Cultured Rat Bone Marrow Research Articles

Ti3C2Tx@PLGA/Icaritin microspheres-modified PLGA/β-TCP scaffolds modulate Icaritin release to enhance bone regeneration through near-infrared response

Porous poly (lactic-co-glycolic acid)/β-tricalcium phosphate/Icaritin (PLGA/β-TCP/ICT, PTI) scaffold is a tissue engineering scaffold based on PLGA/β-TCP (PT) containing Icaritin, the main active ingredient of the Chinese medicine Epimedium. Due to its excellent mechanical properties and osteogenic effect, PTI scaffold has the potential to promote bone defect repair. However, the release of ICT from the scaffolds is difficult to control. In this study, we constructed Ti3C2Tx@PLGA/ICT microspheres (TIM) and evaluated their characterization as well as ICT release under near-infrared (NIR) irradiation. We utilized TIM to modify the PT scaffold and performed biological experiments. First, we cultured rat bone marrow mesenchymal stem cells on the scaffold to assess biocompatibility and osteogenic potential under on-demand NIR irradiation. Subsequently, to evaluate the osteogenic properties of TIM-modified scaffoldin vivo, the scaffold was implanted into a femoral condyle defect model. TIM have excellent drug-loading capacity and encapsulation efficiency for ICT, and the incorporation of Ti3C2Txendows TIM with photothermal conversion capability. Under 0.90 W cm-2NIR irradiation, the temperature of TIM maintained at 42.0 ± 0.5 °C and the release of ICT was accelerated. Furthermore, while retaining its original properties, the TIM-modified scaffold was biocompatible and could promote cell proliferation, osteogenic differentiation, and biomineralizationin vitro, as well as the osteogenesis and osseointegrationin vivo, and its effect was further enhanced through the modulation of ICT release under NIR irradiation. In summary, TIM-modified scaffold has the potential to be applied in bone defects repairing.

In-Vitro Degradation Behaviors and Biocompatibility of Sodium Alginate/Platelet-Rich Plasma-Sr2+ Porous Microcarriers

Porous microcarriers as cell carriers have attracted extensive research interest in tissue engineering. In this work sodium alginate (SA) and sodium alginate/platelet-rich plasma (SA/PRP) porous microcarriers cross-linked by SrCl2 (SA-Sr2+ and SA/PRP-Sr2+) were prepared using an emulsion method combined with a freeze-drying method. The in-vitro degradation behaviors of the SA-Sr2+ and SA/PRP-Sr2+ porous microcarriers in phosphate-buffered saline (PBS) were investigated. The cell proliferation ability and osteogenic activity of the SA-Sr2+ and SA/PRP-Sr2+ porous microcarriers were investigated by culturing rat bone marrow mesenchymal stem cells (rBMSCs). During the degradation process, the degradation behaviors, including changes of the pH of the PBS and the weight loss and morphology, of both the SA-Sr2+ and SA/PRP-Sr2+ porous microcarriers showed a similar change. After 6 wk of degradation, parts of both the SA-Sr2+ and SA/PRP-Sr2+ porous microcarriers collapsed. The SA/PRP-Sr2+ porous microcarriers showed higher cell proliferation ability and osteoinductive ability than the SA-Sr2+ porous microcarriers during a culture time of 14 days.

Preparation and Properties of Sodium Alginate/Silk Fibroin Microcarriers

Injectable porous microcarriers, with excellent biocompatibility, biodegradability, high cell seeding density and simple implantable procedures in the body, have been widely used in bone tissue engineering. In this work sodium alginate/silk fibroin (SA/SF) porous microcarriers were prepared cross-linked by SrCl2 or CaCl2 using emulsion method combined with freeze-drying method. The morphology and structure of the microcarriers were characterized by scanning electron microscopy (SEM). The cell proliferation ability and osteogenic activity of the microcarriers were investigated by culturing rat bone marrow mesenchymal stem cells (rBMSCs). The SA/SF microcarriers showed an interconnective porous structure with an average particle size of 254.3 ± 50.0 μm and an average pore size of 42.0 ± 5.3 μm. The SA/SF microcarriers cross-linked with SrCl2 showed higher cell proliferation ability and osteoinductive ability than those cross-linked with CaCl2. These features indicated that the designed SA/SF microcarriers are ideal for tissue engineering.

