Postmortem chondrocyte viability in porcine articular cartilage: Influence of time, temperature, and burial under winter conditions.

The aim of the present study was to investigate the effects of time, temperature, and burial in a natural environment on the viability of chondrocytes in porcine femoral condyles using confocal laser scanning microscopy. Hind trotters from 10 pigs were buried or left unburied. Samples were collected daily and stained with a combination of vital dyes (calcein-AM and ethidium homodimer-1). The chondrocytes showed an intense staining corresponding to their vitality. In the first 3 days, viability decreased slowly and showed no statistical difference between buried and unburied samples. After the first 3 days, it decreased rapidly, with the viability of the buried samples being 66% on day 4, decreasing to 25% on day 8 and to 16% on day 10, while in the unburied samples it decreased to 43% on day 4, 13% on day 8 and 5% on day 10. Our results indicate a time, temperature, and burial dependent decrease in chondrocyte viability and suggest the use of chondrocyte viability as a marker for estimating PMI in both the natural environment and in animals, as well as its potential use in humans.

Sphenoidal Foramen Ovale in the Slovenian Population: An Anatomical Evaluation with Clinical Correlations.

The foramen ovale (FO) is a crucial feature of the skull base, serving as a passage for clinically important neurovascular structures. The present study aimed to provide a comprehensive morphometric and morphologic analysis of the FO and highlight the clinical significance of the anatomical characterization. A total of 267 FO were analyzed in skulls obtained from deceased inhabitants of the Slovenian territory. The anteroposterior (length) and the transverse (width) diameters were measured using a digital sliding vernier caliper. Dimensions, shape, and anatomical variations of FO were analyzed. The mean length and width of the FO were 7.13 and 3.71 mm on the right side and 7.20 and 3.88 mm on the left side. The most frequently observed shape was oval (37.1%), followed by almond (28.1%), irregular (21.0%), D-shaped (4.5%), round (3.0%), pear-shaped (1.9%), kidney-shaped (1.5%), elongated (1.5%), triangular (0.7%), and slit-like (0.7%). In addition, marginal outgrowths (16.6%) and several anatomical variations were noted, including duplications, confluences, and obstruction due to a complete (5.6%) or incomplete (8.2%) pterygospinous bar. Our observations revealed substantial interindividual variation in the anatomical characteristics of the FO in the studied population, which could potentially impact the feasibility and safety of neurosurgical diagnostic and therapeutic procedures.

Exploration of macromolecular phenotype of human skeletal muscle in diabetes using infrared spectroscopy.

Introduction: The global burden of diabetes mellitus is escalating, and more efficient investigative strategies are needed for a deeper understanding of underlying pathophysiological mechanisms. The crucial role of skeletal muscle in carbohydrate and lipid metabolism makes it one of the most susceptible tissues to diabetes-related metabolic disorders. In tissue studies, conventional histochemical methods have several technical limitations and have been shown to inadequately characterise the biomolecular phenotype of skeletal muscle to provide a holistic view of the pathologically altered proportions of macromolecular constituents. Materials and methods: In this pilot study, we examined the composition of five different human skeletal muscles from male donors diagnosed with type 2 diabetes and non-diabetic controls. We analysed the lipid, glycogen, and collagen content in the muscles in a traditional manner with histochemical assays using different staining techniques. This served as a reference for comparison with the unconventional analysis of tissue composition using Fourier-transform infrared spectroscopy as an alternative methodological approach. Results: A thorough chemometric post-processing of the infrared spectra using a multi-stage spectral decomposition allowed the simultaneous identification of various compositional details from a vibrational spectrum measured in a single experiment. We obtained multifaceted information about the proportions of the different macromolecular constituents of skeletal muscle, which even allowed us to distinguish protein constituents with different structural properties. The most important methodological steps for a comprehensive insight into muscle composition have thus been set and parameters identified that can be used for the comparison between healthy and diabetic muscles. Conclusion: We have established a methodological framework based on vibrational spectroscopy for the detailed macromolecular analysis of human skeletal muscle that can effectively complement or may even serve as an alternative to histochemical assays. As this is a pilot study with relatively small sample sets, we remain cautious at this stage in drawing definitive conclusions about diabetes-related changes in skeletal muscle composition. However, the main focus and contribution of our work has been to provide an alternative, simple and efficient approach for this purpose. We are confident that we have achieved this goal and have brought our methodology to a level from which it can be successfully transferred to a large-scale study that allows the effects of diabetes on skeletal muscle composition and the interrelationships between the macromolecular tissue alterations due to diabetes to be investigated.

