Strength, power and aerobic capacity of transgender athletes: a cross-sectional study.

OBJECTIVE: The primary objective of this cross-sectional study was to compare standard laboratory performance metrics of transgender athletes to cisgender athletes. METHODS: 19 cisgender men (CM) (mean±SD, age: 37±9 years), 12 transgender men (TM) (age: 34±7 years), 23 transgender women (TW) (age: 34±10 years) and 21 cisgender women (CW) (age: 30±9 years) underwent a series of standard laboratory performance tests, including body composition, lung function, cardiopulmonary exercise testing, strength and lower body power. Haemoglobin concentration in capillary blood and testosterone and oestradiol in serum were also measured. RESULTS: In this cohort of athletes, TW had similar testosterone concentration (TW 0.7±0.5 nmol/L, CW 0.9±0.4 nmol/), higher oestrogen (TW 742.4±801.9 pmol/L, CW 336.0±266.3 pmol/L, p=0.045), higher absolute handgrip strength (TW 40.7±6.8 kg, CW 34.2±3.7 kg, p=0.01), lower forced expiratory volume in 1 s:forced vital capacity ratio (TW 0.83±0.07, CW 0.88±0.04, p=0.04), lower relative jump height (TW 0.7±0.2 cm/kg; CW 1.0±0.2 cm/kg, p<0.001) and lower relative V̇O2max (TW 45.1±13.3 mL/kg/min/, CW 54.1±6.0 mL/kg/min, p<0.001) compared with CW athletes. TM had similar testosterone concentration (TM 20.5±5.8 nmol/L, CM 24.8±12.3 nmol/L), lower absolute hand grip strength (TM 38.8±7.5 kg, CM 45.7±6.9 kg, p=0.03) and lower absolute V̇O2max (TM 3635±644 mL/min, CM 4467±641 mL/min p=0.002) than CM. CONCLUSION: While longitudinal transitioning studies of transgender athletes are urgently needed, these results should caution against precautionary bans and sport eligibility exclusions that are not based on sport-specific (or sport-relevant) research.

Engineering of TIMP-3 as a LAP-fusion protein for targeting to sites of inflammation.

Tissue inhibitor of metalloproteinase (TIMP)-3 is a natural inhibitor of a range of enzymes that degrade connective tissue and are involved in the pathogenesis of conditions such as arthritis and cancer. We describe here the engineering of TIMP-3 using a novel drug-delivery system known as the 'LAP technology'. This involves creating therapeutic proteins in fusion with the latency-associated peptide (LAP) from the cytokine TGF-? to generate proteins that are biologically inactive until cleavage of the LAP to release the therapy. LAP-TIMP-3 was successfully expressed in mammalian cells and the presence of the LAP resulted in a 14-fold increase in the quantity of recombinant TIMP-3 produced. LAP-TIMP-3 was latent until release from the LAP by treatment with matrix metalloproteinase when it could inhibit proteases of the adamalysins and adamalysins with thrombospondin motifs families, but not matrix metalloproteinases, indicating that this version of TIMP-3 is a more specific inhibitor than the native protein. There was sufficient protease activity in synovial fluid from human joints with osteoarthritis to release TIMP-3 from the LAP fusion. These results demonstrate the potential for development of TIMP-3 as a novel therapy for conditions where upregulation of catabolic enzymes are part of the pathology.

Suppression of mammalian bone growth by membrane transport inhibitors.

