Establishment of a 3D esthetic analysis workflow on 3D virtual patient and preliminary evaluation.

BACKGROUND: In esthetic dentistry, a thorough esthetic analysis holds significant role in both diagnosing diseases and designing treatment plans. This study established a 3D esthetic analysis workflow based on 3D facial and dental models, and aimed to provide an imperative foundation for the artificial intelligent 3D analysis in future esthetic dentistry. METHODS: The established 3D esthetic analysis workflow includes the following steps: 1) key point detection, 2) coordinate system redetermination and 3) esthetic parameter calculation. The accuracy and reproducibility of this established workflow were evaluated by a self-controlled experiment (n = 15) in which 2D esthetic analysis and direct measurement were taken as control. Measurement differences between 3D and 2D analysis were evaluated with paired t-tests. RESULTS: 3D esthetic analysis demonstrated high consistency and reliability (0.973 < ICC < 1.000). Compared with 2D measurements, the results from 3D esthetic measurements were closer to direct measurements regarding tooth-related esthetic parameters (P<0.05). CONCLUSIONS: The 3D esthetic analysis workflow established for 3D virtual patients demonstrated a high level of consistency and reliability, better than 2D measurements in the precision of tooth-related parameter analysis. These findings indicate a highly promising outlook for achieving an objective, precise, and efficient esthetic analysis in the future, which is expected to result in a more streamlined and user-friendly digital design process. This study was registered with the Ethics Committee of Peking University School of Stomatology in September 2021 with the registration number PKUSSIRB-202168136.

CDC20 promotes bone formation via APC/C dependent ubiquitination and degradation of p65.

The E3 ubiquitin ligase complex CDC20-activated anaphase-promoting complex/Cyclosome (APC/CCDC20 ) plays a critical role in governing mitotic progression by targeting key cell cycle regulators for degradation. Cell division cycle protein 20 homolog (CDC20), the co-activator of APC/C, is required for full ubiquitin ligase activity. In addition to its well-known cell cycle-related functions, we demonstrate that CDC20 plays an essential role in osteogenic commitment of bone marrow mesenchymal stromal/stem cells (BMSCs). Cdc20 conditional knockout mice exhibit decreased bone formation and impaired bone regeneration after injury. Mechanistically, we discovered a functional interaction between the WD40 domain of CDC20 and the DNA-binding domain of p65. Moreover, CDC20 promotes the ubiquitination and degradation of p65 in an APC11-dependent manner. More importantly, knockdown of p65 rescues the bone loss in Cdc20 conditional knockout mice. Our current work reveals a cell cycle-independent function of CDC20, establishes APC11CDC20 as a pivotal regulator for bone formation by governing the ubiquitination and degradation of p65, and may pave the way for treatment of bone-related diseases.

DUSP5 promotes osteogenic differentiation through SCP1/2-dependent phosphorylation of SMAD1.

Dual-specificity phosphatases (DUSPs) are defined by their capability to dephosphorylate both phosphoserine/phosphothreonine (pSer/pThr) and phosphotyrosine (pTyr). DUSP5, a member of DUSPs superfamily, is located in the nucleus and plays crucially regulatory roles in the signaling pathway transduction. In our present study, we discover that DUSP5 significantly promotes osteogenic differentiation of mesenchymal stromal cells (MSCs) by activating SMAD1 signaling pathway. Mechanistically, DUSP5 physically interacts with the phosphatase domain of small C-terminal phosphatase 1/2 (SCP1/2, SMAD1 phosphatases) by the linker region. In addition, we further confirm that DUSP5 activates SMAD1 signaling through a SCP1/2-dependent manner. Specifically, DUSP5 attenuates the SCP1/2-SMAD1 interaction by competitively binding to SCP1/2, which is responsible for the SMAD1 dephosphorylation, and thus results in the activation of SMAD1 signaling. Importantly, DUSP5 expression in mouse bone marrow MSCs is significantly reduced in ovariectomized (OVX) mice in which osteogenesis is highly passive, and overexpression of Dusp5 via tail vein injection reverses the bone loss of OVX mice efficiently. Collectively, this work demonstrates that the linker region of DUSP5 maybe a novel chemically modifiable target for controlling MSCs fate choices and for osteoporosis treatment.

Interdisciplinary 3D digital treatment simulation before complex esthetic rehabilitation of orthodontic, orthognathic and prosthetic treatment: workflow establishment and primary evaluation.

