Chondrocytes within osteochondral grafts are more resistant than osteoblasts to tissue culture at 37°C

It is proposed that an ideal osteochondral allograft for cartilage repair consists of a devitalized bone but functional cartilage. The different modes of nutrient supply in vivo for bone (vascular support) and cartilage (diffusion) suggest that a modulation of storage conditions could differentially affect the respective cells, resulting in the proposed allograft. For this purpose, osteochondral tissues from porcine humeral heads were either cultured at 37°C for up to 24 hr or stored at 4°C for 24 hr, the temperature at which osteochondral allografts are routinely stored. Functionality of the cells was assessed by in situ hybridization for transcripts encoding collagen types I and II. At 37°C, a time-dependent significant reduction of the bone surface covered with functional cells was observed with only 5% ± 5% coverage left at 24 hr compared with 41% ± 10% at 0 hr. Similarly, cartilage area containing functional cells was significantly reduced from 84% ± 7% at 0 hr to 70% ± 3% after 24 hr. After 24 hr at 4°C, a significantly reduced amount of functional cells covering bone surfaces was observed (27% ± 5%) but not of cells within the cartilage (79% ± 8%). In the applied experimental setup, bone cells were more affected by tissue culture at 37°C than cartilage cells. Even though chondrocytes appear to be more sensitive to 37°C than to 4°C, the substantially reduced amount of functional bone cells at 37°C warrants further investigation of whether a preincubation of osteochondral allografts at 37°C--prior to regular storage at 4°C--might result in an optimized osteochondral allograft with devitalized bone but viable cartilage.

THA performed using conventional and navigated tissue-preserving techniques

Less invasive methods of performing total hip arthroplasty have been considered controversial after increased complication rates and component malpositioning were reported. A new method of performing total hip arthroplasty through an incision in the superior capsule, posterior to the abductors and anterior to the posterior capsule, was developed with the aim of producing a technique that maintained the joint stability of the transgluteal exposure and the rapid abductor recovery of the posterior exposure. We assessed the recovery and complications of this technique performed with surgical navigation. The study group was compared with similar subjects who had conventional total hip arthroplasty, without surgical navigation, using the transgluteal exposure. There were 185 consecutive total hip arthroplasties in the study group and 189 nonconsecutive historical total hip arthroplasties in the control group. The two groups were controlled for complexity and had no differences in body mass index, gender, diagnosis, operative side, bilateral operations, and previous surgeries. Patients were evaluated for clinical recovery and perioperative complications at 9 and 24 weeks. The study group recovered faster at both followup examinations. The study group had fewer perioperative and postoperative complications compared with the control group. Accuracy of component positioning was not compromised compared to the control group. Less invasive surgery with the philosophy of maximally preserving the abductors, posterior capsule, and short rotators may result in a safer operation with faster recovery than traditional techniques.

Differential response of porcine osteoblasts and chondrocytes in cell or tissue culture after 5-aminolevulinic acid-based photodynamic therapy

OBJECTIVE: Outcome in osteochondral allografting is limited by the immunological incompatibility of the grafted tissue. Based on a resistance of chondrocytes to photodynamic therapy in cell culture it is proposed that 5-aminolevulinic acid-based photodynamic therapy (5-ALA-PDT) might be used to inactivate bone while maintaining viability of chondrocytes and thus immunomodulate bone selectively. METHODS: Chondrocytes and osteoblasts from porcine humeral heads were either isolated (cell culture) or treated in situ (tissue culture). To quantify cytotoxic effects of 5-ALA-PDT (0-20J/cm(2), 100mW/cm(2)) an (3-(4,5-dimethylthiazol-2-yl)-2,5-di-phenyltetrazolium bromide) (MTT)-assay was used in cell culture and in situ hybridization in tissue culture to assess metabolic active cells (functional osteoblasts: colalpha(1)(I) mRNA, functional chondrocytes: colalpha(1)(II) mRNA). RESULTS: In cell culture, survival after 5-ALA-PDT was significantly higher for chondrocytes (5J/cm(2): 87+/-12% compared to untreated cells) than for osteoblasts (5J/cm(2): 12+/-11%). In tissue culture, the percentage of functional chondrocytes in cartilage showed a decrease after 5-ALA-PDT (direct fixation: 92+/-2%, 20J/cm(2): 35+/-15%; P<0.0001). A significant decrease in the percentage of bone surfaces covered by functional osteoblasts was observed in freshly harvested (31+/-3%) compared to untreated tissues maintained in culture (11+/-4%, P<0.0001), with no further decrease after 5-ALA-PDT. CONCLUSION: Chondrocytes were more resistant to 5-ALA-PDT than osteoblasts in cell culture, while in tissue culture a loss of functional chondrocytes was observed after 5-ALA-PDT. Since osteoblasts - but not chondrocytes - were sensitive to the tissue culture conditions, devitalized bone with functional cartilage might already be achieved by applying specific tissue culture conditions even without 5-ALA-PDT.

