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Background The survival of engineered cardiac muscle ‘grafts’ to the epicardium is limited by vascularization post‐transplantation in rat models. In this article, we describe the methodology of a novel rat model that allows for the transplantation of an engineered cardiac muscle flap (ECMF) onto the epicardium. Materials and Methods A total of 40 rats were used. Twenty‐four neonatal rats were used to harvest cardiomyocytes. At week 1, ECMF were generated by seeding cardiomyocytes into the arteriovenous loop (AVL) tissue engineering chamber implanted into the right groin of adult rats (n = 8). At week 6, the ECMF were harvested based on a pedicle along the femoral‐iliac‐abdominal vessel and anastomosed to the neck vessels of the recipient syngeneic adult rats (n = 8). The flaps were delivered into the thoracic cavity and onto the epicardium. The transplanted flaps were harvested at week 10. Survival of the flaps was assessed by the patency of anastomoses and viability of the cardiomyocytes through histological analysis (hematoxylin and eosin [H&E], desmin, and von Willebrand factor [vWF] immunostaining). Results Six out of 8 rats survived the transplantation procedure. These remaining 6 recipient rats survived until harvest time point at 4 weeks post‐transplantation. The mean area of the flap was 46.7mm2. Six out of 6 flaps harvested at week 10 showed viable cardiomyocytes using desmin immunostaining and vascular channels were seen at the interface between flap and epicardium. Conclusion This is a technically feasible model that will be useful for future assessment of different cardiac stem cell implants and their functional significance in rat heart models.

Supplemental Digital Content is available in the text., Background: Cancer patients often require radiotherapy (RTx) to enhance their survival. Unfortunately, RTx also damages nearby healthy non-cancer tissues, leading to progressive fibrotic soft-tissue injury, consisting of pain, contracture, tissue-breakdown, infection, and lymphoedema. Mechanisms underlying the clinically observed ability of fat grafting to ameliorate some of these effects, however, are poorly understood. It was hypothesized that RTx significantly alters fibroblast cell function and the paracrine secretome of adipose-derived stem cells (ADSC) may mitigate these changes. Methods: To investigate cellular changes resulting in the fibrotic side-effects of RTx, cultured normal human dermal fibroblasts (NHDF) were irradiated (10Gy), then studied using functional assays that reflect key fibroblast functions, and compared with unirradiated controls. RNA-Seq and targeted microarrays (with specific examination of TGFβ) were performed to elucidate altered gene pathways. Finally, conditioned-media from ADSC was used to treat irradiated fibroblasts and model fat graft surgery. Results: RTx altered NHDF morphology, with cellular functional changes reflecting transition into a more invasive phenotype: increased migration, adhesion, contractility, and disordered invasion. Changes in genes regulating collagen and MMP homeostasis and cell-cycle progression were also detected. However, TGFβ was not identified as a key intracellular regulator of the fibroblast response. Finally, treatment with ADSC-conditioned media reversed the RTx-induced hypermigratory state of NHDF. Conclusions: Our findings regarding cellular and molecular changes in irradiated fibroblasts help explain clinical manifestations of debilitating RTx-induced fibrosis. ADSC-secretome-mediated reversal indicated that these constituents may be used to combat the devastating side-effects of excessive unwanted fibrosis in RTx and other human fibrotic diseases.

For decades, plastic surgeons have spent considerable effort exploring anatomical regions for free flap design. More recently, tissue-engineering approaches have been utilised in an attempt to grow transplantable tissue flaps in vivo. The aim of this study was to engineer a fat flap with a vascular pedicle by combining autologous fat grafts and a novel acellular hydrogel (Adipogel) in an established tissue-engineering model comprising a chamber and blood vessel loop. An arteriovenous loop was created in the rat groin from the femoral vessels and positioned inside a perforated polycarbonate chamber. In Group 1, the chamber contained minced, centrifuged autologous fat; in Group 2, Adipogel was added to the graft; and in Group 3, Adipogel alone was used. Constructs were histologically examined at 6 and 12 weeks. In all groups, new tissue was generated. Adipocytes, although appearing viable in the graft at the time of insertion, were predominantly nonviable at 6 weeks. However, by 12 weeks, new fat had formed in all groups and was significantly greater in the combined fat/Adipogel group. No significant difference was seen in final construct total volume or construct neovascularisation between the groups. This study demonstrated that a pedicled adipose flap can be generated in rats by combining a blood vessel loop, an adipogenic hydrogel, and a lipoaspirate equivalent. Success appears to be based on adipogenesis rather than on adipocyte survival, and consistent with our previous work, this adipogenesis occurred subsequent to graft death and remodelling. The regenerative process was significantly enhanced in the presence of Adipogel.

Hair follicle cells contribute to wound healing, skin circulation, and skin diseases including skin cancer, and hair transplantation is a useful technique to study the participation of hair follicle cells in skin homeostasis and wound healing. Although hair follicle transplantation is a well-established human hair-restoration procedure, follicular transplantation techniques in animals have a number of shortcomings and have not been well described or optimized. To facilitate the study of follicular stem and progenitor cells and their interaction with surrounding skin, we have established a new murine transplantation model, similar to follicular unit transplantation in humans. Vibrissae from GFP transgenic mice were harvested, flip-side microdissected, and implanted individually into needle hole incisions in the back skin of immune-deficient nude mice. Grafts were evaluated histologically and the growth of transplanted vibrissae was observed. Transplanted follicles cycled spontaneously and newly formed hair shafts emerged from the skin after 2 weeks. Ninety percent of grafted vibrissae produced a hair shaft at 6 weeks. After pluck-induced follicle cycling, growth rates were equivalent to ungrafted vibrissae. Transplanted vibrissae with GFP-positive cells were easily identified in histological sections. We established a follicular vibrissa transplantation method that recapitulates human follicular unit transplantation. This method has several advantages over current protocols for animal hair transplantation. The method requires no suturing and minimizes the damage to donor follicles and recipient skin. Vibrissae are easier to microdissect and transplant than pelage follicles and, once transplanted, are readily distinguished from host pelage hair. This facilitates measurement of hair growth. Flip-side hair follicle microdissection precisely separates donor follicular tissue from interfollicular tissue and donor cells remain confined to hair follicles. This makes it possible to differentiate migration of hair follicle cells from interfollicular epidermis in lineage tracing wound experiments using genetically labeled donor follicles.

