A delivery system of linezolid to enhance the MRSA osteomyelitis prognosis: in vivo experimental assessment.

Staphylococcus aureus, a major responsible microorganism of osteomyelitis, represents a challenge to treat because of the poor penetration of antibiotics in bone and increasing minimum inhibitory concentrations (MICs) to glycopeptides. The calcium-deficient apatites (CDA), closer to the biological components found in bone and other calcified tissues, have osteoconductive properties. So, to process severe osseous infections, CDA can be used to deliver in the infectious site antibiotics like linezolid. The acute experimental osteomyelitis due to methicillin-resistant Staphylococcus aureus (MRSA) was induced in rabbit's femurs and surgery mimicking human procedures was performed at day three after inoculation. Animals were randomly assigned to treatment groups: L((IV)) [4-day linezolid IV infusion, human-equivalent dose of 10 mg/kg/12 h], L((CDA50%)) (100 mg CDA with linezolid 500 µg/mg) and L((CDA50%)) + L((IV)). Surviving bacteria were counted in bone marrow (BM) and bone (Bo) at day 3 (before treatment), day 7 (4-day treatment) or day 17 (14-day treatment). L(iv) was effective after a 4-day treatment with a log(10)CFU/g decrease of -2.63 ± 1.92 and -2.17 ± 1.58 in bone marrow and bone, respectively. CDA loaded with linezolid enhance the efficacy of the IV linezolid regimen by more than one log(10)CFU/g.

Bone texture analysis of human femurs using a new device (BMA™) improves failure load prediction.

UNLABELLED: We measured bone texture parameters of excised human femurs with a new device (BMA™). We also measured bone mineral density by DXA and investigated the performance of these parameters in the prediction of failure load. Our results suggest that bone texture parameters improve failure load prediction when added to bone mineral density. INTRODUCTION: Bone mineral density (BMD) is a strong determinant of bone strength. However, nearly half of the fractures occur in patients with BMD which does not reach the osteoporotic threshold. In order to assess fracture risk properly, other factors are important to be taken into account such as clinical risk factors as well as macro- and microarchitecture of bone. Bone microarchitecture is usually assessed by high-resolution QCT, but this cannot be applied in routine clinical settings due to irradiation, cost and availability concerns. Texture analysis of bone has shown to be correlated to bone strength. METHODS: We used a new device to get digitized X-rays of 12 excised human femurs in order to measure bone texture parameters in three different regions of interest (ROIs). We investigated the performance of these parameters in the prediction of the failure load using biomechanical tests. Texture parameters measured were the fractal dimension (Hmean), the co-occurrence matrix, and the run length matrix. We also measured bone mineral density by DXA in the same ROIs as well as in standard DXA hip regions. RESULTS: The Spearman correlation coefficient between BMD and texture parameters measured in the same ROIs ranged from -0.05 (nonsignificant (NS)) to 0.57 (p = 0.003). There was no correlation between Hmean and co-occurrence matrix nor Hmean and run length matrix in the same ROI (r = -0.04 to 0.52, NS). Co-occurrence matrix and run length matrix in the same ROI were highly correlated (r = 0.90 to 0.99, p < 0.0001). Univariate analysis with the failure load revealed significant correlation only with BMD results, not texture parameters. Multiple regression analysis showed that the best predictors of failure load were BMD, Hmean, and run length matrix at the femoral neck, as well as age and sex, with an adjusted r (2) = 0.88. Added to femoral neck BMD, Hmean and run length matrix at the femoral neck (without the effect of age and sex) improved failure load prediction (compared to femoral neck BMD alone) from adjusted r (2) = 0.67 to adjusted r (2) = 0.84. CONCLUSION: Our results suggest that bone texture measurement improves failure load prediction when added to BMD.

In vivo assessment of the antimicrobial activity of a calcium-deficient apatite vancomycin drug delivery system in a methicillin-resistant Staphylococcus aureus rabbit osteomyelitis experimental model.

The antimicrobial activities of calcium-deficient apatite loaded with different concentrations (25, 100, and 500 microg/mg) of vancomycin as a filling biomaterial were evaluated in a methicillin-resistant Staphylococcus aureus (MRSA) rabbit acute osteomyelitis model. Bacterial counts in bone, bone marrow, and joint fluid samples treated with forms of the apatite were compared to those in tissue samples receiving a constant intravenous vancomycin infusion after 4 days. This study demonstrates that using a calcium-deficient apatite loaded with vancomycin dramatically decreases the bacterial counts in bone and marrow.

