Calcium phosphate cements for bone engineering and their biological properties

Calcium phosphate cements （CPCs） are frequently used to repair bone defects. Since their discovery in the 1980s, extensive research has been conducted to improve their properties, and emerging evidence supports their increased application in bone tissue engineering. Much effort has been made to enhance the biological performance of CPCs, including their biocompatibility, osteoconductivity, osteoinductivity, biodegradability, bioactivity, and interactions with cells. This review article focuses on the major recent developments in CPCs, including 3D printing, injectability, stem cell delivery, growth factor and drug delivery, and pre- vascularization of CPC scaffolds via co-culture and tri-culture techniques to enhance angiogenesis and osteogenesis.

REINFORCEMENT OF CALCIUM PHOSPHATE CEMENTS WITH PHOSPHORYLATED CHITIN

Phosphorylated chitins (P-chitins) as the additives of calcium phosphate cements (CPCs) were prepared by the phosphorylation of chitin with phosphorus pentoxide in methanesulfonic acid. Their physical properties and effects on CPCs from monocalcium phosphate monohydrate (MCPM) and calcium oxide (CaO) or dicalcium phosphate dihydrate (DCPD) and calcium hydroxide [Ca(OH)(2)] were investigated. Addition of P-chitin (M-w = 2.60 x 10(4); degree of substitution, DS = 0.68) to the liquid phase in amounts up to 3 wt.% for MCPM and CaO cements or 1.5 wt.% for DCPD and Ca(OH)(2) cements could enhance the mechanical strength considerably, while little influence on the setting time was observed. However, further addition of P-chitin will cause no setting.

Effect of Additives on the Morphology of the Hydrated Product and Physical Properties of a Calcium Phosphate Cement

The morphology of a hydrated calcium phosphate cement （CPC） doped with several normally used additives was investigated by scanning electron microscopy （SEM） and the compressive strength of the cement was determined in this study. The hydrated products of CPC without additives was rod-like hydroxyapatite （HA） grains with around 2-5 μm in length and 100 nm in width. The addition of Sr obviously decreased the crystal size of the rod-like grains. CPCs containing carbonate, collagen and gelatin showed flake-like crystal morphology. Crylic acid-containing CPC presented flocculus-like structure. And malic acid-containing CPC exhibited oriented flake-like structure. The X-ray diffraction （XRD） analysis showed that the additives used in this study did not alter the hydration products of the cement. The compressive strength tests indicated that the compressive strength of the cement with rod-like morphology HA crystals was much higher than that of the cement with flake-like morphology HA crystals, and the cement with oriented flake-like morphology HA crystals exhibited the poorest compressive strength.

The In-situ Reinforcement of Calcium Phosphate Cement and Its Micro-structural Analysis

Carbon nanotubes （ CNTs ） and polyacrylic acid were employed to modify the setting process and hydration products of β-TCP/TTCP calcium phosphate cement. The micro-structure of hydration product and the fashion of how additives and hydration particles interconnected were investigated. With the modification effect of CNTs , the setting particles and CNTs got winded and interconnected and thus made the composite more compact and denser.

The Effect of Premixed Schedule on the Crystal Formation of Calcium Phosphate Cement-chitosan Composite with Added Tetracycline

In this study, calcium phosphate cements （CPC） were prepared by mixing cement powders of tetracalcium phosphate （TTCP） with a cement liquid of phosphate acid saline solution. Tetracycline （TTC）-CPC, chitosan-CPC and chitosan-TTC-CPC were investigated with different premixed schedule. It was demonstrate that both TTC and chitosan worked on the phase transition and crystal characteristics. TTCP mixed with phosphate acid saline solution had similar features of Fourier transform-infrared spectrometry （FT-IR） no matter it was mixed with chitosan or TTC or both. TTC premixed with cement liquid or powder had significant different features of FT-IR and 876 cm-1 seemed to be a special peak for TTC when TTC was premixed with cement liquid. This was also supported by XRD analysis, which showed that TTC premixed with cement liquid improved phase transition of TTCP to OCP. Chitosan, as organic additive, regulates the regular crystal formation and inhibits the phase transition of TTCP to OCP, except when it is mingled with cement liquid premixed with TTC in field scanning electron microscope. It was concluded that the premixed schedule influences the crystal formation and phase transition, which may be associated with its biocompatibility and bioactivities in vivo.

