Mode of Vascularization of Control and Basic Fibroblast Growth Factor-Stimulated Prefabricated Skin Flaps

Objective To reconstruct soft tissue defect of the elbow combined with brachial artery injury using anterolateral thigh flap that used the transverse branch of the lateral femoral circumflex artery as the vessel pedicle, and evaluate the clinical results. Methods Eight cases of brachial artery injury along with soft tissue defect at the elbow were treated between March 2000 and February 2008. Primary repair of the tissue defect was done by free transfer of the anterolateral thigh flap using the transverse branch of the lateral femoral circumflex artery as the vessel pedicle. The transverse branch of the lateral femoral circumflex artery was simultaneously inserted between the two ends of the injured brachial artery (3 to 7 cm defect). The areas of the skin defect ranged from 12 cm×6 cm to 20 cm×10 cm. Results Limbs and flaps of the 8 cases all survived. Postoperative follow up ranged from 10 months to 21 months. There were no obvious bulkiness and scar contracture of the tlaps. The average ROM of the elbows was 105°(95° to 125°). In all 8 cases, the pulse strength of the distal ulnar and radial arteries was the same as that of the uninjured side. Ultrasound detection of the brachial artery revealed blood flow of the injured side similar to that of at the uninjured fide. Conclusion When there exists vascular variation of the descending branch of the lateral femoral circumflex artery during dissection of the anterolateral thigh flap, the transverse branch of the hteral femoral circumflex artery can be used as the vessel pedicle of the flap. Made as a flow-through flap, this flap can be transferred to recommit soft tissue defects of the elbow combined with brachial artery defect. Key words: Wounds and injuries; Brachial artery; Surgical flans; Microsurgery

Current techniques of free muscle transplantation with microvascular anastomosis have the disadvantages of limitation of available donor sites, loss of donor muscle function, and the possibility of donor site defect or deformity. Using modifications of a "secondary tissue flap" created by neovascularization of a given tissue, neovascularized muscle flaps with pedicle vessels were created by wrapping external abdominal oblique muscles around the superficial inferior epigastric blood vessels. Different pedicle types were examined to explore possible differences in neovascularization. In 60 rats, four groups of neovascularized muscle flaps were created and transplanted as free flaps, with a high degree of success. After transplantation, neovascularization of the flaps was evaluated by histology and latex casts of vascular tree of the muscle flaps. Degree of neovascularization differed, depending on the type of vascular pedicle used to create the flap.

Free flaps have a distinct role in a select group of patients with large abdominal wall defects. They offer a completely autologous reconstructive solution in a single stage for difficult abdominal wounds for which pedicled flaps would be inadequate. From 1996 to 2005, five patients with complex abdominal wall defects underwent reconstruction using free flaps. All patients had multiple comorbidities, making the use of alloplastic materials relatively contraindicated. Flaps used included a free radial forearm flap in one patient, a tensor fasciae latae myocutaneous flap in two patients, a free anterolateral thigh myocutaneous flap in one patient, and free conjoined tensor fasciae latae and anterolateral thigh myocutaneous flaps in the last patient. The mean defect size was 470 cm (range, 136 to 875 cm). The femoral artery and long saphenous vein reliably provided recipient vessels in cases for which suitable vessels could not be located within the abdomen. A temporary arteriovenous shunt of the long saphenous vein to the femoral artery could be created. This was later divided to provide a recipient artery and vein. Flap complications were wound edge necrosis, hematoma, infection, and venous thrombosis. All were successfully managed and there were no flap failures. The average length of hospitalization was 64 days (range, 41 to 128 days). Lateral thigh flaps based on the lateral circumflex femoral system are our preferred donor site. A large amount of soft tissue, strong fascia, and innervated muscle are available, enabling single-stage autologous reconstruction of the entire anterior abdominal wall. Free flaps offer a reliable single-stage solution to complex abdominal wall defects. With these techniques, even the most challenging defects can be reconstructed with completely autologous tissue.

