Tissue Sterilization Research Articles

Treatment of Tuberculosis with Rifamycin-containing Regimens in Immune-deficient Mice

Daily rifapentine plus isoniazid-pyrazinamide in mice infected with Mycobacterium tuberculosis produces cure in 3 months. Whether cure corresponds to latent infection contained by host immunity or true tissue sterilization is unknown. To determine the length of treatment with rifapentine-isoniazid-pyrazinamide or rifampin-isoniazid-pyrazinamide needed to prevent relapse in immune-deficient mice. Aerosol-infected BALB/c and nude mice were treated 5 days per week with either 2 months of the rifapentine-based regimen followed by rifapentine-isoniazid up to 12 months or the same regimen with rifampin instead of rifapentine. Cultures of lung homogenates were performed during the first 3 months and then every 3 months. Relapse rates were assessed after 3, 6, 9, and 12 months of treatment in BALB/c (± 1 mo of cortisone) and nude mice. All rifapentine-treated mice were lung culture-negative at 3 months but 13% of BALB/c that received cortisone and 73% of nude mice relapsed. After 6, 9, and 12 months of treatment no mouse relapsed. Rifampin-treated BALB/c mice remained culture positive at 3 months. All were culture negative at 6, 9, and 12 months. None, including those receiving cortisone, relapsed. Rifampin-treated nude mice harbored more than 4 log(10) lung cfu at Month 2 and approximately 6 log(10) cfu with isoniazid resistance at Month 3. A supplementary experiment demonstrated that 7 days a week treatment did not prevent isoniazid resistance, whereas addition of ethambutol did. In nude mice, sterilization of tuberculosis is obtained with rifapentine-containing treatment, whereas failure with development of isoniazid resistance is obtained with rifampin-containing treatment.

Preservation of human artery function following prolonged cold storage with a new solution

Blood vessels are an important tissue for allogenic vessel replacement surgery, which is needed for example following infection of artificial grafts. For tissue banking, European legislation requires evidence of tissue sterility with assays performed over 1 week. Currently, used cold storage solutions do not protect vascular function longer than 2 days. This does not allow completion of microbiological testing. This discrepancy has almost completely stopped vessel banking in Europe. We compared the recently developed storage solution TiProtec (Dr F Köhler Chemie, Bensheim, Germany) with traditionally used histidine-tryptophan-ketoglutarate (HTK) solution, 0.9% NaCl, and phosphate-buffered saline (physiological saline solution [PSS]) solution for extended cold (4°C) storage up to 25 days. Isolated rings of human internal mammary artery were studied with respect to several parameters of vessel function, including vessel tone development, endothelium-dependent and endothelium-independent relaxation, and tissue reductive capacity. Vessels stored in NaCl or PSS for ≥10 hours failed to develop tone after rewarming. Mammary arteries stored in HTK for 4 hours at 4°C initially showed a well-preserved vessel function with respect to vessel tone development, as well as endothelial and smooth muscle dilatative function. However, following 4 days of cold storage, vessel tone development and dilatative responses were significantly impaired. In contrast, arteries stored in TiProtec showed full preservation of vessel tone as well as endothelial and smooth muscle function after 4 days of cold storage. Even after 10 days of cold storage, endothelium-dependent relaxation was approximately 50% of control, and smooth muscle function was fully preserved. Over 2 weeks, tissue reductive capacity was significantly better maintained after cold storage in TiProtec compared with vessels stored in NaCl. In contrast to traditional HTK, NaCl, or PSS storage, TiProtec solution offers an excellent potential for prolonged cold storage of human arteries, which may close the existing gap between legal requirements for tissue banking and current cold preservation methods.

Live Pig Skin Tissue and Wound Toxicity of Cold Plasma Treatment

Cold atmospheric pressure plasmas have emerged as a promising new tool for medical applications. Compared to conventional thermal plasma, such as arc coagulators and desiccators, cold plasma can be more selective in its application and may be used for effective sterilization of skin and wound tissue, wound healing and tissue regeneration, cancer treatment and blood coagulation. One of the key questions that has to be answered before these plasma technologies are introduced in medical practice is the safety of plasma treatment of living tissues, i.e. toxic dose levels of plasma exposure should be determined. It is well established that porcine (pig) skin closely resembles human skin; hence we evaluated the potential toxic effects of plasma treatment on intact and wounded skin in a Yorkshire pig model. Varying doses of Floating Electrode Dielectric Barrier Discharge (FE-DBD) and microsecond Pin-to-Hole Spark Discharge (PHD) plasmas were applied to determine a dosage regime where tissue damage occurs.

