A simple method to isolate structurally and chemically intact brain vascular basement membrane for neural regeneration following traumatic brain injury.

BACKGROUND: The brain vascular basement membrane (brain-VBM) is an important component of the brain extracellular matrix, and the three-dimensional structure of the cerebrovascular network nested with many cell-adhesive proteins may provide guidance for brain tissue regeneration. However, the potential of ability of brain-VBM to promote neural tissue regeneration has not been examined due to the technical difficulty of isolating intact brain-VBM. METHODS: The present study developed a simple, effective method to isolate structurally and compositionally intact brain-VBM. Structural and component properties of the brain-VBM were characterized to confirm the technique. Seed cells were cocultured with brain-VBM in vitro to analyze biocompatibility and neurite extension. An experimental rat model of focal traumatic brain injury (TBI) induced by controlled cortical impact were conducted to further test the tissue regeneration ability of brain-VBM. RESULTS: Brain-VBM isolated using genipin showed significantly improved mechanical properties, was easy to handle, supported high cell viability, exhibited strong cell adhesive properties, and promoted neurite extension and outgrowth. Further testing of the isolated brain-VBM transplanted at lesion sites in an experimental rat model of focal TBI demonstrated considerable promise for reconstructing a complete blood vessel network that filled in the lesion cavity and promoting repopulation of neural progenitor cells and neurons. CONCLUSION: The technique allows isolation of intact brain-VBM as a 3D microvascular scaffold to support brain tissue regeneration following TBI and shows considerable promise for the production of naturally-derived biomaterials for neural tissue engineering.

Comparing the Characteristics of Amniotic Membrane-, Endometrium-, and Urinary-Derived ECMs and Their Effects on Endometrial Regeneration in a Rat Uterine Injury Model.

The decellularized extracellular matrices (d-ECMs) currently utilized to repair endometrial injuries are derived from three tissue sources, the endometrium (dE-ECM), placental amniotic membrane (dA-ECM), and urinary (dU-ECM). Notably, the structures of dU-ECM and dE-ECM are similar. These d-ECMs are derived from different tissues, and their specific roles in endometrial injury repair remain unclear. This study aimed to analyse the characteristics of the tissue microstructures and compositions to confirm specific differences among the three ECM types. And using a rat model of endometrial injury, the effects of all the matrices after implantation in vivo on the promotion of endometrial regeneration were analysed. After decellularization, dE-ECM had more residual active factors than the other two ECM types, while dA-ECM had significantly less DNA, α-Gal antigen components and extracellular matrix components than the other two groups. Although the three ECMs had no effect on the proliferation of stromal cells in vitro, dA-ECM may have increased the sensitivity of stromal cells to oestradiol (E2) responses. In vivo experiments confirmed the promotional effect of dA-ECM on endometrial regeneration. For example, the endometrial thickness, collagen deposition, endometrial tissue regeneration, vascular regeneration and pregnancy outcomes were significantly better in this group than in the other two groups. These findings might be associated with the excellent immune tolerance of dA-ECM. Therefore, when selecting a d-ECM for the treatment of endometrial injury, dE-ECM, which has the strongest tissue specificity, is not the preferred choice. Controlling the inflammatory responses in local lesions at the early stage may be a prerequisite for ECMs to exert their functions.

3D-printed hydrogel scaffold-loaded granulocyte colony-stimulating factor sustained-release microspheres and their effect on endometrial regeneration.

After injury, the endometrium cannot self-repair or regenerate because damage to the uterine basal layer often leads to intrauterine adhesions (IUAs), which can cause serious problems such as infertility and recurrent miscarriage. At present, no clinically effective method is available for the treatment of IUAs. With its advantages of being individualized and precise, three-dimensional (3D) bioprinting technology has been used to regenerate various damaged tissues and organs. Granulate colony-stimulating factor (G-CSF) clearly plays a positive role in endometrial regeneration, but precise and individualized drug applications are a prerequisite for improving the therapeutic effect of G-CSF. This study utilized a 3D-printed hydrogel in combination with a sustained-release microsphere (SRM) system to prepare a 3D-printed G-CSF-SRM system (3D microsphere) in vitro. The system advantageously allowed the spatial control of drug distribution and structural individualization. In addition to being long-acting and having a sustained release, the 3D microspheres increased the local concentration of G-CSF. Using a Sprague-Dawley rat IUA model, we confirmed that the 3D microspheres promoted local endometrial regeneration, significantly suppressed endometrium tissue fibrosis, and improved endometrial cell (epithelial and stromal cell) and vascular regeneration. The 3D microspheres significantly improved the endometrial receptivity and restored the pregnancy function of the damaged endometrium. We believe that the 3D-printed G-CSF-SRM hydrogel scaffold design concept may be used to develop a more precise and individualized treatment method for the structural and functional repair of damaged endometrial tissues.

