Treatment with synchronized radiofrequency and facial muscle stimulation: Histologic analysis of human skin for changes in collagen and elastin fibers.

BACKGROUND: Skin's exposure to intrinsic and extrinsic factors causes age-related changes, leading to a lower amount of dermal collagen and elastin. AIM: This study investigated the effects of a novel facial muscle stimulation technology combined with radiofrequency (RF) heating on dermal collagen and elastin content for the treatment of facial wrinkles and skin laxity. METHODS: The active group subjects (N = 6) received four 20-min facial treatments with simultaneous RF and facial muscle stimulation, once weekly. The control subject (N = 1) was untreated. Skin biopsies obtained at baseline, 1-month and 3-month follow-up were evaluated histologically to determine collagen and elastin fibers content. A group of independent aestheticians evaluated facial skin appearance and wrinkle severity. Patient safety was followed. RESULTS: In the active group, collagen-occupied area reached 11.91 ± 1.80 × 106 µm2 (+25.32%, p < 0.05) and 12.35 ± 1.44 × 105 µm2 (+30.00%, p < 0.05) at 1-month and 3-month follow-up visits. Elastin-occupied area at 1-month and 3-month follow-up was 1.64 ± 0.14 × 105 µm2 (+67.23%, p < 0.05), and 1.99 ± 0.21 × 105 µm2 (+102.80%, p < 0.05). In the control group, there was no significant difference (p > 0.05) in collagen and elastin fibers. Active group wrinkle scores decreased from 5 (moderate, class II) to 3 (mild, class I). All subjects, except the control, improved in appearance posttreatment. No adverse events or side effects occurred. CONCLUSION: Decreased dermal collagen and elastin levels contributes to a gradual decline in skin elasticity, leading to facial wrinkles and unfirm skin. Study results showed noticeable improvement in facial appearance and increased dermal collagen and elastin content subsequent to simultaneous, noninvasive RF, and facial muscle stimulation treatments.

Upregulation of Skin-Aging Biomarkers in Aged NHDF Cells by a Sucrose Ester Extract from the Agroindustrial Waste of Physalis peruviana Calyces.

As part of a search for new sustainable plant sources of valuable compounds, the EtOAc extract of the discarded calyces of Physalis peruviana fruit was selected for its significant antiaging activity. Eight new sucrose esters (SEs), named peruvioses F-M (1-8), along with three known SEs, peruvioses A (9), peruviose B (10), and nicandrose D (11), were isolated. Their structures were elucidated by comprehensive analyses of their NMR and MS data. A global fragmentation pattern of these SEs was established from their MS data. The SE extract (SEE) at a concentration of 0.5 mg L-1 upregulated multiple skin-aging biomarkers, namely, collagen I, elastin, and fibrillin-1, in aged normal human dermal fibroblast cells. A 36% increase in collagen I was observed. The elastin and fibrillin-1 contents were fully recovered, and an increase of at least 10% in the production of elastin was observed.

Humanizing Miniature Hearts through 4-Flow Cannulation Perfusion Decellularization and Recellularization.

Despite improvements in pre-clinical drug testing models, predictability of clinical outcomes continues to be inadequate and costly due to poor evidence of drug metabolism. Humanized miniature organs integrating decellularized rodent organs with tissue specific cells are translational models that can provide further physiological understanding and evidence. Here, we evaluated 4-Flow cannulated rat hearts as the fundamental humanized organ model for cardiovascular drug validation. Results show clearance of cellular components in all chambers in 4-Flow hearts with efficient perfusion into both coronary arteries and cardiac veins. Furthermore, material characterization depicts preserved organization and content of important matrix proteins such as collagens, laminin, and elastin. With access to the complete vascular network, different human cell types were delivered to show spatial distribution and integration into the matrix under perfusion for up to three weeks. The feature of 4-Flow cannulation is the preservation of whole heart conformity enabling ventricular pacing via the pulmonary vein as demonstrated by noninvasive monitoring with fluid pressure and ultrasound imaging. Consequently, 4-Flow hearts surmounting organ mimicry challenges with intact complexity in vasculature and mechanical compliance of the whole organ providing an ideal platform for improving pre-clinical drug validation in addition to understanding cardiovascular diseases.

