Ingeniería tisular del tejido óseo. Diseño y desarrollo de materiales híbridos biológicamente activos basados en vitrocerámicas para sustitución ósea / Bone tissue engineering. Design and development of biologically active vitroceramic-based hybrid materials to be used as bone substitutes

Objetivo: Describir el desarrollo y caracterización de un material vitrocerámico y la respuesta inicial de células madre mesenquimales adultas (MSC-A) aisladas de la medula ósea. Material y metodología: El material se obtuvo por calentamiento de un vidrio 55SiO2-41CaO-4P2O5 (mol/%) por el método sol-gel. Las células se aislaron por aspirados directos de cresta ilíaca de pacientes adultos jóvenes. Se estudió el grado de adherencia, proliferación y diferenciación a osteoblastos de las MSC-A sembradas sobre el material. La diferenciación celular se evaluó mediante la producción de osteocalcina y la pérdida del marcador mesenquimal CD90. La proliferación celular sobre el sustrato se realizó mediante el ensayo de reducción de sales de tetrazolio. El material sembrado se implantó en un defecto crítico realizado en fémur de conejo para valorar su capacidad osteorregeneradora, y se observó mediante TAC. Resultados: Las MSC-A se adhirieron, expandieron, proliferaron y produjeron matriz extracelular mineralizada sobre el material durante el tiempo en cultivo, al mismo tiempo que mostraron fenotipo osteoblástico, e incrementaron la producción de osteocalcina y la pérdida de expresión de CD90. El material se reabsorbió parcialmente al final del estudio. Conclusión: El material es citocompatible, osteoconductor, bioactivo, con capacidad de promover la diferenciación de MSC-A a osteoblastos y la neoformación ósea después de su implantación en asociación con MSC-A; es una matriz adecuada para la regeneración del tejido óseo (AU)

Purpose: To describe the development and characterization of a vitroceramic material as well as the initial response of adult mesenchymal stem (MSCs-A) isolated from bone marrow. Material and methodology: The material was obtained by heating glass with composition in mol% 55SiO2-41CaO-4P2O5 by a sol gel method. Cells were isolated from direct iliac crest aspirates from young adult patients. An analysis was performed of the degree of adhesion, proliferation and osteoblastic differentiation of MSCs-A seeded onto the material. Cell differentiation was evaluated through the production of osteocalcin and the loss of the CD90 mesenchymal marker. Cell proliferation on the substrate was performed using the tetrazolium salt reduction method. The seeded material was implanted in a critical defect caused in a rabbit femur in order to determine its osteogenerating capacity; CT observations were carried out. Results: MSCs-A se bound to the material, expanded, proliferated and produced mineralized extracellular matrix on the material during the culture period. At the same time, they showed an osteoblastic phenotype, increasing osteocalcin production and losing CD90 expression. The material was partially resorbed at the end of the study. Conclusion: The material is cytocompatible, osteoconductive, bioactive and has a capacity to promote osteoblastic differentiation of MSCs-A as well as new bone formation following its implantation in association with MSCs-A; an appropriate matrix for bone tissue regeneration (AU)

In vitro behaviour of adult mesenchymal stem cells seeded on a bioactive glass ceramic in the SiO(2)-CaO-P(2)O(5) system.

