Biochemical parameters of the metabolism of the intercellular substance of connective tissue, correlating with morphometric signs of long bone lesions in an open aseptic fracture]

The aim of the research was experimental study of dynamics and correlative dependences between biochemical connective tissue matrix metabolism indices (blood serum collagenase, cathepsin B, elastase, antielastase activity, hydroxyproline fractions and glycosaminoglycans concentration) and tissue damage morphometric indices after long bone aseptic osteotomy of rat in terms 3 h-60 days. The most strong and significant correlation was found between cathepsin B, elastase activity indices and dimensions of bone marrow ischemic damage focuses and summarised periosteal regenerates volume in bone fragments.     

Subcutaneous tissue fibroblast cytoskeletal remodeling induced by acupuncture: evidence for a mechanotransduction-based mechanism

Acupuncture needle rotation has been previously shown to cause specific mechanical stimulation of subcutaneous connective tissue. This study uses acupuncture to investigate the role of mechanotransduction-based mechanisms in mechanically-induced cytoskeletal remodeling. The effect of acupuncture needle rotation was quantified by morphometric analysis of mouse tissue explants imaged with confocal microscopy. Needle rotation induced extensive fibroblast spreading and lamellipodia formation within 30 min, measurable as an increased in cell body cross sectional area. The effect of rotation peaked with two needle revolutions and decreased with further increases in rotation. Significant effects of rotation were present throughout the tissue, indicating the presence of a response extending laterally over several centimeters. The effect of rotation with two needle revolutions was prevented by pharmacological inhibitors of actomyosin contractility (blebbistatin), Rho kinase (Y-27632 and H-1152), and Rac signaling. The active cytoskeletal response of fibroblasts demonstrated in this study constitutes an important step in understanding cellular mechanotransduction responses to externally applied mechanical stimuli in whole tissue, and supports a previously proposed model for the mechanism of acupuncture involving connective tissue mechanotransduction.     

Mechanical loading history and skeletal biology

A comprehensive theory which relates tissue mechanical stresses to many features of skeletal morphogenesis, growth, regeneration, maintenance and degeneration is reviewed. The theory considers the repeated or intermittent mechanical forces which constitute the loading history on the chondro-osseous skeleton. The results of numerous mechanical stress analyses indicate that the local tissue stress history plays a major role in controlling connective tissue biology. The strong influence of mechanical energy in ontogenesis implies a comparably strong influence in phylogenesis. The fact that the mechanical stress histories in skeletal tissues are directly related to the force of gravity suggests that the life forms that have evolved on Earth are closely tied to our gravitational field.     

Dynamic fibroblast cytoskeletal response to subcutaneous tissue stretch ex vivo and in vivo

Cytoskeleton-dependent changes in cell shape are well-established factors regulating a wide range of cellular functions including signal transduction, gene expression, and matrix adhesion. Although the importance of mechanical forces on cell shape and function is well established in cultured cells, very little is known about these effects in whole tissues or in vivo. In this study we used ex vivo and in vivo models to investigate the effect of tissue stretch on mouse subcutaneous tissue fibroblast morphology. Tissue stretch ex vivo (average 25% tissue elongation from 10 min to 2 h) caused a significant time-dependent increase in fibroblast cell body perimeter and cross-sectional area (ANOVA, P < 0.01). At 2 h, mean fibroblast cell body cross-sectional area was 201% greater in stretched than in unstretched tissue. Fibroblasts in stretched tissue had larger, "sheetlike" cell bodies with shorter processes. In contrast, fibroblasts in unstretched tissue had a "dendritic" morphology with smaller, more globular cell bodies and longer processes. Tissue stretch in vivo for 30 min had effects that paralleled those ex vivo. Stretch-induced cell body expansion ex vivo was inhibited by colchicine and cytochalasin D. The dynamic, cytoskeleton-dependent responses of fibroblasts to changes in tissue length demonstrated in this study have important implications for our understanding of normal movement and posture, as well as therapies using mechanical stimulation of connective tissue including physical therapy, massage, and acupuncture. mechanotransduction; connective tissue; tensegrity; musculoskeletal manipulations; acupuncture     

Electrical Impedance of Acupuncture Meridians: The Relevance of Subcutaneous Collagenous Bands

Background

The scientific basis for acupuncture meridians is unknown. Past studies have suggested that acupuncture meridians are physiologically characterized by low electrical impedance and anatomically associated with connective tissue planes. We are interested in seeing whether acupuncture meridians are associated with lower electrical impedance and whether ultrasound-derived measures – specifically echogenic collagenous bands - can account for these impedance differences.