Tissue Transglutaminase Impairs HTR-8/SVneo Trophoblast Cell Invasion via the PI3K/AKT Signaling Pathway.

OBJECTIVES: The pathogenesis of preeclampsia (PE) is associated with impaired trophoblast invasion, which results in placental insufficiency. Our earlier studies demonstrated that tissue transglutaminase (tTG) is highly expressed in human PE serum. However, whether tTG participates in trophoblast invasion remains unclear. The aim of the present study was to determine the role and mechanism of tTG in regulating matrix metalloproteinase (MMP)-2/MMP-9 expression to reduce trophoblast invasiveness in PE. METHODS: HTR-8/SVneo cells were transfected with a lentivirus vector and small interfering RNA targeting tTG. The protein level was detected by Western blotting. Cell proliferation and apoptosis were assessed by MTS and flow cytometry assays, respectively. Cell invasion was investigated by Transwell assay. In addition, the influence of tTG on PI3K and AKT mRNA levels in HTR-8/SVneo cells was evaluated using reverse transcription-quantitative PCR. RESULTS: tTG-overexpression inhibited HTR-8/SVneo cell proliferation and invasion and promoted apoptosis. In addition, upregulation of tTG induced an increase of PI3K and phosphorylated AKT and a decrease of MMP-2 and MMP-9 expression. tTG-knockdown significantly promoted the proliferation and invasion of HTR-8/SVneo cells and inhibited the apoptosis. Furthermore, the PI3K expression level was reduced, and the MMP-2/MMP-9 protein levels were increased. CONCLUSION: Taken together, the present study demonstrated that tTG-overexpression inhibited HTR-8/SVneo cell invasion via reducing the expression of MMP-2 and MMP-9 by activating PI3K/AKT signaling pathway, which may lead to the occurrence or development of PE. The present data provide new insights into modulation of tTG expression as a potential therapeutic target for PE.

Associations between first-trimester intrauterine hematoma and twin pregnancy outcomes: a retrospective cohort study.

BACKGROUND: In recent years, first-trimester intrauterine hematoma (IUH) has become increasingly common in twin pregnancy. The majority of studies on IUH have excluded twin pregnancies, and others did not differentiate between singleton and twin pregnancies. The impact of IUH on twin pregnancy is unclear. Therefore, the primary objective of our study was to examine associations between first-trimester IUH and pregnancy outcomes in twin pregnancies. METHODS: The data of 1020 twin pregnancies in women who received a routine examination from January 2014 to December 2018 were reviewed. We compared baseline data and pregnancy outcomes between those with and without IUH. Multivariable logistic regression analysis was used to adjust for possible confounding factors. RESULTS: A total of 209 patients (21.3%) developed IUH in the first trimester. First-trimester IUH was significantly associated with increased odds of miscarriage (adjusted odds ratio 14.27, 95% CI 8.25-24.70) and vanishing twin syndrome (adjusted odds ratio 3.26, 95% CI 1.11-4.61). However, there were no differences in the rates of stillbirth, preeclampsia, preterm labor (< 34 weeks), low birth weight, postpartum hemorrhage or fetal distress between the two groups. Maternal age, previous preterm birth, chorionicity in twins and the gestational week at first ultrasound did not differ between the two groups. The women with IUH had high rates of previous miscarriage (46.73% vs 38.37%, p = 0.01), assisted conception (48.56% vs 32.60%, p < 0.001) and accompanied vaginal bleeding (67.46% vs 13.43%, p < 0.001). According to the logistic regression analyses, these characteristics were not associated with pregnancy loss or vanishing twin syndrome. No IUH characteristics, including volume, largest diameter, or the presence of vaginal bleeding, were associated with pregnancy loss or vanishing twin syndrome before 20 weeks of gestation (P > 0.05). CONCLUSION: In women with twin pregnancy, the presence of IUH in the first trimester was associated with the loss of one or both fetuses before 20 weeks of gestation. However, previous miscarriage, the conception method, the IUH size and the presence of vaginal bleeding were not independently associated with miscarriage or vanishing twin syndrome.

