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.     

Phylogenetic analysis of platelet-derived growth factor by radio- receptor assay

Competition between 125I-labeled platelet-derived growth factor (PDGF) and unlabeled PDGF forms the basis of a specific "radio-receptor assay" for quantifying PDGF in clotted blood serum. Human clotted blood serum contains 15 ng/ml of PDGF by radio-receptor assay; this corresponds to a PDGF content of approximately 7.5 x 10(-5) pg per circulating platelet, a figure which is corroborated by purification data. Clotted blood sera from mammals, lower vertebrates and marine invertebrates were screened for homologues of human PDGF by radio-receptor assay. All tested specimens from phylum Chordata contain a mitogenic agent that competes with human PDGF for receptor binding. Sera from tunicates down on the chordate line of evolution and sera from all tested animals on the arthropod line of development were negative. The phylogenetic distribution of PDGF homologue does not correlate with platelet distribution since platelets and their precursor cell--the bone marrow megacaryocyte--are unique to the mammalian hematopoietic system. One anatomical feature appearing coordinately with PDGF on the vertebrate line of development is a pressurized circulatory system. The coincidental appearance of these features may lend support to the hypothesis that PDGF plays a role in maintenance and repair of the vascular lining in vivo.     

A Simple and Efficient Method for DNA Purification from Samples of Highly Clotted Blood

Rapid purification of DNA from samples of highly clotted blood is a challenging problem due to the difficulty in recovering and dispersing blood clots. We developed a new method for discarding the serum-separator gel and rapidly dispersing the blood clots. A special disposable tip was inserted into the serum-separator gel so that the serum-separator gel could be discarded. The blood clot obtained was dispersed into small pieces through a copper mesh (pore size, 250 μm) in a special dispersing instrument by centrifugation. After lysis of red blood cells and white blood cells, genomic DNA was concentrated and desalted by isopropanol precipitation. The mean yield of DNA purified from a 0.3-ml blood clot was 22.70 μg in 173 samples of clotted blood cryopreserved for 1 month, and 19.02 μg in 1,372 samples of clotted blood cryopreserved for >6 months. DNA samples were successfully performed through polymerase chain reaction, real time polymerase chain reaction, and melt curve analysis. Their quality was comparable with that purified directly from EDTA-anticoagulated blood. The new method overcomes the difficulties in recovering and dispersing blood clots, allowing efficient purification of DNA from samples of highly clotted blood.     

The use of autologous platelet-rich plasma (platelet gel) and autologous platelet-poor plasma (fibrin glue) in cosmetic surgery

The purpose of this study was to evaluate a new technique of harvesting and preparing autologous platelet gel and autologous fibrin glue (body glue) and to evaluate their effectiveness in stopping capillary bleeding in the surgical flaps of patients undergoing cosmetic surgery. A convenience sample of 20 patients ranging from 25 to 76 years of age undergoing cosmetic surgery involving the creation of a surgical flap were included in the study. The types of surgical procedures included face lifts, breast augmentations, breast reductions, and neck lifts. Platelet-poor and platelet-rich plasma were prepared during the procedure from autologous blood using a compact, tabletop, automated autologous platelet concentrate system (SmartPReP, Harvest Autologous Hemobiologics, Norwell, Mass.). The platelet-poor and platelet-rich plasma were combined with a thrombin-calcium chloride solution to produce autologous fibrin glue and autologous platelet gel, respectively. Capillary bed bleeding was present in all cases and effectively sealed within 3 minutes following the application of platelet gel and fibrin glue. The technique for making the solution and for evaluating its effectiveness in achieving and maintaining hemostasis during cosmetic surgical procedures is described. Autologous platelet gel and fibrin glue prepared by the automated concentrate system are compared with autotransfusor-prepared platelet gel and Tisseel (Baxter Healthcare Corp.), a commercially prepared fibrin sealant preparation.     