Efficacy of mesotherapy in facial rejuvenation: a histological and immunohistochemical evaluation.

BACKGROUND: Mesotherapy, commonly known as "biorejuvenation" or "biorevitalization", is a technique used to rejuvenate the skin by means of a transdermal injection of a multivitamin solution and natural plant extracts that are thought to improve the signs of skin aging. OBJECTIVES: This prospective study aimed to evaluate the clinical effect of mesotherapy applied to periorbital wrinkles and to quantitatively evaluate histological changes in the skin occurring in response to the same treatment. METHODS: Six volunteers with Fitzpatrick skin types III or IV and Glogau class I-III wrinkles were subjected to a three-month course of mesotherapy injections in the periocular area (six sessions administered at two-week intervals). Standard photographs and skin biopsies were obtained from the treatment area at baseline, at the end of treatment, and at three months post-treatment. Quantitative evaluation of collagen types I, III, and VII, newly synthesized collagen, total elastin, and tropoelastin was performed using a computerized morphometric analysis. RESULTS: The clinical evaluation of volunteers at baseline, end of treatment, and three months post-treatment revealed no significant differences. Histological and immunostaining analysis of collagen types I, III, and VII, newly synthesized collagen, total elastin, and tropoelastin showed no statistically significant changes (P > 0.05) after mesotherapy injection. CONCLUSIONS: The present study indicates that mesotherapy for skin rejuvenation does not result in statistically significant histological changes or clinical improvement.

Changes in the composition of the thoracic aortic wall in spontaneously hypertensive rats treated with losartan or spironolactone.

1. In the present study, we compared the elastin and collagen content of thoracic aortic medial and adventitial layers from Wistar-kyoto (WKY) and spontaneously hypertensive rats (SHR). In addition, the effects of losartan, an angiotensin II receptor antagonist, and spironolactone, a mineralocorticoid receptor antagonist, on collagen and elastin content were determined. 2. Prehypertensive (4-week-old) and hypertensive (16-week-old) SHR were randomly divided into three groups treated with either 0.9% NaCl, losartan (20 mg/kg per day) or spironolactone (200 mg/kg per day). Prehypertensive and hypertensive SHR were treated for 12 and 16 weeks, respectively. Age-matched WKY rats were not treated with NaCl, losartan or spironolactone and served as the control group. 3. The medial and adventitial layers of the thoracic aorta were composed mainly of elastin and collagen, respectively, in both SHR and WKY rats. Compared with WKY rats, SHR exhibited greater collagen and elastin content in the media, but decreased collagen and elastin content in the adventitial layer. Both medial and adventitial collagen and elastin content increased significantly with age in both strains and was greater in 32-week-old rats compared with 16-week-old rats. Spironolactone treatment decreased collagen content in the media of thoracic aortas from prehypertensive SHR, whereas losartan decreased collagen content in the media of aortas from hypertensive SHR. In contrast, neither spironolactone nor losartan had any effect on adventitial collagen content in prehypertensive and hypertensive SHR. Medial collagen and elastin were positively related to pulse pressure (PP), but there was no correlation between adventitial mass or collagen content and PP or mean arterial pressure in untreated and treated SHR and WKY rats. 4. In conclusion, the composition of the medial and adventitial layers of the thoracic aorta differs and treatment of SHR with losartan and spironolactone decreases collagen content when delivered at the hypertensive or prehypertensive stage, respectively. However, neither drug has any effect on adventitial collagen content in SHR.

Marginal vitamin A deficiency affects lung maturation in rats from prenatal to adult stage.