Promoting osseointegration of titanium implants through magnesium- and strontium-doped hierarchically structured coating

This study aimed to investigate the effect of magnesium (Mg) and strontium (Sr) surface modification on osseointegration of titanium (Ti) implants with a hierarchically structured coating. Mg- and Sr-doped hierarchical surface coating has been fabricated on Ti implant through the hydrothermal method. Scanning electron microscopy (SEM), atomic force microscopy (AFM), energy dispersive spectrometer (EDS), inductively coupled plasma/optical emission spectroscopy (ICP-OES) and contact-angle measurement have been applied to characterize the surface coating. The biocompatibility and osseointegration of Ti implants with Mg- and Sr-dropped hierarchical coating have been assessed in vivo using a rat tibia implantation model. The proposed mechanism that improves osteointegration of Mg- and Sr-doped coating Ti implant has been evaluated by culturing rat bone marrow stromal cells (BMSCs) alone or co-culturing Schwann cells (SCs) and BMSCs on the coating surfaces. Our results proved that both Mg- and Sr-doped hierarchical coating could promote osseointegration with Mg exerted a stronger effect in vivo. The in vitro outcomes showed that Mg-doped coating enhances BMSCs adhesion, while the Sr-doped surface promotes osteogenic differentiation of BMSCs. Furthermore, when BMSCs were cultured with SCs, Mg-dropped surfaces accelerated not only the adhesion but also osteogenic differentiation of BMSCs. Conclusively, the hierarchical surface coating with trace element modification can provide a strong guarantee for endosseous applications of titanium (Ti) implants, and Mg-doped hierarchical coating is more effective in promoting osseointegration than Sr modification.

Influence of Culture Period on Osteoblast Differentiation of Tissue-Engineered Bone Constructed by Apatite-Fiber Scaffolds Using Radial-Flow Bioreactor.

With the limitation of autografts, the development of alternative treatments for bone diseases to alleviate autograft-related complications is highly demanded. In this study, a tissue-engineered bone was formed by culturing rat bone marrow cells (RBMCs) onto porous apatite-fiber scaffolds (AFSs) with three-dimensional (3D) interconnected pores using a radial-flow bioreactor (RFB). Using the optimized flow rate, the effect of different culturing periods on the development of tissue-engineered bone was investigated. The 3D cell culture using RFB was performed for 0, 1 or 2 weeks in a standard medium followed by 0, 1 or 2 weeks in a differentiation medium. Osteoblast differentiation in the tissue-engineered bone was examined by alkaline phosphatase (ALP) and osteocalcin (OC) assays. Furthermore, the tissue-engineered bone was histologically examined by hematoxylin and eosin and alizarin red S stains. We found that the ALP activity and OC content of calcified cells tended to increase with the culture period, and the differentiation of tissue-engineered bone could be controlled by varying the culture period. In addition, the employment of RFB and AFSs provided a favorable 3D environment for cell growth and differentiation. Overall, these results provide valuable insights into the design of tissue-engineered bone for clinical applications.

Regional gene therapy for bone healing using a 3D printed scaffold in a rat femoral defect model.

At the present time there are no consistently satisfactory treatment options for some challenging bone loss scenarios. We have previously reported on the properties of a novel 3D-printed hydroxyapatite-composite material in a pilot study, which demonstrated osteoconductive properties but was not tested in a rigorous, clinically relevant model. We therefore utilized a rat critical-sized femoral defect model with a scaffold designed to match the dimensions of the bone defect. The scaffolds were implanted in the bone defect after being loaded with cultured rat bone marrow cells (rBMC) transduced with a lentiviral vector carrying the cDNA for BMP-2. This experimental group was compared against 3 negative and positive control groups. The experimental group and positive control group loaded with rhBMP-2 demonstrated statistically equivalent radiographic and histologic healing of the defect site (p > 0.9), and significantly superior to all three negative control groups (p < 0.01). However, the healed defects remained biomechanically inferior to the unoperated, contralateral femurs (p < 0.01). When combined with osteoinductive signals, the scaffolds facilitate new bone formation in the defect. However, the scaffold alone was not sufficient to promote adequate healing, suggesting that it is not substantially osteoinductive as currently structured. The combination of gene therapy with 3D-printed scaffolds is quite promising, but additional work is required to optimize scaffold geometry, cell dosage and delivery.

