Generation and functional characterization of ovine bone marrow-derived macrophages.

A method for the culturing and propagation of ovine bone marrow-derived macrophages (BMM) in vitro is described. Bone marrow cells from sterna of freshly slaughtered sheep were cultured in hydrophobic (teflon foil) bags in the presence of high serum concentrations (20% autologous serum and 20% fetal calf serum). During an 18 day culture period in the absence of added conditioned medium, and without medium change, a strong enrichment of mononuclear phagocytes was achieved. Whereas the number of macrophages increased four to fivefold during this time, granulocytes, lymphoid cells, stem cells and undifferentiated progenitor cells were reduced to less than 3% of their numbers at Day 0. This resulted in BMM populations of 94 +/- 3% purity. These cells had morphological and histochemical characteristics of differentiated macrophages, and they performed functions similar to those of non-activated, unprimed human monocyte-derived macrophages. Thus, they avidly ingested erythrocytes coated with IgG of heterologous or homologous origin. They expressed a modest level of procoagulant activity, but upon triggering with lipopolysaccharide (LPS), a marked increase in cell-associated procoagulant activity was observed. LPS triggering promoted the secretion of interleukin-1, as evidenced by measurement of murine thymocyte costimulatory activity, and transforming growth factor-beta. Using the mouse L929 cell cytotoxicity assay as an indication of tumor necrosis factor (TNF) activity, no TNF activity was detected in the same supernatants, a result possibly due to species restriction. BMM generated low levels of O2- upon triggering with phorbol 12-myristate 13-acetate (PMA). On the other hand, no O2- production was observed upon stimulation with zymosan opsonized with ovine or human serum. Using luminol-enhanced chemiluminescence (CL) as a more sensitive indicator of an oxidative burst, both PMA or zymosan were able to trigger CL, but the response was subject to partial inhibition by sodium azide, an inhibitor of myeloperoxidase. This points to non-macrophage cells contributing also to the CL response, and is consistent with the view that unprimed BMM elicit a low oxidative burst upon triggering with strong inducers of a burst. Our functional characterization now allows us to apply priming and activation protocols and to relate their effect to functional alterations.

Culture of ovine bone marrow-derived macrophages and evidence for serum factors distinct from M-CSF contributing to their propagation in vitro.

An in vitro system allowing the culture of ovine bone marrow-derived macrophages (BMMs) is described. Bone marrow (BM) cells from the sternum of 4- to 9-month-old sheep were cultured in liquid suspension in hydrophobic bags with medium containing 20% autologous serum and 20% fetal calf serum (FCS). Cells with macrophage characteristics were positively selected and increased four- to five-fold between day (d) 0 and d18. Granulocytes and cells of lymphoid appearance including progenitor cells were negatively selected and were diminished 50-fold during this 18-d culture. The addition of macrophage colony-stimulating factor (M-CSF)-containing supernatants to liquid cultures did not significantly improve the yield of BMM in 18-d cultures. In contrast, cell survival at d6 and macrophage cell yield at d18 depended on the concentration and source of serum in the culture medium. FCS and 1:1 mixtures of FCS and autologous serum were superior to autologous serum alone. Analysis of growth requirements of ovine BMMs suggested that they are under more complex growth control than their murine counterparts. In an [3H]thymidine incorporation assay with BM cells collected at different times of culture, d3 or d4 BM cells responded to human recombinant M-CSF, human recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF), bovine GM-CSF, murine M-CSF or murine M-CSF-containing supernatants, and bovine interleukin 1 beta (IL-1 beta) in decreasing order of magnitude. Likewise, pure murine BMM populations harvested at d6 responded to homologous GM-CSF, IL-3, and human or murine M-CSF. FCS did not stimulate the proliferation of murine BMMs (d6) and of ovine BM cells (d3 or d4). In contrast, ovine BM cells harvested at d12 responded to FCS by proliferation in a dose-dependent manner but failed to proliferate in the presence of human or murine M-CSF or M-CSF-containing supernatants of mouse and sheep fibroblasts containing mouse macrophage growth-promoting activity. Likewise, various cytokine-containing supernatants and recombinant cytokines (murine IL-3, murine and human GM-CSF, murine and bovine IL-1 beta) did not promote proliferation of ovine d12 BM cells to an extent greater than that achieved with 15% FCS alone. Thus, ovine BMM proliferation is under the control of at least two factors acting in sequence, M-CSF and an unidentified factor contained in FCS. The ovine BMM culture system may provide a model for the analysis of myelomonocytopoiesis in vitro.