Microstructure and Mechanical Properties of Calcium Phosphate Cement/Gelatine Composite Scaffold with Oriented Pore Structure for Bone Tissue Engineering

The macroporous calcium phosphate（CPC） cement with oriented pore structure was prepared by freeze casting. SEM observation showed that the macropores in the porous calcium phosphate cement were interconnected aligned along the ice growth direction. The porosity of the as-prepared porous CPC was measured to be 87.6% by Archimede＇s principle. XRD patterns of specimens showed that poorly crystallized hydroxyapatite was the main phase present in the hydrated porous calcium phosphate cement. To improve the mechanical properties of the CPC scaffold, the 15% gelatine solution was infiltrated into the pores under vacuum and then the samples were freeze dried to form the CPC/gelatine composite scaffolds. After reinforced with gelatine, the compressive strength of CPC/gelatine composite increased to 5.12 MPa, around fifty times greater than that of the unreinforced macroporous CPC scaffold, which was only 0.1 MPa. And the toughness of the scaffold has been greatly improved via the gelatine reinforcement with a much greater fracture strain. SEM examination of the specimens indicated good bonding between the cement and gelatine. Participating the external load by the deformable gelatine, patching the defects of the CPC pores wall, and crack deflection were supposed to be the reinforcement mechanisms. In conclusion, the calcium phosphate cement/gelatine composite with oriented rmre structure nrenared in this work might be a potential scaffold for bone tissue engineerinm

Evaluation of Calcium Phosphate Cement As a Root Canal Sealer Filling Material

Calcium phosphate cement for root end sealing was obtained by in mixing alpha-tricalcium phosphate and additives with an aqueous solation of citric. Powder and liquid were mixed at a ratio of 1.25g/mL. The biocompatibility of this material was investigated primarily by subcutaneous implantation tests. Then calcium phosphate cement was used to fill three adult dogs' root canal, both calcium hydroxide paste and hydroxyapatite paste as control. The animals were killed at 4, 12, 20 weeks postoperatively respectively. The effects of different materials on the apical closure, restoration of periapical tissues and adaptability to the dentinal surface were examined by optical and electronic microscope. The observation at 20 weeks shows that the calcium phosphate cement has the potentialities of being a root canal sealer filling material available for pulpless teeth with open-apex and destructive periapical tissue.

Preparation of a Novel Calcium Phosphate Cement Using N-methylene Phosphonic Chitosan as a Gelling Agent

A modified chitosan （ N-methylene phosphonic Chitosan, NMPC） was synthesized to improve solubility and ability to bind calcium ion. The properties of the raw material chitosan and its derivative NMPC were characterised using FTIR , ^1H- NMR . The aim of this study was to enhance the compressive CPC by reinforcing with NMPC. A formulation consisting of CPC powder , buffer solution and gelling agent was used for preparation of the CPC. CPC powder coasisted of tetracalcium phosphate（ TTCP ） and dicalcium phosphate anhydrous （ DCPA ）. NMPC which acted as the gelling ageut was dissohed into KH2PO4-Na2 HPO4 buffer solution. Each specimen in the mold was sandciched between two fritted glass sides and kept for 24 hours. Compressive strengths were determined, the setting product was identified using X-ray diffraction and scanning electron microscopy was used to investigate the hydroxyapatite particles size and porosity. The experimental results showed that the dominating influence on the compressive strengths of CPC-AMPC was the HA panicle size, its uniformity and appropriate porosity.

Microstructure and Mechanical Properties of Calcium Phosphate Cement/gelatine Composite Scaffold with Oriented Pore Structure for Bone Tissue Engineering

In this study,the macroporous calcium phosphate cement with oriented pore structure was prepared by freeze casting.SEM observation showed that the macropores in the porous calcium phosphate cement were interconnected aligned along the ice growth direction.The porosity of the as-prepared porous CPC was measured to be 87.6% by Archimede's principle.XRD patterns of specimens showed that poorly crystallized hydroxyapatite was the main phase present in the hydrated porous calcium phosphate cement.To improve the mechanical properties of the CPC scaffold,the 15% gelatine solution was infiltrated into the pores under vacuum and then the samples were freeze dried to form the CPC/gelatine composite scaffolds.After reinforced with gelatine,the compressive strength of CPC/gelatin composite increased to 5.12 MPa,around 50 times greater than that of the unreinforced macroporous CPC scaffold,which was only 0.1 MPa.And the toughness of the scaffold has been greatly improved via the gelatine reinforcement with a much greater fracture strain.SEM examination of the specimens indicated good bonding between the cement and gelatine.In conclusion,the calcium phosphate cement/gelatine composite with oriented pore structure prepared in this study might be a potential scaffold for bone tissue engineering.

