Polytetrafluoroethylene (PTFE) as a Suture Material in Tendon Surgery.

With the evolution of suture materials, there has been a change in paradigms in primary and secondary tendon repair. Improved mechanical properties allow more aggressive rehabilitation and earlier recovery. However, for the repair to hold against higher mechanical demands, more advanced suturing and knotting techniques must be assessed in combination with those materials. In this protocol, the use of polytetrafluoroethylene (PTFE) as a suture material in combination with different repair techniques was investigated. In the first part of the protocol, both linear tension strength and elongation of knotted against not-knotted strands of three different materials used in flexor tendon repair were evaluated. The three different materials are polypropylene (PPL), ultra-high molecular weight polyethylene with a braided jacket of polyester (UHMWPE), and polytetrafluoroethylene (PTFE). In the next part (ex vivo experiments with cadaveric flexor tendons), the behavior of PTFE using different suture techniques was assessed and compared with PPL and UHMWPE. This experiment is comprised of four steps: harvesting of the flexor tendons from fresh cadaveric hands, transection of the tendons in a standardized manner, tendon repair by four different techniques, mounting, and measurement of the tendon repairs on a standard linear dynamometer. The UHMWPE and PTFE showed comparable mechanical properties and were significantly superior to PPL in terms of linear traction strength. Repairs with four- and six-strand techniques proved stronger than two-strand techniques. Handling and knotting of PTFE are a challenge due to very low surface friction but fastening of the four- or six-strand repair is comparatively easy to achieve. Surgeons routinely use PTFE suture material in cardiovascular surgery and breast surgery. The PTFE strands are suitable for use in tendon surgery, providing a robust tendon repair so that early active motion regimens for rehabilitation can be applied.

Individualized Wound Closure-Mechanical Properties of Suture Materials.

Wound closure is a key element of any procedure, especially aesthetic and reconstructive plastic surgery. Therefore, over the last decades, several devices have been developed in order to assist surgeons in achieving better results while saving valuable time. In this work, we give a concise review of the literature and present a biomechanical study of different suturing materials under mechanical load mimicking handling in the operating theatre. Nine different suture products, all of the same USP size (4-0), were subjected to a standardized crushing load by means of a needle holder. All materials were subjected to 0, 1, 3 and 5 crushing load cycles, respectively. The linear tensile strength was measured by means of a universal testing device. Attenuation of tensile strength was evaluated between materials and between crush cycles. In the pooled analysis, the linear tensile strength of the suture materials deteriorated significantly with every cycle (p < 0.0001). The suture materials displayed different initial tensile strengths (in descending order: polyglecaprone, polyglactin, polydioxanone, polyamid, polypropylene). In comparison, materials performed variably in terms of resistance to crush loading. The findings were statistically significant. The reconstructive surgeon has to be flexible and tailor wound closure techniques and materials to the individual patient, procedure and tissue demands; therefore, profound knowledge of the physical properties of the suture strands used is of paramount importance. The crushing load on suture materials during surgery can be detrimental for initial and long-term wound repair strength. As well as the standard wound closure methods (sutures, staples and adhesive strips), there are promising novel devices.

Bone Allograft and Locking Plate for Severe Proximal Humeral Fractures: Early and Late Outcomes.