This study describes a free flow-through flap model in the rat for use in the evaluation of the physiologic and hemodynamic characteristics of this type of flap in clinical practice. The rat is a preferred animal model because it is inexpensive, readily available, and reliable. There is no free flow-through flap model available for laboratory animals, although this model gained popularity in clinical use recently. Twenty Wistar rats weighing 200-250 g were used in our experiment. In 5 rats, the vascular anatomy of the groin and proximal thigh region was determined by anatomic dissection. The experimental design consisted of two groups. In the experimental group (N = 5), a flow-through epigastric skin flap was harvested based on the femoral artery, preparing both its proximal and distal stumps. The flap was transferred to the contralateral groin, and end-to-end vascular anastomosis was performed between a proximal and distal stump of the femoral arteries of the flap and recipient site. The proximal stump of the femoral vein of the flap was anastomosed to the femoral vein of the recipient site. The control group was divided into two subgroups. In all control group rats (N = 10), the flap was harvested in the same manner, and transferred to the contralateral groin, but standard free-flap procedure with one artery and one vein anastomosis was performed in 5 rats (conventional free-flap subgroup), and anastomosis was not performed between the flap and the recipient site in the remaining 5 rats (graft subgroup). Survival of the flap was evaluated on postoperative day 7 by direct observation, and microangiography was performed to delineate the vascularity of the flow-through flap. The results showed that all flaps survived in the experimental group and the conventional free-flap subgroup of the control group, whereas in the graft subgroup, all flaps underwent total necrosis. The authors conclude that the flow-through epigastric flap for the rat is a simple and reliable model for future physiologic and pharmacologic studies.

The development of drug delivery systems has enabled the release of multiple bioactive molecules. The efficacy of nerve conduits coated with dual controlled release of stromal cell-derived factor-1 (SDF-1) and basic fibroblast growth factor (bFGF) for peripheral nerve regeneration was investigated. Sixty-two C57BL6 mice were used for peripheral nerve regeneration with a nerve conduit (inner diameter, 1 mm, and length, 7 mm) and an autograft. The mice were randomized into five groups based on the different repairs of nerve defects. In the group of repair with conduits alone (n = 9), a 5-mm sciatic nerve defect was repaired by the nerve conduit. In the group of repair with conduits coated with bFGF (n = 10), SDF-1 (n = 10), and SDF-1/bFGF (n = 10), it was repaired by the nerve conduit with bFGF gelatin, SDF-1 gelatin, and SDF-1/bFGF gelatin, respectively. In the group of repair with autografts (n = 10), it was repaired by the resected nerve itself. The functional recovery, nerve regeneration, angiogenesis, and TGF-β1 gene expression were assessed. In the conduits coated with SDF-1/bFGF group, the mean sciatic functional index value (-88.68 ± 10.64, p = .034) and the axon number (218.8 ± 111.1, p = .049) were significantly higher than the conduit alone group, followed by the autograft group; in addition, numerous CD34-positive cells and micro vessels were observed. TGF-β1 gene expression relative values in the conduits with SDF-1/bFGF group at 3 days (7.99 ± 5.14, p = .049) significantly increased more than the conduits alone group. Nerve conduits coated with dual controlled release of SDF-1 and bFGF promoted peripheral nerve regeneration.

The aims of this study were to develop an endovascular delivery system containing gelatin hydrogels for the controlled release of basic fibroblast growth factor (bFGF) with the use of polyethylene terephthalate fiber coils and to analyze whether such a system would promote healing in an experimental aneurysm. Carotid aneurysms were constructed in 66 rabbits with venous pouches. The polyethylene terephthalate fiber coils coated with and without gelatin hydrogels with different water volumes containing 0, 10, 50, and 100 microg bFGF were implanted into the aneurysms. Histological specimens were harvested at 1, 2, and 3 weeks and at 6 months after implantation. A histological evaluation was performed while the area occupied by the fibrosis in the aneurysms was calculated. Three weeks after the application of the coils coated with gelatin hydrogels (95 vol%) containing 100 microg bFGF, all aneurysmal orifices were completely closed with neointima. When the coils coated with gelatin hydrogel (98 vol%) containing 100 microg bFGF were used, the orifices in three of the six aneurysms were closed. In contrast, the orifice of the aneurysm was not obliterated when other materials were used. After implanting the coils coated with gelatin hydrogel (95 vol%) containing 100 microg bFGF more than 3 weeks later, the aneurysm was histologically suffused with fibrous tissue, and the area occupied by fibrosis was significantly larger than that observed in the other groups (P < 0.05). Local, controlled release of sufficient amounts of bFGF with polyethylene terephthalate fiber coils coated with gelatin hydrogel accelerated the organization of aneurysms.