Acute Cartilage Injury Induced by Trans-Articular Sutures.

OBJECTIVE: To determine the extent of acute cartilage injury by using trans-articular sutures. METHODS: Five different absorbable sutures, monofilament polydioxanone (PDS) and braided polyglactin (Vicryl), were compared on viable human osteochondral explants. An atraumatic needle with 30 cm of thread was advanced through the cartilage with the final thread left in the tissue. A representative 300 µm transversal slice from the cartilage midportion was stained with Live/Dead probes, scanned under the confocal laser microscope, and analyzed for the diameters of (a) central "Black zone" without any cells, representing in situ thread thickness and (b) "Green zone," including the closest Live cells, representing the maximum injury to the tissue. The exact diameters of suture needles and threads were separately measured under an optical microscope. RESULTS: The diameters of the Black (from 144 to 219 µm) and the Green zones (from 282 to 487 µm) varied between the different sutures (P < 0.001). The Green/Black zone ratio remained relatively constant (from 1.9 to 2.2; P = 0.767). A positive correlation between thread diameters and PDS suturing material, toward the Black and Green zone, was established, but needle diameters did not reveal any influence on the zones. CONCLUSIONS: The width of acute cartilage injury induced by the trans-articular sutures is about twice the thread thickness inside of the tissue. Less compressible monofilament PDS induced wider tissue injury in comparison to a softer braided Vicryl. Needle diameter did not correlate to the extent of acute cartilage injury.

The Influence of a Single Intra-Articular Lidocaine Injection on the Viability of Articular Cartilage in the Knee.

OBJECTIVE: To evaluate the in vivo effect of a single intra-articular injection of local anesthetic (LA) lidocaine on the viability of articular cartilage in the intact or osteoarthritic (OA) human knees, and to measure the synovial postinjection concentration of lidocaine in the knee. DESIGN: This study includes 3 interconnected experiments: (A) Synovial LA concentration measurement after a 2% lidocaine injection before knee arthroscopy in 10 patients by liquid chromatography-tandem mass spectrometry (LC-MS/MS). (B) Human osteochondral explants (N = 27) from intact knees procured at autopsies were incubated for different time intervals (30 minutes, 2 hours, 24 hours) with 2% lidocaine, 0.04% lidocaine (measured), or culture medium (control), and later evaluated for cell viability by LIVE/DEAD staining. (C) Ten out of 19 matched patients scheduled for knee replacement received a single intra-articular injection of 2% lidocaine approximately 30 minutes prior to the procedure; 9 patients served as control. Osteochondral samples with OA changes were harvested during surgery and analyzed for chondrocyte viability by LIVE/DEAD staining. RESULTS: (A) The synovial LA concentration was significantly lower than the primary concentration injected: average 0.23 mg/mL (0.02%), highest measured 0.37 mg/mL (0.04%). (B) In vitro exposure to a reduced LA concentration had no significant influence on chondrocyte viability in intact cartilage explants (24-hour averages: control, 93%; 0.04% lidocaine, 92%; 2% lidocaine, 79%). (C) Viability of chondrocytes in OA knees was similar between 2% lidocaine injection (85%) and control (80%). CONCLUSIONS: A single intra-articular knee injection of 2% lidocaine did not influence the chondrocyte viability neither in healthy nor in OA cartilage. A fast postinjection reduction of synovial LA concentration (more than 40 times) is the most likely protective mechanism.

Safranin O without fast green is the best staining method for testing the degradation of macromolecules in a cartilage extracellular matrix for the determination of the postmortem interval.

Methods for the determination of the postmortem interval (PMI) include methods that monitor the postmortem changes of cells and molecules in different tissues. The rate of pathological degradation of macromolecules in the extracellular matrix (ECM) of hyaline cartilage could be verified by assessing the intensity of collagen and proteoglycan (PG) staining. In the presented in vitro pilot study, this methodology was used for the first time to determine PMI. The osteochondral samples of three donors were stored at 11 °C and 35 °C and analyzed on day 1, day 12, and day 36 postmortem. The intensity of staining using Masson's trichrome and Sirius red for collagen, and Alcian blue and Safranin O dyes for PG was estimated ten times according to the modified Bern grading scale. Statistical analysis showed that the Safranin O without Fast green method is the most appropriate (raters agreement 0.5541) for up to 36 days postmortem, and that the influence of time is more important (p = 0.023) than the influence of temperature (p = 0.061) on the degradation of the ECM macromolecules. The described method, which is simple and can be performed in any histological laboratory, should be verified in corpore conditions, on a large number of donors, and using an objective method for assessing the intensity of cartilage macromolecule staining for PMI determination.

