Distraction force promotes the osteogenic differentiation of Gli1+ cells in facial sutures via primary cilia-mediated Hedgehog signaling pathway.

BACKGROUND: Trans-sutural distraction osteogenesis (TSDO) involves the application of distraction force to facial sutures to stimulate osteogenesis. Gli1+ cells in the cranial sutures play an important role in bone growth. However, whether Gli1+ cells in facial sutures differentiate into bone under distraction force is unknown. METHODS: 4-week-old Gli1ER/Td and C57BL/6 mice were used to establish a TSDO model to explore osteogenesis of zygomaticomaxillary sutures. A Gli1+ cell lineage tracing model was used to observe the distribution of Gli1+ cells and explore the role of Gli1+ cells in facial bone remodeling. RESULTS: Distraction force promoted bone remodeling during TSDO. Fluorescence and two-photon scanning images revealed the distribution of Gli1+ cells. Under distraction force, Gli1-lineage cells proliferated significantly and co-localized with Runx2+ cells. Hedgehog signaling was upregulated in Gli1+ cells. Inhibition of Hedgehog signaling suppresses the proliferation and osteogenesis of Gli1+ cells induced by distraction force. Subsequently, the stem cell characteristics of Gli1+ cells were identified. Cell-stretching experiments verified that mechanical force promoted the osteogenic differentiation of Gli1+ cells through Hh signaling. Furthermore, immunofluorescence staining and RT-qPCR experiments demonstrated that the primary cilia in Gli1+ cells exhibit Hedgehog-independent mechanosensitivity, which was required for the osteogenic differentiation induced by mechanical force. CONCLUSIONS: Our study indicates that the primary cilia of Gli1+ cells sense mechanical stimuli, mediate Hedgehog signaling activation, and promote the osteogenic differentiation of Gli1+ cells in zygomaticomaxillary sutures.

Human umbilical cord mesenchymal stem cell-derived exosomes combined with gelatin methacryloyl hydrogel to promote fractional laser injury wound healing.

To investigate whether human umbilical cord mesenchymal stem cell-derived exosomes combined with gelatin methacryloyl (GelMA) hydrogel are beneficial in promoting healing of laser-injured skin wounds in mice. Supernatants of cultured human umbilical cord mesenchymal stem cells (HUC-MSCs) were collected to obtain human umbilical cord MSC-derived exosomes (HUC-MSCs-Exos), which were combined with GelMA hydrogel complex to treat a mouse fractional laser injury model. The study was divided into PBS group, EX (HUC-MSCs-Exos) group, GEL (GelMA hydrogel) group and EX+GEL (HUC-MSCs-Exos combined with GelMA hydrogel) group. The healing of laser-injured skin in each group was observed by gross view and dermatoscopy, and changes in skin structure, angiogenesis and proliferation-related indexes were observed during the healing process of laser-injured skin in each group. The results of the animal experiments showed that the EX and GEL groups alone and the EL+EX group exhibited less inflammatory response compared to the PBS group. The EX and GEL groups showed marked tissue proliferation and favourable angiogenesis, which promoted the wound healing well. The GEL+EX group had the most significant promotion of wound healing compared to the PBS group. qPCR results showed that the expression levels of proliferation-related factors, including KI67 and VEGF and angiogenesis-related factor CD31, were significantly higher in the GEL+EX group than in the other groups, with a time-dependent effect. The combination of HUC-MSCs-Exos and GelMA hydrogel is beneficial in reducing the early inflammatory response of laser-injured skin in mice and promoting its proliferation and angiogenesis, which in turn promotes wound healing.

The SHH-GLI1 pathway is required in skin expansion and angiogenesis.

