DPSC-Derived Extracellular Vesicles Promote Rat Jawbone Regeneration.

Repair and functional reconstruction of large jawbone defects remain one of the challenges in the field of head and neck surgery. The recent progress in tissue engineering technologies and stem cell biology has significantly promoted the development of regenerative reconstruction of jawbone defects. The multiple trophic activities of extracellular vesicles (EVs) produced by mesenchymal stem cells (MSCs) may play a critical role in their therapeutic effects. Accumulating evidence has shown the promise of dental pulp stem cells (DPSCs) in bone regeneration, but less is known about the regenerative effects of DPSC-EVs on jawbone defects. The purpose of this study is to explore the osteogenic effects of DPSC-EVs on jawbone marrow-derived MSCs (JB-MSCs) in vitro and their osteoinductive effects in a mandibular bone defect model in rats. Our results showed that JB-MSCs could efficiently uptake DPSC-EVs, which in turn significantly promoted the expression of osteogenic genes, such as runt-related transcription factor 2 (RUNX2), alkaline phosphatase (ALP), and osteocalcin (OCN), as well as the osteogenic differentiation capability of JB-MSCs. Meanwhile, we found that the pro-osteogenic effect in vitro induced by DPSC-EVs was comparable to that induced by BMP-2 (bone morphogenetic protein 2), currently the only Food and Drug Administration-approved osteoinductive growth factor. In vivo, animals that were locally treated with DPSC-EVs laden with a commercially available collagen membrane exhibited a relatively fast wound closure and increased new bone density at the mandible defects. Our results provide evidence for the osteogenic and osteoinductive effects of DPSC-EVs on jawbone regeneration. Due to the accessibility, rapid proliferation, and osteogenic propensity of DPSCs, DPSC-EVs may represent a safe cell-free therapeutic approach for craniofacial bone regeneration.

A modified in vitro larvae migration inhibition assay using rumen fluid to evaluate Haemonchus contortus viability.

Anthelmintic effects of plant secondary compounds may be occurring in the rumen, but in vitro larvae migration inhibition (LMI) methods using rumen fluid and forage material have not been widely used. Forage material added to an in vitro system can affect rumen pH, ammonia N, and volatile fatty acids, which may affect larvae viability (LV). Validating a LMI assay using rumen fluid and a known anthelmintic drug (Ivermectin) and a known anthelmintic plant extract (Quebracho tannins; QT) is important. Rumen fluid was collected and pooled from 3 goats, mixed with buffer solution and a treatment (1 jar/treatment), and placed into an anaerobic incubator for 16h. Ensheathed larvae (<3 months old) were then anaerobically incubated with treatment rumen fluid for 2, 4, or 16h depending on the trial. Larvae (n=15-45) were then transferred onto a screen (n=4-6 wells/treatment) within a multi-screen 96-well plate that contained treatment rumen fluid. Larvae were incubated overnight and those that passed through the 20-µm screen were considered viable. Adding dry or fresh juniper material reduced (P<0.05) pH, ammonia N, and isobutyric, butyric, isovaleric, and valeric acids, and increased (P<0.001) acetic, propionic, and total VFA. Including 4.5% (w/v) polyethylene glycol (PEG) in rumen fluid mixture with or without forage material reduced (P<0.01) LV. However, LV was similar at all PEG concentrations tested (0-2%, w/v; 89.4, 78.9, 76.5, 75.5, and 77.5% viable). Q. tannin concentrations from 0 to 1.2% (w/v) quadratically reduced (P<0.001) LV; 89.4, 65.5, 22.8, and 9.2%. Ivermectin concentrations from 0 to 15µg/mL quadratically reduced (P<0.001) LV; 90.2, 82.6, 73.6, 66.3, 51.9, 56.5, 43.5, 41.9, 29.3, and 19.9% viable, respectively. Effects of altering in vitro rumen fluid pH, ammonia N, and VFA and using PEG when evaluating LV need to be further investigated. In vitro rumen fluid assays using QT and Ivermectin resulted in decreased LV, validating the efficacy of this technique for measuring Haemonchus contortus larval viability.

