Bone Marrow Stromal Cell Cultures Research Articles

Gender-Specific Differences in the Skeletal Response to Continuous PTH in Mice Lacking the IGF1 Receptor in Mature Osteoblasts.

The primary goal of this study was to determine whether the IGF1R in mature osteoblasts and osteocytes was required for the catabolic actions of continuous parathyroid hormone (cPTH). Igf1r was deleted from male and female FVN/B mice by breeding with mice expressing cre recombinase under control of the osteocalcin promoter ((0CN) Igfr1(-/-) ). Littermates lacking the cre recombinase served as controls. PTH, 60 μg/kg/d, was administered continuously by Alzet minipumps for 4 weeks. Blood was obtained for indices of calcium metabolism. The femurs were examined by micro-computed tomography for structure, immunohistochemistry for IGF1R expression, histomorphometry for bone formation rates (BFR), mRNA levels by qPCR, and bone marrow stromal cell cultures (BMSC) for alkaline phosphatase activity (ALP(+) ), mineralization, and osteoblast-induced osteoclastogenesis. Whereas cPTH led to a reduction in trabecular bone volume/tissue volume (BV/TV) and cortical thickness in the control females, no change was found in the control males. Although trabecular BV/TV and cortical thickness were reduced in the (0CN) Igfr1(-/-) mice of both sexes, no further reduction after cPTH was found in the females, unlike the reduction in males. BFR was stimulated by cPTH in the controls but blocked by Igf1r deletion in the females. The (0CN) Igfr1(-/-) male mice showed a partial response. ALP(+) and mineralized colony formation were higher in BMSC from control males than from control females. These markers were increased by cPTH in both sexes, but BMSC from male (0CN) Igfr1(-/-) also were increased by cPTH, unlike those from female (0CN) Igfr1(-/-) . cPTH stimulated receptor activator of NF-κB ligand (RANKL) and decreased osteoprotegerin and alkaline phosphatase expression more in control female bone than in control male bone. Deletion of Igf1r blocked these effects of cPTH in the female but not in the male. However, PTH stimulation of osteoblast-driven osteoclastogenesis was blocked by deleting Igfr1 in both sexes. We conclude that cPTH is catabolic in female but not male mice. Moreover, IGF1 signaling plays a greater role in the skeletal actions of cPTH in the female mouse than in the male mouse, which may underlie the sex differences in the response to cPTH.

Knockout of Nuclear High Molecular Weight FGF2 Isoforms in Mice Modulates Bone and Phosphate Homeostasis

We previously reported that targeted overexpression of the fibroblast growth factor 2 (FGF2) high molecular weight (HMW) isoforms in osteoblastic lineage cells in mice resulted in phenotypic changes, including dwarfism, rickets, osteomalacia, hypophosphatemia, increased serum parathyroid hormone, and increased levels of the phosphatonin FGF23 in serum and bone. This study examined the effects of genetically knocking out the FGF2HMW isoforms (HMWKO) on bone and phosphate homeostasis. HMWKO mice were not dwarfed and had significantly increased bone mineral density and bone mineral content in femurs and lumbar vertebrae when compared with the wild-type (WT) littermates. Micro-computed tomography analysis of femurs revealed increased trabecular bone volume, thickness, number, and connective tissue density with decreased trabecular spacing compared with WT. In addition, there was significantly decreased cortical porosity and increased cortical thickness and sub-periosteal area in femurs of HMWKO. Histomorphometric analysis demonstrated increased osteoblast activity and diminished osteoclast activity in the HMWKO. In vitro bone marrow stromal cell cultures showed there was a significant increase in alkaline phosphatase-positive colony number at 1 week in HMWKO. At 3 weeks of culture, the mineralized area was also significantly increased. There was increased expression of osteoblast differentiation marker genes and reduced expression of genes associated with impaired mineralization, including a significant reduction in Fgf23 and Sost mRNA. Normal serum phosphate and parathyroid hormone were observed in HMWKO mice. This study demonstrates a significant negative impact of HMWFGF2 on biological functions in bone and phosphate homeostasis in mice.

