Chemistry,ultrastructure and porosity of monophasic and biphasic bone forming materials derived from marine algae

The research on bioceramics during the last decades has proved that the bioactivity of inorganic bone grafts depends fundamentally on an optimal combination of chemistry and structural porosity. This study presents a comparison of a resorbable monophasic hydroxyapatite (HA) and several newly developed resorbable biphasic hydroxyapatite – ß‐tricalcium phosphate (HA/TCP) composites both derived from naturally grown red marine algae with respect to the phase composition, microstructure and porosity. The highly porous three dimensional mineral scaffold of the native alga is maintained in the final products all investigated materials and possesses a pronounced interconnecting microporous structure. There are generally high values of specific porosity calculated for all tested materials: 1.07 cm³/g for pure phycogenic HA and between 0.65 cm³/g and 1.04 cm³/g for phycogenic biphasic HA/TCP composites with various HA/TCP ratios. The ultrastructure of the phycogenic HA/TCP composites changes significantly with the building and the increase of the ß‐TCP phase due to the bigger polyedric ß‐TCP crystals compared to the finer polycrystalline HA. Despite these structural changes the interconnected porous scaffold is kept throughout the production process. In all investigated materials the porosity is mainly based on pores with pore sizes between 1 and 10 μm in diameter, which is given by the structure of the natural alga. The specific chemistry combined with the structural porosity is decisive for the high in‐vivo bioactivity of the studied materials.     

Effective formulations for the preparation of calcium phosphate bone cements

In the system CaO–P₂O₅–H₂O 13 different solids with varying Ca/P ratios are known. In addition calcium phosphates containing other biocompatible constituents like Na, or K, or Mg or Cl or carbonate, are known. Therefore, a large number of combinations of such compounds is possible which might result in the formation of calcium phosphate cements upon mixing with water. However, the number of calcium phosphates possibly formed by precipitation at room or body temperatures is limited to 12, which should limit the number of suitable combinations. In this study more than 450 different combinations of reactants have been investigated. The results were evaluated on the basis of the following criteria: (a) was the intended reaction product formed? (b) was the final setting time shorter than 60 min? (c) was the compressive strength after soaking for 1 day in Ringer's solution at 37°C higher than 2 MPa? We found that 15 formulations satisfied all of these criteria. The distribution of cements synthesized in this way was 3 DCPD type, 3 CMP type, 6 OCP type and 3 CDHA type cements. The DCPD type cements were acidic during setting and remained that for a long time afterwards. CDHA type cements were neutral or basic during setting, and remained neutral after completion of the reaction. The OCP type cements were neutral both during and after setting. Two CMP type cements were basic both during and after setting. In this study compressive strengths were found up to 90 MPa. Also, in the literature values up to 90 MPa have been reported for this type of cement. Taking into account the excellent biocompatibility and the good osteoconductivity of calcium phosphates and the fact that these calcium phosphate cements can be injected into the site of operation, it may be expected that these materials will become the materials of choice for bone replacement and augmentation. Their suitability for the fixation of metal endoprostheses for joint replacement should be investigated as well.     

Preparation of porous apatite granules from calcium phosphate cement

A versatile method for preparing spherical, micro- and macroporous (micro: 2–10 and macro: 150–550 μm pores), carbonated apatitic calcium phosphate (Ap-CaP) granules (2–4 mm in size) was developed by using NaCl crystals as the porogen. The entire granule production was performed between 21 and 37 °C. A CaP cement powder, comprising α-Ca₃(PO₄)₂ (61 wt.%), CaHPO₄ (26%), CaCO₃ (10%) and precipitated hydroxyapatite, Ca₁₀(PO₄)₆(OH)₂ (3%), was dry mixed with NaCl crystals varying in size from 420 μm to 1 mm. Cement powder (35 wt.%) and NaCl (65 wt.%) mixture was kneaded with an ethanol–Na₂HPO₄ initiator solution, and the formed dough was immediately agitated on an automatic sieve shaker for a few minutes to produce the spherical granules. Embedded NaCl crystals were then leached out of the granules by soaking them in deionized water. CaP granules were micro- and macroporous with a total porosity of 50% or more. Granules were composed of carbonated, poorly crystallized, apatitic CaP phase. These were the first spherical and porous CaP granules ever produced from a self-setting calcium phosphate cement. The granules reached their final handling strength at the ambient temperature through the cement setting reaction, without having a need for sintering. Certain commercial equipment, instruments or materials are identified in this paper to foster understanding. Such identification does not imply recommendation or endorsement by the author, nor does it imply that the equipment or materials identified are necessarily the best available for the purpose.     