A systematic review assessing soft tissue augmentation techniques

AIM: The aim of the present review was to systematically assess the dental literature in terms of soft tissue grafting techniques. The focused question was: is one method superior over others for augmentation and stability of the augmented soft tissue in terms of increasing the width of keratinized tissue (part 1) and gain in soft tissue volume (part 2). METHODS: A Medline search was performed for human studies focusing on augmentation of keratinized tissue and/or soft tissue volume, and complemented by additional hand searching. Relevant studies were identified and statistical results were reported for meta-analyses including the test minus control weighted mean differences with 95% confidence intervals, the I-squared statistic for tests of heterogeneity, and the number of significant studies. RESULTS: Twenty-five (part 1) and three (part 2) studies met the inclusion criteria; 14 studies (part 1) were eligible for comparison using meta-analyses. An apically positioned flap/vestibuloplasty (APF/V) procedure resulted in a statistically significantly greater gain in keratinized tissue than untreated controls. APF/V plus autogenous tissue revealed statistically significantly more attached gingiva compared with untreated controls and a borderline statistical significance compared with APF/V plus allogenic tissue. Statistically significantly more shrinkage was observed for the APF/V plus allogenic graft compared with the APF/V plus autogenous tissue. Patient-centered outcomes did not reveal any of the treatment methods to be superior regarding postoperative complications. The three studies reporting on soft tissue volume augmentation could not be compared due to lack of homogeneity. The use of subepithelial connective tissue grafts (SCTGs) resulted in statistically significantly more soft tissue volume gain compared with free gingival grafts (FGGs). CONCLUSIONS: APF/V is a successful treatment concept to increase the width of keratinized tissue or attached gingiva around teeth. The addition of autogenous tissue statistically significantly increases the width of attached gingiva. For soft tissue volume augmentation, only limited data are available favoring SCTGs over FGG.

Comparison of mesencephalic free-floating tissue culture grafts and cell suspension grafts in the 6-hydroxydopamine-lesioned rat

Ventral mesencephalon (VM) of fetal rat and human origin grown as free-floating roller-tube (FFRT) cultures can survive subsequent grafting to the adult rat striatum. To further explore the functional efficacy of such grafts, embryonic day 13 ventral mesencephalic tissue was grafted either after 7 days in culture or directly as dissociated cell suspensions, and compared with regard to neuronal survival and ability to normalize rotational behavior in adult rats with unilateral 6-hydroxydopamine (6-OHDA) lesions. Other lesioned rats received injections of cell-free medium and served as controls. The amphetamine-induced rotational behavior of all 6-OHDA-lesioned animals was monitored at various time points from 18 days before transplantation and up to 80 days after transplantation. Tyrosine hydroxylase (TH) immunostaining of the histologically processed brains served to assess the long-term survival of grafted dopaminergic neurons and to correlate that with the behavioral effects. Additional cultures and acutely prepared explants were also fixed and stored for histological investigation in order to estimate the loss of dopaminergic neurons in culture and after transplantation. Similar behavioral improvements in terms of significant reductions in amphetamine-induced rotations were observed in rats grafted with FFRT cultures (127%) and rats grafted with cell suspensions (122%), while control animals showed no normalization of rotational behavior. At 84 days after transplantation, there were similar numbers of TH-immunoreactive (TH-ir) neurons in grafts of cultured tissue (775 +/- 98, mean +/- SEM) and grafts of fresh, dissociated cell suspension (806 +/- 105, mean +/- SEM). Cell counts in fresh explants, 7-day-old cultures, and grafted cultures revealed a 68.2% loss of TH-ir cells 7 days after explantation, with an additional 23.1% loss after grafting, leaving 8.7% of the original number of TH-ir cells in the intracerebral grafts. This is to be compared with a survival rate of 9.1% for the TH-ir cells in the cell-suspension grafts. Immunostaining for the calcium-binding proteins calretinin, calbindin, and parvalbumin showed no differences in the neuronal expression of these proteins between the two graft types. In conclusion, we found comparable dopaminergic cell survival and functional effects of tissue-culture grafts and cell-suspension grafts, which currently is the type of graft most commonly used for experimental and clinical grafting. In this sense the result is promising for the development of an effective in vitro storage of fetal nigral tissue, which at the same time would allow neuroprotective and neurotrophic treatment prior to intracerebral transplantation.

