Effect of two cleaning processes for bone allografts on gentamicin impregnation and in vitro antibiotic release

Bone allografts are a useful and sometimes indispensable tool for the surgeon to repair bone defects. Microbial contamination is a major reason for discarding allografts from bone banks. To improve the number of safe allografts, we suggest chemical cleaning of the grafts followed by antibiotic impregnation. Comparison of two chemical cleaning processes for bone allografts aiming for antibiotic impregnation and consequently delivery rates in vitro. Bone chips of 5–10 mm were prepared from human femoral heads. Two cleaning methods (cleaning A and cleaning B) based on solutions containing hydrogen peroxide, paracetic acid, ethanol and biological detergent were carried out and compared. After the cleaning processes, the bone chips were impregnated with gentamicin. Bacillus subtilis bioassay was used to determine the gentamicin release after intervals of 1–7 days. Differences were compared with non-parametric Mann–Whitney U tests. The zones of inhibition obtained from the bone grafts cleaned with both cleaning processes were similar between the groups. The concentration of the released antibiotic was decreasing gradually over time, following a similar pattern for both groups. The cleaning procedure A as well as the cleaning procedure B for bone allografts allowed the impregnation with gentamicin powder in the same concentrations in both groups. The delivery of gentamicin was similar for both groups. Both cleaning procedures were easy to be carried out, making them suitable for routine use at the bone banks.     

Mechanical strength of bone allografts subjected to chemical sterilization and other terminal processing methods

Infectious disease transmission through the use of human donor allografts can be a catastrophic complication in an otherwise straightforward surgical procedure. The use of bone allograft in reconstructive orthopedic surgeries is increasing, yet severe complications, including death, can result if the transplanted tissues transmit a communicable disease to the tissue recipient. The BioCleanse((R)) tissue sterilization process is a fully automated, low-temperature chemical sterilization process that renders allograft tissue sterile. The purpose of this study was to evaluate the effect of a chemical tissue sterilization process on the mechanical strength of cortical bone allografts prior to implantation. Cylindrical cortical bone specimens were harvested from seven human cadaver donors and treated either by: chemical sterilization alone; chemical sterilization and terminal sterilization by gamma irradiation; chemical sterilization, lyophilization, terminal sterilization by STERRAD and rehydration; or untreated. The specimens were tested to failure in axial compression, diametral compression, shear, or bending. There were no significant differences in ultimate stress, strain, or fracture energy between the chemically sterilized and control groups in any of the testing modes.     

The effect of chemical cleansing procedures combined with peracetic acid-ethanol sterilization on biomechanical properties of cortical bone.

Peracetic acid-ethanol sterilization (PES) with a preceding delipidation step is an effective sterilization method for allograft bone, but its influence on biomechanical properties of bone has not been studied. The aim of this study was to evaluate the effects of different incubation times of water, hydrogen peroxide and alcohol cleansing procedures combined with PES on biomechanical properties of freeze-dried cortical bone. These effects were studied by performing three-point bending tests on cortical samples. The lyophilized cortical samples were rehydrated prior to mechanical testing. The bending strength and the absorbed energy of the processed cortical samples were increased slightly but the Young's modulus was decreased compared to unprocessed samples. However, when the residual moisture content of the processed cortical samples was reduced from <5% to 0% all the biomechanical properties studied were significantly decreased. Hexane elution was used to determine the residual fat content of the processed cortical bone. Reducing the incubation time in cleansing had no effect on the residual fat content of the bone samples. Our in vitro study indicates that the cleansing procedure proposed combined with PES affects the biomechanical properties of cortical bone only on a limited scale.     

