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Purpose:Osteonecrosis is a devastating complication of high-dose corticosteroid therapy in patients with cancer. Core decompression for prevention of bone collapse has been recently combined with the delivery of autologous concentrated bone marrow aspirates. The purpose of our study was to develop an imaging test for the detection of transplanted bone marrow cells in osteonecrosis lesions.Experimental Design:In a prospective proof-of-concept clinical trial (NCT02893293), we performed serial MRI studies of nine hip joints of 7 patients with osteonecrosis before and after core decompression. Twenty-four to 48 hours prior to the surgery, we injected ferumoxytol nanoparticles intravenously to label cells in normal bone marrow with iron oxides. During the surgery, iron-labeled bone marrow cells were aspirated from the iliac crest, concentrated, and then injected into the decompression track. Following surgery, patients received follow-up MRI up to 6 months after bone marrow cell transplantation.Results:Iron-labeled cells could be detected in the access canal by a dark (negative) signal on T2-weighted MR images. T2* relaxation times of iron-labeled cell transplants were significantly lower compared with unlabeled cell transplants of control patients who were not injected with ferumoxytol (P = 0.02). Clinical outcomes of patients who received ferumoxytol-labeled or unlabeled cell transplants were not significantly different (P = 1), suggesting that the added ferumoxytol administration did not negatively affect bone repair.Conclusions:This immediately clinically applicable imaging test could become a powerful new tool to monitor the effect of therapeutic cells on bone repair outcomes after corticosteroid-induced osteonecrosis.

Figure S1 shows that quantity of transplanted cells may predict outcome of osteonecrosis.

Current treatment of chronic wounds has been critically limited by various factors, including bacterial infection, biofilm formation, impaired angiogenesis, and prolonged inflammation. Addressing these challenges, we developed a multifunctional wound dressing-based three-pronged approach for accelerating wound healing. The multifunctional wound dressing, composed of nanofibers, functional nanoparticles, natural biopolymers, and selected protein and peptide, can target multiple endogenous repair mechanisms and represents a promising alternative to current wound healing products.

Background: The transplantation of mesenchymal stem cells (MSCs) into cartilage defects has led to variable cartilage repair outcomes. Previous in vitro studies have shown that ascorbic acid and reduced iron independently can improve the chondrogenic differentiation of MSCs. However, the combined effect of ascorbic acid and iron supplementation on MSC differentiation has not been investigated. Purpose: To investigate the combined in vivo effects of ascorbic acid and a US Food and Drug Administration (FDA)–approved iron supplement on MSC-mediated cartilage repair in mature Göttingen minipigs. Study Design: Controlled laboratory study. Methods: We pretreated bone marrow–derived MSCs with ascorbic acid and the FDA-approved iron supplement ferumoxytol and then transplanted the MSCs into full-thickness cartilage defects in the distal femurs of Göttingen minipigs. Untreated cartilage defects served as negative controls. We evaluated the cartilage repair site with magnetic resonance imaging at 4 and 12 weeks after MSC implantation, followed by histological examination and immunofluorescence staining at 12 weeks. Results: Ascorbic acid plus iron–pretreated MSCs demonstrated a significantly better MOCART (magnetic resonance observation of cartilage repair tissue) score (73.8 ± 15.5), better macroscopic cartilage regeneration score according to the International Cartilage Repair Society (8.6 ± 2.0), better Pineda score (2.9 ± 0.8), and larger amount of collagen type II (28,469 ± 21,313) compared with untreated controls (41.3 ± 2.5, 1.8 ± 2.9, 12.8 ± 1.9, and 905 ± 1326, respectively). The obtained scores were also better than scores previously reported in the same animal model for MSC implants without ascorbic acid. Conclusion: Pretreatment of MSCs with ascorbic acid and an FDA-approved iron supplement improved the chondrogenesis of MSCs and led to hyaline-like cartilage regeneration in the knee joints of minipigs. Clinical Relevance: Ascorbic acid and iron supplements are immediately clinically applicable. Thus, these results, in principle, could be translated into clinical applications.

Recent evidence of gadolinium deposition in the brain has raised safety concerns. Iron oxide nanoparticles are re-emerging as promising alternative MR contrast agents, because the iron core can be metabolized. However, long-term follow up studies of the brain after intravenous iron oxide administration have not been reported thus far. In this study, we investigated, if intravenously administered ferumoxytol nanoparticles are deposited in porcine brains. Methods: In an animal care and use committee-approved prospective case-control study, ten Gottingen minipigs received either intravenous ferumoxytol injections at a dose of 5 mg Fe/kg (n=4) or remained untreated (n=6). Nine to twelve months later, pigs were sacrificed and the brains of all pigs underwent ex vivo MRI at 7T with T2 and T2*-weighted sequences. MRI scans were evaluated by measuring R2* values (R2*=1000/T2*) of the bilateral caudate nucleus, lentiform nucleus, thalamus, dentate nucleus, and choroid plexus. Pig brains were sectioned and stained with Prussian blue and evaluated for iron deposition using a semiquantitative scoring system. Data of ferumoxytol exposed and unexposed groups were compared with an unpaired t-test and a Mann-Whitney U test. Results: T2 and T2* signal of the different brain regions was not visually different between ferumoxytol exposed and unexposed controls. There were no significant differences in R2* values of the different brain regions in the ferumoxytol exposed group compared to controls (p>0.05). Prussian blue stains of the same brain regions, scored according to a semiquantitative score, were not significantly different either between the ferumoxytol exposed group and unexposed controls (p>0.05). Conclusions: Our study shows that intravenous ferumoxytol doses of 5-10 mg Fe/kg do not lead to iron deposition in the brain of pigs. We suggest iron oxide nanoparticles as a promising alternative for gadolinium-enhanced MRI.

