Background

Endothelial colony-forming cells (ECFCs) have been implicated in the process of vascularization, which includes vasculogenesis and angiogenesis. Vasculogenesis is a de novo formation of blood vessels, and is an essential physiological process that occurs during embryonic development and tissue regeneration. Angiogenesis is the growth of new capillaries from pre-existing blood vessels, which is observed both prenatally and postnatally. The placenta is an organ composed of a variety of fetal-derived cells, including ECFCs, and therefore has significant potential as a source of fetal ECFCs for tissue engineering.

Aim

To investigate the possibility of isolating clonal ECFCs from human early gestation chorionic villi (CV-ECFCs) of the placenta, and assess their potential for tissue engineering.

Methods

The early gestation chorionic villus tissue was dissociated by enzyme digestion. Cells expressing CD31 were selected using magnetic-activated cell sorting, and plated in endothelial-specific growth medium. After 2-3 wks in culture, colonies displaying cobblestone-like morphology were manually picked using cloning cylinders. We characterized CV-ECFCs by flow cytometry, immunophenotyping, tube formation assay, and Dil-Ac-LDL uptake assay. Viral transduction of CV-ECFCs was performed using a Luciferase/tdTomato-containing lentiviral vector, and transduction efficiency was tested by fluorescent microscopy and flow cytometry. Compatibility of CV-ECFCs with a delivery vehicle was determined using an FDA approved, small intestinal submucosa extracellular matrix scaffold.

Results

After four passages in 6-8 wks of culture, we obtained a total number of 1.8 × 10⁷ CV-ECFCs using 100 mg of early gestational chorionic villus tissue. Immunophenotypic analyses by flow cytometry demonstrated that CV-ECFCs highly expressed the endothelial markers CD31, CD144, CD146, CD105, CD309, only partially expressed CD34, and did not express CD45 and CD90. CV-ECFCs were capable of acetylated low-density lipoprotein uptake and tube formation, similar to cord blood-derived ECFCs (CB-ECFCs). CV-ECFCs can be transduced with a Luciferase/tdTomato-containing lentiviral vector at a transduction efficiency of 85.1%. Seeding CV-ECFCs on a small intestinal submucosa extracellular matrix scaffold confirmed that CV-ECFCs were compatible with the biomaterial scaffold.

Conclusion

In summary, we established a magnetic sorting-assisted clonal isolation approach to derive CV-ECFCs. A substantial number of CV-ECFCs can be obtained within a short time frame, representing a promising novel source of ECFCs for fetal treatments.

Objective

To reconstruct the auricle using a porous, hollow, three-dimensional (3D)-printed mold and autologous diced cartilage mixed with platelet-rich plasma (PRP).

Methods

Materialise Magics v20.03 was used to design a 3D, porous, hollow auricle mold. Ten molds were printed by selective laser sintering with polyamide. Cartilage grafts were harvested from one ear of a New Zealand rabbit, and PRP was prepared using 10 mL of auricular blood from the same animal. Ear cartilage was diced into 0.5- to 2.0-mm pieces, weighed, mixed with PRP, and then placed inside the hollow mold. Composite grafts were then implanted into the backs of respective rabbits (n = 10) for 4 months. The shape and composition of the diced cartilage were assessed histologically, and biomechanical testing was used to determine stiffness.

Results

The 3D-printed auricle molds were 0.6-mm thick and showed connectivity between the internal and external surfaces, with round pores of 0.1 to 0.3 cm. After 4 months, the diced cartilage pieces had fused into an auricular shape with high fidelity to the anthropotomy. The weight of the diced cartilage was 5.157 ± 0.230 g (P > 0.05, compared with preoperative). Histological staining showed high chondrocyte viability and the production of collagen II, glycosaminoglycans, and other cartilaginous matrix components. In unrestricted compression tests, auricle stiffness was 0.158 ± 0.187 N/mm, similar to that in humans.

Conclusion

Auricle grafts were constructed successfully through packing a 3D-printed, porous, hollow auricle mold with diced cartilage mixed with PRP. The auricle cartilage contained viable chondrocytes, appropriate extracellular matrix components, and good mechanical properties.

Levels of evidence

NA. Laryngoscope, 129:2467-2474, 2019.

Background: IMD-0354 is a kind of hydrophobic small molecule inhibitor of IKKβ, which can effectively inhibit the NF-κB pathway. Besides, IMD-0354 can inhibit a variety of tumor cells in culture, but its poor water solubility and low utilization have limited its clinical application. Methods: In this study, IMD-0354 was synthesized through esterifying the folate acid (FA) conjugated dextran (Dex) as well as the lauryl alcohol (LA). Results:The particle (IMD/FA-Dex-LA) size was 212.13±10.62nm, the encapsulation efficiency was 89.27±6.51%, and the drug loading was 4.25±0.42%. Cell viability studies indicated that the IMD/FA-Dex-LA effectively inhibited survival of B16F10 cells in culture. Meanwhile, Western Blotting results showed that the nuclear transport of NF-κB was reduced after blocking the IKK pathway, which would thereby suppress melanoma cell division and proliferation. Moreover, subcutaneous tumor implantation experiment revealed that, the drug-loading complex had an obvious effect on suppressing melanoma cells. Findings of this study demonstrated that the IMD-0354 loaded FA-Dex-LA was more effective than IMD-0354 alone. Conclusion: In summary, FA-Dex-LA has been successfully synthesized in this study, which can serve as a carrier for hydrophobic drug. Further, it is believed the FA-Dex-LA can potentially applied in cancer treatment.