We have characterized the importance of size, sulfation, and anticoagulant activity of heparin in release of basic fibroblast growth factor (bFGF) from the subendothelial extracellular matrix (ECM) and the luminal surface of the vascular endothelium. For this purpose, 125I-bFGF was first incubated with ECM and confluent endothelial cell cultures, or administered as a bolus into the blood of rats, the immobilized 125I-bFGF was then subjected to release by various chemically modified species of heparin and size-homogeneous oligosaccharides derived from depolymerized heparin. Both totally desulfated and N-desulfated heparin failed to release the ECM-bound bFGF. Likewise, substitution of N-sulfate groups of heparin and low molecular weight heparin (fragmin) by acetyl or hexanoyl residues resulted in an almost complete inhibition of bFGF release by these polysaccharides. The presence of O-sulfate groups in heparin increased but was not critical for release of ECM-bound bFGF. Similar structural requirements were identified for release of 125I-bFGF bound to low-affinity sites on the surface of vascular endothelial cells. Oligosaccharides derived from depolymerized heparin and containing as little as 8-10 sugar units were, on a weight basis, equivalent to whole heparin in their ability to release bFGF from ECM. Low-sulfate oligosaccharides were less effective releasers of bFGF as compared to medium- and high-sulfate fractions of the same size oligosaccharides. Heparin fractions with high and low affinity to antithrombin III exhibited a similar high bFGF-releasing activity despite a 200-fold difference in their anticoagulant activities.(ABSTRACT TRUNCATED AT 250 WORDS)

Introduction: This study evaluated the effects of low-level laser therapy (LLLT) on human skin fibroblasts (HSFs) that have been cultured in high glucose concentration media. Materials and methods: HSFs were cultured under physiological glucose condition medium, and then cultured in high glucose concentration medium (15 mM/L) for 1 or 2 weeks prior to LLLT. Experimental HSFs were irradiated with three energy densities (0.5, 1, and 2 J/cm2) once daily for three consecutive days. Release of interleukin-6 (IL-6) and basic fibroblast growth factor (bFGF) was evaluated using the enzyme-linked immunosorbent assay (ELISA) method. Results: Statistical analysis showed three doses of 0.5 (p = 0.049), 1 (p = 0.027), and 2 J/cm2 (p = 0.004) stimulated the release of IL-6 in HSFs cultured in high glucose concentration medium compared with that of non-irradiated HSFs that were cultured in the same medium. LLLT with 2 J/cm2 induced the release of bFGF from HSFs cultured in high glucose concentration medium for 1 or 2 weeks (both p = 0.04). Conclusion: Our study showed that LLLT stimulated the release of IL-6 and bFGF from HSFs cultured in high glucose concentration medium. LLLT was more effective in releasing IL-6 and bFGF while HSFs which were cultured in physiologic glucose concentration medium during laser irradiation.

Surgical errors in the length and orientation of a flap pedicle can result in kinks, twists, or tension on the pedicle vessels. When these errors occur together with the microvascular repairs associated with free flap transfer, thrombosis and flap failure may be the outcome. A model was designed to simulate these events. The rabbit central ear artery and vein were dissected and all other tissue connections were severed in a near-complete amputation. The artery was transected, shortened by 2 mm, and repaired standardly. The ear was then rotated 360 degrees, twisting the vein around the repaired artery. The ear cartilage was shortened by 6 mm, then sutured, and the skin was closed. In a series of 20 ears, 95% necrosed, 18 from arterial thrombosis and 1 from venous thrombosis. Several other groups were used in which aspects of the basic model were modified to determine the factors influencing failure. When the arterial anastomosis was not performed and the vascular pedicle was rotated with cartilage shortening, all ears survived; when the cartilage was not resected in this paradigm, leaving tension on the pedicle, 50% of the ears necrosed from venous thrombosis. In counterpoint, when the artery was repaired without twisting the pedicle, all ears survived, whether or not the cartilage was shortened. When the artery was repaired without shortening it and with rotation of the ear, 50% of the ears necrosed when the cartilage was shortened, 40% from venous thrombosis and 10% from arterial thrombosis; all ears necrosed (of venous thrombosis) when the cartilage was not shortened.(ABSTRACT TRUNCATED AT 250 WORDS)