Incorporation of sol–gel bioactive glass into PLGA improves mechanical properties and bioactivity of composite scaffolds and results in their osteoinductive properties

In this study, 3D porous bioactive composite scaffolds were produced and evaluated for their physico-chemical and biological properties. Polymer poly-L-lactide-co-glycolide (PLGA) matrix scaffolds were modified with sol–gel-derived bioactive glasses (SBGs) of CaO–SiO2–P2O5 systems. We hypothesized that SBG incorporation into PLGA matrix would improve the chemical and biological activity of composite materials as well as their mechanical properties. We applied two bioactive glasses, designated as S2 or A2, differing in the content of SiO2 and CaO (i.e. 80 mol% SiO2, 16 mol% CaO for S2 and 40 mol% SiO2, 52 mol% CaO for A2). The composites were characterized for their porosity, bioactivity, microstructure and mechanical properties. The osteoinductive properties of these composites were evaluated in human bone marrow stromal cell (hBMSC) cultures grown in either standard growth medium or treated with recombinant human bone morphogenetic protein-2 (rhBMP-2) or dexamethasone (Dex). After incubation in simulated body fluid, calcium phosphate precipitates formed inside the pores of both A2-PLGA and S2-PLGA scaffolds. The compressive strength of the latter was increased slightly compared to PLGA. Both composites promoted superior hBMSC attachment to the material surface and stimulated the expression of several osteogenic markers in hBMSC compared to cells grown on unmodified PLGA. There were also marked differences in the response of hBMSC to composite scaffolds, depending on chemical compositions of the scaffolds and culture treatments. Compared to silica-rich S2-PLGA, hBMSC grown on calcium-rich A2-PLGA were overall less responsive to rhBMP-2 or Dex and the osteoinductive properties of these A2-PLGA scaffolds seemed partially dependent on their ability to induce BMP signaling in untreated hBMSC. Thus, beyond the ability of currently studied composites to enhance hBMSC osteogenesis, it may become possible to modulate the osteogenic response of hBMSC, depending on the chemistry of SBGs incorporated into polymer matrix.

ERRγ is not required for skeletal development but is a RUNX2-dependent negative regulator of postnatal bone formation in male mice.

To assess the effects of the orphan nuclear Estrogen receptor-related receptor gamma (ERRγ) deficiency on skeletal development and bone turnover, we utilized an ERRγ global knockout mouse line. While we observed no gross morphological anomalies or difference in skeletal length in newborn mice, by 8 weeks of age ERRγ +/− males but not females exhibited increased trabecular bone, which was further increased by 14 weeks. The increase in trabecular bone was due to an increase in active osteoblasts on the bone surface, without detectable alterations in osteoclast number or activity. Consistent with the histomorphometric results, we observed an increase in gene expression of the bone formation markers alkaline phosphatase (Alp) and bone sialoprotein (Bsp) in bone and increase in serum ALP, but no change in the osteoclast regulators receptor activator of NF-κB ligand (RANKL) and osteoprotegerin (OPG) or the resorption marker carboxy-terminal collagen crosslinks (CTX). More colony forming units-alkaline phosphatase and -osteoblast (CFU-ALP, CFU-O respectively) but not CFU-fibroblast (CFU-F) formed in ERRγ +/− versus ERRγ +/+ stromal cell cultures, suggesting that ERRγ negatively regulates osteoblast differentiation and matrix mineralization but not mesenchymal precursor number. By co-immunoprecipitation experiments, we found that ERRγ and RUNX2 interact in an ERRγ DNA binding domain (DBD)-dependent manner. Treatment of post-confluent differentiating bone marrow stromal cell cultures with Runx2 antisense oligonucleotides resulted in a reduction of CFU-ALP/CFU-O in ERRγ +/− but not ERRγ +/+ mice compared to their corresponding sense controls. Our data indicate that ERRγ is not required for skeletal development but is a sex-dependent negative regulator of postnatal bone formation, acting in a RUNX2- and apparently differentiation stage-dependent manner.

Mesenchymal stem cell therapy for immune-modulation: the donor, the recipient, and the drugs in-between.

Adoptive transfer of cultured bone marrow stromal cells (mesenchymal stem cells also known as MSCs) is a promising new way to aid tissue regeneration and treat a wide variety of diseases where regulation of inflammatory responses is derailed. Although significant advances have been made in the field, pinpointing important mechanistic details about how MSCs function in vitro and in vivo, there are still many unanswered questions that need to be addressed before welcoming MSCs in the therapeutic arsenal of immune mediated diseases. In this viewpoint, we highlight and discuss a few factors that we believe are critical in terms of therapeutic success employing cultured MSCs. Selecting the right donor population, choosing the best culture conditions and picking the patient population that is most likely to give a favourable therapeutic response is just as important as considering interactions between MSCs and the combination of drugs in the recipient's body. Given the complexity of MSC-host interactions, it is also imperative to develop screening tools that account for as many variables as possible and predict precisely the in vivo response rates before MSCs enter the body. To achieve this, a multidisciplinary approach is required with comprehensive knowledge of basic MSC biology, immunology, pharmacology and good clinical practice.

