Bi-directional interactions of prostate cancer cells and bone marrow endothelial cells in three-dimensional culture

BACKGROUND: Prostate cancer preferentially metastasizes to bone, yet little is known about the cellular and molecular factors that support this growth. Endothelial cells are likely the initial contact for circulating prostate cells entering the bone microenvironment. METHODS: Using co-culture and conditioned media experiments, we studied cellular and molecular interactions of prostate cancer cells of varying aggressiveness (PC-3 and LNCaP) with bone marrow endothelial (HBME-1) cells in collagen gels. RESULTS: In co-culture, HBME-1 cells stimulated proliferation ( approximately 90% increase) and migration of the more aggressive PC-3 cell line, while having little effect on LNCaP cell proliferation or migration. Concomitantly, HBME-1 cell growth was inhibited by both PC-3 and LNCaP cells and their conditioned media. Additionally, HBME-1 cells underwent significant morphological changes in co-culture, forming large, branching, cord-like structures, which mimic angiogenesis. Prostate cancer cell conditioned media induced a similar effect on HBME-1 cells. In comparison, conditioned media from PC-3 cells also inhibited growth of non-bone marrow-derived endothelial cells, but did not affect their morphology. CONCLUSIONS: Significant bi-directional interactions, including secreted factors and direct cellular interactions, exist between bone marrow endothelial cells and highly metastatic prostate cancer cells, and may underlie the propensity for prostate cancer to metastasize to the bone.     

Human mesenchymal tumour-associated macrophages differentiate into osteoclastic bone-resorbing cells

The cellular mechanisms which account for the formation of osteoclasts and bone resorption associated with enlarging benign and malignant mesenchymal tumours of bone are uncertain. Osteoclasts are marrow-derived, multinucleated, bone-resorbing cells which express a macrophage phenotype. We have determined whether tumour-associated macrophages (TAMs) isolated from benign and malignant mesenchymal tumours are capable of differentiating into osteoclasts. Macrophages were cultured on both coverslips and dentine slices for up to 21 days with UMR 106 osteoblastic cells in the presence of 1,25 dihydroxyvitamin D3 (1,25(OH)2D3) and human macrophage colony-stimulating factor (M-CSF) or, in the absence of UMR 106 cells, with M-CSF and RANK ligand. In all tumours, the formation of osteoclasts from CD14-positive macrophages was shown by the formation of tartrate-resistant-acid-phosphatase and vitronectin-receptor-positive multinucleated cells which were capable of carrying out lacunar resorption. These results indicate that the tumour osteolysis associated with the growth of mesenchymal tumours in bone is likely to be due in part to the differentiation of mononuclear phagocyte osteoclast precursors which are present in the TAM population of these lesions.     

Epithelial cell lines that induce bone formation in vivo produce alkaline phosphatase-enriched matrix vesicles in culture.

Hypertrophic chondrocytes and osteoblasts produce alkaline phosphatase (ALPase)-enriched matrix vesicles in vivo and in vitro and, along with certain epithelial cell lines and osteoblast precursors, induce bone when implanted in mesenchymal tissues. This study examined whether ALPase-enriched matrix vesicle production in vitro was a general property of cells that induce bone in vivo. Epithelial cell lines FL, WISH, and OK 16; connective tissue cell lines HEPM 1 and HEPM 2; neonatal rat muscle cells; rat costochondral chondrocytes; and human fibroblasts were implanted intramuscularly into nude mice. The FL and WISH cells produced tumors and induced large islands of bone with focal areas of cartilage immediately adjacent to the tumors. The chondrocytes formed cartilage nodules but did not induce bone, indicating that the ability of the cells to form a solid mass was not an a priori requirement for bone formation. No other cell type produced tumors or nodules or induced bone formation, although connective tissue cells have been shown to induce chondrogenesis in vitro and osteogenesis in vivo. Only matrix vesicles from normal chondrocytes, FL, WISH, and OK16 cultures exhibited enriched ALPase-specific activity. Matrix vesicles from FL and WISH cultures exhibited ALPase specific activities similar to those isolated from osteoblast or chondrocyte cultures. These data suggest that the ability to produce ALPase-enriched matrix vesicles in culture may be associated with the ability of cells to induce bone or cartilage in vivo.     

