Normothermic Machine Perfusion Systems: Where Do We Go From Here?

Normothermic machine perfusion (NMP) aims to preserve organs ex vivo by simulating physiological conditions such as body temperature. Recent advancements in NMP system design have prompted the development of clinically effective devices for liver, heart, lung, and kidney transplantation that preserve organs for several hours/up to 1 d. In preclinical studies, adjustments to circuit structure, perfusate composition, and automatic supervision have extended perfusion times up to 1 wk of preservation. Emerging NMP platforms for ex vivo preservation of the pancreas, intestine, uterus, ovary, and vascularized composite allografts represent exciting prospects. Thus, NMP may become a valuable tool in transplantation and provide significant advantages to biomedical research. This review recaps recent NMP research, including discussions of devices in clinical trials, innovative preclinical systems for extended preservation, and platforms developed for other organs. We will also discuss NMP strategies using a global approach while focusing on technical specifications and preservation times.

Decellularization Methods of Ovary in Tissue Engineering.

The ovaries or female gonads are situated in the ovarian fossa of the abdominal cavity. These are paired, almond-shaped organs measuring about 3.5 cm long and 1.5 cm thick and exist out of a central medullary zone and a peripheral cortex that are enclosed in a fibrous capsule called the tunica albuginea. The ovaries serve 2 main functions, the first one being the production of female gametes called oocytes (oogenesis). Interestingly, the number of primary oocytes that reside in the ovary is determined at birth. About 400 oocyte-containing follicles successfully go through all the developmental stages from this limited pool during folliculogenesis throughout the female reproductive life. In this process, primordial follicles grow and advance until forming a mature or Graafian follicle; during ovulation, secondary oocytes are released and the remaining follicular wall collapses and forms the highly vascularized corpus luteum or luteal gland. This ovarian cycle is regulated by several hormones secreted from the adenohypophysis and lasts about 28 days. During this cycle, the ovaries also serve as endocrine glands and produce female sex hormones such as estrogens and progesterone (steroidogenesis), influencing the growth and development of tissues sensitive to these hormones such as the endometrium. Hence, the endometrial cycle goes synchronized with the ovarian cycle.

Decellularization Methods of Uterus in Tissue Engineering.

A new field of investigation which aims to design tissues and organs similar to their native origin has been developed recently, named as regenerative medicine (tissue engineering and bio-engineering). Uterus is the main organ for regeneration and contributes in the fertility. At an ultimate level, the uterus plays a role in embryo implantation, sperm migration and fetal nutrition. Uterine congenital anomalies, attained uterine lesions and immune system disorders may affect such uterine functions preventing successful pregnancy. Due to following reasons, it is essential to consider regenerative medicine as a new approach for the treatment of uterine dysfunctions to overcome the failures that cannot be treated with clinical medication.

Decellularization Methods of Vagina and Cervix in Tissue Engineering.

The vagina is a fibromuscular elastic tubular tract that connects the cervix with the outer genitals and has an important function discharging uterine secretions, sexual intercourse and acts as the passage for the full-term fetus. Currently, a new field of investigation which aims to design tissues and organs similar to their native origin has been developed recently and was named regenerative medicine (tissue engineering and bioengineering). Malformations in cervix tissue represent a hard challenge for medicine. Experts in bioengineering have tried to reconstruct vaginas or cervix with the aim to achieve cervicovaginal disorders, most of them with congenital cause. However, only few research groups have launched themselves upon the decellularization. The aim of this chapter is investigating the decellularization methods for cervix and vaginal tissues.

Bioengineered endometrial hydrogels with growth factors promote tissue regeneration and restore fertility in murine models.

