Osteogenesis and Degradation Behavior of rhBMP-27/β-Tricalcium Phosphate Porous Composite Materials

Ultrafine-tricalcium phosphate(β-TCP)powders with good crystalline structure were produced by a new process through bone tissue engineering approach rorous β-TCPcermic was combined with recombined human bone morphogenetic proteins-2(rhBMP-2)to develop a novel composite material ,osteogenesis capacity of the composite was investigated intramuscularly in rat with histological analyses and SEM examination pureβ-TCP porous carmic wsa investigated as the control results show that the compostie materials possess good bilcompatibility biodegradation and strong osteogenesis capacity through inductive process after implantation material degradation began from 2 weeks post-implantation accompanying with the changing o pore structure with the enwrapping and separation fo materials by hyperplatic mesenchymal cells and fibroblast and with the phagocytose reaction of multinucleated giant cells early in 72h immature cartilage could be found within novel composite mature lamellar bone was induced to generate after 3 weeks with strong osteoinduction capacity and controlable bildegradation the novel rhBMP-2\β-TCP porous ceramic is expected to be a promising bone grafting substitute for bone tissue engineering

Hydroxyapatite/β-tricalcium Phosphate Composite for Guiding Bone Tissue Growth into a Titanium Tube in 8 mm Dog Tibia Cavity Defects

We developed a fixation method and evaluate bone regrowth in the cavities of a Ф4 mm× 8 mm titanium（Ti）tube through porous hydroxyapatite（HAP）/β-tricalcium phosphate（β-TCP）composite filling（group A）,chitosan/calcium phosphate composite filling（group B）,and HAP particle modification（group C）.After 2 and 5 months of implantation in dog tibia defects,new bone formation in the three groups was studied by histology and histomorphometry.Group A displayed the most bone regenerated area in both 2 and 5 months post-operation.The chitosan/calcium phosphate composite in group B mostly degraded 2 months after implantation,leading to fibrous tissue invasion after 5 months.By contrast,less bone formation was observed in group C.These results indicated that filling the cavities of metalprostheses with a porous HAP/β-TCP composite can be used for stable long-term fixation in clinicalsettings.

Three-dimensional printing of β-tricalcium phosphate/calcium silicate composite scaffolds for bone tissue engineering

Bioactive scaffolds with interconnected porous structures are essential for guiding cell growth and new bone formation. In this work, we successfully fabricated three-dimensional （3D） porous β-tricalcium phosphate （β-TCP）/calcium silicate （CS） composite scaffolds with different ratios by 3D printing technique and further investigated the physiochemical properties, in vitro apatite mineralization properties and degradability of porous β-TCP/CS scaffolds. Moreover, a series of in vitro cell experiments including the attachment, proliferation and osteogenic differentiation of mouse bone marrow stromal cells were conducted to testify their biological performances. The results showed that 3D printed β-TCP/CS scaffolds possessed of controllable internal porous structures and external shape. Furthermore, the introduction of CS decreased the shrinkage of scaffolds and improved the in vitro apatite formation activity and degradation rate. Meanwhile, compared with pure β- TCP scaffold, the β-TCP/CS composite scaffolds were more conducive to promote cell adhesion, spread and osteogenesis differentiation. However, when the content of CS was increased to 45%, the ions dissolution rate of the composite scaffolds was so high that leaded to the increase in pH value, which inhibited the proliferation of cells. Our results suggested that the introduction of appropriate CS into β-TCP bioceramic is an effective strategy to prepare bioactive 3D printed bioceramic scaffolds for hard tissue regeneration.

