the-arteriovenous-av-loop-small-animal-model-to-study-angiogenesis

The Arteriovenous (AV) Loop in a Small Animal Model to Study Angiogenesis and Vascularized Tissue Engineering

A functional blood vessel network is a prerequisite for the survival and growth of almost all tissues and organs in the human body. Moreover, in ...

University Hospital of Erlangen, Friedrich-Alexander University of Erlangen-Nürnberg (FAU)

Treatment of complex bone defects in which vascular supply is insufficient is still a challenge. To overcome the limitations from autologous grafts, a sheep model has been established recently, which is characterized by the development of an independent axial vascularization of a bioartificial construct, permitting microsurgical transplantation. To engineer independently axially vascularized bone tissue in the sheep arteriovenous (AV)-loop model, mesenchymal stem cells (MSCs), without and in combination with recombinant human bone morphogenetic protein-2 (rhBMP-2), were harvested and directly autotransplanted in combination with β-tricalcium phosphate-hydroxyapatite (β-TCP-HA) granules into sheep in this study. After explantation after 12 weeks, histological and immunohistochemical evaluation revealed newly formed bone in both groups. An increased amount of bone area was obtained using directly autotransplanted MSCs with rhBMP-2 stimulation. Osteoblastic and osteoclastic cells were detected adjacent to the newly formed bone, revealing an active bone remodelling process. Directly autotransplanted MSCs can be found close to the β-TCP-HA granules and are contributing to bone formation. Over time, magnetic resonance imaging (MRI) and micro-computed tomography (μCT) imaging confirmed the dense vascularization arising from the AV-loop. This study shows de novo engineering of independently axially vascularized transplantable bone tissue in clinically significant amounts, using directly autotransplanted MSCs and rhBMP-2 stimulation in about 12 weeks in the sheep AV-loop model. This strategy of engineering vascularized transplantable bone tissue could be possibly transferred to the clinic in the future in order to augment current reconstructive strategies.

Engineering axially vascularized bone in the sheep arteriovenous-loop model.

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.

New aspects on efficient anticoagulation and antiplatelet strategies in sheep.

Comments on: "In vivo bone regeneration using tubular perfusion system bioreactor cultured nanofibrous scaffolds" vascularization--one challenge of tissue engineering.

New therapeutic strategies are required for critical size bone defects, because the gold standard of transplanting autologous bone from an unharmed area of the body often leads to several severe side effects and disadvantages for the patient. For years, tissue engineering approaches have been seeking a stable, axially vascularized transplantable bone replacement suitable for transplantation into the recipient bed with pre-existing insufficient conditions. For this reason, the arteriovenous loop model was developed and various bone substitutes have been vascularized. However, it has not been possible thus far to engineer a primary stable and axially vascularized transplantable bone substitute. For that purpose, a primary stable silica-embedded nanohydroxyapatite (HA) bone substitute in combination with blood, bone marrow, expanded, or directly retransplanted mesenchymal stem cells, recombinant human bone morphogenetic protein 2 (rhBMP-2), and different carrier materials (fibrin, cell culture medium, autologous serum) was tested subcutaneously for 4 or 12 weeks in the sheep model. Autologous serum lead to an early matrix change during degradation of the bone substitute and formation of new bone tissue. The best results were achieved in the group combining mesenchymal stem cells expanded with 60 μg/mL rhBMP-2 in autologous serum. Better ingrowth of fibrovascular tissue could be detected in the autologous serum group compared with the control (fibrin). Osteoclastic activity indicating an active bone remodeling process was observed after 4 weeks, particularly in the group with autologous serum and after 12 weeks in every experimental group. This study clearly demonstrates the positive effects of autologous serum in combination with mesenchymal stem cells and rhBMP-2 on bone formation in a primary stable silica-embedded nano-HA bone grafting material in the sheep model. In further experiments, the results will be transferred to the sheep arteriovenous loop model in order to engineer an axially vascularized primary stable bone replacement in clinically relevant size for free transplantation.

Autologous serum improves bone formation in a primary stable silica-embedded nanohydroxyapatite bone substitute in combination with mesenchymal stem cells and rhBMP-2 in the sheep model.

