A novel early precursor cell population from rat bone marrow promotes angiogenesis in vitro.

BACKGROUND: Some studies demonstrated therapeutic angiogenesis attributable to the effects of endothelial progenitor cells (EPC), others have reported disappointing results. This may be due to the fact that EPC populations used in these contradictory studies were selected and defined by highly variable and differing experimental protocols. Indeed, the isolation and reliable characterization of ex vivo differentiated EPC raises considerable problems due to the fact there is no biomarker currently available to specifically identify EPC exclusively. On the other hand traditional differentiation of primary immature bone marrow cells towards the endothelial lineage is a time-consuming process of up to 5 weeks. To circumvent these shortcomings, we herein describe a facile method to isolate and enrich a primary cell population from rat bone marrow, combining differential attachment methodology with cell sorting technology. RESULTS: The combination of these techniques enabled us to obtain a pure population of early endothelial precursor cells that show homogenous upregulation of CD31 and VEGF-R2 and that are positive for CD146. These cells exhibited typical sprouting on Matrigel™. Additionally, this population displayed endothelial tube formation when resuspended in Matrigel™ as well as in fibrin glue, demonstrating its functional angiogenic capacity. Moreover, these cells stained positive for DiI-ac-LDL and FITC-UEA, two markers that are commonly considered to stain differentiating EPCs. Based upon these observations in this study we describe a novel and time-saving method for obtaining a pure endothelial precursor cell population as early as 2-3 weeks post isolation that exhibits endothelial abilities in vitro and which still might have retained its early endothelial lineage properties. CONCLUSION: The rapid isolation and the high angiogenic potential of these syngeneic cells might facilitate and accelerate the pre-vascularization of transplanted tissues and organs also in a human setting in the future.

PHDs inhibitor DMOG promotes the vascularization process in the AV loop by HIF-1a up-regulation and the preliminary discussion on its kinetics in rat.

BACKGROUND: The Arterovenous Loop (AV Loop) model is a vascularization model in tissue engineering research, which is capable of generating a three dimensional in vivo unit with cells as well as the supporting vessels within an isolation chmaber. In our previous studies the AV loop in the isolation chamber was discovered to undergo hypoxia, characterized by Hypoxia Inducible Factor (HIF) up-regulation. The vascularization followed the increase of HIF-α temporally, while it was spatially positively correlated with the HIF-α level, as well. This study aims to prove that HIF-1a up-regulation is the stimulus for vascularization in the AV loop model. METHOD: The AV loop model in rats was created by interposing a femoral vein graft into the distal ends of the contralateral femoral artery and vein, and the loop was embeded in fibrin matrix and fixed in isolation chamber. PHD (prolyl hydroxylases) inhibitor DMOG (Dimethyloxallyl Glycine) was applied systemically in the rats in 40 mg/KG at day 0 and day 3 (DMOG-1), or in 15 mg/KG at day 8, day10 and day12 (DMOG-2). Two weeks later the specimens were explanted and underwent morphological and molecular evaluations. RESULTS: Compared to the control group, in the DMOG-2 group the HIF-1α positive rate was siginicantly raised as shown in immunohistochemistry staining, accompanied with a smaller cross section area and greater vessel density, and a HIF-1α accumulation in the kidney. The mRNA of HIF-1α and its angiogenic target gene all increased in different extends. Ki67 IHC demostrate more positive cells. There were no significant change in the DMOG-1 group. CONCLUSION: By applying DMOG systemically, HIF-1α was up-regulated at the protein level and at the mRNA level, acompanied with angiogenic target gene up-regulateion, and the vascularization was promoted correspondingly. DMOG given at lower dosage constantly after one week tends to have better effect than the group given at larger dosage in the early stage in this model, and promotes cell proliferation, as evidenced by Ki67 IHC. Thus, this study proves that HIF-1a up-regulation is the stimulus for vascularization in the AV loop model and that the process of the vessel outgrowth can be controlled in the AV Loop model utilizing this mechanism.

Cancer research by means of tissue engineering--is there a rationale?

