In Process Citation

Abstract Background: Allogeneic bone transplantation is at risk of infection, and established disinfection methods typically compromise bone quality. High hydrostatic pressure (HHP) is well established for disinfection in food technology, and also it does protect biomechanical and biological properties of bone. This study is the first investigation of HHP regarding disinfection of bone biopsies. Materials and methods: Bone biopsies of 34 patients with chronic infections were subjected to HHP and assessed for persisting bacterial growth. In series 1, bone biopsies were proceeded directly to HHP (10 min; maximal pressure P(max) 600 MPa). In series 2, HHP was applied after 5-day incubation in growth media (10 min or 2x30 min; P(max) 600 MPa). Furthermore, HHP-induced changes of bacterial morphology on artificially infected bone samples were evaluated by scanning electron microscopy (SEM). Results: For series 1, 71% of the bone samples were sterilised by HHP (n=17), compared to 38% of the untreated control samples, which were obtained during the same surgery (n=8). For series 2, after prior incubation, HHP disinfected 7% of the bone specimens (n=55), all control samples showed bacterial growth (n=33). Destruction of cell wall integrity of Gram-negative strains was observed by SEM. Conclusion: The effectiveness of HHP for bone disinfection should be improved by optimising treatment parameters. Infections with barosensitive Gram-negative bacteria or yeast might represent possible clinical indications.     

Successful disinfection of femoral head bone graft using high hydrostatic pressure

The current standard for sterilization of potentially infected bone graft by gamma irradiation and thermal or chemical inactivation potentially deteriorates the biomechanical properties of the graft. We performed an in vitro experiment to evaluate the use of high hydrostatic pressure (HHP); which is widely used as a disinfection process in the food processing industry, to sterilize bone grafts. Four femoral heads were divided into five parts each, of which 16 were contaminated (in duplicate) with 10⁵–10⁷ CFU/ml of Staphylococcus epidermidis, Bacillus cereus, or Pseudomonas aeruginosa or Candida albicans, respectively. Of each duplicate, one sample was untreated and stored similarly as the treated sample. The remaining four parts were included as sterile control and non-infected control. The 16 parts underwent HHP at the high-pressure value of 600 MPa. After HHP, serial dilutions were made and cultured on selective media and into enrichment media to recover low amounts of microorganism and spores. Three additional complete femoral heads were treated with 0, 300 and 600 MPa HHP respectively for histological evaluation. None of the negative-control bone fragments contained microorganisms. The measured colony counts in the positive-control samples correlated excellent with the expected colony count. None of the HHP treated bone fragments grew on culture plates or enrichment media. Histological examination of three untreated femoral heads showed that the bone structure remained unchanged after HHP. Sterilizing bone grafts by high hydrostatic pressure was successful and is a promising technique with the possible advantage of retaining biomechanical properties of bone tissue.     

超高压对植物乳杆菌能量代谢影响的研究

以植物乳杆菌ATCC8014为试材,研究超高压对其能量代谢的影响。建立了用氯化碘硝基四唑紫测定ATCC8014的INT代谢还原活性的比色法。用比色法测定了超高压对ATCC8014的INT代谢还原活性与葡萄糖利用的影响。试验结果表明,150~250MPa作用15min在MRS琼脂培养基上随着压力的增大菌落数显著降低,INT代谢还原活性降低显著,葡萄糖的利用变化不明显;超过300MPa后,葡萄糖的利用才显著降低;400MPa处理15min,尽管在MRS琼脂培养基上菌落数低于检测限,INT代谢还原活性为0%,而葡萄糖的利用能力仍为对照组的56.1%,超高压作用下ATCC8014的灭活与INT代谢还原活性的降低的相关性较好。说明ATCC8014的细胞膜上参与葡萄糖的吸收和运输的酶、糖酵解的酶与调节系统比三羧酸循环的酶与调节系统较耐压。三羧酸循环比糖酵解对超高压敏感,三羧酸循环的抑制是超高压灭活其的重要原因,这为了探讨超高压杀灭植物乳杆菌的机制提供了一定的理论依据。     

High hydrostatic pressure inactivated human tumour cells preserve their immunogenicity.

