The impact of VEGF and bFGF on vascular stereomorphology in the context of angiogenic neo-arborisation after vascular induction.

The aim of this in vivo study was to gather quantitative information on the three-dimensional morphology of a new vascular network under the influence of angioactive growth factors. For this purpose, the arteriovenous loop model was used in 10 Lewis rats to generate a bioartificial vascular assembly by means of vascular induction. In this model, an isolated organoid is created in the medial thigh of the animal by methods of tissue engineering. A fibrin gel containing vascular endothelial growth factor (VEGF(165)) and basic fibroblastic growth factor (bFGF) was used as a matrix in the effect group (GF+). Fibrin matrices devoid of growth factors were used as controls (GF-). A microvascular replica of the organoid was created by means of corrosion casting and the network was investigated on stereo-paired images obtained by scanning electron microscopy. Vectors of intercapillary and interbranching distances as well as the diameter of the pores in the intussusceptive events diameter and the ratio of sprouting versus intussusceptive angiogenic events were compared in the two groups. The results were highly significant. In the GF+ group there were more profound three-dimensional morphological traits of angiogenesis, whereas advanced neovascularisation in the phase of remodelling was demonstrated by a higher incidence of intussusception, compared to control. These results illustrate the importance of morphological studies with focus on the generation of three-dimensional vascular networks.

De novo generation of an axially vascularized processed bovine cancellous-bone substitute in the sheep arteriovenous-loop model.

BACKGROUND/AIMS: The aim of this study was to generate an axially vascularized bone substitute. The arteriovenous (AV)-loop approach in a large-animal model was applied in order to induce axial vascularization in a clinically approved processed bovine cancellous bone (PBCB) matrix of significant volume with primary mechanical stability and to assess the course of increasing axial vascularization. METHODS: PBCB constructs were implanted into 13 merino sheep together with a microsurgically created AV loop in an isolation chamber. The vascularization process was monitored by sequential magnetic resonance imaging (MRI) scans. Explants were subjected to micro-computed tomography (micro-CT) analysis, histomorphometry and immunohistochemistry for CD31 and CD45. RESULTS: Increasing axial vascularization in PBCB constructs was quantified by histomorphometry and visualized by micro-CT scans. Intravital sequential MRI scans demonstrated a significant progressive increase in perfused volume within the matrices. Immunohistochemistry confirmed endothelial lining of newly formed vessels. CONCLUSION: This study demonstrates successful axial vascularization of a clinically approved, mechanically stable bone substitute with a significant volume by a microsurgical AV loop in a large-animal model. Thus microsurgical transplantation of a tissue-engineered, axially vascularized and mechanically stable bone substitute with clinically relevant dimensions may become clinically feasible in the future.

Regenerative medicine: then and now--an update of recent history into future possibilities.

The fields of tissue engineering (TE) and regenerative medicine (RegMed) are yet to bring about the anticipated therapeutic revolution. After two decades of extremely high expectations and often disappointing returns both in the medical as well as in the financial arena, this scientific field reflects the sense of a new era and suggests the feeling of making a fresh start although many scientists are probably seeking reorientation. Much of research was industry driven, so that especially in the aftermath of the recent financial meltdown in the last 2 years we have witnessed a biotech asset yard sale. Despite any monetary shortcomings, from a technological point of view there have been great leaps that are yet to find their way to the patient. RegMed is definitely bound to play a major role in our life because it embodies one of the primordial dreams of mankind, such as: everlasting youth, flying, remote communication and setting foot on the moon. The Journal of Cellular and Molecular Medicine has been at the frontier of these developments in TE and RegMed from its beginning and reflects recent scientific advances in both fields. Therefore this review tries to look at RegMed through the keyhole of history which might just be like looking 'back to the future'.

Evaluation of blood vessel ingrowth in fibrin gel subject to type and concentration of growth factors.