Tissue engineering (TE) has evoked new hopes for the cure of organ failure and tissue loss by creating functional substitutes in the laboratory. Besides various innovations in the context of Regenerative Medicine (RM), TE also provided new technology platforms to study mechanisms of angiogenesis and tumour cell growth as well as potentially tumour cell spreading in cancer research. Recent advances in stem cell technology--including embryonic and adult stem cells and induced pluripotent stem cells--clearly show the need to better understand all relevant mechanisms to control cell growth when such techniques will be administered to patients. Such TE-Cancer research models allow us to investigate the interactions that occur when replicating physiological and pathological conditions during the initial phases of replication, morphogenesis, differentiation and growth under variable given conditions. Tissue microenvironment has been extensively studied in many areas of TE and it plays a crucial role in cell signalling and regulation of normal and malignant cell functions. This article is intended to give an overview on some of the most recent developments and possible applications of TE and RM methods with regard to the improvement of cancer research with TE platforms. The synthesis of TE with innovative methods of molecular biology and stem-cell technology may help investigate and potentially modulate principal phenomena of tumour growth and spreading, as well as tumour-related angiogenesis. In the future, these models have the potential to investigate the optimal materials, culture conditions and material structure to propagate tumour growth.

Severe soft tissue infection caused by a non-beta-hemolytic Streptococcus pyogenes strain harboring a premature stop mutation in the sagC gene.

We recovered a non-beta-hemolytic Streptococcus pyogenes strain from a severe soft tissue infection. In this isolate, we detected a premature stop codon within the sagC gene of the streptolysin S (SLS) biosynthetic operon. Reintroduction of full-length sagC gene on a plasmid vector restored the beta-hemolytic phenotype to our clinical isolate, indicating that the point mutation in sagC accounted for loss of hemolytic activity. To the best of our knowledge, this is the first report to demonstrate that a severe soft tissue infection can be caused by a non-beta-hemolytic S. pyogenes strain lacking a functional SagC.

New aspects on efficient anticoagulation and antiplatelet strategies in sheep.

BACKGROUND: After addressing fundamental questions in preclinical models in vitro or in small animals in vivo, the translation into large animal models has become a prerequisite before transferring new findings to human medicine. Especially in cardiovascular, orthopaedic and reconstructive surgery, the sheep is an important in vivo model for testing innovative therapies or medical devices prior to clinical application. For a wide variety of sheep model based research projects, an optimal anticoagulation and antiplatelet therapy is mandatory. However, no standardised scheme for this model has been developed so far. Thus the efficacy of antiplatelet (acetylsalicylic acid, clopidogrel, ticagrelor) and anticoagulant (sodium enoxaparin, dabigatran etexilate) strategies was evaluated through aggregometry, anti-factor Xa activity and plasma thrombin inhibitor levels in sheep of different ages. RESULTS: Responses to antiplatelet and anticoagulant drugs in different concentrations were studied in the sheep. First, a baseline for the measurement of platelet aggregation was assessed in 20 sheep. The effectiveness of 225 mg clopidogrel twice daily (bid) in 2/5 sheep and 150 mg bid in 3/5 lambs could be demonstrated, while clopidogrel and its metabolite carboxylic acid were detected in every plasma sample. High dose ticagrelor (375 mg bid) resulted in sufficient inhibition of platelet aggregation in 1/5 sheep, while acetylsalicylic acid did not show any antiplatelet effect. Therapeutic anti-factor Xa levels were achieved with age-dependent dosages of sodium enoxaparin (sheep 3 mg/kg bid, lambs 5 mg/kg bid). Administration of dabigatran etexilate resulted in plasma concentrations similar to human ranges in 2/5 sheep, despite receiving quadruple dosages (600 mg bid). CONCLUSION: High dosages of clopidogrel inhibited platelet aggregation merely in a low number of sheep despite sufficient absorption. Ticagrelor and acetylsalicylic acid cannot be recommended for platelet inhibition in sheep. Efficient anticoagulation can be ensured using sodium enoxaparin rather than dabigatran etexilate in age-dependent dosages. The findings of this study significantly contribute to the improvement of a safe and reliable prophylaxis for thromboembolic events in sheep. Applying these results in future translational experimental studies may help to avoid early dropouts due to thromboembolic events and associated unnecessary high animal numbers.

