Presence Of Osteogenic Factors Research Articles

Exposure to Random Positioning Machine Alters the Mineralization Process and PTX3 Expression in the SAOS-2 Cell Line.

Bone loss is among the most frequent changes seen in astronauts during space missions. Although weightlessness is known to cause high bone resorption and a rapid decrease in bone minerals and calcium, the underlying mechanisms are not yet fully understood. In our work, we investigated the influence of random positioning machine (RPM) exposure on the mineralization process in the SAOS-2 cell line, in osteogenic and non-osteogenic conditions, by examining changes in their mineralizing capacity and in the expression of PTX3, a positive regulator of bone mineralization. We analyzed cell viability by MTS assay and the mineralization process after staining with Toluidine Blue and Alizarin Red, while PTX3 expression was investigated by immunocytochemistry and western blotting analysis. Our results showed that RPM exposure increased cells’ viability and improved their mineralizing competence when not treated with osteogenic cocktail. In contrast, in osteogenic conditions, cells exposed to RPM showed a reduction in the presence of calcification-like structures, mineral deposits and PTX3 expression, suggesting that the effects of RPM exposure on mineralizing matrix deposition depend on the presence of osteogenic factors in the culture medium. Further studies will be needed to clarify the role of potential mineralization markers in the cellular response to the simulated biological effects of microgravity, paving the way for a new approach to treating osteoporosis in astronauts exposed to spaceflight.

Icaritin Enhancing Bone Formation Initiated by Sub-Microstructured Calcium Phosphate Ceramic for Critical Size Defect Repair

Adequate bone tissue regeneration has been challenging to achieve at critical-sized bone defects caused by disease. Bone tissue engineering using a combination of scaffolds and bioactive factors provides new hope for the treatment of this extreme condition. Icaritin, a herb-derived chemical, has shown its ability to enhance bone formation both in vitro and in vivo, and it has been found that sub-micron surface structure instructs bone formation in calcium phosphate ceramics (CaPs). Here, we evaluated the possibility of using a submicron surface structured CaP ceramic as the carrier of icaritin for bone tissue regeneration in critical-sized bone defects. Icaritin, an herb-derived chemical, was loaded into a submicron surface structured porous calcium phosphate ceramic (Ø12.8 × 3 mm) to get samples with 0, 10, 50, 250, and 1,250 µg icaritin per CaP disc (M0, M10, M50, M250, M1250 groups, respectively). In vitro evaluation with the certain dosages correlated to those released from the samples showed a dose-dependent enhancement of osteogenic differentiation and mineralization of human bone marrow stromal cells with the presence of osteogenic factors in the culture medium, indicating icaritin is an osteopromotive factor. After intramuscular implantation of the samples in dogs for 8 weeks, a dose-dependent of bone formation was seen with enhanced bone formation at the dosage of 50 and 250 µg. To evaluate the in vivo osteogenic potentials of icaritin-containing CaP ceramic scaffolds in the orthopedic site, a 12.8 mm calvarial defect model in rabbits was established. Micro-computed tomography (micro-CT) and histology results at weeks 4, 8 and 12 post-surgery showed more newly formed bone in M250 group, with correspondingly more new vessel ingrowth. The results presented herein suggested that being osteopromotive, icaritin could enhance bone formation initiated by sub-microstructured CaP ceramics and the CaP ceramics scaffold incorporating icaritin is a promising biomaterial for the treatment of critical-sized defect.

Adipose-Derived Stromal Vascular Fraction/Xenohybrid Bone Scaffold: An Alternative Source for Bone Regeneration.