A new method of culturing rat bone marrow endothelial progenitor cells in vitro.

Endothelial progenitor cells (EPCs) play an important role in the re-endothelialization of ischemic cerebrovascular disease. However, the current acquisition method has some deficiencies. This study aimed to design a new and practical method for obtaining EPCs. Bone marrow was obtained autologously from the right tibia of living rats. Briefly, the right tibia bone was carefully exposed and two holes (1 mm in diameter) were made in the tuberosity and lower one-third of the tibia, respectively. A PE-50 catheter and syringe (5 mL) were inserted through the holes to aspirate the bone marrow. Bone marrow mononuclear cells (BMMCs) were isolated by density-gradient centrifugation with Ficoll and counted. Adherent cell culture continued for 2 weeks, and the medium was replaced every 3 days. During the first days of culture, adherent cells formed a monolayer, consisting predominantly of small-sized cells. Single large cells with endothelial morphology were observed. On day 4, the nonadherent cells were removed, and the adherent cells were left for further culture. On day 6-7, a proliferating population of round cells formed clusters in the culture chamber, and morphological analysis revealed a homogeneous population of colony-forming units (CFUs). Large, flat cells with endothelial morphology sprouted from the CFUs, which had nearly disappeared by day 14 of culture. The adherent cells were positive for CD133 and vascular endothelial growth factor receptor 2 (VEGFR2), internalized acetylated low-density lipoprotein, and bound ulex europaeus-agglutinin-I, but were negative for CD45, which correlated with the endothelial morphology and ability to form capillaries of EPCs. Our results are direct evidence that mononuclear cells (MCS) from living rat bone marrow can be used to culture EPCs in vitro under certain culture conditions, providing a new method for the further study of autologous EPC transplantation.

Icariin Accelerates Fracture Healing via Activation of the WNT1/β-catenin Osteogenic Signaling Pathway.

Icariin has been shown to enhance bone formation. The present study aimed to investigate whether icariin also promotes bone fracture healing and its mechanisms. First, we isolated and cultured rat bone marrow stromal cells (rBMSCs) with icariincontaining serum at various concentrations (0%, 2.5%, 5% and 10%) and then measured alkaline phosphatase (ALP) activity and the expression of Core-binding factor, alpha 1 (Cbfα1), bone morphogenetic protein-2 (BMP-2) and bone morphogenetic protein-4 (BMP-4) in the rBMSCs. Second, we established a model of fracture healing in rats and performed gavage treatment for 20 days. Then, we detected bone biochemical markers (ELISA kits) in the serum, fracture healing (digital radiography, DR), and osteocalcin expression (immunohistochemistry). Icariin treatment increased ALP activity and induced the expression of Cbfα1, BMP-2 and BMP-4 in rBMSCs in a dose-dependent manner. In addition, Icariin increased the serum levels of osteocalcin (OC), bone-specific alkaline phosphatase (BAP), N-terminal telopeptides of type I collagen (NTX-1), C-terminal telopeptide of type I collagen (CTX-1) and tartrate-resistant acid phosphatase 5b (TRACP-5b); promoted osteocalcin secretion at the fracture site; and accelerated fracture healing. Icariin can promote the levels of bone-formation markers, accelerate fracture healing, and activate the WNT1/β-catenin osteogenic signaling pathway.

Development of Nontoxic Biodegradable Polyurethanes Based on Polyhydroxyalkanoate and L-lysine Diisocyanate with Improved Mechanical Properties as New Elastomers Scaffolds.

