LGR5 Modulates Differentiated Phenotypes of Chondrocytes Through PI3K/AKT Signaling Pathway.

BACKGROUND: Tissue engineering is increasingly viewed as a promising avenue for functional cartilage reconstruction. However, chondrocyte dedifferentiation during in vitro culture remains an obstacle for clinical translation of tissue engineered cartilage. Re-differentiated induction have been employed to induce dedifferentiated chondrocytes back to their original phenotype. Regrettably, these strategies have been proven to be only moderately effective. METHODS: To explore underlying mechanism, RNA transcriptome sequencing was conducted on primary chondrocytes (P0), dedifferentiated chondrocytes (P5), and redifferentiated chondrocytes (redifferentiation-induction of P5, P5.R). Based on multiple bioinformatics analysis, LGR5 was identified as a target gene. Subsequently, stable cell lines with LGR5 knocking-down and overexpression were established using P0 chondrocytes. The phenotypic changes in P1 and P5 chondrocytes with either LGR5 knockdown or overexpression were assessed to ascertain the potential influence of LGR5 dysregulation on chondrocyte phenotypes. Regulatory mechanism was then investigated using bioinformatic analysis, protein-protein docking, immunofluorescence co-localization and immunoprecipitation. RESULTS: The current study found that dysregulation of LGR5 can significantly impact the dedifferentiated phenotypes of chondrocytes (P5). Upregulation of LGR5 appears to activate the PI3K/AKT signal via increasing the phosphorylation levels of AKT (p-AKT1). Moreover, the increase of p-AKT1 may stabilize ß-catenin and enhance the intensity of Wnt/ß-catenin signal, and help to restore the dedifferentated phenotype of chondrocytes. CONCLUSION: LGR5 can modulate the phenotypes of chondrocytes in P5 passage through PI3K/AKT signaling pathway.

Atypical mandibulofacial dysostosis with microcephaly diagnosed through the identification of a novel pathogenic mutation in EFTUD2.

BACKGROUND: Mandibulofacial dysostosis with microcephaly (MFDM, OMIM# 610536) is a rare monogenic disease that is caused by a mutation in the elongation factor Tu GTP binding domain containing 2 gene (EFTUD2, OMIM* 603892). It is characterized by mandibulofacial dysplasia, microcephaly, malformed ears, cleft palate, growth and intellectual disability. MFDM can be easily misdiagnosed due to its phenotypic overlap with other craniofacial dysostosis syndromes. The clinical presentation of MFDM is highly variable among patients. METHODS: A patient with craniofacial anomalies was enrolled and evaluated by a multidisciplinary team. To make a definitive diagnosis, whole-exome sequencing was performed, followed by validation by Sanger sequencing. RESULTS: The patient presented with extensive facial bone dysostosis, upward slanting palpebral fissures, outer and middle ear malformation, a previously unreported orbit anomaly, and spina bifida occulta. A novel, pathogenic insertion mutation (c.215_216insT: p.Tyr73Valfs*4) in EFTUD2 was identified as the likely cause of the disease. CONCLUSIONS: We diagnosed this atypical case of MFDM by the detection of a novel pathogenetic mutation in EFTUD2. We also observed previously unreported features. These findings enrich both the genotypic and phenotypic spectrum of MFDM.

Profiling of Long Non-Coding RNAs in Auricular Cartilage of Patients with Isolated Microtia.

Introduction: Microtia is the second most common maxillofacial birth defect worldwide. However, the involvement of long non-coding RNAs (lncRNAs) in isolated microtia is not well understood. This study aimed at identifying lncRNAs that regulate the expression of genes associated with isolated microtia. Methods: We used our microarray data to analyze the expression pattern of lncRNA in the auricular cartilage tissues from 10 patients diagnosed with isolated microtia, alongside 15 control subjects. Five lncRNAs were chosen for validation using real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Results: We identified 4651 differentially expressed lncRNAs in the auricular cartilage from patients with isolated microtia. By Gene Ontology/Kyoto Encyclopedia of Genes and Genomes pathway (GO/KEGG) analysis, we identified 27 differentially expressed genes enriched in pathways associated with microtia. In addition, we predicted 9 differentially expressed genes as potential cis-acting targets of 12 differentially expressed lncRNAs. Our findings by qRT-PCR demonstrate significantly elevated expression levels of ZFAS1 and DAB1-AS1, whereas ADIRF-AS1, HOTAIRM1, and EPB41L4A-AS1 exhibited significantly reduced expression levels in the auricular cartilage tissues of patients with isolated microtia. Conclusions: Our study sheds light on the potential involvement of lncRNAs in microtia and provides a basis for further investigation into their functional roles and underlying mechanisms.

