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Monocyte chemoattractant protein (MCP-1) plays a key role in the pathogenesis of obesity. It is an inflammogen that attracts monocytes to the adipose tissue and promotes their differentiation to macrophages. These tissue macrophages produce a plethora of pro-adipogenic cytokines, one of which is nitric oxide (NO). NO is produced by the inducible nictric oxide synthase (iNOS) and is suspected to be a key factor in neoadipogenesis. In this study, we mimicked the processes of fat formation in a murine adipose tissue engineering chamber model. The aim was to analyse how the pathology as found in obesity can be exploited for soft tissue reconstruction based on adipose tissue enigneering. MCP-1 and the yeast-derived inflammogen Zymosan A (Zy) were evaluated for their adipogenic potential while aminoguanidine (AG) was applied as specific iNOS inhibitor. A plastic chamber (5 mm lang, 42 µl) filled with Matrigelℳ and fibroblast growth factor 2 was implanted around a vascular pedicle but otherwise isolated from the surrounding tissue. The following conditions were tested: 1) control (Matrigelℳ, bFGF only), 2) Matrigelℳ, bFGF, MCP-1 (0.05 ng/ml), 3) Matrigelℳ, bFGF, Zy (0.02 og/ml), 4) Matrigelℳ, bFGF, Zy, AG (40 µg/ml). Constructs were harvested after 6 weeks. Absolute volumes of fat tissue were calculated from the proportion of fat (%) and the wet volume. Matrigel-FGF-2 matrix alone was not sufficient to drive substantial adipogenesis (16.15 ± 5.03 % and 1.55 ± 0.63 µl adipose tissue). Zy- and MCP-1-supplemented chambers revealed dose-dependent neo-adipogenesis. Zy-treated constructs produced 44.4 ± 5.3 % fat (p < 0.005) and volumes of 5.93 ± 1.05 µl adipose tissue (p < 0.005). MCP-1 induced 40.57 ± 3.65 % (p = 0.005) and 5.05 ± 0.92 µl adipose tissue (p = 0.0085). AG addition decreased percentage and volume of fat significantly (19.73 ± 3.13 % (p < 0.005), 2.32 ± 0.54 µl (p < 0.05)). Histologically, all explanted MCP-1 and Zy samples showed healthy adipose tissue with infiltration of macrophages. The model presented here reveals that mimicking of the processes in obesity enables neoadipogenesis in an adipogenic matrix. Key factors in this setting are macrophages and the inflammation which they generate through their NO production. A better understanding of the processes in obesity and the Metabolic Syndrome will help to develop and test new, promising approaches in adipose tissue engineering.

Adipose tissue forms when basement membrane extract (Matrigel) and fibroblast growth factor-2 (FGF-2) are added to our mouse tissue engineering chamber model. A mouse tumor extract, Matrigel is unsuitable for human clinical application, and finding an alternative to Matrigel is essential. In this study we generated adipose tissue in the chamber model without using Matrigel by controlled release of FGF-2 in a type I collagen matrix. FGF-2 was impregnated into biodegradable gelatin microspheres for its slow release. The chambers were filled with these microspheres suspended in 60 microL collagen gel. Injection of collagen containing free FGF-2 or collagen containing gelatin microspheres with buffer alone served as controls. When chambers were harvested 6 weeks after implantation, the volume and weight of the tissue obtained were higher in the group that received collagen and FGF-2 impregnated microspheres than in controls. Histologic analysis of tissue constructs showed the formation of de novo adipose tissue accompanied by angiogenesis. In contrast, control groups did not show extensive adipose tissue formation. In conclusion, this study has shown that de novo formation of adipose tissue can be achieved through controlled release of FGF-2 in collagen type I in the absence of Matrigel.

The efficacy of end-to-side repair as a method of nerve reconstruction has been questioned, and most studies that characterize the mode of re-innervation are marred by inappropriate experimental design and lack quantitative analysis. This makes characterization of re-innervating neurons confusing and consequently controversy remains as to the extent and source of reinnervating axons. In an experimental brachial plexus rat model, we transected the musculocutaneous nerve, labeled its neuron pool with Fast-Blue and joined the distal stump to the side of the intact ulnar nerve, or to the proximal stump of the divided ulnar nerve, to characterize neurons that reinnervate the recipient nerve. Tetramethyl-rhodamine dextran (TMRD) or fluoro-gold was used to map the reinnervating motor and sensory neurons at 12 weeks post-transection. No neurons originally labeled from musculocutaneous nerve were subsequently labeled with TMRD or fluoro-gold, showing that this original neuron pool does not contribute to re-innervation of the distal musculocutaneous nerve, but that reinnervation occurs solely by ulnar nerve motor and sensory axons. In the end-to-side group, 16.4% of the motor and 7% of the sensory donor ulnar nerve neurons re-innervated the musculocutaneous nerve exclusively, and a further 10% motor and 11.6% sensory innervated the musculocutaneous nerve by collateral sprouting of their axons. This compared to re-innervation by 62.6% of motor and 70.4% of ulnar nerve sensory neurons in the positive control that underwent end-to-end repair. Our results confirm the concept of collateral sprouting and support the use of end-to-side repair.