Avulsion, epidermal marsupialization, and infection cause failure at the skin-material interface. A robust interface would permit implantable robotics, prosthetics, and other medical devices; reconstruction of surgical defects, and long-term access to blood vessels and body cavities. Torus-shaped cap-scaffold structures were designed to work in conjunction with negative pressure to address the three causes of failure. Six wounds were made on the backs of each of four 3-month old pigs. Four unmodified (no caps) scaffolds were implanted along with 20 cap-scaffolds. Collagen type 4 was attached to 21 implants. Negative pressure then was applied. Structures were explanted and assessed histologically at day 7 and day 28. At day 28, there was close tissue apposition to scaffolds, without detectable reactions from defensive or interfering cells. Three cap-scaffolds explanted at day 28 showed likely attachment of epidermis to the cap or cap-scaffold junction, without deeper marsupialization. The combination of toric-shaped cap-scaffolds with negative pressure appears to be an intrinsically biocompatible system, enabling a robust skin-material interface. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 1307-1318, 2017.

'Off-the-shelf' tissue-engineered skin alternatives for epidermal and dermal skin layers are available; however, no such alternative for the subdermal fat layer exists. Without this well-vascularized layer, skin graft take is variable and grafts may have reduced mobility, contracture and contour defects. In this study a novel adipose-derived acellular matrix (Adipogel) was investigated for its properties to generate subdermal fat in a rat model. In a dorsal thoracic site, a 1 × 1 cm Adipogel implant was inserted within a subdermal fat layer defect. In a dorsal lumbar site, an Adipogel implant was inserted in a subfascial pocket. Contralateral control defects remained empty. At 8 weeks wound/implant sites were evaluated histologically, immunohistochemically and morphometrically. Identifiable thoracic Adipogel implants lost volume in vivo over 8 weeks. Neovascularization and adipogenesis were evident within implants and adipocyte percentage volume was 33.07 ± 6.55% (mean ± SEM). A comparison of entire cross-sections of thoracic wounds demonstrated a significant increase in total wound fat in Adipogel-implanted wounds (37.19 ± 4.48%, mean ± SEM) compared to control (16.53 ± 4.60%; p = 0.0092), indicating that some Adipogel had been completely converted to normal fat. At the lumbar site, Adipogel also lost volume, appearing flattened, although fat generation and angiogenesis occurred. At both sites macrophage infiltration was mild, whilst many infiltrating cells were PDGFRβ-positive mesenchymal cells. Adipogel is adipogenic and angiogenic and is a promising candidate for subcutaneous fat regeneration; it has the potential to be a valuable adjunct to wound-healing therapy and reconstructive surgery practice. Copyright © 2015 John Wiley & Sons, Ltd.

The extracellular matrix (ECM) Matrigel™ has frequently and successfully been used to generate new adipose tissue experimentally, but is unsuitable for human application. This study sought to compare the adipogenic potential of a number of alternative, biologically derived or synthetic ECMs with potential for human application, with and without growth factors and a small fat autograft. Eight groups, with six severe combined immunodeficient (SCID) mice per group, were created with bilateral chambers (silicone tubes) implanted around the epigastric vascular pedicle, with one chamber/animal containing a 5mg fat autograft. Two animal groups were created for each of four ECMs (Matrigel™, Myogel, Cymetra® and PuraMatrix™) which filled the bilateral chambers. One group/ECM had no growth factors added to chambers whilst the other group had growth factors (GFs) (vascular endothelial growth factor-A (VEGF-A) plus fibroblast growth factor-2 (FGF-2) plus platelet-derived growth factor-BB (PDGF-BB)) added to both chambers. At 6weeks, chamber tissue was morphometrically assessed for percent and absolute adipose tissue volume. Overall, the triple GF regime significantly increased percent(∗) and absolute(#) adipose tissue volume (p

Poor vascularization remains a key limiting factor in translating advances in tissue engineering to clinical applications. Vascular pedicles (large arteries and veins) isolated in plastic chambers are known to sprout an extensive capillary network. This study examined the effect vascular pedicles and scaffold architecture have on vascularization and tissue integration of implanted silk scaffolds. Porous silk scaffolds with or without microchannels are manufactured to support implantation of a central vascular pedicle, without a chamber, implanted in the groin of Sprague Dawley rats, and assessed morphologically and morphometrically at 2 and 6 weeks. At both time points, blood vessels, connective tissue, and an inflammatory response infiltrate all scaffold pores externally, and centrally when a vascular pedicle is implanted. At week 2, vascular pedicles significantly increase the degree of scaffold tissue infiltration, and both the pedicle and the scaffold microchannels significantly increase vascular volume and vascular density. Interestingly, microchannels contribute to increased scaffold vascularity without affecting overall tissue infiltration, suggesting a direct effect of biomaterial architecture on vascularization. The inclusion of pedicles and microchannels are simple and effective proangiogenic techniques for engineering thick tissue constructs as both increase the speed of construct vascularization in the early weeks post in vivo implantation.

The ability to generate controlled amounts of adipose tissue would greatly ease the burden on hospitals for reconstructive surgery. We have previously shown that a tissue engineering chamber containing a vascular pedicle was capable of forming new fat; however, further refinements are required to enhance fat formation. The development and maintenance of engineered adipose tissue requires a suitable source of growth factors and a suitable scaffold. A hydrogel derived from adipose tissue may fulfil this need. Subcutaneous fat was processed into a thermosensitive hydrogel we refer to as adipose-derived matrix (ADM). Protein analysis revealed high levels of basement membrane proteins, collagens and detectable levels of growth factors. Adipose-derived stem cells exposed to this hydrogel differentiated into adipocytes with >90% efficiency and in vivo testing in rats showed significant signs of adipogenesis after 8 weeks. ADM's adipogenic properties combined with its simple gelation, relatively long shelf life and its tolerance to multiple freeze-thaw cycles, makes it a promising candidate for adipose engineering applications.