Role of Glucocorticoid-induced Leucine Zipper (GILZ) in Bone Acquisition

Glucocorticoids (GCs) have both anabolic and catabolic effects on bone. However, no GC anabolic effect mediator has been identified to date. Here we show that targeted expression of glucocorticoid-induced leucine zipper (GILZ), a GC anti-inflammatory effect mediator, enhances bone acquisition in mice. Transgenic mice, in which the expression of GILZ is under the control of a 3.6-kb rat type I collagen promoter, exhibited a high bone mass phenotype with significantly increased bone formation rate and osteoblast numbers. The increased osteoblast activity correlates with enhanced osteogenic differentiation and decreased adipogenic differentiation of bone marrow stromal cell cultures in vitro. In line with these changes, the mRNA levels of key osteogenic regulators (Runx2 and Osx) increased, and the level of adipogenic regulator peroxisome proliferator-activated receptor (PPAR) γ2 decreased significantly. We also found that GILZ physically interacts with C/EBPs and disrupts C/EBP-mediated PPARγ gene transcription. In conclusion, our results showed that GILZ is capable of increasing bone acquisition in vivo, and this action is mediated via a mechanism involving the inhibition of PPARγ gene transcription and shifting of bone marrow MSC/progenitor cell lineage commitment in favor of the osteoblast pathway.

Toxicity Study Of gelatin-chitosan Films with Bone Marrow Stromal Cells Cultured in Rat

Background :Gelatin and chitosan are known as biodegradable and biocompatible biopolymers.These biopolymers have recently receivedincreasingly more attention for tissueengineering.The aim of this study was to evaluate the toxicity effects of gelatin-chitosan film on bone marrow stromal cell(BMSCs) culture in rat. Materials & Methods: First, gelatin- chitosan composites film were prepared by solution mixing, followed by film casting of both biopolymers in glacial acetic acid.After two passage of BMSCs culture, the cells were cultured in four plate groups including: control plates have no any film,gelatinplates,chitosan plates and gelatin- chitosan plates.Theproliferation,differentiation,viability and apoptosis rates of BMSCs were studied duringthesecond,fourthand sixth days.The activity of superoxidedismutase(SOD),catalase(CAT)and glutathione(GSH) and malondialdehyde(MDA) levels were determined by using biochemical methods. Results:The results showed that the mean BMSCs proliferation significantly reduced in chitosan group compared to control group (p<0.05),but in gelatin and gelatin- chitosan groups were similar to control group.Mean percentage of BMSCs apoptosis in all groups except chitosan group were similar to control group. Mean percentage of BMSCs viability at all groups was similar to control group except chitosan film. In addition, no significant changes were observed in CAT activity and GSH and MDA levels in comparison with control group.After72hours,SOD activity in gelatin-chitosan group was significantly reduced compared to other groups. No cell differentiation was detected in all groups. Conclusion: Results of proliferation, differentiation, apoptosis and antioxidantactivity in cultured BMSCs on a gelatin–chitosanfilm showed that gelatin-chitosanfilm can be used in tissue engineering and cell therapy as a good model of a biodegradable scaffold. Keywords: Antioxidant system, Bone Marrow StromalCells, Cell Proliferation andDifferentiation, Gelatin-Chitosan Film.

Silver Nanoparticles and Growth Factors Incorporated Hydroxyapatite Coatings on Metallic Implant Surfaces for Enhancement of Osteoinductivity and Antibacterial Properties

Research on incorporation of both growth factors and silver (Ag) into hydroxyapatite (HA) coatings on metallic implant surfaces for enhancing osteoinductivity and antibacterial properties is a challenging work. Generally, Ag nanoparticles are easy to agglomerate and lead to a large increase in local Ag concentration, which could potentially affect cell activity. On the other hand, growth factors immobilization requires mild processing conditions so as to maintain their activities. In this study, bone morphology protein-2 (BMP-2) and Ag nanoparticle contained HA coatings were prepared on Ti surfaces by combining electrochemical deposition (ED) of Ag and electrostatic immobilization of BMP-2. During the ED process, chitosan (CS) was selected as the stabilizing agent to chelate Ag ions and generate Ag nanoparticles that are uniformly distributed in the coatings. CS also reduces Ag toxicity while retaining its antibacterial activity. Afterwards, a BMP/heparin solution was absorbed on the CS/Ag/HA coatings. Consequently, BMP-2 was immobilized on the coatings by the electrostatic attraction between CS, heparin, and BMP-2. Sustained release of BMP-2 and Ag ions from HA coatings was successfully demonstrated for a long period. Results of antibacterial tests indicate that the CS/Ag/HA coatings have high antibacterial properties against both Staphylococcus epidermidis and Escherichia coli. Osteoblasts (OB) culture reveals that the CS/Ag/HA coatings exhibit good biocompatibility. Bone marrow stromal cells (BMSCs) culture indicates that the BMP/CS/Ag/HA coatings have good osteoinductivity and promote the differentiation of BMSCs. Ti bars with BMP/CS/Ag/HA coatings were implanted into the femur of rabbits to evaluate the osteoinductivity of the coatings. Results indicate that BMP/CS/Ag/HA coatings favor bone formation in vivo. In summary, this study presents a convenient and effective method for the incorporation of growth factors and antibacterial agents into HA coatings. This method can be utilized to modify a variety of metallic implant surfaces.

