Attempt to treat congenital pseudarthrosis of the tibia with mesenchymal stromal cell transplantation

Background aimsCongenital pseudarthrosis of the tibia (CPT) caused by neurofibromatosis type 1 (NF1) is a refractory disease occurring in childhood. We present two cases that had failed all earlier treatment attempts and, as a last treatment attempt, the patients were chosen to receive mesenchymal stromal cell (MSC) transplantation prior to amputation.MethodsThe MSC from bone marrow (BM) were harvested from the iliac crest and cultured in osteoinductive medium for 3 weeks. The cultured MSC were injected in solution into BM canals of the tibia and around the resection line or bone defect in a 3-dimensional collagen sponge scaffold. After the MSC transplantation, the patients were monitored during a 10-month follow-up period. In both cases, bone formation at the pseudarthrosis site was observed and two of three treated bone defects healed. For clinical reasons not related to cell transplantation, such as new infection and pseudarthrosis and severe shortening of the leg, both extremities were finally amputated and bone samples were analyzed to evaluate MSC therapy effect and safety.ResultsMSC transplantation normalized bone remodeling, promoted bone resorption and improved the overall structure of bone. The number of osteoclasts in the cortical bone was 2-fold higher compared with the monitored situation before MSC transfer. In addition, the mineral content of the bone improved after transplantation. We could see no sign of aberrant bone formation or malignant transformation.ConclusionsOur data suggest that MSC transplantation is a possibility for treatment of CPT caused by NF1 in less severe cases without adjunct defects.     

Intramyocardial and intracoronary autologous bone marrow-derived mesenchymal stromal cell treatment in chronic severe dilated cardiomyopathy

Background aimsMesenchymal stromal cells (MSC) may improve cardiac function following myocardial infarction. MSC can differentiate into cardiomyocytes and endothelial cells while exerting additional paracrine effects. There is limited information regarding the efficacy of route for MSC treatment of severe dilated cardiomyopathy (DCM). The aim of this study was to demonstrate the clinical safety, feasibility and efficacy of direct intramyocardial and intracoronary administration of autologous bone marrow-derived MSC treatment for no-option patients with chronic severe refractory DCM.MethodsTen symptomatic patients with DCM and refractory cardiac function, despite maximum medical therapy, were selected. Five had ischemic DCM deemed unlikely to benefit from revascularization alone and underwent bypass operations with concurrent intramyocardial MSC injection (group A). Two patients had previous revascularization and three had non-ischemic DCM and received intracoronary MSC injection (group B).ResultsGroup A and B patients received 0.5–1.0 × 10⁶ and 2.0–3.0 × 10⁶ MSC/kg body weight, respectively. All patients remained alive at 1 year. There were significant improvements from baseline to 6 and 12 months in left ventricular ejection fraction and other left ventricular parameters. Scar reduction was noted in six patients by 12 months.ConclusionsAutologous bone marrow MSC treatment is safe and feasible for treating chronic severe refractory DCM effectively, via intracoronary or direct intramyocardial administration at prescribed doses.     

Cancellous bone allograft seeded with human mesenchymal stromal cells: a potential good manufacturing practice-grade tool for the regeneration of bone defects

Background aimsCombining autologous bone precursor cells with cancellous bone allograft (CBA) offers an appealing strategy for skeletal regeneration. In this context, multipotent mesenchymal stromal cells (MSC) provide an excellent cell source because they are readily harvested from donors, expanded and differentiated in vitro. The aim of this study was to evaluate the proliferation, morphology, osteogenic differentiation and stem cell-related gene expression during static long-term ex vivo cultivation using human MSC and CBA under good manufacturing practice (GMP)-conforming conditions.MethodsMSC were isolated from healthy donors (n = 5) and cultivated on peracetic acid-sterilized CBA in the presence of 10% human platelet-rich plasma without osteogenic supplements. Total protein content, cell-specific alkaline phosphatase (ALP) activity and osteogenic marker gene expression levels were assessed. Stem cell-related gene expression was compared with MSC monolayer cultivation using microarray analysis. Furthermore, cellular distribution and morphology within the porous CBA were visualized by histology and scanning electron microscopy.ResultsEffective adhesion, spreading, proliferation and intercellular contact of human MSC within the pores of CBA were observed during the study (≤42 days). Cell-specific ALP activity peaked after 3 weeks of cultivation. Gene expression of early, intermediate and late osteogenic marker genes was detectable during long-term cultivation. Microarray-based annotation and biologic interaction network data analysis indicated that expression levels of genes encoding crucial differentiation-regulating proteins and extracellular matrix components involved in the process of osteogenesis were induced in CBA-cultivated MSC.ConclusionsMSC-vitalized CBA offers an attractive GMP-grade bone-filling material. Further research is warranted to evaluate its bone-healing potential in vivo.     

