Study protocol to investigate biomolecular muscle profile as predictors of long-term urinary incontinence in women with gestational diabetes mellitus.

BACKGROUND: Pelvic floor muscles (PFM) and rectus abdominis muscles (RAM) of pregnant diabetic rats exhibit atrophy, co-localization of fast and slow fibers and an increased collagen type I/III ratio. However, the role of similar PFM or RAM hyperglycemic-related myopathy in women with gestational diabetes mellitus (GDM) remains poorly investigated. This study aims to assess the frequency of pelvic floor muscle disorders and pregnancy-specific urinary incontinence (PS-UI) 12 months after the Cesarean (C) section in women with GDM. Specifically, differences in PFM/RAM hyperglycemic myopathy will be evaluated. METHODS: The Diamater is an ongoing cohort study of four groups of 59 pregnant women each from the Perinatal Diabetes Research Centre (PDRC), Botucatu Medical School (FMB)-UNESP (São Paulo State University), Brazil. Diagnosis of GDM and PS-UI will be made at 24-26 weeks, with a follow-up at 34-38 weeks of gestation. Inclusion in the study will occur at the time of C-section, and patients will be followed at 24-48 h, 6 weeks and 6 and 12 months postpartum. Study groups will be classified as (1) GDM plus PS-UI; (2) GDM without PS-UI; (3) Non-GDM plus PS-UI; and (4) Non-GDM without PS-UI. We will analyze relationships between GDM, PS-UI and hyperglycemic myopathy at 12 months after C-section. The mediator variables to be evaluated include digital palpation, vaginal squeeze pressure, 3D pelvic floor ultrasound, and 3D RAM ultrasound. RAM samples obtained during C-section will be analyzed for ex-vivo contractility, morphological, molecular and OMICS profiles to further characterize the hyperglycemic myopathy. Additional variables to be evaluated include maternal age, socioeconomic status, educational level, ethnicity, body mass index, weight gain during pregnancy, quality of glycemic control and insulin therapy. DISCUSSION: To our knowledge, this will be the first study to provide data on the prevalence of PS-UI and RAM and PFM physical and biomolecular muscle profiles after C-section in mothers with GDM. The longitudinal design allows for the assessment of cause-effect relationships between GDM, PS-UI, and PFMs and RAMs myopathy. The findings may reveal previously undetermined consequences of GDM.

Maternal protein malnutrition: effects on prostate development and adult disease.

Well-controlled intrauterine development is an essential condition for many aspects of normal adult physiology and health. This process is disrupted by poor maternal nutrition status during pregnancy. Indeed, physiological adaptations occur in the fetus to ensure nutrient supply to the most vital organs at the expense of the others, leading to irreversible consequences in tissue formation and differentiation. Evidence indicates that maternal undernutrition in early life promotes changes in key hormones, such as glucocorticoids, growth hormones, insulin-like growth factors, estrogens and androgens, during fetal development. These alterations can directly or indirectly affect hormone release, hormone receptor expression/distribution, cellular function or tissue organization, and impair tissue growth, differentiation and maturation to exert profound long-term effects on the offspring. Within the male reproductive system, maternal protein malnutrition alters development, structure, and function of the gonads, testes and prostate gland. Consequently, these changes impair the reproductive capacity of the male offspring. Further, permanent alterations in the prostate gland occur at the molecular and cellular level and thereby affect the onset of late life diseases such as prostatitis, hyperplasia and even prostate cancer. This review assembles current thoughts on the concepts and mechanisms behind the developmental origins of health and disease as they relate to protein malnutrition, and highlights the effects of maternal protein malnutrition on rat prostate development and homeostasis. Such insights on developmental trajectories of adult-onset prostate disease may help provide a foundation for future studies in this field.

Morphological changes in rat rectus abdominis muscle induced by diabetes and pregnancy.

