Human Skin as an Ex Vivo Model for Maintaining Mycobacterium leprae and Leprosy Studies.

The in vitro cultivation of M. leprae has not been possible since it was described as causing leprosy, and the limitation of animal models for clinical aspects makes studies on leprosy and bacteria-human host interaction a challenge. Our aim was to standardize the ex vivo skin model (hOSEC) to maintenance and study of M. leprae as an alternative animal model. Bacillary suspensions were inoculated into human skin explants and sustained in DMEM medium for 60 days. Explants were evaluated by RT-PCR-16SrRNA and cytokine gene expression. The viability and infectivity of bacilli recovered from explants (D28 and D60) were evaluated using the Shepard's model. All explants were RT-PCR-16SrRNA positive. The viability and infectivity of recovered bacilli from explants, analyzed after 5 months of inoculation in mice, showed an average positivity of 31%, with the highest positivity in the D28 groups (80%). Furthermore, our work showed different patterns in cytokine gene expression (TGF-ß, IL-10, IL-8, and TNF-α) in the presence of alive or dead bacilli. Although changes can be made to improve future experiments, our results have demonstrated that it is possible to use the hOSEC to maintain M. leprae for 60 days, interacting with the host system, an important step in the development of experimental models for studies on the biology of the bacillus, its interactions, and drug susceptibility.

The role of biopsy in diagnosing infection after hip and knee arthroplasty: a meta-analysis.

INTRODUCTION: Early diagnosis of periprosthetic hip and knee infection still represents a major challenge, as no single test can achieve ideal results. Currently, multiple preoperative indicators were performed to diagnose periprosthetic joint infection (PJI) to confirm or exclude infection in the early stage. However, the diagnostic value of biopsy-related tests in diagnosing periprosthetic hip and knee infection remains unclear. MATERIALS AND METHODS: Publications in PubMed, Embase, and the Web of Science databases were searched systematically until October 2020. Inclusion and exclusion criteria were used for screening biopsy-related studies of the diagnosis of periprosthetic hip and knee infection. RESULTS: Three biopsy-related tests were identified in 14 articles and further analyzed in the present meta-analysis. The combined method had the highest value for the area under the curve (0.9805), followed by histology (0.9425) and microbiological tests (0.9292). In the subgroup, statistical differences were identified in sensitivity and specificity for PJI diagnosis between the synovial fluid culture and biopsy culture group, as well as in the biopsy-related combined method and serum C-reactive protein. CONCLUSIONS: Biopsy culture does not appear to be advantageous compared to synovial fluid culture in the preoperative diagnosis of periprosthetic hip and knee infection. In contrast, combined biopsy microbial culture with histology analysis shows great potential in improving the preoperative diagnosis of PJI. The standard procedure of biopsy needs to be further explored. Further research is required to verify our results.

Ovarian Tissue Culture to Preserve Fertility in Transgender Male Patients after Hormonal Treatment / Cultura de tecido ovariano para preservação da fertilidade em pacientes transexuais masculinos após tratamento hormonal

Abstract Objective To evaluate the reproductive and histological characteristics of fresh cultured ovarian tissue from transgender male patients. Methods An in vitro pilot study in which samples were collected during sex reassignment surgery for transgender male patients. The ovarian cortex was cut into fragments of 2 mm, 3mm, and 4 mm, and placed in a 96-well plate suitable for cultivation at days 0, 2, 4, 6, and 8, when the histology was analyzed. Results Stromal hyperplasia was observed in all samples, and it was not associated with the obtainment of primordial or primary follicles. Peripheral reduction in cell count was also a recurrent finding. Primordial and primary follicles were identified with a heterogeneous pattern in fragments from the same patient and from different patients, and follicles in more advanced stages of development (secondary and antral) were not found. There was an association between the diameter of the ovarian fragments and the identification of primary follicles (p=0.036). The number of days in culture was associated with histological signs of tissue damaging in the fragments (p=0.002). The total number of follicles identified in the samples with 2mm in diameter was significantly lower than in those that measured 4mm in diameter (p=0.031). Conclusion A diameter of 4mm is suitable for ovarian tissue culture with the benefit of ease of handling. Even after prolonged exposure to testosterone, the ovarian fragments presented primordial and primary follicles, maintaining viability throughout the days they were exposed to the culture. Freezing the ovarian cortex of transgender patients who will undergo surgery for gender reassignment would be an interesting option, in the future, for the preservation of fertility.

