Prefabrication of bone by use of a vascularized periosteal flap and bone morphogenetic protein.

The purpose of this pilot study was to prefabricate a vascularized bone graft by using a vascularized periosteal flap containing osteoprogenitor cells, a structural matrix, and recombinant human bone morphogenetic protein-2 (rhBMP-2). In a rat model, a periosteal flap vascularized by the saphenous artery and vein was dissected off the medial surface of the tibia. This flap consisted of three layers-periosteum, muscle, and fascia-and was tubed on itself to form a watertight chamber that was then transferred on its vascular pedicle to the groin. A total of 78 vascularized periosteal chambers were constructed in 39 animals and divided into 10 groups. In group 1, the periosteal chamber was left empty. Groups 2, 3, and 4 consisted of the periosteal flap and rhBMP-2, but in group 3, the proximal vascular pedicle was ligated, and in group 4, the flap was harvested without the periosteal layer and turned inside out. Groups 5 through 10 consisted of the vascularized periosteal flap containing several different structural matrices (calcium alginate spheres, polylactic acid, or demineralized bone matrix) with or without rhBMP-2. Animals were killed at 2, 4, or 8 weeks in each group. The presence and density of any new bone formation was evaluated both radiologically and histologically. Significant bone formation was seen only in those periosteal flaps containing rhBMP-2 and either the calcium alginate or polylactic acid matrix. New bone formation increased both radiologically and histologically from 2 weeks to 8 weeks only in the periosteal flaps containing the polylactic acid matrix and rhBMP-2. This preliminary study therefore suggests that four factors-blood supply, osteoprogenitor cells in the periosteal layer, a biodegradable matrix, and rhBMP-2-are required for optimal prefabrication of a vascularized bone graft.     

Influences of vascularization and osteogenic cells on heterotopic bone formation within a madreporic ceramic in rats

Research in biomaterials for bone reconstruction has led to elaborate osteogenic composites that combine porous ceramics with bone marrow stromal cells. The aim of this study was to evaluate the influence of direct vascularization of such composites on osteogenesis and the ability to produce a vascularized bone substitute transplant in an ectopic muscular site. Sixty-four coralline biomaterials were implanted in 32 Fisher rats under four conditions: (1) alone (reference group M, n = 16), (2) coated with bone marrow stromal cells (group MC, n = 16), (3) combined with a vascular pedicle (group MV, n = 16), or (4) coated with bone marrow stromal cells and combined with a vascular pedicle (MCV group, n = 16). The number of vessels in the pores (vessel-pore ratio) of the implants and the proportion of pores showing bone ingrowth (bone-pore ratio) were measured at 2, 4, 6, and 8 weeks on four implants of each group. Compared with the reference group, angiogenesis was higher when the biomaterial was combined with a vascular pedicle or was coated with osteoprogenitor cells. The association of both vascular pedicle and osteoprogenitor cells increased vascularization by 60 percent (p = 0.003) and osteogenesis by 62 percent (p < 0.001). A combination of both vascular pedicle and bone marrow osteoprogenitor cells in coralline implants enhances neovascularization and osteogenesis after implantation in ectopic intramuscular sites to a greater extent than either does alone.     

Microvascular A-V shunts and the growth of autologous tissue flaps in Millipore chambers.

An animal model was developed using the male Sprague-Dawley rat to establish a protocol and design criteria for the growth of autologous tissue based on a microvascular pedicle. It became apparent that growth within the chamber depended heavily on membrane porosity and the presence of the microarteriovenous shunt. Different membrane porosities ranging from 0.0, 0.25, 1.2, to 8.0 microns were evaluated (n = 48). Optimal growth occurred with the 1.2-microns and the 8.0-microns micropore Millipore. Growth within the chamber consisted of a radial deposition of collagen and neovascularization originating from the arteriovenous (A-V) anastomosis. In contrast, control chambers (no A-V anastomosis), with the preceding range in membrane porosity, experienced little to no cell growth. In addition, the majority of A-V shunts did not remain patent in chambers having 0.0-microns porosity or 0.25-microns porosity. Thus it is apparent that a strong relationship exists between membrane porosity, patency, and in situ vascularization allowing for the proliferation of cells and collagen.     

