负载抗菌药物的骨水泥的药物释放实验研究

目的 揭示负载抗菌药物的骨水泥的药物释放浓度、持续时间、药物残留程度及低于最小有效抑菌浓度的释放时间,为合理应用抗菌药物提供理论依据。方法 通过对负载盐酸万古霉素和亚胺培南西司他丁钠的骨水泥进行体外洗提实验,采用高效液相色谱法检测分析两种负载抗菌药物的骨水泥的药物释放浓度、释放持续时间和药物残留程度。结果 ①负载抗菌药物的骨水泥第一天药物释放量最大,后逐渐持平,且释放时间较长,释放浓度较低;②截止到第7周盐酸万古霉素、亚胺培南西司他丁释放量分别只占总负载量的15%、25%;③盐酸万古霉素第1周后释放浓度低于最小有效抑菌浓度,亚胺培南西司他丁钠第7周后释放浓度低于最小有效抑菌浓度。结论 载抗菌药物骨水泥存在长时间药物残留现象,是导致持续低于最小有效抑菌浓度的主要原因。此种现象不但对细菌起不到抑菌作用,而且是诱导细菌耐药性的危险因素。     

Controlled release of gentamicin from calcium phosphate-poly(lactic acid-co-glycolic acid) composite bone cement

Modification of a self setting bone cement with biodegradable microspheres to achieve controlled local release of antibiotics without compromising mechanical properties was investigated. Different biodegradable microsphere batches were prepared from poly(lactic-co-glycolic acid) (PLGA) using a spray-drying technique to encapsulate gentamicin crobefate varying PLGA composition and drug loading. Microsphere properties such as surface morphology, particle size and antibiotic drug release profiles were characterized. Microspheres were mixed with an apatitic calcium phosphate bone cement to generate an antibiotic drug delivery system for treatment of bone defects. All batches of cement/microsphere composites showed an unchanged compressive strength of 60 MPa and no increase in setting time. Antibiotic release increased with increasing drug loading of the microspheres up to 30% (w/w). Drug burst of gentamicin crobefate in the microspheres was abolished in cement/microsphere composites yielding nearly zero order release profiles. Modification of calcium phosphate cements using biodegradable microspheres proved to be an efficient drug delivery system allowing a broad range of 10-30% drug loading with uncompromised mechanical properties.     

The release of gentamicin from acrylic bone cements in a simulated prosthesis-related interfacial gap

Gentamicin is added to polymethylmethacrylate bone cements in orthopedics as a measure against infection in total joint arthroplasties. Numerous studies have been published on gentamicin release from bone cements, but none have been able to estimate the local concentrations in the prosthesis-related interfacial gap, present after implantation. The aim of this study was to develop a method allowing determination of antibiotic release in such a gap. Two-hundred-micrometer-wide gaps with a volume of 6 microl and a surface area of 0.6 cm2 were created by inserting stainless-steel strips in gentamicin-loaded bone cement plugs prior to polymerization. After hardening, the gap surface was exposed to 6 microl or 10 ml of phosphate-buffered saline. Within 2 h, gentamicin concentrations in the gaps reached around 4000 microg/ml for 4 different CMW and Palamed cements and 2500 microg/ml for Palacos R. Concentrations measured in the larger volume were several hundred times lower than in the gaps. This simulated prosthesis-related interfacial gap model offers new insights in the clinical efficacy of antibiotic-loaded bone cements. It is demonstrated that concentrations up to 1000-fold the antibiotic resistance levels for most bacterial strains causing implant infection can be achieved in a realistic in vitro model.     

