Hydroxyapatite Granules Research Articles

Osteopontin and bone repair in rabbit tibial defect

Human osteopontin (OPN) was produced by a recombinant technique. A circular defect was created in each tibia of 30 adult rabbits. The animals were divided in six equal groups. Four animals of each group were randomly chosen as experimental group in which OPN coated hydroxyapatite granules (OPN-HA) and non-coated hydroxyapatite (HA) granules were inserted alternatively in created defects. One animal of each group was used as control animal to observe the spontaneous healing process of the defects. The created defects in these animals did not receive any implants. The animals were sacrificed after 1, 2, 6, 12, 18 and 30 weeks. The histological sections were magnified and scanned digitally. In the first measurement, the total amount of bone formation in the entire defect was calculated. In a second measurement, new bone formation was quantified at the edges and at the centre of the defects. The total amount of bone formation in OPN treated defects was significantly higher, whereas no significant difference of bone formation was observed at the edges or at the centre of the defects. A careful interpretation of these results suggests that human OPN might stimulate bone formation in the rabbit tibia.

Characterization of established cementoblast-like cell lines from human cementum-lining cells in vitro and in vivo

To study cellular characteristics of human cementoblasts using a cellular model is important for understanding the mechanisms of homeostasis and regeneration of periodontal tissues. However, at present no immortalized human cementoblast cell line has been established due to limitation of the life span. In the present study, therefore, we attempted to establish human cementoblast-like cell lines by transfection with telomerase catalytic subunit hTERT gene. Two stable clones (HCEM-1 and -2) with high telomerase activity were obtained and they grew over 200 population doublings without significant growth retardation. The expression of mRNA for differentiation markers, type I collagen, alkaline phosphatase (ALP), runt-related transcription factor 2, osteocalcin, bone sialoprotein and cementum-derived protein was revealed in these clones by RT-PCR. Moreover, these cells showed high ALP activity and calcified nodule formation in vitro. Interestingly, HCEM-2 showed cementum like formation on the surface of hydroxyapatites granules by subcutaneous transplantation into immunodeficient mice with hydroxyapatite granules. Thus, we established human cementoblast-like cell lines. We suggest that HCEM cell lines can be useful cell models for investigating the characteristics of human cementoblasts.

Particle Size of Hydroxyapatite Granules Calcified from Red Algae Affects the Osteogenic Potential of Human Mesenchymal Stem Cells in vitro

Hydroxyapatite (HA) microparticles as a carrier in an injectable tissue-engineered bone filler are considered promising candidates for the treatment of small bone defects in the craniomaxillofacial region. HA granules calcified from red algae, varying in size, were evaluated in vitro for their suitability to be used as a carrier for human mesenchymal stem cells (hMSCs). Three groups of granules were produced in grain sizes of 10–100, 200–500 and 600–1,000 µm. After seeding and culturing hMSCs under osteogenic differentiation conditions onto HA particles for 3, 6 and 9 days, cellular proliferation (tetrazolium salt, XTT), alkaline phosphatase (ALP)-specific activity and total protein synthesis were investigated. The osteoblastic phenotype of the cells was evaluated by assaying the bone-specific genes osteocalcin, osteopontin and collagen type I. XTT assay revealed significantly higher (p < 0.01) proliferation of cells grown on the smallest grain size after 9 days of culture. Regarding ALP-specific activity, significantly higher levels of activity were detected in cells grown on the smallest grain size. Different grain sizes had no significant effects on the secretion of osteocalcin and osteopontin. Collagen type I production was significantly higher (p < 0.05) in cells grown on the biggest grain size in comparison with the two other grain sizes. These results show that the particle size of HA microparticles affects the osteogenic potential of cultured hMSCs and lead to the conclusion that particle size has differential effects on ALP-specific activity and collagen type I production.

Initial Stability of the Stem Fixed by Impaction Grafting Technique with Hydroxyapatite Granules in Revision THA

In revision THA with bone defects, impaction bone grafting is one proposed solution. Morsellized allografts are commonly used, however allograft has some disadvantages. We tried to use hydroxyapatite (HA) granules in stead of morsellized allografts. Three types of HA granules were prepared. To facilitate the manipulation of HA granules, we tried to add arginine amino acid as glue. These granules were impacted into the plastic femoral canal and collarless polished tapered stem was fixed with PMMA bone cement. Cyclic compression test and torsional test were performed using an Instron type mechanical tester. Mechanical stability of the stem fixed with HA granules for impaction grafting was superior to morsellized allografts. Adding arginine amino acid to HA granules, impaction technique became easier and the initial stability of the stem was improved.