The urethral muscle of diabetic pregnant rats is affected by long-term mild diabetes and short-term severe diabetes, which plays a crucial role in the pathogenesis of pelvic floor disorders. We hypothesized that muscles outside the pelvis are subject to similar changes. The current study aimed at analyzing the effects of long-term mild and short-term severe diabetes on the structure and ultrastructure of fiber muscles and collagen in rats' rectus abdominis (RA) muscle. Therefore, the RA muscle of virgin, pregnant, long-term mild diabetic, short-term severe diabetic, long-term mild diabetic pregnant and short-term severe diabetic pregnant 3-month-old Wistar rats were collected. The structure was analyzed by picrosirius red staining, immunohistochemistry for fast and slow muscle fibers and transmission electron microscopy. We investigated two levels of STZ- induced diabetes: long-term mild diabetes (blood glucose level: 120-200 mg/dL) and short-term severe diabetes (blood glucose level >300 mg/dL). Long-term mild diabetic pregnant and short-term severe diabetic pregnant rats had decreased fast fibers and increased slow fibers, disrupted areas of sarcomere, intermyofibrillar mitochondria and myelin figures in the RA muscle. Both groups enabled us to analyze the specific influence of pregnancy, separately from diabetes. The current study demonstrated that diabetes and pregnancy induced intramuscular transformation and reorganization of RA muscle with a switch of fiber type adjusting their architecture according to intensity and duration of hyperglycemic insult within pregnancy.

Improved tympanic membrane regeneration after myringoplastic surgery using an artificial biograft.

Tympanic membrane perforations are due to common otologic problems. The current treatments to heal tympanic membrane perforation, such as myringoplasty, have some disadvantages, including the need for autologous grafting, which is rapidly absorbed by the organism before perforation recovery is complete. To improve the structural and functional tympanic membrane healing after surgery, we propose a new branch of artificial grafts. In this study, we report the development of artificial grafts using electrospun bioabsorbable polymers. Polymers such as poly (l-lactic acid) and poly (lactic-co-glycolic acid) acted as the scaffold for cell growth in a co-culture of fibroblasts and keratinocytes. This co-culture promoted the growth of an epithelial-equivalent tissue over the electrospun scaffold, which was used as an alternative graft in myringoplasty. The in vivo study was performed in Sprague Dawley rats. Ear endoscopy was performed 30days after surgery and showed that tympanic membrane perforations treated with artificial grafts healed naturally, completely and with the possibility of maintaining their actual functionality. In conclusion, our study described a new artificial graft created specifically to fulfill the requirements of perforated tympanic membrane healing processes, which are compatibility, proper durability and less intense side effects following myringoplasty.

Implante de condrócitos homólogos em defeitos osteocondrais de cães: padronização da técnica e avaliação histopatológica / Homologous articular chondrocytes implantation in osteochondral defects of dogs: technique and histopathological evaluation standardization

Padronizou-se a metodologia para cultura de condrócitos em cães e avaliou-se seu implante em lesões osteocondrais, utilizando-se a membrana biossintética de celulose (MBC) como revestimento. Dez cães, adultos e clinicamente sadios, foram submetidos à artrotomia das articulações fêmoro-tíbio-patelares. Defeitos de 4mm de diâmetro e profundidade foram induzidos no sulco troclear de ambos os membros. MBC foi aplicada na base e na superfície das lesões. Os defeitos do membro direito foram preenchidos com condrócitos homólogos cultivados formando o grupo-tratado (GT); os do membro esquerdo, sem implante celular, foram designados grupo-controle (GC). A evolução pós-operatória foi analisada com especial interesse nos processos de reparação da lesão, por meio de histomorfometria e imuno-histoquímica para colágeno tipo II e sulfato de condroitina. A cultura de condrócitos homólogos apresentou alta densidade e taxa de viabilidade. Observou-se integridade do tecido neoformado com a cartilagem adjacente na avaliação histológica, em ambos os grupos. Na imuno-histoquímica, verificou-se predomínio de colágeno tipo II no GT. Morfometricamente, não houve diferença significativa entre o tecido fibroso e o fibrocartilaginoso entre os grupos. A cultura de condrócitos homólogos de cães foi exequível. O tecido neoformado apresentou qualidade discretamente superior associado ao implante homólogo de condrócitos, contudo não promoveu reparação por cartilagem hialina.

