To investigate the state and significance of bone marrow angiogenesis in hematological diseases, bone marrow microvascular density (BM-MVD) in plastic-embedded section was examined using acetone-fixed bone marrow tissues embedded in glycol-methacrylate (GMA) resin and by the method of immunohistochemistry. The results showed that bone marrow MVD increased greatly in newly diagnosed hematological malignancies before treatment. BM-MVD in patients with acute leukemia decreased down to the normal range as the controls at the time of complete remission. In the non-remission group, BM-MVD decreased less, but when relapsed it increased again up to the same range as the newly diagnosed hematological malignancies, significant increase of BM-MVD was found in patients with anemia, but in less degree than that in hematological malignancies. It is concluded that bone marrow angiogenesis plays a key role in the pathogenesis and development of hematological malignancy. Antiangiogenic therapy may be able to constitute a novel strategy for the treatment of hematological malignancies including leukemia.
Acute Disease ; Bone Marrow ; blood supply ; pathology ; Hematologic Diseases ; blood ; pathology ; Humans ; Leukemia ; blood ; pathology ; Microcirculation ; Neovascularization, Pathologic ; blood ; pathology

OBJECTIVETo evaluate the therapeutic effect of bone morphogenetic protein 2 (BMP-2) gene modified tissue engineering bone (GMB) combined with vascularized periosteum in the reconstruction of segmental bone defect.

METHODSAdenovirus carrying BMP-2 gene (Ad-BMP-2) was transfected into the isolated and cultured rabbit bone marrow stromal cells (MSCs). The transfected MSCs were seeded on bovine cancellous bone scaffolds (BCB) to construct gene modified tissue engineering bone (GMB). The bilateral rabbits radial defects (2.5 cm long) were created as animal model. The rabbits were divided into five groups to reconstruct the defects with CMB combined with vascularized periosteum (group A); or GMB combined with vascular bundle implantation (group B); or GMB combined with free periosteum (group C); or GMB only (group D); or BCB scaffolds only (group E). Angiogenesis and osteogenesis were observed by X-ray, histological examination, biomechanical analysis and capillary ink infusion.

RESULTSIn group A, the grafted GMB was revascularized rapidly. The defect was completely reconstructed at 8 weeks. The mechanism included both intramemerbrane and endochondral ossification. In group B, the vascular bundle generated new blood vessels into the grafted GMB, but the osteogenesis process was slow in the central zone, which healed completely at 12 weeks. In group C, the free graft of periosteum took at 4 weeks with angiogenesis. The thin extremal callus was formed at 8 weeks and the repairing process almost finished at 12 weeks. Better osteogenesis was found in group D than in group E, due to the present of BMP2 gene-transfected MSCs. The defects in group D were partial repaired at 12 weeks with remaining central malunion zone. The defects in group E should nonunion at 12 weeks with only fibre tissue.

CONCLUSIONSBMP-2 gene modified tissue engineering bone combined with vascularized periosteum which provides periosteum osteoblasts as well as blood supply, has favorable ability of osteogenesis, osteoinduction and osteoconduction. It is an ideal method for the treatment of segmental bone defect.

Animals ; Bone Marrow Cells ; cytology ; Bone Morphogenetic Protein 2 ; genetics ; Bone Regeneration ; Bone Substitutes ; Bone Transplantation ; methods ; Bone and Bones ; pathology ; Cattle ; Mesenchymal Stromal Cells ; cytology ; Periosteum ; blood supply ; transplantation ; Rabbits ; Surgical Flaps ; blood supply ; Tissue Engineering ; methods ; Tissue Scaffolds ; Transfection

OBJECTIVETo study whether tissue engineered bone can repair the large segment bone defect of large animal or not. To observe what character the fascia flap played during the osteanagenesis and revascularization process of tissue engineered bone.

