Rat Bone Marrow Cells Research Articles

ラット骨髄細胞に対するエムドゲイン^[○!R]由来合成ペプチドの生物学的影響(大阪歯科大学大学院歯学研究科博士(歯学)学位論文内容要旨および論文審査結果要旨の公表)

ラット骨髄細胞に対するエムドゲイン^[○!R]由来合成ペプチドの生物学的影響(大阪歯科大学大学院歯学研究科博士(歯学)学位論文内容要旨および論文審査結果要旨の公表)

Вплив трансплантації культур клітин на перебіг експериментального цукрового діабету у тварин

As we know from the literature data, type 1 diabetes mellitus occurs due to the loss of β-cells of island of Langerhans, manufacturing insulin, by the organism, which causes its deficiency. The treatment of this type of diabetes, even at early stages, lies in the substitution therapy together with a careful control of blood glucose which may last for life. It is for this reason that the cellular technologies as an alternative method of treatment of this pathology are growing more and more relevant.Objective of the study: to study the impact of allogenic transplantation of the cultures of bone marrow, lipid tissue and pancreas cells on the course of experimentally generated diabetes mellitus in animals.Task: 1. To obtain the cultures of bone marrow, lipid tissue and pancreas cells in rats. 2. To compare the impact of allogenic transplantation of the cultures of bone marrow, lipid tissue and pancreas cells in the course of experimentally generated diabetes mellitus in rats. 3. To study the impact of pancreas cells culture on the course of experimental diabetes mellitus in cats.The studies were conducted using clinically healthy animals (30 males of white non-pedigree rats with body weight of 200–250 g, aged 4–5 months; 9 white non-pedigree junior rats aged 12 days; 4 mongrel cats aged 15–17 months) and missed fetuses of cats remained after obstetric aid.Alloxan diabetes was generated by means of single subcutaneous injection of alloxane monohydrate in the dose of 150 mg/kg in the form of 5% solution on citrate buffer (pH 4.5) after preliminary 24-hour absolute diet with free access to water.The cultures of bone marrow and pancreas cells were obtained from the bone marrow of tubular bones and pancreas of puppies aged 12 days correspondingly, lipid tissue – from rats aged 4–5 months. The culture of cats’ pancreas cells was obtained from the pancreas of cat fetuses. Cell culture process was carried out according the standard method in CO2- incubator. Glucose level in blood serum was determined by means of electrochemical analysis.The results of the study. The authors studied the impact of allogenic transplantation cells culture of bone marrow, lipid tissue and pancreas on the course of experimentally generated diabetes mellitus in rats. The study revealed the decrease of glucose level in the blood of the animals under investigation at cell material transplantation. The most efficient culture for experimental diabetes mellitus therapy is the culture of pancreas cells which has become the cause of its further study in cats.During allogenic transplantation cells culture of pancreas in cats the authors observed abrupt decrease of glucose level in the blood of the animals under investigation immediately after cell transplantation with the further approximation to the initial state.The experimental models presented the positive effect of cell culture transplantation providing the grounds for the further implementation of this diabetes mellitus therapy method in the clinical practice.

Bioactivity of NANOZR Induced by Alkali Treatment.

In recent years, zirconia has been a recognized implant material in clinical dentistry. In the present study, we investigated the performance of an alkali-modified ceria-stabilized tetragonal ZrO2 polycrystalline ceramic-based nanostructured zirconia/alumina composite (NANOZR) implant by assessing surface morphology and composition, wettability, bovine serum albumin adsorption rate, rat bone marrow (RBM) cell attachment, and capacity for inducing bone differentiation. NANOZR surfaces without and with alkali treatment served as the control and test groups, respectively. RBM cells were seeded in a microplate with the implant and cultured in osteogenic differentiation medium, and their differentiation was evaluated by measuring alkaline phosphatase (ALP) activity, osteocalcin (OCN) production, calcium deposition, and osteogenic gene expression. The alkali-treated NANOZR surface increased ALP activity, OCN production, calcium deposition, and osteogenesis-related gene expression in attached RBM cells. These data suggest that alkali treatment enhances the osteogenesis-inducing capacity of NANOZR implants and may therefore improve their biointegration into alveolar bone.

