Aileen Crawford

Synovial fluid mesenchymal stem cells in health and early osteoarthritis: Detection and functional evaluation at the single‐cell level

OBJECTIVE Arthritic synovial fluid (SF) contains mesenchymal stem cells (MSCs), which could simply reflect their shedding from diseased joint structures. This study used the bovine model to explore SF MSCs in health and enumerated them at the earliest stages of human osteoarthritis (OA) in radiographically normal joints. METHODS Clonogenicity and multipotentiality of normal bovine SF MSCs were compared with donor-matched bone marrow (BM) MSCs at the single-cell level. The colony-forming unit-fibroblastic assay was used for MSC enumeration. The XTT assay was employed to assess cell proliferation, and flow cytometry was used to investigate the marker phenotype of bovine and human SF MSCs. RESULTS Single MSCs were present in normal bovine SF, and 96% of them were able to expand at least 1 million-fold. These cells were CD271-, multipotential, considerably more clonogenic, and less adipogenic than matched BM MSCs. In both pellet assays and on polyglycolic acid scaffolds, SF clones displayed consistent chondrogenic differentiation, while BM clones were variable. MSCs were present in arthroscopically normal human joints and were increased 7-fold in early OA (P = 0.034). Their numbers correlated with numbers of free microscopic synovial tissue fragments (r = 0.826, P < 0.0001). OA SF had a growth-promoting effect on synovial MSCs. CONCLUSION This study confirms the presence of MSCs in normal SF and shows their numerical increase in early human OA. SF MSCs are likely to originate from synovium. These findings provide a platform for the exploration of the potential role of SF MSCs in joint homeostasis and for investigation of their utility in novel joint regeneration strategies.

Skeletal tissue engineering using silk biomaterials

Silks have been proposed as potential scaffold materials for tissue engineering, mainly because of their physical properties. They are stable at physiological temperatures, flexible and resist tensile and compressive forces. Bombyx mori (silkworm) cocoon silk has been used as a suture material for over a century, and has proved to be biocompatible once the immunogenic sericin coating is removed. Spider silks have a similar structure to silkworm silk but do not have a sericin coating. This paper provides a general overview on the use of silk protein in biomaterials, with a focus on skeletal tissue engineering. Copyright

Evaluation of extracellular matrix formation in polycaprolactone and starch-compounded polycaprolactone nanofiber meshes when seeded with bovine articular chondrocytes

Cartilage defects are a major health problem. Tissue engineering has developed different strategies and several biomaterial morphologies, including natural-based ones, for repairing these defects. We used electrospun polycaprolactone (PCL) and starch-compounded PCL (SPCL) nanofiber meshes to evaluate extracellular matrix (ECM) formation by bovine articular chondrocytes (BACs). The main aim of this work was to evaluate the suitability of PCL and SPCL nanofiber meshes in chondrocyte cultures, and their capability to produce ECM when seeded onto these nanostructured materials. The effect of culture conditions (static vs dynamic) on ECM formation was also assessed. BACs were seeded onto PCL and SPCL nanofiber meshes using a dynamic cell-seeding procedure and cultured under static or dynamic conditions for 4 weeks. Constructs were characterized using scanning electron microscopy, histology, immunolocalization of collagen types I and II, and glycosaminoglycan (GAG) quantification. Results show an extensive cell colonization of the entire nanofiber mesh, for both materials, and that chondrocytes presented typical spherical morphology. Some degree of cell infiltration inside the nanofiber meshes was noticeable for both materials. ECM formation and GAG were detected throughout the materials, evidencing typical construct maturation. PCL and SPCL nanofiber meshes are suitable as supports for ECM formation and therefore are adequate for cartilage tissue-engineering approaches.