Bone lengthening during skeletal growth is driven primarily by the controlled enlargement of growth plate (GP) chondrocytes. The cellular mechanisms are unclear but membrane transporters are probably involved. We investigated the role of the Na(+)/H(+) antiporter (NHE1) and anion exchanger (AE2) in bone lengthening and GP chondrocyte hypertrophy in Sprague-Dawley 7-day-old rat (P7) bone rudiments using the inhibitors EIPA (5-(N-ethyl-N-isopropyl)amiloride) and DIDS (4,4-diidothiocyano-2,2-stilbenedisulphonate), respectively. We have also determined cell-associated levels of these transporters along the GP using fluorescent immunohistochemistry (FIHC). Culture of bones with EIPA or DIDS inhibited rudiment growth (50% at approx. 250 and 25 µM, respectively). Both decreased the size of the hypertrophic zone (P < 0.05) but had no effect on overall length or cell density of the GP. In situ chondrocyte volume in proliferative and hypertrophic zones was decreased (P < 0.01) with EIPA but not DIDS. FIHC labeling of NHE1 was relatively high and constant along the GP but declined steeply in the late hypertrophic zone. In contrast, AE2 labeling was relatively low in proliferative zone cells but increased (P < 0.05) reaching a maximum in the early hypertrophic zone, before falling rapidly in the late hypertrophic zone suggesting AE2 might regulate the transition phase of chondrocytes between proliferative and hypertrophic zones. The inhibition of bone growth by EIPA may be due to a reduction to chondrocyte volume set-point. However the effect of DIDS was unclear but could result from inhibition of AE2 and blocking of the transition phase. These results demonstrate that NHE1 and AE2 are important regulators of bone growth.

The effects of physiological and injurious hydrostatic pressure on murine ex vivo articular and growth plate cartilage explants: an RNAseq study.

Introduction: Chondrocytes are continuously exposed to loads placed upon them. Physiological loads are pivotal to the maintenance of articular cartilage health, while abnormal loads contribute to pathological joint degradation. Similarly, the growth plate cartilage is subject to various loads during growth and development. Due to the high-water content of cartilage, hydrostatic pressure is considered one of the main biomechanical influencers on chondrocytes and has been shown to play an important role in the mechano-regulation of cartilage. Methods: Herein, we conducted RNAseq analysis of ex vivo hip cap (articular), and metatarsal (growth plate) cartilage cultures subjected to physiological (5 MPa) and injurious (50 MPa) hydrostatic pressure, using the Illumina platform (n = 4 replicates). Results: Several hundreds of genes were shown to be differentially modulated by hydrostatic pressure, with the majority of these changes evidenced in hip cap cartilage cultures (375 significantly upregulated and 322 downregulated in 5 MPa versus control; 1022 upregulated and 724 downregulated in 50 MPa versus control). Conversely, fewer genes were differentially affected by hydrostatic pressure in the metatarsal cultures (5 significantly upregulated and 23 downregulated in 5 MPa versus control; 7 significantly upregulated and 19 downregulated in 50 MPa versus control). Using Gene Ontology annotations for Biological Processes, in the hip cap data we identified a number of pathways that were modulated by both physiological and injurious hydrostatic pressure. Pathways upregulated in response to 50 MPa versus control, included those involved in the generation of precursor metabolites and cellular respiration. Biological processes that were downregulated in this tissue included ossification, connective tissue development, and chondrocyte differentiation. Discussion: Collectively our data highlights the divergent chondrocyte phenotypes in articular and growth plate cartilage. Further, we show that the magnitude of hydrostatic pressure application has distinct effects on gene expression and biological processes in hip cap cartilage explants. Finally, we identified differential expression of a number of genes that have previously been identified as osteoarthritis risk genes, including Ctsk, and Chadl. Together these data may provide potential genetic targets for future investigations in osteoarthritis research and novel therapeutics.

The osmotic sensitivity of rat growth plate chondrocytes in situ; clarifying the mechanisms of hypertrophy.

Bone elongation is predominantly driven by the volume expansion of growth plate chondrocytes. This mechanism was initially believed to be "hypertrophy", describing a proportional increase of cell water and organelles. However, morphometrical analysis subsequently assumed the increase to be "swelling", resulting in a disproportionate increase of cell water (osmotically active fraction). Histological approaches were performed on fixed tissue, and for the "swelling" assumption to be valid, the osmotic sensitivity of living cells before and during volume increase should differ. To test this, analysis of images acquired by 2-photon laser scanning microscopy (2PLSM) were used to determine the osmotic sensitivity, and osmotically active/inactive proportions of in situ chondrocytes from 15 living rat growth plates exposed to varying media osmolarities ( approximately 0-580 mOsm). The dimensions of cell volume swelling in hypotonic media were different to the preferential lengthening seen in vivo, confirming the complexity of directional cell volume increase. Boyle-van't Hoff analysis of cell volume over the range of media osmolarity indicated no significant difference (Student's t-test) in the osmotically inactive fraction, 39.5 +/- 2.9% and 47.0 +/- 4.3% (n = 13) for proliferative and hypertrophic zones, respectively, or the sensitivity of volume to changes in media osmolarity (proliferative 15.5 +/- 0.8 and hypertrophic zone 15.5 +/- 1.2%volume . Osm). The osmotic fractions did not change as chondrocytes progress from proliferative to hypertrophic regions of the growth plate. Our data suggest cell volume increase by hypertrophy may play a greater role in cell enlargement than swelling, and should be re-evaluated as a mechanism responsible for growth plate chondrocyte volume increase and hence bone elongation.

