Rabbit Osteoblasts Research Articles

Silk Fibroin/Hydroxyapatite Coating Improved Osseointegration of Porous Titanium Implants under Diabetic Conditions via Activation of the PI3K/Akt Signaling Pathway.

The application of three-dimensional printed porous titanium implants (TIs) is compromised in patients suffering from diabetes mellitus (DM), which disturbs the normal process of implant osseointegration, resulting in fixation failure. It was possibly because of reactive oxygen species (ROS) overproduction at the bone-implant interface. A silk fibroin-based hydroxyapatite (SF/HA) hybrid material emerged as a novel biological material for accelerating new bone formation. We proposed that the SF/HA hybrid coated titanium implant (SHT) could mitigate DM-mediated impaired osseointegration, which had never been reported previously. To test this assumption and further elucidate the mechanisms, primary rabbit osteoblasts were seeded on TIs or SHTs and cultured with normal serum, diabetic serum (DS), DS + N-acetyl-L-cysteine (NAC) (a potent ROS inhibitor), and DS + LY294002 (a specific PI3K/Akt inhibitor) for osteoblast behavior examinations. An animal study was performed on diabetic rabbits implanted with the two kinds of implants for osseointegration tests. DM-mediated ROS overproduction caused osteoblastic biological dysfunctions and apoptotic injury, associated with suppression of PI3K/Akt signaling in osteoblasts cultured on a TI substrate. Of note, the SHT substrate significantly suppressed ROS overproduction under diabetic conditions, improved osteoblast functional recovery including ameliorative osteoblast adhesion and morphology, improved cellular proliferation and differentiation, and abrogated apoptosis, which exhibited the same effect as NAC administration on the TI. The in vitro results were further corroborated in vivo by enhanced osteogenesis and osseointegration of SHTs in diabetic rabbits. Moreover, the aforesaid promotive effects afforded by the SF/HA coating were totally abolished with administration of LY294002 for blocking PI3K/Akt signaling. The above results collectively demonstrated that the SF/HA hybrid coating significantly ameliorated DM-mediated impaired osseointegration of the TI via reactivation of the ROS-mediated PI3K/Akt signaling pathway. The hybrid coating elicited a novel surface biofunctionalization strategy to attain favorable clinical performance of TI in diabetics.

Facilitated and Controlled Strontium Ranelate Delivery Using GCS-HA Nanocarriers Embedded into PEGDA Coupled with Decortication Driven Spinal Regeneration.

Background and PurposeStrontium ranelate (SrR) is an oral pharmaceutical agent for osteoporosis. In recent years, numerous unwanted side effects of oral SrR have been revealed. Therefore, its clinical administration and applications are limited. Hereby, this study aims to develop, formulate, and characterize an effective SrR carrier system for spinal bone regeneration.MethodsHerein, glycol chitosan with hyaluronic acid (HA)-based nanoformulation was used to encapsulate SrR nanoparticles (SrRNPs) through electrostatic interaction. Afterward, the poly(ethylene glycol) diacrylate (PEGDA)-based hydrogels were used to encapsulate pre-synthesized SrRNPs (SrRNPs-H). The scanning electron microscope (SEM), TEM, rheometer, Fourier-transform infrared spectroscopy (FTIR), and dynamic light scattering (DLS) were used to characterize prepared formulations. The rabbit osteoblast and a rat spinal decortication models were used to evaluate and assess the developed formulation biocompatibility and therapeutic efficacy.ResultsIn vitro and in vivo studies for cytotoxicity and bone regeneration were conducted. The cell viability test showed that SrRNPs exerted no cytotoxic effects in osteoblast in vitro. Furthermore, in vivo analysis for new bone regeneration mechanism was carried out on rat decortication models. Radiographical and histological analysis suggested a higher level of bone regeneration in the SrRNPs-H-implanted groups than in the other experimental groups.ConclusionLocal administration of the newly developed formulated SrR could be a promising alternative therapy to enhance bone regeneration in bone-defect sites in future clinical applications.