Histological evaluations of apatite-fiber scaffold cultured with mesenchymal stemcells by implantation at rat subcutaneoustissue.

There is a strong impetus for the development of alternative treatments for bone disease that avoid the complications associated with autografts and allografts. To address this, we previously developed porous apatite-fiber scaffolds (AFSs) which have three-dimensional interconnected pores, and constructed tissue-engineered bone by culturing rat bone marrow cells (RBMCs) using AFSs in a radial-flow bioreactor (RFB). To generate additional baseline data for the development of tissue-engineered bone constructed for clinical application using a RFB, we cultured RBMCs using AFSs under static conditions (hereafter, RBMC AFS culture), and monitored RBMC growth and differentiation characteristics in vitro, and two weeks after subcutaneous inoculation into recipient rats. RBMCs were seeded to AFSs and growth, differentiation and calcification were monitored in vitro and in vivo by histological evaluation using hematoxylin eosin, alkaline phosphatase and alizarin red S stains. RBMCs in/on AFSs proliferated and differentiated normally in vitro and in vivo, and calcification was evident two weeks after subcutaneous AFS culture implantation. Histological assays revealed that AFSs and RBMC AFS cultures were biocompatible, and did not induce inflammation or immunological rejection in vivo. These findings suggest that AFSs are a conducive microenvironment for bone regeneration and are well tolerated in vivo. The results provide valuable baseline data for the design of implant studies using tissue-engineered bone constructed by RFB.

The study of bone marrow cells in rats according to their age and caloric intake

The bone marrow is a central organ of haemopoiesis and a source of pluripotent hemopoietic stem cells. The age-related changes in bone marrow induce the disorder of coordination between parenchyma and stroma of the organ that negatively influences proliferative potential and differentiation of the cells. That is why it is urgent to study the age-related changes of bone marrow and influence of caloric intake limitation on it.Aim. The aim of presented work was the study of proliferative potential, index of viability and percentage ratio of the bone marrow cells in rats depending on their age and caloric intake.Methods. The following methods were used in the study: selection and cultivation of the bone marrow stem cells, assessment of proliferative potential and viability of cells, analysis of percentage ratio of cellular types.Result. It was established, that the number of cells in bone marrows increases with age. The limitation of caloric intake favors the decrease of cells number in bone marrow despite the rats’ age. The received data demonstrated that the limitation of caloric intake increases the proliferative potential of the bone marrow only in old animals. It was established, that the viability of bone marrow cells was high during all period of cultivation. The morphological population of bone marrow cells in rats of different age was heterogenic, not depending on the conditions of their feeding. It was demonstrated, that the limitation of feeding caused the increase of the number of non-differentiated blasts in bone marrow of old animals that can be connected with the activation of the processes of self-activation of stem cells pool and non-differentiated predecessors of immune system cells. The morphological analysis of the bone marrow cells culture of all animal groups despite the age and nutrition regime demonstrated that the cultivation favors the selective decrease of cells heterogeneity. At that the cells of erythroid and megakaryocytic sprouts were absent in cellular cultures.Conclusions. As a result, the limitation of caloric intake of old rats favors the restoration or activation of proliferative potential of immunocompetent cell of bone marrow in vitro system

3D-Printed Bioactive Ca3SiO5 Bone Cement Scaffolds with Nano Surface Structure for Bone Regeneration.

