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PubMed2004

Design and fabrication of standardized hydroxyapatite scaffolds with a defined macro-architecture by rapid prototyping for bone-tissue-engineering research.

This investigation describes the production and characterization of calcium phosphate scaffolds with defined and reproducible porous macro-architectures and their preliminary in vitro and in vivo bone-tissue-engineered response. Fugitive wax molds were designed and produced using a rapid prototyping technique. An aqueous hydroxyapatite slurry was cast in these molds. After sintering at 1250 degrees C and then cleaning, dimensional and material characterizations of the scaffolds were performed. The resulting scaffolds represented the design, and their dimensions were remarkably consistent. A texture inherent to the layer-by-layer production of the mold was impressed onto the vertical surfaces of the scaffolds. The surface roughness (R(a)) of the textured surfaces was significantly greater than that of the nontextured surfaces. Material analyses revealed a beta-TCP phase in addition to hydroxyapatite for the molded ceramics. Non-molded control ceramics exhibited only hydroxyapatite. Thirty scaffolds were seeded with culture-expanded goat bone-marrow stromal cells (BMSCs) and implanted subcutaneously in nude mice for 4 or 6 weeks. Histology revealed mineralized bone formation in all the scaffolds for both implantation periods. After 4 weeks, bone was present primarily as a layer on scaffold surfaces. After 6 weeks, the surface bone formation was accompanied by bone budding from the surface and occasional bridging of pores. This budding and bridging bone formation almost always was associated with textured scaffold surfaces. However, the area percentage of bone in pores was similar for the 4- and 6-week implantation periods.

Animals↗
PubMed2004

Collagen-hydroxyapatite composite prepared by biomimetic process.

A novel bone graft substitute comprising a porous, collagenous scaffold was biomimetically coated with hydroxyapatite using a simulated body fluid solution chemistry method. The scaffold had a porosity of approximately 85%, with pore sizes between 30 microm and 100 microm. Glutaraldehyde vapor was used to stabilize the collagenous scaffold, giving a significantly increased thermal stability over an unstabilized scaffold, as shown by differential scanning calorimetry. A thin layer (<10 microm) of crystalline hydroxyapatite was deposited onto the stabilized collagenous scaffold by soaking the collagenous construct in simulated body fluid in the presence of calcium silicate glass. The presence of crystalline hydroxyapatite was confirmed by X-ray diffraction, energy-dispersive X-ray spectroscopy, and scanning electron microscopy. In vitro cytotoxicity testing of the composite construct using L-929 fibroblasts (ISO 10993-5) and rabbit periosteal cells revealed a cytocompatible material that supported cellular attachment and proliferation.

Animals↗
PubMed2005

Effects of synergistic reinforcement and absorbable fiber strength on hydroxyapatite bone cement.

Approximately a million bone grafts are performed each year in the United States, and this number is expected to increase rapidly as the population ages. Calcium phosphate cement (CPC) can intimately adapt to the bone cavity and harden to form resorbable hydroxyapatite with excellent osteoconductivity and bone-replacement capability. The objective of this study was to develop a strong CPC using synergistic reinforcement via suture fibers and chitosan, and to determine the fiber strength-CPC composite strength relationship. Biopolymer chitosan and cut suture filaments were randomly mixed into CPC. Both suture filaments and composite were immersed in a physiological solution. After 1-day immersion, cement flexural strengths (mean +/- SD; n = 6) were: (2.7 +/- 0.8) MPa for CPC control; (11.2 +/- 1.0) MPa for CPC-chitosan; (17.7 +/- 4.4) MPa for CPC-fiber composite; and (40.5 +/- 5.8) MPa for CPC-chitosan-fiber composite. They are significantly different from each other (Tukey's at 0.95). The strength increase from chitosan and fiber together in CPC was much more than that from either fiber or chitosan alone. The composite strength became (9.8 +/- 0.6) MPa at 35-day immersion and (4.2 +/- 0.7) MPa at 119 days, comparable to reported strengths for sintered porous hydroxyapatite implants and cancellous bone. After suture fiber dissolution, long macropore channels were formed in CPC suitable for cell migration and tissue ingrowth. A semiempirical relationship between suture fiber strength S(F) and composite strength S(C) were obtained: S(C) = 14.1 + 0.047 S(F), with R = 0.92. In summary, this study achieved substantial synergistic effects by combining random suture filaments and chitosan in CPC. This may help extend the use of the moldable, in situ hardening hydroxyapatite to moderate stress-bearing orthopedic applications. The long macropore channels in CPC should be advantageous for cell infiltration and bone ingrowth than conventional random pores and spherical pores.

