Geraldine M. Mitchell

An arteriovenous loop in a protected space generates a permanent, highly vascular, tissue-engineered construct.

A major obstacle to 3‐dimensional tissue engineering is incorporation of a functional vascular supply to support the expanding new tissue. This is overcome in an in vivo intrinsic vascularization model where an arteriovenous loop (AVL) is placed in a noncollapsible space protected by a polycarbonate chamber. Vascular development and hypoxia were examined from 3 days to 112 days by vascular casting, morphometric, and morphological techniques to understand the models vascular growth and remodeling parameters for tissue engineering purposes. At 3 days a fibrin exudate surrounded the AVL, providing a scaffold to migrating inflammatory, endothelial, and mesenchymal cells. Capillaries formed between 3 and 7 days. Hypoxia and cell proliferation were maximal at 7 days, followed by a peak in percent vascular volume at 10 days (23.2±03.14% compared with 3.5±92.68% at 3 days, P<0.001). Maximal apoptosis was observed at 112 days. The protected space and spontaneous microcirculatory development in this model suggest it would be applicable for in vivo tissue engineering. A temporal window in a period of intense angiogenesis at 7 to 10 days is optimal for exogenous cell seeding and survival in the chamber, potentially enabling specific tissue outcomes to be achieved.—Lokmic, Z., Stillaert, F., Morrison, W. A., Thompson, E. W., Mitchell, G. M. An arteriovenous loop in a protected space generates a permanent, highly vascular, tissue‐engineered construct. FASEB J. 21, 511–522 (2007)

Engineering the Microcirculation

The ultimate survival of tissue-engineered constructs in vivo depends on the provision of an adequate blood supply to the engineered tissue and the capacity of the engineered microcirculation to connect with the existing recipient circulation. Techniques for the vascularization of tissue-engineered constructs can be broadly grouped into in vitro and in vivo approaches that rely on the presence of a pro-angiogenic microenvironment. Significant advances have been made in resolving the problem of microcirculatory network formation for large 3-dimensional constructs; however, issues concerning construct-host vessel connection, expansion of vascular volume accompanying growing tissue, and prevention of premature or excessive vascular regression remain to be resolved. This review provides an overview of current approaches to creating microcirculatory networks with respect to the cells involved, growth factors, growth factor delivery systems, and scaffold properties required to engineer a permanent microcirculatory network for tissue-engineered constructs. In addition, the review examines concerns related to vascular remodeling and regression reported in some tissue-engineering models.

MicroRNAs in Insulin Resistance and Obesity

MicroRNAs (miRNAs) are a class of short, single-stranded non-protein coding gene products which can regulate the gene expression through post-transcriptional inhibition of messenger RNA (mRNA) translation. They are known to be involved in many essential biological processes including development, insulin secretion, and adipocyte differentiation. miRNAs are involved in complex metabolic processes, such as energy and lipid metabolism, which have been studied in the context of diabetes and obesity. Obesity, hyperlipidemia (elevated levels of blood lipids), and insulin resistance are strongly associated with the onset of type 2 diabetes. These conditions are also associated with aberrant expression of multiple essential miRNAs in pancreatic islets of Langerhans and peripheral tissues, including adipose tissue. A thorough understanding of the physiological role these miRNAs play in these tissues, and changes to their expression under pathological conditions, will allow researchers to develop new therapeutics with the potential to correct the aberrant expression of miRNAs in type 2 diabetes and obesity.

Three Dimensional Collagen Scaffold Promotes Intrinsic Vascularisation for Tissue Engineering Applications

Here, we describe a porous 3-dimensional collagen scaffold material that supports capillary formation in vitro, and promotes vascularization when implanted in vivo. Collagen scaffolds were synthesized from type I bovine collagen and have a uniform pore size of 80 μm. In vitro, scaffolds seeded with primary human microvascular endothelial cells suspended in human fibrin gel formed CD31 positive capillary-like structures with clear lumens. In vivo, after subcutaneous implantation in mice, cell-free collagen scaffolds were vascularized by host neovessels, whilst a gradual degradation of the scaffold material occurred over 8 weeks. Collagen scaffolds, impregnated with human fibrinogen gel, were implanted subcutaneously inside a chamber enclosing the femoral vessels in rats. Angiogenic sprouts from the femoral vessels invaded throughout the scaffolds and these degraded completely after 4 weeks. Vascular volume of the resulting constructs was greater than the vascular volume of constructs from chambers implanted with fibrinogen gel alone (42.7±5.0 μL in collagen scaffold vs 22.5±2.3 μL in fibrinogen gel alone; p<0.05, n = 7). In the same model, collagen scaffolds seeded with human adipose-derived stem cells (ASCs) produced greater increases in vascular volume than did cell-free collagen scaffolds (42.9±4.0 μL in collagen scaffold with human ASCs vs 25.7±1.9 μL in collagen scaffold alone; p<0.05, n = 4). In summary, these collagen scaffolds are biocompatible and could be used to grow more robust vascularized tissue engineering grafts with improved the survival of implanted cells. Such scaffolds could also be used as an assay model for studies on angiogenesis, 3-dimensional cell culture, and delivery of growth factors and cells in vivo.