Resumo Objetivo Avaliar as características reprodutivas e histológicas de tecido ovariano cultivado a fresco de pacientes transexuais masculinos. Métodos Estudo experimental in vitro e piloto, no qual amostras foram coletadas durante a cirurgia de redesignação de sexo para pacientes transexuais masculinos. O córtex ovariano foi cortado em fragmentos de 2mm, 3mm, e 4mm, e colocado em placa de 96 poços própria para cultivo nos dias 0, 2, 4, 6 e 8, quando a histologia foi analisada. Resultados Hiperplasia estromal foi observada em todas as amostras, e não esteve associada à obtenção de folículos primordiais ou primários. A redução periférica no número de células também foi um achado recorrente. Folículos primordiais e primários foramidentificados com padrão heterogêneo emfragmentos domesmo paciente e em fragmentos de pacientes diferentes, não sendo encontrados folículos em estágios mais avançados de desenvolvimento (secundários e antrais). Houve associação entre o diâmetro dos fragmentos ovarianos e a identificação dos folículos primários (p=0,036). O número de dias de cultura esteve associado a sinais histológicos de lesão tecidual nos fragmentos (p=0,002). O número total de folículos identificados nas amostras de 2mm de diâmetro foi significativamente menor do que nas de 4mm de diâmetro (p=0,031). Conclusão O diâmetro de 4mm parece ser mais adequado para a cultura de tecido ovariano com a vantagem de fácil manejo. Mesmo após exposição prolongada à testosterona, os fragmentos ovarianos apresentavam folículos primordiais e primários, e manteve a viabilidade ao longo dos dias de exposição à cultura. No futuro, o congelamento da cortical do ovário de pacientes transgêneros que se submeterão à cirurgia de redesignação sexual poderia ser uma opção interessante para a preservação da fertilidade.

The challenge of ovarian tissue culture: 2D versus 3D culture.

BACKGROUND: Cryopreservation of ovarian tissue is a powerful technique for preserving female fertility, as it can restore fertility and endocrine function. To increase the longevity of the transplant and decrease the risk of reimplantation of neoplastic cells, several studies have been carried out with culture of ovarian tissue. The aim of this study was to compare a conventional (2D) culture with an alginate matrix three-dimensional (3D) model for ovarian tissue culture. RESULTS: The ovarian tissue culture within the alginate matrix (3D) was similar to 2D culture, regarding follicular density and cell apoptosis in follicles and stroma. The proliferation rate remained stable in both models for follicles, but for stromal cell proliferation it decreased only in 3D culture (p = 0.001). At 24 h of culture, cytotoxicity was lower in the 3D model (p = 0.006). As culture time increased, cytotoxicity seemed similar. Degradation of the tissue was suggested by the histological score analysis of tissue morphology after 72 h of culture. Tissue injury was greater (p = 0.01) in 3D culture due to higher interstitial oedema (p = 0.017) and tissue necrosis (p = 0.035). CONCLUSION: According to our results, 3D culture of ovarian tissue has no advantage over 2Dculture; it is more time consuming and difficult to perform and has worse reproducibility.

Microdroplets-on-chip: A review.

Single-cell analysis serves as an important approach to study cell functions and interactions. Catering to the demand of Big Data Era, fast reactions for single cells and paralleled high-throughput analysis have become an urgent need. Microdroplet in microfluidics has advantages of modularity and integrity, as well as high throughput and sensitivity, which present great potential in the field of single-cell analysis. This review is carried out on three aspects to introduce microdroplet chips for single-cell analysis: droplet formation, droplet detection and practical functions. Structures of droplet formation are categorized into three types, including T-shaped channel, flow-involved channel and three-dimensional micro-vortice. The detection methods, including fluorescence, Raman spectroscopy, mass spectroscopy and electrochemical detection, are summarized from applications. Both pros and cons for existing techniques are reviewed and discussed. The functions of microdroplets-on-chip cover cell culture, nucleic acid test and cell identification. For each field, principles/mechanisms and/or schematic images are laconically introduced. Microdroplet in microfluidics has become a major research direction in single-cell analysis. With updated methods of droplet formation such as inertial ordering and micro-vortice, microdroplets-based biochips will expect high throughput detection and high-accuracy trace detection for clinical diagnosis in the near future.

