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Injuries to articular cartilage are frequently difficult to repair, in part because of the poor regenerative capacity of this tissue. To date, no successful system for complete regeneration of the most challenging cartilage defects has been demonstrated. The aim of this work was to develop functionalised hydrogels at the nanoscale by Layer-by-Layer (LbL) assembly to promote cartilage healing. Hydrogels, based on sodium alginate (NaAlg) and gelatin (G), were prepared by an external gelation method consisting of CaCl2 diffusion and genipin addition for G crosslinking. Successively, hydrogels were coated with G to obtain a positive charge on the surface, then functionalised by LbL assembly to create 16 nanolayers, based on poly(styrene sulfonate)/poly(allyl amine) (PSS/PAH), including a specific peptide sequence (CTATVHL) and transforming growth factors β1 (TGF-β1). Physico-chemical properties were evaluated by XPS, ATR-FTIR and rheological analyses while in vitro cytocompatibility was studied using bovine articular chondrocytes (BAC). XPS spectra showed N1s and S2p peaks, indicating that PAH and PSS have been introduced with success. ATR-FTIR indicated the specific PAH and PSS absorption peaks. Finally, the biomolecule incorporation influenced positively the processes of BAC adhesion and proliferation, and glycosamynoglycan secretion. The functionalised alginate-based hydrogels described here are ideally suited to chondral regeneration in terms of their integrity, stability, and cytocompatibility.

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. © 2015 The Authors. Journal of Tissue Engineering and Regenerative Medicine Published by John Wiley & Sons Ltd.

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.

Poly[2-ethyl(2-pyrrolidone) methacrylate] and hyaluronic acid hydrogels were synthesized via free-radical polymerization of 2-ethyl(2-pyrrolidone) methacrylate, hyaluronic acid and different crosslinkers. The ability of these hydrogels to induce apatite formation by incubating in simulated body fluid was investigated. The effect of hyaluronic acid content, crosslinkers and immersion time on mineralization behaviour and interface properties as well as the metabolic activity of different cultured cells were also determined. The bioactivity of the poly[2-ethyl(2-pyrrolidone) methacrylate] and hyaluronic acid hydrogels along with cell viability data indicated their potential application in bone tissue engineering.

Three novel borosilicate bioactive glasses (BBGs) of general formula of 0.05Na2O·0.35x·0.20B2O3·0.40SiO2 (molar ratio, where x = MgO or CaO or SrO) were prepared and used to investigate the effect of crystallization on their properties including cytotoxicity. The three postmelt compositions were determined using X-ray fluorescence spectroscopy, and crystallization events were studied using differential thermal analysis and X-ray diffraction. This information was used to determine heat treatments to prepare glass-ceramics by controlled crystallization. X-ray diffraction analysis and Fourier transform infrared spectroscopy showed that, after higher heat treatment temperatures (800–900 °C), borosilicate bioactive glass-ceramics (BBGCs) contained mainly borate and silicate crystalline phases. Specifically, BBG-Mg, BBG-Ca, and BBG-Sr glass-ceramics detected the presence of magnesium silicate-Mg2(SiO3)2 and magnesium borate-Mg2B2O5; wollastonite-2M-CaSiO3 and calcium borate-Ca(BO2)2; and strontium silicate-SrSiO3 and strontium borate-Sr2B2O5, respectively. In vitro cytotoxicity tests were performed using the mouse fibroblast cell line (L929). Glass and glass ceramic at concentrations lower than 50 mg/mL did not exhibit any level of cytotoxicity when compared with the control. However, quantitative evaluation indicated that greater cell growth occurred in the presence of materials with crystalline phases. Control of BBGs crystallization may therefore be used to influence the biocompatibility of these glass-ceramic systems., Portuguese Foundation for Science and Technology (Ph.D. Grant BD/73162/2010), European Union’s Seventh Framework Programme (FP7/2007-2013) under Grant No. REGPOT-CT2012-31633-POLARIS and UK EPSRC Centre for Innovative Manufacturing of Medical Devices-MeDe Innovation (EPSRC Grant EP/K029592/1)