Silicate bioactive materials have been widely studied for bone regeneration because of their eminent physicochemical properties and outstanding osteogenic bioactivity, and different methods have been developed to prepare porous silicate bioactive ceramics scaffolds for bone-tissue engineering applications. Among all of these methods, the 3D-printing technique is obviously the most efficient way to control the porous structure. However, 3D-printed bioceramic porous scaffolds need high-temperature sintering, which will cause volume shrinkage and reduce the controllability of the pore structure accuracy. Unlike silicate bioceramic, bioactive silicate cements such as tricalcium silicate (Ca3SiO5 and C3S) can be self-set in water to obtain high mechanical strength under mild conditions. Another advantage of using C3S to prepare 3D scaffolds is the possibility of simultaneous drug loading. Herein, we, for the first time, demonstrated successful preparation of uniform 3D-printed C3S bone cement scaffolds with controllable 3D structure at room temperature. The scaffolds were loaded with two model drugs and showed a loading location controllable drug-release profile. In addition, we developed a surface modification process to create controllable nanotopography on the surface of pore wall of the scaffolds, which showed activity to enhance rat bone-marrow stem cells (rBMSCs) attachment, spreading, and ALP activities. The in vivo experiments revealed that the 3D-printed C3S bone cement scaffolds with nanoneedle-structured surfaces significantly improved bone regeneration, as compared to pure C3S bone cement scaffolds, suggesting that 3D-printed C3S bone cement scaffolds with controllable nanotopography surface are bioactive implantable biomaterials for bone repair.

Порівняльна характеристика генетичної стабільності культур клітин жирової тканини та кісткового мозку щурів на ранніх пасажах

From literary sources, we received a number of controversial data regarding the risks of neoplastic transformation of stem cells in vitro. Inasmuch as today bone marrow has been sufficiently studied as a source of adult donor stem cells, and adipose tissue is an alternative source for obtaining cell material (from which it may be obtained using less invasive methods and in much greater quantity), determination of genetic stability of these particular cell cultures currently is the most relevant issue. Comparison of genetic stability of rat bone marrow and adipose cell cultures from the first to the sixth passages. Materials and methods: 3 non-linear 4 months old male rats and 9 non-linear 12 days old infant rats were used in the experiment. Adipose cell culture was obtained from subcutaneous fat using the standardized method in our own modification. Bone marrow cell culture was obtained from the rat femurs, tibiae, and humeri using the standardized method. Obtained cell mass was cultivated in a standard culture medium in a СО2 incubator at 37 ºС and with 5% СО2 concentration. Cytogenetic analysis was carried out on 30 metaphase plates from each passage. Cells from the first to the sixth passage we used in the research. A modification of the standard cytogenetic method was used to obtain chromosome preparations. In the preparations prepared in an aforementioned way, numerical chromosome abnormalities, like aneuploidy, polyploidy, were determined, as well as the number of binucleate cells, micronucleus cells, mitotic and apoptotic indexes. Here, we provide the comparison of genetic stability indicators for rat bone marrow and adipose cell cultures throughout the in vitro cultivation thereof. Changes in the genetic apparatus were identified in both bone marrow and adipose tissue cell cultures. Karyotype analysis of rat bone marrow and adipose cell cultures showed that, given the conditions that we used for the cultivation, the number of aneuploidies and polyploidies varies from one passage to another; however it does not go beyond the limits of spontaneous mutagenesis typical for mammals. Based on the results of cytogenetic evaluation of the culture, it was determined that the number of cells with micronuclei and the binucleate cell remains within the normal range from the first to the sixth passages. The number of cells with aneuploidy and micronuclei in adipose cell culture on all passages was lower than in bone marrow cell culture; this indicates its higher genetic stability in terms of these indicators. The highest mitotic index was determined on the first passage with its following decrease throughout the cultivation in inverse proportion to apoptotic index. The obtained data on the genetic stability of both adipose and bone marrow cell cultures are within normal range typical for mammals in terms of all studied indicators. This allows for the following studies relating to the transplantation with minimal risk of neoplastic transformation of the said cultures.