Autologous therapeutic cells are typically harvested and transplanted in one single surgery. This makes it impossible to label them with imaging biomarkers through classical transfection techniques in a laboratory. To solve this problem, we developed a novel microfluidic device, which provides highly efficient labeling of therapeutic cells with imaging biomarkers through mechanoporation. Methods: Studies were performed with a new, custom-designed microfluidic device, which contains ridges, which compress adipose tissue-derived stem cells (ADSCs) during their device passage. Cell relaxation after compression leads to cell volume exchange for convective transfer of nanoparticles and nanoparticle uptake into the cell. ADSCs were passed through the microfluidic device doped with iron oxide nanoparticles and 18F-fluorodeoxyglucose (FDG). The cellular nanoparticle and radiotracer uptake was evaluated with DAB-Prussian blue, fluorescent microscopy, and inductively coupled plasma spectrometry (ICP). Labeled and unlabeled ADSCs were imaged in vitro as well as ex vivo in pig knee specimen with magnetic resonance imaging (MRI) and positron emission tomography (PET). T2 relaxation times and radiotracer signal were compared between labeled and unlabeled cell transplants using Student T-test with p 1 pg iron per cell) and 18F-FDG uptake (61 mBq/cell), with a labeling efficiency of 95%. The labeled ADSCs could be detected with MRI and PET imaging technologies: Nanoparticle labeled ADSC demonstrated significantly shorter T2 relaxation times (24.2±2.1 ms) compared to unlabeled cells (79.6±0.8 ms) on MRI (p

Purpose: Osteonecrosis is a devastating complication of high-dose corticosteroid therapy in patients with cancer. Core decompression for prevention of bone collapse has been recently combined with the delivery of autologous concentrated bone marrow aspirates. The purpose of our study was to develop an imaging test for the detection of transplanted bone marrow cells in osteonecrosis lesions. Experimental Design: In a prospective proof-of-concept clinical trial (NCT02893293), we performed serial MRI studies of nine hip joints of 7 patients with osteonecrosis before and after core decompression. Twenty-four to 48 hours prior to the surgery, we injected ferumoxytol nanoparticles intravenously to label cells in normal bone marrow with iron oxides. During the surgery, iron-labeled bone marrow cells were aspirated from the iliac crest, concentrated, and then injected into the decompression track. Following surgery, patients received follow-up MRI up to 6 months after bone marrow cell transplantation. Results: Iron-labeled cells could be detected in the access canal by a dark (negative) signal on T2-weighted MR images. T2* relaxation times of iron-labeled cell transplants were significantly lower compared with unlabeled cell transplants of control patients who were not injected with ferumoxytol (P = 0.02). Clinical outcomes of patients who received ferumoxytol-labeled or unlabeled cell transplants were not significantly different (P = 1), suggesting that the added ferumoxytol administration did not negatively affect bone repair. Conclusions: This immediately clinically applicable imaging test could become a powerful new tool to monitor the effect of therapeutic cells on bone repair outcomes after corticosteroid-induced osteonecrosis.

PURPOSE: To evaluate, if clinically translatable ferumoxytol nanoparticles can be used for in vivo detection and quantification of stem cell transplants with magnetic particle imaging (MPI). PROCEDURES: Mesenchymal stem cells (MSCs) were labeled with ferumoxytol or ferucarbotran and underwent MPI, magnetic resonance imaging (MRI), prussian blue staining and inductively coupled plasma (ICP) spectrometry. Unlabeled, ferumoxytol, and ferucarbotran-labeled MSCs were implanted in calvarial defects of eight mice and underwent MPI, MRI and histopathology. The iron concentration calculated according to the MPI signal intensity and T2 relaxation times of the three different groups were compared using an analysis of variance (ANOVA) with Bonferroni correction, and a p

To evaluate whether the extent of restricted diffusion and 2-deoxy-2-[18F] fluoro-d-glucose ([18F]FDG) uptake of pediatric rhabdomyosarcomas (RMS) on positron emission tomography (PET)/magnetic resonance (MR) images provides prognostic information. In a retrospective, IRB-approved study, we evaluated [18F]FDG PET/CT and diffusion-weighted (DW) MR imaging studies of 21 children and adolescents (age 1–20 years) with RMS of the head and neck. [18F]FDG PET and DW MR scans at the time of the initial tumor diagnosis were fused using MIM software. Quantitative measures of the tumor mass with restricted diffusion, [18F]FDG hypermetabolism, or both were dichotomized at the median and tested for significance using Gray’s test. Data were analyzed using a survival analysis and competing risk model with death as the competing risk. [18F]FDG PET/MR images demonstrated a mismatch between tumor areas with increased [18F]FDG uptake and restricted diffusion. The DWI, PET, and DWI + PET tumor volumes were dichotomized at their median values, 23.7, 16.4, and 9.5 cm3, respectively, and were used to estimate survival. DWI, PET, and DWI + PET overlap tumor volumes above the cutoff values were associated with tumor recurrence, regardless of post therapy COG stage (p = 0.007, p = 0.04, and p = 0.07, respectively). The extent of restricted diffusion within RMS and overlap of hypermetabolism plus restricted diffusion predict unfavorable clinical outcomes.

Purpose To evaluate if the formation of a protein corona around ferumoxytol nanoparticles can facilitate stem cell labeling for in vivo tracking with magnetic resonance (MR) imaging. Materials and Methods Ferumoxytol was incubated in media containing human serum (group 1), fetal bovine serum (group 2), StemPro medium (group 3), protamine (group 4), and protamine plus heparin (group 5). Formation of a protein corona was characterized by means of dynamic light scattering, ζ potential, and liquid chromatography-mass spectrometry. Iron uptake was evaluated with 3,3'-diaminobenzidine-Prussian blue staining, lysosomal staining, and inductively coupled plasma spectrometry. To evaluate the effect of a protein corona on stem cell labeling, human mesenchymal stem cells (hMSCs) were labeled with the above formulations, implanted into pig knee specimens, and investigated with T2-weighted fast spin-echo and multiecho spin-echo sequences on a 3.0-T MR imaging unit. Data in different groups were compared by using a Kruskal-Wallis test. Results Compared with bare nanoparticles, all experimental groups showed significantly increased negative ζ values (from -37 to less than -10; P = .008). Nanoparticles in groups 1-3 showed an increased size because of the formation of a protein corona. hMSCs labeled with group 1-5 media showed significantly shortened T2 relaxation times compared with unlabeled control cells (P = .0012). hMSCs labeled with group 3 and 5 media had the highest iron uptake after cells labeled with group 1 medium. After implantation into pig knees, hMSCs labeled with group 1 medium showed significantly shorter T2 relaxation times than hMSCs labeled with group 2-5 media (P = .0022). Conclusion The protein corona around ferumoxytol nanoparticles can facilitate stem cell labeling for clinical cell tracking with MR imaging.

