Arterial remodeling is a process by which arteries respond to sustained changes in their mechanical environment. This process occurs in a way such that an artery's local mechanical environment (circumferential, shear, and axial stress) is maintained at a homeostatic level. However, most studies utilize a methodology that controls the global parameters (pressure, flow rate, and axial stretch). This approach often confounds the results since the actual drivers of remodeling are not independently isolated. This research involved developing a methodology and system capable of independently controlling each of the local parameters and examining the effect of axial stress on remodeling. An organ culture system capable of monitoring and controlling the three global parameters and calculating the cross sectional geometry was developed. This combination of hardware was incorporated into LabVIEW which afforded the user the ability to define desired values for the local mechanical parameters. Porcine common carotid arteries were cultured for seven days in this system under physiologically normal circumferential and shear stresses and a constant axial stress of either 150 kPa or 300 kPa. Material response, general arterial morphology, tissue viability, and collagen synthesis were examined in order to gauge the effectiveness of the organ culture system and assess any arterial remodeling. The results of this study demonstrate the ability of the organ culture system in achieving and maintaining target values of stress throughout the culture period. Cell viability, general arterial morphology, and collagen synthesis rates were maintained for all arteries. The elevated axial stress appeared to cause a softening of the artery in both the axial and circumferential direction. It was hypothesized that this softening was the result of a changing collagen structure. Additional softening seen in arteries was attributed to the effects of the culture system.

Thesis (M. S.)--Chemical and Biomolecular Engineering, Georgia Institute of Technology, 2005. / Dr. Athanassios Sambanis, Committee Member ; Dr. Timothy M. Wick, Committee Member ; Dr. Ajit P.Yoganathan, Committee Chair. Includes bibliographical references.

Abstract For decades, the mammalian kidney has served as a model system for studying developmental processes, such as induced epithelialization, branching morphogenesis, and cell differentiations. The possibility to recapitulate and follow the renal organogenesis ex vivo in organ culture set-ups has provided a large amount of molecular and cellular information about sequential events during development. However, certain limitations remain when combining traditional organ culture set-ups with modern molecular technology. This thesis seeks to address these disadvantages. In the experimental part of the thesis, the traditional organ culture set-ups were studied, modified, and optimized to meet the needs of functional genetic screening. First, the traditional transfilter- induced nephrogenesis was characterized with a panel of nephron segment specific markers to reveal the differentiation level of in vitro developing mouse renal tissue. A comprehensive genome wide time course microarray analysis was also performed to in vitro- induced metanephric mesenchyme. Next, to improve the accessibility of genetic tools into the three- dimensional organ in culture, the classic kidney culture set-ups were modified to tolerate dissociation and re-aggregation before the induction of nephrogenesis. This step was achieved with the aid of preservative growth factors offering a 24- hour window to manipulate the genetic and cellular composition of the explant. The dissociation and re-aggregation per se had not particular effect on the progress of the nephron differentiation. Demonstrations of the addition and removal of cells, as well as a virus vector mediated gene knock in and knock down are presented. The gene expression data, together with the novel organ manipulation and culture techniques presented in this thesis, provide a useful guide and specific tools to further characterize the details of nephron development and differentiation in functional manner. / Tiivistelmä Nisäkkäiden munuainen on toiminut vuosikymmeniä mallielimenä tutkittaessa kehitysbiologisia tapahtumasarjoja, kuten epitelisaatiota, haaroittumismorfologiaa sekä solujen erilaistumista. Munuaisaihioita voidaan viljellä laboratorio-olosuhteissa, jolloin kehityksen aikaisia muutoksia päästään seuraamaan lähes reaaliaikaisesti. Perinteisten kudosviljelytekniikoiden tarjoamat mahdollisuudet solujen molekulaariseen muokkaukseen ovat kuitenkin varsin rajalliset. Tässä väitöskirjassa esitettävät tulokset pyrkivät osaltaan vähentämään näitä rajoitteita. Väitöskirjan kokeellisessa osassa tarkastellaan lähemmin klassista munuaiskudosviljelyä sekä esitetään siihen tehtyjä optimointeja, joiden avulla kudosviljelyä pyritään hyödyntämään geenien toiminnan tutkimuksessa. Aluksi perinteisellä tavalla reikäisen kalvon läpi indusoitu nefroni karakterisoitiin tarkasti hyödyntäen useita erilaistumista osoittavia merkkimolekyylejä. Lisäksi samalla tekniikalla tuotettujen munuaiskudosviljelmien geeniekspressiota tutkittiin mikrosiruanalyysillä. Klassisia kudosviljelytekniikoita muokattiin soveltuvammaksi moderneille geneettisille työkaluille. Munuaiskudos hajotettiin ensin solususpensioksi, jonka jälkeen solut muodostivat uudelleen kolmiulotteisen, kudosmaisen rakenteen. Hyödyntämällä suojaavia kasvutekijöitä, hajotus kyettiin tekemään jo ennen nefronien muodostumisen alkua. Näin saavutettin 24 tunnin aikaikkuna indusoimattoman kudoksen geneettiselle muokkaukselle. Väitöskirjassa esitelläänkin demonsrtaatiot solujen lisäämisestä ja poistamisesta sekä virusvälitteisestä geenin aktivoinnista ja hiljennyksestä hyödyntäen uutta kudosmanipulaatio ja –vilejelytekniikkaa. Nefronin kehityksen aikaisen geeniekspression kartoitus sekä tässä tutkimuksessa kehitetyt uudet kudosmanipulaatio ja -viljelytekniikat tarjoavat yhdessä työkaluja molekyylitason yksityiskohtaiseen tutkimiseen.