During the last decades, a range of excellent and promising strategies in Bone Tissue Engineering have been developed. However, the remaining major problem is the lack of vascularization. In this study, extrinsic and intrinsic vascularization strategies were combined for acceleration of vascularization. For optimal biomechanical stability of the defect site and simplifying future transition into clinical application, a primary stable and approved nanostructured bone substitute in clinically relevant size was used. An arteriovenous (AV) loop was microsurgically created in sheep and implanted, together with the bone substitute, in either perforated titanium chambers (intrinsic/extrinsic) for different time intervals of up to 18 weeks or isolated Teflon(®) chambers (intrinsic) for 18 weeks. Over time, magnetic resonance imaging and micro-computed tomography (CT) analyses illustrate the dense vascularization arising from the AV loop. The bone substitute was completely interspersed with newly formed tissue after 12 weeks of intrinsic/extrinsic vascularization and after 18 weeks of intrinsic/extrinsic and intrinsic vascularization. Successful matrix change from an inorganic to an organic scaffold could be demonstrated in vascularized areas with scanning electron microscopy and energy dispersive X-ray spectroscopy. Using the intrinsic vascularization method only, the degradation of the scaffold and osteoclastic activity was significantly lower after 18 weeks, compared with 12 and 18 weeks in the combined intrinsic-extrinsic model. Immunohistochemical staining revealed an increase in bone tissue formation over time, without a difference between intrinsic/extrinsic and intrinsic vascularization after 18 weeks. This study presents the combination of extrinsic and intrinsic vascularization strategies for the generation of an axially vascularized bone substitute in clinically relevant size using a large animal model. The additional extrinsic vascularization promotes tissue ingrowth and remodeling processes of the bone substitute. Extrinsic vessels contribute to faster vascularization and finally anastomose with intrinsic vasculature, allowing microvascular transplantation of the bone substitute after a shorter prevascularization time than using the intrinsic method only. It can be reasonably assumed that the usage of perforated chambers can significantly reduce the time until transplantation of bone constructs. Finally, this study paves the way for further preclinical testing for proof of the concept as a basis for early clinical applicability.

Acceleration of vascularized bone tissue-engineered constructs in a large animal model combining intrinsic and extrinsic vascularization.

Comment on "Microsurgical Techniques Used to Construct the Vascularized and Neurotized Tissue Engineered Bone".

Preventing ischemia-related cell damage is a priority when preserving tissue for transplantation. Perfusion protocols have been established for a variety of applications and proven to be superior to procedures used in clinical routine. Extracorporeal perfusion of muscle tissue though cumbersome is highly desirable since it is highly susceptible to ischemia-related damage. To show the efficacy of different perfusion protocols external field stimulation can be used to immediately visualize improvement or deterioration of the tissue during active and running perfusion protocols. This method has been used to show the superiority of extracorporeal perfusion using porcine rectus abdominis muscles perfused with heparinized saline solution. Perfused muscles showed statistically significant higher ability to exert force compared to nonperfused ones. These findings can be confirmed using Annexin V as marker for cell damage, perfusion of muscle tissue limits damage significantly compared to nonperfused tissue. The combination of extracorporeal perfusion and external field stimulation may improve organ conservation research.

Assessing viability of extracorporeal preserved muscle transplants using external field stimulation: a novel tool to improve methods prolonging bridge-to-transplantation time.

Comment on 'Basic concepts regarding fractures healing and the current options and future directions in managing bone fractures'.

Research and ideas for potential applications in the field of Tissue Engineering (TE) and Regenerative Medicine (RM) have been constantly increasing over recent years, basically driven by the fundamental human dream of repairing and regenerating lost tissue and organ functions. The basic idea of TE is to combine cells with putative stem cell properties with extracellular matrix components, growth factors and supporting matrices to achieve independently growing tissue. As a side effect, in the past years, more insights have been gained into cell-cell interaction and how to manipulate cell behavior. However, to date the ideal cell source has still to be found. Apart from commonly known various stem cell sources, telocytes (TC) have recently attracted increasing attention because they might play a potential role for TE and RM. It becomes increasingly evident that TC provide a regenerative potential and act in cellular communication through their network-forming telopodes. While TE in vitro experiments can be the first step, the key for elucidating their regenerative role will be the investigation of the interaction of TC with the surrounding tissue. For later clinical applications further steps have to include an upscaling process of vascularization of engineered tissue. Arteriovenous loop models to vascularize such constructs provide an ideal platform for preclinical testing of future therapeutic concepts in RM. The following review article should give an overview of what is known so far about the potential role of TC in TE and RM.