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and trade one defect for another. Tissue engineering holds the promise to address this increasing demand. However, most tissue engineering strategies fail to generate stable and functional tissue substitutes because of poor vascularization. This paper focuses on an in vivo tissue engineering chamber model of intrinsic vascularization where a perfused artery and a vein either as an arteriovenous loop or a flow-through pedicle configuration is directed inside a protected hollow chamber. In this chamber-based system angiogenic sprouting occurs from the arteriovenous vessels and this system attracts ischemic and inflammatory driven endogenous cell migration which gradually fills the chamber space with fibro-vascular tissue. Exogenous cell/matrix implantation at the time of chamber construction enhances cell survival and determines specificity of the engineered tissues which develop. Our studies have shown that this chamber model can successfully generate different tissues such as fat, cardiac muscle, liver and others. However, modifications and refinements are required to ensure target tissue formation is consistent and reproducible. This article describes a standardized protocol for the fabrication of two different vascularized tissue engineering chamber models in vivo.

Cardiac tissue engineering offers the prospect of a novel treatment for acquired or congenital heart defects. Previously, our studies have shown a significant mass of vascularized cardiac tissue can be generated using a vascularized tissue engineering chamber approach in nude rats. In this present study, syngeneic rats were investigated as an animal model for cardiac tissue engineering using the arteriovenous loop (AVL) chamber in the presence of a functional immune system. Neonatal cardiomyocytes implanted into the AVL chamber survived and assembled into a contractile flap confirming the basic features we previously showed in growing a cardiac construct. There was no significant loss of the assembled cardiac muscle from immune response. The engineered cardiac muscle flaps (ECMFs) formed were transplanted to the neck vessels of the same animal using a microsurgical technique, and all transplanted tissues remained contractile. The cardiac muscle volume of the control and transplant groups was estimated with histomorphometry using desmin and α-sarcomeric actin immunostaining, and there were no significant differences between the two groups. Finally, utilizing a novel model of transplantation, the ECMFs were transplanted to the heart of a recipient syngeneic rat as a vascularized tissue. The cardiac muscle within the transplanted ECMF was shown to survive and remain contractile for the 4-week post-transplantation period, and importantly, the cardiomyocytes retained the elongated, striated appearance of a mature phenotype. This study demonstrated the proof of concept for transplanting tissue-engineered cardiac muscle as a vascularized cardiac construct.

Mammographic density (MD) is the area of breast tissue that appears radiologically white on mammography. Although high MD is a strong risk factor for breast cancer, independent of BRCA1/2 mutation status, the molecular basis of high MD and its associated breast cancer risk is poorly understood. MD studies will benefit from an animal model, where hormonal, gene and drug perturbations on MD can be measured in a preclinical context. High and low MD tissues were selectively sampled by stereotactic biopsy from operative specimens of high-risk women undergoing prophylactic mastectomy. The high and low MD tissues were transferred into separate vascularised biochambers in the groins of SCID mice. Chamber material was harvested after 6 weeks for histological analyses and immunohistochemistry for cytokeratins, vimentin and a human-specific mitochondrial antigen. Within-individual analysis was performed in replicate mice, eliminating confounding by age, body mass index and process-related factors, and comparisons were made to the parental human tissue. Maintenance of differential MD post-propagation was assessed radiographically. Immunohistochemical staining confirmed the preservation of human glandular and stromal components in the murine biochambers, with maintenance of radiographic MD differential. Propagated high MD regions had higher stromal (p = 0.0002) and lower adipose (p = 0.0006) composition, reflecting the findings in the original human breast tissue, although glands appeared small and non-complex in both high and low MD groups. No significant differences were observed in glandular area (p = 0.4) or count (p = 0.4) between high and low MD biochamber tissues. Human mammary glandular and stromal tissues were viably maintained in murine biochambers, with preservation of differential radiographic density and histological features. Our study provides a murine model for future studies into the biomolecular basis of MD as a risk factor for breast cancer.

The histological fate of abdominal dermis-fat grafts implanted into the temporomandibular joint (TMJ) following condylectomy was studied. 21 rabbits underwent left TMJ discectomies and condylectomies; 6 were controls (Group A; no graft used); 15 (Group B) had autogenous abdominal grafts transplanted into the left TMJ. Animals were killed after 4, 12 and 20 weeks. Specimens of the TMJ were histologically and histomorphometrically evaluated. At 4 weeks, fat necrosis was clear in all specimens. The dermis component survived and formed cysts with no necrosis. By 12 weeks, viable fat deposits appeared with no evidence of necrotic fat. At 20 weeks, large amounts of viable fat were present in Group B specimens. Group A had no fat, although the missing condyles regenerated. In the presence of viable fat, Group B showed little condyle regeneration 20 weeks after condylectomy. Non-vascularised fat grafts do not survive transplantation, but stimulate neoadipogenesis. The fate of the dermis component of the graft is independent of the fat component. Fat in the joint space disrupts the regeneration of a new condylar head. Neoadipogensis inhibits growth of new bone and cartilage. This has clinical implications for TMJ ankylosis management and preventing heterotopic bone formation around prosthetic joints.