Background

Hemophilia A (HA) is an X-linked recessive disorder caused by mutations in the Factor VIII (FVIII) gene leading to deficient blood coagulation. As a monogenic disorder, HA is an ideal target for cell-based gene therapy, but successful treatment has been hampered by insufficient engraftment of potential therapeutic cells.

Methods

In this study, we sought to determine whether co-transplantation of endothelial colony-forming cells (ECFCs) and placenta-derived mesenchymal stromal cells (PMSCs) can achieve long-term engraftment and FVIII expression. ECFCs and PMSCs were transduced with a B domain deleted factor VIII (BDD-FVIII) expressing lentiviral vector and luciferase, green fluorescent protein or Td-Tomato containing lentiviral tracking vectors. They were transplanted intramuscularly into neonatal or adult immunodeficient mice.

Results

In vivo bioluminescence imaging showed that the ECFC only and the co-transplantation groups but not the PMSCs only group achieved long-term engraftment for at least 26 weeks, and the co-transplantation group showed a higher engraftment than the ECFC only group at 16 and 20 weeks post-transplantation. In addition, cell transplantation at the neonatal age achieved higher engraftment than at the adult age. Immunohistochemical analyses further showed that the engrafted ECFCs expressed FVIII, maintained endothelial phenotype, and generated functional vasculature. Next, co-transplantation of ECFCs and PMSCs into F8 knock-out HA mice reduced the blood loss volume from 562.13 ± 19.84 μl to 155.78 ± 44.93 μl in a tail-clip assay.

Conclusions

This work demonstrated that co-transplantation of ECFCs with PMSCs at the neonatal age is a potential strategy to achieve stable, long-term engraftment, and thus holds great promise for cell-based treatment of HA.

Glycolysis was reported to have a positive correlation with radioresistance. Our previous study found that the miR-33a functioned as a tumor suppressor in malignant melanoma by targeting hypoxia-inducible factor1-alpha (HIF-1α), a gene known to promote glycolysis. However, the role of miR-33a-5p in radiosensitivity remains to be elucidated. We found that miR-33a-5p was downregulated in melanoma tissues and cells. Cell proliferation was downregulated after overexpression of miR-33a-5p in WM451 cells, accompanied by a decreased level of glycolysis. In contrast, cell proliferation was upregulated after inhibition of miR-33a-5p in WM35 cells, accompanied by increased glycolysis. Overexpression of miR-33a-5p enhanced the sensitivity of melanoma cells to X-radiation by MTT assay, while downregulation of miR-33a-5p had the opposite effects. Finally, in vivo experiments with xenografts in nude mice confirmed that high expression of miR-33a-5p in tumor cells increased radiosensitivity via inhibiting glycolysis. In conclusions, miR-33a-5p promotes radiosensitivity by negatively regulating glycolysis in melanoma.

Growing evidence suggests that long non-coding RNAs NORAD and miR-205 play a significant role in regulating cancer progression and metastasis. In this study, high expression of NORAD was firstly observed in melanoma tissues and human malignant melanoma cell lines, our aim was to study the interaction of them in the process of invasion and migration of malignant melanoma cells. NORAD, miR-205, and EGLN2 mRNA level in MM cells was detected by qRT-PCR. In situ hybridization (ISH) was performed to detect NORAD expression in MM tissues specimens. Effects of NORAD and miR-205 on Prolyl hydroxylase 2 (EGLN2) expression was explored by western blot in MM cells line. Dual-luciferase reporter assay was performed to verify the interaction relationship between NORAD and miR-205, as well as, miR-205 and EGLN2. Transwell assay was conducted to explore the effects of NORAD and miR-205 in vitro. Xenografts in nude mice experiment were used to confirm the role of NORAD and miR-205 in vivo. In vitro, NORAD knockdown significantly inhibited migration and invasion of malignant melanoma cells and elevated the expression of miR-205, there was an interaction between miR-205 and NORAD in the RNA-induced silencing complex. Upregulation of miR-205 induced significant inhibition of migratory and invasive ability compared with the scrambled control. However, downregulating NORAD largely reversed this effect. Furthermore, the regulatory effects of miR-205 on EGLN2 levels and the induction of endoplasmic reticulum stress were reversed by NORAD. In vivo, deletion of miR-205 induced tumor growth in nude mice. NORAD may play critical roles in tumorigenesis and progression of malignant melanoma by regulating of the miR-205-EGLN2 pathway, and may serve as a new therapeutic target.

