Assessment of the efficacy of intra-articular platelet rich plasma treatment in an ACLT experimental model by dynamic contrast enhancement MRI of knee subchondral bone marrow and MRI T2∗ measurement of articular cartilage.

OBJECTIVE: The vascularization of subchondral bone plays a significant role in the progression of knee osteoarthritis (OA). Treatment with platelet-rich plasma (PRP) has positive effects on cartilage lesions. However, PRP's efficacy for subchondral bone marrow lesions and the relationship of these lesions to cartilage are still undiscovered. Therefore, our aims were first to longitudinally investigate the change in subchondral flow by dynamic contrast enhanced MRI and degeneration of cartilage by MRI T2∗ in an anterior cruciate transection rodent (ACLT) model, and second to examine changes in parameters after intra-articular PRP injection. DESIGN: A 32-week investigation in 18 rats allocated to sham-control, ACLT with normal saline injection (ACLT + NS), and ACLT with PRP injection groups ended with histological evaluation. Another rat was used as a donor of allogenic PRP. RESULTS: Compared to the sham-control group, the ACLT + NS group had higher subchondral blood volume A (0.051, 95% confidence interval: 0.009, 0.092) and lower venous washout kel (-0.030: -0.055, -0.005) from week 4; lower permeability kep from week 18 (-0.954: -1.339, -0.569); higher cartilage T2∗ values (1.803: 1.504, 2.102) reflecting collagen loss beginning at week 10. For the PRP treatment group, subchondral bone marrow A and cartilage T2∗ decreased from week 10. Histological results confirmed and were correlated with the MRI findings. CONCLUSION: Subchondral hyper-perfusion plays a vital role in the pathogenesis of OA and was associated with cartilage degeneration. The efficacy of PRP can be observed from reduced perfusion and MRI T2∗ values.

Study of prognostic significance of marrow angiogenesis assessment in patients with de novo acute leukemia.

BACKGROUND: Angiogenesis is the highly ordered formation of new blood vessels from pre-existing vessels. It is seen throughout growth, in wound healing, menses, and is important in cancer, where pro- and antiangiogenic signals can be released by cancer cells, endothelial cells, stromal cells, blood, and the extracellular matrix. Aim of the study is to use standardized method for counting blood vessels to verify the significance and prognostic value of assessing marrow angiogenesis at diagnosis of de novo acute leukemia. SUBJECTS AND METHODS: The study included 70 newly diagnosed acute leukemia cases and a control group composed of 35 bone marrow biopsy sections obtained from breast cancer patients. Examination of CD34 immunohistochemically stained sections for the assessment of marrow angiogenesis by quantification of its microvessel density (MVD). RESULTS: MVD was significantly increased in acute leukemia patients in comparison to control group (P-value <0.001). Increased MVD was associated with unfavorable outcome. CONCLUSION: The study demonstrated an evidence of increased angiogenesis in acute leukemia detected by high bone marrow MVD which may play a significant role in leukemic process. Understanding its role may help in designing new therapeutic strategies for acute leukemia.

AngioMap is a novel image analysis algorithm for assessment of plasma cell distribution within bone marrow vascular niche.

The ability to characterize distribution of neoplastic hematopoietic cells and their progenitors in their native microenvironment is emerging as an important challenge and potential therapeutic target in many disease areas, including multiple myeloma. In multiple myeloma, bone marrow (BM) angiogenesis is typically increased and microvessel density is a known indicator of poor prognosis. However, the difficulty of consistently measuring 3D vessels from 2D cut sections has previously limited the study of spatial distribution of plasma cells (PC) and their interaction with BM microenvironment. The aim of the study is to report a novel method to study myeloma cells spatial distribution within their hematopoietic niche context using readily available tissue sections and standard histology approaches. We utilized a novel whole-tissue image analysis approach to identify vessels, and then applied computational grown regions extended out from each vessel at 15, 35, 55, 75, and 100 µm to identify the spatial distribution of PC on CD34/CD138 double-stained core biopsy slides. Percent PC to total cells (TC) was significantly higher at <15 µm distance compared with those at 16 to 35, 36 to 55, 56 to 75, and 76 to 100 µm distance (P=0.0001). Similarly, PC/TC at <35 µm region was significantly higher compared with 36 to 55 (P=0.0001), 56 to 75 (P≤0.0001), and 76 to 100 (P=0.0002) µm distances. The mean PC/TC differences in the spatial gradient of 36 to 55, 56 to 75, and 76 to 100 µm distance regions were not significant. Our findings suggest possible preferential advantage to neoplastic PC in the proximity of blood vessels compared with other hematopoietic marrow cells. We demonstrate the feasibility of analyzing the spatial distribution of PC, and possibly other hematopoietic/stem cells in their microenvironment, as characterized by the distance to vessels in BM using a novel image analysis approach.