Pretreatment of nerve grafts with resveratrol improves axonal regeneration following replantation surgery for nerve root avulsion injury in rats.

BACKGROUND: Replantation of the avulsed nerve root has been proposed for the treatment of severe brachial plexus injury for several decades. However, due to the complexity of the technique and limited functional improvement, practical applications are yet to be implemented. OBJECTIVE: In the present study, we investigated the effect of pretreatment with resveratrol on nerve autografts used for replantation surgery in a rat model of nerve root avulsion. METHODS: Resveratrol pretreatment was performed using an explant culture technique. Two surgical procedures were performed. During the first surgery, Sprague-Dawley rats were subjected to left C6 nerve root avulsion, and nerves were harvested for autografting. The harvested grafts were explant-cultured for 1 week. A second procedure was performed to replant the C6 nerve root using the explant-cultured nerve graft 1 week after the first procedure. Histological and immunohistochemical analyses were performed 8 weeks after the second procedure. We first compared findings between explant-cultured nerve grafts and fresh nerve grafts, following which we compared findings between explant-cultured grafts pretreated with and without resveratrol. Changes induced within nerve grafts by 1 week of explant culture with or without resveratrol were investigated in vitro. RESULTS: There was no significant difference in outcomes between 1 week-explant-cultured and fresh nerve grafts. Addition of resveratrol to the explant culture medium resulted in a significant increase in the number and myelin thickness of regenerated axons, and in the number of regenerating motor neurons in the C6 spinal cord segment. In vitro analyses revealed that nerve grafts pretreated with resveratrol exhibited significant increases in glial cell line-derived neurotrophic factor (GDNF) expression and the number of dedifferentiated Schwann cells. CONCLUSIONS: Resveratrol may promote axonal regeneration following replantation surgery for the treatment of nerve root avulsion injury; however, further studies are required to verify these findings in humans.

[Oral mucosa analog allografts in non-consanguineous rats].

INTRODUCTION: Although there are therapeutic options for the treatment of oral mucosa defects, the need for functional, anatomical and aesthetically similar substitutes persists, as well as for solutions to reduce autologous grafts morbidity. OBJECTIVE: To determine clinical and histological compatibility of equivalent oral mucosa allografts generated through tissue engineering in non-consanguineous rats. MATERIALS AND METHODS: We used a sample of oral mucosa from Sprague Dawley rats to obtain a fibroblast culture and a keratinocytes and fibroblasts co-culture. In both cases, we used a commercial collagen membrane as "scaffold". After ten weeks of culture, we grafted the resulting membranes into four Wistar rats. The first phase of the study was the development of the oral mucosa equivalents generated by tissue engineering. Then, we implanted them in immunocompetent Wistar rats, and finallywe evaluated the clinical and histological features of the allografts. RESULTS: In vivo evaluation of mucosal substitutes showed a correct integration of artificial oral mucosa in immunocompetent hosts, with an increase in periodontal biotype and the creation of a zone with increased keratinization. Histologically, the tissue was similar to the control oral mucosa sample with no inflammatory reaction nor clinical or histological rejection signs. CONCLUSION: The equivalent oral mucosa allografts generated by tissue engineering showed clinical and histological compatibility.

Aloinjertos análogos de mucosa bucal en ratas no consanguíneas / Oral mucosa analog allografts in non-consanguineous rats

Resumen Introducción: A pesar de que existen opciones terapéuticas para el tratamiento de defectos de la mucosa bucal, persiste la necesidad de encontrar sustitutos funcionales, anatómicos y estéticamente similares al tejido que se va a reemplazar, así como soluciones que reduzcan la morbilidad de los injertos autólogos. Objetivo: Determinar la compatibilidad clínica e histológica de aloinjertos equivalentes de mucosa bucal elaborados mediante ingeniería tisular en ratas no consanguíneas. Materiales y métodos: Se utilizó una muestra de mucosa bucal de ratas Sprague Dawley para la obtención de un cultivo de fibroblastos y otro de queratinocitos y fibroblastos. En ambos casos, se usó una membrana de colágeno comercial como soporte. Después de diez semanas de cultivo, las membranas resultantes se injertaron en cuatro ratas Wistar. La primera fase del estudio consistió en la elaboración de los tejidos análogos de mucosa bucal mediante ingeniería tisular, los cuales se implantaron en ratas Wistar inmunocompetentes; posteriormente, se evaluaron las características clínicas e histológicas del aloinjerto. Resultados: La evaluación in vivo de los tejidos análogos demostró que se habían integrado correctamente en los huéspedes inmunocompetentes, y se había logrado el aumento del biotipo periodontal y la creación de una zona con mayor queratinización. Desde el punto de vista histológico, el tejido adquirió características similares a las de la muestra de mucosa bucal de control, sin ningún tipo de reacción inflamatoria ni signos clínicos o histológicos de rechazo. Conclusión: Hubo compatibilidad clínica e histológica de los aloinjertos equivalentes de mucosa bucal obtenidos mediante ingeniería tisular.