A novel method of spontaneous generation of new adipose tissue from an existing fat flap is described. A defined volume of fat flap based on the superficial inferior epigastric vascular pedicle in the rat was elevated and inset into a hollow plastic chamber implanted subcutaneously in the groin of the rat. The chamber walls were either perforated or solid and the chambers either contained poly(D,L-lactic-co-glycolic acid) (PLGA) sponge matrix or not. The contents were analyzed after being in situ for 6 weeks. The total volume of the flap tissue in all groups except the control groups, where the flap was not inserted into the chambers, increased significantly, especially in the perforated chambers (0.08 +/- 0.007 mL baseline compared to 1.2 +/- 0.08 mL in the intact ones). Volume analysis of individual component tissues within the flaps revealed that the adipocyte volume increased and was at a maximum in the chambers without PLGA, where it expanded from 0.04 +/- 0.003 mL at insertion to 0.5 +/- 0.08 mL (1250% increase) in the perforated chambers and to 0.16 +/- 0.03 mL (400% increase) in the intact chambers. Addition of PLGA scaffolds resulted in less fat growth. Histomorphometric analysis rather than simple hypertrophy documented an increased number of adipocytes. The new tissue was highly vascularized and no fat necrosis or atypical changes were observed.

The gastrocnemius muscle is versatile in flap repair of defects from anterior knee to lower leg. We describe a case of extensor reconstruction using an extended medial gastrocnemius myocutaneous flap. A medial gastrocnemius flap was raised distal to its musculotendinous insertion. At its point of rotation, it was anchored to the anterior periosteum of the tibia, hence obliterating dead space and recreating the extensor mechanism of the knee. The patient had no flap loss and was able to ambulate independently. He showed no extensor lag. Our flap is unique in that it is the only medial gastrocnemius flap that has shown no extensor lag as well as complete flexion of the knee following a disrupted entire quadriceps mechanism. It is also the first myocutaneous gastrocnemius flap that has been described for this purpose.

Engineering adipogenic tissue in vivo requires the concomitant induction of angiogenesis to generate a stable long-term three-dimensional construct. Histioconductive tissue engineering strategies have been used. The disadvantage of using biodegradable scaffolds is a delayed angiogenic induction resulting in ischemic necrosis of the central cell population in the scaffold. We evaluated an histioinductive approach for adipose tissue engineering by combining essential key components for adipogenic induction: (1) a precursor cell source, (2) a vascular pedicle, (3) a supportive matrix, and (4) a chamber to preserve space for the new tissue to develop. We observed concomitant adipogenic and angiogenic induction after 6 weeks in three-dimensional adipose tissue constructs.

An in vivo murine vascularized chamber model has been shown to generate spontaneous angiogenesis and new tissue formation. This experiment aimed to assess the effects of common biological scaffolds on tissue growth in this model. Either laminin-1, type I collagen, fibrin glue, hyaluronan, or sea sponge was inserted into silicone chambers containing the epigastric artery and vein, one end was sealed with adipose tissue and the other with bone wax, then incubated subcutaneously. After 2, 4, or 6 weeks, tissue from chambers containing collagen I, fibrin glue, hyaluronan, or no added scaffold (control) had small amounts of vascularized connective tissue. Chambers containing sea sponge had moderate connective tissue growth together with a mild "foreign body" inflammatory response. Chambers containing laminin-1, at a concentration 10-fold lower than its concentration in Matrigel, resulted in a moderate adipogenic response. In summary, (1) biological hydrogels are resorbed and gradually replaced by vascularized connective tissue; (2) sponge-like matrices with large pores support connective tissue growth within the pores and become encapsulated with granulation tissue; (3) laminin-containing scaffolds facilitate adipogenesis. It is concluded that the nature and chemical composition of the scaffold exerts a significant influence on the amount and type of tissue generated in this in vivo chamber model.

The effect of adipose tissue on inductive adipogenesis within Matrigel (BD Biosciences) was assessed by using a murine chamber model containing a vascular pedicle. Three-chamber configurations that varied in the access to an adipose tissue source were used, including sealed- and open-chamber groups that had no access and limited access, respectively, to the surrounding adipose tissue, and a sealed-chamber group in which adipose tissue was placed as an autograft. All groups showed neovascularization, but varied in the amount of adipogenesis seen in direct relation to their access to preexisting adipose tissue: open chambers showed strong adipogenesis, whereas the sealed chambers had little or no adipose tissue; adipogenesis was restored in the autograft chamber group that contained 2- to 5-mg fat autografts. These showed significantly more adipogenesis than the sealed chambers with no autograft ( p < 0.01). Autografts with 1mg of fat were capable of producing adipogenesis but did so less consistently than the larger autografts. These findings have important implications for adipose tissue engineering strategies and for understanding de novo production of adipose tissue.

