Study of gelatinized marrow stroma osteoblasts and true bone ceramic active bone

Trueboneceramic(TBC)isacomparativelygoodframematerialinbonetissueengineeringbecauseofitsadvantagesofnaturalbonetrabeculestructure, easydegradation, nonimmunogenicity, easymanufacture, abundantresources, andlowcost, etc1.Owingtoitsfragileness, nonductility, andasmoothsurfaceunfitforseedcelladhesion, itisstilldifficultforwidespreadclinicaluse. Bytakingadvantageofthepeculiaritiesofsodiumalginatethatwilltransformfromliquidstateintogelatinationstateandproducelateralconjunctionwhencombiningwithbiva…     

The effects of low-level laser irradiation on differentiation and proliferation of human bone marrow mesenchymal stem cells into neurons and osteoblasts—an in vitro study

Bone marrow-derived mesenchymal stem cells (BMSCs) are promising for use in regenerative medicine. Several studies have shown that low-level laser irradiation (LLLI) could affect the differentiation and proliferation of MSCs. The aim of this study was to examine the influence of LLLI at different energy densities on BMSCs differentiation into neuron and osteoblast. Human BMSCs were cultured and induced to differentiate to either neuron or osteoblast in the absence or presence of LLLI. Gallium aluminum arsenide (GaAlAs) laser irradiation (810 nm) was applied at days 1, 3, and 5 of differentiation process at energy densities of 3 or 6 J/cm² for BMSCs being induced to neurons, and 2 or 4 J/cm² for BMSCs being induced to osteoblasts. BMSCs proliferation was evaluated by MTT assay on the seventh day of differentiation. BMSCs differentiation to neurons was assessed by immunocytochemical analysis of neuron-specific enolase on the seventh day of differentiation. BMSCs differentiation to osteoblast was tested on the second, fifth, seventh, and tenth day of differentiation via analysis of alkaline phosphatase (ALP) activity. LLLI promoted BMSCs proliferation significantly at all energy densities except for 6 J/cm² in comparison to control groups on the seventh day of differentiation. LLLI at energy densities of 3 and 6 J/cm² dramatically facilitated the differentiation of BMSCs into neurons (p < 0.001). Also, ALP activity was significantly enhanced in irradiated BMSCs differentiated to osteoblast on the second, fifth, seventh, and tenth day of differentiation (p < 0.001 except for the second day). Using LLLI at 810 nm wavelength enhances BMSCs differentiation into neuron and osteoblast in the range of 2–6 J/cm², and at the same time increases BMSCs proliferation (except for 6 J/cm²). The effect of LLLI on differentiation and proliferation of BMSCs is dose-dependent. Considering these findings, LLLI could improve current in vitro methods of differentiating BMSCs prior to transplantation.     

Biocompatibility of Poly-ε-caprolactone-hydroxyapatite composite on mouse bone marrow-derived osteoblasts and endothelial cells

Background  

Tissue-engineered bone may be developed by seeding the cells capable of both osteogenesis and vascularization on biocompatible composite scaffolds. The current study investigated the performance of mice bone marrow-derived osteogenic cells and endothelial cells as seeded on hydroxyapatite (HA) and poly-ε-caprolactone (PCL) composite scaffolds.     

The effects of GM1 and bFGF synergistically inducing adultrat bone marrow stromal cells to form neural progenitor cells and their differentiation

Objective: To investigate the effects of GM1 on inducing adult rat bone marrow stromal cells (MSCs) to form neural progenitor cells and their differentiation. Methods: Purified MSCs were induced by different components of basic fibroblast growth factor (bFGF) alone, GM1 alone or combination of bFGF with GM1. After 3 days‘ incubation, fibronectin and collagen I were detected with immunocytochemistry, and nestin was detected with immunofluorescence. Neuron-specific enolase ( NSE ), glial fibrillary acidic protein ( GFAP ) and galactose cerebroside ( GalC ) were detected with immunocytochemistry after 7 days‘ incubation. Results: After induction with bFGF alone or combination of bFGF and GM1, some MSCs exhibited the pbenotypes of neural progenitor cells, and then neurons and astrocytes. In these two groups, the positive cells for fibronectin and collagen I decreased markedly after 3 days‘ induction. At the same time, the positive cells for nestin increased markedly. After 7 days‘ induction, NSE and GFAP-positive cells increased significantly. Furthermore, the addition of bFGF and GM1 caused the maximal variation. However, addition of GM1 alone had no inductive effects. Conclusions: Combination of bFGF with GM1 may synergistically promote the transformation of MSCs and differentiation into neurons and astrocyte-like cells. The results suggest a promising route for the application of MSCs.     

