Fibroblast growth factor-2 and vascular endothelial growth factor mediated augmentation of angiogenesis and bone formation in vascularized bone allotransplants.

We previously demonstrated recipient-derived neoangiogenesis to maintain viability of living bone allogeneic transplants without long-term immunosuppression. The effect of cytokine delivery to enhance this process is studied. Vascularized femur transplantation was performed from Dark Agouti to Piebald Virol Glaxo rats. Poly(d,l-lactide-co-glycolide) microspheres loaded with buffer (N = 11), basic fibroblast growth factor (FGF2) (N = 10), vascular endothelial growth factor (VEGF) (N = 11), or both (N = 11) were inserted intramedullarly alongside a recipient-derived arteriovenous bundle. FK-506 was administered for 2 weeks. At 18 weeks, bone blood flow, microangiography, histologic, histomorphometric, and alkaline phosphatase measurements were performed. Bone blood flow was greater in the combined group than control and VEGF groups (P = 0.04). Capillary density was greater in the FGF2 group than in the VEGF and combined groups (P < 0.05). Bone viability, growth, and alkaline phosphatase activity did not vary significantly between groups. Neoangiogenesis in vascularized bone allotransplants is enhanced by angiogenic cytokine delivery, with results using FGF2 that are comparable to isotransplant from previous studies. Further studies are needed to achieve bone formation similar to isotransplants.

The role of fabricated chimeric free flaps in reconstruction of devastating hand and forearm injuries.

Devastating hand and forearm injuries almost exclusively need free flap transfer if reconstruction is attempted. Early active and passive motion is only possible with aggressive, early, and comprehensive reconstruction. Despite recent advances in compound flaps, in selected cases it might be wise to harvest several smaller flaps and microsurgically combine them to one "chain-linked" flap "system." Four microsurgically fabricated chimeric free flaps were used in four patients for complex hand and forearm injuries. The combinations were sensate anterolateral thigh (ALT) flap plus sensate extended lateral arm flap (2x), ALT plus free fibula, and ALT plus functional musculocutaneous gracilis muscle. All flaps survived completely. Functional rehabilitation was possible immediately after flap transfer. There were no donor-site complications except two widened scars. The microsurgical fabrication of chimeric free flaps, as well established in head and neck reconstruction, can be successfully adapted to massive hand injuries as well. Individual placement of selected tissue components, early comprehensive reconstruction, and reduction of the number of operations are beneficial in cases that need more than one free flap.

Surgical angiogenesis: a new approach to maintain osseous viability in xenotransplantation.