Purpose: Stem cell transplants are an effective approach to repair large bone defects. However, comprehensive techniques to monitor the fate of transplanted stem cells in vivo are lacking. Such strategies would enable corrective interventions at an early stage and greatly benefit the development of more successful tissue regeneration approaches. In this study, we designed and synthesized a dual-modality imaging probe (Feru-AFC) that can simultaneously localize transplanted stem cells and diagnose immune rejection-induced apoptosis at an early stage in vivo. Methods: We used a customized caspase-3 cleavable peptide-dye conjugate to modify the surface of clinically approved ferumoxytol nanoparticles (NPs) to generate the dual-modality imaging probe with fluorescence “light-up” feature. We labeled both mouse mesenchymal stem cells (mMSCs, matched) and pig mesenchymal stem cells (pMSCs, mismatched) with the probe and transplanted the labeled cells with biocompatible scaffold at the calvarial defects in mice. We then employed intravital microscopy (IVM) and magnetic resonance imaging (MRI) to investigate the localization, engraftment, and viability of matched and mismatched stem cells, followed by histological analyses to evaluate the results obtained from in vivo studies. Results: The Feru-AFC NPs showed good cellular uptake efficiency in the presence of lipofectin without cytotoxicity to mMSCs and pMSCs. The fluorescence of Feru-AFC NPs was turned on inside apoptotic cells due to the cleavage of peptide by activated caspase-3 and subsequent release of fluorescence dye molecules. Upon transplantation at the calvarial defects in mice, the intense fluorescence from the cleaved Feru-AFC NPs in apoptotic pMSCs was observed with a concomitant decrease in the overall cell number from days 1 to 6. In contrast, the Feru-AFC NP-treated mMSCs exhibited minimum fluorescence and the cell number also remained similar. Furthermore, in vivo MRI of the Feru-AFC NP-treated mMSC and pMSCs transplants could clearly indicate the localization of matched and mismatched cells, respectively. Conclusions: We successfully developed a dual-modality imaging probe for evaluation of the localization and viability of transplanted stem cells in mouse calvarial defects. Using ferumoxytol NPs as the platform, our Feru-AFC NPs are superparamagnetic and display a fluorescence “light-up” signature upon exposure to activated caspase-3. The results show that the probe is a promising tool for long-term stem cell tracking through MRI and early diagnosis of immune rejection-induced apoptosis through longitudinal fluorescence imaging.

Superparamagnetic iron oxide (SPIO) nanoparticles have been intensively studied for the development of contrast agents in MRI. First-generation SPIO nanoparticles had diagnostic capabilities only, whereas a new generation of SPIO nanoparticle has multifunctional characteristics for combined therapeutic and diagnostic applications. These theranostic nanoparticles hold great potential for image-guided cancer therapies. In particular, polymer-coated theranostic SPIO nanoparticles have enjoyed increasing attention as a result of good biocompatibility, biodegradability and versatile functionality endowed by polymeric matrices. This review provides an overview of recently developed polymer-coated multifunctional SPIO nanoparticles for cancer theranostics and discusses current challenges and future perspectives.

BACKGROUND: Cartilage repair outcomes of matrix-associated stem cell implants (MASIs) in patients have been highly variable. Conventional MRI cannot help distinguish between grafts that will and grafts that will not repair the underlying cartilage defect until many months after the repair. PURPOSE: To determine if ferumoxytol nanoparticle labeling could be used to depict successful or failed MASIs compared with conventional MRI in a large-animal model. MATERIALS AND METHODS: Between January 2016 and December 2017, 10 Göttingen minipigs (n = 5 male; n = 5 female; mean age, 6 months ± 5.1; age range, 4–20 months) received implants of unlabeled (n = 12) or ferumoxytol-labeled (n = 20) viable and apoptotic MASIs in cartilage defects of the distal femur. All MASIs were serially imaged with MRI on a 3.0-T imaging unit at week 1 and weeks 2, 4, 8, 12, and 24, with calculation of T2 relaxation times. Cartilage regeneration outcomes were assessed by using the MR observation of cartilage repair tissue (MOCART) score (scale, 0–100), the Pineda score, and histopathologic quantification of collagen 2 production in the cartilage defect. Findings were compared by using the unpaired Wilcoxon rank sum test, a linear regression model, the Fisher exact test, and Pearson correlation. RESULTS: Ferumoxytol-labeled MASIs showed significant T2 shortening (22.2 msec ± 3.2 vs 27.9 msec ± 1.8; P < .001) and no difference in cartilage repair outcomes compared with unlabeled control MASIs (P > .05). At week 2 after implantation, ferumoxytol-labeled apoptotic MASIs showed a loss of iron signal and higher T2 relaxation times compared with ferumoxytol-labeled viable MASIs (26.6 msec ± 4.9 vs 20.8 msec ± 5.3; P = .001). Standard MRI showed incomplete cartilage defect repair of apoptotic MASIs at 24 weeks. Iron signal loss at 2 weeks correlated with incomplete cartilage repair, diagnosed at histopathologic examination at 12–24 weeks. CONCLUSION: Ferumoxytol nanoparticle labeling can accelerate the diagnosis of successful and failed matrix-associated stem cell implants at MRI in a large-animal model. © RSNA, 2019 Online supplemental material is available for this article. See also the editorial by Sneag and Potter in this issue.

Until now, the Food and Drug Administration (FDA)-approved iron supplement ferumoxytol and other iron oxide nanoparticles have been used for treating iron deficiency, as contrast agents for magnetic resonance imaging and as drug carriers. Here, we show an intrinsic therapeutic effect of ferumoxytol on the growth of early mammary cancers, and lung cancer metastases in liver and lungs. In vitro, adenocarcinoma cells co-incubated with ferumoxytol and macrophages showed increased caspase-3 activity. Macrophages exposed to ferumoxytol displayed increased mRNA associated with pro-inflammatory Th1-type responses. In vivo, ferumoxytol significantly inhibited growth of subcutaneous adenocarcinomas in mice. In addition, intravenous ferumoxytol treatment before intravenous tumour cell challenge prevented development of liver metastasis. Fluorescence-activated cell sorting (FACS) and histopathology studies showed that the observed tumour growth inhibition was accompanied by increased presence of pro-inflammatory M1 macrophages in the tumour tissues. Our results suggest that ferumoxytol could be applied ‘off label’ to protect the liver from metastatic seeds and potentiate macrophage-modulating cancer immunotherapies.