Fatal overdose with a combination of SNRIs venlafaxine and duloxetine.

Drugs for the treatment of depressive disorders, including SNRIs (serotonin noradrenaline reuptake inhibitors) venlafaxine and duloxetine, are widely prescribed as they have a high therapeutic to toxicity ratio. In rare cases, adverse effects may be severe, usually due to iatrogenic, accidental or intentional self-overdose that cause the excessive accumulation of serotonin and noradrenaline in synaptic clefts. Lethal intoxication with a combination of venlafaxine and duloxetine (postmortem blood concentrations 24 mg/L and 0.97 mg/L, respectively) without co-ingested substances, comorbidities or injuries that could have an unknown contribution to a fatal outcome is presented for the first time in the following case report, with a comprehensive clinical history, and complete results of the performed analyses. The cause of death was a serotonin syndrome that progressed to death in approximately six hours and 15 min after the suicidal ingestion of venlafaxine and duloxetine. Despite the high therapeutic to toxicity ratio SNRIs, which are reserved for patients with severe forms of depressive disorders and a higher suicidal tendency, they should be cautiously prescribed and handed over in smaller packages to make them easier to follow, and thus avoid accumulation within the patient's reach.

Testicular morphology in hypogonadotropic hypogonadism after the abuse of anabolic steroids.

Hypogonadism in men results from the failure of the testes to produce physiological levels of testosterone and a normal number of spermatozoa due to a disruption of the hypothalamic-pituitary-testicular axis. An example of secondary hypogonadism as a result of anabolic steroid abuse is presented with the case report of a man who committed suicide after a history of aggressive behavior and physical abuse of his wife. The autopsy revealed shrunken testicles, with more than 30% of parenchymatous sclerosis, absent spermatogenesis, and very few Leydig cells detected only by immunohistochemistry. A low-specific immunochemical analysis revealed a very high level of total testosterone (serum 109.8 nmol/L; urine 81.2 nmol/L). A more accurate analysis confirmed an overdose of synthetic anabolic steroids. Doctors in different medical areas should be alerted to chronic abusers of synthetic anabolic steroids among the growing number of recreational athletes.

Central nervous system granulomastous phlebitis with limited extracranial involvement of the heart and lungs: An autopsy case.

Primary angiitis of the central nervous system is a rare condition, usually with an insidious onset. There is a wide variety of histological types (granulomatous, lymphocytic or necrotizing vasculitis) and types of vessel involved (arteries, veins or both). Most cases are idiopathic. We describe a first case of idiopathic granulomatous central nervous system phlebitis with additional limited involvement of the heart and lung, exclusively affecting small and medium sized veins in a 22-year-old woman, presenting as a sub acute headache. The reasons for this peculiar limitation of inflammation to the veins and the involvement of the heart and lungs are unknown.

Cartilage: a new parameter for the determination of the postmortem interval?

The determination of the time of death or the postmortem interval (PMI) is one of the most important and frequently asked questions in forensic medicine. The methods used for PMI determination are based largely on early and late postmortem changes. The determination of the PMI during the late postmortem changes is based primarily on a subjective assessment and is less precise due to the lack of objective methods. Different studies have presented a gradual decrease in chondrocytes' viability but these researches did not answer the question whether we can use the decrease of chondrocytes' viability for an objective PMI determination. The structure and anatomical location of the cartilage together with its mechanical, physical and chemical properties enable chondrocytes to survive for several weeks after the individual's death, and give cartilage the attributes of a compartment. Therefore, cartilage could be a new parameter for PMI determination. This idea had been partially confirmed by a few in vitro studies. The next step in testing this idea should be an extensive in corpore study.

Viability of human articular chondrocytes harvested postmortem: changes with time and temperature of in vitro culture conditions.

