Multiple proliferation signaling pathways are modulated by octacalcium phosphate in osteoblasts.

Octacalcium phosphate (OCP), a type of bioactive ceramics, may be associated with dentine, tooth apatite, and especially bone generation, and promotes wound healing after fracture. Recently, commercial bone grafting products containing a large amount of OCP material have been released because OCP can be synthesized in large quantities. It is reported to increase cell proliferation, but the interaction between OCP and cell signaling pathways is still unclear. In this study, first, we demonstrated OCP mediated cell signaling pathways with only purified OCP materials. OCP regulated P38, JNK (c-Jun N-terminal kinase), Src, and AKT (protein kinase B) signaling pathways. OCP crystals appeared in the characteristic ribbon shape but varied by several tens of micrometers in size. The X-ray diffraction pattern was the same as previously reported. We studied two concentrations of OCP (10 mg/ml and 20 mg/ml) to understand whether the effect of OCP on cell signaling pathways is dose dependent. We confirmed that OCP treatment affected cell proliferation and alkaline phosphatase and disrupted Src phosphorylation but did not change the total protein level. P38 phosphorylation was activated with OCP treatment and inhibited by SB203580, but P38 total protein level did not change. OCP inhibited JNK phosphorylation signaling, whereas PD98509 inhibited JNK phosphorylation with or without OCP. Interestingly, the AKT total level decreased after OCP treatment, but AKT phosphorylation increased considerably. Our results demonstrate that OCP materials modulate cell signaling pathways and increase cell proliferation.

Development of Cellular Signaling Pathways by Bioceramic Heat Treatment (Sintering) in Osteoblast Cells.

Bioceramics are calcium-phosphate-based materials used in medical and dental implants for replacing or repairing damaged bone tissues; however, the effect of bioceramic sintering on the intracellular signaling pathways remains unknown. In order to address this, we analyzed the impact of sintering on the cell signaling pathways of osteoblast cells using sintered and non-sintered hydroxyapatite (HA) and beta-tricalcium phosphate (ß-TCP). X-ray diffraction indicated that only the morphology of HA was affected by sintering; however, the sintered bioceramics were found to have elevated the calcium concentrations in relation to the non-sintered variants. Both bioceramics inhibited the JNK signaling pathway; the sintered HA exhibited half the value of the non-sintered variant, while the sintered ß-TCP rarely expressed a p-JNK value. The total Src and Raptor protein concentrations were unaffected by the sintering, while the p-Src concentrations were decreased. The p-EGFR signaling pathway was regulated by the non-sintered bioceramics, while the p-p38 concentrations were reduced by both the sintered ß-TCP and HA. All of the bioceramics attenuated the total AKT concentrations, particularly the non-sintered HA, and the AKT phosphorylation concentration, except for the non-sintered ß-TCP. Thus, the sintering of bioceramics affects several intracellular signaling pathways. These findings may elucidate the bioceramic function and expand their application scope as novel substrates in clinical applications.

Analysis of the inorganic component of autogenous tooth bone graft material.

This study was performed to identify the calcium phosphate minerals, chemical element and Ca/P ratio and to examine the surface structure of autogenous tooth bone grafting material (AutoBT) which recently developed and applied clinically as a bone graft materials. The analytical results showed that AutoBT is composed of low-crystalline hydroxyapatite (HA) and possibly other calcium phosphate minerals, which is similar to the minerals of human bone tissues. And the dental crown portion was composed of high-crystalline calcium phosphate minerals (mainly HA) with higher Ca/P ratio while the root portion was mainly composed of low-crystalline calcium phosphates with relatively low Ca/P ratio.

Effect of different bone substitutes on the concentration of growth factors in platelet-rich plasma.

This study is conducted to determine the effect of different kinds of bone substitutes and collagen on the concentration of platelet-derived growth factor (PDGF) and transforming growth factor beta-1 (TGF beta-1) in platelet-rich plasma (PRP). PRP is treated with thrombin, hydroxyapatite (HA), and thrombin, HA alone, collagen-grafted HA, calcium metaphosphate (CMP), and collagen-grafted CMP. The concentrations of PDGF-AB and TGF beta-1 are measured. After PRP treated with HA and CMP, the concentrations of PDGF and TGF beta-1 are not significantly different from the concentration of them in PRP alone. The concentrations of PDGF in PRP with collagen-grafted HA and collagen-grafted CMP are significantly higher than that of PRP with HA and CMP. The concentrations of PDGF and TGF beta-1 in PRP with collagen-grafted CMP are higher than with collagen-grafted HA. The results of multiple regression analysis show that PDGF increased with the use of collagen and thrombin, and is higher in native whole blood with higher platelet counts. However, PDGF decreased with the use of HA. In conclusion, HA and CMP do not seem to be able to activate platelets by themselves. However, if they had collagen grafted onto them, they could activate platelets and release growth factors.

