In vitro and in vivo biological performance of hydroxyapatite from fish waste.

The aim of this study was to evaluate biocompatibility of hydroxyapatite (HAP) from fish waste using in vitro and in vivo assays. Fish samples (whitemouth croaker - Micropogonias furnieri) from the biowaste was used as HAP source. Pre-osteoblastic MC3T3-E1 cells were used in vitro study. In addition, bone defects were artificially created in rat calvaria and filled with HAP in vivo. The results demonstrated that HAP reduced cytotoxicity in pre-osteoblast cells after 3 and 6 days following HAP exposure. DNA concentration was lower in the HAP group after 6 days. Quantitative RT-PCR did not show any significant differences (p > 0.05) between groups. In vivo study revealed that bone defects filled with HAP pointed out moderate chronic inflammatory cells with slight proliferation of blood vessels after 7 and 15 days. Chronic inflammatory infiltrate was absent after 30 days of HAP exposure. There was also a decrease in the amount of biomaterial, being followed by newly formed bone tissue. All experimental groups also demonstrated strong RUNX-2 immoexpression in the granulation tissue as well as in cells in close contact with biomaterial. The number of osteoblasts inside the defect area was lower in the HAP group when compared to control group after 7 days post-implantation. Similarly, the osteoblast surface as well as the percentage of bone surface was higher in control group when compared with HAP group after 7 days post-implantation. Taken together, HAP from fish waste is a promising possibility that should be explored more carefully by tissue-engineering or biotechnology.

Characterization and Cytotoxicity Evaluation of a Marine Sponge Biosilica.

Bone fractures characterize an important event in the medical healthcare, being related to traumas, aging, and diseases. In critical conditions, such as extensive bone loss and osteoporosis, the tissue restoration may be compromised and culminate in a non-union consolidation. In this context, the osteogenic properties of biomaterials with a natural origin have gained prominence. Particularly, marine sponges are promising organisms that can be exploited as biomaterials for bone grafts. Thus, the objectives of this study were to study the physicochemical and morphological properties of biosilica (BS) from sponges by using scanning electron microscopy, Fourier-transform infrared, X-ray diffraction (SEM, FTIR and XRD respectively), mineralization, and pH. In addition, tests on an osteoblast precursor cell line (MC3T3-E1) were performed to investigate its cytotoxicity and proliferation in presence of BS. Bioglass (BG) was used as gold standard material for comparison purposes. Sponge BS was obtained, and this fact was proven by SEM, FTIR, and XRD analysis. Calcium assay showed a progressive release of this ion from day 7 and a more balanced pH for BS was maintained compared to BG. Cytotoxicity assay indicated that BS had a positive influence on MC3T3-E1 cells viability and qRT-PCR showed that this material stimulated Runx2 and BMP4 gene expressions. Taken together, the results indicate a potential use of sponge biosilica for tissue engineering applications.

In vivo evaluation of shark teeth-derived bioapatites.

OBJECTIVE: The present work proposes the shark teeth as a new source of bioapatites for bone filler applications in maxillary sinus elevation, periodontal regeneration or implants placement. This abundant fishing by-product provides an improved hydroxyapatite (HA) with fluorine contributions. The in vivo evaluation of osteointegration and bone mineral density levels promoted by these marine bioapatites was the main objective. MATERIALS AND METHODS: Marine bioapatite granules of two sizes (1 mm, <20 µm) were obtained and characterized (XRD, SEM, ICP-OES) to determine morphology and composition. In vivo evaluation was performed, after bioapatites implantation in critical defects of parietal bone of 25 rats, for 3 weeks. Commercial synthetic HA/ßTCP (60/40%) material and unfilled defects were used as controls. Radiology, micro-CT, histology and quantification of bone mineral density are presented. RESULTS: These marine bioapatites presented a globular porous morphology. A biphasic composition ~70% apatitic (HA, apatite-CaF, fluorapatite) and ~30% non-apatitic phase (whitlockite, tricalcium bis(orthophosphate)), with contributions of F (1.0 ± 0.5%wt), Na (0.9 ± 0.2%wt) and Mg (0.65 ± 0.04%wt) was confirmed. After implantation period, higher osteointegration of 1-mm marine bioapatites than commercial synthetic granules was observed, together with bone formation from the defect surroundings but also at central area (potential osteoinductive properties). New bone cells penetrated inside pores and inter-granular cavities. Higher bone mineral density, in both 1-mm and <20-µm granules, than on commercial synthetic graft was determined, being significant in 1-mm bioapatites (a P < 0.05). CONCLUSION: Shark teeth bioapatites were successfully validated as new functionally efficient bone filler in rat model, promoting significantly increased bone mineral density than synthetic control.

