Wound closure by means of free flap and arteriovenous loop: Development of flap autonomy in the long-term follow-up.

Free flaps in combination with arterial reconstruction by means of arteriovenous loops or bypass have, meanwhile, been established as a therapeutic option in defect reconstruction for areas without recipient vessels. Our aim was to analyse the long-term performance, flap autonomy, and the flap perfusion. Patients receiving this combined reconstruction at a single-centre institution were included. During follow-up examination, the patency of arterial reconstruction was investigated by duplex ultrasound. Flap micro-circulation was assessed by laser Doppler flowmetry and white light tissue spectrometry (O2C) as well as by indocyanine green fluorescence angiography. Twenty-three patients could be clinically followed up. Duplex ultrasound showed, in four cases, arterial pedicle occlusion in spite of vital flap. Comparison of the O2C perfusion parameters between flaps with occluded pedicles and those with intact inflow showed no significant difference (parameters sO2: P = .82; Flow: P = .31). Similar results were obtained by fluorescence angiography; no significant difference could be detected between both groups (parameters Ingress P = .13; Ingressrate P = .54). Combined vascular reconstruction with free tissue transfer is associated with a good long-term outcome and wound closure. Even after flap transplantation to areas with critical tissue perfusion, the flap can develop autonomy and thus survive after pedicle occlusion.

Efficacy of the Arteriovenous Loop for Free Flap Reconstruction in Patients with Complex Limb Trauma: Case Series and Literature Review.

Background and objectives: Complex limb traumas are commonly treated with microsurgical reconstruction and free flaps. However, complications are frequent in patients affected by a previous trauma or comorbidity, atheromasia and a single valid vessel. Free flap reconstruction is indeed a challenging procedure in complex injuries, which may increase the risk of limb ischemia. The Arteriovenous loop (AVL) technique may be considered an efficient alternative treatment. We herein report our procedure and previous research regarding the AVL method using a two-step reconstruction in cases of complex high-energy limb injuries. Materials and Methods: In this single center retrospective cohort study, all the patients from 2014 to 2018 who underwent to AVL reconstruction were assessed. A total of six patients were included in the study for traumatic limb trauma. The two-stage technique was performed each time. The age and sex of patient, the time between stage one and two, the length of AVL loop and rate of free flap success were evaluated. Results: A total of seven AVL reconstructions were performed. The mean age of patients was 36 years old. Eight free flaps were performed; six free flaps were transferred to the vascular loops. The average time between stage one and two was 13 days. The mean length of the pedicle was 25 cm for the upper limb and 33.7 cm for the lower limb. All the free flaps successfully take root. In one case, a surgical revision was required the second day post-operatory due to venous congestion. Conclusions: AVL is a useful and safe technique in microsurgical reconstruction which will prevent vascular complications. Our investigations suggest the efficacy and feasibility of a two-step intervention in acute post-traumatic events. A single-step procedure should be preferred in chronic situation and oncologic reconstruction.

The Arteriovenous Loop: Engineering of Axially Vascularized Tissue.

BACKGROUND: Most of the current treatment options for large-scale tissue defects represent a serious burden for the patients, are often not satisfying, and can be associated with significant side effects. Although major achievements have already been made in the field of tissue engineering, the clinical translation in case of extensive tissue defects is only in its early stages. The main challenge and reason for the failure of most tissue engineering approaches is the missing vascularization within large-scale transplants. SUMMARY: The arteriovenous (AV) loop model is an in vivo tissue engineering strategy for generating axially vascularized tissues using the own body as a bioreactor. A superficial artery and vein are anastomosed to create an AV loop. This AV loop is placed into an implantation chamber for prevascularization of the chamber inside, e.g., a scaffold, cells, and growth factors. Subsequently, the generated tissue can be transplanted with its vascular axis into the defect site and anastomosed to the local vasculature. Since the blood supply of the growing tissue is based on the AV loop, it will be immediately perfused with blood in the recipient site leading to optimal healing conditions even in the case of poorly vascularized defects. Using this tissue engineering approach, a multitude of different axially vascularized tissues could be generated, such as bone, skeletal or heart muscle, or lymphatic tissues. Upscaling from the small animal AV loop model into a preclinical large animal model could pave the way for the first successful attempt in clinical application. Key Messages: The AV loop model is a powerful tool for the generation of different axially vascularized replacement tissues. Due to minimal donor site morbidity and the possibility to generate patient-specific tissues variable in type and size, this in vivo tissue engineering approach can be considered as a promising alternative therapy to current treatment options of large-scale defects.