Microemulsion Synthesis of Mesoporous β-tricalcium Phosphate Powder with a Novel System

The synthesis of mesoporous β-tricalcium phosphate(β-TCP)powder was performed by using the microemulsion approach,with hexadecyltrimethyl ammonium bromide(CTAB)/cyclohexane/n-octyl alcohol microemulsion system.The influences of different pH values and calcination temperatures on the phase composition of the β-TCP powder were studied.The in vitro proliferation of bone marrow mesenchymal stem cells(BMSCs)in the suspensions of β-TCP powders with meso-structure was studied.The phase composition,mesoporous structure,powder morphology,cell morphology and the optical density(OD)were characterized through X-ray diffraction(XRD),field emission scanning electron microscopy(FESEM),Fourier transform infrared(FTIR)spectroscopy,Nadsorption-desorption isotherms,inverted phase contrast microscopy and Multiskan spectrum,respectively.The mesoporous β-TCP powder with specific surface area of 12.85 m^(2)/g and the average pore size 7.11 nm was obtained through the microemulsion approach(100 g/L CTAB/250 mL/L cyclohexane/250 mL/L n-octyl alcohol)with a controlled pH of 7.0,after calcinating the powder at 800℃.It was confirmed that mesoporous β-TCP powder benefits the activity of BMSCs more than the non-mesoporous β-TCP powder.

Local Application of Alendronate on <i>β</i>-Tricalcium Phosphate Accelerated Induction of Osteogenesis with Formation of Giant Osteoclast-Like Cell

Intrinsic osteoinductivity—the ability to induce bone formation in ectopic sites without addition of osteogenic factors has been reported in various porous materials. Tartrate-resistant acid phosphatase (TRAP)-positive osteoclast-like cells are thought to play an important role in material-induced osteoinduction. To investigate the influence of osteoclastic activity on intrinsic osteoinduction, we loaded alendronate (10–2 , 10–4 , and 10–6 M) onto porous β-tricalcium phosphate (β-TCP) blocks to inhibit osteoclastic activity, and evaluated osteoinductivity by implantation of the blocks into the dorsal muscles of adult beagle dogs. Alendronate-loaded porous β-TCP blocks increased both speed and amount of osteoinduction, as measured 4 weeks after implantation, with the 10–4 M alendronate-loaded β-TCP being especially active. This finding indicates that β-TCP loaded with 10–4 M alendronate might prove crucial in providing the desirable balance between the degradation rate of bone scaffolds and their osteoinductive replacement. Thus, material-induced osteoinduction may be controlled by local application of alendronate, establishing alendronate loading as a promising therapeutic approach.

Microstructure and mechanical properties analysis of β-tricalcium phosphate/carbon nanotubes scaffold based on rapid prototyping

β-TCP ceramic scaffolds were fabricated with selective laser sintering (SLS) in this work. Carbon nanotubes (CNTs) were mixed with porous β-TCP matrix to enhance the mechanical performance of the bone tissue engineering scaffolds. Scaffold reconstruction and microstructure analysis were fulfilled based on micro-computed tomography (Micro-CT) scanning data. Results show that the strength of scaffold mixed with 0.2% CNTs reaches 0.819 MPa which has been improved by 85.7% compared with that without CNTs. Micro-CT analysis shows that the scaffold has a good interconnectivity, and pore size mainly distributes in the two regions of 60-340 μm and 500-620 μm.

Synthesis of Biphasic Calcium Phosphate by Hydrothermal Route and Conversion to Porous Sintered Scaffold

Biphasic calcium phosphate (BCP) consisting of hydroxyapatite (HA) and β-tricalcium phosphate (β-TCP) was successfully synthesized by new hydrothermal route using β-TCP as precursor. The X-ray diffraction analysis of as-synthesized powder indicated that β-TCP had been transformed into HA phase and amount of HA formed gradually increased with prolonged time. The results revealed that the recent technique may be able to control the composition of the obtained BCP which would influence the bioresorbability. Porous body of BCP was prepared by impregnation of polymeric sponge template with the slurry of the powder followed by sintering. The X-ray diffraction of porous product revealed that the composition of β-TCP increased after sintering indicating that HA had been decomposed. Porous BCP obtained from the recent technique possessed both macro and micropores structure which are useful for rapid tissue formation. Besides, the recent porous fabrication technique yielded porous BCP which preserved the sponge template morphology, enabling it to fabricate porous material with controlled pores structure.