Study on Gelatin/Hydroxyapatite/Chitosan Material Modified with Osteoblast for Bone Bioengineering

Designing of a suitable ‘cell-material’ construct has enabled the regeneration of bone. It was in accordance with the increasing demand for the repair of defective and/or diseased bone tissue. In this research, the relative behavior of the osteoblast-seeded materials was demonstrated. It enabled the selection of the cell-material system for rabbit bone bioengineering studies. Significant role of polypeptide-polymer-ceramic-cell material in bone tissue engineering was shown. The properties of natural polymer-based scaffold were attributed to their origin and chemical modifications. In this study, the lyophilized osteoblast-material conjugate was designed and characterized extensively. The same was done by physicochemical measurement, for surface and core morphology analysis. The atomic force microscopy (AFM) and transmission electron microscopy (TEM) data from these conjugates have shown surface parameters. The surface suitable for cell attachment and proliferation along with uniform interconnected porous morphology was selected. The calcium to phosphate proportion-based energy-dispersive X-ray was done. It defines the inorganic content in these composites. The crystal spots were found from the selected area electron diffraction pattern. It had supported the insights from the physicochemical measurements. AFM and TEM micrographs have shown surface and core morphology with median surface roughness of 14.96 nm and uniform porous architecture, respectively. Fourier transmission Infra-red spectroscopy and X-ray diffraction had confirmed the formation of mineral deposits within the scaffolds. The subsequent in vitro study has revealed that among the biomaterials, ‘gelatin in hydroxyapatite-coated chitosan matrix’ has prominence over ‘gelatin-hydroxyapatite.’ It was confirmed only after seeding them with the rabbit ‘iliac crest-derived’ osteoblast. Two types of rabbit osteoblast derivatives were used. They are the osteoblast from the bone tissue (rT) and osteoblast obtained after Mesenchymal stem cell (MSCs) differentiation. Bone marrow was the source of MSCs. This ‘rT-seeded’ biomaterial was found appropriate for bone bioengineering applications.

Puerarin facilitates osteogenesis in steroid-induced necrosis of rabbit femoral head and osteogenesis of steroid-induced osteocytes via miR-34a upregulation

The present study investigated the effect of puerarin on promoting the osteogenesis in steroid-induced necrosis of the femoral head (SONFH). New Zealand rabbits were administrated with horse serum and methylprednisolone (MPS) for establishing SONFH in vivo model, which was then treated with puerarin treatment. Histo-morphological changes in the femoral head were examined by hematoxylin-eosin staining. Osteoblasts were isolated from healthy rabbits and treated by individual or combined administration of dexamethasone and puerarin. Osteoblast viability was measured by CCK-8 assay. Mineralized nodule formation was evaluated by alizarin red assay. Expressions of RUNX family transcription factor 2 (RUNX2), Type-I collagen α 1 (COL1A1), ALP and miR-34a in the femoral head were determined by qRT-PCR and Western blot. Puerarin attenuated the effect of SONFH on promoting histopathological abnormalities and counteracted SONFH inhibition on the expressions of ALP, RUNX2, COL1A1 and miR-34a in the rabbits. Rabbit osteoblasts were successfully isolated, as they showed red mineralized nodules. Dexamethasone exposure decreased osteoblast viability, which was increased by puerarin treatment. Furthermore, puerarin treatment attenuated dexamethasone-induced inhibition on the viability, osteoblastic differentiation, and the expressions of ALP, RUNX2, COL1A1 and miR-34a in the osteoblasts. Puerarin facilitated osteogenesis of steroid-induced necrosis of rabbit femoral head and osteogenesis of steroid-induced osteocytes via miR-34a upregulation.

Evaluation of Acellular Bovine Cancellous Bone Matrix Seeded with Foetal Rabbit Osteoblasts for Segmental Bone Defects in Rabbits

The present work was assessed the restoration of segmental bone gap defect in rabbits by application of composite bone grafts. Composite bone grafts were prepared by seeding of rabbit foetal osteoblast on the bovine acellular cancellous bone matrix and evaluated in the segmental gap defect of 20 mm in rabbits. Thirty six adult New Zealand White rabbits of either sex were allotted to three groups of 12 each. Autograft (group A), Acellular cancellous bone matrix (group B) and composite graft seeded with fetal osteoblasts (group C) were implanted in the defects. Radiography, gross observations and histopathology at different intervals were done to evaluate healing. It was concluded that the seeded foetal osteoblasts in the composite grafts augment regeneration of the new bone leading to better integration of graft in host in comparison to bovine acellular cancellous bone matrix graft.

Osteoblast studied on gelatin based biomaterials in rabbit Bone Bioengineering.