To investigate the role of GLI1 on skin proliferation and neovascularization during skin expansion in mice. We constructed GLI1-cre/R26-Tdtomato and GLI1-cre/R26-mtmg gene-tagged skin expansion mouse models. Using a two-photon in vivo imaging instrument to observe the changes in the number and distribution of GLI1(+) cells during the expansion process and to clarify the spatial relationship between GLI1(+) cells and blood vessels during the expansion process. In vitro proliferation assays were performed to further validate the effects of SHH (sonic hedgehog) and its downstream component GLI1 on cell proliferation viability. Finally, qRT-PCR was used to verify the changes in proliferation, angiogenesis-related factors, SHH signalling pathway-related factors, and the role of GLI1 cells in the process of skin expansion in mice. The number of GLI1(+) cells increased during dilation and were attached to the outer membrane of the vessel. The epidermis was thickened and the dermis thinned after the dilated skin was taken, while the epidermal thickening was suppressed and the dermis became thinner after the GLI1 cells were inhibited. The non-inhibited group showed a significant increase in PCNA positivity with prolonged dilation compared to the GANT61(GLI specificity inhibitor) inhibited group; CD31 immunofluorescence showed a significant increase in the number of dilated skin vessels and a significant decrease in the number of vessels after treatment with GANT61 inhibitor. In vitro proliferation results showed that SHH signalling activator significantly increased the proliferation viability of GLI1(+) hair follicle mesenchymal stem cells, while GNAT61 significantly inhibited the proliferation viability of GLI1(+) hair follicle mesenchymal stem cells. GLI1 is necessary for proliferation and neovascularization in expansion skin of mice through activation of the SHH signalling pathway.

Exosomes of Adipose Tissue-Derived Stem Cells Promote Wound Healing by Sponging miR-17-5p and Inducing Autophagy Protein Ulk1.

BACKGROUND: Wound healing undergoes intricate phases: hemostasis, inflammation, proliferation, and remodeling. Stem cell therapy based on adipose tissue-derived stem cell exosomes (ADSCs-exo) is considered a potential effective treatment for accelerating wound healing. However, the molecular mechanisms of wound healing using ADSCs-exo remain largely unknown. METHODS: Circular wounds, 1 × 1 cm, were generated on C57BL/6 mice, followed by OriCell C57BL/6 mouse adipose-derived mesenchymal stem cell suspension treatment, and wound area was measured and recorded at days 0, 7, and 21, respectively. A comprehensive transcriptome profiling of skin wounds was conducted in the mouse model. Importantly, the authors also examined autophagy and cell migration in mouse keratinocytes treated with ADSCs-exo. Further competing endogenous RNA networks were also used to reveal the relationship between Neat1 and Ulk1 . RESULTS: Mouse keratinocytes treated with ADSCs-exo showed significant up-regulation of pathways related to wound healing, including response to virus, bacterium, immune system, and wounding. Activated autophagy was detected, which significantly promoted the wound repair of mice. Competing endogenous RNA networks uncovered that Neat1 induces the expression of Ulk1 and thus up-regulates autophagic activity to promote wound repair through sponging miR-17-5p. CONCLUSIONS: Collectively, these results reveal a novel molecular mechanism that the autophagy pathway enhanced by the Neat1 /miR-17-5p/ Ulk1 axis can promote the wound healing and suggest that long noncoding RNA Neat1 loaded by ADSCs-exo might be a potential therapeutic target for skin nonhealing wounds. CLINICAL RELEVANCE STATEMENT: This study may provide new clues for the applications of ADSCs-exo in skin wounds and promote the development of ADSCs-exo therapy in clinical treatment of skin wounds.

Four cases of abdominal expander implantation in adult chronic osteomyelitis of lower extremity with soft tissue defect and literature review.

In this study, we intend to explore the clinical efficacy of abdominal expander implantation combined with flap technique in adult chronic osteomyelitis of lower limb with soft tissue defects, and to provide the basis for the promotion of the technique in clinical practice. Four patients diagnosed with chronic osteomyelitis of lower extremity with soft tissue defect were enrolled in this prospective study. Evaluation indicators included state of flap survival, state of abdominal incision, surrounding of abdominal wound scar, satisfaction of the patient, state of flap survival half a year after surgery, whether the 3D prosthesis is successfully implanted and limb movement. Four patients had complete flap survival, two of whom had a small amount of skin graft survival disorder in the vascular pedicle area, which improved after 1 month of dressing change. The expander had an average expansion time of 31.5 days, an average water injection of 525 mL, and an average skin volume taken of 159 cm2 . No incision exudation, incision dehiscence, subcutaneous exudate and other complications occurred. The mean Vancouver Scar Scale score at 3 months after surgery was 3.5 (range from 3 to 6) points. Four patients showed good flap survival at six-month follow-up. 3D printed prosthesis were all successfully implanted. The treatment of adult chronic osteomyelitis of lower extremities with lower abdominal implantable expander combined with flap technique can effectively increase the skin harvesting area, reduce the suture tension of abdominal skin harvesting area and the scar hyperplasia of abdominal skin harvesting area.