Different studies of long-term chondrocytes viability have shown a gradual reduction as a function of time and ambient temperature. The aim of our in vitro study was to establish chondrocyte postmortem viability curves for 4°C, 11°C, 23°C, 35°C during 63 days after the donors' death. Osteochondral cylinders were procured from the knees of 16 male donors (20-47 years), stored in preservation media that was not changed, and analyzed in 3-day intervals using a confocal laser scanning microscope. A significant influence of time on viability was found from Day 9 (p = 0.0029) and onwards (p < 0.0001). The lowest overall chondrocyte viability was at 35°C, followed by 4°C (p < 0.0001). The conditions used in this in vitro analysis suggest that similar viabilities may occur while in situ in the decedent. Further studies of chondrocyte viability from individuals with known postmortem intervals may show premise to help evaluate time since death in the late postmortem interval.

Cryopreservation with glycerol improves the in vitro biomechanical characteristics of human patellar tendon allografts.

PURPOSE: To evaluate the in vitro biomechanical characteristics of patellar tendon ligaments (BTB) when stored as fresh frozen or as glycerol cryopreserved allografts. METHODS: Seventy patellar tendons were harvested from 35 cadaveric human donors and randomly assigned into seven groups. Grafts in group FRESH were mechanically tested within 2 h of harvesting. FROZ-3, FROZ-6, and FROZ-9 were deep-frozen to -80 °C for 3, 6, and 9 months, respectively. Grafts in groups CRYO-3, CRYO-6, and CRYO-9 were initially incubated with 10% glycerol in a phosphate-buffered saline for 1 h and then stored in glycerol solution (10% glycerol in PBS) at -80 °C for 3, 6, and 9 months, respectively. Grafts were mechanically tested with two cycling modes (50-250 °N and 150-500 °N) and then loaded to failure. RESULTS: Cryopreserved grafts demonstrated more consistent results and expressed lower elongation rates after both cycling loading protocols compared to their frozen counterparts at all storage times. During load-to-failure analysis, ultimate stiffness levels were predominantly higher (23.9-61.5%) in cryopreserved grafts compared with frozen grafts, and ultimate stress levels were 26% (13.3-47.7%) higher, regardless of the storage time. Moreover, cryopreserved grafts revealed similar ultimate elongation and uniformly higher ultimate stiffness and ultimate stress levels compared to fresh grafts. CONCLUSION: The results of this in vitro study demonstrated superior mechanical properties of cryopreserved grafts compared to frozen grafts within a preservation period of 9 months. Cryopreservation with glycerol solution might be used to further improve the quality of preserved soft-tissue allografts.

The optimal combination of cartilage source and apparatus for long-term in vitro chondrocyte viability analysis.

Most studies of long-term chondrocytes survival were for tissue banks. They showed a gradual reduction in the viable chondrocytes percentage as a function of time and ambient temperature, but the samples were harvested under optimal conditions. The aim of our study was to determine the most reliable combination of cartilage source and assay for the in vitro postmortem chondrocyte viability analysis in the conditions that imitate a dead body. Osteochondral cylinders were procured from femoral condyles and talar trochleas of three male donors and stored in the cell culture media at 4 ± 2°C and 23 ± 2°C. The samples were analyzed by a cell viability analyzer and a confocal laser scanning microscope (CLSM) initially 24-36 h after death and then in 4-week intervals. The results reconfirmed the significant influence of time (p = 0.0002), but not of the temperature (p = 0.237). The largest reproducibility was presented for the knee joint and the CLSM.

Comparison of articular and auricular cartilage as a cell source for the autologous chondrocyte implantation.

Articular (medial femoral condyle) and auricular cartilage (anithelix) was compared as a cell source for the autologous joint repair. Cells isolated from five human cadaveric donors were cultured parallel in the monolayer cultures and in the 3D alginate hydrogel constructs for 1 week. Cell morphology was controlled by the fluorescent microscopy and gene expressions of type I collagen (COL1), type II collagen (COL2), aggrecan (AGR), versican (VER), and elastin (ELS) were analyzed by the real-time polymerase chain reaction. COL1 and ELS, predominant in the phenotype of auricular biopsy, were statistically lower in the articular biopsies. Even though COL2 and AGR decreased in monolayers of both cell sources, the dedifferentiation process affected auricular cells intensely. Cells embedded in the alginate hydrogel directly after the isolation did not exhibit the dedifferentiated phenotype. Additionally, COL1, COL2, AGR, and VER were comparable between the two sources. ELS however, remained higher in the auricular cells regardless of the culture type. The study indicates that auricular chondrocytes cultured in a 3D environment immediately after the isolation have a neo-cartilage potential for the articular surface reconstruction.