Evaluation of cartilage repair tissue after matrix-associated autologous chondrocyte transplantation using a hyaluronic-based or a collagen-based scaffold with morphological MOCART scoring and biochemical T2 mapping: preliminary results

In cartilage repair, bioregenerative approaches using tissue engineering techniques have tried to achieve a close resemblance to hyaline cartilage, which might be visualized using advanced magnetic resonance imaging.

Infection of organotypic slice cultures from rat central nervous tissue with Neospora caninum: an alternative approach to study host-parasite interactions

Neospora caninum is an apicomplexan parasite which has emerged as an important cause of bovine abortion worldwide. Abortion is usually triggered by reactivation of dormant bradyzoites during pregnancy and subsequent congenital infection of the foetus, where the central nervous system appears to be most frequently affected. We here report on an organotypic tissue culture model for Neospora infection which can be used to study certain aspects of the cerebral phase of neosporosis within the context of a three-dimensionally organised neuronal network. Organotypic slice cultures of rat cortical tissue were infected with N. caninum tachyzoites, and the kinetics of parasite proliferation, as well as the proliferation-inhibitory effect of interferon-gamma (IFN-gamma), were monitored by either immunofluorescence, transmission electron microscopy, and a quantitative PCR-assay using the LightCycler instrument, respectively. In addition, the neuronal cytoskeletal elements, namely glial acidic protein filaments as well as actin microfilament bundles were shown to be largely colocalising with the pseudocyst periphery. This organotypic culture model for cerebral neosporosis provides a system, which is useful to study the proliferation, ultrastructural characteristics, development, and the interactions of N. caninum within the context of neuronal tissue, which at the same time can be modulated and influenced under controlled conditions, and will be useful in the future to gain more information on the cerebral phase of neosporosis.

Stochastic amplitude-modulated stretching of Rabbit flexor digitorum profundus tendons reduces stiffness compared to Cyclic Loading but does not affect Tenocyte Metabolism

Background It has been demonstrated that frequency modulation of loading influences cellular response and metabolism in 3D tissues such as cartilage, bone and intervertebral disc. However, the mechano-sensitivity of cells in linear tissues such as tendons or ligaments might be more sensitive to changes in strain amplitude than frequency. Here, we hypothesized that tenocytes in situ are mechano-responsive to random amplitude modulation of strain. Methods We compared stochastic amplitude-modulated versus sinusoidal cyclic stretching. Rabbit tendon were kept in tissue-culture medium for twelve days and were loaded for 1h/day for six of the total twelve culture days. The tendons were randomly subjected to one of three different loading regimes: i) stochastic (2 – 7% random strain amplitudes), ii) cyclic_RMS (2–4.42% strain) and iii) cyclic_high (2 - 7% strain), all at 1 Hz and for 3,600 cycles, and one unloaded control. Results At the end of the culture period, the stiffness of the “stochastic” group was significantly lower than that of the cyclic_RMS and cyclic_high groups (both, p < 0.0001). Gene expression of eleven anabolic, catabolic and inflammatory genes revealed no significant differences between the loading groups. Conclusions We conclude that, despite an equivalent metabolic response, stochastically stretched tendons suffer most likely from increased mechanical microdamage, relative to cyclically loaded ones, which is relevant for tendon regeneration therapies in clinical practice.

Epizootiologic Investigations of Selected Abortive Agents in Free-Ranging Alpine Ibex (Capra Ibex Ibex) in Switzerland