This work describes the evaluation of a glass ceramic (55S41C4P-1300) as a potential substrate for bone tissue engineering. For that purpose, the capacity of mesenchymal stem cells (MSCs), isolated from rabbit bone marrow, to adhere, proliferate and differentiate into osteoblast (OBs) with or without 55S41C4P-1300 was investigated. Two types of culture medium, i.e. growth medium (GM) and osteogenic medium (OM), were evaluated. The bioactive 55S41C4P-1300, containing pseudowollastonite, wollastonite, tricalcium phosphate and crystoballite as crystalline phases, was obtained by heat treatment of a sol-gel glass (55SiO(2), 41CaO, 4P(2)O(5) (mol.%)) at 1300 degrees C. The results showed that the MSCs adhered, spread, proliferated and produced mineralized extracellular matrix on 55S41C4P-1300 regardless of the culture medium used. As the same time, they showed an osteoblastic phenotype, and this phenomenon was accompanied by the gradual diminution of the marker CD90 expression. The 55S41C4P-1300 was able to induce the differentiation of MSCs into OBs in the same way as OM without glass ceramic. This effect increased with the combination of 55S41C4P-1300 with OM. The glass ceramic evaluated in this work is bioactive, cytocompatible and capable of promoting the differentiation of MSCs into OBs. For that reason, it could be regarded as a suitable matrix in tissue engineering for bone tissue regeneration.

Estradiol and testosterone concentrations increase with fasting in weaned pups of the northern elephant seal (Mirounga angustirostris).

Although neonatal development is generally associated with increased levels of circulating testosterone (T) and estradiol (E2), food deprivation may inhibit steroidogenesis. Therefore, these potentially conflicting stimuli were examined in fasting weaned northern elephant seal (Mirounga angustirostris) pups by measuring serum concentrations of T, E2, progesterone (P4), and luteinizing hormone (LH) by either radioimmunoassay (P4, LH) or enzymeimmunoassay (T, E2). Blood samples were obtained from 20 male and 20 female pups at both early (<1 wk postweaning) and late (6-8 wk postweaning) periods during their natural postweaning fast. T in males (early: 2.9 +/- 0.4 ng/mL; late: 16 +/- 2 ng/mL; P < 0.0001) and E2 in females (early: 42 +/- 6 pg/mL; late: 67 +/- 5 pg/mL; P < 0.01) increased between the two measurement periods, while P4 (early: 2.5 +/- 0.3 ng/mL; late: 2.1 +/- 0.3 ng/mL; P > 0.05) did not. LH increased (early: 46 +/- 4 pg/mL; late: 65 +/- 6 pg/mL; P < 0.05) in males but not in females (early: 69 +/- 9 pg/mL; late: 65 +/- 6 pg/mL; P > 0.05). Increases in LH and T suggest that LH may stimulate T secretion. Alternatively, relatively low concentrations of LH in both males and females may reflect negative feedback inhibition imposed by elevated T and E2 concentrations. Despite the inherent postweaning fast, concentrations of T and E2 increased, suggesting that they may be critical for the continued development of pups. Therefore, compensatory mechanisms may exist that alleviate the fasting-induced inhibition of gonadal steroidogenesis during neonatal development in elephant seal pups.

GH and ghrelin increase with fasting in a naturally adapted species, the northern elephant seal (Mirounga angustirostris).

After nursing, pups of the northern elephant seal (Mirounga angustirostris) are approximately 46% body fat and rely almost entirely on the oxidation of their large fat stores to sustain their metabolism for the ensuing 8-12 week postweaning fast, which is a natural component of their life history. Thus, fasting pups provide an ideal opportunity to examine the hormonal alterations associated with prolonged food deprivation in a naturally adapted model. Cortisol, ghrelin, glucagon, growth hormone (GH), insulin-like growth factor-I (IGF-I), insulin, blood urea nitrogen (BUN), glucose and non-esterified fatty acids (NEFA) were examined in 20 male and 20 female pups blood sampled early (<1 week postweaning) and late (6-8 weeks postweaning) during the fast. Mean cortisol, ghrelin, GH, and glucagon increased 1.8-, 1.8-, 1.4-, and 2.3-fold between early and late periods, while mean IGF-I and insulin decreased 97% and 38%, respectively. NEFA increased 2.3-fold, while BUN and glucose decreased 46% and 11%, respectively. NEFA was significantly and positively correlated with cortisol and GH; individually; however, when the relationship was examined as a multiple regression the correlation improved suggesting that cortisol and GH act synergistically to promote lipolysis during the fast. GH and BUN were negatively and significantly correlated between early and late fasting suggesting that GH may promote protein sparing as well. The decrease in glucose may be responsible for stimulating glucagon, resulting in the maintenance of relative hyperglycemia. The increases in cortisol, ghrelin, glucagon, and GH suggest that these hormones may be integral in mediating the metabolism of seal pups during prolonged fasting.

