Combinatorial morphogenetic and mechanical cues to mimic bone development for defect repair.

Endochondral ossification during long bone development and natural fracture healing initiates by mesenchymal cell condensation, directed by local morphogen signals and mechanical cues. Here, we aimed to mimic development for regeneration of large bone defects. We hypothesized that engineered human mesenchymal condensations presenting transforming growth factor-ß1 (TGF-ß1) and/or bone morphogenetic protein-2 (BMP-2) from encapsulated microparticles promotes endochondral defect regeneration contingent on in vivo mechanical cues. Mesenchymal condensations induced bone formation dependent on morphogen presentation, with BMP-2 + TGF-ß1 fully restoring mechanical function. Delayed in vivo ambulatory loading significantly enhanced the bone formation rate in the dual morphogen group. In vitro, BMP-2 or BMP-2 + TGF-ß1 initiated robust endochondral lineage commitment. In vivo, however, extensive cartilage formation was evident predominantly in the BMP-2 + TGF-ß1 group, enhanced by mechanical loading. Together, this study demonstrates a biomimetic template for recapitulating developmental morphogenic and mechanical cues in vivo for tissue engineering.

Critical Airway Compromise due to a Massive Vagal Schwannoma.

We describe the case of a 37-year-old man with a slowly enlarging neck lump and compressive symptoms. He presented to a separate institution 10 years prior where an observational approach was advocated. Following preoperative investigations and embolization, an 11cm vagal schwannoma was excised and vagus nerve was sacrificed. Although conservative management is appropriate for a select patient population, surgical excision is treatment of choice for cervical neurogenic tumours and paraganglionomas and must be considered in young patients or rapidly expanding tumours to avoid compressive symptoms, as in this case.

Antimicrobial peptide LL-37 on surfaces presenting carboxylate anions.

Antimicrobial peptides (AMPs) are part of the immune system in a wide range of organisms. They generally carry positive charges under physiological conditions, allowing them to accumulate on the negatively charged bacterial membrane as the first step of bactericidal action. The concentration range of AMPs necessary for rapid killing of bacteria tested in vitro is much higher than levels found at epithelial surfaces and body fluids in vivo, and close to the a level that is toxic to the host cells. It is likely that AMPs in vivo are localized and act cooperatively to enhance antimicrobial activity, while the global concentration is low thus demonstrating low toxicity to host cells. Herein we employed well-defined mixed self-assembled monolayers (SAMs) to localize LL-37, one of the most studied AMPs, via electrostatic interactions. We systematically varied the surface density of LL-37, and found that the immobilized AMPs not only attracted bacteria Pseudomonas aeruginosa to the surface, but also killed nearly all bacteria when above a threshold density. More significantly, the AMPs displayed low toxicity to human corneal epithelial cells. The results indicated that localization of AMPs on suitable polyanion substrates facilitated the bactericidal activity while minimizing the cytotoxicity of AMPs.

The effect of elevated extracellular glucose on migration, adhesion and proliferation of SV40 transformed human corneal epithelial cells.

PURPOSE: To examine the effect of elevated extracellular glucose, thus simulating diabetes, on migration, adhesion and proliferation of SV40 transformed human corneal epithelial (HCE) cells. METHODS: HCE cells were maintained in serum supplemented media containing 5 mM, 17.5 mM or 38 mM D-glucose. Cell migration was determined using Blind well chambers fitted with fibronectin/collagen I coated filters. In adhesion experiments, cells were allowed to adhere to extracellular matrix protein-coated wells for 90 min at 37 degrees C. Non-adherent cells were removed by washing, then the fluorochrome calcein-AM was added to quantitate the number of attached cells. Proliferation was determined by plating the cells at low density, then quantitating viable cells with calcein-AM 5 to 7 days later. RESULTS: Raising extracellular glucose from 5 mM to 17.5 mM significantly increased cell migration by 42%. When glucose was further raised to 38 mM, migration was not significantly different from that in 5 mM glucose. Adhesion to fibronectin and collagen I (but not IV) was significantly increased (62% and 32% respectively) when cells were cultured in 17.5 mM glucose. Similarly, proliferation was increased by 44%. Adhesion and proliferation tended to be decreased at 38 mM compared to 17.5 mM glucose, but not significantly so. In the presence of 5 mM glucose and mannitol (12.5 mM or 33 mM), neither migration, adhesion nor proliferation were significantly different from that in 5 mM glucose alone. CONCLUSION: Elevated extracellular glucose modulates migration, adhesion and proliferation of HCE cells. The effects are dependent on the concentration of glucose and are not due to changes in osmolality since mannitol failed to produce similar results. Our in vitro findings suggest that high-glucose effects may directly contribute to the etiology of impaired corneal wound healing in diabetes.