UNLABELLED: Mild or marginal vitamin A deficiency (MVAD) is still a serious and widespread public health problem in pregnant women and children in developing countries. This study investigated rat lung maturation from prenatal to adult stage during pregnancy and postnatal MVAD and the recovery after postnatal vitamin A supplementation (VAS). Adult female rats and their offspring were randomized into three groups. 1. CONTROL: the mothers and offspring received a normal diet.2. MVAD: The mothers and offspring received a MVAD diet.3. VAS: the mothers received MVAD diet till parturition, and then received the normal diet. The offspring of the VAS group were given low-dose vitamin A from postnatal day 1 to day 7 and received the normal diet after weaning. The lung development, structure, and collagen and elastic fiber of offspring were monitored by morphometric analysis at age 1 d, 2 and 8 wk, respectively. Lower body weight, lung weight, reduced numbers of alveoli and total alveolar surface area as well as increased alveoli septa thickness was observed in MVAD compared to that in the control animals. Increased collagen deposits and decreasing elastic fiber were found in MVAD rats. However, all of these were significantly improved in VAS-treated animals. These data suggest that the rat lung is sensitive to MVAD during the developing stage. Early postnatal vitamin A supplementation can partially restore the normal lung structure.

Biomimetic regeneration of elastin matrices using hyaluronan and copper ion cues.

Current efforts to tissue engineer elastin-rich vascular constructs and grafts are limited because of the poor elastogenesis of adult vascular smooth muscle cells (SMCs) and the unavailability of appropriate cues to upregulate and enhance cross-linking of elastin precursors (tropoelastin) into organized, mature elastin fibers. We earlier showed that hyaluronan (HA) fragments greatly enhance tropo- and matrix-elastin synthesis by SMCs, although the yield of matrix elastin is low. To improve matrix yields, here we investigate the benefits of adding copper (Cu(2+)) ions (0.01 M and 0.1 M), concurrent with HA (756-2000 kDa), to enhance lysyl oxidase (LOX)-mediated elastin cross-linking machinery. Although absolute elastin amounts in test groups were not different from those in controls, on a per-cell basis, 0.1 M of Cu(2+) ions slowed cell proliferation (5.6 +/- 2.3-fold increase over 21 days vs 22.9 +/- 4.2-fold for non-additive controls), stimulated synthesis of collagen (4.1 +/- 0.4-fold), tropoelastin (4.1 +/- 0.05-fold) and cross-linked matrix elastin (4.2 +/- 0.7-fold). LOX protein synthesis increased 2.5 times in the presence of 0.1 M of Cu(2+) ions, and these trends were maintained even in the presence of HA fragments, although LOX functional activity remained unchanged in all cases. The abundance of elastin and LOX in cell layers cultured with 0.1 M of Cu(2+) ions and HA fragments was qualitatively confirmed using immunoflourescence. Scanning electron microscopy images showed that SMC cultures supplemented with 0.1 M of Cu(2+) ions and HA oligomers and large fragments exhibited better deposition of mature elastic fibers ( approximately 1 mum diameter). However, 0.01 M of Cu(2+) ions did not have any beneficial effect on elastin regeneration. In conclusion, the results suggest that supplying 0.1 M of Cu(2+) ions to SMCs to concurrently (a) enhance per-cell yield of elastin matrix while allowing cells to remain viable and synthetic and not density-arrested in long-term culture because of their moderating effects on otherwise rapid cell proliferation and (b) provide additional benefits of enhanced elastin fiber formation and cross-linking within these tissue-engineered constructs.

[Effects of rejuvenation by intense pulsed light and basic mechanism thereof].

OBJECTIVE: To evaluate the effects of rejuvenation by intense pulsed light (IPL) and the mechanism thereof. METHODS: Fifty-eight patients with photo aging were treated with IPL of single, double, or triple pulse pattern for 3 - 5 times with the intervals of 3 - 4 weeks. Three weeks after the last treatment, photography was conducted and the pictures underwent grading by the physicians and patients according to blind method. Skin specimens of the posteroinferior ear lobe or the nape were obtained from 4 patients to undergo HE staining, Uana orcein staining of elastin, immunohistochemical staining for collagenous fibers of types I and III, and transmission electron microscopy was conducted in 2 of the 4 patients. Skin digitalized image analysis was conducted on 34 female patients to measure and analyze the depth and width of dermatographs, roughness of skin. RESULTS: After the third treatment, the wrinkles and skin texture of 62.1% of the patients showed improvement, and 84.60% of the pigmented lesions and 81.25% of the vascular lesions showed improvement. Pathology showed that type I and type III collagen increased while elastin decreased, and the fibers were orderly re-arranged. Transmission electron microscopy showed that after treatment the fibroblasts increased in number and became more active in secretion and there were more collagen fibers orderly re-arranged in the stroma. Digitalized image analysis showed significant improvement in skin smoothness, depth, arithmetic average roughness and average roughness of skin texture (all P < 0.01). CONCLUSION: IPL is effective to improve the skin texture. The mechanism may be the increasing of the activity of the fibroblasts, and hyperplasia and re-arrangement of collagen and elastin.