Coulter counter-based evaluation of sperm volume to assess sperm viability of bull semen and application to X/Y sperm sorting.

The Coulter Counter Hypo-Osmotic Swelling test (CC-HOS) was developed to provide insight into the membrane integrity (relative volume shift Vr) of sperm necessary for fertilization, and to identify the optimum buffer needed for the X/Y chromosome sorting process. Using the CC-HOS test on neat bovine semen, the mean relative volume shift Vr for July and August was 1.20 and 1.14, respectively, whereas mean Vr values ranged from 1.32 to 1.41 during September to November. There was an inverse relationship between Vr magnitude and environmental temperature; we inferred that this enhanced sperm viability during autumn relative to summer. A method was developed to measure the dynamics of volume change of sperm in the buffer (pH 6.5) used for the X/Y chromosome sorting process. When exposed to the buffer (4 mM K+, 153 mM Na+, 140 mM Cl(-)), sperm from Bull C had a mean modal volume of 22.8+/-0.2 fL during a 0-300 s time interval, which did not significantly vary from sperm volumes (21.88+/-0.66 fL for Bull A and 22.46+/-0.38 fL for Bull B) noted in isotonic Isoton II solution. However, when exposed to lower ionic concentrations (2 mM K+, 62 mM Na+, 47 mM Cl-), the mean volume of Bull C sperm increased to 29.2+/-1.5 fL and exhibited slower rates toward stabilized volumes relative to higher ionic concentration buffers. Utilization of volume swelling measurements for measuring the impact of ion concentrations in X/Y chromosome sorting process buffers illustrated the importance of its application for emerging sperm-based biotechnologies.

Hypoxia-selective antitumor agents. 16. Nitroarylmethyl quaternary salts as bioreductive prodrugs of the alkylating agent mechlorethamine.

Nitrobenzyl quaternary salts of nitrogen mustards have been previously reported as hypoxia-selective cytotoxins. In this paper we describe the synthesis and evaluation of a series of heterocyclic analogues, including pyrrole, imidazole, thiophene, and pyrazole examples, chosen to cover a range of one-electron reduction potentials (from -277 to -511 mV) and substitution patterns. All quaternary salt compounds were less toxic in vitro than mechlorethamine, and all were more toxic under hypoxic than aerobic conditions, although the differentials were highly variable within the series. The most promising analogue, imidazole 2, demonstrated DNA cross-linking selectively in hypoxic RIF-1 cells, and was active in vivo in combination with radiation or cisplatin. However, 2 also produced unpredictable toxicity in vivo, suggestive of nonspecific nitrogen mustard release, and this has restricted further development of these compounds as hypoxia-selective cytotoxins.

Hypoxia-dependent retinal toxicity of bioreductive anticancer prodrugs in mice.

The bioreductive anticancer prodrug CI-1010 ((2R)-1-[(2-bromoethyl)amino]-3-(2-nitro-1H-imidazol-1-yl)-2-propanol hydrobromide) is an alkylating nitroimidazole which shows selective toxicity against hypoxic cells in murine tumors, but causes extensive apoptosis in the outer retina in rodents and monkeys. This irreversible retinal toxicity has terminated preclinical development of CI-1010. We have investigated whether such toxicity is due to physiological hypoxia in the retina, and whether it is a general feature of hypoxia-selective bioreductive drugs. Retinal damage was quantified by morphometric analysis of histological sections following treatment of female C57Bl6 mice. Both CI-1010 and tirapazamine (TPZ, 1,2,4-benzotriazin-3-amine 1,4-dioxide), a bioreductive drug in Phase III clinical trial, caused a time and dose-dependent loss of photoreceptor cells of the outer retina following administration of single intraperitoneal doses. The lesion caused by TPZ was qualitatively similar to that with CI-1010, but was less severe at equivalent fractions of the maximum tolerated dose (as defined by lethality). With both bioreductive drugs, lesion severity was increased if animals breathed 10% O(2) for 3 h after drug administration, while breathing 95% O(2)/5% CO(2) was protective. Other hypoxia-selective bioreductive drugs tested (the quinone porfiromycin, the anthraquinone N-oxide AQ4N and the nitrogen mustard prodrugs SN 23816 and SN 25341) did not cause retinal damage at their maximum tolerated doses. This study suggests that the retinal toxicity of bioreductive drugs might be avoided by manipulation of tissue hypoxia using 95% O(2)/5% CO(2), although this intervention could suppress antitumor activity. The finding that not all bioreductive drugs cause retinal toxicity suggests this toxicity can be avoided through appropriate drug design.

