Bone Cell Cultures Research Articles

Overexpression of VEGF Induces Bone Formation in the Model of Transplantation of Cultured Bone Cells

Overexpression of VEGF Induces Bone Formation in the Model of Transplantation of Cultured Bone Cells

The osteogenic response of mesenchymal stromal cells to strontium-substituted bioactive glasses.

Bioactive glasses are known to stimulate bone healing, and the incorporation of strontium has the potential to increase their potency. In this study, calcium oxide in the 45S5 bioactive glass composition was partially (50%, Sr50) or fully (100%, Sr100) substituted with strontium oxide on a molar basis. The effects of the substitution on bioactive glass properties were studied, including density, solubility, and in vitro cytotoxicity. Stimulation of osteogenic differentiation was investigated using mesenchymal stromal cells obtained from rat bone marrow. Strontium substitution resulted in altered physical properties including increased solubility. Statistically significant reductions in cell viability were observed with the addition of bioactive glass powders to culture medium. Specifically, addition of ≥ 13.3 mg/ml of 45S5 bioactive glass or Sr50, or ≥ 6.7 mg/ml of Sr100, resulted in significant inhibition. Real‐time PCR analyses detected the upregulation of genes associated with osteoblastic differentiation in the presence of all bioactive glass compositions. Some genes, including Alpl and Bglap, were further stimulated in the presence of Sr50 and Sr100. It was concluded that strontium‐substituted bioactive glasses promoted osteogenesis in a differentiating bone cell culture model and, therefore, have considerable potential for use as improved bioactive glasses for bone tissue regeneration. © 2015 The Authors. Journal of Tissue Engineering and Regenerative Medicine Published by John Wiley & Sons Ltd.

Piezoelectric poly(vinylidene fluoride) microstructure and poling state in active tissue engineering

Tissue engineering strategies rely on suitable membranes and scaffolds, providing the necessary physicochemical stimuli to specific cells. This review summarizes the main results on piezoelectric polymers, in particular poly(vinylidene fluoride), for muscle and bone cell culture. Further, the relevance of polymer microstructure and surface charge on cell response is demonstrated. Together with the necessary biochemical cues, the proper design of piezoelectric polymers can open the way to novel and more reliable tissue engineering strategies for cells in which electromechanical stimuli are present in their environment.

Up-regulation of BMP2/4 signaling increases both osteoblast-specific marker expression and bone marrow adipogenesis in Gja1Jrt/+ stromal cell cultures.

Gja1(Jrt)/+ mice carry a mutation in one allele of the gap junction protein α1 gene (Gja1), resulting in a G60S connexin 43 (Cx43) mutant protein that is dominant negative for Cx43 protein production of <50% of wild-type (WT) levels and significantly reduced gap junction formation and function in osteoblasts and other Cx43-expressing cells. Previously we reported that Gja1(Jrt)/+ mice exhibited early-onset osteopenia caused by activation of osteoclasts secondary to activation of osteoblast lineage cells, which expressed increased RANKL and produced an abnormal resorption-stimulating bone matrix high in BSP content. Gja1(Jrt)/+ mice also displayed early and progressive bone marrow atrophy, with a significant increase in bone marrow adiposity versus WT littermates but no increase in adipose tissues elsewhere in the body. BMP2/4 production and signaling were increased in Gja1(Jrt)/+ trabecular bone and osteogenic stromal cell cultures, which contributed to the up-regulated expression of osteoblast-specific markers (e.g., Bsp and Ocn) in Gja1(Jrt)/+ osteoblasts and increased Pparg2 expression in bone marrow-derived adipoprogenitors in vitro. The elevated levels of BMP2/4 signaling in G60S Cx43-containing cells resulted at least in part from elevated levels of cAMP. We conclude that up-regulation of BMP2/4 signaling in trabecular bone and/or stromal cells increases osteoblast-specific marker expression in hyperactive Gja1(Jrt)/+ osteoblasts and may also increase bone marrow adipogenesis by up-regulation of Pparg2 in the Cx43-deficient Gja1(Jrt)/+ mouse model.

3D porous calcium-alginate scaffolds cell culture system improved human osteoblast cell clusters for cell therapy.

