Bioactivation of calcium deficient hydroxyapatite with foamed gelatin gel. A new injectable self-setting bone analogue

An alternative approach to bone repair for less invasive surgical techniques, involves the development of biomaterials directly injectable into the injury sites and able to replicate a spatially organized platform with features of bone tissue. Here, the preparation and characterization of an innovative injectable bone analogue made of calcium deficient hydroxyapatite and foamed gelatin is presented. The biopolymer features and the cement self-setting reaction were investigated by rheological analysis. The porous architecture, the evolution of surface morphology and the grains dimension were analyzed with electron microscopy (SEM/ESEM/TEM). The physico-chemical properties were characterized by X-ray diffraction and FTIR analysis. Moreover, an injection test was carried out to prove the positive effect of gelatin on the flow ensuing that cement is fully injectable. The cement mechanical properties are adequate to function as temporary substrate for bone tissue regeneration. Furthermore, MG63 cells and bone marrow-derived human mesenchymal stem cells (hMSCs) were able to migrate and proliferate inside the pores, and hMSCs differentiated to the osteoblastic phenotype. The results are paving the way for an injectable bone substitute with properties that mimic natural bone tissue allowing the successful use as bone filler for craniofacial and orthopedic reconstructions in regenerative medicine.     

可注射磷酸钙骨水泥及其应用

可注射磷酸钙骨水泥作为一种新型人工骨替代材料,以其良好的生物相容性和骨传导性被广泛应用于临床骨缺损和牙缺损的修复.本文介绍了可注射磷酸钙骨水泥的种类和特性,指出了存在的问题和应用前景.     

用于骨缺损修复的新型可注射自固化复合材料

硫酸钙骨水泥具有良好的骨传导性,但降解速率快、生物活性差的缺点限制了其临床应用.本文将β-磷酸三钙纳米颗粒(粒径43.8±9.0 nm)和半水硫酸钙颗粒(粒径5–21μm)混合作为固相,与液相聚乙烯醇溶液(5 wt.%)按优化重量比混匀,制备了可注射自固化复合材料.该材料具有合理的自固化时间(11.7–19.2 min)及适宜的压缩强度(2.28–6.33 MPa).同时,利用镁颗粒作为成孔剂,制备出大孔径(大于100μm)的多孔支架.体外细胞实验显示,MC3T3-E1细胞伸展良好,表现出大量的板状伪足和伸展的丝状伪足,表明该复合材料无细胞毒性.将可注射复合材料植入比格犬股骨髁缺损区,10个月后骨缺损愈合良好,表明该材料具有良好的骨缺损修复潜力.     

Tissue reaction against a self-setting calcium phosphate cement set in bone or outside the organism

Calcium phosphate cements are able to set in situ when injected into bone tissue. We evaluated the tissue reaction occurring when a DCPD-based calcium phosphate cement was either set within the bone or implanted when already set. The samples were implanted in rabbit condyles and examined histologically after 8 and 16 weeks. The relative bone surface, the fibrous capsule around the implants and the implant section surface were measured. Solid material seemed to be better tolerated than paste implants. More bone was found at the solid implant contact whatever the implantation time and the solid material degraded much less rapidly. In conclusion, the physico-chemical modification of the biological environment occurring during setting increases the foreign body reaction against the material.     

一种可注射可降解磷酸钙骨水泥的结构与性能

通过采用部分结晶磷酸钙和磷酸氢钙制备了新型可注射可降解磷酸钙骨水泥.研究表明:该材料具备优良的可注射性能,并通过添加变性淀粉,显著改善了材料的抗溃散性能,骨水泥的水化产物是直径和长度在分别约为100和1000nm左右的棒状类骨羟基磷灰石.所研制的骨水泥在体温(37℃)条件下凝结较快,而在室温(25℃)和冷藏温度(5℃)可在较长时间保持不固化,这就为骨水泥的临床应用提供了很有利的条件.体外溶血试验、体外细胞毒试验、热原性试验、小鼠的急性毒性试验、微核试验、豚鼠的致敏性试验、小鼠的肌内埋植试验及兔的骨内埋植试验等一系列毒性及生物相容性试验表明该材料无毒副作用,具有良好的生物相容性.复合rhBMP-2的可注射磷酸钙骨水泥植入猕猴椎体后的近远期影像学和组织学观察表明,骨水泥可降解且降解和新骨长入基本同步.     