Treatment of critical bone defects using calcium phosphate cement and mesoporous bioactive glass providing spatiotemporal drug delivery

Calcium phosphate cements (CPC) are currently widely used bone replacement materials with excellent bioactivity, but have considerable disadvantages like slow degradation. For critical-sized defects, however, an improved degradation is essential to match the tissue regeneration, especially in younger patients who are still growing. We demonstrate that a combination of CPC with mesoporous bioactive glass (MBG) particles led to an enhanced degradation in vitro and in a critical alveolar cleft defect in rats. Additionally, to support new bone formation the MBG was functionalized with hypoxia conditioned medium (HCM) derived from rat bone marrow stromal cells. HCM-functionalized scaffolds showed an improved cell proliferation and the highest formation of new bone volume. This highly flexible material system together with the drug delivery capacity is adaptable to patient specific needs and has great potential for clinical translation.

Local treatment of osteoporosis with alendronate-loaded calcium phosphate cement

Background A new treatment strategy is to target specific areas of the skeletal system that are prone to clinically significant osteoporotic fractures.We term this strategy as the ＂local treatment of osteoporosis＂.The study was performed to investigate the effect of alendronate-loaded calcium phosphate cement （CPC） as a novel drug delivery system for local treatment of osteoorosis.Methods An in vitro study was performed using CPC fabricated with different concentrations of alendronate （ALE,0,2,5,10 weight percent （wt％））.The microstructure,setting time,infrared spectrum,biomechanics,drug release,and biocompatibility of the composite were measured in order to detect changes when mixing CPC with ALE.An in vivo study was also performed using 30 Sprague-Dawley rats randomly divided into six groups：normal,Sham （ovariectomized （OVX） ＋ Sham）,CPC with 2％ ALE,5％ALE,and 10％ ALE groups.At 4 months after the implantation of the composite,animals were sacrificed and the caudal vertebrae （levels 4-7） were harvested for micro-CT examination and biomechanical testing.Results The setting time and strength of CPC was significantly faster and greater than the other groups.The ALE release was sustained over 21 days,and the composite showed good biocompatibility.In micro-CT analysis,compared with the Sham group,there was a significant increase with regard to volumetric bone mineral density （BMD） and trabecular number （Tb.N） in the treated groups （P ＜0.05）.Trabecular spacing （Tb.Sp） showed a significant increase in the Sham group compared to other groups （P ＜0.01）.However,trabecular thickness （Tb.Th） showed no significant difference among the groups.In biomechanical testing,the maximum compression strength and stiffness of trabecular bone in the Sham group were lower than those in the experimental groups.Conclusions The ALE-loaded CPC displayed satisfactory properties in vitro,which can reverse the OVX rat vertebral trabecular bone microarchitecture and biomechanical properties in vivo.

Reconstruction of orbital defect in rabbits with composite of calcium phosphate cement and recombinant human bone morphogenetic protein-2

Background Calcium phosphate cement （CPC） is a biocompatible and osteoconductive bone substitute, and recombinant human bone morphogenetic protein-2 （rhBMP-2） has strong osteoinductibility, therefore we developed a composite bone substitute with CPC and rhBMP-2 and evaluate its reconstruction effect in rabbit orbital defect.Methods Thirty-six rabbits were randomly divided into two groups and a 5 mmx5 mmx2 mm bone defect in the infraorbital rim was induced by surgery in each orbit （72 orbits in all）. The orbital defects were treated with pure CPC or composite of CPC and rhBMP-2. The osteogenesis ability of different bone substitute was evaluated by gross observation, histological examination, histomorphometrical evaluation, compressive load-to-failure testing, and scanning electron microscope （SEM）.Results Gross observation showed that both bone substitutes were safe and effective for reconstruction of orbital defect. However, histological examination, histomorphometrical evaluation and SEM showed that CPC/rhBMP-2 group had faster speed in new bone formation and degradation of substitute material than CPC group. Compressive load-to-failure testing showed that CPC/rhBMP-2 group had stronger compressive strength than CPC group at every stage with significant difference （P ＜0.05）.Conclusion Composite of CPC/rhBMP-2 is an ideal bioactive material for repairing orbital defect, with good osteoconductibility and osteoinductibility.