3D Bioprinting a human iPSC-derived MSC-loaded scaffold for repair of the uterine endometrium.

Common events in the clinic, such as uterine curettage or inflammation, may lead to irreversible endometrial damage, often resulting in infertility in women of childbearing age. Currently, tissue engineering has the potential to achieve tissue manipulation, regeneration, and growth, but personalization and precision remain challenges. The application of "3D cell printing" is more in line with the clinical requirements of tissue repair. In this study, a porous grid-type human induced pluripotent stem cell-derived mesenchymal stem cell (hiMSC)-loaded hydrogel scaffold was constructed using a 3D bioprinting device. The 3D-printed hydrogel scaffold provided a permissive in vitro living environment for hiMSCs and significantly increased the survival duration of transplanted hiMSCs when compared with hiMSCs administered locally in vivo. Using an endometrial injury model, we found that hiMSC transplantation can cause early host immune responses (the serological immune response continued for more than 1 month, and the local immune response continued for approximately 1 week). Compared with the sham group, although the regenerative endometrium failed to show full restoration of the normal structure and function of the lining, implantation of the 3D-printed hiMSC-loaded scaffold not only promoted the recovery of the endometrial histomorphology (endometrial tissue and gland regeneration) and the regeneration of endometrial cells (stromal cells and epithelial cells) and endothelial cells but also improved endometrial receptivity functional indicators, namely, pinopode formation and leukemia inhibitory factor and αvß3 expression, which partly restored the embryo implantation and pregnancy maintenance functions of the injured endometrium. These indicators were significantly better in the 3D-printed hiMSC-loaded scaffold group than in the unrepaired (empty) group, the hiMSCs alone group and the 3D scaffold group, and the empty group showed the worst repair results. Our study confirm that the 3D-printed hiMSC-loaded hydrogel scaffold may be a promising material for endometrial repair.

Investigation of the effects of laminin present in the basal lamina of the peripheral nervous system on axon regeneration and remyelination using the nerve acellular scaffold.

This study aimed to investigate the effects of laminin (LN) located in the basal lamina, which are important components of the peripheral nervous system-extracellular matrix, on axon regeneration and remyelination. Nerve acellular scaffolds (NASs) (S-untreated) were prepared using the acellular technique. The active component LN in the NASs was blocked (S-LN- ) or upregulated (S-LN+ ); S-LN+ contained seven times more LN than did the S-untreated group. The adhesion capacity of Schwann cells (SCs) to the three types of NAS (S-untreated, S-LN- , and S-LN+ ) was assessed in vitro. Our results showed that the adhesion of SCs to the NASs was significantly reduced in the S-LN- group, whereas no difference was observed between the S-LN+ and S-untreated groups. The pretreated NASs were used to repair nerves in a nerve injury mouse model with the animals divided into four groups (S-LN- group, S-untreated group, S-LN+ group, and autograft group). Two weeks after surgery, although there was no difference in the S-LN- group, S-untreated group and S-LN+ group, the newly formed basal lamina in the S-LN- group were significantly lower than those in the other two groups. Four weeks after surgery, the S-LN+ group had higher numbers of newly generated axons and their calibers, more myelinated fibers, thicker myelin sheaths, increased myelin basic protein expression, and improved recovery of neural function compared to those of the S-LN- and S-untreated groups, but all of these parameters were significantly worse than those of the autograft group. Downregulation of the LN level in the NAS leads to a reduction in all of the above parameters.

Comparison of the Effects of BMSC-derived Schwann Cells and Autologous Schwann Cells on Remyelination Using a Rat Sciatic Nerve Defect Model.

Schwann cells (SCs) are primarily responsible for the formation of myelin sheaths, yet bone marrow mesenchymal stem cell (BMSC)-derived SCs are often used to replace autologous SCs and assist with the repair of peripheral nerve myelin sheaths. In this study, the effects of the two cell types on remyelination were compared during the repair of peripheral nerves. Methods: An acellular nerve scaffold was prepared using the extraction technique. Rat BMSCs and autologous SCs were extracted. BMSCs were induced to differentiate into BMSC-derived SCs (B-dSCs) in vitro. Seed cells (BMSCs, B-dSCs, and autologous SCs) were cocultured with nerve scaffolds (Sca) in vitro. Rats with severed sciatic nerves were used as the animal model. A composite scaffold was used to bridge the broken ends. After surgery, electrophysiology, cell tracking analyses (EdU labeling), immunofluorescence staining (myelin basic protein (MBP)), toluidine blue staining, and transmission electron microscopy were conducted to compare remyelination between the various groups and to evaluate the effects of the seed cells on myelination. One week after transplantation, only a small number of B-dSCs expressed MBP, which was far less than the proportion of MBP-expressing autologous SCs (P<0.01) but was higher than the proportion of BMSCs expressing MBP (P<0.05). Four weeks after surgery, the electrophysiology results (latency time, conductive velocity and amplitude) and various quantitative indicators of remyelination (thickness, distribution, and the number of myelinated fibers) showed that the Sca+B-dSC group was inferior to the Sca+autologous SC group (P<0.05) but was superior to the Sca+BMSC group (P<0.05). Conclusions: Within 4 weeks after surgery, the use of an acellular nerve scaffold combined with B-dSCs promotes remyelination to a certain extent, but the effect is significantly less than that of the scaffold combined with autologous SCs.