Renal effects of fresh water-induced hypo-osmolality in a marine adapted seal.

With few exceptions, marine mammals are not exposed to fresh water; however quantifying the endocrine and renal responses of a marine-adapted mammal to the infusion of fresh water could provide insight on the evolutionary adaptation of kidney function and on the renal capabilities of these mammals. Therefore, renal function and hormonal changes associated with fresh water-induced diuresis were examined in four, fasting northern elephant seal ( Mirounga angustirostris) (NES) pups. A series of plasma samples and 24-h urine voids were collected prior to (control) and after the infusion of water. Water infusion resulted in an osmotic diuresis associated with an increase in glomerular filtration rate (GFR), but not an increase in free water clearance. The increase in excreted urea accounted for 96% of the increase in osmotic excretion. Following infusion of fresh water, plasma osmolality and renin activity decreased, while plasma aldosterone increased. Although primary regulators of aldosterone release (Na(+), K(+) and angiotensin II) were not significantly altered in the appropriate directions to individually stimulate aldosterone secretion, increased aldosterone may have resulted from multiple, non-significant changes acting in concert. Aldosterone release may also be hypersensitive to slight reductions in plasma Na(+), which may be an adaptive mechanism in a species not known to drink seawater. Excreted aldosterone and urea were correlated suggesting aldosterone may regulate urea excretion during hypo-osmotic conditions in NES pups. Urea excretion appears to be a significant mechanism by which NES pups sustain electrolyte resorption during conditions that can negatively affect ionic homeostasis such as prolonged fasting.

A new perspective on adiposity in a naturally obese mammal.

Many mammals seasonally reduce body fat due to inherent periods of fasting, which is associated with decreased leptin concentrations. However, no data exist on the correlation between fat mass (FM) and circulating leptin in marine mammals, which have evolved large fat stores as part of their adaptation to periods of prolonged fasting. Therefore, FM was estimated (by tritiated water dilution), and serum leptin and cortisol were measured in 40 northern elephant seal (Mirounga angustirostris) pups early (<1 wk postweaning) and late (6-8 wk postweaning) during their natural, postweaning fast. Body mass (BM) and FM were reduced late; however, percent FM (early: 43.9 +/- 0.5, late: 45.5 +/- 0.5%) and leptin [early: 2.9 +/- 0.1 ng/ml human equivalents (HE), late: 3.0 +/- 0.1 ng/ml HE] did not change. Cortisol increased between early (9.2 +/- 0.5 microg/dl) and late (16.3 +/- 0.9 microg/dl) periods and was significantly and negatively correlated with BM (r = 0.426; P < 0.0001) and FM (r = 0.328; P = 0.003). FM and percent FM were not correlated (P > 0.10) with leptin at either period. The present study suggests that these naturally obese mammals appear to possess a novel cascade for regulating body fat that includes cortisol. The lack of a correlation between leptin and FM may reflect the different functions of fat between terrestrial and marine mammals.

Effects of prolonged fasting on plasma cortisol and TH in postweaned northern elephant seal pups.

Northern elephant seal (Mirounga angustirostris) pups rely on the oxidation of fat stores as their primary source of energy during their 8- to 12-wk postweaning fast; however, potential endocrine mechanisms involved with this increased fat metabolism have yet to be examined. Therefore, 15 pups were serially blood sampled in the field during the first 7 wk of their postweaning fast to examine the changes in plasma concentrations of cortisol and thyroid hormones (TH), which are involved in fat metabolism in other mammals. Cortisol increased, indicating that it contributed to an increase in lipolysis. Increased total triiodothyronine (tT(3)) and thyroxine (tT(4)) may not reflect increased thyroid gland activity, but rather alterations in hormone metabolism. tT(3)-to-tT(4) ratio decreased, suggesting a decrease in thyroxine (T(4)) deiodination, whereas the negative correlation between total proteins and free T(4) suggests that the increase in free hormone is attributed to a decrease in binding globulins. Changes in TH are most similar to those observed during hibernation than starvation in mammals, suggesting that the metabolic adaptations to natural fasting are more similar to hibernation despite the fact these animals remain active throughout the fasting period.

