Maintenance of adult stem cells from human minor salivary glands via the Wnt signaling pathway.

BACKGROUND: Xerostomia is a salivary gland dysfunction that negatively impacts the life quality of patients; however, there is no effective treatment for xerostomia. Bioengineered organs, generated using stem cells obtained from newborn salivary glands and ligated injury models, are a new organ transplantation strategy that could be feasible for xerostomia treatment. Reconstruction of salivary gland organoids by seed cells obtained from human minor salivary glands will offer theoretical fundaments and technology support for clinical application and organ regeneration research. Herein, we aimed to propose a new method for culturing and enriching adult human minor salivary gland stem cells in vitro in a three-dimensional (3D) environment via Wnt signaling activation. METHODS: Obtained and characterized human minor salivary gland stem cells (hMSGSCs) with self-organization ability were 3D-cultured to generate organoids. We examined hMSGSCs proliferation and colony formation using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays. Telomerase reverse transcriptase staining, flow cytometry, immunofluorescence assay, RNA isolation, RT-PCR, and qPCR were performed to assess hMSGSCs structure and the function of reconstructive organoids in vitro. RESULTS: hMSGSCs showed typical epithelial-like characteristics, such as positive for CD49f and cell KRT expression. hMSGSCs served as adult stem cells in salivary glands and could differentiate into acinar and duct cells. Upon the addition of Noggin, CHIR99021, and Wnt3A to the 3D culture system, hMSGSCs showed higher LGR5 expression and decreased AMY1B and MUC5B expression. Therefore, the Wnt and bone morphogenetic protein (BMP) pathways are important in regulating hMSGSCs self-organization and differentiation. CONCLUSIONS: We showed that the stem cell properties of hMSGSCs in a 3D culture system can be maintained by activating the Wnt signaling pathway and inhibiting the BMP signaling pathway. Our findings contribute new insights on salivary gland organoid generation in vitro.

Classification of Alar Dynamic Aesthetic in an Asian Female Population: Experts or Automatic Algorithms?

AIM: To provide referenced classifications of alar dynamic aesthetics from both subjective and objective perspectives for determining proper surgical strategies in alarplasty. METHODS: A total of 150 healthy Asian female participants were instructed to perform two standardized facial movements including a resting pose and a maximum smile while taking care not to show their teeth. The participants were recorded using a dynamic three-dimensional surface imaging system. Frames depicting the resting position and the alar maximum enlargement during the smile were exported separately for anthropometric analysis and classification. The alar dynamic aesthetic was assessed through measurement of the anthropomorphic changes comparing the resting and maximum smile statuses and then transformed into quantitative analysis through the algorithm [Formula: see text]. Subjective classification and evaluation of the subject cosmetic deficiencies and proposals for therapeutic interventions to improve the subjects' alar dynamic aesthetic were performed by three senior plastic surgeons through visualization of the resting and smiling images. The surgeons were asked to divide and classify the subjects into three groups (Class I, Class II and Class III) according to the surgeons' perceptions of degree of the subjects' deficiencies in alar dynamic aesthetic. The more deficiency there was in the aesthetic, the higher the class that the subject was assigned into. The surgeons were presented with the full set of images of the patients on two separate occasions each three months apart, to assess interobserver reliability. Clustering analysis, which is based on machine learning, was applied for objective classification of the images. RESULTS: According to the senior plastic surgeon experts' subjective classification, the subjects' alar flaring mobility was judged as follows: Class I (6.78 ± 3.84%), Class II (10.35 ± 4.18%), and Class III (18.68 ± 4.15%), while alar base mobility was judged as Class I (12.71 ± 7.57%), Class II (20.06 ± 10.06%), and Class III (30.86 ± 13.20%). By clustering analysis, alar flaring mobility was determined to be Class I (7.01 ± 3.51%), Class II (11.18 ± 4.76%), and Class III (12.72 ± 5.66%), while alar base mobility was Class I (9.07 ± 4.23%), Class II (21.88 ± 4.25%), and Class III (38.59 ± 7.08%). No statistical significance was found in the distribution and assignment of classes between the two methodologies. CONCLUSION: Classifications of alar dynamic aesthetics could arouse attention to facial dynamic aesthetics and provide referenced quantitative parameters for plastic surgeons to determine appropriate treatments for alarplasty. For patients with Class I mobility, treatments are not recommended, while minimally invasive treatments can be deemed to be optional for patients with Class II alar mobility to potentially improve alar dynamic aesthetics. For patients with Class III alar mobility, surgical treatments are strongly recommended as options. Combing subjective classification with automated algorithms can provide a novel perspective and improve reliability for facial aesthetic classification analysis. LEVEL OF EVIDENCE IV: This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors   www.springer.com/00266 .