Quantitative assessment of microcirculation and diffusion in the bone marrow of osteoporotic rats using VCT, DCE-MRI, DW-MRI, and histology.

BACKGROUND: Etiologic and pathophysiologic role of functional bone marrow processes is not fully understood especially in the case of osteoporosis. PURPOSE: To investigate the role of vascularization and diffusion in rat models of osteoporosis through a cross-correlation between non-invasive in-vivo imaging and invasive ex-vivo imaging of bone, bone marrow, and in particular of microcirculation. MATERIAL AND METHODS: Osteoporosis was induced in rats by combining ovariectomy (OVX) with calcium and Vitamin D3 deficiency, or with glucocorticoid (dexamethasone). For comparison, controls underwent a sham surgery. In in-vivo investigations, animals (n = 36) were examined by volumetric CT (VCT) and MRI at 1, 3, or 12 months post surgery. Using VCT, bone morphology was monitored and relative bone density r within pelvis was extracted. With DCE-MRI and DW-MRI, parameters A (amplitude), Kep (exchange rate constant), and ADC (apparent diffusion coefficient) were acquired for regions of lumbar vertebrae, pelvis, and femur. In ex-vivo investigations, selective histological sections of pelvis were either stained with hematoxylin and eosin (HE stain) for quantifying vessel size and density or immunostained for collagen IV and α-smooth muscle actin to assess vessel maturity (SMA/collagen IV ratio). RESULTS: After 12 months, decrease in DCE-MRI parameter Kep was found in all locations of osteoporotic rats (strongest in femur and lumbar vertebrae) while no significant differences were seen for parameter A and DW-MRI parameter ADC. Furthermore, vessel rarefication and maturation were observed on the histological level in animals with osteoporotic phenotype. In particular in the pelvis, the osteoporotic individuals (irrespective of the osteoporosis inducers applied) exhibited decreased Kep, significantly reduced vessel density, significantly increased vessel maturity, as well as statistically unaltered A, ADC, and vessel diameter. CONCLUSION: Changes in microcirculation but not diffusion in the bone marrow of osteoporotic rats are detected by DCE-MRI and DW-MRI due to vessel rarefication and maturation.

Assessment of bone marrow fibrosis and angiogenesis in monitoring patients with multiple myeloma.

The aim of our study was to emphasize the importance of accurate and standardized techniques for detailed monitoring of the microenvironment in multiple myeloma (MM). Bone marrow fibrosis, angiogenesis, and plasma cell infiltrates in bone marrow biopsy (BMB) samples at the time of diagnosis and on completion of therapy were analyzed for 42 patients with newly diagnosed MM. Computerized image analysis was used for all slides stained with anti-CD138 and anti-CD34. The patients with fibrosis in pretreatment BMB samples had significantly higher microvessel density (MVD) and plasma cell infiltrates. In posttreatment BMB samples, nonresponders had a significantly higher frequency and grade of fibrosis and higher values of MVD, total vascular area, and plasma cell percentage. The overall survival of nonresponders and patients with increased marrow fibrosis in posttreatment BMB samples was significantly shorter. The obtained results confirm that complex morphologic examination of the bone marrow microenvironment during the monitoring of MM can provide better prognostic significance.

Inability of immunomorphometric assessment of angiogenesis to distinguish primary versus secondary myelofibrosis.