Vascularized acellular dermal matrix island flaps for the repair of abdominal muscle defects

The potential widespread use of tissue-engineered matrices in soft-tissue reconstruction has been limited by the difficulty in fabricating and confirming a functional microcirculation. Acellular dermal matrix placed in a soft-tissue pocket acts as a scaffold to be incorporated by the host's fibrovascular tissue. A new method for noninvasive real-time observation of functional microvascular networks using orthogonal polarization spectral (OPS) imaging has recently been reported. Arterioles, venules, and capillaries can be directly visualized, and the movement of individual blood cells through them can be observed. The present study was performed to investigate the use of prefabricated acellular dermal matrix with an arteriovenous unit for the repair of abdominal muscle defects. OPS imaging was used to determine the presence of a functional microcirculation in the neovascularized matrix. In Sprague-Dawley rats, vascularized matrix was prefabricated by placing the superficial epigastric artery and vein on a 2-cm x 2-cm implant-type acellular dermal matrix in the thigh. Three weeks after implantation, the matrix-arteriovenous unit was elevated as an axial-type flap and a 2-cm x 2-cm full-thickness block of abdominal muscle immediately superior to the inguinal ligament was resected. Additional procedures were performed according to group: no repair (group 1, n = 20); repair with nonvascularized acellular dermal matrix (group 2, n = 20); repair with devascularized acellular dermal matrix (group 3, = 20); and repair with vascularized acellular dermal matrix (group 4, n = 20). OPS imaging (field of view, 1 mm in diameter; scan depth range, 0.2 mm) was performed on both sides of each flap on a total of 10 random distal regions before and after pedicle transection in group 3 and with the pedicle preserved in group 4. Hernia rate and duration of survival were compared for 21 days. OPS imaging showed directional blood cell movement through the capillary network in all areas scanned in group 4. No microvascular perfusion was observed after pedicle transection in group 3. Hernia rates of 100, 80, 90, and 0 percent were seen in groups 1, 2, 3, and 4, respectively. Median survival times of 9, 11.5, 9, and 21 postoperative days were noted in groups 1, 2, 3, and 4, respectively. Histopathologic analysis with factor VIII revealed full-thickness infiltration of the matrix by endothelial cells, signifying newly formed blood vessels. Repair of abdominal muscle defects using vascularized acellular dermal matrix resulted in no hernia and survival of all animals for the duration of study. However, repairs using avascular or devascularized matrix resulted in significant rates of hernia and decreased survival. Acellular dermal matrix can be prefabricated into vascularized tissue using an arteriovenous unit and used successfully to repair abdominal muscle defects. OPS imaging allowed for high-contrast direct visualization of microcirculation in previously acellular tissue following prefabrication with an arteriovenous unit.     

Prefabricated vascularized bone flap: a tissue transformation technique for bone reconstruction

In this study, an attempt was made to transform a muscle vascularized pedicle raised on host vessels into a vascularized bone flap, using recombinant human bone morphogenetic protein 2 (rhBMP-2). The purpose of this study was to produce new bone vascularized in nature to increase the survival rate of the subsequently grafted bone and to fabricate the newly formed bone into the desired shape. Silicone molds in the shape of a rat mandible were used to deliver rat bone matrix impregnated with or without rhBMP-2. A muscle pedicle the same size as the mold was raised on the saphenous vessels in the rat thigh and then sandwiched in the center of the silicone molds. The molds were sliced in half and each section was filled with rat bone matrix that was impregnated either with 25 microg of rhBMP-2 for the experimental group or with diluting material alone for the control group. The sandwiched flaps were then secured by tying them to the adjacent muscles and were harvested at 2 and 4 weeks after surgery. Three and six rats were used in the control and experimental groups at each time point, respectively. Bone formation was assessed in the ex vivo specimens by macroscopic, radiologic, and histologic evaluation. Macroscopically, the continuation of the vascular pedicle was clearly visible for both the control and experimental muscle flaps. However, no evidence of muscle-tissue transformation was observed in the control flaps, whereas all the flaps treated with rhBMP-2 produced new bone that replicated the shape of the mold exactly and had saphenous vessels supplying the newly formed bone. This study demonstrates that this experimental model has the potential to be therapeutically applied for effective bone reconstruction.     

