Gwendolen C. Reilly

Intrinsic extracellular matrix properties regulate stem cell differentiation

One of the recent paradigm shifts in stem cell biology has been the discovery that stem cells can begin to differentiate into mature tissue cells when exposed to intrinsic properties of the extracellular matrix (ECM), such as matrix structure, elasticity, and composition. These parameters are known to modulate the forces a cell can exert upon its matrix. Mechano-sensitive pathways subsequently convert these biophysical cues into biochemical signals that commit the cell to a specific lineage. Just as with well-studied growth factors, ECM parameters are extremely dynamic and are spatially- and temporally-controlled during development, suggesting that they play a morphogenetic role in guiding differentiation and arrangement of cells. Our ability to dynamically regulate the stem cell niche as the body does is likely a critical requirement for developing differentiated cells from stem cells for therapeutic applications. Here, we present the emergence of stem cell mechanobiology and its future challenges with new biomimetic, three-dimensional scaffolds that are being used therapeutically to treat disease.

BMP responsiveness in human mesenchymal stem cells.

Bone morphogenetic proteins (BMPs) are well known to induce bone formation in animal models and can promote osteogenesis in cultures of multipotential mesenchymal stem cells (MSC) isolated from rat and mouse bone marrow. However, clinical trials of BMPs suggest that BMPs are relatively ineffective inducers of osteogenesis in humans. Recent studies from our lab indicate that when human bone marrow MSC are placed in primary culture, osteogenesis can be induced by dexamethasone (Dex), but not by BMP-2, -4, or -7. We have therefore investigated components of BMP signaling pathways in human MSC. First passage cells, derived from the bone marrow of patients undergoing hip replacement surgery, were cultured with ascorbate phosphate and treated with 100 nM dexamethasone (Dex), 100 ng/ml BMP, or both. After 6 days, alkaline phosphatase activity of cell extracts was measured, and RNA was extracted for RT-PCR analysis of mRNA levels. Among human MSC samples from more than a dozen patients, only one patient sample showed significantly elevated alkaline phosphatase after exposure to BMP; the rest responded to Dex but not BMP. Analysis of mRNA from cultured human MSC indicated that, while Dex treatment caused increased levels of mRNA for alkaline phosphatase, BMP did not. Noggin is a BMP-binding protein that is upregulated by BMPs. BMP-treated human MSC cultures that did not show increased alkaline phosphatase did express elevated levels of noggin mRNA, indicating that the cells are capable of some BMP response. Our results suggest that BMP signaling in mesenchymal stem cells utilizes more than one system for transcriptional activation. The inability of most human MSC to activate transcription of the alkaline phosphatase gene implies that a defect exists in the system required for induction of the osteoblast phenotype.

Shear stress induces osteogenic differentiation of human mesenchymal stem cells

AIM To determine whether fluid flow-induced shear stress affects the differentiation of bone marrow-derived human mesenchymal stem cells (hMSCs) into osteogenic cells. MATERIALS & METHODS hMSCs cultured with or without osteogenic differentiation medium were exposed to fluid flow-induced shear stress and analyzed for alkaline phosphatase activity and expression of osteogenic genes. RESULTS Immediately following shear stress, alkaline phosphatase activity in osteogenic medium was significantly increased. At days 4 and 8 of culture the mRNA expression of bone morphogenetic protein-2 and osteopontin was significantly higher in hMSCs subjected to shear stress than those cultured in static conditions. However, hMSCs cultured in osteogenic differentiation medium were less responsive in gene expression of alkaline phosphatase and bone morphogenetic protein-2. CONCLUSION These data demonstrate that shear stress stimulates hMSCs towards an osteoblastic phenotype in the absence of chemical induction, suggesting that certain mechanical stresses may serve as an alternative to chemical stimulation of stem cell differentiation.

Effect of formaldehyde fixation on some mechanical properties of bovine bone.

The risk of infection of investigators working on the biomechanics of human bone from a variety of modern pathogens including the human immunodeficiency virus or the hepatitis B virus has increased recently. New safety procedures are needed to reduce that risk. The procedure we follow in our laboratory employs brief (< 3 h) fixation in formaldehyde, and we report here the effects it has on some mechanical properties of bovine bone. Results in quasistatic loading tests were almost unaffected by our fixation protocol, but a significant decrease in impact strength was found. These results indicate that there may be some interaction between fixation and strain rate dependent effects and, therefore, some caution is needed when using common biomechanical measurement methods on fixed bone material.