Research Progress on Preparation, Mechanism, and Clinical Application of Nanofat.

Autologous adipose tissue is an ideal soft tissue filling material in theory, which has the advantages of easy access, comprehensive source, and high biocompatibility and is now widely used in clinical practice. Based on the above benefits of autologous fat, autologous fat grafting is an essential technique in plastic surgery. Conventional macrofat is used to improve structural changes after soft tissue damage or loss caused by various causes such as disease, trauma, or aging. Due to the large diameter of particles and to avoid serious complications such as fat embolism, blunt needles with larger diameters (2 mm) are required, making the macrofat grafting difficult to the deep dermis and subdermis. Nanofat grafting is a relatively new technology that has gained popularity in cosmetic surgery in recent years. Nanofat is produced by mechanical shuffling and filtration of microfat, which is harvested by liposuction. The harvesting and processing of nanofat are cost-effective as it does not require additional equipment or culture time. Unlike microfat, nanofat particles are too small to provide a notable volumizing effect. Studies have shown that nanofat contains abundant stromal vascular fraction cells and adipose-derived stem cells, which help reconstruct dermal support structures, such as collagen, and regenerate healthier, younger-looking skin. Moreover, the fluid consistency of nanofat allows application in tissue regeneration, such as scars, chronic wounds, and facial rejuvenation. This article reviews the current research progress on the preparation, mechanism, and clinical application of nanofat.

Clinical study on orthopaedic treatment of chronic osteomyelitis with soft tissue defect in adults.

To investigate the clinical application value of different flap transfer and repair techniques in adult patients with chronic osteomyelitis of limbs complicated with soft tissue defects. According to the characteristics and defects of 21 cases, different plastic surgery was applied, including debridement, negative pressure device, and tissue flap to cover wound. Among 21 cases of chronic osteomyelitis complicated with local soft tissue defect, 15 patients were repaired with sural neurotrophic musculocutaneous flap transfer, 2 patients were repaired with medial plantar skin flap transfer, 2 patients were repaired with ilioinguinal skin flap transfer, 1 patient was repaired with z-forming wound, and 1 patient was repaired with soleus muscle flap combined with full-thickness skin graft. All the 21 patients underwent bone cement implantation after dead bone osteotomy. Among them, 19 patients underwent bone cement replacement with 3D prosthesis within 6 months to 1 year after surgery, and 2 patients carried bone cement for a long time. Early intervention, thorough debridement, removal of necrotic or infection, and then selecting the appropriate wound skin flap coverage are important means of guarantee slow osteomyelitis wound healing and for providing a possible way to permanent prosthesis implantation subsequently.

Island sternocleidomastoid myocutaneous flap for posterior pharyngeal wall defect repair after anterior cervical spine surgery.

Injuries and tumours of the cervical spine represent therapeutic challenges to the treating surgeon due to the complex anatomical relationships and biomechanical features. The anterior cervical midline (ACM) and anterior cervical retropharyngeal (ACR) approaches are effective and safe surgical approaches for certain cervical spine lesions, such as cervical spine neoplasms, atlantoaxial subluxation, and certain odontoid fractures. Posterior pharyngeal wall defects (PPWDs) is one of the most frequently encountered surgical morbidities after anterior cervical spine surgery (ACSS). However, limited information has been published concerning effective approaches for PPWD reconstruction after ACSS. The manuscript aimed to describe a novel application of the island sternocleidomastoid myocutaneous flap (ISMF) in the management of PPWDs after ACSS, including surgery with the ACM approach and ACR approach. From April 2015 to November 2019, the clinical data of three patients with PPWDs repaired using the ISMF in Peking university third hospital were retrospectively analysed. The observational indexes are as follows: postoperative survival of the flap, wound healing 2 weeks after surgery, eating and pronunciation function 2 months after surgery. The above indexes of these three cases recovered well. Three patients did not have any persistent PPWD after repair with the ISMF and did not require any further surgical procedures related to the cervical spine.

Clinical significance of the secondary pedicle amputation of the repair of distal defects with pedicled axial flap.