Quantitative Framework Fabrication with Autogenous Costal Cartilage in Microtia Reconstruction.

Hearing improvement is another basic requirement for microtia patients in addition to aesthetic needs. This quantitative framework fabrication method can reduce the learning curve, obtain satisfactory aesthetic results with few complications, and reserve a certain space for future canalplasty. Laryngoscope, 134:148-153, 2024.

Long-Term Effectiveness of Ear Molding and Factors Affecting Outcomes.

BACKGROUND: The EarWell System offers a correction opportunity for infants born with ear anomalies. However, the long-term effectiveness of ear molding remains unclear. This study aimed to explore the long-term effectiveness of this novel technique and to determine the risk factors for recurrence. METHODS: This retrospective, population-based cohort study was performed from 2017 through 2021. Infants who completed ear molding therapy and were followed up for longer than 6 months were enrolled. The main outcomes were immediate and long-term efficacy, which were graded by two blinded plastic surgeons. RESULTS: A total of 226 infants (334 ears) were recruited. The most common anomalies were helical deformities [113 ears (33.8%)], and the rarest were cryptotia [five ears (1.5%)] and conchal crus [five ears (1.5%)]. The age at initiation of treatment was a factor affecting both immediate ( P = 0.004) and long-term effectiveness ( P = 0.009). The type of anomaly also influenced long-term molding outcomes. For cup ears, the success rate of long-term outcomes (76.0%) was significantly lower than that of immediate outcomes (98.7%) ( P < 0.001). Prominent ear, cup ear, and microtia were found to be the most likely to relapse during long-term follow-up. The results of logistic regression also demonstrated age, duration time, and the type of anomaly to be risk factors of ear molding effects. CONCLUSIONS: The EarWell System was shown to be a secure and effective method for treatment of congenital ear anomalies. Some infants' ear anomalies recurred after successful immediate results. The age at initiation of treatment and the type of anomaly were predictors of long-term outcomes. CLINICAL QUESTION/LEVEL OF EVIDENCE: Risk, III.

The circular RNA expression profile of human auricle cartilage and the role of circCOL1A2 in isolated microtia.

Microtia is one of the most common craniofacial birth defects worldwide, and its primary clinical manifestation is auricle deformity. Epigenetic factors are known to contribute to the etiology of microtia, yet the involvement of circular RNAs (circRNAs) in human auricle development and their association with microtia remains poorly understood. In this study, we aimed to analyze differentially expressed circRNAs and explore their functional implications in isolated microtia. By employing circRNA microarray analysis and bioinformatics approaches, we identified 340 differentially expressed circRNAs in auricle cartilage of patients with isolated microtia, comprising 152 upregulated and 188 downregulated circRNAs. A circRNA-mRNA co-expression network was constructed, followed by gene ontology analysis and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis. Subsequently, we selected four significantly upregulated circRNAs from the co-expression network based on their association with cartilage development and validated their expressions in 30 isolated microtia and 30 control clinical auricle cartilage samples. Among these circRNAs, circCOL1A2, the most significantly upregulated circRNA, was selected as a representative circRNA for investigating its role in isolated microtia. Overexpression of circCOL1A2 significantly inhibited chondrocyte proliferation and chondrogenic differentiation of human mesenchymal stem cells. Additionally, circCOL1A2 upregulated Dermatan Sulfate Epimerase Like (DSEL) expression by sponging miR-637 through the competing endogenous RNA (ceRNA) mechanism. Notably, the downregulation of DSEL attenuated the inhibitory effect of circCOL1A2 overexpression on cell proliferation and chondrogenic differentiation. Collectively, these findings highlight the involvement of circCOL1A2 in the pathogenesis of isolated microtia and emphasize the potential significance of dysregulated circRNAs in disease development.