Successful human long-term application of in situ bone tissue engineering.

Tissue Engineering (TE) and Regenerative Medicine (RM) have gained much popularity because of the tremendous prospects for the care of patients with tissue and organ defects. To overcome the common problem of donor-site morbidity of standard autologous bone grafts, we successfully combined tissue engineering techniques for the first time with the arteriovenous loop model to generate vascularized large bone grafts. We present two cases of large bone defects after debridement of an osteomyelitis. One of the defects was localized in the radius and one in the tibia. For osseus reconstruction, arteriovenous loops were created as vascular axis, which were placed in the bony defects. In case 1, the bone generation was achieved using cancellous bone from the iliac crest and fibrin glue and in case 2 using a clinically approved ß-tricalciumphosphate/hydroxyapatite (HA), fibrin glue and directly auto-transplanted bone marrow aspirate from the iliac crest. The following post-operative courses were uneventful. The final examinations took place after 36 and 72 months after the initial operations. Computer tomogrphy (CT), membrane resonance imaging (MRI) and doppler ultrasound revealed patent arterio-venous (AV) loops in the bone grafts as well as completely healed bone defects. The patients were pain-free with normal ranges of motion. This is the first study demonstrating successfully axially vascularized in situ tissue engineered bone generation in large bone defects in a clinical scenario using the arteriovenous loop model without creation of a significant donor-site defect utilizing TE and RM techniques in human patients with long-term stability.

[Vascularloops in reconstructive microsurgery: A review of the literature]. / Les boucles vasculaires en microchirurgie réparatrice : revue de la littérature.

The success of free tissue transfer depends on the quality of vascular micro-anastomosis and recipient vessels. Adequate recipient vessels are sometimes not available near the recipient site for they can be either destroyed or of poor quality (radiotherapy, traumatism). In such cases, good quality recipient vessels are at a distance from the reconstructed site. If this distance is important flap pedicle lengthening implies - for the artery, for the vein or for both flap artery and vein. This lengthening can be carried out in two manners - by interpositional vein grafts (VG) or by a vascular loop (VL) in one or two stages. The aim of this study was to review the utilisation of VL and their type since their introduction in the clinical practice of reconstructive microsurgery. Two main types of VL are used - BV by VG and VL "in situ". Both of them can be carried out in one or two stages. Each of these techniques has its advantages and disadvantages. The overall data from the literature shows that VL are indicated in cases where both artery and vein are damaged or destroyed. There is not enough evidence concerning the VL in one or two stages but there are some tendencies in favour of the VL in one stage. The technique of VL seems to be more avantageous over the interpositional VG but with a smaller success rate compared to free-flaps with direct anastomosis to recipient vessels. Further studies are necessary to investigate these controversial questions.

Hypoxia as a stimulus for tissue formation: The concept of organogenesis in microsurgically vascularized tissue engineering constructs.