Fibrin gel-immobilized VEGF and bFGF efficiently stimulate angiogenesis in the AV loop model

The modulation of angiogenic processes in matrices is of great interest in tissue engineering. We assessed the angiogenic effects of fibrin-immobilized VEGF and bFGF in an arteriovenous loop (AVL) model in 22 AVLs created between the femoral artery and vein in rats. The loops were placed in isolation chambers and were embedded in 500 microL fibrin gel (FG) (group A) or in 500 microL FG loaded with 0.1 ng/microL VEGF and 0.1 ng/microL bFGF (group B). After two and four weeks specimens were explanted and investigated using histological, morphometrical, and ultramorphological [scanning electron microscope (SEM) of vascular corrosion replicas] techniques. In both groups, the AVL induced formation of densely vascularized connective tissue with differentiated and functional vessels inside the fibrin matrix. VEGF and bFGF induced significantly higher absolute and relative vascular density and a faster resorption of the fibrin matrix. SEM analysis in both groups revealed characteristics of an immature vascular bed, with a higher vascular density in group B. VEGF and bFGF efficiently stimulated sprouting of blood vessels in the AVL model. The implantation of vascular carriers into given growth factor-loaded matrix volumes may eventually allow efficient generation of axially vascularized, tissue-engineered composites.     

Differential effects of interferon gamma and alpha on in vitro model of angiogenesis.

The formation of blood vessels in vivo (angiogenesis) is an important process and is usually initiated in response to injury, tumor growth, or normal tissue development. We have studied the effect of human interferon (IFN) alpha (alpha) and gamma (gamma) on the capillary-like network formation in an in vitro model of angiogenesis using human umbilical vein endothelial cells (HUVEC). When HUVEC cells are plated on Matrigel (reconstituted basement membrane matrix enriched in laminin), a network of capillary like structures (endotubes) rapidly forms. IFN-alpha enhanced the tube formation in a dose-dependent manner, whereas IFN-gamma significantly inhibited the tube formation. In addition, both the enhancement and inhibition of angiogenesis by IFN-alpha and gamma was found to be greater if the cells were pretreated with IFN for 12 hr before plating on the Matrigel. These results suggest that IFN may play an important role in several vascular processes including early stages of wound healing, recanalization of thrombi, tumor growth, metastasis, normal growth, and development.     

Visfatin promotes angiogenesis by activation of extracellular signal-regulated kinase 1/2

Adipose tissue is highly vascularized and requires the angiogenic properties for its mass growth. Visfatin has been recently characterized as a novel adipokine, which is preferentially produced by adipose tissue. In this study, we report that visfatin potently stimulates in vivo neovascularization in chick chorioallantoic membrane and mouse Matrigel plug. We also demonstrate that visfatin activates migration, invasion, and tube formation in human umbilical vein endothelial cells (HUVECs). Moreover, visfatin evokes activation of the extracellular signal-regulated kinase 1/2 (ERK1/2) in endothelial cells, which is closely linked to angiogenesis. Inhibition of ERK activation markedly decreases visfatin-induced tube formation of HUVECs and visfatin-stimulated endothelial cell sprouting from rat aortic rings. Taken together, these results demonstrate that visfatin promotes angiogenesis via activation of mitogen-activated protein kinase ERK-dependent pathway and suggest that visfatin may play important roles in various pathophysiological angiogenesis including adipose tissue angiogenesis.     

Peripheral neovascularization of muscle and musculocutaneous flaps in the pig.

Late loss of free muscle flaps following surgical or accidental trauma to the dominant vascular pedicle has been reported. In this study, time-dependent ligation of the dominant vascular pedicle was undertaken in denervated latissimus dorsi musculocutaneous or muscle-only island flaps in the pig. Muscle flaps were covered with a skin graft, and silicon rubber sheets were inserted between the flaps and their bases to simulate a poorly vascularized bed. Hemodynamic and viability studies were then performed using intravenous fluorescein (skin viability), tetrazolium blue (muscle viability), and radiolabeled 15-micron microspheres (capillary blood flow). Blood flow did not change in acutely raised musculocutaneous flaps (n = 10) but was significantly elevated in acutely raised muscle-only flaps (n = 10), suggesting that the skin paddle may steal blood flow from the underlying muscle in musculocutaneous flaps. Peripheral neovascularization at 1 day to 8 weeks was assessed (n = 30). Viability increased during the first week of revascularization and was not different in musculocutaneous and muscle-only flaps. Revascularization of muscle-only flaps was enhanced compared with musculocutaneous flaps in the 2- to 8-week period.     