Effect of water glass coating of tricalcium phosphate granules on in vivo bone formation.

BACKGROUND: Recently, some authors introduced a water glass (WG, sodium-silicate glass; Na2O·SiO2·nH2O) coating over tricalcium phosphate (TCP) bioceramic to modulate its resorption rate and enhance the bone cell behaviors. In this study, four different types of granular samples were prepared to evaluate the ability of new bone formation in vivo using micro-computed tomography and histology. METHODS: Four types sample groups: group A (pure HA as a negative resorption control); group B (pure TCP as a positive resorption control); group C (WG-coated TCP as an early resorption model); and group D (same as group C but heat-treated at 500°C as a delayed resorption model). Cylindrical tube-type carriers with holes were fabricated with HA by extrusion and sintering. Each carrier was filled densely with each granular sample. Four types of tubes were implanted into the medial femoral condyle and medial tibial condyle of New Zealand White rabbits. RESULTS: The HA group (A) showed the lowest amount of new bone formation. All the TCP sample groups (B, C, and D) showed more new bone formation. On the other hand, among the TCP groups, group C (early resorption model) showed slightly more bone formation. The amount of residual bioceramics was most abundant in the HA group (A). All the TCP sample groups showed less residual bioceramics than group A. Among the TCP groups, group C showed slightly more residual bioceramics. Group B showed the lowest amount of residual bioceramics. CONCLUSIONS: The WG-coated TCP sample (group C) is the best bone substitute candidate because of its proper biodegradation rate and the Si ions release because the WG-coated layer reduces the material resorption and enhances the new bone formation. That is, the WG-coated TCP is believed to be the best material for the application of an artificial bone substitute material.

Preparation and characterization of anodized titanium surfaces and their effect on osteoblast responses.

In this study, titanium (Ti) surface was modified by anodizing with a mixture of beta-glycerophosphate sodium and calcium (Ca) acetate, and the anodized surfaces were characterized by scanning electron microscopy, X-ray diffraction, and electron probe microanalysis. In vitro osteoblast response to anodized oxide was also evaluated. The anodic oxide produced was observed to have interconnected pores (0.5-2 microm in diameter) and intermediate roughness (0.60-1.00 microm). In addition, anodic oxide was observed to have amorphous and anatase oxide. Calcium and phosphorus ions were deposited on the Ti oxide during anodization. Osteoblast differentiation, as indicated by alkaline phosphatase production, was enhanced on anodized surfaces. It was thus concluded from this study that Ca phosphate can be deposited on Ti surfaces by anodization. It was also concluded that the phenotypic expression of osteoblast was enhanced by the presence of Ca phosphate and higher roughness on anodized Ti surfaces.

A poly(lactic acid)/calcium metaphosphate composite for bone tissue engineering.

A new method to prepare PLA/CMP (poly-L-lactide/calcium metaphosphate) composite scaffolds was developed for effective bone tissue engineering. This novel sintering method is composed of pressing the mixture of PLA, CMP, and salt particles at 150 MPa for 3 min followed by heat treatment at 210 degrees C for 30 min. The scaffolds had a homogeneously interconnected porous structure without a skin layer, and they exhibited a narrower pore size distribution and higher mechanical strength in comparison with scaffolds made by a solvent casting method. The scaffolds were seeded by osteoblasts and cultured in vitro or implanted into nude mice subcutaneously for up to 5 weeks. The number of cells attached to and proliferated on the scaffolds at both in vitro and in vivo was in the order of; PLA by novel sintering < PLA/CMP by solvent casting < PLA/CMP by novel sintering. In addition, the alkaline phosphatase activity of and calcium deposition in the scaffolds explanted from mice were enhanced significantly for the scaffolds by novel sintering compared to them by solvent casting. The in vitro results agreed well with the in vivo data. Such a superior characteristic of the novel sintering method should have resulted from the fact that the CMP particles could contact directly with cells/tissues to stimulate the cell proliferation and osteogenic differentiation, while the CMP particles would be coated by polymers and hindered to interact with cells/tissues in the case of a solvent casting method. As the novel sintering method does not use any solvents it offers another advantage to avoid problems associated with solvent residue.

