Uterine fibroid cell cytoskeletal organization is affected by altered G protein-coupled estrogen receptor-1 and phosphatidylinositol 3-kinase signaling.

OBJECTIVE: To determine whether cyclic strain affects fibroid cell cytoskeletal organization, proliferation, and collagen synthesis differently than myometrial cells. DESIGN: A basic science study using primary cultures of patient-matched myometrial and fibroid cells. SETTING: Academic laboratory. PATIENT(S): Premenopausal women undergoing myomectomy or hysterectomy for the treatment of symptomatic uterine fibroids. INTERVENTION(S): Application of uniaxial strain patterns mimicking periovulation, menses, or dysmenorrhea using the Flexcell tension system or static control. Secondarily, inhibition of G protein-coupled estrogen receptor-1 and phosphatidylinositol 3-kinase. MAIN OUTCOME MEASURE(S): Cell alignment, cell number, and collagen content. RESULT(S): Menses-strained cells demonstrated the most variation in cell alignment, cell proliferation, and procollagen content between myometrial and fibroid cells. Procollagen content decreased in myometrial cells with increasing strain amplitude and decreasing frequency. G protein-coupled estrogen receptor-1 inhibition decreases cellular alignment in the presence of strain. CONCLUSION(S): Mechanotransduction affecting cytoskeletal arrangement through the G protein-coupled estrogen receptor-1-phosphatidylinositol 3-kinase pathway is altered in fibroid cells. These results highlight the importance of incorporating mechanical stimulation into the in vitro study of fibroid pathology.

Innervation of an Ultrasound-Mediated PVDF-TrFE Scaffold for Skin-Tissue Engineering.

In this work, electrospun polyvinylidene-trifluoroethylene (PVDF-TrFE) was utilized for its biocompatibility, mechanics, and piezoelectric properties to promote Schwann cell (SC) elongation and sensory neuron (SN) extension. PVDF-TrFE electrospun scaffolds were characterized over a variety of electrospinning parameters (1, 2, and 3 h aligned and unaligned electrospun fibers) to determine ideal thickness, porosity, and tensile strength for use as an engineered skin tissue. PVDF-TrFE was electrically activated through mechanical deformation using low-intensity pulsed ultrasound (LIPUS) waves as a non-invasive means to trigger piezoelectric properties of the scaffold and deliver electric potential to cells. Using this therapeutic modality, neurite integration in tissue-engineered skin substitutes (TESSs) was quantified including neurite alignment, elongation, and vertical perforation into PVDF-TrFE scaffolds. Results show LIPUS stimulation promoted cell alignment on aligned scaffolds. Further, stimulation significantly increased SC elongation and SN extension separately and in coculture on aligned scaffolds but significantly decreased elongation and extension on unaligned scaffolds. This was also seen in cell perforation depth analysis into scaffolds which indicated LIPUS enhanced perforation of SCs, SNs, and cocultures on scaffolds. Taken together, this work demonstrates the immense potential for non-invasive electric stimulation of an in vitro tissue-engineered-skin model.

A bioreactor for studying negative pressure wound therapy on skin grafts.

Negative pressure wound therapy (NPWT) has become the prevailing standard of care for treating complex soft tissue wounds and is now being considered for use in alternative applications including improving skin graft take. While it is generally agreed that negative pressure leads to improved wound healing, universal consensus on its optimal application is not supported in the literature. We describe the design and validation of a bioreactor to determine the prospective benefits of NPWT on skin grafts and engineered skin substitutes (ESS). Clinically relevant pressures were applied, and the native human skin was able to withstand greater negative pressures than the engineered substitutes. Both skin types were cultured under static, flow-only, and -75 mm Hg conditions for 3 days. While it remained intact, there was damage to the epidermal-dermal junction in the ESS after application of negative pressure. The normal skin remained viable under all culture conditions. The engineered skin underwent apoptosis in the flow-only group; however, the application of negative pressure reduced apoptosis. Vascular endothelial growth factor levels were significantly higher in the normal flow-only group, 152.0 ± 75.1 pg/mg protein, than the other culture conditions, 81.6 ± 35.5 pg/mg for the static and 103.6 ± pg/mg for the negative pressure conditions. The engineered skin had a similar trend but the differences were not significant. This bioreactor design can be used to evaluate the impacts of NPWT on the anatomy and physiology of skin to improve outcomes in wounds after grafting with normal or engineered skin.