A review of osteoinductive testing methods and sterilization processes for demineralized bone

Allogeneic demineralized bone has been used extensively as a clinical graft material because it has osteoinductive and osteoconductive properties. Concerns over processing and terminal sterilization procedures that may reduce performance have led clinicians to call for assurances of product potency. There is extensive experience on effects of demineralized bone in animal and cell culture models with the possibility for future evidence-based standards for release of products. Evaluation of the current state of knowledge leads to the fact that we cannot conclude that performance of different lots of demineralized bone allografts in in vivo or in vitro test systems can be used as a measure of clinical performance. It may be possible to adopt an osteoinductivity standard for release-to-market, but it should be followed by clinical monitoring and further research.Presented in part at the 27th Annual Meeting of the American Association of Tissue Banks, San Diego, CA, August 24, 2003.     

Interfacial Strength of Cement Lines in Human Cortical Bone

In human cortical bone, cement lines (or reversal lines) separate osteons from the interstitial bone tissue, which consists of remnants of primary lamellar bone or fragments of remodeled osteons. There have been experimental evidences of the cement line involvement in the failure process of bone such as fatigue and damage. However, there are almost no experimental data on interfacial properties of cement lines in human cortical bone. The objective of this study is to design and assemble a precision and computer controlled osteon pushout microtesting system, and to experimentally determine the interfacial strength of cement lines in human cortical bone by performing osteon pushout tests. Thirty specimens were prepared from humeral diaphyses of four human subjects. Twenty specimens were tested under the condition of a small hole in the supporting plate, in which the cement line debonding occurred. The cement line interfacial strength ranged from 5.38 MPa to 10.85 MPa with an average of 7.31±1.73 MPa. On the other hand, ten specimens were tested under the condition of a large hole in the supporting plate, in which the shear failure inside osteons was observed. The specimens tested under the condition of the large hole resulted in an average shear strength of 73.71±15.06 MPa, ranging from 45.97 MPa to 93.74 MPa. Therefore, our results suggest that the cement line interface between osteon and interstitial bone tissue is weaker than that between bone tissue lamellae.     

Effects of <Superscript>60</Superscript>Co gamma radiation dose on initial structural biomechanical properties of ovine bone—patellar tendon—bone allografts

Gamma radiation is established as a procedure for inactivating bacteria, fungal spores and viruses. Sterilization of soft tissue allografts with high dose ⁶⁰Co gamma radiation has been shown to have adverse effects on allograft biomechanical properties. In the current study, bone-patellar tendon-bone (BPTB) allografts from 32 mature sheep were divided into two treatment groups: low-dose radiation at 15 kGy (n = 16) and high-dose radiation at 25 kGy (n = 16) with the contralateral limb serving as a 0 kGy (n = 32) non-irradiated control. Half of the tendons from all treatment groups were biomechanically tested to determine bulk BPTB mechanical properties, cancellous bone compressive properties, and interference screw pull-out strength. The remaining tissues were prepared, implanted, and mechanically tested in an acute in vitro anterior crucial ligament (ACL) reconstruction. Low-dose radiation did not adversely affect mechanical properties of the tendon allograft, bone, or ACL reconstruction compared to internal non-irradiated control. However, high-dose radiation compromised bulk tendon load at failure and ultimate strength by 26.9 and 28.9%, respectively (P < 0.05), but demonstrated no negative effect on the cancellous bone compressive properties or interference screw pull-out strength. Our findings suggest that low dose radiation (15 kGy) does not compromise the mechanical integrity of the allograft tissue, yet high dose radiation (25 kGy) significantly alters the biomechanical integrity of the soft tissue constituent.     

Characterization of the mechanical properties of bovine cortical bone treated with a novel tissue sterilization process

Over the past decade chemical processing and engineering of musculoskeletal tissue (tendon and bone) has improved dramatically. The use of bone allograft and xenograft in reconstructive orthopedic and maxillofacial surgeries is increasing, yet severe complications can occur if the material is contaminated in any way. A novel tissue sterilization process, BioCleanse^®, has been developed to clean and sterilize musculoskeletal tissue for implantation. The present study was designed to determine the effect of this novel cleaning process on the biomechanical properties of bovine cortical bone prior to implantation. The mechanical properties of treated bovine bone material were compared to human samples with respect to failure under compression, shear and three-point bending. The data demonstrate that bovine bone treated with the novel sterilization procedure has favorable biomechanical properties compared to that of human bone treated in a similar fashion.     