The Effect of Fixation Materials on the Long-Term Stability of Cartilage Framework for Microtia Reconstruction.

Objective: To observe the effect of fixation materials on the long-term stability of the cartilage framework in auricular reconstruction. Methods: Consecutive patients who underwent the first stage of auricular reconstruction by the same surgical team from September 2018 to March 2021 were included. Those with braided absorbable suture, polypropylene suture, and titanium wire were defined as Groups A, B, and C, respectively. Six months later, when the patients underwent the second stage of surgery, absorption and deformation of the framework were assessed. Results: A total of 604 patients (622 ears) were included. The most common occurrence was spot absorption in the antihelix and was observed in 34.7%, 17.4%, and 22.8% ears in the A, B, and C groups, respectively (p < 0.05). There were also significant differences regarding severe absorption and severe deformation. The histological examination suggested that those in Group A had more T cells and macrophages around the suture than those in Groups B and C. Conclusion: Absorbable suture seems to correlate with higher risk of cartilage absorption and helix displacement. Titanium wire had the lowest rate of helix pop-out or fracture, which means the best stability.

Changes of Age-related Auricular Cartilage Plasticity and Biomechanical Property in a Rabbit Model.

OBJECTIVES: Ear molding is an emerging technique that can correct auricular deformities. Treatment initiation time is the most important prognostic determinant of ear molding. Here, we aimed to examine why auricular cartilage plasticity appeared to diminish with age. Thus, we characterized age-related changes in the biomechanical, biochemical, and morphological properties of auricular cartilage. METHODS: New Zealand rabbits were used as the experimental animal. We examined immature [postnatal 0 day (P0), 5 days (P5), 15 days (P15)], young [2 months (2M)], and mature [6 months (6M)] rabbits. Rabbits' ears were splinted and folded using adhesive fixation strips. Folding duration ranged from 1 day to 5 days to 10 days. Photographs were taken to calculate the retained fold angle. Cartilage morphology and extracellular matrix (ECM) content were examined histologically (using hematoxylin-eosin, Safranin O, elastic Van Gieson, and Masson's trichrome). Water content, DNA content, and cell density were also analyzed. Biomechanical properties were measured using a Nano indenter. RESULTS: Immature ears had smaller angles after strip removal, and the angled deformation lasted a longer time. Cartilage matrix compositions, including glycosaminoglycan (GAG), elastin fiber, and collagen, increased over development. The water content, DNA content, and cell density decreased with age. Young's modulus was significantly higher in mature cartilage. CONCLUSIONS: Here, we successfully established an animal model of ear molding and demonstrated that immature cartilage was associated with better plasticity. We also found that the cartilage's biomechanical property increased with the accumulation of ECM. The biomechanical change could underlie age-related shape plasticity. LEVEL OF EVIDENCE: NA Laryngoscope, 133:88-94, 2023.

Using ear molding to correct auricular helix adhesion deformity.

Objectives: This study examined the effectiveness of Byrd's EarWell system for the treatment of auricular helix adhesion. Methods: The newborns with helix adhesion were treated with ear molding. The photos of pinna were taken before, during, and after the treatment. The immediate and long-term outcomes, as well as the complications, were assessed by two independent plastic surgeons. Study design: A retrospective study. Data on family history, neonatal weight, gestational age, delivery method, laterality, gender, age of initiating treatment, medical comorbidities, duration of treatment, and follow-up time were collected. Study site and period: From 2019 to 2021, infants treated with the EarWell System in the Eye and ENT Hospital of Fudan University were enrolled in this study. Results: A total of 46 newborns (66 ears) with helix adhesion were included. The average onset time of treatment was 4.57 ± 3.63 weeks. The average duration of treatment was 7.40 ± 2.05 weeks. 97.0% ears' (64/66) immediate results were excellent or good. During long-term follow-up, 95.5% ears achieved excellent or good outcomes. Age of initiation treatment significantly affected immediate (p = 0.001) and long-term (p = 0.004) outcomes. Conclusions: EarWell System was an effective method to correct auricular helix adhesion. Using this approach, patients with helix adhesion could avoid surgeries. Age of initiation treatment was the predictor of successful correction.