Abstract Introduction: Although there are therapeutic options for the treatment of oral mucosa defects, the need for functional, anatomical and aesthetically similar substitutes persists, as well as for solutions to reduce autologous grafts morbidity. Objective: To determine clinical and histological compatibility of equivalent oral mucosa allografts generated through tissue engineering in non-consanguineous rats. Materials and methods: We used a sample of oral mucosa from Sprague Dawley rats to obtain a fibroblast culture and a keratinocytes and fibroblasts co-culture. In both cases, we used a commercial collagen membrane as "scaffold". After ten weeks of culture, we grafted the resulting membranes into four Wistar rats. The first phase of the study was the development of the oral mucosa equivalents generated by tissue engineering. Then, we implanted them in immunocompetent Wistar rats, and finally we evaluated the clinical and histological features of the allografts. Results: In vivo evaluation of mucosal substitutes showed a correct integration of artificial oral mucosa in immunocompetent hosts, with an increase in periodontal biotype and the creation of a zone with increased keratinization. Histologically, the tissue was similar to the control oral mucosa sample with no inflammatory reaction nor clinical or histological rejection signs. Conclusion: The equivalent oral mucosa allografts generated by tissue engineering showed clinical and histological compatibility.

[Preclinical models in head and neck tumors : Evaluation of cellular and molecular resistance mechanisms in the tumor microenvironment]. / Präklinische Modelle für Kopf-Hals-Tumoren : Aufklärung zellulärer und molekularer Resistenzmechanismen im Gewebekontext.

Because head and neck squamous cell carcinomas (HNSCC) are characterized by a distinct intertumorigenic and intratumorigenic heterogeneity, they often show substantial differences in the response to established therapy strategies. At present, a multitude of biologics and new pharmacological compounds for targeted therapies are available that allow more efficient and less toxic treatment. There is increasing pressure to establish predictive assays not only for ex ante analysis of the individual patient response to combined chemoradiotherapy and targeted therapies but also for investigation of the efficacy of new drugs. In this respect it is essential to maintain the pathophysiological tissue composition as it is known that paracrine tumor-stroma cell interactions may influence tumor reactivity to treatment. More complex models for individualized sensitivity testing have recently been described and the results are promising to pave the way for personalized cancer therapy. This review article focuses on different systems for maintaining the tumor microenvironment and hence the individual cellular composition, such as 3D organotypic models, organotypic multicellular spheroids, patient-derived xenografts and ex vivo tissue cultures and discusses the advantages and disadvantages in terms of translation into clinical application.

Estudio preliminar del tratamiento de úlceras crónicas con equivalentes cutáneos obtenidos por ingeniería de tejidos / Preliminary Study of the Treatment of Chronic Skin Ulcers with Tissue-engineered Skin Equivalents

Las úlceras de las extremidades inferiores son afecciones de incidencia y prevalencia elevada, cuya cronicidad y persistencia en hacer recidivas son dos de las características evolutivas más relevantes. A pesar de un tratamiento etiológico correcto, a lo cual se suma una gran variedad de apósitos disponibles, el porcentaje de curación y la velocidad de cicatrización siguen siendo bajos. Por tanto se realizó un estudio con el objetivo de obtener un sustituto de piel para su aplicación en la clínica. Se aislaron y cultivaron queratinocitos, que fueron sembrados a una alta densidad, en un sustrato optimizado de fibrina con fibroblastos. De esta forma se desarrolló un nuevo equivalente cutáneo más práctico y de bajo costo, cuyo transplante es directo y de fácil aplicación. Antes de comenzar los estudios clínicos para evaluar sus ventajas y su alcance, se hizo una prueba preliminar con el fin de ver si los cambios efectuados en la técnica estándar no impedían su viabilidad y su acción. A tal fin fueron seleccionaron seis pacientes con ulceras crónicas, sin curación mediante todo tratamiento, a quienes se les aplicaron los nuevos equivalentes. Los pacientes tratados mostraron que los equivalentes eran viables, efectivos, de fácil manipulación y de rápida acción sobre los tejidos afectados. Todos los pacientes que completaron el procedimiento se recuperaron, fue excelente ejemplo un paciente con una lesión crónica de seis meses de evolución que había sido refractaria a los múltiples tratamientos convencionales empleados.