PHYTOHORMONE INDUCED CHANGES ON BONE HISTOLOGY IN NORMAL AND DIABETIC MALE RAT

<p><strong>Objective: </strong>To study the effect of brasslinolide keto isoform 28-homocastasterone in diabetic male rat bone marrow cells, bone histological changes, electrolytes and alkaline phosphatase activity in rat blood.</p><p><strong>Methods: </strong>Diabetes was induced in the group (n=6) of rats with a single peritoneal injection of streptozotocin (60 mg/kg bwt). With a treatment schedule of 15 consecutive days, control (n=6) and diabetic rats received 666µg/kg bwt of 28-homocastasterone. Circulating blood cell count, cell indices, bone marrow cells, bone histology, electrolytes Na<sup>+</sup>, K<sup>+</sup>, Cl<sup>-</sup>, P, Ca<sup>2+</sup>and alkaline phosphatase activity was assessed.</p><p><strong>Results: </strong>Cytological examination showed an increased erythrocyte progenitor and megakaryocyte cell lineage along with improved osteoblastic and bone histomorphology in the treated group was noted. A significant reduction in electrolytes level (p<0.05) and increased in alkaline phosphatase activity (p<0.05) was noted in treated groups.</p><strong>Conclusion: </strong>It is suggested that brassinosteroid keto isoform 28-homocastasterone exhibits a hematopoietic effect in diabetic rat and improves bone histology while reducing hyperglycemic damage in bone.<p> </p>

Chemotherapeutical effects of the herbal medicine Uncaria tomentosa (Willd.) DC.

The use of medicinal plants dates back to the beginning of humanity, and today their application as complementary therapy has been widely disseminated as an alternative to conventional therapy. The medicinal plant named Uncaria tomentosa (Willd.) DC. (known as cat's claw) is a common woody vine of the Amazon forest that has traditionally been used in the treatment of arthritis because of its anti-inflammatory properties. This study aimed to evaluate the cytotoxic, mutagenic, and antimutagenic potentials of this medicinal plant. The biological activities of U. tomentosa were determined on bone marrow cells of Wistar rats that were treated in vivo. For the cytotoxic and mutagenic analyses, aqueous plant extract solutions were administered by gavage (1, 2, or 3 mg/mL) for 24 h (an acute treatment) or 7 days (a subchronic treatment). For the antimutagenic analyses, aqueous plant extract solutions (1 mg/mL) were administered by gavage before (pretreatment), simultaneous to (simultaneous treatment), or after (post-treatment), the administration of cyclophosphamide (1.5 mg/mL). U. tomentosa did not show any cytotoxic or mutagenic effects in any of the cytological or chromosomal analyses. Besides, the antimutagenic tests showed that the plant extracts displayed antimutagenic activities, which significantly reduced the percentages of the chromosomal aberrations that were induced by cyclophosphamide at 53.91, 58.60, and 57.03%, respectively, for the simultaneous treatment, pretreatment, and post-treatment. The results suggested a safe use of this herbal medicine that is available free of charge from the Brazilian Public Health System for the treatment of arthritis. This medicinal plant can also effectively contribute to improving the quality of life and the recovery of people undergoing chemotherapeutical treatments.

In vivo antimutagenic activity of the medicinal plants Pfaffia glomerata (Brazilian ginseng) and Ginkgo biloba.

Complementary and alternative therapies, including the use of medicinal plants, have become almost standard among the world's population. Pfaffia glomerata (PG), popularly known as Brazilian ginseng, is widely used as a restorer of vital functions, increasing mental balance, and is used for the treatment of diabetes and rheumatism. Ginkgo biloba (GB) is one of the oldest known gymnosperms, whose leaves are widely used for its potentiating action on the nervous system. The biological activities of these plants were determined on bone marrow cells of Wistar rats treated in vivo. For cytotoxic and mutagenic acute analysis, plant extracts were administered by gavage at concentrations of 0.15, 1.5, and 15 mg PG/mL water and 1, 2, and 3 mg GB/mL water. For antimutagenic analysis, plant extracts aqueous solution (PG, 1.5 mg/mL or GB, 2 mg/mL) were administered by gavage before (pretreatment), simultaneous to (simultaneous treatment), or after (post-treatment) the administration of cyclophosphamide (1.5 mg/mL, intraperitoneally). Both plant extracts have no cytotoxic or mutagenic potential, and they significantly reduce the percentage of chromosomal aberrations induced by the cyclophosphamide given simultaneously (PG, 87%; GB, 75%), pretreatment (PG, 98%, GB, 78%) and post-treatment (PG, 99%, GB, 75%). This beneficial antimutagenic property of the medicinal plants P. glomerata and G. biloba presented here, with no cytotoxic or mutagenic activity, can efficiently contribute to improvements in quality of life and recovery for people undergoing chemotherapeutic treatment, or those looking for health and preventive habits.

Cell Therapy for Liver Disease Using Bioimaging Rats.