Isolation, culture, and identification of ceruminous gland cells.

External auditory canal (EAC) stenosis or atresia usually requires a skin graft to repair, but due to the lack of a graft containing functional glands, postoperative complications such as infection and eczema are common. The aim of this study was to isolate and characterize seed cells for the construction of tissue engineered EAC skin containing ceruminous gland by isolating and cultivating cells of ceruminous gland. In this study, EAC skin samples were harvested from adult goats for ceruminous gland cell isolation. Cell morphology and proliferation rates, expression of CK7, CK8, CK18, and CK19 (glandular cell specific-markers), and secretion of ß-defensin-1, lysozyme, and polysaccharides were evaluated at different passages to verify the presence of ceruminous gland cells and determine whether function and proliferation potential were maintained. Ceruminous glands were successfully isolated and extracted from goat EAC skin. Furthermore, the isolated glandular cells maintained robust proliferation potential, exhibited high expression of CK7, CK8, CK18, and CK19, and vigorously secreted ß-defensin-1, lysozyme, and polysaccharides in this culture system. However, expression of glandular cell specific-markers and secretory function gradually declined with increasing passage number, indicating dedifferentiation of the subcultured ceruminous gland cells after five passages. In conclusion, ceruminous glands were successfully isolated, cultured, and expanded from goat EAC skin using the serumcontaining culture system. Importantly, the isolated glandular cells retained robust proliferation potential and maintained their phenotype and function in early passages (P1-P3), indicating the method's potential application for ceruminous gland regeneration.

Reconstruction of Distal Nasal Defects With a Large Postauricular Skin-Fat-Fascia Composite Graft.

BACKGROUND: Composite grafts have previously been reported to achieve a good outcome for nasal defect repair, but composite grafts have greater metabolic needs than simple skin. Therefore, the traditionally recommended size of a composite graft for nasal reconstruction is less than 1.5 cm in diameter. However, the distal nose is generally well supplied with blood vessels, which might support the use of larger composite grafts in such a highly vascularized recipient site. The aim of the article is to investigate whether a large skin-fat-fascia composite graft (larger than 2.0 cm) is viable for the repair of partial-thickness nasal defects. METHODS: From October 2017 to December 2019, 13 patients with partial-thickness nasal defects underwent nasal reconstruction using a large postauricular skin-fat-fascia composite graft. Cases were followed up for 3 to 14 months postoperatively. The aesthetic outcome was evaluated in comparison with preoperative digital images. RESULTS: Skin-fat-fascia composite grafts survived without graft necrosis, dermal fibrosis, or skin contraction in all cases. Favorable aesthetic outcomes were obtained in all patients, and no further revision surgery was need. CONCLUSIONS: A postauricular composite graft larger than 2.0 cm is a safe and effective reconstruction approach for partial-thickness nasal defects. This technique offers significant advantages in terms of no additional facial scar, no visible asymmetry on the face, no additional surgery for revision, and with mild scar in the donor site of the postauricular region.

Comparative study of the external auditory canal in humans and large mammals.

Given the limited source of human external auditory canal (EAC) skin, animal experiments remain an important approach for studying functional EAC reconstruction. However, differences between humans and animals in terms of the general EAC structure, histological characteristics of EAC skin, and cell markers of its specific glands in cartilaginous EAC skin remain unknown. We compared the characteristics of the EAC between humans and large animals, as a basis for appropriate animal model selection. Temporal bone computed tomography was used to compare the EACs of humans, goats, pigs, and dogs. EAC skin samples were harvested and their histological characteristics evaluated. The skin's ultrastructure and the histological structure of specific glands and cell markers related to cell phenotype and function were further identified. The EAC structure in goats was similar to that in humans in terms of diameter, length, and cartilaginous segment ratio of the EAC, while that of pigs and dogs differed markedly. Furthermore, histological evaluation showed that there were abundant ceruminous and sebaceous glands in the goat's cartilaginous skin, while dogs and pigs showed notably fewer of these glands in cartilaginous skin than humans. Nevertheless, ceruminous glands in all species studied showed similar expression of cell biomarkers and secretion function. Goats might have advantages in terms of surgery and reconstruction of the functional EAC skin compared to dogs and pigs and can be a useful candidate for ceruminous gland cell sources.