Hyaluronic acid facilitates bone repair effects of calcium phosphate cement by accelerating osteogenic expression

Calcium phosphate cements(CPC)are widely anticipated to be an optimum bone repair substitute due to its satisfied biocompatibility and degradability,suitable to be used in minimally invasive treatment of bone defects.However the clinical application of CPC is still not satisfied by its poor cohesiveness and mechanical properties,in particular its osteoinductivity.Hyaluronic acid reinforced calcium phosphate cements(HA/CPC)showed extroadinary potential not only enhancing the compressive strength of the cements but also significantly increasing its osteoinductivity.In our study,the compressive strength of HA/CPC increased significantly when the cement was added 1%hyaluronic acid(denoted as 1-HA/CPC).In the meantime,hyaluronic acid obviously promoted ALP activity,osteogenic related protein and mRNA expression of hBMSCs(human bone marrow mesenchymal stem cells)in vitro,cement group of HA/CPC with 4%hyaluronic acid adding(denoted as 4-HA/CPC)showed optimal enhancement in hBMSCs differentiation.After being implanted in rat tibial defects,4-HA/CPC group exhibited better bone repair ability and bone growth promoting factors,comparing to pure CPC and 1-HA/CPC groups.The underlying biological mechanism of this stimulation for HA/CPC may be on account of higher osteogenic promoting factors secretion and osteogenic genes expression with hyaluronic acid incorporation.These results indicate that hyaluronic acid is a highly anticipated additive to improve physicochemical properties and osteoinductivity performance of CPCs for minimally invasive healing of bone defects.

Sudoku of porous, injectable calcium phosphate cements – Path to osteoinductivity

With the increase of global population,people’s life expectancy is growing as well.Humans tend to live more active lifestyles and,therefore,trauma generated large defects become more common.Instances of tumour resection or pathological conditions and complex orthopaedic issues occur more frequently increasing necessity for bone substitutes.Composition of calcium phosphate cements(CPCs)is comparable to the chemical structure of bone minerals.Their ability to self-set and resorb in vivo secures a variety of potential applications in bone regeneration.Despite the years-long research and several products already reaching the market,finding the right properties for calcium phosphate cement to be osteoinductive and both injectable and suitable for clinical use is still a sudoku.This article is focused on injectable,porous CPCs,reviewing the latest developments on the path toward finding osteoinductive material,which is suitable for injection.

In vitro and in vivo analysis of the biocompatibility of two novel and injectable calcium phosphate cements

Calcium phosphate cements(CPCs)have been widely used as bone graft substitutes for many years.The aim of this study was to evaluate the biocompatibility of two novel injectable,bioactive cements:b-tricalcium phosphate(b-TCP)/CPC and chitosan microsphere/CPC in vitro and in vivo.This was accomplished by culturing mouse pre-osteoblastic cells(MC3T3-E1)on discs and pastes of CPCs.Cell growth,adhesion,proliferation and differentiation were assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and alkaline phosphatase assays as well as by scanning electron microscopy and fluorescence.The effect of CPC paste curing was also evaluated.Implantation of two materials into the muscle tissue of rabbits was also studied and evaluated by histological analysis.Cell analysis indicated good biocompatibility in vitro.The fluorescence assay suggested that the cured material discs had no obvious effect on cell growth,while the curing process did.Histological examination showed no inflammatory cell infiltration into soft tissue.These data suggest that b-TCP/CPC and chitosan microsphere/CPC composites may be promising injectable material for bone tissue engineering.

The Release Behavior,Biocompatibility and Physical Properties of Aid-loaded Strontium Doped Calcium Phosphate Cement

The effect of concurrent attendance of two inhibitors of bone degradation,namely Alendronate(Aid)sodium trihydrate and Strontium(Sr),on Calcium Phosphate Cement(CPC)characteristics was explored.To this aim,5 wt%Strontium and 21 mM Alendronate sodium trihydrate were used in calcium phosphate cement and setting time,ion and drug release were analyzed.RAW264.7 and G cell were cultured on cement samples and Tartrate-Resistant Acid Phosphatase(TRAP),Alkaline phosphatase(ALP)activity and MTT assay were studied.The results of structural analysis indicated that 21 mM Aid did not let the cement set.Therefore,colloidal silica was added to the cement formula and successfully decreased the setting time.In vitro tests showed Sr-loaded sample had a greater inhibitory effect on biocompatibility of G cells than Aid-loaded and Sr-Ald-loaded samples.In addition,the findings about osteoblast MTT and ALP activity indicated that Sr was more effective in osteogenic activity of G cells.The simultaneous presence of Aid and Sr in Calcium Phosphate Cement(CPC)was not as effective in its biocompatibility as the presence of Sr alone.