Two-versus one photon excitation laser scanning microscopy: critical importance of excitation wavelength.

It is often anticipated that two-photon excitation (TPE) laser scanning microscopy should improve cell survival and tissue penetration relative to conventional one-photon excitation (OPE) confocal scanning laser microscopy (CLSM). However few studies have directly compared live cell imaging using one- vs two-photon laser scanning microscopy. We have used calcein-loaded in situ chondrocytes within cartilage as a model for quantitatively comparing these techniques. TPE reduced photo-bleaching and improved cell viability compared to OPE. Using improved detection sensitivity coupled with increased tissue penetration of the near infra-red TPE laser, it was possible to capture images deeper within the cartilage. However, the advantages of TPE vs OPE were strongly dependent on excitation wavelength. We conclude that optimising TPE conditions is essential for realizing the full benefits of this approach.

Abnormal human chondrocyte morphology is related to increased levels of cell-associated IL-1ß and disruption to pericellular collagen type VI.

Early osteoarthritis (OA) is poorly understood, but abnormal chondrocyte morphology might be important. We studied IL-1ß and pericellular collagen type VI in morphologically normal and abnormal chondrocytes. In situ chondrocytes within explants from nondegenerate (grade 0/1) areas of human tibial plateaus (n = 21) were fluorescently labeled and visualized [2-photon laser scanning microscopy (2PLSM)]. Normal chondrocytes exhibited a "smooth" membrane surface, whereas abnormal cells were defined as demonstrating ≥1 cytoplasmic process. Abnormal chondrocytes were further classified by number and average length of cytoplasmic processes/cell. IL-1ß or collagen type VI associated with single chondrocytes were visualized by fluorescence immuno-histochemistry and confocal laser scanning microscopy (CLSM). Fluorescence was quantified as the number of positive voxels (i.e., 3D pixels with fluorescence above baseline)/cell. IL-1ß-associated fluorescence increased between normal and all abnormal cells in the superficial (99.7 ± 29.8 [11 (72)] vs. 784 ± 382 [15 (132)]; p = 0.04, positive voxels/cell) and deep zones (66.5 ± 29.4 [9 (64)] vs. 795 ± 224 [9 (56)]; p = 0.006). There was a correlation (r(2) = 0.988) between the number of processes/cell (0-5) and IL-1ß, and an increase particularly with short processes (≤5 µm; p = 0.022). Collagen type VI coverage and thickness decreased (p < 0.001 and p = 0.005, respectively) with development of processes. Abnormal chondrocytes in macroscopically nondegenerate cartilage demonstrated a marked increase in IL-1ß and loss of pericellular type VI collagen, changes that could lead to cartilage degeneration.

A key role for membrane transporter NKCC1 in mediating chondrocyte volume increase in the mammalian growth plate.