Manganese-enhanced magnetic resonance imaging (MEMRI) was used to investigate retrograde axonal tracing in the rat sciatic nerve model to assess its potential to examine peripheral nerve injury. The right sciatic nerve was exposed and crushed. After each recovery period, the distal part of the right sciatic nerve was injected with manganese (400 mM, 15 microl). After allowing 3 days for manganese transport the animals were subsequently scanned to visualize the sciatic nerve and its corresponding spinal cord and dorsal root ganglia with T1-weighted MRI. Thirty-four animals were randomly divided into 4 experimental groups according to their recovery period post-crush injury: 3 days (n=6), 2 weeks (n=6), 4 weeks (n=6) and 12 weeks (n=6); and two control groups: a non-crushed group (n=6) and a nerve cut group (n=4). In the no-injury group, the right sciatic nerve tract including its corresponding spinal cord and dorsal root ganglia showed significant T1 signal enhancement. In the animals with crush injury, the MR signal intensity was significantly reduced proximal to the injured site but gradually reappeared with increasing recovery period. The signal intensity of the sciatic tract was compared to the results of behavioral functional testing, retrograde axonal tracing with neural tracer fluorogold and histomorphometric analysis of the distal nerve. Significant correlations were observed between the MR signal intensity and the behavioral functional test (r=0.50, p

Reconstruction after resection of a large oropharyngeal tumour poses a difficult challenge. This case illustrates an extension of the previously described facial artery musculomucosal (FAMM) flap, whereby a defect of the soft palate is replaced with a similar trilaminar structure in the form of irradiated, redundant lower lip. This allows not only the treatment of the palatal defect, but also management of dribbling secondary to a marginal mandibular palsy.

The aim of this study was to assess the quality-of-life (QoL) outcomes of patients who had undergone temporomandibular joint (TMJ) discectomy with dermis-fat grafting compared with a cohort of closely matched patients who had not had surgery.A cross-sectional study of 61 patients was undertaken. All patients completed a TMJ surgery-specific QoL questionnaire. They were divided into 2 groups according to whether they had undergone TMJ discectomy (postsurgical group, n = 32) or not (presurgical group, n = 29). The 2 groups were closely matched for age, gender, clinical presentation, and radiologic diagnoses of Wilkes stage IV TMJ internal derangement.Post-TMJ surgery patients showed statistically significant decreases in pain levels (P.05), diet and chewing (P.01), mood (P.01), anxiety (P.01), and general health (P.05) compared with the presurgical patients. However, there were no statistically significant differences between the pre- and post-TMJ surgery groups in terms of speech, level of activity, recreation, and general well-being.The results of this study suggest that TMJ discectomy with dermis-fat grafting appears to have a positive QoL effect in terms of reducing pain levels and improving diet and chewing, mood, anxiety, and general health in patients with Wilkes stage IV TMJ internal derangement.

Transplantation of islets into the portal vein of diabetic patients has emerged as a promising procedure for the treatment of type 1 diabetes. However, shortages of donors and adverse effects leading to graft impairment and/or rejection have prevented this procedure from achieving widespread clinical application. The aim of this study was to develop a method that could support the survival and function of transplanted islets using a prevascularized tissue engineering chamber. Islets were transplanted into tissue engineering chambers established on the epigastric pedicle in the groin of diabetic mice. Islets were transplanted at the time of chamber implantation or with 21 days prevascularization of the chamber. Transplantation of islets into prevascularized chambers into diabetic RIP-K(b) mice resulted in a significant reduction in blood glucose levels that became evident in the third week and improved glycemic control as measured by a glucose tolerance test. This study highlights that islet survival and function are potentiated by allowing a period of prevascularization within tissue engineering chambers before islet transplantation. This novel prevascularized chamber may be an improved method of islet transplantation. It can be easily accessed for islet seeding, easily retrieved, and transplanted to alternative anatomical sites by microvascular methods.

Previously we described a subcutaneous arteriovenous loop (AVL)-based tissue engineering chamber system, which contains an intrinsic circulation circuit created by joining the proximal ends of the femoral artery and vein with a venous graft. We showed that nicotinamide adenine dinucleotide phosphate (NADPH) oxidase was involved in mediating neovascularization inside the chamber. However, the role of NADPH oxidase in tissue formation in the chamber is unknown. In this study, we examined the effects of gp91ds-tat, a peptidyl inhibitor of NADPH oxidase, on the growth of engineered tissue blocks, using a rat chamber model. Chambers containing the AVL were filled with Matrigel mixed with gp91ds-tat (100 microM) or the scrambled control peptide. At 14 days, in control chambers, most of the Matrigel was replaced by granulation tissues; gp91ds-tat treatment significantly reduced the level of reactive oxygen species and retarded the tissue formation process. Although the total number of blood vessels per unit cellularized area was not different between two groups, most vessels in gp91ds-tat-treated tissues had smaller lumens as compared to control. The total area occupied by vessel lumens was much less in gp91ds-tat-treated tissues (10.3 +/- 1.3% in control vs. 1.7 +/- 0.5% in gp91ds-tat group; p < 0.001). In vitro, gp91ds-tat treatment reduced proliferation and migration of cultured microvascular endothelial cells. Our data suggest that inhibition of NADPH oxidase function retards tissue formation in the tissue engineering chamber, which may be related to the malformed new blood vessels in the engineered tissue.

The purpose of this study was to investigate the radiological fate of the dermis-fat graft within the temporomandibular joint (TMJ) using magnetic resonance imaging (MRI). Fifteen patients with dermis-fat grafts placed in 17 TMJs following discectomy for severe internal derangement were divided into 3 equal groups according to the time lapse between TMJ surgery and the MRI investigation: 0-6 months, 7-23 months and 2 or more years. The radiological presence of fat was found within the joint or surrounding the condyle in all 17 operated joints. The interpositional material found within the radiologically defined joint space was mainly grey (Grade 3, 12 joints), suggesting tissue change to other than fat, i.e. scar or granulation tissue. Two joints showed interpositional material entirely composed of fat (Grade 1), while 3 joints showed heterogeneous material composed of fat interspersed with grey tissue (Grade 2). There was no statistically significant difference in size of fat graft between the time intervals studied. Fat was present in similar quantities within or surrounding all joints regardless of the time lapse since surgery. Intermittent compressive forces of the joint may act as a negative influence on the growth and maintenance of fat tissue within the joint space itself.