High hydrostatic pressure (HHP) is an established method to inactivate biomolecules and microoganisms. It is routinely used for the sterilization of foodstuff. Recently, new applications as inactivation of microorganisms and tumour cells for bone transplants or for cancer vaccines have emerged. Characterization of the HHP-induced cellular responses are a prerequisite for its clinical use. To this end, we investigated the fate of human cells after HHP by cytofluorometry. We observed that the induction by HHP of cell death is time- and pressure-dependent. Surprisingly, an HHP-treatment of 100 MPa did not reduce viability at any time point. Pressures from 150 to 250 MPa-induced programmed cell death in most cells. However, survivors were observed in long term culture experiments under these conditions. Pressures above 300 MPa immediately induced cell death by necrosis and completely inactivated the cells. In contrast to inactivation by other necrosis inducing treatments like heat, freeze/thaw, or chemical agents, HHP avoids generation of Maillard products and disintegration and lysis of the cells. Instead HHP generates a gelatinised mixture of antigens captured in a distinct and robust particle and maintains their humoral immunogenicity. The high viscosity of the internal matrix of a pressurised cell is reflected by the slow penetration of the low molecular compound propidium iodide and limits the bleeding of antigen before uptake by antigen presenting cells. Taken together, HHP is an alternative method for the inactivation of mammalian cells in clinical settings.     

Effects of high hydrostatic pressure on bacterial growth on human ossicles explanted from cholesteatoma patients

Background

High hydrostatic pressure (HHP) treatment can eliminate cholesteatoma cells from explanted human ossicles prior to re-insertion. We analyzed the effects of HHP treatment on the microbial flora on ossicles and on the planktonic and biofilm states of selected isolates.

Methodology

Twenty-six ossicles were explanted from cholesteatoma patients. Five ossicles were directly analyzed for microbial growth without further treatment. Fifteen ossicles were cut into two pieces. One piece was exposed to HHP of 350 MPa for 10 minutes. Both the treated and untreated (control) pieces were then assessed semi-quantitatively. Three ossicles were cut into two pieces and exposed to identical pressure conditions with or without the addition of one of two different combinations of antibiotics to the medium.Differential effects of 10-minute in vitro exposure of planktonic and biofilm bacteria to pressures of 100 MPa, 250 MPa, 400 MPa and 540 MPa in isotonic and hypotonic media were analyzed using two patient isolates of Staphylococcus epidermidis and Neisseria subflava. Bacterial cell inactivation and biofilm destruction were assessed by colony counting and electron microscopy.

Principal Findings

A variety of microorganisms were isolated from the ossicles. Irrespective of the medium, HHP treatment at 350 MPa for 10 minutes led to satisfying but incomplete inactivation especially of Gram-negative bacteria. The addition of antibiotics increased the efficacy of elimination. A comparison of HHP treatment of planktonic and biofilm cells showed that the effects of HPP were reduced by about one decadic logarithmic unit when HPP was applied to biofilms.High hydrostatic pressure conditions that are suitable to inactivate cholesteatoma cells fail to completely sterilize ossicles even if antibiotics are added. As a result of the reduced microbial load and the viability loss of surviving bacteria, however, there is a lower risk of re-infection after re-insertion.     

The effects of high hydrostatic pressure treatment on the flavor and color of grated ginger

High hydrostatic pressure (HHP) was applied to grated ginger in order to inactivate quality-degrading enzymes in a non-thermal manner. The effects of HHP treatment on the flavor and the color of the grated ginger were investigated just after treatment and during storage. After HHP treatment (400 MPa, 5 min), geraniol dehydrogenase (GeDH) was inactivated to less than 5%, but the activity of polyphenol oxidase (PPO) was reduced only to 37%. Heat treatment (100 °C, 10 min) inactivated GeDH to 43% and PPO to about 10%. In storage, the reduction of geranial, neral, and citronellal to the corresponding alcohols was observed in the untreated and the heat-treated ginger, while it was not in the HHP-treated grated ginger. In the HHP-treated sample, terpene aldehydes almost disappeared without the formation of the corresponding alcohols. Browning was not observed immediately after HHP treatment, while it was complete in the heat-treated sample. The color change during storage appeared to reflect the residual activity of PPO.     

Hydrostatic pressure induced death of mammalian cells engages pathways related to apoptosis or necrosis.

We investigated the response to high hydrostatic pressure (HHP) of mammalian cells, since HHP is proposed to be suitable to inactivate mammalian cells in biopharmaceutics and patient's material. We observed that cells were not restricted in their viability by pressures up to 100 MPa. Mammalian cells die when treated with pressures of 200 MPa or more. But the effects of 200, 300 or 400 MPa do not follow the same pattem. At 200 MPa, cells die in a way that is related to apoptosis. Some apoptotic characteristics like phosphatidylserine (PS) exposure and morphological alterations appear very fast. Other features like a higher exposure of intracellular NPn ligands and pronounced degradation of DNA and lectin ligands are unique features of HHP induced apoptosis. Cells treated with 300 and 400 MPa die immediately following a unique necrotic pathway, since treated cells harbour high DNA and glycoprotein degrading activities.     