Human bone marrow mesenchymal cells express NG2: possible increase in discriminative ability of flow cytometry during mesenchymal stromal cell identification

Background aimsMesenchymal stromal cells (MSC) exhibit non-specific hematopoietic cell and/or stromal cell markers (e.g. CD73, CD105 and CD166) that have been used to identify MSC by flow cytometry. Because a neural glial antigen, NG2 (a progenitor cell marker in the central nervous system), is expressed by several tissue cells originating in the mesenchyme but not hematopoietic cells, it might be useful for isolating and identifying MSC. We investigated NG2 expression on culture-expanded MSC by flow cytometry.MethodsHuman bone marrow (BM) samples taken from 12 donors were cultured for MSC to be used in up to nine serial passages. Using flow cytometry, the neural glial antigen NG2 and commonly used MSC markers CD73, CD105 and CD166, were analyzed on the surface of culture-expanded MSC. The multipotential differentiation of the MSC was examined by adipogenic and osteogenic induction.ResultsThe percentage of cells positive for NG2 was similar to the percentages of cells positive for CD73, CD105 and CD166 in all passages of BM samples. The mean fluorescent intensities of NG2 did not change with culture passage. The MSC was successfully differentiated into adipogenic and osteogenic lines. The cells showed no karyotypic abnormalities.ConclusionsNG2 seems to be a promising marker for investigating the biology of MSC.     

Rat marrow-derived mesenchymal stem cells developed in a medium supplemented with the autologous versus bovine serum

The controversial effect of autologous serum (AS) on human mesenchymal stem cells (MSC) was studied in rat MSC culture. Rat bone marrow cells were plated in a medium containing either FBS (fetal bovine serum) or AS were cultured to passage 3, during which the population doubling number (PDN) of both cultures were measured and compared statistically. The number of viable cells, the cell colonogic activity, and cell growth rate were also compared. In addition, mineralization in the osteogenic cultures from each system was measured. Our data indicated that AS enriched medium provided a microenvironment in which growth rate as well as bone differentiation of the isolated MSCs were significantly higher than in FBS enriched medium.     

Collagen-Hydroxyapatite Scaffolds Induce Human Adipose Derived Stem Cells Osteogenic Differentiation In Vitro

Mesenchymal stem cells (MSCs) play a crucial role in regulating normal skeletal homeostasis and, in case of injury, in bone healing and reestablishment of skeletal integrity. Recent scientific literature is focused on the development of bone regeneration models where MSCs are combined with biomimetic three-dimensional scaffolds able to direct MSC osteogenesis. In this work the osteogenic potential of human MSCs isolated from adipose tissue (hADSCs) has been evaluated in vitro in combination with collagen/Mg doped hydroxyapatite scaffolds. Results demonstrate the high osteogenic potential of hADSCs when cultured in specific differentiation induction medium, as revealed by the Alizarin Red S staining and gene expression profile analysis. In combination with collagen/hydroxyapatite scaffold, hADSCs differentiate into mature osteoblasts even in the absence of specific inducing factors; nevertheless, the supplement of the factors markedly accelerates the osteogenic process, as confirmed by the expression of specific markers of pre-osteoblast and mature osteoblast stages, such as osterix, osteopontin (also known as bone sialoprotein I), osteocalcin and specific markers of extracellular matrix maturation and mineralization stages, such as ALPL and osteonectin. Hence, the present work demonstrates that the scaffold per se is able to induce hADSCs differentiation, while the addition of osteo-inductive factors produces a significant acceleration of the osteogenic process. This observation makes the use of our model potentially interesting in the field of regenerative medicine for the treatment of bone defects.     

