Engineered hsa-miR-455-3p-Abundant Extracellular Vesicles Derived from 3D-Cultured Adipose Mesenchymal Stem Cells for Tissue-Engineering Hyaline Cartilage Regeneration.

Efforts are made to enhance the inherent potential of extracellular vesicles (EVs) by utilizing 3D culture platforms and engineered strategies for functional cargo-loading. Three distinct types of adipose mesenchymal stem cells-derived EVs (ADSCs-EVs) are successfully isolated utilizing 3D culture platforms consisting of porous gelatin methacryloyl (PG), PG combined with sericin methacryloyl (PG/SerMA), or PG combined with chondroitin sulfate methacryloyl (PG/ChSMA). These correspond to PG-EVs, PG/SerMA-EVs, and PG/ChSMA-EVs, respectively. Unique microRNA (miRNA) profiles are observed in each type of ADSCs-EVs. Notably, PG-EVs encapsulate higher levels of hsa-miR-455-3p and deliver more hsa-miR-455-3p to chondrocytes, which results in the activation of the hsa-miR-455-3p/PAK2/Smad2/3 axis and the subsequent hyaline cartilage regeneration. Furthermore, the functionality of PG-EVs is optimized through engineered strategies, including agomir/lentivirus transfection, electroporation, and Exo-Fect transfection. These strategies, referred to as Agomir-EVs, Lentivirus-EVs, Electroporation-EVs, and Exo-Fect-EVs, respectively, are ranked based on their efficacy in encapsulating hsa-miR-455-3p, delivering hsa-miR-455-3p to chondrocytes, and promoting cartilage formation via the hsa-miR-455-3p/PAK2/Smad2/3 axis. Notably, Exo-Fect-EVs exhibit the highest efficiency. Collectively, the 3D culture conditions and engineered strategies have an impact on the miRNA profiles and cartilage regeneration capabilities of ADSCs-EVs. The findings provide valuable insights into the mechanisms underlying the promotion of cartilage regeneration by ADSCs-EVs.

Semi-synthesis and structural elucidation of brevicanines A-D, four new C19-diterpenoid alkaloids with rotameric phenomenon from Aconitum brevicalcaratum.

Four new C19-diterpenoid alkaloids brevicanines A-D (1-4) with rotameric phenomenon were isolated from Aconitum brevicalcaratum. They all possessed an unusual axial chiral phenyl-quinazoline side chain and their structures were elucidated by extensive spectroscopic analysis and chemical methods. Meanwhile, brevicanines A and B were semi-synthesized from their parent compound scaconine to further confirm their structures. Variable-temperature NMR spectroscopy was also used to investigate the atropisomers of brevicanine A, in which two sets of signals in 1H NMR spectra were observed at room temperature and coalesced over 140 °C. It's the first time to determine the atropisomeric preference of diterpenoid alkaloids.

Differential expression of long noncoding RNAs in congenital microtia.

OBJECTIVE: To analyse lncRNA expression profiles in microtia using bioinformatics analysis. METHODS: We examined lncRNA expression profiles in residual ear cartilage and normal ear cartilage from individual congenital microtia patients. RESULTS: The gene chips used in this study included 30586 lncRNAs and 26109 mRNA probes. Intotal, 180 lncRNAs with differential expression weredetected in the residual ear cartilage compared with the normal cartilage, including 74 up-regulated and 106down-regulated lncRNAs. Signalling pathway analysis highlighted glyceride metabolism, osteoclast differentiation, andtumour growth. The results of qRT-PCR analysis were consistent with those of themicroarray. CONCLUSION: Differential expression of lncRNAs occurs in microtia. These lncRNAs and related signalling pathways may play an important role in the occurrence and development ofmicrotia.

κ-carrageenan induces the disruption of intestinal epithelial Caco-2 monolayers by promoting the interaction between intestinal epithelial cells and immune cells.

κ-carrageenan (κ-CGN) is an important food additive that has been demonstrated to induce colitis in animal models. In the present study, the effects of κ-CGN were assessed using an in vitro co-culture system that contained intestinal epithelial Caco-2 cells and activated macrophage-like THP-1 cells. The results demonstrated that in single cultures of Caco-2 and THP-1 cells treated with κ-CGN, the cytotoxicity and the secretion levels of IL-1ß, IL-6 and TNF-α were low. In the co-culture system, however, κ-CGN treatment resulted in apoptosis and reduced the transepithelial electrical resistance of the Caco-2 cell monolayers. The secretion levels of TNF-α, IL-1ß and IL-6 from the two cell types were increased significantly by κ-CGN treatment. Furthermore, pretreatment of the co-culture system with anti-TNF receptor 1 antibody suppressed the κ-CGN-induced apoptosis and attenuated the changes in the levels of IL-6 and IL-1ß in the Caco-2 monolayers. This study indicated that κ-CGN-induced TNF-α secretion is the main contributor to cellular damage in Caco-2 monolayers exposed to κ-CGN.