Nano-Bio Fusion Science Opened and Created with Plasmas

Recent years have brought about a rapid rise in research on applications in biological and medical fields including cell and tissue inactivation, sterilization and disinfection, blood coagulation, therapy, surgery, pharmaceuticals, biosensing, and biochips by exploiting low-pressure, atmospheric-pressure, and solution plasma processes. Many of the basic constituent materials of biological organisms are nanometer-scale in size, thus presenting the possibility of a growing field of science and technology focused on a fusion between “bio” and “nano” mechanisms; we are now in the germinal stage of a new era of next-generation applied research in the biological and medical applications of nanocarbons typified by fullerenes and carbon nanotubes. In this context, the authors have proposed and implemented research utilizing nanoscopic processes in advanced gas, liquid, and gas-liquid interfacial plasmas, directed toward the creation of synthetic materials and devices composed of nanocarbons, nanoparticles, and biomolecules. This research is positioned to advance the establishment of nano-bio fusion science opened and created with plasmas to construct next-generation nanobio and medical systems relating to nanobio-electronics devices, intracellular nanoengineering, and nanomedicine.

A Review of the International Atomic Energy Agency (IAEA) Code of Practice for the Radiation Sterilisation of Tissue Allografts

The code of practice adopted by the IAEA sets out the main requirements to ensure that the radiation sterilisation of tissues produces sterilised tissue allografts suitable for safe clinical use. The IAEA main contribution to the process of tissue sterilisation is the development of the guideline in using radiation for tissue. Sterilising tissue grafts using ionising radiation which is being used in many countries, offers a clear advantage in terms of safety compared with other sterilisation techniques for health care products and medical devices.

Anterior Cruciate Ligament Allograft Surgery

Background: There has been much controversy regarding the effects of sterilization techniques and graft history on the structural integrity of allograft ligaments used in reconstruction of the anterior cruciate ligament. The purpose of this review was to comprehensively examine anterior cruciate ligament allograft studies published in the last decade to evaluate the extent to which tissue source, tissue processing techniques, and donor age are reported. Methods: The authors reviewed 202 articles published from 1999 through November 2009, of which 68 retrospective, prospective, biomechanical, and histology studies evaluating 4689 allografts were included. Results: The majority of studies do not accurately report key elements of graft history including tissue bank, processing or sterilization technique, or donor age. Underreporting was particularly prominent in clinical studies as compared with basic science studies. Limited reporting of allograft processing, allograft source, and donor age raises serious questions regarding the generalizability of published studies of allograft tissue for anterior cruciate ligament reconstruction. Conclusion/Clinical Relevance: Orthopaedic surgeons who use allograft tissue must understand the tissue treatments being used (ie, preservation methods, chemical and sterilization processes) to best inform their patients regarding the risks, benefits, and long-term outcomes when such tissues are used. A complete understanding of the implanted tissue, as well as surgeon and recipient demographics, is necessary to ensure appropriate and predictable long-term outcomes.

Preparation of Rat Tail Tendons for Biomechanical and Mechanobiological Studies

Rat tail tendons (RTTs) are a common biological model used in experimental in vitro studies in the fields of tendon physiology and tendinopathy. Working with those tissues is challenging because they are very fragile, and until now there was no rigorously detailed protocol for their isolation. Faced with these challenges, we have developed methods and instruments to facilitate manipulation of RTTs and control tissue viability, sterility and integrity. This article describes the experimental procedures used to prepare RTTs for biomechanical and mechanobiological studies. Our work is divided into four main steps: extraction, cross-sectional area measurement, rinsing and loading into the bioreactor chamber. At each step, all procedures, materials and manipulations are presented in detail so that they can be easily reproduced. Moreover, the specific instruments developed are presented: a manipulation plate used to segregate RTTs, an optic micrometer to position the tissue during the cross-sectional area measurement and an anchoring system to attach the RTTs onto a bioreactor. Finally, we describe the results obtained after multiple tests to validate our methods. The viability, sterility and integrity evaluations demonstrate that our procedures are sufficiently rigorous for manipulations of fragile tissues such as rat tail tendons.