Study on injectable and degradable cement of calcium sulphate and calcium phosphate for bone repair

Injectable calcium sulphate/phosphate cement (CSPC) with degradable characteristic was developed by introduction of calcium sulphate (CS) into calcium phosphate cement (CPC). The setting time, compressive strength, composition, degradation, cells and tissue responses to the CSPC were investigated. The results show that the injectable CSPC with optimum L/P ratio exhibited good injectability, and had suitable setting time and mechanical properties. Furthermore, the CSPC had good degradability and its degradation significantly faster than that of CPC in Tris–HCl solution. Cell culture results indicate that CSPC was biocompatible and could support MG63 cell attachment and proliferation. To investigate the in vivo biocompatibility and osteogenesis, the CSPC were implanted in the bone defects of rabbits. Histological evaluation shows that the introduction of CS into CPC enhanced the efficiency of new bone formation, and CSPC exhibited good biocompatibility, degradability and osteoconductivity with host bone in vivo. It can be concluded that the injectable CSPC had a significant clinical advantage over CPC, and might have potential to be applied in orthopedic, reconstructive and maxillofacial surgery, especially for minimally invasive techniques.     

Fabrication of calcium phosphate-calcium sulfate injectable bone substitute using chitosan and citric acid

In this study, an injectable bone substitute (IBS) consisting of citric acid, chitosan solution as the liquid phase and tetra calcium phosphate (TTCP), dicalcium phosphate anhydrous (DCPA) and calcium sulfate hemihydrate (CSH) powders as the solid phase was prepared. Four groups containing different percentages (0–30%) of calcium sulfate hemihydrate (CSH, CaSO₄ · 0.5H₂O) were investigated. Initial setting times for IBS with CSH were longer than those without CSH. The setting times for all compositions were in the range of 25–45 min. The injectability was improved by the addition of CSH in the present system. Scanning electron microscopy images showed that fiber-like crystallization appeared in the cements. The enhancement of crystallinity was confirmed by XRD profiles where the peak intensity of HAp increased with incubation time and the addition of CSH. Also, the compressive strength increased with the addition of CSH. The maximum compressive strength obtained for IBS was with 20% CSH after 28-day incubation in 100% humidity at 37°C.     

Optimisation of the mechanical and handling properties of an injectable calcium phosphate cement

Calcium phosphate cements have the potential to be successful in minimally invasive surgical techniques, like that of vertebroplasty, due to their ability to be injected into a specific bone cavity. These bone cements set to produce a material similar to that of the natural mineral component in bone. Due to the ceramic nature of these materials they are highly brittle and it has been found that they are difficult to inject. This study was carried out to determine the factors that have the greatest effect on the mechanical and handling properties of an apatitic calcium phosphate cement with the use of a Design of Experiments (DoE) approach. The properties of the cement were predominantly influenced by the liquid:powder ratio and weight percent of di-sodium hydrogen phosphate within the liquid phase. An optimum cement composition was hypothesised and tested. The mechanical properties of the optimised cement were within the clinical range for vertebroplasty, however, the handling properties still require improvement.     

Preparation and properties of calcium sulfate bone cement incorporated with silk fibroin and Sema3A-loaded chitosan microspheres

To search for new bioactive materials which can be used as the substitute of bone repairing and drug carriers, Sema3A-loaded chitosan microspheres (SLCM) and silk fibroin (SF) were mixed with calcium sulfate cement (CSC). SEM, particle size analysis and swelling rate determination were performed to study properties of the microspheres. The drug loading, encapsulation efficiency and drug release rate were determined by ELISA. Microspheres with different SLCM weight contents (0.5%, 1% and 5%) were prepared to determine which one has the strongest mechanical properties and the appropriate setting time. It was revealed that CSC/SF/0.5SLCM has satisfactory mechanical properties, and its in vitro biocompatibility was assessed by MTS. Chitosan microspheres (5--18 μm) were globular, the surface was smooth, and the swelling rate is (77.02±5.57)%. With this formula, the setting time was increased with the addition of SLCM in CSC/SF, and the cumulative drug release rate is 44.62% in 28 d. XRD results demonstrate that the main component is calcium sulfate. Also it was found that CSC/SF/0.5SLCM supports the growth of MC3T3 cells. Thus the preparation of CSC/SF/0.5SLCM was reliable, and the products had good structures, physical properties and biocompati-bility, appearing to be a promising bone substitute material.     