Microstructure and properties of biodegradable β-TCP reinforced Mg-Zn-Zr composites

Magnesium alloys have good biocompatibility, but their mechanical properties and corrosion resistance may not be satisfied for using as degradable materials within bone due to its high corrosion rate in the physiological environment. Nano β-TCP particles were added into Mg-Zn-Zr alloy to improve its microstructure and the properties. As-extruded Mg-3Zn-0.8Zr alloy and Mg-3Zn-0.8Zr/xβ-TCP （x=0.5%, 1.0% and 1.5%） composites were respectively fabricated. The grains of Mg-Zn-Zr/β-TCP composites were significantly refined. The results of the tensile tests indicate that the ultimate tensile strength and the elongation of composites were improved with the addition of β-TCP. The electrochemical test result in simulation body fluid shows that the corrosion resistance of the composites was strongly enhanced comparing with that of the alloy. The corrosion potential of Mg-3Zn0.8-Zr/1.0β-TCP composite is 1.547 V and its corrosion current density is 1.20×10 6 A/cm 2 .

In Vitro Characterizations of PLLA/β-TCP Porous Matrix Materials and RMSC-PLLA-β-TCP Composite Scaffolds

To develop a novel degradable poly (L-lactic acid)/β-tricalcium phosphate (PLLA/β-TCP) bioactive materials for bone tissueengineering, β-TCP powder was produced by a new wet process. Porous scaffolds were prepared by three steps, i.e. solventcasting, compression molding and leaching stage. Factors influencing the compressive strength and the degradation behaviorof the porous scaffold, e.g. weight fraction of pore forming agent-sodium chloride (NaCl), weight ratio of PLLA: β-TCP,the particle size of β-TCP and the porosity, were discussed in details. Rat marrow stromal cells (RMSC) were incorporatedinto the composite by tissue engineering approach. Biological and osteogenesis potential of the composite scaffold weredetermined with MTT assay, alkaline phosphatase (ALP) activity and bone osteocalcin (OCN) content evaluation. Resultsshow that PLLA/β-TCP bioactive porous scaffold has good mechanical and pore structure with adjustable compressive strengthneeded for surgery. RMSCs seeding on porous PLLA/β-TCP composite behaves good seeding efficacy, biocompatibility andosteoinductive potential. Osteoprogenitor cells could well penetrate into the material matrix and begin cell proliferation andosteogenic differentiation. Osseous matrix could be formed on the surface of the composite after culturing in vitro. It isexpected that the PLLA/β-TCP porous composites are promising scaffolds for bone tissue engineering in prosthesis surgery.

Corrosion behaviour of PEEK or β-TCP-impregnated Ti6Al4V SLM structures targeting biomedical applications

Ti6Al4V cellular structures were produced by selective laser melting(SLM)and then filled either with beta-tricalcium phosphate(β-TCP)or PEEK(poly-ether-ether-ketone)through powder metallurgy techniques,to improve osteoconductivity and wear resistance.The corrosion behavior of these structures was explored considering its importance for the long-term performance of implants.Results revealed that the incorporation of open cellular pores induced higher electrochemical kinetics when being compared with dense structures.The impregnation ofβ-TCP and PEEK led to the creation of voids or gaps between the metallic matrix and the impregnated material which also influenced the corrosion behavior of the cellular structures.

In Vitro Dissolution Kinetics of α-TCP Cement Containing Tetracycline Hydrochloride

In vitro dissolution kinetics of a-tricalcium phosphate （α-TCP） cement and α-TCP cement containing tetracycline Hydrochloride（TTCH） were studied in the present paper. It shows that dissolution process of α-TCP cement and TTCH-loaded α-TCP cement accords with Avrami dissolution kinetics model： x=1-exp（-kt^n）, and Avrami constant n is 0.5 and 0.4 respectively, which means dissolution process is diffusion control. Apparent dissolution activation energy of α-TCP cement and TTCH-loaded α-TCP cement is about 9.87 kJ/moland 7.17 kJ/mol respectively. Comparing with α-TCP cement, activation energy and Avrami constant of TTCH-loaded α-TCP cement decrease slightly, but its [Ca^2＋] solubility decreases from 40 ppm to 11.5 ppm, which could result from the change of interracial property and morphology of hydrated apatite crystal caused by absorption of TTCH on the apatite.