Improved Malar Projection with Transconjunctival Hydroxyapatite Granules

Augmentation of the zygomatic body enhances appearance and provides a more youthful look. Porous hydroxyapatite granules offer an alternative to alloplastic implants. Hydroxyapatite granules were placed by means of a transconjunctival approach into subperiosteal malar pockets (n = 8). From the preoperative, postoperative, and 1-year follow-up lateral views, malar projection was measured as the right angle distance from the point of malar prominence to the nasale-subnasale line. Patients were either very satisfied (six of eight) or satisfied (two of eight). Malar projection was significantly improved postoperatively and was maintained after 1 year. This technique, performed for cosmetic and reconstructive indications, resulted in measurable improvement in malar projection, minimal complications, and optimal patient satisfaction.

Porous Granules of β-Tricalcium Phosphate Composed of Rod-Shaped Particles

Porous granules of β-tricalcium phosphate (β-Ca3(PO4)2; β-TCP) were prepared from porous hydroxyapatite granules with calcium deficient composition synthesized by hydrothermal method. The β-TCP granules were composed of rod-shaped particles of about 10-20 µm in length. Rod-shaped particles were locked together to make micro-pores, and the size of micro-pores formed by tangling of rod-shaped particles was about 0.1-0.5 µm. The granule size, particle size, and micro-pore size could be controlled by our unique method. The porous granules of β-TCP must be suitable for the bone graft material and for scaffold of cultured bone.

Control of bisphosphonate release using hydroxyapatite granules

The efficacy of hydroxyapatite (HAp) as a carrier was investigated to establish a method of local administration of bisphosphonates (Bps), which has currently been administered systemically. HAp granules (300-500 microm in size) with different physicochemical features were prepared by altering the sintering temperature. To ascertain the physicochemical properties of the HAp granules, their crystallinity was assessed using X-ray diffraction, the surface morphology was examined under scanning electron microscopy, and the specific surface area and calcium dissolution were evaluated. Different Bps-HAp composites were subsequently prepared and the concentration of Bps released from these composites was measured. The influence of Bps-HAp composites on the rate of osteoclast survival was also evaluated. The results revealed that (1) HAp solubility depends on the sintering temperature; (2) The concentration of released Bps could be controlled by regulating the sintering temperature of HAp as a carrier; and (3) Bps released from Bps-HAp composites reduced the number of osteoclasts. These findings indicated that Bps-HAp composites could be locally administered as a drug delivery system to areas with bone resorption.

Treatment of Kienböck’s disease with cultured stem cell-seeded hybrid tendon roll interposition arthroplasty: experimental study

BackgroundSeveral procedures that consist of excision arthroplasty, intercarpal arthrodesis with or without excision of the lunate, proximal row carpectomy, and joint leveling procedures are used to treat advanced Kienböck’s disease. However, no reconstructive surgery for a collapsed lunate has been established. The aim of this study was to develop a method of tendon roll interposition arthroplasty using a tissue-engineering technique to improve the clinical results of the tendon roll interposition arthroplasty for treating advanced Kienböck’s disease. MethodsTwenty-four Japanese white rabbits were used for this study as three treatment models of Kienböck’s disease. The lunate of the right paw was excised, and then one of three kinds of tendon roll was interposed into the excision space as follows: group A (controls), a conventional tendon roll made of autologous Achilles tendon; group B (cored tendon roll) a tendon roll with a core of the collagen-ceramic composite; and group C (hybrid tendon roll), a scaffold seeded with cultured cells was rolled in the Achilles tendon. In all groups the right wrist was immobilized with cast 6weeks after surgery. Wrist radiography was evaluated 0, 4, 6, and 12weeks after surgery; specimens were sectioned and evaluated histologically 12weeks after surgery. ResultsIn group C the small shadow of the hydroxyapatite granule gradually increased until 12weeks after surgery, whereas in group B the shadow of the core gradually decreased. The deficit spaces of the lunate remained radiographically lucent for 12weeks after surgery in group A. Histological findings revealed new bone formation at the center and stained cartilage matrix at the peripheral of the hybrid tendon roll group (group C) but not in group A or B. ConclusionsRadiological and histological examinations proved that it is possible to make new hybrid tendon rolls using this method, as osteogenesis in the center and cartilage matrix in the peripheral of the tendon roll were revealed.