[Effect of vacuum drainage on the blood circulation of expanded flap in ear reconstruction].

OBJECTIVE: To investigate the effect of vacuum drainage on blood circulation of expanded skin flap, so as to confirm suitable negative pressure for ear reconstruction. METHODS: 21 patients (Auricular Reconstructive Center of Plastic Surgery Hospital, PUMC) were randomly divided into A, B, C 3 groups to receive vacuum drainage of: -20, -40 and -60 kPa, respectively. The length and width of reconstructed ears,which represented the swelling degree, were measured instantly and 5, 10 days postoperatively. The changes were calculated and reflected venous drainage of flaps. The temperature of proximal (concha) and distal (helix) part of the expanded flaps, which represented the arterial blood supply of flaps, was detected by infrared thermography after operation. RESULTS: Five days after operation [(1.48 +/- 0.47) mm, (0.36 +/- 0.06) mm] and immediately [(1.67 +/- 0.69) mm, (0.40 +/- 0.16) mm)] , the changes of the length and width of reconstructed ear in group B was significantly low, while there was no statistically difference between group A and C (P > 0.05). The data between 10 days and 5 days after operation showed the same result. Along with the increase of the negative pressure, the swelling degree of reconstructed ears decreased firstly and then increased reversely ( P < 0.01 ). The flap temperature of reconstructed ears increased (P < 0.01), especially in proximal (concha) part (P < 0.01) , when the negative pressure increased (0 to -60 kPa). CONCLUSIONS: Vacuum drainage has effect on both the venous drainage and blood supply. Negative pressure of -40 kPa can improve both the blood supply and venous drainage. It should be as reference for clinical application.

[Correction of cryptotia using crescent flap with skin-grafting].

OBJECTIVE: To investigate the therapeutic effect of crescent flap with skin-grafting for the correction of cryptotia. METHODS: Between the helix and scalp, we designed a crescent flap with the pedicle above the helix. Another affiliated triangular flap was formed below the crescent flap. Skin-grafting was performed after the transposition of two flaps. RESULTS: From Jan. 2008 to Mar. 2013, 16 cases with cryptotia (19 ears) were treated by this method. The ears were re-positioned to normal location with auriculocephalic sulcus. No vascular crisis was happened. The patients were followed-up for 3 months to 2 years with satisfactory result and no recurrence. CONCLUSIONS: This method of crescent flap with skin-grafting is suitable for cryptotia at any grade. It is designed brilliantly with no inconspicuous scar.

[Reconstruction of traumatic ear defect by retro-auricular expanded flap and cartilage graft].

OBJECTIVE: To investigate the therapeutic effect of retro-auricular expanded flap and cartilage graft for reconstruction of traumatic ear defect. METHODS: From Aug. 2008 to Aug. 2010, 10 cases of traumatic ear defects were treated with retro-auricular expanded flap and cartilage graft. The expanders (volume, 50 ml) were implanted subcutaneously at retro-auricular area on the first stage. Then the expansion began at 1 week after operation until the volume reached 60 ml. On the second stage, the ear defects were reconstructed with the expanded flaps, rib cartilage framework, as well as skin graft. RESULTS: All the wounds healed primarily without any complication. The patients were followed up for 6 months to 2 years with satisfactory cosmetic results. Good symmetry was achieved. CONCLUSIONS: It is an effective and reliable method to reconstruct traumatic ear defect by retro-auricular expanded flap and cartilage graft.

[Ear reconstruction after rupture of postauricular expanded flap].

OBJECTIVE: To summarize the principles of ear reconstruction after rupture of postauricular expanded flap. METHODS: The rupture of the postauricular expanded flap was graded as I-IV degree. For flap rupture of I and II degree, the ear reconstruction was performed immediately with autogenous cartilage framework. For flap rupture of III degree, topical and systemic treatment should be done to relieve the redness and swelling. Then the ear reconstruction was performed. For flap rupture of IV degree, the expanded postauricular flap was smoothened. Ear reconstruction was performed 3 months later. RESULTS: There were 67 cases with flap rupture, including 43 cases as I and II degree, 17 cases as III degree and 7 cases as IV degree. Ear reconstruction with autogenous cartilage framework was performed in 43 cases, with Medpor framework in 17 cases. Flap smoothing and secondary ear reconstruction with autogenous cartilage framework was performed in 7 cases. The results were satisfactory with complications in 4 cases (7%), which resolved after treatment. Primary healing was achieved in all the patients. CONCLUSIONS: Good result could be achieved in ear reconstruction, even after flap rupture. Proper treatment should be adopted according to the rupture degree.