Ferumoxytol nanoparticles are being used clinically for the treatment of anemia and molecular imaging in patients. It is well documented that while most patients tolerate ferumoxytol well, a small percentage of patients (i.e., 0.01%) develop severe allergic reactions. The purpose of our proof-of-concept study was to determine whether patients with or without hypersensitivity reactions have specific protein corona profiles around ferumoxytol nanoparticles. In a retrospective, institutional review board approved pilot study, we enrolled 13 pediatric patients (5 girls, 8 boys, mean age 16.9 ± 8.2 years) who received a ferumoxytol-enhanced magnetic resonance imaging and who did (group 1, n = 5) or did not (group 2, n = 8) develop an allergic reaction. Blood samples of these patients were incubated with ferumoxytol, and the formation of a hard protein corona around ferumoxytol nanoparticles was measured by dynamic light scattering, zeta potential, and liquid chromatography–mass spectrometry. We also performed in vitro immune response analyses to randomly selected coronas from each group. Our results provide preliminary evidence that ex vivo analysis of the biomolecular corona may provide useful and predictive information on the possibility of severe allergic reactions to ferumoxytol nanoparticles. In the future, patients with predisposition of an allergic reaction to ferumoxytol may be diagnosed based on the proteomic patterns of the corona around ferumoxytol in their blood sample.

"Off the shelf" allogeneic stem cell transplants and stem cell nano-composites are being used for the treatment of degenerative bone diseases. However, major and minor histocompatibility antigens of therapeutic cell transplants can be recognized as foreign and lead to their rejection by the host immune system. If a host immune response is identified within the first week post-transplant, immune modulating therapies could be applied to prevent graft failure and support engraftment. Ferumoxytol (Feraheme™) is an FDA approved iron oxide nanoparticle preparation for the treatment of anemia in patients. Ferumoxytol can be used "off label" as an magnetic resonance (MR) contrast agent, as these nanoparticles provide measurable signal changes on magnetic resonance imaging (MRI). In this focused review article, we will discuss three methods to localize and identify innate immune responses to stem cell transplants using ferumoxytol-enhanced MRI, which are based on tracking stem cells, tracking macrophages or detecting mediators of cell death: (a) monitor MRI signal changes of ferumoxytol-labeled stem cells in the presence or absence of innate immune responses, (b) monitor influx of ferumoxytol-labeled macrophages into stem cell implants, and (c) monitor apoptosis of stem cell implants with caspase-3 activatable nanoparticles. These techniques can detect transplant failure at an early stage, when immune-modulating interventions can potentially preserve the viability of the cell transplants and thereby improve bone and cartilage repair outcomes. Approaches 1 and 2 are immediately translatable to clinical practice. This article is categorized under: Diagnostic Tools > in vivo Nanodiagnostics and Imaging Nanotechnology Approaches to Biology > Cells at the Nanoscale Diagnostic Tools > Biosensing.

CD47 monoclonal antibodies (mAbs) activate tumor-associated macrophages (TAMs) in sarcomas to phagocytose and eliminate cancer cells. Though CD47 mAbs have entered clinical trials, diagnostic tests for monitoring therapy response in vivo are currently lacking. Ferumoxytol is an FDA-approved iron supplement which can be used “off label” as a contrast agent: the nanoparticle-based drug is phagocytosed by TAM and can be detected with magnetic resonance imaging (MRI). We evaluated if ferumoxytol-enhanced MRI can monitor TAM response to CD47 mAb therapy in osteosarcomas. Forty-eight osteosarcoma-bearing mice were treated with CD47 mAb or control IgG and underwent pre- and post-treatment ferumoxytol-MRI scans. Tumor enhancement, quantified as T2 relaxation times, was compared with the quantity of TAMs as determined by immunofluorescence microscopy and flow cytometry. Quantitative data were compared between experimental groups using exact two-sided Wilcoxon rank-sum tests. Compared to IgG-treated controls, CD47 mAb-treated tumors demonstrated significantly shortened T2 relaxation times on ferumoxytol-MRI scans (p p p

PURPOSE: To evaluate whether the extent of restricted diffusion and 2-deoxy-2-[(18)F] fluoro-D- glucose ([(18)F]FDG) uptake of pediatric rhabdomyosarcomas (RMS) on positron emission tomography (PET)/magnetic resonance (MR) images provides prognostic information. PROCEDURE: In a retrospective, IRB-approved study, we evaluated [(18)F]FDG PET/CT and diffusion-weighted (DW) MR imaging studies of 21 children and adolescents (age 1–20 years) with RMS of the head and neck. [(18)F]FDG PET and DW MR scans at the time of the initial tumor diagnosis were fused using MIM software. Quantitative measures of the tumor mass with restricted diffusion, [(18)F]FDG hypermetabolism, or both were dichotomized at the median and tested for significance using Gray’s test. Data were analyzed using a survival analysis and competing risk model with death as the competing risk. RESULTS: [(18)F]FDG PET/MR images demonstrated a mismatch between tumor areas with increased [(18)F]FDG uptake and restricted diffusion. The DWI, PET, and DWI + PET tumor volumes were dichotomized at their median values, 23.7, 16.4, and 9.5 cm(3), respectively, and were used to estimate survival. DWI, PET, and DWI + PET overlap tumor volumes above the cutoff values were associated with tumor recurrence, regardless of post therapy COG stage (p = 0.007, p = 0.04, and p = 0.07, respectively). CONCLUSION: The extent of restricted diffusion within RMS and overlap of hypermetabolism plus restricted diffusion predict unfavorable clinical outcomes.

PURPOSE: While imaging matrix-associated stem cell transplants aimed for cartilage repair in a rodent arthritis model, we noticed that some transplants formed locally destructive tumors. The purpose of this study was to determine the cause for this tumor formation in order to avoid this complication for future transplants. PROCEDURES: Adipose-derived stem cells (ADSC) isolated from subcutaneous adipose tissue were implanted into 24 osteochondral defects of the distal femur in ten athymic rats and two immunocompetent control rats. All transplants underwent serial magnetic resonance imaging (MRI) up to 6 weeks post-transplantation to monitor joint defect repair. Nine transplants showed an increasing size over time that caused local bone destruction (group 1), while 11 transplants in athymic rats (group 2) and 4 transplants in immunocompetent rats did not. We compared the ADSC implant size and growth rate on MR images, macroscopic features, histopathologic features, surface markers, and karyotypes of these presumed neoplastic transplants with nonneoplastic ADSC transplants. RESULTS: Implants in group 1 showed a significantly increased two-dimensional area at week 2 (p = 0.0092), 4 (p = 0.003), and 6 (p = 0.0205) compared to week 0, as determined by MRI. Histopathological correlations confirmed neoplastic features in group 1 with significantly increased size, cellularity, mitoses, and cytological atypia compared to group 2. Six transplants in group 1 were identified as malignant chondrosarcomas and three transplants as fibromyxoid sarcomas. Transplants in group 2 and immunocompetent controls exhibited normal cartilage features. Both groups showed a normal ADSC phenotype; however, neoplastic ADSC demonstrated a mixed population of diploid and tetraploid cells without genetic imbalance. CONCLUSIONS: ADSC transplants can form tumors in vivo. Preventive actions to avoid in vivo tumor formations may include karyotyping of culture-expanded ADSC before transplantation. In addition, serial imaging of ADSC transplants in vivo may enable early detection of abnormally proliferating cell transplants.

