Ultrastructural Analysis on the Osteogenesis and Transformation of Calcium Phosphate Ceramics in Vivo

To study the osteogenesis and transformation process of calcium phosphate bioceramic in vivo, biodegradable porous β-tricalcium phosphate ceramics (β-TCP, φ5×8 mm) were implanted in the tibia of rabbits. β-TCP ceramics with surrounding bone tissue were retrieved and observed by SEM, TEM and EPMA every month after implantation. The results showed that osteogenesis was active and β-TCP ceramics bonded to bones directly. The new bones were forming and maturing as materials were continuously degrading, and materials were finally replaced by new bone. Parts of the materials were degraded, absorbed and recrystallized, while the rest were dispersed to the spongy bone and the Haversian lamella in an irregular arrangement, becoming incorporated into bone formation directly by remodeling the structure. Some β-TCP crystals cleaved along its (001) rhombohedral plane and formed lath-like crystals in vivo.

Protein Adsorption of Calcium Phosphate Ceramics in vitro

In order to provide valuable information for the design of new calcium phosphate bone repair materials,bone tissue engineering scaffold materials, and other dinical application, the interaction between calcium phosphate materials and proteins were investigated. The adsorption of the calcium phosphate ceramic to the protein was investigated by using FT-IR, XPS, SEM, and SDS-PAGE. As the results shown, the proteins were strongly adsorbed adsorbed the CPC, and a shift of the feature peak of the protein and also a chemical shift in the Ca2p and O1s bind energy of CPC was observed. This indicated that the acidic amino-group and alkaline amino- residue on the proteins＇ surface bonded to the Ca^2＋ in the β- TCP crystal by ionic bond and the proteins＇ alkaline amino groups to the oxygen in PO4^3＋ by hydrogen bond and electrostatic attraction. The adsorption mechanism of the protein in the CPC can be described as three adsorption layers ： irreversible chemical adsorption layer, physical adsorption layer and biomineralized adsorption layer.

One-step co-doping of ZnO and Zn^(2+)in osteoinductive calcium phosphate ceramics with synergistic antibacterial activity for regenerative repair of infected bone defect

How to endow bone grafts with long-term antibacterial activity and good bone regenerative ability to achieve the regenerative repair of infected bone defects has been the focus of the clinical treatment of osteomyelitis.The present study introduced a novel one-step route to realizing the co-doping of zinc oxide(ZnO)and zinc ion(Zn^(2+))in biphasic calcium phosphate(BCP)ceramics to utilize their synergistic antibacterial.Compared with the conventional BCP ceramics(BCP-Ca),the ZnO/Zn^(2+)co-doping ones(BCP-Zn)possessed strong antibacterial ability on E.coli and S.aureus as well as stimulated the osteogenic differentiation of bone marrow mesenchymal stem cells(BMSCs)effectively.The synergistic antibacterial mechanism of ZnO and Zn^(2+)was also investigated.BCP-Zn showed excellent osteoinductivity and angiogenesis at three months postoperatively in the canine intramuscular implantation model.Moreover,BCP-Zn exhibited excellent anti-infective ability and bone regenerative repair compared to BCP-Ca and control groups in the infected bone defect model of rat femur.Collectively,these findings suggest that the simultaneous introduction of ZnO/Zn^(2+)could have immense potential to expand the application of osteoinductive BCP ceramics in the regenerative repair of infected bone defects.

The material and biological characteristics of osteoinductive calcium phosphate ceramics

The discovery of osteoinductivity of calcium phosphate(Ca-P)ceramics has set an enduring paradigm of conferring biological regenerative activity to materials with carefully designed structural characteristics.The unique phase composition and porous structural features of osteoinductive Ca-P ceramics allow it to interact with signaling molecules and extracellular matrices in the host system,creating a local environment conducive to new bone formation.Mounting evidence now indicate that the osteoinductive activity of Ca-P ceramics is linked to their physicochemical and three-dimensional structural properties.Inspired by this conceptual breakthrough,many laboratories have shown that other materials can be also enticed to join the rank of tissue-inducing biomaterials,and besides the bones,other tissues such as cartilage,nerves and blood vessels were also regenerated with the assistance of biomaterials.Here,we give a brief historical recount about the discovery of the osteoinductivity of Ca-P ceramics,summarize the underlying material factors and biological characteristics,and discuss the mechanism of osteoinduction concerning protein adsorption,and the interaction with different types of cells,and the involvement of the vascular and immune systems.