Guanylate-binding protein 1 expression from embryonal endothelial progenitor cells reduces blood vessel density and cellular apoptosis in an axially vascularised tissue-engineered construct.

BACKGROUND: Guanylate binding protein-1 (GBP-1) is a large GTPase which is actively secreted by endothelial cells. It is a marker and intracellular inhibitor of endothelial cell proliferation, migration, and invasion. We previously demonstrated that stable expression of GBP-1 in murine endothelial progenitor cells (EPC) induces their premature differentiation and decreases their migration capacity in vitro and in vivo. The goal of the present study was to assess the antiangiogenic capacity of EPC expressing GBP-1 (GBP-1-EPC) and their impact on blood vessel formation in an axially vascularized 3-D bioartificial construct in vivo. RESULTS: Functional in vitro testing demonstrated a significant increase in VEGF secretion by GBP-1-EPC after induction of cell differentiation. Undifferentiated GBP-1-EPC, however, did not secrete increased levels of VEGF compared to undifferentiated control EPC expressing an empty vector (EV-EPC). In our In vivo experiments, we generated axially vascularized tissue-engineered 3-D constructs. The new vascular network arises from an arterio-venous loop (AVL) embedded in a fibrin matrix inside a separation chamber. Total surface area of the construct as calculated from cross sections was larger after transplantation of GBP-1-EPC compared to control EV-EPC. This indicated reduced formation of fibrovascular tissue and less resorption of fibrin matrix compared to constructs containing EV-EPC. Most notably, the ratio of blood vessel surface area over total construct surface area in construct cross sections was significantly reduced in the presence of GBP-1-EPC. This indicates a significant reduction of blood vessel density and thereby inhibition of blood vessel formation from the AVL constructs caused by GBP-1. In addition, GBP-1 expressed from EPC significantly reduced cell apoptosis compared to GBP-1-negative controls. CONCLUSION: Transgenic EPC expressing the proinflammatory antiangiogenic GTPase GBP-1 can reduce blood vessel density and inhibit apoptosis in a developing bioartificial vascular network and may become a new powerful tool to manipulate angiogenetic processes in tissue engineering and other pathological conditions such as tumour angiogenesis.

Endothelial progenitor cells are integrated in newly formed capillaries and alter adjacent fibrovascular tissue after subcutaneous implantation in a fibrin matrix.

Vascularization of bioartificial matrices is crucial for successful tissue engineering. Endothelial progenitor cells (EPC) have shown vascularization potential in ischemic conditions and may also support blood vessel formation in tissue-engineered matrices. The aim of our study was to investigate the impact of a well-characterized murine embryonal EPC line (T17b-EPC) on vascularization and fibrovascular granulation tissue formation after suspension in a fibrine matrix followed by subcutaneous implantation in a separation chamber in rats. EPC were fluorescently labelled in vitro prior to implantation. After 3, 7 or 14 days, animals were killed followed by explantation and histological analysis of the constructs. Before the end of the experiment, Bandeirea Simplicifolia lectin was intravenously injected to mark the vascular ingrowth into the implanted constructs. The transplanted cells were histologically detected at all time-points and located almost exclusively within the fibrin matrix at day 3 but the number of cells in the clot continuously decreased over day 7 to day 14. Conversely, cells were detected within the newly formed granulation tissue in increasing numbers from day 3 over day 7 to day 14. Transplanted cells were also found in the intermuscular septa. Cell viability was confirmed by use of an EPC clone expressing ß-galactosidase. Fluorescence microscopy demonstrated integration of the transplanted cells in newly formed blood vessels within the fibrovascular granulation tissue adjacent to the fibrin clot. Presence of cells in the fibrin clot lead to thicker granulation tissue and an increased blood vessel diameter compared to cell-free controls. Organ standard controls showed presence of the transplanted cells in spleens at day 14 after transplantation. In summary, EPC exhibited biological activity after subcutaneous implantation in a fibrin matrix by migration from the fibrin clot into the granulation tissue and along intermuscular septae, undergoing differentiation into mature endothelial cells and integration into newly formed blood vessels and altering fibrovascular granulation tissue development. EPC may hold promise to modulate blood vessel formation in bioartificial matrices.