Bone Substitutes↗
PubMed2006

Effects of in vitro bone formation on the mechanical properties of a trabeculated hydroxyapatite bone substitute.

This study was designed to test the hypothesis that the mechanical properties of a trabecular bone substitute can be enhanced through in vitro tissue formation. Our specific objectives were to (1) determine the effects of in vitro marrow stromal cell-mediated tissue deposition upon a trabeculated hydroxyapatite scaffold on the strength and toughness of the resulting bone substitute; and (2) identify and characterize regions of newly deposited matrix and mineral. This work provides a basis for future investigations aimed at transforming a brittle hydroxyapatite scaffold into an osteoinductive, biomechanically functional implant through in vitro bone deposition. As hypothesized, the mechanical properties of the trabecular bone substitutes were significantly enhanced by in vitro tissue formation. As a result of cell seeding and a 5 week culture protocol, mean strength increased by 85% (p = 0.008) and energy to fracture increased by 130% (p = 0.003). Accompanying the enhancement of mechanical properties was the deposition of significant amounts of bone matrix and mineral. Fluorescence imaging, scanning electron microscopy, electron probe microanalysis, and nanoindentation confirmed the presence of bonelike mineral with Ca/P ratio, modulus, and hardness similar to that within human and rat trabecular bone tissue. This new mineralization was found to exist within a newly deposited parallel-fibered matrix both encasing and bridging between scaffold trabeculae. Taken as a whole, our results establish the feasibility of the production of an osteoinductive hydroxyapatite-based trabecular bone substitute with mechanical properties enhanced through in vitro bone deposition.

Animals↗
PubMed2006

Influence of hydroxyapatite microstructure on human bone cell response.

Microstructure of calcium phosphate ceramics has been shown to influence long-term in vitro cellular events like proliferation and differentiation, and to favor bone integration in vivo. As long-term cellular events are known to be dependent of early cell adhesion events, we decided to study the in vitro influence of the microstructure of a microporous hydroxyapatite (mHA) and a nonmicroporous hydroxyapatite (pHA) ceramic on serum protein adsorption and SaOs-2 human bone cells attachment after 30 min, 1, 4, and 24 h and cell growth after 96 h. Plastic coverslips were used as controls. Hydroxyapatite composition of mHA and pHA was confirmed by X-ray diffraction and Fourier transform infra-red spectroscopy. The surface energies of ceramics were calculated from contact-angle measurements in di-iodomethane, water or complete culture medium. The total surface energy was 44.8 mJ/m(2) for pHA and 48.7 mJ/m(2) for plastic. The contact-angle measurement was impossible on mHA likely because they displayed 12% of open microporosity, pHA ceramic exhibiting only closed pores (2.5%). Moreover, the roughness amplitude was largely higher on mHA (Sa = 4.35 microm) than on pHA (Sa = 0.065 microm) and plastic (Sa = 0.042 microm). Three different techniques were used to evaluate protein adsorption on the ceramics. SDS-PAGE of desorbed proteins demonstrated that more proteins desorbed from mHA (66.02 microg/m(2)) than from pHA (17.2 microg/m(2)) or plastic (0.08 microg/m(2)). A new method was used to evaluate in situ the quantity of adsorbed total proteins: the temperature-programmed desorption (TPD) analysis coupled with mass spectrometry. The TPD analysis confirmed that 10-fold more proteins adsorbed on mHA compared with those on pHA. A direct immunolabeling on ceramics revealed than more fibronectin and serum albumin adsorbed on microporous ceramic than on dense ceramic. The morphology of SaOs-2 cells was the same on all the substrates after 30 min. At later time points, cell morphology on mHA was radically different than on other surfaces, with the particularity of the cytoplasmic edge that appeared undistinguishable from the surface. Only the extremity of the cells and lamellipodia were visible. Cells seemed like "adsorbed" by the mHA surface, whereas on plastic and pHA surfaces the cells displayed classical aspects of polygonal spreading. The cells displayed on mHA the highest initial attachment potential after 30 min, 1, 4, 24 h but the lower proliferation potential after four days. This study confirms that a microporous ceramic surface can modulate the adsorption of proteins and further the adhesion and proliferation of human bone cells.