Preparation of Rat Tail Tendons for Biomechanical and Mechanobiological Studies

Rat tail tendons (RTTs) are a common biological model used in experimental in vitro studies in the fields of tendon physiology and tendinopathy. Working with those tissues is challenging because they are very fragile, and until now there was no rigorously detailed protocol for their isolation. Faced with these challenges, we have developed methods and instruments to facilitate manipulation of RTTs and control tissue viability, sterility and integrity. This article describes the experimental procedures used to prepare RTTs for biomechanical and mechanobiological studies. Our work is divided into four main steps: extraction, cross-sectional area measurement, rinsing and loading into the bioreactor chamber. At each step, all procedures, materials and manipulations are presented in detail so that they can be easily reproduced. Moreover, the specific instruments developed are presented: a manipulation plate used to segregate RTTs, an optic micrometer to position the tissue during the cross-sectional area measurement and an anchoring system to attach the RTTs onto a bioreactor. Finally, we describe the results obtained after multiple tests to validate our methods. The viability, sterility and integrity evaluations demonstrate that our procedures are sufficiently rigorous for manipulations of fragile tissues such as rat tail tendons.

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.

The Effects of Various Cleaning and Sterilization Processes on Allograft Bone Incorporation

Although bone allografts have a long history of safe and effective clinical use, there is great variability in tissue processing and sterilization processes. Chemical and gamma radiation sterilization are commonly performed on allografts, and numerous animal and clinical studies have examined their integration into host bone. The objective of this literature review was to assess the effects of various cleaning and sterilization processes on the incorporation of allograft bone.

Transfer of Escherichia coli O157:H7 from soil, water, and manure contaminated with low numbers of the pathogen to lettuce plants.

The sources of contamination of leafy greens remain unclear, but it is evident that contaminated water, soil amendments, and wildlife likely contribute. The objective of the present study was to determine transfer of low numbers of Escherichia coli O157:H7 from soil, manure-amended soil, and water to growing lettuce plants. Lettuce plants, young (12 days of age) or mature (30 days of age), were grown in soil, manure-amended soil, or irrigated with water containing 10(1), 10(2), 10(3), or 10(4) CFU E. coli O157:H7 per g or ml. Harvested plants were processed to determine whether E. coli O157:H7 was associated with the entire plant or within internal locations. Young plants (12 days) were harvested at 1, 10, 20, and 30 days postexposure. No samples were positive for E. coli O157:H7 after direct plating of serial dilutions. Enrichment of all samples from young plants exposed to contaminated soil, manure-amended soil, and irrigation water demonstrated that approximately 21% (113 of 552) of plants were positive for E. coli O157:H7. Approximately 30% (36 of 120) of the mature plants initially irrigated with or grown in contaminated soil (including manure-amended soil) for 15 days were positive for E. coli O157:H7. Based on sterilization of surface tissue, E. coli O157:H7 was in protected locations of lettuce tissue. The results suggest that lettuce exposed to, and grown in the presence of, low numbers of E. coli O157:H7 may become contaminated and thus present a human health risk.

Status of the reproductive system of bony fish from the Teterev River and the Kiev Reservoir 20 years after the Chernobyl accident

The status of the reproductive system is investigated in five species of Teleostei from the Teterev River and the Kiev Reservoir (Ukraine), descendants of F42–F7 generations of fish exposed to radiation from the accident at the Chernobyl nuclear power plant. Twenty years after the accident, a wide range of morphofuinctional anomalies of this system is present in fish. The most significant of them are: asymmetry and anomalous morphology of gonads, proliferation of connective tissue—sterilization, mass destruction of follicular and sex cells of various developmental stages, and hermaphroditism. Among the investigated species, disturbances of reproductive glands were preset to the highest extent in pike Esox lucius and crucian carp Carassius carassius. In the series of investigated generations the maximum frequency of occurrence of gonad anomalies was noted in F4 and F5, due to prolonged mutagenesis (Dubinin, 1986).

Micropropagation of Populus trichocarpa ‘Nisqually-1’: the genotype deriving the Populus reference genome

Populus serves as a model tree for biotechnology and molecular biology research due to the availability of the reference genome sequence of Populustrichocarpa (Torr. & Gray) genotype ‘Nisqually-1’. However, ‘Nisqually-1’ has been shown to be very recalcitrant to micropropagation, regeneration and transformation. In this study, a highly efficient micropropagation protocol from greenhouse-grown shoot tips of ‘Nisqually-1’ was established. The optimal micropropagation protocol involves growing in vitro shoots in plant growth regulator-free Murashige and Skoog (MS) basal medium supplemented with 3% sucrose, 0.3% Gelrite® and 5–10 g L−1 of activated charcoal. Plants grown on this medium were significantly longer, and contained significantly higher concentrations of chlorophyll. This highly effective protocol provides a consistent supply of quality leaf and stem materials throughout the year for transformation experiments and other in vitro manipulations, therefore eliminating inconsistency due to seasonal and greenhouse environmental variations and the need for repetitive tissue sterilization.