Our aim was to quantitatively assess the angiogenetic effects of VEGF and bFGF immobilized in a fibrin-based drug delivery system in a suitable subcutaneous rat model. After evaluation of a suitable implantation technique (6 rats), four teflon isolation chambers containing fibrin gel matrices were implanted subcutaneously in an upside-down fashion on the back of 30 Lewis rats. The matrices consisted of 500 microl fibrin gel with two different fibrinogen concentrations (10 mg/ml or 40 mg/ml fibrinogen) and 2 I.U./ml thrombin and contained VEGF and bFGF in five different concentrations (0 to 250 ng/ml each). At 3, 7 and 14 days after implantation, matrices were explanted and subjected to histological and morphometrical analysis. At 1 week, the volume of the fibrin clots was significantly smaller in the 100 and 250 ng/ml VEGF and bFGF groups in comparison to lower concentrated growth factors. At 1 and 2 weeks, the use of growth factors in low concentrations (25 ng/ml VEGF and bFGF) significantly increased the amount of fibrovascular tissue, average fraction of blood vessels and number of blood vessels at the matrix-host interface in comparison to growth factor-free controls. Higher concentrations were neither associated with further increase of tissue formation nor with increased sprouting of blood vessels in this model. This study demonstrates that fibrin gel-immobilized angioinductive growth factors efficiently stimulate generation of fibrovascular tissue and sprouting of blood vessels in a newly developed subcutaneous upside-down isolation chamber model with an optimum between 25 and 100 ng/ml.

Regression and persistence: remodelling in a tissue engineered axial vascular assembly.

In later stages of vasculoangiogenesis a vascular network is going through a metamorphosis for optimal perfusion and economy of energy. In this study we make a quantitative approach to phenomena of remodelling in a bioartificial neovascular network and suggest variance of calibre as a parameter of neovascular maturation. For this study, 18 male Lewis rats were subjected to the AV loop operation in combination with a hard porous biogenic matrix and an isolation chamber. The animals were allocated into three groups for different explantation intervals set to 2, 4 and 8 weeks, respectively. Collective attributes like vascular density, percent fractional area and variance of calibre were evaluated for a predefined region of interest (ROI). Late morphogenesis was evaluated by means of scanning electron microscopy. After the fourth week the absolute number of vessels within the ROI decreased (P < 0.03) whereas, on the contrary, the fractional area of all segments increased (P < 0.02). The variance in calibre was significantly increased in the 8-week group (P < 0.05). Lymphatic growth after week 4, early pericyte migration as well as intussusceptive angiogenesis were identified immunohistologically. Phenomena of remodelling were evaluated quantitatively in a neovascular network and variance could be proposed as a parameter of net vascular maturation.

[Prevascularisation strategies in tissue engineering]. / Prävaskularisationsstrategien im Tissue Engineering.

INTRODUCTION: Experiments on animals have underlined the importance of vascularisation for biointegration and functionality of any given tissue engineering device. The aim of this investigation was to dissect the angiogenetic process in the frame of axial neovascularisation of a xenogenic solid matrix. The ultimate goal of this series of studies is the application of cells onto a prevascularised matrix, with the ambition to enhance cell survival after transplantation in vivo. MATERIALS AND METHODS: We performed a study in the rat with different vascular configurations in an isolation chamber. A disc-formed biogenic hard matrix (9 x 5 mm) was encased into an isolation chamber made of Teflon. In group 1, an arteriovenous fistula (AV loop) between the femoral vessels was microsurgically constructed and was placed around the matrix (n = 15). In group 2, the vascular carrier had the form of an arteriovenous ligated pedicle (n = 15). Evaluation intervals were two, four and eight weeks after implantation. The modes of evaluation included histology, scanning electron microscopy of corrosion casts as well as intravital micro-magnetic resonance imaging (MRI). RESULTS: The arteriovenous loop as vascular carrier revealed a higher capacity for angiogenesis over the bundle configuration. The neo-fibrovascular tissue displayed minimal inflammatory elements but dense vascularisation. Scanning electron microscopy demonstrated a vivid angiogenesis with rapid evolution of the vascular bead into mature, hierarchically organised network. Micro-MRI could be used for serial investigation in terms of flow measurements and detection of thrombosis. DISCUSSION: The presence of a vascular bed prior to cell transplantation might protect against hypoxia-induced cellular death, especially at central portions of the matrix, and therefore ensure physiological function of the device. The generation of vascularised bioartificial tissue substitutes might offer new modalities of surgical reconstruction for use in reparative medicine.