Adipose tissue-derived stem cells (ASCs) are a promising tool for the treatment of bone diseases or skeletal lesions, thanks to their ability to potentially repair damaged tissue. One of the major limitations of ASCs is represented by the necessity to be isolated and expanded through in vitro culture; thus, a strong interest was generated by the adipose stromal vascular fraction (SVF), the noncultured fraction of ASCs. SVF is a heterogeneous cell population, directly obtained after collagenase treatment of adipose tissue. In order to investigate and compare the bone-regenerative potential of SVF and ASCs, they were plated on SmartBone®, a xenohybrid bone scaffold, already used in clinical practice with successful results. We showed that SVF plated on SmartBone, in the presence of osteogenic factors, had better osteoinductive capabilities than ASCs, in terms of differentiation into bone cells, mineralization, and secretion of soluble factors stimulating osteoblasts. Indeed, we observed an increasing area of new tissue over time, with and without OM. These data strongly support an innovative idea for the use of adipose SVF and bone scaffolds to promote tissue regeneration and repair, also thanks to an easier cell management preparation that allows a potentially larger use in clinical applications.

Nonmulberry Silk Braids Direct Terminal Osteocytic Differentiation through Activation of Wnt-Signaling.

Silk polymers can regulate osteogenesis by mimicking some features of the extracellular matrix of bone and facilitate mineralized deposition on their surface by cultured osteoprogenitors. However, terminal differentiation of these mineralizing osteoblasts into osteocytic phenotypes has not yet been demonstrated on silk. Therefore, in this study we test the hypothesis that flat braids of natively (nonregenerated) spun nonmulberry silk A. mylitta, possessing mechanical stiffness in the range of trabecular bone, can regulate osteocyte differentiation within their 3D microenvironment. We seeded human preosteoblasts onto these braids and cultured them under varied temperatures (33.5 and 39 °C), soluble factors (dexamethasone, ascorbic acid, and β-glycerophosphate), and cytokine (TGF-β1). After 1 week, cell dendrites were conspicuously evident, confirming osteocyte differentiation, especially, in the presence of osteogenic factors and TGF-β1 expressing all characteristic osteocyte markers (podoplanin, DMP-1, and sclerostin). A. mylitta silk braids alone were sufficient to induce this differentiation, albeit only transiently. Therefore, we believe that the combinatorial effect of A. mylitta silk (surface chemistry, braid rigidity, and topography), osteogenic differentiation factors, and TGF-β1 were critical in stabilizing the mature osteocytic phenotype. Interestingly, Wnt signaling promoted osteocytic differentiation as evidenced by the upregulated expression of β-catenin in the presence of osteogenic factors and growth factor. This study highlights the role of nonmulberry silk braids in regulating stable osteocytic differentiation. Future studies could benefit from this understanding of the signaling mechanisms associated with silk-based matrices in order to develop 3D in vitro bone model systems.

In Vivo Osteogenic Differentiation of Human Dental Pulp Stem Cells Embedded in an Injectable In Vivo-Forming Hydrogel.

In this study, human dental pulp stem cells (hDPSCs) are examined as a cellular source for bone tissue engineering using an in vivo-forming hydrogel. The hDPSCs are easily harvested in large quantities from extracted teeth. The stemness of harvested hDPSCs indicates their relative tolerance to ex vivo manipulation in culture. The in vitro osteogenic differentiation of hDPSCs is characterized using Alizarin Red S (ARS), von Kossa (VK), and alkaline phosphatase (ALP) staining. The solution of hDPSCs and a methoxy polyethylene glycol-polycaprolactone block copolymer (PC) is easily prepared by simple mixing at room temperature and in no more than 10 s it forms in vivo hydrogels after subcutaneous injection into rats. In vivo osteogenic differentiation of hDPSCs in the in vivo-forming hydrogel is confirmed by micro-computed tomography (CT), histological staining, and gene expression. Micro-CT analysis shows evidence of significant tissue-engineered bone formation in hDPSCs-loaded hydrogel in the presence of osteogenic factors. Differentiated osteoblasts in in vivo-forming hydrogel are identified by ARS and VK staining and are found to exhibit characteristic expression of genes like osteonectin, osteopontin, and osteocalcin. In conclusion, hDPSCs embedded in an in vivo-forming hydrogel may provide benefits as a noninvasive formulation for bone tissue engineering applications.