Skin redraping for correction of lower eyelid epiblepharon combined with medial epicanthal fold: a retrospective analysis of 286 Asian children.

OBJECTIVE: This study was performed to evaluate the clinical effect of skin redraping on lower eyelid epiblepharon accompanied by a medial epicanthal fold. METHODS: This retrospective case series involved 572 eyes of 286 patients who underwent skin redraping surgery to treat lower eyelid epiblepharon accompanied by a medial epicanthal fold from January 2015 to May 2019. The postoperative surgical results were classified as "good", "fair" and "poor". The incision scars were assessed using the Vancouver scar scale. The patients' subjective satisfaction and incidence of complications were also documented. RESULTS: The mean patient age at the time of surgery was 6.9 ± 3.6 years (3-12 years), and the mean follow-up time was 32.6 ± 13.5 months (6-58 months). The clinical symptoms and severity of keratopathy were improved postoperatively. "Good" surgical outcomes were obtained in all patients, the mean Vancouver scar scale score was 1.1 ± 0.3, and hypertrophic scar formation did not occur. A total of 272 patients and their guardians were "very satisfied" with the cosmetic outcomes. CONCLUSION: Skin redraping was effective and endurable in the treatment of lower eyelid epiblepharon accompanied by a medial epicanthal fold. The postoperative scars were slight and nearly invisible, and no cases of recurrence were observed in this study.

Modified Maximal Levator Palpebrae Superioris Shortening in Correcting Congenital Severe Ptosis in Children.

OBJECTIVE: This study aims to evaluate the clinical effect of modified maximal levator palpebrae superioris shortening method for severe congenital ptosis. METHODS: A retrospective case series was performed including 66 eyes from 62 patients who underwent modified maximal levator palpebrae superioris shortening surgery to treat severe congenital ptosis between February 2015 and November 2018. Preoperative and postoperative margin reflex distance 1 and levator muscle function were recorded. The surgical results were graded as good, satisfied, and poor for functional and cosmetic improvement of the eyelids, and the incidence of complications was also documented. RESULTS: The mean patient age at the time of surgery was 4.6 ± 1.8 years (2-9 years), and the mean follow-up time was 36.3 ± 14.1 (12-55 months). A mean significant improvement in margin reflex distance 1 and levator function after operation was noted (P < 0.01). The eyelid height and symmetry were satisfied in 59 patients, with success rate of 95.2%. For the patients in the levator function (≤2 mm) group, the success rate was 87.5%. Moreover, the levator function (≤2 mm) group had a higher rate of poor results than levator function (2-4 mm) group (12.5% vs 2.2%). Overcorrection (6.5%) and eyelid fold deformity (11.3%) were the most frequent postoperative complications. CONCLUSION: Modified maximal levator palpebrae superioris shortening was effective and endurable in the treatment of severe congenital ptosis with poor levator function, including in patients whose levator function was less than 2 mm.

Phenotypic redifferentiation of dedifferentiated microtia chondrocytes through a three-dimensional chondrogenic culture system.

Chondrocytes from microtia patients are a valuable cell source for the tissue-engineering of auricles. However, dedifferentiation of microtia chondrocytes remains an obstacle for clinical translation. Strategies, such as three-dimensional (3D) culture systems, and the use of chondrogenic growth factors, have successfully induced redifferentiation of dedifferentiated chondrocytes from healthy individuals. However, it remains unknown whether these strategies are similarly effective for microtia patient-derived chondrocytes, which may carry genomic defects. To address this issue, dedifferentiated microtia chondrocytes (DMCs) were cultured in a 3D chondrogenic culture system for 4-8 weeks to investigate their redifferentiated properties and to generate redifferentiated microtia chondrocytes (RMCs). To predict the degree and course of redifferentiation, RMCs at different time points were harvested and examined for cell morphology, cell proliferation, type II collagen expression at passaging, and chondrogenic capacity. We show that a 3D chondrogenic culture system can effectively induce DMCs to become redifferentiated, functional chondrocytes, enabling them to regenerate mature cartilage. Furthermore, RMCs achieved their full original function after culture in the chondrogenic culture system for 6-8 weeks. Interestingly, redifferentiation of microtia chondrocytes exhibited a time-dependent trend. Although the primary mechanism by which the 3D chondrogenic culture system regulated the transition of DMCs into RMCs remains unknown, the current study provides deeper insight into microtia chondrocytes and promotes clinical translation of tissue-engineered auricles.