Directly auto-transplanted mesenchymal stem cells induce bone formation in a ceramic bone substitute in an ectopic sheep model.

Bone tissue engineering approaches increasingly focus on the use of mesenchymal stem cells (MSC). In most animal transplantation models MSC are isolated and expanded before auto cell transplantation which might be critical for clinical application in the future. Hence this study compares the potential of directly auto-transplanted versus in vitro expanded MSC with or without bone morphogenetic protein-2 (BMP-2) to induce bone formation in a large volume ceramic bone substitute in the sheep model. MSC were isolated from bone marrow aspirates and directly auto-transplanted or expanded in vitro and characterized using fluorescence activated cell sorting (FACS) and RT-PCR analysis before subcutaneous implantation in combination with BMP-2 and ß-tricalcium phosphate/hydroxyapatite (ß-TCP/HA) granules. Constructs were explanted after 1 to 12 weeks followed by histological and RT-PCR evaluation. Sheep MSC were CD29(+), CD44(+) and CD166(+) after selection by Ficoll gradient centrifugation, while directly auto-transplanted MSC-populations expressed CD29 and CD166 at lower levels. Both, directly auto-transplanted and expanded MSC, were constantly proliferating and had a decreasing apoptosis over time in vivo. Directly auto-transplanted MSC led to de novo bone formation in a heterotopic sheep model using a ß-TCP/HA matrix comparable to the application of 60 µg/ml BMP-2 only or implantation of expanded MSC. Bone matrix proteins were up-regulated in constructs following direct auto-transplantation and in expanded MSC as well as in BMP-2 constructs. Up-regulation was detected using immunohistology methods and RT-PCR. Dense vascularization was demonstrated by CD31 immunohistology staining in all three groups. Ectopic bone could be generated using directly auto-transplanted or expanded MSC with ß-TCP/HA granules alone. Hence BMP-2 stimulation might become dispensable in the future, thus providing an attractive, clinically feasible approach to bone tissue engineering.

Role of guanylate binding protein-1 in vascular defects associated with chronic inflammatory diseases.

Rheumatic autoimmune disorders are characterized by a sustained pro-inflammatory microenvironment associated with impaired function of endothelial progenitor cells (EPC) and concomitant vascular defects. Guanylate binding protein-1 (GBP-1) is a marker and intracellular regulator of the inhibition of proliferation, migration and invasion of endothelial cells induced by several pro-inflammatory cytokines. In addition, GBP-1 is actively secreted by endothelial cells. In this study, significantly increased levels of GBP-1 were detected in the sera of patients with chronic inflammatory disorders. Accordingly we investigated the function of GBP-1 in EPC. Interestingly, stable expression of GBP-1 in T17b EPC induced premature differentiation of these cells, as indicated by a robust up-regulation of both Flk-1 and von Willebrand factor expression. In addition, GBP-1 inhibited the proliferation and migration of EPC in vitro. We confirmed that GBP-1 inhibited vessel-directed migration of EPC at the tissue level using the rat arterio-venous loop model as a novel quantitative in vivo migration assay. Overall, our findings indicate that GBP-1 contributes to vascular dysfunction in chronic inflammatory diseases by inhibiting EPC angiogenic activity via the induction of premature EPC differentiation.

Development of a regulatable oncolytic herpes simplex virus type 1 recombinant virus for tumor therapy.