[Experimental research on DNA methylation profile in congenital microtia].

OBJECTIVE: To screen for abnormal methylation in CpG islands and CpG sites through whole genome of congenital microtia to identify their associated genes. To discuss the relationship between abnormal methylation level of genes and the etiology of congenital microtia. METHODS: Residual ear cartilage of 50 patients with microtia was collected with ear cartilage of 34 patients without ear malformations as control. Nimblegen CpG promoter array was chosen to screen the 28,226 CpG islands in the whole genome of both experimental and control groups. The genes with differential methylated CpG islands were selected. SpectroCHIP array was chosen to detect the methylation level of each CpG site in abnormal methyletion CpG islands of both experimental and control groups. The CpG sites with differential methylation level were selected. RESULTS: There were 36 CpG islands with differential methylated level in whole genome between experimental group and control group, among which 29 CpG islands were connected with 29 named genes. In the abnormal methylated CpG islands of COL18A1, MYH14, RBMY1A1 and ZIC3, 6 differentially methylated CpG sites were found with statistical significance. The methylation level of these 6 CpG sites in experimental group and control group were COL18A1_2_CpG_170.9783 +/- 0.0235 and 0.9526 +/- 0.0589; MYH14_CpG_170.9600 +/- 0.0414 and 0.9284 +/- 0.0655; RBMY1A1_1_CpG_3.40.9966 +/- 0.0055 and 0.9914 +/- 0.0069; RBMY1A1_1_CpG_130.9648 +/- 0.0118 and 0.9757 +/- 0.0127; ZIC3_3_CpG_150.0867 +/- 0.0212 and 0.0543 +/- 0.0399; ZIC3_2_CpG_270.3775 +/- 0.1816 and 0.472 3 +/- 0.0439. CONCLUSIONS: The DNA methylation profile of the entire genome is initially established. The abnormal methylated CpG islands of COL18A1, MYH14, RBMY1A1 and ZIC3 might be related to the pathogenesis of microtia.

[Sectional anatomical analysis of auricular and middle ear malformation in patients with microtia].

OBJECTIVE: To analyze the sectional anatomical features of auricular and middle ear malformation in patients with microtia so as to improve the clinical classification and the instruction of surgery. METHODS: From Jun. to Dec. 2009, 36 cases with microtia were selected in the center of auricular reconstruction in Plastic Surgery Hospital, including 22 cases of unilateral microtia and 14 cases of bilateral microtia. 22 patients with unilateral microtia were studied with the contralateral healthy ears as controls. Spiral CT was performed for high-resolution scan of the temporal bone. The coronal, sagittal and 3D reconstruction images were created with Mimic software. Several distances and degrees were measured. RESULTS: The patients were classified by Max classification. The anteroposterior diameter and the vertical diameter of tympanic cavity were (7.75 +/- 1.92) mm and (14.66 +/- 4.75) mm for type I; (6.17 +/- 2.56) mm and(14.35 +/- 5.12) mm for type II; (6.31 +/- 3.40) mm and (9.97 +/- 4.36) mm for type III (P = 0.001). The mastoid pneumatization degree for type I, II, III were 13.33%, 13.64%, 30.77% in sclerotic type, 13.33%, 18.18%, 7.69% in diploe type, 0, 9.09%, 38.46% in composite type, 73.33%, 59.09%, 23.08% in pneumatic type (chi2 = 24.11, P = 0.002). The cover of fenestra vestibuli by facial nerve was 21.43%, 47.62%, 54.55% (chi2 = 23.44, P = 0.002) for type I, II, III. There was a statistical difference between the microtia group and the control group. CONCLUSIONS: According to the Max classification, the middle ear malformation changed along the auricular malformation. The anatomical variations was complicated in type II microtia, which should be sub-classified.

[Application of bi-pedicled frontal expanded flap for cervical cicatricial contracture].