Osteogenesis Capacity of a Novel BMP/α-TCP Bioactive Composite Bone Cement

To improve the osteogenesis ability of a-tricalcium phosphate (α-TCP) bone cement, a novel BMP/ α-TCP composite bone cement was prepared. By measuring the setting time and compressive strength, the hydration characteristic of bone cement was evaluated. Animal experiments including histological observation, radiographic investigation as well as digital image analyses reveal the difference of osteogenesis ability among BMP,a-TCP bone cement and BMP/α-TCP composite bone cement. Results show that α-TCP bone cement possesses excellent hydration and setting properties as well as high mechanical property. Comparison experiments show that BMP/ α-TCP composite bone cement has a stronger osteogenesis ability. The gross observation of the implant site does not exhibit any inflammation or necrosis. Histological analyses reveal that the material has good osteointegration with host bone, and new bone formation is detected within the materials, which are degrading. Strong osteogenesis ability of the composite is due to not only the excellent osteoconductive potential but also the osteoinductive potential contributed by active BMP releasing and the material degradation. Large skull defect could be well-healed by filling BMP/α-TCP composite bone cement. This novel material proves itself to be an absorbable and bioactive bone cement with an osteogenesis ability. Key words α-tricalcium phosphate (α-TCP) - bone morphogenetic proteins (BMP) - bone cement - osteogenesis - osteoinductivity - bone tissue engineering Funded by 863 Hi-Tech Research and Development Program of China (2002AA326080) and the Fund for Outstanding Young Teacher of the Education Ministry of China(2002123)

Effect of minor amounts of β-calcium pyrophosphate and hydroxyapatite on the physico-chemical properties and osteoclastic resorption of β-tricalcium phosphate cylinders

β-Tricalcium Phosphate(β-TCP),one of the most used bone graft substitutes,may contain up to 5 wt%foreign phase according to standards.Typical foreign phases includeβ-calcium pyrophosphate(β-CPP)and hydroxyapatite(HA).Currently,the effect of small amounts of impurities on β-TCP resorption is unknown.This is surprising since pyrophosphate is a very potent osteoclast inhibitor.The main aim of this study was to assess the effect of small β-CPP fractions(<1 wt%)on the in vitro osteoclastic resorption of β-TCP.A minor aim was to examine the effect of β-CPP and HA impurities on the physico-chemical properties of β-TCP powders and sintered cylinders.Twenty-six batches of β-TCP powder were produced with a Ca/P molar ratio varying between 1.440 and 1.550.Fifteen were further processed to obtain dense and polished β-TCP cylinders.Finally,six of them,with a Ca/P molar ratio varying between 1.496(1 wt% β-CPP)and 1.502(1 wt% HA),were incubated in the presence of osteoclasts.Resorption was quantified by white-light interferometry.Osteoclastic resorption was significantly inhibited by β-CPP fraction in a linear manner.The presence of 1% β-CPP reduced β-TCP resorption by 40%,which underlines the importance of controllingβ-CPP content when assessing β-TCP biological performance.

Heterotopic bone formation in the musculus latissimus dorsi of sheep usingβ-tricalcium phosphate scaffolds:evaluation of different seeding techniques

Osseous reconstruction of large bone defects remains a challenge in oral and maxillofacial surgery.In addition to autogenous bone grafts,which despite potential donor-site mobility still represent the gold standard in reconstructive surgery,many studies have investigated less invasive alternatives such as in vitro cultivation techniques.This study compared different types of seeding techniques on pureβ-tricalcium phosphate scaffolds in terms of bone formation and ceramic resorption in vivo.Cylindrical scaffolds loaded with autologous cancellous bone,venous blood,bone marrow aspirate concentrate or extracorporeal in vitro cultivated bone marrow stromal cells were cultured in sheep on a perforator vessel of the musculus latissimus dorsi over a 6-month period.Histological and histomorphometric analyses revealed that scaffolds loaded with cancellous bone were superior at promoting heterotopic bone formation and ceramic degradation,with autogenous bone and bone marrow aspirate concentrate inducing in vivo formation of vital bone tissue.These results confirm that autologous bone constitutes the preferred source of osteoinductive and osteogenic material that can reliably induce heterotopic bone formation in vivo.