Physical and pharmacologic restraints in long-term care facilities.

This study examined the effects of education on the attitudes and practices of long-term care staff toward use of restraints. The intervention, a 1-day educational seminar, used a collaborative team of speakers from the Utah Survey Agency and medical professions. Seminar goals were threefold: first, to provide information about best practices for managing behaviors of individuals with dementia in long-term care settings; second, to provide an explanation of the Omnibus Budget Reconciliation Act regulations pertaining to restraint use; and third, to present alternative strategies to link best practice guidelines to the provision of care. Results showed significant changes in participants' attitudes toward use of restraints. Participants reported replicating the seminar for nursing home staff, revisiting facility policies on restraints, and modifying resident care plans.

Phenotypic responses of mouse mammary tumours and normal mammary epithelium cultured in collagen gels: correlation with tumour type and progression.

Mammary tumours in female BR6/Icrf mice and the corresponding contralateral normal mammary glands were disaggregated with collagenase and the epithelial structures released ('organoids') separated from other cellular components by filtration. The organoids were established in primary culture in a collagen matrix and the outgrowths obtained were studied by light microscopy and time-lapse cinemicroscopy. The pattern of three-dimensional outgrowths produced was found to be specific to the original tissue. Organoids from normal tissue formed a characteristic outgrowth designated Pattern A. Normal tissue from pregnant mice formed an additional characteristic outgrowth (Pattern A') which has not been described previously. Pregnancy-dependent tumours produced a distinctive phenotypic outgrowth designated Pattern D, whereas pregnancy-independent tumours gave a different distinctive Pattern B as well as a unique specific outgrowth designated Pattern C. Outgrowths of Pattern D from a pregnancy-dependent tumour were removed from culture and implanted into a syngeneic female mouse. Tumours arising in the host were found to be pregnancy-independent and showed phenotypic outgrowths in subsequent culture of pregnancy-independent Patterns B and C. The results show that the type of outgrowths in these cultures correlates with the biology of the tissue in vivo and that changes in tumour progression in vivo are accompanied by alterations in phenotypic outgrowths in culture.

Reduction of metastasis in a murine mammary tumour model by heparin and polyinosinic-polycytidylic acid.

A murine mammary tumour model has been used to test the efficacy of a combination of heparin and the interferon inducer, poly I:C on spontaneous metastasis from a s.c. primary tumour and on experimental metastasis following i.v. injection of tumour cells. This treatment has no effect on the growth of primary tumours, but lung metastases arising from these tumours were reduced. When tumour cells were injected i.v. the number of lung colonies was significantly reduced and survival time extended. Short-term treatment did not prevent the subsequent growth of extravasated, but dormant tumour cells, although mice treated for 8 or 12 weeks survived at least 6 months without any sign of lung colonies. Several mechanisms may contribute to the overall effect of this treatment; a reduction in the mitotic indices of lung colonies (observed in poly I:C treated mice) and also NK cells appeared to be important for the effectiveness of poly I:C since the reduction in experimental metastasis was abrogated by concomitant treatment with anti-asialo GM1 serum.

Interferons alpha and gamma differ in their ability to cause tumour stasis and regression in vivo.