The bone-derived-osteoblast seeded biomaterials scaffold in tissue engineering, have displayed prominence in the treatment of the osseous medical condition. In vitro osteogenesis of rabbit osteoblast cell (rOb) from bone tissue (rT) and MSC-derived rOb from bone marrow (rM) on Gelatin-Hydroxyapatite (HG) based biomaterials was investigated. In this work, lyophilized biomaterial was prepared by the addition of amorphous chitosan ('C') to 'H' dispersed in 'G' matrix, to find its role in biomaterials biocompatibility. Isolated rOb seeded biomaterials were studied using CLSM and flow cytometry for proliferation potential. The biomaterial's core and surface morphology was studied from SEM-EDX and AFM respectively. Upon co-culture with HCG, rT over rM showed rabbit bone extracellular matrix (ECM) mimicking properties both in in vitro studies and biomaterials micro architecture. The in vitro metabolic behaviour was studied by Alamar Blue (AB) assay, DNA content using Hoechst 33258, potency via the activity of Alkaline Phosphatase (ALP), Calcium's relative content by Alizarin Red S (ARS) assay. A novel combination of biomaterials-cell interaction was observed when rT was co-cultured with HCG and proved effective in osteogenesis with regard to Bone Bioengineering.

Biocompatibility and Osteogenic Capacity of Mg-Zn-Ca Bulk Metallic Glass for Rabbit Tendon-Bone Interference Fixation.

Mg-based alloys have great potential for development into fixation implants because of their highly biocompatible and biodegradable metallic properties. In this study, we sought to determine the biocompatibility of Mg60Zn35Ca5 bulk metallic glass composite (BMGC) with fabricated implants in a rabbit tendon–bone interference fixation model. We investigated the cellular cytotoxicity of Mg60Zn35Ca5 BMGC toward rabbit osteoblasts and compared it with conventional titanium alloy (Ti6Al4V) and polylactic acid (PLA). The results show that Mg60Zn35Ca5 BMGC may be classed as slightly toxic on the basis of the standard ISO 10993-5. We further characterized the osteogenic effect of the Mg60Zn35Ca5 BMGC extraction medium on rabbit osteoblasts by quantifying extracellular calcium and mineral deposition, as well as cellular alkaline phosphatase activity. The results of these tests were found to be promising. The chemotactic effect of the Mg60Zn35Ca5 BMGC extraction medium on rabbit osteoblasts was demonstrated through a transwell migration assay. For the in vivo section of this study, a rabbit tendon–bone interference fixation model was established to determine the biocompatibility and osteogenic potential of Mg60Zn35Ca5 BMGC in a created bony tunnel for a period of up to 24 weeks. The results show that Mg60Zn35Ca5 BMGC induced considerable new bone formation at the implant site in comparison with conventional titanium alloy after 24 weeks of implantation. In conclusion, this study revealed that Mg60Zn35Ca5 BMGC demonstrated adequate biocompatibility and exhibited significant osteogenic potential both in vitro and in vivo. These advantages may be clinically beneficial to the development of Mg60Zn35Ca5 BMGC implants for future applications.

Ophiopogonin D improves osteointegration of titanium alloy implants under diabetic conditions by inhibition of ROS overproduction via Wnt/β-catenin signaling pathway

A high failure rate of titanium implants in diabetic patients has been indicated in clinical evidences. Excessive oxidative stress at the bone-implant interface plays an important role in the impaired osteointegration under diabetic conditions. While the underlying mechanisms remain unknown and the targeted treatments are urgently needed. Ophiopogonin D (OP-D), isolated from Chinese herbal Radix Ophiopogon japonicus, is generally reported to be a potent antioxidant agent. In the present study, we hypothesized that OP-D exerted promotive effects on osteointegration against oxidative stress, and investigated the underlying mechanisms associated with alteration of Wnt/β-catenin signaling pathway. Rabbit osteoblasts incubated on titanium alloy implant were co-cultured with normal serum (NS), diabetic serum (DS), DS + OP-D, DS + NAC (a potent ROS inhibitor) and DS + OP-D + Dkk1 (a Wnt inhibitor) for examinations of osteoblast behaviors. For in vivo study, titanium alloy implants were implanted into the femoral condyle defects on diabetic rabbits. Results demonstrated that diabetes-induced oxidative stress resulted in osteoblast dysfunctions and apoptotic injury at the bone-implant interface, concomitant with the inactivation of Wnt/β-catenin signaling. Importantly, OP-D administration attenuated oxidative stress, directly reactivating Wnt/β-catenin signaling. Osteoblast dysfunctions were thus reversed as evidenced by improved osteoblast adhesion, proliferation and differentiation, and ameliorated apoptotic injury, exerting similar effects to NAC treatment. In addition, the positive effects afforded by OP-D were confirmed by improved osteointegration and oetogenesis within the titanium alloy implants in vivo by Micro-CT and histological analyses. Furthermore, the pro-osteogenic effects of OP-D were almost completely abolished by the Wnt inhibitor Dkk1. These results demonstrated, for the first time, OP-D administration alleviated the damaged osteointegration of titanium alloy implants under diabetic conditions by means of inhibiting oxidative stress via a Wnt/β-catenin-dependent mechanism. The OP-D administration would become a reliable treatment strategy for implant failure therapy in diabetics due to the optimal anti-oxidative and pro-osteogenic properties.

