Maximilian Ackermann

Angiogenesis in Wounds Treated by Microdeformational Wound Therapy

BACKGROUND Mechanical forces play an important role in tissue neovascularization and are a constituent part of modern wound therapies. The mechanisms by which vacuum assisted closure (VAC) modulates wound angiogenesis are still largely unknown. OBJECTIVE To investigate how VAC treatment affects wound hypoxia and related profiles of angiogenic factors as well as to identify the anatomical characteristics of the resultant, newly formed vessels. METHODS Wound neovascularization was evaluated by morphometric analysis of CD31-stained wound cross-sections as well as by corrosion casting analysis. Wound hypoxia and mRNA expression of HIF-1α and associated angiogenic factors were evaluated by pimonidazole hydrochloride staining and quantitative reverse transcription-polymerase chain reaction (RT-PCR), respectively. Vascular endothelial growth factor (VEGF) protein levels were determined by western blot analysis. RESULTS VAC-treated wounds were characterized by the formation of elongated vessels aligned in parallel and consistent with physiological function, compared to occlusive dressing control wounds that showed formation of tortuous, disoriented vessels. Moreover, VAC-treated wounds displayed a well-oxygenated wound bed, with hypoxia limited to the direct proximity of the VAC-foam interface, where higher VEGF levels were found. By contrast, occlusive dressing control wounds showed generalized hypoxia, with associated accumulation of HIF-1α and related angiogenic factors. CONCLUSIONS The combination of established gradients of hypoxia and VEGF expression along with mechanical forces exerted by VAC therapy was associated with the formation of more physiological blood vessels compared to occlusive dressing control wounds. These morphological changes are likely a necessary condition for better wound healing.

External volume expansion increases subcutaneous thickness, cell proliferation, and vascular remodeling in a murine model.

Background: Fat grafting is a powerful tool for soft-tissue reconstruction; however, the science behind recipient bed preparation has not been thoroughly explored. External volume expansion using suction before fat grafting has been used clinically to improve reliability and consistency of graft survival. The authors developed a murine model to investigate the underlying mechanism of external volume expansion. Methods: The authors created an external volume expansion device using a soft-silicone dome connected to a vacuum source (25 mmHg) to treat the dorsum of mice, and the response was compared with treatment with an occlusive dressing. Treated areas were monitored with magnetic resonance imaging. Remodeling of microvasculature was studied with corrosion casting on day 7. Effects on tissue thickness, number of adipocytes, cell proliferation, and blood vessel density were analyzed at 28 days. Results: Macroscopic analysis showed tissue swelling at sites treated with the external volume expansion device by 21 days, without skin damage. On day 28, external volume expansion increased the thickness of the subcutaneous fat layer twofold, consistent with magnetic resonance imaging observations. The proliferation rate in the subcutaneous layer of expansion-treated areas increased twofold, with a net 2.2-fold increase in number of adipocytes in columns; remodeling of the vessels network occurred, with reorientation and increase of vessel diameters shown by corrosion casting and 1.9-fold augmentation of vessels density. Conclusions: External volume expansion applied to mouse integument induces highly proliferative and vascularized subcutaneous tissue. Recipient-site preparation using external volume expansion devices may be a promising tool to enhance cell and tissue engraftment.

Biomechanical and histological evaluation of abdominal wall compliance with intraperitoneal onlay mesh implants in rabbits: A comparison of six different state-of-the-art meshes

BACKGROUND An ideal prosthetic mesh for incisional hernia repair should mimic the anisotropic compliance of the abdominal wall, and at lower loads should exhibit higher distensibility without impairment of safety at higher loads. This study evaluated the biomechanical properties of six meshes in a rabbit model. METHODS New Zealand white rabbits were used for this study. Two meshes of the same brand (Ethicon Physiomesh™, Bard Composix(®) L/P, Gore Dualmesh(®), Bard Sepramesh(®), Ethicon Proceed(®) or Parietex™ Composite) were implanted into each animal for assessment of intra-abdominal hernia repair, with a total of ten meshes per group. Twelve weeks after implantation, the abdominal walls with ingrown meshes were harvested and examined biomechanically with a plunger test. The mesh-tissue compliance was evaluated by the forces exerted at given displacements and also described through a simple mathematical approximation. Abdominal wall samples were collected for histopathology, cell turnover and morphometry. RESULTS No mesh-related complications were seen. The adhesion score was significantly higher in Bard Composix(®) L/P and Ethicon Proceed(®) meshes. Significant shrinkage was seen in Gore Dualmesh(®) and Parietex™ Composite meshes. Physiomesh™ exhibited the highest compliance during plunger testing, characterized by lower, more physiological reaction forces against tissue displacement than the competitor meshes. In contrast, the safety modulus was comparable in all groups. Histology showed less collagen and less foreign body reaction in the Physiomesh™ samples contributing to patients comfort. CONCLUSION In terms of safety, this study showed no superiority of any single mesh. The comfort modulus however differed, being lowest in the newly developed Physiomesh™.