BACKGROUND Early failure of osteosyntheses is common even with use of locking plates. In patients with comminuted fractures and epiphyseal osseous defects, we performed a series of osteosyntheses by locking plate in combination with an allograft bone augmentation. Because of encouraging short-term results in the literature, we assumed that the method could be a potential alternative to a reverse shoulder prosthesis. MATERIAL AND METHODS Twenty-six patients with a dislocated proximal humeral fracture (Neer IV/V/VI) were studied. A lyophilized allogeneic bone graft was used to reinforce the humeral head fragments before locking plate osteosynthesis. The outcomes of fractures were assessed with Disabilities of the Arm, Shoulder and Hand (DASH) and Constant-Murley (Constant) scores, range of motion, a visual analog scale, and with radiological testing. The Constant-Murley scores were the endpoint of our study. RESULTS The Neer classification of the fractures was type IV in 4 patients, type V in 20 patients, and type VI in 2 patients. The mean DASH score was 52.85 (range, 4.17-79.3) and the mean Constant score was 39.26 (range, 17-88). We observed late necrosis of the humeral head in 15 of 24 patients (62.5%), although early radiological follow-up showed that the humeral head had been anatomically reconstructed. CONCLUSIONS Long-term follow-up demonstrated inferior functional results, as displayed by poor Constant scores. There was a high incidence of necrosis, in spite of initial anatomical reconstruction. Biointegration of the allogeneic bone graft and revascularization of the humeral head fragments could be impaired in geriatric patients who have gross dislocation. Therefore, augmentation of the humeral head with allogeneic bone grafts cannot be recommended in these patients.

Polytetrafluoroethylene (PTFE) suture vs fiberwire and polypropylene in flexor tendon repair.

BACKGROUND: In this study, we evaluate the value of novel suture material based on monofilamentous-extruded polyfluoroethylene (PTFE) compared to polypropylene (PPL) and Fiberwire (FW). MATERIALS AND METHODS: 60 flexor tendons were harvested from fresh cadaveric upper extremities. 4-0 sutures strands were used in the PPL, FW and PTFE group. Knotting properties and mechanical characteristics of the suture materials were evaluated. A 4-strand locked cruciate (Adelaide) or a 6-strand (M-Tang) suture technique was applied as core sutures for a tendon repair. Two-way ANOVA tests were performed with the Bonferroni correction. RESULTS: Stable knotting was achieved with 5 throws with the PPL material, 7 throws for FW and 9 throws for PTFE. In the PPL group, linear tensile strength was 45.92 ± 12.53 N, in the FW group 80.11 ± 18.34 N and in the PTFE group 76.16 ± 29.10 N. FW and PTFE are significantly stronger than PPL but show no significant difference among each other. Similar results were obtained in the subgroup comparisons for different repair techniques. The Adelaide and the M-Tang knotting technique showed no significant difference. CONCLUSION: Fiberwire showed superior handling and knotting properties in comparison to PTFE. However, PTFE allows easier approximation of the stumps. In both, M-Tang and Adelaide repairs, PTFE was equal to FW in terms of repair strength. Both PTFE and FW provide for a robust tendon repair so that early active motion regimens for rehabilitation can be applied.

Can systemically administered antibiotics be detected in wound tissues and surfaces under negative pressure wound therapy?

In this study, we evaluated a new aspect of negative pressure wound therapy (NPWT) as an analytical tool for pharmacokinetic studies. Twenty-one patients with soft tissue defects scheduled to receive NPWT were included in this study. Concomitant to NPWT, all patients received intravenous moxifloxacin (MX). At different time intervals, blood plasma levels of MX were sampled and compared with synchronous concentrations of MX in the exudate obtained from the NPWT drainage system. Serial measurements were performed upon initiation of the therapy as well as in the steady state (after 5 days). At steady state, wound tissue was obtained intraoperatively. High-performance liquid-chromatography (HPLC) was used for analysis. At 1 hour post-administration, the exudate/plasma levels (mg/L) were 1.92/3.07; at 12 hours, 0.80/1.14; at 24 hours, 0.26/0.43; and at 120 hours (steady state), 0.42/0.47. There was a correlation between exudate and plasma levels reaching approximately 0.75. Until now, methods for pharmacokinetic studies concerning interstitial fluid are difficult to apply in the clinical context. The presented method showed limitations, but we believe that, after methodological improvements, measurements of substances in the interstitial fluid by means of NPWT are feasible.

Flexor tendon repair with a polytetrafluoroethylene (PTFE) suture material.