Immunocytochemical detection and phenotypic characterization of micrometastatic tumour cells in bone marrow of patients with prostate cancer

Monoclonal antibodies (mAbs) specific for cytokeratins are potent probes for the identification of disseminated individual epithelial tumour cells in mesenchymal organs such as bone marrow. We have used a monoclonal antibody (mAB) against cytokeratin 18 (CK18) for the detection of individual metastatic tumour cells in bone marrow aspirates from 84 patients with carcinoma of the prostate. CK18+cells were detected in a sensitivity of 1 per 8×10⁵ marrow cells using the alkaline phosphatase anti-alkaline phosphatase (APAAP) system for staining. We were able to detect CK18+tumour cells in the marrow of 33% of patients with stage N0M0 prostate cancers. The incidence of CK18+cells showed a significant correlation with established risk factors, such as local tumour extent, distant metastases and tumour differentiation. For further characterization of such cells in patients with prostate cancer, we developed an immunocytochemical procedure for simultaneous labelling of cytokeratin component no. 18 (CK18) and prostate-specific antigen (PSA). In a first step, cells were incubated with a murine mAb against PSA, followed by goldconjugated goat anti-mouse antibodies. In a second step, a biotinylated mAb to CK18 was applied as primary antibody and subsequently incubated with complexes of streptavidin-conjugated alkaline phosphatase, which were developed with Newfuchsin substrate. The binding of gold-labelled antibodies was visualized by silver enhancement. CK18+cells co-expressing PSA were found in bone marrow aspirates from 5 out of 14 patients with carcinomas of the prostate. The specificity of CK18 for epithelial tumour cells in bone marrow was supported by negative staining of 12 control aspirates from patients with benign prostatic hyperplasia (BPH). Thus the prostatic origin of CK+cells in bone marrow of patients with prostate cancer has been directly demonstrated for the first time in this work. In conclusion, the approaches presented appear to be reliable methods of identifying and phenotyping individual prostatic carcinoma cells and may help to identify those patients with prostate cancer who are at high risk of relapse.     

VEGF producing bone marrow stromal cells (BMSC) enhance vascularization and resorption of a natural coral bone substitute

Bone graft substitutes often exhibit poor bone regeneration in large defects because of inadequate vascularization. Studies have shown that if blood supply is compromised, application of osteogenic factors alone could not induce successful healing. This study was to evaluate the effects of vascular endothelial growth factor, which combined with a coralline scaffold, on vascularization, scaffold resorption and osteogenesis in a rabbit radius critical size defect model. The scaffold was either coated with a control-plasmid DNA (group 1), coated with VEGF-plasmid DNA (group 2), loaded with mesenchymal stem cells (BMSC) transfected with control plasmid (group 3) or with both stem cells and the VEGF plasmid (group 4). X-rays were taken every 4 weeks up to week 16, when animals were euthanized. The volume of new bone was measured by mu-CT scans and blood vessels were counted after anti-CD31 staining of endothelial cells. The results from the solitary VEGF- and VEGF-transfected cells (groups 2 and 4) demonstrated significantly enhanced vascularization, osteogenesis and resorption of the carrier when compared to the control group. The highest degree of osteogenesis was found when the carrier was loaded with BMSC (group 3), whereas VEGF-transfected cells led to the highest vascularization and fastest resorption of the bone substitute. Additionally, VEGF-transfected BMSC led to a more homogenous vascularization of the defect. The results indicate that VEGF can be a helpful factor to improve healing in large bone defects, in which bone substitutes will otherwise not be vascularized and replaced by fresh bone.     

Risk factors for coronary artery disease, circulating endothelial progenitor cells, and the role of HMG-CoA reductase inhibitors