Impaired differentiation potential of human trabecular bone mesenchymal stromal cells from elderly patients

Background aimsAdvances in bone tissue engineering with mesenchymal stromal cells (MSC) as an alternative to conventional orthopedic procedures has opened new horizons for the treatment of large bone defects. Bone marrow (BM) and trabecular bone are both sources of MSC. Regarding clinical use, we tested the potency of MSC from different sources.MethodsWe obtained MSC from 17 donors (mean age 64.6 years) by extensive washing of trabecular bone from the femoral head and trochanter, as well as BM aspirates of the iliac crest and trochanter. The starting material was evaluated by histologic analysis and assessment of colony-forming unit–fibroblasts (CFU-F). The MSC populations were compared for proliferation and differentiation potential, at RNA and morphologic levels.ResultsMSC proliferation potential and immunophenotype (expression of CD49a, CD73, CD90, CD105, CD146 and Stro-1) were similar whatever the starting material. However, the differentiation potential of MSC obtained by bone washing was impaired compared with aspiration; culture-amplified cells showed few Oil Red O-positive adipocytes and few mineralized areas and formed inconsistent Alcian blue-positive high-density micropellets after growth under adipogenic, osteogenic and chondrogenic conditions, respectively. MSC cultured with 1 ng/mL fibroblast growth factor 2 (FGF-2) showed better differentiation potential.ConclusionsTrabecular bone MSC from elderly patients is not good starting material for use in cell therapy for bone repair and regeneration, unless cultured in the presence of FGF-2.     

Dexamethasone has variable effects on mesenchymal stromal cells

Background aimsDexamethasone (Dex) is a potent synthetic member of the glucocorticoid class of steroid drugs. Frequently, Dex has been used to enhance osteogenic, chondrogenic and adipogenic differentiation of mesenchymal stromal cells (MSC). Recently, Dex was applied to promote MSC proliferation, because of the rare frequency of MSC in bone marrow, and could protect the cells from apoptosis. The effects of Dex on MSC cytobiology behavior needs to be investigated.MethodsMSC were obtained from human umbilical cord. The surface phenotype and functional characterization of MSC cultured with different concentrations of Dex were investigated, in comparison with a control group, including MSC proliferation, apoptosis, cytokine expression and immunosuppression.ResultsDifferent concentrations of Dex exerted diverse effects on MSC proliferation and apoptosis. Dex was also able to affect the pattern of cytokine expression of MSC. Furthermore, Dex impaired immunosuppression of MSC on peripheral blood mononuclear cells.ConclusionsA low dose of Dex favors MSC expansion in vitro, and protects against apoptosis. It is not suitable for MSC to be pre-treated with Dex when they are to be used to treat immunologic disease. However, when MSC are applied to promote angiogenesis, it is beneficial for them to be pre-treated with 10^?9 mol/L Dex.     

Articular cartilage-derived cells hold a strong osteogenic differentiation potential in comparison to mesenchymal stem cells in vitro

Cartilaginous matrix-degenerative diseases like osteoarthritis (OA) are characterized by gradual cartilage erosion, and also by increased presence of cells with mesenchymal stem cell (MSC) character within the affected tissues. Moreover, primary chondrocytes long since are known to de-differentiate in vitro and to be chondrogenically re-differentiable. Since both findings appear to conflict with each other, we quantitatively assessed the mesenchymal differentiation potential of OA patient cartilage-derived cells (CDC) towards the osteogenic and adipogenic lineage in vitro and compared it to that of MSC isolated from adipose tissue (adMSC) of healthy donors. We analyzed expression of MSC markers CD29, CD44, CD105, and CD166, and, following osteogenic and adipogenic induction in vitro, quantified their expression of osteogenic and adipogenic differentiation markers. Furthermore, CDC phenotype and proliferation were monitored. We found that CDC exhibit an MSC CD marker expression pattern similar to adMSC and a similar increase in proliferation rate during osteogenic differentiation. In contrast, the marked reduction of proliferation observed during adipogenic differentiation of adMSC was absent in CDC. Quantification of differentiation markers revealed a strong osteogenic differentiation potential for CDC, however almost no capacity for adipogenic differentiation. Since in the pathogenesis of OA, cartilage degeneration coincides with high bone turnover rates, the high osteogenic differentiation potential of OA patient-derived CDC may affect clinical therapeutic regimens aiming at autologous cartilage regeneration in these patients.     