Axial vascularization of tissue constructs is essential to maintain an adequate blood supply for a stable regeneration of a clinically relevant tissue size. The versatility of the arterio-venous loop (AVL) has been previously shown in various small and large animal models as well as in clinical reports for bone regeneration. We have previously demonstrated the capability of the AVL to induce axial vascularization and to support the nourishment of tissue constructs in small animal models after applying high doses of ionizing radiation comparable to those applied for adjuvant radiotherapy after head and neck cancer. We hypothesize that this robust ability to induce regeneration after irradiation could be related to a state of hypoxia inside the constructs that triggers the HIF1 (hypoxia induced factor 1) - SDF1 (stromal derived factor 1) axis leading to chemotaxis of progenitor cells and induction of tissue regeneration and vascularization. We analyzed the expression of HIF1 and SDF1 via immunofluorescence in axially vascularized bone tissue engineering constructs in Lewis rats 2 and 5 weeks after local irradiation with 9Gy or 15Gy. We also analyzed the expression of various genes for osteogenic differentiation (collagen 1, RUNX, alkaline phosphatase and osteonectin) via real time PCR analysis. The expression of HIF1 and SDF1 was enhanced two weeks after irradiation with 15Gy in comparison to non-irradiated constructs. The expression of osteogenic markers was enhanced at the 5-weeks time point with significant results regarding collagen, alkaline phosphatase and osteonectin. These results indicate that the hypoxia within the AVL constructs together with an enhanced SDF1 expression probably play a role in promoting tissue differentiation. The process of tissue generation triggered by hypoxia in the vicinity of a definite vascular axis with enhanced tissue differentiation over time resembles hereby the well-known concept of organogenesis in fetal life.

Elevated Shear Stress Modulates Heterogenous Cellular Subpopulations to Induce Vascular Remodeling.

Rationale: Elevated shear stress (ESS) induces vascular remodeling in veins exposed to arterial blood flow, which can lead to arteriovenous (AV) fistula failure. The molecular mechanisms driving remodeling have not been comprehensively examined with a single-cell resolution before. Objective: Using an in vivo animal mode, single-cell RNA sequencing, and histopathology, we precisely manipulate blood flow to comprehensively characterize all cell subpopulations important during vascular remodeling. Methods: AV loops were created in saphenous vessels of rats using a contralateral saphenous vein interposition graft to promote ESS. Saphenous veins with no elevated shear stress (NSS) were anastomosed as controls. Findings: ESS promoted transcriptional homogeneity, and NSS promoted considerable heterogeneity. Specifically, ESS endothelial cells (ECs) showed a more homogeneous transcriptional response promoting angiogenesis and upregulating endothelial-to-mesenchymal transition inhibiting genes (Klf2). NSS ECs upregulated antiproliferation genes such as Cav1, Cst3, and Btg1. In macrophages, ESS promoted a large homogeneous subpopulation, creating a mechanically activated, proinflammatory and thus proangiogenic myeloid phenotype, whereas NSS myeloid cells expressed the anti-inflammatory and antiangiogenetic marker Mrc1. Conclusion: ESS activates unified gene expression profiles to induce adaption of the vessel wall to hemodynamic alterations. Targeted depletion of the identified cellular subpopulations may lead to novel therapies to prevent excessive venous remodeling, intimal hyperplasia, and AV fistula failure.

In Vivo Engineering and Transplantation of Axially Vascularized and Epithelialized Flaps in Rats.

The arteriovenous loop (AVL) model allows the in vivo engineering of axially vascularized flaps, the so-called AVL flaps. Although AVL flaps can be transplanted microsurgically to cover tissue defects, they lack an epithelial layer on the surface. Therefore, the objective of this study was to engineer axially vascularized AVL flaps with an accompanying epithelial layer for local defect reconstruction. In this study, AVLs were established in 20 male Lewis rats. Minimally invasive injection of keratinocytes onto the surface of the AVL flaps was performed on postoperative day (POD) 21. AVL flaps were explanted from 12 rats on POD 24 or POD 30, then the epithelium formed by the keratinocytes on the surface of the flaps was evaluated using immunofluorescence staining. In six other rats, the AVL flap was locally transposed to cover a critical defect in the rats' leg on POD 30 and explanted for analysis on POD 40. In two control rats, sodium chloride was applied instead of keratinocytes. These control flaps were also transplanted on POD 30 and explanted on POD 40. Our results revealed that 3 days after keratinocyte application, a loose single-layered epithelium was observed histologically on the AVL flaps surface, whereas after 9 days, a multilayered and structured epithelium had grown. The epithelium on the transplanted AVL flaps showed its physiological differentiation when being exposed to an air-liquid interface. Histologically, a layered epithelium identical to the rats' regular skin was formed. In the sodium chloride control group, no epithelium had been grown. This study clearly demonstrates that axially vascularized AVL flaps can be processed in the subcutaneous chamber by minimally invasive injection of keratinocytes. Thus, AVL flaps with an intact epithelial layer were engineered and could be successfully transplanted for local defect coverage in a small animal model.

