Biomedical subjects
A Gigante
Publications and source records attributed to A Gigante.
Expression of NGF, Trka and p75 in human cartilage.
Nerve growth factor (NGF) exerts its action through two types of receptor: high-affinity tyrosine kinase A receptor (trkA) and low-affinity p75 receptor. NGF has a neurotrophic role in central and peripheral nervous system development, but there is also clear evidence of its involvement in the developing skeleton. The aim of the present immunohistochemical study was to investigate the expression and distribution of NGF, trkA, and p75 in normal cartilaginous tissues from adult subjects: articular and meniscal cartilage of the knee, cartilage from the epiglottis, and intervertebral disc tissue. Detection of NGF mRNA was also performed by in situ hybridization. Immunoreaction for NGF and the two receptors in articular chondrocytes, chondrocyte-like cells of meniscus and annulus fibrosus, and chondrocytes of the epiglottis demonstrated that they are all expressed in hyaline, fibrous and elastic cartilaginous tissues, suggesting that they could be involved in cartilage physio-pathology.
Patellar tendon healing after removal of its central third. A morphologic evaluation in rabbits.
The central third of the patellar tendon of the right knee was removed in 40 adult New Zealand White (NZW) rabbits. The animals were divided into two groups of 20 animals each. In group 1 the tendon defect was left open and in group 2 the tendon defect was closed. The peritenon was closed in all cases. The animals were killed after 2 weeks, 1, 3, 6 and 9 months. Operated and contralateral normal tendons of each knee underwent a gross, histologic, immunohistochemical and morphometric analysis. Gross examination revealed that in the closed group there was a greater shortening of the patellar tendon but the difference between the two groups was not statistically significant. The width, thickness and cross-sectional area increased progressively in both groups and were greater in the closed group. Histological examination showed that at 1 month in both groups the harvest site was filled with a loose unoriented fibrovascular tissue. In group 1, large fatty inclusions could be seen in the deep layer, that were still present in the 3-, 6- and 9-month samples. At 3 months in group 1 the newly formed tissue was immature and rich in elastic fibres, as confirmed by immunohistochemical analysis, whereas in group 2 it was more compact and oriented. In group 1 there was in many cases an ossification on the lower pole of the patella, which was not seen in group 2. At 9 months a well-organized fibrous tissue could be seen in both groups at the harvest site. Histomorphometric analysis confirmed the better orientation and the greater density of the newly formed tissue in the closed group as compared to the open group, and the greater number of elastic fibres in the open group, even after 9 months.
Autologous chondrocyte implantation: cells phenotype and proliferation analysis.
The phenotype and proliferation of human chondrocytes in culture were analyzed before they were implanted as autologous graft in cartilage lesions. During ten autologous chondrocyte implantations in articular cartilage lesions of the knee in ten patients, small amounts of cells to be implanted were collected and analyzed by morphology, cytochemistry (alcian blue, safranin-O), and immunocytochemistry (antibodies anti-S100 protein, anti-collagen types I and II, anti-chondroitin-S). In four cases the cells were also cultured for 3 weeks. At 1, 10, and 20 days of culture cells were incubated with 1 microCi/ml [3H]thymidine for proliferation analysis. In all cases the cells showed the morphological appearance of mature chondrocytes, stained positively for alcian blue and safranin-O, and revealed a strong immunoreaction for S-100 protein, type II collagen, and chondroitin-S but not for type I collagen. Radioisotope assay of chondrocyte proliferation at 1, 10, and 20 days of culture revealed a progressive increase in [3H]thymidine incorporation. These findings indicate that the cells before autologous implantation maintain their differentiated phenotype of mature chondrocytes and proliferate greatly. This analysis is an essential step preceding wider use of this treatment in humans. However, other biological aspects of the autologous chondrocyte graft remain to be elucidated.
Characterization of cultured human ligamentum flavum cells in lumbar spine stenosis.
To investigate the pathogenesis of the degenerative changes of the ligamentum flavum occurring in lumbar spine stenosis, yellow ligament cells from patients with lumbar spine stenosis were cultured for the first time and subjected to biochemical, histochemical and immunohistochemical study. Stenotic ligamentum flavum (SLF) cells were seen to express high levels of alkaline phosphatase (ALP) activity and to produce a matrix rich in type I and III collagen, fibronectin and osteonectin. The matrix mineralized only following beta-glycerophosphate (betaGP) and ascorbic acid supplementation. Stimulation with human parathyroid hormone (PTH) increased intracellular cAMP concentration. These findings indicate that there was significant evidence of osteoblast-like activity in these cells. SLF cells also stained for S100 protein, type II and type X collagen, and co-localized type II collagen and ALP labelling, reflecting the presence of hypertrophic chondrocyte-like cells. Cultures from control patients showed neither osteoblastic nor chondrocytic features: they expressed type I and type III collagen and fibronectin, but did not stain for osteonectin, nor were bone-like calcifications observed in presence or absence of betaGP. Normal ligamentum flavum (NLF) cells did not synthesized S100 protein or type II or type X collagen, and showed a weaker response to PTH stimulation. Our data demonstrated the presence of hypertrophic chondrocytes with an osteoblast-like activity in the ligamentum flavum of patients with spinal stenosis suggesting that they could have a role in the pathophysiology of the heterotopic ossification of ligamentum flavum (OLF) in lumbar spine stenosis.
