Development of dual-compartment perfusion bioreactor for serial coculture of hepatocytes and stellate cells in poly(lactic-co-glycolic acid)-collagen scaffolds.

An important challenge in liver tissue engineering is to overcome the rapid loss of hepatocyte functions. In vivo, hepatocytes are compact polyhedral cells with round nuclei; however, they readily loss many of their differentiated functions in vitro. To overcome this challenge, we have established a new perfusion bioreactor that consists of two compartments which enabled the serial coculture of hepatocytes and hepatic stellate cells-T6 without direct contact between each other. Three dimensional scaffolds were utilized in the bioreactor as physical anchors for cells. The scaffolds consist of collagen grafted poly(lactic-co-glycolic acid) microfibers and cross-linked collagen sponges between microfibers for additional cellular support and adhesion. The advantages of this new bioreactor are enabling cell culture in three dimensional organization and controlling the culture parameters of the supporting cells independently from the hepatocytes. The results showed that the hepatocytes exhibited much higher levels of the differentiated functions such as albumin secretion, urea synthesis, and cytochrome P450 enzymatic activity when compared with the monoculture system where hepatocytes alone were cultured. This perfusion bioreactor system has potential applications in the development of bioartificial liver devices or cell-based tissue constructs transplantation therapies.

The response of the sebaceous glands of female rats ovariectomized at birth to testosterone.

Newborn Sprague-Dawley rats were ovariectomized within 24 h of birth and control animals were sham-operated. Immediately, one-half of the rats in each group received a single injection of 500 micrograms testosterone propionate. The other half acted as a vehicle control. Normal male and female rats were also included for comparison. The secretion rate of sebum was measured at the age of 80 days from the amount of skin-surface lipids that could be extracted with acetone and which had been produced during 2 days. Male rats secreted more sebum than female rats. In female rats, irrespective the presence or absence of ovaries, when treated with a single injection of testosterone propionate at birth, although secreted significantly more sebum than vehicle control groups, the increase was still far below the level normally found in the male. On the other hand, implantation of testosterone pellet at the age of 4 weeks in neonatally ovariectomized-androgenized female rats greatly increased sebum secretion to an extent far exceeding that of the normal male rats. It is concluded that testosterone can directly increase sebum secretion in the female rats irrespective the presence or absence of ovaries when given at birth and reinforcement at about puberty produces a full effect of secretion.

Studies on the critical period of neonatal life for the differentiation of sebum secretion in rats.

Sprague-Dawley rats were castrated at the age of 1, 3, 5, 10 or 21 days. A control group of male and female rats were also included for comparison. The secretion rate of sebum was assessed from the amount of skin-surface lipids that could be extracted by acetone and which had been produced during 2 days. Sebum production increased with age, particularly in male rats. Castration of male rats at the age of 1, 3, 5, or 10 days showed no differences from female rats in sebum secretion when measured at the age of 80 days. On the other hand, male rats that had been castrated at the age of 21 days secreted significantly more sebum than female rats.

Combined effects of neonatal thyroxine treatment and neonatal castration on sebum production in adult male rats.

Newborn Sprague-Dawley rats were castrated within 24 h of birth and control animals were sham-operated. Intact female rats were also included for comparison. One-half of the rats in each group was treated with 28 micrograms thyroxine (t4) daily for the first week of life. The secretion rate of sebum was measured at the age of 80 days from the amount of skin-surface lipids that could be extracted with acetone and which had been produced during 2 days. Castration of rats at birth decreased the rate of sebum secretion and neonatal castration plus neonatal thyrotoxicosis produced an even lower level of sebum which more nearly approached that seen in female rats with neonatal T4 treatment. It was concluded that a reduction in the response of the sebaceous glands in adult rats made thyrotoxic with high doses of T4 in the early stage of life is not due to a decreased secretion of gonadal steroids.

Effect of neonatal castration on liver tumor induction by N-2-fluorenylacetamide in suckling BALB/c mice.

BALB/c mice were castrated at 2 days of age and control animals were sham-operated. Untreated male and female mice were also included for comparison. One-half of the mice in each group were fed on alternate days with 1.5% N-2-fluorenylacetamide suspended in 1% gelatine by stomach tube beginning at 1 week of age for a total of 14 feedings. The experiment was terminated when the mice reached one year old. Approximately 30% of the male mice, but none of the females, developed liver tumors in groups fed carcinogen. This male predominance in the incidence of hepatocellular carcinomas was completely abolished when the animals were castrated neonatally. No lesions were observed in the liver of mice treated with gelatine suspension alone except one with neoplastic nodule. Although it has been observed that liver tumors are produce more readily in younger than in older mice, the present investigation shows that male hormonal environment during early life is more important than the age on the development of liver tumors initiated by carcinogen.

Effect of ovariectomy at birth on the regulation of sebaceous glands in rats.