Prolonged fasting increases the response of the renin-angiotensin-aldosterone system, but not vasopressin levels, in postweaned northern elephant seal pups.

The 8- to 12-week postweaning fast exhibited by northern elephant seal pups (Mirounga angustirostris) occurs without any apparent deleterious effects on fluid and electrolyte homeostasis. However, during the fast the role of vasopressin (AVP) has been shown to be inconclusive and the involvement of the renin-angiotensin-aldosterone system (RAAS) has yet to be examined. To examine the effects of prolonged fasting on these osmoregulatory hormones, 15 postweaned pups were serially blood-sampled during the first 49 days of their fast. Fasting did not induce significant changes in ionic or osmotic concentrations, suggesting electrolyte homeostasis. Total proteins were reduced by day 21 of fasting and remained depressed, suggesting a lack of dehydration. Aldosterone and plasma renin activity exhibited a correlated, linear increase over the first 49 days of the fast, suggesting an active RAAS. Aldosterone exhibited a parabolic trend over the fast with a peak at day 35, suggesting a shift in the sensitivity of the kidney to aldosterone later in the fast. AVP was elevated at day 49 only, but concentrations were relatively low. RAAS was modified during the postweaning fast in pups and appears to play a significant role in the regulation of electrolyte and, most likely, water homeostasis during this period.

Effects of hypoxia and hypercapnia on aerobic metabolic processes in northern elephant seals.

An hypoxia-induced metabolic down-regulation has been implicated as an important protective mechanism against tissue deoxygenation in mammals. Whether the same response to hypoxia occurs in northern elephant seals was studied. The effects of hypercapnia were also examined to determine whether the reduced ventilatory response of seals to CO2 is associated with an analogous protective metabolic down-regulation. Thirty three seals (7-300-days-old) were studied using open-flow respirometry with simultaneous monitoring of apnea frequencies and heart rates. Hypoxia (11% O2) and hypercapnia (7% CO2) caused increases in metabolism of up to 38% with corresponding decreases in the percent time spent apneic (%AP) and increases in heart rate. The metabolic, breathing and heart rate responses to altered inspired gases were independent of age. Metabolism was strongly negatively correlated with %AP suggesting that elevated metabolism during hypoxia and hypercapnia exposure is attributable to decreases in %AP. In young elephant seals metabolic down-regulation is not an automatic protective response to experimentally-imposed hypoxia or hypercapnia.

Ontogeny of humoral immunity in northern elephant seal (Mirounga angustirostris) neonates.

Northern elephant seal (NES) serum concentrations of total immunoglobulin (Ig) G, an IgG sub-class, and an IgM-like protein were determined by capture immunoassay using three monoclonal antibodies with specificities for Ig of members of the Phocidae pinniped family. These assays were calibrated for use with NES sera using affinity column purified Ig. Concentrations of these Ig populations were estimated in adult female sera sampled at two time points during the lactation period, as well as sera from their pups collected during the first 5 weeks after birth. In pups, concentrations of the IgM-like protein was found to increase rapidly post-partum. In some individuals, values reached mean concentrations within 10-14 days. In addition, rapid increases in pup total IgG and IgG sub-class concentrations were also observed. Collectively, these findings suggest that the majority of post-partum increases in serum Ig can be accounted for by de-novo synthesis.

Plasma melatonin concentration in neonatal northern elephant seals, Mirounga angustirostris.