The aim of the study is to standardize the methodology to achieve canine chondrocytes culture, and evaluate its implant on osteochondral defects made in the femoral trochlear sulcus of dogs, using the cellulose biosynthetic membrane (CBM) as coating. Ten healthy adult dogs without locomotor disorders were used. All animals were submitted to arthrotomy of stifle joints and defects of four millimeters in diameter x four millimeters deep were done in the femoral trochlear sulcus of both limbs. CBM were applied in the lesion base and surface of all limbs. In the treated group (TG), defects of the right limb were filled with cultivated homologous chondrocytes, and in control group (CG), defects of the left limb were left without cellular implant. Postoperative follow up was done by histomorphometry and Collagen type II and anti-chondroitin sulfate immunohistochemistry. The homologous chondrocytes culture showed high density and viability rate. Upon immunohistochemistry the predominance of type II collagen in extracellular matrix of TG was verified. However, no significant statistical difference was observed between the groups upon histomorphometry analysis of fibrous and fibrocartilaginous tissues. Canine homologous chondrocytes culture was practicable. Neoformed tissue showed slightly higher quality in TG, but without promoting repair by the hyaline cartilage.

Plasma rico em plaquetas no tratamento de lesões condrais articulares induzidas experimentalmente em equinos: avaliação clínica, macroscópica, histológica e histoquímica / Platelet-rich plasma in the treatment of articular chondral defects experimentally induced in horses: clinical, macroscopic, histopathological and histochemical evaluation

Estudou-se a eficácia do plasma rico em plaquetas (PRP) no tratamento de lesões condrais articulares, experimentalmente induzidas em equinos. Para isso, foi induzida uma lesão condral, na tróclea medial femoral dos dois membros pélvicos de quatro animais. Após 30 dias da indução, as oito articulações foram divididas em dois grupos. Os animais do grupo 1 receberam o tratamento intralesional e intra-articular com PRP, e os do grupo 2 foram tratados apenas com solução fisiológica. As avaliações clínicas, constituídas de exames de claudicação e análises do líquido sinovial, foram realizadas antes da indução da lesão - tempo zero -, quinzenalmente, até 120 dias e aos 150 dias. Avaliações macroscópicas, histológicas e histoquímicas foram realizadas no tempo zero e aos 150 dias. Os equinos do grupo 1 apresentaram melhora do grau de claudicação em relação aos do grupo 2. Os exames macroscópicos, histológicos e histoquímicos revelaram melhor tecido de reparação igualmente no grupo 1. Concluiu-se que a administração de PRP apresentou efeitos benéficos no tratamento de lesões condrais experimentais de equinos.

The use of platelet-rich plasma (PRP) in the treatment of articular cartilage defects induced experimentally in horses was studied. For this purpose, both patellofemoral joints of four animals were approached through arthroscopic surgery to perform a cartilage defect on the medial femoral trochlea. After 30 days of induction the eight joints were divided into two groups. Group 1 animals received intralesional and intra-articular treatment with PRP and Group 2 animals were treated only with saline solution. The clinical assessments, constituted by lameness signs and synovial fluid analysis, were performed before induction of injury (time zero) and every 15 days in 120 days, with last analysis on day 150. The macroscopic and morphologic analysis were performed at 0 and 150 days. During the experiment Group 1 animals showed improvement in lameness when compared to Group 2 animals. The macroscopic and morphological analysis showed a better tissue repair in the treated horses. Thus, the implantation of autologous PRP showed beneficial effects in the treatment of chondral lesions, experimentally induced in horses.