An Endochondral Ossification-Based Approach to Bone Repair: Chondrogenically Primed Mesenchymal Stem Cell-Laden Scaffolds Support Greater Repair of Critical-Sized Cranial Defects Than Osteogenically Stimulated Constructs In Vivo.

The lack of success associated with the use of bone grafts has motivated the development of tissue engineering approaches for bone defect repair. However, the traditional tissue engineering approach of direct osteogenesis, mimicking the process of intramembranous ossification (IMO), leads to poor vascularization. In this study, we speculate that mimicking an endochondral ossification (ECO) approach may offer a solution by harnessing the potential of hypertrophic chondrocytes to secrete angiogenic signals that support vasculogenesis and enhance bone repair. We hypothesized that stimulation of mesenchymal stem cell (MSC) chondrogenesis and subsequent hypertrophy within collagen-based scaffolds would lead to improved vascularization and bone formation when implanted within a critical-sized bone defect in vivo. To produce ECO-based constructs, two distinct scaffolds, collagen-hyaluronic acid (CHyA) and collagen-hydroxyapatite (CHA), with proven potential for cartilage and bone repair, respectively, were cultured with MSCs initially in the presence of chondrogenic factors and subsequently supplemented with hypertrophic factors. To produce IMO-based constructs, CHA scaffolds were cultured with MSCs in the presence of osteogenic factors. These constructs were subsequently implanted into 7 mm calvarial defects on Fischer male rats for up to 8 weeks in vivo. The results demonstrated that IMO- and ECO-based constructs were capable of supporting enhanced bone repair compared to empty defects. However, it was clear that the scaffolds, which were previously shown to support the greatest cartilage formation in vitro (CHyA), led to the highest new bone formation (p < 0.05) within critical-sized bone defects 8 weeks postimplantation. We speculate this to be associated with the secretion of angiogenic signals as demonstrated by the higher VEGF protein production in the ECO-based constructs before implantation leading to the greater blood vessel ingrowth. This study thus demonstrates the ability of recapitulating a developmental process of bone formation to develop tissue-engineered constructs that manifest appreciable promise for bone defect repair.

A computer-designed scaffold for bone regeneration within cranial defect using human dental pulp stem cells.

A computer-designed, solvent-free scaffold offer several potential advantages such as ease of customized manufacture and in vivo safety. In this work, we firstly used a computer-designed, solvent-free scaffold and human dental pulp stem cells (hDPSCs) to regenerate neo-bone within cranial bone defects. The hDPSCs expressed mesenchymal stem cell markers and served as an abundant source of stem cells with a high proliferation rate. In addition, hDPSCs showed a phenotype of differentiated osteoblasts in the presence of osteogenic factors (OF). We used solid freeform fabrication (SFF) with biodegradable polyesters (MPEG-(PLLA-co-PGA-co-PCL) (PLGC)) to fabricate a computer-designed scaffold. The SFF technology gave quick and reproducible results. To assess bone tissue engineering in vivo, the computer-designed, circular PLGC scaffold was implanted into a full-thickness cranial bone defect and monitored by micro-computed tomography (CT) and histology of the in vivo tissue-engineered bone. Neo-bone formation of more than 50% in both micro-CT and histology tests was observed at only PLGC scaffold with hDPSCs/OF. Furthermore, the PLGC scaffold gradually degraded, as evidenced by the fluorescent-labeled PLGC scaffold, which provides information to tract biodegradation of implanted PLGC scaffold. In conclusion, we confirmed neo-bone formation within a cranial bone defect using hDPSCs and a computer-designed PLGC scaffold.