Methods/Results

In 28 healthy subjects, we assessed electrical impedance of skin and underlying subcutaneous connective tissue using a four needle-electrode approach. The impedances were obtained at 10 kHz and 100 kHz frequencies and at three body sites - upper arm (Large Intestine meridian), thigh (Liver), and lower leg (Bladder). Meridian locations were determined by acupuncturists. Ultrasound images were obtained to characterize the anatomical features at each measured site. We found significantly reduced electrical impedance at the Large Intestine meridian compared to adjacent control for both frequencies. No significant decrease in impedance was found at the Liver or Bladder meridian. Greater subcutaneous echogenic densities were significantly associated with reduced impedances in both within-site (meridian vs. adjacent control) and between-site (arm vs. thigh vs. lower leg) analyses. This relationship remained significant in multivariable analyses which also accounted for gender, needle penetration depth, subcutaneous layer thickness, and other ultrasound-derived measures.

Conclusion/Significance

Collagenous bands, represented by increased ultrasound echogenicity, are significantly associated with lower electrical impedance and may account for reduced impedances previously reported at acupuncture meridians. This finding may provide important insights into the nature of acupuncture meridians and the relevance of collagen in bioelectrical measurements.     

The unfolded protein response and its relevance to connective tissue diseases

The unfolded protein response (UPR) has evolved to counter the stresses that occur in the endoplasmic reticulum (ER) as a result of misfolded proteins. This sophisticated quality control system attempts to restore homeostasis through the action of a number of different pathways that are coordinated in the first instance by the ER stress-senor proteins IRE1, ATF6 and PERK. However, prolonged ER-stress-related UPR can have detrimental effects on cell function and, in the longer term, may induce apoptosis. Connective tissue cells such as fibroblasts, osteoblasts and chondrocytes synthesise and secrete large quantities of proteins and mutations in many of these gene products give rise to heritable disorders of connective tissues. Until recently, these mutant gene products were thought to exert their effect through the assembly of a defective extracellular matrix that ultimately disrupted tissue structure and function. However, it is now becoming clear that ER stress and UPR, because of the expression of a mutant gene product, is not only a feature of, but may be a key mediator in the initiation and progression of a whole range of different connective tissue diseases. This review focuses on ER stress and the UPR that characterises an increasing number of connective tissue diseases and highlights novel therapeutic opportunities that may arise.     

Dynamic morphometric characterization of local connective tissue network structure in humans using ultrasound

Background  

In humans, connective tissue forms a complex, interconnected network throughout the body that may have mechanosensory, regulatory and signaling functions. Understanding these potentially important phenomena requires non-invasive measurements of collagen network structure that can be performed in live animals or humans. The goal of this study was to show that ultrasound can be used to quantify dynamic changes in local connective tissue structure in vivo. We first performed combined ultrasound and histology examinations of the same tissue in two subjects undergoing surgery: in one subject, we examined the relationship of ultrasound to histological images in three dimensions; in the other, we examined the effect of a localized tissue perturbation using a previously developed robotic acupuncture needling technique. In ten additional non-surgical subjects, we quantified changes in tissue spatial organization over time during needle rotation vs. no rotation using ultrasound and semi-variogram analyses.     