Advances in stem cell research suggest that cell therapy is a potential alternative to liver transplantation. The use of individualized and minimally invasive cell therapy is desirable to avoid rejection and reduce patient burden. While allo-hepatocyte transplantation has been performed for metabolic hepatic disease, auto-bone marrow transplantation (BMT) has shifted toward mesenchymal stem cells (MSCs) transplantation for liver cirrhosis. In this article, an overview of cell transplantation research for liver disease is provided through our recent rat studies. We have developed various kinds of rat imaging models and have evaluated the effect of cell therapy for liver disease. Bone marrow cells (BMCs) of the Alb-DsRed2 rat were transplanted via the portal vein (PV) in acute and chronic liver damage models. The number of Alb-DsRed2+ albumin-producing cells increased, and the size of the cells increased in the chronic liver damage model as well as in the acute liver damage model. Luciferase transgenic (luc-Tg) rat hepatocytes were transplanted into the hepatectomized LEW rat via the PV. Luminescence intensity lasted for 2 months in the hepatectomized rat. BMCs obtained from green fluorescent protein (GFP) Tg rats were transplanted repeatedly via the PV using an implanted catheter with a port. Repeated BMT via the PV reduced the liver fibrosis. Adipocyte-derived MSCs from the luc-Tg rat were transplanted into the hepatectomized rat model via the PV after ischemic reperfusion. MSCs inhibited hepatocyte apoptosis and promoted liver regeneration. Transplanting the optimal number of cells by an effective and safe way is important for clinical application. Bioimaging rats are a powerful tool for cell transplantation research because it makes observation of the in vivo kinetics of transplanted cells possible. Cell transplantation research using bioimaging rats contributes greatly to evaluating effective methods of cell therapy.

ASSESSMENT OF GENOTOXIC POTENTIAL OF HYDROGEN CYANAMIDE USING CHROMOSOMAL ABERRATION AND MICRONUCLEUS ASSAYS IN RAT BONE MARROW

Despite its widespread use in agriculture, data about cyanamide-induced genotoxicity are scanty, and its genotoxic potential was considered equivocal in vitro. Hence, the current study was carried out to evaluate the in vivo genotoxic potential of hydrogen cyanamide to complement the earlier in vitro research work. Thirty adult male rats were randomly assigned into 3 equal groups. Group I rats served as a control. Animals of groups II and III received hydrogen cyanamide in a dose of 10 and 20 mg/kg body weight/day respectively. All animals were treated by oral gavage, once daily, for 28 days. As end points, chromosomal aberrations, micronucleated polychromatic erythrocytes, mitotic index and polychromatic/normochromatic cell ratio in the animals’ bone marrow cells were analyzed. Negative results were obtained regarding the presence of chromosome aberrations and micronuclei in each of the lowdose and high-dose cyanamide-treated rats compared to the controls. Also, cyanamide treatment did not reduce the mitotic index or polychromatic/normochromatic cell ratio in the exposed animals. This study found that hydrogen cyanamide exposure had no genotoxic potential in rat bone marrow cells in the doses used and under the conditions of the performed assays.

Potential anti-genotoxic effect of sodium butyrate to modulate induction of DNA damage by tamoxifen citrate in rat bone marrow cells.

Sodium butyrate (SB) is one of the histone deacetylase inhibitors (HDACi's) that is recently evidenced to have a prooxidant activity and an ability to reduce hydrogen peroxide-induced DNA damage. Since the majority of estrogen receptor positive breast cancer patients are treated with tamoxifen citrate (TC), which exerts well established oxidative and genotoxic effects, thus the basic objective of this study is to determine whether SB could ameliorate or curate tamoxifen citrate-induced oxidative DNA damage and genotoxic effect in vivo through up-regulation of some antioxidant enzymes. The individual and combined effects of SB and TC have been examined on rat bone marrow cells, using Micronucleus assays (MN), Comet assay, DNA fragmentation, expression of some antioxidant genes using Real time-PCR and finally, oxidative stress analysis. SB significantly increased the mitotic activity (P<0.05), while TC induced marked micronuclei and oxidative DNA damage, in the SB post-treatment group, the combination of SB (300mg/kg) and TC (40mg/kg) was able to decrease the induction of MN and oxidative DNA damage through up-regulation of Cat, Sod and Gpx1 genes significantly at (P<0.05) more efficiently than that in the SB pre-treatment one. Therefore, we postulate that SB can be used therapeutically in combination with TC treatment to modulate TC genotoxic effect by reducing its oxidative stress, and thus being an appropriate agonist agent to combine with TC than each compound alone.