Age-related orthopedic disorders and bone defects have become a critical public health issue, and cell-based therapy is potentially a novel solution for issues surrounding bone tissue engineering and regenerative medicine. Long-term cultures of primary bone cells exhibit phenotypic and functional degeneration; therefore, culturing cells or tissues suitable for clinical use remain a challenge. A platform consisting of human osteoblasts (hOBs), calcium-alginate (Ca-Alginate) scaffolds, and a self-made bioreactor system was established for autologous transplantation of human osteoblast cell clusters. The Ca-Alginate scaffold facilitated the growth and differentiation of human bone cell clusters, and the functionally-closed process bioreactor system supplied the soluble nutrients and osteogenic signals required to maintain the cell viability. This system preserved the proliferative ability of cells and cell viability and up-regulated bone-related gene expression and biological apatite crystals formation. The bone-like tissue generated could be extracted by removal of calcium ions via ethylenediaminetetraacetic acid (EDTA) chelation, and exhibited a size suitable for injection. The described strategy could be used in therapeutic application and opens new avenues for surgical interventions to correct skeletal defects.

Incorporation of uranium into a biomimetic apatite: physicochemical and biological aspects.

Bone is the main target organ for the storage of several toxic metals, including uranium. But the mode of action of uranium on bones remains poorly understood. To better assess the impact of uranium on bone cells, synthetic biomimetic apatites encompassing a controlled amount of uranium were prepared and analyzed. This study revealed the physicochemical impact of uranium on apatite mineralization: the presence of the metal induces a loss of crystallinity and a lower mineralization rate. The prepared samples were then used as substrates for bone cell culture. Osteoblasts were not sensitive to the presence of uranium in the support, whereas previous results showed a deleterious effect of uranium introduced into a cell culture solution. This work should therefore have some original prospects within the context of toxicological studies concerning the effect of metallic cations on bone cell systems.

Bone cells in cultures on nanocarbon-based materials for potential bone tissue engineering: A review (Phys. Status Solidi A 12∕2014)

Bone cells in cultures on nanocarbon-based materials for potential bone tissue engineering: A review (Phys. Status Solidi A 12∕2014)

Tumor-expressed adrenomedullin accelerates breast cancer bone metastasis.

IntroductionAdrenomedullin (AM) is secreted by breast cancer cells and increased by hypoxia. It is a multifunctional peptide that stimulates angiogenesis and proliferation. The peptide is also a potent paracrine stimulator of osteoblasts and bone formation, suggesting a role in skeletal metastases—a major site of treatment-refractory tumor growth in patients with advanced disease.MethodsThe role of adrenomedullin in bone metastases was tested by stable overexpression in MDA-MB-231 breast cancer cells, which cause osteolytic bone metastases in a standard animal model. Cells with fivefold increased expression of AM were characterized in vitro, inoculated into immunodeficient mice and compared for their ability to form bone metastases versus control subclones. Bone destruction was monitored by X-ray, and tumor burden and osteoclast numbers were determined by quantitative histomorphometry. The effects of AM overexpression on tumor growth and angiogenesis in the mammary fat pad were determined. The effects of AM peptide on osteoclast-like multinucleated cell formation were tested in vitro. A small-molecule AM antagonist was tested for its effects on AM-stimulated ex vivo bone cell cultures and co-cultures with tumor cells, where responses of tumor and bone were distinguished by species-specific real-time PCR.ResultsOverexpression of AM mRNA did not alter cell proliferation in vitro, expression of tumor-secreted factors or cell cycle progression. AM-overexpressing cells caused osteolytic bone metastases to develop more rapidly, which was accompanied by decreased survival. In the mammary fat pad, tumors grew more rapidly with unchanged blood vessel formation. Tumor growth in the bone was also more rapid, and osteoclasts were increased. AM peptide potently stimulated bone cultures ex vivo; responses that were blocked by small-molecule adrenomedullin antagonists in the absence of cellular toxicity. Antagonist treatment dramatically suppressed tumor growth in bone and decreased markers of osteoclast activity.ConclusionsThe results identify AM as a target for therapeutic intervention against bone metastases. Adrenomedullin potentiates osteolytic responses in bone to metastatic breast cancer cells. Small-molecule antagonists can effectively block bone-mediated responses to tumor-secreted adrenomedullin, and such agents warrant development for testing in vivo.Electronic supplementary materialThe online version of this article (doi:10.1186/s13058-014-0458-y) contains supplementary material, which is available to authorized users.