Adsorption↗
PubMed2007

Morphological and X-ray diffraction studies of crystalline hydroxyapatite-reinforced polycaprolactone.

Morphological and mechanical properties of hydroxyapatite (HAP)-reinforced polycaprolactone (PCL) were studied. The objective was to examine how morphological features alter the bulk mechanical properties in our laboratory-synthesized HAP-reinforced PCL. HAP crystals were synthesized by hydrolysis of mixtures of calcium and phosphate salts in the laboratory with wet chemical methods. The properties of the commercially available hydroxyapatite (HAP(1)) are compared with that of laboratory-synthesized hydroxyapatite (HAP(2)). The HAP crystals and composition were characterized using X-ray diffraction, scanning electron microscopy, transmission electron microscopy, and Fourier transform infrared spectrometry (FTIR). The HAP(1) and HAP(2) crystals were dispersed into polymers to examine the mechanical behavior of bioactive composites, and the interfacial interactions between the polymer and HAP crystals are addressed. The FTIR results confirmed that the two forms of HAP crystals are consistent in terms of the functional chemical groups. The wide angle X-ray diffraction study was performed to determine the crystallinity of the bioactive composites. It was observed that the crystallinty of HAP-filled PCL steadily increased as the filler concentration increased. Generally, HAP(2) has a particle size considerably smaller than HAP(1) and the composite derived had higher modulus than conventional HAP-filled polymers. This increase in modulus is attributed to better interfacial interaction. Bioresorbability tests performed on HAP particles found that the synthesized HAP had higher resorption rates. It is clear that the mechanical properties are influenced by the particle size and therefore by the processing method used.

Animals↗
PubMed2006

A solid-state NMR investigation of the structure of nanocrystalline hydroxyapatite.

Nanocrystalline hydroxyapatite (HAp) prepared by a precipitation route was investigated. The X-ray diffraction (XRD) powder patterns of the elongated nanocrystals with a typical diameter of about 10 nm and length of 30-50 nm (by transmission electron microscopy (TEM)) revealed the presence of HAp with significantly broadened XRD reflections. However, Ca deficiency was found, as the Ca/P ratio was 1.5 only (so-called calcium-deficient hydroxyapatite (CDHA)), and not 1.67. This Ca deficiency of nanocrystalline HAp is explained using NMR. It is shown unambiguously that (i) the nanocrystals consist of a crystalline core and a (disordered) surface region with a relative phosphate content of about 1:1, (ii) the crystalline core is HAp, and (iii) the surface region is dominated by hydrogen phosphate anions (with no hydroxyapatite-like structural motif) and structural water (hydrate). From the relative phosphate content and taking into account the crystal shape, the thickness of the surface layer along the main crystal axis could be estimated to be about 1 nm, and the average chemical composition of the surface layer has been determined. Finally, a Ca/P ratio of 1.52 was estimated from the NMR data that compares well with the value of 1.51 from chemical analysis. The important consequences are that the surface of nanocrystalline HAp has nothing in common with the bulk composition and that the chemistry of such materials (e.g. the binding of protein molecules to phosphate surfaces) must be reconsidered.

Calcium↗
PubMed1995

Three-phase bone scintigraphy of hydroxyapatite ocular implants.