Ulcers of the lower extremities have high prevalence and incidence. Their chronicity and frequent recurrences are two of their most important characteristics. Despite proper etiologic treatment and the great variety of dressings available, the healing rate and recovery speed remain low. Therefore, we conducted a study to obtain skin substitutes for clinical application by isolating and cultivating keratinocytes, which were seeded on a fibroblast-containing fibrin gel at high density. Hence, a new practical and inexpensive skin equivalent of straightforward and easy-to-perform transplantation was developed. Before starting the clinical trials to evaluate its advantages and applicability, we conducted a preliminary test to determine if the changes to the standard technique affected its feasibility and action. For this purpose, we implanted the new equivalents into six patients with chronic ulcers unresponsive to treatment. Outcomes in these patients showed that skin equivalents were feasible, effective, easy to handle and had rapid action on target tissues. All patients who completed the procedure recovered. An excellent example was a patient with a chronic lesion refractory to multiple conventional treatments that had been present for six months.

Biaxial stress relaxation of semilunar heart valve leaflets during simulated collagen catabolism: Effects of collagenase concentration and equibiaxial strain state.

Heart valve leaflet collagen turnover and remodeling are innate to physiological homeostasis; valvular interstitial cells routinely catabolize damaged collagen and affect repair. Moreover, evidence indicates that leaflets can adapt to altered physiological (e.g. pregnancy) and pathological (e.g. hypertension) mechanical load states, tuning collagen structure and composition to changes in pressure and flow. However, while valvular interstitial cell-secreted matrix metalloproteinases are considered the primary effectors of collagen catabolism, the mechanisms by which damaged collagen fibers are selectively degraded remain unclear. Growing evidence suggests that the collagen fiber strain state plays a key role, with the strain-dependent configuration of the collagen molecules either masking or presenting proteolytic sites, thereby protecting or accelerating collagen proteolysis. In this study, the effects of equibiaxial strain state on collagen catabolism were investigated in porcine aortic valve and pulmonary valve tissues. Bacterial collagenase (0.2 and 0.5 mg/mL) was utilized to simulate endogenous matrix metalloproteinases, and biaxial stress relaxation and biochemical collagen concentration served as functional and compositional measures of collagen catabolism, respectively. At a collagenase concentration of 0.5 mg/mL, increasing the equibiaxial strain imposed during stress relaxation (0%, 37.5%, and 50%) yielded significantly lower median collagen concentrations in the aortic valve (p = 0.0231) and pulmonary valve (p = 0.0183), suggesting that relatively large strain magnitudes may enhance collagen catabolism. Collagen concentration decreases were paralleled by trends of accelerated normalized stress relaxation rate with equibiaxial strain in aortic valve tissues. Collectively, these in vitro results indicate that biaxial strain state is capable of affecting the susceptibility of valvular collagens to catabolism, providing a basis for further investigation of how such phenomena may manifest at different strain magnitudes or in vivo.

Usefulness of Skin Explants for Histologic Analysis after Fractional Photothermolysis.

BACKGROUND: Fractional laser resurfacing treatment has been extensively investigated and is widely used. However, the mechanism underlying its effects is poorly understood because of the ethical and cosmetic problems of obtaining skin biopsies required to study the changes after laser treatment. OBJECTIVE: To evaluate the usefulness of human skin explants for the investigation of fractional photothermolysis. METHODS: Full-thickness discarded skin was treated in 4 ways: no treatment (control), fractional carbon dioxide laser, fractional Er:YAG laser, and fractional 1,550-nm erbium-doped fiber laser. Both treated and non-treated skin samples were cultured ex vivo at the air-medium interface for 7 days. Frozen tissue was sectioned and stained with hematoxylin & eosin for histologic examination and nitro blue tetrazolium chloride for viability testing. RESULTS: Skin explants cultured for up to 3 days exhibited histologic changes similar to those observed in in vivo studies, including microscopic treatment zones surrounded by a thermal coagulation zone, re-epithelialization, and formation of microscopic epidermal necrotic debris. However, the explant structure lost its original form within 7 days of culture. The viability of skin explants was maintained for 3 days of culture but was also lost within 7 days. CONCLUSION: The skin explant model may be a useful tool for investigating the immediate or early changes following fractional photothermolysis, but further improvements are required to evaluate the long-term and dermal changes.