The in vitro elution of gentamicin sulfate from a commercially available gentamicin-loaded acrylic bone cement, VersaBond AB

The present study was designed to yield results that would be used to contribute to the ongoing debate about the mechanism of the in vitro elution of an antibiotic from an antibiotic-loaded acrylic bone cement. To this end, the elution rates (R) of gentamicin sulfate (expressed as a weight percentage of the initial mass of the antibiotic in the specimen, normalized with respect to the duration of the test) from statically loaded (STATIC) and dynamically loaded (+/-10 MPa; 2 Hz; until fracture; DYNAMIC) specimens fabricated from a commercially available acrylic bone cement (VersaBond AB), in phosphate-buffered saline solution at 37 degrees C, were obtained with the use of a spectrophotometric method. There was evidence of microcracking in the fracture surfaces of DYNAMIC specimens, but no such evidence in the case of STATIC specimens. The surface area of the DYNAMIC specimens, during the tensile phase of the cyclical loading, was estimated to be about 3% larger than for the STATIC specimens (1742 mm(2) versus 1696 mm(2)). The bulk porosities P of the specimens in both sets were also determined and found to not be statistically different, with P for the STATIC and DYNAMIC specimens being 8.55 +/- 0.10 and 8.88 +/- 0.18%, respectively. At the end of the test period, R was found to be 0.36 +/- 0.20 and 1.28 +/- 0.14 wt %/day for the STATIC and DYNAMIC specimens, respectively. It is suggested that the present results provide support for the postulate that the elution mechanism of gentamicin in this cement is a surface phenomenon.     

Study the effect of polymerization temperature in the release of antibiotic from bone cement

Antibiotic-loaded bone cement is widely used to treat musculoskeletal infections. Here we tested whether the curing temperature affected pore size of the cement and antibiotic release. Bone cement containing vancomycin was cured at 3 temperatures (50, 25 and 0°C). The solidified vancomycin-loaded cement was stored for 1-week at 25 and 5°C and then assayed for antibiotic release. We found that a significantly higher proportion of vancomycin was released from cement cured at 0°C versus that cured at 50°C, and that lower storage temperature also increased antibiotic release.     

Release of gentamicin sulphate from a modified commercial bone cement. Effect of (2-hydroxyethyl methacrylate) comonomer and poly(N-vinyl-2-pyrrolidone) additive on release mechanism and kinetics

The influence of the (2-hydroxyethyl methacrylate) (HEMA) monomer addition as a comonomer to the cement liquid component and of a polymer, poly(N-vinyl-2-pyrrolidone) (PVP) to the solid component of a standard CMW-1 bone cement on gentamicin sulphate (GS) on its drug release properties have been studied. The addition of HEMA modifies the habit of the delivery curves. The incorporation of PVP into the cement matrix, apparently, did not very much modify the shape of the HEMA modified cement release curves, but led to a remarkable increase of the maximum amount of GS released. This effect was proportional to the PVP concentration incorporated. From the matrix composition and SEM data, a model based on the morphology of the matrix has been proposed. The cumulative amount of GS released by each slab Mt is most adequately fitted and represented by the equation Mt = c + at 1/2 + b[1 - exp(-nt)], which corroborates that the release occurs according to the model proposed. by means of three discrete mechanisms, namely: (i) a short-term initial elution due to the imperfections in the poly(methyl methacrylate) covering of the most external GS beads, burst effect by the buffer solution; (ii) followed by a fracture by stress cracking in an active media of the coated GS beads located on the external surface of the matrix where water molecules enter to dissolve GS molecules releasing them into the buffer solution by a diffusion-controlled process; and (iii) a third process in which the buffer solution penetrates into the internal voids and cracks creating a series of channels in a labyrinthic structure, which may facilitate the access of water molecules to the plastic-coated GS beads within the bulk matrix. This third process is enhanced by the incorporation of PVP beads as dissolved molecules within the matrix. This water-soluble additive is leachable, generating a highly porous structure in the cement. This HEMA and PVP modified cement may be used as a drug delivery system to modulate the GS release rate.     

