyses, which were confirmed by subsequent cell-based assays, indicated that chlorate predominantly disrupted the cell cycle and actin cytoskeleton and upregulated cholesterol synthesis, without affecting cell migration or attachment. Furthermore, we observed that disruption of the functions of the proteoglycan syndecan-4 replicated phenotypes induced by chlorate, implicating a primary role for this proteoglycan in providing bioactivity for these cells.
Bone tissue homeostasis relies upon the ability of cells to detect and interpret extracellular signals that direct changes in tissue architecture. This study utilized a four-point bending model to create both fluid shear and strain forces...
moreBone tissue homeostasis relies upon the ability of cells to detect and interpret extracellular signals that direct changes in tissue architecture. This study utilized a four-point bending model to create both fluid shear and strain forces (loading) during the time-dependent progression of MC3T3-E1 preosteoblasts along the osteogenic lineage. Loading was shown to increase cell number, alkaline phosphatase (ALP) activity, collagen synthesis, and the mRNA expression levels of Runx2, osteocalcin (OC), osteopontin, and cyclo-oxygenase-2. However, mineralization in these cultures was inhibited, despite an increase in calcium accumulation, suggesting that loading may inhibit mineralization in order to increase matrix deposition. Loading also increased fibroblast growth factor receptor-3 (FGFR3) expression coincident with an inhibition of FGFR1, FGFR4, FGF1, and extracellular signal-related kinase (ERK)1/2 phosphorylation. To examine whether these loading-induced changes in cell phenotype and FGFR expression could be attributed to the inhibition of ERK1/2 phosphorylation, cells were grown for 25 days in the presence of the MEK1/2 inhibitor, U0126. Significant increases in the expression of FGFR3, ALP, and OC were observed, as well as the inhibition of FGFR1, FGFR4, and FGF1. However, U0126 also increased matrix mineralization, demonstrating that inhibition of ERK1/2 phosphorylation cannot fully account for the changes observed in response to loading. In conclusion, this study demonstrates that preosteoblasts are mechanoresponsive, and that long-term loading, whilst increasing proliferation and differentiation of preosteoblasts, inhibits matrix mineralization. In addition, the increase in FGFR3 expression suggests that it may have a role in osteoblast differentiation.
Osteogenic differentiation is coordinated by the exposure of cells to temporal changes in a combination of growth factors and elements within the extracellular matrix (ECM). Many of the key proteins that drive these changes share the...
moreOsteogenic differentiation is coordinated by the exposure of cells to temporal changes in a combination of growth factors and elements within the extracellular matrix (ECM). Many of the key proteins that drive these changes share the property of being dependent on ECM glycosaminoglycans (GAGs) for their activity. Here, we examined whether GAGs isolated from proliferating, differentiating and mineralizing MG-63 osteosarcoma cells differed in their physical properties, and thus in their capacities to coordinate the osteogenic cascade both in human MG-63 osteosarcoma cells and primary human mesenchymal stem cells (hMSCs). Our results show that the size distribution of GAGs, the expression of GAG-carrying proteoglycan cores and the expression of enzymes involved in their modification systematically change as MG-63 cells mature in culture. When dosed back onto cells exogenously in soluble form, GAGs regulated MG-63 survival and growth in a dose-dependent manner, but not differentiation in either cell type. In contrast, hMSCs aggregated into distinct colonies when grown on GAG-coated substrates, while MG-63 cells did not. Heparin-coated substrates improved hMSC viability without inducing aggregation. These results suggest a complex role for GAGs in coordinating the emergence of the osteoblast phenotype, and provide further evidence for the use of heparans in bone tissue repair applications.
The transcription factor Runx2 can be controlled by a number of upstream regulators involved in intracellular signalling, including the activation ERK1/2 signaling by fibroblast growth factor-2 (FGF-2). FGFs interact with their cell...
moreThe transcription factor Runx2 can be controlled by a number of upstream regulators involved in intracellular signalling, including the activation ERK1/2 signaling by fibroblast growth factor-2 (FGF-2). FGFs interact with their cell surface receptors (FGFRs) through an obligate cross-binding interaction with heparan sulfate proteoglycan (HSPG) co-receptors; exogenous HS sugar chains have been shown to potently modulate changes in cell phenotype depending on the stage of tissue differentiation when the HS is harvested, suggesting that HS chain structure and function varies depending on the stage of cell maturity. This study examined the potential of bone-derived heparan sulfate (HS), harvested from differentiating osteoblasts, for the enhancement of preosteoblast growth and differentiation. HS was harvested from conditioned media, cell surface and matrix compartments of postconfluent (differentiating) MC3T3-E1 osteoblasts and dosed back onto preconfluent MC3T3-E1 cells. We show that HS can increase the expression Runx2, ALP, and OPN in preosteoblast cells, suggesting the potential for exogenous HS to shift cells from proliferative to differentiative phenotypes. In line with their structural differences, only HS released into the media was found to co-stimulate the mitogenic effect of FGF-2, whilst exogenous application of all the HSs together with FGF-2 served to increase the expression of OPN. Only the application of cell surface-derived HS triggered a synergistic increase in FGFR1 expression together with FGF-2, although all three HS preparations could trigger transient increases in PI3K, ERK1/2, and stat3 phosphorylation levels. These findings demonstrate that the compartmentally distinct HS species expressed by differentiating MC3T3-E1 cells act in complex ways to coordinate the extracellular conditions that lead to osteoblast differentiation, with the cell surface species coordinating the FGF response.