Recent studies suggest that postnatal neovascularization relies not exclusively on sprouting of preexisting vessels ("angiogenesis"), but also involves the contribution of bone marrow-derived circulating endothelial progenitor cells (EPCs). EPCs can be isolated from peripheral blood or bone marrow mononuclear cells, CD34(+) or CD133(+) hematopoietic progenitors. Infusion of EPCs was shown to promote postnatal neovascularization of ischemic tissue after myocardial infarction in animal models and initial clinical trials. Moreover, circulating endothelial precursor cells can home to denuded arteries after balloon injury and contribute to endothelial regeneration, thereby limiting the development of restenosis. Thus, circulating endothelial cells may exert an important function as endogenous repair mechanism to maintain the integrity of the endothelial monolayer and to promote ischemia-induced neovascularization. However, risk factors for coronary artery disease, such as diabetes, hypercholesterolemia, and hypertension are associated with impaired number and function of EPC in patients with coronary artery disease. Therapeutically, the reduction of EPC number and the decreased functional activity in patients with coronary artery disease was counteracted by 3-hydroxy-3-methylglutaryl coenzymeA (HMG-CoA) reductase inhibitors (statins), vascular endothelial growth factor (VEGF), estrogen, or exercise. At the molecular level, these factors are well established to activate the phosphatidyl-inositol-3-kinase (PI3K)-Akt-dependent activation of the endothelial nitric oxide synthase (eNOS), suggesting that the PI3K-Akt-eNOS signaling pathway may be involved in the transduction of atheroprotective factors. Taken together, the balance of atheroprotective and proatherosclerotic factors may influence EPC levels and their functional capacity to improve neovascularization and endothelial regeneration.     

骨髓CD34+细胞人工血管内皮化实验研究

目的探讨骨髓CD34^ 细胞种植的人工血管内皮化和通畅性。方法取犬骨髓,经免疫磁珠分离出CD34^ 细胞,内皮细胞生长因子(vascular endothelial grow factor,VEGF)诱导并扩增;种植聚四氟乙烯(polytetra fluoro ethylene,PTFE)人工血管,再植入犬的下腔静脉和腹主动脉。结果骨髓分离的细胞经流式细胞仪鉴定为CD34^ 细胞,CD34^ 细胞经VEGF诱导培养后经免疫细胞化学和透射电镜检测证实为内皮细胞。内皮化人工血管植入动脉闭塞率0,狭窄率12．5％;人工血管植入静脉闭塞率12．5％,狭窄率25．0％。结论骨髓经免疫磁珠可分离出CD34^ 细胞,CD34^% 细胞经VEGF诱导可定向分化为内皮细胞。骨髓CD34^ 细胞经体外培养种植于PTFE人工血管,获得理想的内皮化和通畅率。     

Neutrophil-induced transmigration of tumour cells treated with tumour-conditioned medium is facilitated by granulocyte-macrophage colony-stimulating factor.

OBJECTIVE: To investigate the effect of different cytokines that are present in tumour-conditioned medium on human neutrophil (PMN)-induced tumour cell transmigration. DESIGN: Laboratory study. SETTING: University hospital, Ireland. MATERIAL: Isolated human PMN and cultured human breast tumour cell line, MDA-MB-231. Interventions: Human PMN treated with either tumour-conditioned medium or different media neutralised with monoclonal antibodies (MoAb), and MDA-MB-231 cells were plated on macrovascular and microvascular endothelial monolayers in collagen-coated transwells to assess migration of tumour cells. MAIN OUTCOME MEASURES: Cytokines present in tumour-conditioned medium, PMN cytocidal function and receptor expression, and tumour cell transmigration. RESULTS: tumour-conditioned medium contained high concentrations of granulocyte-macrophage colony-stimulating factor (GM-CSF), vascular endothelial growth factor (VEGF), and interleukin 8 (IL-8), but not granulocyte colony-stimulating factor (G-CSF) and interleukin 3 (IL-3). Anti-GM-CSF MoAb significantly reduced PMN-induced transmigration of tumour cells treated with tumour-conditioned medium (p < 0.05), whereas anti-VEGF and anti-IL-8 MoAbs did not affect their migration. In addition, anti-GM-CSF MoAb, but not anti-VEGF or anti-IL-8 MoAb, reduced PMN CD11b and CD18 overexpression induced by tumour-conditioned medium (p < 0.05). CONCLUSION: These results indicate that the GM-CSF that is present in tumour-conditioned medium may be involved, at least in part, in alterations in PMN function mediated by the medium and subsequently PMN-induced transmigration of tumour cells.     