In vivo osteogenic differentiation of human turbinate mesenchymal stem cells in an injectable in situ-forming hydrogel

Human turbinate mesenchymal stromal cells (hTMSCs) are an alternate source of adult stem cells for regenerative medicine. In this work, we demonstrated that hTMSCs are easily harvested from turbinate tissue using a minimal surgical procedure. hTMSCs showed positive expression of mesenchymal stem cell markers and proliferated at a high rate. The specific surface proteins of harvested hTMSCs were relatively tolerant of ex vivo manipulation in culture. hTMSCs exhibited osteogenic differentiation in vitro in the presence of osteogenic factors. To examine osteogenic differentiation of hTMSCs in vivo in an injectable hydrogel, cells were incorporated into a methoxy polyethylene glycol–polycaprolactone block copolymer (MPEG–PCL (MP)) solution simply by mixing. hTMSC-loaded MP solutions exhibited a temperature-dependent solution-to-gel phase transition. The hTMSC attached and grew well on in vitro- and in vivo-formed MP hydrogels. hTMSC-loaded MP solutions formed a hydrogel almost immediately upon injection into animals and the cells remained viable, even after 12 weeks. Injected hTMSCs in in situ-formed MP hydrogels differentiated into osteogenic cells, mainly in the presence of osteogenic factors. Differentiated osteoblasts were identified by Alizarin Red S, von Kossa, and alkaline phosphatase (ALP) staining, and osteonectin, osteopontin, and osteocalcin mRNA expression. To the best of our knowledge, this is the first study to show hTMSCs undergoing osteogenic differentiation in in vivo-formed MP hydrogels. In conclusion, hTMSCs could serve as adult stem cell sources and, when embedded in an in situ-formed hydrogel, may provide numerous benefits as a noninvasive alternative for bone tissue engineering applications.

Trabecular titanium can inducein vitroosteogenic differentiation of human adipose derived stem cells without osteogenic factors

Trabecular Titanium (TT) is an innovative highly porous structure that imitates the morphology of trabecular bone with good mechanical properties. Adipose-derived stem cells are a multipotent cell population that can be used in regenerative medicine, in particular, for bone therapeutic applications. The ability of TT to induce the osteogenic differentiation of human adipose derived stem cells (hASCs) in the absence of osteogenic factors was evaluated using molecular biological, biochemical, and immunohistochemical methods. At 7 and 21 days from differentiation, the hASCs grown on TT scaffolds showed similar expressions of alkaline phosphatase (ALP) and Runx-2 both in the presence and in the absence of osteogenic factors, as well as at transcript and protein levels. hASCs cultured on monolayer in the presence of the medium obtained from the wells where hASCs/scaffold constructs were cultured in the absence of osteogenic factors differentiated towards the osteogenic phenotype: their gene and protein expression of ALP and Runx-2 was similar to that of the same cells cultured in the presence of osteogenic factors, and significantly higher than that of the ones cultured in growth medium.

Engineered 3D Silk‐Based Metastasis Models: Interactions Between Human Breast Adenocarcinoma, Mesenchymal Stem Cells and Osteoblast‐Like Cells

AbstractBone metastasis occurs in 70% of breast cancer patients and is a frequent cause of morbidity in cancer patients. A delicate balance exists in the bone microenvironment, but the functional dynamics underlying the tumor cell‐microenvironment interactions remain poorly understood. 3D in vitro model systems of metastasis can throw new light on this phenomenon. Silk protein fibroin scaffolds, are cytocompatible for 3D cancer cell culture. They are structurally more resistant to protease degradation than other native biomaterials making these matrices suitable for cancer modeling. In this report, human breast adenocarcinoma cells, human osteoblast like cells and mesenchymal stem cells are co‐cultered. Cancer cells and osteoblast‐like cells are found to interact through secreted products. Decreased population of osteoblast‐like cells and mineralization of extracellular matrix are observed as a result of co‐culture. Significantly increased migration of breast cancer cells is observed in the bone‐like constructs than in non‐seeded scaffolds. The co‐culture constructs show significant increase in drug resistance, invasiveness and angiogenicity. Co‐culture of breast cancer cells with osteoblast like cells and mesenchymal stem cells also indicate that the interaction of cancer cells with bone microenvironment varies with spatial organization, presence of osteogenic factors as well as stromal cell type. Here, results show that 3D in vitro co‐culture models is possibly a better system to study and target cancer progression.