PUBLISHED, The aim of this study was to determine whether or not biochemical markers can be used as surrogate measures for the mechanical quality of tissue engineered cartilage. The biochemical composition of tissue engineered cartilage constructs were altered by varying either (i) the initial cell seeding density of the scaffold (seeding density protocol) or (ii) the length of time the engineered tissue was cultured (culture period protocol). The aggregate or Young?s moduli of the constructs were measured (by confined or unconfined compression respectively), and compared with the composition of the extracellular matrix by quantitative measurement of the glycosaminoglycan (GAG), hydroxyproline, collagen I and collagen II and collagen cross-links. The aggregate modulus correlated positively with both GAG and collagen II content, but not with collagen I content. Young?s modulus correlated positively with GAG, collagen II and collagen I content, and the ratio of mature to immature cross-links, but not with hydroxyproline content. These results suggested that hydroxyproline may be an unreliable indicator of mechanical quality of tissue engineered cartilage, and that a measure of collagen II and GAG content is required to predict the biomechanical quality of tissue engineered cartilage., This work was funded by the European Union (EU) under the SCAFCART project (contract G5RD-CT-1999-00050). APH is funded in part by an endowed chair from the UK Arthritis Research Campaign.

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 ( T crit ), independent of the overall composition of the material, at approximately 32°C. The change in swelling at T crit is a function of the amount of polyNIPAM in the system. However, there is a much smaller change in the surface contact angles at T crit . 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 T crit to induce a change in the conformation of the polyNIPAM chain at T crit . The importance of the crosslink hydrogel component is illustrated by significant changes in cell attachment/cell viability as the crosslink density is changed.

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

For guided tissue regeneration (GTR) membrane, synchronization of the membrane biodegradation rate and tissue regeneration rate is important. Besides, the major reason for GTR membrane failure in clinical application is infection which can be prevented by loading anti-bacterial drug. To realize the consistency in membrane degradation rate and tissue regeneration rate of the anti-infective membrane, we developed metronidazole-loaded electrospun poly(ɛ-caprolactone)-gelatin nanofiber membranes with different poly(ɛ-caprolactone)/gelatin ratios (95:5, 90:10, 80:20, 70:30, 60:40, and 50:50). Homogeneous nanofibers were successfully fabricated. The mechanical strength of the membranes increased with the poly(ɛ-caprolactone) content, while the hydrophilicity decreased. The controlled and sustained release of metronidazole from all the membranes prevented the colonization of anaerobic bacteria. At all poly(ɛ-caprolactone)/gelatin ratios, all the membranes presented good biocompatibility while the increase of gelatin content resulted in enhanced cell adhesion and proliferation. Subcutaneous implantation in rabbits for 8 months demonstrated that all the membranes showed good biocompatibility without infection. Both in vitro and in vivo results showed that the biodegradation rate of the membranes was accelerated with the increase of gelatin content. The biodegradation rate and biocompatibility of the membranes can be adjusted by changing the PCL/gelatin ratio. The optimal membrane can be chosen based on the patient and tissue type to realize the synchronization of membrane degradation with tissue regeneration for the best treatment effect.

We read with interest an article by Ando et al. [1] entitled “Clonal analysis of synovial fluid stem cells to characterize and identify stable mesenchymal stromal cell/mesenchymal progenitor cell phenotypes in a porcine model: a cell source with enhanced commitment to the chondrogenic lineage.” The authors isolated clonal populations of mesenchymal stromal cells (MSCs) from pig synovial fluid (SF) and demonstrated good chondrogenic potential of 18 isolated clones. The authors concluded that the inherent chondrogenic phenotype of SF MSCs may be optimal for cartilage repair. Interestingly, the data of Ando et al. closely follow and support original work in the field of SF MSCs, which was unfortunately overlooked by this report. In 2000, Marinova-Mutafchieva et al. [2] showed the presence of CD44-positive, highly proliferative fibroblastic cells in arthritic SF, and, 4 years later, we demonstrated their tri-potentiality toward osteogenesis, adipogenesis and chondrogenesis at the single-cell level [3]. On average, single-cellederived SFMSCs had high levels of chondrogenicity and low levels of osteogenicity [3]. Furthermore, in this article, we showed increased numbers of MSCs in the SF of patients with osteoarthritis (OA) compared with rheumatoid arthritis; this was later explored by Tokyo [4] and Calgary groups [5] who furnished further evidence that OA may be associated with endogenous repair processes leading to an expansion of MSCs in the joint space. In our subsequent report (2008), we explored the nature of SF MSCs in health with the use of a bovine model and in early human OA [6]. We showed that in the bovine, clonally derived SF MSCs had a more