Vascular smooth muscle cells display a certain level of phenotype plasticity. Under specific conditions fully differentiated cells are able to undergo dedifferentiation and to restart growth and proliferation. An organ culture method is a useful technique for the analysis of dedifferentiation of vascular smooth muscle cells, because it provides an opportunity for studying the changes in cell phenotype. The aim of this study was to investigate the basic contractile characteristics in rat femoral arteries cultured for different time periods (from one to three days). In addition, the effects of fetal bovine serum (FBS), that contains various growth factors and other biological active molecules, on contractile function were studied. We also tried to attenuate cell dedifferentiation by lowering the calcium influx, because calcium is an important second messenger participating in cell growth and proliferation. To achieve this goal we used cultivation with nifedipine, a voltage-dependent calcium channel inhibitor. The cultivation without FBS slightly decreased arterial contractility, whereas the cultivation with FBS decreased arterial contractility considerably. The major change in contractility of arteries cultivated with FBS occurred approximately within 24 hours of cultivation. The cultivation with...

Retinoic acid, suspended in cottonseed oil, was administered via gavage to pregnant mice (ICR strain) on day 11 (E 11) of gestation at doses of either 20, 40, or 80 mg/kg. Fetuses were examined for external malformations on day 17 (E 17). Retinoic acid treatment induced micromelia (with the elimination of several long bones at higher doses) and digital defects (ectrodactyly and syndactyly) in a dose‐dependent manner in fetuses examined on day 17. Hindlimbs were affected more than forelimbs. In another group of experiments, limbs exposed to retinoic acid treatment in utero on E 11 were cultured on E 12 and maintained for 3 days in submerged culture. Cultured limbs were examined qualitatively for digital and long bone defects, and image analysis of the area and form of bone anlagen of cultured limbs was used to quantitatively evaluate the teratogenic potential of retinoic acid. The qualitative evaluation indicated that the retinoic acid‐induced effects obtained in vivo and with pretreated, cultured limbs were essentially the same, except that the severity of regional effects changed as a result of culture. The incidence of ectrodactyly was higher with cultured limbs than with E 17 fetal limbs, but fewer cultured limbs were missing long bones. These results suggest that culturing limbs, after they have been pretreated in utero, modifies their response to a teratogen and demonstrates that the paw skeleton is extremely sensitive to teratogen treatment under these experimental conditions. Therefore, care must be exercised when attempting to compare in vivo and in vitro teratogenic data. This study also clearly demonstrates the power and usefulness of image analysis for quantitative evaluation of both the area and form of a cultured specimen such as the developing limb bud. Quantitative, image analysis of cultured limbs showed a dose‐dependent decrease in area of both fore‐ and hindlimbs. The effect was most severe in hindlimbs. In the forelimb, the paw was affected more than the long bones; as the dose increased, this disparity of effect also increased. With the hindlimb, a greater effect on the paw occurred only at 80 mg/kg. Computing the soft tissue/bone ratio illustrated that retinoic acid had a greater effect on chondrogenic tissue than on soft tissue.