Platelet concentrate as an additive to bone allografts: a laboratory study using an uniaxial compression test

Chemical cleaning procedures of allografts are destroying viable bone cells and denaturing osteoconductive and osteoinductive proteins present in the graft. The aim of the study was to investigate the mechanical differences of chemical cleaned allografts by adding blood, clotted blood; platelet concentrate and platelet gel using a uniaxial compression test. The allografts were chemically cleaned, dried and standardized according to their grain size distribution. Uniaxial compression test was carried out for the four groups before and after compacting the allografts. No statistically significant difference was found between native allografts, allografts mixed with blood, clotted blood, platelet concentrate and platelet concentrate gel regarding their yield limit after compaction. The authors recommend to chemical clean allografts for large defects, optimize their grain size distribution and add platelet concentrate or platelet rich plasma for enhancing as well primary stability as well bone ingrowth.     

Solvent-dehydrated calvarial allografts in craniofacial surgery

Craniofacial surgery almost always requires the use of bone grafting. Although autografts are the standard procedure for bone grafting, it is sometimes not possible to harvest bone, and autografts have particular risks. The use of allograft bone provides a reasonable alternative to meet the need for graft material. Solvent dehydration is a multistage procedure in which human cadaveric bone is processed by osmotic exchange baths and gamma sterilization. This processing avoids the risk of infection transmission, decreases antigenicity, and does not weaken the mechanical properties of the bone. Solvent-dehydrated, gamma-irradiated human calvarial bone allografts were used for reconstruction of craniofacial deformities in 24 patients between 1988 and 2002. Resorption of the allografts and results of the surgical intervention were evaluated with plain radiographs and three-dimensional computed tomography 12 months after surgery, in 21 patients. Serologic tests for human immunodeficiency virus-1 antibody, hepatitis B surface antigen, and hepatitis C antigen were also performed. Biopsy specimens were taken from the allografts. Average follow-up in this group was 30 months (range, 8 to 60 months), and results of serologic tests were negative in all patients. Seventy-one percent of the patients (15 of 21) showed no resorption, with partial and complete allograft fusion. One patient had nearly total graft loss and the remaining five patients had 10 to 25 percent graft resorption. Rigid fixation of the allograft, contact with the dura and periosteum, and prevention of dead spaces around the allograft are the most important factors in achieving a satisfactory result. In solvent-dehydrated bone allografts, sterilization and antigenic tissue cleaning are achieved after several steps with a minimal dose of radiation. The result is a nonantigenic, sterile mechanical scaffold that can tolerate external forces. Although autografts are the standard in craniofacial surgery, solvent-dehydrated calvarial bone allografts produced successful results in selected cases.     

Development of a novel method for the strengthening and toughening of irradiation-sterilized bone allografts

Reconstruction of large skeletal defects is a significant and challenging issue. Bone allografts are often used for such reconstructions. However, sterilizing bone allografts by using γ-irradiation, damages collagen and causes the bone to become weak, brittle and less fatigue resistant. In a previous study, we successfully protected the mechanical properties of human cortical bone by conducting a pre-treatment with ribose, a natural and biocompatible agent. This study focuses on examining possible mechanisms by which ribose might protect the bone. We examined the mechanical properties, crosslinking, connectivity and free radical scavenging potentials of the ribose treatment. Human cortical bone beams were treated with varying concentration of ribose (0.06–1.2 M) and γ-irradiation before testing them in 3-point bending. The connectivity and amounts of crosslinking were determined with Hydrothermal-Isometric-Tension testing and High-Performance-Liquid-Chromatography, respectively. The free radical content was measured using Electron Paramagnetic Resonance. Ribose pre-treatment improved the mechanical properties of irradiation sterilized human bone in a pre-treatment concentration-dependent manner. The 1.2 M pre-treatment provided >100% of ultimate strength of normal controls and protected 76% of the work-to-fracture (toughness) lost in the irradiated controls. Similarly, the ribose pre-treatment improved the thermo-mechanical properties of irradiation-sterilized human bone collagen in a concentration-dependent manner. Greater free radical content and pentosidine content were modified in the ribose treated bone. This study shows that the mechanical properties of irradiation-sterilized cortical bone allografts can be protected by incubating the bone in a ribose solution prior to irradiation.     