Inbred Sprague-Dawley rats were ovariectomized within 24 h of birth. Sham-operated and untreated male and female rats were also included for comparison. At the age of 80 days, the secretion rate of sebum was assessed from the amount of skin-surface lipids that could be extracted by acetone and which had been produced during 2 days. There were no differences in sebum secretion, when expressed both in relationship to body weight and to surface area of the body, when rats ovariectomized at birth, sham-operated and intact female rats were compared. The removal of the ovaries within 24 h of birth increased the body weight, however. Although the weights of the pituitary, thyroid and adrenal glands, when corrected for body weight, were significantly smaller in rats ovariectomized at birth than in the sham-operated or intact female rats, there were no statistically significant differences in the absolute weights of these endocrine organs between neonatally spayed rats and sham-operated animals. The uterus became atrophic in the absence of ovaries. It has been concluded that the presence or absence of ovaries in the early stage of life had no effect on the activity of the sebaceous glands.

Role of the gonads in the regulation of sebaceous glands in rats: comparison of the effects of castration at and after birth.

Sprague-Dawley rats were castrated either within 24 h of birth or at 4 weeks of age. Control animals were sham operated. Intact female rats were also included for comparison. Sebum production was assessed at 80 days of age by measuring the amount of skin-surface lipids that could be extracted with acetone and which had been produced during 2 days. The removal of the testes at birth reduced the activity of the sebaceous glands to level more nearly approaching that seen in the female rats whereas castration at 4 weeks of age only partially decreased the rate of sebum secretion so that it was intermediate between the male and female rats. The weights of the pituitary gland, thyroid and adrenal glands increased after castration but there were no differences between rats castrated at birth and those castrated at 4 weeks of age except in the weight of the thyroid gland. It would appear that the role of the testes in the control of the activity of the sebaceous glands is a sequential event which has already started at birth.

Effect of neonatal administration of thyroxine on the rate of sebum production in rats.

Newborn Sprague-Dawley rats of both sexes were treated with 28 micrograms thyroxine (T4) daily for the first week of life. At the age of 80 days, the secretion rate of sebum was measured from the amount of skin-surface lipids extractable by acetone and which had been produced during 2 days. Treatment with such excess amounts of T4 during the early postnatal period significantly reduced the production of sebum in both male and female rats when compared with control rats and with rats deprived of food early in life. The thyroid, the pituitary gland, the testes and the seminal vesicles were significantly smaller but the weights of the ovaries and uteri remained relatively unaffected. There was a similar ratio of sex difference in the rate of sebum secretion irrespective of treatment. It is suggested that a reduction of sebaceous response in rats made thyrotoxic with large doses of T4 early in life was probably due to a decreased secretion of thyroid hormone which is required to maintain normal activity of the sebaceous glands.

Oxygen consumption in the rat following neonatal thyrotoxicosis.

Adult rats made thyrotoxic with large doses of thyroxine during the neonatal period had a lower oxygen uptake as compared with neonatal controls and those of neonatal calorically-deprivated ones.

Inhibitory effect of hypothalamic lesions on liver tumor induction by N-2-fluorenylacetamide in male rats.

Bilateral electrolytic lesions were placed in the median eminence area of the hypothalamus in 12-week-old male Wistar rats. Sham-operated and untreated control rats were also included. Two weeks later, one-half of them were given 0.03% N-2-fluorenylacetamide incorporated into the diet for 16 weeks with adequate resting periods in between. The animals were killed 34 weeks after the last carcinogen feeding. The results show that lesions in the hypothalamus effectively inhibited liver tumor formation (0 of 16, 0%). In contrast, the incidence of hepatocellular carcinomas in sham-operated rats was 38.5% (5 of 13), and that of untreated controls was 42.9% (6 oactive thyroid glands, and shorter nasoanal lengths were observed in rats with lesions in the hypothalamus irrespective of carcinogen treatment. It is apparent from these data that lesions in the median eminence area of the hypothalamus inhibit the induction of liver carcinogenesis with N-2-fluorenylacetamide in male rats.

The effect of neonatal administration of sex hormones on ribonucleic acid metabolism in the liver of male and female rats.

Fifteen minutes after the intraperitoneal injection of (32)P labelled phosphate, normal adult male rats show a higher incorporation of isotope into their liver nuclear RNA than do females. A single injection of testosterone into neonatal female rats causes a higher uptake of (32)P in adult life, while a single injection of oestradiol into male neonates lowers the incorporation in adult life. Gonadectomy at 4 weeks of age has only a small effect on the subsequent incorporation of (32)P into nuclear RNA either in control rats or in rats injected with sex hormones immediately after birth, showing that this effect of liver metabolism is mainly determined by the hormanal pattern at about the time of birth. The possible relevance of this sex difference in RNA metabolism to the different sex incidence of spontaneous or induced liver cancer is discussed.