The recruitment of bone marrow-derived cells to skin wounds is independent of wound size

Wounded skin recruits progenitor cells, which repair the tissue defect. These cells are derived from stem cells in several niches in the skin. In addition, bone marrow-derived cells (BMDCs) are recruited and contribute to wound repair. We hypothesized that larger wounds recruit more cells from the bone marrow. Wild-type rats were lethally irradiated and transplanted with bone marrow cells from green fluorescent protein (GFP)-transgenic rats. Seven weeks later, 4, 10, and 20 mm wounds were created. The wound tissue was harvested after 14 days. The density of GFP-positive cells in the wounds and the adjacent tissues was determined, as well as in normal skin from the flank. Bone marrow-derived myofibroblasts, activated fibroblasts, and macrophages were also quantified. After correction for cell density, the recruitment of BMDCs (23±11%) was found to be independent of wound size. Similar fractions of GFP-positive cells were also detected in nonwounded adjacent tissue (29±11%), and in normal skin (26±19%). The data indicate that BMDCs are not preferentially recruited to skin wounds. Furthermore, wound size does not seem to affect the recruitment of BMDCs.

Comparative analysis of canine monocyte- and bone-marrow-derived dendritic cells

Dendritic cells (DC) represent a heterogeneous cell family of major importance for innate immune responses against pathogens and antigen presentation during infection, cancer, allergy and autoimmunity. The aim of the present study was to characterize canine DC generated in vitro with respect to their phenotype, responsiveness to toll-like receptor (TLR) ligands and T-cell stimulatory capacity. DC were derived from monocytes (MoDC) and from bone marrow hematopoietic cells cultured with either Flt3-ligand (FL-BMDC) or with GM-CSF (GM-BMDC). All three methods generated cells with typical DC morphology that expressed CD1c, CD11c and CD14, similar to macrophages. However, CD40 was only found on DC, CD206 on MPhi and BMDC, but not on monocytes and MoDC. CD1c was not found on monocytes but on all in vitro differentiated cells. FL-BMDC and GM-BMDC were partially positive for CD4 and CD8. CD45RA was expressed on a subset of FL-BMDC but not on MoDC and GM-BMDC. MoDC and FL-DC responded well to TLR ligands including poly-IC (TLR2), Pam3Cys (TLR3), LPS (TLR4) and imiquimod (TLR7) by up-regulating MHC II and CD86. The generated DC and MPhi showed a stimulatory capacity for lymphocytes, which increased upon maturation with LPS. Taken together, our results are the basis for further characterization of canine DC subsets with respect to their role in inflammation and immune responses.

Bone marrow-derived cells in palatal wound healing

OBJECTIVE: Myofibroblasts are responsible for contraction and scarring after cleft palate repair. This leads to growth disturbances in the upper jaw. We hypothesized that cells from the bone marrow are recruited to palatal wounds and differentiate into myofibroblasts. METHODS: We transplanted bone marrow from green fluorescent protein (GFP)-transgenic rats into lethally irradiated wild-type rats. After recovery, experimental wounds were made in the palatal mucoperiosteum, and harvested 2 weeks later. GFP-expressing cells were identified using immunostaining. Myofibroblasts, activated fibroblasts, endothelial cells, and myeloid cells were quantified with specific markers. RESULTS: After transplantation, 89 ± 8.9% of mononuclear cells in the blood expressed the GFP and about 50% of adherent cells in the bone marrow. Tissue obtained during initial wounding contained only minor numbers of GFP-positive cells, like adjacent control tissue. Following wound healing, 8.1 ± 5.1% of all cells in the wound area were positive, and 5.0 ± 4.0% of the myofibroblasts, which was significantly higher than in adjacent tissue. Similar percentages were found for activated fibroblasts and endothelial cells, but for myeloid cells it was considerably higher (22 ± 9%). CONCLUSIONS: Bone marrow-derived cells contribute to palatal wound healing, but are not the main source of myofibroblasts. In small wounds, the local precursor cells are probably sufficient to replenish the defect.

Cardiovascular changes after PMMA vertebroplasty in sheep: the effect of bone marrow removal using pulsed jet-lavage