In early pregnancy, fetal skin wounds can heal quickly and undergo a transition period from scarless healing to scar formation. The aim of the present study was to identify potential biomarkers associated with scarless repair of cleft lips, in order to determine the intrinsic factors leading to scar formation in embryonic tissue. A stable model of cleft lip was established using microsurgery by constructing a wedge‑shaped cleft lip‑like defect in fetal rats at gestational age (GA) 16.5 and GA18.5. The GA16.5 and GA18.5 groups were used to model scarless healing and scar formation, respectively. The fetuses were returned to the uterus following surgery, then removed 72 h after the procedure. Macroscopic observation of the cleft defect and histological examination were carried out. Reverse transcription‑quantitative (RT‑q) PCR and parallel reaction monitoring (PRM) were used to detect mRNA and protein expression levels, respectively. The upper‑left lip completely healed 72 h after surgery in the GA16.5 group of fetal rats. However, this was not the case in the GA18.5 group. Histological examination indicated new follicles visible under the epidermis of the scarless group (GA16.5). Scarring was visible on the upper‑left cleft lip wound of the fetal rats in the GA18.5 group. The expression of some growth and pro‑inflammatory factors, including TNF‑α, were also different between two groups. Label‑free quantification was used to identified differentially expressed proteins and five differentially expressed proteins (Smad4, Fabp5, S100a4, S100a8 and S100a9) were identified. The relative expression of these molecules at the mRNA and protein levels were measured using RT‑qPCR and PRM. These molecules may represent potential biomarkers for the scarless repair of fetal rat cleft lip wounds.

Diabetic ischemic wound treatment remains a critical clinical challenge. Neovascularization plays a significant role in wound healing during all stages of the tissue repair process. Strategies that enhance angiogenesis and neovascularization and improve ischemic pathology may promote the healing of poor wounds, particularly diabetic wounds in highly ischemic conditions. We previously identified a cyclic peptide LXW7 that specifically binds to integrin αvβ3 on endothelial progenitor cells (EPCs) and endothelial cells (ECs), activates vascular endothelial growth factor (VEGF) receptors, and promotes EC growth and maturation. In this study, we designed and synthesized a multi-functional pro-angiogenic molecule by grafting LXW7 and collagen-binding peptides (SILY) to a dermatan sulfate (DS) glycosaminoglycan backbone, named LXW7-DS-SILY, and further employed this multi-functional molecule to functionalize collagen-based extracellular matrix (ECM) scaffolds. We confirmed that LXW7-DS-SILY modification significantly promoted EPC attachment and growth on the ECM scaffolds in vitro and supported EPC survival in vivo in the ischemic environment. When applied in an established Zucker Diabetic Fatty (ZDF) rat ischemic skin flap model, LXW7-DS-SILY-functionalized ECM scaffolds loaded with EPCs significantly improved wound healing, enhanced neovascularization and modulated collagen fibrillogenesis in the ischemic environment. Altogether, this study provides a promising novel treatment to accelerate diabetic ischemic wound healing, thereby reducing limb amputation and mortality of diabetic patients.

The lack of vascularization associated with deep burns delays the construction of wound beds, increases the risks of infection, and leads to the formation of hypertrophic scars or disfigurement. To address this challenge, we have fabricated a multi-functional pro-angiogenic molecule by grafting integrin αvβ3 ligand LXW7 and collagen-binding peptide (SILY) to a dermatan sulfate (DS) glycosaminoglycan backbone, named LXW7-DS-SILY (LDS), and further employed this to functionalize collagen-based Integra scaffolds. Using a large deep burn wound model in C57/BLK6 mice (8-10 weeks old, 26-32g, n = 39), we demonstrated that LDS-modified collagen-based Integra scaffolds loaded with endothelial cells (ECs) accelerate wound healing rate, re-epithelialization, vascularization, and collagen deposition. Specifically, a 2 cm × 3 cm full-thickness skin burn wound was created 48 h after the burn, and then wounds were treated with four groups of different dressing scaffolds, including Integra + ECs, Integra + LDS, and Integra + LDS + ECs with Integra-only as the control. Digital photos were taken for wound healing measurement on post-treatment days 1, 7, 14, 21, 28, and 35. Post-treatment photos revealed that treatment with the Intgera + LDS + ECs scaffold exhibited a higher wound healing rate in the proliferation phase. Histology results showed significantly increased re-epithelialization, increased collagen deposition, increased thin and mixed collagen fiber content, increased angiogenesis, and shorter wound length within the Integra + LDS + ECs group at Day 35. On Day 14, the Integra + LDS + ECs group showed the same trend. The relative proportions of collagen changed from Day 14 to Day 35 in the Integra + LDS + ECs and Integra + ECs groups demonstrated decreased thick collagen fiber deposition and greater thin and mixed collagen fiber deposition. LDS-modified Integra scaffolds represent a promising novel treatment to accelerate deep burn wound healing, thereby potentially reducing the morbidity associated with open burn wounds. These scaffolds can also potentially reduce the need for autografting and morbidity in patients with already limited areas of harvestable skin.