Kartogenin Induced Adipose-Derived Stem Cell Exosomes Enhance the Chondrogenic Differentiation Ability of Adipose-Derived Stem Cells.

Objective: The objective of this study is to explore the effect of kartogenin (KGN-)-pretreated adipose-derived stem cell-derived exosomes (ADSC-EXOs) on the chondrogenic differentiation ability of ADSCs. Methods: Adipose-derived stem cells (ADSCs) were treated with different doses of KGN, and exosomes (EXOs) were extracted. EXOs were then identified using an electron microscope (EM), nanoparticle tracking analyzer, nanoparticle tracking analysis software, and exosomal protein markers. EXOs were labeled with the fluorescent dye PKH67 and their uptake by cells was evaluated. A cell counting kit-8 (CCK-8) assay, flow cytometry, clonogenic assay, and a cell scratch assay were used to detect the abilities of proliferation, apoptosis, clone formation, and migration of ADSCs, respectively. Subsequently, Alcian blue staining and toluidine blue staining were used to detect the chondrogenic differentiation ability of ADSCs in each group. Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot (WB) techniques were used to detect the expression of chondrogenic differentiation-related genes. Results: In this study, ADSCs and KGN-induced ADSC-EXOs were successfully extracted and isolated. EXOs and ADSCs coculturing results showed that KGN-induced ADSC-EXOs can significantly promote proliferation, clone formation, migration, and chondrogenic differentiation of ADSCs and inhibit apoptosis. In addition, KGN-induced ADSC-EXOs can increase the expression of chondrogenic-related genes in ADSCs (Aggrecan, Collagen III, Collagen II, and SOX9), and can significantly decrease the expression of chondrolysis-related genes (MMP-3, ADAMTS4, and ADAMTS5). Conclusion: KGN-induced ADSC-EXOs can enhance the chondrogenic differentiation ability of ADSCs by promoting cell proliferation and migration while inhibiting cell apoptosis. KGN treatment can also increase the expression of chondrogenic differentiation-related genes and decrease the expression of chondrolysis-related genes. These results provide a new approach to cartilage repair and regeneration.

Posttranscriptional control of PLOD1 in adipose-derived stem cells regulates scar formation through altering macrophage polarization.

BACKGROUND: The level of cutaneous scar formation is a critical parameter to determine the success of skin wound healing. Adipose-derived mesenchymal stem cells (AMSCs) have been applied to improve treatment of cutaneous injury with the purpose of reducing scar formation. METHODS: The levels of procollagen-lysine 1,2-oxoglutarate 5-dioxygenase 1 (PLOD1) were assessed at scar sites. Then, PLOD1 in AMSCs was depleted by either expression of a PLOD1-specific short-hair interfering RNA (shPLOD1) or by expression of microRNA-449 (miR-449) that targets and suppresses protein translation of PLOD1 through 3 prime untranslated region (3'-UTR) interfering. For induction of skin injury, a blade cut of 1.5-cm long and 2-mm thick was made on the middle back of the mice. Transplantation of either AMSCs-shPLOD1 or AMSCs-miR-449 into the injured region of the mice was performed via tail vein injection. The fibrosis as well as underlying mechanisms were assessed. RESULTS: The AMSCs expressed high levels of PLOD1, a potent stimulator of fibrosis. We knocked down PLOD1 in AMSCs by expression of either shPLOD1 or miR-449. Transplantation of either AMSCs-shPLOD1 or AMSCs-miR-449 significantly reduced the fibrotic process in the injured region of the mice to a similar degree. Mechanistically, transplantation of either AMSCs-shPLOD1 or AMSCs-miR-449 shifted macrophage polarization from M2 to M1-like and reduced both reactive oxygen species (ROS) and activation of myofibroblasts from fibroblasts. CONCLUSIONS: Suppression of PLOD1 levels in AMSCs either directly by shPLOD1 or indirectly by miR-449 may substantially improve the anti-fibrotic potential of AMSCs during wound healing, likely through altering macrophage polarization.

Microvascular Ear Replantation: A Multicenter Study of 22 Patients with Complete Ear Amputation.