Retinoids increase lung elastin expression but fail to alter morphology or angiogenesis genes in premature ventilated baboons.

Retinoids regulate elastin synthesis by alveolar myofibroblasts and affect angiogenesis pathways, both of which are processes critical for alveolar development. Retinoids accelerate alveolarization in rodents and are now used therapeutically in premature infants at risk of bronchopulmonary dysplasia (BPD). This study examined the effects of retinoid supplementation on alveolar elastin expression and deposition and angiogenesis-related signaling in a primate model of BPD. Premature baboons delivered at 125 d of gestation after maternal steroid treatment were given surfactant and ventilated with minimal supplemental oxygen for 14 d with (n = 5) and without (n = 5) supplemental vitamin A (5000 U/kg/d) and compared with 140-d unventilated controls. Ventilatory efficiency index (VEI) and oxygenation index (OI) were not statistically different between ventilated treatment groups. Expression of vascular endothelial growth factor A (VEGF-A), fms-related tyrosine kinase 1 (Flt-1), and tyrosine kinase with immunoglobulin-like and EGF-like domains 1 (TIE-1) was repressed by premature delivery and mechanical ventilation and was not altered by retinoid supplementation. Retinoid supplementation did not enhance alveolar angiogenesis. Elastin expression was repressed by premature delivery and extended ventilation, and retinoid supplementation increased elastin expression specifically in alveolar myofibroblasts within alveolar walls. These results suggest that the small decrease in mortality among premature infants receiving retinoid supplementation may not be mediated through enhanced alveolar development.

[Qualitative change in the elastin from the calcified portion of human artery].

To examine the qualitative changes of elastin and the aorta related to calcification of human arteries, biochemical properties were measured, including calcium (Ca), phosphorus (P) and magnesium (Mg) contents in the aorta or in the elastin fraction in calcification, cholesterol content in atherosclerosis, desmosine content of cross-link, free thiol contents (free SH/total SH) and hydrophobic properties in the elastin fraction from the calcified portion, adjacent sites and another normal artery. The results from different sites of the calcified abdominal artery are as follows: The contents of Ca, P and Mg in aorta and the elastin fraction from the calcification site were higher than those at other sites. Moreover, Ca in the aorta and elastin fraction correlated positively with P and Mg. The content of cholesterol in the calcification site was the same as at other sites and did not correlate with Ca, P or Mg. The content of desmosine in the calcification site was significantly lower than that in different sites. In addition, its content was negatively associated with Ca and P in the elastin fraction and with the aortic Mg. The content of free thiol in the calcification site was similar to the other sites and correlated negatively with Ca and P in the aorta. The hydrophobicity in the calcification was similar to that at other sites, and was negatively associated with Ca and Mg in the elastin fraction.

Elastin-associated microfibrils (10 nm) in a three-dimensional fibroblast culture.

The purpose of this study is to present a three-dimensional dermal fibroblast model. Skin fibroblasts cultured in this system deposit large amounts of collagen and microfibrils. Fibroblasts were seeded onto a nylon filtration mesh and incubated in the presence or absence of ascorbic acid. Collagen fibril formation was found in the presence of ascorbic acid whereas microfibril formation was seen independent of ascorbic acid supplementation. Immunoelectron microscopy revealed that microfibrils were labeled with fibrillin at 67 nm periodicity. Isolated microfibrils studied by rotary shadowing had a beaded appearance consisting of beads linked to each other by a filamentous structure. The spaces between the beads ranged from 10.00-33.33 nm, suggesting that these microfibrils may have an extension-contraction mechanism. Furthermore, the size and spacing of the beads were similar to that seen in microfibrils from tissues (measured after rotary shadowing). Fibroblasts cultured in a three-dimensional mesh represent an effective in vitro model with which to study microfibril formation.