Hydrothermal treatment of Ti surface to enhance the formation of low crystalline hydroxyl carbonate apatite.

BACKGROUND: Ti and its alloys have been widely used as orthopedic and dental implants due to their outstanding mechanical properties and biocompatibility. However, long time is required to form bond between Ti implant and surrounding tissues. Therefore, these implants necessitate surface treatment such as mechanical/chemical treatment and coating of bioactive materials for improving the osseointegration. RESULTS: This study was focused on the calcium-phosphate (Ca-P) coating on machined Ti, blasted-Ti (B-Ti), and blasted-NaOH-etched-Ti (BNH) surfaces by hydrothermal method to evaluate the ability of HA formation. Nanostructured morphology was created by NaOH etching on blasted-Ti surface. XRD analysis confirmed the existence of sodium titanate phase on such samples. Rutile and anatase phases along with hydroxyapatite were observed after hydrothermal treatment in Ca-P solution. Substantial hydroxyapatite together with TiO2 was observed during hydrothermal treatment at 200°C for 12 hrs. Blasted-NaOH-etched samples (BNH-Ti) revealed appreciable bone-like apatite formation as compared to machined-Ti and blasted-Ti (B-Ti) surfaces. However, maximum HA formation was confirmed on Ca-P coated-BNH samples (BNHA-Ti-200-12) by XRD and ICP analysis. CONCLUSION: Multistep surface treatment adopted in current study would be effective to enhance HA formation on Ti surface.

BMP-2 Grafted nHA/PLGA Hybrid Nanofiber Scaffold Stimulates Osteoblastic Cells Growth.

Biomaterials play a pivotal role in regenerative medicine, which aims to regenerate and replace lost/degenerated tissues or organs. Natural bone is a hierarchical structure, comprised of various cells having specific functions that are regulated by sophisticated mechanisms. However, the regulation of the normal functions in damaged or injured cells is disrupted. In order to address this problem, we attempted to artificially generate a scaffold for mimicking the characteristics of the extracellular matrix at the nanoscale level to trigger osteoblastic cell growth. For this purpose, we have chemically grafted bone morphogenetic protein (BMP-2) onto the surface of L-glutamic acid modified hydroxyapatite incorporated into the PLGA nanofiber matrix. After extensive characterization using various spectroscopic techniques, the BMP-g-nHA/PLGA hybrid nanofiber scaffolds were subjected to various in vitro cytocompatibility tests. The results indicated that BMP-2 on BMP-g-nHA/PLGA hybrid nanofiber scaffolds greatly stimulated osteoblastic cells growth, contrary to the nHA/PLGA and pristine PLGA nanofiber scaffold, which are used as control. These results suggest that BMP-g-nHA/PLGA hybrid nanofiber scaffold can be used as a nanodrug carrier for the controlled and targeted delivery of BMP-2, which will open new possibilities for enhancing bone tissue regeneration and will help in the treatment of various bone-related diseases in the future.

Cellular biocompatibility and stimulatory effects of calcium metaphosphate on osteoblastic differentiation of human bone marrow-derived stromal cells.

In the present study, the in vitro biocompatibility of calcium metaphosphate (CMP) with human bone marrow stromal cells (HBMSCs) and its effect on osteoblastic differentiation have been investigated. Powder and disk forms of CMP do not exert a cytotoxic effect on the HBMSCs undergoing osteoblastic differentiation. In addition, the HBMSCs adhere to the surface of the CMP disk as successfully as to the culture plate or hydroxyapatite (HA) disk. The HBMSCs adhered to either the HA or CMP disk display an undistinguishable actin arrangement and cellular phenotypes, indicating that the CMP does not disrupt normal cellular responses. An analysis of the differentiation of the HBMSCs cultured on culture plate, the HA and the CMP disk shows that three matrices are capable of supporting osteoblastic differentiation of the HBMSCs as accessed by alkaline phosphatase (ALP) staining. Further molecular analysis of osteoblastic differentiation of HBMSCs reveals that the CMP disk has a better ability than the HA disk to induce an expression of osteoblast-related genes, including ALP, osteoprotegerin (OPG), a decoy receptor for RANK ligand, and osteopontin (OPN), a non-collagenous bone matrix protein. The results demonstrate that, in addition to favorable biocompatibility, the CMP can stimulate osteoblastic differentiation of the HBMSCs in vitro.