Different Roles of GNAS and cAMP Signaling During Early and Late Stages of Osteogenic Differentiation

Progressive osseous heteroplasia (POH) and fibrous dysplasia (FD) are genetic diseases of bone formation at opposite ends of the osteogenic spectrum: imperfect osteogenesis of the skeleton occurs in FD, while heterotopic ossification in skin, subcutaneous fat, and skeletal muscle forms in POH. POH is caused by heterozygous inactivating germline mutations in GNAS, which encodes G-protein subunits regulating the cAMP pathway, while FD is caused by GNAS somatic activating mutations. We used pluripotent mouse ES cells to examine the effects of Gnas dysregulation on osteoblast differentiation. At the earliest stages of osteogenesis, Gnas transcripts Gsα, XLαs and 1A are expressed at low levels and cAMP levels are also low. Inhibition of cAMP signaling (as in POH) by 2',5'-dideoxyadenosine enhanced osteoblast differentiation while conversely, increased cAMP signaling (as in FD), induced by forskolin, inhibited osteoblast differentiation. Notably, increased cAMP was inhibitory for osteogenesis only at early stages after osteogenic induction. Expression of osteogenic and adipogenic markers showed that increased cAMP enhanced adipogenesis and impaired osteoblast differentiation even in the presence of osteogenic factors, supporting cAMP as a critical regulator of osteoblast and adipocyte lineage commitment. Furthermore, increased cAMP signaling decreased BMP pathway signaling, indicating that G protein-cAMP pathway activation (as in FD) inhibits osteoblast differentiation, at least in part by blocking the BMP-Smad pathway, and suggesting that GNAS inactivation as occurs in POH enhances osteoblast differentiation, at least in part by stimulating BMP signaling. These data support that differences in cAMP levels during early stages of cell differentiation regulate cell fate decisions. Supporting information available online at http:/www.thieme-connect.de/ejournals/toc/hmr.

Detection of hydroxyapatite in calcified cardiovascular tissues

ObjectiveThe objective of this study is to develop a method for selective detection of the calcific (hydroxyapatite) component in human aortic smooth muscle cells in vitro and in calcified cardiovascular tissues ex vivo. This method uses a novel optical molecular imaging contrast dye, Cy-HABP-19, to target calcified cells and tissues. MethodsA peptide that mimics the binding affinity of osteocalcin was used to label hydroxyapatite in vitro and ex vivo. Morphological changes in vascular smooth muscle cells were evaluated at an early stage of the mineralization process induced by extrinsic stimuli, osteogenic factors and a magnetic suspension cell culture. Hydroxyapatite components were detected in monolayers of these cells in the presence of osteogenic factors and a magnetic suspension environment. ResultsAtherosclerotic plaque contains multiple components including lipidic, fibrotic, thrombotic, and calcific materials. Using optical imaging and the Cy-HABP-19 molecular imaging probe, we demonstrated that hydroxyapatite components could be selectively distinguished from various calcium salts in human aortic smooth muscle cells in vitro and in calcified cardiovascular tissues, carotid endarterectomy samples and aortic valves, ex vivo. ConclusionHydroxyapatite deposits in cardiovascular tissues were selectively detected in the early stage of the calcification process using our Cy-HABP-19 probe. This new probe makes it possible to study the earliest events associated with vascular hydroxyapatite deposition at the cellular and molecular levels. This target-selective molecular imaging probe approach holds high potential for revealing early pathophysiological changes, leading to progression, regression, or stabilization of cardiovascular diseases.