Collagen and elastin changes in D-penicillamine-induced pseudoxanthoma elasticum-like skin.

A patient with cystinuria who was treated with large doses of D-penicillamine for 19 years developed skin abnormalities resembling those seen in pseudoxanthoma elasticum. Biochemical and histological examination of the dermis showed that the collagen content, the ratio of the major genetic forms of collagen and the distribution of collagen types was normal. Light microscopy demonstrated the presence of vastly increased amounts of elastin in the dermis, and the individual elastin fibres were shown by electron microscopy to be abnormal; chemical analysis showed the elastin to be poorly cross-linked. Some of the collagen also appeared structurally abnormal, and biochemically resembled that seen in the dermis of a young child with respect to cross-linking and hexosyl-lysine content. The therapy led to an increased deposition of collagen and elastin fibres which appeared abnormal, and resulted in an increase in total skin surface area. These data indicate that D-penicillamine was not fully effective in inhibiting collagen and elastin cross-linking, and appeared to prevent or inhibit the natural maturation process of the collagen.

Biochemical defect in chick lung resulting from copper deficiency.

Lung tissue from groups of chicks raised 23 d on diets deficient in copper had a higher proportion of 0.15 M NaCl-extractable collagen but lower aldehyde-reacting compounds and elastin than controls the same age. Although the lungs from deficient chicks weighed less, the ratio of lung to body weight for the two groups stayed relatively constant throughout the 23-d period. Lysyl oxidase activity in lungs of deficient chicks was strongly suppressed. Feeding the deficient chicks the diet with copper supplements restored lysyl oxidase activity in lung. This showed the close and specific control of lung lysyl oxidase by copper. The impairment in cross-linking through lysyl oxidase failure is implied from these studies. Clearly, copper is needed for the normal development of lung.

Serum testosterone, testicular connective tissue protein and testicular histology in rats treated with hyperbaric oxygen.

This study was done to ascertain whether hyperbaric oxygenation really affects testicular activity in rats. We evaluated serum testosterone concentration, testicular content of connective tissue protein and histological analysis of testes in each rat treated with 2 or 3 atmospheric pressures associated with oxygen saturation, together or singly for 90 minutes daily for 58 days. There were no significant differences in serum testosterone concentrations between the examined groups. The amounts of collagen, non-collagenous protein or elastin in testis were similar in all groups except one set of determinations. In addition, no prominent histological differences were noted in the testes of the animals. Thus, the pressure environment of this study, which can be regarded as almost the same as that of the hyperbaric oxygenation chamber in clinical use, appears to be nontoxic to rat testes.

Lung collagen and elastin after ozone exposure in vitamin B-6-deficient rats.

The effects of vitamin B-6 deficiency and ozone exposure on selected features of connective tissue metabolism in lung were investigated in groups of weanling male rats fed one of three diets: B-6-supplemented, fed ad lib; B-6-deficient, fed ad lib; or B-6-supplemented, restricted to the food intake of deficient rats for 5 weeks. Also, perinatal rat pups were studied that were nursed from dams fed one of the 3 diets from parturition to day 15 of lactation. During the final week of each experiment, half of the rats in each of the groups were exposed to 0.64 ppm of ozone (23.5 h per day). The collagen and elastin content, collagen synthesis rate, total protein synthesis rate, and lysyloxidase activity of lungs were measured. Perinatal pups rendered vitamin B-6-deficient were particularly sensitive to ozone exposure (65% died as compared to fewer than 5% of the ad lib or food-restricted controls). When L-proline incorporation into collagen and total protein was investigated using lung minces, food restriction and B-6-deficiency resulted in about one-half the incorporation normally observed. Total lung lysyl oxidase activity was also decreased in B-6-deficient and food-restricted rats compared to B-6-supplemented rats fed ad lib. Exposure to ozone resulted in increased lysyl oxidase activity and collagen synthesis in lungs from B-6-supplemented rats, but such responses were not observed in B-6-deficient or food-restricted (FR) rats exposed to ozone.