Land crabs with smooth lungs: Grapsidae,Gecarcinidae, and Sundathelphusidae ultrastructure and vasculature

The highly terrestrial grapsids and gecarcinids and the amphibious sundathelphusids all have large, expanded branchial chambers. The lining of the branchial chambers is smooth and well vascularized, and it functions as a lung. The respiratory membrane and the cuticle lining the lung are extremely thin (200–350 nm). The blood vessels within the lung are formed from connective tissue cells supported by collagen fibres and lined by a basal lamina. The major vessels in the lung are embedded deep in the branchiostegite and lie just beneath the thick outer carapace. These vessels branch towards the respiratory membrane, where they eventually lose their connective tissue coverings to form thin, flattened lacunae directly below the respiratory epithelium. The lacunae (exchange sites) are bordered by specialized connective tissue cells, which either bear microvilli on their apical surface (fimbriated cells) or are very smooth. The respiratory circulation in the lung is very complex, with two portal systems present between the afferent and efferent systems, producing a total of three lacunal exchange beds. Portal systems increase the surface area available for gas exchange. The major distributing vessel in the lung is the branchiostegal vein, which runs along the inner margin of the branchiostegite. The main venous supplies come anteriorly from the infraorbital and ventral sinuses and posteriorly from the procardial sinus. The main collecting vessel is the pulmonary vein, which arises anteriorly and which runs around the ventral perimeter of the branchiostegite before emptying into the pericardial sinus. © 1993 Wiley-Liss, Inc.     

Optimized extraction of glycosaminoglycans from normal and osteoarthritic cartilage for glycomics profiling

Articular cartilage is a highly specialized smooth connective tissue whose proper functioning depends on the maintenance of an extracellular matrix consisting of an integrated assembly of collagens, glycoproteins, proteoglycans (PG), and glycosaminoglycans. Isomeric chondroitin sulfate glycoforms differing in position and degree of sulfation and uronic acid epimerization play specific and distinct functional roles during development and disease onset. This work introduces a novel glycosaminoglycan extraction method for the quantification of mixtures of chondroitin sulfate oligosaccharides from intact cartilage tissue for mass spectral analysis. Glycosaminoglycans were extracted from intact cartilage samples using a combination of ethanol precipitation and enzymatic release followed by reversed-phase and strong anion exchange solid-phase extraction steps. Extracted chondroitin sulfate glycosaminoglycans were partially depolymerized using chondroitinases, labeled with 2-anthranilic acid-d(4) (2-AA) and subjected to size exclusion chromatography with online electrospray ionization mass spectrometric detection in the negative ion mode. The method presented herein enabled simultaneous determination of sulfate position and uronic acid epimerization in juvenile bovine and adult human cartilage samples. The method was applied to a series of 13 adult human cartilage explants. Standard deviation of the mean for the measurements was 1.6 on average. Coefficients of variation were approximately 4% for all compositions of 40% or greater. These results show that the new method has sufficient accuracy to allow determination of topographical distribution of glycoforms in connective tissue.     

Fibroblast spreading induced by connective tissue stretch involves intracellular redistribution of α- and β-actin

Mechanical stretching of connective tissue occurs with normal movement and postural changes, as well as treatments including physical therapy, massage and acupuncture. Connective tissue fibroblasts were recently shown to respond actively to short-term mechanical stretch (minutes to hours) with reversible cytoskeletal remodeling, characterized by extensive cell spreading and lamellipodia formation. In this study, we have examined the effect of tissue stretch on the distribution of α- and β-actin in subcutaneous tissue fibroblasts ex vivo. Normal fibroblasts uniformly exhibited α-smooth muscle actin (α-SMA) immunoreactivity. Unlike cultured fibroblasts and smooth muscle cells, α-SMA in these fibroblasts was not in F-actin form (indicated by lack of phalloidin co-localization) nor was it organized into distinct stress fibers. The lack of stress fibers and fibronexus was confirmed by electron microscopy, indicating that these cells were not myofibroblasts. In unstretched tissue, the pattern of α-actin was diffuse and granular. With tissue stretch (30 min), α-actin formed a star-shaped pattern centered on the nucleus, while β-actin extended throughout the cytoplasm including lamellipodia and cell cortex. This dual response pattern of α- and β-actin may be an important component of cellular mechanotransduction mechanisms relevant to physiologic and therapeutic mechanical forces applied to connective tissue.     