The effects of α-zearalanol on the proliferation of bone-marrow-derived mesenchymal stem cells and their differentiation into osteoblasts

The aim of this study was to explore the effects of α-zearalanol (α-ZAL) on the proliferation of mouse bone-marrow-derived mesenchymal stem cells (BMSCs) and their differentiation into osteoblasts. Six- to eight-week-old BALB/C mice were used either as recipients or as bone marrow donors. BMSCs were isolated and collected using a differential adhesion method, with use of 10 % fetal bovine serum and Iscove’s modified Dulbecco’s medium. After the third generation, the BMSCs were randomly placed into the following subgroups: a control group, an osteogenic medium (OM) group, a 17β-estradiol group, an α-ZAL 10^?7 mol/L group, an α-ZAL 10^?6 mol/L group, and an α-ZAL 10^?5 mol/L group. Flow cytometry was used to identify the BMSCs collected from the bone marrow. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide test was performed, and markers of the osteoblasts were measured in the different subgroups. In addition, expression of osteoprotegerin and expression of receptor activator of nuclear factor κB ligand were examined using Western blot. In contrast to the control and OM groups, BMSCs in the α-ZAL groups exhibited long fusiform shapes, and contact inhibition was observed when the cells were closely packed. After induction, the BMSCs grew well and exhibited triangular, star, polygonal, or irregular shapes. Clumps and multiple cells were evident. The trends of the proliferation and differentiation for the control, OM, 17β-estradiol, and α-ZAL groups were similar. Compared with the control and OM groups, in the α-ZAL groups the expression levels of alkaline phosphatase, procollagen type I N-terminal propeptide, bone morphogenetic protein 2, and osteocalcin were significantly increased (p < 0.05). In addition, α-ZAL inhibited osteoclastogenesis by increasing the expression of osteoprotegerin and decreasing the expression of nuclear factor κB ligand. In conclusion, α-ZAL can increase the proliferation of BMSCs and their differentiation into osteoblasts and can effectively suppress osteoclastogenesis.     

Impacts of age and gender on bone marrow profiles of BMP7, BMPRs and Stro-1⁺ cells in patients with total hip replacement

Purpose  

A successful osseointegration relies on the interplay of implant surface and surrounding bone marrow cells. This study was undertaken to investigate the impact of age and gender on the bone marrow composition.     

Differentiation of adult human bone marrow mesenchymal stem cells into Schwann-like cells in vitro

Bonemarrowmesenchymalstemcells, alsocalledbonemarrowstromalcells (BMSCs), areisolatedfrombonemarrow, andtheycanmultiplyinvitroanddifferentiateintoosteogeniccells,chondrocytes, adipocytes, musclecellsandneuralcells.1 5 RecentstudieshavedemonstratedthatBMSCsfromadultratscoulddifferentiateintoSchwann likecellsinspecificconditions.6, 7 BMSCsmayhavepotentialapplicationforautologousneuraltransplantation. Inthisstudy, wetrytoinvestigatethedifferentiativecapabilityofadulthumanBMSCsintoSchwann l…     

Retinaldehyde dehydrogenase 1 deficiency inhibits PPARγ-mediated bone loss and marrow adiposity