Hydroxyapatite ocular implants are replicas of lamellar bone tissue derived from the exoskeleton of a reef-building coral by a hydrothermal chemical exchange reaction. Attached to the eye muscles, they act as a passive framework for fibrovascular ingrowth and can be drilled to hold the visible part of the artificial eye and allow synchronous eye movement. Fibrovascular ingrowth has to be confirmed by bone scintigraphy before the drilling procedure. This study monitored the vascular ingrowth into the implant in ten patients over 12 months to establish a clinically feasible imaging protocol. Tracer accumulation was monitored visually and quantitatively in dynamic and single-photon emission tomography (SPET) scans after the intravenous administration of 600 MBq of 99mTc-DPD. The implants showed no tracer accumulation in the arterial or blood pool phase. Accordingly, dynamic scintigraphy can be omitted from the imaging protocol. Delayed tracer accumulation appeared no earlier than 2 and no later than 6 months after surgery. Planar scintigraphy is not recommended as high-resolution SPET is necessary to separate the implant from the surrounding bone. We conclude that imaging can be confined to high-resolution SPET 3 h after tracer injection, no earlier than 3 months after surgery. The vascularized hydroxyapatite orbital implant is an important in vivo model for bone-seeking agents to study their uptake kinetics independently of any soft tissue and bone disease. Our results provide evidence that in normal bones the chemical absorption of 99mTc-DPD into the crystalline structure of hydroxyapatite is the only quantitatively relevant uptake mechanism.

Diphosphonates↗
PubMed1995

Human polymorphonuclear leukocyte phagocytosis of crystalline cholesterol, bilirubin, and calcium hydroxyapatite in vitro.

We tested the hypothesis that human polymorphonuclear leukocytes (PMNs) phagocytize crystalline cholesterol, bilirubin, or calcium hydroxyapatite in vitro and in the process release oxygen metabolites and enzymes involved in the inflammatory process. Chemiluminescence (CL), elicited by the respiratory burst (release and activation of oxygen metabolites and enzymes) of PMNs during phagocytosis of a target particle, was used to quantitate PMN phagocytosis of each crystal. Significant CL (P < 0.05) was observed with cholesterol concentrations of 1.3-5.3 mg/ml and the dose-response was linear (r > or = 0.95). With bilirubin, significant CL was observed with concentrations of 0.07-0.33 mg/ml. The response to calcium hydroxyapatite was variable. Human PMNs phagocytize cholesterol, bilirubin, and to a lesser extent, calcium hydroxyapatite. PMN chemiluminescence was associated with phagocytosis, indicating that inflammatory substances are being released in the process. These results support the concept that crystals that occur in the gallbladder may initiate gallbladder inflammation.

Bilirubin↗
PubMed1993

[Effect of synthetic hydroxyapatite ceramics on long-term cultures of isolated chondrocytes].

This study deals with the influence of hydroxyapatite-ceramic on long-term cultures of rabbit articular chondrocytes. By additioni of granules of this material into the culture wells improval of cell proliferation by factor 12 over a period of twelve weeks is found. More important is the maintaining of the status of differentiation of the chondrocytes, documented by immunoidentification of the expressed collagen I production over collagen II as marker for chondrocytes. In controls without addition of hydroxyapatite-ceramic there is a dynamic increase of collagen I production over the culture period and morphological alteration of the cell shape leading to fibroblast-like cells. As this dedifferentiation is the main problem concerning chondrocytes long-term cultures, the addition of HAC into the culture medium is a simple and effective method enhancing the productivity of the culture system. Moreover this experimental design with chondrocytes as cells familiar with mineralized matrix gives an important proof of the excellent cytocompatibility of the hydroxyapatite-ceramic used in this study.

Animals↗
PubMed1997

[Hydroxyapatite ceramics in clinical application. Histological findings in 23 patients].

Based on the histological findings of 23 patients who had received implants of the bovine hydroxyapatite ceramic Endobon for a period of up to 16 months, the biocompatibility, nature and extent of osseointegration as well as the resorption and degradation behaviour of the ceramic were investigated. The investigation material consisted mainly of small fragments that had been retrieved during revision operations that were indicated for other reasons. The results confirm the good tolerability and suitability that have been systematically investigated in experimental studies and described for hydroxyapatite ceramic as bone substitute in a vital cancellous bone bed that is not exposed to excessive strain (due to its brittle character). The importance of fulfilling certain requirements in order to achieve a successful result, such as stable implantation in a well vascularized, infection-free bone bed also with a minimization of the contact with local connective tissue has been further substantiated. Good success has been achieved by simultaneous loading with autogenous bone marrow as is practised by many ceramic users. In some cases a widening of intergrain boundaries as well as partial dissociation of superficial hydroxyapatite crystallites were observed in the implant surface.

