Search

This in vivo study investigated the influence of two calcium phosphate particle sizes (40-80 microm and 200-500 microm) on the cellular degradation activity associated with the bone substitution process of two injectable bone substitutes (IBS). The tested biomaterials were obtained by associating a biphasic calcium phosphate (BCP) ceramic mineral phase and a 3% aqueous solution of a cellulosic polymer (hydroxypropylmethylcellulose). Both were injected into osseous defects at the distal end of rabbit femurs for 2- and 3-week periods. Quantitative results for tartrate-resistant acid phosphatase (TRAP) cellular activity, new bone formation, and ceramic resorption were studied for statistical purposes. Positive TRAP-stained degradation cells were significantly more numerous for IBS 40-80 than IBS 200-500, regardless of implantation time. BCP degradation was quite marked during the first 2 weeks for IBS 40-80, and bone colonization occurred more extensively for IBS 40-80 than for IBS 200-500. The resorption-bone substitution process occurred earlier and faster for IBS 40-80 than IBS 200-500. Both tested IBS displayed similar biological efficiency, with conserved in vivo bioactivity and bone-filling ability. Differences in calcium phosphate particle sizes influenced cellular degradation activity and ceramic resorption but were compatible with efficient bone substitution.

Calcium phosphate materials have been increasingly employed in orthopedic and dental applications in recent years and are now being developed for use in noninvasive surgery or as carriers for drug delivery systems. We developed an injectable bone substitute (IBS) constituted of biphasic calcium phosphate and a hydrosoluble polymer as a carrier. In vivo biocompatibility and biofunctionality of IBS were tested in rabbits using implants in osseous and nonosseous areas. The results obtained demonstrated that the concept of IBS, a filler without initial mechanical properties but able to be rapidly resorbed and replaced by newly formed bone, can be applied to new surgical applications in orthopedic surgery, maxillofacial surgery, and dentistry for pulp capping and root filling.

We studied a new injectable biomaterial for bone and dental surgery consisting of a hydrophilic polymer as matrix and bioactive calcium phosphate (CaP) ceramics as fillers. This material is composed of complex fluids whose flow is determined by the laws of rheology. We investigated the macromolecular effects on this composite in a tube. The stability of the polymer and the mixture is essential to the production of a ready-to-use injectable biomaterial. These flow properties are necessary to obtain CaP bioactivity in a dental canal or bone defect during percutaneous surgery. Macromolecules provide spaces between CaP ceramic granules and facilitate the role of the biological agents of bone substitution.

Calcium-phosphate ceramics used in surgery, as bone-bonding materials, are currently available in different forms (blocks, granules, etc.). However, progress in noninvasive surgery has favored the development of injectable composite materials associating a polymeric and a dusty mineral phase. The purpose of this study was the in vivo evaluation of biphasic calcium phosphate of various grains sizes, to elucidate the role of granulometries in ceramic degradation/resorption, bone ingrowth, and inflammatory reactions. Three particle sizes were compared: 10-20, 80-100, and 200-400 microm. The 10-20-microm powders provided the best bone ingrowth, with a higher resorption/degradation rate in conjunction with stronger early inflammatory reactions. The 200-400-microm powders showed higher bone ingrowth than 80-100-microm ones, indicating that properties of cell recruitment for osseous apposition and mechanical support for bone bonding may both play a role in both ingrowth mechanisms. Our results suggest that the strong inflammatory reaction in 10-20-microm granulated powders was due to a faster reversal of the resorption/apposition sequence in bone. This may have resulted from massive release of bone ingrowth factors, which implies that the brief inflammatory process observed in the early stages of implantation was favorable to the osteoconduction process.

There is increasing evidence that noncollagenous matrix proteins initiate bone mineralization in vivo. Fibronectin, which is present during the early phases of mineralization, may contribute to this process in bone tissues. In this context, the mineralization potential of fibronectin was tested in an agarose gel precipitation system and a metastable calcium phosphate solution. The protein inhibited the precipitation of calcium phosphate crystals in solution but had no apparent effect in gel. Conversely, fibronectin stimulated crystal formation when apatite powder was used to seed crystal growth in gel. Although these results in vitro do not clearly indicate that fibronectin is involved in the mineralization process, they are consistent with in vivo events. Free fibronectin (e.g. in biological fluids) could inhibit crystal growth but might also activate the mineralization process when absorbed on apatite powder in a bone environment and areas of ectopic mineralization.