Effect of Natural and 24-h Illumination on Mesenchymal Stem Cells.

We studied the effect 24-h illumination on quantitative and qualitative parameters of the bone marrow cells in Wistar rats. It was shown that desynchronosis reduced the release of nucleated cells from the femoral bone, while melatonin weakened this effect. The number of bone marrow mesenchymal stromal cells was resistant to circadian rhythm disturbances, while proliferation depended on glucose concentration in the medium.

ФЕНОТИПОВІ ТА МОРФОЛОГІЧНІ ЗМІНИ КУЛЬТУРИ КЛІТИН КІСТКОВОГО МОЗКУ ЩУРІВ В ПРОЦЕСІ ЇХ КУЛЬТИВУВАННЯ

Bone marrow is the only adult tissue which normally consists of immature undifferentiated and low differentiated cells which called stem cells and they are similar in structure to embryonic stem cells. But literature data analysis doesn't give an unambiguous answer regarding phenotypic and morphological changes of bone marrow cells culture of rats during their in vitro cultivation which necessitated further research.Investigate phenotypic and morphological changes of bone marrow cells culture of rats during their in vitro cultivation from first to fourth passage.We were used in these research bone marrow cells of rats from the first to the fourth passages. Microscopic analysis and evaluation morphological changes of bone marrow cells culture of rats during cultivation were carried out using inverted microscope Axiovert 40. Control of changes phenotype was performed by detecting CD markers (CD10, CD38, CD34, CD45, CD48, CD54, CD56, CD66e, CD96, CD227, CD326, pan–keratin). The evaluation was performed by the semi– quantitative method (H–Score).The research of primary culture of rat bone marrow cells showed that it morphologically heterogeneous, noted the small number of cells polygonal shape, surrounded by the fibroblast cells. During the cultivation cell culture becomes more homogenous at the expense of fibroblast–like cells. As a result of occurred the transition process from heterogeneous culture in zero passage to the most homogeneous culture in 4 passage. Immunophenotyping population of cell culture derived from rat bone marrow, revealed a high level of expression of pan–keratin; moderate level – CD34, CD48, CD66e, CD95; low level – CD38, CD45, CD56, CD227, CD326; lack of expression – CD10, CD54. Change of the expression of surface markers varies in each passage CD48, CD66e, CD95 increased significantly; CD38, SD45, SD326, pan–keratin reduced significantly. The markers CD34, CD 56, CD 227 were expressed on the one level from the first to the fourth passage. The expression of the CD10, CD54 markers during the study period was not identified.

Maxillofacial bone regeneration with osteogenic matrix cell sheets: An experimental study in rats

ObjectiveRegeneration of maxillofacial bone defects, characterized by relatively small but complicated shapes, poses a significant clinical challenge. Osteogenic matrix cell sheets (OMCSs) have osteogenic ability and good shaping properties and may be ideal graft materials. Here, we assessed whether implantation of OMCSs could be used to repair maxillofacial bone defects. DesignWe adopted a rat mandibular symphysis model. The rat mandible is formed by a paired bone and the central portion consisting of fibrous tissue. There is no bone tissue at the site; accordingly, this site was interpreted as a physiological bone gap and was used for evaluation. Rat bone marrow cells were cultured in medium containing dexamethasone and ascorbic acid phosphate to create OMCSs. The OMCSs were implanted into the rat mandibular symphysis without a scaffold. Microcomputed tomography and histological analyses were conducted after 2, 4, and 8 weeks. ResultsTwo weeks after implantation, microcomputed tomography images and histological sections showed some sparse granular calcification tissue within the bone gap at the mandibular symphysis. At 4 weeks, the calcification tissue spread, and the gap of the mandibles were continued. At 8 weeks, this continuous new bone tissue was matured. The experimental group showed abundant new bone tissue in the group with OMCS implantation, but not in the group with sham implantation. ConclusionsOur present results indicated that use of OMCSs may be an optimal approach towards achieving maxillofacial regeneration.

