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¯.

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.

Vessel grafts for tissue engineering revisited-Vessel segments show location-specific vascularization patterns in ex vivo ring assay.

OBJECTIVE: Transplantation of prefabricated tissue-engineered flaps can be a potential alternative for healing large tissue defects. Providing adequate vascular supply for an engineered tissue construct is one of the key points in establishing successful tissue engineering-based treatment approaches. In tissue engineering-based vascularization techniques like the arteriovenous loop, vascular grafts with high angiogenic potential can help to enhance neovascularization and tissue formation. Therefore, our study aimed to compare the angiogenic potential of vascular grafts from different locations in the rat. METHODS: The angiogenic activity was investigated by an ex vivo vessel outgrowth ring assay using 1-mm height vascular segments embedded in fibrin for 2 weeks. RESULTS: Maximum vessel outgrowth was observed on Days 10-12. Upper extremity vessels exhibited stronger outgrowth than lower extremity vessels. Moreover, arterial vessels demonstrated higher angiogenic potential compared with venous vessels. CONCLUSION: Collectively, our ex vivo findings suggest that upper extremity arterial vessels have a higher angiogenic capacity, which could be used to improve neovascularization and tissue formation in tissue engineering.

The bone is the major source of high circulating intact fibroblast growth factor-23 in acute murine polymicrobial sepsis induced by cecum ligation puncture.

Fibroblast growth factor-23 (FGF23), a bone-produced hormone, plays a critical role in mineral homeostasis. Human diseases associated with excessive intact circulating FGF23 (iFGF23) result in hypophosphatemia and low vitamin D hormone in patients with normal kidney function. In addition, there is accumulating evidence linking FGF23 with inflammation. Based on these studies and the frequent observation of hypophosphatemia among septic patients, we sought to elucidate further the relationship between FGF23 and mineral homeostasis in a clinically relevant murine polymicrobial sepsis model. Medium-severity sepsis was induced by cecum ligation puncture (CLP) in adult CD-1 mice of both sexes. Healthy CD-1 mice (without CLP) were used as controls. Forty-eight hours post-CLP, spontaneous urine was collected, and serum, organs and bones were sampled at necropsy. Serum iFGF23 increased ~20-fold in CLP compared to control mice. FGF23 protein concentration was increased in the bones, but not in spleen or liver of CLP mice. Despite the ~20-fold iFGF23 increase, we did not observe any significant changes in mineral homeostasis or parathyroid hormone levels in the blood of CLP animals. Urinary excretion of phosphate, calcium, and sodium remained unchanged in male CLP mice, whereas female CLP mice exhibited lower urinary calcium excretion, relative to healthy controls. In line with renal FGF23 resistance, expression of phosphate-, calcium- and sodium-transporting proteins did not show consistent changes in the kidneys of male and female CLP mice. Renal expression of the co-receptor αKlotho was downregulated in female, but not in male CLP mice. In conclusion, our data demonstrate that the dramatic, sex-independent rise in serum iFGF23 post-CLP was mainly caused by an upregulation of FGF23 secretion in the bone. Surprisingly, the upsurge in circulating iFGF23 did not alter humoral mineral homeostasis in the acutely septic mice. Hence, the biological function of elevated FGF23 in sepsis remains unclear and warrants further studies.

Watching the Vessels Grow: Establishment of Intravital Microscopy in the Arteriovenous Loop Rat Model.

Tissue engineering in reconstructive surgery seeks to generate bioartificial tissue substitutes. The arteriovenous (AV) loop allows the generation of axially vascularized tissue constructs. Cellular mechanisms of this vascularization process are largely unclear. In this study, we developed two different chamber models for intravital microscopy and imaging of the AV loop in the rat. Multiple design variations were implanted and the stability of the chamber and AV loop patency was tested in vivo. Our novel chamber facilitates repetitive observation of the AV loop using fluorescence-enhanced intravital microscopy. This technique can be used for daily evaluation of leukocyte-endothelial cell interactions, vascularization, and tissue formation in the AV loop model on 14 consecutive days. Therefore, our newly developed model for intravital microscopy will provide better understanding of cellular and molecular processes in tissue engineering in the AV loop. Moreover, it supports initiation of the novel approaches for therapeutic applications. Impact statement In the Arteriovenous (AV) loop, axially vascularized tissue can be generated and modified using different tissue engineering approaches. Cellular mechanisms of this vascularization process are largely unclear. We managed to develop an intravital microscopy model for long-term observation of intravascular and perivascular events in the AV loop. Leukocyte-endothelial cell interactions, vascularization, and tissue formation in the AV loop can now be evaluated on a day-to-day basis. This will provide better understanding of cellular and molecular processes happening during tissue engineering within the AV loop.

Actually Seeing What Is Going on - Intravital Microscopy in Tissue Engineering.

Intravital microscopy (IVM) study approach offers several advantages over in vitro, ex vivo, and 3D models. IVM provides real-time imaging of cellular events, which provides us a comprehensive picture of dynamic processes. Rapid improvement in microscopy techniques has permitted deep tissue imaging at a higher resolution. Advances in fluorescence tagging methods enable tracking of specific cell types. Moreover, IVM can serve as an important tool to study different stages of tissue regeneration processes. Furthermore, the compatibility of different tissue engineered constructs can be analyzed. IVM is also a promising approach to investigate host reactions on implanted biomaterials. IVM can provide instant feedback for improvising tissue engineering strategies. In this review, we aim to provide an overview of the requirements and applications of different IVM approaches. First, we will discuss the history of IVM development, and then we will provide an overview of available optical modalities including the pros and cons. Later, we will summarize different fluorescence labeling methods. In the final section, we will discuss well-established chronic and acute IVM models for different organs.