The local Shwartzman reaction (LSR) is an inflammatory response in the skin that is considered a model for skin necrosis associated with meningococcemia. We tested the hypothesis that ischemia can act as the provocative agent to produce this response. In eight rats, bilateral inferior epigastric flaps were outlined. Within each flap, three injection sites were marked. Site 1 had 0.1 mL of endotoxin injected 24 h before surgery. The other two sites had either endotoxin or saline injected immediately before surgery. Both flaps were raised on their pedicle and one side rendered ischemic for 6 h and then reperfused. Animals were killed either 30 min or 48 h later and the tissue from each site examined. After 48 h of reperfusion, necrosis was grossly visible at the site of 24-h preischemia injection of endotoxin in three of four rats. No abnormalities were present at the other injection sites. Microscopically, all 24-h-delayed injection sites showed hemorrhage into all layers of the skin after both 30 min and 48 h of reperfusion. No hemorrhage was present at the other sites. These findings may serve as a potential model for the skin necrosis seen in meningococcemia.

Recurrent massive epistaxes due to heriditary haemorrhagic telangectasia were managed in two patients by ablation of the mucosa of the nasal and maxillary cavities and resurfacing with radial forearm fasciocutaneous free flaps. Both patients had undergone multiple procedures attempting to control the epistaxes prior to the definitive procedures described. Both patients remained epistaxis free postoperatively and retained their nasal airways. Olfactory function was preserved in the second patient due to preservation of the superior turbinate and cribriform olfactory mucosa.

Tissue engineering is a multidisciplinary field with the potential to replace tissues lost as a result of trauma, cancer surgery, or organ dysfunction. The successful production, integration, and maintenance of any tissue-engineered product are a result of numerous molecular interactions inside and outside the cell. We consider the essential elements for successful tissue engineering to be a matrix scaffold, space, cells, and vasculature, each of which has a significant and distinct molecular underpinning (Fig. 1). Our approach capitalizes on these elements. Originally developed in the rat, our chamber model (Fig. 2) involves the placement of an arteriovenous loop (the vascular supply) in a polycarbonate chamber (protected space) with the addition of cells and an extracellular matrix such as Matrigel or endogenous fibrin (34, 153, 246, 247). This model has also been extended to the rabbit and pig (J. Dolderer, M. Findlay, W. Morrison, manuscript in preparation), and has been modified for the mouse to grow adipose tissue and islet cells (33, 114, 122) (Fig. 3)...

The authors previously described induction of spontaneous tissue generation by implanting a collagen matrix and a ligated pedicle (arteriovenous bundle) into a hollow porous chamber in vivo in the rabbit. They hypothesized that increased tissue volume could be obtained by the application of basic fibroblast growth factor (bFGF) and/or by increasing the chamber size and porosity.In rabbits, a saphenous arteriovenous pedicle and a collagen sponge were inserted into a porous chamber in the groin. Small-volume pore chambers (experiment 1, n = 7) and larger-volume, wider pore chambers (experiment 2, n = 13) were compared, and each was compared with and without bFGF. An additional three flaps of experiment 2 with bFGF were skin grafted, microsurgically transplanted to the ear, and evaluated at 6 months for stability.All patent chambers grew tissue; chambers with bFGF were almost filled, and those without were only half-filled. Histomorphometric analysis confirmed a significant difference. The larger-volume, larger-pore chambers produced more than twice the volume of tissue as the smaller chambers did, and this was significant. Tissue volume in both the control and bFGF groups of experiment 2 was significantly greater than that in the respective groups of experiment 1. Histology, angiography, and scanning electron microscopy confirmed greater vascularity in the bFGF groups and demonstrated vascular connections penetrating the chamber pores linking with angiogenic sprouts, probably from the vasa vasorum of the pedicle, to contribute to new growth. Transplanted flaps survived and appeared normal 6 months later.Patent pedicles, bFGF, large pore size, and larger-volume chambers all seemed to contribute to increased tissue growth in this model. The tissue is stable long term.

