An intersegmental reflex between the copulatory swimmerets of the male squat lobster,Galathea strigosa

The structure of the sexually modified 1st 2 pairs of swimmerets of the male squat lobster Galathea strigosa are described, and a reflex involving these swimmerets is characterised. The endopodite of the 1st swimmeret forms a partially rolled lamina which hooks around the “bottle brush” end of the endopodite of the 2nd swimmeret. Mechanical stimulation of a sensitive region on the endopodite of each 1st swimmeret causes retraction of both 2nd swimmerets, inducing the “bottle brush” to push up within the endopodite of the 1st swimmeret. The reflex is thought to be involved in sperm transfer during copulation. Neural information travels between the 1st and 2nd swimmerets in the interganglionic connective ipsilateral to the stimulus.     

Effects of different concentrations of serum on cartilage growth in an organ culture system

Summary The purpose of the present study was to examine the effects of various concentrations of serum on the behavior of neonatal condylar cartilage when cultured in an organ culture system. Mandibular condylar cartilages were obtained from newborn ICR mice, of which the zone of undifferentiated chondroprogenitor cells along with a few layers of young cartilage cells were cultivated at the medium-air interface. The incubation medium included fetal bovine serum at concentrations ranging from 0 to 10%, and the explants were kept in vitro up to 10 d. The serum-free medium maintained the chondrogenic expression, and the overall size of the cartilagenous protion of the explants increased with the decrease of the concentrations of serum in the medium. When explants were labeled with [³H]thymidine and were then processed for autoradiography, the peak of labeling was noticed at 48 h, a feature that recapitulated itself in all cultures (73, 140, 175, 201, and 129 labeled cells per chondroprogenitor zone in explants grown in 0, 1, 2.5, 5, and 10%, respectively). It can be concluded that serum-free medium maintains the chondrogenic phenotype of condylar cartilage in vitro. This study was supported in part by a research grant from the Gesellschaft fur Biotechnologische Forschung mbH, Braunschweig-Stockheim, Federal Republic of Germany.     

Separation and characterization of chondroitin 6-sulfotransferase and chondroitin 4-sulfotransferase from chick embryo cartilage

Two distinct sulfotransferases (chondroitin 6-sulfotransferase and chondroitin 4-sulfotransferase), which catalyzed transfer of sulfate to position 6 and position 4 of acetylgalactosamine residues of chondroitin, were extracted from epiphyseal cartilage of 14-day-old chick embryos and separated by gel chromatography on Sephacryl S-200 in the presence of 3 M guanidine-HCl. When the enzyme solutions containing 3 M guanidine-HCl were dialyzed against 0.02 M Tris-HCl, pH 7.2, containing 10% glycerol, chondroitin 4-sulfotransferase became almost insoluble, whereas chondroitin 6-sulfotransferase remained soluble. Endogenous acceptors for sulfate transfer were completely removed from both enzyme preparations. Addition of basic proteins and polyamines as well as Mn²⁺ to the incubation medium caused a stimulation of both sulfotransferases; the stimulation of chondroitin 6-sulfotransferase with these cations was higher than that of chondroitin 4-sulfotransferase. The K_m values for 3′-phosphoadenylyl sulfate of both enzymes were much smaller in the presence of protamine or spermine than in the presence of Mn²⁺. The two sulfotransferases differed in the requirement for sulfhydryl compounds; in the absence of sulfhydryl compounds, the activity of chondroitin 4-sulfotransferase was very low, whereas the activity of chondroitin 6-sulfotransferase was essentially unaffected. These observations indicate that at least two sulfotransferases are involved in the biosynthesis of chondroitin sulfate, and suggest that the production of the isomers of chondroitin sulfate in chondrocytes is affected by various factors such as the intracellular concentration of sulfhydryl compounds and basic substances.     