A nontoxic and biodegradable polyurethane was prepared, characterized, and evaluated for biomedical applications. Stretchable, biodegradable, and biocompatible polyurethanes (LPH) based on L-lysine diisocyanate (LDI) with poly(ethylene glycol) (PEG) and polyhydroxyalkanoates(PHA) of different molar ratios were synthesized. The chemical and physical characteristics of the LPH films are tunable, enabling the design of mechanically performance, hydrophilic, and biodegradable behavior. The LPH films have a Young’s modulus, tensile strength, and elongation at break in the range of 3.07–25.61 MPa, 1.01–9.49 MPa, and 102–998%, respectively. The LPH films demonstrate different responses to a change of temperature from 4 to 37 °C, with the swelling ratio for the same sample at equilibrium varying from 184% to 151%. In vitro degradation tests show the same LPH film has completely different degradation morphologies in pH of 3, 7.4, and 11 phosphate buffered solution (PBS). In vitro cell tests show feasibility that some of the LPH films are suitable for culturing rat bone marrow stem cells (rBMSCs), for future soft-tissue regeneration. The results demonstrate the feasibility of the LPH scaffolds for many biomedical applications.

Effect of Zinc Supplementation on Renal Anemia in 5/6-Nephrectomized Rats and a Comparison with Treatment with Recombinant Human Erythropoietin.

Anemia is a severe complication in patients with chronic kidney disease (CKD). Treatment with exogenous erythropoietin (EPO) can correct anemia in many with CKD. We produced 5/6-nephrectomized rats that became uremic and anemic at 25 days post surgery. Injection of the anemic 5/6-nephrectomized rats with 2.8 mg zinc/kg body weight raised their red blood cell (RBC) levels from approximately 85% of the control to 95% in one day and continued for 4 days. We compared the effect of ZnSO4 and recombinant human erythropoietin (rHuEPO) injections on relieving anemia in 5/6-nephrectomized rats. After three consecutive injections, both the ZnSO4 and rHuEPO groups had significantly higher RBC levels (98 ± 6% and 102 ± 6% of the control) than the saline group (90 ± 3% of the control). In vivo, zinc relieved anemia in 5/6-nephrectomized rats similar to rHuEPO. In vitro, we cultured rat bone marrow cells supplemented with ZnCl2, rHuEPO, or saline. In a 4-day suspension culture, we found that zinc induced erythropoiesis similar to rHuEPO. When rat bone marrow cells were supplement-cultured with zinc, we found that zinc stimulated the production of EPO in the culture medium and that the level of EPO produced was dependent on the concentration of zinc supplemented. The production of EPO via zinc supplementation was involved in the process of erythropoiesis.

Differentiation Potential and Tumorigenic Risk of Rat Bone Marrow Stem Cells Are Affected By Long-Term In Vitro Expansion

Objective:Mesenchymal stem cells (MSCs) have the capacity for extensive expansion and adipogenic, osteogenic, chondrogenic, myogenic, and neural differentiation in vitro. The aim of our study was to determine stemness, differentiation potential, telomerase activity, and ultrastructural characteristics of long-term cultured rat bone marrow (rBM)-MSCs.Materials and Methods:rBM-MSCs from passages 3, 50, and 100 (P3, P50, and P100) were evaluated through immunocytochemistry, reverse transcription-polymerase chain reaction, telomerase activity assays, and electron microscopy.Results:A dramatic reduction in the levels of myogenic markers actin and myogenin was detected in P100. Osteogenic markers Coll1, osteonectin (Sparc), and osteocalcin as well as neural marker c-Fos and chondrogenic marker Coll2 were significantly reduced in P100 compared to P3 and P50. Osteogenic marker bone morphogenic protein-2 (BMP2) and adipogenic marker peroxisome proliferator-activated receptor gamma (Pparγ) expression was reduced in late passages. The expression of stemness factor Rex-1 was lower in P100, whereas Oct4 expression was decreased in P50 compared to P3 and P100. Increased telomerase activity was observed in long-term cultured cells, signifying tumorigenic risk. Electron microscopic evaluations revealed ultrastructural changes such as smaller number of organelles and increased amount of autophagic vacuoles in the cytoplasm in long-term cultured rBM-MSCs.Conclusion:This study suggests that long-term culture of rBM-MSCs leads to changes in differentiation potential and increased tumorigenic risk.