As in vivo tissue engineering of complex tissues and organs progresses, there is a need for an independently vascularized, alterable, and recoverable model. Current models of islet cell transplantation (release into the portal venous system, placement under the renal capsule, and microencapsulation) lack these qualities. We have developed a model of angiogenesis and spontaneous tissue generation in the rat that lends itself as a potential platform for tissue engineering. In this experiment, we examined the effectiveness of such a model in addressing some of the shortcomings of endocrine pancreatic transplantation. An arteriovenous loop was created in the groins of five adult inbred Sprague-Dawley rats, and placed within polycarbonate chambers. Isolated pancreatic islet cell clusters were placed within the chambers, suspended in a matrix of Matrigel®. The chambers were recovered at 3 weeks, and the newly generated tissue was processed for histologic and immunohistochemical analysis. By 3 weeks, spontaneous generation of angiogenesis and collagen matrix and deposition of a collagen matrix was observed. Surviving islet cells were identified by histology and their viability was confirmed via immunohistochemistry for insulin and glucagon. This study demonstrates the ability to maintain viability and functionality of transplanted islet cells on a tissue-engineered platform with an independent vascular supply. The model provides the ability to alter the graft environment via matrix substitution, cellular coculture, and administration of growth factors. The transplanted tissues are recoverable without animal sacrifice and are microsurgically transferable. This model may provide an in vivo culture platform for the study of islet transplantation.

We report three clinical cases in which Dupuytren’s disease was triggered by surgical trauma. All patients developed the contracture between 3 weeks and 3 months after operation for unrelated / pathology of the hand. They had significant swelling of the hand postoperatively, preventing full mobilization. They did not have a strong diathesis for the disease. Since the appearance of the contracture, they have not developed the disease in the contralateral hand or anywhere else in the body. In one case, a similar operation on the contralateral hand has not provoked onset of the disease.

Five cases of spontaneous thrombosis of a palmar digital vein are presented. The patients, all female, complained of a tender and unsightly lump over the digital palmar surface at the proximal interphalangeal joint level. Two fingers were affected in one patient. None could recall any history of trauma. In four cases a surgical excision was carried out. Histology confirmed the intraoperative findings of thrombosis of a superficial digital vein in every case. All patients are free from recurrence at a mean follow-up of 2.5 years.

Plantar fibromatosis is a recognised form of Dupuytren's disease. Two cases are presented in which there were flexion contractures of the toes in the involved feet. This is an extremely rare form of this plantar affliction.

This study aimed to explore the hypothesis that activated complement components contribute significantly to I/R (ischaemia/reperfusion) injury in skeletal muscle. After 50, 70 and 90 min of tourniquet ischaemia and 24 h of reperfusion, viability of the medial gastrocnemius muscle in CBA-C57BL/6 wild-type mice, assessed histochemically by reduction of NBT (Nitro Blue Tetrazolium) dye, was 60, 21 and 8% respectively. Skeletal muscle viability after 70 min of ischaemia and 24 h of reperfusion in transgenic mice expressing a combination of human CD46, CD55 and CD59, all inhibitors of complement activation, was 45% compared with 24% in ischaemic reperfused wild-type mice (P=0.008; n=6 per group). Muscle from sham-treated transgenic mice and wild-type littermates had no significant loss of viability relative to normal contralateral gastrocnemius muscle. A significant reduction in myeloperoxidase activity (a measure of neutrophil infiltration), xanthine oxidase activity (a source of free radicals) and water content (a measure of oedema) was observed in ischaemic reperfused muscle from transgenic mice compared with ischaemic reperfused wild-type muscle (P

Background Ischaemia reperfusion (IR) injury of skeletal muscle, is a significant cause of morbidity following trauma and surgical procedures, in which muscle fibre types exhibit different susceptibilities. The relative degree of mast cell mediated injury, within different muscle types, is not known. Methods In this study we compared susceptibility of the fast-twitch, extensor digitorum longus (EDL), mixed fast/slow-twitch gastrocnemius and the predominately slow-twitch soleus, muscles to ischemia reperfusion (IR) injury in four groups of mice that harbour different mast cell densities; C57/DBA mast cell depleted (Wf/Wf), their heterozygous (Wf/+) and normal littermates (+/+) and control C57BL/6 mice. We determined whether susceptibility to IR injury is associated with mast cell content and/or fibre type and/or mouse strain. In experimental groups, the hind limbs of mice were subjected to 70 minutes warm tourniquet ischemia, followed by 24 h reperfusion, and the muscle viability was assessed on fresh whole-mount slices by the nitroblue tetrazolium (NBT) histochemical assay. Results Viability was remarkably higher in the Wf/Wf strain irrespective of muscle type. With respect to muscle type, the predominately slow-twitch soleus muscle was significantly more resistant to IR injury than gastrocnemius and the EDL muscles in all groups. Mast cell density was inversely correlated to muscle viability in all types of muscle. Conclusion These results show that in skeletal muscle, IR injury is dependent upon both the presence of mast cells and on fibre type and suggest that a combination of preventative therapies may need to be implemented to optimally protect muscles from IR injury.