Composites between hydroxyapatite and poly(ε-caprolactone) synthesized in open system at room temperature

The hydroxyls present on the surface of hydroxyapatite (HA) granules, annealed at 700 composite function, 900 composite function and 1,100 composite function C, are able to initiate the polymerization of epsilon-caprolactone (CL), not only at 185 composite function C under vacuum, but also at room temperature in open system. A polymer layer ionically linked to the substrate is formed on HA surface, enhancing the compatibility between the organic phase and the inorganic one in composite biomaterials. We studied the characteristics of the polymer, produced by the reaction carried out at room temperature in open system, as well as the percentages of the poly(epsilon-caprolactone) (PCL) ionically bonded to the HA structure and of the "free" one. Both percentages appear very dependent on the annealing temperature; in particular, HA annealed for 1 h at 1,100 composite function C is the most efficient initiator of the reaction leading to ionically bonded PCL. The percentages of "free" polymer are much higher than at 185 composite function C under vacuum. Its formation is attributed to the role of water in opening the CL rings, and to the presence of CO(3) (2-) and HPO(4) (2-) ions in the HA annealed at lower temperatures. The presence of water appears to be the limiting factor for the production of PCL not bonded to the HA structure.

Hydroxyapatite scaffolds for bone tissue engineering made by 3D printing

Nowadays, there is a significant need for synthetic bone replacement materials used in bone tissue engineering (BTE). Rapid prototyping and especially 3D printing is a suitable technique to create custom implants based on medical data sets. 3D printing allows to fabricate scaffolds based on Hydroxyapatite with complex internal structures and high resolution. To determine the in vitro behaviour of cells cultivated on the scaffolds, we designed a special test-part. MC3T3-E1 cells were seeded on the scaffolds and cultivated under static and dynamic setups. Histological evaluation was carried out to characterise the cell ingrowth. In summary, the dynamic cultivation method lead to a stronger population compared to the static cultivation method. The cells proliferated deep into the structure forming close contact to Hydroxyapatite granules.

Effect of carbonate substitution on the ultrastructural characteristics of hydroxyapatite implants

Carbonate ion substitution has been shown to be beneficial for increasing the amount of in vivo osseointegration to hydroxyapatite (HA). Nevertheless, mechanisms by which carbonate ions increase in vivo bioactivity are not fully understood. Sintered granules of HA and carbonate-substituted hydroxyapatite (CHA) were implanted for 6 and 12 weeks in an ovine model. Samples containing the bone-implant interface were prepared for transmission electron microscopy (TEM) and TEM was used to compare the in vivo reactivity of sintered granules of HA and CHA. The current findings demonstrated that CHA (1.2 and 2.05 wt.%) is more soluble than pure HA in vivo. More dissolution was observed from the CHA, at the bone-implant interface and within the implant, when compared to pure HA. A less crystalline phase was formed between the 2.05 wt.% CHA and bone at 12 weeks in vivo. Bone surrounding both the pure HA and 1.2 wt.% CHA was relatively disorganised at 12 weeks. In comparison, bone surrounding the 2.05 wt.% CHA was considerably more organised and in many regions collagen fibrils were present. Despite increased quality of bone surrounding 2.05 wt.% CHA, compared to 1.2 wt.% CHA, the amount of dissolution from both materials was similar.

Preparation of microstructured hydroxyapatite microspheres using oil in water emulsions

Hydroxyapatite (HAP) microspheres with peculiar spheres-in-sphere morphology were prepared by using oil-in-water emulsions and solvent evaporation technique. Ethylene vinyl acetate co-polymer (EVA) was used as the binder material. Preparation of HAP/EVA microspheres was followed by the thermal debinding and sintering at 1150°C for 3 h to obtain HAP microspheres. Each microsphere of 100–1000 μm was in turn composed of spherical hydroxyapatite granules of 2–15 (μm size which were obtained by spray drying the precipitated HAP. The parameters such as percentage of initial HAP loading, type of stabilizer, concentration of stabilizer, stirring speed and temperature of microsphere preparation were varied to study their effect on the particle size and geometry of the microspheres obtained. It was observed that these parameters do have an effect on the size and shape of the microspheres obtained, which in turn will affect the sintered HAP microstructure. Of the three stabilizers used viz. polyoxyethylene(20) sorbitan monopalmitate (Tween-40), sodium laurate and polyvinyl alcohol (PVA), only PVA with a concentration not less than 0.1 wt% showed controlled stabilization of HAP granules resulting in spherical microspheres of required size. Morphologically better spherical microspheres were obtained at 20°C. Increasing the stirring speed produced smaller microspheres. Smaller microspheres having size < 50 μm were obtained at a stirring speed of 1500 ±50 rpm. A gradual decrease in pore size was observed in the sintered microspheres with increase in HAP loading.