A routine method for contrasting elastin at the ultrastructural level.

A reliable, simple, and inexpensive method for ultrastructural investigation of elastin is described. This method uses uranyl acetate dissolved in absolute methanol, followed by an optional lead citrate counterstain. The procedure was tested on a number of animal and human tissues that had been fixed and processed differently.

A copper-deficient, zinc-supplemented diet produces emphysema in pigs.

A mild form of emphysema was produced in pigs raised on a copper-deficient, zinc-supplemented diet. The copper-requiring enzyme, lysyl oxidase, catalyzes the cross-linking of tropoelastin into mature elastin. Zinc further inhibits the activity of lysyl oxidase. Lungs from animals raised on copper-deficient, zinc-supplemented diets of demonstrate perforations in alveolar walls and diminished amounts of elastin bronchi and pulmonary arteries. Mean linear intercepts are greater and alveolar internal surface areas are less than those in control animals, fulfilling the generally accepted definition of emphysema. Physiologic confirmation is provided by a leftward shift of the saline volume-pressure curves when compared with those in control animals. Ultrastructurally, the alveolar walls are effaced and pores of Kohn are enlarged. There are areas in which elastin is absent leaving remnant microfibrils, and there are other changes consistent with active elastin synthesis. Biochemical data demonstrate no difference in elastin content as micrograms/ml of fat-free dry weight but do demonstrate increased collagen content in experimental animal lungs compared with that in control lungs. Ultrastructural similarities to enzyme-induced models of emphysema suggest the presence of elastin degradation in our model. We speculate that although the copper-deficient, zinc-supplemented state may stimulate protein synthesis in general, elastin is being degraded by endogenous means, but collagen is not.

[Proportions of various types of collagen present in medium-sized arteries]. / Proportions des différents types de collagène présents dans les artères de taille moyenne.

A technique using alternatively dithiothreitol reduction, sonication and pepsin digestions permits the extraction of 75% of the insoluble collagen from human common iliac arteries. The pattern of the collagen types obtained by acrylamide gel electrophoresis is far different from that of the aortic wall: type I averages 44 +/- 14% of the total, type III 48.5 +/- 14% and a supplementary fraction, probably belonging to type V, 9 +/- 5% of the total in females and 6 +/- 2% in males.

Copper salts for growth stimulation and alleviation of aortic rupture losses in turkeys.

Approximately 4000 Large White, day-old turkey poults were used in a series of eight experiments to study the effects of dietary copper supplementation. The hens were grown to 15 or 16 weeks and toms to 24 weeks of age. Either copper as the oxide or sulfate (hydrate) at 120 ppm stimulated growth up to 10% with a concurrent reduction in feed requirement. Levels of 60 ppm were not adequate, whereas 240 ppm were not toxic. There was a greater response to copper in the presence of 4-nitrophenylarsonic acid (4-nitro) or with low protein diets. In two of the eight studies copper enhanced aortic elastin content. Incidence of aortic rupture was greater in the presence of 4-nitro and was reduced by one-half through copper supplementation. Liver copper was not altered by 60 or 120 ppm Cu; 240 ppm caused a significant increase.

Influence of D-penicillamine on the formation of elastin and its cross-links.

The amount of insoluble elastin and its content of desmosine cross-links were investigated in aortas of chick embryos, to which D-penicillamine was administered on the 6th or 14th--16th day of incubation. D-Penicillamine was shown to alter the formation and maturation of elastin. Using lower doses (less than 50 mg) the weight of pooled aortic elastin is higher as compared with controls (related to 1 mg of elastin or to total weight of elastin). Increased isodesmosine:desmosine ratio in these samples indicates that this elastin is very young. On the other hand, a high dose of D-penicillamine (100 mg) decreased the content of elastin and also of its desmosine cross-links. The authors explain their findings by counteraction of two factors due to administration of penicillamine: the increased solubility of "insoluble elastin", and the decreased cross-link formation.