Strategic considerations on the configuration of free flaps and their vascular pedicles combined with Ilizarov distraction in the lower extremity

For the injury of the lower leg associated with both bone and soft-tissue defect, the combined free flap and the Ilizarov distraction method were described as a useful treatment modality. During the procedure of distraction, however, revisions were frequently needed to change the pin position or to change the flap configuration. In case of flap ischemia, distraction should be delayed or abandoned. Then, a vascularized bone transfer might be necessary. To avoid these complications and achieve safe distraction, the configuration of the flap with its vascular pedicle should be carefully planned in terms of the future bony lengthening procedures and the concomitant soft-tissue changes of the lower leg. According to the response of local tissue to the distraction process, the lower limb can be divided into four compartments (active mobile, passive mobile, receptive, and restrictive). The configuration of the transferred free flap with its vascular pedicle can be classified into five types. To minimize the undue forces to the vascular pedicle and reduce the possibility of vascular compromise, the transferred free flap should have the configuration that its vascular pedicle lies in the territory of the mobile compartment. In performing free-tissue transfer combined with the Ilizarov method in the lower extremity, the configuration of the flap with its vascular pedicle should be carefully planned, and the characteristics of lower leg tissue should be kept in mind during the distraction.     

A novel non-containing-nitrogen bisphosphonate inhibits both in vitro and in vivo angiogenesis

Bisphosphonates (BP) are powerful inhibitors of bone resorption and are widely used in the treatment of patients with metastasis-induced osteolysis. In the present study, we show that a novel non-nitrogen-containing BP (BP7033) that exhibits antitumor activity is a potent inhibitor of both in vivo and in vitro angiogenesis. When administered to mice, BP7033 inhibited tumoral angiogenesis (65% at 0.06mg/injection) as well as tumor growth (65% at 0.006mg/injection) in a tumor model of A431 cells xenografted in nude mice, with no sign of toxicity. Additionally, in vivo angiogenesis induced by vascular endothelial growth factor-containing Matrigel implants was reduced by 90% in the presence of BP7033 (0.6mg/plug). In vitro, BP7033 inhibited proliferation of human umbilical vein endothelial cells (HUVEC) (IC(50) value 3x10(-4) M) and completely prevented the formation of capillary-like tubules by HUVEC in Matrigel. Moreover, treatment of A431 cells by BP7033 induced an inhibition of Ras processing and a decrease in the secretion of both vascular endothelial growth factor and matrix metalloproteinase-2, two well-known stimulators of the proliferation and migration of endothelial cells. These findings indicate that this new BP compound has marked antiangiogenic properties and thus represents a promising candidate for treatment of malignant diseases with an angiogenic component.     

Flow-through anterior thigh flaps with a short pedicle for reconstruction of lower leg and foot defects

New flow-through perforator flaps with a large, short vascular pedicle are proposed because of their clinical significance and a high success rate for reconstruction of the lower legs. Of 13 consecutive cases, the authors describe two cases of successful transfer of a new short-pedicle anterolateral or anteromedial thigh flow-through flap for coverage of soft-tissue defects in the legs. This new flap has a thin fatty layer and a small fascial component, and is vascularized with a perforator originating from a short segment of the descending branch of the lateral circumflex femoral system. The advantages of this flap are as follows: flow-through anastomosis ensures a high success rate for free flaps and preserves the recipient arterial flow; there is no need for dissecting throughout the lateral circumflex femoral system as the pedicle vessel; minimal time is required for flap elevation; there is minimal donor-site morbidity; and the flap is obtained from a thin portion of the thigh. Even in obese patients, thinning of the flap with primary defatting is possible, and the donor scar is concealed. This flap is suitable for coverage of defects in legs where a single arterial flow remains. It is also suitable for chronic lower leg ulcers, osteomyelitis, and plantar coverage.     