Prolonged viability of human organotypic skin explant in culture method (hOSEC)

Abstract BACKGROUND: Currently, the cosmetic industry is overwhelmed in keeping up with the safety assessment of the increasing number of new products entering the market. To meet such demand, research centers have explored alternative methods to animal testing and also the large number of volunteers necessary for preclinical and clinical tests. OBJECTIVES: This work describes the human skin ex-vivo model (hOSEC: Human Organotypic Skin Explant Culture) as an alternative to test the effectiveness of cosmetics and demonstrate its viability through cutaneous keratinocytes' proliferative capacity up to 75 days in culture. METHODS: The skin explants obtained from surgeries were cultured in CO2-humid incubator. After 1, 7, 30 and 75 days in culture, skin fragments were harvested for analysis with histomorphological exam (HE staining) on all days of follow-up and immunohistochemistry for Ck5/6, Ck10 and Ki-67 only on the 75th day. RESULTS: On the 7th day, the epidermis was perfect in the dermoepidermal junction, showing the viability of the model. On the 30th day, the epidermis was thicker, with fewer layers on the stratum corneum, although the cutaneous structure was unaltered. On the 75th day, the skin became thinner but the dermoepidermal junctions were preserved and epidermal proliferation was maintained. After the 75th day on culture, the skin was similar to normal skin, expressing keratinocytes with Ck5/6 on supra-basal layers; Ck10 on differentiated layers; and viability could be assessed by the positivity of basal cells by Ki-67. CONCLUSION: The hOSEC model seems a good alternative to animal testing; it can be used as a preclinical test analogous to clinical human skin test with similar effectiveness and viability proven by immunohistological analyses. .

Usefulness of Skin Explants for Histologic Analysis after Fractional Photothermolysis

BACKGROUND: Fractional laser resurfacing treatment has been extensively investigated and is widely used. However, the mechanism underlying its effects is poorly understood because of the ethical and cosmetic problems of obtaining skin biopsies required to study the changes after laser treatment. OBJECTIVE: To evaluate the usefulness of human skin explants for the investigation of fractional photothermolysis. METHODS: Full-thickness discarded skin was treated in 4 ways: no treatment (control), fractional carbon dioxide laser, fractional Er:YAG laser, and fractional 1,550-nm erbium-doped fiber laser. Both treated and non-treated skin samples were cultured ex vivo at the air-medium interface for 7 days. Frozen tissue was sectioned and stained with hematoxylin & eosin for histologic examination and nitro blue tetrazolium chloride for viability testing. RESULTS: Skin explants cultured for up to 3 days exhibited histologic changes similar to those observed in in vivo studies, including microscopic treatment zones surrounded by a thermal coagulation zone, re-epithelialization, and formation of microscopic epidermal necrotic debris. However, the explant structure lost its original form within 7 days of culture. The viability of skin explants was maintained for 3 days of culture but was also lost within 7 days. CONCLUSION: The skin explant model may be a useful tool for investigating the immediate or early changes following fractional photothermolysis, but further improvements are required to evaluate the long-term and dermal changes.

Fatty Acid Composition of Tissue Cultured Breast Carcinoma and the Effect of Stearoyl-CoA Desaturase 1 Inhibition.