Effect of substance P, insulin-like growth factor-1 and vasoactive intestinal polypeptide on corneal re-epithelialization in galactosemic rats.

PURPOSE: The purpose of the study was to examine the effect of topical application of substance P (SP), insulin-like growth factor-1 (IGF-1) and vasoactive intestinal polypeptide (VIP) on corneal re-epithelialization in galactosemic rats. METHODS: Experimental galactosemia was induced by feeding a diet containing 30% galactose for 4-6 months. The corneal epithelium was debrided bi-laterally by scraping with a blunted scalpel blade. One eye (control) received only a saline solution whilst the other eye received a solution of SP and/or IGF-1 or VIP. A single drop of control or test solution was administered 4 times daily until wound closure. Corneas were stained with fluorescein and videotaped under ultraviolet illumination at regular time intervals after debridement. After digitizing the video image, the wound area was calculated using an image analysis program (NIH Image). RESULTS: Corneal re-epithelialization was significantly delayed in galactosemic compared to normal animals. Rates of healing were 1.3 +/- 0.07 mm2/hour and 1.02 +/- 0.02 mm2/hour for normal and galactosemic animals, respectively. Topical application of SP in concentrations ranging from 25 pg/ml up to 250 microg/ml had no significant effect on the rate of corneal re-epithelialization. Similarly, IGF-1 (1 microg/ml) or VIP (1 microg/ml) when applied alone did not affect re-epithelialization. Furthermore, resurfacing of the debrided area was not affected by co-application of SP (250 microg/ml) and IGF-1 or VIP. CONCLUSIONS: Independent or combined topical application of SP, VIP or IGF-1 at the concentrations tested, does not modulate corneal epithelial wound healing in rats with galactosemia induced by 30% galactose.

Time course of action of pertussis toxin to block the inhibition of stimulated insulin release by norepinephrine.

Male Sprague-Dawley rats were injected ip with 1 microgram pertussis toxin (PTX)/100 g BW. The rats were killed 24, 48, and 72 h after injection, and their pancreases were removed. At each time point, insulin secretion by isolated islets was measured under basal and glucose-stimulated conditions and in the absence and presence of norepinephrine. cAMP levels were measured under basal and forskolin-stimulated conditions in the absence and presence of norepinephrine. PTX-induced ADP ribosylation of Gi/Go proteins in vivo was monitored by ADP ribosylation in vitro using PTX and 32P-labeled NAD and also by Western blotting. At 24 h, 1 microM norepinephrine inhibited glucose-stimulated insulin secretion by 92% in the control islets, but by only 53% in the PTX-treated islets; at 48 h, norepinephrine still inhibited secretion (by 40%) in the PTX-treated islets; at 72 h, the inhibitory effect of norepinephrine was abolished. Therefore, contrary to recent suggestions, all of the effect of norepinephrine to inhibit insulin release is PTX sensitive. The effects of PTX on the ability of norepinephrine to lower cAMP levels were similar to those observed for the inhibition of insulin release. PTX partially blocked the effect of norepinephrine to lower cAMP levels at 24 and 48 h, and the block was complete after 72 h. The extent of the in vivo ADP ribosylation of the Gi/Go proteins, monitored at each time point by in vitro [32P]ADP-ribosylation and Western blotting, demonstrated a profound ADP ribosylation at 48 and 72 h. As detected by Western blotting, the rates of ADP ribosylation by PTX and the onset of decreased expression varied among the different G-proteins. G alpha o was virtually eliminated after only 24 h of PTX treatment. G alpha i2 was markedly affected by 48 h; G alpha i3 was little affected until 72 h.