Effect of n-Butanol on Chromosomal Damage in Mice Bone Marrow Cells

Background: n-Butanol is a four-carbon alcohol used widely in foods, cosmetics industries, biology and chemistry research laboratories, and other fields. Long time-effects of inhalation or consumption of small amounts of Butanol on human health are still unknown. On the other hand, numerous reports about the development of n-Butanol toxicity are available. The main objective of the study was to investigate the effects of inhaled and oral administration of n-Butanol as a long-term in vivo investigation. M aterials and Methods: Small white laboratory, male mice (20-30 g) were used in 11 groups (n=4) including experimental 1 to 6, 1 to 4 control A” and positive control groups. Experimental groups 1-3, for 10, 20, and 40 days; 5 hours a day were inside a box with ventilation facilities exposed to air saturated with n-Butanol vapor. Experimental groups 4 to 6, received water containing n-Butanol 0.2%, 1% and 5% for 10 days. Control groups B, 1 to 3 was placed for 10, 20, and 40 days inside a similar box exposed to normal air, respectively. Control group B 4 received water without any particular substance for 10 days. The positive control group received 30µl subcutaneous vinblastine. Bone marrow cells were extracted 24 hours after treatments and stained by May-Grunwald-Giemsa staining and the number of micronucleus was counted. Vinblastine, as a positive control, increased an average of micronucleus numbers significantly compared to control group (P<0.001). R es ults: n-Butanol inhalation caused no significant difference in 1-3 experimental groups in the average numbers of micronucleus compared to control group, even in the 40 days treatment group, average numbers of micronucleus was decreased comparing to control group (P<0.05). Also, oral administration of 0.2% and 1% n-Butanol had no effect on the average micronucleus numbers compared to the control group, while oral administration of 5% n-Butanol caused even decrease in average numbers of micronucleus compared to control group (P<0.05). Conclusion: n-Butanol inhalation may not cause chromosome damages in rat bone marrow cells that probably is due to its very fast metabolism and decomposition in the body. Therefore, the amount of n-Butanol in the systemic circulation and tissues is very low and, probably, the damaging potential is decreased.

453. Tumorigenic Potential in Clones Derived from Rat Bone Marrow Cells

Therapy using different stem cell populations has recently emerged as a complementary treatment for several diseases, and a wide variety of stem cell lineages have been explored. Mesenchymal stem cells (MSCs) represent an attractive option for use in cellular therapy because of their accessible nature and abundance in various tissues, such as bone marrow (BM) and adipose tissue. While MSCs are routinely isolated from BM, it is now evident that MSCs can also be isolated from many other tissues. These cells can be divided, based on their source of origin, into two broad categories: marrow-derived and non-marrow-derived. Although MSCs isolated from different sources share similar surface antigens and exhibit similar classical differentiation potential (bone, fat, and cartilage), these cells still exhibit heterogeneity in their phenotype and biological properties, which apparently depends on their tissue of origin and microenvironmental niche. Self-renewal is a property shared among cancer cells and stem cells. Stem cells can certainly be genetically susceptible and acquire mutations through the cell-culture process; even so, the potential tumorigenic risk of using MSCs from a heterogeneous cellular mixture has not yet been properly evaluated. Some cells derived from BM can present some markers associated with cancer (e.g., CD117 and CD34, which are always found in a gastrointestinal stromal tumor (GIST) diagnosis, the most common mesenchymal tumor of the gastrointestinal tract. Nonetheless, the presence of both markers on BM with tumorigenic potential has not been sufficiently explored yet. Interestingly, some subpopulations isolated from BM supernatant express CD34 and CD117 (c-Kit). CD117 is a tyrosine-kinase receptor activated by stem cell factor. Upon stimulation, CD117 activates multiple signaling pathways, including ERK and mTOR, which ultimately lead the activation of NFk-B; this nuclear factor is coincidently upregulated in various tumors. The goal of this work was to identify a direct correlation between immunophenotype and possible tumorigenic potential in a specific clone of adherent c-kit+/ CD34+ cells isolated by cell sorting from the supernatant of unfractionated rat bone marrow (SNBMC). We evaluated SNBMC tumorigenic potential in vivo and in vitro. We subcutaneously injected SNBMC CD117/CD34-positive cells and heterogeneous SNBMC (unsorted) into the bilateral flank of 55 male nude mice. We measured tumor size weekly for a month, and we found that tumors were present in 20% of the mice treated with unsorted cells and in 100% of the mice that received SNBMC CD117/CD34-positive cells. All of the tumors were solid neoplasias with small, undifferentiated cells and frequent mitotic figures; intratumoral necrotic areas were abundant, suggesting a rapid proliferation rate. Immunohistochemistry staining revealed intense immunoreactivity for cytokeratin, and we suggested a diagnosis of anaplastic carcinoma. Flow cytometry analysis from tumor-isolated cells revealed a loss of MSC surface markers such as CD90 and CD73; however CD117 and CD34 were preserved. Finally the participation of NFk-B in cell proliferation was evaluated by transfection with an IkB-α adenovirus and a significantly reduced proliferation rate was found. Nonetheless, given the increasing interest in using MSCs for regenerative medicine applications, the safety of administering MSCs to humans remains to be carefully evaluated in light of the potential role of MSCs in driving tumorigenicity, particularly cells expressing CD117/CD34. Understanding the tumorigenic process in a future will be important to determining the role of these markers on the molecular level.