The dissolution-precipitation process for calcium-phosphate ceramics in contact with biological fluid was studied by incubating blocks of biphasic calcium phosphate composed of hydroxyapatite (HA) and beta-tricalcium phosphate (beta-TCP) in different solutions: ionic simulated body fluid (SBF) without protein or SBF that contained various proteins and macromolecules separately (fibronectin, vitronectin, albumin, and poly-L-glutamic acid). Transmission electron microscopy studies revealed that apatite-precipitated microcrystals appeared around ceramic crystals as a result of secondary nucleation; microcrystals were in continuity with the lattice planes of the HA crystals but in a different direction from that of beta-TCP; the size of the precipitates was smaller when fibronectin, vitronectin, and poly-(L-glutamic acid) were present in SBF as compared to SBF without protein; and fibronectin and vitronectin initiated crystal nucleation in the void spaces between the ceramic crystals.

Fourier-transform infrared microspectroscopy (FT-IRM) was used to study bone mineralization processes in an in vivo model and in enamel in osteogenesis imperfecta. Finally, the ability of FT-IRM to map new bone formed in implanted macroporous calcium phosphate biomaterial from sections was reported for the first time. FT-IRM allowed the correlation of the microstructure of bone formation in the in vivo model with modifications in carbonate and phosphate environments of the mineral phases during maturation. FR-IRM analysis on enamel sections revealed changes in the mineral environment of carbonate and phosphate ions and probably in the size of enamel crystals. These modifications contributed to the fragility of enamel in osteogenesis imperfecta. The infrared functional group imaging of a part of implanted biomaterial and the bone ingrowth provided the visualization of chemical modifications occurring in biomaterial implants at 20 microns spatial resolution. The use of FT-IRM, in conjunction with appropriate sampling methods and data analysis should provide further insight into the molecular structure of mineral phases of calcified tissues and help to elucidate mineralization processes, skeletal disorders and properties of the biomaterials used as bone substitute.

The development of calcium phosphate ceramics and other related biomaterials for bone graft involved a better control of the process of biomaterials resorption and bone substitution. The bioactive concept was developed for biphasic calcium phosphate ceramics (BCP). An optimum balance of the more stable phase of HA and more soluble TCP was obtained for controlling gradual dissolution in the body, seeding new bone formation as it releases calcium and phosphate ions into the biological medium. The bone/material interface and the events occurring in the development of this dynamic interface such as cellular response, biodegradation or bioresorption of the materials and their transformation to carbonate hydroxyapatite (CHA) were described. These processes were observed in both bulk samples, implant coating and injectable bone substitute (IBS).

Hydroxypropylmethylcellulose (HPMC) of high-viscosity grade is used as a ligand for a bioactive calcium phosphate ceramic (the filler) in a ready-to-use injectable sterilized biomaterial for bone and dental surgery. Application of physico-chemical methods such as XPS, NMR, or Raman spectroscopy encounters difficulties when used to study such a multiphased material. This paper reports on the application of FT-IR microspectroscopy (FT-IRM) for the investigation of inorganic and organic phases of the rough composite and separated phases obtained by mechanical or chemical extraction methods. A comparison of FT-IRM with the conventional KBr pellet method was made and indicates that the macro and micro FT-IR methods are complementary: the former revealed new chemical groups not visualized with the KBr method whereas the latter detected the major compound of the blend. FT-IR microspectroscopy was revealed to be a powerful method of analysis that is complementary to other existing spectroscopic methods. Moreover, it is expected to be a useful tool in the study of biomaterials in biological samples.

Calcium phosphate ceramics recently have been used for administering therapeutic agents in bone. The present work investigated the efficacy of macroporous biphasic calcium phosphate (MBCP) implants as a matrix for local delivery of human growth hormone (hGH). An initial study showed that the release of 5 microg of hGH loaded onto MBCP cylinders was rapid during the first 48 h and sustained for a total of 11 days. The biological integrity of hGH (88.2%) was checked using a specific bioassay (cellular proliferation of hGH-sensitive Nb2 cells) in comparison with a radioimmunoassay to calculate the proportion of bioactive hGH released. MBCP cylinders then were loaded with 1, 10, and 100 microg of hGH and implanted into rabbit femurs (n = 16) to determine hGH effects on bone ingrowth and ceramic resorption, as evaluated by scanning electron microscopy and image analysis. Results indicated that hGH increased bone ingrowth and ceramic resorption significantly in comparison with contralateral and control implants. Biochemical parameters monitored in rabbit plasma showed that hGH did not produce detectable systemic effects. Thus the use of MBCP appears to be effective for local delivery of hGH and for increasing bone ingrowth.