The windblown hand - diagnosis, clinical picture and pathogenesis.

The syndrome of the windblown hand deformity is a complex constellation of malformations affecting not only the head and the feet but also the hands in a quite distinct manner. In the hand, it involves congenital bilateral flexion contracture with ulnar deviation of the metacarpophalangeal joints. The thumb is characteristically adducted (reaching the palm; "thumb-in-palm deformity") with flexion of the MP joint and hyperextension of the IP joint. The etiology is basically unknown. We present two theories based on knowledge derived from the disciplines of evolution biology and embryology. We believe that the atavistic appearance of phylogenetically primitive muscle groups in conjunction with an impaired rotation of the extremities during embryological development account for this malformation syndrome.

[Breast-reduction surgery--a long-term survey of indications and outcomes]. / Leichter--freier--unbeschwerter. Das neue Leben mit der kleineren Brust.

Between 1986 and 2003, breast-reduction surgery was performed in a total of 814 women. The indication was established on the basis of physical complaints, chronic back pain, stiff neck or recurrent intertrigo in the foldbeneath the breasts. A proportion of the patients were interviewed postoperatively using a questionnaire, to determine the impact of the operation on their quality of life. 91% of those surveyed reported a postoperative improvement in the perception of their own body, and 80% were satisfied with the reduced size of their breasts. In conclusion, in the hands of an experienced breast surgeon, breast-reduction surgery for the proper indication results in a reliable and safe diminishment in breast size and tightening of slack tissue, leading to a significant enhancement in the patient's quality of life.

[Bioreactors in regenerative medicine--from a technical device to a reconstructive alternative?]. / Bioreaktoren in der Regenerativen Medizin--Vom technischen Apparat zur rekonstruktiven Alternative?

With the advent of the era of tissue engineering (TE), experimental settings have been developed that allow for a defined environment with optimised cell growth conditions and/or the production of specific substitutes. These isolated systems have been termed "bioreactors". By translating the principles of bioreactors into an in vivo context, advances in biomaterial sciences and cell biology have been merged into an integrative research concept. Even today, in the age of regenerative Medicine (RegMed) the transfer of experimental in vitro findings into a clinical in vivo approach still remains a vast challenge. In order to fulfil these specific requirements bioreactors had to be defined anew. Latest advances in areas like reconstructive medicine (the arteriovenous loop as a means of organogenesis) or modern wound management (topical negative pressure therapy as a perfusion bioreactor) give new impulses towards the translation of Reg-Med concepts into the clinical routine.

The venous graft as an effector of early angiogenesis in a fibrin matrix.

The arteriovenous loop (AV loop) model is gaining importance as a means of initiating and sustaining perfusion in tissue engineering constructs in vivo. This study represents an attempt to dissect the morphology of early arterialization and angiogenesis in the AV loop in a fibrin matrix with special focus on the interpositional venous graft (IVG) segment. An AV loop was constructed in 30 rats using the femoral vessels and an IVG. The AV loop was encased in an isolation chamber filled with a fibrin matrix. Evaluation methods included scanning electron microscopy (SEM) of corrosion casts, immune histology and micro magnetic resonance angiography (MRA). Direct luminal neovascular sprouting was evident between day 10 and day 14 from the vein and the IVG but not from the arterial segment. Arterialization of the IVG manifested itself on the corrosion casts as a gradual reduction in luminal caliber with onset after day 7. Microdissection of the microvascular replicas could demonstrate for the first time the presence of direct luminal sprouts from the IVG. MRA was used to display the shunt pattern of perfusion in the patent AV loop. From the three segments of the vascular axis in the AV loop the IVG is the most versatile for applications in the clinical as well as the experimental setting. Kinetics of angiogenesis warrant further investigation in the IVG.