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 impact of delayed fibrinopeptide-A release on fibrin structure. Studies of an abnormal fibrinogen

The impact of delayed fibrinopeptide-A release on polymerization and structure of fibrin gels was studied utilizing a heterozygously transmitted variant fibrinogen. An arginine to histidine substitution at position 16 of the alpha chain of the abnormal fibrinogen delayed release of an abnormal fibrinopeptide-A (A) by thrombin and completely blocked release of A by reptilase. When clotted with thrombin, patient fibrin formed more slowly than normal fibrin, but clottability was normal and gel fiber mass/length ratios were decreased less than 10%. Gels formed with reptilase clotted slowly, demonstrated reduced clottability, but had normal fiber mass/length ratios. Reptilase clotted the normal but not the variant component of the patient fibrinogen. Thrombin-induced cleavage of fibrinopeptide-B prior to A occurred in these experiments, but polymerization of this species beyond trimers has been reported to be minimal under the conditions used. With time, A is removed by thrombin resulting in the slow production of normal fibrin monomer from the abnormal component. These monomers subsequently polymerize. The minimal change in gel fiber size caused by slow A release implies that fibrin fiber size is primarily a function of ionic environment and not of the sequence of peptide release.     

Platelet size distribution in mice following immunothrombopenia and vincristine administration

In the present study platelet size distribution was investigated after induction of immunothrombocytopenia by rabbit-anti-mouse-platelet-serum (RAMPS) and after vincristine-induced thrombocytopenia. The platelet size distribution after a single dose of RAMPS showed a shift to larger volumes at day 1 and 2, and a decrease to slightly smaller volumes than normal at day 8. These differences, however, were not statistically significant. After vincristine administration, a dose-dependent increase of the platelet size distribution was demonstrated, which lasted from day 1-7. It is suggested that in immune-induced thrombocytopenia the young platelets released from bone marrow megakaryocytes are not exclusively large platelets. On the other hand the early appearance of large platelets after vincristine administration points to a toxic or segregating effect of vincristine on circulating platelets. Therefore, in our experiments, the platelet size is not suitable for the differentiation of young and old platelets.     

Effects of Hydrogen Peroxide Cleaning Procedures on Bone Graft Osteoinductivity and Mechanical Properties

Bone allografts are frequently used during orthopaedic trauma cases or other reconstructive procedures. Most allografts are processed and cleaned before use. Our goals were to determine if an improved cleaning procedure compromises the strength or osteoinductivity of a graft. We compared our improved cleaning procedure to our standard cleaning procedure on cortical bone allograft. The cleaning procedures are generally composed of a series of chemical steps with nonionic detergents, hydrogen peroxide, and alcohol under time and temperature control, subjected to ultrasonic agitation. We tested the compressive strength, impact strength, and shear strength following the standard and improved cleaning procedures. Osteoinductivity was tested in 4 groups, using the improved cleaning procedure with four different hydrogen peroxide cleaning times: 0, 1, 3, and 5 h. Osteoinductivity was evaluated in vivo, using a 28-day implant in the hamstring muscle of an athymic, nude mouse. Results demonstrated that osteoinductivity is maintained with cleaning in hydrogen peroxide for up to 1 h, and that compressive strength, impact strength, and shear strength were all unaffected by the improved cleaning procedure. The improved cleaning procedure therefore did not compromise the strength or osteoinductivity of cortical bone allografts in comparison to the standard procedure.     

Detection of hepatitis B virus in bone allografts from donors with occult hepatitis B infection

The implementation of nucleic acid testing in donor screening has improved the safety of tissue allografts. Although infectious disease transmission can be considered a rare event, the detection of occult hepatitis B infection remains challenging. The studies concerning this risk are mainly based on testing blood specimens. This work shows the correlation between results of samples obtained from donor blood and the corresponding tissue washing solution. Hepatitis B virus deoxyribonucleic acid was detected both in bone allografts from donors with serological profiles associated to active hepatitis B infection and occult hepatitis B infection. These results suggest that hepatitis B virus seems to concentrate in bone marrow even when a low viral load is present in peripheral blood. Even detection at molecular level is not enough to avoid the risk of hepatitis B virus transmission and a multiparametrical evaluation is required in tissue donor screening. The role of clinicians in recognition and reporting of allograft-associated infections is a major concern for the acquisition of experience to be applied in risk control of disease transmission.     

Structural changes in platelets in alloxan diabetes as parameters of their activation in the vascular bed and morphologic features]

The present study shows that a stable level of blood platelet number persists in diabetic rats (following 15 and 60 days after experimental alloxan injection) Unusual platelet megaforms are seen accumulating (up to 36%), having no size analogs in control animals. A quantitative electron-microscopic analysis demonstrated that the enlargements of diabetic platelets was accompanied by their rounding in shape and by a more frequent (by 2.0-2.6 times) generation of surface pseudopodia. The share of platelets increased in two independent fractions, with considerable deflexion over the normal range limit variations in the number, size and general volume of their alpha-granules (cutting down) or dense (increase) bodies. At the same time a common decrease in these indexes for different types of grains in platelets of various dimensions occurred. The integral picture of the platelet granular apparatus (image of granular apparatus), that demonstrates quantitative proportions between the number and size indexes of different grain types, is found distorted in diabetic rats.     