The development of pineal function in northern elephant seals was examined in an attempt to understand the physiological basis for previously observed high daytime levels of melatonin in neonatal southern elephant seals. Pineal glands from four northern elephant seal pups, estimated age less than 1 week, weighted 3.0 +/- 0.80 g, which was significantly less than that previously found in southern elephant seals (4.6 +/- 0.35 g). Midday concentrations of plasma melatonin in pups averaged more than 3000 pmol/l in the first 5 days post-partum, but declined rapidly to less than 400 pmol/l after day 9. Daytime melatonin levels in northern elephant seals tended to be lower than in southern elephant seals, although they were very high compared with other species. A circadian cycle of plasma melatonin concentration was observed in newborn northern elephant seals, with levels of 3000-5000 pmol/l during the day, rising to more than 10,000 pmol/l late in the dark phase. Soon after weaning at 4 weeks of age, daytime and night-time levels were in the range 60-100 pmol/l and 100-400 pmol/l, respectively. When approximately 10 weeks old, most samples were in the range 100-400 pmol/l with no discernible difference between day and night levels. The results do not support the hypothesis that the pineal gland is involved in thermogenesis in new-born southern elephant seals. Instead, the very active pineal gland may contribute to energy conservation, by lowering body temperature, particularly at night. As physical insulation is acquired by the deposition of blubber, the mechanism is not required and melatonin falls to adult levels.

Skeletal development in newborn and weanling northern elephant seals.

The neonatal period in the northern elephant seal (Mirounga angustirostris) differs markedly from that of most mammals. An intense 1-mo nursing period is followed by 2-3 mo during which seal pups consume neither food nor water. Because skeletal development in other species is rapid during this period, we determined the capability of elephant seals to continue skeletal growth under fasting conditions. We analyzed indirect markers of bone turnover [serum osteocalcin and alkaline phosphatase activity and urinary calcium and hydroxyproline (Hyp) excretion] in samples obtained sequentially from live pups throughout the fast. Serum osteocalcin was low at the beginning of the fast, peaked between 17 and 42 days, and then decreased to initial levels after 10 wk. Alkaline phosphatase activity increased linearly through 9 wk of fasting. Urinary calcium was relatively constant through 9 wk but decreased after 10 wk, whereas free and total Hyp decreased linearly. Clearance of 3H-labeled Hyp showed that virtually all (> 99%) free Hyp was oxidized and that only bound Hyp, for which there is no degradative mechanism, was excreted. Kinetics of 45Ca clearance, a direct measurement of mineral turnover, was virtually identical to that for young humans. Histomorphometry of ulnae obtained from pups which died of natural causes (usually trauma inflicted by adult males) indicated active surfaces (osteoid plus eroded) between 30 and 60% of total surface of highly porous bone containing < 60% bone area/tissue area. Thus all indicators of skeletal activity confirmed that young elephant seals continue skeletal development, notwithstanding the prolonged period during which they take in no food or water.

Casein secretion by mammary gland epithelia from collagen gel cultures and lactating glands.

Amino acid incorporation experiments show that epithelial cells from lactating mouse mammary glands and from collagen gel culture both synthesize and secrete four principal phosphocaseins (p45, p40, p27, and p23 kD). In both cases, however, the casein production is largely dominated by the p27 species. The average percentage distribution of the above casein species in medium from cultured epithelia is approximately 13%, 6%, 68%, and 14%, respectively; for milk the distribution is approximately 23%, 7%, 54%, and 16%. The predominance of the p27 species is not a consequence of extensive extracellular differential degradation of the secreted caseins since no significant casein degradation was observed in culture medium, either in contact or isolated from epithelial cell monolayers. Synthesis and secretion of all the caseins by cultured epithelia is dependent upon insulin, prolactin, and hydrocortisone. Presumably some intracellular events result in the secretion of p27 as the principal casein in mouse milk. Apparently, some selection factor(s) operate to make p27 a major nitrogenous nutritional component for a newborn mouse. In addition, on a quantitative basis, the relative levels of various caseins secreted by epithelia from lactating mammary glands is essentially duplicated by epithelia in collagen gel culture.