Plasma rico em plaquetas no tratamento de lesões condrais articulares induzidas experimentalmente em equinos: avaliação clínica, macroscópica, histológica e histoquímica / Platelet-rich plasma in the treatment of articular chondral defects experimentally induced in horses: clinical, macroscopic, histopathological and histochemical evaluation

Estudou-se a eficácia do plasma rico em plaquetas (PRP) no tratamento de lesões condrais articulares, experimentalmente induzidas em equinos. Para isso, foi induzida uma lesão condral, na tróclea medial femoral dos dois membros pélvicos de quatro animais. Após 30 dias da indução, as oito articulações foram divididas em dois grupos. Os animais do grupo 1 receberam o tratamento intralesional e intra-articular com PRP, e os do grupo 2 foram tratados apenas com solução fisiológica. As avaliações clínicas, constituídas de exames de claudicação e análises do líquido sinovial, foram realizadas antes da indução da lesão - tempo zero -, quinzenalmente, até 120 dias e aos 150 dias. Avaliações macroscópicas, histológicas e histoquímicas foram realizadas no tempo zero e aos 150 dias. Os equinos do grupo 1 apresentaram melhora do grau de claudicação em relação aos do grupo 2. Os exames macroscópicos, histológicos e histoquímicos revelaram melhor tecido de reparação igualmente no grupo 1. Concluiu-se que a administração de PRP apresentou efeitos benéficos no tratamento de lesões condrais experimentais de equinos.

The use of platelet-rich plasma (PRP) in the treatment of articular cartilage defects induced experimentally in horses was studied. For this purpose, both patellofemoral joints of four animals were approached through arthroscopic surgery to perform a cartilage defect on the medial femoral trochlea. After 30 days of induction the eight joints were divided into two groups. Group 1 animals received intralesional and intra-articular treatment with PRP and Group 2 animals were treated only with saline solution. The clinical assessments, constituted by lameness signs and synovial fluid analysis, were performed before induction of injury (time zero) and every 15 days in 120 days, with last analysis on day 150. The macroscopic and morphologic analysis were performed at 0 and 150 days. During the experiment Group 1 animals showed improvement in lameness when compared to Group 2 animals. The macroscopic and morphological analysis showed a better tissue repair in the treated horses. Thus, the implantation of autologous PRP showed beneficial effects in the treatment of chondral lesions, experimentally induced in horses.

MMP-2 and TIMP-2 in the prostates of male and female mongolian gerbils: effects of hormonal manipulation.

TIMPs in the prostates of male and female gerbils and evaluated the effects of testosterone on the expression of these enzymes. Ventral prostates from male gerbils that were either intact or had been castrated for 7 or 21 days, along with prostates from female gerbils that were either intact or had been treated with testosterone for 7 or 21 days, were submitted to histological, stereological and immunohistochemical analyses. Stereology of prostatic components showed significant alterations of tissue compartments in the ventral male prostate after castration, especially after 21 days, with a significant increase in stroma. Administration of testosterone led to disorganization in the female prostate, with a significant increase in collagen fibers and smooth muscle cells after 21 days, along with the development of epithelial lesions such as PINs. MMP-2 increased after 21 days of castration in males; however, the TIMP-2 immunoreaction for this group was weak or absent. In females, the expression of MMP-2 appeared to decrease after 7 days of treatment with testosterone, but after 21 days, both epithelium and stroma showed a stronger reaction for MMP-2 than the controls. The expression of TIMP-2 in the treated females was similar to its expression in the castrated males. We conclude that the distribution of MMPs and TIMPs in both male and female prostates is altered by androgen manipulation, but the mechanism of stromal regulation appears to be distinct between genders because both the lack of T in castrated males and the excess levels of T in treated females lead to the same effect.

Matrix metalloproteinase (MMP)-2 and MMP-9 activity and localization during ventral prostate atrophy and regrowth.