Structural organization of the orbicular muscle of the mouth in children with a congenital cleft of the upper lip undergoing electrostimulation

At light optic and electron microscopical levels with application of morphometric analysis the mouth orbicular muscle has been studied in 6-8-month-old children with a complete unilateral cleft lip. The muscle is characterized by distinctly manifested signs of hypertrophy: high contents of the connective tissue, poor capillarization, presence of focal destructive-degenerative changes in the muscle fibers; they result from decreased function of the muscle activity. Preoperative physiotherapeutic treatment with pulsed low-frequency electrical current stimulates development of the muscle tissue. In the muscle specific share of muscle fibers increases, and contents of the connective tissue decreases, respectively, indices of capillarization improve, mitochondrial apparatus of the muscle fibers becomes more powerful.     

X-irradiation reduces lesion scarring at the contusion site of adult rat spinal cord

Spinal cord injury (SCI) results in cell death and tissue destruction, and ultimately cavitation followed by the formation of lesion scars at the injury site. The lesion scars include an astrocytic component (glial scar) and a fibroblastic component (connective tissue scar). The purpose of the present study is to determine if X-irradiation could minimize the formation of lesion scars and reduce the levels of chondroitin sulfate proteoglycans (CSPGs) in the contusion SCI model of the adult rat. Two weeks after SCI, a connective tissue scar formed at the injury site consisting primarily of fibroblasts and exhibits strong CSPG immunoreactivity. The fibroblasts might originate from the connective tissue of pia mater or arachnoid mater. At the same time, reactive astrocytes in the spared tissue accumulate surrounding the lesion cavity to form a thick glial scar with significant enhancement of glial fibrillary acidic protein (GFAP) and CSPG immunoreactivity. After X-irradiation (40 Gy) of the injury site 2 days post-injury, that results in an attenuated dose to the lesion, the connective tissue scar was not observed, and accordingly, almost no CSPG immunoreactivity was detected at this area. Meanwhile, the glial scar and its CSPG immunoreactivity were prominently reduced. X-irradiation did not show significant improvement in locomotor recovery, but resulted in a slight delay of body weight recovery following injury. This preparative treatment could be used to reduce secondary scarring in the lesion resulting in an enriched site for further treatment such as growth related transplantation.     

Basement membranes as the regulatory system between epithelial cell connections and connective tissue

Basement membranes develop by an interaction between epithelium and underlying mesenchymal. Basement membranes are to an great extend involved in the metabolic induced informational exchange between these 2 compartments. Informational selectivity is not only given with the construction of basement membranes. By redox systems like ascorbic acid/dehydroascorbic acid, preferentially located in basement membranes, the capability exists of scavenging free oxygen radicals on the border line between epithelia and connective tissue. This seems to be a very important factor in coupling the biorhythms between epithelia and connective tissue.     

Regional differences in renewal rates of fibroblasts in young adult female mice

Summary Young adult female mice were given a total of 90 intraperitoneal injections each of (methyl-³H)thymidine (³HTdR) at intervals of 8 h over 30 days to establish renewal rates of fibroblasts in various locations. Radioautographs prepared from punch biopsy material of the ears, taken repeatedly during the labeling procedure, revealed an approximately linear increase of labeling indices of dermal fibroblasts with time. Labeling indices of fibroblasts at the end of repetitive injections of ³HTdR differed depending on the site and/or type of connective tissues examined. Low values were obtained for fibroblasts in the leptomeninx (3.9%), the tracheal wall (4.4%), the achilles tendon (5.8%) and the dermis of the ear (6.5%), while higher values were registered for fibroblasts located in the lower half of the abdominal dermis (12.3%), peritendinous sheaths (12.6%), the interstitial connective tissue of the thigh muscles (12.9%), the submucosa of the colon (23%), the fibrous capsule of the adrenals (25.7%), and the upper half of the abdominal dermis (26%). These regional differences, with the exception of the skin and possibly of the tracheal wall, did not correlate with local temperature. Possible additional factors influencing fibroblast renewal rates may include the type of connective tissue, the degree of vascularization, mechanical stress and hormonal action. Estimated turnover times, based on the assumptions of a DNA synthesis time of 6 h or more and a linear increase of labeling indices as a function of time of repetitive labeling, range from about 90 to more than 700 days. The higher values approximate the median life span of the mouse strain used.This work was supported by the Swiss National Science Foundation     