Blood chemistry homeostasis during prolonged fasting in the northern elephant seal.

Serum electrolytes, 3 enzymes, and 11 metabolites were monitored for 32-68 days in weaned, naturally fasting elephant seal pups. Serum glucose, urea nitrogen and creatinine levels declined as the fast progressed, whereas total protein, albumin, and globulin levels remained nearly constant. By contrast, triglycerides, cholesterol, uric acid, and bilirubin were quite variable and no definite trends were apparent. Alkaline phosphatase activity appeared to increase during fasting, while serum glutamic oxalacetic transaminase and lactate dehydrogenase remained fairly uniform. Comparisons of averaged blood chemistry values from singly and multiply sampled fasting pups to that of four nursing pups showed significant differences in the levels of blood urea nitrogen, creatinine, cholesterol, bilirubin, and albumin, but sampling uncertainty limited physiological interpretation. Electrolyte levels in all animals were maintained within narrow limits under all conditions with little interindividual variation. These results further document the remarkable homeostasis achieved during prolonged fasting in elephant seals and support the hypothesis that fat is the primary energy substrate during the protracted natural fasts characteristic of this species.

Intracellular sodium accumulation and transmitter release at crustacean neuromuscular junctions.

Since the intracellular concentrations of Na+ and Ca2+ are relatively low and the cell's ability to actively expel or sequester these ions is limited, prolonged, high-frequency impulse activity at many crustacean axon terminals can result in significant increases in [Na+]i and [Ca2+]i. These changes parallel increases in transmitter output and may persist for several seconds (short-term facilitation, or STF) or even for many hours (long-term facilitations, or LTF). These two phenomena appear to be qualitatively distinct processes. The unique properties of LTF include: 1) the need for Na+ (but not Ca2+) in the extracellular media; 2) long decay times (from minutes to hours); 3) acceleration of devolpment and retardation of decay under conditions that inhibit the membrane Na+-K+ pump, e.g., cardiac glycosides, low [K+]o. The subcellular mechanism(s) linking Na+ accumulation to increased transmitter release are unknown but may involve alterations in [Ca2+]i, either through release from intracellular stores or through increased membrane conductance. Sodium-induced modifications in transmitter synthesis, storage, or availability may also be involved. Long-term functional changes in synaptic efficacy like LTF may provide important clues to more general questions of nervous system plasticity.

Depressant action of lithium at the crayfish neuromuscular junction: pre- and postsynaptic effects.

1. The effects of replacement of external sodium ions with lithium have been studied at the excitatory neuromuscular junction of the crayfish. 2. Intracellularly recorded excitatory junctional potentials fall 45% in amplitude in the first 10 min after lithium substitution, and fail irreversibly in 48--120 min. 3. The quantal content of extracellularly recorded excitatory junctional potentials declines 25--70% within the first 10 min of Li+ exposure. During the next 40--120 min the nerve terminal potentials and quantal release at individual synapses fail simultaneously and irreversibly. 4. The mean amplitude of the spontaneous miniature excitatory junctional potentials (m.e.j.p.s) is reduced 13% by Li+ substitution, but recovers upon restoration of sodium. The mean frequency of m.e.j.p.s rises steadily during Li+ exposure, and continues to increase after reintroduction of Na+. 5. The postsynaptic response to iontophoretically applied L-glutamate falls 35--40% in 10 min, but never falls below 45% of the control level. The effect on the glutamate response is completely reversible with sodium restoration. 6. The effective resistance of the postsynpatic cells is unaffected or only increased slightly by lithium substitution. 7. Thus, the primary mechanism of transmission block by lithium appears to be decreased transmitter release and inexcitability of presynaptic terminals, probably as a result of intracellular accumulation of lithium.