Oncolytic viruses are genetically modified viruses that preferentially replicate in host cancer cells, leading to the production of new viruses and, ultimately, cell death. Currently, no oncolytic viruses that are able to kill only tumor cells while leaving normal cells intact are available. Using T-REx (Invitrogen, Carlsbad, CA) gene switch technology and a self-cleaving ribozyme, we have constructed a novel oncolytic HSV-1 recombinant, KTR27, whose replication can be tightly controlled and regulated by tetracycline in a dose-dependent manner. Infection of normal replicating cells as well as multiple human cancer cell types with KTR27 in the presence of tetracycline led to 1,000- to 250,000-fold-higher progeny virus production than in the absence of tetracycline, while little viral replication and virus-associated cytotoxicity was observed in infected growth-arrested normal human cells. We show that intratumoral inoculation with KTR27 markedly inhibits tumor growth in a xenograft model of human non-small-cell lung cancer in nude mice. It is shown further that replication of KTR27 in the inoculated tumors can be efficiently controlled by local codelivery of tetracycline to the target tumors at the time of KTR27 inoculation. Collectively, KTR27 possesses a unique pharmacological feature that can limit its replication to the targeted tumor microenvironment with localized tetracycline delivery, thus minimizing unwanted viral replication in distant tissues following local virotherapy. This regulatory mechanism would also allow the replication of the virus to be quickly shut down should adverse effects be detected.

Myogenic differentiation of mesenchymal stem cells co-cultured with primary myoblasts.

TE (tissue engineering) of skeletal muscle is a promising method to reconstruct loss of muscle tissue. This study evaluates MSCs (mesenchymal stem cells) as new cell source for this application. As a new approach to differentiate the MSCs towards the myogenic lineage, co-cultivation with primary myoblasts has been developed and the myogenic potential of GFP (green fluorescent protein)-transduced rat MSC co-cultured with primary rat myoblasts was assessed by ICC (immunocytochemistry). Myogenic potential of MSC was analysed by ICC, FACS and qPCR (quantitative PCR). MSC-myoblast fusion phenomena leading to hybrid myotubes were evaluated using a novel method to evaluate myotube fusion ratios based on phase contrast and fluorescence microscopy. Furthermore, MSC constitutively expressed the myogenic markers MEF2 (myogenic enhancer factor 2) and α-sarcomeric actin, and MEF2 expression was up-regulated upon co-cultivation with primary myoblasts and the addition of myogenic medium supplements. Significantly higher numbers of MSC nuclei were involved in myotube formations when bFGF (basic fibroblast growth factor) and dexamethasone were added to co-cultures. In summary, we have determined optimal co-culture conditions for MSC myogenic differentiation up to myotube formations as a promising step towards applicability of MSC as a cell source for skeletal muscle TE as well as other muscle cell-based therapies.

Hyaluronan-based heparin-incorporated hydrogels for generation of axially vascularized bioartificial bone tissues: in vitro and in vivo evaluation in a PLDLLA-TCP-PCL-composite system.

Smart matrices are required in bone tissue-engineered grafts that provide an optimal environment for cells and retain osteo-inductive factors for sustained biological activity. We hypothesized that a slow-degrading heparin-incorporated hyaluronan (HA) hydrogel can preserve BMP-2; while an arterio-venous (A-V) loop can support axial vascularization to provide nutrition for a bio-artificial bone graft. HA was evaluated for osteoblast growth and BMP-2 release. Porous PLDLLA-TCP-PCL scaffolds were produced by rapid prototyping technology and applied in vivo along with HA-hydrogel, loaded with either primary osteoblasts or BMP-2. A microsurgically created A-V loop was placed around the scaffold, encased in an isolation chamber in Lewis rats. HA-hydrogel supported growth of osteoblasts over 8 weeks and allowed sustained release of BMP-2 over 35 days. The A-V loop provided an angiogenic stimulus with the formation of vascularized tissue in the scaffolds. Bone-specific genes were detected by real time RT-PCR after 8 weeks. However, no significant amount of bone was observed histologically. The heterotopic isolation chamber in combination with absent biomechanical stimulation might explain the insufficient bone formation despite adequate expression of bone-related genes. Optimization of the interplay of osteogenic cells and osteo-inductive factors might eventually generate sufficient amounts of axially vascularized bone grafts for reconstructive surgery.

T17b murine embryonal endothelial progenitor cells can be induced towards both proliferation and differentiation in a fibrin matrix.