In the early 2000s, several colonies of Alpine ibex (Capra ibex ibex) in Switzerland ceased growing or began to decrease. Reproductive problems clue to infections with abortive agents might have negatively affected recruitment. We assessed the presence of selected agents of abortion in Alpine ibex by serologic, molecular, and culture techniques and evaluated whether infection with these agents might have affected population densities. Blood and fecal samples were collected from 651 ibex in 14 colonies throughout the Swiss Alps between 2006 and 2008. All samples were negative for Salmonella. spp., Neospora caninum, and Bovine Herpesvirus-1. Antibodies to Coxiella burnetii, Leptospira spp., Chlamydophila abortus, Toxoplasma gondii, and Bovine Viral Diarrhea virus were detected in at least one ibex. Positive serologic results for Brucella spp. likely were false. Overall, 73 samples (11.2%) were antibody-positive for at least one abortive agent. Prevalence was highest for Leptospira spp. (7.9%, 95% CI=5.0-11.7). The low prevalences and the absence of significant differences between colonies with opposite population trends suggest these pathogens do not play a significant role in the population dynamics of Swiss ibex. Alpine ibex do not seem to be a reservoir for these abortive agents or an important source of infection for domestic livestock in Switzerland. Finally, although interactions on summer pastures occur frequently, spillover from infected livestock to free-ranging ibex apparently is uncommon.

N-(3-Cyanophenyl)-2-phenylacetamide, an effective inhibitor of morbillivirus-induced membrane fusion with low cytotoxicity

Based on the structural similarity of viral fusion proteins within the family Paramyxoviridae, we tested recently described and newly synthesized acetanilide derivatives for their capacity to inhibit measles virus (MV)-, canine distemper virus (CDV)- and Nipah virus (NiV)-induced membrane fusion. We found that N-(3-cyanophenyl)-2-phenylacetamide (compound 1) has a high capacity to inhibit MV- and CDV-induced (IC(50) muM), but not NiV-induced, membrane fusion. This compound is of outstanding interest because it can be easily synthesized and its cytotoxicity is low [50 % cytotoxic concentration (CC(50)) >/= 300 muM], leading to a CC(50)/IC(50) ratio of approximately 100. In addition, primary human peripheral blood lymphocytes and primary dog brain cell cultures (DBC) also tolerate high concentrations of compound 1. Infection of human PBMC with recombinant wild-type MV is inhibited by an IC(50) of approximately 20 muM. The cell-to-cell spread of recombinant wild-type CDV in persistently infected DBC can be nearly completely inhibited by compound 1 at 50 muM, indicating that the virus spread between brain cells is dependent on the activity of the viral fusion protein. Our findings demonstrate that this compound is a most applicable inhibitor of morbillivirus-induced membrane fusion in tissue culture experiments including highly sensitive primary cells.

Ex vivo assessment of chemotherapy-induced apoptosis and associated molecular changes in patient tumor samples

BACKGROUND: There are inherent conceptual problems in investigating the pharmacodynamics of cancer drugs in vivo. One of the few possible approaches is serial biopsies in patients. However, this type of research is severely limited by methodological and ethical constraints. MATERIALS AND METHODS: A modified 3-dimensional tissue culture technique was used to culture human tumor samples, which had been collected during routine cancer operations. Twenty tumor samples of patients with non-small cell lung cancer (NSCLC) were cultured ex vivo for 120 h and treated with mitomycin C, taxotere and cisplatin. The cytotoxic activity of the anticancer agents was quantified by assessing the metabolic activity of treated tumor cultures and various assays of apoptosis and gene expression were performed. RESULTS: The proliferative activity of the tissue was maintained in culture as assessed by Ki-67 staining. Mitomycin C, cisplatin and taxotere reduced the metabolic activity of the tumor tissue cultures by 51%, 29% and 20%, respectively, at 120 h. The decrease in metabolic activity corresponded to the induction of apoptosis as demonstrated by the typical morphological changes, such as chromatin condensation and nuclear fragmentation. In addition, activated caspase-3 could be verified in apoptotic cells by immunohistochemistry. To verify functional aspects of apoptosis, the induction of chemotherapy-induced cell death was inhibited with the caspase inhibitor z-VAD.fmk. RNA was extracted from the tissue cultures after 120 h of ex vivo drug treatment and was of sufficient quality to allow quantitative PCR. CONCLUSION: The 3-dimensional ex vivo culture technique is a useful method to assess the molecular effects of pharmacological interventions in human cancer samples in vitro. This culture technique could become an important tool for drug development and for the prediction of in vivo drug efficacy.

