gms | German Medical Science

GMS German Plastic, Reconstructive and Aesthetic Surgery – Burn and Hand Surgery

Deutsche Gesellschaft der Plastischen, Rekonstruktiven und Ästhetischen Chirurgen (DGPRÄC)
Deutsche Gesellschaft für Verbrennungsmedizin (DGV)

ISSN 2193-7052

Optimization of vascularisation in axially vascularised matrices in tissue engineering

Optimierung der Vaskularisation von axial durchbluteten Matrizes im Tissue Engineering

Review Article

Search Medline for

  • corresponding author Andreas Arkudas - Department of Plastic and Hand Surgery, University Hospital of Erlangen, Friedrich-Alexander-University of Erlangen-Nuernberg, Erlangen, Germany
  • author Ulrich Kneser - Department of Plastic and Hand Surgery, University Hospital of Erlangen, Friedrich-Alexander-University of Erlangen-Nuernberg, Erlangen, Germany; Department of Hand, Plastic and Reconstructive Surgery – Burn Center, BG Trauma Center Ludwigshafen, Department of Plastic Surgery, University of Heidelberg, Germany
  • author Raymund E. Horch - Department of Plastic and Hand Surgery, University Hospital of Erlangen, Friedrich-Alexander-University of Erlangen-Nuernberg, Erlangen, Germany

GMS Ger Plast Reconstr Aesthet Surg 2014;4:Doc03

doi: 10.3205/gpras000022, urn:nbn:de:0183-gpras0000222

Published: March 6, 2014

© 2014 Arkudas et al.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( You are free: to Share – to copy, distribute and transmit the work, provided the original author and source are credited.


Nowadays, autologous tissue transplantation represents the gold standard for the reconstruction of large tissue defects. To minimize the resulting donor side morbidity and to overcome the limitations regarding shape and volume of bone grafts research has focused on tissue engineering of axially vascularised tissue constructs. We have evaluated different strategies to improve the vascularisation of bioartificial transplantable tissue volumes. First we used angiogenic growth factors such as VEGF and bFGF in the subcutaneous and arteriovenous loop model in the rat to increase vessel ingrowth. Afterwards we combined the intrinsic vascular pathway of the arteriovenous loop model with an additional extrinsic vessel ingrowth by using a porous titanium chamber. Transplanted cells also showed an increased survival in the AV loop model compared to subcutaneously implanted matrices. In this manuscript we show the evolution of different newly developed methods to improve vascularisation of axially vascularised matrices in tissue engineering.

Keywords: tissue engineering, vascularisation, growth factors, VEGF, bFGF, AV loop model


Für die Therapie angeborener und erworbener Gewebedefekte, z.B. im Bereich des Knochens, stellt heutzutage nach wie vor der autologe Gewebetransfer den Goldstandard dar. Um den hierbei resultierenden Hebedefekt und die Einschränkung hinsichtlich Formbarkeit und Länge/Volumen des verfügbaren Gewebes zu minimieren, steht das Tissue Engineering von axial vaskularisierten Gewebekonstrukten im Fokus des wissenschaftlichen Interesses. Wir untersuchten verschiedene Forschungsansätze, um die Vaskularisation von bioartifiziellen, transplantierbaren Gewebekonstrukten zu verbessern. Es wurden eine Steigerung der Vaskularisation mit Hilfe verschiedener angiogenetischer Wachstumsfaktoren im Subkutanmodell und in einem arteriovenösen Gefäßschleifenmodell in der Ratte gezeigt sowie eine Kombination der arteriovenösen Gefäßschleife mit peripher einsprießenden Gefäßen mit Hilfe einer neuartigen porösen Titankammer nachgewiesen. Mit Hilfe der Gefäßschleife konnte zudem ein erhöhtes Überleben von transplantierten Zellen im Vergleich zu nicht vaskularisierten Matrizes gezeigt werden. Die hier vorgestellte Arbeit zeigt die Entwicklung neuartiger Ansätze zur Optimierung der Vaskularisation von axial durchbluteten Matrizes im Tissue Engineering.