Although calcium phosphate biomaterials often are used as drug delivery systems (DDS) at bone sites, the conditions affecting the loading of the therapeutic agent (TA) have not been well documented. A human growth hormone (hGH) adsorption method was used in this study to investigate the influence of the formulated apatite (AP)-specific area on loading and release. AP powders were formulated with a 200-500 microm granulometry and various specific areas. Two milligrams of hGH in solution were deposited for 24 h at 37 degrees C on 100 mg of AP with different specific areas. The amount of hGH loaded was determined by immunoradiometric assay (IRMA) and eluted stain bioassay (ESTA) using Nb2 lymphoma rat cells. Although loading was not greatly influenced by a specific area between 3 and 25 m2/g, dependency was noted for higher specific areas. Human GH release was measured by IRMA and ESTA over a 33-day period, with half-time release between 25 and 79 h. Comparison of IRMA and ESTA measurements for the hGH amounts loaded showed that hGH biologic activity was conserved. Results indicate that it is feasible to control the quantity of TA loading on AP by modifying specific areas for in vivo applications.

We investigated the influence of natural coral implants used as a bone substitute on the quality of bone ingrowth in rabbits 2, 3, and 6 weeks after implantation. Explants were characterized by transmission electron microscopy and electron diffraction. Bone ingrowth has been previously demonstrated by light microscopy, however, few have been performed in electron microscopy to compare mineralized tissue ingrowth in coral implants which occurs at the expense of calcium carbonate to that of calcium phosphate (CaP) implants. The interface between coral aragonite and mineralized tissue or bone was abrupt, with no invasion of the aragonite structure by newly formed crystals, as occurs in micropores when biphasic CaP (BCP) ceramics were used. The restoring process appears to be different from that induced by BCP implants. Precipitation of needle-like apatite crystals on the CaCO3 implant surface was not observed. Instead, apatitic smooth-shaped crystals formed in aggregates. The coral dissolution process does not release phosphate and so precipitation of apatite does not occur in the micropores of the coral implant, thereby limiting the formation of an apatite layer and hence bone bonding to the outer surface of the implant. In addition, on the outer surface of the implant, close to bone and a phosphorus source, the CaP crystals that do form are in aggregates presumably due to the carbonate and mismatch between the aragonite and the apatite. This seems to result in a delayed bone attachment or weaker bone bonding than CaP implants which encourage an epitaxial biological crystal deposition.

This study investigated the effects of human growth hormone (hGH) on the monocyte/macrophage lineage, the first cell population involved in degradation of calcium phosphate ceramic after in vivo implantation. Monocytes isolated from human blood were cultured on biphasic calcium pellets (200 mg) for 8 days in the presence of lipopolysaccharides (LPS, 0.5 microgram/mL), hGH (10 and 50 ng/mL), or an association of LPS with hGH (10 and 50 ng/mL). Unlike LPS, hGH significantly decreased (about 25%) the total number of lacunae formed by monocytes. However, hGH induced the formation of lacunae with a greater surface area (about a 90% increase) as compared to the control. Finally, intense upmodulation (about a 250% increase) of lacuna surface area was observed in the presence of both soluble factors, suggesting that hGH and LPS act synergistically. In view of the development of a drug delivery system for hGH bone release, this study shows that hGH not only stimulates bone cells implicated in the synthesis of the extracellular matrix but also those involved in the early degradation of calcium phosphate biomaterial.

Calcium-phosphate bone replacement biomaterial has been used as a drug carrier for therapeutic agents. This study investigated the efficacy of local administration of human growth hormone (hGH) by macroporous biphasic calcium phosphate (MBCP) implants in improving the bone substitution qualities of ceramics. hGH release from MBCP implants loaded with 1 microg of hGH was rapid during the first 48 h and then sustained for a total of 9 days. Immunolocalization of hGH in vitro and in vivo by transmission electron microscopy showed its presence inside the material, indicating that it was able to penetrate within the porosity of the ceramic during the adsorption process. MBCP cylinders (6 x 6 mm) were loaded with 0.1, 1, and 10 microg of hGH and implanted into rabbit femurs (n = 40). The effects of locally released hGH on bone ingrowth and ceramic resorption were evaluated by scanning electron microscopy and image analysis. The results indicated that hGH increased bone ingrowth (+65%) and ceramic resorption (+140%) significantly in comparison with control implants and that the increase was dose dependent. Biochemical parameters monitored in rabbit plasma and urine, as well as the absence of any significant difference between contralateral implants and the control, indicated that hGH did not produce detectable systemic effects. Thus, the use of MBCP appears to be effective for local delivery of hGH, resulting in improved bone substitution.