The mechanisms that underlie growth plate chondrocyte volume increase and hence bone lengthening are poorly understood. Many cell types activate the Na-K-Cl cotransporter (NKCC) to bring about volume increase. We hypothesised that NKCC may be responsible for the volume expansion of hypertrophic chondrocytes. Metatarsals/metacarpals from 16 rat pups (P(7)) were incubated in the presence/absence of the specific NKCC inhibitor bumetanide and measurement of whole-bone lengths and histologic analysis of the growth plate were done after 24 hours. Fluorescent NKCC immunohistochemistry was visualised using a confocal laser scanning microscopy on seven rat tibial growth plates (P(7)). Microarray analysis was performed on mRNA isolated from proliferative and hypertrophic zone cells of tibial growth plates from five rats of each of three ages (P(49/53/58)). Exposure to bumetanide resulted in approximately 35% reduction (paired Student's t test, p < .05) of bone growth in a dose-dependent manner; histologic analysis showed that a reduction in hypertrophic zone height was responsible. Quantification of fluorescence immunohistochemistry revealed a significant (paired Student's t test, p < .05) change in NKCC from the intracellular space of proliferative cells to the cytosolic membrane of hypertrophic zone cells. Further, microarray analysis illustrated an increase in NKCC1 mRNA between proliferative and hypertrophic cells. The increase in NKCC1 mRNA in hypertrophic zone cells, its cellular localization, and reduced bone growth in the presence of the NKCC inhibitor bumetanide implicate NKCC in growth plate hypertrophic chondrocyte volume increase. Further investigation is warranted to determine the regulatory control of NKCC in the mammalian growth plate and the possible detrimental effect on bone growth with chronic exposure to loop diuretics.

Chondrocyte death in mechanically injured articular cartilage--the influence of extracellular calcium.

Calcium is thought to be an important regulator of chondrocyte death associated with articular cartilage injury. Our objective was to determine the influence of extracellular calcium on chondrocyte death following mechanical injury. Using a surgically relevant model of sharp mechanical injury (with a scalpel) and confocal laser scanning microscopy (CLSM), in situ chondrocyte death was quantified within the full thickness of articular cartilage as a function of medium calcium concentration and time (2.5 h and 7 days). Exposure of articular cartilage to calcium-free media (approximately 0 mM) significantly reduced superficial zone chondrocyte death after mechanical injury compared with exposure to calcium-rich media (2-20 mM, ANOVA at 2.5 h, p = 0.002). In calcium-rich media, although the extent of chondrocyte death increased with increasing medium calcium concentration, cell death remained localized to the superficial zone of articular cartilage over 7 days (ANOVA, p < 0.05). However, in calcium-free media, there was an increase in chondrocyte death within deeper zones of articular cartilage over 7 days. The early (within hours) chondroprotective effect in calcium-free media suggests that the use of joint irrigation solutions without added calcium may decrease chondrocyte death from mechanical injury during articular surgery. The delayed (within days) increase in chondrocyte death in calcium-free media supports the use of calcium supplementation in media used during cartilage culture for tissue engineering or transplantation.

Osmolarity influences chondrocyte death in wounded articular cartilage.

BACKGROUND: Mechanical injury results in chondrocyte death in articular cartilage. The purpose of the present study was to determine whether medium osmolarity affects chondrocyte death in injured articular cartilage. METHODS: Osteochondral explants (n = 48) that had been harvested from the metacarpophalangeal joints of three-year-old cows were exposed to media with varying osmolarity (0 to 480 mOsm) for ninety seconds to allow in situ chondrocytes to respond to the altered osmotic environment. Explants were then wounded with a scalpel through the full thickness of articular cartilage, incubated in the same media for 2.5 hours, and transferred to 340-mOsm Dulbecco's Modified Eagle Medium (control medium) with further incubation for seven days. The spatial distribution of in situ chondrocyte death, percentage cell death, and marginal cell death at the wounded cartilage edge were compared as a function of osmolarity and time (2.5 hours compared with seven days) with use of confocal laser scanning microscopy. RESULTS: In situ chondrocyte death was mainly localized to the superficial tangential zone of injured articular cartilage for the range of medium osmolarities (0 to 480 mOsm) at 2.5 hours and seven days. Therefore, a sample of articular cartilage from the superficial region (which included the scalpel-wounded cartilage edge) was studied with use of confocal laser scanning microscopy to compare the effects of osmolarity on percentage and marginal cell death in the superficial tangential zone. Compared with the control explants exposed to 340-mOsm Dulbecco's Modified Eagle Medium, percentage cell death in the superficial tangential zone was greatest for explants exposed to 0-mOsm (distilled water) and least for explants exposed to 480-mOsm Dulbecco's Modified Eagle Medium at 2.5 hours (13.0% at 340 mOsm [control], 35.5% at 0 mOsm, and 4.3% at 480 mOsm; p

Sodium-dependent recovery of ionised magnesium concentration following magnesium load in rat heart myocytes.