Poly(2-ethyl-(2-pyrrolidone)methacrylate)-hyaluronic acid hydrogels based on the free radical polymerization of 2-ethyl-(2-pyrrolidone)methacrylate combined with hyaluronic acid, using N,N′-methylenebisacrylamide or triethylene glycol dimethacrylate, as cross-linking agents, were considered for tissue engineering applications. Bovine articular chondrocytes were seeded onto the poly(2-ethyl-(2-pyrrolidone)methacrylate)-hyaluronic acid hydrogels, under orbital agitation, for a total of 40 days. The engineered cell-constructs were characterized according to cell proliferation, morphology and distribution as well as the biochemical composition of the tissue formed. The chondrocytes were found to be attached and presented a typical spherical morphology. Cells were able to proliferate and synthesize a hyaline-like matrix rich in glycosaminoglycans and collagen type II which were mainly located on the superficial area. Increased content of individual components poly(2-ethyl-(2-pyrrolidone)methacrylate) and hyaluronic acid, in triethylene glycol dimethacrylate-cross-linked networks led to enhanced cell distribution and total glycosaminoglycans content, supporting their potential application for the repair of cartilaginous tissues.

Summary Objective Interleukin-1β (IL-1β) is involved in the up-regulation of matrix metalloproteinases (MMPs) leading to cartilage degradation. Cannabinoids are anti-inflammatory and reduce joint damage in animal models of arthritis. This study aimed to determine a mechanism whereby the synthetic cannabinoid WIN-55,212-2 mesylate (WIN-55) may inhibit cartilage degradation. Methods Effects of WIN-55 were studied on IL-1β stimulated production of MMP-3 and -13 and their inhibitors TIMP-1 and -2 in human chondrocytes. Chondrocytes were obtained from articular cartilage of patients undergoing total knee replacement. Chondrocytes were grown in monolayer and 3D alginate bead cultures. Real-time polymerase chain reaction (PCR) was used to determine the gene expression of MMP-3, -13, TIMP-1 and -2 and Enzyme Linked Immunosorbent Assay (ELISA) to measure the amount of MMP-3 and MMP-13 protein released into media. Immunocytochemistry was used to investigate the expression of cannabinoid receptors in chondrocyte cultures. Results Treatment with WIN-55 alone or in combination with IL-1β, decreased or abolished MMP-3, -13, TIMP-1 and -2 gene expression in human chondrocyte monolayer and alginate bead cultures in both a concentration and time dependent manner. WIN-55 treatment alone, and in combination with IL-1β, reduced MMP-3 and -13 protein production by chondrocytes cultured in alginate beads. Immunocytochemistry demonstrated the expression of cannabinoid receptors in chondrocyte cultures. Conclusion Cannabinoid WIN-55 can reduce both basal and IL-1β stimulated gene and protein expression of MMP-3 and -13. However WIN-55 also decreased basal levels of TIMP-1 and -2 mRNA. These actions of WIN-55 suggest a mechanism by which cannabinoids may act to prevent cartilage breakdown in arthritis.

Novel chitosan/polybutylene succinate fibre-based scaffolds (C-PBS) were seeded with bovine articular chondrocytes in order to assess their suitability for cartilage tissue engineering. Chondrocytes were seeded onto C-PBS scaffolds using spinner flasks under dynamic conditions, and cultured under orbital rotation for a total of 6 weeks. Non-woven polyglycolic acid (PGA) felts were used as reference materials. Tissue-engineered constructs were characterized by scanning electron microscopy (SEM), hematoxylin-eosin (H&E), toluidine blue and alcian blue staining, immunolocalization of collagen types I and II, and dimethylmethylene blue (DMB) assay for glycosaminoglycans (GAG) quantification at different time points. SEM showed the chondrocytes' typical morphology, with colonization at the surface and within the pores of the C-PBS scaffolds. These observations were supported by routine histology. Toluidine blue and alcian blue stains, as well as immunohistochemistry for collagen types I and II, provided qualitative information on the composition of the engineered extracellular matrix. More pronounced staining was observed for collagen type II than collagen type I. Similar results were observed with constructs engineered on PGA scaffolds. These also exhibited higher amounts of matrix glycosaminoglycans and presented a central region which contained fewer cells and little matrix, a feature that was not detected with C-PBS constructs., J. T. O. would like to acknowledge the Portuguese Foundation for Science and Technology (FCT) for his grant (SFRH/BD17135/2004). The authors would like to thank Dr. Chris Hill for his help with the SEM analysis. This work was carried out under the scope of the European NoE EXPERTISSUES (NMP3-CT-2004-500283).