Production of Honokiol and Magnolol in Suspension Cultures of Magnolia Dealbata Zucc

Honokiol and magnolol, important anxiolytic and anti-cancer agents, have been produced in cell-suspension cultures of the endangered Mexican plant Magnolia dealbata Zucc. In vitro cultures of the plant were established, and the accumulation of honokiol and magnolol in callus and cell-suspension cultures was measured. Leaf samples were the best explants for callus establishment and metabolite production, and Murashige and Skoog medium supplemented with 1 mg/L 2, 4-dichlorophenoxyacetic acid and 1 mg/L kinetin yielded 2.3 mg/g of honokiol and 5.9 mg/g of magnolol. Bacterial and fungal contamination was inhibited with a multiple-step tissue sterilization procedure. Oxidation was inhibited with 1 g/L activated charcoal. Cell-suspension batch cultures derived from friable callus obtained from leaves of this species were grown for 30 days in shaker flasks containing Murashige and Skoog medium. Throughout the growth cycle, honokiol and magnolol levels, fresh and dry weight, and sucrose uptake were determined. The effects of carbon source concentration on biomass accumulation and the synthesis of bioactive compounds were studied. By using 3 mL of inocula supplemented with 3% (w/v) sucrose, maximum yields of honokiol (8.1 mg/g) and magnolol (13.4 mg/g) were obtained after 25 days. These yields were 300% and 382%, respectively, of the yields of honokiol and magnolol obtained from field-grown plants.

Echinocandin activity againstAspergillusspp. and the importance of pharmacodynamics

Echinocandins represent a safe and well tolerated option for the therapy of invasive aspergillosis in patients who are unable to tolerate other agents or have refractory disease. In vitro and animal model data provide useful insights into the activity, appropriate dose, and potential role of these agents in invasive aspergillosis. These studies reveal a potentially concerning lack of tissue sterilization when echinocandins are used as monotherapy but clearly show improved survival with increasing doses. Furthermore pharmacodynamic studies suggest that echinocandin doses currently in widespread clinical use may optimize outcomes in invasive aspergillosis. A paucity of clinical data exists examining these agents as monotherapy for invasive aspergillosis and virtually no clinical data exists for using these agents as primary therapy. Further data examining the role of echinocandin monotherapy for invasive aspergillosis is unlikely to be forthcoming in the foreseeable future due to several factors including the aforementioned issues, the relatively small number patients with this infection, and the belief that these agents are potentially best used as part of combination therapy regimens for invasive aspergillosis.

Treatment of osteomyelitis by liposomal gentamicin-impregnated calcium sulfate

Traditional therapy of staphylococcal osteomyelitis is ineffective in producing complete sterilization of infected bones due to the formation of the Staphylococcus aureus biofilms. The aim of this study was to develop a new drug-delivery system of antibiotics for treatment of chronic experimental osteomyelitis. In the current work, cationic liposomal gentamicin was prepared and impregnated in calcium sulfate (CS), and tested for anti-biofilm activities in vitro and in vivo. The combination of liposomal gentamicin and CS showed initial burst-release of active liposomal gentamicin and had continuous-release (12 days). Liposomal gentamicin released from CS had the same anti-biofilm activity with the liposomal gentamicin prepared freshly. Meanwhile, both agents were more effective relative to free gentamicin at low drug concentration. Therapeutic trials with antibiotics given intravenously revealed that free gentamicin for 14 days was ineffective in sterilizing bone. Treatment with liposomal gentamicin for 14 days resulted in recovery of 33.3% of treated animals, which was the lower slightly than the result treated with implantation of gentamicin-impregnated CS (66.7%). Complete sterilization of bone tissues on cultures (100% cure) was obtained only in the group of liposomal gentamicin-impregnated CS treated for 14 days. The new drug-delivery system was effective in preventing biofilm infection in a contaminated defect, and it could also be used clinically for bacterial infections in the conditions like plaque formation or in arresting biofilm formation in the implanted devices or dead bone of osteomyelitis.