The mechanical behaviour of intracondylar cancellous bone of the femur at different loading rates

The compressive properties of human cancellous bone of the distal intracondylar femur in its wet condition were determined. Specimens were obtained from six cadaveric femora and were tested at a strain rate of 0.002, 0.10 and 9.16 sec⁻¹. It was found that the compressive strength decreases with an increasing vertical distance from the joint. The highest compressive strength level was recorded in the posterior medial condyle. Correlations among the mechanical properties, the bulk specimen density and the bone mineral content yield (i) highly significant correlations between the compressive strength and the elastic modulus (ii) highly significant correlations between the compressive strength or the modulus of elasticity and the bulk specimen density (iii) a doubtful correlation between the compressive strength and the bone mineral content. All recorded graphs of the impact loaded specimens displayed several well defined stress peaks, unlike the graphs recorded at low loading rates. It can be concluded that upon impact loading the localized trabecular failure which is associated with each peak, does not affect the spongy bone's stress capacity in a detrimental way.     

Compressive axial mechanical properties of rat bone as functions of bone volume fraction,apparent density and micro-ct based mineral density

Mechanical testing has been regarded as the gold standard to investigate the effects of pathologies on the structure–function properties of the skeleton. With recent advances in computing power of personal computers, virtual alternatives to mechanical testing are gaining acceptance and use. We have previously introduced such a technique called structural rigidity analysis to assess mechanical strength of skeletal tissue with defects. The application of this technique is predicated upon the use of relationships defining the strength of bone as a function of its density for a given loading mode. We are to apply this technique in rat models to assess their compressive skeletal response subjected to a host of biological and pharmaceutical stimulations. Therefore, the aim of this study is to derive a relationship expressing axial compressive mechanical properties of rat cortical and cancellous bone as a function of equivalent bone mineral density, bone volume fraction or apparent density over a range of normal and pathologic bones.We used bones from normal, ovariectomized and partially nephrectomized animals. All specimens underwent micro-computed tomographic imaging to assess bone morphometric and densitometric indices and uniaxial compression to failure.We obtained univariate relationships describing 71–78% of the mechanical properties of rat cortical and cancellous bone based on equivalent mineral density, bone volume fraction or apparent density over a wide range of density and common skeletal pathologies. The relationships reported in this study can be used in the structural rigidity analysis introduced by the authors to provide a non-invasive method to assess the compressive strength of bones affected by pathology and/or treatment options.     

Utility of alkaline protease from marine shipworm bacterium in industrial cleansing applications

Summary An alkaline protease, previously isolated from a symbiotic bacterium found in the gland of Deshayes of marine shipworm, was evaluated as a cleansing additive. The protease nearly doubled the cleaning power of a standard phosphate detergent at temperatures up to 50°C as determined by fabric swatch assays. The enzyme was, however, ineffective at 70°C. In both fresh and seawater, it was also an efficient presoak. The cleaning power of a non-phosphate detergent was significantly improved by added protease, independent of the pH range 10 to 12. The enzyme degraded lysozyme, the major protein contaminant of contact lenses, more extensively than subtilisin and was effective in solutions containing hydrogen peroxide, often employed to sterilize lenses. The protease was unusually stable in sodium perborate, as well as hydrogen peroxide, and retained good activity in the presence of sodium hypochloride.     