The death-associated protein kinase 2 is up-regulated during normal myeloid differentiation and enhances neutrophil maturation in myeloid leukemic cells

The death-associated protein kinase 2 (DAPK2) belongs to a family of Ca(2+)/calmodulin-regulated serine/threonine kinases involved in apoptosis. During investigation of candidate genes operative in granulopoiesis, we identified DAPK2 as highly expressed. Subsequent investigations demonstrated particularly high DAPK2 expression in normal granulocytes compared with monocytes/macrophages and CD34(+) progenitor cells. Moreover, significantly increased DAPK2 mRNA levels were seen when cord blood CD34(+) cells were induced to differentiate toward neutrophils in tissue culture. In addition, all-trans retinoic acid (ATRA)-induced neutrophil differentiation of two leukemic cell lines, NB4 and U937, revealed significantly higher DAPK2 mRNA expression paralleled by protein induction. In contrast, during differentiation of CD34(+) and U937 cells toward monocytes/macrophages, DAPK2 mRNA levels remained low. In primary leukemia, low expression of DAPK2 was seen in acute myeloid leukemia samples, whereas chronic myeloid leukemia samples in chronic phase showed intermediate expression levels. Lentiviral vector-mediated expression of DAPK2 in NB4 cells enhanced, whereas small interfering RNA-mediated DAPK2 knockdown reduced ATRA-induced granulocytic differentiation, as evidenced by morphology and neutrophil stage-specific maturation genes, such as CD11b, G-CSF receptor, C/EBPepsilon, and lactoferrin. In summary, our findings implicate a role for DAPK2 in granulocyte maturation.

Immunohistochemical diagnosis of persistent infection with Bovine Viral Diarrhea Virus (BVDV) on skin biopsies

Detection of persistent infection with BovineViral Diarrhea Virus (BVDV) is essential for both epidemiological and clinical reasons. In addition to the classical virological methods such as virus isolation in tissue culture, ELISA and RT-PCR, immunohistochemistry of skin biopsies has become a useful and reliable tool. Assuming that the presence of BVDV antigen in skin structures is restricted to persistent infection, this method could differentiate from transient infection. In order to answer this question, 6 calves were experimentally infected orally with a non-cytopathic genotype 1 BVDV strain belonging to the subtype k.The calves developed fever, mucopurulent nasal discharge, coughing and leucopenia with relative lymphopenia. Immunohistochemistry of skin biopsies taken daily up to day 13-post infection did not reveal any evidence of BVDV infection. BVDV was, however, isolated from blood samples on cell cultures. Anti-NS3-antibody-ELISA and serum neutralization tests showed that all six calves seroconverted. We conclude that in acute BVDV infections, with genotype 1 and the subtypes found in Switzerland (b, e, h and k) viral antigen is not found in epidermal structures of the skin. In contrast, persistently infected animals test positive for BVD viral antigen by immunohistochemistry of the skin.

Magnetic resonance imaging for diagnosis and assessment of cartilage defect repairs

Clinical magnetic resonance imaging (MRI) is the method of choice for the non-invasive evaluation of articular cartilage defects and the follow-up of cartilage repair procedures. The use of cartilage-sensitive sequences and a high spatial-resolution technique enables the evaluation of cartilage morphology even in the early stages of disease, as well as assessment of cartilage repair. Sequences that offer high contrast between articular cartilage and adjacent structures, such as the fat-suppressed, 3-dimensional, spoiled gradient-echo sequence and the fast spin-echo sequence, are accurate and reliable for evaluating intrachondral lesions and surface defects of articular cartilage. These sequences can also be performed together in reasonable examination times. In addition to morphology, new MRI techniques provide insight into the biochemical composition of articular cartilage and cartilage repair tissue. These techniques enable the diagnosis of early cartilage degeneration and help to monitor the effect and outcome of various surgical and non-surgical cartilage repair therapies.

Penetrating keratoplasty and graft rejection in eight horses

The aim of this study was to describe long-term follow-up and difference in immune reactions in the tear film following penetrating keratoplasty (PK) in horses when differently preserved corneas were utilised. This report describes for the first time the use of corneal grafts preserved in tissue culture media in equine PK. Eight experimental horses with normal eyes were included and freshly harvested, frozen or preserved corneal grafts were used for the PK. The graft-taking technique and storage, PK surgery, postoperative treatments and complications are described. The mean postoperative follow-up time was 286 days. Tear film samples taken before and periodically after surgery were measured for IgM, IgG and IgA contents by direct ELISA. All grafts were incorporated into the donor horse but were rejected to some degree. The differently harvested corneal grafts healed in the same manner and looked similar. Preoperatively, the clear corneas meant low risk for graft failure, and the fresh or stored tissues provided intact endothelium, although there were no clear graft sites postoperatively. The presence of IgA, IgG and IgM was demonstrated in the tear film from the early postoperative period. IgG levels were lower than IgA or IgM and had a constant baseline in every case, as IgA and IgM had great variability with time and an individual pattern in each eye.