Effects of different Ca2+ level on fluoride-induced apoptosis pathway of endoplasmic reticulum in the rabbit osteoblast in vitro

In reviewing the literature, the cellular mechanism of fluoride F-induced osteoblast OB cells apoptosis is diverse and perplexing, but detailed regulatory pathway, targets and role of extracellular Ca2+ remains still unclear. Hence, in the present study, we investigated the effects of F (9 mg/L F ion) and different Ca2+ (0.5, 1, 2, 4, 8 mmol/L) levels treatment on the proliferation rate of osteoblast cells, intracellular free Ca2+ ([Ca2+]i) and endoplasmic reticulum (ER) stress apoptosis pathway related gene levels of rabbit OB cells. Our results demonstrated that F exposure had a pronounced negative effect on osteoblast survival, further different Ca2+ levels treatment suggested that low concentration of Ca2+ (0.5–1 mmol/L) relieved the damaged effect, on the contrary, high concentration of Ca2+ (2–8 mmol/L) enhanced the effect. In addition, F significantly increased [Ca2+]i levels and the expression of ER stress-induced cell apoptosis pathway related genes. Treatment with 0.5–1 mmol/L Ca2+ markedly reversed the F-induced harmful effects, but high dose Ca2+ (2–8 mmol/L) enhanced these effects. In summary, 0.5–1 mmol/L Ca2+ can alleviate F-induced OB cells injure through ER stress apoptosis pathway, which provided a dose basis for the future study on the treatment of skeletal fluorosis with Ca2+.

Promotion of osteointegration under diabetic conditions by tantalum coating-based surface modification on 3-dimensional printed porous titanium implants

Clinical evidence indicates a high failure rate for titanium implants (TiI) in diabetic patients, involving the overproduction of reactive oxygen species (ROS) at the implant/bone interface. Tantalum coating on titanium (TaTi) has exerted better tissue integration properties than TiI, but its biological performance under diabetic conditions remains elusive. To investigate whether TaTi may ameliorate diabetes-induced implant destabilization and the underlying mechanisms, primary rabbit osteoblasts cultured on 3-dimensional printed TiI and TaTi were exposed to normal serum (NS), diabetic serum (DS), DS+NAC (a potent ROS inhibitor), and DS+SB203580 (a specific p38 MAPK inhibitor). An in vivo study was performed on diabetic sheep implanted with TiI or TaTi. Diabetes induced mitochondrial-derived ROS overproduction and caused cellular dysfunction and apoptosis, together with the activation of p38 MAPK in osteoblasts on TiI surface. Importantly, TaTi significantly attenuated ROS production and p38 MAPK phosphorylation and exerted more osseointegrative cell behavior than TiI, as shown by improved osteoblast adhesion, increased cell proliferation and differentiation and decreased apoptosis. These results were confirmed in vivo by the enhanced bone healing efficacy of TaTi. Moreover, treatment with NAC or SB203580 on TiI not only inhibited the activation of p38 MAPK but also improved cell function and alleviated apoptotic injury, whereas TaTi combined with NAC or SB203580 failed to further improve osteoblast functional recovery compared with TaTi alone. These results demonstrated that the tantalum coating markedly improved diabetes-induced impaired osteogenesis of TiI, which may be attributed to the suppression of the ROS-mediated p38 MAPK pathway.