The influence of ultrasound on the release of gentamicin from antibiotic-loaded acrylic beads and bone cements

Gentamicin-loaded acrylic beads are loosely placed in infected bone cavities, whereas gentamicin-loaded acrylic bone cement is used as a mechanical filler in bone to anchor prosthetic components. Both drug delivery systems are used to decrease infection rates by gentamicin release. The objective of this study is to investigate the effects of pulsed ultrasound on gentamicin release from both materials. Gentamicin release from gentamicin-loaded beads (Septopal) and from three commercially-available brands of gentamicin-loaded bone cement (CMW 1, Palacos R-G, and Palamed G) was measured after 18 h of exposure in PBS to an ultrasonic field of 46.5 kHz in a 1:3 duty cycle with an average acoustic intensity of 167 mW/cm(2). Samples not exposed to ultrasound were used as controls. Pulsed ultrasound significantly enhanced gentamicin release from gentamicin-loaded beads, whereas gentamicin release from the gentamicin-loaded bone cements was not significantly enhanced. Mercury intrusion porosimetry revealed an increased distribution of pores between 0.1 and 0.01 microm in beads after gentamicin release, while in bone cements no increase in the number of pores was found. Increased gentamicin release in beads due to ultrasound may be explained by micro-streaming in a porous structure, whereas the absence of changes in pore structure after gentamicin release in bone cement is concurrent with the lack of an enhanced release of the antibiotic by ultrasound. As an effective treatment of infections requires high local concentrations of antibiotic, increased gentamicin release due to ultrasound may be of clinical significance, especially since ultrasound has been demonstrated to increase bacterial killing by antibiotics.     

The effect of the extracorporeal shock wave lithotriptor on bone cement

For the purpose of studying its applicability for acrylic cement removal during total hip revision surgery, experiments with an extracorporeal shock wave lithotriptor were carried out. High-energy shock waves (HESW) were focussed on discs of polymethylmethacrylate bone cement. The average discharge was 18.1 kV; the number of shock waves 0, 100, 250, 500, 1000, and 2000; the application rate was 85 shocks/min. Macroscopic or radiographic effects were not in evidence. Microscopically, typical lesions in a small concentric focal area with a diameter of 8.5 (+/- 2.5) mm were found. The individual lesions were smaller than 0.1 mm, and displayed characteristic shapes. The area porosity increased with the number of shocks. The maximal area porosity caused by the HESW, measured by quantitative microscopy, was 4% after 2000 shock waves. The lesions were also studied by scanning electron microscopy. It can be concluded that HESW causes only microscopic lesions on the frontal surface of discs of bone cement, and that these lesions are small compared to the pores normally present in bone cement, when applied clinically.     

降钙素基因相关肽对内皮素释放的影响

本实验在大鼠的整体及离体血管条上观察了降钙素基因相关肽（ＣＧＲＰ）对内皮素（ＥＴ）释放的影响。结果表明：ＣＧＲＰ静脉注射（５μｇ／ｋｇ）能明显降低大鼠内毒素血症时血浆ＥＴ含量，ＣＧＲＰ孵育（１０＾－８ｍｏｌ／Ｌ）离体的大鼠主动脉血管条能有效地抑制凝血酶（ｔｈｒｏｍｂｉｎ）引起的ＥＴ释放，但是ＣＧＲＰ静注及孵育不影响血浆ＥＴ的基础含量及离体血管条ＥＴ的基础释放。结果提示ＣＧＲＰ能抑制病理条件下ＥＴ的     

The effect of protein structure on their controlled release from an injectable peptide hydrogel

Hydrogel materials are promising vehicles for the delivery of protein therapeutics. Proteins can impart physical interactions, both steric and electrostatic in nature, that influence their release from a given gel network. Here, model proteins of varying hydrodynamic diameter and charge are directly encapsulated and their release studied from electropositive fibrillar hydrogels prepared from the self-assembling peptide, MAX8. Hydrogelation of MAX8 can be triggered in the presence of proteins for their direct encapsulation with neither effect on protein structure nor the hydrogel's mechanical properties. Bulk release of the encapsulated proteins from the hydrogels was assessed for a month time period at 37 °C before and after syringe delivery of the loaded gels to determine the influence of the protein structure on release. Release of positively charged and neutral proteins was largely governed by the sterics imposed by the network. Conversely, negatively charged proteins interacted strongly with the positively charged fibrillar network, greatly restricting their release to <10% of the initial protein load. Partition and retention studies indicated that electrostatic interactions dictate the amount of protein available for release. Importantly, when protein encapsulated gels were delivered via syringe, the release profiles of the macromolecules show the similar trends as those observed for non-sheared gels. This study demonstrates that proteins can be directly encapsulated in self assembled MAX8 hydrogels, which can then be syringe delivered to a site where subsequent release is controlled by protein structure.     