Ephrin B2/EphB4 Mediates the Actions of IGF-I Signaling in Regulating Endochondral Bone Formation

Ephrin B2/EphB4 mediates interactions among osteoblasts (OBs), osteoclasts (OCLs), and chondrocytes to regulate their differentiation. We investigated the role of ephrin B2/EphB4 signaling in mediating the anabolic effects of insulin-like growth factor-I (IGF-I) and parathyroid hormone (PTH) on those cells and overall endochondral bone formation. Immunohistochemistry demonstrated that the expression of ephrin B2 in OBs, OCLs, and osteocytes, and the expression of EphB4 in OBs and osteocytes was dramatically decreased in global IGF-I knockout mice. Inactivation of EphB4 by EphB4 small, interfering RNA (siRNA) in cultured bone marrow stromal cells significantly decreased the mRNA levels of OB differentiation markers and abolished the stimulatory effects of IGF-I on these markers. Blocking the interaction of EphB4 and ephrin B2 in the OB-OCL cocultures with the EphB4 specific peptide TNYL-RAW or deletion of ephrin B2 in OCL prior to coculture led to fewer and smaller tartrate-resistant acid phosphatase (TRAP)-positive cells, decreased expression of OB differentiation markers, and blunted response to IGF-I for both OCL and OB differentiation. In the growth plate, both ephrin B2 and EphB4 are expressed in late stage proliferating and prehypertrophic chondrocytes, and their expression was decreased in mice lacking the IGF-I receptor specifically in chondrocytes. In vitro, blocking the interaction of EphB4 and ephrin B2 in chondrogenic ATDC5 cells with TNYL-RAW significantly decreased both basal and IGF1-induced expression of type II and type X collagen. In the cocultures of ATDC5 cells and spleen cells (osteoclast precursors), TNYL-RAW decreased the numbers of TRAP-positive cells and the expression of nuclear factor of activated T cells, cytoplasmic 1 (NFATc1) and receptor activator of NF-κB (RANK), and blocked their stimulation by IGF-I. Our data indicate that IGF-I/IGF-IR signaling promotes OB, OCL, and chondrocyte differentiation via ephrin B2/EphB4 mediated cell-cell communication.

Local transplantation is an effective method for cell delivery in the osteogenesis imperfecta murine model

Osteogenesis imperfecta is a serious genetic disorder that results from improper type I collagen production. We aimed to evaluate whether bone marrow stromal cells (BMSC) delivered locally into femurs were able to engraft, differentiate into osteoblasts, and contribute to formation of normal bone matrix in the osteogenesis imperfect murine (oim) model. Donor BMSCs from bone-specific reporter mice (Col2.3GFP) were expanded in vitro and transplanted into the femoral intramedullary cavity of oim mice. Engraftment was evaluated after four weeks. We detected differentiation of donor BMSCs into Col2.3GFP+ osteoblasts and osteocytes in cortical and trabecular bone of transplanted oim femurs. New bone formation was detected by deposition of dynamic label in the proximity to the Col2.3GFP+ osteoblasts, and new bone showed more organized collagen structure and expression of type I α2 collagen. Col2.3GFP cells were not found in the contralateral femur indicating that transplanted osteogenic cells did not disseminate by circulation. No osteogenic engraftment was observed following intravenous transplantation of BMSCs. BMSC cultures derived from transplanted femurs showed numerous Col2.3GFP+ colonies, indicating the presence of donor progenitor cells. Secondary transplantation of cells recovered from recipient femurs and expanded in vitro also showed Col2.3GFP+ osteoblasts and osteocytes confirming the persistence of donor stem/progenitor cells. We show that BMSCs delivered locally in oim femurs are able to engraft, differentiate into osteoblasts and osteocytes and maintain their progenitor potential in vivo. This suggests that local delivery is a promising approach for introduction of autologous MSC in which mutations have been corrected.