Preparation of highly porous hydroxyapatite from cuttlefish bone

Hydroxyapatite structures for tissue engineering applications have been produced by hydrothermal (HT) treatment of aragonite in the form of cuttlefish bone at 200°C. Aragonite (CaCO₃) monoliths were completely transformed into hydroxyapatite after 48 h of HT treatment. The substitution of CO₃ ²⁻ groups predominantly into the PO₄ ³⁻ sites of the Ca₁₀(PO₄)₆(OH)₂ structure was suggested by FT-IR spectroscopy and Rietveld structure refinement. The intensity of the ν₃PO₄ ³⁻ bands increase, while the intensity of the ν₂CO₃ ²⁻ bands decrease with the duration of HT treatment resulting in the formation of carbonate incorporating hydroxyapatite. The SEM micrographs have shown that the interconnected hollow structure with pillars connecting parallel lamellae in cuttlefish bone is maintained after conversion. Specific surface area (S _BET) and total pore volume increased and mean pore size decreased by HT treatment.     

Calcium-phosphate derived from mineralized algae for bone tissue engineering applications

In this work, several routes are described towards obtaining pure inorganic phases derived from Coralline officinallis red algae. The scanning electron microscopy studies have shown that it becomes possible not only to eliminate the undesired organic phase, but also to preserve or tailor the red algae typical microporosity. X-ray diffraction analysis was used to investigate the phase content of the red algae before and after performing the different treatment routes. Hydroxyapatite nanocrystallites were obtained after converting the coralline calcium carbonate skeleton by means of combining thermal and chemical routes. These results were confirmed by Fourier transform infra-red spectroscopic analysis. The processing routes herein described are very promising in order to design bioceramics of algae origin that might find useful applications as bone fillers and tissue engineering scaffolds.     

Preparation and evaluation of osteoinductive porous biphasic calcium phosphate granules obtained from eggshell for bone tissue engineering

The synthetic bone graft substitutes currently used clinically are osteoconductive but not osteoinductive; their low success rate is thus their biggest disadvantage. The use of biomass raw materials for synthesizing calcium phosphate has gradually attracted increased research attention. In this study, hydroxyapatite powder was prepared through liquid-phase precipitation from eggshell, and porous biphasic calcium phosphate granules (EBGs) were then obtained using a pore former and sintering procedure. The EBGs were discovered to have high biocompatibility and no cytotoxicity. The results of animal experiments showed that the area of new bone growth was high, numerous Haversian canals could be observed, and almost all EBGs were surrounded by new bone tissue, which proved that the EBGs had excellent osteoinductivity. By contrast, numerous fat cells were found in the femoral defect area when a commercial bone graft was employed. Various biological inorganic ions (Mg, Sr, Na, and Fe) originally in the eggshell raw materials were incorporated into the EBGs, and the EBGs exhibited excellent osteogenic abilities. The developed approach provides an economical and feasible solution for the treatment of bone defects.     

Controlled release of local anesthetic from calcium phosphate bone cements

Novel lidocaine containing calcium phosphate bone cements have been developed. Lidocaine release kinetics of these cements have been evaluated.Calcium phosphate cements have a great potential for local drug delivery. Release of local anesthetic, such as lidocaine, at the implant site can be useful for reducing pain immediately after implantation.In this work a local anesthetic – lidocaine hydrochloride – was incorporated into α-tricalcium phosphate cement. Lidocaine release profile was dependent on cement components used. All cements were characterized by an initial burst release, which can be correlated with cement pH values, followed by gradual drug release. Drug release continued for up to 6 days and was slower, if cement pH was higher. Addition of lidocaine hydrochloride accelerated setting and changed microstructure of the set cement.     