Sustained release of Semaphorin 3A from a-tricalcium phosphate based cement composite contributes to osteoblastic differentiation of MC3T3-E1 cells

The reinforcement of calcium phosphate materials with silk fibroin （SF） has been one of the strategies to overcome the brittleness. However, the lack of osteoinductivity may still restrict their further use. This study aimed to investigate the biocompatibility and osteogenesis capacity of a novel Semaphorin 3A-loaded chitosan microspheres/SF/a-tricalcium phosphate composite （Sema3A CMs/SF/a-TCP） in vitro. Sema3A was first incorporated into CMs, and the Sema3A CMs/SF/a-TCP composite was then prepared. The morphology of the CMs was observed using SEM. The in vitro release kinetics, cytotoxicity, and cell compatibility were evaluated, and the real-time quantitative polymerase chain reaction （RT-qPCR） and activity of alkaline phosphatase （ALP） were used to evaluate the osteogenesis capacity of the composite. The in vitro release of Sema3A from the Sema3A CMs/SF/a-TCP composite showed a temporally controlled manner. The extract of the Sema3A CMs/SF/a-TCP composite presented no obvious side effect on the MC3T3-E1 cell proliferation, nor promote cell proliferation. The MC3T3-E1 cells were well-spread and presented an elongated shape on the Sema3A CMs/SF/a-TCP composite surface; the ALP activity and the osteogenic-related gene expression were higher than those seeded on the surface of the CMs/SF/a-TCP and SF/a-TCP composites. In conclusion, Sema3A CMslSF/a-TCP has excellent biocompatibility and contributes to the osteoblastic differentiation of MC3T3-E1 cells.

Sequential Fluorescent Labeling Observation of Maxillary Sinus Augmentation by a Tissue-engineered Bone Complex in Canine Model

Aim To evaluate the effects of maxillary sinus floor elevation by a tissue-engineered bone complex of β-tricalcium phosphate （β-TCP） and autologous osteoblasts in dogs. Methodology Autologous osteoblasts from adult Beagle dogs were cultured in vitro. They were further combined with β-TCP to construct the tissue-engineered bone complex. 12 cases of maxillary sinus floor elevation surgery were made bilaterally in 6 animals and randomly repaired with the following 3 groups of materials： Group A （osteoblasts/D-TCP）; Group B （β-TCP）; Group C （autogenous bone） （n=4 per group）. A polychrome sequential fluorescent labeling was performed post-operatively and the animals were sacrificed 24 weeks after operation for histological observation.Results Our results showed that autologous osteoblasts were successfully expanded and the osteoblastic phenol- types were confirmed by ALP and Alizarin red staining. The cells could attach and proliferate well on the surface of the ~3-TCP scaffold. The fluorescent and histological observation showed that the tissue-engineered bone complex had an earlier mineralization and more bone formation inside the scaffold than β-TCP along or even autologous bone. It had also maximally maintained the elevated sinus height than both control groups. Conclusion Porous 13-TCP has served as a good scaffold for autologous osteoblasts seeding. The tissue-engineered bone complex with β-TCP and autologous osteoblasts might be a better alternative to autologous bone for the clinical edentulous maxillary sinus augmentation.