A study of the extent and pathology of experimental avulsion injury in rabbit arteries and veins

A comparison was made between operating microscope observations and histopathological examination of the ruptured ends of experimentally avulsed rabbit femoral arteries and veins. Under the operating microscope no damage was evident in arteries or veins more than 0.8 cm (on average) from the rupture site, the common lesions being tears, holes, bruising, sleeving and dilatations. In light microscope and electron microscope studies arterial and venous lesions were often noted up to 4 cm from the rupture site both proximally and distally. Severe circumferential skip lesions involving the tunica intima and media in the arteries were noted, and commonly deep clefts also extended through all three tunicae at arterial bifurcations. In avulsed veins complete tears through all tunicae or partial loss of intima and media were observed. The extensive nature of these lesions is the most likely reason for the lower success rate of avulsed digits in replantation surgery.

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.

Time course analysis of hypoxia, granulation tissue and blood vessel growth, and remodeling in healing rat cutaneous incisional primary intention wounds.

Hypoxia and the development and remodeling of blood vessels and connective tissue in granulation tissue that forms in a wound gap following full‐thickness skin incision in the rat were examined as a function of time. A 1.5 cm‐long incisional wound was created in rat groin skin and the opposed edges sutured together. Wounds were harvested between 3 days and 16 weeks and hypoxia, percent vascular volume, cell proliferation and apoptosis, α‐smooth muscle actin, vascular endothelial growth factor‐A, vascular endothelial growth factor receptor‐2, and transforming growth factor‐β1 expression in granulation tissue were then assessed. Hypoxia was evident between 3 and 7 days while maximal cell proliferation at 3 days (123.6±22.2 cells/mm2, p<0.001 when compared with normal skin) preceded the peak percent vascular volume that occurred at 7 days (15.83±1.10%, p<0.001 when compared with normal skin). The peak in cell apoptosis occurred at 3 weeks (12.1±1.3 cells/mm2, p<0.001 when compared with normal skin). Intense α‐smooth muscle actin labeling in myofibroblasts was evident at 7 and 10 days. Vascular endothelial growth factor receptor‐2 and vascular endothelial growth factor‐A were detectable until 2 and 3 weeks, respectively, while transforming growth factor‐β1 protein was detectable in endothelial cells and myofibroblasts until 3–4 weeks and in the extracellular matrix for 16 weeks. Incisional wound granulation tissue largely developed within 3–7 days in the presence of hypoxia. Remodeling, marked by a decline in the percent vascular volume and increased cellular apoptosis, occurred largely in the absence of detectable hypoxia. The expression of vascular endothelial growth factor‐A, vascular endothelial growth factor receptor‐2, and transforming growth factor‐β1 is evident prior, during, and after the peak of vascular volume reflecting multiple roles for these factors during wound healing.

The nature and extent of histopathologic injury in human avulsed arteries and veins and in experimentally avulsed monkey arteries.

&NA; To determine the end point of histopathologic damage in avulsed arteries, the forearm arteries of five monkeys being sacrificed were avulsed longitudinally and samples of proximal and distal arteries prepared for light microscopy and transmission and scanning electron microscopy. A severe and consistent circumferential skip lesion was found on the luminal surface involving the intima and media. In 30 percent of vessels, histopathologic damage extended more than 3.0 cm from the rupture point. Similar circumferential tears occurred on the luminal surface of resected human avulsed arteries collected at the time of replantation surgery. No consistent lesions were noted in resected veins from human avulsed amputations. It is possible that in the human artery (as in the monkey) circumferential lesions frequently extend many centimeters from the rupture point and therefore beyond resection distances. Lesions present in the vessel after resection and microsurgical repair might be the site of thrombosis and subsequent occlusion.

A morphological study of the long-term repair process in experimentally stretched but unruptured arteries and veins

As many avulsion amputations are incomplete and the vessels remain intact, the immediate pathology and long-term repair process (to 3 months post-injury) of experimentally stretched but unruptured rabbit femoral arteries and veins were examined. In stretched arteries, circumferential skip lesions involving endothelium, internal elastic lamina (IEL) and media occurred frequently and often up to 3 cm from the point of stretch. Medial smooth muscle cells (SMC) were significantly damaged or lost at lesions. Macrophages and neutrophils were found in lesions 1-4 days post-injury. Between 2-4 days, lesions were covered by endothelium and synthetic state SMC appeared in the media. At 1 week, a thin neointima (which persisted to 3 months) covered many lesions. The media at lesions gradually filled with SMC but generally remained disorganised even at 3 months post-injury. Stretching caused tears in vein walls, particularly close to the point of injury. There was no evidence of venous damage or repair in specimens examined 3 weeks and 3 months post-injury.

Comparative study of vascularized and nonvascularized tendon grafts for reconstruction of flexor tendons in zone 2: An experimental study in primates

Vascularized tendon grafts were compared with nonvascularized tendon grafts in a primate experimental model. In four monkeys, seven vascularized extensor hallucis longus grafts were placed in the foots digital fibroosseous canal and these were compared with eight nonvascularized tendon grafts similarly placed in the opposite extremities. The juncture techniques and postoperative protocols were identical for both tendon groups. All tendons were explored 5 months after insertion. The vascular pedicle was patent in all vascularized tendons. Three tendon ruptures occurred in nonvascularized tendons and only one rupture occurred in a vascularized tendon. The digits with vascularized tendons demonstrated a significantly better simulated total active motion (117 degrees versus 128 degrees, p less than 0.05) than digits with nonvascularized tendons. The difference was even more significant when a localized tenolysis of the proximal juncture of the tendon graft was performed (215 degrees versus 138 degrees, p less than 0.01). This study supports the concept that vascularized tendon grafts may be advantageous in scarred tendon beds.