BACKGROUND: Large segmental osseous defects are challenging clinical problems. Current reconstructive methods, using non-viable allografts, vascularized autografts or prostheses have significant rates of serious complications and failure. These include infection, stress fracture and non-union (frozen structural allogenic bone); loosening and implant failure (prosthetic replacement); limited availability, poor match of size and shape and donor site morbidity (vascularized autograft bone). In the future, microvascular transplantation of living allogenic or xenogenic bone could solve some of these issues, combining the advantages of living bone autografts (capability of primary osseous healing, remodeling, and fracture resistance) with the ability to match size and shape, provide immediate stability and avoid donor site morbidity. Xenotransplants would be particularly attractive, as they could be readily available, if long-term bone survival could be achieved without unacceptable morbidity. Here, we present a preliminary study to evaluate a new and unique method to maintain xenogenic bone circulation without need for long-term immune modulation that depends upon generation of a neo-angiogenic circulation within the transplanted bone from recipient-derived vessels. Thus, only short-term immunosuppression would be required to achieve bone survival. METHODS: One hundred and forty-one hamster femora were microsurgically transplanted to rats, restoring nutrient vessel circulation with standard microvascular anastomoses. At the same time, a host-derived arteriovenous bundle (AVB) was placed within the medullary canal. Two independent variables were evaluated: use of tacrolimus/cyclophosmamid immunosuppression (IS) and patency of the implanted AVB. Rats were therefore randomized to four groups; group 1-no IS + patent AVB; group 2-no IS + ligated AVB; group 3-IS + patent AVB; group 4-IS + ligated AVB. Rats were sacrificed after 1 or 2 weeks. We evaluated bone blood flow (microsphere entrapment), neoangiogenesis (microangiography and quantification of capillary density), bone necrosis rate (osteocyte counts) and nutrient pedicle rejection (microsurgical anastomotic patency). Statistical Analysis was performed with two-way ANOVA with Bonferroni adjustment. Differences were considered significant when P < 0.05. RESULTS: Capillary density was significantly increased with a patent intramedullary AVB (groups 1/3) compared to groups with ligated AVBs (groups 3/4). Capillary sprouting was predominantly restricted to the endosteal layer. Most nutrient pedicles (78.7%) stayed patent in groups with IS (groups 3 and 4). Consequently, bone blood flow was significantly higher in groups 3 and 4 compared to groups 1 and 2. Similarly, a patent AV bundle improved flow in group 1 when compared to group 2. The bone necrosis rate was not influenced by the presence of patent AVBs but was significantly reduced in groups 3 and 4. CONCLUSIONS: Surgical angiogenesis occurs when patent arteriovenous bundles are placed in the medullary canal of xenogenic bone and leads to increased bone blood flow. Bone viability was not significantly influenced by neoangiogenesis. Although capillary sprouting was restricted to the endosteal layer in this short term study, more complete cortical revascularization might be observed in a long-term study. Such a study should further evaluate whether these new vessels supply sufficient blood flow to maintain long-term bone viability and allow remodeling.

Osteo-periosteal-cutaneous flaps of the medial femoral condyle: a valuable modification for selected clinical situations.

In situations of bony nonunions with poor skin coverage, transplantation of vascularized soft tissue in addition to bone graft is desirable. The use of the corticoperiosteal vascularized bone graft from the medial femoral condyle is well described. There are only anecdotal reports about its use as an osteocutaneous flap. This article presents our results with the use of an osteocutaneous flap from the medial femoral condyle. Between 2004 and 2009, four patients were treated with supracondylar osteocutaneous flaps for bony nonunions (tibia, ankle, calcaneous) with concomitant soft tissue defects. The size of the osseous grafts ranged from 3 x 5 to 6 x 5 cm. The supplying cutaneous vessels were an unnamed perforator of the descending genicular artery (two cases) or the saphenous branch (two cases). The first three cases healed primarily. Bony union was achieved between 32 and 170 days. The follow-up of the fourth case was too short to achieve a bony union. There was no flap loss or surgery-related complications at the donor site. The transfer of free combined vascularized corticoperiosteal-cutaneous flaps seems to be ideally suited for postradiation-induced fractures or chronic nonunions with poor chances of spontaneous healing and a concomitant small skin defect.

Short-term immunosuppression and surgical neoangiogenesis with host vessels maintains long-term viability of vascularized bone allografts.

Currently available methods to reconstruct large skeletal defects have limitations. These include nonunion and stress fractures in structural allografts, and inability to match the size, shape, and/or strength of most recipient sites using vascularized fibular autografts. Prosthetic diaphyseal replacements may loosen or produce periprosthetic fractures. Transplantation of living allogenic bone would enable matching donor bone to the recipient site, combined with the desirable healing and remodeling properties of living bone. We propose a novel method by which the transplantation of such tissue might be done without the risks of life-long immunosuppression, using surgical neoangiogenesis to develop a new host-derived osseous blood supply. We performed vascularized femoral allografts from 86 female Dark Agouti donor rats to male Piebald Virol Glaxo recipients across a major histocompatibility (MHC) barrier. In addition to microvascular reconstruction of the nutrient vessel, we surgically implanted a host arteriovenous (AV) bundle into the medullary canal to promote host vessel neoangiogenesis. Independent variables included patency of the implanted AV bundle, and use of 2 weeks' FK-506 immunosuppression. After 18 weeks, bone blood flow was measured, and neoangiogenic capillary density quantified. Bone blood flow and capillary density were significantly greater in transiently immunosuppressed recipients with a patent AV pedicle. We conclude that neoangiogenesis from implanted host-derived AV-bundles, combined with short-term immunosuppression maintains blood flow in vascularized bone allografts, and offers potential for clinical application.