Promotion of calvarial cell osteogenesis by endothelial cells

Bone development and remodeling are associated with changes in the pattern of vascularization. Here we show that endothelial cells isolated from rat liver or bovine aorta can greatly enhance bone formation when implanted in diffusion chambers with rat fetal calvarial cells. The latter cells are unable to form bone when implanted alone at low initial cell density. The amount of mineralization measured by calcium deposition was 70 times higher in chambers containing calvarial cells mixed with endothelial cells from isologous liver or bovine aorta than in chambers containing endothelial or calvarial cells alone. Alkaline phosphatase activity was increased 20-fold. Calvarial cells in the presence of demineralized bone matrix powder did not form bone when implanted under similar conditions. Endothelial cells implanted alone seemed to enhance neovascularization around the Millipore diffusion chambers.     

BMSCs来源成骨细胞和内皮细胞复合壳聚糖-羟基磷灰石多孔支架构建血管化组织工程骨研究

目的探讨大鼠BMSCs来源的成骨细胞和内皮细胞复合壳聚糖-羟基磷灰石多孔支架植入大鼠桡骨缺损处的成骨作用和成血管作用。方法取分离培养至第3代的SD大鼠BMSCs行成骨和成内皮细胞诱导并鉴定。分别将内皮细胞(A组)、成骨细胞(B组)、混合细胞(成骨细胞和内皮细胞比例为1∶1,C组)均匀滴加于壳聚糖-羟基磷灰石多孔支架上制备3组细胞-支架复合物,MTT检测支架内细胞增殖活性。取2月龄雄性SD大鼠30只,制作大鼠桡骨5 mm长缺损模型并分别植入3组细胞-支架复合物(n=10)。术后4、8、12周分别取移植物行HE染色观察,CD34免疫组织化学染色计数微血管密度,RT-PCR法检测骨桥蛋白(osteopontin,OPN)和骨保护素(osteoprotegrin,OPG)mRNA表达。结果 BMSCs成骨诱导7 d后ALP染色可见细胞质内蓝染颗粒,细胞核呈红染;内皮细胞诱导14 d后,CD34免疫细胞化学染色可见细胞内棕色颗粒。MTT检测示3组细胞活性随时间延长逐渐升高。HE染色示,术后12周A组未见明显类骨质形成,而有较密集的微血管结构及较多纤维组织形成;B、C组可见均质的类骨质,呈条索状和岛状分布,可见大量成骨样细胞存在。术后各时间点A、C组微血管密度均显著高于B组(P<0.05);A组术后12周微血管密度高于C组(P<0.05),其余2个时间点A、C组间差异无统计学意义(P>0.05)。A组3个时间点OPN和OPG mRNA表达水平均较低,与B、C组比较差异有统计学意义(P<0.05);B、C组分别于术后8、12周OPN mRNA表达达峰值,4周时OPG mRNA表达达峰值。结论 BMSCs来源的成骨细胞和内皮细胞按1∶1比例共培养于壳聚糖-羟基磷灰石多孔支架作为组织工程骨移植物,可以促进大鼠桡骨缺损部位骨的形成和血管化,促进骨缺损愈合。     

Warfarin inhibition of metastasis: the role of anticoagulation

Warfarin inhibits metastasis in animal models by mechanisms that remain unclear. A better understanding of this phenomenon may clarify processes underlying metastasis in human cancer. We have studied the effects of warfarin on metastasis in a rat model by intravenous injection of Mtln3 mammary carcinoma cells and subsequent counting of pulmonary seedlings. To determine whether warfarin acts principally on the tumour cells or the host, we pretreated either cells or animals with warfarin before intravenous injection of 10(4) Mtln3 cells. Pretreatment of tumour cells had no effect, whilst pretreatment of the host reduced median seedlings from 67 to 4.5 per animal (P less than 0.005). To determine whether warfarin was acting via its anticoagulant action, we reversed warfarin anticoagulation by intravenous injection of coagulation factors II, VII, IX and X. Restoration of coagulation for 12 h immediately after injection of cells completely reversed the warfarin effect (P less than 0.001), but if the injection of factors was delayed for 12 h it had no effect (P = 0.1753). We conclude that warfarin acts principally on the host, not the tumour cell, and that it acts via its effect on coagulation. The restriction of the effect to the first 12 h after tumour cell injection suggests a mechanism involving intravascular processes such as tumour cell survival or endothelial adhesion.     