Extracellular matrix mineralization in murine MC3T3-E1 osteoblast cultures: An ultrastructural, compositional and comparative analysis with mouse bone

Bone cell culture systems are essential tools for the study of the molecular mechanisms regulating extracellular matrix mineralization. MC3T3-E1 osteoblast cell cultures are the most commonly used in vitro model of bone matrix mineralization. Despite the widespread use of this cell line to study biomineralization, there is as yet no systematic characterization of the mineral phase produced in these cultures. Here we provide a comprehensive, multi-technique biophysical characterization of this cell culture mineral and extracellular matrix, and compare it to mouse bone and synthetic apatite mineral standards, to determine the suitability of MC3T3-E1 cultures for biomineralization studies. Elemental compositional analysis by energy-dispersive X-ray spectroscopy (EDS) showed calcium and phosphorus, and trace amounts of sodium and magnesium, in both biological samples. X-ray diffraction (XRD) on resin-embedded intact cultures demonstrated that similar to 1-month-old mouse bone, apatite crystals grew with preferential orientations along the (100), (101) and (111) mineral planes indicative of guided biogenic growth as opposed to dystrophic calcification. XRD of crystals isolated from the cultures revealed that the mineral phase was poorly crystalline hydroxyapatite with 10 to 20nm-sized nanocrystallites. Consistent with the XRD observations, electron diffraction patterns indicated that culture mineral had low crystallinity typical of biological apatites. Fourier-transform infrared spectroscopy (FTIR) confirmed apatitic carbonate and phosphate within the biological samples. With all techniques utilized, cell culture mineral and mouse bone mineral were remarkably similar. Scanning (SEM) and transmission (TEM) electron microscopy showed that the cultures had a dense fibrillar collagen matrix with small, 100nm-sized, collagen fibril-associated mineralization foci which coalesced to form larger mineral aggregates, and where mineralized sites showed the accumulation of the mineral-binding protein osteopontin. Light microscopy, confocal microscopy and three-dimensional reconstructions showed that some cells had dendritic processes and became embedded within the mineral in an osteocyte-like manner. In conclusion, we have documented characteristics of the mineral and matrix phases of MC3T3-E1 osteoblast cultures, and have determined that the structural and compositional properties of the mineral are highly similar to that of mouse bone.

Earthworm (Pheretima aspergillum) extract stimulates osteoblast activity and inhibits osteoclast differentiation.

BackgroundThe potential benefits of earthworm (Pheretima aspergillum) for healing have received considerable attention recently. Osteoblast and osteoclast activities are very important in bone remodeling, which is crucial to repair bone injuries. This study investigated the effects of earthworm extract on bone cell activities.MethodsOsteoblast-like MG-63 cells and RAW 264.7 macrophage cells were used for identifying the cellular effects of different concentrations of earthworm extract on osteoblasts and osteoclasts, respectively. The optimal concentration of earthworm extract was determined by mitochondrial colorimetric assay, alkaline phosphatase activity, matrix calcium deposition, Western blotting and tartrate-resistant acid phosphatase activity.ResultsEarthworm extract had a dose-dependent effect on bone cell activities. The most effective concentration of earthworm extract was 3 mg/ml, significantly increasing osteoblast proliferation and differentiation, matrix calcium deposition and the expression levels of alkaline phosphatase, osteopontin and osteocalcin. Conversely, 3 mg/ml earthworm extract significantly reduced the tartrate-resistant acid phosphatase activity of osteoclasts without altering cell viability.ConclusionsEarthworm extract has beneficial effects on bone cell cultures, indicating that earthworm extract is a potential agent for use in bone regeneration.

Bone cells in cultures on nanocarbon-based materials for potential bone tissue engineering: A review