Carpal tunnel syndrome in young adults--an ultrasonographic and neurophysiological study.

BACKGROUND: The carpal tunnel syndrome (CTS) is by far the most frequent compression neuropathy and encompasses 45% of all non-traumatic nerve lesions. Women are affected twice as often as men and manifestation usually occurs over the age of 30; 76% of all patients become symptomatic between the age of 40-70 years. In young adults typical diagnostic clues suggestive of a CTS, may be absent leading to misdiagnosis and late treatment. PATIENTS AND METHODS: 30 patients suffering from CTS were subdivided in 2 groups according to age. Patients under the age of 35 were allocated in group A whereas patients over 35 years old were included in group B. The two groups were compared to each other in terms of demographics, clinical signs, electrodiagnosis and ultrasonography of the carpal tunnel. All patients were subjected to a decompression procedure of the median nerve and postoperative alleviation of the symptoms was considered as confirmation of the presence of a CTS. Two characteristic clinical cases of young adults suffering from CTS are demonstrated. RESULTS: The female to male ratio was higher in group A (7:1) than in group B (3:1). Only two (25%) of the patients in group A had a positive Tinel sign but in 7 patients (87.5%) the Phalen test could be readily evoked. In group B a Tinel sign was present in 45% whereas a positive Phalen test was present in 86%. A positive Tinel sign was associated with a distal motor latency beyond 4 ms in the two patients from group A. A distal motor latency was present in 95.2% of the patients in group B. In ultrasound there was an average decrease of 0.557 mm in the thickness of the median nerve throughout its course in the carpal tunnel in group B. In group A, ultrasonography revealed no significant changes in nerve diameter. DISCUSSION: The value of electrophysiological studies as a diagnostic tool in CTS still remains controversial. In young adults phenomena leading to impaired nerve conduction like axonal demyelination of the median nerve might occur later due to a higher regenerative potential. However, symptomatic patients may be strongly hindered in their everyday activities and occupation and should be readily referred to a hand specialist in spite of a negative work-up.

Autonomously vascularized cellular constructs in tissue engineering: opening a new perspective for biomedical science.

In tissue engineering cell cultures play a crucial role besides the matrix materials for the end of substituting lost tissue functions. The cell itself is situated at the cross-roads leading to different orders of scale, from molecule to organism and different levels of function, from biochemistry to macrophysiology. Extensive in vitro investigations have dissected a vast amount of cellular phenomena and the role of a number of bioactive substances has been elucidated in the past. Further, recombinant DNA technologies allow modulation of the expression profiles of virtually all kinds of cells. However, issues of vascularization in vivo limit transferability of these observations and restrict upscaling into clinical applications. Novel in vivo models of vascularization have evolved inspired from reconstructive microsurgical concepts and they encompass axial neovascularization by means of vascular induction. This work represents a brief description of latest developments and potential applications of neovascularization and angiogenesis in tissue engineering.

Tissue engineering of bone: the reconstructive surgeon's point of view.

Bone defects represent a medical and socioeconomic challenge. Different types of biomaterials are applied for reconstructive indications and receive rising interest. However, autologous bone grafts are still considered as the gold standard for reconstruction of extended bone defects. The generation of bioartificial bone tissues may help to overcome the problems related to donor site morbidity and size limitations. Tissue engineering is, according to its historic definition, an "interdisciplinary field that applies the principles of engineering and the life sciences toward the development of biological substitutes that restore, maintain, or improve tissue function". It is based on the understanding of tissue formation and regeneration and aims to rather grow new functional tissues than to build new spare parts. While reconstruction of small to moderate sized bone defects using engineered bone tissues is technically feasible, and some of the currently developed concepts may represent alternatives to autologous bone grafts for certain clinical conditions, the reconstruction of large-volume defects remains challenging. Therefore vascularization concepts gain on interest and the combination of tissue engineering approaches with flap prefabrication techniques may eventually allow application of bone-tissue substitutes grown in vivo with the advantage of minimal donor site morbidity as compared to conventional vascularized bone grafts. The scope of this review is the introduction of basic principles and different components of engineered bioartificial bone tissues with a strong focus on clinical applications in reconstructive surgery. Concepts for the induction of axial vascularization in engineered bone tissues as well as potential clinical applications are discussed in detail.