Endothelial progenitor cells (EPC) may enhance blood vessel formation in a variety of clinical settings such as ischaemia and tumour angiogenesis as well as in tissue-engineered matrices. In the present study, we cultured a murine endothelial progenitor cell line, T17b, in vitro in cell culture as well as in an FDA-approved fibrin matrix and investigated cell proliferation, differentiation and secretion patterns of the angiogenic growth factor VEGF under hypoxia and differentiation. We show that T17b EPC remain viable for at least 8 days in the fibrin matrix where they proliferate and form clusters including lumen-like structures. Proliferation in fibrin clots overlayed with basal medium (BM) was confirmed morphologically and immunohistochemically by positive Ki67 staining, indicating mitotic activity. Significant cell proliferation and Ki-67 expression were absent when cells were incubated with dibutyryl-cAMP and retinoic acid (RA). Incubation with dibutyryl-cAMP and RA stimulated the expression of the EPC differentiation markers von Willebrand Factor (vWF) and VEGF receptor 2 (VEGFR-2), indicating successful differentiation in the fibrin clot. EPC differentiation induced by dibutyryl-cAMP and RA was confirmed in 2-D chamber slide cultures by positive vWF immunostaining, which was absent in BM controls. EPC chamber slides also displayed positive vWF staining when exposed to hypoxia under BM conditions, indicating EPC activation and differentiation could also be induced by hypoxia. Taken together, T17b EPC secrete increased levels of VEGF when submitted to either hypoxia or differentiation and can be differentiated into mature endothelial cells not only in cell and matrigel cultures but also in a fibrin matrix that is FDA approved for clinical application.

Translating tissue engineering technology platforms into cancer research.

Technology platforms originally developed for tissue engineering applications produce valuable models that mimic three-dimensional (3D) tissue organization and function to enhance the understanding of cell/tissue function under normal and pathological situations. These models show that when replicating physiological and pathological conditions as closely as possible investigators are allowed to probe the basic mechanisms of morphogenesis, differentiation and cancer. Significant efforts investigating angiogenetic processes and factors in tumorigenesis are currently undertaken to establish ways of targeting angiogenesis in tumours. Anti-angiogenic agents have been accepted for clinical application as attractive targeted therapeutics for the treatment of cancer. Combining the areas of tumour angiogenesis, combination therapies and drug delivery systems is therefore closely related to the understanding of the basic principles that are applied in tissue engineering models. Studies with 3D model systems have repeatedly identified complex interacting roles of matrix stiffness and composition, integrins, growth factor receptors and signalling in development and cancer. These insights suggest that plasticity, regulation and suppression of these processes can provide strategies and therapeutic targets for future cancer therapies. The historical perspective of the fields of tissue engineering and controlled release of therapeutics, including inhibitors of angiogenesis in tumours is becoming clearly evident as a major future advance in merging these fields. New delivery systems are expected to greatly enhance the ability to deliver drugs locally and in therapeutic concentrations to relevant sites in living organisms. Investigating the phenomena of angiogenesis and anti-angiogenesis in 3D in vivo models such as the Arterio-Venous (AV) loop mode in a separated and isolated chamber within a living organism adds another significant horizon to this perspective and opens new modalities for translational research in this field.

Collagen matrices from sponge to nano: new perspectives for tissue engineering of skeletal muscle.

BACKGROUND: Tissue engineering of vascularised skeletal muscle is a promising method for the treatment of soft tissue defects in reconstructive surgery. In this study we explored the characteristics of novel collagen and fibrin matrices for skeletal muscle tissue engineering. We analyzed the characteristics of newly developed hybrid collagen-I-fibrin-gels and collagen nanofibers as well as collagen sponges and OPLA-scaffolds. Collagen-fibrin gels were also tested with genipin as stabilizing substitute for aprotinin. RESULTS: Whereas rapid lysis and contraction of pure collagen I- or fibrin-matrices have been great problems in the past, the latter could be overcome by combining both materials. Significant proliferation of cultivated myoblasts was detected in collagen-I-fibrin matrices and collagen nanofibers. Seeding cells on parallel orientated nanofibers resulted in strongly aligned myoblasts. In contrast, common collagen sponges and OPLA-scaffolds showed less cell proliferation and in collagen sponges an increased apoptosis rate was evident. The application of genipin caused deleterious effects on primary myoblasts. CONCLUSION: Collagen I-fibrin mixtures as well as collagen nanofibers yield good proliferation rates and myogenic differentiation of primary rat myoblasts in vitro In addition, parallel orientated nanofibers enable the generation of aligned cell layers and therefore represent the most promising step towards successful engineering of skeletal muscle tissue.