Procedure and findings

The reconstruction of large bone defects represents an important research field because nowadays therapy is limited. In the clinical routine autologous tissue transplantation still represents the gold standard for the reconstruction of bony defects [1], [2]. Advantages of the autologous bone transplantation are their given osteoconductive, osteoinductive and osteogenic activity [3], [4]. Depending on the indication the bone graft can be used in different shapes such as chips and blocks [5], [6]. Known disadvantages of the autologous bone transplantation are beside the limitations in form and volume the donor side morbidity including the possibility of infections, hematomas, seromas, chronic pain or paraesthesia at the donor side [7]. Areas of limited vascularisation e.g. caused by irradiation or trauma or large bone defects require microsurgical transfer of vascularised bone grafts such as fibula, iliac crest of scapula bone [8], [9], [10].

Tissue engineering is defined as an interdisciplinary field with the aim to create bioartificial tissue to maintain, improve or substitute organ functions [11]. The engineering of bioartificial bone tissue represents one major research field in the area of tissue engineering. Most tissue engineering concepts lack the initial vascularisation of the constructs, therefore transplantation of cell containing matrices are often limited because of a high initial cell loss. Diffusion can only maintain viability of cells in a range of 200 µm in the surrounding of vessels [12], [13]. Vascularisation is crucial for any successful in vivo tissue engineering model. Vascularisation can be achieved by an extrinsic or intrinsic pathway using a defined vascular axis. The advantage of an intrinsic vascularisation is the possible microsurgical transfer using the vascular pedicle.

We have evaluated different strategies to improve vascularisation of bioartificial transplantable bone constructs. We used the arteriovenous loop model in the rat, first described by Erol and Spira in 1979 [14]. This model relies on an arteriovenous vessel loop, created in the medial thigh of the rat using an interpositional vein graft of the contralateral thigh. Afterwards the AV loop is placed in an isolation chamber containing the tissue matrix. After a defined vascularisation period the constructs containing AV loop and matrix are explanted and vessel ingrowth as well as bone generation are evaluated using histology and/or micro-CT.

Based on the results of other research groups of our laboratory we implanted a processed bovine cancellous bone matrix in the arteriovenous loop model of the rat [15]. In the first operation the AV loop and the matrix were implanted and placed in the chamber and after a vascularisation period fibrin gel immobilized CFDA (carboxyfluorescein diacetate succinimidyl ester) labeled osteoblasts were injected in the matrices. Subcutaneously implanted matrices containing the same matrix and osteoblasts served as a control group in this study. At the explantation time point matrices were perfused using India ink to detect functional vessels in the histological processing. We were able to show an increased survival rate of osteoblasts in the AV loop group compared to the control group. Furthermore bone generation and expression of osteoblast-specific gens were increased in the AV loop group compared to the subcutanous group.

To minimize the time interval between AV loop implantation and cell injection we studied different strategies to increase vascularisation speed of the axially vascularised constructs. Therefore we tested different angiogenic growth factors in a newly developed upside-down subcutaneous implantation technique with the opening of the chamber facing the muscular layer of the back of the rat and not the dorsal skin [16]. Using this technique we were able to maximize the contact area between matrix and host organism and to minimize shear forces leading to a more homogeneous construct area. We evaluated the angiogenic potential of the angiogenic growth factors VEGF165 (vascular endothelial growth factor) and bFGF (basic fibroblast growth factor) in five different concentrations and combinations at the contact area between construct and musculature. We were able to show a significant increase of vessel number and generation of vascularised connective tissue with an optimum between 25 ng/ml and 100 ng/ml VEGF and bFGF.

Based on these results we evaluated the angiogenic effect of fibrin gel immobilized VEGF and bFGF in four different concentrations in the arteriovenous loop model of the rat [17]. The evaluation was based on conventional histology and micro-CT. We detected a significant increase of angiogenesis in the AV loop model at a growth factor concentration of 100 ng/ml each.

Afterwards we applied 100 ng/ml VEGF and bFGF in a hydroxyapatite/tricalciumphosphate bone matrix and implanted the constructs in the AV loop model of the rat [18]. The evaluation was performed using conventional histological techniques and a newly developed automatic three dimensional micro-CT evaluation (in cooperation with A. Hess, Institute of Pharmacology and Toxicology, University of Erlangen-Nuremberg, Erlangen, Germany) (Figure 1 [Fig. 1]). We were able to show an increase of vascularisation speed of the HA/TCP matrix in the AV loop model using the angiogenic growth factor combination.