Background: We aimed to report our experience in treating ear amputations with microvascular replantation, with the largest sample to date. Methods: Twenty-two patients with complete ear amputation underwent microvascular ear replantation at three medical centers between May 2003 and May 2020. Arterial anastomoses, venous anastomoses, or vein graft were performed depending on different situations. Re-exploration was performed in four patients due to venous congestion (n = 3) or arterial compromise (n = 1). Results: Eleven patients had vascular complications (venous congestion: 10, arterial compromise: 1) and four of them required re-exploration. Three ears were completely salvaged (75%) and one case failed. Eighteen (81.8%) replanted ears survived completely, with 15 repaired ears demonstrating a good contour and 3 ears showing atrophy. Three replanted ears sustained partial loss, and one sustained total loss. Three extraordinary cases with the longest ischemic time, smallest tissue size, and youngest age reported thus far all survived and had cosmetically satisfactory appearances. Statistical analysis indicated no significant correlation between replanted ear survival and potentially influential factors, including ischemic time, number of arterial and venous anastomoses, presence of vein graft, and re-exploration. Conclusions: Microvascular replantation for ear amputations achieved excellent results. It may be considered the primary choice for surgeons with microsurgical skill.

Effect of a subset of adipose-derived stem cells isolated with liposome magnetic beads to promote cartilage repair.

This study aimed to investigate the ability of CD146+ subset of ADSCs to repair cartilage defects. In this study, we prepared CD146+ liposome magnetic beads (CD146+ LMB) to isolate CD146+ ADSCs. The cells were induced for chondrogenic differentiation and verified by cartilage-specific mRNA and protein expression. Then a mouse model of cartilage defect was constructed and treated by filling the induced cartilage cells into the damaged joint, to evaluate the function of such cells in the cartilage microenvironment. Our results demonstrated that the CD146+ LMBs we prepared were uniform, small and highly stable, and cell experiments showed that the CD146+ LMB has low cytotoxicity to the ADSCs. ADSCs isolated with CD146+ LMB were all CD146+ , CD105+ , CD166+ and CD73+ . After chondrogenic induction, the cells showed significantly increased expression of cartilage markers Sox9, collagen Ⅱ and aggrecan at protein level and significantly increased Sox9, collagen Ⅱ and aggrecan at mRNA level, and the protein expression and mRNA expression of CD146+ ADSCs group were higher than those of ADSCs group. The CD146+ ADSCs group showed superior tissue repair ability than the ADSCs group and blank control group in the animal experiment, as judged by gross observation, histological observation and histological scoring. The above results proved that CD146+ LMB can successfully isolate the CD146+ ADSCs, and after chondrogenic induction, these cells successfully promoted repair of articular cartilage defects, which may be a new direction of tissue engineering.

Noninvasive Photochemical Sealing for Achilles Tendon Rupture by Combination of Upconversion Nanoparticles and Photochemical Tissue Bonding Technology.

Photochemical tissue bonding (PTB), based on photosensitizer rose bengal (RB) and green light, has been regarded as an effective alternative to surgical suture and has been reported to provide benefits for Achilles tendon repair. Limited to the poor penetration of green light, secondary damage still exists while applying PTB for closed Achilles tendon rupture. This study is aimed at exploring the effects of noninvasive photochemical sealing on Achilles tendon rupture by the combination of PTB and upconversion nanoparticles (UCNPs). The rare-earth UCNPs of NaYF4 : Yb/Er (Y : Yb : Er = 78 : 20 : 2) were fabricated and then loaded into Chitosan/ß-GP hydrogel containing RB to prepare UCNPs@RB/Chitosan/ß-GP hydrogel. The properties of UCNPs and UCNP/Chitosan/ß-GP hydrogel were characterized by TEM, SEM, DLS, and FTIR analysis. The effects of UCNP and PTB combination were evaluated in an Achilles tendon rupture rat model using histological analysis. Bioluminescence imaging of ROS was performed to explore the potential mechanism. UCNPs had a uniform shape with a diameter of 29.7 ± 2.6 nm. The UCNPs@RB/Chitosan/ß-GP hydrogel could upconvert the near-infrared light into green light. The results of histological assessment showed that compared with traditional suture repair, the rats injected with UCNPs@RB/Chitosan/ß-GP hydrogel followed by irradiating with near-infrared light and the rats treated with RB solution followed by irradiating with green light had better effects on Achilles tendon repair. The benefits might be related to the generation of ROS in the PTB process. These findings indicated that the combination of PTB and UCNPs@RB/Chitosan/ß-GP hydrogel could be used as a noninvasive photochemical sealing for Achilles tendon rupture.

A Modified Procedure for Blepharoplasty: Physiological Structure Reconstruction of Upper Eyelids.