Cellular uptake of nanoparticles (NPs) depends on the nature of the nanobio system including the solid nanocomponents ( e. g., physicochemical properties of NPs), nanobio interfaces ( e. g., protein corona composition), and the cellular characteristics ( e. g., cell type). In this study, we document the role of sex in cellular uptake of NPs as an "overlooked" factor in nanobio interface investigations. We demonstrate that cell sex leads to differences in NP uptake between male and female human amniotic stem cells (hAMSCs), with greater uptake by female cells. hAMSCs are one of the earliest sources of somatic stem cells. The experiments were replicated with primary fibroblasts isolated from the salivary gland of adult male and female donors of similar ages, and again the extent of NP uptake was altered by cell sex. However, in contrast to hAMSCs, uptake was greater in male cells. We also found out that female versus male amniotic stem cells exhibited different responses to reprogramming into induced pluripotent stem cells (iPSCs) by the Yamanaka factors. Thus, future studies should consider the effect of sex on the nanobio interactions to optimize clinical translation of NPs and iPSC biology and to help researchers to better design and produce safe and efficient therapeutic sex-specific NPs.

About 43 million individuals in the U.S. encounter cartilage injuries due to trauma or osteoarthritis, leading to joint pain and functional disability. Matrix-associated stem cell implants (MASI) represent a promising approach for repair of cartilage defects. However, limited survival of MASI creates a significant bottleneck for successful cartilage regeneration outcomes and functional reconstitution. We report an approach for noninvasive detection of stem cell apoptosis with magnetic resonance imaging (MRI), based on a caspase-3-sensitive nanoaggregation MRI probe (C-SNAM). C-SNAM self-assembles into nanoparticles after hydrolysis by caspase-3, leading to 90% amplification of (1)H MR signal and prolonged in vivo retention. Following intra-articular injection, C-SNAM causes significant MR signal enhancement in apoptotic MASI compared to viable MASI. Our results indicate that C-SNAM functions as an imaging probe for stem cell apoptosis in MASI. This concept could be applied to a broad range of cell transplants and target sites.

Purpose To determine whether endogenous labeling of macrophages with clinically applicable nanoparticles enables noninvasive detection of innate immune responses to stem cell transplants with magnetic resonance (MR) imaging. Materials and Methods Work with human stem cells was approved by the institutional review board and the stem cell research oversight committee, and animal experiments were approved by the administrative panel on laboratory animal care. Nine immunocompetent Sprague-Dawley rats received intravenous injection of ferumoxytol, and 18 Jax C57BL/6-Tg (Csf1r-EGFP-NGFR/FKBP1A/TNFRSF6) 2Bck/J mice received rhodamine-conjugated ferumoxytol. Then, 48 hours later, immune-matched or mismatched stem cells were implanted into osteochondral defects of the knee joints of experimental rats and calvarial defects of Jax mice. All animals underwent serial MR imaging and intravital microscopy (IVM) up to 4 weeks after surgery. Macrophages of Jax C57BL/6-Tg (Csf1r-EGFP-NGFR/FKBP1A/TNFRSF6) 2Bck/J mice express enhanced green fluorescent protein (GFP), which enables in vivo correlation of ferumoxytol enhancement at MR imaging with macrophage quantities at IVM. All quantitative data were compared between experimental groups by using a mixed linear model and t tests. Results Immune-mismatched stem cell implants demonstrated stronger ferumoxytol enhancement than did matched stem cell implants. At 4 weeks, T2 values of mismatched implants were significantly lower than those of matched implants in osteochondral defects of female rats (mean, 10.72 msec for human stem cells and 11.55 msec for male rat stem cells vs 15.45 msec for sex-matched rat stem cells; P = .02 and P = .04, respectively) and calvarial defects of recipient mice (mean, 21.7 msec vs 27.1 msec, respectively; P = .0444). This corresponded to increased recruitment of enhanced GFP- and rhodamine-ferumoxytol-positive macrophages into stem cell transplants, as visualized with IVM and histopathologic examination. Conclusion Endogenous labeling of macrophages with ferumoxytol enables noninvasive detection of innate immune responses to stem cell transplants with MR imaging. © RSNA, 2017 Online supplemental material is available for this article.

Human mesenchymal stem cells (hMSCs) are a promising tool for cartilage regeneration in arthritic joints. hMSC labeling with iron oxide nanoparticles enables non-invasive in vivo monitoring of transplanted cells in cartilage defects with MR imaging. Since graft failure leads to macrophage phagocytosis of apoptotic cells, we evaluated in vitro and in vivo whether nanoparticle-labeled hMSCs show distinct MR signal characteristics before and after phagocytosis by macrophages. We found that apoptotic nanoparticle-labeled hMSCs were phagocytosed by macrophages while viable nanoparticle-labeled hMSCs were not. Serial MRI scans of hMSC transplants in arthritic joints of recipient rats showed that the iron signal of apoptotic, nanoparticle-labeled hMSCs engulfed by macrophages disappeared faster compared to viable hMSCs. This corresponded to poor cartilage repair outcomes of the apoptotic hMSC transplants. Therefore, rapid decline of iron MRI signal at the transplant site can indicate cell death and predict incomplete defect repair weeks later. Currently, hMSC graft failure can be only diagnosed by lack of cartilage defect repair several months after cell transplantation. The described imaging signs can diagnose hMSC transplant failure more readily, which could enable timely re-interventions and avoid unnecessary follow up studies of lost transplants.