DL-penicillamine induced alteration of elastic fibers of periosteum-perichondrium and associated growth inhibition: an experimental study.
Perichondrium-periosteum, being of collagen and elastic fiber, is regarded as a bone growth regulating factor. The aim of the present study was to investigate the role of collagen and elastic fibers on bone growth, by interfering with the fiber assembly in growing chicks upon administration of DL-penicillamine (DL-PNA). Our findings demonstrated that DL-PNA determined relevant modifications in the perichondrium-periosteum, as shown by histochemical, histomorphometrical,biochemical and ultrastructural analysis. This chemical has been shown to inhibit the formation of desmosine cross-links in elastin and to induce an increase of elastin associated microfibrils. On the contrary, the collagen network and the biochemical collagen markers were not affected. These changes resulted in a dramatically reduced growth of long bones in comparison with control. Perichondrial-periosteal regulation of bone growth may be mediated by mechanical and biological factors. This study demonstrates a microstructural change in the perichondrium-periosteum with decreased elastin and increased elastic microfibrils content in penicillamine treated chicks. The mechanism linking changes in the perichondrium-periosteum with altered growth still needs to be elucidated.
Indoor travel and simple tasks as physical exercise for people with profound multiple disabilities.
2 to 4 30-min. periods of indoor travel and simple tasks were arranged daily as physical exercise for two women with profound mental retardation, deafness, and profound visual impairment. The questions were whether (a) the women could manage this fairly extensive exercise regimen (over about 9 and 4.5 mo.) with support technology and only minimal staff supervision and (b) the exercise could affect aspects of the women's physical condition. Data indicated that both women could successfully manage the exercise regimen with support technology, and the exercise seemed to have some beneficial effects on physical aspects such as bone metabolism and step width.
Elastic fibers of musculoskeletal tissues in bovine Marfan syndrome: a morphometric study.
Bovine Marfan syndrome has all the major pathognomonic, clinical, and pathological features of the human syndrome. To further explore the alterations characterizing Marfan syndrome, musculoskeletal tissues from affected and healthy cattle were subjected to histochemical and histomorphometric analysis. Our findings demonstrated reduced elastic fiber content in the periosteum, capsule, interosseous ligament, and flexor tendons of the metatarsophalangeal joint of affected cattle. The elastic properties of a tissue are supplied by elastic fibers in the extracellular matrix; therefore, their reduced content in articular tissues suggests that these fibers could be functionally incompetent to withstand normal stress, causing a predisposition to joint laxity and dislocation. Moreover, perichondrial-periosteal membranes, which are made of collagen and elastic fibers, are known to affect the growth process of the long bones. The decrease in the number of elastic fibers in these membranes could result in reduced restraint of skeletal growth and explain some skeletal abnormalities of Marfan syndrome (i.e., dolichostenomelic habitus).
N,N-dicarboxymethyl chitosan as delivery agent for bone morphogenetic protein in the repair of articular cartilage.
Bone morphogenetic protein (BMP), associated with N,N-dicarboxymethyl chitosan, is used to induce or facilitate the repair of articular cartilage lesions. This association is intended for the synergistic potentiation of the respective biological effects. Data show that BMP-7 enhances the in vivo proliferation of cells with chondrocytes phenotype in the articular environment, leading to partial healing of the articular surface of the lesions. N,N-dicarboxymethyl chitosan is found to be useful as a molecular carrier or drug delivery agent.
Plantar fibromatosis: an immunohistochemical and ultrastructural study.
The analogies between plantar fibromatosis and Dupuytren's disease (palmar fibromatosis) are well known. The latter is clinically more frequent and has been the object of extensive immunohistochemical and ultrastructural studies, with a view to investigating its pathogenesis. By contrast, such data on plantar fibromatosis are quite scarce. A histochemical, immunohistochemical, and ultrastructural study was performed on nodule tissue from six patients who were subjected to total fasciectomy for plantar fibromatosis. The study of myofibroblasts revealed features suggestive of their fibroblastic origin and evidenced a cytoskeleton and an extracellular filamentous system that could enable myofibroblasts to generate and exert the intracellular forces that contribute to the contraction of the aponeurosis. These aspects are similar to those observed in Dupuytren's disease and seem to lend support to the theory that the two diseases are expressions of the same disorder.