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.

Vascularization is a major hurdle for growing three-dimensional tissue engineered constructs. This study investigated the mechanisms involved in hypoxic preconditioning of primary rat myoblasts in vitro and their influence on local angiogenesis postimplantation. Primary rat myoblast cultures were exposed to 90 min hypoxia at

Background— Cardiac tissue engineering offers the prospect of a novel treatment for acquired or congenital heart defects. We have created vascularized pieces of beating cardiac muscle in the rat that are as thick as the adult rat right ventricle wall. Method and Results— Neonatal rat cardiomyocytes in Matrigel were implanted with an arteriovenous blood vessel loop into a 0.5-mL patented tissue-engineering chamber, located subcutaneously in the groin. Chambers were harvested 1, 4, and 10 weeks after insertion. At 4 and 10 weeks, all constructs that grew in the chambers contracted spontaneously. Immunostaining for α-sarcomeric actin, troponin, and desmin showed that differentiated cardiomyocytes present in tissue at all time points formed a network of interconnected cells within a collagenous extracellular matrix. Constructs at 4 and 10 weeks were extensively vascularized. The maximum thickness of cardiac tissue generated was 1983 μm. Cardiomyocytes increased in size from 1 to 10 weeks and were positive for the proliferation markers Ki67 and PCNA. Connexin-43 stain indicated that gap junctions were present between cardiomyocytes at 4 and 10 weeks. Echocardiograms performed between 4 and 10 weeks showed that the tissue construct contracted spontaneously in vivo. In vitro organ bath experiments showed a typical cardiac muscle length-tension relationship, the ability to be paced from electrical field pulses up to 3 Hz, positive chronotropy to norepinephrine, and positive inotropy in response to calcium. Conclusion— In summary, the use of a vascularized tissue-engineering chamber allowed generation of a spontaneously beating 3-dimensional mass of cardiac tissue from neonatal rat cardiomyocytes. Further development of this vascularized model will increase the potential of cardiac tissue engineering to provide suitable replacement tissues for acquired and congenital defects.

Iatrogenic erectile dysfunction following radical prostatectomy is primarily neurogenic in origin. Attempts to improve postoperative potency aim to preserve or regenerate the autonomic cavernous nerves. Testosterone is integral for erectile function and it has profound positive effects on nerve regeneration. Androgen ablation impairs nerve regeneration. In this animal study we evaluated whether testosterone deprivation effects axonal regeneration in cavernous nerve grafts or the erectile response to cavernous nerve graft stimulation.A total of 45 male Sprague-Dawley rats underwent bilateral cavernous nerve neurotomy, followed by unilateral nerve graft using the genitofemoral nerve. Rats were then randomized to castrate, intact and testosterone treated arms. At 3 months grafts were explored and electrostimulation was performed with intracavernous pressure responses recorded. Grafted nerves were then harvested for immunohistochemical analysis.Univariate analysis demonstrated a significant difference in the maximal intracavernous pressure response between groups. Mean +/- SEM maximal intracavernous pressure was 47 +/- 7.9, 23.7 +/- 4.9 and 59 +/- 7.4 mm Hg in the intact, castrate and testosterone treated groups, respectively (p = 0.003). Total axon counts did not differ between treatment groups with a mean total axon count of 789 +/- 97, 706 +/- 134 and 726 +/- 111, respectively. Castrate animals had lower neuronal nitric oxide synthase axon counts compared to intact animals (p = 0.089). The mean axon count was 480 +/- 78, 285 +/- 53 and 435 +/- 71 in the intact, castrate and testosterone treated groups respectively.Castration resulted in a decreased erectile response to electrostimulation following nerve grafting. This may be due to decreased graft neuronal nitric oxide synthase positive axonal regeneration. This has important implications in patients in whom cavernous nerve grafting could be considered.

We sought to determine whether the zone of stasis of an experimental burn exhibits a defined period of sensitivity to a sublethal exposure to ischemia. A standard burn injury was inflicted on the abdominal skin of rats consisting of two burn areas separated by a 5-mm strip corresponding to the zone of stasis. Either immediately or after a delay of 24, 48, or 72 hours, the area of the burn and interspace was elevated as a flap based on the inferior epigastric vessels and subjected to a 5-hour period of ischemia that in itself is insufficient to cause necrosis. Forty-eight hours later, the flap was harvested to assess the viability of the panniculus carnosus. Necrosis of the panniculus in the interspace was not seen in any animal where burn was not subjected to ischemia (n = 6) or in any flap subjected to a 5-hour period of ischemia immediately (less than 30 minutes) after the burn (n = 7). In contrast, when the period of ischemia was delayed 24 hours after the burn, necrosis of the muscle in the interspace was seen in 5 out of 6 flaps (P = .0047), as it was in rats in which the start of ischemia was delayed 48 hours (n = 2). However, this effect was abolished with a delay of 72 hours (n = 2). These findings show that the zone of stasis displays increased sensitivity to ischemia 24 to 48 hours after burning. Thus, sequential subnecrotic injuries can have a cumulative effect, but a delay between the two is required.

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.

In this study morphologic techniques have been used to detail the angiogenic response that accompanies endochondral fracture healing in a clinically relevant, reproducible rat model. In this displaced fracture, the gap fills with cartilage that later is replaced by bone, via endochondral ossification. A transient periosteal circulation, followed by a permanent medullary circulation accompany this progression. From 2 to 6 weeks, vessels grow out from the periosteal tissue and give rise to vascular buds, which abut directly onto the avascular zone corresponding to the fracture defect. From 3 weeks onwards, a second wave of vessels grows out from the marrow to the cartilage-filled fracture defect, terminating as vascular buds and loops lined by endothelial and perivascular cells. The loops and buds stain strongly for laminin but transmission electron microscopy does not demonstrate an identifiable basement membrane, pointing to a region of active extracellular matrix turnover. These vessels are intimately associated with osteoblasts and newly formed woven bone forming finger-like composite structures that protrude into the mineralized cartilage matrix with which they form a clearly demarcated interface. Invading vessels and woven bone successively replace the cartilage matrix to mediate repair. Both the vascular structures and progression of endochondral ossification observed, closely resemble those described in the normal epiphyseal growth plate, indicating that the fundamental processes are similar. However, there is a difference in the spatial orientation of cells such that the healing front in the fracture model is relatively disorganized, compared to the orderly linear array of cells at the epiphyseal growth plate.