We have studied the antitumour activity of human lymphoblastoid interferon-alpha [HuIFN-alpha(N)] and human recombinant interferon-gamma (rHuIFN-gamma) on 12 early passage (passages 2-7) human tumour xenografts derived from primary malignancies. Systemic daily therapy of established (approx. 0.5 cm diameter) subcutaneous xenografts with HuIFN-alpha(N) resulted in significant tumour stasis, and occasionally regression, in nine of 12 breast, bowel, and ovarian cancers studied. A significant decrease in tumour mitotic index was seen in three HuIFN-alpha(N) sensitive breast tumours. In contrast, none of nine of the same tumours responded significantly to rHuIFN-gamma therapy. Direct administration of rHuIFN-gamma into the tumour did not improve its therapeutic efficacy. However, when tumour cells from xenografts were dissociated and grown as colonies in soft agar, both IFNs, used at doses that are found circulating in vivo after therapy, inhibited colony development in three of three lines tested. Combinations of rHuIFN-gamma and HuIFN-alpha(N) had no synergistic benefit in four of four xenografts studied.

Reinfection of virus free mice with mouse mammary tumour virus.

BR6/Icrf mice carrying a milk-transmitted mammary tumour virus (MMTV) develop tumours after several pregnancies. If the mice are freed from MMTV, no tumours develop. In the experiments described in this paper, MMTV was reintroduced into MMTV-free mice by foster nursing, which was least effective if the pups were exposed to the virus only during the first week of life. Exposure for even a short time after that age led to a tumour incidence similar to that found in normally infected mice. Reinfection was also achieved by injection of MMTV-containing milk into weanling or pregnant mice, and was then transmitted naturally to the next generation.

Comparison of metastatic cell lines derived from a murine mammary tumour, and reduction of metastasis by heparin.

A murine mammary carcinoma, which had a high potential for metastasis to the lungs, was established in culture, and from the parent line several clonally derived variants were isolated, showing different characteristics including metastatic potential. C1, a high metastatic clone, and C2, a low one, were selected for further study. When tumour cells were injected s.c. the growth rates of the resulting tumours were higher when they developed from the parent line (P2) or C1 cells, than from C2 cells. The numbers of lung colonies seen following i.v. inoculation of tumour cells also varied, C2 cells yielding the lowest score. In vitro C1 cells were more efficient at aggregating platelets than C2, an effect reduced by the addition of heparin. In vivo heparin reduced the number of tumour cells arrested in the lungs after i.v. injection, and also the number lung colonies which subsequently became established. The number of metastases which developed following s.c. injection of tumour cells was also reduced by heparin.

Concerted activation of two potential proto-oncogenes in carcinomas induced by mouse mammary tumour virus.

The induction of tumours by retroviruses lacking transduced oncogenes can involve the transcriptional or functional activation of cellular proto-oncogenes by an integrated provirus. Thus, the two cellular genes int-1 and int-2, identified as common targets for activation by mouse mammary tumour virus (MMTV), may constitute previously unrecognized oncogenes. In tumours, proviral insertion at these loci leads to expression of messenger RNAs which are undetectable in normal mammary glands. Here we report that in a survey of the two transcriptional activity and structural integrity of the two int loci in 30 BR6 mouse mammary tumours, around 50% of the tumours expressed both of these genes, in ostensibly monoclonal cell populations. Our data suggest that int-1 and int-2 may act cooperatively in the genesis of mammary carcinomas. However, because three tumours (10%) involved neither gene, and because in five cases activation occurred in the apparent absence of an adjacent provirus, it is clear that other loci and mechanisms contribute to tumorigenesis.

Students' attitudes toward university health services.

This study measured students' attitudes toward a university's student health services (SHS) and identified factors that were related to their attitudes. A questionnaire surveying students' attitudes was administered to a sample of 150 students at the Oxford Campus of the University of Mississippi. Analysis of the data revealed that students' attitudes had a statistically significant correlation with the following variables: perceived medical care cost, amount of health information/education received during medical encounters, time spent in the waiting room of the SHS, sex of the student, and income of the student. There was no significant relationship between attitude and age.