In vitro effects of elastase on periosteum of long bones: an histochemical, immunohistochemical and morphometric study.
The aim of the study was to determine the in vitro effects of porcine pancreatic elastase on the periosteum of long bones and to what extent the effects are selective for the elastic fibres of the tissue. Twenty-eight new-born chicks' tibiae were incubated for 1 or 3 hours in different experimental conditions (PBS, 30 or 60 units (U)/ml of porcine pancreatic elastase) or immediately formalin fixed. The tibiae were then processed for histo-chemical (Verhoeff and van Gieson stain), immunohistochemical (anti-elastin antibody) and histomorphometric analysis. A decrease of periosteal elastic fibres in all the specimens incubated with elastase in comparison with non incubated specimens was evident. The effect of elastase was easily detectable even at the lower concentration (30 U/ml) and at the shorter time of incubation (1 h). The amount of elastic fibres decreased in accordance with the rise of enzyme levels and incubation time, while periosteal collagen fibre content was not substantially modified by elastase activity. Present data are a prerequisite to evaluate the in vitro and in vivo effects of experimental destruction of periosteal elastic fibres by elastase and to assess the role of these fibres in the growth process of long bones.
Distribution of elastic fiber types in the epiphyseal region.
Three types of elastic fibers have been described: mature elastic fibers, elaunin fibers, and oxytalan fibers. To our knowledge, their location in the immature epiphysis has never been previously reported. The aim of the present study was to use histochemistry, immunohistochemistry, and electron microscopy to demonstrate the distribution of each type of elastic fiber in the epiphyseal region of growing humans and rabbits. Histological samples were collected from the knees of 10 immature New Zealand White rabbits and four children of various ages. The Weigert resorcin-fuchsin, Gomori aldehyde-fuchsin, Verhoeff iron haematoxylin, and Fullmer-Lillie methods were used for histochemistry; anti-elastin monoclonal antibodies, for immunohistochemistry; and tannic acid, uranyl acetate, and lead citrate stain, for transmission electron microscopy analysis. Elastic fibers were detected in the perichondrium, the epiphyseal vessels, and the outer and middle zones of Ranvier's groove. Their orientation was longitudinal in the outer zone and circumferential in the middle zone of the groove. Oxytalan fibrils (i.e., bundles of filaments of 10-12 nm in diameter that do not contain elastin) and elaunin fibers (i.e., filaments that cross discontinuous aggregates of elastin) were more plentiful in the middle zone and decreased with age, whereas mature fibers were more numerous in the outer zone and increased with age. This organization of elastic fibers seems to indicate an age-related process of maturation of the elastic network. The contribution of these fibers to the mechanical properties of the epiphyseal plate and to the growth process remains to be determined.
Subungual exostosis of the foot.
Eleven subungual exostoses of the foot (10 on the hallux, 1 on the third toe) were studied. The initial symptom was subungual pain. When a subungual mass of fibrous tissue appeared, the nail was pushed up and in one case the mass became infected. X-rays exhibited a bone mass protruding from the terminal phalanx on the dorsomedial aspect of the toe in all cases. All patients underwent surgical excision of the lesions with partial onychectomy. Three layers were identified in five cases: a cap of fibrous tissue, a middle zone of hyaline cartilage with enchondral ossification, and a deep zone of cancellous bone. In three other cases, the histological pattern was pleomorphic and poorly characterized. The study shows that most subungual bone masses exhibited the pathological features of conventional osteochondromas. Nonetheless, a small number of lesions were pleomorphic and differed from osteochondromas, with abundant fibrous tissue merging irregularly into scattered islets of cartilage that was not organized in columns. Radical excision of the mass achieved complete relief of symptoms and recovery without recurrences in all cases.
Fetal chondral homografts in the repair of articular cartilage defects.
Lesions of the articular cartilage were bilaterally induced in the patella and the femoral condyle of adult NZW rabbits. A fresh fetal chondral homograft was implanted in one side, the untreated side serving as control. Fetal chondral tissue appeared to survive when implanted at the site of articular injury in adult animals, with no histological evidence of cell-mediated immune response. Fetal chondroblasts showed some signs of maturation and induced endochondral ossification at the base of the graft, with sound anchoring of the graft to the host tissue. In most cases, the homograft fused directly with the adjacent articular cartilage restoring the continuing of the articular surface. However, at implantation the graft could not be easily kept in place, and therefore some grafts had poor mechanical stability. Condylar grafts were incorporated better than patellar grafts. The healing process seemed to be related to the mechanical stability of the graft and to the anatomical site of injury.