Feasibility and efficacy of bone tissue engineering using human bone marrow stromal cells cultivated in serum-free conditions

Current standard techniques for bone tissue engineering utilize ex vivo expanded osteogenic cells. However, ex vivo expansion requires serum, which may hinder clinical applications. Here, we report the feasibility and efficacy of bone tissue engineering with human bone marrow stromal cells (BMSCs) expanded in serum-free conditions. Bone marrow was aspirated from 4 healthy donors and adherent cells were cultured in either serum-free medium (STEMPRO^® MSC SFM) or conventional serum-containing medium (α-MEM supplemented with 10% serum). Efficacy of expansion was greater in serum-free medium. Phenotypically, serum-free expanded BMSCs were smaller in cell-size and showed expression of CD105⁺⁺ and CD146^dim. After osteogenic induction, serum-free expanded BMSCs showed lower alkaline phosphatase activity. However, they showed higher responsiveness to induction. In vivo bone-forming ability was also confirmed. In conclusion, bone tissue engineering with serum-free expanded BMSCs is feasible and as efficient as that obtained with BMSCs expanded in conventional serum-containing medium.     

Osteogenic Differentiation of Human Mesenchymal Stem Cells in Mineralized Alginate Matrices

Mineralized biomaterials are promising for use in bone tissue engineering. Culturing osteogenic cells in such materials will potentially generate biological bone grafts that may even further augment bone healing. Here, we studied osteogenic differentiation of human mesenchymal stem cells (MSC) in an alginate hydrogel system where the cells were co-immobilized with alkaline phosphatase (ALP) for gradual mineralization of the microenvironment. MSC were embedded in unmodified alginate beads and alginate beads mineralized with ALP to generate a polymer/hydroxyapatite scaffold mimicking the composition of bone. The initial scaffold mineralization induced further mineralization of the beads with nanosized particles, and scanning electron micrographs demonstrated presence of collagen in the mineralized and unmineralized alginate beads cultured in osteogenic medium. Cells in both types of beads sustained high viability and metabolic activity for the duration of the study (21 days) as evaluated by live/dead staining and alamar blue assay. MSC in beads induced to differentiate in osteogenic direction expressed higher mRNA levels of osteoblast-specific genes (RUNX2, COL1AI, SP7, BGLAP) than MSC in traditional cell cultures. Furthermore, cells differentiated in beads expressed both sclerostin (SOST) and dental matrix protein-1 (DMP1), markers for late osteoblasts/osteocytes. In conclusion, Both ALP-modified and unmodified alginate beads provide an environment that enhance osteogenic differentiation compared with traditional 2D culture. Also, the ALP-modified alginate beads showed profound mineralization and thus have the potential to serve as a bone substitute in tissue engineering.     

Efficient isolation and enrichment of mesenchymal stem cells from bone marrow

Background aimsBone marrow (BM) mesenchymal stromal cells (MSC) have been identified as a source of pluripotent stem cells used in clinical practice to regenerate damaged tissues. BM MSC are commonly isolated from BM by density-gradient centrifugation. This process is an open system that increases the risk of sample contamination. It is also time consuming and requires technical expertise that may result in variability regarding cellular recovery. The BD Vacutainer^® Cell Preparation Tube? (CPT) was conceived to separate mononuclear cells from peripheral blood. The main goal of this study was to verify whether MSC could be isolated from BM using the CPT.MethodsBM was harvested, divided into two equal aliquots and processed using either CPT or a Ficoll-Paque? PREMIUM density gradient. Both methods were compared regarding cell recovery, viability, proliferation, differentiation capacities and the presence of MSC progenitors.ResultsSimilar numbers of mononuclear cells were isolated from BM when comparing the two methods under study. No differences were found in terms of phenotypic characterization, viability, kinetics and lineage differentiation potential of MSC derived by CPT or Ficoll. Surprisingly, a fibroblast–colony-forming unit (CFU-F) assay indicated that, with CPT, the number of MSC progenitors was 1.8 times higher compared with the Ficoll gradient separation.ConclusionsThe CPT method is able to isolate MSC efficiently from BM, allowing the enrichment of MSC precursors.     