METHODS9 Chinese goats were made 2 cm left tibia diaphyseal defect. The repairing effect of the defects was evaluated by ECT, X-ray and histology. 27 goats were divided into three groups: group of CHAP, the defect was filled with coral hydroxyapatite (CHAP); group of tissue engineered bone, the defect was filled with CHAP + bone marrow stroma cells (BMSc); group of fascia flap, the defect was filled with CHAP + BMSc + fascia flap. After finished culturing and inducing the BMSc, CHAP of group of tissue engineered bone and of fascia flap was combined with it. Making fascia flap, different materials as described above were then implanted separately into the defects. Radionuclide bone imaging was used to monitor the revascularization of the implants at 2, 4, 8 weeks after operation. X-ray examination, optical density index of X-ray film, V-G staining of tissue slice of the implants were used at 4, 8, 12 weeks after operation, and the biomechanical character of the specimens were tested at 12 weeks post operation.

RESULTSIn the first study, the defect showed no bone regeneration phenomenon. 2 cm tibia defect was an ideal animal model. In the second study, group of CHAP manifested a little trace of bone regeneration, as to group of tissue engineered bone, the defect was almost repaired totally. In group of fascia flap, with the assistance of fascia flap which gave more chance to making implants to get more nutrient, the repair was quite complete.

CONCLUSIONSThe model of 2 cm caprine tibia diaphyseal defect cannot be repaired by goat itself and can satisfy the tissue engineering's demands. Tissue engineered bone had good ability to repair large segment tibia defect of goat. Fascia flap can accelerate the revascularization process of tissue engineered bone. And by this way, it augment the ability of tissue engineered bone to repair the large bone defect of goat.

Animals ; Bone Marrow Cells ; cytology ; Bone Marrow Transplantation ; Bone Regeneration ; physiology ; Bone Substitutes ; Cells, Cultured ; Durapatite ; Fascia ; transplantation ; Goats ; Implants, Experimental ; Neovascularization, Physiologic ; Osteogenesis ; Random Allocation ; Stromal Cells ; cytology ; Tibia ; blood supply ; injuries ; surgery ; Tibial Fractures ; surgery ; Tissue Engineering

BACKGROUNDBone marrow transplantation (BMT) conditioning procedure is considered as the cause of damage to bone marrow microvasculature and the delay of hematopoiesis recovery. However, hematopoiesis regulation post BMT by vascular endothelial growth factor (VEGF) has not yet been studied. In this study, adenovirus were used to investigate the effects of VEGF gene transfer on preventing damages to bone marrow microenvironment and its promotion of hematopoiesis in post-BMT mice.

METHODSRecombinant adenovirus (Ad)-enhanced green fluorescent protein (EGFP)/hVEGF165 was injected via tail vein into BALB/c mice undergoing syngeneic BMT. During the different phases post BMT, the distribution of adenovirus and the plasma levels of hVEGF were measured as well as the numbers of white blood cells (WBC), platelet (PLT) and red blood cells (RBC) in peripheral blood. At the same time, the mice were injected with Chinese ink via tail vein, following which the tibias were separated and were used for analysis of bone marrow microvasculature surface area and cellularity.

RESULTSSignificant expression of EGFP and hVEGF was observed in multiple organs at different phases post BMT, and the plasma level of hVEGF was up to (866.67 +/- 97.13) pg/ml. The recovery of WBC, PLT and RBC of the group treated with recombinant adenovirus Ad-EGFP/hVEGF165 were significantly more rapid than those of other BMT groups (P < 0.05, respectively). At the 20th day post BMT, the percentage of bone marrow microvasculature surface area in group treated with VEGF [(61.2 +/- 4.0)%] returned to normal level [(62.0 +/- 5.0)%, P > 0.05]. The restoration of hematopoiesis was retarded more than that of microvasculature. The cellularity of bone marrow in each group was still lower than that of normal control [(62.3 +/- 4.0)%, P < 0.05] at the 30th day post BMT, but the percentage in group treated with VEGF at the 20th and 30th days post BMT [(46.5 +/- 5.0)% and (55.1 +/- 4.5)%] exceeded those of other BMT groups (P < 0.05, respectively).

CONCLUSIONVEGF gene transfer mediated by adenovirus may protect the hematopoietic microenvironment to promote the restoration of hematopoiesis in post-BMT mice.