Osteoclasts, the main protagonists involved in bone resorption mechanisms, are generally considered to be of haematopoietic origin, although the exact nature of the primary osteoclastic stem cells is still unknown. In vitro cellular models developed to study the different events of osteoclastic differentiation have revealed that not only several cell types (osteoblasts, monocytes, lymphocytes, etc.) but also many soluble factors (cytokines, hormones, vitamins, ions, etc.) and extracellular matrix elements (osteopontin, osteocalcin, etc.) are involved in osteoclastic differentiation and activation. This article provides an exhaustive review of recent knowledge on the origin of the osteoclast and the main substances involved in the osteoclastogenesis and activation of these cells.

This study investigated the possible role in vitro of insulin-like growth factor I (IGF-I) as a mediator of the effects of growth hormone (GH) on osteoclastic resorption in an unfractioned rabbit bone cell model. After 4 days of rabbit bone cell culture, human GH (hGH) (50 ng/mL) and human IGF-I (hIGF-I) (50 ng/mL) significantly increased the formation of osteoclast-like cells with a lower level than parathyroid hormone (50 ng/mL) or VD3 (10(-8) mol/L). As well as parathyroid hormone and 1-alpha,25-dihydroxyvitamin D3, addition of hGH (1, 10, and 50 ng/mL) and hIGF-I (1, 10, and 50 ng/mL) stimulated the resorption activity of osteoclasts in terms of the percentage of dentin slice surface resorbed, number of lacunae per surface unit, and mean area of lacunae as compared to the control. When neutralizing antiserum against hIGF-I (4 micrograms/mL) was added at the start of culture, the stimulatory effects of hIGF-I and hGH on osteoclastic resorption activity were totally abolished. These results indicate that the effects of GH stimulation on osteoclastic resorption in vitro are mediated via local IGF-I secretion by stromal cells such as osteoblasts. As IGF-I receptors have recently been reported on rabbit osteoclasts, a direct action of IGF-I on mature osteoclasts could be envisaged. Further experiments will be required to determine the real level of IGF-I implicated in the stimulation of bone osteoclastic resorption.

The use of calcium phosphate biomaterials as a bone substitute necessitates the use of normative biocompatibility and biodegradation techniques which must be fast, simple and reproducible. In the present study, we have developed an in vitro model to study and to compare different calcium phosphate ceramics. After activation with 1,25-dihydroxy-vitamin D3 and phorbol 12,13-dibutyrate, the monoblastic U937 cells became multinucleated, expressed tartrate-resistant acid phosphatase and several markers of monocyte/macrophage differentiation. Activated U937 cells did not express the vitronectin receptor (VNR) (as revealed using monoclonal antibodies 23C6 or 13C2) but around 25% of the cells were strongly reactive with 211D, a novel monoclonal antibody that recognizes an osteoclast-specific membrane antigenic determinant. These cells remain active/viable with hydroxyapatite (HA) or beta-tricalcium phosphate (beta-TCP) ceramics. In conclusion, activated U937 cells are good candidates to use in a normative in vitro method to evaluate new biomaterials.

The monocyte is an essential element in cellular immunity. Consequently, its role in the biocompatibility process is important. A model study of the degradation of bioactive ceramics (calcium phosphate) using human peripheral blood monocytes activated by 1,25 (OH)2 VD3 and interferon (IFN)-gamma was implemented. Activated monocytes cultivated on calcium phosphate tablets led to cellular morphological modifications and to changes in the number of nuclei (from d 2 to 14). IFN-gamma promoted adhesion, the appearance of cytoplasmic extensions and multinuclei. Cavity resorption activity on the material was observed simultaneously.

Ruptures of the anterior cruciform ligament is difficult to repair. Ligament prostheses are sometimes useful. We tested a ligament prosthesis which had the original property of combining a polyester braid with a matrix favouring collagen ingrowth. We implanted this prosthesis in the knee joint of 10 sheep after sectioning the anterior cruciform ligament at it origin. The ligaments were recovered after 6 months. We studied the functional and radiographic results. Tolerance was studied in samples of the articular fluid and synovial tissue. We evaluated bone attachment and fibrous growth using micro-radiography and specific coloration techniques. Samples were examined under electron microscope. No rupture was encountered. We observed a satisfactory functional and radiographic result. No intolerance was observed. The neoligament had a bony or mixed attachment in 60% of the cases and was comprised of oriented tissue in 90% of the cases. Vascular invasion was important. These findings are promising and suggest that long-term studies should be undertaken.