Rosalind Romeo

Formation of New Tissue from an Arteriovenous Loop in the Absence of Added Extracellular Matrix

A major requirement for the microsurgical repair of contour defects of the skin, for example, following removal of a skin cancer on the face, is a mass of vascularised subcutaneous tissue. Such tissue can be generated in vivo using basic tissue engineering principles. In previous studies in our laboratory, we have used a model comprising an arteriovenous (AV) shunt loop sandwiched in artificial dermis, placed in a cylindrical plastic growth chamber, and inserted subcutaneously to grow new connective tissue progressively up to 4 weeks. To learn more about the basic growth characteristics with this model, the same AV shunt loop within a chamber without added extracellular matrix was inserted subcutaneously into the groins of rats for 2, 4, or 12 weeks (n = 5 per group). There was a progressive increase in the mass and volume of tissue such that the chamber was two-thirds full after 12 weeks. Histological examination showed that at 2 weeks there was evidence of fibroblast and vascular outgrowth from the AV shunt, with the formation of granulation tissue, surrounded by a mass of coagulated exudate. At 4 weeks the connective tissue deposition was more extensive, with a mass of more mature granulation tissue containing considerable collagen. By 12 weeks there was an extensive, well vascularized mass of mature fibrous tissue. The blood vessels and residual adventitia of the AV shunt were the likely source of growth factors and of the cells which populated the chamber with new maturing connective tissue. A patent AV shunt in an isolated chamber appears to be the minimal requirement for the generation of new vascularized tissue that is potentially suitable for microsurgical transplantation.

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.

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

Targeted disruption of the nitric oxide synthase 2 gene protects against ischaemia/reperfusion injury to skeletal muscle

To provide definitive insight into the complicated roles of the nitric oxide synthase (NOS) enzymes in ischaemia/reperfusion (I/R) injury of skeletal muscle, experiments were undertaken in mice with targeted disruption of the inducible NOS (NOS‐2 KO) isoform, compared with the wild‐type mouse strain. The degree of I/R injury in the NOS‐2 KO mice was attenuated relative to that in the wild‐type strain. After 70 min of ischaemia (24 h reperfusion), nitroblue tetrazolium (NBT) staining of skeletal muscle showed significant necrosis (40%) in wild‐type mice, whilst in NOS‐2 KO mice, ischaemia could be prolonged to 90 min before significant necrosis (38%) was apparent. Specific enzyme activities of the mitochondrial respiratory chain enzymes, measured in skeletal muscle homogenates, suggested that direct inhibition of the enzymes is not causal in the I/R injury. Immunohistological examination of skeletal muscle for NOS‐2 showed its induction selectively in mast cells. In vitro experiments using bone marrow‐derived mast cells showed that NOS‐2 induction was associated with increased degranulation of mast cells. These findings suggest that NO generated by induction of NOS‐2 has a deleterious effect in I/R injury of skeletal muscle and that NO exerts its damaging effect through factors released by degranulation of mast cells. Copyright

Acute inflammation in foetal and adult sheep: the response to subcutaneous injection of turpentine and carrageenan

Because of the small number of inflammatory cells found in skin wounds, it has been considered that foetal sheep are incapable of an adequate inflammatory response. The present study shows that subcutaneous injection of turpentine or carrageenan into foetal sheep consistently excites a severe cellular inflammatory reaction. At 75 days gestation the inflammatory cells are all macrophages. As the foetus develops, polymorphs play an increasing role in the early response to turpentine. By 120 days inflammatory cells are almost all polymorphs as in adult sheep. However the response to carrageenan remains macrophage in type. At all stages of gestation the acute cellular response is followed by development of scar tissue, more pronounced with turpentine than carrageenan. Any proposed intrauterine surgery must allow for the pronounced and prolonged inflammatory response and scar tissue formation caused by persistent inflammatory stimuli in foetal animals.