PURPOSE: Stearoyl-CoA desaturase 1 (SCD1) is a novel therapeutic target in various malignancies, including breast cancer. The present study was designed to investigate the effect of the pharmacologic inhibition of SCD1 on fatty acid composition in tissue explant cultures of human breast cancer and to compare these effects with those in adjacent nonneoplastic breast tissue. METHODS: Paired samples of tumor and adjacent noncancerous tissue were isolated from 12 patients with infiltrating ductal breast cancer. Samples were explant cultured in vitro, exposed to the highly selective SCD1 inhibitor CAY10566, and examined for fatty acid composition by gas liquid chromatography. The cytotoxic and antigrowth effects were evaluated by quantification of lactate dehydrogenase release and by sulforhodamine B (SRB) measurement, respectively. RESULTS: Breast cancer tissue samples were found to have higher levels of monounsaturated fatty acids (MUFA) (p<0.001) and arachidonic acid (20:4n-6, p<0.001) and a lower level of linoleic acid (18:2n-6, p=0.02) than the normal-appearing breast tissues. While exhibiting no evident cytotoxicity, treatment with the SCD1 inhibitor, CAY10566 (0.1-1 µM), for 48 hours significantly increased 18:2n-6 levels in both the tumor and adjacent normal-appearing tissue (approximately 1.2 fold, p<0.05). However, the breast cancer tissue samples showed significant increases in the levels of MUFA and 20:4n-6 compared to the normal-appearing breast tissues (p<0.05). The SRB growth assay revealed a higher rate of inhibition with the SCD1 inhibitor in breast cancer tissues than in normal-appearing tissues (p<0.01, 41% vs. 29%). The SCD1 inhibitor also elevated saturated fatty acid (1.46-fold, p=0.001) levels only in the tumor tissue explant. CONCLUSION: The fatty acid composition and response to SCD1 inhibition differed between the explant cultures from breast cancer and the adjacent normal-appearing tissue. Altered fatty acid composition induced by SCD1 inhibition may also, in addition to Δ9 desaturation, modulate other reactions in de novo fatty acid synthesis and lipogenesis, and subsequently affect the overall survival and progression of breast cancer.

Design and testing of a cyclic stretch and flexure bioreactor for evaluating engineered heart valve tissues based on poly(glycerol sebacate) scaffolds.

Cyclic flexure and stretch are essential to the function of semilunar heart valves and have demonstrated utility in mechanically conditioning tissue-engineered heart valves. In this study, a cyclic stretch and flexure bioreactor was designed and tested in the context of the bioresorbable elastomer poly(glycerol sebacate). Solid poly(glycerol sebacate) membranes were subjected to cyclic stretch, and micromolded poly(glycerol sebacate) scaffolds seeded with porcine aortic valvular interstitial cells were subjected to cyclic stretch and flexure. The results demonstrated significant effects of cyclic stretch on poly(glycerol sebacate) mechanical properties, including significant decreases in effective stiffness versus controls. In valvular interstitial cell-seeded scaffolds, cyclic stretch elicited significant increases in DNA and collagen content that paralleled maintenance of effective stiffness. This work provides a basis for investigating the roles of mechanical loading in the formation of tissue-engineered heart valves based on elastomeric scaffolds.

Fatty Acid Composition of Tissue Cultured Breast Carcinoma and the Effect of Stearoyl-CoA Desaturase 1 Inhibition / 한국유방암학회지

PURPOSE: Stearoyl-CoA desaturase 1 (SCD1) is a novel therapeutic target in various malignancies, including breast cancer. The present study was designed to investigate the effect of the pharmacologic inhibition of SCD1 on fatty acid composition in tissue explant cultures of human breast cancer and to compare these effects with those in adjacent nonneoplastic breast tissue. METHODS: Paired samples of tumor and adjacent noncancerous tissue were isolated from 12 patients with infiltrating ductal breast cancer. Samples were explant cultured in vitro, exposed to the highly selective SCD1 inhibitor CAY10566, and examined for fatty acid composition by gas liquid chromatography. The cytotoxic and antigrowth effects were evaluated by quantification of lactate dehydrogenase release and by sulforhodamine B (SRB) measurement, respectively. RESULTS: Breast cancer tissue samples were found to have higher levels of monounsaturated fatty acids (MUFA) (p<0.001) and arachidonic acid (20:4n-6, p<0.001) and a lower level of linoleic acid (18:2n-6, p=0.02) than the normal-appearing breast tissues. While exhibiting no evident cytotoxicity, treatment with the SCD1 inhibitor, CAY10566 (0.1-1 microM), for 48 hours significantly increased 18:2n-6 levels in both the tumor and adjacent normal-appearing tissue (approximately 1.2 fold, p<0.05). However, the breast cancer tissue samples showed significant increases in the levels of MUFA and 20:4n-6 compared to the normal-appearing breast tissues (p<0.05). The SRB growth assay revealed a higher rate of inhibition with the SCD1 inhibitor in breast cancer tissues than in normal-appearing tissues (p<0.01, 41% vs. 29%). The SCD1 inhibitor also elevated saturated fatty acid (1.46-fold, p=0.001) levels only in the tumor tissue explant. CONCLUSION: The fatty acid composition and response to SCD1 inhibition differed between the explant cultures from breast cancer and the adjacent normal-appearing tissue. Altered fatty acid composition induced by SCD1 inhibition may also, in addition to Delta9 desaturation, modulate other reactions in de novo fatty acid synthesis and lipogenesis, and subsequently affect the overall survival and progression of breast cancer.