OBJECTIVE: To investigate the therapeutic effect of bi-pedicled frontal expanded flap for cervical cicatricial contracture. METHODS: Tissue expanders were implanted under frontal muscle. After expansion, the frontal flaps were designed based on bilateral superficial temporal vessels and were transferred to the neck wound. The cervical spine movement and the mento-cervical angle was measured and analyzed by SPSS16.0 and t test. RESULTS: From September 2006 to May 2010, 7 patients were treated by this method. The range of active cervical movement was improved in all direction. The mento-cervical angle decreased from (152.7 +/- 1.9) to (90.7 +/- 2.2) degrees after operation. The patients were followed up for 5 months to 3 years with satisfactory result and no contracture recurrence. CONCLUSIONS: The bi-pedicled expanded frontal flap has a reliable blood supply and is very suitable for cervical cicatricial contracture with good functional and esthetic results, leaving less morbidity at donor site.

[Application of the remnant in repairing the reconstructed ear in microtia].

OBJECTIVE: To explore the application of the remnant in repairing the reconstructed ear in microtia. METHODS: From October 2000 to July 2006, 1,823 patients with reconstructed ear underwent the second operation for repairing. The remnants were used to reconstruct tragus, antitragus, crus of helix, concha cavum, or relax the adhesion between the reconstructed auricle and cranio wall, or insert into the reconstructed lobule or under the basement of the costal cartilage frame to restore the cranio-auricular angle or to the preauricular subcutaneous to correct the facial local depression. RESULTS: In 1,823 cases, the tragus, antitragus and concha cavum was reconstructeded by remnant auricle in 1,180 cases; helix crus was repaired by remnant auricle in 743 cases; the wound was repaired by remnant auricle flap resulted from relaxing the adhesion between the reconstructed auricle and cranial wall in 68 cases; Subcutaneous and cartilage flap was formed and transferred in 95 cases, and among them the flap was transferred under the basement of the frame to restore the cranio-auricular angle in 77 cases and was transferred to the preauricular subcutaneous to correct the facial local depression in 18 cases; the skin flap was formed by remnant auricle to repair the lobule in 37 cases. All flaps survived completely. The appearance of reconstructed ear was satisfied. CONCLUSION: The remnant is very useful in repairing the reconstructed ear in microtia. It should be preserved propriately in auricular reconstruction.

[Surgical treatment and etiological analysis of polyotia].

OBJECTIVE: To explore the surgical treatment and pathologic mechanism of polyotia. METHODS: The operative methods for polyotia were flexible. The main procedures were aimed at the remodeling of tragus with the redundant tissue. Tissue transplantation was used to correct the depression deformity. Compound auricular tissue transplantation and Z-plasty were used for correction of the malformation at the posterior part of polyotia. RESULTS: From 2003 to 2008, 9 patients with polyotia were treated. The tragus was reconstructed after resection of redundant tissue. The depression deformity was corrected with tissue infilling in 6 cases. The compound auricular tissue transplantation was performed in 4 cases. Z-plasty at the lower ear lobe was performed in 2 cases. The appearance and the location of the reconstructed ears looked symmetric to the healthy ear. CONCLUSIONS: Natural and symmetric appearance of reconstructed ear can be achieved in polyotia. The abnormal migration of neural crest cell might be the pathologic mechanism of polyotia.

[Study of methylation of promoter of EYA1 gene in microtia].

OBJECTIVE: To explore the methylation of CpG islands in promoter of eye absent gene 1 (EYA1) in microtia. METHODS: The methylation of CpG islands in EYA1 gene in 64 microtias and 36 healthy controls were measured using the technique of matrix-assisted laser desorption/ionization-time of flight. RESULTS: The methylation of CpG_Unit3 and CpG_Unit5 of EYA1 gene in microtia were 0.09258 +/- 0.033846 and 0.0922 +/- 0.02379, respectively, which were significantly lower than those in control. CONCLUSIONS: Hypomethylation in microtia may be related to the pathogenesis of the disease.

[Application of quantitative tissue expansion in ear reconstruction].

OBJECTIVE: To explore the application of tissue expander in ear reconstruction and to deal with the complications. METHODS: 50 ml kidney-shape tissue expanders were implanted subcutaneously in the mastoid area. The drainage tube was removed 3 days after operation. The suture was removed 10 days later. Since 7 days after operation, 5 ml NS was injected into the expander every time, three times a week. The total injection volume was about 60 ml. After that, the expander was maintained for one month. RESULTS: From January 1992 to December 2006, 5,248 patients of microtia were treated with 6,252 expander. After the maintaining period, the expanded skin was thin and well-vascularized. The complication rate was 7.79%, including hematoma, malunion and infection. CONCLUSIONS: The quantitative tissue expansion is easily manipulated with few complications. It can provide hairless, thin skin with reliable blood supply for ear reconstruction.

[Fabrication and application of three-dimensional autogenous cartilage framework in auricular reconstruction].