Matrix metalloproteinses (MMPs) are enzymes involved in prostatic development, growth, disease-induced tissue remodelling and secretory fluid. Although the prostate function depends upon androgen regulation, the relationship between MMPs and androgen has not been well established. Here, we evaluated MMP-2 and MMP-9 gelatinolytic activity in association with tissue localization during ventral prostate atrophy and regrowth induced by testosterone replacement (TR). Adult male Wistar rats were divided into three experimental groups: control, castrated (CS) and TR 21 days after castration. Ventral prostate (VP) was excised at 3, 5, 7 and 21 days after castration in CS group, and at 3, 5, 7 and 10 days after TR (4 mg/kg/day) in TR group. The VP was dissected, weighed and processed for histology, immunohistochemistry, ultrastructure and zymography analyses. Castration elicited the typical parenchymal atrophy and stromal condensation. TR induced intense epithelial growth towards the stromal space to restore the prostate histoarchitecture. MMP-2 and MMP-9 immunostaining presented intense reaction in CS and TR groups, mainly in the epithelial and endothelial cells. After TR, a strong immunoreaction for MMP-2 was observed in the activated stromal fibroblasts. Zymography showed that MMP-2 and MMP-9 activity, mainly the active form, increased after castration. In contrast, TR induced an additional increase in MMP-2 activity, but not in MMP-9. In conclusion, the overall behaviour of MMP-2 and MMP-9 within the prostate under androgen handling is highly complex, as each glandular compartment and cell type is affected differently by the androgenic status. Prostate regrowth appears to involve a more effective participation of MMP-2 in both epithelial and stromal compartments, while MMP-9 plays a major role in the late prostate atrophy and early regrowth.

Avaliação das metaloproteinases de matriz -2 e -9 em gatos com desmineralização óssea secundária à tirotoxicose induzida / Evaluation of matrix metalloproteinases -2 and -9 in cats under bone demineralization secondary to induced thyrotoxicosis

Observou-se significativo aumento de atividade das formas ativas das metaloproteinases -2 e -9 em gatos com tirotoxicose induzida e desmineralização óssea. As formas pró e intermediária da metaloproteinase -2 elevaram-se com 14 dias de administração hormonal, porém, posteriormente, houve uma tendência de queda. Observou-se correlação negativa entre a forma ativa das metaloproteinases de matriz -2 e -9 e a densidade mineral óssea da extremidade distal do rádio. Os resultados sugerem aumento da degradação da matriz colágena secundária com a elevação dos hormônios tiroidianos.

Significant increase of activity of active forms of matrix metalloproteinases -2 and -9 in cats under induced thyrotoxicosis and bone demineralization was observed. Pro and intermediated forms of matrix metalloproteinases -2 and -9 increased at 14 days of hormonal treatment, followed by decrease tendency. A negative correlation between active forms of matrix metalloproteinases -2 and -9 and bone mineral density of radius distal extremity was also observed. The results suggest an increase of collagen matrix degradation secondary to high levels of thyroid hormones.

Variations in the glycosaminoglycan content, swelling properties and morphological aspects of different regions of the superficial digital flexor tendon of pigs.

The superficial digital flexor tendon of pigs is a wrap around tendon supporting compressive in addition to the tensional forces in its proximal region, and only tensional forces in the intermediate region. Clear distinctions were observed in the physical properties, composition of proteoglycan and morphology for the different regions. A greater swelling in water and a larger amount of glycosaminoglycan were found in the proximal region compared to the intermediate one. Dermatan sulfate was detected in all regions of this tendon, while chondroitin sulfate was prominent only near the bone. In the toluidine blue stained sections, intense metachromasy was observed in the sites under compressive forces, especially in the portion close to the bone. Chondrocyte like cells were also observed in these arms. Crimp morphology, observed in the intermediate region, exhibited a clearly sloped aspect in relation to the main axis of the tendon. Elastic fibers were found in all regions, and were disposed in different directions in the areas under compressive forces, and parallel to the collagen bundles in the region under tension. These results reinforce the idea that mechanical forces contribute to a differentiated composition and organization of the extracellular matrix of tendons.