Adenoviridae ; genetics ; Animals ; Bone Marrow ; blood supply ; Bone Marrow Transplantation ; Female ; Gene Transfer Techniques ; Genetic Therapy ; Hematopoiesis ; Male ; Mice ; Mice, Inbred BALB C ; Microcirculation ; Vascular Endothelial Growth Factor A ; blood ; genetics

BACKGROUNDMany treatment options for lower limb ischemia are difficult to apply for the patients with poor arterial outflow or with poor general conditions. The effect of medical treatment alone is far from ideal, especially in patients with diabetic foot. A high level amputation is inevitable in these patients. This study aimed to explore the effect of transplantation of autologous bone marrow mononuclear cells on the treatment of lower limb ischemia and to compare the effect of intra-arterial transplantation with that of intra-muscular transplantation.

METHODSIn this clinical trial, 32 patients with lower limb ischemia were divided into two groups. Group 1 (16 patients with 18 affected limbs) received transplantation of autologous bone marrow mononuclear cells by intra-muscular injection into the affected limbs; and group 2 (16 patients with 17 affected limbs) received transplantation of autologous bone marrow mononuclear cells by intra-arterial injection into the affected limbs. Rest pain, coldness, ankle/brachial index (ABI), claudication, transcutaneous oxygen pressure (tcPO(2)) and angiography (15 limbs of 14 patients) were evaluated before and after the mononuclear cell transplantation to determine the effect of the treatment.

RESULTSTwo patients died from heart failure. The improvement of rest pain was seen in 76.5% (13/17) of group 1 and 93.3% (14/15) of group 2. The improvement of coldness was 100% in both groups. The increase of ABI was 44.4% (8/18) in group 1 and 41.2% (7/17) in group 2. The value of tcPO(2) increased to 20 mmHg or more in 20 limbs. Nine of 15 limbs which underwent angiography showed rich collaterals. Limb salvage rate was 83.3% (15/18) in group 1 and 94.1% (16/17) in group 2. There was no statistically significant difference in the effectiveness of the treatment between the two groups.

CONCLUSIONSTransplantation of autologous bone marrow mononuclear cells is a simple, safe and effective method for the treatment of lower limb ischemia, and the two approaches for the implantation, intra-muscular injection and intra-arterial injection, show similar results.

Aged ; Aged, 80 and over ; Blood Gas Monitoring, Transcutaneous ; Bone Marrow Cells ; cytology ; Bone Marrow Transplantation ; Female ; Humans ; Ischemia ; therapy ; Leg ; blood supply ; Leukocytes, Mononuclear ; transplantation ; Male ; Middle Aged ; Transplantation, Autologous

To investigate the role of angiogenesis in multiple myeloma (MM), bone marrow biopsy from 75 adults with newly diagnosed, untreated MM were evaluated. Microvessels were scored in at least 3 areas ( x 200 fields) of the highest microvessel density in representative sections of each bone marrow specimen using immunohistochemistry for CD34. Prognostic variables were also evaluated for the overall survival. Microvessel counts were significantly higher in patients with MM (n=69.42+/-9.67), compared with control (n=26.81+/-2.85). Microvessel density had a weak correlation with percentage of bone marrow plasma cells. By univariate analysis, age, beta2-microglobulin, serum albumin, serum creatinine, serum calcium, hemoglobin, platelet count, and bone marrow plasma cell percentage were correlated with survival. By multivariate analysis, age, serum albumin, serum creatinine, hemoglobin, platelet count and bone marrow plasma cell percentage were correlated with overall survival, whereas microvessel density was not. In summary, microvessel density in bone marrow of MM is significantly increased compared to control, but was not correlated with overall survival. Further studies regarding angiogeneic molecules are needed to determine the functional role of angiogenesis in MM.
Adult ; Aged ; Aged, 80 and over ; Bone Marrow/blood supply ; Endothelial Growth Factors/physiology ; Female ; Hematopoietic Stem Cell Transplantation ; Human ; Lymphokines/physiology ; Male ; Microcirculation ; Middle Age ; Multiple Myeloma/therapy ; Multiple Myeloma/mortality ; Multiple Myeloma/blood supply* ; Neovascularization, Pathologic/physiopathology* ; Survival Rate