The tumor microenvironment, and its heterogeneous composition, is a major contributor to metastasis and often responsible for the varied responsiveness of tumors to therapy. Within the microenvironment, the vasculature plays an important role in regulating the influx of cellular infiltrates that contribute to the local immune response driving either immune suppression or tumor rejection. The role of pre-existing, dermal lymphatic vessels in regulating this local immune response is assumed to be the passive transport of antigen and activated dendritic cells to the sentinel draining lymph node, which leads to the induction of adaptive immunity. However, we recently demonstrated that abnormal lymphatic vessel growth, or tumor-associated lymphangiogenesis, leads to impaired adaptive immunity and limited responsiveness to immunotherapy. This led us to interrogate the immunological implications of tumor-associated lymphangiogenesis as compared to pre-existing dermal lymphatic vessels. We hypothesized that while aberrant lymphatic vessel growth is associated with immune dysfunction, the pre-existing lymphatic vasculature would be necessary for immune induction within the tumor microenvironment. To test this hypothesis we implanted B16 F10 murine melanomas into mice completely lacking dermal lymphatic vessels, K14 VEGFR3-Ig. B16 F10 tumors in K14 VEGFR3-Ig mice demonstrate impaired fluid drainage and dendritic cell transport to the sentinel node. Consequently, this lymph node lacks an inflammatory response and prophylactic dermal vaccination against a model tumor antigen is unable to induce tumor rejection. Surprisingly, however, we also observed impaired local inflammation, characterized by a lack of both inflammatory and suppressive cytokines and the absence of an immune infiltrate. All immune subsets analyzed showed a dramatic reduction in K14 VEGFR3-Ig mice, including suppressive subsets such as regulatory T cells and myeloid derived suppressor cells, which importantly function to inhibit local T cell cytotoxic activity. These changes were not, however, observed systemically and were restricted to the microenvironment of the tumor itself. As a result of the altered tumor microenvironment, which led to improved T cell function, an adoptive T cell therapy was more efficacious in animals lacking dermal lymphatic vessels. This work highlights the important role of the existing lymphatic vasculature in regulating the immune response and suggests that peripheral lymphatic vessels are not only required for lymph node activation but also necessary for the initiation of local, tissue inflammation. This abstract is also presented as Poster A42. Citation Format: Amanda Lund, Marek Wagner, Manuel Fankhauser, Helge Wiig, Melody Swartz. Dermal lymphatic vessels are required for the induction of local inflammation in a B16 F10 murine melanoma. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr PR02. doi:10.1158/1538-7445.CHTME14-PR02

Angiogenesis is a complex process, where several cell types and mediators interact to establish a specific microenvironment suitable for the formation of new capillaries from pre-existing vessels. Such biological processes occur in several physiological conditions, such as embryo development and wound healing, as well as in pathological conditions, including tumours and diabetic retinopathy. T lymphocytes, neutrophils and monocytes fully participate in the angiogenic process by secreting cytokines that may control endothelial cell (EC) proliferation, their survival and apoptosis, as well as their migration and activation. Angiogenesis is the result of a net balance between the activities exerted by positive and negative regulators. This balance is conceptually very similar to that of the Th1/Th2 cells that modulate an appropriate and specific immune response. Th1 or Th2 cytokines may control angiogenesis directly, by acting on cell growth and differentiation, indirectly by inducing the release of other cytokines in the microenvironment, and by modulating the expression of specific receptors, involved in the control of angiogenic processes, such as EC proliferation and migration. In this review we will mainly discuss the role of Th1- and Th2-type cytokines in the angiogenic process, emphasizing the complexity of the cytokine and leukocyte/EC network, and highlighting the care that needs to be taken when designing new therapeutic interventions involving Th1 and Th2 cytokines.