Gi2 and Gi3 proteins mediate the inhibition of adenylyl cyclase by galanin in the RINm5F cell.

Inhibition of adenylyl cyclase activity is one of at least four mechanisms by which the neuropeptide galanin inhibits insulin secretion from pancreatic beta-cells. In a membrane preparation of the insulin-secreting cell line RINm5F, a maximally effective concentration of galanin inhibited forskolin-stimulated adenylyl cyclase activity by 30%. Pretreatment of the cells with pertussis toxin abolished the inhibitory effect of galanin, indicating the involvement of Gi or Go guanine nucleotide binding proteins (G-proteins). Because galanin receptors interact with four G-proteins (Gi1, Gi2, Gi3, and Go1), any or all of these may inhibit adenylyl cyclase. Therefore, to identify the G-protein(s) involved, antibodies raised against various G-protein alpha-subunits were used to block the inhibition of forskolin-stimulated adenylyl cyclase activity by galanin in RINm5F membrane preparations. Antisera AS/7 and EC/2, specific for G alpha i1/alpha i2 and G alpha i3, respectively, were able to significantly attenuate the inhibitory effect of galanin, whereas antisera specific for Go proteins were not. The use of additional antisera specific for the various subtypes of Gi proteins indicated that Gi2 and Gi3, but not Gi1, are involved. Simultaneous application of antisera AS/7 and EC/2 resulted in a greater attenuation of the effect of galanin than application of either antiserum alone. Thus, galanin inhibition of adenylyl cyclase activity in these cells is selectively mediated by two inhibitory G-proteins, Gi2 and Gi3.

Noradrenaline induces supersensitivity of adenylyl cyclase in NG108-15 and HT29-18 cells but not in the beta-cell RINm5F.

In some cells a supersensitive adenylyl cyclase response follows hormonal inhibition of the enzyme. We have compared the effect of pre-treatment with noradrenaline in RINm5F cells to that in cells known to demonstrate supersensitivity. In NG108-15 cells, 12 or 24 hours pre-treatment with 1-10 microM noradrenaline markedly enhanced forskolin (1 microM) stimulated cAMP accumulation compared to that in untreated cells (12 h, 429 +/- 123 vs 72 +/- 8 pmol/mg protein; 24 h 190 +/- 32 vs 82 +/- 10 for treated and untreated cells, respectively). In a second cell, HT29-18, 30 min pre-treatment with 10 microM noradrenaline enhanced forskolin (10 microM) stimulated cAMP accumulation from 47 +/- 13 pmol/mg protein to 405 +/- 109 pmol/mg protein. In contrast, pre-treatment of RINm5F cells with noradrenaline under the same conditions did not enhance the forskolin response. These data indicate that noradrenaline which induces a supersensitive adenylyl cyclase response in NG108-15 and HT29-18 cells does not induce the response in the insulin secreting cell RINm5F.

Sodium fluoride stimulates exocytosis at a late site of calcium interaction in stimulus-secretion coupling: studies with the RINm5F beta cell line.