Cyclic Adenosine Monophosphate Eliminates Sex Differences in Estradiol-Induced Elastin Production from Engineered Dermal Substitutes.

Lack of adult cells' ability to produce sufficient amounts of elastin and assemble functional elastic fibers is an issue for creating skin substitutes that closely match native skin properties. The effects of female sex hormones, primarily estrogen, have been studied due to the known effects on elastin post-menopause, thus have primarily included older mostly female populations. In this study, we examined the effects of female sex hormones on the synthesis of elastin by female and male human dermal fibroblasts in engineered dermal substitutes. Differences between the sexes were observed with 17ß-estradiol treatment alone stimulating elastin synthesis in female substitutes but not male. TGF-ß levels were significantly higher in male dermal substitutes than female dermal substitutes and the levels did not change with 17ß-estradiol treatment. The male dermal substitutes had a 1.5-fold increase in cAMP concentration in the presence of 17ß-estradiol compared to no hormone controls, while cAMP concentrations remained constant in the female substitutes. When cAMP was added in addition to 17ß-estradiol and progesterone in the culture medium, the sex differences were eliminated, and elastin synthesis was upregulated by 2-fold in both male and female dermal substitutes. These conditions alone did not result in functionally significant amounts of elastin or complete elastic fibers. The findings presented provide insights into differences between male and female cells in response to female sex steroid hormones and the involvement of the cAMP pathway in elastin synthesis. Further explorations into the signaling pathways may identify better targets to promote elastic fiber synthesis in skin substitutes.

Skin-Nerve Co-Culture Systems for Disease Modeling and Drug Discovery.

Prominent clinical problems related to the skin-nerve interface include barrier dysfunction and erythema, but it is the symptoms of pain and itch that most often lead patients to seek medical treatment. Tissue-engineered innervated skin models provide an excellent solution for studying the mechanisms underlying neurocutaneous disorders for drug screening, and cutaneous device development. Innervated skin substitutes provide solutions beyond traditional monolayer cultures and have advantages that make them preferable to in vivo animal studies for certain applications, such as measuring somatosensory transduction. The tissue-engineered innervated skin models replicate the complex stratified epidermis that provides barrier function in native skin, a feature that is lacking in monolayer co-cultures, while allowing for a level of detail in measurement of nerve morphology and function that cannot be achieved in animal models. In this review, the advantages and disadvantages of different cell sources and scaffold materials will be discussed and a presentation of the current state of the field is reviewed. Impact statement A review of the current state of innervated skin substitutes and the considerations that need to be addressed when developing these models. Tissue-engineered skin substitutes are customizable and provide barrier function allowing for screening of topical drugs and for studying nerve function.

Protease levels are significantly altered in pediatric burn wounds.

Burn wounds contain high levels of protease activity due to the need to remodel the damaged extracellular matrix proteins. While necessary, excessive protease activity can lead to improper wound healing and is associated with increased contraction and fibrosis. No studies to date have investigated the expression changes of all the collagenases and elastases in burn wounds. The present study compares gene expression changes and changes in collagenase and elastase activity between burn wound eschar and normal skin in a pediatric population. Deidentified pediatric tissues were used for these experiments. Burn wound tissue was excised as part of normal standard care within a week from injury; normal skin was removed during elective plastic surgery procedures. RNA-sequencing was performed and significant results were confirmed with qRT-PCR. Activity assays showed a significant increase in both collagenase and elastase activity in the burn wound tissue compared to the normal skin. Western blotting and substrate zymography of tissue homogenates evaluated the results at the protein levels. Four elastases and three collagenases were determined to be significantly upregulated in the wound tissues by both RNA-sequencing and qRT-PCR. Cathepsin V was the only protease that was significantly downregulated. All but one metalloproteinase studied was significantly upregulated. None of the serine proteases were significantly altered in the wound tissues. In conclusion, matrix metalloproteinases appear to be the most highly elevated proteases after a pediatric burn wound injury, at least within the first 3-7 days. The data warrant further investigation into the effects of MMPs on burn wound healing.