Hypoxic Preconditioning Attenuated Simulated Ischemia/Reperfusion Injury in Cultured Bone Marrow Stromal Cells

Aim: Bone marrow stromal cells (BMSCs) are a promising candidate for cell therapy in ischemic stroke. However, the majority of BMSCs are readily lost after transplantation because of apoptosis due to ischemia-reperfusion (I/R) injury. In the present study, we aimed to evaluate the effects of hypoxia preconditioning (HP) on simulated I/R injury in cultured BMSCs. Methods: Four generations of BMSCs were used as subjects. BMSCs were divided into 6 groups, including normal group, I/R group(I/R followed 0 h HP), 2 h-HP group(I/R followed 2 h HP ), 4 h-HP group(I/R followed 4 h HP), 6 h-HP group (I/R followed 6 h HP) and 8 h-HP group(I/R followed 8 h HP) 2 h-HP group(I/R followed 2 h HP ), 4 h-HP group(I/R followed 4 h HP), 6 h-HP group (I/R followed 6 h HP) and 8 h-HP group(I/R followed 8 h HP). BMSCs were subjected to HP by exposing the cells to hypoxia (2%O2). After 12 h of reoxygenation, BMSCs were suffered from 3 h ischemia (<0.5% O2 and serum deprivation) and subsequent reperfusion (I/R). Cell viability, hypoxia-inducible factor (HIF) 1-α, caspase-3 and apoptosis were tested using MTT, ELISA and immunofluorescence staining respectively. Results: There were no changes in cell viability after 2 h to 8 h HP in cultured BMSCs as compared to the normal group. But HP upregulated the content of HIF-1α in BMSCs and protected the BMSCs from later I/R injury, as evidenced by increased cell viability and decreased Caspase-3 release and apoptosis, especially in 8 h-HP group. Conclusion: HP attenuated the apoptosis induced by I/R injury via co-regulating the expression of HIF-1α and Caspase-3 in BMSCs, and thereby might play a beneficial effect on cell therapy

In vitro induction of alkaline phosphatase levels predicts in vivo bone forming capacity of human bone marrow stromal cells

One of the applications of bone marrow stromal cells (BMSCs) that are produced by ex vivo expansion is for use in in vivo bone tissue engineering. Cultured stromal cells are a mixture of cells at different stages of commitment and expansion capability, leading to a heterogeneous cell population that each time can differ in the potential to form in vivo bone. A parameter that predicts for in vivo bone forming capacity is thus far lacking. We employed single colony-derived BMSC cultures to identify such predictive parameters. Using limiting dilution, we have produced sixteen single CFU-F derived BMSC cultures from human bone marrow and found that only five of these formed bone in vivo. The single colony-derived BMSC strains were tested for proliferation, osteogenic-, adipogenic- and chondrogenic differentiation capacity and the expression of a variety of associated markers. The only robust predictors of in vivo bone forming capacity were the induction of alkaline phosphatase, (ALP) mRNA levels and ALP activity during in vitro osteogenic differentiation. The predictive value of in vitro ALP induction was confirmed by analyzing “bulk-cultured” BMSCs from various bone marrow biopsies. Our findings show that in BMSCs, the additional increase in ALP levels over basal levels during in vitro osteogenic differentiation is predictive of in vivo performance.

Sustained Alterations In Bone Marrow Stromal Cells From Patients With Myeloproliferative Neoplasms (MPN) Contribute To Remodelling Of The Bone Marrow Microenvironment Prior To The Manifestation Of Myelofibrosis