Bone resorption and renal calcium reabsorption in renal cell carcinoma-bearing mice: the effects of bisphosphonate

OBJECTIVE: To examine the contribution of the skeleton and the kidney to the development of humoral hypercalcaemia of malignancy (HHM) in a mouse model of HHM treated with a potent bisphosphonate. MATERIALS AND METHODS: Mice bearing the human RCC cell line RC-9 were treated with bisphosphonate (subcutaneous, 0.25 mg/kg body weight olpadronate) or saline solution. Treatment was initiated at a tumour volume (TV) of approximately 100 mm(3) and 500 mm(3), and the mice were monitored for approximately 4 weeks. Serum calcium and phosphate concentrations and trabecular bone volume (TBV) were assessed during and/or after treatment. RESULTS: Athymic mice implanted with the RCC RC-9, developed severe hypercalcaemia and bone resorption. During tumour growth the mean (sd) serum calcium concentration increased to 4.1 (0.3) mmol/L, and phosphate decreased to 1.6 (0.3) mmol/L, vs 2.3 (0.1) and 2.9 (0.4) mmol/L in controls, respectively. TBV decreased from 8.7 (1.8)% in mice with no tumour, to 5.3 (2.7)% in RC-9-bearing mice. Olpadronate initiated at a Tv of 100 mm(3) prevented the loss of bone induced by RCC RC-9 cells, with a TBV of 12.8 (2.1)%, but the development of hypercalcaemia was unaffected. Olpadronate treatment at a TV of 500 mm(3) did not influence the development of hypercalcaemia and did not protect against bone resorption. Kinetic monitoring showed an identical rate of tumour growth in the presence or absence of bisphosphonate, while under both conditions there was a tumour load-dependent increase in calcium concentration. CONCLUSIONS: Bisphosphonate can prevent parathyroid hormone-related peptide (PTHrP)-mediated bone resorption when administered during the early phase of renal tumour growth, but has no effect on the tumour-induced development of hypercalcaemia, indicating a primary role for renal tubular reabsorption of calcium in the kidney by PTHrP in HHM.     

Galectin-3 enhances proliferation and angiogenesis of endothelial cells differentiated from bone marrow mesenchymal stem cells

Purpose

To observe the effects of galectin-3 on proliferation and angiogenesis of endothelial cells differentiated from bone marrow mesenchymal stem (MSCs).

Methods

Cultured MSCs were isolated from bone marrow of Sprague–Dawley rats and purified by gradient centrifugation with lymphocytes separation medium. Cells of passage 3 were differentiated into endothelial cels by vascular endothelial growth factor and basic fibroblast growth factor. These cells were identified as endothelial cells by immunohistochemistry staining and electronic microscopy after 14 days. The cells were cultivated with the galectin-3 at the concentrations of 0.1, 1, and 5 μg/mL for 24 hours. The proliferation of endothelial cells were measured by 3-(4,5-methylth-iazol-2-yl)-diphenyltetrazolium bromide (MTT) and the cell cycle was investigated by using flow cytometry. The functionality of angiogensis was observed when the cells appeared tube formation in presence of glacetin-3.

Results

The proliferation activity, analyzed by MTT method, in the galectin-3 groups (1 and 5 μg/mL) were 0.3002 ± 0.0159 and 0.3514 ± 0.0133, respectively, which were significantly greater than that in the control group (0.2339 ± 0.0041; P < .05). Flow cytometry detection showed that S phase cells (%) are 29.42 ± 0.45, 34.56 ± 0.82, and 52.58 ± 2.84 in groups of 0.1, 1, and 5 μg/mL, respectively, and G₂M phase cells increased from 4.88 ± 1.12 to 5.26 ± 0.45 with the concentrations of 1 and 5 μg/mL, respectively, which demonstrated significant difference compared with the control group (P < .05). The tubular network formation was lengthened significantly compared with the control group (P < .05).

Conclusion

Galectin-3 can promote the proliferation and angiogenesis of endothelial cells differentiated from bone marrow mesenchymal stem cells.     