No / Although ATP-sensitive potassium (K(ATP)) channel openers, e.g., minoxidil and diazoxide, can induce hair growth, their mechanisms require clarification. Improved drugs are needed clinically. but the absence of a good bioassay hampers research. K(ATP) channels from various tissues contain subtypes of the regulatory sulfonylurea receptor, SUR, and pore-forming, K(+) inward rectifier subunits, Kir6.X, giving differing sensitivities to regulators. Therefore, the in vitro effects of established potassium channel openers and inhibitors (tolbutamide and glibenclamide), plus a novel, selective Kir6.2/SUR1 opener, NNC 55-0118, were assessed on deer hair follicle growth in serum-free median without streptomycin. Minoxidil (0.1-100 microM, p<0.001), NNC 55-0118 (1 mM, p<0.01; 0.1, 10, 100 microM, p<0.001), and diazoxide (10 microM, p<0.01) increased growth. Tolbutamide (1 mM) inhibited growth (p<0.001) and abolished the effect of 10 microM minoxidil, diazoxide and NNC 55-0118; glibenclamide (10 microM) had no effect, but prevented stimulation by 10 microM minoxidil. Phenol red stimulated growth (p<0.001), but channel modulator responses remained unaltered. Thus, deer follicles offer a practical, ethically advantageous in vitro bioassay that reflects clinical responses in vivo. The results indicate direct actions of K(ATP) channel modulators within hair follicles via two types of channels, with SUR 1 and SUR 2, probably SUR2B, sulfonylurea receptors.

BACKGROUND: Alopecia causes widespread psychological distress, but is relatively poorly controlled. The development of new treatments is hampered by the lack of suitable human hair follicle models. Although intermediate and vellus hair follicles are the main clinical targets for pharmacological therapy, terminal hair follicles are more frequently studied as smaller hair follicles are more difficult to obtain. OBJECTIVES: This investigation was designed to quantify in vivo morphological and in vitro behavioural differences in organ culture between matched intermediate and terminal hair follicles, in order to develop a new clinically relevant model system. METHODS: Microdissected terminal and intermediate hair follicles, from the same individuals, were analysed morphometrically (250 follicles; five individuals), or observed and measured over 9 days of organ culture (210 follicles; six individuals). RESULTS: Intermediate hair follicles were less pigmented and smaller, penetrating less below the skin surface (mean +/- SEM) (2.59 +/- 0.07 vs. 3.52 +/- 0.10 mm; P = 0.02), with smaller fibre (0.03 +/- 0.002 vs. 0.07 +/- 0.002 mm), connective tissue sheath (0.24 +/- 0.01 mm vs. 0.33 +/- 0.01 mm), bulb (0.19 +/- 0.01 vs. 0.31 +/- 0.01 mm) and dermal papilla (0.06 +/- 0.002 vs. 0.12 +/- 0.01 mm) diameters (P < 0.001). Intermediate hair follicle bulbs appeared 'tubular', unlike their 'bulbous' terminal follicle counterparts. In organ culture they also grew more slowly (0.044 +/- 0.002 vs. 0.067 +/- 0.003 mm per day; P < 0.001), remained in anagen longer (84 +/- 0.03% vs. 74 +/- 0.03% at day 9; P = 0.012) and produced less hair fibre (0.36 +/- 0.02 vs. 0.50 +/- 0.03 mm; P < 0.001) than terminal follicles. CONCLUSIONS: Smaller intermediate hair follicles showed major morphological differences from terminal follicles in vivo and retained significant, biologically relevant differences in vitro in organ culture. Therefore, intermediate hair follicles offer a novel, exciting, more clinically relevant, albeit technically difficult, model for future investigations into hair growth. This should be particularly important for developing new therapies.