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 put-pose 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.

Restoration of adequate function in patients with metacarpal hands is a major challenge. In 1984, we presented a technique in which the contralateral uninjured ring finger was transferred by microvascular techniques to restore the dual hand functions of pinch and palmar grips. This was performed in combination with thumb reconstruction when necessary. The original 5 patients described in 1984, as well as 2 later cases, were reviewed for a long-term follow-up study (average, 19 y; range, 8-25 y). The average age of the patients at follow-up evaluation was 44 years (range, 13-73 y). Range of motion, grip span, sensibility, and grip strengths were measured. The Disabilities of the Arm, Shoulder, and Hand outcome measure was used to evaluate disability. The data show persistent good function with improving sensibility over the years. The ring finger transfer in reconstruction of transmetacarpal amputation has proven to be a good technique with an overall satisfactory outcome, especially in its ability to restore some capacity for palmar grip, a function that has hitherto been unattainable by other means.

The results of repairing a transected rat musculocutaneous nerve by suturing the distal stump, end to side or end to end, to the ipsilateral ulnar nerve were assessed at 3 months by retrograde labeling and morphologic and physiologic analysis. Unlike most other models of end-to-side repair in which the injured recipient and donor reinnervating nerves have overlapping neuron pools in the spinal cord, in this model the neurons of the injured musculocutaneous and the reinnervating ulnar nerves are located in mutually exclusive segments of the spinal cord. Using retrograde labeling we show that the reinnervating fibers are derived solely from the ulnar nerve pool. Both end-to-side and end-to-end coaptation resulted in reinnervation of the distal musculocutaneous nerve and significant functional reinnervation of its dependent biceps brachii muscle. Although end-to-end coaptation resulted in better axon morphology and muscle function, it resulted in total loss of donor nerve function. By contrast, end-to-side coaptation resulted in good recovery with only minimal donor nerve deficit. These results show that significant functional reinnervation of biceps brachii muscle can occur solely on the basis of collateral sprouting of intact axons from the adjacent ulnar nerve.

We report the case of a transgender patient who was referred to our department for management of chronic strictures of his neophalloplasty. We successfully used a free jejunal flap for a long anterior urethral reconstruction. He achieved a patent and functional urethra with satisfactory urinary control and was happy with the cosmetic outcome.

Death of cells within living tissues may occur by either necrosis or apoptosis. These two processes have been shown to have fundamentally different morphological appearances, mechanisms and significance. For many years it was thought that apoptosis, then termed either karyorhexis or pyknosis, was restricted to the tissue remodelling that occurs during embryonic development and that loss of cells induced by injury, and in particular by ischaemia, invariably occurred by necrosis (Majno & Joris 1995). However in 1971 it was observed that loss of cells in ischaemic atrophy of the liver occurred not by necrosis but in a manner similar to that seen in embryonic development (Kerr 1971). In later studies Kerr and colleagues showed the presence of this form of cell death in many pathological lesions, including neoplasms, and named the phenomenon apoptosis (Kerr 1971; Kerr et al. 1972). It is now known that apoptosis is an active process requiring RNA and protein synthesis and, while the precise mechanism varies in different tissues, many details of its metabolic process have been elucidated (reviewed in Haslett 1997). It has been shown that in apoptosis cleavage of DNA occurs in a characteristic and regulated manner which can be recognized histologically by the TUNEL (terminal deoxynucleotidyl transferase mediated dUPT nick end labelling) technique or biochemically by formation of a ladder pattern in PAGE (Gavrieli et al. 1992). Ischaemia, with or without subsequent reperfusion, is a common and clinically important cause of injury in many tissues. Many aspects of its pathophysiological basis and morphology have been established but only recently has it been recognized that apoptosis may play a role in ischaemic injury (Fernandes & Cotter 1994). Apoptosis has been observed in hypoxic endothelial cells (Gobe et al. 1995), in cells subjected to oxidative stress (Buttke & Sandstrom 1994) and following ischaemia-reperfusion injury to the intestine (Horie et al. 1997), liver (Terao et al. 1997) and heart (Bielawska et al. 1997; Yue et al. 1998). At a cellular level, oxidative stress and altered calcium homeostasis are considered to be mediators of apoptosis, synergistically lowering the mitochondrial membrane potential and decreasing cellular ATP levels, thereby causing cell death (Richter et al. 1996). However no evidence is available of the role of apoptosis in ischaemia-reperfusion injury to skeletal muscle. The aim of the present study was to determine whether apoptotic cells are visible in skeletal muscle during the first 24 h of reperfusion after tourniquet ischaemia of the hind limb of the rat.

This study evaluates tendon coaptation using Nd:YAG laser photoirradiation in an in vivo cockerel model. Using the intervinculum segments of the flexor profundus tendons, experimental transactions were performed. Tendon coaptation was then attempted using laser photoirradiation. Tendons were immediately examined for evidence of stable coaptation. After this assessment, specimens were excised and processed for electron microscopic examination and exposure to trypsin digestion. Despite varying multiple laser parameters, tissue welding was not observed. The subsequent functional and ultrastructural observations of irradiated tendon suggest that these changes are those of simple thermal denaturation. The results of this study suggest that when successful tissue welding has been observed in other tissue types, the mechanism is unlikely to be because of formation of intermolecular collagen bonds as hypothesized. An alternative hypothesis is that laser welding reflects photothermal coagulation of cytoplasmic peptides or nucleic acids liberated at the coaptation interface. This may explain the successful welding of cell-rich tissues such as bowel, vas deferens, and arteries and the observed failure of laser welding in collagen-rich but relatively hypocellular tendon.