Magnetic resonance (MR) imaging of superparamagnetic iron oxide (SPIO)-labeled stem cells offers a noninvasive evaluation of stem cell engraftment in host organs. Excessive cellular iron load from SPIO labeling, however, impairs stem cell differentiation. The purpose of this study was to magnetically label human embryonic stem cells (hESCs) via a reduced exposure protocol that maintains a significant MR signal and no significant impairment to cellular pluripotency or differentiation potential. hESCs were labeled by simple incubation with Food and Drug Administration-approved ferumoxides, using concentrations of 50- 200 µg Fe/ml and incubation times of 3-24 h. The most reduced exposure labeling protocol that still provided a significant MR signal comparable to accepted labeling protocols was selected for subsequent studies. Labeled hESCs were compared to unlabeled controls for differences in pluripotency as studied by fluorescence staining for SSEA-1, SSEA-4, TRA-60, and TRA-81 and in differentiation capacity as studied by quantitative real-time PCR for hOCT4, hACTC1, hSOX1, and hAFP after differentiation into embryoid bodies (EBs). Subsequent MR and microscopy imaging were performed to evaluate for cellular iron distribution and long-term persistence of the label. An incubation concentration of 50 µg Fe/ml and incubation time of 3 h demonstrated a significantly reduced exposure protocol that yielded an intracellular iron uptake of 4.50 ± 0.27 pg, an iron content comparable to currently accepted SPIO labeling protocols. Labeled and unlabeled hESCs showed no difference in pluripotency or differentiation capacity. Ferumoxide-labeled hESCs demonstrated persistent MR contrast effects as embryoid bodies for 21 days. Electron microscopy confirmed persistent lysosomal storage of iron oxide particles in EBs up to 9 days, while additional microscopy visualization confirmed the iron distribution within single and multiple EBs. Labeling hESCs with ferumoxides by this tailored protocol reduces exposure of cells to the labeling agent while allowing for long-term visualization with MR imaging and the retention of cellular pluripotency and differentiation potential.

BACKGROUND AND AIMS Vitamin D deficiency is one of the major health problems and unexpectedly has a high prevalence in sunny countries (e.g. Middle East). In this study we determined the prevalence of vitamin D deficiency in pregnant women and their newborns in Isfahan, a sunny city in Iran. METHODS In a cross-sectional study, 88 newborns born in Beheshty hospital, affiliated to Isfahan University of Medical Sciences (August-September, 2005) and their mothers were studied. Their data were collected by questionnaires and blood sampling was done to measure serum alkaline phosphatase (ALP), calcium, phosphorus, 25 (OH) vitamin D and parathormone (PTH). Vitamin D deficiency defined as levels of 25 (OH) D < 20 and < 12.5 ng/ml for mothers and newborns, respectively and local cut-offs defined as levels in which mean serum PTH started to increase. RESULTS The prevalence of vitamin D deficiency according to 25 (OH) D < 20 ng/ml in mothers and < 12.5 ng/ml in newborns was 5.7% and 4.5%, respectively. According to local cut-offs (35 ng/ml for mothers and 26 ng/ml for newborns) 26.1% of mothers and 53.4% of newborns were vitamin D deficient. CONCLUSION According to local definition, vitamin D deficiency is a health problem in pregnant women and their newborns in this sunny city.

Human induced pluripotent stem cells (hiPSCs) have demonstrated great potential for hyaline cartilage regeneration. However, current approaches for chondrogenic differentiation of hiPSCs are complicated and inefficient primarily due to intermediate embryoid body formation, which is required to generate endodermal, ectodermal, and mesodermal cell lineages. We report a new, straightforward and highly efficient approach for chondrogenic differentiation of hiPSCs, which avoids embryoid body formation. We differentiated hiPSCs directly into mesenchymal stem /stromal cells (MSC) and chondrocytes. hiPSC-MSC-derived chondrocytes showed significantly increased Col2A1, GAG, and SOX9 gene expression compared to hiPSC-MSCs. Following transplantation of hiPSC-MSC and hiPSC-MSC-derived chondrocytes into osteochondral defects of arthritic joints of athymic rats, magnetic resonance imaging studies showed gradual engraftment, and histological correlations demonstrated hyaline cartilage matrix production. Results present an efficient and clinically translatable approach for cartilage tissue regeneration via patient-derived hiPSCs, which could improve cartilage regeneration outcomes in arthritic joints.

About 43 million individuals in the US currently suffer from disabilities due to arthritis. Cartilage defects are the major source of pain in the affected joints. Current treatments, whilst alleviating some of the clinical symptoms, prove insufficient to cure the underlying irreversible cartilage loss. Stem cells represent a unique source for restoration of cartilage defects. Pre-clinical and clinical trials are currently pursued to investigate the potential of various types of stem cells and stem cell derived chondrocytes to repair arthritic joints. A major challenge with all stem cell-mediated tissue regeneration approaches is death of the transplanted cells with clearance by the immune system. Our current inability to diagnose successful or unsuccessful engraftment of transplanted cells non-invasively in vivo represents a major bottleneck for the development of successful stem cell therapies. A large variety of non-invasive Magnetic Resonance (MR) imaging techniques have been developed over the last decade, which enable sensitive in vivo detection of Matrix Associated Stem Cell Implants (MASI) and early diagnosis of related complications. While initially focused on successfully harvesting cellular MR imaging approaches with easily applicable SuperParamagnetic Iron Oxide Nanoparticles (SPIO), our team began to observe details that will facilitate clinical translation. We therefore started a broader effort to define a comprehensive set of novel, clinically applicable imaging approaches for stem cell transplants in patients. We established immediately clinically applicable nanoparticle labeling techniques for tracking stem cell transplants with MR imaging; we have evaluated the long term MR signal effects of iron oxide nanoparticle labeled MASI in vivo; and we have defined distinct signal characteristics of labeled viable and apoptotic MASI. This review article will provide an overview over these efforts and discuss important implications for clinical translation.

Aim: To develop a clinically applicable MRI technique for tracking stem cells in matrix-associated stem-cell implants, using the US FDA-approved iron supplement ferumoxytol. Materials & methods: Ferumoxytol-labeling of adipose-derived stem cells (ADSCs) was optimized in vitro. A total of 11 rats with osteochondral defects of both femurs were implanted with ferumoxytol- or ferumoxides-labeled or unlabeled ADSCs, and underwent MRI up to 4 weeks post matrix-associated stem-cell implant. The signal-to-noise ratio of different matrix-associated stem-cell implant was compared with t-tests and correlated with histopathology. Results: An incubation concentration of 500 µg iron/ml ferumoxytol and 10 µg/ml protamine sulfate led to significant cellular iron uptake, T2 signal effects and unimpaired ADSC viability. In vivo, ferumoxytol- and ferumoxides-labeled ADSCs demonstrated significantly lower signal-to-noise ratio values compared with unlabeled controls (p < 0.01). Histopathology confirmed engraftment of labeled ADSCs, with slow dilution of the iron label over time. Conclusion: Ferumoxytol can be used for in vivo tracking of stem cells with MRI. Original submitted 28 February 2012; Revised submitted 8 November 2012