OBJECTIVE: To explore the fabrication and application of three-dimensional autogenous cartilage framework in auricular reconstruction. METHODS: The process of fabrication of three-dimensional cartilage framework consisted of cartilage harvesting, carving and assembling the cartilage. The rib cartilage was harvested separately. The three-dimensional framework was composed of three main parts:the helix, the base and the pad, at different layer. The framework was fabricated according to the development of rib cartilage and contour and height of the reconstructed ear. RESULTS: From January 1992 to December 2006, 5,248 patients of microtia were treated with 6,252 autogenous cartilage frameworks. CONCLUSIONS: The three-dimensional framework is easily manipulated. The reconstructed ears look natural and had an erect contour. This method can effectively use the cartilage.

[Clinical study of chest contour deformity after harvesting of costal cartilage for total ear reconstruction].

OBJECTIVE: To investigate the chest contour deformities after harvesting of costal cartilage for total ear reconstruction, so as to prevent donor site morbidity. METHODS: 107 cases of microtia who underwent ear reconstruction with costal cartilage during the period from 1994 to 2003, were retrospectively studied. Both the chest depression deformity and the costal arch deformity were evaluated. The possible related factors to the chest contour deformities were analyzed statistically with Stata software. RESULTS: The incidence of chest depression was 54.55% (48/88), including 35 cases of mild depression (39.77%) and 13 cases of severe depression (14.77% ). The incidence of costal arch deformity was 52.77% (46/88), including 33 cases of mild deformity (37.50%) and 13 cases of severe deformity (14.77%). The incidence of both deformities was significantly different at different age (P < 0.01), but not at different follow-up period (P = 0.294 and P = 0.225, respectively). Gender was related to the incidence of chest depression (P < 0.01), but not to the costal arch contour deformity (P = 0.619). Comparing resection of the 6th and 7th rib cartilage with resection of 7th and 8th rib cartilage, the incidence of costal arch deformity was significantly different (P < 0.01), but the incidence of depression deformity was not (P = 0.657). CONCLUSIONS: The incidence of chest deformities increases with early operation time (< 13 years old). The male patients have higher incidence of depression deformity than female. The arch deformity is higher when the 6th and 7th rib cartilage were resected. The follow-up period is not related to the chest deformities.

[Clinical application of three methods for total ear reconstruction].

OBJECTIVE: To investigate the indication and results of three methods for total ear reconstruction. METHODS: 960 cases of total ear defect were treated with skin expansion and autogenous rib cartilage framework (n = 786), or skin expansion and Medpor framework (n = 150), or ear prosthesis (n = 24). The indication and results of the three methods were analysed. RESULTS: Good cosmetic results were achieved with all three methods. Autogenous rib cartilage framework was suitable for patients under 30 years old with unaffected skin at mastoid region. Medpor framework was good for grown-up, especially over 30 years old, or with mild infection at mastoid region. Patients with severe skin injury at mastoid region or unwilling to accept surgery should choose ear prosthesis. CONCLUSIONS: Satisfactory results can be achieved with suitable methods for total ear reconstruction.

[Clinical significance of vascular distribution type of the expanded skin in the mastoid region in congenital microtia].

OBJECTIVE: To classify the blood vessel distribution of the expanded skin in the mastoid region and its relevance to reasonable and reliable design of the expanded flap for auricular reconstruction in congenital microtia. METHODS: The blood vessel distribution of the expanded skin in the mastoid region was observed by light permeation test. The expanded flaps with different ratio of length to width were designed according to their blood vessel distribution types. RESULTS: The vascular distribution of the expanded skin in 403 cases was divided into five types. All the flaps survived completely. CONCLUSIONS: The blood vessel distribution type of the expanded skin in mastoid region has great significance for the design of post-auricular expanded flap in auricular reconstruction.

[Surgical treatment of acquired ear defect].

OBJECTIVE: To explore surgical procedure for the treatment of acquired ear defect. METHODS: The ear reconstruction was carried out by using soft tissue skin expander and autogenous rib cartilage framework, Medpor framework, auricular prosthesis, and so on. RESULTS: The long-term follow-ups showed that the flap of reconstructed ear was ruddy, soft, with normal sensory function. The cartilage framework had no degeneration, absorption and deformation. The Medpor framework had no deformation. The contour of auricular prosthesis was vivid and the implant system was stable. In addition, the reconstructed ears were coincidence with the normal side on location, form and dimension. CONCLUSIONS: It may be a major method for traumatic ear reconstruction that the soft tissue skin expander with autogenous rib cartilage framework could be applied at present time. In some special circumstances, the soft tissue skin expander with Medpor framework and auricular prosthesis may be other helpful choices.