BACKGROUND: Double eyelid operation has become the most popular cosmetic procedure in China, while how to make sure the acquired double eyelids the same as congenital ones is still a problem. OBJECTIVE: This study aimed to explore a design and operation procedure to obtain natural and dynamic double eyelids, which are accordant with the congenital double eyelids both in appearance and movement. METHODS: The authors conducted a retrospective review of 352 patients who underwent our operation from October 2012 to October 2018. The procedures were performed by one senior surgeon. First, design the incision line 7 to 8 mm distance parallel to the ciliary margin. Then, cut down the skin and orbicularis muscle, and dissect the orbicularis myocutaneous flap from the anterior surface of the tarsal plate. Next, expose the orbital septum transversely in the lowest site from lateral to medial direction, and dispose of the fat tissue herniated spontaneously. Subsequently, suture the upper margin of pretarsal orbicularis myocutaneous flap to the levator aponeurosis. Finally, the skin edge of incision was closed interruptedly. The patients were followed up from 6 to 30 months after surgery by 2 surgeons to evaluate the long-term results. RESULTS: A total of 352 patients with 704 eyes were operated on and followed up in our study. There were 29 patients (8.2%) that showed slight asymmetry, and 7 eyes (2.0%) showed shallow creases. Besides that, all the creases were symmetry, parallel and natural. Just as congenital double eyelids, when opening eyes there was no sausagelike appearance, and when closing eyes there were no depressed scars. CONCLUSIONS: By reconstructing the physiological structure of upper eyelids, our double eyelid operation creates dynamic double eyelids, which are accordant with the congenital ones both in appearance and movement.

Combined Transverse Incision and Pouch Incision for the Correction of Medial Epicanthus.

BACKGROUND: The epicanthal fold is a distinct characteristic of the upper eyelid in many Asians. To achieve satisfactory results, epicanthoplasty is usually performed with double eyelid plasty and blepharoptosis. Although many surgical procedures have been reported for the elimination of epicanthal folds, such as recurrence, copious designs, conspicuous scar, and unnatural palpebral contours are challenges to the surgeon and also make patients worried. METHODS: From June 2010 to June 2015, epicanthoplasty was performed for 236 Chinese female patients using transverse incision combined with pouch incision. The transverse straight incision was performed in new inner canthus to the original eanthal corner point, after the original inner canthus corner point was reached, the oblique parallel incision was performed along the lower eyelid, so that full subcutaneous separation was obtained on the upper and lower incision, the malpositioned isomerous orbicular muscle and thickened tissue were released and excised, so that the epicanthus skin was naturally restored, and finally the incision was sutured without tension. The extent of postoperative scarring and improvement of the epicanthal fold were evaluated after surgery. The medial canthal distance was measured preoperatively and 12 months postoperatively. RESULTS: The average intercanthal distance decreased significantly from a mean of 41.68 ±â€Š2.57 mm preoperatively to 37.14 ±â€Š1.94 mm 12 months postoperatively (P < 0.05, paired t-test). And all patients were satisfied with the excellent aesthetic results in terms of an open medial canthus without definite recurrence, hypertrophic scarring, and other complications during the 12-month follow-up period. CONCLUSION: Epicanthoplasty with transverse incision and pouch incision is a simple and effective method for elimination epicanthal folds, resulting in a pleasant visualization, inconspicuous scar. However, its long-term effects require further study.

Comparison of various reagents for preparing a decellularized porcine cartilage scaffold.

Cartilage lesion repair is difficult due to the limited self-repair capability of cartilage and its lack of vascularization. Our previous study established a sandwich model for engineering cartilage with acellular cartilage sheets (ACSs) and chondrocytes. However, there is still debate over which agent achieves the optimal decellularization of cartilage sheets. In addition, changes in the extracellular matrix after decellularization are worth studying. We aimed to determine the optimal decellularization reagents and decellularization time for preparing cartilage sheets. This study compared the effects of 2 extraction chemicals [t-octylphenoxypolyethoxyethanol (Triton X-100) and sodium dodecyl sulfate (SDS)] on cartilage sheets. The sheets were soaked in various concentrations (0.1-2%) of the extraction solutions for various time periods (24-72 h). After the decellularization process with the various treatments, we examined the cell removal and preservation of the matrix components and microstructure to determine which method was the most efficient while inducing minimal damage to the perichondrium. Both protocols achieved decellularization within an acceptable time. DNA analysis showed that the reagent removed nearly all of the DNA from the cartilage sheets. The growth factor contents in the Triton X-100 samples were higher than those in the SDS samples, quantified by enzyme-linked immunosorbent assay (ELISA). Furthermore, Triton X-100 decreased the glycosaminoglycan (GAG) and increased the chondromodulin-I contents compared with SDS. The results of a Cell Counting Kit-8 (CCK-8) assay revealed that the ACSs were not cytotoxic. In conclusion, our results demonstrate that cartilage sheets decellularized by 1% SDS for 24 h or by 2% Triton X-100 for 48 h may be suitable candidate scaffolds for cartilage tissue engineering.

