Maurizio Sabbatini

In vitro mechanical compression induces apoptosis and regulates cytokines release in hypertrophic scars.

Hypertrophic scars resulting from severe burns are usually treated by continuous elastic compression. Although pressure therapy reaches success rates of 60–85% its mechanisms of action are still poorly understood. In this study, apoptosis induction and release of interleukin‐1β (IL‐1β) and tumor necrosis factor‐α (TNF‐α) were evaluated in normal (n = 3) and hypertrophic (=7) scars from burns after in vitro mechanical compression. In the absence of compression (basal condition) apoptotic cells, scored using terminal deoxyribonucleotidyl transferase assay, were present after 24 hours in the derma of both normal scar (23 ± 0.4% of total cell) and hypertrophic scar (11.3 ± 1.4%). Mechanical compression (constant pressure of 35 mmHg for 24 hours) increased apoptotic cell percentage both in normal scar (29.5 ± 0.4%) and hypertrophic scar (29 ± 1.7%). IL‐1β released in the medium was undetectable in normal scar under basal conditions while in hypertrophic scar the IL‐1β concentration was 3.48 ± 0.2 ng/g. Compression in hypertrophic scar‐induced secretion of IL‐1β twofold higher compared to basal condition. (7.72 ± 0.2 ng/g). TNF‐α basal concentration measured in normal scar medium was 8.52 ± 4.01 ng/g and compression did not altered TNF‐α release (12.86 ± 7.84 ng/g). TNF‐α basal release was significantly higher in hypertrophic scar (14.74 ± 1.42 ng/g) compared to normal scar samples and TNF‐α secretion was diminished (3.52 ± 0.97 ng/g) after compression. In conclusion, in our in vitro model, mechanical compression resembling the clinical use of elastocompression was able to strongly increase apoptosis in the hypertrophic scar derma as observed during granulation tissue regression in normal wound healing. Moreover, the observed modulation of IL‐1β and TNF‐α release by mechanical loading could play a key role in hypertrophy regression induced by elastocompression. (WOUND REP REG 2003;11:331–336)

The pattern of c-Fos immunoreactivity in the hindbrain of the rat following stomach distension

It has been previously shown that the walls of the stomach contain vagal and splanchnic afferents, connected to low and high threshold (LT and HT) gastric receptors, that convey physiological and noxious information to areas of the hindbrain involved mainly in the control of gastrointestinal function. Because distension of the stomach also reflexly increases the sympathetic drive to the cardiovascular system, the present study was planned to examine the pattern of activation of all nuclei encountered throughout the hindbrain in response to gastric distension. In anaesthetized rats, the stimulus was controlled by employing different transmural pressures and frequencies of distension, and c-Fos immunohistochemistry was used to characterize neuronal activation. Low intensity stimulation induced c-Fos expression in the cranial part of nucleus of solitary tract (NTS), the nucleus ambiguus (NA), the lateral reticular area (LRt) and the ventrolateral medulla (RVL/CVL). At low frequency of stimulation c-Fos positive nuclei (p.n.) were found in NTS only. At high frequency of stimulation an increase in c-Fos immunoreactivity was found. High intensity stimulation induced c-Fos expression in area postrema (AP), the lateral vestibular nucleus (LVe) and the caudal part of the NTS. At low frequency, only the number of c-Fos p.n. was increased. Increasing the frequency of stimulation induced c-Fos expression in further nuclei such as the parabrachial nucleus (PBN), the inferior olive subnuclei (IOn), the oral part of spinal trigeminal nucleus (Sp5O) and locus coeruleus (LC). At higher frequencies c-Fos immunoreactivity decreased in NTS and LRt, disappeared in VLM and increased in NA. Thus stomach distension activated several neuronal excitatory and inhibitory circuits that are involved in the control of gastrointestinal function as well as in cardiovascular, respiratory and pain regulation. The differences in c-Fos immunoreactivity induced by changing the distension patterns suggested interactions between groups of vagal and splanchnic afferents.