Varying Negative Pressure Wound Therapy Acute Effects on Human Split-Thickness Autografts.

Over 6.5 million people in the United States suffer from traumatic, burn, acute, and chronic wounds yearly. When reconstruction is required, split and full-thickness autografts are a first line of treatment intervention. Negative pressure wound therapy (NPWT) is gaining traction as an adjunct modality to improve graft survival, yet the specifics on what settings to apply topically over the graft is unsubstantiated and associated with morbidities. This study was performed in an effort to understand initial changes in wound and graft healing with a long-term goal of surface pressure optimization. Excess skin from elective procedures from six human subjects was trimmed to 0.012 inch in order represent a split-thickness autografts. These grafts were treated continuously with either -75 mm Hg (n = 4), -125 mm Hg (n = 4), or no pressure (n = 4) for 3 hours. Six skin grafts were treated with no sponge or pressure control (n = 6). RNAseq was performed on all treatment groups and compared with no pressure control. Significant gene expression changes with a subset focusing on inflammatory, cellular/extracellular matrix proliferation and angiogenic mediators and having greater than 2-fold were confirmed with immunohistochemistry staining. There are 95 significant gene transcription differences among all treatment groups. NPWT leads to significantly increased gene expression of FGFR1, ET-1, and 22 Keratin proteins. Between -75 and -125 mm Hg groups, there are 19 significant gene changes. Proinflammatory genes S100A8 and Tenacin C (TNC) demonstrate an 8.8- and 9.1-fold change, respectively, and is upregulated in -125 mm Hg group and downregulated in -75 mm Hg group. Fibrinogen genes fibrinogen gamma chain and fibrinogen alpha chain had respective log2-fold changes of -7.9 and -7.4 change between treatment groups and were downregulated in -125 mm Hg group and upregulated in -75 mm Hg group. There are varying effects of surface pressures on human split-thickness autografts during the imbibition time period. NPWT may improve cellular migration, proliferation, and angiogenesis over controls. Human skin grafts respond differently to -125 and -75 mm Hg within 3 hours of NPWT treatment. The results suggest -75 mm Hg leads to less inflammation and increased fibrinogen production compared with the -125 mm Hg group, at least initially. Reducing "time to heal" with NPWT is critical to successful outcomes and quality of life within young patients who often experience pain/discomfort when treated at the current standard pump settings. The results from this study and continued investigation may quickly translate to the clinical setting by finding the ideal pressure setting utilized in an effort to reduce NPWT length of treatment, improve patient comfort, satisfaction, and psychosocial well-being.

Urinary neurotrophic peptides in postmenopausal women with and without overactive bladder.

AIMS: The aim of this study was to compare the expression of urinary nerve growth factor (NGF), brain derived neurotrophic factor (BDNF), substance P (SP), and calcitonin-gene related peptide (CGRP) in women with and without overactive bladder (OAB). We sought to determine factors associated with higher expression of these neuropeptides. METHODS: Participants with OAB and age-matched controls were enrolled. Symptom severity was assessed with validated questionnaires. Urinary neurotrophin levels, symptom scores, and clinical data were compared between the groups. Multivariate analysis determined independent factors associated with urinary neurotrophin levels. RESULTS: Sixty-seven women (38 OAB, 29 controls) were included. Women with OAB and controls were similar in age, race, body mass index, parity, and smoking status. Women with OAB were more likely to report a history of pelvic pain and pelvic surgery. Neurotrophic factor levels normalized to urinary creatinine did not differ between the groups. Increasing age was associated with greater urinary levels of BDNF and NGF (ß = 0.23, 95%CI 0.11-0.34 and 0.75, 95%CI 0.17-1.33, respectively, P < 0.02). Higher urinary NGF was associated with increasing BMI (ß = 0.81, 95%CI 0.05-1.57, P = 0.04) while pain was associated with elevated urinary SP (ß = 0.21, 95%CI 0.09-0.33, P = 0.001). CONCLUSIONS: Our data does not support a relationship between urinary neurotrophin levels and OAB in age-matched postmenopausal women. Further research is necessary to elucidate the role of urinary neurotrophins in the diagnosis and management of OAB. Neurourol. Urodynam. 36:740-744, 2017. © 2016 Wiley Periodicals, Inc.