[Modified gloving technique for vacuum therapy in the hand]. / Modifizierte "Handschuhtechnik" zur Vakuumtherapie an der Hand.

INTRODUCTION: During the last 10 years sub-atmospheric pressure dressings (Topical Negative Pressure Therapy = TNP) has become a well accepted standard therapeutic modality in the management of acute and chronic wounds. However, in the hand and the fingers TNP treatment is not as an established method as elsewhere in the body. This is mostly due to difficulties in the technique of sealing wounds near the interdigital folds, especially when the interdigital folds are affected themselves. MATERIALS AND METHODS: Over a period of 36 months we treated 9 extensive open wounds of the hand with the TNP dressing procedure. To optimize the effective sealing procedure several methods of closure were applied: sterile vinyl gloves, split V.A.C. gel-straps and the "Sandwich"-principle. RESULTS: In all cases we were able to achieve a tight vacuum sealing with the use of our vinyl or latex free surgical glove as an aid to cover the interdigital spaces. There was a rapid and complete remission of the symptoms in terms of edema, redness, tenderness and range of motion. The indication spectrum encompassed infection control, temporary coverage of exposed bone or tendons, intermittent irrigation with local antiseptics as well as wound preconditioning before skin grafting or flap coverage. For the definite closure or coverage a second operation was necessary in all cases. CONCLUSIONS: The TNP-dressing can be securely achieved even in wounds encompassing the interdigital folds or adjacent to the in digital spaces with a modified surgical gloving technique to aid the standard sealing foil. It is a suitable principle for the management of severe hand infections with a reduction of clinical symptoms, reduction of the frequency of dressing changes compared to open approaches and a significant amelioration in the quality of life both for patients and health care personnel.

[Vacuum-assisted closure as temporary coverage in the "problem zone hand"]. / Die Vakuumtherapie als temporäre Deckung an der "Problemzone Hand".

INTRODUCTION: Vacuum therapy of hand defect problematic due to the interdigital folds and in particular due to the small surface. As a temporary cover in staged procedures, e. g. following excision of tumor-suspicious skin alterations at the hand, and/or at the fingers, the application of vacuum therapy results not only in a sterile, temporary cover of the wound, furthermore it promotes wound conditioning and reduction of the wound area as well as lymph and edema reduction. PATIENTS AND METHODS: A 75 year old female patient presented herself with a skin alteration at the right dorsal ring finger and the tentative diagnosis of a Morbus Bowen like lesion was raised. Due to the unclear histological status, a staged procedure with excision of the tumour and temporary defect coverage by means of vacuum therapy until final histological evaluation was indicated. We accomplished the radical excision of the unclear skin tumour at the proximal phalanx under preservation of the synovial sheath of the tendon. Subsequently, the wound was temporarily closed with vacuum therapy. Histological examination revealed the complete removal of a solar keratosis. Finally, after 7 days of vacuum therapy as a sterile dressing, with obvious reduction of wound area and simultaneous decrease of edema, the defect was finally covered with a reversed cross finger flap from the adjacent middle finger. The donor site was covered with a full thickness skin graft harvested from the right forearm. The further course was without complication after flap dissection and complete healing. DISCUSSION: Vacuum therapy represents a usefull procedure for temporary wound coverage. Especially at hand defects, when nerves, tendons or bones following trauma or staged procedures are exposed, vacuum therapy can be successfully applied utilizing the special surgical "hand glove" technique.

A new approach to tissue engineering of vascularized skeletal muscle.