Growth cartilage calcification and formation of bone trabeculae are late and dissociated events in the endochondral ossification of Rana catesbeiana.

Endochondral ossification in the growth cartilage of long bones from the bullfrog Rana catesbeiana was examined. In stage-46 tadpoles and 1-year-old animals, the hypertrophic cartilage had a smooth contact with the bone marrow and the matrix showed no calcification or endochondral bone formation. In spite of showing no aspects of calcification, the chondrocytes exhibited alkaline phosphatase activity and some of them died by apoptosis. However, matrix calcification and endochondral ossification were observed in 2-year-old bullfrogs. Calcium deposits appeared as isolated or coalesced spherical structures in the extracellular matrix of hypertrophic cartilage. Bone trabeculae were restricted to the central area at the sites where the hypertrophic cartilage surface was exposed to the bone marrow. Cartilage matrix calcification and the formation of bone trabeculae were not dependent on each other. Osteoclasts were involved in calcified matrix resorption. These results demonstrate that the calcification of hypertrophic cartilage and the deposition of bone trabeculae are late events in R. catesbeiana and do not contribute to the development and growth of long bones in adults. These processes may play a role in reinforcing bony structures as the bullfrog gains weight in adulthood. In addition, the deposition of bone trabeculae is not dependent on cartilage matrix calcification.

Regional adaptations in three rat tendons.

Although detailed histological and immunocytochemical studies have been published for the rat calcanear tendon (CT), little is known of the structure, composition and biomechanics of the deep (DFT) and superficial (SFT) flexor tendons. In this study, we examined the structural specialization of these three tendons in 90-day-old rats by applying histochemical and biochemical assays to different tendon regions (proximal, intermediate and distal regions of the DFT and SFT, and proximal and distal regions of the CT). There were regional differences in tissue structure, glycosaminoglycan type and content, swelling properties and in the amount and distribution of elastic fibers. Dermatan sulfate occurred in all regions, but chondroitin sulfate predominated in the intermediate region of the DFT and in the distal region of the CT. These two chondroitin sulfate-bearing regions showed swelling in water, while all other regions lost fluid in water. Fibrocartilaginous sites were observed on the CT, one at the insertion to the bone and another distally at the innermost area of the tendon. The intermediate region of the DFT showed round cells disposed in lacunae, while the proximal and distal regions were typically fibrous. The intermediate region of the SFT showed a wavy array of collagen bundles but neither toluidine blue staining in the matrix nor round cells. Elastic fibers were present in each region of the three tendons, but were more prominent in the intermediate zone of the SFT. These results demonstrate regional variation in the three tendons. Tendon differentiation may occur by an increase in the number of elastic fibers and by variations in the arrangement of collagen fibers, without fibrocartilage formation.

Collagen fiber reorganization in the rat ventral prostate following androgen deprivation: a possible role for smooth muscle cells.

BACKGROUND: Stroma plays an essential role in glandular function in different systems. In the prostate, it is responsible for the development and maintenance of the differentiated state of the epithelium. The marked reduction in the epithelial compartment of the prostate gland following castration is followed by a similarly important reorganization of the stroma. In this work, we characterized the reorganization of collagen fibers in the ventral prostate of castrated rats. METHODS: Histochemical tests and immunohistochemistry for type I and III collagens plus confocal microscopy of triple-labeled (collagen III, actin, and DNA) tissue sections were employed. RESULTS: We showed that collagen fibers are composed of type I and type III collagens and that they are progressively concentrated around the epithelial structures (ducts and acini) and become increasingly undulated and folded. Double-labeling of collagen fibers and F-actin demonstrated that smooth muscle cells (SMC) are intimately associated with collagen fibers. CONCLUSIONS: The results demonstrated a marked reorganization of the collagen fibers, and suggest an active role of the SMC in the reorganization of the fibrillar components of the stroma.