Development of bisphosphonates controlled delivery systems for bone implantation: influence of the formulation and process used on in vitro release.

The present study investigates the development of controlled drug delivery devices by association of bisphosphonates (BPs) with calcium-deficient apatite (CDA) to obtain a prolonged drug delivery. In a first part, we studied the microencapsulation of methylene bisphosphonic acid, our model of BPs, in biodegradable PLGA by the double emulsion (w/o/w) solvent evaporation/extraction process. Secondly, we associated BPs, either in a free form or microencapsulated, with calcium phosphate biomaterials. The association of free BPs with CDA was performed by isostatic compression at 80 MPa and we tested the interest of adding a binder, HPMC, in the formulation to reinforce the association. In parallel, microparticles were associated with calcium-deficient apatite, either by simple mixture or by isostatic compression. To compare the different formulations, in vitro dissolution studies were performed. All the formulations tested appear to be efficient to produce BPs loaded biomaterials able to deliver the drug slowly and at a constant rate. The slowest release rate (2.7% in 14 days) was obtained with the blend of microencapsulated BPs with CDA.

In vitro characterisation of calcium phosphate biomaterials loaded with lidocaine hydrochloride and morphine hydrochloride.

Calcium phosphate substitutes drug delivery systems are well known substances used in minor bone void-filling to release their therapeutic agent in situ. Few studies associating anaesthetics and analgesics have been performed to date. The aim of this work was to study the association of the analgesic, morphine, and the local anaesthetic, lidocaine, with a calcium deficient apatite matrix. Three types of biomaterials i.e. powders, granules and blocks, were prepared by isostatic compression, wet granulation and a combination of the two, evaluated and compared. The chemical structure of the associated therapeutic agent was studied and the characteristics of the drug delivery systems were appraised in terms of drug release. The integrity of the lidocaine hydrochloride structure, as determined by RMN (1)H, was confirmed regardless of the formulation technique used (isostatic compression or wet granulation). However, analyses of morphine hydrochloride by RMN (1)H revealed slight structural modifications. The association and formulation techniques that were used made it possible to obtain an in vitro release time varying from 1 to 4 days for lidocaine hydrochloride and from 1 to 3 days for morphine hydrochloride.

Implants delivering bisphosphonate locally increase periprosthetic bone density in an osteoporotic sheep model. A pilot study.

It is a clinical challenge to obtain a sufficient orthopaedic implant fixation in weak osteoporotic bone. When the primary implant fixation is poor, micromotions occur at the bone-implant interface, activating osteoclasts, which leads to implant loosening. Bisphosphonate can be used to prevent the osteoclastic response, but when administered systemically its bioavailability is low and the time it takes for the drug to reach the periprosthetic bone may be a limiting factor. Recent data has shown that delivering bisphosphonate locally from the implant surface could be an interesting solution. Local bisphosphonate delivery increased periprosthetic bone density, which leads to a stronger implant fixation, as demonstrated in rats by the increased implant pullout force. The aim of the present study was to verify the positive effect on periprosthetic bone remodelling of local bisphosphonate delivery in an osteoporotic sheep model. Four implants coated with zoledronate and two control implants were inserted in the femoral condyle of ovariectomized sheep for 4 weeks. The bone at the implant surface was 50% higher in the zoledronate-group compared to control group. This effect was significant up to a distance of 400mum from the implant surface. The presented results are similar to what was observed in the osteoporotic rat model, which suggest that the concept of releasing zoledronate locally from the implant to increase the implant fixation is not species specific. The results of this trial study support the claim that local zoledronate could increase the fixation of an implant in weak bone.

Biphasic calcium phosphate: a comparative study of interconnected porosity in two ceramics.

Interconnection, one of the main structural features of macroporous calcium-phosphate ceramics, contributes to the biological and physicochemical properties of bone substitutes. As no satisfactory method exists for evaluating this feature, analysis was performed to determine the permeability, tortuosity, and equivalent diameter of interconnecting channels, that is the parameters that appear to be representative of the way pores are linked. The testing of two ceramics with similar porosity levels revealed important differences in all three interconnection parameters. One ceramic showed poor permeability, corresponding to a small equivalent diameter for interconnecting channels in conjunction with a high tortuosity factor, while the other displayed high permeability, a large diameter for interconnecting channels, and a low tortuosity factor. The methodology used, which can be applied to the quantification of interconnection in all calcium-phosphate ceramics, constitutes the first step in a complete study of the role of this feature in cellular colonization of the ceramic, matrix dissolution, and drug release from the calcium-phosphate matrix.