Preparation of a novel anorganic bovine bone xenograft with enhanced bioactivity and osteoconductivity.

A novel anorganic bovine bone xenograft with enhanced bioactivity and osteoconductivity was prepared by an ion substitution method using sodium hypochlorite. Bovine bone granules were defatted, washed, and then soaked in sodium hypochlorite solution at room temperature. Subsequently, the granules were dried and then heat-treated at 1000°C with sodium hypochlorite. As a control, bovine bone granules were prepared with the same conditions but without sodium hypochlorite treatment. Phase, functional group, and elemental analyses by XRD, FTIR, and EPMA showed that the granules heat-treated without and with sodium hypochlorite were pure hydroxyapatite and sodium-chlorine-bearing hydroxyapatite, respectively. After soaking in simulated body fluid (SBF) for 1 week, low crystalline hydroxyl carbonate apatite fully covered the surface of sodium-chlorine-bearing hydroxyapatite, whereas it formed little on the hydroxyapatite surface. After soaking in SBF and deionized water, ICP-AES and IC analyses showed that the dissolutions of calcium, sodium, chlorine, and hydroxyl ions from sodium-chlorine-bearing hydroxyapatite notably increased compared with those from hydroxyapatite. This resultantly increased the ionic activity product of apatite in SBF and induced new formation of low crystalline hydroxyl carbonate apatite. The cytotoxicity test by BCA assay showed that there were no statistically significant differences between hydroxyapatite and sodium-chlorine-bearing hydroxyapatite. In addition, sodium-chlorine-bearing hydroxyapatite showed better osteoconductivity in the calvarial defects of New Zealand white rabbits within 4 weeks compared with that of hydroxyapatite. The results suggest that this novel anorganic bovine bone xenograft possesses encouraging potential for use as a bone grafting material due to better bioactivity and osteoconductivity than hydroxyapatite.

Highly sinterable calcium phosphate fabricated by using starfish bone.

Nano-sized calcium phosphate powders were simply synthesized by using dried starfish bone. The calcined bone was mixed with phosphoric acid and the dried mixtures were heated for synthesis. The powder compacts were fully sintered at 1100 degrees C for 1 h. The densified samples showed CaO-free calcium phosphate phase and non-uniform, over sized grains.

Fossils as candidate material for orthopedic applications.

Ceramic powders from fossil deposits were thoroughly characterized from the material point of view and sintered to produce massive components. The raw material, a mixture of apatite minerals, feldspars, and quartz, seems ideally suitable to obtain a biologically compatible glass ceramic. Preliminary in vitro tests of proliferation and adhesion of MG63 human osteoblast-like cell line on a selected sample are encouraging. Results are correlated with sintering conditions and phase composition: the fossil can be sintered to almost full density at temperatures as low as 900 °C and seems to quickly promote cell activation with respect to hydroxylapatite.

Evaluation of bone growth using artificial bone substitute (osteoset) and platelet gel mixtures: a preliminarily study in dogs.

Platelet gels (PG), activated by bovine thrombin (BT), have increasingly been used in orthopedic surgery. However, BT may induce immunological reactions and carry potential viral and prion risks. To avoid these side effects, thrombin derived from human plasma (human thrombin, HT) is becoming the preferred platelet activator to prepare PG. However, limited experience and data on the clinical benefits of HT-generated PG (HTPG) in orthopedic surgery is reported. Consequently, we designed and performed a series of studies in dogs to compare the impacts of promotion of bone growth by an artificial bone substitute (Osteoset) in combination with HTPG or without it in the spinal repair experiments. X-ray observations and histological studies were performed at predetermined periods post-operation. The preliminary results revealed the preparation of HTPG was easy and required less than 30 minutes. HTPG was capable of embedding the artificial bone substitute Osteoset to prepare a sticky and easily manipulated composite for the application into spinal defect. We found HTPG exhibited enhancement of grafting capacity in consolidation of bone mass. After 12 weeks, tissue reconstruction reached approximately 80% of the injury defects when treated by HTPG/Osteoset combination, but only 30 approximately 40% in the absence of HTPG. The physiological activity of artificial bone substitute combined with PG activated by HT may therefore open beneficial prospects for more successful and safer bone formation in spine procedures in the near future.