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

The histochemical structure of the deep fascia and its interface with the underlying muscle was examined in ten pigs. This structure was also evaluated after it had been raised as a fascial flap and in another site after the underlying muscle surface had been disrupted. The deep fascial is a simple structure of densely-packed collagen bundles and elastin fibres, and has hyaluronic acid concentrated on its inner surface, which is in contact with the underlying muscle. There is no specialised lining of this surface of the fascia to account for its gliding properties. The post-surgical specimens demonstrated preservation of the structure of the interface between fascia and muscle, including the retention of the hyaluronic acid lining, if the epimysium was intact. However, if the epimysium was disrupted, the structure of the interface was obliterated.

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.

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.

The ability of heat stress to improve the survival of ischemic-reperfused skeletal muscle in vivo was investigated. Ischemia-reperfusion was applied using the rat hindlimb tourniquet model. The viability of ischemic-reperfused muscle (11 +/- 1%) was increased by prior mild heat stress (86 +/- 2%). To investigate whether heat shock protein 70 (Hsp 70) expression in the muscle of the heated limb was responsible for this protection, the survival of Hsp 70-expressing transduced myoblasts and myocytes was measured after exposure to mediators of ischemia-reperfusion injury. Survival was improved in Hsp 70-positive myoblasts but not in myocytes, suggesting that the mechanism of protection conferred by heat stress in vivo cannot be explained by the expression of Hsp 70 in myocytes and may involve a more complex mechanism. In conclusion, prior heat stress is effective in protecting mature skeletal muscle in vivo against necrosis after ischemia-reperfusion and has potential for use in microsurgical procedures requiring tourniquet applications.

To determine the role of mast cells in ischaemia-reperfusion (IR) injury to skeletal muscle, W(f)/W(f) 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

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. Muscle flaps (n = 6 per group) were subjected to variable periods of cold storage (0, 1, 2, 3, or 4 days) and 24-h normothermic reperfusion. Muscle viability, as determined by nitroblue tetrazolium (NBT) histochemical staining of viable mitochondria and supported by histological examination, was 100%, 26%, 11%, 4%, and 1%, respectively. Using 24-h cold storage/24-h reperfusion as the experimental conditions, groups of muscle flaps (n = 5 per group) were perfused immediately before cold storage with either modified, colloid-free University of Wisconsin (UW) solution, a solution described by Kohout et al. (Br J Plast Surg 1995;48:132–144) or normal saline. Perfusion with modified UW solution or Kohout's solution increased survival to 33% (P < 0.05) and 28% (not statistically significant), respectively, compared with the 19% viability of separate groups of nonperfused or saline-perfused controls. These findings indicate that cold-stored skeletal muscle is highly susceptible to cold IR injury and that the viability can be increased by prior perfusion with a tissue preservation solution such as modified UW solution. © 2000 Wiley-Liss, Inc. MICROSURGERY 20:343–349 2000

The present experiment was designed to investigate the possibility of prefabricating a tissue flap in a rat by combining an arteriovenous (A-V) shunt loop with artificial skin dermis (AS). The A-V fistula loop was constructed between the right femoral artery and vein by the interposition of a vein graft and the loop was wrapped with a folded sheet of AS and buried beneath the inguinal skin. In the control group the folded sheet of AS was inserted without a vessel loop and embedded in the inguinal region as in the experimental group. There were three experiments. In experiment 1, the total volume of the generated tissue formed within the AS was calculated after 4 weeks in the experimental and control groups. In experiment 2, the AS in the experimental group was harvested at 2 (group 1) and 4 (group 2) weeks after insertion to assess the change in morphology over time. In experiment 3, full thickness skin grafts were placed over the generated tissue of the experimental groups to investigate the possibility of creating skin flaps. The total volume of tissue generated in the experimental group was significantly greater than in the control group (P0.01). Histological and carbon injection studies suggest that the new capillary bed is derived from the graft loop vessels and tissue generation and organisation of the AS were further advanced in group 2 than in group 1. The skin grafts placed over the tissues generated showed complete survival and could be raised as island flaps in both groups.

Leukemia inhibitory factor (LIF), a cytokine which has neurotrophic and myotrophic activities, has been shown to enhance nerve regeneration and consequent return of muscle function in the entubulation model of sciatic nerve repair. Fibronectin (FN) and laminin (LN) are two extracellular matrix (ECM) components that, when combined, promote axon growth in the entubulation model. The aim of this study was to determine the optimal LIF dose and the efficacy of FN plus LN administered either alone or simultaneously with the optimal LIF dose. We found that at 12 weeks following nerve repair, a single 10 ng LIF dose produced the largest medial gastrocnemius (MG) muscle mass (P < 0.0001) and maximum force contraction (P < 0.001). The diameter of the axons in the FN plus LN group were significantly greater than for saline (P < 0.001) and the LIF dose groups (P < 0.01). When 10 ng LIF was combined with FN plus LN, the MG muscle mass was significantly greater than the optimal LIF dose (P < 0.05), suggesting an additive effect. Our findings support the view that combinations of factors, which perhaps act on complementary mechanisms for nerve regeneration, will be required to maximally potentiate nerve regeneration and return of muscle function after nerve injury.