Premixed injectable calcium phosphate cement with excellent suspension stability

Premixed injectable calcium phosphate cement (p-ICPC) pastes have advantages over aqueous injectable calcium phosphate cement (a-ICPC) because p-ICPC remain stable during storage and harden only after placement into the defect. This paper focused on the suspension stability of p-ICPC paste by using fumed silica as a stabilizing agent and propylene glycol (PEG) as a continuous phase. Multiple light scanning techniques were first applied to evaluate the suspension stability. The results indicated that fumed silica effectively enhanced the suspension stability of p-ICPC pastes. The stabilizing effect of fumed silica results from the network structure formed in PEG because of its thixotropy. The p-ICPC could be eventually hydrated to form hydroxyapatite under aqueous circumstances by the unique replacement between water and PEG. p-ICPC (1) not only possesses proper thixotropy and compressive strength but has good injectability as well. p-ICPC (1) was cytocompatible and had no adverse effect on the attachment and proliferation of MG-63 cells in vitro. These observations may have applicability to the development of other nonaqueous injectable biomaterials for non-immediate filling and long-term storage.     

Physical properties and self-setting mechanism of calcium phosphate cements from calcium bis-dihydrogenophosphate monohydrate and calcium oxide

An apatitic calcium phosphate cement was developed from calcium bis-dihydro-genophosphate monohydrate (or monocalcium phosphate monohydrate, MCPM) and calcium oxide (CaO). The powder had a Ca/P molar ratio of 1.67, and the liquid was either pure water or 0.25 M–1 M sodium phosphate buffer, pH 7.4. The influence of the powder-to-liquid (P/L) ratio on the setting time and the mechanical strength were studied. The best results were obtained for the 1 M phosphate buffer with a P/L ratio of 1.53; the setting time was 7 min and the compressive strength was 25 MPa after 24 h and 33 MPa after 11 d. The mechanism and kinetics of the setting reaction were investigated by X-ray diffraction, differential scanning calorimetry, 31P magic angle spinning–nuclear magnetic resonance and infrared spectrometry. The setting reaction was found to be biphasic: in the first step, during the mixing time, MCPM reacted with CaO immediately to give calcium hydrogenophosphate dihydrate (or dicalcium phosphate dihydrate, DCPD) which, in the second step, reacted more slowly with the remaining CaO to give hydroxyapatite. The conversion of the starting materials to hydroxyapatite was complete within 24 h when the liquid was water, but was slower and incomplete with the phosphate buffers. Of the starting materials, 30% remained after 3 d. © 1999 Kluwer Academic Publishers.     

Preparation and characterization of injectable calcium phosphate cement paste modified by polyethylene glycol-6000

An ICPC with high structure recoverability and paste stability was successfully developed directly incorporating PEG-6000 into the liquid phase of CPC. The rheological behavior of ICPC was investigated with rheometric scientific ARES902-30004 controlled strain rheometer. Novel approaches of flow rate, shear thinning index (SI), shear stress slowdown (Δτ) and thixotropy loop area have been applied to assess the injectability and structure recoverability of the ICPC paste. The addition of PEG-6000 to ICPC resulted in a thixotrophic structure with shortened setting time, slightly increased viscosity, larger thixotropic hysteresis loop area and lower Δτ, with the improvement largely dependent on the PEG-6000 content. With acceptable injectability and shortened setting time, ICPC (1%) showed the lowest Δτ and the highest SI, endowing the paste good structure recoverability and paste stability. The ICPC (1%) was bioactive and facilitated cell attachment and proliferation. The optimized ICPC (1%) paste with a relatively good structure stability and paste stability may serve as a good candidate for tooth root-canal fillings and percutaneous vertebroplasty in microinvasive surgery.     

