Seeding density is a crucial determinant for the in vivo vascularisation capacity of adipose tissue-derived microvascular fragments.

Adipose tissue-derived microvascular fragments (ad-MVF) represent effective vascularisation units for the seeding of dermal substitutes. However, particularly in case of extensive skin defects, the required amounts of donor fat tissue for the harvesting of ad-MVF may not always be available. Therefore, we herein determined the lowest ad-MVF density needed to induce a sufficient vascularisation and incorporation of seeded implants. Collagen-glycosaminoglycan matrices (Integra®; diameter: 4 mm) were seeded with 15,000 (HD), 10,000 (MD) and 5,000 (LD) ad-MVF and implanted into full-thickness skin defects within mouse dorsal skinfold chambers, to analyse their in vivo vascularisation and incorporation. Intravital fluorescence microscopy showed a comparable vascularisation of HD and MD ad-MVF-seeded Integra®, which was significantly higher when compared to LD ad-MVF-seeded Integra®. As assessed by photoacoustic imaging, this was associated with an increased oxygenation of the implants. Additional histological and immunohistochemical analyses revealed an enhanced cellular infiltration, collagen content, microvessel density and epithelialisation of HD and MD ad-MVF-seeded Integra®, indicating a better incorporation compared to LD ad-MVF-seeded implants. These findings demonstrate that 80,000 ad-MVF/cm² is the least required density to guarantee an effective vascularisation of the dermal substitute.

Heparin-protamine does not aggravate local LPS-provoked leukocytic inflammation in vivo.

OBJECTIVE: Secondary complications involving inflammation limit postoperative results in cardiac surgery. Because heparin-protamine can elicit inflammatory reactions, this study evaluates in vivo whether treatment with heparin-protamine aggravates local endotoxin-induced injury. METHODS: Mice received intravenous injections of either heparin-protamine, protamine alone or PBS for controls, before local air pouch challenge with LPS. Leukocytes recruited within the air pouches were collected and analyzed by flow cytometry. RESULTS: LPS provoked a local leukocytic infiltration in a dose- and time-dependent manner with significantly elevated numbers of 1.75 +/- 0.29 x 10 (6) cells after four hours compared to non-LPS-stimulated controls (0.55 +/- 0.08 x 10 (6) cells). Recruited cells comprised of 74 +/- 4 % PMNLs and 26 +/- 4 % MNLs. The largest fraction of MNLs was positive for the T cell-specific marker CD90.2 (59 +/- 6 %). B cells were only rarely observed (4 +/- 1 %). In non-LPS-challenged air pouches, heparin-protamine provoked a leukocytic infiltration, which was comparable to that observed after LPS (1.51 +/- 0.22 x 10 (6) cells). However, neither heparin-protamine nor protamine alone aggravated the LPS-mediated leukocyte recruitment (2.25 +/- 0.25 x 10 (6) and 1.77 +/- 0.23 x 10 (6) cells). Neither treatment influenced the distribution of leukocyte subpopulations compared to PBS-treated controls. Furthermore, surface expression of CD11a and CD11b on blood leukocytes did not differ between the groups, indicating that protamine does not increase the activation of circulating leukocytes during LPS-induced local inflammation. CONCLUSIONS: Our data indicate that heparin-protamine, although pro-inflammatory in nature, does not aggravate local inflammation provoked by LPS. Thus, enhanced inflammation during the perioperative course of cardiac surgery patients seems not to be attributable to the intraoperative use of heparin-protamine.

Impact of leukocytes and platelets in mediating hepatocyte apoptosis in a rat model of systemic endotoxemia.

Apoptotic hepatocytes have been demonstrated to represent an important signal for transmigration of leukocytes sequestered in sinusoids during endotoxemia in vivo. Beside leukocytes, platelets and their adhesion to endothelial cells and leukocytes have been implicated in inflammatory liver injury. Using in vivo multifluorescence microscopy, we examined the possibility that hepatocellular apoptosis causes both leukocytes and platelets to colocalize within the sinusoidal microvasculature of endotoxemic livers. We further addressed the issue whether cellular colocalization with apoptotic hepatocytes is cause or consequence of apoptosis. Intraperitoneal exposure of rats with LPS (5 mg/kg) induced liver injury after 6 and 16 h, as given by nutritive perfusion failure (20 +/- 2 and 21 +/- 2%), intrahepatic leukocyte (60 +/- 10 and 121 +/- 48 cells/mm(2)), and platelet (12 +/- 4 and 34 +/- 4 cells/mm(2)) accumulation as well as parenchymal cell apoptosis (4 +/- 1 and 11 +/- 2 cells/mm(2)) and caspase cleavage (4.7 +/- 2.4- and 7.0 +/- 3.0-fold increase; P < 0.05 vs. saline-exposed controls). Higher doses of LPS (10 mg/kg ip) further increased intrahepatic leukocyte and platelet accumulation but not the extent of parenchymal apoptosis. Detailed spatial analysis revealed colocalization of leukocytes (range 12-24%) but barely of platelets (<6%) with apoptotic hepatocytes in all endotoxemic groups studied. It is of interest, however, that platelets were found at increasing rates in colocalization with leukocytes at 6 and 16 h after LPS exposure (5 mg/kg LPS: 7 +/- 3 and 25 +/- 6%; 10 mg/kg LPS: 11 +/- 4 and 14 +/- 1%). Platelet-leukocyte events significantly correlated with the extent of caspase cleavage as an indicator of tissue apoptosis (P < 0.05; r = 0.82). Blockade of apoptosis by a pan-caspase inhibitor caused a significant reduction of leukocyte adherence and platelet-leukocyte colocalization on LPS exposure. On the other hand, leukocytopenic animals revealed reduced hepatocyte apoptosis, although values still exceeded those of controls, and in leuko- and thrombocytopenic animals, hepatocyte apoptosis was found reduced to control values. Taken together, LPS-associated hepatocyte apoptosis seems to be initiated by circulating blood cells that become adherent within the liver but might also contribute to further sustain the inflammatory cell-cell response.

Effects of human alpha-interferon on granulocyte-macrophage progenitor cells (CFU-GM) in vitro.

Two preparations of human interferon (IFN)-alpha were assessed for their influence on granulocyte-macrophage progenitor cells (CFU-GM) in vitro. Both highly purified human IFN-alpha Ly and recombinant IFN-alpha 2a suppressed CFU-GM colony formation in a dose-dependent manner using low-density bone-marrow target cells. Suppression of CFU-GM colony formation was accompanied by an increase in clusters. However, depletion of monocytes, T lymphocytes and B lymphocytes from low-density bone-marrow cells resulted in insensitivity of progenitor cells to IFN-alpha. These results demonstrate that the effects of human IFN-alpha on myeloid progenitor cells (CFU-GM) are mediated by accessory cells within the bone marrow.