Tissue-engineered endometrial model for the study of cell-cell interactions.

Endometrial stromal and epithelial cell cross talk is known to influence many of the dynamic changes that occur during the menstrual cycle. We modified our previous model and embedded telomerase-immortalized human endometrial stromal cells and Ishikawa adenocarcinoma epithelial cells in a collagen-Matrigel hydrogel to create a tissue-engineered model of the endometrium. Comparisons of single and cocultured cells examined communication between endometrial stromal and epithelial cells, which were cultured with 0 or 10 nmol/L 17ß estradiol; conditioned medium was used to look at the production of paracrine factors. Using this model, we were able to identify the changes in interleukin 6 (IL-6) and active matrix metalloproteinase 2, which appear to be due to paracrine signaling and differences in transforming growth factor ß1 (TGF-ß1) that do not appear to be due to paracrine signaling. Moreover, IL-6, TGF-ß1, and DNA content were also affected by the presence of estradiol in many of the tissues. These results indicate that paracrine and endocrine signaling are involved in human endometrial responses and support the use of coculture models to further investigate cell-cell and cell-matrix interactions.

A tissue-engineered human endometrial stroma that responds to cues for secretory differentiation, decidualization, and menstruation.

OBJECTIVE: To show the responsiveness of tissue-engineered human endometrial stroma to combinations of hormones that mimic the secretory and menstrual phases of the cycle. DESIGN: In vitro experimental study. SETTING: University uterine biology research laboratory. PATIENT(S): None. INTERVENTION(S): Telomerase immortalized human endometrial stromal cells cultured in monolayers (two-dimensional, 2D) or encapsulated in a collagen I hydrogel (three-dimensional, 3D) to create a simplified tissue-engineered stroma were exposed to hormone treatments mimicking early and late secretory phases, decidualization, and steroid withdrawal conditions to recapitulate menstruation. MAIN OUTCOME MEASURE(S): Morphologic and biochemical markers of decidualization and collagenase activity. RESULT(S): The 3D tissue can manifest changes in morphology and biochemical markers of decidualization similar to 2D culture and characteristic of endometrial stroma in vivo. Unlike 2D culture, the 3D tissue responded to steroid withdrawal by increased collagenase activity and tissue breakdown. CONCLUSION(S): Three-dimensional tissue-engineered endometrial stroma can mimic secretory and menstrual phases of the cycle and may be useful for studying uterine receptivity and menstruation in a physiological endocrine environment.

Cyclic strain improves strength and function of a collagen-based tissue-engineered vascular media.

Tissue-engineered blood vessels may provide a solution to the lack of suitable blood vessels for coronary and peripheral vessel bypass grafting. Cyclic strain can be used to provide a more physiological environment that may result in tissue that more closely resembles native artery. In this study, cyclic strain is applied to a collagen-based, tissue-engineered vascular medium. An increased culture time was used to allow the tissue to adhere to the silastic sleeve and to eliminate longitudinal compaction. Cyclic strain improved tissue strength through increased collagen content as well as some radial tissue compaction. Mechanical stimulation promoted a more contractile phenotype and led to a greater contractile response to the vasoconstrictor endothelin-1.

Tissue engineering of a collagen-based vascular media: Demonstration of functionality.

The property of vasoactivity is important for both resistance vessels and larger arteries. Evaluation of smooth muscle cell phenotype is often done in place of functional testing in engineered tissues, assuming a direct correlation between cell phenotype and tissue contractile force. In this study we look at a large panel of vasoactive agents to determine the functionality of our collagen-based tissue. The engineered vascular media elicited a measurable change in force in response to seven of the nine agents used. As part of this characterization, TGF-ß1 and TNF-α were used to promote a more contractile and synthetic cell phenotype respectively. Both smooth muscle α-actin and vasoconstriction were evaluated in ring sections. Due to large differences in cell-compaction and cell distribution in the tissues, no correlation was found between α-actin expression and contractile strength. This highlights the need for functional testing of engineered tissue and the importance of cell-matrix interactions in vasoactivity.