Long non-coding RNA SCARNA2 induces cutaneous squamous cell carcinoma progression via modulating miR-342-3p expression.

BACKGROUND: Long non-coding RNAs (lncRNAs) play important roles in the progression of tumors. However, the function and expression of SCARNA2 in cutaneous squamous cell carcinoma (cSCC) is still unreported. METHODS: A quantitative polymerase chain reaction was applied to study the expression of SCARNA2 and miR-342-3p. Cell counting kit-8, flow cytometry and transwell assays were performed to study cell growth, cycle and cell invasion. RESULTS: We found that SCARNA2 expression is up-regulated in cSCC cell lines and SCARNA2 expression is higher in cSCC tissues than in adjacent non-tumor specimens. Ectopic expression of SCARNA2 promoted cell growth, cell cycle and invasion in SCC13 cells. In addition, the data indicate that miR-342-3p expression is down-regulated in cSCC cell lines and miR-342-3p is down-regulated in cSCC tissues compared to adjacent non-tumor specimens. We showed that the SCARNA2 expression is negatively associated with miR-342-3p in cSCC. Moreover, we noted that SCARNA2 sponges miR-342-3p expression in cSCC cells. Overexpression of SCARNA2 suppressed the miR-342-3p expressed in SCC13 cells. We found that elevated expression of SCARNA2 promotes cell growth, cell cycle and invasion via regulating miR-342-3p expression in SCC13 cells. CONCLUSIONS: These data suggest that SCARNA2 acts in an oncogenic role and may be a potential target for cSCC.

Dietary Calcium Intake and HPV Infection Status Among American Women: A Secondary Analysis from National Health and Nutrition Examination Survey (NHANES) Data Set of 2003 - 2016.

BACKGROUND The evidence on the link of dietary calcium (DCa) to human papillomavirus (HPV) infection is limited. Thus, this research was conducted to explore whether DCa is independently associated with HPV infection status in American women with age of 18 to 59 years old. MATERIAL AND METHODS We performed a secondary analysis from the National Health and Nutrition Examination Survey (NHANES) data set including 7 cycles from 2003 to 2016. A total of 13 475 selected participants were used for data analysis. The interested independent and the outcome variable were DCa and HPV infection status (HPV infection; HPV subtype). Sociodemographic, dietary, laboratory, questionnaire, and physical examination data were covariates. Weighted binary logistic regression and generalized additive model (GAM) were used for the investigation of both linear and non-linear relationships between DCa and HPV infection status. RESULTS Weighted multivariable binary logistic regression indicated DCa was not associated with HPV infection and subtype (OR: 0.93; 95% CI: 0.82-1.05 for HPV infection; OR: 1.09; 95% CI: 0.93-1.28 for HPV subtype). For HPV infection, a non-linear correlation was detected, whose inflection points were 9.78 of log2 DCa. The OR values and the confidence intervals on both sides of inflection point were 0.83 (95% CI: 0.70-0.98) and 1.18 (95% CI: 0.91-1.52), respectively. CONCLUSIONS At the range of 3.32-9.78 of log2 calcium intake, DCa intake was negatively correlated with HPV infection. After this interval, DCa intake was not associated with the risk of HPV infection.

A moldable thermosensitive hydroxypropyl chitin hydrogel for 3D cartilage regeneration in vitro and in vivo.

