Key Engineering Materials Vols. 361-363

Abstract: In order to develop bone substitute with osteogenic properties, a novel strategy of grafting cyclo-DfKRG peptides to enhance cells adhesion and osteointegration of hydroxyapatite (HA) implants was developed. Objectives of the study were (1) to evaluate the osteogenic properties of HA implants grafted with RGD peptides and (2) to compare histomorphometry and micro-computed tomography ((CT) with bone quantification. Pure HA grafted or not with cyclo- DfKRG peptides and autologous stromal cells was implanted in femoral condyle on 2 groups (2 and 4 weeks) of 6 New Zealand rabbits. Measurements concerning bone reconstruction and material structure were obtained with a (CT and the results were compared to those obtained after histomorphometry. Finally, it appears that grafting cyclo-DfKRG on HA implants enhances nonsignificantly the rate of bone formation, and a high correlation of the results was found comparing histomorphometry and (CT analysis.

Abstract: Osteoblasts were perceived as pivotal cells, recognized as the cells that control both the formative and the resorptive phases of the bone remodeling cycle. Osteoblasts were an essential requirement for osteoclastogenesis though expressing or secreating bioactive osteoclast-differentiation-regulatory proteins, osteoclast differentiation factor (ODF)was the most important factor among these, ODF participate nearly in every step of differentiation and activation of osteoclasts. In addition, intercellular adhesion molecule-1 (ICAM-1)and its receptors LFA-1 play a role in osteoclast development by affecting adhesion between stromal cells and osteoclast progenitors before the occurrence of ODF-ODF receptor signaling. However, it is not clear about the relationship between ODF, ICAM-1 expression of osteoblasts and differentiation state of osteoblasts. So,the aim of this study was to investgate whether the expression of ODF, ICAM-1 depended on the stage of osteoblastic differentiation from rat bone marrow mesenchymal stem cells(rBMSCs). The viability of rBMSCs is reduced significantly by osteogenic inducement as differentiating into osteoblasts, ALPase activity of OS-treated rBMSCs was enhanced obviously within 9 days , declined subsequently and recovered nearly the original level at day 14. Expression of ODF is enhanced with osteogenic differentiation guadully. whereas, expression of ICAM-1 is activated at OS-treated day 6, then keeping at a stable level. This study indicated that rBMSCs undergoing osteogenic inducement was an ideal model for studying the differentiation and maturation of osteoblasts. During the early stage of differentiation along osteoblasts from stem cells to osteocytes, rBMSCs or Osteoprogenitor react somewhat differently from osteoblasts, suggesting the ability of osteoblasts to regulating differentiation and maturation of osteoclasts have been improved with osteogenic culture.

Abstract: Recently, nanomaterials have received considerable attention because of their potential applications in the biomedical field. In the present study, we investigated the effects of nano-sized calcium metaphosphate (CMP) particles (50 nm) compared with micro-sized CMP particles (200-500 nm and 10 μm) on the proliferation and osteoblastic differentiation of human bone marrow stem cells (BMSCs). BMSCs were challenged with CMP particles with different sizes for 3, 5, and 7 days. An analysis of the proliferation revealed that the nano-sized CMP particles (50 nm) stimulated the proliferation of BMSCs up to 27.79% compared to the untreated control. This stimulatory effect of the nano-sized CMP particle was dose-dependent. CMP particles appeared to adhere on the surface of BMSCs but this did not cause distinguishable morphological changes. Moreover, all CMP particles (50 nm to 10 μm) were capable of stimulating an osteoblastic differentiation of BMSCs as accessed by alkaline phosphatase (ALP) and von Kossa stainings. Further molecular analysis revealed that all the CMP particles induced an expression of osteoblast-related genes such as osteocalcin (OC) and collagen I (Col I). Taken together, our data demonstrate that nano-sized CMP particles have the potential to stimulate the proliferation and osteoblastic differentiation of BMSCs.

Abstract: The aim of this study is to analyse the influence of differently fabricated HA-scaffolds on bone marrow stromal cells. Therefore, three methods were used (a polyurethane (PU)-replica technique, the dispense-plotting and a negative mould technique) to produce porous hydroxyapatite (HA) ceramics. The different HA-scaffolds were then cultivated with an osteoblastic precursor cell line. In our study, highest cell proliferation and differentiation was achieved by using (PU)-replica technique. However, this study shows also that all three types of scaffolds are suitable for tissue engineering applications and as bone substitute material. The knowledge about the influence of pore size and geometry on the cell behaviour will help to tailor scaffolds, by different 3D fabrication methods, for the needs of tissue engineering laboratories or patients.