Heparan sulfate proteoglycans (HSPGs) are key components of the extracellular matrix that mediate cell proliferation, invasion, and cellular signaling. The biological functions of HSPGs are linked to their co-stimulatory effects on...
moreHeparan sulfate proteoglycans (HSPGs) are key components of the extracellular matrix that mediate cell proliferation, invasion, and cellular signaling. The biological functions of HSPGs are linked to their co-stimulatory effects on extracellular ligands (e.g., WNTs) and the resulting activation of transcription factors that control mammalian development but also associated with tumorigenesis. We examined the expression profile of HSPG core protein syndecans (SDC1-4) and glypicans (GPC1-6) along with the enzymes that initiate or modify their glycosaminoglycan chains in human breast cancer (HBC) epithelial cells. Gene expression in relation to cell proliferation was examined in the HBC cell lines MCF-7 and MDA-MB-231 following treatment with the HS agonist heparin. Heparin increased gene expression of chain initiation and modification enzymes including EXT1 and NDST1, as well as core proteins SDC2 and GPC6. With HS/Wnt interactions established, we next investigated WNT pathway components and observed that increased proliferation of the more invasive MDA-MB-231 cells is associated with activation of the Wnt signaling pathway. Specifically, there was substantial upregulation (>5-fold) of AXIN1, WNT4A, and MYC in MDA-MB-231 but not in MCF-7 cells. The changes in gene expression observed for HSPG core proteins and related enzymes along with the associated Wnt signaling components suggest coordinated interactions. The influence of HSPGs on cellular proliferation and invasive potential of breast cancer epithelial cells are cell and niche specific. Further studies on the interactions between HSPGs and WNT ligands may yield clinically relevant molecular targets, as well as new biomarkers for characterization of breast cancer progression.
The glycosaminoglycan sugar heparan sulfate (HS) is an attractive agent for the repair of bone defects due to its ability to regulate endogenous growth factors. The sustained delivery of HS to the localized wound site over the period of...
moreThe glycosaminoglycan sugar heparan sulfate (HS) is an attractive agent for the repair of bone defects due to its ability to regulate endogenous growth factors. The sustained delivery of HS to the localized wound site over the period of healing which can last for over 1 month may prove advantageous for its therapeutic use. In this study we investigated the encapsulation of HS by the water-in oil-in water (W(1)/O/W(2)) technique in polycaprolactone (PCL) microcapsules as a prolonged delivery device. Encapsulation efficiencies of 70% could be achieved by using a 1:1 mixture of dichloromethane (DCM) and acetone as the solvent in the organic phase, while DCM alone gave poor encapsulation. Although addition of polyvinyl alcohol (PVA) to the drug phase did not affect the size or drug loading of the microcapsules, it did however produce a large change in the morphology and drug distribution, which resulted in different release rates. Release from capsules made with PVA in the drug phase reached 60% after 40 days, while those made with water in the drug phase completed release after 20 days. In vitro biocompatibility studies were performed and detected no increase in cell death in human mesenchymal stem cells (hMSC) or induction of an inflammatory response in macrophages after exposure to release products from HS-loaded microcapsules. The released HS retained its ability to increase the proliferation of hMSC after the encapsulation process. These results indicate that encapsulation of HS by the W(1)/O/W(2) method creates a promising device for the repair of bone tissue.
Human mesenchymal stem cells (hMSCs) have attracted much attention for tissue repair and wound healing because of their self-renewal capacity and multipotentiality. In order to mediate an effective therapy, substantial numbers of cells...
moreHuman mesenchymal stem cells (hMSCs) have attracted much attention for tissue repair and wound healing because of their self-renewal capacity and multipotentiality. In order to mediate an effective therapy, substantial numbers of cells are required, which necessitates extensive sub-culturing and expansion of hMSCs. Throughout ex vivo expansion, the cells undergo telomere shortening, and critically short telomeres can trigger loss of cell viability. Telomeres are nucleoprotein structures that cap the ends of chromosomes, and serve to protect the DNA from the degradation which occurs due to the end-replication problem in all eukaryotes. As hMSCs have only a finite ability for self-renewal like most somatic cells, assaying for telomere length in hMSCs provides critical information on the replicative capacity of the cells, an important criterion in the selection of hMSCs for therapy. Telomere length is generally quantified by Southern blotting and fluorescence in situ hybridization, and more recently by PCR-based methods. Here we describe the quantification of hMSC telomere length by real-time PCR; our results demonstrate the effect of telomere shortening on the proliferation and clonogenicity of hMSCs. Thus, this assay constitutes a useful tool for the determination of relative telomere length in hMSCs.