We aimed to explore the clinical significance of the secondary pedicle amputation of the repair of distal defects with pedicled axial flap. Five patients who underwent pedicled axial flap transfer to repair a large area of skin and soft tissue defects in our hospital were included in this retrospective study. Detailed information including general data and clinical data, such as preoperative complication, type of primary wound, the distance between the primary wound and the donor site (cm), postoperative complications, and types of axial flap were collected. The patients had good joint movement at 6 months after pedicle amputation. At 48 hours after transplantation, except for the last patient (NO.5), there were no obvious complications such as blood supply disorder, infection, and incision dehiscence of the patients, and the flaps survived well. Just after pedicle amputation, 3 and 6 months after pedicle amputation, the flaps survived well with good local morphology. Forty-eight hours after operation, part of the distal flap in the last patient (NO.5) was necrotic. After 6 months of pedicle amputation, part of the flap was transferred to the distal wound again. At 6 months after pedicle amputation, these patients could accept local scars even though the scar of the last patient was obvious. The secondary pedicle amputation of the repair of distal defects with axial flap could avoid the compression of the vascular pedicle in the subcutaneous tunnel between the donor site and the primary wound, which may ensure the bold supply and increase the survival rate of the flap.

Chondrocyte Spheroids Laden in GelMA/HAMA Hybrid Hydrogel for Tissue-Engineered Cartilage with Enhanced Proliferation, Better Phenotype Maintenance, and Natural Morphological Structure.

Three-dimensional cell-laden tissue engineering has become an extensive research direction. This study aimed to evaluate whether chondrocyte spheroids (chondro-spheroids) prepared using the hanging-drop method could develop better cell proliferation and morphology maintenance characteristics, and be optimized as a micro unit for cartilage tissue engineering. Chondro-spheroids were loaded into a cross-linkable hybrid hydrogel of gelatin methacrylate (GelMA) and hyaluronic acid methacrylate (HAMA) in vivo and in vitro. Cell proliferation, aggregation, cell morphology maintenance as well as cartilage-related gene expression and matrix secretion in vitro and in vivo were evaluated. The results indicated that compared with chondrocyte-laden hydrogel, chondro-spheroid-laden hydrogel enhanced proliferation, had better phenotype maintenance, and a more natural morphological structure, which made it appropriate for use as a micro unit in cartilage tissue engineering.

Sutural fibroblasts exhibit the function of vascular endothelial cells upon mechanical strain.

Midfacial hypoplasia is a type of facial dysplasia. The technique of trans-sutural distraction osteogenesis promotes midface growth so as to ameliorate this symptom. In the process of distraction osteogenesis, the fiber matrix in the suture acts as a mechanical sensor. Compared with osteogenesis, the formation of collagen fibers by fibroblasts is significant in the early stage of sutural distraction. However the transformation of fibroblasts during sutural bone formation induced by tensile force is poorly characterized. Here, we used single-cell RNA sequencing to define the cell classification of the zygomatic maxillary suture and the changes of cell clusters in the suture before and after seven-day distraction. We identified twenty-nine cell subsets spanning monocyte/macrophages, neutrophils, red blood cells, B cells and fibroblasts. Compared with the control group, Monocle analysis revealed the emergence of a unique fibroblast subset (Cdh5+, Col4a1+, Fat1-, and Acta2-) (cluster 27) that expressed vascular endothelial cell genes within the distracted zygomatic maxillary suture. We constructed the differentiation trajectories of the fibroblast population (cluster 23, 27) in the suture before and after distraction. In addition, we clarified that a subset of fibroblasts (cluster 27) lost expression of Fat1, an upregulator of the Hippo pathway, and upregulated Cyr61, a downstream gene of the Hippo pathway, during the distraction process. Further enrichment analysis suggests that cells of the new subset (cluster 27) are undergoing conversion of their identity into a vascular endothelial cell-like state in response to mechanical stimulation, associated with upregulation of angiogenesis genes along the single-cell trajectory. Further immunofluorescence staining confirmed this phenomenon. A combined general transcriptome RNA sequencing data analysis demonstrated that the fibroblasts expressed a number of extracellular matrix-related genes under mechanical strain. These data together provide a new view of the role of fibroblasts in tension-induced sutural angiogenesis via interaction with the Hippo pathway.