Vascularized bone allotransplantation: current state and implications for future reconstructive surgery.

This article focuses on current advances in musculoskeletal tissue allotransplantation, including strategies for maintaining tissue viability in the face of histocompatibility mismatch and resulting acute and chronic rejection responses. In particular, it introduces a novel concept developed in the authors' laboratory and currently under evaluation that may obviate the problem of chronic rejection. The authors have used therapeutic angiogenesis to develop a host-derived neoangiogenic circulation that maintains blood flow regardless of rejection. The replacement of the allogeneic vessels together with bone remodeling from host-derived cells eventually may largely replace the allogeneic osteocytes and bone with native bone.

Transcardiopulmonary vs pulmonary arterial thermodilution methods for hemodynamic monitoring of burned patients.

The objective of this study was to validate a new method of transcardiopulmonary thermodilution for assessment of cardiac index (CI), stroke volume index (SVI), systemic vascular resistance index (SVRI) and additional parameters such as intrathoracic blood volume index and extravascular lung water index (EVLWI) by comparison with conventional pulmonary artery catheter values in a severely burned population. The pulmonary artery measurements were performed continuously with the Vigilance system, and the transcardiopulmonary thermodilution with the PiCCO(R) system. One hundred thirteen measurements with each system on up to six consecutive days were taken in 14 severely burned patients (average TBSA, 49.6%; average ABSI, 10.3), aged 21 to 61 years (mean, 42.2 years) and compared intraindividually. An excellent correlation between the two methods was shown for CI (r = 0.80) and its derived parameters SVI and SVRI in states of low to normal cardiac output. The correlation was poor for cardiac indices greater then 5.5 up to their maximum values (r = 0.46). No correlation between index of oxygenation (PaO2/FiO2) vs EVLW I was observed. There was no difference between survivors and nonsurvivors, and between patients with and without inhalation injury in EVLWI. The method of transcardiopulmonary thermodilution is suitable to assess SVI, CI and SVRI under the special pathophysiologic condition of a major burn for low to normal cardiac output states. It is less reliable when cardiac output is high. The lower cost and less invasive nature are the advantages of the system compared with use of the pulmonary artery catheter. The role of intrathoracic blood volume index and EVLWI in cardiopulmonary monitoring of severely burned patients remains to be further determined.

Bilateral double free-flaps for reconstruction of extensive chest wall defect.

A 44-year-old woman presented with ulcerating stage IV breast cancer involving the chest wall. En bloc resection of the second to seventh ribs on the right side, parts of the second to eighth ribs on the left side, the sternum, the chest wall muscles, and skin was completed with immediate reconstruction using bilateral double free flaps consisting of anterior lateral thigh and tensor fascia latae elevated on the lateral circumflex femoral system and creating recipient vessels with an arteriovenous loop.

Living bone allotransplants survive by surgical angiogenesis alone: development of a novel method of composite tissue allotransplantation.

