Francis W. Wang

Preliminary report on the biocompatibility of a moldable, resorbable, composite bone graft consisting of calcium phosphate cement and poly(lactide-co-glycolide) microspheres ☆

We have assessed the biocompatibility of a new composite bone graft consisting of calcium phosphate cement (CPC) and poly(lactide‐co‐glycolide) (PLGA) microspheres (approximate diameter of 0.18–0.36 mm) using cell culture techniques. CPC powder is mixed with PLGA microspheres and water to yield a workable paste that could be sculpted to fit the contours of a wound. The cement then hardens into a matrix of hydroxyapatite microcrystals containing PLGA microspheres. The rationale for this design is that the microspheres will initially stabilize the graft but can then degrade to leave behind macropores for colonization by osteoblasts. The CPC matrix could then be resorbed and replaced with new bone. In the present study, osteoblast‐like cells (MC3T3‐E1 cells) were seeded onto graft specimens and evaluated with fluorescence microscopy, environmental scanning electron microscopy and the Wst‐1 assay (an enzymatic assay for mitochondrial dehydrogenase activity). Cells were able to adhere, attain a normal morphology, proliferate and remain viable when cultured on the new composite graft (CPC–PLGA) or on a control graft (CPC alone). These results suggest that our new cement consisting of CPC and PLGA microspheres is biocompatible. This is the first time that a ‘polymer‐in‐mineral’ (PLGA microspheres dispersed in a CPC matrix) cement has been formulated that is moldable, resorbable and that can form macropores after the cement has set.

A Role for γ/δ T Cells in a Mouse Model of Fracture Healing

OBJECTIVE Fractures can initiate an immune response that disturbs osteoblastic and osteoclastic cellular homeostasis through cytokine production and release. The aim of our study was to investigate gamma/delta T cells, innate lymphocytes known to be involved in tissue repair, as potential cellular components of the osteoimmune systems response to an in vivo model of bone injury. The absence of such cells or their effector cytokines influences the fate of other responder cells in proliferation, differentiation, matrix production, and ultimate callus formation. METHODS Tibia fractures were created in 60 gamma/delta T cell-deficient mice (also called delta T cell receptor [TCR]-knockout mice) and 60 control C57BL/6 mice. Analysis included radiographs, basic histology, mechanical testing, flow cytometry, and immunohistochemical localization of gamma/delta TCR-positive subsets from control animals and of CD44 expression from both groups, as well as enzyme-linked immunosorbent assay for the effector cytokines interleukin-2 (IL-2), interferon-gamma (IFNgamma), and IL-6. RESULTS Animals deficient in gamma/delta T cells demonstrated more mature histologic elements and quantitative increases in the expression of major bone (bone sialoprotein) and cartilage (type II collagen) matrix proteins and in the expression of bone morphogenetic protein 2 at a critical reparative phase. Moreover, only gamma/delta T cell-deficient animals had a decrease in the osteoprogenitor antiproliferative cytokines IL-6 and IFNgamma at the reparative phase. The result was improved stability at the repair site and an overall superior biomechanical strength in gamma/delta T cell-deficient mice compared with controls. CONCLUSION The evidence for a role of gamma/delta T cells in the context of skeletal injury demonstrates the importance of the immune systems effect on bone biology, which is relevant to the field of osteoimmunology, and offers a potential molecular platform from which to develop essential therapeutic strategies.

Novel fluorescence method for cure monitoring of epoxy resins

Abstract The fluorescence spectra of organic dyes dissolved in epoxy resins are sensitive to local viscosity. The excimer forming dyes are particularly useful as probes since the monomer emission can be used as an internal standard in the measurement. In this case, the probability of excimer formation is related to molecular mobility and hence to the microviscosity. This approach has been demonstrated on epoxy resins. In another approach, trace amounts of 1-(4-dimethylaminophenyl)-6-phenyl-1,3,5-hexatriene (DMA-DPH) and 9,10-diphenylanthracene (DPA) are added to an epoxy resin. The fluorescence intensity of DMA-DPH increases with the increase in local viscosity while the fluorescence intensity of DPA is insensitive to local viscosity and can be used as an internal standard. The ratio of the fluorescence intensities of DMA-DPH and of DPA has been measured to monitor the cure of epoxy resins.

Novel excimer fluorescence method for monitoring polymerization: 1. Polymerization of methyl methacrylate

Abstract An excimer is formed by the association of an excited molecule with another molecule in its ground state. Such an excimer is characterized by a broad structureless fluorescence which is shifted to longer wavelengths compared to the fluorescence spectrum of the isolated molecule. Intramolecular excimer fluorescence has been observed in solutions of pyrene-labelled alkanes such as 1,3-bis-(1-pyrene)propane and 1,10-bis-(1-pyrene)decane. We have measured the solvent-viscosity dependence of the intensity ratio F M F D for solutions of these pyrene-labelled alkanes in mixed solvents made of ethyl acetate and glycerol tripropionate. Here FM and FD are, respectively, the fluorescence intensity of the unassociated pyrene groups and that of the intramolecularly formed pyrene excimers. We have found that for each of the two pyrene-labelled alkanes, the ratio F M F D increases with the increase in solvent viscosity. Further, we have shown that by adding a trace amount of 1,3-bis-(1-pyrene)propane or 1,10-bis-(1-pyrene)decanee to a polymerizing system, we can measure the ratio F M F D to monitor in situ the polymerization reaction. polymerization;

