OBJECTIVE: To determine the effect of clinically used zoledronate (ZOL) and docetaxel on breast cancer cells and bone biology under both bone remodeling stages and the rate of tumor dissemination and state of dormancy. METHODS: The effect of clinically used zoledronate (ZOL) was examined on MDA-MB-231 and MDA-BO cells in a roller tube system under bone resorption and formation conditions. Three groups; calvaria alone, calvarial co-cultured with tumor cells, and calvaria with tumor cells treated with four repeat doses of 2 µM of ZOL were cultured for 8, 14 and 20 days. The formation groups were supplemented with 150 µg/ml ascorbic acid. Cell counts were performed on trypsinized calvaria harvested at 2, 8 and 14 days. Media was changed every 2 days and the changed media was re-seeded in a 24-well for 20 days. To test the impact of chemotherapy agents on cancer-bone metastasis the effect of 10 µM of docetaxel was tested on breast cancer cells under formation and resorption conditions using the above design. Tumor burden was assessed at 8 days. RESULTS: Tumor burden: no statistically significant difference between ZOL treated and untreated groups under resorption and formation conditions in both cell lines. Exposure to docetaxel revealed that ~30% of the cells were affected by chemotherapy in formation model, while ~70% was affected in resorption model in both cell types. Dissemination model: the dissemination rate for MDA and BO cells under formation condition is significantly less than for resorption conditions. Fluorescent microscopy: MDA and BO tumor-calvaria were treated with Ki 67 antibody showed that under bone resorption conditions the cancer-bone cells colony were predominantly in proliferation stage while under formation conditions cancer cells were in dormancy. Confocal microscopy: observation confirmed the relation of the mode of cancer cell attachment to bone endosteal cell layer with the dormancy and cell proliferation states. CONCLUSIONS: Both cancer cell lines showed resistance to ZOL under formation and resorption conditions. Drug resistance to docetaxel was more evident under formation condition, where cells are dormant and not proliferating. The dissemination rate is significantly higher in resorption condition, suggesting that cells in formation are dormant with lower dissemination rate. / 2019-07-23T00:00:00Z

Background: An ex vivo experimental strategy replicating in vivo intestinal development would provide an accessible setting to study normal and dysmorphic biology, and would be a test bed for tissue engineering. Previous studies implicated transforming growth factor β1 (TGFβ1) in postnatal gut maturation and regeneration following injury, but its potential role in intestinal development is poorly understood. I firstly hypothesised that embryonic small intestine is able to heal after physical injury. To test this idea, I aimed to create an organ culture model using explants of embryonic jejunum. I secondly hypothesised that TGFβ1 affects embryonic small intestine growth and differentiation. Accordingly, I aimed to use the same organ culture model to determine potential effects of exogenous TGFβ1.Methods. Segments of mouse embryonic jejunum were isolated by dissection and placed on semipermeable platforms. They were fed with defined, serum free, media, in some cases supplemented with TGFβ1. Growth, differentiation and healing of explants were characterized and quantified using a battery of techniques that included whole mount imaging, histology, immunostaining and RNA arrays. TGFβ1 was measured in amniotic fluid by enzyme-linked immunosorbent assay. Groups were compared by statistical tests. Results: After three days of culture, jejunal rudiments differentiated from simple tubes into a more complex structures containing smooth muscle surrounding newly formed villi. Pairs of rudiments, linked by a thread, fused and formed a continuous single lumen, as assessed by trajectories of fluorescent dextrans injected into their distal ends. Functional continuity was confirmed by spontaneous waves of peristalsis crossing the point of fusion. In vivo, TGFβ receptors I and II were detected in embryonic longitudinal smooth muscle cells and, in organ culture, exogenous TGFβ1 induced differentiation of longitudinal smooth muscle. Microarray profiling showed that TGFβ1 increased smooth muscle associated transcripts in a dose-dependent manner. TGFβ1 protein was detected in amniotic fluid at a time when the embryonic small intestine was physiologically herniated. Conclusion: Embryonic jejunal segments can fuse to form a single functional organ when aided by a mechanical manipulation. By analogy with the requirement for exogenous TGFβ1 for smooth muscle differentiation in culture, the TGFβ1 protein that I demonstrated to be present in the amniotic fluid may enhance intestinal development when it is physiologically herniated in early gestation. Future studies of embryonic intestinal cultures should add TGFβ1 in the defined media to produce a more faithful model of in vivo muscle differentiation. In future, this model could be used to test whether other growth factors enhance intestinal growth, and so pave the way to novel biological treatments for short bowel syndrome, a devastating disease with a high mortality.