A novel bioactive nerve conduit for the repair of peripheral nerve injury

The use of a nerve conduit provides an opportunity to regulate cytokines,growth factors and neurotrophins in peripheral nerve regeneration and avoid autograft defects.We constructed a poly-D-L-lactide(PDLLA)-based nerve conduit that was modified using poly{(lactic acid)-co-[(glycolic acid)-alt-(L-lysine)]}andβ-tricalcium phosphate.The effectiveness of this bioactive PDLLA-based nerve conduit was compared to that of PDLLA-only conduit in the nerve regeneration following a 10-mm sciatic nerve injury in rats.We observed the nerve morphology in the early period of regeneration,35 days post injury,using hematoxylin-eosin and methylene blue staining.Compared with the PDLLA conduit,the nerve fibers in the PDLLA-based bioactive nerve conduit were thicker and more regular in size.Muscle fibers in the soleus muscle had greater diameters in the PDLLA bioactive group than in the PDLLA only group.The PDLLA-based bioactive nerve conduit is a promising strategy for repair after sciatic nerve injury.

Preparation of Filling Porous Osteoconduction Materials and Its Animal Experiment Study

Titanium rods were processed into implant samples with cavity and groove in which was filled with HAP/β-TCP porous osteoconduction composite materials in order to increase the mechanical stability of the implant in vivo.The phase compositions of the composite was characterized by X-ray diffraction （XRD） and scanning electron microscopy（SEM）.Histological evaluation showed that the biogradable composite could enhanced the ability of new bone formation.The composite can conduct new bone tissue growing into the cavities gradually after implanted into animal,and then achieve mechanical fixation.The filling biogradable compound exhibited excellent biocompatibility,which combined with the new bone tissues tightly without inflammation and loosing.

3D-printed strontium-incorporatedβ-TCP bioceramic triply periodic minimal surface scaffolds with simultaneous high porosity,enhanced strength,and excellent bioactivity

In bone tissue engineering,scaffolds with excellent mechanical and bioactive properties play prominent roles in space maintaining and bone regeneration,attracting increasingly interests in clinical practice.In this study,strontium-incorporatedβ-tricalcium phosphate(β-TCP),named Sr-TCP,bioceramic triply periodic minimal surface(TPMS)structured scaffolds were successfully fabricated by digital light processing(DLP)-based 3D printing technique,achieving high porosity,enhanced strength,and excellent bioactivity.The Sr-TCP scaffolds were first characterized by element distribution,macrostructure and microstructure,and mechanical properties.Notably,the compressive strength of the scaffolds reached 1.44 MPa with porosity of 80%,bringing a great mechanical breakthrough to porous scaffolds.Furthermore,the Sr-TCP scaffolds also facilitated osteogenic differentiation of mouse osteoblastic cell line(MC3T3-E1)cells in both gene and protein aspects,verified by alkaline phosphatase(ALP)activity and polymerase chain reaction(PCR)assays.Overall,the 3D-printed Sr-TCP bioceramic TPMS structured scaffolds obtained high porosity,boosted strength,and superior bioactivity at the same time,serving as a promising approach for bone regeneration.

Preparation of Biodegradable Mg/β-TCP Biofunctional Gradient Materials by Friction Stir Processing and Pulse Reverse Current Electrodeposition

Mg alloys,as a new generation of biodegradable bone implant materials,are facing two tremendous challenges of enhancing strength and reducing degradation rate in physiological environment to meet clinical needs.In this study,tricalcium phosphate(β-TCP)particles were dispersed in Mg–2 Zn–0.46 Y–0.5 Nd alloy by friction stir processing(FSP)to produce Mg-based functional gradient materials(Mg/β-TCP FGM).On the surface of Mg/β-TCP FGM,the hydroxyapatite(HA)coating was prepared by electrodeposition.The effects of FSP and electrochemical parameter on the microstructure,microhardness,bonding strength and corrosion performance of the Mg/β-TCP FGM were investigated.After four passes of FSP,a uniform and fine-grained structure was formed in Mg/β-TCP and the microhardness increased from 47.9 to 76.3 HV.Compared to the samples withoutβ-TCP,the bonding strength of the Mg/β-TCP FGM increased from 23.1±0.462 to 26.3±0.526 MPa and the addition of degradableβ-TCP contributed to the in situ growth of HA coating.The thickness of HA coating could be dominated by controlling the parameters of electrodeposition.According to the results of immersion tests and electrochemical tests in simulated body fluid,it indicated that the degradation rate of the Mg/β-TCP FGM could be adjusted.