Thrombospondin-1 inhibits ossification of tissue engineered cartilage constructed by ADSCs.

Cartilage tissue engineering provides a new method in the treatment of cartilage defects, and adipose derived stem cells seem to be an ideal seed cell in cartilage tissue engineering because of its characteristics. However, ossification after in vivo implantation of tissue engineered cartilage remains a challenge. Thrombospondin-1 which has been reported to have an inhibitory effect on angiogenesis, may play an important role in inhibiting the ossification of tissue engineered cartilage constructed by adipose derived stem cells. Therefore, the effect of thrombospondin-1 in inhibiting the ossification of tissue engineered cartilage was evaluated in this study. Lentivirus vectors carrying thrombospondin-1 cDNA were transfected into adipose derived stem cells, and the transfected cells were used in the experiments. The expression of thrombospondin-1 was evaluated by quantitative reverse transcriptase-polymerase chain reaction and western blot, and the effects of thrombospondin-1 over-expression on angiogenesis were analyzed by angiogenesis assays. The quality of tissue engineered cartilage and the degree of ossification were assessed by biomechanical and molecular biology methods. The results showed that thrombospondin-1 infected cells have a high expression of thrombospondin-1 in mRNA and protein level, which inhibited the tube formation of endothelial cells, indicating the anti-angiogenic effects. Gene expression analyses in vitro showed that thrombospondin-1 inhibits the osteogenic differentiation of adipose derived stem cells significantly, and the results of in vivo study revealed that thrombospondin-1 significantly inhibits the expression of osteogenic genes. Compared to that in the control group, tissue engineered cartilage constructed by thrombospondin-1 transfected adipose derived stem cells in vivo showed a higher GAG content and lower compressive modulus, which indicating lower level of ossification. In conclusion, the current study indicated that the anti-angiogenic factor thrombospondin-1 suppresses the osteogenic differentiation of adipose derived stem cells in vitro, and inhibits ossification of tissue engineered cartilage constructed by adipose derived stem cells in vivo.

The application of autologous platelet­rich plasma gel in cartilage regeneration.

Cartilage defect caused by disease or trauma remains a challenge for surgeons, owning to the limited healing capacity of cartilage tissues. Cartilage tissue engineering provides a novel approach to address this issue, and appears promising for patients with cartilage defects. The cell scaffold, as one of the three key elements of tissue engineering, plays an important role in cartilage tissue engineering. Platelet­rich plasma (PRP), which is a fraction of the plasma containing multiple growth factors, has become a major research focus in the context of its use as a bioactive scaffold for tissue engineering. Therefore, we investigated the value of using PRP scaffolds combined with chondrocytes in cartilage tissue engineering. In this study, we examined the levels of growth factors in PRP, and the effects of PRP on cell proliferation and matrix synthesis in rabbit chondrocytes cultured in PRP. Short-term in vitro culture followed by long­term in vivo implantation was performed to evaluate the chondrogenesis of neocartilage in vivo. The results show that PRP may provide a suitable environment for the proliferation and maturation of chondrocytes, and can be used as a promising bioactive scaffold for cartilage regeneration.

Local injection of botulinum toxin A: an alternative therapy for axillary osmidrosis.

The objective of this study was to investigate the efficacy of local injection of botulinum toxin A for treating axillary osmidrosis. One hundred and fifty patients with axillary osmidrosis were randomly divided to receive botulinum toxin A injection treatment (50 U of botulinum toxin A was injected intracutaneously into 6-20 different sites within each axilla, n = 74) or surgical excision of the apocrine glands (n = 76). The patients were followed up for 1-3 months to analyze the therapeutic effect and complications of the two methods. The curative effect in patients with mild and moderate axillary osmidrosis was not significantly different between the botulinum toxin A injection group and operation group. However, for patients with severe axillary osmidrosis, surgery treatment seemed to be superior to botulinum toxin A treatment (P = 0.005). There was also no significant difference in the modified Dermatology Life Quality Index between the two treatments. Two cases showed complications related to hemorrhage and incision infection in the operation group. In conclusion, local injection of botulinum toxin A is a safe, fast and effective treatment for mild and moderate axillary osmidrosis, but the long-term effect remains to be further investigated.