Fixation of porous calcium phosphate with expanded bone marrow cells using an autologous plasma clot

Multipotent human mesenchymal stromal cells (hMSCs) are currently the most promising cell type for regenerative medicine and tissue engineering. For clinical applications with special focus on fracture repair a method to deliver in vitro‐expanded bone marrow cells to the fracture site is presented. Autologous blood plasma clotting was used to fix expanded cells and synthetic tricalcium phosphate particles. A jelly‐like but solid composite matrix was obtained which could easily be handled by the surgeon. The majority of embedded cells remained viable after the plasma gelation process, as was determined by fluorescence microscopy. Experimental cell culture over a period of 15 days showed proliferating mesenchymal stromal cells within the plasma clot.     

Controlled release of gentamicin from calcium phosphate/alginate bone cement

Calcium phosphate cement (CPC) is a good biomaterial for bone defect repair and as a delivery system for active agents. The aim of this study was to explore the physicochemical properties and in vitro soaking and release behaviors of gentamicin-loaded CPC with and without alginate; in particular, for biocompatibility. MTT colorimetric assay and RT-PCR were used to detect U2OS cell viability and level of cyclooxygenase-2 (COX-2), respectively. As a result, the setting time increased after the addition of 0.5% alginate and 5% gentamicin, reaching 19 min—significantly higher than the 8 min taken by the CPC control, demonstrating the adverse effect of alginate and gentamicin on the setting reaction of CPC. Gentamicin might reduce the diametral tensile strength, while alginate did not affect the strength. The rate of gentamicin release from CPC can be extended by the presence of alginate. The addition of gentamicin did not show signs of impaired cell viability, but alginate enhanced the cell viability. COX-2 expression of U2OSs cultured in the alginate-containing cement extract was about one-third level of the cement extract without alginate. Alginate-containing CPC is not only useful as a reservoir for antibiotic delivery but it also helps stimulate bone regeneration.     

Static and dynamic cultivation of bone marrow stromal cells on biphasic calcium phosphate scaffolds derived from an indirect rapid prototyping technique

The adequate regeneration of large bone defects is still a major problem in orthopaedic surgery. Synthetic bone substitute materials have to be biocompatible, biodegradable, osteoconductive and processable into macroporous scaffolds tailored to the patient specific defect. Hydroxyapatite (HA) and tricalcium phosphate (TCP) as well as mixtures of both phases, biphasic calcium phosphate ceramics (BCP), meet all these requirements and are considered to be optimal synthetic bone substitute materials. Rapid prototyping (RP) can be applied to manufacture scaffolds, meeting the criteria required to ensure bone ingrowth such as high porosity and defined pore characteristics. Such scaffolds can be used for bone tissue engineering (BTE), a concept based on the cultivation of osteogenic cells on osteoconductive scaffolds. In this study, scaffolds with interconnecting macroporosity were manufactured from HA, TCP and BCP (60 wt% HA) using an indirect rapid prototyping technique involving wax ink-jet printing. ST-2 bone marrow stromal cells (BMSCs) were seeded onto the scaffolds and cultivated for 17 days under either static or dynamic culture conditions and osteogenic stimulation. While cell number within the scaffold pore system decreased in case of static conditions, dynamic cultivation allowed homogeneous cell growth even within deep pores of large (1,440 mm³) scaffolds. Osteogenic cell differentiation was most advanced on BCP scaffolds in both culture systems, while cells cultured under perfusion conditions were generally more differentiated after 17 days. Therefore, scaffolds manufactured from BCP ceramic and seeded with BMSCs using a dynamic culture system are the method of choice for bone tissue engineering.     

Lead and copper removal from aqueous solutions by porous glass derived calcium hydroxyapatite

A porous glass was prepared by sintering Na₂O-CaO-B₂O₃ glass powder with powdered sodium chloride. Subsequently, the sodium chloride was dissolved in water resulting in a highly porous material. A sample was prepared consisting of 60 vol% glass and 40 vol% salt which both had particle sizes <100 and 400-500 μm, respectively. The sample was sintered at a temperature of 580 °C for 15 min which resulted in an optimum structure with 57.5% porosity and 0.944 MPa compressive strength. Amorphous calcium hydroxyapatite was formed by immersing these materials in 0.1 M K₂HPO₄ solutions at room temperature for 1 day. The porous glass derived hydroxyapatite matrix was then processed for removing lead and copper ions from aqueous solutions. The results showed that the glass derived calcium hydroxyapatite matrix effectively immobilizes lead and copper ions in solution. The adsorption mechanism was investigated by the X-ray Diffraction (XRD) and Scanning Electron Microscopy including Energy Dispersive X-Ray Spectrometry (SEM-EDX).     