BACKGROUND: Segmental bone defects pose reconstructive challenges. Composite tissue allotransplantation offers a potential solution but requires long-term immunosuppression with attendant health risks. This study demonstrates a novel method of composite-tissue allotransplantation, permitting long-term drug-free survival, with use of therapeutic angiogenesis of autogenous vessels to maintain circulation. METHODS: Ninety-three rats underwent femoral allotransplantation, isotransplantation, or allografting. Group-1 femora were transplanted across a major histocompatibility complex barrier, with microsurgical pedicle anastomoses. The contralateral saphenous artery and vein (termed the AV bundle) of the recipient animal were implanted within the medullary canal to allow development of an autogenous circulation. In Group 2, allotransplantation was also performed, but with AV bundle ligation. Group 3 bones were frozen allografts rather than composite-tissue allotransplantation femora, and Group 4 bones were isotransplants. Paired comparison allowed evaluation of AV bundle effect, bone allogenicity (isogeneic or allogeneic), and initial circulation and viability (allotransplant versus allograft). Two weeks of immunosuppression therapy maintained blood flow initially, during development of a neoangiogenic autogenous blood supply from the AV bundle in patent groups. At eighteen weeks, skin grafts from donor, recipient, and third-party rats were tested for immunocompetence and donor-specific tolerance. At twenty-one weeks, bone circulation was quantified and new bone formation was measured. RESULTS: Final circulatory status depended on both the initial viability of the graft and the successful development of neoangiogenic circulation. Median cortical blood flow was highest in Group 1 (4.6 mL/min/100 g), intermediate in Group 4 isotransplants (0.4 mL/min/100 g), and absent in others. Capillary proliferation and new bone formation were generally highest in allotransplants (15.0%, 6.4 µm³/µm²/yr) and isotransplants with patent AV bundles (16.6%, 50.3 µm³/µm²/yr) and less in allotransplants with ligated AV bundles (4.4%, 0.0 µm³/µm²/yr) or allografts (8.1%, 24.1 µm³/µm²/yr). Donor and third-party-type skin grafts were rejected, indicating immunocompetence without donor-specific tolerance. CONCLUSIONS: In the rat model, microvascular allogeneic bone transplantation in combination with short-term immunosuppression and AV bundle implantation creates an autogenous neoangiogenic circulation, permitting long-term allotransplant survival with measurable blood flow.

Arteriovenous loops in microsurgical free tissue transfer in reconstruction of central sternal defects.

OBJECTIVE: In some patients with chest wall defects, free tissue transfer is indicated. Complications arise if multiple operations have left the trunk devoid of recipient vessels. In such patients, an arteriovenous loop between the cephalic vein and the thoracoacromial artery can be used. METHODS: A review of all our patients who underwent chest wall reconstruction with a cephalic vein-thoracoacromial artery loop between 2000 and 2009 was performed (n = 29, 19 women and 10 men). The mean age was 64.9 years. Underlying causes were sternal osteomyelitis (n = 20), tumor (n = 4), and osteoradionecrosis (n = 5). All patients were in American Society of Anesthesiologists classes III and IV. Flap selection, intraoperative and postoperative complications, operative time, time of ventilatory support, mean hospital stay, and midterm survival were recorded. RESULTS: Twenty-five patients received a tensor fascia lata flap, 2 a vertical rectus myocutaneuos flap, and 2 a deep inferior epigastric perforator flap. Mean duration of surgery was 6.8 hours (4.7-10.5 hours). Two transplanted tissue flaps died and/or had to be removed and 4 were revised successfully. Seven patients had wound complications such as infection or prolonged wound healing. Mean time for ventilator support was 93.6 hours (4-463 hours). The median intensive care unit time was 11 days and the overall hospital stay 27.4 days (11-102 days). One-year survival in the whole group was 69.8%. CONCLUSIONS: The concept of arteriovenous loops allows creation of neovessels at the recipient site and has proven to be a superb tool to facilitate free tissue transfer or to provide an exit strategy in situations with unexpected vascular problems at the recipient site.

Augmentation of surgical angiogenesis in vascularized bone allotransplants with host-derived a/v bundle implantation, fibroblast growth factor-2, and vascular endothelial growth factor administration.