Wear of retrieved UHMWPE hip liners.

After the gamma-irradiation sterilization, the most widely used orthopaedic grade polymer bearing liner material for the total joint replacement, ultra-high molecular weight polyethylene (UHMWPE), degrades through the progressive in vivo oxidation. The oxidative degradation makes UHMWPE brittle and leads to reduction of its mechanical properties. In this study, the effect of the in vivo post-irradiation ageing time on the wear of UHMWPE was investigated. Twelve retrieved polyethylene hip liners implanted for 3-16 years and then stored in the air for 1.5-8 years were used. Two types of the pin-on-disk wear testing were conducted. The uni-directional repeat pass rotating and the linear reciprocating wear testing were done with stainless steel disks against stationary polyethylene pins under 4MPa at 1Hz with bovine serum lubrication. Wear of the retrieved polyethylene hip liners does not have significant correlation with the in vivo or total ageing time. The linear reciprocal sliding motion generated a more pronounced wear than the uni-directional repeat pass sliding motion. This indicates that the kinematic motion significantly affects the wear of aged UHMWPE, having a brittle, white band region.

Hydroxyapatite and calcium phosphate coatings on aluminium oxide orbital implants.

BACKGROUND: Hydroxyapatite and calcium phosphate have been used as bone graft substitutes as they facilitate and promote tissue ingrowth. We carried out a study to examine uncoated and coated aluminium oxide (alumina) spherical orbital implants and assess whether the coatings influence fibrovascular ingrowth. METHODS: The aluminium oxide spheres (three coated with hydroxyapatite, three coated with calcium metaphosphate and three uncoated) were manufactured at the School of Materials Engineering, Yeungnam University, Kyongsan, Kyongbuk, Korea. The implants were examined macroscopically and with scanning electron microscopy and were analysed chemically by means of x-ray powder diffraction and x-ray fluorescence spectrophotometry. Implantation of three hydroxyapatite-coated, three calcium metaphosphate-coated and three uncoated aluminium oxide spheres was done in nine adult male New Zealand albino rabbits. Implant vascularization was evaluated at 4, 8 and 12 weeks by means of histopathological sectioning. RESULTS: All three types of implant had multiple interconnected pores. The coatings increased the size of the trabeculae from 150 microm to 300 microm. As a result, the pores appeared slightly smaller but still ranged in size from 300 microm to 750 microm, compared to 400 microm to 800 microm in the uncoated implants. The coatings also increased the weight of the implants slightly. The implants were all strong mechanically. They were made up primarily of aluminium oxide. The coated implants contained significant amounts of calcium oxide (a contaminant). There was no clinical difference in the socket response between the three groups. Histopathologically, fibrovascularization occurred uniformly throughout each implant at 4, 8 and 12 weeks after implantation. INTERPRETATION: The hydroxyapatite and calcium metaphosphate coatings did not appear to facilitate or inhibit fibrovascular ingrowth at 4, 8 and 12 weeks. Longer-term studies are need to determine whether the coatings play a role in long-term acceptance and retention of the implants.

Comparative characteristics of porous bioceramics for an osteogenic response in vitro and in vivo.

Porous calcium phosphate ceramics are used in orthopedic and craniofacial applications to treat bone loss, or in dental applications to replace missing teeth. The implantation of these materials, however, does not induce stem cell differentiation, so suitable additional materials such as porous calcium phosphate discs are needed to influence physicochemical responses or structural changes. Rabbit adipose-derived stem cells (ADSC) and mouse osteoblastic cells (MC3T3-E1) were evaluated in vitro by the MTT assay, semi-quantitative RT-PCR, and immunoblotting using cells cultured in medium supplemented with extracts from bioceramics, including calcium metaphosphate (CMP), hydroxyapatite (HA) and collagen-grafted HA (HA-col). In vivo evaluation of the bone forming capacity of these bioceramics in rat models using femur defects and intramuscular implants for 12 weeks was performed. Histological analysis showed that newly formed stromal-rich tissues were observed in all the implanted regions and that the implants showed positive immunoreaction against type I collagen and alkaline phosphatase (ALP). The intramuscular implant region, in particular, showed strong positive immunoreactivity for both type I collagen and ALP, which was further confirmed by mRNA expression and immunoblotting results, indicating that each bioceramic material enhanced osteogenesis stimulation. These results support our hypothesis that smart bioceramics can induce osteoconduction and osteoinduction in vivo, although mature bone formation, including lacunae, osteocytes, and mineralization, was not prominent until 12 weeks after implantation.