Enhancement of hydroxyapatite‐mediated three‐dimensional‐like proliferation of mouse fibroblasts by heat treatment: Effects of heat shock‐induced p38 MAPK pathway

Regulation of the biocompatibility of compositional hydroxyapatite (HA) with cells is affected by various environmental factors. The aim of this study was to determine whether the p38 mitogen-activated protein kinase (MAPK) pathway has a key role in enhancement of HA-mediated three-dimensional (3D)-like proliferation of mouse fibroblasts after heat treatment. C3H10T1/2 mouse fibroblasts were cultured with HA granules for 10 weeks after heat treatment at 44 degrees C for 5, 10, 20, and 30 min. The mean rate of formation of 3D-like proliferation patterns by cells heat treated for 20 min was only 2.1-fold higher than that by untreated cells, but the mean rates of formation of 3D-like proliferation patterns by cells heat treated for 5 and 10 min were significantly higher (3.7- and 3.3-fold higher, respectively) than that by untreated cells (p < 0.01). Western blot analysis demonstrated that phosphorylation of p38 MAPK was markedly increased by heat treatment at 44 degrees C for 5 and 10 min. In addition, the activation of heat shock-induced p38 MAPK was markedly reduced by treatment at 44 degrees C for 30 min. We concluded that 3D-like proliferation of heat-treated cells was induced by activation of p38 MAPK. The results of this study should be useful for further studies aimed at elucidation of regulation of the biocompatibility of compositional HA with cells.

Development of a system to adsorb drugs onto calcium phosphate materials

Several studies were carried out in order to reduce the systemic use of antibiotics due to the high concentration required to provide the minimum inhibitory concentration (MIC) at infected sites. The aim of this study was to develop a system of drug adsorption onto commercial hydroxyapatite (HA, Ca10(PO4)6(OH)2) and glass reinforced hydroxyapatite (GR-HA) granules. The drug will then be released for the local treatment of periodontitis. The antibiotics used in this study were metronidazole, a specific antibiotic indicated for the systemic treatment of periodontitis, and ampicillin, a wide spectrum antibiotic. UV spectroscopy was used to measure the amount of drug adsorbed onto HA and GR-HA granules. Results showed that metronidazole was unable to adsorb on the material's surface, as opposed to ampicillin which adsorbed both onto HA and GR-HA. Preliminary release kinetics studies were carried out using a flow through dissolution system. Results are discussed in terms of the influence of the different surface characteristics of the materials on the adsorption processes.

Radiological outcome of tibial plateau fractures treated with percutaneously introduced synthetic porous Hydroxyapatite granules

Synthetic bone-graft substitutes are increasingly being used in clinical orthopaedic practice. In India, Sree Chitra Tirunal Institute of Medical Sciences and Technology has pioneered the research and development of indigenous ceramic bone substitutes. The Chitra HAP is a synthetic porous Hydroxyapatite that has completed pre-market trials according to prescribed format and has also undergone limited clinical trials in humans. This study is a clinical trial to ascertain the use of the product in the treatment of cancellous impaction fractures. Twenty-eight tibial plateau fractures were treated by a standard surgical protocol using the HAP for cancellous bone augmentation. The results were evaluated by serial radiography at specified intervals up to 1 year. At 1 year the HAP appeared fully integrated with the host bone radiologically and the articular subsidence had ceased. The synthetic HAP remained radio-opaque even at 1 year. No adverse effects were noticed due to the synthetic material. It was concluded that Chitra porous HAP is a safe and useful bone graft substitute for cancellous bone augmentation in fractures.

In vivo assessment of hydroxyapatite and silicate-substituted hydroxyapatite granules using an ovine defect model

Phase pure hydroxyapatite (HA) and two silicate-substituted hydroxyapatites (0.8 and 1.5 wt% Si, or 2.6 and 4.9 wt% SiO4) were prepared by aqueous precipitation methods. The filter-cakes of HA and silicate-substituted hydroxyapatite (SiHA) compositions were processed into granules 1.0-2.0 mm in diameter and sintered at 1200 degrees C for 2 h. The sintered granules underwent full structural characterisation, prior to assessment in an ovine defect model by implantation for a period of 6 and 12 weeks. The results indicate that HA and SiHA implants were well accepted by the host tissue, with no evidence of inflammation. New bone formation was observed directly on the surfaces and in the spaces between the granular implants. Quantitative histomorphometry as determined by the percentage of bone ingrowth and bone coverage for both SiHA implant compositions was significantly greater than that for phase pure HA. These findings indicate that the in vivo bioactivity of hydroxyapatite was significantly improved by the incorporation of silicate ions into the HA structure, making SiHA ceramics attractive alternatives to conventional HA materials for use as bone graft substitute ceramics.