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 (W f /W f ) mice implicate mast cells in this pathology, they do not exclude a contribution by other deficiencies expressed in W f /W f 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 W f /W f littermates. After 12 weeks, the hind limbs of W f /W f , engrafted W f /W f 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 W f /W f mice subjected to IR have a significantly greater proportion of viable fibres than normal littermates subjected to identical injury (78.975.2 vs 27.273.7%, respectively). When W f /W f IR-resistant mice were engrafted with BMMC from normal littermates and subjected to IR, the proportion of viable muscle fibres was significantly reduced (78.975.2 vs 37.076.5%). Thus, engraftment of BMMC into W f /W f mice restores the susceptibility of skeletal muscle to IR injury irrespective of other abnormalities in W f /W f 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. Laboratory Investigation (2004) 84, 1103–1111, advance online publication, 7 June 2004; doi:10.1038/labinvest.3700126

This study was designed to clarify which vascular carrier, the arteriovenous shunt loop or the arteriovenous bundle, has more potential as a vascular carrier for an artificial skin flap in rats. An arteriovenous shunt loop was constructed between the femoral artery and vein using an interpositional artery (group I) or vein (group II) graft. For arteriovenous bundle groups, the femoral artery and vein were used and subdivided into two groups: distal ligation type (group III) and flow-through type (group IV). The vascular pedicle was wrapped with an artificial dermis and implanted beneath the inguinal skin for 4 weeks. For the control group, a folded sheet of artificial dermis without any vascular carrier was embedded. In experiment 1, the volumes of generated tissue within the artificial dermis were measured in the experimental and control groups (n = 5 in each group). In experiment 2, the origin of new blood vessels sprouting from the arteriovenous shunt loop and arteriovenous bundle were evaluated histologically. The volume of generated tissue in the shunt groups was significantly greater than that in the bundle groups (p < 0.01). However, the bundle groups also showed a great potential for producing new tissue. Serial histological studies showed that new capillaries were derived not only from the vasa vasorum of the femoral vessels but directly from the femoral vein in both the shunt and the bundle groups. This "sprouting" was extensively exhibited in the group III. Although the arteriovenous shunt loop showed a greater potential for producing new tissue and capillaries, the distal ligation type of bundle was thought to be an effective and practical vascular carrier for producing a tissue-engineered skin flap.

The free osteocutaneous fibula flap is the most commonly transferred vascularized bone flap used today. The associated donor site morbidity is well-reported, including vague pain which usually presents late, and is associated with few objective clinical findings. The authors report a case in which the pain had an identifiable cause; they suggest how this can be avoided.

We compared the efficacy with which the fluorescent tracers Fast Blue (FB) and Diamidino Yellow (DY) retrogradely label neutrons. Trace crystals were applied to the sciatic nerve exclusively (single label) or serially (double label). Unbiased cell counts showed that FB and DY label similar numbers of motoneurons (P=1.00, df 5) or DRG neurons (P=0.95, df 5) when applied exclusively. Plotting of motoneurons revealed a similar pattern of distribution of FB and DY labeled neurons. When the tracers were applied serially, 79% of labeled motoneurons and 77% of labeled DRG neurons were double-labeled irrespective of which tracer was applied first. Equal proportions of the remaining labeled neurons were single-labeled with FB or DY. These data show that FB and DY label equal numbers of motor and sensory neurons of the sciatic nerve following exclusive or serial application of tracers. These findings support the use of FB and DY together in serial fluorescent labeling experiments.

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.

We examined the role of endothelin in ischaemia/reperfusion injury in skeletal muscle, using the endothelin receptor antagonist Bosentan. In the rat hindlimb tourniquet ischaemia model, one hindlimb was rendered ischaemic for 2 h at 36 degrees C, then blood flow was re-established for either 24 h to assess muscle survival or 1.5 h for a study of capillary perfusion. In the first set of rats, the gastrocnemius muscle was removed from the postischaemic limb and assessed for viability histochemically using the nitro blue tetrazolium stain. Tissue water content (a measure of oedema) and myeloperoxidase activity (a measure of neutrophil accumulation) were also assessed in the ischaemic muscle, the contralateral non-ischaemic muscle and the lungs. In the second set of rats, the hind limb was infused with India ink after 2-h ischaemia and 1.5-h reperfusion and the muscle was harvested, fixed and cleared. In control rats, muscle viability was 17+/-2% (S.E.M.). In rats treated with Bosentan (10 mg/kg, i.p.) 30 min before release of the tourniquet, muscle viability (48+/-7%) was significantly increased compared to the control group (P0.01). Bosentan treatment had no significant effect on tissue water content or myeloperoxidase activity in the ischaemic muscle, the contralateral non-ischaemic muscle or the lung. Immunoreactive endothelin levels in serum increased to a peak at 90 min of reperfusion and returned to control levels by 24-h reperfusion. India ink studies demonstrated a significantly increased functional capillary density in postischaemic Bosentan-treated muscles compared with postischaemic control muscles (P0.05). These results suggest that endothelin plays an important role in the necrosis which results from a period of ischaemia and reperfusion in skeletal muscle, by mediating a decrease in postischaemic microvascular perfusion.