Implants of composite scaffolds of PLA/HA coated with polypyrrole by plasma for bone neo-tissue in rabbit

Event Abstract Back to Event Implants of composite scaffolds of PLA/HA coated with polypyrrole by plasma for bone neo-tissue in rabbit Maria Flores1, Roberto Olayo2, Juan Morales2, Atlántida M. Raya3 and Diego R. Esquiliano3 1 Universidad Autonoma Metropolitana, Biomedical Engineering, Mexico 2 Universidad Autonoma Metropolitana, Physics, Mexico 3 Child Hospital of México Federico Gómez, Tissue Engineering, Mexico Introduction: Composites of hydroxyapatite (HA) and biodegradable polymers had been tasted as scaffolds for bone implants with several problems to solve, two of this problems are the cell adhesion and morphology, pore size and accessibility of the HA are important and than the cell interaction with bought materials must be good[1]-[3]. In this work we study a scaffold made by electrospinning from a suspension of HA in polylactic acid (PLA) solutions, to made a material with large porosity and a large surface area. We increased the surface cell interaction by plasma polymerization of pyrrole[4],[5]. Scaffolds were tested with rabbit osteoblasts in vitro and in vivo, showing excellent bone neo-tissue formation. Materials and Methods: Electrospinning, was used to manufacture porous matrices. Solutions of polylactic acid and two different concentrations of hydroxyapatite, 18wt% and 36wt% were electrospunned. Samples were characterized by scanning electron microscopy (SEM) and probed as a support for cell multiplication of mesenchymal stem cells (MSC´s) differentiated into osteoblasts. Cells were obtained by a biopsy of rabbit’s femur marrow. The differentiation evaluation of MSC´s was performed using antibodies and flow cytometry, also by observing alkaline phosphatase in cells. To further induce cell adhesion and stimulation, a thin layer of polypyrrole doped with iodine (PPy-I), by plasma polymerization, was deposited in the matrices. Samples of PLA/HA/PPy-I underwent in vitro cell culture. Culture was maintained in vitro for 7, 14, 21, and 28 days. At day 7, cells were implanted in vivo, in the back of a rabbit (subcutaneous) for 30 days. Their cell viability was characterized by MTT cell proliferation assay in both cultures. Results: Fibrous porous matrices were obtained of PLA/HA and PLA/HA/PPy-I fiber diameters varied from 800 nm up to 50 µm and pore size from 10 µm to 100 µm which allows passage of osteoblasts and culture medium within the matrices, as the diameter of osteoblasts is 10 μm. The differentiation of mesenchymal stem cells into osteogenic lineage was confirmed. Matrices showed good cell adhesion and proliferation in vitro and in vivo culture. Discussion: In the matrices of interest with osteoblasts at day 7 of culture, we can see the beginning of the adhesion of these bone cells. Figure 1. a) SEM of scaffolds at 7 day of in vitro culture of osteoblasts. a) PLA/HA(18 wt%), b) PLA/HA(18 wt%)/PPy-I, c) PLA/HA(36 wt%) , d) PLA/HA(36 wt%)/PPy-I Such cells tend to be in the hydroxyapatite rich areas, adherence is enhanced in the matrices with PPy-I. Also, samples were compared at 7, 14, 21, and 28 days, and, in this case MTT assay showed that samples with PPy-I and 36wt% of hydroxyapatite presented higher cell viability. Figure 2. Results of MTT assay When implanted in vivo samples were implanted for 30 days and there was no rejection of the material by the animal, there was vascularization and tissue formation in the matrix, indicating that the material effectively allowed the adhesion and proliferation of cells. Conclusion: This study confirms that the technique of electrospinning produces porous matrices of PLA/HA. Neotissue is achieved using PLA/HA and PLA/HA/Ppy-I. Better viability was observed when Ppy-I was added to the scaffolds. Increasing the concentration of HA further improved cell viability. The authors thank the support to CONACYT (Instituto Nacional de Ciencia y Tecnología) for the scholarship.

Insulin improves osteogenesis of titanium implants under diabetic conditions by inhibiting reactive oxygen species overproduction via the PI3K-Akt pathway.

Clinical evidence indicates that insulin therapy improves implant survival rates in diabetic patients; however, the mechanisms responsible for this effect are unknown. Here, we test if insulin exerts anti-oxidative effects, thereby improving diabetes-associated impaired osteoblast behavior on titanium implants. To test this hypothesis, we cultured primary rabbit osteoblasts in the presence of titanium implants and studied the impact of treatment with normal serum (NS), diabetic serum (DS), DS + insulin, DS + tempol (a superoxide dismutase mimetic), DS + insulin + tempol, and DS + insulin + wortmannin. We analyzed cell function, apoptosis, and reactive oxygen species (ROS) production in osteoblasts following the various treatments. Treatment with DS induced osteoblast dysfunction, evidenced by impaired cell attachment and morphology, decreased cell proliferation and ALP activity, and decreased expression of osteogenesis-related genes. We also observed a significant increase in apoptosis. Importantly, treatment with DS resulted in increased production of ROS in osteoblasts. In contrast, treatment with insulin inhibited ROS production, alleviated cell dysfunction, and decreased apoptosis of osteoblasts on the implants. Scavenging ROS with tempol also attenuated cell dysfunction. Compared to insulin treatment alone, the combination of insulin and tempol failed to further improve osteoblast functional recovery. Moreover, the anti-oxidative and pro-osteogenic effects afforded by insulin were almost completely abolished by the phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin. These results demonstrate, for the first time, that insulin treatment alleviates the impaired osteogenesis of titanium implants under diabetic conditions by inhibiting ROS overproduction via a PI3K/Akt-dependent mechanism. Both the anti-oxidative and metabolic properties of insulin should make it a viable therapeutic option to combat diabetic implant failure.