Abstract: Porous hydroxyapatite (HA) scaffolds were processed in hyaluronic acid solution. Bone marrow cells obtained from the bone shaft of femurs of Fischer 344 rats at 1×106/ml concentration were seeded in pores of the scaffolds. The scaffolds were implanted in the dorsal subcutaneous tissue of rats for 2, 4, 6 or 8 weeks. Removed HA scaffolds at 2 and 4 week after dorsal subcutaneous implantation were histologically examined. At all experimental periods, osteocalcin in the scaffold was immunochemically measured for the quantitative analysis of osteogenesis by bone marrow cells in the porous HA scaffolds. Moreover, value of alkaline phosphatase (ALP) activity in the scaffolds was measured. Osteocalcin measured in scaffolds without bone marrow cells was 1.3 ng in an average and the ALP activity was 62.2 μmol at 4 week. In hyaluronic acid processed scaffold with bone marrow cells, quantity of osteocalcin increased from 1.6 ng at 2 week to 2.2 ng at 4 week after implantation of the scaffold. Histologically, many pores containing bone in the scaffolds immersed in hyaluronic acid solution were detected. Significant difference of the quantity of osteocalcin was recognized between 2 and 4 week implantation. There was no significant difference in the quantity of osteocalcin between the scaffolds implanted for 4 and 8 weeks. Value of ALP activity of the scaffold implanted for 4 weeks showed significant difference comparing with that implanted for 6 and 8 weeks. From the results of this study, quantitative increase of the bone formation in the pores of HA scaffolds would be able to observe from 6 to 8 weeks after implantation on the scaffolds by immersion in hyaluronic acid solution

Abstract: The purpose of this study was to estimate influence of lysine for osteogenesis in the porous hydroxyapatite (HA) scaffolds with bone marrow cells. The HA scaffolds were soaked in 100mM concentration of lysine solution. They were kept in bone marrow cell suspension at 1×106 cells/ml density. Another HA scaffolds without immersion in lysine solution were kept in the cell suspension at 1×106 or 1×107 cells/ml density. They were respectively implanted into dorsal subcutis of rats for 4 weeks. Serially sectioned paraffin specimens were made and observed histologically. In several sections, total pores and ones with bone were counted. Many pores containing bone were found in1×107 cells/ml concentration group. The significant difference was between 1×107 cells/ml group, the lysine group, and 1×106 cells/ml group. Although more bone formation was seen in lysine group than in 1×106 cells/ml group. There was no significant difference between the groups. Concentration of lysine to add in culture medium or scaffold should be improved respectively.

Abstract: A glass (G) and a glass-ceramic (GC) of different composition were selected and studied to realize a biocompatible e/o bioactive material with antibacterial properties through the introduction of silver ions. The glass was produced in bulk form, instead glass-ceramic powders were sintered to abtain massive samples, which are characterized in terms of biocompatibility and subjected to ion- exchange technique [1] to allow the silver ions introduction and modify only the external surface layer of the materials, thus maintaining unchanged the bulk characteristics. The obtained samples were completely characterized to verify if the silver introduction leads to structural, morphological or in vitro behaviour change; silver release test was also carried out as well as antibacterial test with Staphylococcus Aureus and cytotoxicity test on human cells.

Abstract: Apatite nuclei were synthesized by raising pH of simulated body fluid (SBF). The apatite nuclei were attached to surfaces of Ag microspheres. Hydroxyapatite (HAp) was induced from the apatite nuclei and spread over whole surface area of the Ag microspheres in SBF, then encapsulated Ag microspheres with HAp were fabricated. The encapsulated Ag microspheres with HAp were soaked in saline and changes in Ag ion concentration in saline were measured. The encapsulated Ag microspheres with HAp showed sustained-release of Ag ion.

Abstract: Despite systemic prophylaxis, infection rates after orthopedic surgery can reach more than 1%. A new HAP/TCP bone substitute loaded with 125 mg of gentamicin was designed for prophylactic use. Its aim was to enhance the efficacy of systemic prophylactic treatments by increasing the local antibiotic concentration. For prophylactic applications, release had to take place within 48 hours not to select antibiotic-resistant bacterial strains. The purpose of this study was to investigate the releasing mechanisms of gentamicin from the porous HAP/TCP matrix. The release rate of gentamicin trough the porosities of the bone substitute was investigated in vitro, in 0.9% sodium chloride solution. The rate appeared to be related to the bone substitute volume and fit classical diffusion laws. All the gentamicin was released in less than 48 hours: this rate corresponds to the recommendations for the prophylactic use of antibiotics.

Abstract: Cancer cells synthesize abnormal proteins and peptides which are associated to heat shock proteins being overproduced by these cells due to the stress induced by the particular biology of cancer tissue. We have purified on hydroxylapatite powder heat shock proteins using the HAparticles as purification bed, vectors for the proteins and vaccination adjuvant. The powder make possible that the purified HSPs and their associated peptides are transfected to the antigen presenting cells and presented to the T cells for the destruction of the cancer cells bearing the antigens.