Adolescent↗
PubMed2006

The fixation strength of hydroxyapatite-coated Schanz screws and standard stainless steel Schanz screws in lower extremity lengthening : a comparison based on a new torque value index: the fixation index.

INTRODUCTION: Lengthening procedures are often complicated by loosening of pins. It has been reported that coating with hydroxyapatite improves fixation and reduces the rate of pin-track infection. MATERIALS AND METHODS: We compared 47 hydroxyapatite-coated Schanz screws (HA screws) in 12 monolateral fixators mounted at the University Hospital Hamburg-Eppendorf with 45 standard stainless steel screws in 9 monolateral fixators mounted at the St Josefs-Hospital Wiesbaden by measuring the insertion and extraction torque values. The average implantation period was 7 months for the hydroxyapatite-coated screws and 5.4 months for the uncoated screws. We established the quotient of the maximum extraction torque over insertion torque which shows the change in the fixation strength with respect to time, the fixation index. It eliminates the influence of the varying pin-bone contact. RESULTS: There was no significant difference in the rate of infection. In the Schanz screws without signs of infection the index was 1.92 for the HA screws and 0.76 for the stainless steel screws (P = 0.0002) giving evidence of the improvement of the fixation by the coating. CONCLUSION: HA coating resulted in improved fixation of Schanz screws in bone and may be useful in prolonged external fixation of the lower leg. The fixation index proved to be a simple tool for the evaluation of the fixation strength of Schanz screws.

Bone Lengthening↗
PubMed2001

Correction of skull defects using hydroxyapatite cement (HAC)--evidence derived from animal experiments and clinical experience.

BACKGROUND: The development of satisfactory cranioplasty material and technique has been a continuing bio-engineering challenge. Cranial defects resulting from trauma, tumour or infection are most frequently reconstructed with nonviable alloplastic materials. At present, all synthetic or biological materials in the use for human cranioplasty are more or less ideal. METHODS: The in vivo properties of a fully resorbable bony substitute--hydroxyapatite cement (HAC, BoneSource) are described in clinical investigations and animal experiments. HAC is prepared from calcium phosphate precursors which are hydrated and harden endothermically at 37 degrees C to form hydroxyapatite. Bone formation and resorption characteristics of HAC are examined in an adult minipig cranial defect model. FINDINGS: Cranial bone integrity has been restored in ten of eleven patients. Radiographic examination 6 months after surgery reveal a successful reconstruction of the skull defects. Sections of the cranial defect site from animals sacrificed at 12, 18 and 40 weeks demonstrate that new bone formation proceeds in HAC filled osseous defects. Histomorphological evaluation of HAC resorption and new bone formation indicates that HAC is nearly completely resorbed within 40 weeks and replaced by new bone with no loss in size or volume. INTERPRETATION: Hydroxyapatite cement (HAC) has an excellent biocompatibility (non-immunogenic and non-toxic), seems to be an optimal implant for cranial reconstruction and provides a biological scaffold for bone formation. However, further studies need to be conducted to determine the long-term stability of HAC.

Adolescent↗
PubMed2002

Hydroxyapatite ceramics as a carrier of gene-transduced bone marrow cells.

The aim of this study was to develop an efficient exogenous gene delivery system using cultured marrow cells and porous hydroxyapatite ceramics. Bone marrow cells were obtained from the femoral shaft of a Fischer 344 rat and cultured in a medium containing 15% fetal bovine serum until confluent. After trypsinization, cells were subcultured at a cell density of 1 x 10(4) cells/cm2 in the presence of fetal bovine serum. The subcultured bone marrow cells were infected with recombinant retroviruses carrying the lacZ gene. The retrovirus infection was performed seven times from day 1 to day 7 during the culturing procedure. Cells expressing the lacZ gene were stained blue with the X-gal staining and represented approximately 80%. Composites of virus-infected bone marrow cells and hydroxyapatite ceramics were implanted at the subcutaneous site of recipient Fischer 344 rats. Four weeks after the implantation the ceramics were harvested. The histological sections of the ceramics showed abundant bone formation in the pores of the ceramics and obviously blue-stained osteoblasts and osteocytes. Other cell types that were stained blue were some fibroblastic cells and endothelial cells in the newly formed capillaries. These findings indicate that osteoblasts and osteocytes in the newly formed bone were derived from the cultured bone marrow cells, and therefore gene transfection by retroviruses did not disturb the bone formation process. Because of the durability of the newly formed bone tissue, creating composites of cultured bone marrow cells and hydroxyapatite ceramics might be an ideal method for exogenous gene transfection.