In Vivo Evaluation of a Novel Oriented Scaffold-BMSC Construct for Enhancing Full-Thickness Articular Cartilage Repair in a Rabbit Model

Tissue engineering (TE) has been proven usefulness in cartilage defect repair. For effective cartilage repair, the structural orientation of the cartilage scaffold should mimic that of native articular cartilage, as this orientation is closely linked to cartilage mechanical functions. Using thermal-induced phase separation (TIPS) technology, we have fabricated an oriented cartilage extracellular matrix (ECM)-derived scaffold with a Young''s modulus value 3 times higher than that of a random scaffold. In this study, we test the effectiveness of bone mesenchymal stem cell (BMSC)-scaffold constructs (cell-oriented and random) in repairing full-thickness articular cartilage defects in rabbits. While histological and immunohistochemical analyses revealed efficient cartilage regeneration and cartilaginous matrix secretion at 6 and 12 weeks after transplantation in both groups, the biochemical properties (levels of DNA, GAG, and collagen) and biomechanical values in the oriented scaffold group were higher than that in random group at early time points after implantation. While these differences were not evident at 24 weeks, the biochemical and biomechanical properties of the regenerated cartilage in the oriented scaffold-BMSC construct group were similar to that of native cartilage. These results demonstrate that an oriented scaffold, in combination with differentiated BMSCs can successfully repair full-thickness articular cartilage defects in rabbits, and produce cartilage enhanced biomechanical properties.     

Injurious mechanical compression of bovine articular cartilage induces chondrocyte apoptosis

A bovine cartilage explant system was used to evaluate the effects of injurious compression on chondrocyte apoptosis and matrix biochemical and biomechanical properties within intact cartilage. Disks of newborn bovine articular cartilage were compressed in vitro to various peak stress levels and chondrocyte apoptotic cell death, tissue biomechanical properties, tissue swelling, glycosaminoglycan loss, and nitrite levels were quantified. Chondrocyte apoptosis occurred at peak stresses as low as 4.5 MPa and increased with peak stress in a dose-dependent manner. This increase in apoptosis was maximal by 24 h after the termination of the loading protocol. At high peak stresses (>20 MPa), greater than 50% of cells apoptosed. When measured in uniaxial confined compression, the equilibrium and dynamic stiffness of explants decreased with the severity of injurious load, although this trend was not significant until 24-MPa peak stress. In contrast, the equilibrium and dynamic stiffness measured in radially unconfined compression decreased significantly after injurious stresses of 12 and 7 MPa, respectively. Together, these results suggested that injurious compression caused a degradation of the collagen fibril network in the 7- to 12-MPa range. Consistent with this hypothesis, injurious compression caused a dose-dependent increase in tissue swelling, significant by 13-MPa peak stress. Glycosaminoglycans were also released from the cartilage in a dose-dependent manner, significant by 6- to 13-MPa peak stress. Nitrite levels were significantly increased above controls at 20-MPa peak stress. Together, these data suggest that injurious compression can stimulate cell death as well as a range of biomechanical and biochemical alterations to the matrix and, possibly, chondrocyte nitric oxide expression. Interestingly, chondrocyte programmed cell death appears to take place at stresses lower than those required to stimulate cartilage matrix degradation and biomechanical changes. While chondrocyte apoptosis may therefore be one of the earliest responses to tissue injury, it is currently unclear whether this initial cellular response subsequently drives cartilage matrix degradation and changes in the biomechanical properties of the tissue.     

Biomechanical assessment of tissue retrieved after in vivo cartilage defect repair: tensile modulus of repair tissue and integration with host cartilage

Failure to restore the mechanical properties of tissue at the repair site and its interface with host cartilage is a common problem in tissue engineering procedures to repair cartilage defects. Quantitative in vitro studies have helped elucidate mechanisms underlying processes leading to functional biomechanical changes. However, biomechanical assessment of tissue retrieved from in vivo studies of cartilage defect repair has been limited to compressive tests. Analysis of integration following in vivo repair has relied on qualitative histological methods. The objectives of this study were to develop a quantitative biomechanical method to assess (1) the tensile modulus of repair tissue and (2) its integration in vivo, as well as determine whether supplementation of transplanted chondrocytes with IGF-I affected these mechanical properties. Osteochondral blocks were obtained from a previous 8 month study on the effects of IGF-I on chondrocyte transplantation in the equine model. Tapered test specimens were prepared from osteochondral blocks containing the repair/native tissue interface and adjacently located blocks of intact native tissue. Specimens were then tested in uniaxial tension. The tensile modulus of repair tissue averaged 0.65 MPa, compared to the average of 5.2 MPa measured in intact control samples. Integration strength averaged 1.2 MPa, nearly half the failure strength of intact cartilage samples, 2.7 MPa. IGF-I treatment had no detectable effects on these mechanical properties. This represents the first quantitative biomechanical investigation of the tensile properties of repair tissue and its integration strength in an in vivo joint defect environment.     