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 use of frozen sectioning is a convenient and rapid means of observing the results obtained using fluorescent retrograde tracers. Quantitation of these results using the biased stereological methods currently available can be associated with large errors. A recently developed stereological tool, the optical dissector, provides unbiased and efficient results, however, the requirements for its use in frozen sections has not previously been established. In this study, a comparison was made of neuron numbers, estimated using the optical dissector method, in the motoneuron pool retrogradely labelled from the rat sciatic nerve with either Fast Blue or Tetramethylrhodamine dextran (fluoro-ruby) in methacrylate embedded and frozen spinal cord specimens. Despite over 50% shrinkage in the frozen sections, compared with virtually no shrinkage in the methacrylate sections, no significant difference in labelled motoneuron numbers was observed, provided this shrinkage was taken into account. Correction for section shrinkage is therefore essential in order to use the optical dissector with confidence to count fluorescent labelled neurons in frozen tissue.

Storage of skin flaps in the cold before replantation increases their tolerance to ischemic damage. Rat epigastric skin flaps were perfused immediately before 2 days of cold ischemia with 3 ml of normal saline containing either 10 U per milliliter of heparin (group 1, N = 11) or normal saline (group 2, N = 10), or stored without perfusion (group 3, N = 6), and replanted. Flap viability was assessed 7 days later. The mean flap survival in groups 1, 2, and 3 was 73% (p < 0.01 compared with group 2), 10%, and 33% respectively. Intravascular fibrin deposits were detected histochemically 5 minutes before reperfusion in nonperfused flaps and 5 minutes after reperfusion in saline-perfused flaps, but not in flaps perfused with heparinized saline. Angiography revealed evidence of no reflow in the first 5 minutes of reperfusion in saline-perfused flaps, but normal blood flow in heparinized saline-perfused flaps. Tissue water content, myeloperoxidase activity, and hydroperoxide levels after 1 and 24 hours of reperfusion were not significantly different in flaps perfused with heparinized saline and normal saline. These findings indicate that in skin flaps perfused before ischemia, flaps perfused with heparinized saline survive significantly better than flaps perfused with normal saline. They also survive better than nonperfused flaps but the improvement is not significant.

The aim of this study was to investigate the effect of the cytokine, r-metHuG-CSF, in a rat model of ischaemia-reperfusion (IR) injury and the pathophysiological mechanism involved. The administration of r-metHuG-CSF (20 (g/kg, s.c.) 4 h prior to either 100 min or 2 h of tourniquet ischaemia to the upper thigh significantly improved the viability of skeletal muscle after 24 h reperfusion compared with saline-treated rats (P < 0.05). Administration of r-metHuG-CSF earlier (24 h before ischaemia) or later (immediately before ischaemia) had no protective effect. At the dose used, r-metHuG-CSF caused a three-fold increase in the level of circulating blood neutrophils and a modest but significant increase in the neutrophil content of ischaemic muscle after 24 h reperfusion. Reduction of neutrophils to 1.4% of normal levels by cyclophosphamide (150 mg/kg, i.p.) prior to injury had no significant effect on the survival of muscle subjected to 2 h ischaemia and 24 h reperfusion or on the protective effect of r-metHuG-CSF. IR injury to skeletal muscle was accompanied by a time-dependent increase in plasma TNFalpha levels during the first 8 h of reperfusion and the increase was reduced significantly by pretreatment with r-metHuG-CSF. However, a similar time-dependent increase in plasma nitrite/nitrate levels was unaffected by pretreatment with r-metHuG-CSF. These findings suggest that the protective effect of r-metHuG-CSF may be mediated by the attenuated release of TNFalpha and indicate that the level of neutrophils in either blood or injured tissue does not influence significantly the viability of rat muscle after IR injury.

The survival of skeletal muscle myoblasts in culture after exposure either to a donor of NO, sodium nitroprusside (SNP), or ethanamine, 2,2′-(hydroxynitrosohydrazono)bis-(DETA NONOate), or to a donor of both NO and O − 2 , 3-morpholinosydnonimine hydrochloride (SIN-1), was investigated. SIN-1 reduced clonogenic survival markedly but donors of NO alone did not. The injurious effect of SIN-1 was prevented by oxyhemoglobin or by uric acid but not by superoxide dismutase. The exposure of myoblasts to authentic peroxynitrite (ONOO − ) or to DETA NONOate in the presence of an O − 2 -generating system did not reduce their survival. The results show that NO or ONOO − alone is not detrimental to myoblast survival and suggest that SIN-1 toxicity is, at least in part, mediated by H 2 O 2 in this myoblast culture system.