BACKGROUND: There is a consensus that after a flexor tendon repair an aggressive rehabilitation protocol with early active motion can improve functional outcome, provided that the combination of material and suturing technique can meet the higher biomechanic demands. Bearing this in mind we evaluated a polytetrafluoroethylene (PTFE) suture (SERAMON®, Serag-Wiessner) as a possible material for flexor tendon repair. MATERIALS AND METHODS: 40 flexor tendons were harvested from fresh cadaveric upper extremities. 3-0 and 5-0 strands were used both in the polypropylene (PPL) as well as in the PTFE group. In the first phase of the study, we evaluated knotting properties and mechanical characteristics of the suture materials themselves. In the second phase, a 2-strand Kirchmayr-Kessler suture technique was applied for a core suture of a flexor tendon (n = 16). In the third phase, we performed a tendon repair including an epitendinous running suture with 5-0 PPL or 5-0 PTFE material (n = 22). One way ANOVA tests were performed. RESULTS: The linear loading strength of single strand knotted PPL 3-0 was 19.87 ± 0.59 N. The linear loading strength of knotted PTFE 3-0 was 32.47 ± 1.67 N. For PPL 3-0 maximum linear strength was achieved with five knots, for PTFE 3-0 with eight knots. When a Kirchmayr-Kessler core-only repair was performed, then in the PPL group the loading strength of the repaired tendon was 30.74 ± 9.77 N. In the PTFE group the loading strength was 23.74 ± 5.6 N (p = 0.10). However, all repairs in the PTFE group failed due to cheese wiring. When a Kirchmayr-Kessler core and epitendinous repair technique was used, then in the PPL group the loading strength of the repaired tendon was 49.90 ± 16.05 N. In the PTFE group the loading strength was 73.41 ± 19.81 N (p = 0.006). CONCLUSION: PTFE demonstrates superior strength properties in comparison to PPL for flexor tendon repairs. However, standard 2 strand techniques have proved inadequate to bear the higher biomechanic demands.

[The Value of Endoscopic Decompression in Surgical Therapy of Carpal Tunnel Syndrome]. / Stellenwert der endoskopischen Karpaltunneldekompression.

Carpal tunnel syndrome is one of the most common diseases in hand surgery. The gold standard in therapy is the surgical release of the carpal tunnel. We provide a brief update on the relevant pathogenesis, diagnosis and therapy and discuss questions related to minimal invasive decompression of the median nerve. Together with a review of the current literature, we report on our experiences in minimally invasive carpal tunnel release via a monoportal endoscopic access in over 700 cases. In conclusion, the endoscopic technique provides superior convalescence and patient safety is comparable to open methods. In addition, advantages and disadvantages of the various techniques are discussed.

Flow Induced Microvascular Network Formation of Therapeutic Relevant Arteriovenous (AV) Loop-Based Constructs in Response to Ionizing Radiation.

BACKGROUND The arteriovenous (AV) loop model enables axial vascularization to gain a functional microcirculatory system in tissue engineering constructs in vivo. These constructs might replace surgical flaps for the treatment of complex wounds in the future. Today, free flaps are often exposed to high-dose radiation after defect coverage, according to guideline-oriented treatment plans. Vascular response of AV loop-based constructs has not been evaluated after radiation, although it is of particular importance. It is further unclear whether the interposed venous AV loop graft is crucial for the induction of angiogenesis. MATERIAL AND METHODS We exposed the grafted vein to a single radiation dose of 2 Gy prior to loop construction to alter intrinsic and angio-inductive properties specifically within the graft. Vessel loops were embedded in a fibrin-filled chamber for 15 days and radiation-induced effects on flow-mediated vascularization were assessed by micro-CT and two-dimensional histological analysis. RESULTS Vessel amount was significantly impaired when an irradiated vein graft was used for AV loop construction. However, vessel growth and differentiation were still present. In contrast to vessel density, which was homogeneously diminished in constructs containing irradiated veins, vessel diameter was primarily decreased in the more peripheral regions. CONCLUSIONS Vascular luminal sprouts were significantly diminished in irradiated venous grafts, suggesting that the interposing vein constitutes a vital part of the AV loop model and is essential to initiate flow-mediate angiogenesis. These results add to the current understanding of AV loop-based neovascularization and suggest clinical implications for patients requiring combined AV loop-based tissue transfer and adjuvant radiotherapy.