Impacts of treatment parameters on the inactivation of Campylobacter jejuni by high pressure: a statistical study of main effects and interactions

Aim:  The influence of environmental (temperature and pH) and biological (strain) parameters on the inactivation of Campylobacter jejuni by high hydrostatic pressure (HHP) was investigated.
Methods and Results:  Two clinical strains harvested in stationary phase were pressurized at 20°C and 37°C within a range of 50–400 MPa, in a phosphate (pH 7·0) or a citrate phosphate buffer (pH 5·6), for 10 min. Treatment efficiencies were determined by logarithmic comparisons of culturable cells on blood agar before and after treatment. Results were statistically compared using an anova of culturable cells after treatment to evaluate the effect of all factors. At least a 7-log reduction in cell numbers was observed for both strains. The pH and the strains had no effect on HHP treatment at 20°C while at 37°C, both pH and strain influenced significantly the HHP treatment on C. jejuni .
Conclusions:  The pressure efficacy on C. jejuni eradication was affected by both environmental and biological factors.
Significance and Impact of the Study:  Depending on the treatment conditions, C. jejuni sensitivity to HHP can significantly vary. The determination of the inactivation treatment by HPP has to be normalized considering the interaction of environmental and biological factors.     

Characterization of high hydrostatic pressure-injured Bacillus subtilis cells*

High hydrostatic pressure (HHP) affects various cellular processes. Using a sporulation-deficient Bacillus subtilis strain, we characterized the properties of vegetative cells subjected to HHP. When stationary-phase cells were exposed to 250 MPa of HHP for 10 min at 25 °C, approximately 50% of cells were viable, although they exhibited a prolonged growth lag. The HHP-injured cells autolyzed in the presence of NaCl or KCl (at concentrations ≥100 mM). Superoxide dismutase slightly protected the viability of HHP-treated cells, whereas vegetative catalases had no effect. Thus, unlike HHP-injured Escherichia coli, oxidative stress only slightly affected vegetative B. subtilis subjected to HHP.     

Streaming potential-based arthroscopic device is sensitive to cartilage changes immediately post-impact in an equine cartilage injury model

Models of post-traumatic osteoarthritis where early degenerative changes can be monitored are valuable for assessing potential therapeutic strategies. Current methods for evaluating cartilage mechanical properties may not capture the low-grade cartilage changes expected at these earlier time points following injury. In this study, an explant model of cartilage injury was used to determine whether streaming potential measurements by manual indentation could detect cartilage changes immediately following mechanical impact and to compare their sensitivity to biomechanical tests. Impacts were delivered ex vivo, at one of three stress levels, to specific positions on isolated adult equine trochlea. Cartilage properties were assessed by streaming potential measurements, made pre- and post-impact using a commercially available arthroscopic device, and by stress relaxation tests in unconfined compression geometry of isolated cartilage disks, providing the streaming potential integral (SPI), fibril modulus (Ef), matrix modulus (Em), and permeability (k). Histological sections were stained with Safranin-O and adjacent unstained sections examined in polarized light microscopy. Impacts were low, 17.3?±?2.7 MPa (n?=?15), medium, 27.8?±?8.5 MPa (n?=?13), or high, 48.7?±?12.1 MPa (n?=?16), and delivered using a custom-built spring-loaded device with a rise time of approximately 1 ms. SPI was significantly reduced after medium (p?=?0.006) and high (p<0.001) impacts. Ef, representing collagen network stiffness, was significantly reduced in high impact samples only (p?相似文献   

Depth- and strain-dependent mechanical and electromechanical properties of full-thickness bovine articular cartilage in confined compression.