The following materials based on four allotrope types of nanocarbons were investigated: (1) fullerene C60 and hybrid C60/Ti films, (2) composites of synthetic polymers and carbon nanotubules (i.e., carbon nanohorns and carbon nanotubes), (3) graphene-based materials (films and three-dimensional scaffolds), and (4) nanocrystalline diamond-based materials (films and nanofibrous scaffolds loaded with nanodiamond particles). In general, all these substrates provided a good support for colonization with human osteoblast-like cells of the lines MG-63, Saos-2 and U-2 OS, primary osteoblasts, and also human mesenchymal stem cells (hMSC). In the case of fullerenes C60, this was true for aged, i.e., 1-year-old, films. Fresh films, i.e., 1-week-old, had a decreased number of initially adhering cells, with less spreading, growth, metabolic activity and viability, though no DNA damage was detected. In the case of C60/Ti composite films, both fresh and aged films supported cell colonization well. The improved cell performance was attributed to structural changes in fullerene molecules, such as fragmentation, oxidation and polymerization, which occur during aging or co-deposition of C60 and Ti. The addition of single-wall carbon nanohorns or multi-wall carbon nanotubes to a terpolymer of polytetrafluoroethylene, polyvinyldifluoride and polypropylene (PTFE/PVDF/PP) markedly improved the adhesion and growth of bone cells, while no significant changes in cell behavior were found on polysulfone after it had been enriched with the carbon nanotubules mentioned here. Graphene-based films and scaffolds stimulated the adhesion and osteogenic differentiation of bone-forming cells even in the absence of cell adhesion-mediating molecules and differentiation factors in the cell culture medium. Nanocrystalline diamond films proved to be excellent substrates for cell adhesion, growth and osteogenic differentiation, and this cell behavior was further improved by boron doping (concentration of 133–6700 ppm) or by oxygen termination of these films. The addition of diamond nanoparticles to nanofibrous poly(lactide-co-glycolide) (PLGA) scaffolds increased the proliferation of hMSC and supported the adhesion and growth of MG-63 cells in an extent similar to cell culture polystyrene. However, on nanofibrous poly(L-lactide) scaffolds with diamond nanoparticles, the growth of MG-63 cells decreased with increasing nanoparticle concentration.

The stabilization of electrospun chitosan nanofibers by reversible acylation

Chitosan nanofibers fabricated by electrospinning are contaminated by acidic anions from the acid spinning solution, leading to instability of the nanofibers in aqueous solutions, and the traditional fiber treatment method will also lead to the deterioration of the nanostructure. Here we demonstrate a novel approach to removing the acidic anions with full preservation of the nanofibrous structure. The as-spun nanofibers are first protected (stabilized) by reversible acylation. Second, contaminants are then eliminated by hydrolysis; finally, acylation is reversed. Chemical analysis showed the removal of the acidic anions and the graft and removal of acyl groups. Morphological analysis showed that the reversibly acylated fibers had diameters <150 nm and nanofiber structure was maintained after immersion in aqueous solution. The membranes also were compatible with bone cells in culture. The resultant pure chitosan nanofibers show excellent stability in aqueous solution and exhibit broad potential in biomedical applications.

Evaluating the bone tissue regeneration capability of the Chinese herbal decoction Danggui Buxue Tang from a molecular biology perspective.

Large bone defects are a considerable challenge to reconstructive surgeons. Numerous traditional Chinese herbal medicines have been used to repair and regenerate bone tissue. This study investigated the bone regeneration potential of Danggui Buxue Tang (DBT), a Chinese herbal decoction prepared from Radix Astragali (RA) and Radix Angelicae Sinensis (RAS), from a molecular biology perspective. The optimal ratio of RA and RAS used in DBT for osteoblast culture was obtained by colorimetric and alkaline phosphatase (ALP) activity assays. Moreover, the optimal concentration of DBT for bone cell culture was also determined by colorimetric, ALP activity, nodule formation, Western blotting, wound-healing, and tartrate-resistant acid phosphatase activity assays. Consequently, the most appropriate weight ratio of RA to RAS for the proliferation and differentiation of osteoblasts was 5 : 1. Moreover, the most effective concentration of DBT was 1,000 μg/mL, which significantly increased the number of osteoblasts, intracellular ALP levels, and nodule numbers, while inhibiting osteoclast activity. Additionally, 1,000 μg/mL of DBT was able to stimulate p-ERK and p-JNK signal pathway. Therefore, DBT is highly promising for use in accelerating fracture healing in the middle or late healing periods.

Determining a Clinically Relevant Strategy for Bone Tissue Engineering: An “All-in-One” Study in Nude Mice