Novel calcium phosphate composite bone cement: strength and bonding properties

A new high-strength cement prepared from calcium phosphate and calcium aluminate has been developed and was evaluated for potential use in bone and joint repair applications. Cement specimens were aged under simulated physiological conditions. The compressive strength of the cement was determined at time intervals 1 h after setting up to 52 weeks. A compressive strength of 111.6±12.9 MPa was measured at 4 weeks, with the cement attaining 64% of this maximum strength within 4 h of preparation. Compressive strength greater than 90 MPa was maintained up to 52 weeks. The strength of the cement–prosthesis interface was studied using a pull-out test. Polished, 316L stainless steel rods were implanted in canine cadaver femurs to simulate a cemented hip prosthesis. At 4, 24 h, and 60 days post implantation, the force required to displace the rod was measured. Mean interfacial shear strengths of 1.17±0.25, 1.11±0.21, and 1.11±0.32 MPa were observed at respective time-periods.     

Improved mechanical properties of acrylic bone cement with short titanium fiber reinforcement

Acrylic bone cements are widely used in total joint arthroplasties to grout the prosthesis to bone. The changes in the tensile properties and fracture toughness of polymethylmethacrylate (PMMA) bone cements obtained by the addition of control and heat treated short titanium fibers are studied. Heat treatment of titanium fibers is conducted to precipitate titania particles on the fiber surface, which may improve the biocompatibility of the metal. Control (non-heat treated) and heat treated short titanium fibers (250 μm long and 20μm diameter) were used as reinforcements at 3 volume %. X-ray diffraction indicated the presence of a rutile form of titania due to the heat treatments. Results indicate that the tensile and fracture properties of unfilled bone cement were improved by the addition of control and heat-treated fibers. The fracture properties of bone cements reinforced with control titanium fibers were at least 10% higher than those reinforced with heat treated titanium fibers. Therefore, we recommend further studies on the use of non-heat treated titanium fibers to reinforce acrylic bone cement.     

Improved mechanical properties of acrylic bone cement with short titanium fiber reinforcement

Acrylic bone cements are widely used in total joint arthroplasties to grout the prosthesis to bone. The changes in the tensile properties and fracture toughness of polymethylmethacrylate (PMMA) bone cements obtained by the addition of control and heat treated short titanium fibers are studied. Heat treatment of titanium fibers is conducted to precipitate titania particles on the fiber surface to improve the biocompatibility of the metal. Control and heat treated short titanium fibers (250 μ long and 20 μ diameter) were used as reinforcements at 3 volume %. X-ray diffraction indicated the presence of a rutile form of titania due to the heat treatments. The tensile and fracture properties were improved by the addition of fibers. Bone cements reinforced with titanium fibers heated at 550^∘C for 1 h followed by 800^∘C for 30 minutes show the largest increase in fracture toughness along with the smallest changes in elastic modulus and needs to be further investigated.     

Production of in-situ macropores in an injectable calcium phosphate cement by introduction of cetyltrimethyl ammonium bromide

In the present study, cetyltrimethyl ammonium bromide (CTAB) was introduced to an injectable calcium phosphate cement (CPC) to produce macropores during the setting process to accelerate the absorbing ability in vivo. The effects of CTAB on the rheological properties, injectability, setting time, compressive strength, phase evolution, microstructure and degradation rate of CPC were studied. The results showed that the addition of CTAB increased the viscosity and yield stress, and decreased the injectability of the cement pastes. The macroporosity and total porosity increased and the compressive strength of the cement obviously decreased with the increase of CTAB. The macroporosity of the CPC prepared at 5 mM CTAB solution reached 44.2 +/- 2.5% and the mass loss of the cement increased almost 50% as compared with the cement without CTAB. Considering the injectability, compressive strength and degradation rate of CPC, the preferred CTAB concentration was 5 mM. The injectable CPC with macropores is promising to be used in minimally invasive approach.     