Nanophase hydroxyapatite and poly(lactide-co-glycolide) composites promote human mesenchymal stem cell adhesion and osteogenic differentiation in vitro

Human mesenchymal stem cells (hMSCs) typically range in size from 10 to 50 μm and proteins that mediate hMSC adhesion and differentiation usually have a size of a few nanometers. Nanomaterials with a feature size smaller than 100 nm have demonstrated the unique capability of promoting osteoblast (bone forming cell) adhesion and long-term functions, leading to more effective bone tissue regeneration. For new bone deposition, MSCs have to be recruited to the injury or disease sites and then differentiate into osteoblasts. Therefore, designing novel nanomaterials that are capable of attracting MSCs and directing their differentiation is of great interest to many clinical applications. This in vitro study investigated the effects of nanophase hydroxyapatite (nano-HA), nano-HA/poly(lactide-co-glycolide) (PLGA) composites and a bone morphogenetic protein (BMP-7) derived short peptide on osteogenic differentiation of hMSCs. The short peptide was loaded by physical adsorption to nano-HA or by dispersion in nanocomposites and in PLGA to determine their effects on hMSC adhesion and differentiation. The results showed that the nano-HA/PLGA composites promoted hMSC adhesion as compared to the PLGA controls. Moreover, nano-HA/PLGA composites promoted osteogenic differentiation of hMSCs to a similar extent with or without the presence of osteogenic factors in the media. In the MSC growth media without the osteogenic factors, the nanocomposites supported greater calcium-containing bone mineral deposition by hMSC than the BMP-derived short peptide alone. The nanocomposites provided promising alternatives in controlling the adhesion and differentiation of hMSCs without osteogenic factors from the culture media, and, thus, should be further studied for clinical translation and the development of novel nanocomposite-guided stem cell therapies.

Isolation of progenitor cells from cord blood using adhesion matrices

The aim of this study was to develop optimal conditions for selective adhesion and isolation of mesenchymal progenitor cells (MPCs) from cord blood and to determine their potential for osteogenic differentiation. Mononuclear cells (MNCs) were isolated by Ficoll-Paque gradient and plated onto 48-well culture plates precoated with: human or bovine collagen type I, human collagen type IV, fibronectin or matrigel. Cultures were incubated in alphaMEM containing fetal calf serum. Viability of the adherent cells was determined by alamarBlue(R) assay after 2, 3, and 4 weeks. After 4 weeks in culture, cells were typsinized and replated. Primary cultures were analyzed by histochemistry and third passage cells by FACS. Isolated fibroblast-like cells were cultured in the presence of osteogenic factors and differentiation determined by Alizarin Red S staining, RT-PCR and electron dispersive spectroscopy (EDS). MNCs adhered to all types of matrices with the greatest adhesion rates on fibronectin. These cells were CD45(+), CD105(+), CD14(+), CD49a(+), CD49f(+), CD44(+) and CD34(-). The highest incidence of progenitor cells (PC) was observed on fibronectin and polystyrene. Passages were CD45(-), CD14(-), CD34(-) and weakly CD105(+). Primary cultures expressed endothelial/macrophage RNA markers whether cultured on fibronectin or polystyrene and these markers decreased upon passage. The best osteogenic differentiation was observed in MPCs cultured in osteogenic medium containing vitamin D(3) and FGF9. These cells expressed the bone-related mRNA, collagen type I, core binding factor I (Cbfa I), osteocalcin and osteopontin. EDS of deposits produced by these cells demonstrated a calcium/phosphate ratio parallel to hydroxyapatite. It was concluded that fibronectin increased adhesion rates and isolation potential of cord blood mesenchymal progenitor cells.