Thermally responsive polymeric hydrogel brushes: synthesis, physical properties and use for the culture of chondrocytes

Hydrogel brushes are materials composed of a water-swollen network, which contains polymer chains that are grafted with another polymer. Using a thermally responsive polymer, poly(N-isopropyl acrylamide) (polyNIPAM), as the graft component we are able to maintain the critical solution temperature (Tcrit), independent of the overall composition of the material, at approximately 32°C. The change in swelling at Tcrit is a function of the amount of polyNIPAM in the system. However, there is a much smaller change in the surface contact angles at Tcrit. PolyNIPAM-based materials have generated considerable interest, as ‘smart’ substrates for the culture of cells and here, we show the utility of hydrogel brushes in cell culture. Chondrocytes attached to the hydrogel brushes and yielded viable cell cultures. Moreover, the chondrocytes could be released from the hydrogel brushes without the use of proteases by reducing the temperature of the cultures to below Tcrit to induce a change in the conformation of the polyNIPAM chain at Tcrit. The importance of the crosslink hydrogel component is illustrated by significant changes in cell attachment/cell viability as the crosslink density is changed.

Preparation and in vivo efficient anti-infection property of GTR/GBR implant made by metronidazole loaded electrospun polycaprolactone nanofiber membrane.

Infection is the major reason of GTR/GBR membrane failure in clinical application. In this work, we developed GTR/GBR nanofiber membranes with localized drug delivery function to prevent infection. Metronidazole (MNA), an antibiotic, was successfully incorporated into electrospun polycaprolactone (PCL) nanofibers at different concentrations (0, 1, 5, 10, 20, 30, and 40 wt% polymer). To obtain the optimum anti-infection membrane, we systematically investigated the physical-chemical and mechanical properties of the nanofiber membranes with different drug contents. The interaction between PCL and MNA was identified by molecular dynamics simulation. MNA released in a controlled, sustained manner over 2 weeks and the antibacterial activity of the released MNA remained. The incorporation of MNA improved the hydrophilicity and in vitro biodegradation rate of PCL nanofibers. The nanofiber membranes allowed cells to adhere to and proliferate on them and showed excellent barrier function. The membrane loaded with 30% MNA had the best comprehensive properties. Analysis of subcutaneous implants demonstrated that MNA-loaded nanofibers evoked a less severe inflammatory response than pure PCL nanofibers. These results demonstrate the potential of MNA-loaded nanofiber membranes as GTR/GBR membrane with antibacterial and anti-inflammatory function for extensive biomedical applications.

Rat osteogenic sarcoma cells: Comparison of the effects of prostaglandins E1, E2, I2 (prostacyclin), 6-keto F1α and thromboxane B2 on cyclic AMP production and adenylate cyclase activity

Abstract The effects of prostaglandins (PGs) E 1 , E 2 , I 2 (prostacyclin), 6-keto F 1α and thromboxane (Tx) B 2 were compared in freshly isolated cells from a rat osteogenic sarcoma and in membranes from cultured cells of the same tumour. Cyclic AMP production was measured in cells and adenylate cyclase activity was measured in cell membranes. In both systems PGI 2 was less potent than either of the PGEs, and both TxB 2 and 6-keto PGF 1α were only weak agonists. These effects on bone-derived cells suggest that PGI 2 is unlikely to be a potent bone resorbing agent. Resitance experiments suggested that all the PGs share the same receptor site which appears distinct from the site of action of parathyroid hormone.

The osteogenic response of mesenchymal stromal cells to strontium-substituted bioactive glasses

Bioactive glasses are known to stimulate bone healing, and the incorporation of strontium has the potential to increase their potency. In this study, calcium oxide in the 45S5 bioactive glass composition was partially (50%, Sr50) or fully (100%, Sr100) substituted with strontium oxide on a molar basis. The effects of the substitution on bioactive glass properties were studied, including density, solubility, and in vitro cytotoxicity. Stimulation of osteogenic differentiation was investigated using mesenchymal stromal cells obtained from rat bone marrow. Strontium substitution resulted in altered physical properties including increased solubility. Statistically significant reductions in cell viability were observed with the addition of bioactive glass powders to culture medium. Specifically, addition of ≥ 13.3 mg/ml of 45S5 bioactive glass or Sr50, or ≥ 6.7 mg/ml of Sr100, resulted in significant inhibition. Real‐time PCR analyses detected the upregulation of genes associated with osteoblastic differentiation in the presence of all bioactive glass compositions. Some genes, including Alpl and Bglap, were further stimulated in the presence of Sr50 and Sr100. It was concluded that strontium‐substituted bioactive glasses promoted osteogenesis in a differentiating bone cell culture model and, therefore, have considerable potential for use as improved bioactive glasses for bone tissue regeneration.