PurposeAutologous bone grafting (BG) remains the standard reconstruction strategy for large craniofacial defects. Calcium phosphate (CaP) biomaterials, such as biphasic calcium phosphate (BCP), do not yield consistent results when used alone and must then be combined with cells through bone tissue engineering (BTE). In this context, total bone marrow (TBM) and bone marrow-derived mesenchymal stem cells (MSC) are the primary sources of cellular material used with biomaterials. However, several other BTE strategies exist, including the use of growth factors, various scaffolds, and MSC isolated from different tissues. Thus, clinicians might be unsure as to which method offers patients the most benefit. For this reason, the aim of this study was to compare eight clinically relevant BTE methods in an “all-in-one” study.MethodsWe used a transgenic rat strain expressing green fluorescent protein (GFP), from which BG, TBM, and MSC were harvested. Progenitor cells were then mixed with CaP materials and implanted subcutaneously into nude mice. After eight weeks, bone formation was evaluated by histology and scanning electron microscopy, and GFP-expressing cells were tracked with photon fluorescence microscopy.Results/ConclusionsBone formation was observed in only four groups. These included CaP materials mixed with BG or TBM, in which abundant de novo bone was formed, and BCP mixed with committed cells grown in two- and three-dimensions, which yielded limited bone formation. Fluorescence microscopy revealed that only the TBM and BG groups were positive for GFP expressing-cells, suggesting that these donor cells were still present in the host and contributed to the formation of bone. Since the TBM-based procedure does not require bone harvest or cell culture techniques, but provides abundant de novo bone formation, we recommend consideration of this strategy for clinical applications.

New vitamin D less-calcemic analog affect human bone cell line and cultured vascular smooth muscle cells similar to other less-calcemic analogs

Primary cultures of human bone and vascular cells respond to vitamin D treatment by modulation of cell proliferation measured by DNA synthesis (DNA) and energy metabolism measured by creatine kinase specific activity (CK) via binding to vitamin D receptors (VDR) which are expressed in these cells. Vitamin D compounds also modulate the response to estradiol-17β (E2) and the expression mRNAs of estrogen receptors (ERα and ERβ), VDR, 25-hydroxy vitamin D3 1-α hydroxylase (1OHase) and lipoxygenases (12LO and 15LO). We now compared our newly synthesized analog: 1α,25-dihydroxy-9-methylene-19-norvitamin D3 JK152 (JK), on bone and vascular cells compared to other analogs. Human bone cell line SaOS2 respond to JK by increased DNA and stimulated CK dose-dependently, similar to the less-calcemic analogs CB 1093 (CB) and EB 1089 (EB). JK also up-regulated the response to E2 in terms of DNA and CK. JK inhibited DNA synthesis and increased CK in primary human vascular smooth muscle cells (VSMC) dose-dependently similar to EB and CB. JK up regulated the response to E2 in terms of CK with no effect on DNA. JK similar to CB and EB stimulated mRNA expression of VDR and ERα, 12LO and 15LO, with no effect on ERβ and 1OHase mRNA expression in SaOS2 measured by real time PCR. Similar treatments of VSMC with JK, CB and EB stimulated 12LO and 15LO, VDR and ERα mRNA expression with no effect on ERβ and 1OHase mRNA expression. The results presented here demonstrate that the new vitamin D less-calcemic analog JK is similar to other analogs in its effects on human cultured cells and therefore may be used in combined hormone replacement treatment (HRT) both in vitro and in vivo.

Development of polycaprolactone porous scaffolds by combining solvent casting, particulate leaching, and polymer leaching techniques for bone tissue engineering

AbstractSodium chloride and polyethylene glycol (PEG) were used as water‐soluble porogens for the formation of porous polycaprolactone (PCL) scaffolds. The main purpose was to prepare and evaluate in vitro efficacy of highly interconnected, three‐dimensional, porous polymeric scaffolds, as obtained from the combined particulate and polymer leaching techniques. Microscopic analysis confirmed the high interconnectivity of the pores and relatively uniform pore size of 378–435 μm. The PCL scaffolds were further characterized for their density and pore characteristics, water absorption and flow behaviors, and mechanical properties and the potential for their use as bone scaffolding materials was evaluated in vitro using mouse calvaria‐derived preosteoblastic cells (MC3T3‐E1). Evidently, the use of PEG as the secondary porogen not only improved the interconnectivity of the pore structures but also resulted in the PCL scaffolds that exhibited much better support for the proliferation and differentiation of the cultured bone cells. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 102A: 3379–3392, 2014.