Exploration of the effect of mmu-miR-142-5p on osteoblast and the mechanism.

The objective of this study is to explore the effect of mmu-miR-142-5p on osteoblast and the mechanism. Neonatal New Zealand rabbits were selected to collect and culture primary osteoblasts after being killed. The agonists agomiR-142-5p, agomiR-NC and inhibitors antagomiR-142-5p, antagomiR-NC were transfected to establish mmu-miR-142-5p osteoblast model. 48 h after transfection, total RNA was extracted, ELISA was used to detect ALP level, Western Blot was used to detect HSP27 level, and RT-PCR was used to detect levels of RunX2 and OC. Rabbit osteoblasts showed triangle shape and grew adhering to the wall, cytoplasm extended and protruded. As the culture time prolonged, the cell volume was increased, and the amount showed exponential growth. After transfection, the abundance was significantly increased in agonist group and significantly decreased in inhibitor group, indicating that exogenous agonist can successfully up-regulate mmu-miR-142-5p level and exogenous inhibitor can down-regulate mmu-miR-142-5p level in osteoblast. Compared with control group, ALP level was significantly increased in agonist group and decreased in inhibitor group. Transfection of exogenous agonist can up-regulate HSP27 level in osteoblast, and transfection of exogenous inhibitor can decrease HSP27 level in osteoblast; however, there was no effect on HSP27 mRNA level. RunX2 mRNA and OC mRNA were significantly increased in agonist group and decreased in inhibitor group. Transfection of exogenous nucleic acid agomiR-142-5p can up-regulate the abundance and activity of mmu-miR-142-5p, and transfection of antagomiR-142-5p can down-regulate the abundance and activity of mmu-miR-142-5p, indicating that mmu-miR-142-5p can effectively helps the mineralization of osteoblast.

Effect of concentrations of magnesium ions on behavior of fibroblasts and osteoblasts

Objective To find out the safe and optimal concentrations of magnesium ions for the growth of fibroblasts and osteoblasts,with the intention of using the data to control the degradation rate of medical magnesium implants.Methods Tendon and skull cells were isolated from adult New Zealand rabbits for primary culture.After subculture and identification,rabbit fibroblasts were prepared for culture in the medium containing magnesium ions at concentrations of 0,20,50,70,100,110,120,130,140 and 150 mmol/L respectively while rabbit osteoblasts in the medium containing magnesium ions at concentrations of 0,30,40,50,60,70,80,90,100,110 and 120 mmol/L respectively.Growth of the 2 kinds of cells in different concentrations of magnesium ions was observed and compared.Results The fibroblasts grew well in a range of concentrations from 0 mmol/L to 110 mmol/L.The cell viability was the highest at the concentration of 70 rmnol/L and significantly higher than those at other concentrations (P ＜ 0.05).The osteoblasts grew well in a range of concentrations from 0 mmol/L to 70 mmol/L.The cell viability was the highest at the concentration of 30 mmol/L and significantly higher than those at other concentrations (P ＜0.05).Conclusions For rabbit fibroblasts the safe range of concentrations of magnesium ions is from 0 mmol/L to 110 mmol/L and the optimal concentration 70 mmol/L.For rabbit osteoblasts the safe range of concentrations of magnesium ions is from 0 mmol/L to 70 mmol/L and the optimal concentration 30 mmol/L.These data provide evidence for better controlling the in vivo degradation rate of medical magnesium implants,for we can regulate the growth of osteoblasts and fibroblasts through regulating the concentrations of magnesium ions. Key words: Magnesium; Fibroblasts; Osteoblasts

Effect of reactive oxygen species overproduction on osteogenesis of porous titanium implant in the present of diabetes mellitus