Xenogeneic acellular nerve scaffolds supplemented with autologous bone marrow-derived stem cells promote axonal outgrowth and remyelination but not nerve function.

Autologous nerves, artificial scaffolds or acellular nerve scaffolds are commonly used in bridging treatment for peripheral nerve defects. Xenogeneic acellular nerve scaffolds and allogeneic cellular nerve scaffolds have the same structural characteristics. Due to the wider source of raw materials, these latter scaffolds have high-potential value for applications. However, whether their heterogeneity will affect nerve regeneration is unknown. The current study evaluated the efficiency of xenogeneic acellular nerve scaffolds (XANs) combined with 5-ethynyl-2'-deoxyuridine (EdU)-labeling of autologous bone marrow-derived stem cells (BMSCs) for repair of a 1.5 cm gap in rat sciatic nerves. XANs from rabbit tibial nerves were prepared, the structure and components of the scaffolds were evaluated after completely removing the cellular components. Animals were divided into four groups based on graft: the simple XAN group, the XAN + BMSC group, the XAN + Media (from BMSC culture) group, and the autograft group. Serological immune tests showed that XANs induce an immune response in the first 2 weeks after transplantation. Moreover, cell tracking revealed that the proportion of EdU+ cells decreased over time, as shown by the measures at 2 days (70%), 4 days (20%), and 8 days (even <3%) postoperatively. Nerve functional analyses revealed that in contrast to the autograft group results, the XAN-BMSC, XAN + Media, and XAN groups did not exhibit good restoration of the sciatic functional index (SFI) or electrophysiological results (the peak action potential amplitudes) 12 weeks, postoperatively. However, the XAN-BMSC and autograft groups demonstrated greater remyelination and increased axon numbers and myelin thickness than the XAN + Media and XAN groups 12 weeks, postoperatively (p < .05). In conclusion, in the early stage of transplantation, XANs induce a certain degree of inflammation. Although the combination of XANs with autologous BMSCs enhanced the number of regenerated axons and the remyelination, the combination did not effectively improve the recovery of nervous motor function. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 3065-3078, 2018.

Intrauterine hematomas in the second and third trimesters associated with adverse pregnancy outcomes: a retrospective study.

OBJECTIVE: To carry out a retrospective study of the clinical features of patients with intrauterine hematoma in the second and third trimesters, and discuss the risk factors for poor pregnancy outcomes. METHODS: A total of 398 patients who underwent routine examination in our hospital from January 2011 to December 2015 were classified into normal pregnancy (NP) group (N = 265) and adverse pregnancy (AP) group (N = 133), according to their pregnancy outcomes. Maternal clinical demographics, gestational age, location of hematoma, volume of hematoma, and accompanying contraction and vaginal bleeding were recorded. RESULTS: The average age of pregnant women in the NP and AP groups was 28.25 ± 4.06 and 29.5 ± 5.06 years, respectively (p = 0.007). Gestational age at first detection of hematoma was 15.11 ± 5.13 weeks in the NP group compared with 21.22 ± 8.25 weeks in the AP group (p < 0.001). In the AP group, the incidence of retroplacental hematoma (54.1%) and palpable contractions (62.8%) was significantly higher than in the NP group (25.7% and 12.1%, respectively; p < 0.01). However, similar maternal parity or history of delivery, volume of hematoma and incidence of vaginal bleeding were found. CONCLUSION: Intrauterine hematoma in the second and third trimesters is a sign of pathological pregnancy, resulting in adverse outcomes. Maternal age, gestational age at first diagnosis, location of hematoma and accompanying contraction are risk factors for poor pregnancy outcomes.