Extracellular matrix (ECM) hydrogels obtained from decellularized tissues are promising biocompatible materials for tissue regeneration. These biomaterials may provide important options for endometrial pathologies such as Asherman's syndrome and endometrial atrophy, which lack effective therapies thus far. First, we performed a proteomic analysis of a decellularized endometrial porcine hydrogel (EndoECM) to describe the specific role of ECM proteins related to regenerative processes. Furthermore, we investigated the ability of a bioengineered system-EndoECM alone or supplemented with growth factors (GFs)-to repair the endometrium in a murine model of endometrial damage. For this model, the uterine horns of female C57BL/6 mice were first injected with 70% ethanol, then four days later, they were treated with: saline (negative control); biotin-labeled EndoECM; or biotin-labeled EndoECM plus platelet-derived GF, basic fibroblast GF, and insulin-like GF 1 (EndoECM+GF). Endometrial regeneration and fertility restoration were evaluated by assessing the number of glands, endometrial area, cell proliferation, neaoangiogenesis, reduction of collagen deposition, and fertility restoration. Interestingly, regenerative effects such as an increased number of endometrial glands, increased area, high cell proliferative index, development of new blood vessels, reduction of collagen deposition, and higher pregnancy rate occurred in mice treated with EndoECM+GF. Thus, a bioengineered system based on EndoECM hydrogel supplemented with GFs may be promising for the clinical treatment of endometrial conditions such as Asherman's syndrome and endometrial atrophy. STATEMENT OF SIGNIFICANCE: In the last years, the bioengineering field has developed new and promising approaches to regenerate tissues or replace damaged and diseased tissues. Bioengineered hydrogels offer an ideal option because these materials can be used not only as treatments but also as carriers of drugs and other therapeutics. The present work demonstrates for the first time how hydrogels derived from pig endometrium loaded with growth factors could treat uterine pathologies in a mouse model of endometrial damage. These findings provide scientific evidence about bioengineered hydrogels based on tissue-specific extracellular matrix offering new options to treat human infertility from endometrial causes such as Asherman's syndrome or endometrial atrophy.

Improved Models of Human Endometrial Organoids Based on Hydrogels from Decellularized Endometrium.

Organoids are three-dimensional (3D) multicellular tissue models that mimic their corresponding in vivo tissue. Successful efforts have derived organoids from primary tissues such as intestine, liver, and pancreas. For human uterine endometrium, the recent generation of 3D structures from primary endometrial cells is inspiring new studies of this important tissue using precise preclinical models. To improve on these 3D models, we decellularized pig endometrium containing tissue-specific extracellular matrix and generated a hydrogel (EndoECM). Next, we derived three lines of human endometrial organoids and cultured them in optimal and suboptimal culture expansion media with or without EndoECM (0.01 mg/mL) as a soluble additive. We characterized the resultant organoids to verify their epithelial origin, long-term chromosomal stability, and stemness properties. Lastly, we determined their proliferation potential under different culture conditions using proliferation rates and immunohistochemical methods. Our results demonstrate the importance of a bioactive environment for the maintenance and proliferation of human endometrial organoids.

Stem Cells and the Endometrium: From the Discovery of Adult Stem Cells to Pre-Clinical Models.

Adult stem cells (ASCs) were long suspected to exist in the endometrium. Indeed, several types of endometrial ASCs were identified in rodents and humans through diverse isolation and characterization techniques. Putative stromal and epithelial stem cell niches were identified in murine models using label-retention techniques. In humans, functional methods (clonogenicity, long-term culture, and multi-lineage differentiation assays) and stem cell markers (CD146, SUSD2/W5C5, LGR5, NTPDase2, SSEA-1, or N-cadherin) facilitated the identification of three main types of endogenous endometrial ASCs: stromal, epithelial progenitor, and endothelial stem cells. Further, exogenous populations of stem cells derived from bone marrow may act as key effectors of the endometrial ASC niche. These findings are promoting the development of stem cell therapies for endometrial pathologies, with an evolution towards paracrine approaches. At the same time, promising therapeutic alternatives based on bioengineering have been proposed.

A Natural Xenogeneic Endometrial Extracellular Matrix Hydrogel Toward Improving Current Human in vitro Models and Future in vivo Applications.

Decellularization techniques support the creation of biocompatible extracellular matrix hydrogels, providing tissue-specific environments for both in vitro cell culture and in vivo tissue regeneration. We obtained endometrium derived from porcine decellularized uteri to create endometrial extracellular matrix (EndoECM) hydrogels. After decellularization and detergent removal, we investigated the physicochemical features of the EndoECM, including gelation kinetics, ultrastructure, and proteomic profile. The matrisome showed conservation of structural and tissue-specific components with low amounts of immunoreactive molecules. EndoECM supported in vitro culture of human endometrial cells in two- and three-dimensional conditions and improved proliferation of endometrial stem cells with respect to collagen and Matrigel. Further, we developed a three-dimensional endometrium-like co-culture system of epithelial and stromal cells from different origins. Endometrial co-cultures remained viable and showed significant remodeling. Finally, EndoECM was injected subcutaneously in immunocompetent mice in a preliminary study to test a possible hypoimmunogenic reaction. Biomimetic endometrial milieus offer new strategies in reproductive techniques and endometrial repair and our findings demonstrate that EndoECM has potential for in vitro endometrial culture and as treatment for endometrial pathologies.