Aggrecan is one of the two major constituents of articular cartilage, and during diseases such as osteoarthritis (OA) it is subject to degradation by proteolytic enzymes. The primary proteases responsible for aggrecan cleavage are the aggrecanases, identified as members of the ADAMTS family of proteases, which are upregulated in response to inflammatory stimuli. It is uncertain which of the six aggrecanases (ADAMTS-1, -4, -5, -8, -9 and -15) are primarily responsible for the degradation of aggrecan in human cartilage. Here we show that four of the six aggrecanases are expressed in immortalized chondrocyte cell-lines and can be upregulated in response to inflammatory cytokines. Using RNA interference, we demonstrate robust knock-down of ADAMTS-5 and -9 expression in these cells and, by culturing them on three-dimensional (3D) scaffolds, show that reduction in expression of ADAMTS-5 enzyme results in an increase in matrix deposition. These data suggest that the quality of tissue-engineered cartilage matrix might be improved by targeted depletion of aggrecanase expression. Moreover, this work also provides further evidence that ADAMTS-5 may be a therapeutic target in the treatment of arthritic disease.

Naturally derived polymers have been extensively used in scaffold production for cartilage tissue engineering. The present work aims to evaluate and characterize extracellular matrix (ECM) formation in two types of chitosan-based scaffolds, using bovine articular chondrocytes (BACs). The influence of these scaffolds’ porosity, as well as pore size and geometry, on the formation of cartilagineous tissue was studied. The effect of stirred conditions on ECM formation was also assessed. Chitosan-poly(butylene succinate) (CPBS) scaffolds were produced by compression moulding and salt leaching, using a blend of 50% of each material. Different porosities and pore size structures were obtained. BACs were seeded onto CPBS scaffolds using spinner flasks. Constructs were then transferred to the incubator, where half were cultured under stirred conditions, and the other half under static conditions for 4 weeks. Constructs were characterized by scanning electron microscopy, histology procedures, immunolocalization of collagen type I and collagen type II, and dimethylmethylene blue assay for glycosaminoglycan (GAG) quantification. Both materials showed good affinity for cell attachment. Cells colonized the entire scaffolds and were able to produce ECM. Large pores with random geometry improved proteoglycans and collagen type II production. However, that structure has the opposite effect on GAG production. Stirred culture conditions indicate enhancement of GAG production in both types of scaffold., M.L. Alves da Silva would like to acknowledge the Portuguese Foundation for Science and Technology (FCT) for her grant (SFRH/BD/28708/2006), Marie Curie Actions-ALEA JACTA EST (MEST-CT-2004-008104), European NoE EXPERTISSUES (NMP3-CT-2004-500283), IP GENOSTEM (LSHB-CT-2003-503161) and CARTISCAFF (POCTI/SAUIBMA/58982)

Introduction The morphology of a substrate on which cells are seeded is known to affect cells in multiple ways including morphology, proliferation differentiation and matrix production. However, it is difficult to characterise the effects of mechanical loading on cells seeded in scaffolds of different morphology but identical chemistry. The aim of this project is to create polyurethane foam and fibre scaffolds from the same linear polyurethane and to analyse the effect of scaffold morphology on mechanical properties, cell viability, and cell morphology. The current work aims to produce scaffolds of sufficient mechanical strength for mechanical stimulation experiments. Materials and Methods Foams were fabricated from a polypropylene oxide polyol and a toluene diisocyanate (TDI) by the direct foaming technique. Fibres were fabricated by dissolving the foam overnight in a 20wt% solution of dimethylacetamide under stirring conditions. The fibres were spun using an electrospinning rig with a 15kV electric field, 30cm working distance, flow rate of 3.5ml/hr and collected on a rotating drum. MC-3T3-E1 osteoblastic cells were seeded onto 2cm diameter, thin discs of each scaffold type at a density of 100 x 10 cells per sample. The seeded scaffolds were then left for 1hr to allow cell attachment before immersion in 3ml of media and culture for 21 days with a media change every 3 days. The distribution of viable cells was analysed by an assay of metabolic activity (MTS). Results Both foam and fibre morphologies are suitable for support of cell growth over medium term culture periods (21 days). Cell viability analysis showed no significant difference between the two morphologies. Microscopy analysis indicated cell attachment and survival within the pores of the foam scaffold with cells adhering to the struts. Both scaffolds can be cut into disk or dumbbell shapes suitable for mechanical stimulation in compression or tension respectively.