Tissue engineering skin flaps: which vascular carrier, arteriovenous shunt loop or arteriovenous bundle, has more potential for angiogenesis and tissue generation?

This study was designed to clarify which vascular carrier, the arteriovenous shunt loop or the arteriovenous bundle, has more potential as a vascular carrier for an artificial skin flap in rats. An arteriovenous shunt loop was constructed between the femoral artery and vein using an interpositional artery (group I) or vein (group II) graft. For arteriovenous bundle groups, the femoral artery and vein were used and subdivided into two groups: distal ligation type (group III) and flow-through type (group IV). The vascular pedicle was wrapped with an artificial dermis and implanted beneath the inguinal skin for 4 weeks. For the control group, a folded sheet of artificial dermis without any vascular carrier was embedded. In experiment 1, the volumes of generated tissue within the artificial dermis were measured in the experimental and control groups (n = 5 in each group). In experiment 2, the origin of new blood vessels sprouting from the arteriovenous shunt loop and arteriovenous bundle were evaluated histologically. The volume of generated tissue in the shunt groups was significantly greater than that in the bundle groups (p < 0.01). However, the bundle groups also showed a great potential for producing new tissue. Serial histological studies showed that new capillaries were derived not only from the vasa vasorum of the femoral vessels but directly from the femoral vein in both the shunt and the bundle groups. This "sprouting" was extensively exhibited in the group III. Although the arteriovenous shunt loop showed a greater potential for producing new tissue and capillaries, the distal ligation type of bundle was thought to be an effective and practical vascular carrier for producing a tissue-engineered skin flap.     

Free anterolateral thigh adipofascial perforator flap

The anterolateral thigh adipofascial flap is a vascularized flap prepared from the adipofascial layer of the anterolateral thigh region. It is a perforator flap based on septocutaneous or musculocutaneous perforators of the lateral circumflex femoral system. With methods similar to those used for the free anterolateral thigh flap, only the deep fascia of the anterolateral thigh and a 2-mm-thick to 3-mm-thick layer of subcutaneous fatty tissue above the fascia were harvested. In 11 cases, this flap (length, 5 to 11 cm; width, 4 to 8 cm) was used for successful reconstruction of extremity defects. Split-thickness skin grafts were used to immediately resurface the adipofascial flaps for eight patients, and delayed skin grafting was performed for the other three patients. The advantage of the anterolateral thigh adipofascial flap is its ability to provide vascularized, thin, pliable, gliding coverage. In addition, the donor-site defect can be closed directly. Other advantages of this flap, such as safe elevation, a long wide vascular pedicle, a large flap territory, and flow-through properties that allow simultaneous reconstruction of major-vessel and soft-tissue defects, are the same as for the conventional anterolateral thigh flap. The main disadvantage of this procedure is the need for a skin graft, with the possible complications of subsequent skin graft loss or hyperpigmentation.     

Effect of vascular delay on viability, vasculature, and perfusion of muscle flaps in the rabbit

The delay procedure is known to augment pedicled skin or muscle flap survival. In this study, we set out to investigate the effectiveness of vascular delay in two rabbit muscle flap models. In each of the muscle flap models, a delay procedure was carried out on one side of each rabbit (n = 20), and the contralateral muscle was the control. In the latissimus dorsi flap model, two perforators of the posterior intercostal vessels were ligated. In the biceps femoris flap model, a dominant vascular pedicle from the popliteal artery was ligated. After the 7-day delay period, the bilateral latissimus dorsi flaps (based on the thoracodorsal vessels) and the bilateral biceps femoris flaps (based on the sciatic vessels) were elevated. Animals were divided into three groups: part A, assessment of muscle flap viability at 7 days using the tetrazolium dye staining technique (n = 7); part B, assessment of vascular anatomy using lead oxide injection technique (n = 7); and part C, assessment of total and regional capillary blood flow using the radioactive microsphere technique (n = 6). The results in part A show that the average viable area of the latissimus dorsi flap was 96 +/- 0.4 percent (mean +/- SEM) in the delayed group and 84 +/- 0.7 percent (mean +/- SEM) in the control group (p < 0.05, n = 7), and the mean viable area of the biceps femoris flap was 95 +/- 2 percent in the delayed group and 78 +/- 5 percent in the control group (p < 0.05, n = 7). In part B, it was found that the line of necrosis in the latissimus dorsi flap usually appeared at the junction between the second and third vascular territory in the flap. Necrosis of the biceps femoris flap usually occurred in the third territory, and occasionally in both the second and the third territories. In Part C, total capillary blood flow in delayed flaps (both the latissimus dorsi and biceps femoris) was significantly higher than that in the control flaps (p < 0.05). Increased regional capillary blood flow was found in the middle and distal regions, compared with the control (p < 0.05, n = 6). In conclusion, ligation of either the dominant vascular pedicle in the biceps femoris muscle flap or the nondominant pedicle in the latissimus dorsi muscle flap in a delay procedure 1 week before flap elevation improves capillary blood flow and muscle viability. Vascular delay prevents distal flap necrosis in two rabbit muscle flap models.     