Defined serum-free media for in vitro expansion of adipose-derived mesenchymal stem cells

BackgroundThere is a growing interest in mesenchymal stem cells (MSCs) because they are regarded as good candidates for cell therapy. Adipose tissue represents an easily accessible source to derive mesenchymal stem cells (Ad-MSCs) non-invasively in large numbers. The aim of this study was to evaluate a defined serum-free medium for in vitro expansion of MSCs as a prerequisite for their clinical use.MethodsAdipose tissue was isolated from healthy donors. Cells were isolated and expanded for five passages in serum-free medium (Mesencult-XF) and Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (DMEM-FBS). MSC morphology, marker expression, viability, population doubling time and differentiation potential toward osteogenic and adipogenic lineages were evaluated. Bone marrow MSCs were included as controls.ResultsAd-MSCs cultured in Mesencult-XF had shorter population doubling time (33.3 ± 13.7 h) compared with those cultured in DMEM-FBS (54.3 ± 41.0 h, P < 0.05). Ad-MSCs cultured in Mesencult-XF displayed a stable morphology and surface marker expression and a higher differentiation potential in comparison to Ad-MSCs cultured in DMEM-FBS.ConclusionsThe defined serum-free and xeno-free Mesencult-XF media appear to be a good choice for Ad-MSCs, but it is not as good in supporting culture of bone marrow MSCs when the cells are to be used for clinical purposes.     

富血小板纤维蛋白联合人工骨粉在口腔种植引导性骨再生中的临床应用价值

目的:探讨富血小板纤维蛋白(PRF)联合人工骨粉(Bio-Oss骨粉)在口腔种植引导性骨再生中的临床应用价值。方法:选取2017年6月到2018年6月期间在我院接受口腔种植引导性骨再生手术治疗的患者80例,根据随机数字表法分为对照组(40例)和研究组(40例),对照组采用Bio-Oss骨粉联合Bio-Gide生物膜进行干预,研究组采用Bio-Oss骨粉联合PRF进行干预。比较两组患者的组织愈合情况、出血指数、探诊深度、附着丧失、植体周围的骨密度、植骨高度、成骨厚度和术后并发症。结果:研究组的组织愈合优良率为95.00%,高于对照组的80.00%,差异有统计学意义(P0.05)。研究组患者的出血指数、探诊深度、附着丧失均低于对照组,种植体周围的骨密度、成骨厚度均明显大于对照组,植骨高度高于对照组,差异有统计学意义(P0.05)。两组患者的并发症发生率比较差异无统计学意义(P0.05)。结论:PRF联合Bio-Oss骨粉可有效增加种植区骨量和种植体周围的骨密度,促进骨缺损再生和组织愈合,且无明显的并发症,在口腔种植引导性骨再生中有较好的临床应用价值。     

K562 chronic myeloid leukemia cells modify osteogenic differentiation and gene expression of bone marrow stromal cells

Bone marrow (BM) microenvironment plays an important role in normal and malignant hematopoiesis. As a consequence of interaction with the leukemic cells, the stromal cells of the bone marrow become deregulated in their normal function and gene expression. In our study, we found that mesenchymal stem cells (MSC) from BM of chronic myeloid leukemia (CML) patients have defective osteogenic differentiation and on interaction with K562 CML cells, the normal MSC showed reduced osteogenic differentiation. On interaction with K562 cells or its secreted factors, MSC acquired phenotypic abnormalities and secreted high levels of IL6 through NFκB activation. The MSC derived secreted factors provided a survival advantage to CML cells from imatinib induced apoptosis. Thus, a therapy targeting stromal cells in addition to leukemia cells might be more effective in eliminating CML cells.     

Repair of long-bone pseudoarthrosis with autologous bone marrow mononuclear cells combined with allogenic bone graft

Background aimsLong-bone pseudoarthrosis is a major orthopedic concern because of numerous factors such as difficulty of the treatment, high recurrence, high costs and the devastating effects on the patients' quality of life, which sometimes ends in amputation. Although the “gold standard” for the treatment of this pathology is autologous bone grafting, which has high osteogenic, osteoconductive and osteoinductive properties, this treatment presents some restrictions such as the limited amount of bone that can be taken from the patient and donor site morbidity. Bone marrow mononuclear cells (BM-MNCs) comprise progenitor and stem cells with pro-angiogenic and pro-osteogenic properties. Allogenic cancellous bone graft is a natural and biodegradable osteoconductive and osteoinductive scaffold. Combination of these two components could mimic the advantages of autologous bone grafting while avoiding its main limitations.MethodsLong-bone pseudoarthrosis was treated in seven patients with autologous BM-MNCs from iliac crest combined with frozen allogenic cancellous bone graft obtained from the tissue bank.ResultsAll patients showed complete bone consolidation 5.3 ± 0.9 months (range, 2–9 months) after cell transplantation. Moreover, limb pain disappeared in all of them. The mean follow-up was 35.8 ± 4.6 months after transplantation (range, 24–51 months) without pseudoarthrosis recurrence or pain reappearing.ConclusionsCombination of autologous BM-MNCs and allogenic bone graft could constitute an easy, safe, inexpensive and efficacious attempt to treat long-bone pseudoarthrosis and non-union by reproducing the beneficial properties of autologous bone grafting while restricting its disadvantages.     