Variation of mechanical behavior of β-TCP/collagen two phase composite scaffold with mesenchymal stem cell in vitro

The primary aim of this study is to characterize the variational behavior of the compressive mechanical property of bioceramic-based scaffolds using stem cells during the cell culture period. β-Tricalcium phosphate (TCP)/collagen two phase composites and β-TCP scaffolds were fabricated using the polyurethane template technique and a subsequent freeze-drying method. Rat bone-marrow mesenchymal stem cells (rMSCs) were then cultured in these scaffolds for up to 28 days. Compression tests of the scaffolds with rMSCs were periodically conducted. Biological properties, such as the cell number, alkaline phosphatase (ALP) activity, and gene expressions of osteogenesis, were evaluated. The microstructural change due to cell growth and the formation of extracellular matrices was examined using a field-emission scanning electron microscope. The compressive property was then correlated with the biological properties and microstructures to understand the mechanism of the variational behavior of the macroscopic mechanical property. The porous collagen structure in the β-TCP scaffold effectively improved the structural stability of the composite scaffold, whereas the β-TCP scaffold exhibited structural instability with the collapse of the porous structure when immersed in a culture medium. The β-TCP/collagen composite scaffold exhibited higher ALP activity and more active generation of osteoblastic markers than the β-TCP scaffold.

Controlled release of BMP-2 by chitosan/γ-PGA polyelectrolyte multilayers coating on titanium alloy promotes osteogenic differentiation in rat bone-marrow mesenchymal stem cells

The excellent biocompatibility and mechanical properties of titanium alloys have led to their wide-scale application in orthopedic implants. Appropriate surface treatment of these alloys can greatly improve cellular attachment as well as interactions between implants and tissue. This paper reports on the preparation of polyelectrolyte multilayers (PEM) coating comprising chitosan and gamma-poly(glutamic acid) on a Ti6Al4V substrate via spin-coating. Bone morphogenetic protein 2 was then loaded directly into the coating. Following sample preparation, we performed in vitro studies using SD rat bone-marrow mesenchymal stem cells (rBMSCs) to investigate the biological effects of the coating. Our results demonstrate that this chitosan/γ-PGA PEM coating is highly stable and does not have any adverse effects on rBMSCs. The proposed coating proved highly effective in carrying and enabling the gradual release of BMP-2. At day 14, the coated samples presented osteogenic differentiation far exceeding that of the non-coated samples. After co-culturing for 21 days, the calcium content in the PEM coating group was double that of the non-coated group, which is a clear indication of enhanced cellular mineralization.