Comparison of three multi-cryoprotectant loading protocols for vitrification of porcine articular cartilage

Vitrification is a cryopreservation technique for the long-term storage of viable tissue, but the success of this technique relies on multiple factors. In 2012, our group published a working vitrification protocol for intact human articular cartilage and reported promising chondrocyte recovery after using a four-step multi-cryoprotectant (CPA) loading method that required 570 min. However, this protocol requires further optimization for clinical practice. Herein, we compared three multi-step CPA loading protocols to investigate their impact on chondrocyte recovery after vitrification of porcine articular cartilage on a bone base, including our previous four-step protocol (original: 570 min), and two shorter three-step protocols (optimized: 420 min, and minimally vitrifiable: 310 min). Four different CPAs were used including glycerol, dimethyl sulfoxide, ethylene glycol and propylene glycol. As vitrification containers, two conical tubes (50 ml and 15 ml) were evaluated for their heat transfer impact on chondrocyte recovery after vitrification. Osteochondral dowels were cored into two diameters of 10.0 mm and 6.9 mm with an approximately 10-mm thick bone base, and then allocated into the twelve experimental groups based on CPA loading protocol, osteochondral dowel size, and vitrification container size. After vitrification at −196 °C and tissue warming and CPA removal, samples in all groups were assessed for both chondrocyte viability and metabolic activity. The optimized protocol proposed based on mathematical modelling resulted in similar chondrocyte recovery to our original protocol and it was 150 min shorter. Furthermore, this study illustrated the role of CPA permeation (dowel size) and heat transfer (container size) on vitrification protocol outcome.     

Grading cartilage damage with diffuse reflectance spectroscopy: Optical markers and mechanical properties

Osteoarthritis (OA) is one of the most common joint diseases worldwide. Unfortunately, clinical methods lack the ability to detect OA in the early stages. Timely detection of the knee joint degradation at the level of tissue changes can prevent its progressive damage. Here, diffuse reflectance spectroscopy (DRS) in the NIR range was used to obtain optical markers of the cartilage damage grades and to assess its mechanical properties. It was observed that the water content obtained by DRS strongly correlates with the cartilage thickness (R = .82) and viscoelastic relaxation time (R = .7). Moreover, the spectral parameters, including water content (OH-band), protein content (CH-band), and scattering parameters allowed for discrimination between the cartilage damage grades (10⁻⁴ < P ≤ 10⁻³). The developed approach may become a valuable addition to arthroscopy, helping to identify lesions at the microscopic level in the early stages of OA and complement the surgical analysis.     

Development of chondrocyte-laden alginate hydrogels with modulated microstructure and properties for cartilage regeneration

Alginate hydrogel is an attractive biomaterial for cell microencapsulation. The microarchitecture of hydrogels can regulate cellular functions. This study aims to investigate the applicability of sodium citrate buffer (SCB) as a culture medium supplement for modulating the microstructure of alginate microbeads to provide a favorable microenvironment for chondrogenic induction. The chondrocyte-laden microbeads, with and without TGF-β3 incorporation, were produced through an encapsulator. The obtained small-sized microbeads (~300 μm) were exposed to a treatment medium containing SCB, composed of varied concentrations of sodium citrate (1.10–1.57 mM), sodium chloride (3.00–4.29 mM), and ethylenediaminetetraacetic acid (0.60–0.86 mM) to partially degrade their crosslinked structure for 3 days, followed by culture in a normal medium until day 21. Scanning electron microscope micrographs demonstrated a loose hydrogel network with an enhanced pore size in the SCB-treated microbeads. Increasing the concentration of SCB in the treatment medium reduced the calcium content of the microbeads via a Na⁺/Ca²⁺ exchange process and improved the water absorption of the microbeads, resulting in a higher swelling ratio. All the tested SCB concentrations were non-cytotoxic. Increases in aggrecan and type II collagen gene expression and their corresponding extracellular matrix accumulation, glycosaminoglycans, and type II collagen were vividly detected in the TGF-β3-containing microbeads with increasing SCB concentrations in the treatment medium. Our findings highlighted that the combination of SCB treatment and TGF-β3 incorporation in the chondrocyte-laden microbeads is a promising strategy for enhancing cartilage regeneration, which may contribute to a versatile application in cell delivery and tissue engineering.     

Fibroblast dedifferentiation as a determinant of successful regeneration

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Development of a monoclonal anti-ADAMTS-5 antibody that specifically blocks the interaction with LRP1

The potent aggrecanase ADAMTS-5 is constitutively secreted by chondrocytes, but it is rapidly endocytosed in normal cartilage via the cell surface endocytic receptor LRP1. Therefore it is difficult to detect the total ADAMTS-5 activity produced. In this study, we isolated a monoclonal anti-ADAMTS-5 antibody 1B7 that blocks LRP1-mediated internalization without affecting the aggrecanolytic activity. Addition of 1B7 to cultured human chondrocytes revealed the full aggrecanolytic activity of ADAMTS-5 generated by the cells. 1B7 is a useful tool to estimate the ADAMTS-5 activity and to identify its potential roles in the tissues.