Effects of gamma irradiation on mechanical properties of defatted trabecular bone allografts assessed by speed-of-sound measurement

New sterilization methods for human bone allografts may lead to alterations in bone mechanical properties, which strongly influence short- and medium-term outcomes. In many sterilization procedures, bone allografts are subjected to gamma irradiation, usually with 25 KGy, after treatment and packaging. We used speed-of-sound (SOS) measurements to evaluate the effects of gamma irradiation on bone. All bone specimens were subjected to the same microbial inactivation procedure. They were then separated into three groups, of which one was treated and not irradiated and two were exposed to 10 and 25 KGy of gamma radiation, respectively. SOS was measured using high- and low-frequency ultrasound beams in each orthogonal direction. SOS and Young modulus were altered significantly in the three groups, compared to native untreated bone. Exposure to 10 or 25 KGy had no noticeable effect on the study variables. The impact of irradiation was small compared to the effects of physical or chemical defatting. Reducing the radiation dose used in everyday practice failed to improve graft mechanical properties in this study.     

Various Effects of Sandblasting of Dental Restorative Materials

Background

Sandblasting particles which remain on the surfaces of dental restorations are removed prior to cementation. It is probable that adhesive strength between luting material and sandblasting particle remnants might exceed that with restorative material. If that being the case, blasting particles adhere to sandblasted material surface could be instrumental to increasing adhesive strength like underlying bonding mechanism between luting material and silanized particles of tribochemical silica coating-treated surface. We hypothesize that ultrasonic cleaning of bonding surfaces, which were pretreated with sandblasting, may affect adhesive strength of a resin luting material to dental restorative materials.

Methods

We therefore observed adhesive strength of resin luting material to aluminum oxide was greater than those to zirconia ceramic and cobalt-chromium alloy beforehand. To measure the shear bond strengths of resin luting material to zirconia ceramic and cobalt-chromium alloy, forty specimens of each restorative material were prepared. Bonding surfaces were polished with silicon abrasive paper and then treated with sandblasting. For each restorative material, 40 sandblasted specimens were equally divided into two groups: ultrasonic cleaning (USC) group and non-ultrasonic cleaning (NUSC) group. After resin luting material was polymerized on bonding surface, shear test was performed to evaluate effect of ultrasonic cleaning of bonding surfaces pretreated with sandblasting on bond strength.

Results

For both zirconia ceramic and cobalt-chromium alloy, NUSC group showed significantly higher shear bond strength than USC group.

Conclusions

Ultrasonic cleaning of dental restorations after sandblasting should be avoided to retain improved bonding between these materials.     

Acanthamoeba adherence to contact lenses, removal by rinsing procedures, and survival to some ophthalmic products.

Unworn soft and rigid gas-permeable contact lenses were inoculated with an Acanthamoeba keratitis strain to study the protozoon's ability to adhere. Furthermore, the efficacy of the rinsing in saline on acanthamoeba removal was evaluated, as well as the amebicidal activity of five commercial cleaning/disinfecting products: hydrogen peroxide, chlorhexidine, polyaminopropyl biguanide-poloxamine, thimerosal-polyquaternium, and thimerosal-chlorhexidine. Microscopic count of cells showed that Acanthamoeba trophozoites and cysts adhered to all types of contact lenses. A significantly greater adherence of trophozoites than cysts was recorded. The rinsing in saline using a flow-method was significantly more effective than the immersion-method, particularly in removing trophozoites from rigid gas-permeable lenses. The cleaning/disinfecting solutions tested were ineffective in removing or in affecting the viability of all Acanthamoeba trophozoites or cysts in the 17 hours allotted for the experiment. The need for a better care in mechanical and physical hygiene procedures is stressed.     

Sensitivity of multiple damage parameters to compressive overload in cortical bone

Damage accumulation plays a key role in weakening bones prior to complete fracture and in stimulating bone remodeling. The goal of this study was to characterize the degradation in the mechanical properties of cortical bone following a compressive overload. Longitudinally oriented, low-aspect ratio specimens (n=24) of bovine cortical bone were mechanically tested using an overload-hold-reload protocol. No modulus reductions greater than 5% were observed following overload magnitudes less than 0.73% strain. For each specimen, changes in strength and Poisson's ratio were greater (p=0.02) than that in modulus by 10.8- and 26.6-fold, respectively, indicating that, for the specimen configuration used in this study, longitudinal elastic modulus is one of the least sensitive properties to a compressive overload. Residual strains were also proportionately greater by 6.4-fold (p=0.01) in the transverse than axial direction. These results suggest that efforts to relate microcrack density and morphology to changes in compressive mechanical properties of cortical bone may benefit from considering alternative parameters to modulus reductions.     