Development of Decellularized Oviductal Hydrogels as a Support for Rabbit Embryo Culture.

The oviducts (fallopian tubes in mammals) function as the site of fertilization and provide necessary support for early embryonic development, mainly via embryonic exposure to the tubal microenvironment. The main objective of this study was to create an oviduct-specific extracellular matrix (oviECM) hydrogel rich in bioactive components that mimics the native environment, thus optimizing the developmental trajectories of cultured embryos. Rabbit oviducts were decellularized through SDS treatment and enzymatic digestion, and the acellular tissue was converted into oviductal pre-gel extracellular matrix (ECM) solutions. Incubation of these solutions at 37 °C resulted in stable hydrogels with a fibrous structure based on scanning electron microscopy. Histological staining, DNA quantification and colorimetric assays confirmed that the decellularized tissue and hydrogels contained no cellular or nuclear components but retained important components of the ECM, e.g. hyaluronic acid, glycoproteins and collagens. To evaluate the ability of oviECM hydrogels to maintain early embryonic development, two-cell rabbit embryos were cultured on oviECM-coated surfaces and compared to those cultured with standard techniques. Embryo development was similar in both conditions, with 95.9% and 98% of the embryos reaching the late morula/early blastocyst stage by 48 h under standard culture and oviECM conditions, respectively. Metabolomic analysis of culture media in the presence or absence of embryos, however, revealed that the oviECM coating may include signalling molecules and release compounds beneficial to embryo metabolism.

Comparison of different sources of platelet-rich plasma as treatment option for infertility-causing endometrial pathologies.

OBJECTIVE: To study the effect of human plasma from different sources, namely, umbilical cord blood and adult blood platelet-rich plasma (PRP), on the regeneration of endometrial damage. DESIGN: Composition analysis, in vitro approaches, and a preclinical murine model using plasma to promote endometrial regeneration. SETTING: Hospital and university laboratories. PATIENT(S)/ANIMAL(S): Adult plasma from four Asherman syndrome/endometrial atrophy patients and one fertile woman, commercial umbilical cord plasma, and uterine-damaged NOD/SCID mice model were used. INTERVENTION(S): Endometrial stromal cells from primary culture and an endometrial stem cell line were cultured in vitro, and uterine-damaged NOD/SCID mice were treated with plasma samples from several origins. MAIN OUTCOME MEASURE(S): To investigate the possible beneficial effects of PRP from Asherman syndrome/endometrial atrophy patients. To test if plasma from human umbilical cord blood had a stronger effect than adult PRP in endometrial regeneration. To demonstrate if PRP from Asherman syndrome/endometrial atrophy patients was as effective as PRP from a healthy woman and could therefore be used for autologous treatment. RESULT(S): All plasma samples contained molecules with a high potential for regeneration (stem cell factor, platelet-derived growth factor BB, thrombospondin-1, von Willebrand factor). Furthermore, the highest increase in in vitro proliferation and migration rate was found when endometrial stromal cells were treated with umbilical cord plasma; adult PRP also revealed a significant increment. In the mouse model, a higher expression of Ki67 and Hoxa10 in the endometrium was detected after applying adult PRP, and the proteomic analysis revealed a specific protein expression profile depending on the treatment. The damaged uterine tissue showed more proregenerative markers after applying umbilical cord plasma (Stat5a, Uba3, Thy1) compared with the other treatments (nonactivated umbilical cord plasma, activated adult PRP, and no treatment). CONCLUSION(S): Human PRP possesses regeneration properties usable for endometrial pathologies. Besides that, these regenerative effects seem to be more apparent when the source of obtaining is umbilical cord blood.

Tissue-specific decellularized endometrial substratum mimicking different physiological conditions influences in vitro embryo development in a rabbit model.

