Tadalafil treatment for fetuses with early-onset growth restriction: a protocol for a multicentre, randomised, placebo-controlled, double-blind phase II trial (TADAFER IIb).

INTRODUCTION: TheTADAlafil treatment for Fetuses with early-onset growth Restriction: multicentrer, randomizsed, phase II trial (TADAFER II) study showed the possibility of prolonging the pregnancy period in cases of early-onset fetal growth restriction; however, it was an open-label study. To establish further evidence for the efficacy of tadalafil in this setting, we planned a multicentre, randomised, placebo-controlled, double-blind trial. METHODS AND ANALYSIS: This trial will be conducted in 180 fetuses with fetal growth restriction enrolled from medical centres in Japan; their mothers will be randomised into three groups: arm A, receiving two times per day placebo; arm B, receiving one time per day 20 mg tadalafil and one time per day placebo and arm C, receiving 20 mg two times per day tadalafil. The primary endpoint is the prolongation of gestational age at birth, defined as days from the first day of the protocol-defined treatment to birth. To minimise bias in terms of fetal baseline conditions and timing of delivery, a fetal indication for delivery as in TADAFER II will be established in this trial. The investigator will evaluate fetal baseline conditions at enrolment and decide the timing of delivery based on this indication. ETHICS AND DISSEMINATION: This study has been approved by Mie University Hospital Clinical Research Review Board on 22 July 2019 (S2018-007). Written informed consent will be obtained from all mothers before recruitment. Our findings will be widely disseminated through peer-reviewed publications. TRIAL REGISTRATION: jRCTs041190065.

Prenatal Identification of Confined Placental Mosaicism in Pregnant Women with Fetal Growth Restriction.

We examined the influence of confined placental mosaicism (CPM) as a cause of fetal growth restriction (FGR), and whether CPM can be screened using cell-free DNA (cfDNA) analysis of the maternal plasma. We analyzed cfDNA in the maternal plasma of 40 FGR cases with an estimated fetal weight of less than - 2.0 SD using massively parallel sequencing to detect chromosomal aberrations. Fetal and placental genotyping was performed to confirm CPM cases. cfDNA analyses of maternal plasma detected suspected CPM cases with chromosomal aneuploidy or copy number variations in 5 of 40 cases (12.5%). For 4 cases in which the entire placenta consisted of cells with chromosomal abnormalities, fetal growth was severely restricted. CPM can be screened by cfDNA analysis in maternal plasma, accounting for approximately 10% of the causes of moderate or severe FGR, and the higher the proportion of abnormal karyotype cells in the placenta, the more severe the placental dysfunction and FGR.

Exogenous Lysyl Oxidase-Like 2 and Perfusion Culture Induce Collagen Crosslink Formation in Osteogenic Grafts.

Lysyl oxidase (LOX)-mediated collagen crosslinking can regulate osteoblastic phenotype and enhance mechanical properties of tissues, both areas of interest in bone tissue engineering. The objective of this study is to investigate the effect of lysyl oxidase-like 2 (LOXL2) on osteogenic differentiation of mesenchymal stem cells (MSCs) cultured in perfusion bioreactors, enzymatic collagen crosslink formation in the extracellular matrix (ECM), and mechanical properties of engineered bone grafts. Exogenous LOXL2 to MSCs seeded in composite scaffolds under perfusion culture for up to 28 days is administered. Constructs treated with LOXL2 appear brown in color and possess greater DNA content and osteogenic potential measured by a twofold increase in bone sialoprotein gene expression. Collagen expression of LOXL2-treated scaffolds is lower than untreated controls. Functional outputs such as calcium deposition, osteocalcin expression, and compressive modulus are unaffected by LOXL2 supplementation. Excitingly, LOXL2-treated constructs contain 1.8- and 1.4-times more pyridinoline (PYD) crosslinks per mole of collagen and per wet weight, respectively, than untreated constructs. Despite these increases, compressive moduli of LOXL2-treated constructs are similar to untreated constructs over the 28-day culture duration. This is the first report of LOXL2 application to engineered, three-dimensional bony constructs. The results suggest a potentially new strategy for engineering osteogenic grafts with a mature ECM by modulating crosslink formation.

Bioreactor culture duration of engineered constructs influences bone formation by mesenchymal stem cells.