Successful transplantation of three tissue-engineered cell types using capsule induction technique and fibrin glue as a delivery vehicle

Recent advances in cell biology and tissue engineering have used various delivery vehicles for transplanting varying cell cultures with limited success. These techniques are frequently complicated by tissue necrosis, infection, and resorption. The purpose of this study was to investigate whether urothelium cells, tracheal epithelial cells, and preadipocytes cultured in vitro could be successfully transplanted onto a prefabricated capsule surface by using fibrin glue as a delivery vehicle, with the ultimate goal for use in reconstruction. In the first step of the animal study, tissue specimens (bladder urothelium, tracheal epithelial cells, epididymal fat pad) were harvested for in vitro cell culturing, and a silicone block was implanted subcutaneously or within the anterior rectus sheath to induce capsule formation. After 6 to 10 days, when primary cultures were confluent, the animals were re-anesthetized, the newly formed capsule pouches were incised, and the suspensions of cultured urothelia cells (n = 40), tracheal epithelial cells (n = 32), and preadipocytes (n = 40) were implanted onto the capsule surface in two groups, one using standard culture medium as a delivery vehicle and the second using fibrin glue. Histologic sections were taken, and different histomorphologic studies were performed according to tissue type. Consistently in all animals, a highly vascularized capsule was induced by the silicon material. In all animals in which the authors used fibrin glue as a delivery vehicle, they could demonstrate a successful reimplantation of cultured urothelium cells, tracheal epithelial cells, or preadipocytes. Their animal studies showed that capsule induction in combination with fibrin glue as a delivery vehicle is a successful model for transplantation of different in vivo cultured tissue types.     

Cooperation between osteoblastic cells and endothelial cells enhances their phenotypic responses and improves osteoblast function

Osteogenesis requires close co-operation with angiogenesis to create vascularized bone tissue. In this study, an indirect co-culture model using osteoblasts (OBs), primary endothelial cells (ECs) and Matrigel interlayer was established to understand the impact of each cell type on the other. ECs synergistically enhanced osteoblastic gene expression by OBs, while OBs were capable of supporting tubule-like structures formed by ECs on Matrigel, enhancing mean tubule length from 146.5 ± 23.5 μm in ECs alone to 192 ± 28.6 μm in co-culture (p < 0.05). Similar improvements were noted in terms of tubule number. An applicability study of the co-culture model to bone tissue engineering, performed on a biopolymer fibrous membrane, showed substantially enhanced deposition of calcified nodules. These results demonstrate the efficacy of co-culture with ECs to improve osteogenesis for bone tissue engineering.     

Inhibition of Hyaluronic Acid Synthesis Suppresses Angiogenesis in Developing Endometriotic Lesions

Background

The development and long-term survival of endometriotic lesions is crucially dependent on an adequate vascularization. Hyaluronic acid (HA) through its receptor CD44 has been described to be involved in the process of angiogenesis.

Objective

To study the effect of HA synthesis inhibition using non-toxic doses of 4-methylumbelliferone (4-MU) on endometriosis-related angiogenesis.