Construction of a NT-3 sustained-release system cross-linked with an acellular spinal cord scaffold and its effects on differentiation of cultured bone marrow mesenchymal stem cells.

This study sought to promote the adhesion, proliferation and differentiation of rat bone marrow mesenchymal stem cells by constructing a neurotrophin-3 (NT-3) sustained-release system cross-linked with an acellular spinal cord scaffold. 1-Ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDC) chemistry combined with chemical extraction was used to construct an acellular spinal cord scaffold. The decellularization completion was validated. An EDC cross-linking method was used to construct the NT-3 cross-linked acellular spinal scaffold. ELISA was used to verify sustained release of NT-3; the dorsal root ganglion method was used to verify the biological activity of the sustained-release NT-3. DAPI staining was used to confirm the adhesion of the cultured rat bone marrow mesenchymal stem cells (P3) to the NT-3 scaffold, and cell counting kit-8 (CCK-8) analysis was used to verify the cellular proliferation after 24 h and 48 h of culture. Immunohistochemistry was used to confirm the differentiation of the bone marrow cells into neuron-like cells. An NT-3 sustained-release system cross-linked to an acellular spinal cord scaffold was successfully constructed. Sustained-release NT-3 could persist for 35 days and had biological activity for at least 21 days. It could promote the adhesion, proliferation and differentiation of rat bone marrow mesenchymal stem cells. As a composite scaffold, an NT-3 sustained-release system cross-linked with an acellular spinal cord scaffold has potential applications for tissue engineering.

Valence State Manipulation of Cerium Oxide Nanoparticles on a Titanium Surface for Modulating Cell Fate and Bone Formation.

Understanding cell–biomaterial interactions is critical for the control of cell fate for tissue engineering and regenerative medicine. Here, cerium oxide nanoparticles (CeONPs) are applied at different Ce4+/Ce3+ ratios (i.e., 0.46, 1.23, and 3.23) to titanium substrate surfaces by magnetron sputtering and vacuum annealing. Evaluation of the cytotoxicity of the modified surface to cultured rat bone marrow mesenchymal stem cells (BMSCs) reveals that the cytocompatibility and cell proliferation are proportional to the increases in Ce4+/Ce3+ ratio on titanium surface. The bone formation capability induced by these surface modified titanium alloys is evaluated by implanting various CeONP samples into the intramedullary cavity of rat femur for 8 weeks. New bone formation adjacent to the implant shows a close relationship to the surface Ce4+/Ce3+ ratio; higher Ce4+/Ce3+ ratio achieves better osseointegration. The mechanism of this in vivo outcome is explored by culturing rat BMSCs and RAW264.7 murine macrophages on CeONP samples for different durations. The improvement in osteogenic differentiation capability of BMSCs is directly proportional to the increased Ce4+/Ce3+ ratio on the titanium surface. Increases in the Ce4+/Ce3+ ratio also elevate the polarization of the M2 phenotype of RAW264.7 murine macrophages, particularly with respect to the healing‐associated M2 percentage and anti‐inflammatory cytokine secretion. The manipulation of valence states of CeONPs appears to provide an effective modulation of the osteogenic capability of stem cells and the M2 polarization of macrophages, resulting in favorable outcomes of new bone formation and osseointegration.

Histological evaluations of apatite-fiber scaffold cultured with mesenchymal stemcells by implantation at rat subcutaneoustissue.