An outcome analysis and long-term viability of cryopreserved cultured epidermal allografts: assessment of the conservation of transplantable human skin allografts

PURPOSE: To assess the viability of cultured epithelium and preserved by freezing for periods varying from one month to one year. METHODS: Samples of cultured epithelium were incubated in cryoprotectant medium (Group A), packed in aluminum envelopes and packed in polystyrene boxes. The boxes were subjected to a temperature of-70ºC. After freezing for a period of time ranging from one to 12 months, cultured epithelial samples were assessed for their viability by vital staining (Trypan blue) and metabolic analysis based on glucose consumption and lactate production. Samples of not frozen cultured epithelium (Group B) were also tested for viability and the results obtained were used as comparison parameter for the variation of viability. RESULTS: Statistical analysis between the group A and B indicate that the mean age of the donors (p=0.51) and the culture time (p=1.18) showed no statistical difference. In 30 days we obtained 37% of the original viability of cultured epithelium, 25% at six months and one year, less than 15%. This trend was confirmed statistically with a reduction of approximately 1.8% of the original viability epithelium cultured every 30 days of storage. In the analysis by lactate production, similar results were observed. In the analysis by the glucose consumption results were not significant. The viability indices show statistically significant difference between the group A and B (p<0.0001). CONCLUSIONS: Although cryopreserved cultured epithelium showed significant reduction of viability, all samples remained viable. It was also found that the viability of cryopreserved cultured epithelial decreased as a function of storage time.

Desmoplastic small round cell tumor (DSRCT) xenografts and tissue culture lines: Establishment and initial characterization.

Desmoplastic small round cell tumor (DSRCT) is an extremely rare and aggressive neoplasm, which mainly affects young males and generally presents as a widely disseminated tumor within the peritoneal cavity. Due to the rarity of the tumor, its younger and overall healthier patient population (compared with other tumor types) and the fact that it lacks definitive histological and immunohistological features, the diagnosis of DSRCT may be frequently delayed or the tumor may be entirely misdiagnosed as a different type of abdominal sarcoma. The present study aimed to rectify the lack of models that exist for this rare neoplasm, through the development of several DSRCT tissue cultures and xenograft lines. Samples were received from surgeries and biopsies from patients worldwide and were immediately processed for xenograft development in nude mice. Tumor tissues were minced and fragments were injected into the dorsal flanks of nude mice. Of the 14 samples received, nine were established into xenograft lines and five into tissue culture lines. Xenografts displayed the microscopic histology of their parent tumors and demonstrated two different growth rates among the established xenograft lines. Overall, the establishment of these xenograft and tissue culture lines provides researchers with tools to evaluate DSRCT responses to chemotherapy and to investigate DSRCT-specific signaling pathways or mechanisms.

Enhanced synthesis of andrographolide by Aspergillus niger and Penicillium expansum elicitors in cell suspension culture of Andrographis paniculata (Burm. f.) Nees.

BACKGROUND: Andrographis paniculata (Burm. f.) Nees is an important medicinal plant which has enormous applications in pharmaceutical industries. Cell suspension culture of Andrographis paniculata (Burm. f.) Nees. was treated with Aspergillus niger and Penicillium expansum elicitors to enhance the synthesis of andrographolide, the bioactive constituent of A. paniculata. RESULT: The elicitation treatment with fungal elicitors (A. niger and P. expansum) was observed to be most suitable for eliciting andrographolide production in the culture. The quantification of andrographolide was done using High Performance Liquid Chromatography (HPLC) technique. A. niger extract (1.5 ml with10 days treatment duration) revealed 6.94 fold increase in andrographolide content (132 µg) which was higher than the control (19 µg). P. expansum elicitor (0.6% with 8 days treatment duration) could reveal 6.23 fold enhancement in andrographolide content (81.0 µg) over control (13 µg). CONCLUSION: The results obtained reveal that the longer treatment duration is most favorable for the elicitation of andrographolide using both the fungal elicitors.