A Novel Window into Angiogenesis-Intravital Microscopy in the AV-Loop-Model.

Due to the limitations of current in vivo experimental designs, our comprehensive knowledge of vascular development and its implications for the development of large-scale engineered tissue constructs is very limited. Therefore, the purpose of this study was to develop unique in vivo imaging chambers that allow the live visualization of cellular processes in the arteriovenous (AV) loop model in rats. We have developed two different types of chambers. Chamber A is installed in the skin using the purse sting fixing method, while chamber B is installed subcutaneously under the skin. Both chambers are filled with modified gelatin hydrogel as a matrix. Intravital microscopy (IVM) was performed after the injection of fluorescein isothiocyanate (FITC)-labeled dextran and rhodamine 6G dye. The AV loop was functional for two weeks in chamber A and allowed visualization of the leukocyte trafficking. In chamber B, microvascular development in the AV loop could be examined for 21 days. Quantification of the microvascular outgrowth was performed using Fiji-ImageJ. Overall, by combining these two IVM chambers, we can comprehensively understand vascular development in the AV loop tissue engineering model¯.

Arteriovenous Vascular Loops in Latissimus Free Flap Reconstruction of Cervical and Cervicothoracic Spine Wounds.

Introduction Wound dehiscence is the most common complication after spinal fusion procedures, resulting in an increase in mortality rate and hospital length of stay. Reconstruction of these wounds presents a challenge, as the spine is dependent on these implants for stability and must be maintained throughout the wound dehiscence treatment protocol. We describe a method for extending the thoracodorsal pedicle with an arteriovenous loop to permit an increased excursion of the latissimus dorsi muscle in patients with exposed implants and present the results of this procedure. Materials and Methods A retrospective review of patients treated with a latissimus free flap with saphenous vein pedicle extension for posterior spinal wounds from 2010 to 2020 were reviewed. Patient charts were reviewed for demographic information including comorbidities, previous spine operations, wound size and location, and postoperative complications including total flap loss, flap dehiscence, and need for secondary surgery. Results Six patients were identified who underwent a total of eight extended pedicle free flaps. Mean age was 64.8 years with a mean follow-up of 12.3 months (range, 6-20 months). Four wounds were in the cervicothoracic region with two wounds in the cervical region. Mean number of previous spine surgeries was 3.5 (range, 2-4). Mean wound size was 189 cm 2 with a mean vein graft length of 28 cm. Wound coverage was successful in five of six patients. Major complications occurred in five of six patients. Total flap loss occurred in two patients (33%) and both underwent a second extended latissimus flap from the contralateral side. Three patients developed postoperative flap dehiscence which resolved with regular dressing changes. Conclusion Extended pedicle latissimus flaps are an effective treatment for posterior spine wounds but are associated with a high complication rate, secondary to medically complex patients with multiple prior surgeries. Careful patient selection is critical for success.

Acellular Human Placenta Small-Diameter Vessels as a Favorable Source of Super-Microsurgical Vascular Replacements: A Proof of Concept.

In this study, we aimed to evaluate the human placenta as a source of blood vessels that can be harvested for vascular graft fabrication in the submillimeter range. Our approach included graft modification to prevent thrombotic events. Submillimeter arterial grafts harvested from the human placenta were decellularized and chemically crosslinked to heparin. Graft performance was evaluated using a microsurgical arteriovenous loop (AVL) model in Lewis rats. Specimens were evaluated through hematoxylin-eosin and CD31 staining of histological sections to analyze host cell immigration and vascular remodeling. Graft patency was determined 3 weeks after implantation using a vascular patency test, histology, and micro-computed tomography. A total of 14 human placenta submillimeter vessel grafts were successfully decellularized and implanted into AVLs in rats. An appropriate inner diameter to graft length ratio of 0.81 ± 0.16 mm to 7.72 ± 3.20 mm was achieved in all animals. Grafts were left in situ for a mean of 24 ± 4 days. Decellularized human placental grafts had an overall patency rate of 71% and elicited no apparent immunological responses. Histological staining revealed host cell immigration into the graft and re-endothelialization of the vessel luminal surface. This study demonstrates that decellularized vascular grafts from the human placenta have the potential to serve as super-microsurgical vascular replacements.