Mesenchymal stromal cell transplantation in amyotrophic lateral sclerosis: a long-term safety study

Background aimsMesenchymal stem cells/marrow stromal cells (MSC) represent a promising tool for stem cell-based clinical trials in amyotrophic lateral sclerosis (ALS). We present the results of long-term monitoring of 19 ALS patients enrolled in two phase I clinical trials of autologous MSC transplantationMethodsNineteen patients (11 male and eightfemale) with ALS were enrolled in two consecutive phase I clinical trials. The patients were followed-up for 6–9 months and then treated with autologous MSC isolated from bone marrow and implanted into the dorsal spinal cord with a surgical procedure. The patients were monitored regularly before and after transplantation with clinical, psychological and neuroradiologic assessments every 3 months, at the tertiary referral ALS center in Novara (Italy), until deathResultsFollow-up brain magnetic resonance imaging (MRI) revealed no structural changes (including tumor formation) relative to the baseline throughout the follow-up. There was no deterioration in the psychosocial status and all patients coped well. No clear clinical benefits were detected in these patients but the recruitment and selection of appropriate patients into larger trials will be needed to test the efficacy of the treatmentConclusionsThis study is the first to show the safety of MSC transplantation in the central nervous system during a follow-up of nearly 9 years, and is in support of applying MSC-based cellular clinical trials to neurodegenerative disorders.     

CD200 expression in human cultured bone marrow mesenchymal stem cells is induced by pro‐osteogenic and pro‐inflammatory cues

Similar to other adult tissue stem/progenitor cells, bone marrow mesenchymal stem/stromal cells (BM MSCs) exhibit heterogeneity at the phenotypic level and in terms of proliferation and differentiation potential. In this study such a heterogeneity was reflected by the CD200 protein. We thus characterized CD200^pos cells sorted from whole BM MSC cultures and we investigated the molecular mechanisms regulating CD200 expression. After sorting, measurement of lineage markers showed that the osteoblastic genes RUNX2 and DLX5 were up‐regulated in CD200^pos cells compared to CD200^neg fraction. At the functional level, CD200^pos cells were prone to mineralize the extra‐cellular matrix in vitro after sole addition of phosphates. In addition, osteogenic cues generated by bone morphogenetic protein 4 (BMP4) or BMP7 strongly induced CD200 expression. These data suggest that CD200 expression is related to commitment/differentiation towards the osteoblastic lineage. Immunohistochemistry of trephine bone marrow biopsies further corroborates the osteoblastic fate of CD200^pos cells. However, when dexamethasone was used to direct osteogenic differentiation in vitro, CD200 was consistently down‐regulated. As dexamethasone has anti‐inflammatory properties, we assessed the effects of different immunological stimuli on CD200 expression. The pro‐inflammatory cytokines interleukin‐1β and tumour necrosis factor‐α increased CD200 membrane expression but down‐regulated osteoblastic gene expression suggesting an additional regulatory pathway of CD200 expression. Surprisingly, whatever the context, i.e. pro‐inflammatory or pro‐osteogenic, CD200 expression was down‐regulated when nuclear‐factor (NF)‐κB was inhibited by chemical or adenoviral agents. In conclusion, CD200 expression by cultured BM MSCs can be induced by both osteogenic and pro‐inflammatory cytokines through the same pathway: NF‐κB.     

Endothelial cells influence the osteogenic potential of bone marrow stromal cells

Background  

Improved understanding of the interactions between bone cells and endothelial cells involved in osteogenesis should aid the development of new strategies for bone tissue engineering. The aim of the present study was to determine whether direct communication between bone marrow stromal cells (MSC) and human umbilical vein endothelial cells (EC) could influence the osteogenic potential of MSC in osteogenic factor-free medium.