Ultrastructure of normal rabbit mesentery

The ultrastructure of the mesentery was examined by electron microscope (EM) after in vivo fixation in 7 normal rabbits. In 1, the fixation was performed after intravenous injection of an electron-dense tracer (iron dextran) and intraperitoneal infusion of dialysate 4.25% for 1 h. We studied morphometric data of what is considered to be the active peritoneal dialysis membrane, i.e. capillary endothelial cells, interstitium and mesothelial cells. The mesothelial cells are flattened and overlapping with tight junctions between them. They lie on a continuous basement membrane and they contain numerous intracytoplasmic vesicles, separated or in clusters. The mesenteric microvessels were mainly true capillaries of continuous type and postcapillary venules. Capillary lymphatics and larger lymphatic channels (lacunae) seem to be more extensive than the blood capillaries and venules. The endothelial cells contain many vesicles. The interstitium consists of bundles of collagen, fibroblasts and occasional macrophages. The electron-dense tracer was found in the vesicles of the mesothelial cells suggesting that vesicular transport may play an important part in the transportation of at least molecules of a certain size.     

Bmp2 conditional knockout in osteoblasts and endothelial cells does not impair bone formation after injury or mechanical loading in adult mice

Post-natal osteogenesis after mechanical trauma or stimulus occurs through either endochondral healing, intramembranous healing or lamellar bone formation. Bone morphogenetic protein 2 (BMP2) is up-regulated in each of these osteogenic processes and is expressed by a variety of cells including osteoblasts and vascular cells. It is known that genetic knockout of Bmp2 in all cells or in osteo-chondroprogenitor cells completely abrogates endochondral healing after full fracture. However, the importance of BMP2 from differentiated osteoblasts and endothelial cells is not known. Moreover, the importance of BMP2 in non-endochondral bone formation such as intramembranous healing or lamellar bone formation is not known. Using inducible and tissue-specific Cre-lox mediated targeting of Bmp2 in adult (10–24 week old) mice, we assessed the role of BMP2 expression globally, by osteoblasts, and by vascular endothelial cells in endochondral healing, intramembranous healing and lamellar bone formation. These three osteogenic processes were modeled using full femur fracture, ulnar stress fracture, and ulnar non-damaging cyclic loading, respectively. Our results confirmed the requirement of BMP2 for endochondral fracture healing, as mice in which Bmp2 was knocked out in all cells prior to fracture failed to form a callus. Targeted deletion of Bmp2 in osteoblasts (osterix-expressing) or vascular endothelial cells (vascular endothelial cadherin-expressing) did not impact fracture healing in any way. Regarding non-endochondral bone formation, we found that BMP2 is largely dispensable for intramembranous bone formation after stress fracture and also not required for lamellar bone formation induced by mechanical loading. Taken together our results indicate that osteoblasts and endothelial cells are not a critical source of BMP2 in endochondral fracture healing, and that non-endochondral bone formation in the adult mouse is not as critically dependent on BMP2.     

Multifocal spinal angiosarcoma after chordotomy

Summary A male aged 60 is reported who, 5 years after chordotomy at the Th2/3 level, developed acute paraplegia of the legs and a sensory transverse lesion due to an extradural tumour of the Th 1–4 level. Neuropathology revealed transverse necrosis of the thoracic spinal cord (Th 1–4) due to an intradural tumour at the upper thoracic and lumbar spinal levels, the thoracic malignancy arising at the site of the previous chordotomy, with a suture being observed within the tumour mass. Histology, positive immunostaining of tumour cells with Factor VIII antigen, a specific marker of endothelial cells and the ultrastructural demonstration of Weibel-Palade bodies in endothelial cells of the tumour vessels suggested a malignant mesenchymal tumour of angiosarcoma type presumably arising from the spinal meninges, and broadly invading the spinal cord. The usual relation of this extremely rare intraspinal vasoformative malignancy to previous chordotomy is discussed.     

Visualizing mineral binding and uptake of bisphosphonate by osteoclasts and non-resorbing cells