Our objectives were to investigate regulation of intracellular ionised Mg2+ concentration ([fMg2+]i) in cardiac muscle and cardiac Na+/Mg2+ antiport stoichiometry. [fMg2+]i was measured at 37 degrees C in isolated rat ventricular myocytes with mag-fura-2. Superfusion of myocytes with Na+ and Ca2+ free solutions containing 30 mM Mg2+ for 15 min more than doubled [fMg2+]i from its basal level (0.75 mM). Re-addition of Na+ caused [fMg2+]i to fall exponentially with time to basal level, the rate increasing linearly with [Na+]. Log(recovery rate) increased linearly with log([Na+]), the slope of 1.06 (95% confidence limits, 0.94-1.17) suggesting one Na+ ion is exchanged for each Mg2+. [fMg2+]i recovery was complete even if the membrane potential was depolarised to 0 mV or if superfusate [Mg2+] was increased to 3 mM. Recovery was rapid in normal Tyrode (0.3 min(-1)) with a Q10 of 2.2. It was completely inhibited by 200 microM imipramine but was unaffected by 20 microM KB-R7943 or 1 microM SEA0400, suggesting the Na+ /Ca2+ antiporter is not involved. Membrane depolarisation by increasing superfusate [K+] to 70 mM, or voltage clamp to 0 mV, increased recovery rate in Na+ containing solutions more than threefold. We conclude [fMg2+]i recovery is by Mg2+ efflux on a 1 Na+:1 Mg2+ antiport.

Loading rat heart myocytes with Mg2+ using low-[Na+] solutions.

The objective of our study was to investigate how Mg2+ enters mammalian cardiac cells. During this work, we found evidence for a previously undescribed route for Mg2+ entry, and now provide a preliminary account of its properties. Changes in Mg2+ influx into rat ventricular myocytes were deduced from changes in intracellular ionized Mg2+ concentration ([fMg2+]i) measured from the fluorescence of mag-fura-2 loaded into isolated cells. Superfusion of myocytes at 37 degrees C with Ca2+-free solutions with both reduced [Na+] and raised [Mg2+] caused myocytes to load with Mg2+. Uptake was seen with solutions containing 5 mm Mg2+ and 95 mm Na+, and increased linearly with increasing extracellular [Mg2+] or decreasing extracellular [Na+]. It was very sensitive to temperature (Q(10) > 9, 25--37 degrees C), was observed even in myocytes with very low Na+ contents, and stopped abruptly when external [Na+] was returned to normal. Uptake was greatly reduced by imipramine or KB-R7943 if these were added when [fMg2+]i was close to the physiological level, but was unaffected if they were applied when [fMg2+]i was above 2 mm. Uptake was also reduced by depolarizing the membrane potential by increasing extracellular [K+] or voltage clamp to 0 mV. We suggest that initial Mg2+ uptake may involve several transporters, including reversed Na+-Mg2+ antiport and, depending on the exact conditions, reversed Na+-Ca2+ antiport. The ensuing rise of [fMg2+]i, in conjunction with reduced [Na+], may then activate a new Mg2+ transporter that is highly sensitive to temperature, is insensitive to imipramine or KB-R7943, but is inactivated by depolarization.

Passive osmotic properties of in situ human articular chondrocytes within non-degenerate and degenerate cartilage.

Osteoarthritis is characterized by many factors, including proteoglycan loss, decreased collagen stiffness, and increased cartilage hydration. Chondrocyte swelling also occurs, and correlates with the degree of osteoarthritis, however, the cause is unknown but might be related to alterations to their passive osmotic properties. We have used two-photon confocal laser scanning microscopy to measure the passive osmotic characteristics of in situ chondrocytes within relatively non-degenerate and degenerate human tibial plateau cartilage, and in chondrocytes isolated from relatively non-degenerate cartilage. Explants with bone attached were taken from a total of 42 patients undergoing arthroplasty and graded macroscopically and microscopically into two groups, grade 0 + 1 and grade 2 + 3. There was a significant increase in cartilage hydration between these two groups (P < 0.05), however, there was no change when medium osmolarity was varied over approximately 0-480 mOsm. The passive osmotic behavior of in situ chondrocytes (at 4 degrees C) was identical over a range of culture medium osmolarities ( approximately 0-515 mOsm), however, the maximum swelling of cells within degenerate cartilage and isolated chondrocytes was greater compared to those in non-degenerate cartilage. The swelling in the majority of in situ chondrocytes was accounted for by the reduced interstitial osmolarity occurring with cartilage degeneration. There was, however, a small population of in situ chondrocytes whose volume was in excess (>/=2,500 microm(3)) of that predicted from the decreased interstitial osmotic pressure. These results show that for the majority of cells studied, the differences in passive chondrocyte volume between relatively non-degenerate, degenerate, and isolated cells were entirely accounted for by changes to the extracellular osmolarity (180-515 mOsm).