PurposeTo determine whether intravenous ferumoxytol can be used to effectively label mesenchymal stem cells (MSCs) in vivo and can be used for tracking of stem cell transplants.Materials and methodsThis study was approved by the institutional animal care and use committee. Sprague-Dawley rats (6-8 weeks old) were injected with ferumoxytol 48 hours prior to extraction of MSCs from bone marrow. Ferumoxytol uptake by these MSCs was evaluated with fluorescence, confocal, and electron microscopy and compared with results of traditional ex vivo-labeling procedures. The in vivo-labeled cells were subsequently transplanted in osteochondral defects of 14 knees of seven athymic rats and were evaluated with magnetic resonance (MR) imaging up to 4 weeks after transplantation. T2 relaxation times of in vivo-labeled MSC transplants and unlabeled control transplants were compared by using t tests. MR data were correlated with histopathologic results.ResultsIn vivo-labeled MSCs demonstrated significantly higher ferumoxytol uptake compared with ex vivo-labeled cells. With electron microscopy, iron oxide nanoparticles were localized in secondary lysosomes. In vivo-labeled cells demonstrated significant T2 shortening effects in vitro and in vivo when they were compared with unlabeled control cells (T2 in vivo, 15.4 vs 24.4 msec; P < .05) and could be tracked in osteochondral defects for 4 weeks. Histologic examination confirmed the presence of iron in labeled transplants and defect remodeling.ConclusionIntravenous ferumoxytol can be used to effectively label MSCs in vivo and can be used for tracking of stem cell transplants with MR imaging. This method eliminates risks of contamination and biologic alteration of MSCs associated with ex vivo-labeling procedures.

Tissue regeneration using progenitor cell-based therapy has the potential to aid in the healing of a diverse range of pathologies, ranging from short-gut syndrome to spinal cord lesions. However, there are numerous hurdles to be overcome prior to the widespread application of these cells in the clinical setting. One of the primary barriers to effective stem cell therapy is the hostile environment that progenitor cells encounter in the clinical injury wound setting. In order to promote cellular survival, stem cell differentiation, and participation in tissue regeneration, relevant cells and delivery scaffolds must be paired with strategies to prevent cell death to ensure that these cells can survive to form de novo tissue. The Bcl-2 protein is a prosurvival member of a family of proteins that regulate the mitochondrial pathway of apoptosis. Using several strategies to overexpress the Bcl-2 protein, we demonstrated a decrease in the mediators of apoptosis in vitro and in vivo. This was shown through the use of two different clinical tissue repair models. Cells overexpressing Bcl-2 not only survived within the wound environment at a statistically significantly higher rate than control cells, but also increased tissue regeneration. Finally, we used a nonintegrating minicircle technology to achieve this in a potentially clinically applicable strategy for stem cell therapy.

Gadolinium (Gd) based contrast agents are commonly used in magnetic resonance imaging (MRI) diagnosis. These agents possess relatively low contrast efficiency, requiring the administration of large doses of the contrast agent leading to concerns over safety. Self-forming gadolinium chelate nanoparticles (Gd NPs) provide a unique means to create higher efficiency contrast agents by bringing the gadolinium atoms in close proximity to each other, resulting in enhanced relaxivity. The self-assembly process can be activated through a number of mechanisms including changes in pH, the presence of light or any number of enzymatic reactions. These nanoparticles can be used to target and identify specific molecular events and even diseases such as cancer. The Gd NPs are comprised of an organic matrix that both chelates the Gd ions and allows for the self-assembly reaction to occur. As a result, the Gd NPs form as amorphous nanoparticles with a low overall concentration of Gd atoms. This makes it very difficult to identify and characterize the Gd NPs using transmission electron microscopy (TEM) alone. Both energy dispersive x-ray spectroscopy (EDS) and electron energy loss spectroscopy (EELS) can be utilized to identify Gd and confirm the presence of Gd NPs locate them in vitro. Gadolinium has a characteristic x-ray peak at 6.056 keV for EDS analysis and a characteristic energy loss peak at ~148 eV for EELS analysis. Gd NPs were imaged using an FEI Titan TEM with Cs image correction operated at 80 kV. The nanoparticles shown in figure 1(A) are very nondescript, appearing as dark blobs on the holey carbon film substrate. EELS, shown in figure 1(B), was utilized to confirm the presence of Gd indicating that these nanoparticles are indeed Gd NPs. Compared with inorganic Au-Si core shell nanoparticles shown in figure 1(C) these nanoparticles are very non uniform without distinct features that would make them identifiable in vitro. Figure 1 (A) TEM image of Gd NPs showing that they are non-uniform and non-descript. (B) EELS spectra confirming the presence of Gd in the nanoparticles. (C) Inorganic Au-Si core-shell nanoparticles highlighting the difference between amorphous organic nanoparticles ... Gd NPs treated with lipofectamine, to induce uptake, were incubated with rat adipose derived stem cells (rADSC). The cells were then fixed, stained, dehydrated and embedded following standard protocols. 150 nm thick sections were cut and placed on 100 mesh formvar coated Cu grids and imaged using the FEI Tecnai F20 TEM operated at 200 kV. Gd NPs were expected to be located in the vesicles of the cells. Figure 2(A) shows a vesicle with a number of nanoparticles inside. The darker nanoparticles originally thought to be Gd NPs, figure 2(B), are actually comprised largely of iron oxide. The lighter nanoparticles, shown in figure 2(C), are Gd NPs as confirmed by EDS in figure 3. Figure 2 TEM images of (A) a vesicle inside a stem cell showing a large amount of nanoparticulate. The darker nanoparticles (B) are comprised largely of iron oxide while some of the lighter na noparticles (C) are Gd NPs. Figure 3 EDS spectrum taken from the nanoparticle shown in figure 2(C) showing the presence of Gd in the nanoparticle. Locating and characterizing Gd NPs is a difficult task in the TEM requiring the use of advanced analytical techniques including EDS and EELS. With the addition of these tools, it is possible to locate and identify Gd NPs inside stem cells.

To date, several stem cell labeling protocols have been developed, contributing to a fast growing and promising field of stem cell imaging by MRI (magnetic resonance imaging). Most of these methods utilize iron oxide nanoparticles (MION, SPIO, USPIO, VSIOP) for cell labeling, which provide negative (dark) signal effects on T2-weighted MR images. The following protocol describes stem cell labeling techniques with commercially available gadolinium chelates, which provide positive contrast on T1-weighted MR images, which can be advantageous for specific applications.