Clinically, the displacement of intravertebral fat into the circulation during vertebroplasty is reported to lead to problems in elderly patients and can represent a serious complication, especially when multiple levels have to be treated. An in vitro study has shown the feasibility of removing intravertebral fat by pulsed jet-lavage prior to vertebroplasty, potentially reducing the embolization of bone marrow fat from the vertebral bodies and alleviating the cardiovascular changes elicited by pulmonary fat embolism. In this in vivo study, percutaneous vertebroplasty using polymethylmethacrylate (PMMA) was performed in three lumbar vertebrae of 11 sheep. In six sheep (lavage group), pulsed jet-lavage was performed prior to injection of PMMA compared to the control group of five sheep receiving only PMMA vertebroplasty. Invasive recording of blood pressures was performed continuously until 60 min after the last injection. Cardiac output and arterial blood gas parameters were measured at selected time points. Post mortem, the injected cement volume was measured using CT and lung biopsies were processed for assessment of intravascular fat. Pulsed jet-lavage was feasible in the in vivo setting. In the control group, the injection of PMMA resulted in pulmonary fat embolism and a sudden and significant increase in mean pulmonary arterial pressure. Pulsed jet-lavage prevented any cardiovascular changes and significantly reduced the severity of bone marrow fat embolization. Even though significantly more cement had been injected into the lavaged vertebral bodies, significantly fewer intravascular fat emboli were identified in the lung tissue. Pulsed jet-lavage prevented the cardiovascular complications after PMMA vertebroplasty in sheep and alleviated the severity of pulmonary fat embolism.

Micromass co-culture of human articular chondrocytes and human bone marrow mesenchymal stem cells to investigate stable neocartilage tissue formation in vitro

Cell therapies for articular cartilage defects rely on expanded chondrocytes. Mesenchymal stem cells (MSC) represent an alternative cell source should their hypertrophic differentiation pathway be prevented. Possible cellular instruction between human articular chondrocytes (HAC) and human bone marrow MSC was investigated in micromass pellets. HAC and MSC were mixed in different percentages or incubated individually in pellets for 3 or 6 weeks with and without TGF-beta1 and dexamethasone (±T±D) as chondrogenic factors. Collagen II, collagen X and S100 protein expression were assessed using immunohistochemistry. Proteoglycan synthesis was evaluated applying the Bern score and quantified using dimethylmethylene blue dye binding assay. Alkaline phosphatase activity (ALP) was detected on cryosections and soluble ALP measured in pellet supernatants. HAC alone generated hyaline-like discs, while MSC formed spheroid pellets in ±T±D. Co-cultured pellets changed from disc to spheroid shape with decreasing number of HAC, and displayed random cell distribution. In -T-D, HAC expressed S100, produced GAG and collagen II, and formed lacunae, while MSC did not produce any cartilage-specific proteins. Based on GAG, collagen type II and S100 expression chondrogenic differentiation occurred in -T-D MSC co-cultures. However, quantitative experimental GAG and DNA values did not differ from predicted values, suggesting only HAC contribution to GAG production. MSC produced cartilage-specific matrix only in +T+D but underwent hypertrophy in all pellet cultures. In summary, influence of HAC on MSC was restricted to early signs of neochondrogenesis. However, MSC did not contribute to the proteoglycan deposition, and HAC could not prevent hypertrophy of MSC induced by chondrogenic stimuli.

Intraarterial administration of bone marrow mononuclear cells in patients with critical limb ischemia: a randomized-start, placebo-controlled pilot trial (PROVASA)

Critical limb ischemia due to peripheral arterial occlusive disease is associated with a severely increased morbidity and mortality. There is no effective pharmacological therapy available. Injection of autologous bone marrow-derived mononuclear cells (BM-MNC) is a promising therapeutic option in patients with critical limb ischemia, but double-blind, randomized trials are lacking.

Influence of defective bone marrow osteogenesis on fracture repair in an experimental model of senile osteoporosis

http://www.ncbi.nlm.nih.gov/pubmed/20014309

Differential response of Human Bone Marrow Stromal Cells to either TGF-β1 or to rhGDF-5

Cell therapy along with growth factor injection is currently widely investigated to restore the intervertebral disc. However, there is increasing evidence that transplanted unconditioned bone marrow-derived stromal cells (BMSCs) cannot thrive in the intervertebral disc "niche". Moreover, uncertainty exists with respect to the cell phenotype that would be suitable to inject. The intervertebral disc cell phenotype only recently has been started to be characterised using transcriptomics profiling. Recent findings suggest that cytokeratin 19 (KRT-19) could be used as a potential candidate marker for the intervertebral disc, or more specifically the nucleus pulposus cell (NPC) phenotype. We present in vitro cell culture data using alginate bead culture of primary human BMSCs exposed to the standard chondrogenic stimulus, transforming growth factor beta-1 (TGF-β), the growth and differentiation factor 5 and/or bovine NPCs to induce a potential "discogenic" pathway. Chondrogenic induction via TGF-β pathway provoked down-regulation of KRT-19 gene expression in four out of five donors after 18 days of culture, whereas KRT-19 expression remained unchanged in the "discogenic" groups. In addition, the ratio of aggrecan/collagen II gene expression showed a remarkable difference (of at least 3 magnitudes) between the chondrogenic stimulus (low ratio) and the discogenic stimulus (high ratio). Therefore, KRT-19 and aggrecan/collagen II ratio may be potential markers to distinguish chondrogenic from "discogenic" differentiation.