Fluorescence monitoring of polarity change and gelation during epoxy cure

Abstract The fluorescence spectrum of 1-(4-dimethylaminophenyl)-6-phenyl-1,3,5-hexatriene (DMA-DPH) dissolved in a stoichiometric mixture of diglycidyl ether of bisphenol A and diethylene triamine was measured as a function of cure time at various cure temperatures. The frequency of the fluorescence maximum for DMA-DPH increased during the curing reactions because of the change in the polarity of the epoxy resin. In an isothermal cure, the fluorescence frequency increased linearly with the cure time until the gelation occurred. The total change in fluorescence frequency that occurred from the beginning of the isothermal cure to the gelation time was 1000 cm −1 and was independent of the cure temperature, implying that the chemical structure of the infinite network at the gelation time was independent of the cure temperature. The rate constant, K T , for the polarity change during an isothermal cure of the epoxy resin, defined as the rate constant for the linear increase in fluorescence frequency, was determined. The activation energy of K T was estimated to be 60 kJ mol −1 .

Changes in Conformation of Human Serum Albumin (HSA) and Gamma Globulins (γG) upon Adsorption to Polystyrene and Poly(styrene/acrolein) Latexes: Studies by Fluorescence Spectroscopy

Changes of human serum albumin (HSA) and gamma globulins (γG), labelled with 1-pyrene-carboxaldehyde (PCA) and/or with 1,3-bis(1- pyrene)-propane (BPP), resulting from interactions with polystyrene (PS) and poly(styrene/acrolein) (PSA) latexes, were investigated by fluorescence spectros copy. The proteins in solution readily exchanged with the adsorbed proteins. The fluorescence spectra of the PCA label and BPP probe, incorporated into the protein macromolecules, indicate that the protein macromolecules undergo sig nificant conformational changes on contact with the surface of the latex par ticles, and that these changes are not reversible. The internal fluidity for desorbed protein macromolecules is lower than before the interaction with the latex particles. Moreover, due to the conformational changes the PCA labels, formerly present in the hydrophilic and hydrophobic protein regions, became located predominantly in the latter. The differences in the emission spectra for the labelled proteins before attachment to the latex particles and after desorp tion were used to study the kinetics of the protein conformational changes. The dependence of the overall rate constants for protein conformational rearrange ments on the latex concentration was investigated.

Self-diffusion in concentrated polystyrene solutions measured by fluorescence recovery after photobleaching

Abstract A polystyrene polymer of narrow molecular weight distribution was carboxylated, then reduced, and finally esterified with NBD-aminohexanoic acid [6-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-aminohexanoic acid]. The self-diffusion of the NBD-labelled polystyrene polymer in concentrated solutions of the unlabelled polystyrene polymer was measured by the method of fluorescence recovery after photobleaching over a concentration range from 0.017 g/ml to 0.41 g/ml at room temperature. In the semi-dilute region, the concentration dependence of diffusion coefficient was found to be in agreement with the predictions of scaling concepts.

Reduction of sulphur from polysulphidic model compounds by the hyperthermophilic archaebacterium Pyrococcus furiosus

Abstract Several polysulphidic model compounds were studied for their reducibility by a hyperthermophilic archaebacterium, Pyrococcus furiosus . Model compounds that may represent forms of organic sulphur in coal were selected for study including model compounds with bulky alkyl and aryl groups surrounding the sulphur atoms and polymeric polysulphides. P. furiosus reduced these organic polysulphides to hydrogen sulphide with varying efficiency. The use of complex model compounds may be more realistic than simple model compounds for evaluating the potential for microbial removal of organic sulphur from coal.

Process Monitoring of Polymer Matrix Composites Using Fluorescence Probes

On-line process monitoring and control are prerequisites for more efficient and reliable manufacture of polymer matrix composites. The cure process involves complex chemical and physical changes that must be adequately controlled to consistently produce high quality products. Of various changes that occur, the viscosity is particularly important as it influences fiber wetting, uniformity of resin distribution and consolidation of the reinforcement plies. In the usual cure cycle, the viscosity of the resin initially decreases as the temperature of the resin rises owing to the higher temperature of the autoclave and heat released by the exothermic chemical reactions of the cross-linking process. Ultimately, the growing molecular weight of the polymer increases the viscosity beyond the range where flow will occur. If the resin viscosity becomes too low during this period, excess resin will seep out of the part producing an inferior product owing to resin deficiency. On the other hand, if the viscosity increases too fast the flow may be insufficient to achieve good consolidation of the plies.

Cure Monitoring for Polymer Matrix Composites

Polymer matrix composites are desirable engineering materials owing to their high strength and stiffness as well as corrosion resistance. The strength and stiffness achieved in these materials on a per unit mass basis (specific properties) surpasses that for metals by factors of up to 51. This has led to use of polymer composites in weight sensitive applications ranging from commercial and military aircraft to automobiles. Currently, the wider application of these materials is limited by product variability and the labor intensive, time consuming manufacturing processes. These factors are particularly troublesome in large volume production where the trend is towards more rapid processing that tends to exacerbate the problem of high rejection ratios. Recent advances in automated, computer controlled manufacture have been encouraging, although limited by the lack of suitable process monitoring.