Schwann cells play a major role in promoting nerve survival and regeneration after injury. Their activities include providing neurotrophic factors and increasing the production of extracellular matrix components and cell surface adhesion molecules to promote axon regeneration. Following nerve transection, leukemia inhibitory factor (LIF) is up-regulated by Schwann cells at the injury site. LIF receptors are also up-regulated at the nerve injury site, but their cellular localization and function have not been fully characterized. We demonstrate that Schwann cells express mRNAs for LIF and the LIF receptor components LIF receptor subunit beta and glycoprotein 130 in vitro. We also show that although LIF is not required for the genesis of Schwann cells, it can potentiate the survival of differentiated Schwann cells in the context of neuregulin support. Not only does exogenous LIF promote survival under these conditions, but addition of the soluble LIF receptor (LIF binding protein) and anti-LIF antibodies significantly reduced cell survival, suggesting that LIF exerts autocrine effects. These results suggest that Schwann cell survival following nerve injury is potentially modulated by LIF.

The potential use of the anti-inflammatory glucocorticoid, dexamethasone, to treat ischaemic skin flaps prior to reperfusion was investigated. Island flaps were raised in rats, subjected to arteriovenous ischaemia for 2 h, normal blood flow for 24 h, secondary venous ischaemia for 4 h and secondary reperfusion for 7 days. This sequence mimics the clinical sequence of free flap transfer followed by a subsequent venous thrombosis. Groups of 10 rats were administered with an intraperitoneal dose of either saline (controls) or dexamethasone (2.5 mg/kg) 30 min before the end of the venous ischaemia. Compared with viability of 70.0% in controls, dexamethasone treatment increased viability significantly to 92.0% (P0.01). In skin flap tissue harvested at 24 h reperfusion, dexamethasone treatment resulted in significant attenuation of tissue water content, tissue myeloperoxidase activity and tissue hydroperoxide levels. Thus the protective effect of this agent may be explained by the combined reduction of oedema formation, neutrophil infiltration and free radical production, respectively. We conclude that a single systemic dose of dexamethasone prior to reperfusion may be beneficial in treating skin flaps that have undergone secondary venous ischaemia.

In previous studies we have shown that 80-100% of rabbit femoral vascular autografts cold-stored at 4 degrees C for 3 weeks remain patent 3 weeks after reinsertion in the femoral artery. The present study reports the effect on graft patency of increasing either the period of cold storage prior to reinsertion or the duration of reperfusion to 6 months. Rabbit femoral blood vessels were cold-stored (CS) at 4 degrees C for varying periods. CS autografts were reinserted into the contralateral leg for 3 weeks or 6 months. Graft patency was determined and grafts examined by histological, immunohistochemical and electron microscopical techniques. Six months after reinsertion patency of 4-week CS arterial and 1-week CS venous grafts was 40% and 27% respectively, very much lower than the 80-100% seen after 3 weeks reperfusion. Arterial grafts CS for 6 months had a patency rate of 70% after 3 weeks reperfusion but 0% after 6 months. Morphological examination suggests that the delayed failure of cold-stored vascular grafts is caused by thrombus superimposed on intimal hyperplasia within the graft.Cold-stored vascular grafts are useful prostheses when only 3-4 weeks graft patency is required. They are not suitable for use when long-term graft patency is needed.

The outcome of end-to-side repair of peripheral nerves was investigated. The sciatic nerve in 10 male Sprague-Dawley rats was dissected to its tibioperoneal junction. The nerve to the medial gastrocnemius muscle, branching from the tibial nerve, was ligated at its origin and divided. Its distal end was sutured to an epineurial window on the side of the intact tibial nerve 1 cm distally. At 12 weeks, physiologic evaluation of the medial gastrocnemius muscle and analyses of histologic preparations of nerve and muscle were performed. The results showed that reinnervation successfully occurred in 8 rat media gastrocnemii. The mean weight of the reinnervated medial gastrocnemius was 73% of the contralateral normal muscle, the mean muscle fiber cross-sectional area of the reinnervated muscle was 72%, and the force of contraction was 60%. Analyses of histologic preparations revealed evidence of myelinated axons in the medial gastrocnemius nerve and no evidence of damage to axons of the donor tibial nerve.