Devitrification of ionomer glass and its effect on the in vitro biocompatibility of glass-ionomer cements

The effects of devitrification of an ionomer glass with a molar composition 4.5SiO(2).3Al(2)O(3).1.5P(2)O(5).3CaO.2CaF(2) on cement formation and in vitro biocompatibility were investigated. Differential thermal analysis was used to study the phase evolution in the glass, and to determine the heat treatments for production of glass-ceramics. X-ray diffraction patterns from glass frit heat-treated at 750 degrees C for 2h contained peaks corresponding to apatite (JCPDS 15-876), whereas for samples heat-treated at 950 degrees C for 2h apatite and mullite (JCPDS 15-776) were the major phases detected. Transmission electron microscopy (TEM) confirmed that apatite and apatite-mullite phases were present after heat treatments at 750 degrees C and 950 degrees C respectively. Glass and glass-ceramics were ground to prepare <45microm powders and glass ionomer cements were produced using a ratio of 1g powder: 0.2g PAA: 0.3g 10% m/v tartaric acid solution in water. In vitro biocompatibility was evaluated using cultured rat osteosarcoma (ROS) cells. Scanning electron microscopy (SEM) showed that cells colonised the surfaces of cements prepared using untreated ionomer glass and glass crystallised to form apatite (750 degrees C/2h). However, quantitative evaluation using MTT and total protein assays indicated that more cell growth occurred in the presence of cements prepared using ionomer glasses crystallised to apatite than cements prepared using untreated glass. The least cell growth and respiratory activity was observed on cements made with crystallised glass containing both apatite and mullite. It was concluded that the controlled devitrification of ionomer glasses could be used to produce GIC bone cements with improved biocompatibility.

Fabrication and evaluation of electrospun PCL–gelatin micro-/nanofiber membranes for anti-infective GTR implants

Infection is the major reason for GTR/GBR membrane failure in clinical applications. In this work, we developed GTR/GBR membranes with localized drug delivery function to prevent infection. Hierarchical membranes containing micro- and nano-fibers were fabricated. The effects of the incorporation of gelatin and loading content of metronidazole (MNA) (0, 5, 10, 20, 30, and 40 wt% polymer) on the properties of the electrospun membranes were investigated. The interaction between PCL and MNA was identified by molecular dynamics simulation. MNA was released in a controlled manner, and the antibacterial activity of the released MNA remained. The incorporation of gelatin and MNA improved the hydrophilicity, biocompatibility, and in vitro biodegradation rate of PCL nanofibers. The electrospun membranes allowed cells to adhere to and proliferate on them and showed excellent barrier function. The membrane loaded with 30% MNA had the best comprehensive properties. Subcutaneous implantation results demonstrated that MNA-loaded membranes evoked a less severe inflammatory response than pure PCL nanofibers. These results demonstrated the potential of MNA-loaded membranes as GTR/GBR membranes with antibacterial and anti-inflammatory functions for biomedical applications.

Influence of the extracellular matrix on the frictional properties of tissue-engineered cartilage

Low-friction surfaces are critical for efficient joint articulation. The tribological properties of articular cartilage have been studied extensively in native tissue and joints. Despite their importance, very few studies have examined the frictional properties of tissue-engineered cartilage. We have therefore reviewed the relationship between composition, structure and friction in tissue-engineered cartilage.