Isolation of progenitor cells from cord blood using adhesion matrices

The aim of this study was to develop optimal conditions for selective adhesion and isolation of mesenchymal progenitor cells (PCs) from cord blood and to determine their potential for osteogenic differentiation. Mononuclear cells (MNCs) were isolated by Ficoll-Paque gradient and plated onto 48-well culture plates precoated with: human or bovine collagen type I, human collagen type IV, fibronectin or matrigel. Cultures were incubated in alphaMEM containing fetal calf serum. Viability of the adherent cells was determined by alamarBlue(R) assay after 2, 3, and 4 weeks. After 4 weeks in culture, cells were typsinized and replated. Primary cultures were analyzed by histochemistry and third passage cells by FACS. Isolated fibroblast-like cells were cultured in the presence of osteogenic factors and differentiation determined by Alizarin Red S staining, RT-PCR and electron dispersive spectroscopy (EDS). MNCs adhered to all types of matrices with the greatest adhesion rates on fibronectin. These cells were CD45(+), CD105(+), CD14(+), CD49a(+), CD49f(+), CD44(+) and CD34(-). The highest incidence of PCs was observed on fibronectin and polystyrene. Passages were CD45(-), CD14(-), CD34(-) and weakly CD105(+). Primary cultures expressed endothelial/macrophage RNA markers whether cultured on fibronectin or polystyrene and these markers decreased upon passage. The best osteogenic differentiation was observed in MPCs cultured in osteogenic medium containing Vit D(3) and FGF9. These cells expressed the bone-related mRNA, collagen type I, core binding factor I (Cbfa I), osteocalcin and osteopontin. EDS of deposits produced by these cells demonstrated a calcium/phosphate ratio parallel to hydroxyapatite. It was concluded that fibronectin increased adhesion rates and isolation potential of cord blood mesenchymal progenitor cells.

The Proteasome Inhibitor Bortezomib Affects Osteoblastogenesis and Bone Formation In Vitro and In Vivo in Multiple Myeloma Patients.

It is well established that osteoblast formation and function are profoundly impaired in multiple myeloma (MM) patients. Osteoblastic cells also regulate myeloma cell growth and increasing bone formation result in a reduction of tumoral burden in mice. Recent data suggest that ubiquitin-proteasome pathway, the major cellular degradative system and therapeutic target in myeloma cells, also regulates osteoblast differentiation. Further it has been demonstrated that different proteasome inhibitors may stimulate bone formation in mice. Finally, preliminary observations obtained in MM patients treated with the proteasome inhibitor Bortezomib show an increase of bone specific alkaline phosphatase in responder patients as compared to non-responder ones. Currently it is not know whether the proteasome inhibitor Bortezomib may have a direct effect on osteoblast and bone formation in vitro human cultures and in vivo in MM patients.To clarify this issue first we checked the effect of Bortezomib either on osteoblast differentiation and formation or on osteoblast proliferation, survival and function. In long-term human BM cultures we found that Bortezomib did not reduce the number of both early bone marrow (BM) osteoblast progenitors Colony Forming Unit-Fibroblast (CFU-F) and late ones Colony Forming Bone nodules (CFU-OB). On the other hand we found that Bortezomib (2–3 nM) significantly induced osteoblast phenotype in human mesenchymal cells incubated in presence of osteogenic factors. A stimulatory effect on osteoblast markers was observed after 24 hours of Bortezomib treatment. Consistently we found that Bortezomib significantly increased the activity of the transcription factor Runx2/Cbfa1 in human osteoblast progenitors without affecting the canonical WNT signaling pathway checked by the evaluation of nuclear and cytoplasmatic active beta-catenin levels. Using the human osteoblast like cells MG-63 and immortalized normal osteoblasts (HOBIT) we found that Bortezomib at concentration ranging between 2nM and 5nM did not inhibit osteoblast proliferation or induce osteoblast apoptosis. Similarly, Bortezomib did not affect the expression of osteoblast markers, Runx2/Cbfa1 activity and WNT signaling in both MG-63 and HOBIT cells.To extent our in vitro observation we have evaluated the potential effect of Bortezomib in vivo in MM patients. Bone histomorphometry as well as immunostainig for Runx2/Cbfa1 and beta-catenin was performed on BM biopsies obtained from 15 MM patients before and after 6–8 cycles of Bortezomib administrated in mono-therapy. A significant increase in the number of osteoblastic cells X mm2 of bone tissue and in the number of Runx2/Cbfa1 positive osteoblastic cells was observed only in responder patients showing an early increase of the serum alkaline phosphatase.In conclusion our data indicate that Bortezomib may increase osteoblast differentiation in human mesenchymal cells without affecting the proliferation, survival and function of mature osteoblasts. In vivo and in vitro observations support the hypothesis that both direct and indirect effects on bone formation process could occur during Bortezomib treatment.