Because of poor self-repair capacity, the repair of cartilage defect is always a great challenge in clinical treatment. In vitro cartilage regeneration provides a potential strategy for functional reconstruction of cartilage defect. Hydrogel has been known as an ideal cartilage regeneration scaffold. However, to date, in vitro cartilage regeneration based on hydrogel has not achieved satisfactory results. The current study explored the feasibility of in vitro 3D cartilage regeneration based on a moldable thermosensitive hydroxypropyl chitin (HPCH) hydrogel and its in vivo fate. The thermosensitive HPCH hydrogel was prepared and characterized. Goat auricular chondrocytes were encapsulated into the HPCH hydrogel to form a chondrocyte-hydrogel construct. The constructs were injected subcutaneously into nude mice or molded into different shapes for in vitro chondrogenic culture followed by in vivo implantation. The results demonstrated that the HPCH hydrogel possessed satisfactory gelation properties (gelation time < 18 s at 37 °C), biocompatibility (cell amount almost doubled within one week), and the ability to be applied as an injectable hydrogel for cartilage regeneration. All the constructs of in vitro culture basically maintained their original shapes (in vitro to initial: 110.8%) and displayed typical cartilaginous features with abundant lacunae and cartilage specific matrix deposition. These in vitro samples became more mature with prolonged in vivo implantation and largely maintained the original shape (in vivo to in vitro: 103.5%). These results suggested that the moldable thermosensitive HPCH hydrogel can serve as a promising scaffold for cartilage regeneration with defined shapes in vitro and in vivo. STATEMENT OF SIGNIFICANCE: Because of avascular and non-nervous characteristic of cartilage, in vitro regeneration plays an important role in reconstructing cartilage function. Hydrogel has been known as an ideal cartilage regeneration scaffold. However, to date, in vitro cartilage regeneration based on hydrogel has not achieved satisfactory results. The current study demonstrated that the chondrocyte-hydrogel construct generated by high density of chondrocytes encapsulated into a thermosensitive HPCH hydrogel could successfully regenerate in vitro typical cartilage-like tissue with defined shapes and further mature to form homogeneous cartilage with their original shapes after in vivo implantation. The current study indicated that the moldable thermosensitive HPCH hydrogel could serve as a promising scaffold for in vitro and in vivo cartilage regeneration with different shapes.

Regeneration of trachea graft with cartilage support, vascularization, and epithelization.

The repair and functional reconstruction of long-segment tracheal defects is always a great challenge in the clinic. Finding an ideal substitute for tracheal transplantation is the only way to solve this problem. The current study proposed a series of novel strategies for constructing a bionic living trachea substitute. For the issue of tubular cartilage support, cartilage sheet technique based on high-density culture of chondrocytes was adopted to avoid the inflammatory reaction triggered by the materials and thus formed mature cartilage-like tissue in autologous goat model. For the issue of epithelialization, the autologous transplantation of oral mucosal epithelium was used to realize mucosa coverage of the constructed trachea lumen. Finally, the flat trapezius fascia flap with double blood supply was separated by microsurgical techniques to achieve stable pre-vascularization of both the regenerated cartilage and the grafted epithelium simultaneously. By integrating the above strategies, the vascularized and epithelialized tracheal substitute with tubular cartilage support was successfully constructed in a goat model. The reconstructed trachea possessed a multiple layer structure of muscle-cartilage-fascia-mucosa comparable to the native trachea, and thus might realize stable survival and long-term airway function maintenance, providing a promising tracheal substitute for the repair and permanent functional reconstruction of long-segment tracheal defects. STATEMENT OF SIGNIFICANCE: The repair of long-segment tracheal defects is always a great challenge in the clinic. Finding an ideal substitute for tracheal transplantation is the only way to solve this problem. In the current study, by technical integration of cartilage regeneration, microsurgery, and oral mucosa transplantation, a complex tracheal substitute with satisfactory vascularization, epithelialization, and tubular cartilage support was successfully constructed in a goat autologous model. The reconstructed trachea substitute possessed a multiple layer structure of muscle-cartilage-fascia-mucosa exactly similar to native trachea, and thus might realize stable survival and long-term airway function maintenance. The current study provides feasible strategies and ideal tracheal substitutes for permanent functional reconstruction of long-segmental trachea defects.