No changes of peripheral insulin resistance in polycystic ovary syndrome after long-term reduction of endogenous androgens with leuprolide.
The aim of this study was to investigate the relationship between plasma insulin levels, peripheral insulin sensitivity and androgen secretion in ten patients with polycystic ovary syndrome and in six obese women as compared with six normal-weight control subjects. During a euglycemic-hyper-insulinemic clamp no significant change of testosterone, androstenedione or dehydroepiandrosterone sulfate plasma levels was observed in the two groups of patients or in the control subjects; insulin sensitivity was clearly reduced and was similar in polycystic ovary patients and in obese women, in spite of the different plasma androgen levels. A long-term (5 months) androgen suppression with the gonadotropin-releasing hormone agonist leuprolide was not able to improve significantly the insulin sensitivity. These results demonstrate that the short-term hyperinsulinemia achieved with the clamp technique does not affect androgen secretion and that insulin resistance, measured with the same technique, is not influenced by long-term suppression of plasma androgen levels in polycystic ovary syndrome.
No changes of insulin sensitivity in cystic fibrosis patients with different degrees of glucose tolerance: an epidemiological and longitudinal study.
Plasma glucose and insulin responses to oral glucose and insulin sensitivity by the euglycemic hyperinsulinemic clamp technique were investigated in 30 cystic fibrosis patients with normal fasting blood glucose levels and normal (N = 12), impaired (N = 12) or diabetic (N = 6) glucose tolerance, and in 12 control subjects. In a subgroup of 10 cystic fibrosis patients with non-diabetic glucose tolerance both oral glucose tolerance test and clamp were performed again 48-52 months later. Following oral glucose, glycemic responses were higher in cystic fibrosis patients than in controls, whereas insulin responses were reduced significantly only in the patients with diabetic glucose tolerance. Insulin sensitivity did not differ significantly in the patient subgroups with different degrees of glucose tolerance and in controls. In the 10 patients who underwent a 4-year follow-up, insulin responses to oral glucose decreased significantly, whilst insulin sensitivity did not change substantially. Insulin sensitivity persisted unmodified even in the patients with deteriorating glucose tolerance. No correlation were observed between metabolic data and clinical status of patients. In conclusion, in cystic fibrosis subjects with fasting euglycemia and different degrees of glucose tolerance: (i) insulin sensitivity is not impaired; (ii) eventually changes of glucose tolerance over time are not associated with modifications of insulin sensitivity; (iii) insulin secretion deteriorates over time even in the patients with stable glucose tolerance; (iv) eventual deterioration of both glucose tolerance and insulin secretion is not linked to a worsening of either nutritional or clinical parameters.
Age-related distribution of elastic fibers in the rabbit knee.
The distribution and histochemical properties of elastic fibers in the knee of newborn, young, adult, and old New Zealand White rabbits were studied. In the growing rabbit, elastic fibers, especially oxytalan fibers, are abundant in the perichondrium and the fibrous layer of the periosteum. They are homogeneously distributed in tendons, ligaments, and menisci, and are absent in the epiphyseal cartilage and growth plate and at the enthesis. In mature rabbits, the articular capsule, the synovial membrane, the peritenon, the periosteum, the perimysium, and the perivascular connective tissue are rich with elastic fibers. They are rarer in the tendon and are absent at the enthesis, in bone and articular cartilage. In the meniscus, the fibers are distributed centripetally. The distribution of elastic fibers is probably related to the different functional role and biomechanical behavior of each tissue. Finally, the type of elastic fiber in the various tissues changes with age; younger animals have more oxytalan fibers than adult animals. A gradual process of maturation and specialization of the network of elastic fibers takes place with age.
The effect of intra-articular ACL reconstruction on the growth plates of rabbits.
We performed intra-articular reconstruction of the anterior cruciate ligament (ACL) with the semitendinosus tendon placed in 2 mm diameter tunnels in 21 skeletally immature rabbits. The operation caused 11% damage to the physis of the femur on the frontal plane and 3% of its cross-sectional area but no alteration of growth or axial deviation of the bone resulted. In the tibia, the operation caused 12% damage to the physis in the frontal plane and 4% of the cross-sectional area. Two tibiae developed valgus deformities and one was shortened. Histological examination showed no areas of epiphysiodesis. There was no abnormality of growth-plate thickness in the two cases of tibia valga. Osseous metaplasia in the grafted tendons did not occur. The results suggest the need for careful evaluation of the percentage of damage to the growth plate before using intra-articular methods for reconstruction of the anterior cruciate ligament in adolescents.