Adsorption behavior of antimicrobial peptide histatin 5 on PMMA

Adsorption of antimicrobial peptide histatin 5 on a poly(methyl methacrylate) denture base may serve to prevent biofilm formation, leading to a reduction of denture-induced stomatitis. This study focused on adsorption behavior of histatin 5 onto PMMA surfaces modified using a cold plasma technique and the effectiveness of histatin 5 adsorption for reducing Candida albicans biofilm formation by the quartz crystal microbalance-dissipation (QCM-D) technique. PMMA spin-coated specimens were treated with oxygen (O(2)) plasma using a plasma surface modification apparatus. The amount of histatin 5 adsorbed onto the PMMA treated with O(2) plasma is more than six times greater than that adsorbed onto untreated PMMA. The degree of histatin 5 adsorption had a negative correlation with the contact angle, whereas that of zeta-potential showed no significant correlation. XPS analysis revealed that the introduction of the carboxyl and O(2) functional groups were observable on the O(2) plasma-treated PMMA. Increased surface hydrophilicity and the formation of the carboxyl could be responsible for histatin 5 adsorption on plasma-treated PMMA. There is no significant difference between histatin-adsorbed PMMA and control PMMA for C. albicans initially attached. On the contrary, the amount of C. albicans colonization on histatin-adsorbed PMMA was significantly less than the control.     

The effect of porosity on drug release kinetics from vancomycin microsphere/calcium phosphate cement composites

The influence of porosity on release profiles of antibiotics from calcium phosphate composites was investigated to optimize the duration of treatment. We hypothesized, that by the encapsulation of vancomycin-HCl into biodegradable microspheres prior admixing to calcium phosphate bone cement, the influence of porosity of the cement matrix on vancomycin release could be reduced. Encapsulation of vancomycin into a biodegradable poly(lactic co-glycolic acid) copolymer (PLGA) was performed by spray drying; drug-loaded microparticles were added to calcium phosphate cement (CPC) at different powder to liquid ratios (P/L), resulting in different porosities of the cement composites. The effect of differences in P/L ratio on drug release kinetics was compared for both the direct addition of vancomycin-HCl to the cement liquid and for cement composites modified with vancomycin-HCl-loaded microspheres. Scanning electron microscopy (SEM) was used to visualize surface and cross section morphology of the different composites. Brunauer, Emmett, and Teller-plots (BET) was used to determine the specific surface area and pore size distribution of these matrices. It could be clearly shown, that variations in P/L ratio influenced both the porosity of cement and vancomycin release profiles. Antibiotic activity during release study was successfully measured using an agar diffusion assay. However, vancomycin-HCl encapsulation into PLGA polymer microspheres decreased porosity influence of cement on drug release while maintaining antibiotic activity of the embedded substance.     

Characterization and antimicrobial evaluation of SpPR-AMP1, a proline-rich antimicrobial peptide from the mud crab Scylla paramamosain

Antimicrobial peptide (AMP) is an important molecule in the innate immune system. Here, we report the cloning and functional studies of proline-rich AMPs (PR-AMPs) from the three species of mud crab: Scylla paramamosain, S. serrata, and the swimming crab Portunus pelagicus. The deduced peptides revealed that they contain the putative signal peptides and encode for mature peptides, which contain sequence architecture similar to a 6.5-kDa proline-rich AMP of the shore crab, Carcinus maenas which showed similarity with the bactenecin7. Tissue distribution analysis indicated that the SpPR-AMP1 was expressed in a wide range of adult tissues, with the highest expression levels in the crab hemocyte. Challenge experiments showed that the levels of SpPR-AMP1 mRNA expression were up-regulated in the hemocyte after peptidoglycan stimulation. To evaluate the biological properties of mature SpPR-AMP1, peptides were chemically synthesized and recombinantly expressed. SpPR-AMP1 showed strong antibacterial activity against both Gram-positive bacteria Micrococcus luteus and Gram-negative bacteria Vibrio harveyi. The results indicate that the SpPR-AMP1 plays a role in crab immunity.     

