Delivery of demineralized bone matrix powder using a thermogelling chitosan carrier.

Demineralized bone matrix (DBM) powder is widely used for bone regeneration due to its osteoinductivity and osteoconductivity. However, difficulties with handling, its tendency to migrate from graft sites, and lack of stability after surgery can sometimes limit the clinical utility of this material. In this work, the possibility of using a thermogelling chitosan carrier to deliver DBM powder was assessed. The DBM-thermogelling putty improved handling and formed a gel-like composite in situ at body temperature within a clinically relevant time period. The properties of the formed composite, including morphology, porosity, mechanical properties, equilibrium swelling as well as degradability, are significantly influenced by the ratio of DBM to thermogelling chitosan. The in vitro study showed that the alkaline phosphatase activity of C2C12 cells encapsulated in the composite was steadily increased with culture time. The in vivo study showed that increased DBM content in the DBM-thermogelling chitosan induced ectopic bone formation in a nude rat model. The diffusion of growth factor from the DBM-thermogelling chitosan as well as the host-implant interactions are discussed.

Enzymatic crosslinking and degradation of gelatin as a switch for bone morphogenetic protein-2 activity.

Current therapies for tissue regeneration rely on the presence or direct delivery of growth factors to sites of repair. Bone morphogenetic protein-2 (BMP-2), combined with a carrier (usually collagen), is clinically proven to induce new bone formation during spinal fusion and nonunion repair. However, due to BMP-2's short half-life and its diffusive properties, orders of magnitude above physiological levels are required to ensure effectiveness. In addition, a high dose of this multifunctional growth factor is known to induce adverse effects in patients. To circumvent these challenges, we proposed and tested a new approach for BMP-2 delivery, by controlling BMP activity via carrier binding and localized proteolysis. BMP-2 was covalently bound to gelatin through site-specific enzymatic crosslinking using a microbial transglutaminase. Binding of BMP-2 to gelatin can completely switch off BMP-2 activity, as evidenced by loss of its transdifferentiating ability toward C2C12 promyoblasts. When gelatin sequestered BMP-2 is incubated with either microbial collagenase or tissue-derived matrix metalloproteinases, BMP-2 activity is fully restored. The activity of released BMP-2 correlates with the protease activity in a dose- and time-dependent manner. This observation suggests a novel way of delivering BMP-2 and controlling its activity. This improved delivery method, which relies on a physiological feedback, should enhance the known potential of this and other growth factors for tissue repair and regeneration.

The effect of three hemostatic agents on early bone healing in an animal model.

BACKGROUND: Resorbable bone hemostasis materials, oxidized regenerated cellulose (ORC) and microfibrillar collagen (MFC), remain at the site of application for up to 8 weeks and may impair osteogenesis. Our experimental study compared the effect of a water-soluble alkylene oxide copolymer (AOC) to ORC and MFC versus no hemostatic material on early bone healing. METHODS: Two circular 2.7 mm non-critical defects were made in each tibia of 12 rabbits. Sufficient AOC, ORC or MFC was applied to achieve hemostasis, and effectiveness recorded. An autologous blood clot was applied to control defects. Rabbits were sacrificed at 17 days, tibiae excised and fixed. Bone healing was quantitatively measured by micro-computed tomography (micro-CT) expressed as fractional bone volume, and qualitatively assessed by histological examination of decalcified sections. RESULTS: Hemostasis was immediate after application of MFC and AOC, after 1-2 minutes with ORC, and >5 minutes for control. At 17 days post-surgery, micro-CT analysis showed near-complete healing in control and AOC groups, partial healing in the ORC group and minimal healing in the MFC group. Fractional bone volume was 8 fold greater in the control and AOC groups than in the MFC group (0.42 ± 0.06, 0.40 ± 0.03 vs 0.05 ± 0.01, P < 0.001) and over 1.5-fold greater than in the ORC group (0.25 ± 0.03, P < 0.05). By histology, MFC remained at the application site with minimal healing at the defect margins and early fibrotic tissue within the defect. ORC-treated defects showed partial healing but with early fibrotic tissue in the marrow space. Conversely, control and AOC-treated defects demonstrated newly formed woven bone rich in cellular activity with no evidence of AOC remaining at the application site. CONCLUSIONS: Early healing appeared to be impaired by the presence of MFC and impeded by the presence of ORC. In contrast, AOC did not inhibit bone healing and suggest that AOC may be a better bone hemostatic material for procedures where bony fusion is critical and immediate hemostasis required.

Cell delivery using an injectable and adhesive transglutaminase-gelatin gel.