The potential role of telocytes in Tissue Engineering and Regenerative Medicine.

In the field of free flap transfer in reconstructive surgery, the trans- or replanted tissue always undergoes cell damage during ischemia to a more or less strong extent. In previous studies we already showed that conserving muscle transplants by means of extracorporeal perfusion over a period of 6 hours by using a crystalloid solution for perfusion. However, we observed significant edema formation. In this study we aimed at reducing the edema formation by using an iso-oncotic colloid as perfusion solution. This way we want to evaluate a possible new application of hydroxyl-ethyl starch in an extracorporeal setup to exploit potential benefits of the colloid.Examined parameters include the muscles' functionality with external field stimulation, histological examination and edema formation. Perfused muscles showed a statistically significant higher ability to exert force compared to nonperfused ones. These findings can be confirmed using Annexin V as marker for cell damage, perfusion of muscle tissue limits damage significantly compared to nonperfused tissue. Substituting the electrolyte perfusion solution with a colloidal one shows the tendency to reduce the edema formation but without statistical significance.

Hydroxyethyl starch solutionfor extracorporeal tissue perfusion.

There is a need to establish more cell lines from breast tumors in contrast to immortalized cell lines from metastatic effusions in order to represent the primary tumor and not principally metastatic biology of breast cancer. This investigation describes the simultaneous isolation, characterization, growth and function of primary mammary epithelial cells (MEC), mesenchymal cells (MES) and adipose derived stem cells (ADSC) from four normal breasts, one inflammatory and one triple-negative ductal breast tumors.

Article	Year
Engineering axially vascularized bone in the sheep arteriovenous-loop model. Journal of tissue engineering and regenerative medicine\| PubMed ID: 22438065	2013
New aspects on efficient anticoagulation and antiplatelet strategies in sheep. BMC veterinary research\| PubMed ID: 24088206	2013
Comments on: "In vivo bone regeneration using tubular perfusion system bioreactor cultured nanofibrous scaffolds" vascularization--one challenge of tissue engineering. Tissue engineering. Part A\| PubMed ID: 24325477	2014
Autologous serum improves bone formation in a primary stable silica-embedded nanohydroxyapatite bone substitute in combination with mesenchymal stem cells and rhBMP-2 in the sheep model. International journal of nanomedicine\| PubMed ID: 25429218	2014
Acceleration of vascularized bone tissue-engineered constructs in a large animal model combining intrinsic and extrinsic vascularization. Tissue engineering. Part A\| PubMed ID: 25760576	2015
Comment on "Microsurgical Techniques Used to Construct the Vascularized and Neurotized Tissue Engineered Bone". BioMed research international\| PubMed ID: 26064942	2015
Assessing viability of extracorporeal preserved muscle transplants using external field stimulation: a novel tool to improve methods prolonging bridge-to-transplantation time. Scientific reports\| PubMed ID: 26145230	2015
Comment on 'Basic concepts regarding fractures healing and the current options and future directions in managing bone fractures'. International wound journal\| PubMed ID: 26663542	2016
The potential role of telocytes in Tissue Engineering and Regenerative Medicine. Seminars in cell & developmental biology\| PubMed ID: 26805441	2016
Hydroxyethyl starch solutionfor extracorporeal tissue perfusion. Clinical hemorheology and microcirculation\| PubMed ID: 26890243	2016
Selective isolation and characterization of primary cells from normal breast and tumors reveal plasticity of adipose derived stem cells. Breast cancer research : BCR\| PubMed ID: 26968831	2016

Annika Weigand

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