Adhesion of Bacillus spores and Escherichia coli cells to inert surfaces: role of surface hydrophobicity

The ability of bacterial spores and vegetative cells to adhere to inert surfaces was investigated by means of the number of adherent spores (Bacillus cereus and Bacillus subtilis spores) and Escherichia coli cells and their resistance to cleaning or rinsing procedures (adhesion strength). Six materials (glass, stainless steel, polyethylene high density (PEHD), polyamide-6, polyvinyl chloride, and Teflon) were tested. Slight differences in the number of adherent spores (less than 1 log unit) were observed between materials, but a higher number of adherent E. coli cells was found on the hydrophobic materials PEHD and Teflon. Conversely, the resistance of both B. cereus and B. subtilis spores to a cleaning procedure was significantly affected by the material. Hydrophobic materials were harder to clean. The topography parameter derived from the Abbott-Firestone curve, RVK, and, to a lesser extent, the widely used roughness parameters RA (average roughness) and Rz (maximal roughness), were related to the number of adherent cells. Lastly, the soiling level as well as the adhesion strength were shown to depend largely on the microorganism. The number of adhering B. cereus hydrophobic spores and their resistance to a cleaning procedure were found to be 10 times greater than those of the B. subtilis hydrophilic spores. Escherichia coli was loosely bound to all the materials tested, even after 24 h biofilm formation.     

Effect of hydrogen peroxide on human tendon allograft

Bacterial contamination of tendon allografts at the completion of processing has historically been about 2 %, with tendons that are found to be culture positive being discarded. Treatment of tendon allograft with hydrogen peroxide at the beginning of tissue processing may reduce bacterial contamination, however, the potential side effects of hydrogen peroxide treatment include hydrolysis of the collagen and this may alter the mechanical properties of the graft. Pairs of human tendons were used. One was washed in 3 % hydrogen peroxide for 5 min and the untreated tendon was used as a control. The ultimate tensile strength of the tendons was determined using a material testing machine. A freeze clamp technique was used to hold the tendons securely at the high loads required to cause tendon failure. There was no statistical difference in the ultimate tensile strength between the treated and untreated tendons. Mean strength ranged from Extensor Hallucis Longus at 588 Newtons to Tibialis Posterior at 2,366 Newtons. Hydrogen peroxide washing may reduce bacterial contamination of tendon allograft and does not affect the strength of the tendon.     

Effect of radiofrequency electromagnetic field exposure on in vitro models of neurodegenerative disease

In this work we tested viability, proliferation, and vulnerability of neural cells, after continuous radiofrequency (RF) electromagnetic fields exposure (global system for mobile telecommunications (GSM) modulated 900 MHz signal at a specific absorption rate (SAR) of 1 W/kg and maximum duration 144 h) generated by transverse electromagnetic cells. We used two cellular systems, SN56 cholinergic for example, SN56 cholinergic cell line and rat primary cortical neurons, and well‐known neurotoxic challenges, such as glutamate, 25‐35AA beta‐amyloid, and hydrogen peroxide. Exposure to RF did not change viability/proliferation rate of the SN56 cholinergic cells or viability of cortical neurons. Co‐exposure to RF exacerbated neurotoxic effect of hydrogen peroxide in SN56, but not in primary cortical neurons, whereas no cooperative effects of RF with glutamate and 25‐35AA beta‐amyloid were found. These data suggest that only under particular circumstances exposure to GSM modulated, 900 MHz signal act as a co‐stressor for oxidative damage of neural cells. Bioelectromagnetics 30:564–572, 2009. © 2009 Wiley‐Liss, Inc.