There is a strong impetus for the development of alternative treatments for bone disease that avoid the complications associated with autografts and allografts. To address this, we previously developed porous apatite-fiber scaffolds (AFSs) which have three-dimensional interconnected pores, and constructed tissue-engineered bone by culturing rat bone marrow cells (RBMCs) using AFSs in a radial-flow bioreactor (RFB). To generate additional baseline data for the development of tissue-engineered bone constructed for clinical application using a RFB, we cultured RBMCs using AFSs under static conditions (hereafter, RBMC AFS culture), and monitored RBMC growth and differentiation characteristics in vitro, and two weeks after subcutaneous inoculation into recipient rats. RBMCs were seeded to AFSs and growth, differentiation and calcification were monitored in vitro and in vivo by histological evaluation using hematoxylin eosin, alkaline phosphatase and alizarin red S stains. RBMCs in/on AFSs proliferated and differentiated normally in vitro and in vivo, and calcification was evident two weeks after subcutaneous AFS culture implantation. Histological assays revealed that AFSs and RBMC AFS cultures were biocompatible, and did not induce inflammation or immunological rejection in vivo. These findings suggest that AFSs are a conducive microenvironment for bone regeneration and are well tolerated in vivo. The results provide valuable baseline data for the design of implant studies using tissue-engineered bone constructed by RFB.

Bone marrow mesenchymal stem cell‑derived extracellular vesicles improve the survival of transplanted fat grafts.

Autologous fat grafting is a promising surgical technique for soft tissue augmentation, reconstruction and rejuvenation. However, it is limited by the low survival rate of the transplanted fat, due to the slow revascularization of such grafts. Previous studies have demonstrated that bone marrow mesenchymal stem cell-derived extracellular vesicles (BMSC-EVs) are proangiogenic. The present study aimed to investigate whether BMSC-EVs could improve the survival of transplanted fat grafts. Extracellular vesicles were isolated from the supernatant of cultured rat bone marrow mesenchymal stem cells, and characterized by flow cytometry and scanning electron microscopy. Their proangiogenic potential was measured in vitro using tube formation and cell migration assays. Subsequently, human fat tissue grafts, alongside various concentrations of BMSC-EVs, were subcutaneously injected into nude mice. A total of 12 weeks following transplantation, the mice were sacrificed and the grafts were harvested. The grafts from the experimental group had a higher survival rate and an increased number of vessels compared with grafts from the control group, as demonstrated by tissue volume, weight and histological analyses. Reverse transcription-quantitative polymerase chain reaction analysis indicated that the expression levels of proangiogenic factors were increased in the experimental group compared with in the control group, thus suggesting that BMSC-EVs may promote neovascularization by stimulating the secretion of proangiogenic factors. The present study is the first, to the best of our knowledge, to demonstrate that supplementation of fat grafts with BMSC-EVs improves the long-term retention and quality of transplanted fat.

Spreading and proliferation of cultured rat bone marrow stromal cells on the surface of bioactive glass ceramics

Spreading and proliferation of cultured rat bone marrow stromal cells on the surface of bioactive glass ceramics

Culturing bone marrow cells with dexamethasone and ascorbic acid improves osteogenic cell sheet structure.

ObjectivesTo assess the structure and extracellular matrix molecule expression of osteogenic cell sheets created via culture in medium with both dexamethasone (Dex) and ascorbic acid phosphate (AscP) compared either Dex or AscP alone.MethodsOsteogenic cell sheets were prepared by culturing rat bone marrow stromal cells in a minimal essential medium (MEM), MEM with AscP, MEM with Dex, and MEM with Dex and AscP (Dex/AscP). The cell number and messenger (m)RNA expression were assessed in vitro, and the appearance of the cell sheets was observed after mechanical retrieval using a scraper. β-tricalcium phosphate (β-TCP) was then wrapped with the cell sheets from the four different groups and subcutaneously implanted into rats.ResultsAfter mechanical retrieval, the osteogenic cell sheets from the MEM, MEM with AscP, and MEM with Dex groups appeared to be fragmented or incomplete structures. The cell sheets cultured with Dex/AscP remained intact after mechanical retrieval, without any identifiable tears. Culture with Dex/AscP increased the mRNA and protein expression of extracellular matrix proteins and cell number compared with those of the other three groups. More bridging bone formation was observed after transplantation of the β-TCP scaffold wrapped with cell sheets cultured with Dex/AscP, than in the other groups.ConclusionsThese results suggest that culture with Dex/AscP improves the mechanical integrity of the osteogenic cell sheets, allowing retrieval of the confluent cells in a single cell sheet structure. This method may be beneficial when applied in cases of difficult tissue reconstruction, such as nonunion, bone defects, and osteonecrosis.Cite this article: M. Akahane, T. Shimizu, T. Kira, T. Onishi, Y. Uchihara, T. Imamura, Y. Tanaka. Culturing bone marrow cells with dexamethasone and ascorbic acid improves osteogenic cell sheet structure. Bone Joint Res 2016;5:569–576. DOI: 10.1302/2046-3758.511.BJR-2016-0013.R1.