Immunologic analyses of bovine bone treated with a novel tissue sterilization process

This study evaluates the efficacy of removal of xeno-antigens from bovine bone using a patented BioCleanse process for decellularization of allograft tissues for clinical implantation. BioCleanse deploys a combination of chemicals and several high pressure rinses to achieve standardized sterility assurance levels. This method produces sterile grafts without reducing allograft bone biomechanical properties and effectively removes cells, lipids, and other sources of antigenic material from human allografts for clinical use. In this investigation, BioCleanse is evaluated for its potential in removing xenograft antigens from bovine bone grafts followed by immunologic evaluation in the subcutaneous pouch of immunocompetent rats. The alpha-galactosyl (alpha-gal) epitope with the structure Galalpha1-3Galbeta1-4GlcNAc-R constitutes a critical component of xenoantigens and its removal using BioCleanse from bovine bone was compared with tissue levels of unprocessed bone. The relative degree of antigen removal was also determined through measuring the pro-inflammatory cytokine, tumor necrosis factor (TNF)-alpha, and through the use of histologic grading of cellular infiltrates into bone. Compact cortical bone inhibited immune cell migration but cancellous bone demonstrated cellular increase and bone resorption in the untreated control group. The alpha-gal xenoantigen level was significantly lower in both cortical (P < 0.001) and cancellous bone (P < 0.001) compared with controls. TNF-alpha levels were significantly (P < 0.001) reduced compared with untreated controls when human acute monocytic leukemia cells were exposed to cortical or cancellous bone. BioCleanse effectively removed xenoantigens and inflammatory markers justifying a follow up study in primates to determine these benefits in a model that is primed with preformed xeno-antibodies responsible for hyperacute rejection in hard tissues.

The impact of the International Atomic Energy Agency (IAEA) program on radiation and tissue banking in Uruguay: development of tissues quality control and quality management system in the National Multi-Tissue Bank of Uruguay

BNOT was created and regulated in 1977 and started its operation in 1978 according to the Decree No. 86/1977. By the Decree 248/005 is transformed in the National Institute of Donation and Transplantation of Cells, Tissues and Organs (Instituto Nacional de Donación y Trasplante de Células, Tejidos y Organos--INDT). The organisation has been operating within the State University Medical School and the Public Health Secretary and it is the governmental organisation responsible for the regulation, policy and management of donation and transplantation in Uruguay. By the Decree 160/2006 is responsible for human cells and tissues regulation too. The participation of the INDT in the IAEA program facilitated the introduction of the radiation sterilisation technique for the first time in the country. The radiation sterilisation of tissues processed by INDT (ex BNOT), was initially carried out in the 60 Cobalt Industrial Plant in the National Atomic Energy Commission of Argentina and now is carried out in INDT, using a Gamma Cell 220 Excel, which was provided by the IAEA through the national project URU/7/005. The results of the implementation of tissues, quality control and quality management system, are showed.

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 ® 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 impact of the International Atomic Energy Agency (IAEA) program on radiation and tissue banking in Mexico

Tissue banking started in Mexico in 1948-1949, when two bone banks were established, one at the Infantile Hospital of Mexico and other at the Central Military Hospital. Mexico has benefited for the implementation of the IAEA program since through it has been able to settle down and to consolidate the Tissue Bank at the Instituto Nacional de Investigaciones Nucleares ININ (National Institute for Nuclear Research). This is the only bank in Latin America that has a Quality Management System in force, certified under ISO 9001:2000 since August 1, 2003. The first tissue processed was amnion. The main products of the BTR are amnion and pig skin. Both are biological tissues which their main use is as a wound dressing in patients with burns, scars, diabetic ulcers, epidermolysis bullosa, damaged ocular surface, etc. The General Health Law, published in 1984 and reformed in June 19, 2007, describes the procedure for the disposal of organs, tissues and human cadavers in its fourteenth title and in the Regulation for Sanitary Control. During the period 2001-2005, the ININ Tissue Bank produced 292 sterilised tissues (amnion, 86,668 cm(2), and frozen pig skin, 164,220 cm(2), at an estimated cost of 1,012,668 Mexican pesos. Until 2006, one hundred eighty five (185) patients have been treated with the use of sterilised tissues produced by the ININ Tissue Bank. The radiation source used for sterilisation of tissues is an industrial Cobalt-60 irradiator model JS-6500 AECL, which belongs to ININ. This equipment is located in other building, close to the BTR, in the Centro Nuclear de México "Dr. Nabor Carrillo Flores" (Nuclear Center of Mexico). Until 2006, six hospitals use in a routine way the sterilised tissues produced by the ININ Tissue Bank, for the treatment of burns originated by diverse agents like flame, electricity, liquids in boil, chemical reagents, as well as for the reconstruction of the ocular surface. Two of these hospitals treat patients of very low economic incomes, mainly needy individuals, who cannot afford to pay this type of treatments in other hospitals due to their high cost. The results obtained up to now are highly promising.