PPARγ, a ligand-activated nuclear receptor, regulates fundamental aspects of bone homeostasis and skeletal remodeling. PPARγ-activating anti-diabetic thiazolidinediones in clinical use promote marrow adiposity, bone loss, and skeletal fractures. As such, delineating novel regulatory pathways that modulate the action of PPARγ, and its obligate heterodimeric partner RXR, may have important implications for our understanding and treatment of disorders of low bone mineral density. We present data here establishing retinaldehyde dehydrogenase 1 (Aldh1a1) and its substrate retinaldehyde (Rald) as novel determinants of PPARγ–RXR actions in the skeleton. When compared to wild type (WT) controls, retinaldehyde dehydrogenase-deficient (Aldh1a1^−/−) mice were protected against bone loss and marrow adiposity induced by either the thiazolidinedione rosiglitazone or a high fat diet, both of which potently activate the PPARγ–RXR complex. Consistent with these results, Rald, which accumulates in vivo in Aldh1a1^−/− mice, protects against rosiglitazone-mediated inhibition of osteoblastogenesis in vitro. In addition, Rald potently inhibits in vitro adipogenesis and osteoclastogenesis in WT mesenchymal stem cells (MSCs) and hematopoietic stem cells (HSCs) respectively. Primary Aldh1a1^−/− HSCs also demonstrate impaired osteoclastogenesis in vitro compared to WT controls. Collectively, these findings identify Rald and retinoid metabolism through Aldh1a1 as important novel modulators of PPARγ–RXR transactivation in the marrow niche.     

Biochemical markers of bone turnover in diabetes patients—a meta-analysis,and a methodological study on the effects of glucose on bone markers

Summary

This study examined whether markers of bone turnover differ between individuals with and without diabetes. Bone markers showed heterogeneity between studies and were discrepant for markers of bone creation and markers of bone degradation. Bone markers may be of lesser value in diabetes due to heterogeneity.

Introduction

The aim of this meta-analysis was to compare existing literature regarding changes in bone markers among diabetics compared to healthy controls. To exclude that blood glucose levels among diabetes patients could influence the assays used for determining bone turnover markers, a methodological study was performed.

Methods

Medline at Pubmed Embase, Cinahl, Svemed+, Cochrane library, and Bibliotek.dk was searched in August 2012. The studies should examine biochemical bone turnover among diabetes patients in comparison to controls in an observational design. In the methodological study, fasting blood samples were drawn from two individuals. Glucose was added to the blood samples in different concentrations and OC, CTX, and procollagen type 1 amino terminal propeptide were measured after 0, 1, 2, and 3 h.

Results

Twenty-two papers fulfilled the criteria for the meta-analysis. From the pooled data in the meta-analysis, the bone markers osteocalcin (OC) (?1.15 ng/ml [?1.78,-0.52]) and C-terminal cross-linked telopeptide (CTX) (?0.14 ng/ml [?0.22, ?0.05]) were significantly lower among diabetes patients than non-diabetes patients, however other markers did not differ. All markers displayed very high heterogeneity by I2 statistics. In the methodological study, the addition of glucose did not significantly change the bone markers neither by level of glucose nor with increasing incubation time.

Conclusion

The dissociative pattern of biochemical bone markers of bone formation and bone resorption present in diabetes patients is thus not caused by glucose per se but may be modulated by unknown factors associated with diabetes mellitus.     

The biomechanics of point contact-dynamic compression plate and its effects on bone perfusion

Objective: To compare the mechanical properties of point contact-dynamic compression plate (PC-DCP) and its effects on cortical bone perfusion with that of dynamic compression plates (DCP) in goat tibiae. Methods: Twenty pairs of matched fresh goat tibiae were used. A transverse fracture model was established. The fractures with a 3mm interspace between the fracture ends were subject to fixations with the DCPs and the PC-DCPs respectively, then the four-points bending tests and the torsion tests were conducted to compare the mechanical properties of the PC-DCP with that of DCP. Another 13 sexually mature goats underwent fixations with the DCPs and the PC-DCPs, respectively, at the mid-shafts of the intact bilateral tibiae. Ischemic zones were observed at four time points (1 day ,2,6, and 12 weeks after operation) using disulphine blue staining technique. Results: There were no significant differences in mechanical properties, such as bend- and torsion-resistance, between the DCPs and the PC-DCPs. One day, 2, and 6 weeks after operation, on the side of DCP fixation, outer cortical bone ischemia under the plate persisted, and this condition did not reverse until 12 weeks after operation. However, on the side of PC-DCP fixation, cortical bone ischemia occurred only in the periphery of the screw holes and at the contact sites of the PC NUTs 1 day after operation, and it disappeared at 2 weeks after operation. Conclusions: The PC-DCP has similar biomechanical properties of the DCP, byt is less detrimental to local bone blood circulation than the conventional plates.     