Generation of therapeutic angiogenesis to enhance vascularization in the ischemic tissues is a method for treating ischemic tissues in atherosclerotic cardiovascular artery disease. The chemokine stromal cell-derived factor-1 (SDF-1) and its receptor (CXC chemokine receptor 4, CXCR4) play a critical role in the process of post-natal neovascularization. The SDF-1-CXCR4 axis is a potential mechanism for the treatment of ischemic limb. Here, we investigated the role of CXCR4 in bone marrow cells (BMCs) in neovascularization induced by tumor cells and the supernatant of culture media in a murine hind-limb ischemia model which was made by resecting femoral artery and vein. After the injection of mouse melanoma cells B16-F10 (1x10(6) cells in 0.1 mL at the operation day, s.c.) into the abdomen or the cell culture supernatant (0.1 mL/d for 21 d after operation, i.m.) into the ischemic abductor muscle, the CXCR4 positive BMCs were analyzed by flow cytometry. The perfusion of the ischemic limb was evaluated by laser Doppler perfusion imaging (LDPI) on 7, 14 and 21 d after vascular injury operation. Capillary endothelial alkaline phosphatase (AP) was stained to quantify the presence of capillaries, and histological method was used to evaluate the capillary density as a measure of neovascularization in ischemic tissues. The proportions of CXCR4 positive BMCs were notably higher in ischemic limb injected with tumor cells or the supernatant compared to those in the control group (P<0.05). Injection of tumor cells or the supernatant resulted in significantly improved perfusion as measured by LDPI perfusion ratios on 7, 14 and 21 d after femoral artery and vein resection in mice, compared to the controls (P<0.05). Tissue samples harvested from the lower calf muscle at day 21 demonstrated increased capillary densities in mice receiving tumor cells (0.81+/-0.13) or the supernatant (0.63+/-0.05), compared with those in control group (0.44+/-0.09, P<0.05). In conclusion, the injection of B16-F10 tumor cells or the supernatant induces the increase of CXCR4 positive cells in BMCs and the improvement of in vivo neovasculogenesis in mouse ischemic limb.
Animals ; Bone Marrow Cells ; metabolism ; Capillaries ; Cell Culture Techniques ; Chemokine CXCL12 ; metabolism ; Culture Media, Conditioned ; pharmacology ; Disease Models, Animal ; Endothelium, Vascular ; Hindlimb ; blood supply ; Ischemia ; physiopathology ; Melanoma, Experimental ; Mice ; Muscle, Skeletal ; blood supply ; Neovascularization, Physiologic ; Receptors, CXCR4 ; metabolism

BACKGROUNDRandom flap is one kind of the most widely used skin flaps in reconstructive surgery; however, partial necrosis of its distal end remains a significant problem now. The aim of this study was to evaluate the effect of hypoxia preconditioned bone marrow mesenchymal stem cells (HpBMSCs) transplantation on ultra-long random skin flap survival in rats.

METHODSNormoxic bone marrow mesenchymal stem cells (nBMSCs) were cultured under normoxia (20% O2) and HpBMSCs under hypoxia (1% O2) for 48 hours before transplantation. Thirty Sprague-Dawley rats were randomly divided into control group, nBMSCs group and HpBMSCs group with each consisting of 10 rats. Survival area of ultra-long random skin flap on the dorsal of rats was measured seven days after flap surgery and cell transplantation. Cell survival in vivo, microvessel density and vascular endothelial growth factor (VEGF) were evaluated by histological examination and enzyme-linked immunosorbent assay.

RESULTSCompared with other two groups, flap survival area in HpBMSCs group was significantly larger (P < 0.05). Microvessel density in HpBMSCs group (36.20 ± 8.19) was higher than that in nBMSCs group (30.01 ± 5.68) and control group (17.60 ± 4.19) (P < 0.05). VEGF in HpBMSCs group ((300.05 ± 50.41) pg/g) was higher than those in nBMSCs group ((240.55 ± 33.64) pg/g) and control group ((191.65 ± 32.58) pg/g) (P < 0.05).