The Regulation of Osteogenesis Using Electroactive Polypyrrole Films.

To evaluate the effect of electrical conductivity of biomaterials on osteogenesis, polypyrrole (PPy) was fabricated by oxidative chemical polymerization as substrates for cell culture. Through adjusting the concentrations of monomer and initiator, polypyrrole films with different electrical conductivities were fabricated. These fabricated polypyrrole films are transparent enough for easy optical microscopy. Fourier transform infrared spectroscopy, X-ray spectroscopy and four-point probe were used to assess the microstructures, surface chemical compositions and electrical sheet resistance of films, respectively. Results indicate that higher monomer and initiator concentration leads to highly-branched PPy chains and thus promotes the electron mobility and electrical conductivity. Selected polypyrrole films then were applied for culturing rat bone marrow stromal cells. Cell viability and mineralization assays reveal that not only these films are biocompatible, but also capable of enhancing the calcium deposition into the extra cellular matrix by the differentiated cells.

Oxygen delivery augmented bone formation from transplanted bone marrow

Event Abstract Back to Event Oxygen delivery augmented bone formation from transplanted bone marrow Huaifa Zhang1, Daisuke Sato1, Uwe Gbureck2, Benjamin Dalisson1, Simon Tran1 and Jake Barralet1, 3 1 McGill University, Faculty of Dentistry, Canada 2 Julius Maximilians University of Wuerzburg, Department of Functional Materials in Medicine and Dentistry, Germany 3 McGill University, Division of Orthopaedics, Department of Surgery, Faculty of Medicine, Canada Introduction: Bone marrow contains cells that are able to differentiate into bone. Ectopically transplanted bone marrow has also been found to generate bone tissue in vivo[1]. However, hypoxia occurs after tissue transplantation due to the limited blood supply into the transplant. As a result, much of the transplanted marrow tissue dies, thereby reducing the bone regeneration ability of marrow tissue. So far, no effective ways have been reported to reduce cell death in marrow tissue post transplantation. Calcium phosphate scaffolds have been widely used for bone regeneration because of their good osteoconductivity. It has been found that bone formation happens when cultured bone marrow-derived cells within calcium phosphate scaffolds are transplanted[2]. We have developed a self-oxygenating scaffold consisting of metal peroxides and alginate hydrogel. In this work, we encapsulated rat bone marrow into the hydrogel and cultured rat bone marrow under anoxia to determine the potential for survival post implantation. Afterwards, we added the encapsulated rat bone marrow into a 3D printed closed ended calcium phosphate cylindrical scaffold. In the end, we transplanted the scaffold containing marrow tissue subcutaneously in a rat model. Experimental: The bone marrow from rat tibia and femurs was harvested, mixed with sodium alginate and further cross-linked with CaCl2. The encapsulated marrow tissue with and without oxygen release material was cultured under anoxia (95% N2, 5% CO2, 37°C). Cell viability in cultured bone marrow was examined with live/dead and LDH assays. Afterwards, the encapsulated bone marrow was incorporated into the calcium phosphate scaffold and transplanted subcutaneously into rats. Six implants were implanted in each rat (dorsal side), three implants each side. After two and four weeks, the implants were retrieved and fixed. Bone formation in the implanted scaffolds was studied with Micro-CT. Then the scaffolds were embedded in paraffin and resin, respectively, for histological characterization. Result and Discussion: Figure 1 shows the live and dead cells in the marrow. It was apparent that the oxygen release scaffold drastically improved cell viability under anoxia. In the control experiment, most of the cells died after seven days under anoxia, whereas the tissue cultured with oxygen release scaffold exhibited very high cell viability (around 90%). In vivo experimental results show a larger amount of minerals in the presence of oxygen release material compared to the control group (Figure 2). The Micro-CT images clearly show that there were more minerals in the core area of the implants in the experimental group compared to the control group. The newly formed minerals was about 2.2 v/v% and 1 v/v% in experimental group and control group, respectively, after two weeks. Figure 1: Fluorescent images of live/dead assay stained marrow tissue cultured in anoxia with (Left) and without (Right) oxygen release material after seven days. Live cells were stained into green and dead cells into red. The nuclei of all cells were stained into blue. Figure 2 : Micro-CT images of retrieved marrow implants with (Left) and without (Right) oxygen release material after two weeks. Conclusion: To the best of our knowledge, this is the first attempt ever made to sustain bone marrow tissue in vitro using oxygen delivery material. We successfully developed a self-oxygenating scaffold which is capable of improved cell viability in bone marrow tissue under anoxia. Furthermore, the oxygen release scaffold augmented mineral formation after subcutaneous implantation. This is of great clinical interest and offers new prospects in improving clinical applications of bone marrow transplantation. The Natural Sciences and Engineering Research Council of Canada; China Scholarship Council