Hybrid analogs for the production of porous calcium phosphate scaffolds

A polymer–inorganic sol mixture has been used to develop interconnected and highly porous calcium phosphate networks. The inorganic sol was developed by reacting triethyl phosphite and calcium nitrate. The sol was directly added to an aqueous solution of PVA with molecular weights between 40,500 and 155,000 g/mol. This mixture was electrospun at a voltage of 20 kV to produce fibers, whose diameter was less than 1 μm. This electrospun structure was calcined at 600 °C obtain to a highly interconnected sub-micron fibrous network (fiber size ∼ 200 nm) of calcium phosphate. The crystal size is on the order of 30 nm. Micropores could be introduced in each of the fibers by controlling the polymer molecular weight and the sol volume fraction. Such structures can have many potential uses in the repair and treatment of bone defects and in drug delivery.     

Fabrication and mechanical testing of porous calcium phosphate bioceramic granules

Porous hydroxyapatite/tricalcium phosphate (HA/TCP) granules were fabricated by a novel technique of vacuum impregnation of reticulated polyurethane (PU) foams with ceramic slip. The resultant granules had 5-10% interconnected porosity with controlled pore sizes necessary to allow bone ingrowth combined with good mechanical properties. Using PU foams with a different number of pores per inch (ppi), porous HA/TCP granules in the size range of 2-8 mm were successfully manufactured. Dieplunger tests were used to compare the compression and relaxation properties of the granules with those of a commercially available bone graft product, BoneSave. The results of the die-plunger testing showed that the experimental granules were stiffer than the BoneSave materials and had less of a tendency to crumble to powder after testing. This therefore suggests that these experimental granules would be useful for impaction grafting and space filling applications.     

Bioactivity of porous biphasic calcium phosphate enhanced by recombinant human bone morphogenetic protein 2/silk fibroin microsphere

To prepare a bioactive bone substitute, which integrates biphasic calcium phosphate (BCP) and rhBMP-2/silk fibroin (SF) microsphere, and to evaluate its characteristics. Hydroxyapatite and β-tricalcium phosphate were integrated with a ratio of 60–40 %. RhBMP-2/SF (0.5 μg/1 mg) microsphere was prepared, and its rhBMP-2-release kinetics was assed. After joining pore-forming agent (Sodium chloride, NaCl), porous BCP/rhBMP-2/SF were manufactured, and its characteristics and bioactivity in vitro were evaluated. Mean diameter of rhBMP-2/SF microsphere was 398.7 ± 99.86 nm, with a loading rate of 4.53 ± 0.08 %. RhBMP-2 was released in a dual-phase pattern, of which fast-release (nearly half of protein released) focused on the initial 3 days, and slow-release sustained more than 28 days. With the increase in concentration of NaCl, greater was porosity and pore size, but smaller mechanical strength of BCP/rhBMP-2/SF. Material with 150 % (w/v) NaCl had an optimal performance, with a porosity of 78.83 %, pore size of 293.25 ± 42.77μm and mechanical strength of 31.03 MPa. Proliferation of human placenta-derived mesenchymal stem cells (hPMSCs) on leaching extract medium was similar to the normal medium (P = 0.89), which was better than that on control group (P = 0.03). Activity of alkaline phosphatase on BCP/rhBMP-2/SF surface was higher than on pure BCP at each time point except at 1 day (P < 0.05). RhBMP-2 has a burst release on early times and a sustaining release on later times. BCP/rhBMP-2/SF with 150 % (w/v) pore-forming agent has excellent porosity, pore size and mechanical strength. The biomaterial induces proliferation and differentiation hPMSCs effectively.     