We have previously shown experimental transplantation of living allogeneic bone to be feasible without long-term immunosuppression by development of a recipient-derived neoangiogenic circulation within bone. In this study, we examine the role of angiogenic cytokine delivery with biodegradable microspheres to enhance this process. Microsurgical femoral allotransplantation was performed from Dark Agouti to Piebald Virol Glaxo rats. Poly(D,L-lactide-co-glycolide) microspheres loaded with buffer, basic fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), or both, were inserted intramedullarly along with a recipient-derived arteriovenous (a/v) bundle. FK-506 was administered daily for 14 days, then discontinued. At 28 days, bone blood flow was measured using hydrogen washout. Microangiography, histologic, and histomorphometric analyses were performed. Capillary density was greater in the FGF+VEGF group (35.1%) than control (13.9%) (p < 0.05), and a linear trend was found from control, FGF, VEGF, to FGF+VEGF (p < 0.005). Bone formation rates were greater with VEGF (p < 0.01) and FGF+VEGF (p < 0.05). VEGF or FGF alone increased blood flow more than when combined. Histology rejection grading was low in all grafts. Local administration of vascular and fibroblast growth factors augments angiogenesis, bone formation, and bone blood flow from implanted blood vessels of donor origin in vascularized bone allografts after removal of immunosuppression.

Repopulation of vascularized bone allotransplants with recipient-derived cells: detection by laser capture microdissection and real-time PCR.

Mechanisms underlying successful composite tissue transplantation must include an analysis of transplant chimerism, which is little studied, particularly in calcified tissue. We have developed a new method enabling determination of lineage of selected cells in our model of vascularized bone allotransplantation. Vascularized femoral allotransplantation was performed from female Dark Agouti (DA) donor rats to male Piebald Virol Glaxo (PVG) recipients, representing a major histocompatibility mismatch. Four groups differed in use of immunosuppression (+/-2 weeks Tacrolimus) and surgical revascularization, by implantation of either a patent or a ligated saphenous arteriovenous (AV) bundle. Results were assessed at 18 weeks. Bone blood flow was measured by the hydrogen washout technique and transverse specimens were prepared for histology. Real-time PCR was performed on DNA from laser capture microdissected cortical bone regions to determine the extent of chimerism. To do so, we analyzed the relative expression ratio of the sex-determining region Y (Sry) gene, specific only for recipient male rat DNA, to the cyclophilin housekeeper gene. Substantial transplant chimerism was seen in cortical bone of all groups (range 77-97%). Rats without immunosuppression and with a patent AV bundle revealed significantly higher chimerism than those with immunosuppression and a ligated AV bundle, which maintained transplant cell viability. We describe a new method to study the extent of chimerism in rat vascularized bone allotransplants, including a sex-mismatched transplantation model, laser capture microdissection of selected bone regions, and calculation of the relative expression ratio.

Measurement of bone blood flow using the hydrogen washout technique-part II: Validation by comparison to microsphere entrapment.

Accurate and reproducible measurement of bone blood flow has important clinical and experimental applications. Hydrogen washout is simple, safe, and widely used, but its use in bone tissue has not been validated. To this end, we have compared cortical bone blood flow measurements obtained by radioactive-labeled microsphere entrapment with those from hydrogen washout. Blood flow was measured in tibial cortical bone of 12 New Zealand White rabbits by radioactive microsphere entrapment and by hydrogen washout. Besides a control group (n = 6), four animals were treated with systemic epinephrine (0.8 microg/kg/min) (group 2) and two with nitroprusside (100 microg/kg/min) (group 3). Furthermore, nine femora from seven rats were isolated on their vascular pedicles and repeated bone blood flow measurements were made at each location with the hydrogen washout method to confirm reproducibility of blood flow determinations by hydrogen washout. An average flow of 2.3 +/- 2.0 mL/min/100 g was obtained with the microsphere method and 2.0 +/- 0.5 mL/min/100 g with the hydrogen washout method. There was a significant correlation and agreement: R(2) = 0.97 (p < 0.01). No consistent flow variations were found with systemic vasoactive drug administration. Hydrogen washout provided reproducible results and showed high sensitivity to flow changes. Hydrogen washout is both sensitive and reproducible in measuring bone blood flow. Results agree well with flow values obtained by labeled microsphere entrapment.

Measurement of bone blood flow using the hydrogen washout Technique-Part I: quantitative evaluation of tissue perfusion in the laboratory rat.