The efficiency of in vitro isolation and myogenic differentiation of MSCs derived from adipose connective tissue,bone marrow,and skeletal muscle tissue

The objective of the study is to evaluate efficiency of in vitro isolation and myogenic differentiation of mesenchymal stem cells (MSCs) derived from adipose connective tissue (AD-MSCs), bone marrow (BM-MSCs), and skeletal muscle tissue (MC-MSCs). MSCs were isolated from adipose connective tissue, bone marrow, and skeletal muscle tissue of two adult 6-wk-old rats. Cultured MSCs were treated with 5-azacytidine (AZA) to induce myogenic differentiation. Isolated MSCs and differentiated cells were evaluated by immunocytochemistry (ICC), fluorescence-activated cell sorting (FACS), PCR, and RT-PCR. AD-MSCs showed the highest proliferation rate while BM-MSCs had the lowest one. In ICC, isolated MSCs had strong CD90- and CD44-positive expression and negative expression of CD45, CD31, and CD34, while AZA-treated MSCs had strong positive desmin expression. In FACS analysis, AD-MSCs had the highest percentage of CD90- and CD44-positive-expressing cells (99% and 96%) followed by BM-MSCs (97% and 94%) and MC-MSCs (92% and 91%).At 1 wk after incubation with AZA treatment, the peak of myogenin expression reached 93% in differentiated MC-MSCs, 83.3% in BM-MSCs, and 77% in AD-MSCs. MSCs isolated from adipose connective tissue, bone marrow, and skeletal muscle tissue have the same morphology and phenotype, but AD-MSCs were the most easily accessible and had the highest rate of growth on cultivation and the highest percentage of stem cell marker expression. Moreover, although MC-MSCs showed the highest rate of myogenic differentiation potential and expression of myoblast markers, AD-MSCs and BM-MSCs still can be valuable alternatives. The differentiated myoblastic cells could be an available new choice for myoblastic auto-transplantation in regeneration medicine.     

An immunological and morphological study of focal staphylococcal infection against a background of long-term exposure to the herbicide linuron

Small doses of long-term injection of herbicide linuron causes the appearance of secondary immunodeficiency. It is manifested in histological and ultrastructural determination of thymus, severe suppression of immunological indices. Epithelial nurse-cells damage takes place in thymus, the contacts between epithelial cells are destroyed, the reforming of microvessels (their endothelium) occurs which causes the deterioration of vessels penetration and the growth of connective tissue. Against this background a more rapid development of experimental staphylococcus abscesses is observed in animals, the reparation phase being absent.     

A connective tissue tumor in the mussel Mytilus trossulus from a polluted region of Nakhodka Bay, the Sea of Japan

A tumor was found for the first time in a mussel Mytilus trossulus from a heavily polluted area of Nakhodka Bay, Sea of Japan. Tumor cells were found in the connective tissue of different organs and also in gill vessels and hemal sinuses of the visceral mass. They were both attached and diffuse. The tumor was at an advanced stage, replacing the normal connective tissue cells, and formed nodes. The tumor cells were polymorphic, with a high nucleocytoplasmic ratio, and had a prominent nucleolus. The size of their nuclei was three to five times that of the nuclei of agranular hemocytes. The mitotic activity of the tumor cells was more than an order of magnitude higher than in the normal cells: the mean mitotic index was 1.4 +/- 0.5%, ranging from 0.97 to 2.3% in different organs. The mitotic indices in the connective tissue cells of three normal mussels were 0, 0, and 0.12%. A significant proportion (up to 78%) of the mitotic cells were at metaphase. The frequency of abnormal mitoses was 17%. Metaphases with displaced (often multiple) chromosomes constituted 71% of abnormal mitoses; anaphases, 8%; and tri- and tetrapolar mitoses, 11%. The tumor described is similar to diffuse sarcomatoid diseases of mussels from other geographical regions.     