Tissue Engineering of skeletal muscle tissue still remains a major challenge. Every neo-tissue construct of clinically relevant dimensions is highly dependent on an intrinsic vascularisation overcoming the limitations of diffusion conditioned survival. Approaches incorporating the arteriovenous-loop model might bring further advances to the generation of vascularised skeletal muscle tissue. In this study 12 syngeneic rats received transplantaion of carboxy-fluorescine diacetate-succinimidyl ester (CFDA)-labelled, expanded primary myoblasts into a previously vascularised fibrin matrix, containing a microsurgically created AV loop. As control cells were injected into fibrin-matrices without AV-loops. Intra-arterial ink injection followed by explantation was performed 2, 4 and 8 weeks after cell implantation. Specimens were evaluated for CFDA, MyoD and DAPI staining, as well as for mRNA expression of muscle specific genes. Results showed enhanced fibrin resorption in dependence of AV loop presence. Transplanted myoblasts could be detected in the AV loop group even after 8 weeks by CFDA-fluorescence, still showing positive MyoD staining. RT-PCR revealed gene expression of MEF-2 and desmin after 4 weeks on the AVloop side, whereas expression analysis of myogenin and MHC(embryo) was negative. So far myoblast injection in the microsurgical rat AV loop model enhances survival of the cells, keeping their myogenic phenotype, within pre-vascularised fibrin matrices. Probably due to the lack of potent myogenic stimuli and additionally the rapid resorption of the fibrin matrix, no formation of skeletal muscle-like tissue could be observed. Thus further studies focussing on long term stability of the matrix and the incorporation of neural stimuli will be necessary for generation of vascularised skeletal muscle tissue.

The composite vastus medialis-patellar complex osseomuscular flap as a salvage procedure after complex trauma of the knee--an anatomical study and clinical application.

BACKGROUND: In the setting of severe perigenicular trauma or complicated endoprosthetic knee surgery, primary knee fusion may be the last resort for salvage of the limp. In this case, the patella looses its destination as an anterior knee stabilizer and can become a substantial donor of bone substance, especially if osseous defects are involved. PATIENTS AND METHODS: 12 formalin fixated cadavers were studied in terms of vascular anatomy, pedicle reliability, arc of rotation and their relation to sex, age, and height. Moreover, the operation was performed on a suitable patient. RESULTS: The quadriceps with the vastus medialis and the patella can be raised from the tibial tuberosity up to the entrance of the osteoarticular branch of the superficial femoral artery into the vastus medialis muscle ca 16 cm (15-19 cm) from the inferior patellar pole. This distance correlated well to the overall height of the cadavers (P=0.009). The vascular prerequisites were always present. In the clinical case, there was a favorable outcome with knee fusion after 4 months, despite of the lateral condylar defect. DISCUSSION: The composite vastus medialis-patellar complex osseomuscular flap can be safely used as a source of vascularized femoral condyle substitute in the setting of primary knee fusion.

[Plastic surgery coverage in the dorsal trunk area]. / Plastische Deckung im dorsalen Rumpfbereich.

Defects of the trunk are major plastic surgical challenges. In the posterior trunk these defects are congenital or acquired. Most of the defects are due to major tumor resections, rarely after trauma or burns. With regional or local flaps most of the defects can be covered adequately. Presently musculocutaneous and perforator flaps are favoured. An interdisciplinary cooperation is mandatory for a successful result.

[Use of vacuum therapy during defect coverage of the upper extremity with microsurgically grafted arterialized venous flaps]. / Der Einsatz der Vakuumtherapie bei der Defektdeckung an der oberen Extremität mit freien mikrochirurgischen arterialisierten venösen Lappen.

Arterialized venous soft tissue flaps are defined as tissue portions usually harvested from the anterior and distal third of the forearm over a venous pathway to be transplanted to another recipient area. Basically, they are composed of skin, subcutaneous tissue, and subdermal venous plexus. At the recipient site flaps are perfused by a nourishing artery while the other veins are connected to one or more regional veins. The retrograde blood perfusion often results in edema, temporary epidermolysis, or even some degree of skin necrosis that disappear during subsequent treatment. To circumvent these drawbacks grafting of arterialized flaps was combined with local application of vacuum to overcome the venous initial stasis, resulting in improved healing and survival of these flaps.