In the last decades, the decellularization (DC) of organs has become an established technique in the field of regenerative medicine to yield complex and vascularized bioscaffolds. Furthermore, it has been demonstrated in vitro that these decellularized scaffolds retain their native tissue-specificity. This is also the case when this tissue-specific extracellular matrix (ECM) is solubilized and used as hydrogels or coatings to create a biomimetic environment. In this study we investigated if this specificity not only remains when applied to distinct tissues but even more, that these differences can be distinguished within the same tissue at different stages of proliferation. To address this question, a sensitive in vitro animal model was used: rabbit embryos at the third day of development were cultured on coatings made from acellular endometrium that was non-proliferating (non-synchronous, NS) and proliferating (synchronous with the embryo, S) and their development was compared. For this, we obtained whole NS and S rabbit uteri and subjected them to an adapted decellularization protocol. The acellular endometrium was carefully separated by microdissection and converted into a pre-gel solution to be used as hydrogels and coatings for in vitro assays. First, the characteristics of these NS and S hydrogels were investigated by proteomic analysis, electron microscopy and gelling kinetics. When used as substrata for day 3 embryos culture, it became apparent that only the acellular ECM from synchronous endometrial coating achieved similar results to the gold standard culture protocols and conditions, possibly because of the slow release of growth factors present in the synchronous/proliferating endometrium. STATEMENT OF SIGNIFICANCE: It has been shown by in vitro culture of stem cells, progenitor cells and primary culture cells that decellularized tissues retain their specific functions and biochemical and structural compositions. The present work demonstrates that using a mild SDS and perfusion based decellularization (DC) protocol not only effectively decellularize whole rabbit uteri, adding to the growing field of reproductive tissue engineering, but more importantly that the differences in the proliferating endometrium are translated after DC. This implies that DC not only retains the interspecificity of tissues but also the intraspecificity of a developing hormonally stimulated tissue. For the first time, we demonstrate that the coating from decellularized synchronous endometrium acts as a biological support for in vitro embryo development, achieving comparable results with the current gold standard that only uses serum-containing media.

Gender differences on effectiveness of a school-based physical activity intervention for reducing cardiometabolic risk: a cluster randomized trial.

BACKGROUND: Studies that have examined the impact of a physical activity intervention on cardiometabolic risk factors have yielded conflicting results. The objective of this study was to assess the impact of a standardized physical activity program on adiposity and cardiometabolic risk factors in schoolchildren. METHODS: Cluster randomized trial study of 712 schoolchildren, 8-10 years, from 20 public schools in the Province of Cuenca, Spain. The intervention (MOVI-2) consisted of play-based and non-competitive activities. MOVI-2 was conducted during two 90-minute sessions on weekdays and one 150-minute session on Saturday mornings every week between September 2010 and May 2011. We measured changes in adiposity (overweight/obesity prevalence, body mass index [BMI], triceps skinfold thickness [TST], body fat %, fat-free mass, waist circumference) and other cardiometabolic risk factors (LDL-cholesterol, triglycerides/HDL-cholesterol ratio, insulin, C-reactive protein and blood pressure). The analyses used mixed regression models to adjust for baseline covariates under cluster randomization. RESULTS: Among girls, we found a reduction of adiposity in intervention versus control schools, with a decrease in TST (-1.1 mm; 95% confidence interval [CI] -2.3 to -0.7), body fat % (-0.9%; 95% CI -1.3 to -0.4), waist circumference (-2.7 cm; 95% CI -4.5 to -0.9), and an increase in fat-free mass (0.3 kg; 95% CI 0.01 to 0.6). The intervention also led to lower serum LDL-cholesterol and insulin levels. Among boys, a reduction in waist circumference (-1.4 cm; 95% CI -2.6 to -0.1; P = 0.03), and an increase in fat-free mass (0.5 kg; 95% CI 0.2 to 0.9; P = 0.003) was associated with the intervention versus control schools. The prevalence of overweight/obesity or underweight, BMI, and other cardiometabolic risk factors was not modified by the intervention. No important adverse events were registered. CONCLUSIONS: An extracurricular intervention of non-competitive physical activity during an academic year, targeting all schoolchildren regardless of body weight, is a safe and effective measure to reduce adiposity in both genders and to improve cardiometabolic risk profile in girls. TRIAL REGISTRATION: Clinical trials NCT01277224.

Physical activity, fitness, and metabolic syndrome in young adults.