Analysis of vein grafting versus arteriovenous loop in microvascular head and neck reconstruction: Multicenter series of 36 patients.

BACKGROUND: The utilization of an arteriovenous loop is an underreported technique that affords the creation of reliable vascular options. Understanding the efficacy and impacting variables of microvascular reconstruction with an arteriovenous loop can be critical to its use. METHODS: Multi-institutional study of 36 patients who underwent vein grafting or AV loop with free tissue transfer. RESULTS: 58.3% of patients received prior radiation and 38.9% prior flap reconstruction. Flap success for vein grafting was 76% and AV loop was 100% (p = 0.16). Success for the radiated cohort was 90.5% and non-radiated 80% (p = 0.63). Flap success for the radiated, vein grafted patient was 83.3% and 100% flap success rate for radiated, AV loop patient (p = 0.49). Overall flap survival was 83.3% versus 97% overall success rate in the United States. CONCLUSION: The AV loop is a viable modality for vessel-depleted free tissue reconstruction. Radiation and previous surgery do not significantly impact flap success rates.

Head and neck reconstruction in vessel-depleted necks: Case report of a labio-mental and mandibular reconstruction using an arteriovenous loop.

Major defects of the facial structures cause severe functional and esthetic impairment. Difficulty in head and neck reconstruction lies in cases of secondary, tertiary, or further reconstruction. This is not a rare situation for patients who had cancer of the upper airways, since the rate of recurrence, second location, or osteoradionecrosis is high. Multiple surgeries and radiation therapy cause significant fibrosis and scar tissues, making any further reconstruction a major challenge for the surgeon when operating patients with vessel- depleted neck. We report our experience with a clinical case of a patient to whom we performed a double free flap reconstruction anastomosed on a vascular loop in a context of vascular cervical desert. In our case, the use of an arteriovenous loop proved to be a reliable approach for a vessel-depleted free tissue reconstruction. This technique has received insufficient attention, yet it provides a means to establish dependable vascular alternatives.

Microvascular development in the rat arteriovenous loop model in vivo-A step by step intravital microscopy analysis.

The arteriovenous (AV) loop model is a key technique to solve one of the major problems of tissue engineering-providing adequate vascular support for a tissue construct of significant size. However, the molecular and cellular mechanisms of vascularization and factors influencing the generation of new tissue in the AV loop are still poorly understood. We previously established a novel intravital microscopy approach to study these events. In this study, we implanted our observation chamber filled with two types of hydrogels such as fibrin and methacrylate gelatin (GelMA) and performed intravital microscopy (IVM) on days 7, 14, and 21. Initial microvessel formation was observed in GelMA on day 14, while the vessel network showed clear indicators of network rearrangement and maturation on day 21. No visible microvessels were observed in fibrin. The chambers were explanted on day 21. Histological examination revealed higher numbers of microvessels in GelMA compared to fibrin, while the AV loop was thrombosed in all fibrin constructs, possibly due to matrix degradation. GelMA proved to be an ideal matrix for IVM studies in the AV loop model due to its slow degradation and transparency. This IVM model can be employed as a novel tool for live and thus faster comprehension of crucial events in the tissue regeneration process, which can improve tissue engineering application.

An Innovative Arteriovenous (AV) Loop Breast Cancer Model Tailored for Cancer Research.

Animal models are important tools to investigate the pathogenesis and develop treatment strategies for breast cancer in humans. In this study, we developed a new three-dimensional in vivo arteriovenous loop model of human breast cancer with the aid of biodegradable materials, including fibrin, alginate, and polycaprolactone. We examined the in vivo effects of various matrices on the growth of breast cancer cells by imaging and immunohistochemistry evaluation. Our findings clearly demonstrate that vascularized breast cancer microtissues could be engineered and recapitulate the in vivo situation and tumor-stromal interaction within an isolated environment in an in vivo organism. Alginate-fibrin hybrid matrices were considered as a highly powerful material for breast tumor engineering based on its stability and biocompatibility. We propose that the novel tumor model may not only serve as an invaluable platform for analyzing and understanding the molecular mechanisms and pattern of oncologic diseases, but also be tailored for individual therapy via transplantation of breast cancer patient-derived tumors.