OBJECTIVE: To explore the utility of bone marrow (BM) angiogenesis in differentiating primary myelofibrosis (PMF) from secondary myelofibrosis (MF). STUDY DESIGN: CD34 immunostaining was performed on BM biopsies from 21 PMFs, 23 non-PMF myeloproliferative neoplasms (MPN) with associated MF, 20 secondary MF samples, and 10 nonfibrotic controls. Microvessel density (MVD) and microvessel surface area (MSA), along with blood and BM findings were compared between the groups. RESULTS: The post-MPN MF cases included chronic myeloid leukemia-MF and polycythemia vera-MF. Etiologies of secondary MF were metastatic carcinomas, non-MPN hematologic malignancies, tuberculosis, autoimmune MF, and osteopetrosis. Megakaryocytic clustering was the most frequent and intrasinusoidal hematopoiesis the most specific feature of PMF. Higher reticulin grade, collagenization, and osteomyelosclerosis were commoner in PMF. MVD and MSA were significantly increased in fibrotic marrows regardless of etiology. Although mean MVD as well as MSA were highest in PMF, extensive overlaps among groups and marked heterogeneity in the secondary MF group rendered them of limited utility in the differential diagnosis. CONCLUSION: Enhanced angiogenesis is not entirely specific for PMF. Overlaps with secondary MF limits its differential diagnostic utility. Pathogenetically, our findings suggest that enhanced angiogenesis is a secondary paraneoplastic stromal response shared by various unrelated conditions.

Assessment of bone marrow microvessel density in chronic lymphocytic leukemia.

INTRODUCTION: Angiogenesis is a physiologic process of new blood vessels formation mediated by various cytokines called proangiogenic and antiangiogenic factors. Enhancement of angiogenesis in chronic lymphocytic leukemia (CLL) has been recognized more recently. Our study assesses CD34 and von Willebrand factor (vWf) expression and microvessel density (MVD) in the bone marrow of patients with CLL. AIMS: (1) To assess bone marrow MVD in CLL using 2 different monoclonal antibodies, CD34 and vWf; and (2) To examine the possible association of marrow MVD and clinical course, pattern of marrow infiltration, Rai stage, CD38 positivity, and cytogenetic abnormalities detected by fluorescence in situ hybridization. MATERIALS AND METHODS: Bone marrow specimens from 33 patients with CLL and 10 controls were studied. A single microvessel was defined as any vessel with a clear lumen. The screening of the slides was carried out by hotspot method. The slides were initially screened at low power to identify the areas with highest number of microvessel or vascularity hotspot. The count of microvessel in a sufficiently extended field (40x objective lens, 10x ocular lens) was then performed. The mean value of 10 most vascularized areas at 400x field was considered as MVD for a sample. RESULTS: There was a significant difference between MVD counts according to the antibody used. MVD was higher using CD34 versus vWF (CD34: mean +/- SD, 35.91+/-15.7; 95% confidence interval of mean, 30.34-41.48 vessels/field versus vWF: 8.15+/-4.65; 95% confidence interval of mean, 4.11-12.44 vessels/field; P<0.0001]. Bone marrow MVD detected by CD34 was significantly higher in patients with CD38 expression more than 30% (P=0.006) and in patients with unfavorable cytogenetic abnormalities. However, no significant MVD differences were detected between CLL subgroups with regard to clinical course, pattern of marrow infiltration, and Rai stage. Bone marrow MVD in patients with CLL was significantly higher than that in controls (P<0.0001). CONCLUSIONS: MVD assessment using anti-CD34 resulted in higher MVD counts than when using anti-vWF antibody. However, no MVD differences were detected between CLL subgroups subdivided according to the above-mentioned prognostic factors except CD38 expression and genetic abnormalities.

Imaging and quantitative assessment of long bone vascularization in the adult rat using microcomputed tomography.

The objective of this study was to develop and validate a technique for both 3D imaging and quantification of the vascular network of bone tissue in the rat. Five month-old male Wistar rats were divided into tail-suspension (21 days) and control groups. Sixty percent barium sulfate solution was infused into the vena cava. The tibiae were evaluated in 2D and 3D before and after decalcification, using conventional microcomputerized tomography (muCT) at 10 and 5 mum resolution and synchrotron radiation (SR) muCT. The perfusion technique and tomography exhibited excellent bone vasculature imaging. Significant positive correlations were observed between 2D histomorphometric and 3D muCT vascular parameters (P < 0.05). 3DmuCT discriminated significant changes of vessel structures in unloading condition: vessel number decreased by 25%, (P < 0.005), vessel separation increased by 27%, P < 0.01. SRmuCT could image sinusoid clusters in bone. muCT is an accurate and reproducible technique for 3D quantitative evaluation of long bone vascularisation in the rat.