Alveolar Macrophage Dynamics in Murine Lung Regeneration

In most mammalian species, the removal of one lung results in dramatic compensatory growth of the remaining lung. To investigate the contribution of alveolar macrophages (AMs) to murine post‐pneumonectomy lung growth, we studied bronchoalveolar lavage (BAL)‐derived AM on 3, 7, 14 and 21 days after left pneumonectomy. BAL demonstrated a 3.0‐fold increase in AM (CD45+, CD11b−, CD11c+, F4/80+, Gr‐1−) by 14 days after pneumonectomy. Cell cycle flow cytometry of the BAL‐derived cells demonstrated an increase in S + G2 phase cells on days 3 (11.3 ± 2.7%) and 7 (12.1 ± 1.8%) after pneumonectomy. Correspondingly, AM demonstrated increased expression of VEGFR1 and MHC class II between days 3 and 14 after pneumonectomy. To investigate the potential contribution of peripheral blood cells to this AM population, parabiotic mice (wild‐type/GFP) underwent left pneumonectomy. Analysis of GFP+ cells in the post‐pneumonectomy lung demonstrated that by day 14, less than 1% of the AM population were derived from the peripheral blood. Finally, AM gene transcription demonstrated a significant shift from decreased transcription of angiogenesis‐related genes on day 3 to increased transcription on day 7 after pneumonectomy. The increased number of locally proliferating AM, combined with their growth‐related gene transcription, suggests that AM actively participate in compensatory lung growth. J. Cell. Physiol. 227: 3208–3215, 2012.

Restoration of Cerebral and Systemic Microvascular Architecture in APP/PS1 Transgenic Mice Following Treatment with Liraglutide™

Cerebral microvascular impairments occurring in AD may reduce Aβ peptide clearance and impact upon circulatory ultrastructure and function. We hypothesized that microvascular pathologies occur in organs responsible for systemic Aβ peptide clearance in a model of AD and that Liraglutide (Victoza®) improves vessel architecture.

Migration of CD11b+ accessory cells during murine lung regeneration.

In many mammalian species, the removal of one lung leads to growth of the remaining lung to near-baseline levels. In studying post-pneumonectomy mice, we used morphometric measures to demonstrate neoalveolarization within 21 days of pneumonectomy. Of note, the detailed histology during this period demonstrated no significant pulmonary inflammation. To identify occult blood-borne cells, we used a parabiotic model (wild-type/GFP) of post-pneumonectomy lung growth. Flow cytometry of post-pneumonectomy lung digests demonstrated a rapid increase in the number of cells expressing the hematopoietic membrane molecule CD11b; 64.5% of the entire GFP(+) population were CD11b(+). Fluorescence microscopy demonstrated that the CD11b(+) peripheral blood cells migrated into both the interstitial tissue and alveolar airspace compartments. Pneumonectomy in mice deficient in CD11b (CD18(-/-) mutants) demonstrated near-absent leukocyte migration into the airspace compartment (p<.001) and impaired lung growth as demonstrated by lung weight (p<.05) and lung volume (p<.05). Transcriptional activity of the partitioned CD11b(+) cells demonstrated significantly increased transcription of Angpt1, Il1b, and Mmp8, Mmp9, Ncam1, Sele, Sell, Selp in the alveolar airspace and Adamts2, Ecm1, Egf, Mmp7, Npr1, Tgfb2 in the interstitial tissue (>4-fold regulation; p<.05). These data suggest that blood-borne CD11b(+) cells represent a population of accessory cells contributing to post-pneumonectomy lung growth.

Amorphous polyphosphate/amorphous calcium carbonate implant material with enhanced bone healing efficacy in a critical-size defect in rats

In this study the effect of amorphous calcium carbonate (ACC) microparticles and amorphous calcium polyphosphate (polyP) microparticles (termed aCa-polyP-MP) on bone mineral forming cells/tissue was investigated in vitro and in vivo. The ACC particles (termed ACC-P10-MP) were prepared in the presence of Na-polyP. Only the combinations of polyP and ACC microparticles enhanced the proliferation rate of human mesenchymal stem cells (MSCs). Gene expression studies revealed that ACC causes an upregulation of the expression of the cell membrane-associated carbonic anhydrase IX (CA IX; formation of ACC), while the transcript level of the alkaline phosphatase (ALP; liberation of orthophosphate from polyP) changes only relatively little. In contrast, aCa-polyP-MP primarily induces ALP expression. If both components are applied together a strong stimulation of expression of both marker genes is observed. In order to investigate whether ACC also enhances bone regeneration induced by polyP in vivo, the particles were encapsulated into PLGA (poly(d,l-lactide-co-glycolide)) microspheres (diameter ~800 μm) and implanted into rat critical-size calvarial defects. The studies revealed that animals that received aCa-polyP-MP microspheres showed an increased rate of regeneration compared to β-tri-calcium phosphate (β-TCP) controls. This effect is even accelerated if microspheres with both aCa-polyP-MP and ACC-P10-MP (1 : 1 weight ratio) are applied, resulting in an almost complete restoration of the defect area after 12 weeks. qRT-PCR analyses of tissue sections through the regeneration zone with microspheres containing both aCa-polyP-MP and ACC-P10-MP revealed a significantly higher upregulation of expression of the marker genes compared to each of the components alone. The Youngs moduli for microspheres containing aCa-polyP-MP (1.74 MPa) and aCa-polyP-MP/ACC-P10-MP (2.38 MPa) were markedly higher compared to β-TCP-controls (0.63 mPa). Our results show that the combined application of ACC and Ca-polyP (both in the amorphous state) opens new strategies for the development of regenerative implants for the reconstruction of bone defects.