CONCLUSIONHpBMSCs transplantation improves ultra-long random skin flap survival via promoting angiogenesis of more survival cells.

Animals ; Bone Marrow Cells ; cytology ; Cell Hypoxia ; physiology ; Cells, Cultured ; Graft Survival ; Mesenchymal Stem Cell Transplantation ; methods ; Mesenchymal Stromal Cells ; cytology ; Rats ; Rats, Sprague-Dawley ; Skin ; blood supply ; Surgical Flaps ; blood supply

OBJECTIVETo study the bone marrow microvessel density (MVD) and vascular endothelial growth factor (VEGF) expression and their clinical significance in patients with aplastic anemia (AA).

METHODSBone marrow biopsies in 51 newly diagnosed patients with AA were evaluated the MVD and VEGF expression by immunostaining with anti-factor VIII related antigen and VEGF monoclonal antibodies at regular time points after immunosuppressive therapy (IT).

RESULTSThe mean bone marrow MVD in AA group was 5.5 +/- 3.5, being significantly lower than that in normal control group (8.7 +/- 3.4, P < 0.05). MVDs of SAA and NSAA patients were 7.4 +/- 2.9 and 4.3 +/- 3.4, respectively, being significantly different (P < 0.01). The VEGF expression in AA group was significantly lower than that in control group [(6.7 +/- 8.4)% vs (14.7 +/- 6.1)%, P < 0.01], but there was no difference between SAA and NSAA. Bone marrow MVD and VEGF were significantly increased after IT in 22 responded AA patients.

CONCLUSIONBone marrow MVD and VEGF expression are low in AA patients which may be one of pathophysiologic mechanisms of bone marrow failure in AA. Proangiogenic and ameliorating microcirculation agents together with IT might accelerate the recovery of hematopoiesis in AA patients.

Adolescent ; Adult ; Anemia, Aplastic ; metabolism ; pathology ; Bone Marrow ; blood supply ; metabolism ; Child ; Female ; Humans ; Male ; Microvessels ; pathology ; Middle Aged ; Neovascularization, Pathologic ; Vascular Endothelial Growth Factor A ; metabolism

OBJECTIVETo explore the effect of arterial perfusion of marrow multifunctional stem cells (MFSC) in treating femoral head necrosis and its mechanism.

METHODSThe rabbit model of femoral head necrosis was established by large dose of methyl-prednisone through Shwartzman response. Bone marrow was extracted from femoral bone of model rabbit and isolated in vitro for culturing and proliferating MFSC. The experimental rabbits were randomly divided into 4 groups, treated with normal saline (A), Salvia + urokinase (B), MFSC (C) and MFSC + Salvia + urokinase (D), respectively, they were sacrificed in batches at 2 and 4 weeks after treatment, and changes in various parameters, including molybdenum target roentgenogram, routine pathology with HE staining, tetracycline labeled fluorescent microscopy and ultrastructure alteration by scanning electron microscope (SEM), were observed.

RESULTSTypical appearance of femoral head necrosis was shown in the successfully modeled rabbits. Two and 4 weeks after treatment by high selective drug via medial and lateral femoral circumflex arterial perfusion, the X-ray examination showed significant improvement of bone density; pathohistologic manifestation showed decrease of empty bone lacuna, increase of osteoblast and new bone formation; tetracycline fluorescent labeled microscopic picture showed bright fluorescent band of increased osteoblasts in necrosis repairing region with widened border; SEM displayed irregularly arranged fibrosis in necrosis region, abundant organelles in osteoblasts with few empty bone lacuna. The above-mentioned improvement was more significant in rabbits treated by MFSC.

CONCLUSIONHigh selective femoral drug arterial perfusion in treating femoral head necrosis could accelerate the process of revascularization and re-ossification in rabbits. As compared with Salvia, MFSC showed quicker and more potent effect.

Animals ; Bone Marrow Transplantation ; methods ; Femur Head ; blood supply ; Femur Head Necrosis ; chemically induced ; physiopathology ; therapy ; Infusions, Intra-Arterial ; Male ; Methylprednisolone ; Osteogenesis ; Rabbits ; Stem Cell Transplantation ; methods