Composite nanofiber mats consisting of hydroxyapatite and titania for biomedical applications.

Composite nanofiber mats (HA/TiO2) consisting of hydroxyapatite (HA) and titania (TiO2) were fabricated via an electrospinning technique and then collagen (type I) was immobilized on the surface of the HA/TiO2 composite nanofiber mat to improve tissue compatibility. The structure and morphology of the collagen-immobilized composite nanofiber mat (HA/TiO2-col) was investigated using an X-ray diffractometer, electron spectroscopy for chemical analysis, and scanning electron microscope. The potential of the HA/TiO2-col composite nanofiber mat for use as a bone scaffold was assessed by an experiment with osteoblastic cells (MC3T3-E1) in terms of cell adhesion, proliferation, and differentiation. The results showed that the HA/TiO2-col composite nanofiber mats possess better cell adhesion and significantly higher proliferation and differentiation than untreated HA/TiO2 composite nanofiber mats. This result suggests that the HA/TiO2-col composite nanofiber mat has a high-potential for use in the field of bone regeneration and tissue engineering.

Development of a novel bone grafting material using autogenous teeth.

We developed a novel bone grafting material that incorporates autogenous teeth (AutoBT), and provided the basis for its clinical application. AutoBT contains organic and inorganic mineral components and is prepared from autogenous grafting material, thus eliminating the risk of an immune reaction that may lead to rejection. AutoBT was used at the time of implant placement, simultaneously with osteoinduction surgery, and excellent bony healing by osteoinduction and osteoconduction was confirmed.

Morphology and metabolism of Ba-alginate encapsulated hepatocytes with galactosylated poly(allyl amine) and poly(vinyl alcohol) as extracellular matrices.

Lactobionic acid, bearing a beta -galactose group, was coupled with poly(allyl amine) to provide synthetic extracellular matrices together with poly(vinyl alcohol) (PVA). The hepatocytes were encapsulated in Ba-alginate capsules with galactosylated poly(allyl amine) (GA) and PVA as extracellular matrices. From microscopic observation, it was revealed that the microcapsule prepared has a highly porous structure with interconnected pores and pore sizes ranging between 50-150 nm on both the surface and the cross-section. It was found, from the permeability experiment of microcapsules using FITC-dextrans with different molecular weights, that the capsule has a molecular weight cut off (MWCO) of 120 kDa, showing the potential that it can function as an immunoprotecting wall. The hepatocytes, cultured with GA and PVA in the core of the microcapsule, rapidly aggregated within a day, thus resulting in good metabolic functions such as albumin synthesis and ammonia removal.

Total hip arthroplasty with the use of nonmodular cementless acetabular component.

Sixty-seven hips in 63 patients who underwent total hip arthroplasties with the use of the nonmodular cementless acetabular component and alumina-on-polyethylene bearing surface were available for complete clinical and radiographic review at a mean follow-up period of 7 (range 5-9) years. The mean age was 59 years (range 34-75) years. The mean preoperative Harris hip score of 50 points improved to 93 points at final follow-up. One (1.5%) hip required revision for a recurrent dislocation. No component was loose radiographically at final follow-up. The mean linear wear rate was 0.07 (range 0.01-0.23) mm/y. At a mean follow-up of 7 years, there was no aseptic loosening. Further follow-up, however, is necessary to determine the potential advantage of nonmodular acetabular component for the development of pelvic osteolysis.

Surface characteristics and structure of anodic oxide films containing Ca and P on a titanium implant material.

An anodic oxide film that formed on titanium with a mixture of beta-glycerophosphate sodium (beta-GP) and calcium acetate was investigated. The anodic oxide had interconnected pores (ca. 1-2 microm in diameter) and intermediate roughness (0.60-1.50 microm). In addition, it contained a mixture of amorphous, anatase, and rutile oxides. With an increase in the anodizing voltage and/or concentration of calcium incorporated into the oxide, the degree of oxide crystallinity increased. However, with an increase in the concentration of beta-GP, the degree of oxide crystallinity decreased. It was concluded that the surface roughness, oxide crystallinity, and surface composition of the anodic oxide were dependent on the voltage, current density, and concentration of the electrolyte. It was also concluded that the anodized surface could be optimized for maximum osseointegration.