A novel injecable composite cement for vertebroplasty

Event Abstract Back to Event A novel injecable composite cement for vertebroplasty Mehran Dadkhah1, Lucia Pontiroli1, 2, Sonia Fiorilli1, Ion Tcacencu3 and Chiara Vitale-Brovarone1 1 Politecnico di Torino, Department of Applied Science and Technology, Italy 2 University of Leeds, Oral Biology, School of Dentistry, United Kingdom 3 Karolinska Institutet, Department of Dental Medicine, Sweden Introduction: In the past two decades, vertebroplasty has been considered as a safe, efficient and common surgical procedure that plays a main role in the treatment of vertebral compression fractures. This technique has been applied in pain reduction and for preventing further loss of vertebral body height in osteoporosis[1]. Calcium sulphate cements (CSC) are nontoxic, osteoconductive, bioresorbable and have been applied in clinical practice for over 100 years. However, their degradation rate is faster than the formation of new bone, which has limited their application[2],[3]. This study aimed to overcome the drawbacks of calcium sulphate by developing an injectable material containing three phases. Materials and Methods: The injectable cement formulation included: calcium sulphate hemihydrate (CSH-type III), a radiopaque glass ceramic and mesoporous bioactive glass particles. The glass-ceramic particles (mol. composition: SiO2 =57%, CaO=30%, Na2O=6% and ZrO2=7%) were prepared by melting, casting and ceramisation treatment. Mesoporous glass particles were synthesized by spray-drying technique using a water-based synthesis. In order to obtain a cement, the three phases were combined with an optimized liquid to powder ratio, which ensured that the paste remained injectable and completely set in proper time. The new cement was studied in vitro both in simulated body fluid (SBF) and by culturing rat bone marrow stromal cells (BMSC). The behaviour of the calcium sulphate-based composite cement (setting time, injectability, compressive strength) was evaluated with respect to type III calcium sulphate and Cerament® (as a commercial reference). Results: It was found that the new composite cement possessed slightly higher initial (48 min) and final (56 min) setting times, respectively (table 1). Moreover, the injectable cement showed a better compressive strength in both wet (13.8MPa) and dry (18.6MPa) conditions with respect to the commercial reference (table 1). The new cement samples were biodegradable and significantly improved bioactivity during the first week in vitro and this may enhance new bone formation in vivo (figure 1). The in vitro cell culture results proved that the new cement are cytocompatible, and might enhance osteogenic differentiations of the BMSC by increasing alkaline phosphatase activity. The new formulation of the injectable cement for vertebroplasty showed good setting times and compressive strength, and in vitro osteogenic properties which are comparable or better than Cerament®. This composite cement might has significant clinical advantages compared to the traditional CSH. Fig. 1. FESEM micrograph of the composite cement after 24 hr of SBF soaking showing with hydroxyapatite crystals. The research leading to these results has received funding from the European Union Seventh Framework Programme (FP7/2007-2013) under grant agreement n°[280575]-Restoration