Intussusceptive remodeling of vascular branch angles in chemically-induced murine colitis.

Intussusceptive angiogenesis is a developmental process linked to both blood vessel replication and remodeling in development. To investigate the prediction that the process of intussusceptive angiogenesis is associated with vessel angle remodeling in adult mice, we systematically evaluated corrosion casts of the mucosal plexus in mice with trinitrobenzesulfonic acid (TNBS)-induced and dextran sodium sulfate (DSS)-induced colitis. The mice demonstrated a significant decrease in vessel angles in both TNBS-induced and DSS-induced colitis within 4 weeks of the onset of colitis (p<.001). Corrosion casts 28-30 days after DSS treatment were studied for a variety of detailed morphometric changes. The vessel diameter and interbranch distance were significantly increased in the descending colon (p<.05). Also consistent with vessel growth, intervascular distance was decreased in the descending colon (p<.05). In contrast, no statistically significant morphometric changes were noted in the ascending colon. The morphometry of the corrosion casts also demonstrated 1) a similar orientation of the remodeled angles within the XY coordinate plane of the mucosal plexus, and 2) alternating periodicity of remodeled and unremodeled vessel angles. We conclude that inflammation-associated intussusceptive angiogenesis in adult mice is associated with vessel angle remodeling. Further, the morphometry of the vessel angles suggests the influence of blood flow on the location and orientation of remodeled vessels.

Mechanostructural adaptations preceding postpneumonectomy lung growth

ABSTRACT In many species, pneumonectomy results in compensatory growth in the remaining lung. Although the late mechanical consequences of murine pneumonectomy are known, little is known about the anatomic adaptations and respiratory mechanics during compensatory lung growth. To investigate the structural and mechanical changes during compensatory growth, mice were studied for 21 days after left pneumonectomy using microCT and respiratory system impedance (FlexiVent). Anatomic changes after left pneumonectomy included minimal mediastinal shift or chestwall remodeling, but significant displacement of the heart and cardiac lobe. Mean displacement of the cardiac lobe centroid was 5.2 ± 0.8 mm. Lung impedance measurements were used to investigate the associated changes in respiratory mechanics. Quasi-static pressure–volume loops demonstrated progressive increase in volumes with decreased distensibility. Measures of quasi-static compliance and elastance were increased at all time points postpneumonectomy (P < .01). Oscillatory mechanics demonstrated a significant change in tissue impedance on the third day after pneumonectomy. The input impedance on day 3 after pneumonectomy demonstrated a significant increase in tissue damping (5.8 versus 4.3 cmH2O/mL) and elastance (36.7 versus 26.6 cmH2O/mL) when compared to controls. At all points, hysteresivity was unchanged (0.17). We conclude that the timing and duration of the mechanical changes was consistent with a mechanical signal for compensatory growth.

A morphometric study of mechanotransductively induced dermal neovascularization.

Background: Mechanical stretch has been shown to induce vascular remodeling and increase vessel density, but the pathophysiologic mechanisms and the morphologic changes induced by tensile forces to dermal vessels are poorly understood. Methods: A custom computer-controlled stretch device was designed and applied to the backs of C57BL/6 mice (n = 38). Dermal and vascular remodeling was studied over a 7-day period. Corrosion casting and three-dimensional scanning electron microscopy and CD31 staining were performed to analyze microvessel morphology. Hypoxia was assessed by immunohistochemistry. Western blot analysis of vascular endothelial growth factor (VEGF) and mRNA expression of VEGF receptors was performed. Results: Skin stretching was associated with increased angiogenesis as demonstrated by CD31 staining and vessel corrosion casting where intervascular distance and vessel diameter were decreased (p < 0.01). Immediately after stretching, VEGF dimers were increased. Messenger RNA expression of VEGF receptor 1, VEGF receptor 2, neuropilin 1, and neuropilin 2 was increased starting as early as 2 hours after stretching. Highly proliferating epidermal cells induced epidermal hypoxia starting at day 3 (p < 0.01). Conclusions: Identification of significant hypoxic cells occurred after identification of neovessels, suggesting an alternative mechanism. Increased expression of angiogenic receptors and stabilization of VEGF dimers may be involved in a mechanotransductive, prehypoxic induction of neovascularization.