Metastasis and morphology of transplanted BR6 mouse mammary tumours.

BR6 mouse mammary tumours were maintained by serial s.c. transplantation into female or castrated male syngeneic hosts. The tumours could be broadly classified according to their structure, and usually remained stable through many passages. Occasionally, sarcomatous changes were seen (64 out of 800 cases). Pulmonary metastases were found in 5 per cent of mice bearing transplants of well-differentiated acinar tumours, in 34 per cent with tumours of the acinar/ductal type, and in 97 per cent with poorly differentiated tumours. There was a similar correlation between lymph node metastases and degree of primary tumour differentiation, the incidences being 0, 0.5 and 22 per cent respectively. Sarcomatous changes were associated with a reduced incidence of metastasis. Local invasion of muscle or peritoneum occurred in 32 per cent of mice with well-differentiated tumours, and in 59 per cent with less differentiated tumours. Selection for increased metastatic potential was not achieved by transplantation of lung nodules to a subcutaneous site.

Activation of cellular gene by mouse mammary tumour virus may occur early in mammary tumour development.

There is now good evidence that the induction of mammary carcinomas by mouse mammary tumour virus (MMTV) involves provirus activation of specific cellular genes. Thus, a high percentage of virally induced tumours contain an acquired MMTV provirus in either of two defined integration regions, termed int-1 and int-2, and provirus insertion is accompanied by expression of specific RNA transcripts from these regions. We show here that in some recurring, pregnancy-dependent mammary tumours provirus integration within int-2 has already occurred at the earliest appearance of the tumour and may therefore represent an important step in the development of neoplasia. As judged by the distribution of the acquired MMTV proviruses, the tumours recurring at any one site represent the same clonal population of cells as the original tumour and remain clonal during cycles of proliferation and regression, including the transition to hormone-independent status. These data suggest that either the expression of the int-2 locus or the function of this putative oncogene must remain responsive to hormones and that some additional event must be responsible for the transition to autonomous growth.

Proliferative responses of mouse mammary glands to 17 beta-estradiol and progesterone and modification by mouse mammary tumor virus.

The responses of mammary glands of ovariectomized mice to 17 beta-estradiol and progesterone were measured by mitotic and [3H]thymidine-labeled indices in low-tumor C57BL mice and high-tumor BR6 mice. The BR6 mice were subdivided into those carrying murine mammary tumor virus (MuMTV) and those that had been freed from it by foster nursing. In all groups, continuous stimulation by the two hormones administered together resulted in a cell proliferation peak. The magnitude of response varied according to strain and the presence or absence of MuMTV. The maximal effect was seen in BR6 mice carrying the virus. Comparison of percentage of labeled mitoses curves showed that the duration of DNA synthesis was extended in BR6 mice with MuMTV, although not sufficiently to account for the observed differences in labeling indices. The responses to 17 beta-estradiol and progesterone administered separately also varied. Mice carrying the virus were more susceptible to stimulation by progesterone, whereas those without the virus responded more to 17 beta-estradiol. The results suggested that the proliferative response of the mouse mammary gland to ovarian hormones can be modified by mammary tumor virus infection.

Metastasis of mammary tumours in mice: relationship with morphology of primary tumour, and reproductive background of host.

The lungs of BR6 breeding female mice which had developed spontaneous mammary tumours, were examined histologically for the presence of metastases. Metastatic nodules, or tumour cell emboli within blood vessels, were found in the lungs of 3 out of 42 (7 per cent) mice with pregnancy-dependent tumours, and in 33 out of 117 (28 per cent) mice with pregnancy-independent tumours. In general, primary tumours which were well-differentiated and organized were less likely to metastasize than poorly-differentiated ones, but there was an intermediate range within which the likelihood of metastasis could not be predicted. Analysis of the reproductive histories of the mice did not show any significant correlations between factors which might have influenced the growth and progression of the primary tumour and metastatic potential. However, the sustained presence of a high tumour load was associated with an increased probability of metastasis.