The effect of the monomer-to-powder ratio on the material properties of acrylic bone cement

The procedure percutaneous vertebroplasty consists of injecting polymethylmethacrylate cement into vertebral bodies for the treatment of osteoporotic compression fractures and tumors of the spine. Clinicians practicing vertebroplasty commonly alter the mixture of monomer-to-powder recommended by the manufacturer in an effort to decrease viscosity and increase the working time. The purpose of the current study was to measure the effect of varying the monomer-to-powder ratio on the compressive material properties (compressive modulus, yield stress, and ultimate compressive strength) of the cement Simplex P (Stryker-Howmedica-Osteonics, Rutherford, NJ). Cylindrical specimens were prepared using monomer-to-powder ratios of 0.45 to 1.00 mL/g and tested in compression. Peak compressive material properties occurred at the mixture ratio recommended by the manufacturer (0.5 mL/g) but decreased as the ratio of monomer to powder was increased. The material properties of specimens cured for 1 hour were significantly less than those for specimens cured for 24 hours. The monomer-to-powder ratio affects the compressive material properties of cement. The clinical significance of these results with respect to vertebroplasty is yet to be determined.     

Quantitative analysis of monomer vapor release from two-solution bone cement by using a novel FTIR technique

Methyl methacrylate monomer can evaporate from bone cement to reach cytotoxic levels of concentrations in the implant bed of total joint prosthesis. Therefore, this study was performed by using a novel Fourier transform infrared spectroscopy method to quantify the release of monomer vapor from experimental two-solution bone cement in vitro during polymerization, to examine the effect of surface area versus cement mass, and to explore the effect of initiation chemistry. The results revealed that monomer vapor release is a surface phenomenon. In addition, initiation chemistry plays a major role in controlling the reaction time, and therefore heat concentration and dissipation, which resulted in a higher absorbance peak as initiation chemicals concentration increased. It was concluded that using the FTIR to monitor MMA vapors is an effective technique to obtain quantitative information about monomer vapor release from bone cements during polymerization and provides insight on the polymerization kinetics of two-solution acrylic bone cement.     

Exothermal characteristics and release of residual monomers from fiber-reinforced oligomer-modified acrylic bone cement

The aim of this study is to determine the peak temperature of polymerization, the setting time and the release of residual monomers of a modified acrylic bone cement. Palacos R, a commercial bone cement, is used as the main component. The cement is modified by adding short glass fibers and resorbable oligomer fillers, and an additional cross-linking monomer. The test specimens are classified according to the composition of the bone cement matrix (i.e., oligomer-filler, glass-fiber reinforcement, and/or cross-linking monomer). The exothermal characteristics during autopolymerization are analyzed using a transducer connected with a computer. The quantities of residual monomers were analyzed from different test groups using high performance liquid chromatography (HPLC). The DeltaT value for the oligomer filler and the glass-fiber-containing acrylic bone cement is lower than that for the unmodified bone cement (2.1 +/- 0.8 vs. 23.5 +/- 4.2 degrees C). The addition of a cross-linking monomer, EGDMA, shortens the setting time of the autopolymerization of the unmodified bone cement (7.1 +/- 0.9 min vs. 3.3 +/- 0.3 min). The quantity of the residual monomers released is higher in the modified bone cement than that in the unmodified cement. The cement that contains glass fibers and oligomer fillers has a considerably lower exothermal peak, whereas the total quantity of residual monomers released is increased.