Effect of fiber diameter on spreading, proliferation, and differentiation of osteoblastic cells on electrospun poly(lactic acid) substrates

Electrospinning is a promising method to construct fused-fiber biomaterial scaffolds for tissue engineering applications, but the efficacy of this approach depends on how substrate topography affects cell function. Previously, it has been shown that linear, parallel raised features with length scales of 0.5–2 μm direct cell orientation through the phenomenon of contact guidance, and enhance phenotypic markers of osteoblastic differentiation. To determine how the linear, random raised features produced by electrospinning affect proliferation and differentiation of osteoprogenitor cells, poly(lactic acid) and poly(ethylene glycol)-poly(lactic acid) diblock copolymers were electrospun with mean fiber diameters of 0.14–2.1 μm onto rigid supports. MC3T3-E1 osteoprogenitor cells cultured on fiber surfaces in the absence of osteogenic factors exhibited a lower cell density after 7 and 14 days of culture than cells cultured on spin-coated surfaces, but cell density increased with fiber diameter. However, in the presence of osteogenic factors (2 m m β-glycerophosphate, 0.13 m m l-ascorbate-2-phosphate), cell density after 7 and 14 days of culture on fiber surfaces was comparable to or exceeded spin-coated controls, and alkaline phosphatase activity after 14 days was comparable. Examination of cell morphology revealed that cells grown on fibers had smaller projected areas than those on planar surfaces. However, cells attached to electrospun substrates of 2.1 μm diameter fibers exhibited a higher cell aspect ratio than cells on smooth surfaces. These studies show that topographical factors designed into biomaterial scaffolds can regulate spreading, orientation, and proliferation of osteoblastic cells.

Fluid flow stimulates expression of osteopontin and bone sialoprotein by bone marrow stromal cells in a temporally dependent manner

Bone marrow stromal cells (BMSCs) are multipotent progenitor cells with a capacity to form bone tissue in vivo, and to differentiate into the osteoblastic lineage in vitro. Drawing on evidence that bone is mechanosensitive and mechanical stimuli are anabolic, we postulate that proliferation and osteoblastic differentiation of BMSCs may be stimulated by mechanical forces. In this study, BMSCs cultured in the presence of osteogenic factors (dexamethasone, β-glycerophosphate, and ascorbate) were stimulated repeatedly (every second day) with shearing flow (1.6 dyn/cm 2) for 5, 30, or 120 min, and assayed for systematic changes in cell number and phenotypic markers of osteoblastic differentiation. Cells exposed to fluid flow on days 2 and 4 after the addition of osteogenic factors and assayed at day 6 exhibited a modest decrease in cell number and increase in normalized alkaline phosphatase activity, suggesting the detachment of a non-osteogenic subpopulation. Cells exposed to fluid flow on days 6, 8, 10, and 12 and assayed at day 20 demonstrated maximal expression of osteopontin and bone sialoprotein mRNA with 30 min duration of flow. Concurrently, at day 20 expression of the adipogenic marker, lipoprotein lipase, was minimal with a 120-min duration of flow. These results indicate that repeated application of shear stress stimulates late phenotypic markers of osteoblastic differentiation of BMSCs in a manner that depends on the duration of stimulus. Finally, accumulation of prostaglandin E 2 in culture medium in response to shearing flow systematically decreased with repeated exposure to 30 and 120 min of shear stress (from day 6 to day 12), suggesting an adaptation of the cells to fluid flow.