Neoplasia in the connective tissue of the musselMytilus trossulus from polluted areas of Nakhodka Bay, Sea of Japan

A tumor was found for the first time in a musselMytilus trossulus from aheavily polluted area of Nakhodka Bay, Sea of Japan. Tumor cells were found in the connective tissue of different organs and also in gill vessels and hemal sinuses of the visceral mass. They were both attached and diffuse. The tumor was at an advanced stage, replacing the normal connective tissue cells, and formed nodes. The tumor cells were polymorphic, with a high nucleocytoplasmic ratio, and had a prominent nucleolus. The size of their nuclei was three to five times that of the nuclei of agranular hemocytes. The mitotic activity of the tumor cells was more than an order of magnitude higher than in the normal cells: The mean mitotic index was 1.4±0.5%, ranging from 0.97 to 2.3% in different organs. The mitotic indices in the connective tissue cells of three normal mussels were 0, 0, and 0.12%. A significant proportion (up to 78%) of the mitotic cells were at metaphase. The frequency of abnormal mitoses was 17%. Metaphases with displaced (often multiple) chromosomes constituted 71% of abnormal mitoses; anaphases, 8%; and tri- and tetrapolar mitoses, 11%. The tumor described is similar to diffuse sarcomatoid diseases of mussels from other geographical regions.     

DNA synthesis in rat heart cells after injury and the regeneration of myocardia

The regenerative responses of the myocardia of post-natal rats of different age groups (1, 2, 3 and 4 weeks old) to an injury made by a clinical electricator were studied. DNA synthesis and the ultrastructural organization of the cardiac myocytes of the injured myocardia were examined for an evaluation of the potential for regeneration of the developing myocardia. The maximum labeling index of cardiac myocytes was observed in 1-week-old rats showing 8% labeled myocytes 3 days after injury as opposed to 3.2, 2.2 and 0.2% indices in 2-, 3- and 4-week-old rats respectively, 3 days after injury. In subsequent days after injury the labeling indices declined considerably in all age group hearts, and attained values less than 1% labeled myocytes 30 days after injury with the lowest labeling index in the oldest age group heart. When DNA synthesis in uninjured myocardial tissue adjacent to the injured tissue was examined, it was found to be significantly lower than it was in the injured tissue. However, both injured and adjacent uninjured tissue attained a peak in the labeling indices 3 days after injury, with the exception of 3- and 4-week-old uninjured tissue. The overall incorporation of ³H-thymidine into the DNA of heart cells as revealed by scintillation counts showed that the rate of incorporation of the isotope in younger hearts was significantly higher than in the older hearts. Non-muscle cells contributed significantly to the rise of scintillation counts in hearts of all age groups.Ultrastructural analyses of 1- to 4-week-old hearts showed that 24 hr after injury, injured areas of myocardia were heavily crowded with macrophages that surrounded damaged myocytes. Later on, fibroblasts and other non-muscle cells predominated the injury sites along with fibrous connective tissue. Scattered regenerating cardiac myocytes were frequently observed in the injury sites of 1- and 2-week-old hearts 3 days after injury. Myocytes were rare in the corresponding regions of 3- and 4-week-old hearts. Instead abundant non-muscle cells and fibrous connective tissue were predominant. In the fourth and final week of this study, the repaired areas of myocardia in 1- and 2-week-old rats contained more myocytes than those of the 3- and 4-week-old rats, and the repaired zone of the 1-week-old heart contained more myocytes than the repaired areas of the other age groups. These findings suggest that the mammalian myocardia possess an age-dependent potential for regeneration that involves the healing of injury sites with contractile and connective tissues.     

Periodic-Acid-Foot Stain for Connective Tissue

A marked increase in reticular argyrophilia may be obtained in the Foot ammoniated silver carbonate technic by interposing a strong periodic acid oxidation, 4% aqueous for 2 hours at 25-27°C., prior to silvering. Sections so oxidized before the silver bath show a histological picture of connective tissue that is stronger than that given by the original technic. Stroma of lymphoid tissues (but not other types) is further intensified by brief (5-10 sec.) passage through aqueous 1.5% uranium nitrate after oxidation but before silver impregnation. The specific action of periodic acid (cleavage of the 1,2-glycol linkage to produce aldehyde radicals) strengthens the premise that the carbonyl radical plays an important part in the phenomenon of connective tissue argyrophilia.