The role of vein grafts in reconstructive head and neck microsurgery.

OBJECTIVE: Free tissue transfer is widely used for head and neck reconstruction. In certain circumstances, vein grafting is required to elongate free flap pedicles to connect them to appropriate recipient vessels. Because of controversy regarding the use of interposition vein grafts in free tissue reconstruction, this paper reports vein graft indications, techniques, safety, and outcomes for head and neck microvascular surgery. METHODS: Twenty-six patients (23 men and 3 women) who underwent interposition vein grafting concurrent with free tissue transfer were included in this study. The most common reason for head and neck reconstruction with vein graft was tumor recurrence, followed by flap salvage. The interposition vein grafts were applied in two manners as temporary arteriovenous (A-V) loop and conduit to extend the length of the free flap for venous drainage. RESULTS: The most common reconstructions were anterolateral thigh flaps (15 cases), followed by vastus lateralis myocutaneous (3 cases) and radial forearm (2 cases) flaps. The common recipient vessels were superior thyroid artery, superficial temporal artery and external jugular vein. The free flap loss rate was 7.7% with vein grafts and 4.9 without vein grafts (p = 0.380). The free flap complication rate was 50.0% and 16.8% in patients with and without vein grafts, respectively (p < 0.001). Radiation therapy, chemotherapy, prior neck dissection, and prior free flap transfer were more common in the vein graft group (all p < 0.001). The hospital stay was significantly longer for the vein graft group than for the non-vein graft group (29.5 vs. 19.0 days; p = 0.001). CONCLUSION: Overall free flap survival rates of 92.3% and 95.1% in the vein and non-vein graft groups, respectively - indicating the reliability of the vein grafts in challenging head and neck reconstructions, particularly in salvage cases and patients with multiple reconstructions. LEVEL OF EVIDENCE: Level 3.

The Use of Arteriovenous Bundle Interposition Grafts in Microsurgical Reconstruction: A Systematic Review of the Literature.

Microvascular reconstruction frequently requires anastomosis outside of the zone of injury for successful reconstruction. Multiple options exist for pedicle lengthening including vein grafts, arteriovenous loops, and arteriovenous bundle interposition grafts. The authors performed a systematic review of arteriovenous bundle interposition grafts to elucidate indications and outcomes of arteriovenous grafts in microvascular reconstruction. A systematic review of the literature was performed using targeted keywords. Data extraction was performed by two independent authors, and descriptive statistics were used to analyze pooled data. Forty-four patients underwent pedicle lengthening with an arteriovenous graft from the descending branch of the lateral circumflex femoral artery. Most common indications for flap reconstruction were malignancy ( n = 12), trauma ( n = 7), and diabetic ulceration ( n = 4). The most commonly used free flap was the anterolateral thigh flap ( n = 18). There were five complications, with one resulting in flap loss. Arteriovenous bundle interposition grafts are a viable option for pedicle lengthening when free flap distant anastomosis is required. The descending branch of the lateral circumflex femoral artery may be used for a variety of defects and can be used in conjunction with fasciocutaneous, osteocutaneous, muscle, and chimeric free flaps.

A case of an upper anterior abdominal wall extra-skeletal Ewing sarcoma- soft tissue reconstruction with medial intercostal artery perforator flap and free anterolateral thigh fasciocutaneous flap with arteriovenous loop graft.