Cell suspensions of autologous keratinocytes or autologous fibroblasts accelerate the healing of full thickness skin wounds in a diabetic porcine wound healing model.

Autologous dermal fibroblasts may be useful in the treatment of diabetic skin wounds. We hypothesized that cultured fibroblasts or cultured keratinocytes would not only survive in a hyperglycemic wound environment but also enhance the rate of re-epithelialization. We previously developed a new porcine model of delayed cutaneous wound healing in the diabetic pig. Full thickness wounds were created on the dorsum and dressed with polyurethane chambers to keep the wounds wet and to allow for wound fluid monitoring. Suspensions of either autologous fibroblasts or autologous keratinocytes were injected into full thickness wounds and compared with wounds treated in a wet environment in normal saline. Serum glucose and wound fluid glucose concentrations were monitored daily. Wound contraction was monitored and biopsies taken on day 12. Transplantation of suspensions of autologous fibroblasts or autologous keratinocytes enhanced re-epithelialization of cutaneous full thickness wounds. Wounds treated with autologous fibroblasts showed a re-epithelialization rate of 86.75% and wounds treated with autologous keratinocytes showed a re-epithelialization rate of 91.3%. This is compared with a re-epithelialization rate of 56.8% seen in the normal saline treated wounds. While previous studies have shown fibroblasts suspension to have little effect in the treatment of full thickness wounds in nondiabetic wounds, this study shows a clear beneficial effect in the use of fibroblast or keratinocyte suspensions for the cutaneous healing of diabetic wounds in pigs.

De novo generation of axially vascularized tissue in a large animal model.

De novo generation of axially vascularized tissue with clinically relevant dimensions in a large animal model and implementation of clinically established imaging modalities for in vivo evaluation of vascularization. To be used for reconstruction of tissue defects, engineered grafts need to be axially vascularized to enable transplantation without graft loss due to hypoxia. Limitations to dimensions in small animal models had not yet been overcome, which is necessary to yield clinical relevance. Anatomical studies of groin and axillary regions in eight merino sheep were followed by microsurgical creation of an arteriovenous loop (AV-loop), embedded in an isolation chamber filled with fibrin matrix. Constructs were implanted in the groin of six sheep for up to 6 weeks. Course of vascularization in de novo forming tissue was assessed by sequential computed tomography angiography (CTA) and magnetic resonance angiography (MRA) in vivo, as well as by postexplantational micro-computed tomography and histology. A vascular axis was constantly found epifascially at the medial aspect of all sheep's thighs, which was used for AV-loop creation. Patency of AV-loop could be visualized by CTA and MRA scans during 1-6 weeks. Complex 3D-vessel-reconstruction revealed increasing axial vascularization of the fibrin matrix and growing connective tissue within the isolation chamber, which was confirmed by micro-computed tomography and histology postexplantation. De novo formation of axially vascularized tissue was demonstrated for the first time ever in a large animal model, paving the way for the first application of tissue engineering vascularized grafts with clinically relevant dimensions.

Insulin-like growth factor-1 gene therapy and cell transplantation in diabetic wounds.