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 pathological situations such as cancer, vascularization plays a leading role in disease progression. Consequently, there is a strong need for a standardized and well-characterized in vivo model in order to elucidate the mechanisms of neovascularization and develop different vascularization approaches for tissue engineering and regenerative medicine. We describe a microsurgical approach for a small animal model for induction of a vascular axis consisting of a vein and artery that are anastomosed to an arteriovenous (AV) loop. The AV loop is transferred to an enclosed implantation chamber to create an isolated microenvironment in vivo, which is connected to the living organism only by means of the vascular axis. Using 3D imaging (MRI, micro-CT) and immunohistology, the growing vasculature can be visualized over time. By implanting different cells, growth factors and matrices, their function in blood vessel network formation can be analyzed without any disturbing influences from the surroundings in a well controllable environment. In addition to angiogenesis and antiangiogenesis studies, the AV loop model is also perfectly suited for engineering vascularized tissues. After a certain prevascularization time, the generated tissues can be transplanted into the defect site and microsurgically connected to the local vessels, thereby ensuring immediate blood supply and integration of the engineered tissue. By varying the matrices, cells, growth factors and chamber architecture, it is possible to generate various tissues, which can then be tailored to the individual patient's needs.

Flow increase is decisive to initiate angiogenesis in veins exposed to altered hemodynamics.

Exposing a vein to altered hemodynamics by creating an arteriovenous (AV) shunt evokes considerable vessel formation that may be of therapeutic potential. However, it is unclear whether the introduction of oscillatory flow and/or flow increase is decisive. To distinguish between these mechanical stimuli we grafted a femoral vein into the arterial flow pathway of the contralateral limb in rats creating an arterioarterial (AA) loop (n = 7). Alternatively, we connected the femoral artery and vein using the vein graft, whereby we created an AV-loop (n = 27). Vessel loops were embedded in a fibrin filled chamber and blood flow was measured by means of flow probes immediately after surgery (day 0) and 15 days after loop creation. On day 15, animals were sacrificed and angiogenesis was evaluated using µCT and histological analysis. Mean flow increased from 0.5 to 2.4 mL/min and was elevated throughout the cardiac cycle at day 0 in AV-loops whereas, as expected, it remained unchanged in AA-loops. Flow in AV-loops decreased with time, and was at day 15 not different from untreated femoral vessels or AA-loop grafts. Pulsatile flow oscillations were similar in AV-and AA-loops at day 0. The flow amplitude amounted to ~1.3 mL/min which was comparable to values in untreated arteries. Flow amplitude remained constant in AA-loops, whereas it decreased in AV-loops (day 15: 0.4 mL/min). A large number of newly formed vessels were present in AV-loops at day 15 arising from the grafted vein. In marked contrast, angiogenesis originating from the grafted vein was absent in AA-loops. We conclude that exposure to substantially increased flow is required to initiate angiogenesis in grafted veins, whereas selective enhancement of pulsatile flow is unable to do so. This suggests that indeed flow and most likely wall shear stress is decisive to initiate formation of vessels in this hemodynamically driven angiogenesis model.

A histopathologic and immunohistochemical study on liquification of human adipose tissue ex vivo.

BACKGROUND: This preliminary ex vivo study aimed to clarify the pathophysiologic mechanisms of fat tissue depletion by subcutaneous drug application. Therefore, the lipolytic effects of phosphatidylcholine plus deoxycholate (Lipostabil) (L) and of deoxycholate (DC) alone were compared with those of sodium chloride (NaCl) and hydrogen peroxide (H2O2) as control agents. The study enrolled 10 patients receiving abdominoplasty. The treatment periods for each sample and solution were 1, 3, 5, and 7 h. The samples were analyzed morphologically using hematoxylin-eosin (H&E) staining and also immunohistochemically using Caspase 3 and tumor necrosis factor (TNF)-alpha. Morphologic changes were seen best after 5 h of application time. Except for NaCl, all the samples in the H&E staining showed marked damage of adipocyte cell membranes, with the greatest disruption of normal cell architecture after hydrogen peroxide (H2O2) application. Immunohistochemistry using TNF-alpha showed positive results for the deoxycholate and Lipostabil samples and highly positive results for the H2O2 sample. Data from this study indicate that Lipostabil and deoxycholate induce pathways of cell necrosis involving TNF-alpha. These short-term experiments indicate that Lipostabil affects fat tissue in the way of a chemical-toxic destruction rather than via a physiologically induced, programmed cell death.