Observations of microdamage around osteocyte lacunae in bone

Tensile microdamage was examined using laser scanning confocal microscopy in beam specimens of bovine, equine and human long bones loaded in vitro and whole specimens of rat ulnae loaded in vivo. Microcracks were observed to initiate frequently at osteocyte lacunae. The implication is that osteocyte lacunae act as stress concentrating features in bone. This association provides a potential mechanism for the detection of strain and/ or damage by osteocytes in bone.

Decellularization and sterilization of porcine urinary bladder matrix for tissue engineering in the lower urinary tract

BACKGROUND Several synthetic and natural matrices have been described for tissue engineering of bladder but there is little information on the effects of processing on their subsequent mechanical performance or interaction with human cells. AIM Our aim was to assess the effects of delamination, decellularization and sterilization on the mechanical properties of porcine urinary bladder matrix (UBM) and to then assess the ability of the UBM to act as a scaffold for reconstruction with human bladder cells. METHODS A total of 20 porcine bladders were assessed before and after mechanical delamination and four porcine bladders were followed at every stage through a comparison of several decellularization and terminal sterilization methodologies examining histological and mechanical characteristics. The sterile UBM was seeded with normal human urothelial and bladder stromal cells either as a simultaneous coculture, or with stromal cells followed by urothelial cells. RESULTS Mechanical delamination, physical rinsing of the resulting bladder stroma in hypotonic buffer, 0.1% sodium dodecyl sulfate solution and 0.1% peracetic acid resulted in an UBM with acceptable mechanical properties capable of supporting urothelial and bladder stromal cells. Terminal sterilization with ethylene oxide resulted in considerable stiffening of the matrix simultaneous coculture and layered seeding of scaffolds with stromal cells followed by epithelial cells gave similar results with good initial urothelial attachment (followed by loss of cells later) and slow stromal cell penetration. CONCLUSION We describe a decellularized sterilized porcine UBM with acceptable mechanical properties that shows promise as a scaffold for producing an in vitro tissue-engineered bladder patch material for lower urinary tract reconstruction. Future work now needs to focus on the conditions for achieving secure epithelial attachment.

Use of rapidly mineralising osteoblasts and short periods of mechanical loading to accelerate matrix maturation in 3D scaffolds

MLO-A5 cells are a fully differentiated osteoblastic cell line with the ability to rapidly synthesise mineralised extracellular matrix (ECM). We used MLO-A5 cells to develop a system for studying the mechanical modulation of bone matrix formation in 3D using a cyclic compressive loading stimulus. Polyurethane (PU) open cell foam scaffolds were seeded with MLO-A5 cells under static conditions and loaded in compression at 1 Hz, 5% strain in a sterile fluid-filled chamber. Loading was applied for only 2 h per day on days 5, 10 and 15 of culture and cell-seeded scaffolds were assayed on days 10, 15 and 20 of culture. Collagen content as assayed by Sirius red was significantly (2 fold) higher at days 15 and 20 in loaded samples compared with static controls. Calcium content as assayed by alizarin red was significantly (4 fold) higher by day 20. The number of viable cells as assayed by MTS was higher in loaded samples at day 10 but there was no difference by days 15 and 20. Loaded samples also had higher stiffness in compression by the end of the experiment. The mRNA expression of type I collagen, osteopontin and osteocalcin was higher, after a single bout of loading, in loaded than in non-loaded samples as assayed by RT-PCR. In conclusion, mineralisation by fully differentiated osteoblasts, MLO-A5s, was shown to be highly sensitive to mechanical loading, with short bouts of mechanical loading having a strong effect on mineralised matrix production. The 3D system developed will be useful for systematic investigation of the modulators of in vitro matrix mineralisation by osteoblasts in mechanobiology and tissue engineering studies.

Borate Glass Supports the In Vitro Osteogenic Differentiation of Human Mesenchymal Stem Cells