We have used electron paramagnetic resonance to investigate the time course of nitric oxide (NO) generation and its susceptibility to inhibitors of nitric oxide synthase (NOS) in ischemia–reperfusion (IR) injury to rat skeletal musclein vivo.Significant levels of muscle nitroso-heme complexes were detected 24 h postreperfusion, but not after at 0.05, 3, and 8 h of reperfusion. The levels of muscle nitroso-heme complexes were not decreased by the NOS inhibitorN-nitro- l -arginine methyl ester as a single dose (30 mg/kg) prior to reperfusion or as multiple doses continued throughout the reperfusion (total administered, 120 mg/kg) or by the potent NOS inhibitorS-methylisothiourea (3 mg/kg). In contrast, nitroso-heme levels were reduced by the glucocorticoid dexamethasone (2.5 mg/kg). Muscle necrosisin vitrodid not result in the formation of nitroso-heme complexes. The finding that reperfusion after ischemia is necessary for NO formation suggests that an inflammatory pathway is responsible for NOS-independent NO formation in IR injury to skeletal muscle.

The present report introduces a new source of vein graft in rabbit experimentation which is long and of large caliber. The average length of available vein was 13.3 ± 0.9 cm (mean ± SD). The average external diameter of the vein was 1.8 ± 0.2 mm (mean ± SD), 1.4 ± 0.4 mm (mean ± SD), and 1.8 ± 0.2 mm (mean ± SD) at the proximal, middle, and distal portions, respectively. The average number of valves throughout the vein was 7 ± 1. The vein is anatomically termed the lateral saphenous vein distally and the ischiadic vein proximally, and is superficially located on the lateral aspect of the pelvic limb. Therefore, this vein is easily harvested from the lower crus to the upper-thigh, providing a long length of vein graft for experimental study. © 1999 Wiley-Liss, Inc. MICROSURGERY 19:167–170 1999

Two cases are described in which patients presented 16 and 17 years, respectively, after complete or incomplete amputation/replantation of the arm. In case 1, the patient complained of coldness, pain, and tingling in the replanted arm in the previous 24 hours and noticed that his fingers had gone white. Arteriography and subsequent surgery revealed obliteration of the vein graft (inserted in the distal brachial artery) by neointimal thickening and atherosclerotic plaque, which was confirmed in a subsequent morphologic examination. In case 2, the patient presented with discomfort and a pulsatile swelling on the inner aspect of his upper arm. Arteriography and surgery revealed an aneurysm in the previously inserted vein graft in the brachial artery, with some atherosclerotic degeneration. Both vein grafts were successfully replaced with a fresh autologous vein graft and the patients remain well several years later. The 2 cases suggest that as part of replantation surgery of a limb, it is essential to maintain postoperative clinical monitoring for signs of graft degeneration in all patients with long-term vein graft insertion.

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.

We have developed a chamber model of islet engraftment that optimizes islet survival by rapidly restoring islet-extracellular matrix relationships and vascularization. Our aim was to assess the ability of syngeneic adult islets seeded into blood vessel-containing chambers to correct streptozotocin-induced diabetes in mice. Approximately 350 syngeneic islets suspended in Matrigel extracellular matrix were inserted into chambers based on either the splenic or groin (epigastric) vascular beds, or, in the standard approach, injected under the renal capsule. Blood glucose was monitored weekly for 7 weeks, and an intraperitoneal glucose tolerance test performed at 6 weeks in the presence of the islet grafts. Relative to untreated diabetic animals, glycemic control significantly improved in all islet transplant groups, strongly correlating with islet counts in the graft (P

We have developed a model of ischaemia-reperfusion injury in C57BL/6 mice involving ischaemia for 0.5 to 2.5 h with an elastic tourniquet on one hind limb and reperfusion for 24 h, analogous to a well-established model of ischaemia-reperfusion injury in the rat. Viability was assessed in tissue homogenates of the gastrocnemius muscles from the affected and contralateral control limb by a triphenyl tetrazolium chloride dye reaction, measuring the activity of the oxidative mitochondrial enzymes. After 1.5 h ischaemia and 24 h reperfusion, viability in the ischaemic-reperfused limb was 13%, with the control muscle regarded as 100% viable. Significant improvements in viability to 86% (P < 0.05) and 56% (P < 0.05) were achieved, with administration 30 min prior to tourniquet release, of the nitric oxide (NO) synthase inhibitor nitro-L-arginine methyl ester (L-NAME, 30 mg/kg) and the anti-inflammatory glucocorticoid dexamethasone (2.5 mg/kg) respectively, with similar findings in the rat tourniquet model.

The cytokine leukemia inhibitory factor (LIF) favors the survival and growth of axons in vitro and in vivo. Fibronectin has been shown to enhance nerve regeneration when added in combination with various growth factors including LIF. The goal of this study was to evaluate the effect of LIF plus fibronectin on the regeneration of transected nerve and functional recovery of reinnervated skeletal muscle, in one experimental model of peripheral nerve repair, at two recovery times. The rat sciatic nerve was cut at mid-thigh level and a silicone cuff containing either saline (control), LIF, or LIF plus fibronectin (L + F) was used to bridge the proximal and distal nerve stumps leaving a 1 cm gap between them. Rats were then explored at 6 or 12 weeks following the initial surgery. Regenerating nerves were assessed by measuring the diameter of myelinated axons, conduction velocity, and number of myelinated fibers. Muscle reinnervation was assessed by measuring muscle mass, force of contraction, and histologically for changes in muscle fiber type (type I and type II). In this report we demonstrate that at 6 weeks there were significant increases in 1) nerve conduction velocity, 2) myelinated axon diameter, and 3) number of myelinated axons over that of control (saline-treated) animals. Both LIF groups demonstrated a shift in type II muscle fiber area compared to saline-treated controls, with the L + F group having a significant increase in muscle mass. At 12 weeks there was an improved recovery over and above that demonstrated at 6 weeks. Muscle mass was 65% and 42% greater than control for LIF and L + F, respectively. Force of contraction, conduction velocity, myelinated fiber number, and diameter were also significantly greater for both LIF- and L + F- treated rats than saline-treated rats. These results demonstrate that LIF significantly improves the regeneration of damaged peripheral nerves and the preservation of muscle viability, resulting in greatly enhanced recovery of skeletal muscle function. J. Neurosci. Res. 47:208–215, 1997. © 1997 Wiley-Liss, Inc.

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.