Together, the chondrocyte and its pericellular matrix have been collectively termed the chondron. Current opinion is that the pericellular matrix has both protective and signalling functions between chondrocyte and extracellular matrix. Formation of a native chondrocyte pericellular matrix or chondron structure might therefore be advantageous when tissue engineering a functional hyaline cartilage construct. The presence of chondrons has not been previously described in cartilage engineered on a scaffold. In this paper, we describe a modified immunochemical method to detect collagen VI, a key molecular marker for the pericellular matrix, and an investigation of type VI collagen distribution in engineered hyaline cartilage constructs. Cartilage constructs were engineered from adult human or bovine hyaline chondrocytes cultured on sponge or nonwoven fiber based HYAFF 11 scaffolds. Type VI collagen was detected in all constructs, but a distinctive, high-density, chondron-like distribution of collagen VI was present only in constructs exhibiting additional features of hyaline cartilage engineered using nonwoven HYAFF 11. Chondron structures were localized in areas of the extracellular matrix displaying strong collagen II and GAG staining of constructs where type II collagen composed a high percentage (over 65%) of the total collagen.

Adenylate cyclase [ATP pyrophosphate lyase (cyclizing), EC 4.6.1.1] was shown to be present in cultured human articular chondrocytes. Optimal conditions of incubation time, protein and substrate concentrations and pH were determined in whole cell lysates. Maximal activity occurred at pH 8.5 with no decrease in activity up to pH 10.0. Adenylate cyclase activity of particulate membrane preparations was enhanced by the addition of crude cytosol preparations. The prostaglandins E1, E2, F1 alpha, F2 alpha, D2, B1, B2, A1 and A2, as well as adrenaline and isoprenaline, stimulated adenylate cyclase derived from either adult or foetal chondrocytes. No significant stimulation was observed in the presence of human calcitonin or glucagon. Bovine parathyroid hormone always significantly stimulated the adenylate cyclase derived from foetal chondrocytes, but not from adult chondrocytes. Preincubation of the chondrocytes in culture with indomethacin and with or without supernatant medium from cultured mononuclear cells increased the responsiveness of the adenylate cyclase to prostaglandin E1.

Interleukin-1 (IL-1) is the name given to a family of related proteins showing a variety of activities. It was originally shown to be produced by monocytes and macrophages but is now known to be produced by numerous cell types, including synovial cells. From the point of view of arthritis, its most interesting activities are those on connective tissue cells in vitro. These include stimulation of production of prostaglandins, plasminogen activator and metalloproteinases such as collagenase and proteoglycanase. IL-1 is also mitogenic for synoviocytes and bone cells, and can alter rates of production of extracellular matrix constituents. The presence of IL-1 in synovial fluids from rheumatoid and osteoarthritic joints and its actions on connective tissues in vitro suggest that IL-1 may play an important role in the pathogenesis of arthritis. There are several potential cellular sources of IL-1 in the inflamed rheumatoid joint and interactions between these cells, T lymphocytes and plasma cells may continually induce IL-1 so contributing to the chronicity of the disease. The mechanism of action of IL-1 on connective tissue cells is at present uncertain though preliminary studies suggest that IL-1 may induce cellular responses by stimulating phosphoinositide turnover and possibly protein kinase C activity.

1. Homogenates of whole tissues were shown to contain both intracellular and extracellular factors that affected particulate adenylate cyclase activity in vitro. Factors present in the extracellular fluids produced an inhibition of basal, hormone- and fluoride-stimulated enzyme activity but factors present in the cell cytosol increased hormone-stimulated activity with relatively little effect on basal or fluoride-stimulated enzyme activity. 2. The existence of this cytosol factor or factors was investigated using freshly isolated human platelets, freshly isolated rat hepatocytes, and cultured cells derived from rat osteogenic sarcoma, rat calvaria, mouse melanoma, pig aortic endothelium, human articular cartilage chondrocytes and human bronchial carcinoma (BEN) cells. 3. The stimulation of the hormone response by the cytosol factor ranged from 60 to 890% depending on the tissue of origin of the adenylate cyclase. 4. In each case the behaviour of the factor was similar to the action of GTP on that particular adenylate cyclase preparation. 5. No evidence of tissue or species specificity was found, as cytosols stimulated adenylate cyclase from their own and unrelated tissues to the same degree. 6. In the human platelet, the inclusion of the cytosol in the assay of adenylate cyclase increased the rate of enzyme activity in response to stimulation by prostaglandin E1 without affecting the amount of prostaglandin E1 required for half-maximal stimulation or the characteristics of enzyme activation by prostaglandin E.

The effects of prostaglandins (PG's) 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 PGE's, 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 PG's share the same receptor site which appears distinct from the site of action of parathyroid hormone.