DEVELOPMENT OF POLYCAPROLACTONE POROUS SCAFFOLDS BY COMBINING SOLVENT CASTING, PARTICULATE LEACHING, AND POLYMER LEACHING TECHNIQUES FOR BONE TISSUE ENGINEERING

Sodium chloride and polyethylene glycol (PEG) were used as water-soluble porogens for the formation of porous polycaprolactone (PCL) scaffolds. The main purpose was to prepare and evaluate in vitro efficacy of highly interconnected, three-dimensional, porous polymeric scaffolds, as obtained from the combined particulate and polymer leaching techniques. Microscopic analysis confirmed the high interconnectivity of the pores and relatively uniform pore size of 378-435 μm. The PCL scaffolds were further characterized for their density and pore characteristics, water absorption and flow behaviors, and mechanical properties and the potential for their use as bone scaffolding materials was evaluated in vitro using mouse calvaria-derived preosteoblastic cells (MC3T3-E1). Evidently, the use of PEG as the secondary porogen not only improved the interconnectivity of the pore structures but also resulted in the PCL scaffolds that exhibited much better support for the proliferation and differentiation of the cultured bone cells.

Valproate reduces collagen and osteonectin in cultured bone cells

Valproate is a histone deacetylase (HDAC) inhibitor that was introduced more than 40 years ago and is commonly used to treat epilepsy and mood disorders. Its long-term side effects can include bone loss, although the exact mechanism for this is currently unknown. In a previous study, we used iTRAQ labelling and mass spectrometry to profile the effect of valproate on skin fibroblast cells. We found, for the first time, that valproate reduced the amount of two key bone proteins; collagen I and osteonectin (SPARC, BM-40) while over 1000 other proteins remained unchanged (Fuller et al., 2010). We now show that valproate treatment also reduces the protein levels of collagen I and osteonectin in the hFOB1.19 osteoblast-like cell line. Pro-collagen I was reduced by 48% and osteonectin by 25% after 24h exposure to a clinically-relevant concentration of valproate. Collagen I is the main protein component of bone matrix and osteonectin has a major role in bone development and mineralisation, so reduced levels may contribute to bone loss following long-term in vivo exposure to valproate.

Afamin stimulates osteoclastogenesis and bone resorption via Gi-coupled receptor and Ca2+/calmodulin-dependent protein kinase (CaMK) pathways.

Afamin was recently identified as a novel osteoclast-derived coupling factor that can stimulate the in vitro and in vivo migration of preosteoblasts. In order to understand in more detail the biological roles of afamin in bone metabolism, we investigated its effects on osteoclastic differentiation and bone resorption. Osteoclasts were differentiated from mouse bone marrow cells. Tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells were considered as osteoclasts, and the resorption area was determined by incubating the cells on dentine discs. The intracellular cAMP level was determined using a direct enzyme immunoassay. Signaling pathways were investigated using western blot and RT-PCR. Recombinant afamin was administered exogenously to bone cell cultures. Afamin stimulated both osteoclastogenesis and in vitro bone resorption. Consistently, the expressions of osteoclast differentiation markers were significantly increased by afamin. Although afamin mainly affected the late-differentiation stages of osteoclastogenesis, the expression levels of receptor activator of nuclear factor-κB ligand (RANKL)-dependent signals were not changed. Afamin markedly decreased the levels of intracellular cAMP with reversal by pretreatment with pertussis toxin (PTX), a specific inhibitor of Gi-coupled receptor signaling. In addition, PTX almost completely blocked afamin-stimulated osteoclastogenesis. Furthermore, pretreatment with KN93 and STO609 - Ca2+/cal - mo dulin-dependent protein kinase (CaMK) and CaMK kinase inhibitors, respectively - significantly prevented decreases in the intracellular cAMP level by afamin while attenuating afamin-stimulated osteoclastogenesis. Afamin enhances osteoclastogenesis by decreasing intracellular cAMP levels via Gi-coupled receptor and CaMK pathways.

Single and double stage sintering of mechanically alloyed powder for nanostructured Ti6Al4V foams usable in cancellous scaffolds

Mechanical alloying and sintering were used to fabricate nanostructured Ti6Al4V scaffolds of highly controllable pore geometry and fully interconnected porous network. Elemental powders were milled for different periods of time (10, 20, 30, 40 and 60 h), mixed with 40–60 vol.-% of 200–400 μm cuboidal NaCl, compacted at 500–600 MPa and sintered according to single or double stage heat treatment regimes at 790 and 950°C under vacuum. After sintering, the samples were soaked in distilled water to washout the NaCl. Foamy microstructures were obtained showing well shaped biopores and fragmentary embedded micropores. The shape of initial NaCl was copied into the biopores which had highly interconnected architecture considered suitable for cancellous bone tissue adherence and cell culture.