Novel immunostimulatory effects of osteoclasts and macrophages on human γδ T cells

It has been widely reported that T cells are capable of influencing osteoclast formation and bone remodelling, yet relatively little is known of the reciprocal effects of osteoclasts for affecting T cell function and/or activity. In this study we investigated the effects of human osteoclasts on the function of γδ T cells, a subset of non-CD4⁺ T cells implicated in a variety of inflammatory disease states. γδ T cells and CD4⁺ T cells were isolated from peripheral blood of healthy volunteers and were co-cultured with autologous mature osteoclasts (generated by treatment with M-CSF and RANKL) before phenotypical and functional changes in the T cell populations were assessed. Macrophages, osteoclasts, and conditioned medium derived from macrophages or osteoclasts induced activation of γδ T cells, as determined by the expression of the early activation marker CD69. TNFα was a major mediator of this stimulatory effect on γδ T cells. Consistent with this stimulatory effect, osteoclasts augmented proliferation of IL-2-stimulated γδ T cells and also supported the survival of unstimulated γδ and CD4⁺ T cells, although these effects required co-culture with osteoclasts. Co-culture with osteoclasts also increased the proportion of γδ T cells producing IFNγ, but did not modulate IFNγ or IL-17 production by CD4⁺ T cells. We provide new insights into the in vitro interactions between human γδ T cells and osteoclasts/macrophages, and demonstrate that osteoclasts or their precursors are capable of influencing γδ T function both via the release of soluble factors and also through direct cell–cell interactions.     

Influence of cytarabine and cyclophosphamide on the disposition kinetics of cyclosporin A after bone marrow transplantation

The blood concentration of cyclosporin A (CyA) often gradually increases or is unstable in the early period of immunotherapy in bone marrow transplantation patients. In the protocol for bone marrow transplantation, pretreatment with cytarabine (Ara-C) and cyclophosphamide (CPA) is employed. We examined the influence of Ara-C and CPA on the disposition kinetics of CyA, in using rats to define the mechanism for the observation. Rats were intravenously administered daily with Ara-C (120 mg/kg/day) or CPA (60 mg/kg/day) intravenously for 2 days and were then intravenously given CyA (10 mg/kg). The blood concentration of CyA after intravenous administration of CyA at 1 day after the last administration of CPA was significantly lower and the total clearance was significantly larger than those in the vehicle control rats, while the blood concentration and the pharmacokinetic parameters of CyA were unchanged after Ara-C treatment. The expression of mdr1a, mdr1b, and CY3A2 mRNAs, and the levels of the corresponding proteins in the liver were increased after the CPA treatment. These CPA-induced changes were almost fully reversed to the control levels by 2 weeks. Thus, our results indicate that the decrease of blood CyA concentration induced is a consequence of the induction of P-glycoprotein and CYP3A in the liver by the CPA treatment , and these changes are reversed within 2 weeks after the transplantation.     

Comparison of inhibitory effects of warfarin on γ-carboxylation between bone and liver in rats

The purposes of this study were to clarify that warfarin (WF, vitamin K antagonist) levels that inhibit γ-carboxylation are different in liver and bone (experiment 1), and to investigate whether the plasma osteocalcin (OC) level reflects bone OC levels (experiments 2 and 3). Four-week-old male rats were treated with 0.2, 0.4, 0.6, 0.8, 1.0, or 1.2 mg/l of WF solution as drinking water for 4 weeks. Blood coagulation activity, an index of γ-carboxylation of prothrombin in the liver, was significantly decreased in rats receiving 0.8 mg/l or larger doses of WF. A significant decrease of plasma γ-carboxylated OC (GlaOC), an index of γ-carboxylation of OC in bone, was shown in rats receiving 0.2 mg/l or larger doses. Significantly lower OC levels in the femoral diaphysis and metaphysis were shown in the 0.2 mg/l and 0.4 mg/l groups. However, femoral bone mineral density (BMD) values in the WF-treated groups were almost the same as those in the intact group. In experiment 2, we evaluated changes in bone OC levels 4 weeks after discontinuing an 8-week WF treatment. Four-week-old male rats received 0.8 mg/l WF as drinking water for 8 or 12 weeks. Recovery of the OC level after discontinuing the WF treatment was shown in the femoral metaphysis, but not in the diaphysis. In experiment 3, 0.3 mg/kg WF was administrated to 25-week-old male rats three times a week for 8, 12, or 16 weeks. In aged rats, decreased bone OC was shown in the femoral metaphysis, but not in the diaphysis. From these findings, it is suggested that the effects of WF on γ-carboxylation are likely to appear in bone at lower doses than in the liver, that the bone OC level does not always correspond directly to plasma GlaOC, and that the bone OC level is not directly linked with BMD.     