The response of pre-osteoblasts and osteoclasts to gallium containing mesoporous bioactive glasses.

Mesoporous bioactive glasses (MBGs) in the system SiO2-CaO-P2O5-Ga2O3 have been synthesized by the evaporation induced self-assembly method and subsequent impregnation with Ga cations. Two different compositions have been prepared and the local environment of Ga(III) has been characterized using 29Si, 71Ga and 31P NMR analysis, demonstrating that Ga(III) is efficiently incorporated as both, network former (GaO4 units) and network modifier (GaO6 units). In vitro bioactivity tests evidenced that Ga-containing MBGs retain their capability for nucleation and growth of an apatite-like layer in contact with a simulated body fluid with ion concentrations nearly equal to those of human blood plasma. Finally, in vitro cell culture tests evidenced that Ga incorporation results in a selective effect on osteoblasts and osteoclasts. Indeed, the presence of this element enhances the early differentiation towards osteoblast phenotype while disturbing osteoclastogenesis. Considering these results, Ga-doped MBGs might be proposed as bone substitutes, especially in osteoporosis scenarios. STATEMENT OF SIGNIFCANCE: Osteoporosis is the most prevalent bone disease affecting millions of patients every year. However, there is a lack of bone grafts specifically designed for the treatment of bone defects occurred because of osteoporotic fractures. The consequence is that osteoporotic bone defects are commonly treated with the same biomaterials intended for high quality bone tissue. In this work we have prepared mesoporous bioactive glasses doped with gallium, demonstrating osteoinductive capability by promoting the differentiation of pre-osteoblast toward osteoblasts and partial inhibition of osteoclastogenesis. Through a deep study of the local environment of gallium within the mesoporous matrix, this work shows that gallium release is not required to produce this effect on osteoblasts and osteoclasts. In this sense, the presence of this element at the surface of the mesoporous bioactive glasses would be enough to locally promote bone formation while reducing bone resorption.

Biphasic calcium phosphate ceramics for bone reconstruction: A review of biological response.

Autologous bone graft is considered as the gold standard in bone reconstructive surgery. However, the quantity of bone available is limited and the harvesting procedure requires a second surgical site resulting in severe complications. Due to these limits, scientists and clinicians have considered alternatives to autologous bone graft. Calcium phosphates (CaPs) biomaterials including biphasic calcium phosphate (BCP) ceramics have proven efficacy in numerous clinical indications. Their specific physico-chemical properties (HA/TCP ratio, dual porosity and subsequent interconnected architecture) control (regulate/condition) the progressive resorption and the bone substitution process. By describing the most significant biological responses reported in the last 30years, we review the main events that made their clinical success. We also discuss about their exciting future applications as osteoconductive scaffold for delivering various bioactive molecules or bone cells in bone tissue engineering and regenerative medicine. STATEMENT OF SIGNIFICANCE: Nowadays, BCPs are definitely considered as the gold standard of bone substitutes in bone reconstructive surgery. Among the numerous clinical studies in literature demonstrating the performance of BCP, Passuti et al. and Randsford et al. studies largely contributed to the emergence of the BCPs. It could be interesting to come back to the main events that made their success and could explain their large adhesion from scientists to clinicians. This paper aims to review the most significant biological responses reported in the last 30years, of these BCP-based materials. We also discuss about their exciting future applications as osteoconductive scaffold for delivering various bioactive molecules or bone cells in bone tissue engineering and regenerative medicine.

Calcium phosphate drug delivery system: influence of local zoledronate release on bone implant osteointegration.