Vital staining of blood vessels and bile ducts with carboxyfluorescein diacetate succinimidyl ester: a novel tool for isolation of cholangiocytes.

BACKGROUND AND AIM: Current methods for visualization of the blood vasculature, biliary tree and for isolation of vital cholangiocytes are afflicted with a plethora of technical difficulties, especially in mice. In this project, we propose a novel, reliable and straightforward alternative technique for histological demonstration of blood- and biliary systems and derivation of vital cholangiocytes. METHODS: Intravital retrograde perfusion of bile ducts was performed in twenty wild type mice. Liver and gallbladder were exposed by median laparotomy. Using a venous catheter, the gallbladder was cannulated, a few millimeters of the liver edge were cropped to allow free outflow of the perfusate, and carboxyfluorescein diacetate succinimidyl ester (CFDA-SE) solution was retrogradely infused. Thereafter, formaldehyde solution was either injected through the same catheter, or the liver was immediately dissociated into a single-cell suspension for FACS-analysis. Intravital perfusion of the vascular system was performed in ten Lewis rats by direct intra-arterial injection of CFDA-SE into the abdominal aorta. The specificity and sensitivity of CFDA-SE labeling was controlled using Indian ink or cytokeratin 19 immunohistochemistry respectively. RESULTS: Upon histomorphological analysis of cryo- and paraffin sections, strong fluorescence was noted in large and small bile ducts throughout the entire liver and in the vascular system after infusion of the CFDA-SE solution. In preliminary FACS-experiments, we succeeded in separating cholangiocytes based on combined CFDA-SE-staining and cell size. CONCLUSIONS: Visualization of liver architecture and the isolation of cholangiocytes is feasible using a fast and cost-effective method of retrograde perfusion and vital fluorescent labeling of mouse bile duct epithelium and vascular endothelium with CFDA-SE.

Composition of fibrin glues significantly influences axial vascularization and degradation in isolation chamber model.

In this study, different fibrin sealants with varying concentrations of the fibrin components were evaluated in terms of matrix degradation and vascularization in the arteriovenous loop (AVL) model of the rat. An AVL was placed in a Teflon isolation chamber filled with 500 µl fibrin gel. The matrix was composed of commercially available fibrin gels, namely Beriplast (Behring GmbH, Marburg, Germany) (group A), Evicel (Omrix Biopharmaceuticals S.A., Somerville, New Jersey, USA) (group B), Tisseel VH S/D (Baxter, Vienna, Austria) with a thrombin concentration of 4 IU/ml and a fibrinogen concentration of 80 mg/ml [Tisseel S F80 (Baxter), group C] and with an fibrinogen concentration of 20 mg/ml [Tisseel S F20 (Baxter), group D]. After 2 and 4 weeks, five constructs per group and time point were investigated using micro-computed tomography, and histological and morphometrical analysis techniques. The aprotinin, factor XIII and thrombin concentration did not affect the degree of clot degradation. An inverse relationship was found between fibrin matrix degradation and sprouting of blood vessels. By reducing the fibrinogen concentration in group D, a significantly decreased construct weight and an increased generation of vascularized connective tissue were detected. There was an inverse relationship between matrix degradation and vascularization detectable. Fibrinogen as the major matrix component showed a significant impact on the matrix properties. Alteration of fibrin gel properties might optimize formation of blood vessels.