Platelet rich fibrin: a new paradigm in periodontal regeneration

Among the great challenges facing clinical research is the development of bioactive surgical additives regulating inflammation and increasing healing. Although the use of fibrin adhesives and platelet-rich plasma (PRP) is well documented, they have their own limitations. Hence, reconstructive dental surgeons are looking for an “edge” that jump starts the healing process to maximize predictability as well as the volume of regenerated bone. Overcoming the restrictions related to the reimplantation of blood-derived products, a new family of platelet concentrate, which is neither a fibrin glue nor a classical platelet concentrate, was developed in France. This second generation platelet concentrate called platelet-rich fibrin (PRF), has been widely used to accelerate soft and hard tissue healing. Its advantages over the better known PRP include ease of preparation/application, minimal expense, and lack of biochemical modification (no bovine thrombin or anticoagulant is required). This article serves as an introduction to the PRF “concept” and its potential clinical applications with emphasis on periodontal regeneration.     

Structural and hydrodynamic simulation of an acute stenosis-dependent thrombosis model in mice

Platelet activation under blood flow is thought to be critically dependent on the autologous secretion of soluble platelet agonists (chemical activators) such as ADP and thromboxane. However, recent evidence challenging this model suggests that platelet activation can occur independent of soluble agonist signalling, in response to the mechanical effects of micro-scale shear gradients. A key experimental tool utilized to define the effect of shear gradients on platelet aggregation is the murine intravital microscopy model of platelet thrombosis under conditions of acute controlled arteriolar stenosis. This paper presents a computational structural and hydrodynamic simulation of acute stenotic blood flow in the small bowel mesenteric vessels of mice. Using a homogeneous fluid at low Reynolds number (0.45) we investigated the relationship between the local hydrodynamic strain-rates and the severity of arteriolar stensosis. We conclude that the critical rates of blood flow acceleration and deceleration at sites of artificially induced stenosis (vessel side-wall compression or ligation) are a function of tissue elasticity. By implementing a structural simulation of arteriolar side wall compression, we present a mechanistic model that provides accurate simulations of stenosis in vivo and allows for predictions of the effects on local haemodynamics in the murine small bowel mesenteric thrombosis model.     

一种快速、经济提取外周凝血基因组DNA 方法的建立

目的:建立一种经济、快速且高质量提取人体外周凝血DNA的方法。方法:摸索最佳的匀浆条件,对外周凝血块进行匀浆,采用KI法对匀浆液进行基因组DNA的提取,通过凝胶电泳、单重PCR和多重PCR检测凝血基因组DNA的提取产量和质量,并分别与常规的凝血基因组DNA提取方法,即蛋白酶K消化法,以及提取抗凝血基因组DNA的KI法进行比较分析。结果:最佳的匀浆条件为:39000 rmp,15秒。在此条件下提取的基因组DNA完整性好,纯度和产量与蛋白酶K消化法提取凝血DNA和KI法提取抗凝血DNA的结果相比,没有统计学差异。单重PCR和多重PCR也获得了理想的扩增结果。结论:与常规的外周凝血提取方法相比(蛋白酶K消化法),本方法节省了时间和成本,能快速、经济、有效地提取外周凝血基因组DNA,可用于后续的科研和临床诊断需要,解决了部分科研机构血液基因组DNA的样本来源问题。     

An Enzymatic Method to Rescue Mesenchymal Stem Cells from Clotted Bone Marrow Samples

Mesenchymal stem cells (MSCs) - usually obtained from bone marrow - often require expansion culture. Our protocol uses clinical grade urokinase to degrade clots in the bone marrow and release MSCs for further use. This protocol provides a rapid and inexpensive alternative to bone marrow resampling. Bone marrow is a major source of MSCs, which are interesting for tissue engineering and autologous stem cell therapies. Upon withdrawal bone marrow may clot, as it comprises all of the hematopoietic system. The resulting clots contain also MSCs that are lost for expansion culture or direct stem cell therapy. We experienced that 74% of canine bone marrow samples contained clots and yielded less than half of the stem cell number expected from unclotted samples. Thus, we developed a protocol for enzymatic digestion of those clots to avoid labor-intense and costly bone marrow resampling. Urokinase - a clinically approved and readily available thrombolytic drug – clears away the bone marrow clots almost completely. As a consequence, treated bone marrow aspirates yield similar numbers of MSCs as unclotted samples. Also, after urokinase treatment the cells kept their metabolic activity and the ability to differentiate into chondrogenic, osteogenic and adipogenic lineages. Our protocol salvages clotted blood and bone marrow samples without affecting the quality of the cells. This obsoletes resampling, considerably reduces sampling costs and enables the use of clotted samples for research or therapy.     

