Aurora Messina

New murine model of spontaneous autologous tissue engineering, combining an arteriovenous pedicle with matrix materials

The authors previously described a model of tissue engineering in rats that involves the insertion of a vascular pedicle and matrix material into a semirigid closed chamber, which is buried subcutaneously. The purpose of this study was to develop a comparable model in mice, which could enable genetic mutants to be used to more extensively study the mechanisms of the angiogenesis, matrix production, and cellular migration and differentiation that occur in these models. A model that involves placing a split silicone tube around blood vessels in the mouse groin was developed and was demonstrated to successfully induce the formation of new vascularized tissue. Two vessel configurations, namely, a flow-through pedicle (n = 18 for three time points) and a ligated vascular pedicle (n = 18), were compared. The suitability of chambers constructed from either polycarbonate or silicone and the effects of incorporating either Matrigel equivalent (n = 18) or poly(dl-lactic-co-glycolic acid) (n = 18) on angiogenesis and tissue production were also tested. Empty chambers, chambers with vessels only, and chambers with matrix only served as control chambers. The results demonstrated that a flow-through type of vascular pedicle, rather than a ligated pedicle, was more reliable in terms of patency, angiogenesis, and tissue production, as were silicone chambers, compared with polycarbonate chambers. Marked angiogenesis occurred with both types of extracellular matrix scaffolds, and there was evidence that native cells could migrate into and survive within the added matrix, generating a vascularized three-dimensional construct. When Matrigel was used as the matrix, the chambers filled with adipose tissue, creating a highly vascularized fat flap. In some cases, new breast-like acini and duct tissue appeared within the fat. When poly(dl-lactic-co-glycolic acid) was used, the chambers filled with granulation and fibrous tissue but no fat or breast tissue was observed. No significant amount of tissue was generated in the control chambers. Operative times were short (25 minutes), and two chambers could be inserted into each mouse. In summary, the authors have developed an in vivo murine model for studying angiogenesis and tissue-engineering applications that is technically simple and quick to establish, has a high patency rate, and is well tolerated by the animals.

The Influence of Extracellular Matrix on the Generation of Vascularized, Engineered, Transplantable Tissue

Abstract: In a recently described model for tissue engineering, an arteriovenous loop comprising the femoral artery and vein with interposed vein graft is fabricated in the groin of an adult male rat, placed inside a polycarbonate chamber, and incubated subcutaneously. New vascularized granulation tissue will generate on this loop for up to 12 weeks. In the study described in this paper three different extracellular matrices were investigated for their ability to accelerate the amount of tissue generated compared with a no‐matrix control. Poly‐d,l‐lactic‐co‐glycolic acid (PLGA) produced the maximal weight of new tissue and vascularization and this peaked at two weeks, but regressed by four weeks. Matrigel was next best. It peaked at four weeks but by eight weeks it also had regressed. Fibrin (20 and 80 mg/ml), by contrast, did not integrate with the generating vascularized tissue and produced less weight and volume of tissue than controls without matrix. The limiting factors to growth appear to be the chamber size and the capacity of the neotissue to integrate with the matrix. Once the sides of the chamber are reached or tissue fails to integrate, encapsulation and regression follow. The intrinsic position of the blood supply within the neotissue has many advantages for tissue and organ engineering, such as ability to seed the construct with stem cells and microsurgically transfer new tissue to another site within the individual. In conclusion, this study has found that PLGA and Matrigel are the best matrices for the rapid growth of new vascularized tissue suitable for replantation or transplantation.

Generation of a vascularized organoid using skeletal muscle as the inductive source

The technology required for creating an in vivo microenvironment and a neovasculature that can grow with and service new tissue is lacking, precluding the possibility of engineering complex three‐dimensional organs. We have shown that when an arterio‐venous (AV) loop is constructed in vivo in the rat groin, and placed inside a semisealed chamber, an extensive functional vasculature is generated. To test whether this unusually angiogenic environment supports the survival and growth of implanted tissue or cells, we inserted various preparations of rat and human skeletal muscle. We show that after 6 weeks incubation of muscle tissue, the chamber filled with predominantly well‐vascularized recipient‐derived adipose tissue, but some new donor‐derived skeletal muscle and connective tissue were also evident. When primary cultured myoblasts were inserted into the chamber with the AV loop, they converted to mature striated muscle fibers. Furthermore, we identify novel adipogenesis‐inducing properties of skeletal muscle. This represents the first report of a specific three‐dimensional tissue grown on its own vascular supply.

Increasing the volume of vascularized tissue formation in engineered constructs: an experimental study in rats.