Disorders affecting articular cartilage are amongst the most common problems in orthopaedics. Osteoarthritis, the end stage of the disease of articular cartilage, reduces the quality of life for tens of millions of people in the world, and has a profound impact on the economics of industrialized countries. Despite progress in articular cartilage research, the problem is still far from being defeated. Various models e.g. in vitro cartilage explants and in vivo animal models, have been established for cartilage research, but each has its own limitations. Thus, a novel ex vivo isolated joint organ culture model was developed. Bovine metatarsophalangeal joints were chosen as a suitable synovial joint because it consists of a hinge-type joint that is similar to the human knee joint, and has a large cartilage surface that provides enough space for multiple sampling in the same joint. The joints were isolated aseptically and placed into culture media. The viability of chondrocytes, glycosaminoglycan (GAG) content of cartilage matrix, cartilage morphology and water content of matrix were evaluated under different culture conditions, i.e. static, static with flowing media, and dynamic with different durations of the movement period. The model was used to investigate the effect on the sharp scalpel cartilage injury of adding serum to the culture medium by culturing the whole joint explants in serum-supplied or serum-free media. The feasibility of investigating the early phases of chondrocyte implantation in this model was also studied: circular holes of 2.5 mm diameter were created by making a pilot hole with a 2.0 mm drill followed by using a fresh 2.5 mm biopsy punch. Allogeneic isolated chondrocytes at different passages were aggregated as cell pellets and implanted in the holes to evaluate their integration ability and the response from the recipient cartilage. Results from the static model showed that, after 28 days culture, the chondrocytes were still alive with 66.5%, 80.9% and 46.9% viability in the superficial, middle and deep zones, respectively. The GAG content of the static model decreased 19.2% after the first week of culture and then lost another 15.0% during the third week. Paradoxically, at end of the 4th week the GAG level rebounded to some extent and increased 19.0% relative to the previous week. Interestingly, the cell viability of all three zones improved if the culture fluid was flowing as seen with the experiments carried out with stirred media or dynamic movement of the articular surfaces. (e.g. for the stirred media after 28 days of culture the chondrocyte viability was 80.6%, 92.4% and 70.4% for the superficial, middle and deep zones respectively.) The GAG content was maintained at a constant level in the contact area of the dynamic model, but decreased as in the media-stirred model and non-contact area of the dynamic model to a similar extent to that observed with the static model. In the injury model, the GAG content fell approximately 10.8% straight after the scalpel cut, but no further loss was observed if the joint was cultured in the serum-supplied media. In contrast, if the injured joint was cultured in the serum-free media, the GAG content continued to fall week by week and finally dropped by 41.7% at the end of the 4th week. In the chondrocyte implantation model, the majority of the host chondrocytes around the circular defect were alive (78.5 % viability). Viewed from the surface, the dead cells were all within 20 μm from the cut edge. The implanted chondrocytes, which were aggregated as cell pellets, began to transform their shapes and spread to the surrounding surface of the recipient cartilage, but did not appear to integrate with the host tissue during the first 2 weeks of culture. The results supported the validity of this ex vivo joint model and demonstrated that the chondrocytes subjected to flow of the media or dynamic loads survived well over a 4 week period. Of importance was the finding that there was no measured loss of the matrix GAG content when the joints were under dynamic load compared to all of the non-loaded conditions. This whole joint model could be of value in providing a more natural and controllable platform where research involving the normal processes or pathologic mechanisms of articular cartilage can be investigated, as well as the early response to newly developed pharmacological agents and cartilage tissue engineering constructs.

There are well over 100,000 Americans on the kidney transplant list with a median wait time of 3.6 years. However, about 17 American die each day waiting for a kidney transplant, with vascular calcification being one of the most common causes [1, 2]. One vessel that is highly susceptible to vascular calcification is the aorta leading to negative cardiovascular outcomes that are secondary to kidney disease [3]. Therefore, understanding the effects that kidney disease has on disrupting the physiology of the vasculature, and finding potential therapeutic options, are imperative to those waiting for a life-saving kidney transplant. The present study aimed to test two hypotheses: (1) Aortic calcification leads to a decrease in stress in the thoracic and abdominal aorta of a young adult mouse. (2) The attenuated aortic stress seen in aortic calcification is due to the decreased expression of smooth muscle ⍺-actin (SM⍺-Actin). To test these hypotheses, calcification was induced in the ex vivo mouse aorta, followed by histological staining for calcium deposits, immunoblots for SM⍺-Actin, and measurements of aortic stress. The results of this study support the hypotheses in that calcification impairs aortic stress and it does so by decreasing the expression of SM⍺-Actin. The present study is the first to show the effect of phosphate-induced calcification on stress and expression of SM⍺-Actin in an ex vivo mouse aorta. This study is relevant to researchers as it shows key differences between studying vascular calcification in vitro compared to ex vivo. Therefore, investigating the mechanisms of aortic calcification using an ex vivo model, may be more applicable to human patients. / 2024-05-17T00:00:00Z