Polarized M2 macrophages induced by mechanical stretching modulate bone regeneration of the craniofacial suture for midfacial hypoplasia treatment.

The underlying mechanism of the trans-sutural distraction osteogenesis (TSDO) technique as an effective treatment that improves the symptoms of midfacial hypoplasia syndromes is not clearly understood. Increasing findings in the orthopedics field indicate that macrophages are mechanically sensitive and their phenotypes can respond to mechanical cues. However, how macrophages respond to mechanical stretching and consequently influence osteoblast differentiation of suture-derived stem cells (SuSCs) remains unclear, particularly during the TSDO process. In the present study, we established a TSDO rat model to determine whether and how macrophages were polarized in response to stretching and consequently affected bone regeneration of the suture frontal edge. Notably, after performing immunofluorescence, RNA-sequencing, and micro-computed tomography, it was demonstrated that macrophages are first recruited by various chemokines factors and polarized to the M2 phenotype upon optimal stretching. The latter in turn regulates SuSC activity and facilitates bone regeneration in sutures. Moreover, when the activated M2 macrophages were suppressed by pharmacological manipulation, new bone microarchitecture could rarely be detected under mechanical stretching and the expansion of the sutures was clear. Additionally, macrophages achieved M2 polarization in response to the optimal mechanical stretching (10%, 0.5 Hz) and strongly facilitated SuSC osteogenic differentiation and human umbilical vein endothelial cell angiogenesis using an indirect co-culture system in vitro. Collectively, this study revealed the mechanical stimulation-immune response-bone regeneration axis and clarified at least in part how sutures achieve bone regeneration in response to mechanical force.

Extracellular vesicles derived from lipoaspirate fluid promote fat graft survival.

Extracellular vesicles (EVs) are specific subcellular vesicles released by cells under various environmental conditions. Tumescent liposuction is a commonly used procedure in plastic surgery practice. In the present study, we aimed to extract EVs derived from lipoaspirate fluid (LF-EVs) and characterize them using transmission electron microscopy, nanoparticle tracking analysis, and western blotting. The global profiles of proteins and microRNAs from LF-EVs were identified, strongly suggesting a potential regulatory function of LF-EVs. In addition, we investigated the effects and mechanisms of LF-EVs on fat graft survival. Cell functional tests showed that LF-EVs promoted the proliferation, migration, and tube structure formation of human umbilical vein endothelial cells. LF-EVs also promoted the adipogenic differentiation of adipose tissue-derived stem cells. The results of animal experiments showed that the average weights of fat grafts in the LF-EVs-treated group were significantly higher than those in the control group. Histologically, there was less fibrosis, fewer cysts, and increased fat tissue survival in the LF-EVs group. Further investigations of angiogenic and adipogenic factors revealed that LF-EVs also promoted angiogenesis and exerted a pro-adipogenic effect in vivo. Our findings will help to elucidate the functions of LF-EVs and provide a reference dataset for future translational studies.

Hydroxyapatite Nanoparticles Facilitate Osteoblast Differentiation and Bone Formation Within Sagittal Suture During Expansion in Rats.

BACKGROUND: The potential of relapse of craniofacial disharmony after trans-sutural distraction osteogenesis is high due to the failure to produce a stable bone bridge in the suture gap. The aim of this study is to evaluate whether hydroxyapatite nanoparticles (nHAP) have the effect of promoting osteoblast differentiation of suture-derived stem cells (SuSCs) and bone formation in sagittal suture during expansion. METHODS: SuSCs were isolated from sagittal sutures and exposed to various concentrations of nHAP (0, 25, 50, and 100 µg mL-1) to determine the optimal concentration of nHAP in osteoblast differentiation via performing Western Blotting and RT-qPCR. Twenty 4-week-old male Sprague-Dawley rats were randomly assigned into 4 groups: SHAM (sham-surgery), distraction, ACS (absorbable collagen sponge) and ACS+nHAP groups. In the ACS and ACS+nHAP groups, saline solution and nHAP suspended in a saline solution were delivered by ACS placed across the sagittal suture, respectively. In the latter three groups, the suture was expanded for 14 days by 50 g of constant force via a W shape expansion device. Suture gap area, bone volume fraction (BV/TV) and bone mineral density (BMD) of sagittal sutures were assessed via micro-CT, while the mechanical properties of sagittal sutures were evaluated via nanoindentation test. The efficacy of nHAP on bone formation in sagittal suture was also evaluated via BMP-2 immunohistochemistry staining. RESULTS: The expression of osteoblast related genes and proteins induced by 25µg mL-1 nHAP were significantly higher than the other groups in vitro (p<0.05). Furthermore, treating with 25µg mL-1 nHAP in vivo, the suture gap area was significantly reduced when compared with the distraction group. Correspondingly, the BV/TV, BMD, hardness and modulus of sagittal sutures were significantly increased in the ACS+nHAP group (p<0.05). CONCLUSION: The 25µg mL-1 dose of nHAP delivered by ACS can facilitate bone formation into the sagittal suture during expansion via inducing osteoblast differentiation of SuSCs.