Effect of in vitro mechanical compression on Epilysin (matrix metalloproteinase-28) expression in hypertrophic scars

Epilysin, designated matrix metalloproteinase (MMP)‐28, is the newest member of this family of proteases expressed by keratinocytes in response to an injury. MMP‐28′s physiological role and specific substrates are unknown, but its expression pattern suggests that it may serve a role in both tissue homeostasis and wound healing. The aim of this preliminary study was to observe the presence of MMP‐28 protein in normotrophic and hypertrophic scars and to evaluate the effect of in vitro mechanical compression on its expression. Biopsies from normotrophic and hypertrophic scars resulting from burns were divided into two samples, one to be used as control (uncompressed) and the other to be compressed in an oxygenated organ chamber for 24 hours in the presence of a serum‐free medium, using an electromechanical load transducer (stable pressure = 35 mmHg). Analysis of MMP‐28 protein secretion, assessed by Western blot and β‐casein zymography in scar conditioned media, revealed that normotrophic scar did not release MMP‐28 in any condition while hypertrophic scar released active MMP‐28 both in control conditions and after compression. MMP‐28 immunohistochemistry revealed a light protein presence in normotrophic scar keratinocytes and a strong MMP‐28 positivity in hypertrophic scar keratinocytes in control conditions, while compression increased MMP‐28 staining in normotrophic scar and induced a significant reduction of the protein presence in hypertrophic scar keratinocytes. As it has been suggested that MMP‐28 may restructure the skin basal membrane (Saarialho‐Kere et al., 2002), our data indicate that mechanical compression directly acts to modulate the remodeling phase of wound healing, altering release and activity of MMP‐28 in hypertrophic scars.

Activation of caspase-8 triggers anoikis in human neuroblastoma cells

Cells require appropriate interaction with extracellular matrix proteins mediated by integrins to grow, differentiate and survive. Many cell types including nervous cells undergo anoikis, a substrate-dependent apoptosis, when adhesion is impaired. Resistance of tumors to cytotoxic drugs is probably due to disturbed apoptosis programs. The proteolytic enzymes caspases are the main executioners of apoptosis. It was reported that caspase-8 expression is deficient in some neuroblastoma cells. We demonstrated that human neuroblastoma cell line SK-B-BE, differentiated with retinoic acid, expressed caspases 3, 8 and 9. Caspases 8 and 3, but not caspase-9 were activated in SK-N-BE cells cultured in suspension or on aspecific adhesive substrate. Cell positive to caspase-8 were classified into four stages, by morphometric and densitometric parameters. The use of the specific caspase-8 inhibitor Z-IETD-FMK dramatically reduced apoptosis, demonstrating that caspase-8 is the upstream initiator caspase during SK-N-BE cells anoikis. Among matrix proteins, type I collagen is the most effective and fibronectin the least in delaying anoikis. The activation of caspases 8 and 3 by unligated integrins was dependent on the state of neuronal differentiation, since the most differentiated cell was the most vulnerable to anoikis. These data show that activation of caspase-8 is specifically required to promote anoikis in SK-N-BE neuroblastoma cells.

Effects and differentiation activity of IGF-I, IGF-II, insulin and preptin on human primary bone cells

The importance of the complex interrelated regulatory pathways involving IGF factors and pancreatic hormones can be observed in several metabolic diseases, where the deregulation of these factors has a wide impact on bone health. These findings have stimulated us to compare the effect of IGF-I, IGF-II, insulin and preptin on human bone cells. The effect on cell differentiation and cell activity of osteoblasts and osteoclasts has been analysed. We have observed a significant effect by IGF-I, a modest effect by IGF-II and preptin and no effect after insulin administration on human primary osteoblast-like cells. All studied factors have shown an induction on human primary osteoclast differentiation and bone resorption activity, with IGF-I being the most potent factor. We hypothesize that these findings may be on the basis of decreased bone mass density observed in several diseases.

Analysis of nerve supply pattern in human lymphatic vessels of young and old men.

BACKGROUND The present work deals with innervation patterns along collector lymphatic vessels from cervical, mesenteric, and femoral regions, and lymph capillaries in young and elderly subjects. METHODS AND RESULTS Morphological and morphometric analysis of nerve fibers along lymph vessels was performed by immunohistochemistry for PGP 9.5, NPY, TH, ChAT, VIP, SP, and dopamine. Nerves containing NPY and TH were frequent, whereas immunoreactivity for ChAT and VIP were few. SP-positive fibers were widely distributed in the medial and endothelial layers. Dopamine neurotransmitters were observed in a few short nerve fibers. A more diffuse presence of nerve fibers in mesenteric and femoral lymph vessels, compared to cervical ones, was detected. In lymph capillary vessels, a few nerve fibers positive for neuropeptides and neurotransmitters were detected, whereas no dopamine and VIP immunoreactive fibers were detected. A wide reduction of all specific nerve fibers analyzed was detected in lymph vessels from elderly subjects. CONCLUSIONS The presence on lymph vessels of sympathetic and parasympathetic nerve systems can be declared. The differences observed in lymphatic vessel innervation patterns may note the involvement in lymph flow regulation, calling attention in aging, when nerve fibers reduction may cause functional default of lymph vessels.