Controllable release of salmon-calcitonin in injectable calcium phosphate cement modified by chitosan oligosaccharide and collagen polypeptide

The aim of this research is to study the effect of the controlled releasing character of the salmon calcitonin (S-CT) loaded injectable calcium phosphate cement (CPC) modified by adding organic phase, chitosan oligosaccharide (CO) and collagen polypeptide (CP). The uniform design was used to determine the basic formulation with suitable injectable time for clinical application, and then the changes of the physical characters, the controlled releasing character of the modified CPC along with the ratio of the organic phase were also evaluated in vitro. The surface morphous of the modified CPC been implanted in the abdominal cavity or soaked into the serum of rat was also observed by scanning electron microscope (SEM). The result shows that a suitable formulation of modified CPC could be got, and the injectable time is 12 min, the compressive strength is 12 MPa, and the final setting time is 40 min. Comparing with the CPC without organic phase, the releasing rate of S-CT would increase along with the increase of the organic phase after 7th day. Therefore, a novel S-CT loaded bioactive injectable CPC for treating osteoporosis induced bone defect was obtained, and the release of the containing S-CT was controlled easily through adjusting the ratio of CO and CP.     

Influence of curing temperatures on the hydration of calcium aluminate cement/Portland cement/calcium sulfate blends

In this paper, the effects of curing temperature on the hydration of calcium aluminate cement (CAC) dominated ternary binders (studied CAC: Portland cement: calcium sulfate mass ratio were 22.5: 51.7: 25.8) were estimated at 0, 10, 20 and 40 °C, respectively. Both α-hemihydrate and natural anhydrite were employed as the main source of sulfate. The impacts of temperature on the phase assemblages, morphology and pore structure of pastes hydrated up to 3 days were determined by using X-ray diffraction (XRD), backscattered electron imaging (BEI) and mercury intrusion porosimetry (MIP). Results reveal that the main hydration products are firmly related to calcium sulphoaluminate based phases. Increasing temperature would result in a faster conversion from ettringite to plate-like monosulfate for both calcium sulfate doped systems. When the temperature increases to 40 °C, an extraordinary formation of strätlingite (C₂ASH₈) and aluminium hydroxide is observed in anhydrite doped pastes. Additionally, increased temperature exerts different effects on the pore structure, i.e. the critical pore diameter shifts to finer one for pastes prepared with α-hemihydrate, but changes to coarser one for those made with anhydrite. From the mechanical point of view, increased temperature accelerates the 1-day strength development prominently, while exerts marginal influence on the development of 3-day strength.     

Morphological regulation of calcium sulfate hemihydrate from phosphogypsum

The calcium sulfate hemihydrate whiskers were prepared from phosphogypsum at hydrothermal condition. The influences of different organic acid (citric acid and poly (acrylic acid)) on the crystal morphology were detected, respectively. The morphology of crystals can be influenced by adding organic acid. Short and large columnar crystals can be obtained by adding 5 wt.% citric acid, which can be used as cementitious materials. The long columnar crystals with uniform diameter can be obtained by adding poly (acrylic acid), which can be applied in composites.     

Development of a fully injectable calcium phosphate cement for orthopedic and dental applications

A study on the development of a fully injectable calcium phosphate cement for orthopedic and dental applications is presented. The paper describes its characteristic properties including results of biocompatibility studies. A conventional two-component calcium phosphate cement formulation (having a powder part containing dry mixture of acidic and basic calcium phosphate particles and a liquid part containing phosphate solution) is modified with a biocompatible gelling agent, to induce flow properties and cohesion. The quantity of the gelling agent is optimized to get a viscous paste, which is smoothly injectable through an 18-gauge needle, with clinically relevant setting parameters. The new formulation has a setting time of 20 min and a compressive strength of 11 MPa. The X-ray diffraction, Fourier transform infrared spectrometry, and energy dispersive electron microprobe analyses showed the phase to be hydroxyapatite, the basic bone mineral. Scanning electron microscopy revealed a porous structure with particle sizes of a few micrometers. The cement did not show any appreciable dimensional or thermal change during setting. The injectability is estimated by extruding through needle and the cohesive property is assessed by water contact method. The cement passed the in vitro biocompatibility screening (cytotoxicity and haemolysis) tests.