The authors have previously described a model of in vivo tissue generation based on an implanted, microsurgically created vessel loop in a plastic chamber (volume, 0.45 ml) containing a poly(DL-lactic-co-glycolic acid) (PLGA) scaffold. Tissue grew spontaneously in association with an intense angiogenic sprouting from the loop and almost filled the chamber, resulting in a mean amount of tissue in chambers of 0.23 g with no added matrix scaffold and 0.33 g of tissue in PLGA-filled chambers after 4 weeks of incubation. The aim of the present study was to investigate whether a greater volume of tissue could be generated when the same-size vessel loop was inserted into a larger (1.9 ml) chamber. In four groups of five rats, an arteriovenous shunt sandwiched between two disks of PLGA, used as a scaffold for structural support, was placed inside a large polycarbonate growth chamber. Tissue and PLGA weight and volume, as well as histological characteristics of the newly formed tissue, were assessed at 2, 4, 6, and 8 weeks. Tissue weight and volume showed a strong linear correlation. Tissue weight increased progressively from 0.13 +/- 0.04 g at 2 weeks to 0.57 +/- 0.06 g at 6 weeks (p < 0.0005). PLGA weight decreased progressively from 0.89 +/- 0.07 g at 2 weeks to 0.20 +/- 0.09 g at 8 weeks (p < 0.0005). Histological examination of the specimens confirmed increased tissue growth and maturation over time. It is concluded that larger quantities of tissue can be grown over a longer period of time by using larger-size growth chambers.

Myogel, a novel, basement membrane-rich, extracellular matrix derived from skeletal muscle, is highly adipogenic in vivo and in vitro.

Background/Aims: Biological and synthetic scaffolds play important roles in tissue engineering and are being developed towards human clinical applications. Based on previous work from our laboratory, we propose that extracellular matrices from skeletal muscle could be developed for adipose tissue engineering. Methods: Extracellular matrices (Myogels) extracted from skeletal muscle of various species were assessed using biochemical assays including ELISA and Western blotting. Biofunctionality was assessed using an in vitro differentiation assay and a tissue engineering construct model in the rat. Results: Myogels were successfully extracted from mice, rats, pigs and humans. Myogels contained significant levels of laminin α4- and α2-subunits and collagen I compared to Matrigel™, which contains laminin 1 (α1β1γ1) and collagen IV. Levels of growth factors such as fibroblast growth factor 2 were significantly higher than Matrigel, vascular endothelial growth factor-A levels were significantly lower and all other growth factors were comparable. Myogels reproducibly stimulated adipogenic differentiation of preadipocytes in vitro and the growth of adipose tissue in the rat. Conclusions: We found Myogel induces adipocyte differentiation in vitroand shows strong adipogenic potential in vivo, inducing the growth of well-vascularised adipose tissue. Myogel offers an alternative for current support scaffolds in adipose tissue engineering, allowing the scaling up of animal models towards clinical adipose tissue engineering applications.

Inducible nitric oxide synthase (iNOS) activity promotes ischaemic skin flap survival

We have examined the role of nitric oxide (NO) in a model of functional angiogenesis in which survival of a skin flap depends entirely on angiogenesis to provide an arterial blood supply to maintain tissue viability. The different effects of nitric oxide synthase (NOS) inhibitors on rat skin flap survival appeared to be explained on the basis of their NOS isoform selectivity. Skin flap survival was decreased by iNOS‐selective (inducible NOS) inhibitors, S‐methyl‐isothiourea, aminoguanidine and aminoethylthiorea; unaffected by the non‐selective inhibitor nitro‐imino‐L‐ornithine; and enhanced by the cNOS (constitutive NOS, that is endothelial NOS (eNOS) and neuronal NOS (nNOS)) inhibitor, nitro‐L‐arginine methyl ester. Skin flap survival was reduced in mice with targeted disruption of the iNOS gene (iNOS knockout mice), and the administration of nitro‐L‐arginine methyl ester significantly increased flap survival in iNOS knockout mice (P<0.05). iNOS immunoreactivity was identified in mast cells in the angiogenic region. Immunoreactive vascular endothelial growth factor (VEGF) and basic fibroblast growth factor were also localized to mast cells. The combination of interferon‐γ and tumour necrosis factor‐α induced NO production and increased VEGF levels in mast cells cultured from bone marrow of wild‐type, but not iNOS KO mice. The increased tissue survival associated with the capacity for iNOS expression may be related to iNOS‐dependent enhancement of VEGF levels and an ensuing angiogenic response. Our results provide both pharmacological and genetic evidence that iNOS activity promotes survival of ischaemic tissue.

The role of mast cells in ischaemia-reperfusion injury in murine skeletal muscle.