PURPOSE: Our objective was to analyze the association between different intensities of physical activity (PA), physical fitness, and metabolic syndrome (MS) in young adults. METHODS: Cross-sectional study including 275 university students, 18-30 years old, from Cuenca, Spain. We evaluated (a) physical activity using accelerometry, (b) aerobic capacity (VO2max), and (c) muscle strength, by a muscle strength index calculated as the sum of the standardized z score of handgrip dynamometry/weight and standing broad jump. An MS index was estimated by summing standardized z scores of waist circumference, ratio of triglycerides to high-density lipoprotein, mean arterial blood pressure, and HOMA-IR. RESULTS: The mean scores of MS index and HOMAIR were significantly higher and the VO2max significantly lower for individuals who did not perform 20 min or more per week of vigorous physical activity. However, those who performed 250 min/week of moderate physical activity showed no significant differences in either VO2max or the MS index when compared with individuals who did not perform this level of activity. The MS index was lower in those with medium-high levels of aerobic capacity. In addition, individuals with medium-high levels of muscular fitness showed lower waist circumference and a lower MS index. CONCLUSIONS: VO2max and muscle strength are negatively associated with metabolic risk. 20-min/week of vigorous physical activity was associated with lower cardiometabolic risk in young adults; moderate physical activity did not show association with lower cardiometabolic risk.

Protocol of a randomized cluster trial to assess the effectiveness of the MOVI-2 program on overweight prevention in schoolchildren.

INTRODUCTION AND OBJECTIVES: The MOVI physical activity program has been shown to reduce adiposity and to improve serum lipid profiles in schoolchildren. However, MOVI may have not achieved its maximum potential effectiveness, as increased physical activity on weekdays may have been offset by more sedentary behavior at weekends. We therefore developed the MOVI-2 program, which includes physical activity at weekends as well. This paper reports the rationale and methods of a trial to assess the effectiveness of MOVI-2 in preventing overweight and reducing cardiovascular risk in 1200 4th- and 5th-grade primary schoolchildren in Cuenca, Spain. METHODS: Ten schools were randomly assigned to MOVI-2 and 10 schools to the control group. MOVI-2 consisted of recreational physical activity in after-school time, including two 90-min sessions on weekdays and one 150-min session on Saturdays, during each week of one academic year. The control group was expected to follow their usual patterns of physical activity. The primary end points, which were assessed at the start and the end of the MOVI-2 program, were weight and height, waist circumference, skinfold thickness, body fat percentage, blood pressure, lipid profile, and insulin resistance. Secondary end points were physical activity, fitness, health-related quality of life, sleep quality, academic performance, enjoyment with physical activity, and physical self-concept. CONCLUSIONS: This study will assess whether MOVI-2 overcomes some potential limitations of physical activity interventions in children (Clinicaltrials.gov number NCT01277224).

[Validity of a single-factor model underlying the metabolic syndrome in young adults: confirmatory factor analysis]. / Validez de un modelo con un único factor en el síndrome metabólico en adultos jóvenes: análisis factorial confirmatorio.

INTRODUCTION AND OBJECTIVES: To determine by confirmatory factor analysis whether a model of the metabolic syndrome including waist circumference-to-height ratio, as a measure of adiposity, has better goodness of fit than that based on waist circumference alone and, on the basis of the data of the best-fit model, to develop an index of global cardiometabolic risk in young adults. METHODS: Cross-sectional observational study involving 683 university students aged 18 to 30 years, in their first year at the University of Castilla-La Mancha in Spain, during the 2009-10 academic year. We compared the best fit of 2 models of the metabolic syndrome, both of which included the triglyceride-to-high-density lipoprotein cholesterol ratio, HOMA-IR index, and mean arterial blood pressure, but differed in that one of them used waist circumference, whereas the other used the waist circumference-to-height ratio. A metabolic syndrome index (MSI) was constructed and its association with aerobic capacity, daily physical activity and muscle strength was estimated. RESULTS: The single-factor model that included waist circumference was a better indicator of goodness of fit. The MSI was inversely associated with aerobic capacity and muscle strength. CONCLUSIONS: This study confirms that a single factor underlies the concept of metabolic syndrome; including the waist circumference-to-height ratio does not result in improvements over the model in which waist circumference alone is considered, and the development of a quantitative MSI may be useful for the quantification of cardiometabolic risk in clinical practice.