Structure and proteoglycan composition of specialized regions of the elastic tendon of the chicken wing.

The elastic tendon of the avian wing has been described by others as a unique structure with elastic properties due to the predominance of elastic fibers in the midsubstance. Further analyses of the tendon have shown it to possess five anatomically distinct regions. Besides the major elastic region, a distally located fibrocartilage and three tendinous regions are present. The tendinous regions connect: (1) the muscle to the elastic region, (2) the elastic region to the fibrocartilage and (3) the latter to the insertion site. The elastic region possesses thick and abundant elastic fibers and very thin, interconnecting collagen fibers. The collagen fibers in the sesamoid fibrocartilage are thick and interwoven, defining spaces occupied by fibrochondrocytes embedded in a non-fibrillar and highly metachromatic matrix. Biochemical analyses have shown that the fibrocartilage has about tenfold the amount of glycosaminoglycans (GAGs) found in the other regions. The main GAG in this region was chondroitin sulfate (CS) (plus keratan sulfate as detected immunocytochemically), while the other regions showed variable amounts of CS, dermatan sulfate (DS) and heparan sulfate. Further analyses have shown that a large CS-bearing proteoglycan is found in the fibrocartilage. The elastic region possesses two main proteoglycans, a large CS-bearing proteoglycan (which reacted with an antibody against keratan sulfate after chondroitinase ABC treatment) and a predominant DS-bearing proteoglycan, which showed immunoreactivity when assayed with an anti-biglycan antibody. The results demonstrate that the elastic tendon is a complex structure with complex regional structural and compositional adaptations, suited to different biomechanical roles.

The osteochondral ligament: a fibrous attachment between bone and articular cartilage in Rana catesbeiana.

The anuran epiphyseal cartilage shows a lateral expansion that covers the external surface of the bone, besides other features that distinguish it from the corresponding avian and mammalian structures. The fibrous structure that attaches the lateral cartilage to the bone was characterized in this work. It was designated osteochondral ligament (OCL) and presented two main areas. There was an inner area that was closer to the periosteal bone and contained a layer of osteoblasts and elongated cells aligned to and interspersed with thin collagen fibers. The thin processes of the cells in this area showed strong alkaline phosphatase activity. The outer area, which was closer to the cartilage, was rich in blood vessels and contained a few cells amongst thick collagen fibers. TRITC-phaloidin staining showed the cells of the inner area to be rich in F-actin, and were observed to form a net around the cell nucleus and to fill the cell processes which extended between the collagen fibers. Cells of the outer area were poor in actin cytoskeleton, while those associated with the blood vessels showed intense staining. Tubulin-staining was weak, regardless of the OCL region. The main fibers of the extracellular matrix in the OCL extended obliquely upwards from the cartilage to the bone. The collagen fibers inserted into the bone matrix as Sharpey's fibers and became progressively thicker as they made their way through the outer area to the cartilage. Immunocytochemistry showed the presence of type I and type III collagen. Microfibrils were found around the cells and amongst the collagen fibrils. These microfibrils were composed of either type VI collagen or fibrilin, as shown by immunocytochemistry. The results presented in this paper show that the osteochondral ligament of Rana catesbeiana is a complex and specialized fibrous attachment which guarantees a strong and flexible anchorage of the lateral articular cartilage to the periosteal bone shaft, besides playing a role in bone growth.

The epiphyseal cartilage and growth of long bones in Rana catesbeiana.