Cardiovascular effects and c-Fos expression in the rat hindbrain in response to innocuous stomach distension.

The present work was planned to study the effects of non-noxious gastric distension on hemodynamic variables and on cardiovascular hindbrain areas detected by means of c-Fos immunoreactivity, to determine the afferent and central mechanisms involved. In anesthetized rats, innocuous stomach distension increased arterial blood pressure and heart rate and induced c-Fos immunoreactivity within nucleus tractus solitarii, nucleus ambiguus, ventrolateral medulla and lateral reticular nucleus. Bilateral vagotomy abolished the pressor response and c-Fos immunoreactivity in nucleus ambiguus and ventrolateral medulla. Also, c-Fos immunoreactivity was significantly decreased in nucleus tractus solitarii and lateral reticular nucleus. After bilateral splanchnicotomy the pressor and tachycardic responses caused by gastric distension were reduced. c-Fos immunoreactivity in nucleus tractus solitarii, lateral reticular nucleus and nucleus ambiguus was reduced in comparison to the intact rats. In ventrolateral medulla a preferential localization of c-Fos immunoreactivity was found within its caudal portion. It was shown that such gastric distension, known to activate low threshold mechanoreceptors, induced cardiovascular effects via both vagal and splanchnic afferents and involving their central convergence and interaction in modulating the baroreceptor buffer system.

Fibroblast apoptosis and caspase-8 activation in aseptic loosening.

The presence of apoptosis has been investigated in the interface membranes collected during revision surgery of loosened total hip joint arthroplasty (THAs). Terminal deoxyrobonucleotidyl transferase (TdT) assay for apoptotic DNA fragmentation quantification revealed a statistically significant presence of apoptosis in aseptic samples, obtained from both cementless (2.37+/-0.6%) and cemented (12.01+/-1%) prosthesis compared to septic samples where apoptosis was almost absent. Activated caspase-8 immunostaining was almost undetectable in septic samples, while in the aseptic samples active caspase-8 was present weakly in the cementless samples (1.35+/-0.22%) and strongly in the cemented ones (9.0+/-0.40%). The caspase-8 cytoplasmatic staining allowed the morphological recognition of positive cells both as fibroblast-like and immunocompetent cells. In aseptic cemented samples fibroblast-like cells were the most represented subpopulation in the caspase-8 positive population scored (76.6%) compared to the immunocompetent cells (23.4%). Caspase-8 activation is an upstream event in the apoptotic pathway triggered by the activation of cytokines receptors such as TNF-alpha receptor 1 (TNFR-1), and the presence of caspase-8 activation in fibroblast-like cells in the aseptic interface membranes of THAs suggests a possible TNF-alpha dependent apoptosis.

Alveolar bone regeneration in post-extraction socket: A review of materials to postpone dental implant

Tooth extraction usually involves alveolar bone loss and reduction in height and width of the remaining alveolar socket, owing to the physiological bone resorption. This occurrence may perform an inadequate bone profile, that make difficult orthodontic applications, compromising the functional and aesthetic restoration of dental implants. The present review will provide an update on the biological and clinical profile of materials currently in use and those under investigation, in the recovering of bone margins of edentulous sockets.

Overstressed mechanical stretching activates survival and apoptotic signals in fibroblasts.

The interest of scientists in the effects of mechanical stresses on cells is growing, in order to reproduce and understand cell behaviour in an environment closely reproducing physiological conditions. There have been many studies showing that mechanical stimulations are involved in regulating the proliferation, apoptosis and synthesis of proteins and cell morphology. In this study, we have considered the effects of a 20% stretching mechanical stress on MRC5 lung fibroblast cells in order to verify the role of survival/apoptotic pathways. As a survival pathway, the activation of Akt has been studied in association with pro-apoptotic or anti-apoptotic signals such as the Bax/Bcl-2 ratio and cleavage of caspases 3 and 9. Findings have shown the effects of overstressed cellular stretching to be a balance of a cause-and-effect reaction between survival and apoptosis.