Myeloproliferative neoplasms (MPN) are characterized by the loss of normal hematopoiesis and the excessive production and accumulation of non-lymphoid cells and platelets in the BM. Clonal hematopoiesis in MPN is assumed to generate factors which induce profound changes in the non-clonal BM microenvironment. Alterations include massive deposition of extracellular matrix proteins (ECM), progression to bone marrow (BM)-fibrosis and osteosclerosis. We hypothesize that particularly in MPN, alterations in the cross talk between hematopoietic and bone marrow stromal cells (BMSC) play a critical role for an impaired bone marrow microenvironment long before overt signs of myelofibrosis can be detected by conventional methods.To dissect the hematopoiesis supporting capacity and extracellular matrix remodelling of BMSC from patients with MPN, we isolated BMSC from BM of patients with essential thrombocytemia (ET, n=5), polycythemia vera (PV, n=5), chronic myeloid leukemia (CML, n=5) and control BM (n=6). BMSC isolates were taken only from pre-fibrotic MPN patients (bone marrow trephine biopsy reticulin staining graded 0 or 1) and the resulting expansion cultures fullfilled MSC criteria according to the common consensus (Dominici et al., Cytotherapy, 2006; 8(4):315-317). When subjected to myeloid colony forming unit assays, MPN-BMSC conditioned supernatants showed a significantly reduced capacity to stimulate a CFU-GM/G/M growth of non-malignant hematopoietic stem and progenitor cells as compared to control BMSC (control (n=6) vs CML (n=5): p= 0.0032; control vs PV (n=5): p=0.016, control vs ET (n=4) not significant; student«s T- test ). BMSC-dependent matrix remodelling was analysed in a previously established robust matrix remodelling assay in vitro. MPN-BMSC displayed a pronounced increased matrix remodelling capacity compared to control BMSC. Interestingly, among the different MPN subtypes, this effect was highly significant in BMSC derived from patients with ET (Control (n=6) vs. ET (n=5): p<0.001). Furthermore, in vitro ECM production by MPN-BMSC was paralleled by ECM changes observed in matched bone marrow punches as shown by fibronectin immunohistochemistry. Co-expression of the stroma marker CD271 and fibronectin -as shown by confocal microscopy- points towards stroma-mediated ECM production in vivo. As upregulation of fibronectin expression was also detected in reticulin 0 graded BM punches, we hypothesized that fibronectin staining might be a potential marker for pre-fibrotic ECM changes in –so far- reticulin-negative MPN biopsies. To validate this hypothesis, we stained fibronectin in a tissue microarray (TMA), containing primary BM biopsies from patients with ET (n=14), PV (n=14), CML (n=14), MF (n=11) and controls (Non-Hodgkin's lymphoma without bone marrow involvement, n=17). Interestingly, within the reticulin-negative subcohort of pre-fibrotic MPN (n=34), fibronectin stained positive (grade 1 or higher) in 5/7 cases with ET (71%), 6/9 cases with PV (66%) and in 14/14 cases with CML (100%) as well as in all cases with PMF (100%). Furthermore, fibronectin staining correlated significantly with patients' decreased haemoglobin levels as shown by ANOVA analysis of routine clinical parameters (F=5.71; Prob> F 0.0037).We conclude that prior to the manifestation of fibrosis in MPN, BMSC loose their capacity to support non-malignant hematopoiesis and increase their matrix remodelling potential. As to our surprise, these effects are stably maintained in the absence of the malignant hematopoietic clone and since BMSC cultures develop over numerous population doublings in vitro from proliferating mesenchymal precursors, we hypothesize that cell intrinsic effects in BMSC from patients with MPN contribute to the development of myelofibrosis in MPN. [Display omitted] Disclosures:Bruemmendorf: Bristol-Myers Squibb: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Ariad: Consultancy, Honoraria.

Protein Kinase D1 Is Essential for Bone Acquisition during Pubertal Growth

Bone formation and maintenance represents the summation of the balance of local and endocrine hormonal stimuli within a complex organ. Protein kinase D (PKD) is a member of the Ca(2+)/calmodulin-dependent kinase superfamily of serine/threonine kinases and has been described as the crossroads for the bone morphogenetic protein (BMP)-IGF-I signaling axis, which plays a major role in bone formation. The current study exploits the PKD1-deficient mouse model to examine the role of PKD in vivo in the skeleton. Dual-energy x-ray absorptiometry scan analysis of male and female pubescent mice demonstrated significantly decreased bone mineral density in the whole body and femoral bone compartments of PKD1 (+/-) mice, compared with their wild-type littermates. The body weight, nasal-anal length, and percentage body fat of the mice were not significantly different from their wild-type littermates. Cultured bone marrow stromal cells from PKD1 (+/-) mice demonstrated lower alkaline phosphatase activity in early differentiating osteoblasts and decreased mineralized nodule formation in mature osteoblasts. Quantitative RT-PCR analysis of osteoblast differentiation markers and osteoclast markers exhibited lower levels of expression in PKD1 (+/-) male mice than wild type. In female mice, however, only markers of osteoblast differentiation were reduced. PKD1 (+/-) mice also demonstrated a profound reduction in mRNA expression levels of BMP type II receptor and IGF-I receptor and in BMP-7 responsiveness in vitro. Together these data suggest that in mice, PKD1 action contributes to the regulation of osteoblastogenesis by altering gene expression with gender-specific effects on osteoclastogenesis, subsequently affecting skeletal matrix acquisition during puberty.