BACKGROUND: Impaired wound healing is a frequent phenomenon in diabetes mellitus. However, little is known of the fundamental cause of this pathology. The present study examined the effect of human insulin-like growth factor (hIGF)-1 overexpression in combination with autologous cell transplantation to diabetic wounds in a preclinical large-animal model. METHODS: Diabetes was induced in Yorkshire pigs with streptozotocin. Keratinocytes were cultured and transfected with hIGF-1 or LacZ transgene. Plasmids were lipoplexed with either Lipofectin or Lipofectamin 2000. Transgene expression was assessed by enzyme-linked immunosorbent assay or X-gal staining. For in vivo studies, full-thickness wounds were created and dressed with a sealed chamber. Transfected cells were transplanted into the wounds. Wound contraction was monitored and biopsies were obtained for measurement of re-epithelialization. Wound fluid was collected and analysed for IGF-1 concentrations. RESULTS: Quantification showed up to 740 ng/ml IGF-1 in vitro and significantly higher concentrations over 14 days compared to controls for the Lipofectamin 2000 group. Lipofectin-mediated gene transfer showed peak expression on day 2 with 68.5 ng/ml. In vivo, transfected cells showed peak expression of 457 ng/ml at day 1, followed by subsequent decline to 5 ng/ml on day 12 with Lipofectamin 2000. For Lipofectin, no significant IGF-1 expression could be detected. Gene therapy caused significantly faster wound closure (83%) than both controls (native-cell therapy = 57%; control wounds = 32%). CONCLUSIONS: The present study demonstrates that optimized nonviral gene transfer increased IGF-1 expression in diabetic wounds by up to 900-fold. This high IGF-1 concentration in combination with cell therapy improved diabetic wound healing significantly.

Impaired wound healing in an acute diabetic pig model and the effects of local hyperglycemia.

Diabetic wounds result in significant morbidity, prolonged hospitalization, and enormous health-care expenses. Pigs have been shown to have wound healing resembling that in humans. The aim of this study was to develop a large-animal model for diabetic wound healing. Diabetes was induced by streptozotocin injection in Yorkshire pigs. Full-thickness wounds were created and dressed with a sealed chamber. Nondiabetic pigs with or without high glucose wound fluid concentration served as controls. Glucose concentration in serum and wound fluid was measured and collected. Wound contraction was monitored, and biopsies were obtained for measurement of reepithelialization. Wound fluid was analyzed for insulin-like growth factor-1 (IGF-1), platelet-derived growth factor, and transforming growth factor. Glucose concentration in wound fluid initially followed serum levels and then decreased to undetectable on day 9. Reepithelialization was significantly delayed in diabetic pigs. In nondiabetic pigs, wounds treated in a local hyperglycemic environment, and thus excluding the effects of systemic hyperglycemia, showed no difference in wound closure compared with controls. This suggests that delayed wound healing in diabetes is not induced by local high-glucose concentration itself. Analysis of growth factor expression showed a marked reduction in IGF-1 in the diabetic wounds. Diabetic pigs have impaired healing that is accompanied by a reduction of IGF-1 in the healing wound and is not due to the local hyperglycemia condition itself.

Vascularization of the Arteriovenous Loop in a Rat Isolation Chamber Model-Quantification of Hypoxia and Evaluation of Its Effects.

INTRODUCTION: The aim of this study was to analyze the three-dimensional distribution of hypoxia in the arteriovenous (AV) loop model in rats, by examining the distribution of hypoxia-inducible factor-1 alpha (HIF-1α). MATERIALS AND METHODS: AV loops were created from the femoral artery and vein of male Lewis rats and an interpositional graft from the contralateral femoral vein. This AV fistula was embedded in a fibrin-filled isolation chamber and subcutaneously implanted into the thigh. The specimens were harvested after 7 days (n = 4), 10 days (n = 5), and 14 days (n = 4). The fibrin clots were stained for lectin, HIF-1α, and ectodysplasin 1 (ED1). The distribution of positive and negative cells was analyzed in three dimensions and at different points in time. RESULTS: The HIF-1α-positive rate increased from the proximity of the central vessel to the distant regions. From day 7 to 10, we noted a decrease in the HIF-1α-positive rate in the proximity of the vessels and an increase in the periphery. A global decrease in positive cells was seen at day 14. HIF-1α and macrophage (ED1) double staining indicated that macrophages accounted for a significant fraction of the cells. Double staining for endothelium (with lectin) demonstrated that no HIF-1α was detectable in well-vascularized areas. CONCLUSION: In the AV loop model, the HIF-1α-positive cell distribution is highly related to the vascularization process. The onset of rapid vessel outgrowth follows the increase of the HIF-1α rate closely, indicating that HIF-1α may be a driving force for vascularization.