Stimulating Effect of an Arteriovenous Shunt on the in Vivo Growth of Isografted Fibroblasts: A Preliminary Report

Isogenous fibroblasts derived from the skin of inbred Sprague-Dawley rats were cultured in vitro, labeled with bisbenzamide (BB) or carboxyfluorescein diacetate (CFDA), and seeded into polycarbonate growth chambers. After 24 h incubation in vitro, the chambers, either empty or containing an arteriovenous (AV) shunt, were implanted subcutaneously into the inguinal region of Sprague-Dawley rats and examined by fluorescence microscopy 2 or 7 days later. The AV shunt remained patent in all experiments. The density of labeled cells on the chamber surface in all chambers decreased in the first 2 days after insertion. At 7 days, the cell density in the empty chambers had not altered from the 2-day level, but the density in the AV shunt containing chambers had increased to almost three times the day 2 level (p = 0.013). It appears that an AV shunt can induce a significant proliferation of fibroblasts implanted adjacent to it. For at least 7 days after labeling, BB and CFDA provide a simple and effective method of quantitative detection of implanted fibroblasts. It is concluded that nutrients from the AV shunt implanted in a growth chamber result in a significant increase in the number of viable, matrix-synthesizing cells, compared with AV shunt-free controls.

A model of bridging angiogenesis in the rat

A model of angiogenesis has been developed in the rat. The epigastric vascular pedicle was exposed in the groin, a 7 mm segment of epigastric artery was excised leaving the vein intact and, after a variable period of time for angiogenesis to occur between the ends of the artery, a skin flap was elevated on the epigastric vascular pedicle so that it depended completely for its blood supply on bridging angiogenesis across the created gap. Skin flap survival and vessel counts were measured as indices of the angiogenic response. In this model we observed a spontaneous increase in vessel counts between the ends of the artery, and a corresponding increase in skin flap tissue survival until day 10 after which time vessel counts plateaued whilst tissue survival continued to increase until day 14. In the angiogenic pedicle, a time-dependent development of granulation tissue containing numerous macrophages and mast cells, and capillary sprouting were documented. When flap elevation was performed 7 days after arterial excision skin flap survival was 42%. Thus, in this model, 7 days is a suitable interval for the future evaluation of the effects of either pro- or anti-angiogenic agents.

Expression of Leukemia Inhibitory Factor in Human Nerve following Injury

In animal models of peripheral nerve injury, leukemia inhibitory factor (LIF) is normally expressed at very low levels. Following nerve injury, its expression is rapidly increased in the nerve at the injury site and promotes both sensory and motor neuron survival. Once normal nerve function is restored, LIF expression returns to negligible levels. For this reason, LIF is considered to be a peripheral nerve trauma factor. We wished to determine whether LIF is also upregulated in human nerves following trauma and whether it is expressed in neuromas of varying age. Immunohistochemical staining for the presence of LIF was performed on injured and control human nerves from a number of subjects. Results demonstrate that LIF expression is increased in nerves within hours of injury and, in the case of neuroma formation, can persist for several years. LIF immunoreactivity was consistently found in Schwann cells, in peripheral nerve axons, and, at stages when an inflammatory response was present, also in neutrophils, mast cells, macrophages, and blood vessel walls. The level of staining within the connective tissue of injured nerves was elevated compared to control nerves, which may be due to the presence of LIF bound to the soluble secreted form of the LIF receptor. Whether the continued expression of LIF is unhealed injured nerves promotes the development of neuromas remains to be resolved.

Cold storage of rat skeletal muscle free flaps and pre-ischemic perfusion with modified UW solution

We used the rat medial gastrocnemius free flap, based on a pedicle of the femoral artery and vein, in order to test the tolerance of skeletal muscle to cold ischemia‐reperfusion (IR) injury, and to determine whether tolerance can be enhanced by pre‐ischemic perfusion with tissue/organ preservation solutions.

The functional and structural effects of hypothermic storage on ischaemic arterial grafts

The effects of hypothermic ischaemia on blood vessels are unknown. This study aimed to determine the 3 week patency rate and the pathology of 9 experimental groups of hypothermically stored ischaemic arteries and one control group in a rabbit femoral artery model. Ischaemia times were 0 h, 24 h, 1, 2, 4, 6, 8 and 10 weeks (Groups 1-8). Patency was over 80% in all groups after 3 weeks reinsertion. Following reinsertion control grafts maintained normal arterial structure, but cellular degeneration had occurred in all ischaemic grafts and appeared complete after 4 weeks ischaemia. The graft connective tissue framework frequently remained intact. Repair was evident in central graft regions after 2 weeks ischaemia and 3 weeks reinsertion, but occurred only adjacent to the anastomosis in 4-10 week ischaemic arteries. Four week ischaemic arteries (Groups 9 and 10) reinserted for 6 and 12 weeks respectively exhibited near complete repair but patency dropped to 60% in the 12 week group.