Effect of storage temperature on gentamicin release from antibiotic-coated bone chips

Freezing is the most common method for storing bones until use in skeletal reconstruction. However, the effect of freezing on antibiotic delivery from antibiotic-coated bone has not been evaluated. In this study, we compared antibiotic delivery in vitro from gentamicin-coated human bone stored at different temperatures. Bone chips obtained from human femur heads were chemically cleaned and mixed with gentamicin sulfate. Samples were stored for 4 months at ?20 °C, 4 months at ?80 °C, or evaluated immediately without freezing. Antibiotic release from the bone chips was measured using Bacillus subtilis as an indicator strain. Zones of inhibition and rates of gentamicin release were similar in all three groups. Storage at ?20 and ?80 °C for bone allografts has no effect on gentamicin release from chemically cleaned bone chips.     

Albumin-coated structural lyophilized bone allografts: a clinical report of 10 cases

Bone replacement and the use of bone supplementary biological substances have become widespread in clinical practice. Although autografts have excellent properties, their limited availability, difficulties with shaping and donor site morbidity have made allografts a viable and increasingly preferred alternative. The main drawback of allografts is that the preparation destroys osteogenic cells and results in denaturation of osteoinductive proteins. Serum albumin is a well-known constituent of stem cell culture media and we found that lyophilizing albumin onto bone allografts markedly improves stem-cell attachment and bone healing in animal models thus replacing some of the osteoinductive potential. As a first step in the clinical introduction of albumin coated grafts, we aimed to test surgical handling and early incorporation in aseptic revision arthroplasty in humans. We selected patients who needed large structural allografts and the current operation was the last attempt at preserving a moving joint. In a series of 10 cases of hip and knee revision surgery we did not experience any drawbacks of the albumin-coated grafts during handling and implantation. Twelve months radiographic and SPECT-CT follow-up showed that the graft was well received by the host and active remodelling was observed. The lack of graft-related complications and the good 1-year results indicate that controlled trials may be initiated in more common bone grafting indications where long-term effectiveness can be evaluated.     

Design and management of an orthopaedic bone bank in the Netherlands

The design and management of an orthopaedic bone bank is a complex process in which medical organisation and legislation intertwine. Neither in the Netherlands, nor in any other European country, there are official guidelines for the organisation and management of an orthopaedic bone bank. In the Netherlands, the recently modified ‘law of security and quality for using human materials’ (WVKL) dictates requirements for technical and organisational aspects for the use of human tissue and cells. The bone bank procedures include a thorough questionnaire for donor selection, extensive serological, bacteriological and histopathological examination, as well as standard procedures for registration, processing, preservation, storage and distribution of bone allografts. This article describes the organisation of an accredited bone bank and can be used as a proposition for an official guideline or can be useful as an example for other orthopaedic bone banks in Europe.     

Detection of heterogenicity of the lymphoid populations by measuring the diameter of the cells and cell nuclei]

Some parameters of distribution according to the lymphoid cell and its nuclei size in the peripheral blood, bone marrow, thymus and spleen of healthy rats were studied. A comparative assay revealed population homogeneity for the thymus and bone marrow lymphocytes as well as their mean diameter differences. Mixing of these cell types markedly changed the distribution parameters of newly formed population, as shown on the model of the bone marrow lymphoid reaction caused by migration of thymic lymphocytes after 5-fluorouracil use. Preliminary thymectomy excluded migration and homogeneity of the bone marrow lymphocyte size remained unchanged.     

Concentration of high molecular weight solute by swelling of hydrogel

Summary The size exclusion effect produced by the swelling of hydrolyzed polyacrylamide gel was exploited to concentrate a high molecular weight solute in batch. Changing the solvent composition induced gel swelling or shrinking. Several solutes with different molecular weights were used to test the degree of hydrogel exclusion.