Despite total hip replacement (THR) gives generally satisfactory results, the quality of outcome in young patients is markedly decreased compared to the average THR outcome. For this population, pharmacological treatment with bisphosphonate would be beneficial to decrease the peri-implant osteolysis. However, as this population does not necessarily suffer from osteoporosis, a nonsystemic treatment would be preferable. Zoledronate was then grafted to hydroxyapatite (HA) coating of titanium implants. The implants were inserted in rat condyles with various zoledronate concentrations. A positive concentration-dependent effect was observed on the peri-implant bone density and on different histomorphometric parameters. Importantly for the outcome of the implants, the mechanical fixation was increased by the local presence of zoledronate. The obtained results open the way of an easy transformation of currently existing HA-coated implants by grafting bisphosphonate onto the coating in order to increase their service life in the patients.

Adaptive crystal formation in normal and pathological calcifications in synthetic calcium phosphate and related biomaterials.

Mineralization and crystal deposition are natural phenomena widely distributed in biological systems from protozoa to mammals. In mammals, normal and pathological calcifications are observed in bones, teeth, and soft tissues or cartilage. We review studies on the adaptive apatite crystal formation in enamel compared with those in other calcified tissues (e.g., dentin, bone, and fish enameloids) and in pathological calcifications, demonstrating the adaptation of these crystals (in terms of crystallinity and orientation) to specific tissues that vary in functions or vary in normal or diseased conditions. The roles of minor elements, such as carbonate, magnesium, fluoride, hydrogen phosphate, pyrophosphate, and strontium ions, on the formation and transformation of biologically relevant calcium phosphates are summarized. Another adaptative process of crystals in biology concerns the recent development of calcium phosphate ceramics and other related biomaterials for bone graft. Bone graft materials are available as alternatives to autogeneous bone for repair, substitution, or augmentation. This paper discusses the adaptive crystal formation in mineralized tissues induced by calcium phosphate and related bone graft biomaterials during bone repair.

Study of the maturation of the organic (type I collagen) and mineral (nonstoichiometric apatite) constituents of a calcified tissue (dentin) as a function of location: a Fourier transform infrared microspectroscopic investigation.

Fourier transform infrared microspectroscopy (FTIRM) was used to investigate the organic and mineral phases of a calcified tissue (dentin) as a function of its location from predentin toward enamel. Thin dentin slices (decalcified or not) were fixed in formaldehyde and embedded in glycolmethylmethacrylate (GMA). Fixation did not denature collagen, and GMA did not interact with organic or mineral constituents of dentin. The v1v3 PO4 domain was studied in particular in order to estimate mineral maturity and amide I, II, A, and B to obtain data on protein conformation. The results showed that dentin apatite became increasingly mature (stoichiometric) from the mineralization front toward the enamel, especially through loss of HPO4(2-) groups and vacancies. Moreover, collagen fibrils became less and less hydrated, suggesting that intrafibrillar mineralization partially dehydrated the collagen. Combined study of the organic and mineral fractions of calcified tissues may help clarify their relationships in physiological and pathological tissues.

Nuclear magnetic resonance spectroscopy of bone substitutes.

Calcium phosphate bone replacement biomaterials are widely used in different applications. Structure, composition, and organization are, before implantation, analyzed with different methods. Among them, X-ray diffraction is a recognized test. As bioresorption produces more amorphous material, the process is observed and quantified via scanning electron microscopy. Comparatively high-resolution 31P solid-state nuclear magnetic resonance spectroscopy is able to analyze raw ceramics composition and to estimate osteoformation.

Injectable bone substitute using a hydrophilic polymer.

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.

Biphasic calcium phosphate/hydrosoluble polymer composites: a new concept for bone and dental substitution biomaterials.

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.

Short-term effects of mineral particle sizes on cellular degradation activity after implantation of injectable calcium phosphate biomaterials and the consequences for bone substitution.

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.

Modification of gene expression induced in human osteogenic and osteosarcoma cells by culture on a biphasic calcium phosphate bone substitute.