Clinical evidence indicates diabetes as a majorrisk factor for titaniumimplant treatment with high failure rates and poor osteointegration, but the underlying mechanism involved remains elusive.We hypothesize that reactive oxygen species (ROS) overproduction may contribute to the impaired osteogenesis of porous titanium implants (pTi) under diabetic conditions. To test this hypothesis, we culturedprimary rabbit osteoblasts onto pTi and studied the cellular performance when subjected to normal serum (NS), diabetic serum (DS), DS + NAC (a potent ROS inhibitor) and NS + H2O2(an oxidant).In-vivo performance of pTi was investigated by transplanting them intofemoral condyledefects of diabetic rabbits, which received vehicle or NAC treatment respectively.Results showed that diabetic conditions induced significant cellular apoptosis, depressedosteoblast function evidenced by impairedcell attachment and morphology, decreased cell proliferation anddifferentiation, andcompromised in-vivo osteogenesis ofpTi, while cellular ROSgeneration was increased derived from mitochondrial dysfunction. Scavenging ROS with NAC markedly attenuated cell apoptosis and osteoblast dysfunction, and improved bone ingrowth within pTi. Furthermore, treatment withH2O2 exerted similar adverse effect on cellular behavior as diabetes. This study furthers our knowledge on the potential role of ROS overproduction in the diabetes-induced impaired osteogenesis of titanium implants, and indicates anti-oxidative treatment as a promising strategy to promote the treatment efficacy of pTi in diabetic patients.

Osteochondral Interface Generation by Rabbit Bone Marrow Stromal Cells and Osteoblasts Coculture

Physiological osteochondral interface regeneration is a significant challenge. This study aims to investigate the effect of the coculture of chondrogenic rabbit bone marrow stromal cells (rBMSCs) with rabbit osteoblasts in a specially designed two-dimensional (2D)-three-dimensional (3D) co-interface culture to develop the intermediate osteochondral region in vitro. The 2D-3D coculture system was set up by first independently culturing chondrogenic rBMSCs on a scaffold and osteoblasts in cell culture plates, and subsequently placed in contact and cocultured. As control, samples not cocultured with osteoblasts were used. The regulatory effects exerted by osteoblasts on chondrogenic rBMSCs were quantified by real-time polymerase chain reaction. To study the effect of coculture on cells located in different parts of the scaffold, samples were separated into two parts and significantly different gene expression patterns were found between them. In comparison with the control group, a significant moderate downregulation of chondrogenic marker genes, such as Collagen II and Aggrecan was observed. However, the Sox-9 and Collagen I expression increased. More importantly, chondrogenic rBMSCs in the coculture system were shown to form the osteochondral interface layer by expressing calcified cartilage zone specific extracellular matrix marker Collagen X and the hypertrophic chondrocyte marker MMP-13, which were not observed in the control group. Specifically, only the chondrogenic rBMSC layer in contact with the osteoblasts expressed Collagen X and MMP-13, indicating the positive influence of the coculture upon interface formation. Biochemical analyses, histology results, and immunohistochemical staining further supported this observation. In conclusion, this study revealed that specific regulatory stimulations from osteoblasts in the 2D-3D interface coculture system could induce the formation of ostochondral interface for the purpose of osteochondral tissue engineering.

In search of an osteoblast cell model for in vitro research

The process of bone formation, remodelling and healing involves a coordinated action of various cell types. Advances in understanding the biology of osteoblast cells during these processes have been enabled through the use of various in vitro culture models from different origins. In an era of intensive bone tissue engineering research, these cell models are more and more often applied due to limited availability of primary human osteoblast cells. While they are a helpful tool in developing novel therapies or biomaterials; concerns arise regarding their phenotypic state and differences in relation to primary human osteoblast cells. In this review we discuss the osteoblastic development of some of the available cell models; such as primary human, rat, mouse, bovine, ovine and rabbit osteoblast cells; as well as MC3T3-E1, MG-63 and SaOs-2 cell lines, together with their advantages and disadvantages. Through this, we provide suggestions on the selection of the appropriate and most relevant osteoblast model for in vitro studies, with specific emphasis on cell-material based studies.