INTRODUCTION: Extra-skeletal Ewing sarcoma - a rare clinical entity. After the tumour resection, a huge upper anterior abdominal wall defect poses a challenge in soft tissue reconstruction in a thin individual. CASE PRESENTATION: We report an anterior abdominal wall Ewing sarcoma in a 22-year-old gentleman, arising from the left rectus abdominis muscle. After wide local excision, the reconstruction was achieved with free anterolateral thigh(ALT) fasciocutaneous flap with arteriovenous(AV) loop graft, right medial intercostal artery perforator(MICAP) flap and split-thickness skin graft(SSG). DISCUSSION: The goals of anterior abdominal wall reconstruction are to restore the integrity of the abdominal wall, prevent visceral eventration, and provide functional support. In view of the size of the defect, a free ALT flap was harvested and anastomosed to the left deep inferior epigastric bundle with the AV loop graft. Although the current trend is skewed towards the use of biologic mesh, an on-lay prolene mesh was used due to its affordable cost and that the biologic mesh was not available in Malaysia. The options of further reconstruction after the patient developed marginal flap necrosis and surgical site infection were also discussed. Post-operation 3 months, there was denser hair growth on the ALT flap. This finding has never been reported before and warrants further studies. CONCLUSION: The use of combination of various technique, namely free ALT fasciocutaneous flap with AV loop graft, right MICAP flap and SSG in reconstruction ensures a satisfactory functional and aesthetic outcome in the upper anterior abdominal wall reconstruction.

In Vivo Evaluation of Mechanically Processed Stromal Vascular Fraction in a Chamber Vascularized by an Arteriovenous Shunt.

Mechanically processed stromal vascular fraction (mSVF) is a promising source for regenerative purposes. To study the in vivo fate of the mSVF, we herein used a vascularized tissue engineering chamber that insulates the target mSVF from the surrounding environment. In contrast to previous models, we propose an arteriovenous (AV) shunt between saphenous vessels in rats without a venous graft. Mechanical SVF was processed from the fat pads of male Sprague Dawley rats, mixed with a fibrin hydrogel and implanted into an inguinal tissue engineering chamber. An arteriovenous shunt was established between saphenous artery and vein. On the contralateral side, an mSVF-fibrin hydrogel mix without vascular axis served as a non-vascularized control. After two and six weeks, rats were sacrificed for further analysis. Mechanical SVF showed significant numbers of mesenchymal stromal cells. Vascularized mSVF explants gained weight over time. Perilipin and CD31 expression were significantly higher in the mSVF explants after six weeks while no difference in DAPI positive cells, collagen deposition and FABP4 expression was observed. Morphologically, no differentiated adipocytes but a dense cell-rich tissue with perilipin-positive cells was found after six weeks. The phosphorylation of ERK1/2 was significantly enhanced after six weeks while Akt activation remained unaltered. Finally, mSVF explants stably expressed and released VEGF, bFGF and TGFb. Vascularized mSVF is able to proliferate and express adipocyte-specific markers. The AV shunt model is a valuable refinement of currently existing AV loop models in the rat which contributes to the fundamental 3R principles of animal research.

Enhanced vascularization andde novotissue formation in hydrogels made of engineered RGD-tagged spider silk proteins in the arteriovenous loop model.

Due to its low immunogenic potential and the possibility to fine-tune their properties, materials made of recombinant engineered spider silks are promising candidates for tissue engineering applications. However, vascularization of silk-based scaffolds is one critical step for the generation of bioartificial tissues and consequently for clinical application. To circumvent insufficient vascularization, the surgically induced angiogenesis by means of arteriovenous loops (AVL) represents a highly effective methodology. Here, previously established hydrogels consisting of nano-fibrillary recombinant eADF4(C16) were transferred into Teflon isolation chambers and vascularized in the rat AVL model over 4 weeks. To improve vascularization, also RGD-tagged eADF4(C16) hydrogels were implanted in the AVL model over 2 and 4 weeks. Thereafter, the specimen were explanted and analyzed using histology and microcomputed tomography. We were able to confirm biocompatibility and tissue formation over time. Functionalizing eADF4(C16) with RGD-motifs improved hydrogel stability and enhanced vascularization even outperforming other hydrogels, such as fibrin. This study demonstrates that the scaffold ultrastructure as well as biofunctionalization with RGD-motifs are powerful tools to optimize silk-based biomaterials for tissue engineering applications.