To determine the role of mast cells in ischaemia–reperfusion (IR) injury to skeletal muscle, Wf/Wf mast cell‐deficient and their corresponding wild‐type mice were subjected to 70 min tourniquet ischaemia and 24 h reperfusion. As measured by nitroblue tetrazolium (NBT) staining, muscle viability was 9% in wild‐type and 94% in mast cell‐deficient animals (p<0.001). Assay of residual lactate dehydrogenase activity within the injured muscle (p<0.05) and histological examination confirmed the greater muscle necrosis in treated wild‐type than in treated mast cell‐deficient mice. There was no significant difference in the degree of neutrophil infiltration, tissue myeloperoxidase content or water content of IR‐injured muscle in the two mouse phenotypes. To determine further the role of mast cells in IR injury, wild‐type mice were treated 30 min prior to reperfusion with an intraperitoneal dose of either saline or the mast cell‐stabilizing agent lodoxamide trometamol (2.5, 7.5, 25 or 75 mg/kg). Twenty‐four hours after removal of the tourniquet, saline‐treated gastrocnemius muscle had a mean viability of 14% compared with 28% (p<0.05) and 48% (p<0.01) after 25 mg/kg and 75 mg/kg of lodoxamide treatment, respectively. The ability of lodoxamide to stabilize mast cells was confirmed by histological examination. Ischaemic muscle reperfused for 1 h showed much less degranulation of mast cells in mice pretreated with lodoxamide (50 mg/kg) than in saline‐treated controls. These findings suggest that mast cells are a major source of mediators of necrosis in IR injury to skeletal muscle. Copyright

Localization of inducible nitric oxide synthase to mast cells during ischemia/reperfusion injury of skeletal muscle

Nitric oxide contributes to tissue necrosis after ischemia-reperfusion (IR). A biochemical and immunohistochemical study was made of the amounts and localization of both Ca++-independent nitric oxide synthase (NOS) II and Ca++-dependent (NOS I and NOS III) in rat skeletal muscle after ischemia and 0.5, 2, 8, 16, and 24 hours reperfusion. NOS II was not detectable in control muscle or during ischemia, was first detected after 2 hours reperfusion, increased further by 8 hours, and remained elevated at 24 hours. Both NOS II and nitrotyrosine, a marker of peroxynitrite formation, were localized exclusively to mast cells except after 24 hours reperfusion when some macrophages and neutrophils also showed positive immunoreactivity. Mast cells underwent extensive degranulation during reperfusion. NOS I was not detected in injured or control muscle. The level of NOS III, which was localized to the endothelium of blood vessels of all sizes in control muscle, decreased progressively during ischemia and reperfusion to reach undetectable levels after 16 hours reperfusion. These findings indicate that most of the nitric oxide formed during IR injury is generated by NOS II located almost exclusively in mast cells.

Mast cells play a pivotal role in ischaemia reperfusion injury to skeletal muscles

Ischaemia reperfusion (IR) injury is a serious complication of cardiovascular disease, transplantation and replantation surgery. Once established there is no effective method of treatment. Although studies using mast cell-depleted (Wf/Wf) mice implicate mast cells in this pathology, they do not exclude a contribution by other deficiencies expressed in Wf/Wf mice. In order to obtain conclusive evidence for the role of mast cells, we engrafted cultured bone marrow-derived mast cells (BMMC) from normal mice into their Wf/Wf littermates. After 12 weeks, the hind limbs of Wf/Wf, engrafted Wf/Wf and normal littermates were subjected to IR injury. Muscle viability was assessed by both morphology and by nitroblue tetrazolium histochemical assay. Here, we present conclusive evidence for a causal role of mast cells in IR injury. Our data show that muscles from Wf/Wf mice subjected to IR have a significantly greater proportion of viable fibres than normal littermates subjected to identical injury (78.9±5.2 vs 27.2±3.7%, respectively). When Wf/Wf IR-resistant mice were engrafted with BMMC from normal littermates and subjected to IR, the proportion of viable muscle fibres was significantly reduced (78.9±5.2 vs 37.0±6.5%). Thus, engraftment of BMMC into Wf/Wf mice restores the susceptibility of skeletal muscle to IR injury irrespective of other abnormalities in Wf/Wf mice. In this model, the numerical density of mast cells undergoes a significant decrease within 1 h of reperfusion, indicating extensive mast cell degranulation. We conclude that mast cells are pivotal effector cells in the pathogenesis of IR injury of murine skeletal muscle.

Detection of jun but not fos protein during developmental cell death in sympathetic neurons

A large proportion of neurons die during normal development of the nervous system via an active process known as apoptosis. We counted the total number of neurons and apoptotic neurons in the superior cervical ganglion of the GH Wistar rat strain, which possesses a neurotrophic deficit leading to excessive perinatal cell death, and in its normal counterpart (N) by using the optical disector method to quantify the extent of apoptosis during postnatal development. Total neuron numbers fell between postnatal days 3 and 14 by 10 and 40% in N and GH, respectively. In GH ganglia, 1.5% of neurons were apoptotic at any given time, as determined by the presence of condensed chromatin clumps. Some types of cell death have been associated with expression of the immediate‐early genes c‐fos and c‐jun. Therefore, we used histological and immunocytochemical techniques to characterise individual neurons and to detect the products of these immediate‐early genes during developmental cell death. All apoptotic cells were immunopositive for c‐jun protein, whereas no c‐jun protein was detected in nonapoptotic cells. Conversely, members of the fos family of transcription factors were detected in the nucleus of 60% of nonapoptotic cells but in only a minor proportion of cells undergoing apoptosis. These results indicate that c‐jun occurs in neurons that are committed to die. This is the first situation in which the presence of jun protein has been correlated with normal programmed cell death in individual apoptotic neurons.