Bone hybrids made of bioceramics seeded with mesenchymal or osteoblastic cells are very promising alternatives to autologous bone graft. Along this line, the development of in vitro models, dedicated to analyze the influence of these biomaterials on osteogenic cells, will help to improve the performance of these bone substitutes. In the present work we analyzed the effects of a macroporous biphasic calcium phosphate ceramic (BCP, Triosite) on three different human osteosarcoma cell lines and on human primary osteogenic cells and compared this culture substratum to traditional culture on plastic. We showed that all these osteoblastic cells adhere and proliferate on the trabecular BCP blocks, with a different spatial organization for osteosarcoma cells compared to normal osteogenic cells. We also demonstrated that osteoblastic marker genes such as Cbfa1, type I collagen, osteonectin, osteopontin, and osteocalcin were expressed at similar levels by these cells cultured on either substratum, suggesting that adhesion to BCP does maintain the osteoblastic phenotype of these cells. Next, we provided the first evidence of differences of cytokine expression profiles revealed on this Ca-P ceramic as compared to expression in classical culture. These modifications affected the expression of cytokines such as TGF-beta1, G-CSF, and IL-3 and were quantitatively different between osteosarcoma cells and normal osteogenic cells. Given the role of these cytokines in bone biology and in hematopoiesis, these results obtained in vitro suggest that the BCP ceramic studied here could stimulate osteogenesis in vivo by activating cellular processes during bone formation and healing. This study highlights the notion that the nature of the culture substratum must be taken into account when studying bone cell biology in vitro. Owing to the nature and spatial organization of the BCP, our hypothesis is that culture on BCP is closer to the physiological situation than culture on plastic.

Osteoclastic resorption of calcium phosphate ceramics with different hydroxyapatite/beta-tricalcium phosphate ratios.

To study the influence of calcium phosphate ceramic solubility on osteoclastic resorption, neonatal rabbit bone cells were cultured for 2 days on hydroxyapatite (HA), beta-tricalcium phosphate (beta-TCP) and two types of biphasic calcium phosphate (BCP) with HA/beta-TCP ratios of 25/75 and 75/25. Solubility was regulated by varying the ratio of less-soluble HA and more-soluble beta-TCP. After removal of stromal cells by pronase E treatment, ceramic surfaces were observed by scanning electron microscopy. Osteoclasts resorbed BCP most extensively, with an HA/beta-TCP ratio of 25/75, producing typical lobulated, zig-zag track-like resorption lacunae. On pure beta-TCP, which had the highest solubility in acid, osteoclasts formed smaller discontinuous island-like lacunae. The resorption pattern may have been modified by the large number of calcium ions released into the acidic microenvironment at the osteoclast-ceramic interface. No resorption lacunae were found on the other specimens. The extent of osteoclastic resorption of calcium phosphate ceramics might, to a certain degree, be proportional to solubility, although this was not the case when solubility was very high. It would appear that ceramic solubility influences osteoclast resorption activity.

Biodegradation of synthetic biphasic calcium phosphate by human monocytes in vitro: a morphological study.

Biodegradation processes (both intra- and extracellular) occur immediately after implantation of calcium phosphate (CaP) ceramics. Monocytes and macrophages, among the first cells to appear in wound healing, are largely implicated in phagocytosis and may be involved in CaP degradation because of their sensitivity to secreted cytokines. We tested the behaviour of human monocytes placed on the surface of hydroxyapatite (HA) and biphasic calcium phosphate (BCP) tablets in the presence of vitamin D3 (VD3) and interferon gamma (INF gamma). After short-term culture (6 days), morphological events were observed in histological and scanning electron microscopy studies, and degradation lacunae were characterized. There were cell prints but no pits on the HA surface, but pits appeared near cells on the BCP surface. Preincubation of biomaterial in culture medium was essential. Variations in cell morphology were observed in different culture types. In the presence of VD3, degradation was greater than in the control, and cells were more polarized and rounded. With INF gamma, cells were extensively spread out on the sample surface, and the biomaterial seemed to be extracted from the surface by cells. Thus, monocytes are clearly influenced by soluble factors (vitamins, cytokines) and could be key cells in initiating the degradation of biomaterial.

Macroporous biphasic calcium phosphate ceramics: influence of macropore diameter and macroporosity percentage on bone ingrowth.

A total of 60 cylindrical 6 x 6 mm samples of a macroporous biphasic calcium phosphate (MBCP) ceramic were implanted into a distal femoral site in 30 rabbits. These samples represented six kinds of implants with two different macropore diameters and three different macroporosity percentages. Analysis of backscattered electron images of implant surfaces analysed by a factorial design method showed that implants with 565 microm pore size provided more abundant newly formed bone both in peripheral and deep pores than those with 300 microm pore size. No significant differences were found between implants with 40 and 50% macroporosity, suggesting that the influence of macropore size on bone ingrowth was greater than that of macroporosity percentage. MBCP implants with 565 microm pore diameter and 40% macroporosity represented the optimal association for homogeneous and abundant bone ingrowth.