Tissue engineering and regenerative medicine -where do we stand?

Tissue Engineering (TE) in the context of Regenerative Medicine (RM) has been hailed for many years as one of the most important topics in medicine in the twenty-first century. While the first clinically relevant TE efforts were mainly concerned with the generation of bioengineered skin substitutes, subsequently TE applications have been continuously extended to a wide variety of tissues and organs. The advent of either embryonic or mesenchymal adult stem-cell technology has fostered many of the efforts to combine this promising tool with TE approaches and has merged the field into the term Regenerative Medicine. As a typical example in translational medicine, the discovery of a new type of cells called Telocytes that have been described in many organs and have been detected by electron microscopy opens another gate to RM. Besides cell-therapy strategies, the application of gene therapy combined with TE has been investigated to generate tissues and organs. The vascularization of constructs plays a crucial role besides the matrix and cell substitutes. Therefore, novel in vivo models of vascularization have evolved allowing axial vascularization with subsequent transplantation of constructs. This article is intended to give an overview over some of the most recent developments and possible applications in RM through the perspective of TE achievements and cellular research. The synthesis of TE with innovative methods of molecular biology and stem-cell technology appears to be very promising.

Combination of extrinsic and intrinsic pathways significantly accelerates axial vascularization of bioartificial tissues.

BACKGROUND: In this study, the authors present a modification of the arteriovenous loop model that combines extrinsic and intrinsic vascularization modes to enhance vascularization of bioartificial matrices. METHODS: An arteriovenous loop was created in the medial thighs of 24 rats. The loop was placed in a newly developed titanium chamber, which was fabricated with an electron beam melting facility, and was embedded in a hydroxyapatite/ß-tricalcium phosphate/fibrin matrix. At the explantation time points (2, 4, 6, and 8 weeks), constructs were perfused by differently colored dyes to determine the amount of tissue vascularized by either the intrinsic or the extrinsic vascular pathway. Specimens were investigated by means of micro-computed tomography and histologic and morphometric analysis. RESULTS: Although there was an equal number of blood vessels originating from the center and the periphery, 83 percent of all vessels displayed a connection to the arteriovenous loop already at 2 weeks. There was a continuous increase of the relative proportion of vessels connected to the arteriovenous loop over time detectable. At 8 weeks, communications between the newly formed vessels and the arteriovenous loop were visible in 97 percent of all vessels. CONCLUSIONS: This study demonstrates for the first time the enhancement of angiogenesis in an axially vascularized tissue by an additional extrinsic vascular pathway. By 2 weeks, both pathways showed connections, allowing transplantation of the entire construct using the arteriovenous loop pedicle. This approach will allow for reduction of the time interval between arteriovenous loop implantation and transplantation into the defect site and limitation of operative interventions.

Factors influencing successful outcome in the arteriovenous loop model: a retrospective study of 612 loop operations.

The arteriovenous (AV) loop is a complex model and requires advanced microsurgical skills. After several years of studies including characterization of the physiological and molecular phenomena behind the process of neovascularization, we would like to evaluate our experience from a microsurgical point of view. The AV loop operation was performed in 612 male Lewis rats. Species, surgeon, previous microsurgical skills, solid or soft consistency of matrix, weeks from implantation to explantation, experience, and practice of surgeon as well length of operation and outcome (patent versus thrombosed) upon evaluation were analyzed for each loop operation. Previous microsurgical skills, matrix, and explantation interval were not significant predictors. There was a significant correlation of experience, practice, and individual performance of microsurgical technicians with the rate of thrombosis and duration of the procedure. Experimental microsurgical operations on animals are best performed by especially trained technicians, rather than microsurgeons working parallel to their clinical activities. The AV loop is an excellent model in the arena of translational technologies, but due to its complexity is unsuitable for screening purposes. Experience and practice are both significant predictors of successful outcome. Possible applications of the model for research purposes are discussed.

Acute postoperative infection with Aeromonas hydrophila after using medical leeches for treatment of venous congestion.