The effects of PTH,loading and surgical insult on cancellous bone at the bone–implant interface in the rabbit

Enhancing the quantity and quality of cancellous bone with anabolic pharmacologic agents may lead to more successful outcomes of non-cemented joint replacements. Using a novel rabbit model of cancellous bone loading, we examined two specific questions regarding bone formation at the bone–implant interface: (1) does the administration of intermittent PTH, a potent anabolic agent, and mechanical loading individually and combined enhance the peri-implant cancellous bone volume fraction; and, (2) does surgical trauma enhance the anabolic effect of PTH on peri-implant bone volume fraction. In this model, PTH enhanced peri-implant bone volume fraction by 30% in loaded bone, while mechanical loading alone increased bone volume fraction modestly (+ 10%). Combined mechanical loading and PTH treatment had no synergistic effect on any cancellous parameters. However, a strong combined effect was found in bone volume fraction with combined surgery and PTH treatment (+ 34%) compared to intact control limbs. Adaptive changes in the cancellous bone tissue included increased ultimate stress and enhanced remodeling activity. The number of proliferative osteoblasts increased as did their expression of pro-collagen 1 and PTH receptor 1, and the number of TRAP positive osteoclasts also increased. In summary, both loading and intermittent PTH treatment enhanced peri-implant bone volume, and surgery and PTH treatment had a strong combined effect. This finding is of clinical importance since enhancing early osseointegration in the post-surgical period has numerous potential benefits.     

Additive effects of estrogen and mechanical stress on nitric oxide and prostaglandin E2 production by bone cells from osteoporotic donors

Mechanical loading is thought to provoke a cellular response via loading-induced flow of interstitial fluid through the lacuno-canalicular network of osteocytes. This response supposedly leads to an adaptation of local bone mass and architecture. It has been suggested that loss of estrogen during menopause alters the sensitivity of bone tissue to mechanical load, thereby contributing to the rapid loss of bone. The present study aimed to determine whether estrogen modulates the mechanoresponsiveness of bone cells from osteoporotic women. Bone cell cultures from nine osteoporotic women (aged 62–90 years) were pre-cultured for 24 h with 10^–11 mol/l 17-estradiol (E2) or vehicle, and subjected to 1 h of pulsating fluid flow (PFF) or static culture. E2 alone enhanced prostaglandin E₂ (PGE₂) and nitric oxide (NO) production by 2.8-fold and 2.0-fold, respectively, and stimulated endothelial nitric oxide synthase protein expression by 2.5-fold. PFF, in the absence of E2, stimulated PGE₂ production by 3.1-fold and NO production by 3.9-fold. Combined treatment with E2 and PFF increased PGE₂ and NO production in an additive manner. When expressed as PFF-treatment-over-control ratio, the response to fluid shear stress was similar in the absence or presence of E2. These results suggest that E2 does not affect the early response to stress in bone cells. Rather, E2 and shear stress both promote the production of paracrine factors such as NO and PGE₂ in an additive manner.     

Culture of osteoblasts on bio-derived bones

Thegoalofrepairingbonedefectsistofacilitatenewbonesgrowingintotheosseousdefects.Therefore, thematerialsusedforboneregenerationshouldsupporttheattachmentandproliferationofosteoblasts. Avarietyofbio derivedandchemosyntheticmaterialsareavailableforthesurgicaltreatmentofalveolarboneloss.1 Autogenousbonegraftsanddemineralizedfreeze driedboneallografts(DFDBA) fromhumancadavershavebeenusedwithadegreeofsuccess. However, neitherisideal. Autogenousbonegraftsinvolveanadditionalproceduretoharvestthebon…