Afterwards we invented a porous titanium chamber in cooperation with C. Koerner (Institute of Science and Technology of Metals, University of Erlangen-Nuremberg, Germany) in order to combine the intrinsic vascular pathway with an additional extrinsic vascularisation to increase vascularisation of matrices. First we were able to show, that a connection between the intrinsic and extrinsic vascular pathways takes place over time, because otherwise no benefit in vascularisation would be detectable after transplantation using the vascular pedicle of the intrinsic vascular pathway [19]. Two weeks after implantation 83% of all vessels showed a connection with the AV loop whereas after 8 weeks nearly all vessels (97%) were connected with the AV loop. Therefore a transplantation of the entire construct using the vascular pedicle is possible, which is mandatory for later clinical applications.

We also evaluated different fibrin sealants with varying fibrinogen, thrombin, aprotinin and factor XIII concentrations in terms of matrix degradation and vascularisation in the AV loop model of the rat [20]. We found out, that mainly the fibrinogen concentration influences the matrix properties and that there was an inverse relationship between the matrix degradation and vascularisation.


Tissue engineering has arised over the last decades and has the potential to change clinical practice in reconstructive surgery in the future. Besides bone tissue many studies focus on tissue engineering of muscle, fat or skin using a plethora of highly innovative biomaterials combined with stem cells and growth factors. The initial euphoria caused by successful in vitro studies was followed by sober recognition that the transfer of these results into an in vivo environment has proved to be difficult. Vascularisation has emerged to be one of the crucial steps towards clinical application of engineered bioartificial tissues. When in vitro constructs are transferred into an in vivo environment scaffolds have to rely on diffusion and on a newly generated extrinsic vascular network. To overcome the limited initial vascularisation reconstructive surgeons prefer an intrinsic type of vascularisation that could be transferred to the defect site using microsurgical anatomosis leading to an immediate perfusion of the entire scaffolds. Optimized vascularisation techniques may allow the transfer of engineered tissue into the demanding in vivo environment and may help to bridge the gap between bench and bedside.


Competing interests

The authors declare that they have no competing interests.


This study was supported by research grants from Baxter Healthcare Corporation, Xue-Hong and Hans Georg Geis, the University of Erlangen (ELAN Program), the AO Research Fund Grant S-10-36A and the ‘‘Emerging Fields Initiative’’ of the University of Erlangen-Nuremberg.


The authors thank Prof. Peter Greil and Mr. Peter Reinhard for production of the Teflon chambers. This work contains parts of Andreas Arkudas’ habilitation thesis.