Viability and volume of in situ bovine articular chondrocytes-changes following a single impact and effects of medium osmolarity.

OBJECTIVE: Mechanical stress above the physiological range can profoundly influence articular cartilage causing matrix damage, changes to chondrocyte metabolism and cell injury/death. It has also been implicated as a risk factor in the development of osteoarthritis (OA). The mechanism of cell damage is not understood, but chondrocyte volume could be a determinant of the sensitivity and subsequent response to load. For example, in OA, it is possible that the chondrocyte swelling that occurs renders the cells more sensitive to the damaging effects of mechanical stress. This study had two aims: (1) to investigate the changes to the volume and viability of in situ chondrocytes near an injury to cartilage resulting from a single blunt impact, and (2) to determine if alterations to chondrocyte volume at the time of impact influenced cell viability. METHODS: Explants of bovine articular cartilage were incubated with the fluorescent indicators calcein-AM and propidium iodide permitting the measurement of cell volume and viability, respectively, using confocal laser scanning microscopy (CLSM). Cartilage was then subjected to a single impact (optimally 100g from 10 cm) delivered from a drop tower which caused areas of chondrocyte injury/death within the superficial zone (SZ). The presence of lactate dehydrogenase (LDH; an enzyme released following cell injury) was used to determine the effects of medium osmolarity on the response of chondrocytes to a single impact. RESULTS: A single impact caused discrete areas of chondrocyte injury/death which were almost exclusively within the SZ of cartilage. There appeared to be two phases of cell death, a rapid phase lasting approximately 3 min, followed by a slower progressive 'wave of cell death' away from the initial area lasting for approximately 20 min. The volume of the majority (88.1+/-5.99% (n=7) of the viable chondrocytes in this region decreased significantly (P<0.006). By monitoring LDH release, a single impact 5 min after changing the culture medium to hyper-, or hypo-osmolarity, reduced or stimulated chondrocyte injury, respectively. CONCLUSIONS: A single impact caused temporal and spatial changes to in situ chondrocyte viability with cell shrinkage occurring in the majority of cells. However, chondrocyte shrinkage by raising medium osmolarity at the time of impact protected the cells from injury, whereas swollen chondrocytes were markedly more sensitive. These data showed that chondrocyte volume could be an important determinant of the sensitivity and response of in situ chondrocytes to mechanical stress.

Functional analysis of mouse hepatocytes differing in DNA content: volume, receptor expression, and effect of IFNgamma.

Polyploidy and binuclearity are characteristics of the mammalian liver. Increasing polyploidisation occurs with age and after administration of various drugs and chemicals. This study was designed to examine the function of ploidy by addressing several questions: (1) Does the increase in size of polyploid hepatocytes have any physiological function by altering surface receptor expression such as intercellular adhesion molecule-1 (ICAM-1, CD54) or IFNgammaR? and (2) Do polyploid cells respond differently to inflammatory cytokines such as interferon gamma (IFNgamma)? We have developed a method to accurately measure the volume of live isolated hepatocytes using confocal microscopy and image analysis. Using flow cytometry, we have shown that the expression of ICAM-1 increases with increasing DNA content and IFNgammaR is not detectable on isolated mouse hepatocytes. Diploid (2n), tetraploid (4n) and octoploid (8n) hepatocytes were found to be equally susceptible to IFNgamma-induced apoptosis in vitro. Although the function of polyploidy remains unanswered, we have described some of the characteristics of polyploidy in isolated hepatocytes and in vitro.