Background: The usual methods of closure of major chest and abdominal wall defects have significant disadvantages. Skin grafts provide no structural support and result in incisional hernias. Synthetic mesh requires skin cover and is prone to infection and wound breakdown. The tensor fasciae latae (TFL) myocutaneous flap offers skin cover and a semi-rigid fascial layer. We document our unit'experience in pedicled and free TFL flaps. Methods: The TFL flap closure of trunk defects was undertaken in 10 patients between August 1989 and April 1997. All cases were not amenable to primary closure and repair with synthetic mesh or skin grafts. Results: The defect was satisfactorily repaired in all cases without subsequent herniation. The closure techniques using a pedicled TFL flap and a TFL flap for a free-tissue transfer are described. Conclusions: We conclude that the TFL flap is the method of choice for repairs of major truncal defects.

The prefabrication of bone grafts in a rat model was investigated. In 26 Sprague-Dawley rats, free iliac crest bone graft was harvested, bivalved, and reinserted heterotopically into the groin, where it was closed around the mobilized superficial inferior epigastric vascular bundle. In half the animals, the vascular bundle remained in continuity as a flow-through pedicle (group 1); in the remaining animals, the pedicle was ligated and divided distal to the bone graft. All grafts were isolated from other tissues by a silicone sheet envelope. At 3 or 6 weeks, the grafts were re-explored and analyzed by India ink perfusion and histologic examination for evidence of viability and neovascularization. Three weeks after insertion, India ink perfusion of the group 1 and 2 grafts revealed neovascularization extending to the periphery of the graft, and histologic examination showed extensive new bone formation on endosteal, periosteal, and trabecular surfaces of the graft. Six weeks after insertion, creeping substitution had almost completely remodelled the cortical and cancellous bone of both group 1 and 2 grafts to create a viable vascularized bone graft on a pedicle. In 3 control nonvascularized grafts (free iliac cortical bone without an implanted pedicle), all pre-existing bone of the graft was dead 3 weeks after insertion, and only very limited new bone formation was present within the graft.

1. The effects of the nitric oxide synthase (NOS) inhibitors, NG-nitro-l-arginine-methyl ester (l-NAME), nitroiminoethyl-l-ornithine and S-methylisothiourea on skeletal muscle survival following 2 h of tourniquet ischaemia and 24 h of reperfusion were compared with those of the antiinflammatory steroid, dexamethasone. 2. Administration of each of the NOS inhibitors or dexamethasone 30 min before reperfusion reduced the degree of skeletal muscle necrosis 24 h after reperfusion. 3. The influence of timing of drug administration was investigated. l-NAME administered 30 min before reperfusion, at 3 h after reperfusion, but not thereafter, significantly improved muscle survival compared with saline-treated controls. Dexamethasone administered 30 min before, or at 3 or 8 h after reperfusion, but not at 16 h, significantly improved muscle survival, but neither agent had protective effects when administered before ischaemia. 4. After 8 h of reperfusion of ischaemic skeletal muscle, cell-free homogenates contained Ca2+-independent (inducible) NOS activity which was reduced in dexamethasone-treated (2.5 mg/kg) rats. Furthermore, inducible NOS mRNA levels, as detected by reverse transcriptase-PCR, were increased after 8 h of reperfusion in saline, but not in dexamethasone-treated rats. 5. These data suggest a significant deleterious effect of endogenous NO which may be restricted to the first 3 h of the reperfusion phase of ischaemia-reperfusion injury, and raise the possibility of effective treatment of incipient reperfusion injury, even after several hours of reperfusion.

This case report describes the revascularization of the testis in a 21-year-old man after his testicular vessels were inadvertently divided during primary repair of an inguinal hernia. The circumstances in which this occurred and the possible complications are discussed and the need to attempt repair of the divided vessels in such cases is emphasized.

Boosting angiogenesis is a crucial process to enhance tissue growth in tissue engineering (TE). Hepatoma-derived growth factor (HDGF) has been identified as an angiogenic factor, but its involvement in angiogenesis in an arteriovenous loop-based TE chamber developed by the laboratory is unclear. In this study, the authors first examined the effects of HDGF on angiogenic responses in endothelial cells and in a corneal model of neovascularization, and then characterized the expression of HDGF in the TE chamber. HDGF (1-500 ng/mL) induced concentration-dependent angiogenic responses in human endothelial cells in vitro (proliferation, migration, and tube formation). Local application of HDGF stimulated neovascularization in a rat model of corneal angiogenesis. In the TE chamber, there was an increase in blood vessel volume from day 3 to day 14. Immunofluorescence microscopy revealed that HDGF is highly expressed in the neovessels in the chamber. Peak expression of HDGF (day 3) coincided with the infiltration of inflammatory cells, and the mRNA level of endogenous HDGF correlated with that of tumor necrosis factor α (TNFα). In vitro, TNFα stimulated HDGF expression in endothelial cells. The data suggest that HDGF may be involved in angiogenic responses in the TE chamber and the proinflammatory cytokine TNFα may have a pivotal role in stimulating HDGF expression. Enhancing HDGF signaling may be a new approach to extend vascularization for TE. © 2013 National Taiwan University.