GelMA loaded with exosomes from human minor salivary gland organoids enhances wound healing by inducing macrophage polarization.

Non-healing skin wounds pose significant clinical challenges, with biologic products like exosomes showing promise for wound healing. Saliva and saliva-derived exosomes, known to accelerate wound repair, yet their extraction is difficult due to the complex environment of oral cavity. In this study, as a viable alternative, we established human minor salivary gland organoids (hMSG-ORG) to produce exosomes (MsOrg-Exo). In vitro, MsOrg-Exo significantly enhanced cell proliferation, migration, and angiogenesis. When incorporated into a GelMA-based controlled-release system, MsOrg-Exo demonstrated controlled release, effectively improving wound closure, collagen synthesis, angiogenesis, and cellular proliferation in a murine skin wound model. Further molecular analyses revealed that MsOrg-Exo promotes proliferation, angiogenesis and the secretion of growth factors in wound sites. Proteomic profiling showed that MsOrg-Exo's protein composition is similar to human saliva and enriched in proteins essential for wound repair, immune modulation, and coagulation. Additionally, MsOrg-Exo was found to modulate macrophage polarization, inducing a shift towards M1 and M2 phenotypes in vitro within 48 h and predominantly towards the M2 phenotype in vivo after 15 days. In conclusion, our study successfully extracted MsOrg-Exo from hMSG-ORGs, confirmed the effectiveness of the controlled-release system combining MsOrg-Exo with GelMA in promoting skin wound healing, and explored the potential role of macrophages in this action.

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.

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.

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.

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.

Notch signaling inhibition induces G0/G1 arrest in murine Leydig cells.

As a highly evolutionarily conserved signaling pathway, Notch widely participates in cell-fate decisions and the development of various tissues and organs. In male reproduction, research on the Notch signaling pathway has mainly concentrated on germ cells and Sertoli cells. Leydig cells are the primary producers of testosterone and play important roles in spermatogenesis and maintaining secondary sexual characteristics. In this study, we used TM3 cells, a murine adult Leydig cell line, to investigate the expression profiles of Notch receptors and ligands and observe the effect of Notch signaling on the proliferation of TM3 cells. We found that Notch 1-3 and the ligands Dll-1 and Dll-4 were expressed in TM3 cells, Notch 1-3 and the ligand Dll-1 were expressed in testis interstitial Leydig cells, and Notch signaling inhibition suppressed the proliferation of TM3 cells and induced G0/G1 arrest. Inhibition of Notch signaling increased the expression of p21Waf1/Cip1 and p27. Overall, our results suggest that Notch inhibition suppresses the proliferation of TM3 cells and P21Waf1/Cip1 , and p27 may contribute to this process.

Inflammation and angiogenesis in the corpus luteum.

Angiogenesis is a very important process that helps establish and maintain the normal structure and function of the corpus luteum (CL). Early luteal development can be considered a kind of physiological injury with an inflammatory response; therefore, the inflammatory response may play an important role in the luteal angiogenesis. The inflammatory response is companied by activated leukocytes and their mediators. For luteal tissue, numerous activated leukocytes such as macrophages, neutrophils and eosinophils are present in the early luteal phase and are widely involved in neovascularization. The objective of this review is to describe the role of the inflammatory factors in the angiogenesis and to discuss their mechanism. Knowledge of action and mechanism of these inflammatory factors on angiogenic activity will be beneficial for the understanding of luteal function.

Prolyl oligopeptidase regulates progesterone secretion via the ERK signaling pathway in murine luteal cells.

Prolyl oligopeptidase (POP), one of the most widely distributed serine endopeptidases, is highly expressed in the ovaries. However, the physiological role of POP in the ovaries is not clear. In this study, we investigated the significance of POP in the corpus luteum. Murine luteal cells were cultured in vitro and treated with a POP selective inhibitor, (2S)-1[[(2 S)-1-(1-oxo-4-phenylbutyl)-2-pyrrolidinyl carbonyl]-2-pyrrolidinecarbonitrile (KYP-2047). We found that KYP-2047 treatment decreased progesterone secretion. In contrast, POP overexpression increased progesterone secretion. Three essential steroidogenic enzymes, including p450 cholesterol side-chain cleavage enzyme (CYP11A), 3ß-hydroxysteroid dehydrogenase (3ß-HSD), and the steroidogenic acute regulatory protein (StAR), were regulated by POP. Further studies showed that POP overexpression increased ERK1/2 phosphorylation and increased the expression of steroidogenic factor 1 (SF1), while KYP-2047 treatment decreased ERK1/2 phosphorylation and SF1 expression. To clarify the role of ERK1/2 signaling in POP-regulated progesterone synthesis, U0126-EtOH, an inhibitor of the ERK signaling pathway, was used to treat luteal cells. We found that U0126-EtOH decreased progesterone production and the expression of steroidogenic enzymes and SF1. POP overexpression did not reverse the effects of U0126-EtOH. Overall, POP regulates progesterone secretion by stimulating the expression of CYP11A, 3ß-HSD, and StAR in luteal cells. ERK signaling and downstream SF1 expression contribute to this process.

Notch signalling regulates steroidogenesis in mouse ovarian granulosa cells.

The Notch signalling pathway in the mammalian ovary regulates granulosa cell proliferation. However, the effects of Notch signalling on steroidogenesis are unclear. In this study we cultured mouse ovarian granulosa cells from preantral follicles invitro and observed the effect of Notch signalling on steroidogenesis through overexpression, knockdown and inhibition of Notch signalling. Activation of Notch signalling decreased progesterone and oestrogen secretion. In contrast, inhibition of Notch signalling increased the production of progesterone and oestrogen. Expression of the genes for steroidogenic-related enzymes, including 3ß-hydroxysteroid dehydrogenase, p450 cholesterol side-chain cleavage enzyme and aromatase, was repressed after stimulation of Notch signalling. The expression of upstream transcription factors, including steroidogenic factor 1 (SF1), Wilms' tumour 1 (Wt1), GATA-binding protein 4 (Gata4) and Gata6, was also inhibited after stimulation of Notch signalling. Production of interleukin (IL)-6 was positively correlated with Notch signalling and negatively correlated with the expression of these transcription factors and enzymes. In conclusion, Notch signalling regulated progesterone and oestrogen secretion by affecting the expression of upstream transcription factors SF1, Wt1, Gata4 and Gata6, as well as downstream steroidogenic-related enzymes. IL-6, which may be regulated directly by Notch signalling, may contribute to this process. Our findings add to the understanding of the diverse functions of Notch signalling in the mammalian ovary.