Choice of endothelial marker is crucial for assessment of bone marrow microvessel density in chronic lymphocytic leukemia.

Angiogenesis is a potential prognostic factor in chronic lymphocytic leukemia (CLL). Elevated circulating levels of angiogenic factors in CLL have been repeatedly reported. Nevertheless, the issue of bone marrow neovascularization in CLL remains controversial, partly due to limited number of published studies, different methods of assessing microvessel density (MVD) and small patient cohorts. Moreover, there are very scarce data regarding the relationship of marrow angiogenesis to prognostic markers in CLL. Our objectives were: 1. To assess bone marrow MVD in CLL using two different monoclonal antibodies and a reproducible method of MVD quantification; 2. To examine the possible association of marrow MVD and clinical course, pattern of marrow infiltration, Rai stage, cytogenetic abnormalities detected by fluorescence in situ hybridization (FISH), and mutation status of immunoglobulin heavy chain variable region (IgVH). MVD was higher using CD34 vs vWF antibody (p<0.0001). However, no MVD differences were detected between CLL subgroups subdivided according to the above-mentioned prognostic factors. In conclusion, MVD assessment using anti-CD34 resulted in higher MVD counts than when using anti-vWF antibody. No association of MVD with any prognostic factors was observed, possibly due to the limited patient cohort. As the need for bone marrow trephine biopsies in CLL is significantly decreasing, a standardized method of neovascularization assessment is required to enable possible multicentre studies in order to conduct larger investigations and thereby shed more light on the real clinical significance of bone marrow angiogenesis in CLL.

Prognostic value of bone marrow angiogenesis in multiple myeloma: use of light microscopy as well as computerized image analyzer in the assessment of microvessel density and total vascular area in multiple myeloma and its correlation with various clinical, histological, and laboratory parameters.

We studied the prognostic value of parameters of angiogenesis on bone marrow biopsies in newly diagnosed multiple myeloma (MM) patients. Angiogenesis parameters studied were the microvessel count done manually on light microscopy (MVD-A), microvessel count done by using computerized image analyzer (MVD-B), and total vascular area (TVA) measured by computerized image analyzer. One hundred ten newly diagnosed cases of MM treated at Institute Rotary Cancer Hospital, All India Institute of Medical Sciences, were analyzed with respect to clinical features, laboratory findings, histological features, angiogenesis parameters, and responses to the treatment on follow-up. Twenty age- and sex-matched controls were studied for comparing with angiogenesis of the test cases. Bone marrow microvessels were examined using immunohistochemical staining for CD34. MVD-A (range 4.9-85.2; mean 28.2; SD 19.4), MVD-B (range 2.0-26.9; mean 11.7; SD 5.9), and TVA measured in percentage (range 0.1-17.1; mean 2.4; SD 2.5) were measured for test cases (n = 110). Grading of angiogenesis parameters of the test cases were done; such that angiogenesis parameters of controls (taken as baseline) were grade I. There was a statistically highly significant correlation between (MVD-A vs MVD-B, pcc = 0.92; MVD-A vs TVA, pcc = 0.78; MVD-B vs TVA, pcc = 0.76). The myeloma cases had significantly higher angiogenesis parameters when compared with controls (Kruskall-Wallis test, P < 0.001). "Complete responders" (n = 38/110) had significant lower angiogenesis (Mann-Whitney U test, P < 0.001) than "nonresponders" (n = 72/110). On treatment follow-up "rapid disease progressors" had the highest levels of angiogenesis (mean rank for MVD-A = 84.7, MVD-B = 82.1, and TVA = 81.1). On multivariate (logistic regression) analysis, factors found to have independent prognostic significance in complete responders (adjusted odd ratio (95% CI, P value)] were: (a) MVD-B grade I [0.134 (0.10-0.16, P < 0.001)], (b) clinical substage A [0.163 (0.12-0.19, P = 0.008)], (c) Bartl's histological stage II & I [0.262 (0.2-0.32, P = 0.021)], (d) MVD-A grade I [0.28 (0.22-0.36, P = 0.03)], (e) beta2 microglobulin levels less than 3,400 ng/dl [0.31 (0.23-0.42, P = 0.04)]. Kaplan-Meier survival analysis for myeloma-related death (n = 16) shows a mean survival time (in months) of 24.75; SE = 3; 95% CI = 21-28. We conclude that MVD (particularly MVD-B) is a very good predictor for the complete response in patients of MM and should be done routinely on bone marrow biopsies.