Bisphosphonates (BPs) target bone due to their high affinity for calcium ions. During osteoclastic resorption, these drugs are released from the acidified bone surface and taken up by osteoclasts, where they act by inhibiting the prenylation of small GTPases essential for osteoclast function. However, it remains unclear exactly how osteoclasts internalise BPs from bone and whether other cells in the bone microenvironment can also take up BPs from the bone surface. We have investigated this using a novel fluorescently-labelled alendronate analogue (FL-ALN), and by examining changes in protein prenylation following treatment of cells with risedronate (RIS). Confocal microscopic analysis showed that FL-ALN was efficiently internalised from solution or from the surface of dentine by resorbing osteoclasts into intracellular vesicles. Accordingly, unprenylated Rap1A accumulated to the same extent whether osteoclasts were cultured on RIS-coated dentine or with RIS in solution. By contrast, J774 macrophages internalised FL-ALN and RIS from solution, but took up comparatively little from dentine, due to their inability to resorb the mineral. Calvarial osteoblasts and MCF-7 tumour cells internalised even less FL-ALN and RIS, both from solution and from the surface of dentine. Accordingly, the viability of J774 and MCF-7 cells was drastically reduced when cultured with RIS in solution, but not when cultured on dentine pre-coated with RIS. However, when J774 macrophages were co-cultured with rabbit osteoclasts, J774 cells that were adjacent to resorbing osteoclasts frequently internalised more FL-ALN than J774 cells more distant from osteoclasts. This was possibly a result of increased availability of BP to these J774 cells due to transcytosis through osteoclasts, since FL-ALN partially co-localised with trancytosed, resorbed matrix protein within osteoclasts. In addition, J774 cells occupying resorption pits internalised more FL-ALN than those on unresorbed surfaces. These data demonstrate that osteoclasts are able to take up large amounts of BP, due to their ability to release the BP from the dentine surface during resorption. By contrast, non-resorbing cells take up only small amounts of BP that becomes available due to natural desorption from the dentine surface. However, BP uptake by non-resorbing cells can be increased when cultured in the presence of resorbing osteoclasts.     

Microvessel networks in pre-formed in artificial clinical grade dermal substitutes in vitro using cells from haematopoietic tissues

Forming a microcirculation is critical for vascularisation of artificial skin substitutes. One strategy to improve speed of grafting is to pre-form microvascular networks in the substitute before applying to a wound. For clinical application, this requires sufficient functional endothelial cell numbers. In vitro endothelial colony forming cells (ECFCs) derived cells were expanded from cord and adult blood donations and co-cultured with human dermal fibroblasts or bone marrow mesenchymal stem/stromal cells to form microvascular networks in the presence or absence of dermal substitutes which are in clinical use. The number of endothelial cells generated ranged from 1.03×10(9) to 2.18×10(11) from 10 adult blood donations and 1×10(12) to 1.76×10(13) from 6 cord blood units after 50 days in culture. Two adult donations failed to generate ECFCs. Both cord and adult blood cells formed 2D microvascular networks in vitro, although there was a significant difference in the functional capacity of adult and cord blood ECFCs. While co-culture of the latter within dermal substitutes Matriderm or Integra demonstrated the formation of 3D microvascular networks penetrating 100μm, enhanced expansion, while maintaining functional capacity, of adult blood cells is required for fully pre-vascularising the clinical grade acellular dermal substitutes used here prior to applying these to burns.     

Role of myeloid early endothelial progenitor cells in bone formation and osteoclast differentiation in tissue construct based on hydroxyapatite poly(ester‐urethane) scaffolds

Engineering of a vascularized bone construct is a highly challenging task which needs to take into account the impact of different components on the bone regeneration process. Bone repair influencing factors in such constructs range from the material properties and scaffold design, to the interaction of different cell types contributing to bone formation and remodeling or neovascularization, respectively. In this context, early endothelial progenitor cells (EPC), mononuclear cells isolated from the peripheral blood, express the endothelial marker CD31 but also a series of myeloid markers and have been shown to support the formation of vessel‐like structures. These cells are also characterized by a highly adaptable phenotype influenced by other cells creating an instructive niche. The present study was designed to investigate the impact of EPC on bone formation or remodeling using a co‐culture system of outgrowth endothelial cells, mature endothelial cells isolated from the peripheral blood cell cultures, and mesenchymal stem cells grown on hydroxyapatite poly(ester‐urethane) scaffolds. The formation of vessel‐like structures in these constructs was shown by CLSM and immunohistochemistry and further evaluated by real time RT‐PCR. Osteogenic differentiation in these constructs was investigated by von Kossa, Alizarin Red, and real time PCR. Data indicated that osteogenic differentiation occurred within the constructs after 14 days of culture but without a direct influence by EPC in this process. Finally, although we observed a series of osteoclast related makers in the constructs when EPC were included, no indications for an increased osteoclast‐like activity, which might lead to increased bone resorption, were observed. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:1922–1932, 2016.