Analysis of Cell-Seeded 3-Dimensional Bone Constructs Manufactured In Vitro With Hydroxyapatite Granules Obtained From Red Algae

Bone tissue engineering is a promising approach for the treatment of defective or lost bone in the maxillofacial region. Biocompatible and biodegradable scaffolds seeded with living cells are used to create functional tissue for load-bearing bone reconstruction. The aim of this study was to manufacture cell-seeded 3-dimensional bone constructs based on hydroxyapatite ceramic granule calcified from red algae and mesenchymal cambial-layer precursor cells. The ability of these cells to grow on hydroxyapatite ceramic was quantitatively investigated to evaluate 3-dimensional bone constructs for their potential use in bone tissue engineering. Mesenchymal cambial-layer precursor cells were isolated from mandibular periosteum biopsy samples of 3 patients. To manufacture 72 bone constructs, these cells and hydroxyapatite ceramic granules (C GRAFT/Algipore; Clinician's Preference LLC, Golden, CO) were cultivated under osteogenic differentiation conditions in a rotating wall vessel system. After 6 and 21 days, histologic examination and scanning electron microscopy were performed. The absolute DNA content, protein synthesis, and alkaline phosphatase activity were also quantified. The osteoblastic phenotype of the constructs was confirmed by the expression of bone-specific genes (osteocalcin, osteonectin, osteopontin, and core binding factor alpha1) using semiquantitative reverse transcription-polymerase chain reaction and Western blot analysis. Cells within the constructs showed good viability, which was evidenced by an increase in DNA content over the culture period. The decrease in alkaline phosphatase-specific activity could be an indicator of the maturation of cells and the induction of mineralization. The osteoblastic phenotype of the constructs was demonstrated on protein and at the RNA level over the entire culture period. We observed a positive effect of hydroxyapatite ceramic granules on mesenchymal cambial-layer precursor cell behavior in cell-seeded 3-dimensional bone constructs, indicating the potential applicability of C GRAFT/Algipore composites in bone tissue engineering.

Fabricating Hydroxyapatite Spherulites with Characteristic Microporous Structure by Chitin Sol Emulsified in Oil and Gelatinized In Situ

Hydroxyapatite (HA) spherulites had been fabricated successfully through a novel approach including chitin emulsion and geletion processes. The freeze-dried nano-HA powder was firstly dispersed in the chitin solvent before chitin dissolves in its solvent completely. The chitin sol containing nano-HA particles was dropped into oil and emulsified making use of liquids immiscibility effect between oil and chitin sol by stirring. The n-HA/Chitin sol spherulites gelled in situ with the existence of water molecules. Subsequently, the spherular gel granules were rinsed in distilled water to leach the solvent and dried in room circumstance. Finally, special sintering routines were carried to harvest spherular HA granules. The size and porosity of HA spherulites were controlled by the rate of nano-HA to chitin, the chitin concentration in the starting slurry, the stirring rate and the temperature of oil etc. In addition porosifier such as sugar was used in order to adjust the macro- and micro-porous structures in the HA spherulites. The morphological observation showed that the HA spherulites had good sphericity and characteristic microporous structure which were favorable for medical application.

Growth of Normal Human Osteoblast Cells on Hydroxyapatite/Titanium Alloy Composites Formed by Implantation of Hydroxyapatite Granules into Titanium Alloy

On the hydroxyapatite/titanium alloy composite disks, the normal human osteoblast cells were cultured. As a result, the samples were covered with normal human osteoblast cells after 2 hours. Moreover, we did not observe cracks in any of the HA granules, and the normal human osteoblast cells extended between the HA granules and the titanium alloy. From these results, we have concluded that this material has good biocompatibility.

Method for Tailoring and Control the Morphology, Size and Porosity of Calcium Phosphate Granules

Hydroxyapatite (HA) is the calcium-phosphate material with composition closest to that of human bone, what makes it suitable for osseous implant purposes, namely as fillers, spacers and bone grafts substitutes. This study is aimed at the development of a method to produce porous spherical hydroxyapatite granules. The process involves the spraying of a suspension with different amounts of a setting agent to a setting media. The tailor and the control of the morphology, size and porosity of the granules were attained by adjusting the nozzle diameter, the pressure of air flow and the distance between the nozzle and the setting media.