no / Balding causes widespread psychological distress but is poorly controlled. The commonest treatment, minoxidil, was originally an antihypertensive drug that promoted unwanted hair. We hypothesized that another serendipitous discovery, increased eyelash growth side-effects of prostamide F2α-related eyedrops for glaucoma, may be relevant for scalp alopecias. Eyelash hairs and follicles are highly specialized and remain unaffected by androgens that inhibit scalp follicles and stimulate many others. Therefore, we investigated whether non-eyelash follicles could respond to bimatoprost, a prostamide F2α analog recently licensed for eyelash hypotrichosis. Bimatoprost, at pharmacologically selective concentrations, increased hair synthesis in scalp follicle organ culture and advanced mouse pelage hair regrowth in vivo compared to vehicle alone. A prostamide receptor antagonist blocked isolated follicle growth, confirming a direct, receptor-mediated mechanism within follicles; RT-PCR analysis identified 3 relevant receptor genes in scalp follicles in vivo. Receptors were located in the key follicle regulator, the dermal papilla, by analyzing individual follicular structures and immunohistochemistry. Thus, bimatoprost stimulates human scalp follicles in culture and rodent pelage follicles in vivo, mirroring eyelash behavior, and scalp follicles contain bimatoprost-sensitive prostamide receptors in vivo. This highlights a new follicular signaling system and confirms that bimatoprost offers a novel, low-risk therapeutic approach for scalp alopecias.

yes / Purpose.: The aim of this work was to develop an in vitro cornea model to study the effect of proinflammatory cytokines on wound healing. Methods.: Initial studies investigated how to maintain the ex vivo models for up to 4 weeks without loss of epithelium. To study the effect of cytokines, corneas were cultured with the interleukins IL-17A, IL-22, or a combination of IL-17A and IL-22, or lipopolysaccharide (LPS). The effect of IL-17A on wound healing was then examined. Results.: With static culture conditions, organ cultures deteriorated within 2 weeks. With gentle rocking of media over the corneas and carbon dioxide perfusion, the ex vivo models survived for up to 4 weeks without loss of epithelium. The cytokine that caused the most damage to the cornea was IL-17A. Under static conditions, wound healing of the central corneal epithelium occurred within 9 days, but only a single-layered epithelium formed whether the cornea was exposed to IL-17A or not. With rocking of media gently over the corneas, a multilayered epithelium was achieved 9 days after wounding. In the presence of IL-17A, however, there was no wound healing evident. Characterization of the cells showed that wherever epithelium was present, both differentiated cells and highly proliferative cells were present. Conclusions.: We propose that introducing rocking to extend the effective working life of this model and the introduction of IL-17A to this model to induce aspects of inflammation extend its usefulness to study the effects of agents that influence corneal regeneration under normal and inflamed conditions.

Understanding the mechanisms of cardiovascular disease progression is essential in developing novel therapies to combat this disease that contributes to 1 in 3 deaths in the United States every year. Endothelial dysfunction and its effects on vessel growth and remodeling are key factors in the progression and localization of atherosclerosis. Much of our understanding in this area has come from in-vivo and in-vitro experiments however perfused organ culture systems provide an alternative approach. Organ culture systems can provide a more controlled mechanical and biochemical environment compared to in-vivo models. This study focused on furthering development of this organ culture model by introducing a novel device to produce flow waveforms at the high frequencies and low mean flows seen in the mouse model. The device is capable of monitoring pressure, flow, diameter, and nitric oxide release. Each individual mechanism in the system was integrated via a computer using a custom Labview interface. The performance of the device was characterized by developing physiologic, physiologic-oscillatory, low, low-oscillatory waveforms and sinusoidal waveforms at frequencies ranging from 1-10 Hz. Overall this system provides a robust model to test the effects of flow on various biological markers both in real-time and after culture.