MACROPHAGES IN DEGRADATION OF COLLAGEN / HYDROXYLAPATITE(CHA), BETA-TRICALCIUM PHOSPHATE CERAMICS (TCP) ARTIFICIAL BONE GRAFT AN IN VIVO STUDY

The macrophages mediated biodegradation of two biomaterials, collagen / hydroxylapatite (CHA) and beta-tricalcium phosphate ceramics (TCP), was studied in 24 male Kunming mice and 20 male C57BL / 6 mice with histopathologic, histochemical and ultrastructural observation. It was demonstrated that macrophages infiltrated after CHA, TCP were implanted. The macrophages could be differentiated from fibroblasts and the other infiltrated cells for special cellular profile and strong acid phosphatase activity. Morphologically, monocyte macrophages and infused multinuclear giant cell degraded CHA and TCP by phagocytosis and extracellular resorption. The carbonic anhydrase activity of macrophages was demonstrated by histochemical technique. It suggested that macrophages secreted H+ and accomplished the decalcification of calcium phosphate compound of CHA and TCP. We conclude that macrophages are the main mediating cells which degraded CHA and TCP intracellularly and extracellularly.

The Transformation of Calcium Phosphate Bioceramics in Vivo

To study the transformation process of calcium phosphate bioceramic in vivo,biodegradable porous β-tricalcium phosphate ceramics (β-TCP) were used in this experiment. The materials (5×8mm) were implanted in the tibia of rabbits. The β-TCP ceramics with bone tissue were retrieved and treated for histology, and then observed by using a scanning electron microscope (SEM) and an electron probe X-ray microanalyzer (EMPA) every month. The results show that β-TCP ceramics bond to bone directly,new bones are forming and maturing with materials continuous degrading,and the materials are nearly replaced by the formed bone finally.Parts of the materials were degraded,absorpted and recrystallized,the others dispersped on the cancellous bone and the Haversian lamella with an irregular arrangement incorporating in bone formation directly by remodeling structure.

Porous Calcium Phosphate Ceramic Scaffolds for Tissue Engineering

This study examined the biological response of two porous calcium phosphate ceramics, hydroxyapntite （ HA ） and hydroxyapaptite/β-tricalcium phosphate （ HA/β- TCP ） scaffolds. Three different cell types , a human osteoblastic cell line （ HOS ） , primary human osteoblasts （HOB） and human mesenehymal stem cells （MSCs）, were used to examine biocompatibility and osteoinductive capacity. The experimental results showed both materials were highly biocompatible and proliferation was significantly greater on pure HA （ P 〈 0.01 ）, with a peak in proliferation at day 7. Protein levels were significantly higher （ P 〈 0.05） than the control Thermanox（ TMX （ tm） ） for both test materials. Osteoinduction of MSCs was observed on both test materials, with cells seeded on HA/ β-TCP showing greater alkaline phosphatase activity compared to HA alone, indicating an enhancement in osteoinductive property. Both materials show good potential for use as tissue engineered scaffolds.

In Situ Laser Coating of Calcium Phosphate on TC4 Surface for Enhancing Bioactivity

Titanium alloy has been a successful implant material owing to its excellent ratio of strength to weight, toughness, and bio-inert oxide surface. Significant progress has been made in improving the bioactivity of titanium alloy by coating its oxide surface with calcium phosphates. In the present study, in situ coating was reported on Ti6Al4V（TC4） surface with calcium phosphate （Ca-P） bioceramics synthesized and synchronously cladded by laser beam. This coating was grown by first preplacing directly the raw powders, which contain 80% of CaHPO4 · 2H2O, 20% of CaCO3, and dram of rare earth （RE）, on the TC4 surfaces, and then exposing the surfaces to the laser beam with a power density of 12. 73-15.27 MW · m^-2 and a scanning velocity of 10.5 m/s. The resultant coating was characterized using scanning electron microscopy （SEM）, X-ray diffraction （XRD）, thermogravimetric analysis and Different-thermal Scanning （TG-DSC）, and Energy Dispersive X-ray Detection （EDX）. The results show that these laser ceramics include hydroxyapatite （HA）, tricalcium phosphate （TCP）, Ca2 P2 O7, and other Ca-P phases, and the interface between the coating and the TC4 substrate has tighter fixation, in which the chemical bonding is approved. These laser hybrid coatings are useful in enhancing the bioactivity of titanium alloy surfaces.

The Influence of Surface Morphology of Dense Ca-P Ceramics on Apatite Formation in Dynamic SBF

This study aimed at exploring the effect of surface morphology of dense phosphate calcimn （Ca-P） ceramics upon the formation of bone-like apatite in static or dynamic simulated body fluid （SBF）. Dense and sandblasted calcium phosphate ceramics were immersed into dynamic SBF flowing at normal physiological speed of body fluid of skeletal muscle. The changes were characterized using SEM, XPS, IR and XRD. Changes can be observed after the sandblasted surface of dense calcium phosphate ceramics had been immersed in SBF for 14 days. XPS analysis results showed that the flake-like structure was composed of Ca, P, C, O; IR analysis result of surface structure of samples showed that there were specific peaks for CO3^2-; XRD results indicated the decrease in crystallinity and the increase in amorphous structure. The rough surface was advantageous for the formation of bone-like apatite. Increasing the Ca^2＋, HPO4^2- concentration of SBF could also enhance the bonelike apatite formation. All the results demonstrated that local concentration is a key factor affecting nucleation.