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 degrees C. Aragonite (CaCO(3)) monoliths were completely transformed into hydroxyapatite after 48 h of HT treatment. The substitution of CO(3) (2-) groups predominantly into the PO(4) (3-) sites of the Ca(10)(PO(4))(6)(OH)(2) structure was suggested by FT-IR spectroscopy and Rietveld structure refinement. The intensity of the nu(3)PO(4) (3-) bands increase, while the intensity of the nu(2)CO(3) (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.

Electrospun submicron bioactive glass fibers for bone tissue scaffold.

Submicron bioactive glass fibers 70S30C (70 mol% SiO(2), 30 mol% CaO) acting as bone tissue scaffolds were fabricated by electrospinning method. The scaffold is a hierarchical pore network that consists of interconnected fibers with macropores and mesopores. The structure, morphological characterization and mechanical properties of the submicron bioactive glass fibers were studied by XRD, EDS, FIIR, SEM, N(2) gas absorption analyses and nanoindentation. The effect of the voltage on the morphology of electrospun bioactive glass fibers was investigated. It was found that decreasing the applied voltage from 19 to 7 kV can facilitate the formation of finer fibers with fewer bead defects. The hardness and Young's modulus of submicron bioactive glass fibers were measured as 0.21 and 5.5 GPa, respectively. Comparing with other bone tissue scaffolds measured by nanoindentation, the elastic modulus of the present scaffold was relatively high and close to the bone.

Fabrication of bioactive glass-ceramic foams mimicking human bone portions for regenerative medicine.

A technique for the preparation of bioglass foams for bone tissue engineering is presented. The process is based on the in situ foaming of a bioglass-loaded polyurethane foam as the intermediate step for obtaining a bioglass porous monolith, starting from sol-gel synthesized bioglass powders. The obtained foams were characterized using X-ray diffraction analysis, Fourier transform infrared spectroscopy, and field emission scanning electron microscopy observations. The material was assessed by soaking samples in simulated body fluid and observing apatite layer formation. Diagnostic imaging taken from human patients was used to reconstruct a human bone portion, which was used to mould a tailored scaffold fabricated using the in situ foaming technique. The results confirmed that the obtained bioactive materials prepared with three-dimensional processing are promising for applications in reconstructive surgery tailored to each single patient.

Preparation of porous hydroxyapatite with interconnected pore architecture.

Since pore connectivity has significant effects on the biological behaviors of biomedical porous hydroxyapatite (PHA), the preparation of PHA with interconnected pore architecture is of great practical significance. In the present study, PHA with highly interconnected architecture was prepared via a simple burnout route with rod-like urea as the porogen. Microscopy and porosimetry data showed that the as-prepared PHA had open and interconnected pore structure with the average fenestration size of about 120 microm. Open pores occupied up to 98% of the total porosity. The compressive strength and modulus of the as-prepared PHA were respectively 1.3-7.6 MPa and 4.0-10.4 GPa.

Development of graded hydroxyapatite/CaCO(3) composite structures for bone ingrowth.

Ceramic composites composed of constituents with different bone cell reactions present an interesting consideration for a new bone replacement material. The first component of the composite used in this study, hydroxyapatite, is known to be replaced by natural tissue significantly slower than the second, calcium carbonate, which has limited structural stability. A graded hydroxyapatite/calcium carbonate composite with bimodal component distribution was developed using a combined slip infiltration and dip-coating technique from a porous polyurethane sponge replica. A graded hydroxyapatite scaffold with porosities from 5 to 90% was produced and then infiltrated with a calcium carbonate slip and sintered. The resultant composite had improved mechanical properties compared with the monolith as measured by crushing and moduli tests.

Novel tubular composite matrix for bone repair.

Tissue engineering develops organ replacements to overcome the limitations associated with autografts and allografts. The work presented here details the development of biodegradable, porous, three-dimensional polymer-ceramic-sintered microsphere matrices to support bone regeneration. Poly(lactide-co-glycolide)/hydroxyapatite microspheres were formed using solvent evaporation technique. Individual microspheres were placed in a cylindrical mold and sintered at various temperatures. Scaffolds were characterized using scanning electron microscopy, mercury porosimetry, and mechanical testing in compression. After varying the temperature of sintering, a single temperature was selected and the time of sintering was varied. Mechanical testing indicated that as the sintering temperature or time was increased, the elastic modulus, compressive strength, maximum compressive load, and energy at failure significantly increased. Furthermore, increasing the sintering temperature or time resulted in a decreased porosity and the spherical morphology of the microspheres was lost as the microspheres blended together. To more closely mimic the bone marrow cavity observed in native bone tissue, tubular composite-sintered microsphere matrices were formed. These scaffolds demonstrated no statistically significant difference in compressive mechanical properties when compared with cylindrical composite-sintered microsphere matrices of the same dimension. One potential application for these scaffolds is bone regeneration.