Abstract Bioactive ceramics have the ability to bond to surrounding bone and potentially enhance bone in-growth. Silicate based bioactive glasses and glass-ceramics, such as 45S5 bioactive glass, have been widely investigated for bone repair or as scaffolds for cell-based bone tissue engineering. Recent data have demonstrated that silica-free borate glasses also exhibit bioactive behavior and have been shown to convert to calcium phosphate at a remarkably rapid rate. Due to its relative novelty in biological applications, the cytocompatibity of borate glass is largely unknown. The objectives of this study were to investigate the cytocompatibility of borate glass by in vitro cell culture with human mesenchymal stem cells (hMSCs) and hMSC-derived osteoblasts (hMSC-Obs). The choice of hMSCs and hMSC-Obs is based on the rationale that both cell types are preferred cell lineages attached to bone implants. Borate glass particles with diameters of 212–355 μ m were loosely compacted into porous disks (porosity ≈ 40%) followed by sintering at 600°C. Partial or nearly complete conversion of the borate glass to calcium phosphate (Ca-P) material was achieved by soaking the disks for 1 day or 7 days in a 0.25 molar K2HPO4 solution at 37°C and at pH of 9.0. Bone marrow derived hMSCs and hMSC-Obs adhered to the pores of borate glass disks. Upon 2 week incubation of cell-seeded borate glass disks, hMSC-Obs markedly synthesized alkaline phosphatase, an early osteogenic marker. These data provide preliminary evidence of cytocompatibility of borate glass and its role in supporting the osteogenic differentiation of mesenchymal stem cells.

Novel electrospun polyurethane/gelatin composite meshes for vascular grafts

Novel polymeric micro-nanostructure meshes as blood vessels substitute have been developed and investigated as a potential solution to the lack of functional synthetic small diameter vascular prosthesis. A commercial elastomeric polyurethane (Tecoflex® EG-80A) and a natural biopolymer (gelatin) were successfully co-electrospun from different spinnerets on a rotating mandrel to obtain composite meshes benefiting from the mechanical characteristics of the polyurethane and the natural biopolymer cytocompatibility. Morphological analysis showed a uniform integration of micrometric (Tecoflex®) and nanometric (gelatin) fibers. Exposure of the composite meshes to vapors of aqueous glutaraldehyde solution was carried out, to stabilize the gelatin fibers in an aqueous environment. Uniaxial tensile testing in wet conditions demonstrated that the analyzed Tecoflex®–Gelatin specimens possessed higher extensibility and lower elastic modulus than conventional synthetic grafts, providing a closer matching to native vessels. Biological evaluation highlighted that, as compared with meshes spun from Tecoflex® alone, the electrospun composite constructs enhanced endothelial cells adhesion and proliferation, both in terms of cell number and morphology. Results suggest that composite Tecoflex®–Gelatin meshes could be promising alternatives to conventional vascular grafts, deserving of further studies on both their mechanical behaviour and smooth muscle cell compatibility.

Electrospun polyurethane/hydroxyapatite bioactive scaffolds for bone tissue engineering: the role of solvent and hydroxyapatite particles.

Polyurethane (PU) is a promising polymer to support bone-matrix producing cells due to its durability and mechanical resistance. In this study two types of medical grade poly-ether urethanes Z3A1 and Z9A1 and PU-Hydroxyapatite (PU-HA) composites were investigated for their ability to act as a scaffold for tissue engineered bone. PU dissolved in varying concentrations of dimethylformamide (DMF) and tetrahydrofuran (THF) solvents were electrospun to attain scaffolds with randomly orientated non-woven fibres. Bioactive polymeric composite scaffolds were created using 15 wt% Z3A1 in a 70/30 DMF/THF PU solution and incorporating micro- or nano-sized HA particles in a ratio of 3:1 respectively, whilst a 25 wt% Z9A1 PU solution was doped in ratio of 5:1. Chemical properties of the resulting composites were evaluated by FTIR and physical properties by SEM. Tensile mechanical testing was carried out on all electrospun scaffolds. MLO-A5 osteoblastic mouse cells and human embryonic mesenchymal progenitor cells, hES-MPs were seeded on the scaffolds to test their biocompatibility and ability to support mineralised matrix production over a 28 day culture period. Cell viability was assayed by MTT and calcium and collagen deposition by Sirius red and alizarin red respectively. SEM images of both electrospun PU scaffolds and PU-HA composite scaffolds showed differences in fibre morphology with changes in solvent combinations and size of HA particles. Inclusion of THF eliminated the presence of beads in fibres that were present in scaffolds fabricated with 100% DMF solvent, and resulted in fibres with a more uniform morphology and thicker diameters. Mechanical testing demonstrated that the Young׳s Modulus and yield strength was lower at higher THF concentrations. Inclusion of both sizes of HA particles in PU-HA solutions reinforced the scaffolds leading to higher mechanical properties, whilst FTIR characterisation confirmed the presence of HA in all composite scaffolds. Although all scaffolds supported proliferation of both cell types and deposition of calcified matrix, PU-HA composite fibres containing nano-HA enabled the highest cell viability and collagen deposition. These scaffolds have the potential to support bone matrix formation for bone tissue engineering.