INTRODUCTION: Venous convulsion after reconstructive microsurgery procedures is one major complication a surgeon has to deal with. Today, especially in the field of reconstructive microsurgery, medicinal leech therapy enjoys a renaissance. The potential risks such as infections associated with leech therapy are generally underestimated and not sufficiently discussed in literature. METHOD/PATIENTS: We present five male patients with an average age of 47 years. All patients suffered from a trauma incident, which had to be treated as an emergency. Three patients showed, postoperatively, a venous congestion after the reconstructive procedures. Another two patients with flap reconstruction and flap training developed venous problems after 12 and 14 days. In all five cases, the indication was given to use medical leeches (Hirudo medicinalis). In all the patients, a local infection of the injured extremity could be regarded after beginning with the leech treatment. The treatment duration with medical leeches for postoperative venous congestion was an average of 6 days. RESULTS: The reconstructive procedures in all five cases were unfortunately unsuccessful as major local infections were observed. Microbiological analyses showed, in all cases, an infection with Aeromonas hydrophila. CONCLUSION: We recommend making a considered indication for leech therapy, to diagnose wound infections early and to think about prophylactic antibiotics in patients with leech application.

Automatic quantitative micro-computed tomography evaluation of angiogenesis in an axially vascularized tissue-engineered bone construct.

INTRODUCTION: We invented an automatic observer-independent quantitative method to analyze vascularization using micro-computed tomography (CT) along with three-dimensional (3D) reconstruction in a tissue engineering model. MATERIALS AND METHODS: An arteriovenous loop was created in the medial thigh of 30 rats and was placed in a particulated porous hydroxyapatite and beta-tricalcium phosphate matrix, filled with fibrin (10 mg/mL fibrinogen and 2 IU/mL thrombin) without (group A) or with (group B) application of fibrin-gel-immobilized angiogenetic growth factors vascular endothelial growth factor (VEGF¹65) and basic fibroblast growth factor (bFGF). The explantation intervals were 2, 4, and 8 weeks. Specimens were investigated by means of micro-CT followed by an automatic 3D analysis, which was correlated to histomorphometrical findings. RESULTS: In both groups, the arteriovenous loop led to generation of dense vascularized connective tissue with differentiated and functional vessels inside the matrix. Quantitative analysis of vascularization using micro-CT showed to be superior to histological analysis. The micro-CT analysis also allows the assessment of different other, more complex vascularization parameters within 3D constructs, demonstrating an early improvement of vascularization by application of fibrin-gel-immobilized VEGF¹65 and bFGF. CONCLUSIONS: In this study quantitative analysis of vascularization using micro-CT along with 3D reconstruction and automatic analysis exhibit to be a powerful method superior to histological evaluation of cross sections.

Axial vascularization of a large volume calcium phosphate ceramic bone substitute in the sheep AV loop model.

Vascularization still remains an obstacle to engineering of bone tissue with clinically relevant dimensions. Our aim was to induce axial vascularization in a large volume of a clinically approved biphasic calcium phosphate ceramic by transferring the arteriovenous (AV) loop approach to a large animal model. HA/beta-TCP granula were mixed with fibrin gel for a total volume of 16 cm(3), followed by incorporation into an isolation chamber together with an AV loop. The chambers were implanted into the groins of merino sheep and the development of vascularization was monitored by sequential non-invasive magnetic resonance imaging (MRI). The chambers were explanted after 6 and 12 weeks, the pedicle was perfused with contrast agent and specimens were subjected to micro-computed tomography (micro-CT) scan and histological analysis. Sequential MRI demonstrated a significantly increased perfusion in the HA/beta-TCP matrices over time. Micro-CT scans and histology confirmed successful axial vascularization of HA/beta-TCP constructs. This study demonstrates, for the first time, successful axial vascularization of a clinically approved bone substitute with a significant volume in a large animal model by means of a microsurgically created AV loop, thus paving the way for the first microsurgical transplantation of a tissue-engineered, axially vascularized bone with clinically relevant dimensions.