The embryonic mouse inner ear was used as a model with which to study ototoxicity and tissue interactions. The inner ear anlage can be explanted and cultured in vitro from about the 12th gestational day (gd), and will differentiate parallel with the inner ear developing in vivo until a time corresponding to birth (21st gd). During this period the ovoid sac develops into the labyrinth. In the present thesis work, otic anlagen from gd 12, 13, 13.5, 15 and 16 were used. As a rule the explants were kept in culture until a time point equivalent to the 21st gd. Analyses using freeze-fracture technique and transmission electron microscopy showed that in cultured 13th gd otocysts the development of junctional complexes followed the same principal pattern as in vivo. Tight junctions develop into many strands lying parallel to the apical surface of all epithelial cells. Uncoupling of the hair cells occurs with loss of gap junctions. Some tight junctions had an aberrant appearence, with in part very thick strands and strands running at right angles to the apical surface. All aminoglycosides are potentially ototoxic. In the inner ear, outer hair cells of the organ of Corti and vestibular type I hair cells are affected by these antibiotics. The access route to the hair cells and the sites and mechanisms of action of aminoglycosides are not precisely defined. The uptake of tritiated tobramycin in 16th gd inner ears was studied. An initial rapid uptake of the drug, within 10 min, was followed by a slower accumulation, reaching a steady state after 60 min. Most of the tobramycin was bound reversibly, at least after a short period of incubation (2 h). The irreversibly bound fraction was of the same magnitude as the uptake within 10 min. Uptake took place against a concentration gradient. The otocyst can differentiate even without the statoacoustic ganglion. The interaction of the sensory epithelium with the ganglion was investigated by explanting the statoacoustic ganglion without target tissue. Twenty-five percent of the ganglions survived and had outgrowth of neurites but there was no differentiation into either the cochlear or vestibular type of neuron cells. Exposure of cultured otocysts (13 or 13.5 gd) to l-azetidine-2-carboxylic acid, a 1-proline analog that disrupts formation of collagen, resulted in retarded morphogenesis of the labyrinth and a dose- dependent derangement of the basal lamina. The expression of intermediate filaments (IFs) was analysed using monoclonal antibodies. The same IF pattem was found in cultured inner ears as in vivo. Explants were taken on 13th, 15th or 16th gd. Exposure to gentamicin, ethacrynic acid or cisplatin did not alter the IF composition. Cytokeratins (CKs) 8 and 18 were identified in all inner ear epithelia. In addition CKs 7 and 19 were visualized in the epithelia involved in maintaining endolymph homeostasis. The ganglion cells showed coexpression of CK, vimentin and neurofilaments. The elemental composition of the endolymph compartment of 16th gd inner ears cultured for 5 days was studied using energy-dispersive X-ray microanalysis. Na to K ratios characteristic of endolymph were found. / <p>S. 1-34: sammanfattning, s. 37-88: Härtill 6 uppsatser</p> / digitalisering@umu

Distraction osteogenesis (DO) is a surgical procedure in which applied strain stimulates new bone growth; however, the underlying mechanisms by which bone cells respond to load are still uncertain. An organ culture model of DO was developed and validated by using linear distraction on the femoral shafts of 5 day old Wistar rats. Two loading regimes were utilized: distracting the bones for 2 hrs on day 1 (GRP I); distracting the bones for 2 hrs on days 1, 3, and 5 (GRP II). After 1 week in culture, the bones were compared to unloaded contralateral controls and assessed for changes. Structural, dimensional, massing, micro-CT, areal, and viability properties were obtained from testing. Relative to paired controls, distracted bones demonstrated an increase in failure load (9.15% GRP I, 18.85% GRP II), increase in stiffness (31.28% GRP I, 53.21% GRP II), increases in areal and polar moments of inertia, and viability (6.21% GRP I, 13.02% GRP II). Our results suggest that DO can be modeled successfully with an organ culture, and continued use of this system will help to gain insight into the mechanisms and pathways by which distraction osteogenesis occurs.

Hair growth disorders cause significant psychological distress, but are poorly controlled. Since prostaglandin F₂α (PGF₂α) and prostamide F₂α analogue glaucoma treatments cause eyelash growth as side-effects, they may be useful for alopecia. How they function is unknown; possibilities include direct action on hair follicles or stimulating follicular blood flow. It is important to clarify whether scalp follicles can also respond as human follicle response to androgens differ with body site. Therefore, human scalp follicles were grown in vitro in organ culture with PGF₂α, latanoprost, a PGF₂α analogue, and bimatoprost, a prostamide F₂α analogue, with, or without, appropriate antagonists, and the presence of PGF₂α (FP) and prostamide F₂α receptors were investigated using molecular biological and immunohiostochemical methods. Each treatment significantly stimulated follicle growth rate, the percentage of growing follicles, and the amount of hair produced in a dose-responsive manner (10nM-1μM); the receptor antagonists blocked these effects. Immunohistochemistry of frozen scalp sections demonstrated FP protein only in dermal papillae and connective tissue sheaths. RT-PCR identified FP and various prostamide F₂α receptors in anagen follicles and isolated dermal papillae and bulbar connective tissue sheath, but not in bulb matrix or other epithelial tissues. Therefore, isolated human scalp hair follicles can respond biologically to PGF₂α and related pharmaceuticals in organ culture via follicular receptors and express the genes and protein for FP and prostamide F₂α receptors. PGF₂α-related drugs appear to act directly on follicles via receptors in the regulatory dermal papilla. They offer an exciting, novel approach for treating alopecia and merit clinical investigation.