Tailored degradation of biocompatible poly(3-hydroxybutyrate-co-3-hydroxyvalerate)/calcium silicate/poly(lactide-co-glycolide) ternary composites: An in vitro study

Biodegradable materials, which are currently available for bone tissue regeneration, still have limitations regarding their degradation rate, mechanical stability and/or biological response. Thus, a novel generation of materials for bioactive bone scaffolds is needed that triggers hydroxyapatite formation and can be tailored to suit application-specific requirements. In this study we developed ternary bioactive composite materials composed of poly(3-hydroxybutyrate-co-3-hydroxyvalerate), calcium silicate and poly(lactide-co-glycolide) (PHBV/CS/PLGA), which merged the good bioactivity of CS/PHBV composite and the improved degradation velocity of PHBV/PLGA blend. Bioactive character of all composites was proven by formation of hydroxyapatite-like crystals after already one week of incubation in simulated body fluid. Addition of PLGA significantly increased initial ultimate tensile strength (UTS0) and Young's modulus of the ternary composites from 14.3±1.1MPa (binary composite) to 22.3±2.6MPa and 1.23±0.05GPa up to 1.64±0.14GPa, respectively. Furthermore the degradation rate (measured as a decrease of UTS during degradation) could be successfully tailored and was in range of −0.033UTS0 to −0.118UTS0MPa/week. The bioacceptance of the materials was proven in vitro using 2-D (conventional setup) and 3-D (multicellular spheroids) human bone marrow stromal cell cultures.

Preparation method and growth factor content of platelet concentrate influence the osteogenic differentiation of bone marrow stromal cells

Background aimsAn extensive debate about the clinical benefits of autologous platelet concentrates used as a treatment option for patients with orthopedic injuries is ongoing. The aim of this study was to determine whether different compositions of platelet concentrates may affect the osteogenic differentiation of bone marrow stromal cells (BMSC). MethodsPure platelet-rich plasma (P-PRP) and leukocyte-PRP (L-PRP) were characterized for platelet and leukocyte content. As an indicative marker of the delivery of growth factors (GFs), the release of basic fibroblast growth factor (bFGF) from platelet gel (PG) was measured at 1, 18, 48 and 72 h and at 7 d. The ability of different PGs to induce proliferation and differentiation of BMSC was evaluated by using bioactivity assays. ResultsThe platelet recovery was significantly higher in L-PRP, either fresh or frozen. PGs derived from L-PRP and P-PRP showed significant differences in terms of bFGF release and biological activity. bFGF release was faster both in fresh and frozen L-PRP preparations. Moreover, L-PRP samples were able to induce a significantly higher proliferation of BMSC compared with P-PRP or PPP samples. Even though all PG preparations allowed the deposition of mineral nodules in BMSC cultures, the mineralization activity correlated significantly with bFGF levels. ConclusionsThe biological activity of platelet concentrates differs according to preparation technique, which affects platelet and leukocyte content and GF availability. Because GF levels are not always optimal in subjects with defective bone healing, composition and bioactivity of PRP should be analyzed to test the reliability and potential effectiveness of the regenerative treatment.

N-acetyl cysteine as an osteogenesis-enhancing molecule for bone regeneration

Bone regeneration often requires cues from osteogenesis-inducing factors for successful outcome. N-acetyl cysteine (NAC), an anti-oxidant small molecule, possibly modulates osteoblastic differentiation. This study investigated the potential of NAC as an osteogenesis-enhancing molecule in vitro and in vivo. Various concentrations of NAC (0, 2.5, 5.0, and 10 mm) were added to rat bone marrow stromal cell or osteoblastic cell culture in media with or without dexamethasone. The results showed marked enhancement of alkaline phosphatase activity and mineralized matrix formation together with consistent upregulation of bone-related gene markers such as collagen I, osteopontin, and osteocalcin in the osteoblastic culture with addition of 2.5 or 5.0 mm NAC regardless of the presence of dexamethasone. Micro-CT-based analysis and histological observation revealed that addition of NAC to a collagenous sponge implanted in a critical size cortical bone defect (3.0 mm × 5.0 mm) in rat femur yielded acceleration and completion of defect closure, with thick, compact, and contiguous bone after 6 weeks of healing. In contrast, with sponge alone, only sparse and incomplete bone regeneration was observed during the matching healing period. These results indicate that NAC can function as an osteogenesis-enhancing molecule to accelerate bone regeneration by activating differentiation of osteogenic lineages.