Gazdag AR, Lane JM, Glaser D, Forster RA. Alternatives to Autogenous Bone Graft: Efficacy and Indications. J Am Acad Orthop Surg. 1995 Jan;3(1):1-8.
Föll DA, Jokuszies A, Radtke C, Vogt PM. Successful closure of persistent oro-cutaneous fistulas by injection of autologous adipose-derived stem cells: a case report. GMS Ger Plast Reconstr Aesthet Surg. 2013;3:Doc05. DOI: 10.3205/gpras000015 External link
Vaccaro AR. The role of the osteoconductive scaffold in synthetic bone graft. Orthopedics. 2002 May;25(5 Suppl):s571-8.
Kneser U, Schaefer DJ, Polykandriotis E, Horch RE. Tissue engineering of bone: the reconstructive surgeon's point of view. J Cell Mol Med. 2006 Jan-Mar;10(1):7-19. DOI: 10.1111/j.1582-4934.2006.tb00287.x External link
Dasmah A, Thor A, Ekestubbe A, Sennerby L, Rasmusson L. Particulate vs. block bone grafts: three-dimensional changes in graft volume after reconstruction of the atrophic maxilla, a 2-year radiographic follow-up. J Craniomaxillofac Surg. 2012 Dec;40(8):654-9. DOI: 10.1016/j.jcms.2011.10.032 External link
Zeplin PH, Meffert RH, Kuhfuß I. Mid-term results after midcarpal arthrodesis using an iliac crest cortical chip for grade II/III SLAC/SNAC-wrists. GMS Ger Plast Reconstr Aesthet Surg. 2013;3:Doc03. DOI: 10.3205/gpras000013 External link
Younger EM, Chapman MW. Morbidity at bone graft donor sites. J Orthop Trauma. 1989;3(3):192-5. DOI: 10.1097/00005131-198909000-00002 External link
Swartz WM, Banis JC, Newton ED, Ramasastry SS, Jones NF, Acland R. The osteocutaneous scapular flap for mandibular and maxillary reconstruction. Plast Reconstr Surg. 1986 Apr;77(4):530-45. DOI: 10.1097/00006534-198604000-00003 External link
Shea KG, Coleman DA, Scott SM, Coleman SS, Christianson M. Microvascularized free fibular grafts for reconstruction of skeletal defects after tumor resection. J Pediatr Orthop. 1997 Jul-Aug;17(4):424-32. DOI: 10.1097/01241398-199707000-00002 External link
Ozaki T, Hillmann A, Wuisman P, Winkelmann W. Reconstruction of tibia by ipsilateral vascularized fibula and allograft. 12 cases with malignant bone tumors. Acta Orthop Scand. 1997 Jun;68(3):298-301. DOI: 10.3109/17453679708996706 External link
Langer R, Vacanti JP. Tissue engineering. Science. 1993 May;260(5110):920-6. DOI: 10.1126/science.8493529 External link
Folkman J, Hochberg M. Self-regulation of growth in three dimensions. J Exp Med. 1973 Oct;138(4):745-53. DOI: 10.1084/jem.138.4.745 External link
Kneser U, Stangenberg L, Ohnolz J, Buettner O, Stern-Straeter J, Möbest D, Horch RE, Stark GB, Schaefer DJ. Evaluation of processed bovine cancellous bone matrix seeded with syngenic osteoblasts in a critical size calvarial defect rat model. J Cell Mol Med. 2006 Jul-Sep;10(3):695-707. DOI: 10.1111/j.1582-4934.2006.tb00429.x External link
Erol OO, Spira M. New capillary bed formation with a surgically constructed arteriovenous fistula. Surg Forum. 1979;30:530-1.
Arkudas A, Beier JP, Heidner K, Tjiawi J, Polykandriotis E, Srour S, Sturzl M, Horch RE, Kneser U. Axial prevascularization of porous matrices using an arteriovenous loop promotes survival and differentiation of transplanted autologous osteoblasts. Tissue Eng. 2007 Jul;13(7):1549-60. DOI: 10.1089/ten.2006.0387 External link
Arkudas A, Tjiawi J, Saumweber A, Beier JP, Polykandriotis E, Bleiziffer O, Horch RE, Kneser U. Evaluation of blood vessel ingrowth in fibrin gel subject to type and concentration of growth factors. J Cell Mol Med. 2009 Sep;13(9A):2864-74. DOI: 10.1111/j.1582-4934.2008.00410.x External link
Arkudas A, Pryymachuk G, Hoereth T, Beier JP, Polykandriotis E, Bleiziffer O, Horch RE, Kneser U. Dose-finding study of fibrin gel-immobilized vascular endothelial growth factor 165 and basic fibroblast growth factor in the arteriovenous loop rat model. Tissue Eng Part A. 2009 Sep;15(9):2501-11. DOI: 10.1089/ten.tea.2008.0477 External link
Arkudas A, Beier JP, Pryymachuk G, Hoereth T, Bleiziffer O, Polykandriotis E, Hess A, Gulle H, Horch RE, Kneser U. Automatic quantitative micro-computed tomography evaluation of angiogenesis in an axially vascularized tissue-engineered bone construct. Tissue Eng Part C Methods. 2010 Dec;16(6):1503-14. DOI: 10.1089/ten.tec.2010.0016 External link
Arkudas A, Pryymachuk G, Beier JP, Weigel L, Körner C, Singer RF, Bleiziffer O, Polykandriotis E, Horch RE, Kneser U. Combination of extrinsic and intrinsic pathways significantly accelerates axial vascularization of bioartificial tissues. Plast Reconstr Surg. 2012 Jan;129(1):55e-65e. DOI: 10.1097/PRS.0b013e3182361f97 External link
Arkudas A, Pryymachuk G, Hoereth T, Beier JP, Polykandriotis E, Bleiziffer O, Gulle H, Horch RE, Kneser U. Composition of fibrin glues significantly influences axial vascularization and degradation in isolation chamber model. Blood Coagul Fibrinolysis. 2012 Jul;23(5):419-27. DOI: 10.1097/MBC.0b013e3283540c0f External link