Human tibia bone marrow: defining a model for the study of haemodynamics as a function of age by near infrared spectroscopy.

A human model allowing the non-invasive study of bone marrow haemodynamics has been developed. A decrease in postischaemic tissue reperfusion capability (postischaemic hyperaemia) as a function of age (range 25-72 years) was observed both in the human tibia and tibialis anterior muscle. In the tibia bone marrow the reperfusion capability started to decrease after 50 years and was lower than for muscle for all the age range. Mean basal muscle O(2) saturation (80.8% at 25 years) decreases as a function of age (-0.35%+/-0.13% per year) whereas it remains constant for bone marrow (84.8+/-2.8%). A Monte Carlo simulation has been performed to evaluate the accuracy of the derived O(2) saturation measurements and has shown that this parameter is robust even in the presence of substantial noise. It has also been demonstrated that it is necessary to use a multi wavelength NIR spectrometer and a second derivative based fitting algorithm to obtain reliable measurements from the bone marrow, and that the tissue scattering changes occurring during the protocol do not allow the use of the standard near infrared spectroscopy algorithms. The human tibia bone marrow model presented here and the related measurement technique should enable access to new areas of physiological research.

Bronchoalveolar lavage in adult sickle cell patients with acute chest syndrome: value for diagnostic assessment of fat embolism.

Fat embolism of necrotic bone marrow could be a frequent cause of acute chest syndrome (ACS) in sickle cell syndromes (SC), as suggested by postmortem findings. To check this hypothesis in living patients, we evaluated the presence of fatty macrophages recovered by bronchoalveolar lavage (BAL) in ACS. We investigated 20 consecutive cases of ACS by BAL, and identification of alveolar cells containing fat droplets was performed using oil red O (ORO), a specific neutral fat stain. The specificity of the method was determined on control groups, including eight SC patients without acute chest syndrome and 15 non-SC patients. A cut-off of > 5% of alveolar macrophages containing fat droplets was determined from the control groups to assess the diagnosis of fat embolism. In 12 ACS episodes, BAL exhibited > 5% of fatty macrophages, ranging from 10% to 100% (median value 46.5%). In 11 cases, fat embolism was associated with proven (n = 8) or probable (n = 3) bone marrow infraction, which mostly predated ACS. Eight ACS episodes were associated with a low percentage (< or = 5%) of fatty alveolar macrophages and could be related to a cause other than fat embolism in six episodes, such as sepsis, in-situ thrombosis, or rib infarcts generating hypoventilation. This study supports the diagnostic yield of BAL for fat embolism, which can be incriminated in 60% of cases of ACS in this adult population.

Assessment of bone marrow blood flow using positron emission tomography: no relationship with bone marrow cellularity.

Bone marrow blood flow has been assessed using positron emission tomography and the 15O-labelled carbon dioxide steady-state technique. The measurements were performed at the site of the posterior iliac crest. The bone marrow blood flow was 10.0 ml/min/100 cm3 +/- 3.0 (SD) in normal volunteers. It was markedly increased in patients with polycythaemia vera (26.9 +/- 4.6), chronic granulocytic leukaemia (25.2 +/- 3.9) and myelofibrosis (35.1 +/- 7.3). However, bone marrow blood flow did not differ from normal in patients with aplastic anaemia, chronic haemolysis or chronic lymphocytic leukaemia. There was no relationship between bone marrow cellularity and bone marrow blood flow. The data show that bone marrow blood flow is markedly elevated in polycythaemia vera, myelofibrosis and chronic granulocytic leukaemia and suggest that bone marrow cellularity is not a major factor in regulating bone marrow blood flow.