Kurdistan Regional Government/Ministry of Higher Education and Scientific Research

This thesis aimed to verify the causal association between "B. hampsonii" and the re-emergence of mucohaemorrhagic diarrhea in North American swine farms, to investigate the role of the intestinal microbiome as a predisposing factor for infection, to develop a porcine colon in vitro culture model and to apply this model in investigating early host-pathogen interactions. Two infection trials were conducted to determine the pathogenicity of "B. hampsonii" clade II and clade I. Weanling pigs were divided into control (n=6) and inoculated (n=12) groups. In each trial, pigs were inoculated with "B. hampsonii" clade II (tissue homogenate or pure culture) or clade I (pure culture) or sterile culture media. Animals were monitored for clinical signs of diarrhea and upon observation of bloody diarrhea they were necropsied for characterization of lesions. Fecal shedding of "B. hampsonii" was monitored throughout the trials using culture and quantitative real-time PCR. Pre and post-diarrhea fecal samples from the clade II infection trial were used to study the microbiome response to "B. hampsonii" infection and to determine if pre-inoculation microbiome composition differed between pigs that did or did not develop clinical disease. For in vitro model development, numerous factors associated with explant survivability in culture were investigated to develop a protocol for culture of porcine colon explants. The optimized model was used to study the first 12 hours of "B. hampsonii" clade II interaction with the host using a combination of histopathology and gene expression analysis. Pigs inoculated with "B. hampsonii" clade I (9/11) and clade II (9/12 and 8/12 in the tissue homogenate and pure culture experiments, respectively) developed mucohaemorrhagic diarrhea and colitis within 14 days of inoculation. In all trials, mucohaemorrhagic diarrhea was significantly more common in inoculated pigs than controls. No significant differences in richness, diversity or taxonomic composition distinguished the pre-inoculation microbiomes of affected or unaffected clade II inoculated pigs. After the development of diarrhea, the fecal microbiome of diarrheic pigs was more dense and had a had a lower Bacteroidetes:Firmicutes ratio when compared to inoculated but unaffected or control pigs. Cultured porcine colon explants displayed differentiated epithelium and crypts after 5 days in culture, while expressing GAPDH at a constant rate. For explants to thrive in vitro our results suggested the use of distal spiral colon, processed immediately after euthanasia, and cultured in an oxygen-rich gas mix with air-liquid culture interface in media containing antibiotics and antifungals. Explants exposed to "B. hampsonii" for 12 hours had a greater number of necrotic cells and thicker catarrhal exudate than control explants. Interaction of spirochaetes with the epithelium, necrotic cells and crypts was visible under optical microscopy, and a trend of increased expression of IFN-γ and e-cadherin in inoculated explants relative to control explants was observed. Taken together, results of this thesis demonstrate that "B. hampsonii" causes mucohaemorrhagic diarrhea in pigs and modulates their intestinal microbiome. The development of an in vitro infection model that replicates in vivo features facilitated the observation of the initial events in "B. hampsonii" interaction with the colon. When explants were exposed to "B. hampsonii" similar histological lesions to in vivo were observed. This system provides a powerful model for future studies of the pathogenesis of "B. hampsonii" and other enteric pathogens of pigs.

The aortic valve functions in a complex mechanical environment which leads to force dependent cellular and tissue responses. Characterization of these responses provides a fundamental understanding of valve pathogenesis. The aim of this work was to develop an ex vivo organ culture system capable of simulating physiological aortic pressures and flow rates, and study the biological characteristics of native porcine aortic valves cultured in the system. Collagen, sGAG and elastin content of the valve leaflets were measured and cusp morphology, cell phenotype, cell proliferation and apoptosis were examined. Presence of endothelial cells (ECs) on the leaflet surface was also evaluated. The differences in collagen, sGAG and elastin contents were not significant (p greater than0.05) between the cultured and fresh valve leaflets. The cultured valves maintained the structural integrity of the leaflets while preserving the native morphology and cell phenotype. Cell phenotype in leaflets incubated statically under atmospheric conditions decreased compared to fresh and cultured valve leaflets, indicating the importance of mechanical forces in maintaining the natural biology of the valve leaflets. ECs were retained on the surfaces of cultured leaflets with no remodeling of the leaflets. The number of apoptotic cells in the cultured leaflets was significantly (p less than 0.05) less than in the statically incubated leaflets and comparable to fresh leaflets. The sterile ex vivo organ culture system thus maintained the viability and native biological characteristics of the aortic valves that were cultured under dynamic conditions for a period of 48 hours.