CXCL12/CXCR4 signaling in the osteoblast regulates the mesenchymal stem cell and osteoclast lineage populations

The chemokine CXCL12 and its receptor CXCR4 play a key role in regulation of hematopoietic stem cells and cell migratory function during morphogenesis. Osteoblasts express both the ligand and the receptor, but little is known about the role of CXCL12-CXCR4 signaling in maintaining skeletal homeostasis. Using Cre-Lox technology to delete CXCR4 in mature osteoblasts in mice, we show here a significant decrease in bone mass and alterations in cancellous bone structure. CXCR4 gene ablation increased the number of colony-forming units (CFU), CFU-positive for alkaline phosphatase (CFU-AP(+)), and mineralizing nodules in bone marrow stromal cell (BMSC) cultures. The adipocyte precursor population decreased in BMSCs harvested from the KO animals. The nonadherent population of BMSCs harvested from the long bone diaphysis of KO animals formed more osteoclasts, a finding that was associated with increased circulatory levels of pyridinoline, a marker of bone resorption. Our data show that osteoblast-specific CXCR4 deletion has profound effects on the mesenchymal stem cell pool and allocation to the osteoblastic and adipocytic cell lineages. They also show that CXCL12/CXCR4 signaling in the mature osteoblast can feedback to regulate the osteoclast precursor pool size and play a multifunctional role in regulating bone formation and resorption.

ACTH promotes chondrogenic nodule formation and induces transient elevations in intracellular calcium in rat bone marrow cell cultures via MC2-R signaling

Adrenocorticotropic hormone (ACTH) is among several melanocortin peptide hormones that are derived from proopiomelanocortin (POMC). ACTH has been found to enhance osteogenesis and chondrogenesis. We show that, in the presence of dexamethasone, ACTH dose-dependently increases chondrogenic nodule formation in bone marrow stromal cells (BMSC) from the Wistar Kyoto (WKY) rat. The nodules consist in condensed cells highly expressing alkaline phosphatase, Sox9 and type II collagen transcripts and a proteoglycan-rich matrix. Immunoblot analysis of crude membrane fractions has shown that these cells express three melanocortin receptors (MC-R), namely MC2-R, MC3-R and MC5-R and the melanocortin 2-receptor accessory protein (MRAP). To determine which of these receptors mediate ACTH-induced effects, we have used MC-R-specific peptides and the known agonist profiles of the receptors. Neither α-MSH, a strong agonist of MC5-R, nor γ2-MSH, a strong agonist of MC3-R, duplicates ACTH effects in rat BMSC. In addition, calcium flux has been examined as a mechanism for ACTH action at the MC2-R. Consistent with MC2-R and MRAP expression patterns in the BMSC cultures, ACTH-induced transient increases in intracellular calcium are increased with dexamethasone treatment. Neither α-MSH nor γ2-MSH affects calcium flux. Dexamethasone increases MC2-R and MRAP expression and POMC peptide expression and cleavage increasing the production of the lipolytic β-lipotropic hormone product. Therefore, the effects of ACTH in rat BMSC enriched for mesenchymal progenitors are consistent with an MC2-R signaling mechanism, with dexamethasone being capable of regulating components of the melanocortin system in these cells.

Low Oxygen Tension Maintains Multipotency, Whereas Normoxia Increases Differentiation of Mouse Bone Marrow Stromal Cells

Optimization of mesenchymal stem cells (MSC) culture conditions is of great importance for their more successful application in regenerative medicine. O2 regulates various aspects of cellular biology and, in vivo, MSC are exposed to different O2 concentrations spanning from very low tension in the bone marrow niche, to higher amounts in wounds. In our present work, we isolated mouse bone marrow stromal cells (BMSC) and showed that they contained a population meeting requirements for MSC definition. In order to establish the effect of low O2 on cellular properties, we examined BSMC cultured under hypoxic (3% O2) conditions. Our results demonstrate that 3% O2 augmented proliferation of BMSC, as well as the formation of colonies in the colony-forming unit assay (CFU-A), the percentage of quiescent cells, and the expression of stemness markers Rex-1 and Oct-4, thereby suggesting an increase in the stemness of culture when exposed to hypoxia. In contrast, intrinsic differentiation processes were inhibited by 3% O2. Overall yield of differentiation was dependent on the adjustment of O2 tension to the specific stage of BMSC culture. Thus, we established a strategy for efficient BMSC in vitro differentiation using an initial phase of cell propagation at 3% O2, followed by differentiation stage at 21% O2. We also demonstrated that 3% O2 affected BMSC differentiation in p53 and reactive oxygen species (ROS) independent pathways. Our findings can significantly contribute to the obtaining of high-quality MSC for effective cell therapy.