The Z-plasty contributes to the coalescence of a chronic non-healing wound.

This study aimed to explore the treatment effect of Z-plasty on a non-healing wound. A total of 72 patients diagnosed with a chronic non-healing wound in Peking University Third Hospital from November 2009 to August 2019 were retrospectively analysed. Among them, 27 patients were treated with Z-plasty, and 45 patients were treated with the general method. Detailed patient information was retrieved from medical records, including age, gender, body mass index (BMI), alcohol, smoking, and comorbidities (diabetes mellitus, hypertension, heart disease). Surgical parameters included operation time and intraoperative blood loss. Wound swelling, epidermal blisters, wound edge colour, and skin temperature at 1 day after surgery were assessed to evaluate the blood supply of the wound. Surgical complications included infection, haematoma, dehiscence, and non-healing within 2 weeks postoperatively. Student t test (for continuous data) and Chi-square test (for categorical data) were conducted to determine the statistical difference. We found no significant differences in age, gender, BMI, alcohol, smoking, and comorbidities between the two groups. Z-plasty did not show any advantages in the surgical time, invasive blood loss, hospital days, and hospitalisation expenses. The incidence of abnormal wound edge colour with Z-plasty was significantly lower than that with the general treatment (P < .05), and the Z-plasty enables better healing of the patient's wound (P < .05). Z-plasty promoted better recovery of chronic non-healing wounds than direct suturing.

Differentiation potential and mRNA profiles of human dedifferentiated adipose cells and adipose­derived stem cells from young donors.

Dedifferentiated adipose cells (DAs) and adipose­derived stem cells (ADSCs) are two of the primary types of stem cells derived from adipose tissue, which have been reported to possess similar characteristics, but also exhibit unique phenotypic and functional advantages. However, several reports have described inconsistent results regarding their differences in multilineage differentiation function. Moreover, to the best of our knowledge, there are no studies assessing their myogenic ability, or the differences in the transcriptome between the two cell types derived from lipoaspirates via tumescent liposuction from the same donors. The aim of the present study was to compare the properties and expression profiles of these cell types. Subcutaneous adipose tissue of three female patients (aged 23­30 years) with a physiological BMI (19.1­23.9 kg/m2) were obtained during tumescent liposuction of the abdomen or the thigh. The stromal vascular fraction and mature adipocytes were obtained via collagenase digestion, and ADSCs and DAs were cultured successively. To determine the differences between DAs and ADSCs after 6­7 passages, cell proliferation assays, phenotypic assessment, differentiation assays and high­throughput RNA sequencing (seq) were used. Similar cell morphologies, proliferation dynamics, surface markers and transcriptome expression profiles were observed between the DAs and ADSCs. Whilst there were notable individual differences in the osteogenic, lipogenic, chondrogenic and myogenic abilities of the DAs and ADSCs, it was difficult to determine their differentiation potential based only on the cell source. Interestingly, the myogenic ability was relatively stronger in cells with relatively weaker lipogenic ability. Only 186 differentially expressed genes between the two groups were identified using RNAseq. Several of these genes were involved in biological functions such as transcription regulation, protein translation regulation, cytokine interactions and energy metabolism regulation. The results of the present study suggested a similar functional potential of DAs and ADSCs from young donors undergoing tumescent liposuction operation in regeneration areas and the balance of the differentiative ability of the same cell populations. These data may provide a foundation for further clinical administration of stem cells derived from adipose tissues in therapy.