Objective To explore the anatomic basis of the thinning of the free posterior tibial artery perforator flaps and the clinical effect of repairing wound on hand or foot due to trauma. Methods From November, 2016 to December, 2017, 10 cases of lower extremity cadaver specimens perfused with red ralex were dissected, which were perfused through the amputated femoral artery. Five of them were left and the rest were right. All cases were males. The number, diameter, branches and distribution of the perforator was observed. From September, 2012 to September, 2017, there were 13 cases of clinical application, which were 5 cases of hand wound and 8 cases of foot wound. The size of the wound was 3.0 cm × 2.0 cm to 6.0 cm × 4.0 cm, and the flap area was 3.5 cm × 2.2 cm to 6.5 cm × 4.5 cm. The repairing procedure was suitable for the wound associated with tendon, bone, joint capsule exposure. Results The number of posterior tibial artery perforating branches that more than 0.50 mm in diameter was 4 to 6, and the mean diameter was (0.87±0.26) mm. The perforating branch penetrated into the fat layer and was divided into 3 layers of vascular network: deep fat vascular network, superficial fat vascular network and subdermal vascular network. The perforating branch was located according to the positional relationship from deep to shallow, and vessel diameter become smaller step by step. The perforating branch trunk gave off branches to the deep vascular network, and the superficial vascular network had the same origin or shared with the deep blood vessels. The subdermal vascular network issued from the superficial vascular network or directly from the perforating branch trunk. There was no or few communicating branch between the deep vascular network and superficial ones, besides the vessel pedicle. So trimming deep fat layer will not affect the blood supply of superficial vascular network and neither will affect the flap blood supply. Most of the deep fat tissue was trimmed in 13 cases. The superficial and subdermal fat vascular network was preserved, and the same to the trunk and branches of the pedicle. All the flaps survived. Of which, 1 appeared arterial crisis on the 2nd day after operation, and relieved by the local injection of papaverine. There was 1 case of venous crisis on the 3rd day, and improved by stitches, local release of congestion. Followed-up time was ranged from 2 to 12 months. All flaps were soft with good blood supply and good appearance, and did not need a second thinning surgery. Flaps restored the protective feeling 6 months later. Conclusion The microdissection of perforator flap of posterior tibial artery provides a theoretical basis for the perforator flap thinning, and the thinning of perforator flap is a good method to repair the appearance and function of the wound after foot and hand injuries. Key words: Posterior tibial artery perforator flap; Applied anatomy; Wound; Repair; Microsurgical operation

Since the technique of vacuum assisted closure has been clinically introduced, the possibilities in reconstructive head and neck surgery have greatly increased. Preconditioning and prelaminating allows a pedicled or free flap plasty to be epithelialized on both sides before the flap gets transferred to its future location. We describe the technique of preconditioning and prelaminating of pedicled and free microvascular anastomised flap plasties and we report our experience with 5 cases. Our results are compared with data from the literature. The procedures concerned take only little time and the flap gets optimally prepared for its task in view of the postoperatively modified hemodynamics. By preconditioning, the flap to be lifted is prepared for the future centralised blood supply exclusively via the pedicle vessel without leaving the donor side. Thereby the skin island of such flaps can be increased in size over the natural angiosomes without an increased risk of necrotisation. By the technique of prelamination, skin or mucosa tissue may be applied onto both sides of the flap so that it can resist the biochemical and mechanical tasks which will be required after transposition. Especially for the reconstruction of defects in the upper aerodigestive tract this is a great advantage, since the inner side of the flap has to resist saliva which represents one of the most aggressive secretions in the human body.

MUSCULAR URINARY SPHINCTER: ELECTRICALLY STIMULATED MYOPLASTY FOR FUNCTIONAL SPHINCTER RECONSTRUCTION

Growth factors play essential roles as signaling molecules in pulp regeneration. We investigated the effect of a hyaluronic acid (HA)-collagen hybrid hydrogel with controlled release of fibroblast growth factor (FGF)-2 and platelet-derived growth factor (PDGF)-BB on human pulp regeneration. The cell interaction and cytotoxicity of the HA-collagen hybrid hydrogel, the release kinetics of each growth factor, and the effects of the released growth factors on pulp cell proliferation were examined. The vitality of pulp cells was maintained. The amounts of FGF-2 and PDGF-BB released over 7 days were 68% and 50%, respectively. Groups with a different concentration of growth factor (FGF-2: 100, 200, 500, and 1000 ng/mL; PDGF-BB: 10, 50, 100, 200, and 500 ng/mL) were experimented on days 1, 3, 5, and 7. Considering FGF-2 concentration, significantly increased pulp cell proliferation was observed on days 1, 3, 5, and 7 in the 100 ng/mL group and on days 3, 5, and 7 in the 200 ng/mL group. In the case of PDGF-BB concentration, significantly increased pulp cell proliferation was observed at all four time points in the 100 ng/mL group and on days 3, 5, and 7 in the 50, 200, and 500 ng/mL groups. This indicates that the optimal concentration of FGF-2 and PDGF-BB for pulp cell proliferation was 100 ng/mL and that the HA-collagen hybrid hydrogel has potential as a controlled release delivery system for FGF-2 and PDGF-BB.