Factors influencing the drug release from calcium phosphate cements

Thanks to their biocompatibility,biodegradability,injectability and self-setting properties,calcium phosphate cements(CPCs)have been the most economical and effective biomaterials of choice for use as bone void fillers.They have also been extensively used as drug delivery carriers owing to their ability to provide for a steady release of various organic molecules aiding the regeneration of defective bone,including primarily antibiotics and growth factors.This review provides a systematic compilation of studies that reported on the controlled release of drugs from CPCs in the last 25 years.The chemical,compositional and microstructural characteristics of these systems through which the control of the release rates and mechanisms could be achieved have been discussed.In doing so,the effects of(i)the chemistry of the matrix,(ii)porosity,(iii)additives,(iv)drug types,(v)drug concentrations,(vi)drug loading methods and(vii)release media have been distinguished and discussed individually.Kinetic specificities of in vivo release of drugs from CPCs have been reviewed,too.Understanding the kinetic and mechanistic correlations between the CPC properties and the drug release is a prerequisite for the design of bone void fillers with drug release profiles precisely tailored to the application area and the clinical picture.The goal of this review has been to shed light on these fundamental correlations.

Tissue-engineered calcium phosphate cement in rabbit femoral condylar bone defects

Background Calcium phosphate cement （CPC） is a favorable bone-graft substitute, with excellent biocompatibility and osteoconductivity. However, its reduced osteoinductive ability may limit the utility of CPC. To increase its osteoinductive potential, this study aimed to prepare tissue-engineered CPC and evaluate its use in the repair of bone defects. The fate of transplanted seed cells in vivo was observed at the same time. Methods Tissue-engineered CPC was prepared by seeding CPC with encapsulated bone mesenchymal stem cells （BMSCs） expressing recombinant human bone morphogenetic protein-2 （rhBMP-2） and green fluorescent protein （GFP）. Tissue-engineered CPC and pure CPC were implanted into rabbit femoral condyle bone defects respectively. Twelve weeks later, radiographs, morphological observations, histomorphometrical evaluations, and in vivo tracing were performed. Results The radiographs revealed better absorption and faster new bone formation for tissue-engineered CPC than pure CPC. Morphological and histomorphometrical evaluations indicated that tissue-engineered CPC separated into numerous small blocks, with active absorption and reconstruction noted, whereas the residual CPC area was larger in the group treated with pure CPC. In the tissue-engineered CPC group, in vivo tracing revealed numerous cells expressing both GFP and rhBMP-2 that were distributed in the medullar cavity and on the surface of bony trabeculae. Conclusion Tissue-engineered CPC can effectively repair bone defects, with allogenic seeded cells able to grow and differentiate in vivo after transplantation.

Preparation and characterization of a novel injectable strontium-containing calcium phosphate cement with collagen

Purpose： To develop a novel injectable strontium-containing calcium phosphate cement with collagen. Methods： A novel calcium phosphate bone cement （CPC） was prepared with the addition of strontium element, collagenl, and modified starch; the injectability, solidification time, microstructure, phase composition, compressive strength, anti-collapsibility and histological properties of material were evaluated. Results： The results showed that the material could be injected with an excellent performance; the modified starch significantly improved the anti-washout property of cement; with the liquid to solid ratio of 0.3, the largest compressive strength of cement was obtained （48.0 MPa _＋ 2.3 MPa）; histological examination of repair tissue showed that the bone was repaired after 16 weeks; the degradation of cement was consistent with the new bone growth. Conclusion： A novel injectable collagen-strontium-containing CPC with excellent compressive strength and suitable setting time was prepared, with addition of modified starch. The CPC showed a good anti-washout property and the degradation time of the cement met with the new bone growing. This material is supposed to be used in orthopedic and maxillofacial surgery for bone defects.