Materials and Methods

The cytotoxicity of different in vitro doses of 4-MU on endothelial cells was firstly tested by means of a lactate dehydrogenase assay. The anti-angiogenic action of non-cytotoxic doses of 4-MU was then assessed by a rat aortic ring assay. In addition, endometriotic lesions were induced in dorsal skinfold chambers of female BALB/c mice, which were daily treated with an intraperitoneal injection of 0.9% NaCl (vehicle group; n = 6), 20mg/kg 4-MU (n = 8) or 80mg/kg 4-MU (n = 7) throughout an observation period of 14 days. The effect of 4-MU on their vascularization, survival and growth were studied by intravital fluorescence microscopy, histology and immunohistochemistry.

Main Results

Non-cytotoxic doses of 4-MU effectively inhibited vascular sprout formation in the rat aortic ring assay. Endometriotic lesions in dorsal skinfold chambers of 4-MU-treated mice dose-dependently exhibited a significantly smaller vascularized area and lower functional microvessel density when compared to vehicle-treated controls. Histological analyses revealed a downregulation of HA expression in 4-MU-treated lesions. This was associated with a reduced density of CD31-positive microvessels within the lesions. In contrast, numbers of PCNA-positive proliferating and cleaved caspase-3-positive apoptotic cells did not differ between 4-MU-treated and control lesions.

Conclusions

The present study demonstrates for the first time that targeting the synthesis of HA suppresses angiogenesis in developing endometriotic lesions. Further studies have to clarify now whether in the future this anti-angiogenic effect can be used beneficially for the treatment of endometriosis.     

The behavior of fat grafts in recipient areas with enhanced vascularity

Fat grafts are used for soft-tissue augmentation of various anatomic regions, most frequently for the improvement of facial contours. Resorption of the graft is the main problem, and several different procedures have been described to minimize this phenomenon. Using 25 New Zealand rabbits, the behavior of fat grafts in a highly vascularized recipient site was studied. The recipient sites prepared on the backs of the rabbits were divided into four regions. A capsule formation with silicone sheet application was accomplished in two of these recipient areas before the transplantation of the fat grafts. Fat grafts were placed in the other two recipient areas without any prior preparation. We prepared two types of fat tissue; in one the lobular structure was preserved and in the other it was manually crushed and rinsed with lactated Ringer's solution. The fat tissues with preserved lobular structure were placed in area I and area III. Manually crushed and rinsed fat tissues were placed in area II and area IV. In areas III and IV, a capsule formation with silicone sheet had been accomplished 3 weeks before grafting. Biopsy samples were obtained from these sites at the end of the first, third, sixth, and tenth months. Our aim was to observe the histologic fate of fat tissue in different recipient areas. The macroscopic and microscopic evaluation of the fat grafts in areas with silicone sheet indicated significant differences in the resorption time of the fat grafts; however, it was concluded that the significant resorption of the transplanted autologous fat tissue grafts at the end of the first year was an inevitable consequence of fat grafting.     

Vasculogenic and angiogenic potential of adipose stromal vascular fraction cell populations in vitro

Adipose-derived stromal vascular fraction (SVF) is a heterogeneous cell source that contains endothelial cells, pericytes, smooth muscle cells, stem cells, and other accessory immune and stromal cells. The SVF cell population has been shown to support vasculogenesis in vitro as well vascular maturation in vivo. Matrigel, an extracellular matrix (ECM) mixture has been utilized in vitro to evaluate tube formation of purified endothelial cell systems. We have developed an in vitro system that utilizes freshly isolated SVF and ECM molecules both in pure form (fibrin, laminin, collagen) as well as premixed form (Matrigel) to evaluate endothelial tip cell formation, endothelial stalk elongation, and early stages of branching and inosculation. Freshly isolated SVF rat demonstrate cell aggregation and clustering (presumptive vasculogenesis) on Matrigel ECM within the first 36 h of seeding followed by tip cell formation, stalk cell formation, branching, and inosculation (presumptive angiogenesis) during the subsequent 4 days of culture. Purified ECM molecules (laminin, fibrin, and collagen) promote cell proliferation but do not recapitulate events seen on Matrigel. We have created an in vitro system that provides a functional assay to study the mechanisms of vasculogenesis and angiogenesis in freshly isolated SVF to characterize SVF’s blood vessel forming potential prior to clinical implantation.