The measurement of blood flow in bone is of interest in both clinical and experimental settings. Such determinations would ideally provide accurate, quantitative, and reproducible data with relatively simple and safe technology, even in the small bones of experimental animals. Methods that provide absolute or quantitative measurements include positron emission tomography, "isotope fractioning" using radioactive or fluorescent-labeled microspheres, and measurement of the rate of washout of diffusible tracers delivered either by injection or inhalation. In this article, we describe in detail a modification of the original Whiteside hydrogen washout technique, using modern hydrogen sensors, a micromanipulator for probe placement, and custom software that greatly simplifies blood flow determination and is effective in the small bones of experimental animals.

An innovative treatment concept for free flap reconstruction of complex central chest wall defects--the cephalic-thoraco-acromial (CTA) loop.

BACKGROUND: Loco-regional flaps are the method of choice for chest wall reconstruction. However there is a selected group of patients who require free flap reconstruction, when all other options are used up. A small subgroup of these patients was identified where the commonly used recipient vessels (Internal mammary A. + V., Thoraco-dorsal A. + V.) were no longer available. PATIENT AND METHOD: This group comprised 16 seriously ill patients in the period from 2000 to 2004. Underlying diseases were sternum osteomyelitis (10x), tumor (2x), and osteo-radionecrosis (4x). There were 10 women and 6 men with mean age 62.4 years. All patients were classified as ASA III and IV. Fourteen patients received a TFL flap, two patients a vertical rectus myocutaneous flap (VRAM). Recipient vessels were created with a temporary A-V loop between the cephalic vein and the thoraco-acromial artery (CTA-loop). RESULTS: No flap was lost and two had to be revised successfully for thrombosis of the arterial anastomosis. Mean operation time was 6.1 (4.7-8.4) h. Average time for ventilatory support was 56 (4-338) h. Five patients died within 6 months postoperatively due their underlying advanced disease (n = 3) or multiple organ failure (n = 2). CONCLUSION: The new concept of creating recipient vessels for free flap reconstruction of complex thoracic wall defects proved to be safe and reliable. The CTA loop allowed for unhurried flap dissection, best possible flap positioning, and straightforward end-end anastomoses in these seriously sick patients. The outcome with respect to complications and survival justifies the operative effort.

Host-derived angiogenesis maintains bone blood flow after withdrawal of immunosuppression.

A novel method of living bone allotransplantation combining microvascular repair of the nutrient circulation, implantation of host-derived arteriovenous (AV) bundles, and short-term immunosuppression is described. We hypothesized that neoangiogenesis from the implanted vessels would maintain graft viability and circulation after withdrawal of FK506 (Tacrolimus) immunosuppression. Vascularized femoral transplantation was performed between DA and PVG rats. In addition to microsurgical pedicle anastomoses, a saphenous AV bundle from the recipient animal was implanted in the medullary space. Ninety-seven rats were randomly allocated to groups differing in immunosuppression and AV bundle patency. Implanted vessels significantly improved capillary density and bone blood flow in nonimmunosuppressed and immmunosuppressed groups, respectively. A lower incidence of spontaneous AV bundle thrombosis was found with Tacrolimus treatment. More viable osteocytes were seen at 4 weeks when the AV bundle was patent. Further investigations may confirm host-derived neoangiogenesis as an alternative to tolerance induction or immunosuppression in bone allotransplantation.

Free "kite" flap: a new flap for reconstruction of small hand defects.

Small, thin, and pliable flaps are frequently required in hand surgery to reconstruct defects in functionally important areas such as the pulp or the "contact zones" of the digits. The innervated first metacarpal artery flap ("kite" flap) is a reliable procedure to restore sensibility in the thumb and the digits. Four microsurgical (free) kite flaps to the hand were performed between February, 1993 and August, 1999 in male patients. Follow-up examinations were performed in three patients. The static two-point discrimination in the kite flaps ranged from 8 to 15 mm and did not show any difference compared to flaps from the foot described in the literature. Semmes-Weinstein testing results ranged from normal to protective sensation with a normal sharp vs. dull discrimination. A free kite flap provides a valuable, safe alternative to venous flaps or other free flaps for reconstruction of small defects in the hand