Hydroxyapatite formed on/in agarose gel induces activation of blood coagulation and platelets aggregation.

We reported earlier that hydroxyapatite (HA) formed on/in agarose gels (HA/agarose) produced by alternate soaking process is a bone-filling material possessing osteoconductive and hemostatic effects. This process could allow us to make bone-like apatite that was formed on/in organic polymer hydrogel matrices. Here, we investigated the mechanism of hemostasis induced by HA/agarose and found that HA/agarose, but not agarose or HA powder, significantly shortened activated partial thromboplastin time (APTT). While HA/agarose did not show significant platelet aggregation, it markedly enhanced adenosine diphosphate (ADP)-induced platelet aggregation. Moreover, Western blot analysis revealed selective adsorption of vitronectin onto HA/agarose. We also observed marked differences between HA powder and HA/agarose in their XRD patterns. The crystallinity of HA powder was much higher compared to that of HA/agarose. Furthermore, 50-100 nm of tube-form aggregations was observed in HA powder on the other hand 100-200 nm of particles was observed in HA/agarose by SEM observation. Thus 100-200 nm of low crystallized particles on the surface structure of HA/agarose may play an important role in hemostasis. Our results demonstrated a crucial role of HA/agarose in the mechanism of hemostasis and suggested a potential role for HA/agarose as a bone-grafting material.

Transformation of 3DP gypsum model to HA by treating in ammonium phosphate solution.

Three-dimensional printing (3DP) is a CAD/CAM built-up using ink-jet printing technique. Commercially available 3DP system can form only gypsum model and not for bioceramics. On the other hand, transformation of hardened gypsum into hydroxyapatite (HA) by treatment in ammonium phosphate solution was found lately. In the present study, transformation of the 3DP gypsum block to HA was attempted. However, the fabricated 3DP block was soluble in water. To insolubilize, it was heated at 300 degrees C for 10 min, and then, gypsum was transformed to calcium sulfate hemihydrate, CaSO(4) x 0.5H(2)O. The 3D block was immersed in 1M (NH(4))(3)PO(4) x 3H(2)O solution at 80 degrees C for 1-24 h, and the transformation into HA within 4 h was ascertained. A heat-treated plaster of Paris (POP) block was also investigated for comparison. The unheated POP block consisting of gypsum dihydrate took 24 h to complete the transformation, while the heat-treated POP consisting calcium sulfate hemihydrate promoted the transformation into HA; but the transformed thickness in the block was less than the 3DP block. This is probably due to higher solubility of the hemihydrate than gypsum dihydrate. Accelerated transformation of the 3DP block was also caused by its porous structure, which enabled an easy penetration of the phosphate solution. With the present method, it is possible to transform the fabricated gypsum by 3D printing that is adaptive to the osseous defect into HA prostheses or scaffold.

Development and characterization of a porous poly(methyl methacrylate) scaffold with controllable modulus and permeability.

Functional restoration following extensive bone injury often requires bone grafting. The primary source of graft material is either autograft or allograft. The use of both material sources is well established, however both suffer limitations. In response, grafting alternatives are being investigated. This manuscript presents the development of a highly porous scaffold with controllable elastic modulus and permeability for use in tissue grafting and tissue engineering applications that is manufactured from FDA approved poly(methyl methacrylate) (PMMA). Fifteen protocol variations based on the commonly used porogen leaching technique for porous scaffold fabrication were employed to control scaffold pore size, pore interconnectivity, and structural strength. Scaffolds were tested for porosity, permeability, elastic modulus, cell culture compatibility, and fatigue tested in compression. Scaffold permeability ranged from 6.6 x 10(-16) m(2) to 1.4 x 10(-10) m(2), and elastic modulus was adjustable between 14 and 322 MPa; data similar to cancellous bone specimens from a variety of species and anatomic locations. Fatigue evaluations revealed 65% strength maintenance after 80,000 loading cycles, and in vitro culture with marrow-derived stromal cells show no cytotoxic effects based on Live/Dead assay. The scaffolds detailed herein will help broaden the spectrum of available orthopaedic tissue scaffolds for research in this evolving field. , 2007.