The structure of the epiphyseal cartilage of the bullfrog Rana catesbeiana and its role in the growth of long bones were examined. The epiphyseal cartilage was inserted into the end of a tubular bone shaft, defining three regions: articular cartilage, lateral articular cartilage and growth cartilage. Joining the lateral cartilage to the bone was a fibrous layer of periosteum, rich in blood vessels. Osteoblasts with alkaline phosphatase activity were found on the surface of the periosteal bone, which presented a fibrous non-mineralised tip. The growth cartilage was inside the bone. The proliferative chondrocytes presented perpendicular separation of daughter cells and there was no columnar arrangement of the cells. Furthermore, chondrocyte hypertrophy was not associated with either calcification or endochondral ossification, in apparent contrast to the avian and mammalian models. Finally, there was no reinforcement system capable of directing cell volume increase into longitudinal growth. Since bone extension depends on the intramembranous ossification of the periosteum, the growth cartilage is inside and not at the end of the bone and the cells in the growth cartilage show no columnar arrangement and separate in a direction perpendicular to the long bone axis, we conclude that the growth cartilage mainly contributes to the radial expansion of the bone.

Identification and distribution of type VI collagen in tendon fibrocartilages.

Structural and compositional variations are marked in tendon fibrocartilages, which appear at the insertion to bone and in areas subjected to compressive plus frictional loading regime. We are interested in characterizing the extracellular matrix adaptations in these areas, in an attempt to understand cellular responses to biomechanics. In this work, we have applied immunocytochemistry and an ATP treatment for the ultrastructural identification of type VI collagen, to tendons subjected to compressive forces in different species. Immunocytochemistry, after testicular hyaluronidase or pepsin digestion, revealed the presence of type VI collagen in tensional and compressive areas of the plantaris longus tendon of the bullfrog, in the deep flexor tendon of dogs and rabbits, in the calcanear tendon and the suprapatela of rats and in the gastrocnemius tendon of chickens. In each tendon, the tensional region showed a weak reaction, restricted to the collagen fiber surface. However, the compression region was especially rich in type VI collagen, which accumulates in the interfibrillar spaces and is concentrated around the fibrochondrocytes. Intense reaction was also found in the paratenon. The ATP treatment not only allowed for the detection of the typical ladder-like aggregates of type VI collagen in the same areas identified by immunocytochemistry, but also demonstrated that type VI collagen forms a microfibrillar network around the fibrochondrocytes. Besides the function of organizing groups of collagen fibrils, type VI collagen seems to assemble the pericellular matrix in tendon fibrocartilages, perhaps through physical interactions with the large proteoglycans that concentrate in the same area.

The development of the pressure-bearing tendon of the bullfrog, Rana catesbeiana.

The plantaris longus tendon of the bullfrog is a pressure-bearing tendon and develops a fibrocartilage-like arrangement in the area subjected to compressive forces. The fibrocartilage-like tissue shows some distinct aspects of cellular and fibrillar structure and distribution as compared to the mammalian counterparts. In this work, the development of the plantaris longus tendon was assessed by investigating some of its structural, cytochemical and immunocytochemical aspects in developing tadpoles. The pressure-bearing region is structurally distinct from the tension region as early as at stage 35 of larval development. There is little extracellular matrix in both regions, but the former shows round mesenchymal-like cells with many processes and cell junctions, while the latter is populated by fibroblasts. As development proceeds, the cells in the compression region retract the processes, loose the connections to each other, become rounded and produce abundant proteoglycans and some collagen fibers. Progressively, their organelles become localized in a restricted perinuclear area and are surrounded by a constantly increasing amount of vimentin. The fibroblasts of the tension region produce mostly collagen fibrils, which are packed and aligned to each other. These cells become more elongated and show a diminished cytoplasmic area. The results allow for the conclusion that the compression region does not arise by simple hyperplasia of the peripheral layers of a normal tendon, but from a programmed sequence of developmental steps. This assumption is based on the fact that muscle fibers are still developing when the tendon is already showing a differentiated compression region. We further suggest that mechanical stimulation is a secondary factor most likely associated with the maintenance of the differentiated phenotype of this tendon.