New biocomposite [biphasic calcium phosphate/ poly-DL-lactide-co-glycolide/biostimulative agent] filler for reconstruction of bone tissue changed by osteoporosis

Biphasic calcium phosphate-poly-DL-lactide-co-glycolide composite biomaterial with and without biostimulative agents (protein-rich plasma or fibrin) was synthesised in the form suitable for reconstruction of bone defects. The composite used as filler was obtained by precipitation in solvent-non-solvent systems. The material, calcium phosphate granules covered by polymer, was characterised by wide-angle X-ray structural analysis, scanning electron microscopy, infrared spectroscopy and differential scanning calorimetry. Reparation of bone tissue damaged by osteoporosis was investigated in vivo on rats. The method applied enabled production of granules of calcium phosphate-poly-DL-lactide-co- glycolide composite biomaterial of average diameter 150–200 μm. Histological analysis confirmed recuperation of the alveolar bone, which osteoporosis-induced defects were repaired using composite biomaterial. By addition of biostimulative agents, intensity of osteogenesis increases accompanied by the formation of regular, new bone structure.     

Synthesis of calcium phosphate hydrogel from waste incineration fly ash and bone powder

Waste incineration fly ash and bone powder could be successfully recycled into calcium phosphate hydrogel, a type of fast proton conductor. Various properties of the intermediate and calcium phosphate hydrogel from them were characterized and compared with that from calcium carbonate reagent. It was found that the intermediate from the incineration fly ash and calcium phosphate glass was more brittle than that from bone powder and calcium carbonate reagent. The electric conductivity of crystallized hydrogel obtained from all raw materials increases exponentially with temperature. However, the crystallized hydrogel from incineration fly ash has lower electric conductivity and lower crystallinity than that from bone powder and the reagent. Moreover, the difference in electric conductivity between these crystallized hydrogels decreases with temperature. Compared with using the reagent as a raw material, bone powder provides a 25% reduction in the usage of H(3)PO(4) to acquire the crystallized hydrogel which has the highest conductivity. These experimental results suggest that the incineration fly ash and bone powder are useful calcium sources for the synthesis of calcium phosphate hydrogel.     

基于纤维素纤维的多孔碳材料的制备及应用

采用选择性表面溶解（SSD）法将纤维素纤维表面部分溶解,固化后形成多孔结构,最后在Ar气氛中炭化制得多孔碳（HPC-SSD）材料,HPC-SSD材料具有大的比表面积和三维多孔结构。通过SEM、BET、FTIR、XRD及电化学测试,系统地研究了针对纤维素纤维的两种活化预处理方法对HPC-SSD材料的形貌、化学组成、比表面积及电化学性能的影响。通过与纤维素纤维直接炭化所得的多孔碳（HPC）材料的相关性能进行比较发现,HPC-SSD材料的成孔过程更加稳定,有利于大量微孔的形成。采用去离子水→丙酮→二甲基乙酰胺对纤维素纤维进行活化预处理,制得的HPC-SSD材料比电容为226 F·g-1（两电极体系）,是HPC材料的4.5倍,比未经过活化预处理的HPC-SSD材料提高了40%。      

Periodontal regeneration in experimentally-induced alveolar bone dehiscence by an improved porous biphasic calcium phosphate ceramic in beagle dogs

Regeneration of lost periodontium is the focus of periodontal therapy. To achieve the effective regeneration, a number of bone graft substitute materials have been developed. This study aimed to investigate the histological response in alveolar bone dehiscences which were filled with an improved biphasic calcium phosphate (BCP) ceramic with more reasonable pore diameter, pore wall thickness and porosity. Twenty-four alveolar bone dehiscences were made surgically in twelve beagle dogs by reflecting mucoperiosteal flaps on the buccal aspect of bilateral lower second premolars and removing alveolar bone. The left dehiscences were treated with BCP ceramic and the contralaterals were cured with the open flap debridement (OFD) as controls. Three dogs were used at week 4, 12, and 24 respectively. Histological observations were processed through three-dimensional micro-computed tomographic imaging, fluorescence and light microscopy. The histological study indicated that the biphasic ceramic was biocompatible, and regeneration was achieved more effectively through the BCP treatment. There were also arrest of epithelial migration apically and formation of new bone and cementum, as well as proliferation of fibrous connective tissues that became attached to the newly formed cementum at week 24, while there was no significant periodontal regeneration in the OFD group only with epithelial tissue migrating into the dehiscence regions. Clinically speaking, though the surgical location formed a limitation to the application of the improved BCP on the periodontal regeneration, the actual result was positive. It proved that the BCP had biocompatibility and was able to act as a stable scaffold to induce periodontal regeneration effectively.