In the insulin-secreting beta cell line RINm5F, sodium fluoride stimulated exocytosis in a concentration (5-15 mM)- and temperature-dependent manner. Depletion of aluminum with the chelator deferoxamine or addition of aluminum to the buffer failed to affect the NaF-stimulated insulin release. This suggests that stimulation of heterotrimeric G proteins or inhibition of phosphatases or other enzymes by fluoroaluminate, an analog of the phosphate moiety, is not involved in the insulinotropic action of NaF. Removal of extracellular Ca2+ suppressed the NaF-stimulated insulin release. However, nitrendipine, a blocker of L-type voltage-dependent Ca2+ channels, did not inhibit the NaF-stimulated insulin release and NaF did not cause any changes in the cytosolic free calcium concentration ([Ca2+]i). Decreasing [Ca2+]i with thapsigargin or increasing [Ca2+]i with ionomycin or a depolarizing concentration of KCl resulted in suppression or enhancement of NaF-stimulated insulin release, respectively. Furthermore, NaF enhanced Ca(2+)-induced insulin release in electrically permeabilized RINm5F cells. These findings indicate that the effect of NaF on exocytosis is dependent on [Ca2+]i, although NaF itself does not change [Ca2+]i. Inhibitors of protein kinase C, such as staurosporine and bisindolylmaleimide, in concentrations sufficient to block the effects of phorbol esters, did not attenuate the NaF-stimulated insulin release. Neither cellular cAMP content nor [3H]arachidonic acid release was increased by NaF. NaF-stimulated insulin release was synergistically enhanced by the activation of protein kinases A and C. Finally, trifluoperazine, an inhibitor of calmodulin and other Ca(2+)-binding proteins, inhibited the insulinotropic action of NaF in a concentration-dependent manner. Trifluoperazine (50 microM) and W-7 (100 microM) nullified the 10 mM NaF-stimulated insulin release. It is concluded that NaF evokes exocytosis by a novel mechanism of sensitization to Ca2+, possibly on a Ca(2+)-responsive protein that is sensitive to trifluoperazine and W-7, leading to exocytosis. Protein kinases A and C also act at this site or at a more distal point.

Mastoparan stimulates exocytosis at a Ca(2+)-independent late site in stimulus-secretion coupling. Studies with the RINm5F beta-cell line.

Mastoparan, a tetradecapeptide from wasp venom, stimulated exocytosis in a concentration-dependent manner, which was enhanced by pertussis toxin pre-treatment, in the insulin secreting beta-cell line RINm5F. Mastoparan (3-20 microM) also elevated cytosolic free calcium concentration ([Ca2+]i), a rise that was not attenuated by nitrendipine. Divalent cation-free Krebs-Ringer bicarbonate (KRB) medium with 0.1 mM EGTA nullified the mastoparan-induced increase in [Ca2+]i, suggesting that the peptide increased Ca2+ influx but not through the L-type voltage-dependent Ca2+ channel. Depletion of the intracellular Ca2+ pool did not affect the mastoparan-induced elevation of [Ca2+]i. Remarkably, in divalent cation-free KRB medium with 0.1 mM EGTA and 2 microM thapsigargin in which mastoparan reduced [Ca2+]i, the mastoparan-stimulated insulin release was similar to that in normal Ca(2+)-containing KRB medium. Inhibitors of protein kinase C, such as bisindolylmaleimide, staurosporine, and 1-O-hexadecyl-2-O-methyl-rac-glycerol did not suppress the mastoparan-stimulated insulin release. Mastoparan at 10-20 microM did not increase cellular cAMP levels, nor did mastoparan at 5-10 microM affect [3H]arachidonic acid release. In conclusion, although mastoparan increased [Ca2+]i, this increase was not involved in the stimulation of insulin release. Rather, the data suggest that mastoparan directly stimulates exocytosis in a Ca(2+)-independent manner. As GTP-binding proteins (G proteins) are thought to be involved in the process of exocytosis and as mastoparan is known to exert at least some of its effects by activation of G proteins, an action of mastoparan to activate the putative stimulatory Ge (exocytosis) protein is likely.

Inhibition of insulin secretion: a fail-safe system.

Insulin secretion from the beta-cells of the endocrine pancreas is subject to a pot-pourri of stimulatory, modulatory and inhibitory influences. beta-Cell secretion is reduced or blocked by a variety of inhibitors (including galanin, somatostatin and noradrenaline) which reach the cells either via the islet vascular system or are released locally from sympathetic and peptidergic nerves terminating in the pancreas. It is now becoming clear that among these many inhibitors, several have multiple mechanisms by which they inhibit release at the cellular level. Indeed, with multiple inhibitors (some of which are co-secreted) and multiple mechanisms of inhibition, the latter including a late effect in stimulus secretion coupling (perhaps on the exocytotic step per se), inhibition of insulin secretion has the characteristics of a fail-safe system.