Perfusion culture of mesenchymal stem cells (MSCs) seeded in biomaterial scaffolds provides nutrients for cell survival, enhances extracellular matrix deposition, and increases osteogenic cell differentiation. However, there is no consensus on the appropriate perfusion duration of cellular constructs in vitro to boost their bone forming capacity in vivo. We investigated this phenomenon by culturing human MSCs in macroporous composite scaffolds in a direct perfusion bioreactor and compared their response to scaffolds in continuous dynamic culture conditions on an XYZ shaker. Cell seeding in continuous perfusion bioreactors resulted in more uniform MSC distribution than static seeding. We observed similar calcium deposition in all composite scaffolds over 21 days of bioreactor culture, regardless of pore size. Compared to scaffolds in dynamic culture, perfused scaffolds exhibited increased DNA content and expression of osteogenic markers up to 14 days in culture that plateaued thereafter. We then evaluated the effect of perfusion culture duration on bone formation when MSC-seeded scaffolds were implanted in a murine ectopic site. Human MSCs persisted in all scaffolds at 2 weeks in vivo, and we observed increased neovascularization in constructs cultured under perfusion for 7 days relative to those cultured for 1 day within each gender. At 8 weeks post-implantation, we observed greater bone volume fraction, bone mineral density, tissue ingrowth, collagen density, and osteoblastic markers in bioreactor constructs cultured for 14 days compared to those cultured for 1 or 7 days, and acellular constructs. Taken together, these data demonstrate that culturing MSCs under perfusion culture for at least 14 days in vitro improves the quantity and quality of bone formation in vivo. This study highlights the need for optimizing in vitro bioreactor culture duration of engineered constructs to achieve the desired level of bone formation.

Optic nerve atrophy after vitrectomy with indocyanine green-assisted internal limiting membrane peeling in diffuse diabetic macular edema. Adverse effect of ICG-assisted ILM peeling.

PURPOSE: To report anatomic and visual outcomes of vitrectomy and indocyanine green (ICG)-assisted peeling of the retinal internal limiting membrane (ILM) in the treatment of diffuse diabetic macular edema. METHODS: In a retrospective interventional case series, 15 eyes of 11 patients with refractory diffuse diabetic macular edema underwent pars plana vitrectomy with removal of the ILM, which was stained by intravitreal injection of ICG (0.1-0.2 ml of 0.5% ICG), performed by a single surgeon. The patients were followed up for 14-28 months (mean 20.5 months). The main outcome measures were assessment of macular edema by optical coherence tomography and determination of visual acuity and visual field. RESULTS: Intravitreal ICG visualized the ILM to facilitate complete removal of the structure. Qualitative assessment of optical coherence tomography images at the end of follow-up revealed that retinal thickness in the macula appeared nearly normal with or without reappearance of foveal pit in 11 of the 15 eyes (73.3%), decreased in 3 eyes (20.0%), and did not change in 1 eye (6.6%). Best-corrected visual acuity at the end of follow-up improved by 2 lines or more in 4 eyes (26.7%), virtually unchanged in 6 eyes (40.0%), and deteriorated by 2 lines or more in 5 eyes (26.7%). The mean logMAR visual acuity was 0.680 (approximately 12/60) preoperatively and 0.812 (approximately 9/60) postoperatively, the difference being not statistically significant (paired t-test, P=0.445). Seven (46.7%) of the 15 eyes developed optic nerve atrophy that occurred gradually within 6 months after surgery and caused irreversible peripheral visual field defect predominantly affecting the nasal field. CONCLUSION: Intravitreal application of ICG is beneficial in uneventful ILM peeling to help resolution of diffuse diabetic macular edema, but it may potentially damage the optic nerve fibers and lead to unfavorable visual outcomes.

Anatomic and visual outcomes after indocyanine green-assisted peeling of the retinal internal limiting membrane in idiopathic macular hole surgery.

PURPOSE: To report anatomic and visual outcomes after vitrectomy and adjunctive retinal internal limiting membrane (ILM) peeling with and without intravitreal indocyanine green for idiopathic macular hole repair. DESIGN: Retrospective comparative study of consecutive case series. METHODS: Three consecutive groups of idiopathic macular hole cases underwent modifications of surgical technique. Group I (48 eyes of 47 patients) underwent a standard vitrectomy, fluid/gas exchange, and 1 week's face-down positioning, group II (21 eyes of 21 patients) an adjunctive ILM peeling without use of indocyanine green, and group III (28 eyes of 28 patients) an adjunctive peeling of ILM stained with intravitreal application of 0.1 to 0.2 ml of 0.5% indocyanine green dye. RESULTS: Three groups of patients had comparable clinical characteristics as to age, gender, estimated duration of macular hole, preoperative visual acuity, and follow-up time. The rate of macular hole closure after a single surgery, as determined by optical coherence topography was 85.4% in group I, 85.7% in group II, and 100% in group III. Groups I and II showed a statistically significant visual improvement, but group III did not show significant visual acuity improvement as the mean logarithm of the minimal angle of resolution visual acuity was from 0.767 (20/120) preoperatively to 0.691 (20/100) postoperatively (P =.342). Eight cases in group III developed within a few postoperative months of optic disk pallor and irreversible peripheral visual field loss, predominantly affecting the nasal field. CONCLUSIONS: Intravitreal indocyanine green-assisted ILM peeling improves anatomic success in macular hole surgery, but it may potentially lead to unfavorable visual acuity outcome and peripheral visual field loss.