Animals↗
PubMed2005

[Custom moldable hydroxyapatite collagen composite for repair of osseous defects].

AIM: In the following study we evaluated the biological response of a new freely moldable bone substitute in an animal model. MATERIAL AND METHODS: Critically sized defects were created surgically in the lower jaw of ten adult minipigs. The drill defects were filled with hydroxyapatite collagen paste. After observation periods of 1, 3, 6, 12, and 18 months the mandibles were harvested without wound healing defects for histological evaluation of resorption and bone ingrowth with a sawing and grinding technique. RESULTS: The result of the remodeling process was a complete degradation of hydroxyapatite collagen implants after 12-18 months with reorganization of vital trabeculae oriented in a mature pattern. CONCLUSION: The hydroxyapatite collagen cement works as an osteoconductor and shows signs of direct osseointegration and resorption.

Animals↗
PubMed2005

Synthesis and characterization of fluorine-containing hydroxyapatite by a pH-cycling method.

Different fluorine-containing hydroxyapatite (FHA) powders were synthesized through a pH-cycling method by varying sodium fluoride (NaF) concentration in hydroxyapatite (HA) suspensions. The powders were then calcined at 1200 ( composite function)C for 1 h. Both uncalcined and calcined powders were characterized using X-ray diffraction (XRD), Fourier transform infra-red (FTIR), and F-electrode. It was discovered that fluorine incorporation increased with the fluorine content in the initial solution and the number of pH cycles employed. A relatively low fluorine incorporation efficiency, approximately 60%, was attained for most of the FHA samples, and it did not vary significantly after calcination. It was also revealed that the FHA particles produced by the pH-cycling method were inhomogeneous. They were a mixture of hydroxyapatite and F-rich apatite (or FA) particles. After calcination, however, these FHA particles were homogenized and became single phased FHA.

Bone Substitutes↗
PubMed2005

Influence of ferrous iron incorporation on the structure of hydroxyapatite.

Iron is a vital element of cellular function within the body. High concentrations of iron can be found in the kidneys and the circulatory system. In bones and teeth it is present as a trace element. The use of iron-based compounds in combination with hydroxyapatite offers a new alternative for prosthetic devices. This work investigates the synthesis and processing of iron containing apatites as a possible new type of ceramic for biomedical devices. Stoichiometric and calcium deficient iron containing apatites were synthesized by a wet chemical reaction with di-ammonium-hydrogen-phosphate, calcium nitrate and a ferrous iron nitrate solution. A secondary phase of tri-calcium-phosphate (TCP) was observed after heat treatment of iron containing, calcium deficient, hydroxyapatite. The apatite structure was maintained after heat treatment of stoichiometric apatite, synthesized in the presence of iron. Sintering in air produced oxidation of Fe2+ to Fe3+, resulting in the formation of hematite as a secondary phase. The introduction of iron into the synthesis of hydroxyapatite causes: (i) an increase of the a-lattice parameter after synthesis and heat treatment in air; (ii) an increase in the c-lattice parameter after sintering in air.

Bone Substitutes↗
PubMed1978

A quick hydroxyapatite chromatography technique especially adapted for work with DNA networks.

During the renaturation of DNAs large networks build up which cannot be eluted from hydroxyapatite under standard fractionation conditions (60 degrees C, 0.5 M PB). This is a serious problem especially in plant-DNA renaturation studies as hyperpolymers may comprise more than half of the renatured DNA mass even at moderately long initial fragment lengths and low C0t values. Utilizing the acid solubility of hydroxyapatite a method is outlined which will recover the total double-stranded DNA fraction and will prepare the column for the next fractionation in one quick operation. As the method is time saving compared to the standard hydroxyapatite fractionation procedure its general application may prove to be useful.

Biopolymers↗