In this study, we developed an injectable gelatin-transglutaminase (TGase) gel for cell delivery. The procedure provides a minimally invasive approach to deliver cells into tissue in a manner that improves localization. The results indicate gelatin-TGase to be noncytotoxic and to have adhesive properties that help localize and prevent the scattering of the cells after delivery. The in situ crosslinking between gelatin chains and endogenous collagen can create a strong attachment between the gel and tissue extracellular matrix, preventing cells from dissipation. The gelatin-TGase was also shown to maintain the carried cells to be viable and proliferative. Finally, through the adjustment of the enzymatic crosslinker concentration, the release rate of the cells into the surrounding tissue after injection was demonstrated to be controllable.

Beta-endorphin alters electrical activity of gonadotropin releasing hormone neurons located in the terminal nerve of the teleost medaka (Oryzias latipes).

Endogenous opioid peptides (EOPs) are an important class of modulators of the hypothalamo-pituitary axis; treatment with opiates leads to inhibition of GnRH and LH secretion and suppression of reproductive functions. However, little work has been done to investigate the effect of opiates on the electrical activity of GnRH neurons, which ultimately controls GnRH secretion. The purpose of the present study was to investigate the effects of the EOP beta-endorphin on electrical activity of GnRH neurons located in the terminal nerve (TN) associated with the olfactory bulb. We used an excised intact brain preparation from transgenic medaka in which green fluorescent protein (GFP) is genetically expressed in TN-GnRH neurons. These GFP-expressing neurons were then targeted for whole-cell current clamp recordings. Treatment with beta-endorphin led to changes in several characteristics of electrical activity, including depolarization of membrane potential and a decrease in spike amplitude--similar to that observed in response to depolarizing high K(+) treatment. This finding suggests a model in which beta-endorphin depolarizes membrane potential leading to Na(+)-channel inactivation, and subsequent suppression of action-potential amplitude. On the other hand, beta-endorphin had no effect on membrane potential in synaptically isolated GnRH neurons. These results suggest that beta-endorphin is acting indirectly on TN-GnRH neurons to inhibit action potential firing.

Whole-cell electrophysiology of gonadotropin-releasing hormone neurons that express green fluorescent protein in the terminal nerve of transgenic medaka (Oryzias latipes).

Gonadotropin-releasing hormone (GnRH) controls reproduction in vertebrates. Most studies have focused on the population of GnRH neurons in the hypothalamus that ultimately controls gonadal function. However, all vertebrates studied to date have two to three anatomically distinct populations of GnRH neurons that express different forms of this hormone. The purpose of the present study was to develop a new model for studying the population of GnRH neurons in the terminal nerve (TN) associated with the olfactory bulb and then to characterize their pattern of action potential firing to provide a foundation for understanding the role of these neurons in regulating reproduction. A stable line of transgenic medaka (Oryzias latipes) was generated in which a DNA construct containing the salmon GnRH (Gnrh3) promoter linked to green fluorescent protein (GFP) was expressed in TN-GnRH3 neurons. This population of GnRH neurons is located at or near the ventral surface of the brain, making them ideally situated for electrophysiological analysis. Whole-cell and loose-patch recordings in current-clamp mode were performed on these neurons from excised, intact brains of adult males in which afferent and efferent neural connections remained intact. All TN-GnRH3-GFP neurons that we recorded showed a beating pattern of spontaneous action potential firing. Action potentials were blocked by tetrodotoxin, indicating they are generated by a voltage-sensitive Na+ current; however, an oscillation in subthreshold membrane potential persisted. The present results indicate that this transgenic fish will provide an excellent model for studying the cell physiology of an extrahypothalamic population of GnRH neurons.

Rupture of fat cells using laser-generated ultra short stress waves.

BACKGROUND AND OBJECTIVES: Ultrasound-assisted liposuction has been investigated as an aid to breakup fat for its less traumatic removal, and minimizing the post-operative recovery period. This research focused on understanding the mechanism of fat interaction with laser-generated ultra short stress waves with high amplitudes. STUDY DESIGN/MATERIALS AND METHODS: Freshly extracted human fat was secured in an Aluminum (Al) cavity. Sixteen nanoseconds duration stress waves were generated by exfoliating the bottom surface of the Al cavity by focusing 3 nanoseconds-long YAG laser pulses over a 2 mm diameter area at 10 Hz with a maximum pulse energy of 0.95 J. The lipids released due to cell rupture were extracted and measured RESULTS: Four minutes of pulsing released about 0.005 g, which was over 1% of the initial weight of the tissue. In situ temperature rise of only 5 degrees C was measured at the maximum stress wave loading duration of 5 minutes. This was evidenced by histological sections, which showed no burn artifacts. CONCLUSIONS: This research shows that ultra short stress waves can mechanically cavitate fat in vitro without significant damage to adjacent structures, and forms the basis for future clinical work.