Histological assessment in grafts of highly purified beta-tricalcium phosphate (OSferion) in human bones.

Prominent osteoconductive activity and the biodegradable nature of commercially available beta-tricalcium phosphate (beta-TCP, OSferion) have been documented in animal experiments. We analyzed four cases of involving grafted OSferion in human bone with respect to histological features by routine hematoxylin and eosin staining, silver impregnation, immunohistochemistry and in situ hybridization. OSferion affords early bioresorption by osteoclasts, vascular invasion of macropores and osteoblastic cell attachment on the surface on the ceramic surface 14 days after grafting. Prominent bone formation and direct bone connection between preexisting bone and OSferion were evident 28 days after grafting. Nearly the entire TCP surface was covered by lamellar bone; additionally, active osteoblastic lining and attachment of the osteoclast-like giant cells were not observed 72 weeks after grafting. Silver impregnation revealed the presence of collagen fibrils within probable micropores of OSferion.

Fabrication of 1-dimensional porous hydroxyapatite and evaluation of its osteoconductivity.

Porous HA ceramics with 1-dimensional pore channels were fabricated to obtain controllable microstructure. 1-dimensional porous HA was objected to find out the optimum condition of bone ingrowth and also to facilitate the observation of osteocondutive behavior in porous HA. The porous structure was formed by burnt-out of polymeric fibers and the size of pores was determined by the diameter of polymeric fibers. The porosity could be varied by the thickness of HA slurry coated on polymeric fiber and by the thickness of HA tapes inserted between fiber layers. As result, 1-dimensional porous HA ceramics of this study have the uniform interconnection size (50-500 microm) and the linearly open pore structure. The compressive strength of 1-dimensional porous HA was 6-10 MPa similar to that of human cancellous bone. On the in vivo test, oteon-like osteoconduction in pore channel of 1-dimensional porous HA was observed, like what had been found in cortical bones. This osteon-like new bone grew from the surface to the center of pore channels. The 1-dimensional porous HA ceramics prepared in this study were very useful as a model system to observe bone ingrowth in the porous HA implants.

Apatite formation in the reaction-setting mixture of Ca(OH)2--KH2PO4 system.

Development of a new calcium phosphate cement (CPC) system as an alternative to that commonly used, basically consisting of tetracalcium phosphate and dicalcium phosphate self-setting mixtures, could be of interest in achieving special properties of the product. Powder mixtures of Ca(OH)(2) and KH(2)PO(4) were studied to assess their potential for the precipitation of apatite-like phase with the use of potassium phosphate salt solution as the cement liquid. X-ray diffraction and infrared (IR) spectroscopy studies and pH and setting time measurements were performed. The set cement was revealed to consist of a low crystalline carbonate-substituted apatite-like phase. The setting time of the cement was about 5 min. Its dissolution in distilled water led to an increase in solution pH to about 11.5, the pH slowly decreasing to 10.2 at day 10. The results showed the cement to be of an increased carbonate substitution ability compared to the tetracalcium phosphate-dicalcium phosphate anhydrous cement.

Hard tissue remodeling using biofabricated coralline biomaterials.

Biotechnical and biomedical approaches were combined in an attempt to identify potential uses of biofabricated marine carbonate materials in biomedical applications, particularly as biomatrices for remodeling bone and cartilage tissue. After grafting, it is desirable for bone ingrowth to proceed as quickly as possible because the strength of the implanted region depends on a good mechanical bond forming between the implant and surrounding regions in the body. Ingrowth can take place as a result of growth of tissue and cells into the implanted porous material, or it may be promoted by transplanting cells seeded onto such a material. The rate at which ingrowth occurs is dependent on many factors, including pore size and the interconnectivity of the implanted structure. In vivo graftings into osteochondral defects demonstrated that our biofabricated porous material is highly biocompatible with cartilage and bone tissue. The biofabricated matrix was well incorporated into the biphasic osteochondral area. Resorption was followed by bone and cartilage formation, and after 4 months, the biomaterial had been replaced by new tissue. Ossification was induced and enhanced without introduction of additional factors. We believe that this is the first time that such biofabricated materials have been used for biomedical purposes. In face of the obvious environmental disadvantages of harvesting from limited natural resources, we propose the use of bioengineered coralline and other materials such as those cultured by our group under field and laboratory conditions as a possible biomatrix for hard tissue remodeling.