Compression tests have often been performed to assess the biomechanical properties of full-thickness articular cartilage. We tested whether the apparent homogeneous strain-dependent properties, deduced from such tests, reflect both strain- and depth-dependent material properties. Full-thickness bovine articular cartilage was tested by oscillatory confined compression superimposed on a static offset up to 45%. and the data fit to estimate modulus, permeability, and electrokinetic coefficient assuming homogeneity. Additional tests on partial-thickness cartilage were then performed to assess depth- and strain-dependent properties in an inhomogeneous model, assuming three discrete layers (i = 1 starting from the articular surface, to i = 3 up to the subchondral bone). Estimates of the zero-strain equilibrium confined compression modulus (H(A0)), the zero-strain permeability (kp0) and deformation dependence constant (M), and the deformation-dependent electrokinetic coefficient (ke) differed among individual layers of cartilage and full-thickness cartilage. HiA0 increased from layer 1 to 3 (0.27 to 0.71 MPa), and bracketed the apparent homogeneous value (0.47 MPa). ki(p0) decreased from layer 1 to 3 (4.6 x 10(-15) to 0.50 x 10(-15) m2/Pa s) and was less than the homogeneous value (7.3 x 10(-15) m2/Pa s), while Mi increased from layer 1 to 3 (5.5 to 7.4) and became similar to the homogeneous value (8.4). The amplitude of ki(e) increased markedly with compressive strain, as did the homogeneous value: at low strain, it was lowest near the articular surface and increased to a peak in the middle-deep region. These results help to interpret the biomechanical assessment of full-thickness articular cartilage.     

Inactivation of Salmonella Typhimurium and Listeria monocytogenes using high-pressure treatments: destruction or sublethal stress?

AIMS: To investigate potential resuscitation of Listeria monocytogenes and Salmonella Typhimurium after high hydrostatic pressure treatments. METHODS AND RESULTS: Pressure treatments were applied at room temperature for 10 min on bacterial suspensions in buffers at pH 7 and 5.6. Total bacterial inactivation (8 log(10) CFU ml(-1) of bacterial reduction) obtained by conventional plating was achieved regarding both micro-organisms. Treatments at 400 MPa in pH 5.6 and 600 MPa in pH 7 for L. monocytogenes and at 350 MPa in pH 5.6 and 400 MPa in pH 7 for S. Typhimurium were required respectively. A 'direct viable count' method detected some viable cells in the apparently totally inactivated population. Resuscitation was observed for the two micro-organisms during storage (at 4 and 20 degrees C) after almost all treatments. In the S. Typhimurium population, 600 MPa, 10 min, was considered as the treatment achieving total destruction because no resuscitation was observed under these storage conditions. CONCLUSIONS: We suggest a delay before performing counts in treated samples in order to avoid the under-evaluation of surviving cells. SIGNIFICANCE AND IMPACT OF THE STUDY: The resuscitation of pathogen bacteria after physical treatments like high hydrostatic pressure has to be considered from the food safety point of view. Further studies should be performed in food products to study this resuscitation phenomenon.     

A Three-Dimensional Finite Element Model for Biomechanical Analysis of the Hip

The objective of this study was to construct a three-dimensional (3D) finite element model of the hip. The images of the hip were obtained from Chinese visible human dataset. The hip model includes acetabular bone, cartilage, labrum, and bone. The cartilage of femoral head was constructed using the AutoCAD and Solidworks software. The hip model was imported into ABAQUS analysis system. The contact surface of the hip joint was meshed. To verify the model, the single leg peak force was loaded, and contact area of the cartilage and labrum of the hip and pressure distribution in these structures were observed. The constructed 3D hip model reflected the real hip anatomy. Further, this model reflected biomechanical behavior similar to previous studies. In conclusion, this 3D finite element hip model avoids the disadvantages of other construction methods, such as imprecision of cartilage construction and the absence of labrum. Further, it provides basic data critical for accurately modeling normal and abnormal loads, and the effects of abnormal loads on the hip.     

Allogeneic Mesenchymal Stem Cells in Combination with Hyaluronic Acid for the Treatment of Osteoarthritis in Rabbits

Mesenchymal stem cell (MSC)-based therapies may aid in the repair of articular cartilage defects. The purpose of this study was to investigate the effects of intraarticular injection of allogeneic MSCs in an in vivo anterior cruciate ligament transection (ACLT) model of osteoarthritis in rabbits. Allogeneic bone marrow-derived MSCs were isolated and cultured under hypoxia (1% O₂). After 8 weeks following ACLT, MSCs suspended in hyaluronic acid (HA) were injected into the knees, and the contralateral knees were injected with HA alone. Additional controls consisted of a sham operation group as well as an untreated osteoarthritis group. The tissues were analyzed by macroscopic examination as well as histologic and immunohistochemical methods at 6 and 12 weeks post-transplantation. At 6 and 12 weeks, the joint surface showed less cartilage loss and surface abrasion after MSC injection as compared to the tissues receiving HA injection alone. Significantly better histological scores and cartilage content were observed with the MSC transplantation. Furthermore, engraftment of allogenic MSCs were evident in surface cartilage. Thus, injection of the allogeneic MSCs reduced the progression of osteoarthritis in vivo.