PurposeTo develop a clinically applicable imaging technique for monitoring differential migration of macrophages into viable and apoptotic matrix-associated stem cell implants (MASIs) in arthritic knee joints.Materials and methodsWith institutional animal care and use committee approval, six athymic rats were injected with intravenous ferumoxytol (0.5 mmol iron per kilogram of body weight) to preload macrophages of the reticuloendothelial system with iron oxide nanoparticles. Forty-eight hours later, all animals received MASIs of viable adipose-derived stem cells (ADSCs) in an osteochondral defect of the right femur and mitomycin-pretreated apoptotic ADSCs in an osteochondral defect of the left femur. One additional control animal each received intravenous ferumoxytol and bilateral scaffold-only implants (without cells) or bilateral MASIs without prior ferumoxytol injection. All knees were imaged with a 7.0-T magnetic resonance (MR) imaging unit with T2-weighted fast spin-echo sequences immediately after, as well as 2 and 4 weeks after, matrix-associated stem cell implantation. Signal-to-noise ratios (SNRs) of viable and apoptotic MASIs were compared by using a linear mixed-effects model. MR imaging data were correlated with histopathologic findings.ResultsAll ADSC implants showed a slowly decreasing T2 signal over 4 weeks after matrix-associated stem cell implantation. SNRs decreased significantly over time for the apoptotic implants (SNRs on the day of matrix-associated stem cell implantation, 2 weeks after the procedure, and 4 weeks after the procedure were 16.9, 10.9, and 6.7, respectively; P = .0004) but not for the viable implants (SNRs on the day of matrix-associated stem cell implantation, 2 weeks after the procedure, and 4 weeks after the procedure were 17.7, 16.2, and 15.7, respectively; P = .2218). At 4 weeks after matrix-associated stem cell implantation, SNRs of apoptotic ADSCs were significantly lower than those of viable ADSCs (mean, 6.7 vs 15.7; P = .0013). This corresponded to differential migration of iron-loaded macrophages into MASIs.ConclusionIron oxide loading of macrophages in the reticuloendothelial system by means of intravenous ferumoxytol injection can be utilized to monitor differential migration of bone marrow macrophages into viable and apoptotic MASIs in a rat model.

Osteochondral defects due to degenerative or inflammatory arthritis represent one of the major causes of disabilities in the world, leading to annual health care costs in the order of US $95 billion in the United States alone. The pain associated with osteochondral defects leads to secondary effects, such as impaired joint mobility or function, decreasing work productivity, loss of quality of life—and in severe cases, potentially, depression-induced tertiary effects. Unlike other tissues in the body, cartilage cannot regenerate by itself. Therefore, there is an urgentneedfor effectiveapproaches torepair osteochondral defects in order to prevent progression of small defects to chronic disease and to restore larger defects and limit related disabilities. Cartilage repair is one of the most challenging areas in tissue engineering because of its complex and unique structure and exposure to high pressure and motion. The avascular and aneuronal nature of cartilage limits the ability to deliver signaling molecules, growth factors, or cellular components for tissue repair. In addition, mature chondrocytes are not able to migrate to sites of injury and the attraction of other cells (e.g., from the bone marrow or synovium) to cartilage defects is hindered by the complex and tight extracellular cartilage structure. Currently employed clinically applicable osteochondral defect repair strategies are as follows

Objectives The purpose of our study was to assess the chondrogenic potential and the MR signal effects of GadofluorineM-Cy labeled matrix associated stem cell implants (MASI) in pig knee specimen. Materials and Methods Human mesenchymal stem cells (hMSCs) were labeled with the micelle-based contrast agent GadofluorineM-Cy. Ferucarbotran-labeled hMSCs, non-labeled hMSCs and scaffold only served as controls. Chondrogenic differentiation was induced and gene expression and histologic evaluation were performed. The proportions of spindle-shaped vs. round cells of chondrogenic pellets were compared between experimental groups using the Fisher's exact test. Labeled and unlabeled hMSCs and chondrocytes in scaffolds were implanted into cartilage defects of porcine femoral condyles and underwent MR imaging with T1- and T2-weighted SE and GE sequences. Contrast-to-noise ratios (CNR) between implants and adjacent cartilage were determined and analyzed for significant differences between different experimental groups using the Kruskal-Wallis test. Significance was assigned for p0.017). However, hMSC differentiation into chondrocytes was superior for unlabeled and GadofluorineM-Cy-labeled cells compared with Ferucarbotran-labeled cells, as evidenced by a significantly higher proportion of spindle cells in chondrogenic pellets (p

Vitamin D is essential for the maintenance of good health, and vitamin D deficiency has been reported from many countries, including those with a lot of sunshine. This study was conducted to evaluate the vitamin D status in healthy 6- to 7-year-old children in Isfahan, Iran.Five hundred and thirteen healthy children were enrolled. Serum PTH and 25-hydroxyvitamin D (25-OHD) were measured. Dietary vitamin D intake, duration of daily sunlight exposure, and percentage of exposed body surface area were determined. 25-OHD levels20 ng/mL and10 ng/mL were defined as mild and severe vitamin D deficiency, respectively. The ROC curve was utilized to obtain a local cut-off point of vitamin D deficiency.25-OHD was20 ng/mL in 3% and33 ng/mL (local cut-off point of vitamin D deficiency) in 26% of subjects. Duration of sunlight exposure and daily intake of vitamin D had significant effects on serum level of vitamin D.A high prevalence of vitamin D deficiency in Isfahan children was observed in this study. Improvements in duration of sunlight exposure and daily intake of vitamin D can prevent vitamin D deficiency in these children.

Cavernous angioma is a vascular malformation which can be found in any region within the central nervous system.There are few clinical and demographic cavernous angioma studies with large sample sizes. Therefore, the present study was designed to provide further information on the clinical and demographic characteristics of cavernous angioma using a relatively large sample of Persian patients.Patients with cavernous angioma were recruited from the outpatient neurology clinics in Isfahan, Iran, from October 2003 to October 2006.In all cases, the diagnosis of cavernous angioma was based on brain magnetic resonance imaging. There were 35 patients (female/male: 17 : 18) identified with cavernous angioma. The mean age at presentation was 28.8 years. Initial manifestations included seizures in 16, headache in 11 and intracranial hemorrhage in eight patients. During follow-up, all patients experienced seizures and 19 developed headaches. Depression, vertigo, nausea, vomiting, disequilibrium, loss of consciousness and sensorimotor symptoms were also observed.Some of the findings of the present study were in accordance with previous studies. However, more of our patients with positive family history had solitary rather than multiple lesions, and more of our patients had generalized tonic-clonic seizures rather than partial seizures. Moreover, our data demonstrated that if there is a history of cavernous angioma with intracranial hemorrhage in family members, the presenting cavernous angioma patient is more prone to intracranial hemorrhage.