Influence of nicotine on the biological activity of rabbit osteoblasts

To assess the influence of nicotine on the proliferation and gene expression of osteogenic and angiogenic mediators of osteoblasts. Rabbit primary osteoblasts were exposed to various concentrations of nicotine (0.001, 0.1 and 10 μmol/l). The cell proliferation was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. The gene expression of transforming growth factor (TGF)-β(1), bone morphogenetic protein (BMP)-2, platelet-derived growth factor (PDGF)-AA and vascular endothelial growth factor (VEGF) was evaluated using real-time reverse transcription - polymerase chain reaction. The osteoblast proliferation was inhibited by nicotine at the concentration of 0.001-10 μM at 48 and 72 h of culture, but with no significant effect at 24 h. The expression of TGF-β(1), BMP-2, PDGF-AA and VEGF was inhibited by nicotine at the concentrations of 0.1 and 10 μM, but with no significant difference at the low concentration of 0.001 μM. Nicotine suppresses osteoblast proliferation and inhibits the expression of some key osteogenic and angiogenic mediators in the in vitro experimental model. These inhibitory effects of nicotine on the osteoblast activity may reflect, to a certain degree, the overall detrimental effects of tobacco use on the survival rate of dental implants.

Titanium alloy scaffold with controlled internal architecture as an osteoblast carrier to repair rabbit defects

Objective To evaluate effects of titanium alloy scaffolds with controlled internal architecture as an osteoblast carrier on bone response in models of rabbit defects. Methods Electron beam melting process was utilized to fabricate porous titanium alloy scaffolds with fully interconnected and controlled internal pore architecture. After osteoblasts were seeded on the scaffolds and cultured for up to 7 days, the growth of rabbit osteoblasts on the scaffolds was observed by scanning electron microscopy. The experiment was conducted in 4 groups to evaluate the bone formation in vivo: group A (cell/scaffold composite), group B (scaffold only), group C (left empty) and group D (autogenous bone implant) . The scaffolds were transplanted into the rabbit defects after cultured in vitro for 7 days. The animals were sacrificed at 4, 8, and 12 weeks after implantation. Bone formation in the scaffolds was investigated by gross observation, histology and histomorphometry of non-decalcified sections and fluorochrome markers. Results Confluent cell layers could be observed on the scaffold surface and in the internal pores after 7 days of incubation in vitro. New bone growth and revascularization could be observed not only at the margins of the scaffolds, but also inside the central pores of the scaffolds after 12 weeks. New bone formed along the controlled internal channels of the scaffolds. The scaffolds were filled fully with the new bone tissue and blood vessels. More extensive new bone formation was found to originate from the host bone towards the implant in group A than in group B (P ＜0. 05) . Conclusions The controlled scaffolds are well biocompatible enough to accelerate healing of rabbit defects and new bone formation. The controlled honeycomb-like architecture may guide and promote the formation of mineralized tissue. Key words: Scaffold; Titanium; Bone defect; Electron beam melting

Qtracker-labeled rabbit osteoblasts in vitro

Objective To explore the feasibility of labeling in vitro rabbit osteoblasts with Qtracker and the features of Qtracker-labeled rabbit osteoblasts. Methods A healthy male rabbit, 3 months old, weighing 2 kg, was used in this study. After bone marrow was aspirated, bone marrow stromal cells (BMSCs) were isolated and cultured using the adherence method in vitro. The third passage of BMSCs was induced into osteablasts before incubation with Qtracker at concentrations of 1, 2, 4, 8, 16, 32 nmol/10~6 cells (Groups A, B, C, D, E, F respectively). Cells not labeled by Qtracker served as negative control (Group G). The following parameters were measured: induction, differentiation and determination of rabbit osteoblasts; the optimal mass concentration of Qtracker labeling by fluorescence microscopy and flow cytometry; the cell sur-vival rates at various concentrations of Qtraeker labeling by trypan-blue exclusion; Qtracker-labeled cell pro-liferation by MTr. Results The primary and the passage rabbit BMSCs were chiefly of fusiform shape. Rabbit BMSCs differentiated into osteoblasts following induction. The osteoblasts cytoplasm showed green fluorescence under fluorescence microscopy after being labeled by Qtracker. The mean labeling rate increased with the increased concentration of Qtracker, reaching up to (93.58±2.08) % after incubation at 8 nmol/ 10~6 cells by fluorescence microscopy, and (95.24±1.31) % by flow cytometry. There were no significant differences between Groups D, E, F(P>0.05), but significant differences were found between Groups A, B, C and Groups D, E, F (P 0.05) . No significant differences were found in the cell proliferation among various con-centrations (P>0.05). Conclusions Qtraeker can be used as a labeling marker for rabbit osteoblasts. When the concentration is at 8 nmol/10~6 cells, optimal labeling effect can be achieved. Rabbit osteoblasts labeled with Qtracker are of high efficiency and safety. Key words: Bone marrow cells; Osteoblasts; Biological markers