CD301b^(+) macrophages mediate angiogenesis of calcium phosphate bioceramics by CaN/NFATc1/VEGF axis

Calcium phosphate(CaP)bioceramics are important for tissue regeneration and immune response,yet how CaP bioceramics influence these biological processes remains unclear.Recently,the role of immune cells in biomaterial-mediated regeneration,especially macrophages,has been well concerned.CD301b^(+)macrophages were a new subset of macrophages we have discovered,which were required for bioceramics-mediated bone regeneration.Nevertheless,the impact of CD301b^(+)macrophages on angiogenesis,which is a vital prerequisite to bone formation is yet indistinct.Herein,we found that CD301b^(+)macrophages were closely correlated to angiogenesis of CaP bioceramics.Additionally,depletion of CD301b^(+)macrophages led to the failure of angiogenesis.We showed that store-operated Ca^(2+)entry and calcineurin signals regulated the VEGF expression of CD301b^(+)macrophages via the NFATc1/VEGF axis.Inhibition of calcineurin effectively impaired angiogenesis via decreasing the infiltration of CD301b^(+)macrophages.These findings provided a potential immunomodulatory strategy to optimize the integration of angiogenesis and bone tissue engineering scaffold materials.

Preparation and Characterization of Porous Calcium Phosphate Bioceramics

β-tricalcium phosphate （β-TCP） powder and Na20-CaO-MgO-P205 glass binder were synthesized and mixed, and then the biodegradable porous calcium phosphate ceramics were successfully prepared by foaming and sintering at 850℃. The as-prepared ceramics possess a high porosity with partial three-dimension interconnected macro- and micro-pores. As in vitro experiment testified, the calcium phosphate ceramics （CPCs） has good degradability.

Graphene oxide coated three-dimensional printed biphasic calcium phosphate scaffold for angiogenic and osteogenic synergy in repairing critical-size bone defect

The custom-tailored medicine requires a developmental strategy that integrates excellent osteogene-sis with mechanical stability to enhance the reconstruction of the critical-size bone defect(CSBD)and the healing process in weight-bearing bone.We prepared three-dimensional(3D)printed biphasic cal-cium phosphate(BCP)scaffolds composited with nano-graphene oxide(GO).The biological effects of the GO/BCP composite scaffolds could induce the differentiation of rat bone marrow stem cells(BM-SCs)and the migration of human umbilical vein endothelial cells(HUVECs)for bone repair.The proper ratio of GO in the composite scaffold regulated the composites’surface roughness and hydrophilicity to a suitable range for the adhesion and proliferation of BMSCs and HUVECs.Besides,the GO/BCP composite scaffold increased osteogenesis and angiogenesis by activating BMP-2,RUNX-2,Smad1/4,and VEGF.The customized intramedullary nail combined with GO/BCP scaffold was applied to repair CSBD(2.0 cm in length)in a beagle femur model.This fixation strategy was confirmed by finite element analysis.In vivo,the results indicated that the custom-made internal fixation provided sufficient stability in the early stage,ensuring bone healing in a considerable mechanical environment.At 9 months postoperatively,longitudi-nal bony union and blood vessels in osteon were observed in the CSBD area with partial degradation in the 0.3%GO/BCP group.In the three-point bending test,the ultimate load of 0.3%GO/BCP group reached over 50%of the normal femur at 9 months after repair.These results showed a promising application of osteogenic GO/BCP scaffold and custom-made intramedullary nails in repairing CSBD of the beagle femur.This effective strategy could provide an option to treat the clinical CSBD in weight-bearing bones.

Enhanced bone regenerative properties of calcium phosphate ceramic granules in rabbit posterolateral spinal fusion through a reduction of grain size

Osteoinductivity is a crucial factor to determine the success and efficiency of posterolateral spinal fusion(PLF)by employing calcium phosphate(Ca-P)bioceramics.In this study,three kinds of Ca-P ceramics with microscale to nanoscale gain size(BCP-control,BCP-micro and BCP-nano)were prepared and their physicochemical properties were characterized.BCP-nano had the spherical shape and nanoscale gain size,BCP-micro had the spherical shape and microscale gain size,and BCP-control(BAM®)had the irregular shape and microscale gain size.The obtained BCP-nano with specific nanotopography could well regulate in vitro protein adsorption and osteogenic differentiation of MC3T3 cells.In vivo rabbit PLF procedures further confirmed that nanotopography of BCP-nano might be responsible for the stronger bone regenerative ability comparing with BCP-micro and BCP-control.Collectedly,due to nanocrystal similarity with natural bone apatite,BCP-nano has excellent efficacy in guiding bone regeneration of PLF,and holds great potentials to become an alternative to standard bone grafts for future clinical applications.