BACKGROUND: An interdisciplinary treatment simulation and smile design before a complex esthetic rehabilitation is important for clinicians' decision-making and patient motivation. Meanwhile, intervention and interaction are necessary for dental specialists in these complex rehabilitations. However, it is difficult to visualize an interdisciplinary treatment plan by using the conventional method, especially when orthognathic surgery is involved, thus hindering communication between dental specialists. This research aims to establish a 3D digital workflow of interdisciplinary treatment simulation to solve this problem. METHODS: An interdisciplinary 3D digital workflow of simulated treatment plan for complex esthetic rehabilitation was established. Eleven patients were enrolled and illustrated with their treatment plans using 3D treatment simulation, as well as 2D digital smile design (DSD) plus wax-up. Visual analogue scales (VAS) were used to rate the intuitiveness, understanding, and satisfaction or help between the two methods by patients and dental specialists. RESULTS: According to the ratings from the patients, 3D treatment simulation showed obvious advantages in the aspects of intuitiveness (9.7 ± 0.5 vs 6.4 ± 1.4) and treatment understanding (9.1 ± 0.8 vs 6.6 ± 1.5), and the satisfaction rates were also higher (9.0 ± 0.6 vs 7.1 ± 1.8). Dental specialists regarded the 3D digital plans as more intuitive (8.9 ± 0.8 vs 5.9 ± 1.0) and useful to understand the plans from the other specialists (8.9 ± 0.7 vs 6.1 ± 1.0) and helpful to their own treatment plans (8.7 ± 0.9 vs 5.9 ± 1.4). CONCLUSIONS: The interdisciplinary 3D digital treatment simulation helps both patients and dental specialists to improve treatment understanding, and facilitates dental specialists for decision-making before complex esthetic rehabilitation. TRIAL REGISTRATION: This study was registered in the National Clinical Trials Registry under the identification number MR-11-20-002862. This is an observational study in which we did not assign the intervention.

The Current Situation and Future Prospects of Simulators in Dental Education.

The application of virtual reality has become increasingly extensive as this technology has developed. In dental education, virtual reality is mainly used to assist or replace traditional methods of teaching clinical skills in preclinical training for several subjects, such as endodontics, prosthodontics, periodontics, implantology, and dental surgery. The application of dental simulators in teaching can make up for the deficiency of traditional teaching methods and reduce the teaching burden, improving convenience for both teachers and students. However, because of the technology limitations of virtual reality and force feedback, dental simulators still have many hardware and software disadvantages that have prevented them from being an alternative to traditional dental simulators as a primary skill training method. In the future, when combined with big data, cloud computing, 5G, and deep learning technology, dental simulators will be able to give students individualized learning assistance, and their functions will be more diverse and suitable for preclinical training. The purpose of this review is to provide an overview of current dental simulators on related technologies, advantages and disadvantages, methods of evaluating effectiveness, and future directions for development.

MiR-137 knockdown promotes the osteogenic differentiation of human adipose-derived stem cells via the LSD1/BMP2/SMAD4 signaling network.

MicroRNAs are a group of endogenous regulators that participate in several cellular physiological processes. However, the role of miR-137 in the osteogenic differentiation of human adipose-derived stem cells (hASCs) has not been reported. This study verified a general downward trend in miR-137 expression during the osteogenic differentiation of hASCs. MiR-137 knockdown promoted the osteogenesis of hASCs in vitro and in vivo. Mechanistically, inhibition of miR-137 activated the bone morphogenetic protein 2 (BMP2)-mothers against the decapentaplegic homolog 4 (SMAD4) pathway, whereas repressed lysine-specific histone demethylase 1 (LSD1), which was confirmed as a negative regulator of osteogenesis in our previous studies. Furthermore, LSD1 knockdown enhanced the expression of BMP2 and SMAD4, suggesting the coordination of LSD1 in the osteogenic regulation of miR-137. This study indicated that miR-137 negatively regulated the osteogenic differentiation of hASCs via the LSD1/BMP2/SMAD4 signaling network, revealing a new potential therapeutic target of hASC-based bone tissue engineering.

Mitochondrial Phosphoenolpyruvate Carboxykinase Regulates Osteogenic Differentiation by Modulating AMPK/ULK1-Dependent Autophagy.

Mitochondrial phosphoenolpyruvate carboxykinase (PCK2) is a rate-limiting enzyme that plays critical roles in multiple physiological processes. The decompensation of PCK2 leads to various energy metabolic disorders. However, little is known regarding the effects of PCK2 on osteogenesis by human mesenchymal stem cells (hMSCs). Here, we report a novel function of PCK2 as a positive regulator of MSCs osteogenic differentiation. In addition to its well-known role in anabolism, we demonstrate that PCK2 regulates autophagy. PCK2 deficiency significantly suppressed autophagy, leading to the impairment of osteogenic capacity of MSCs. On the other hand, autophagy was promoted by PCK2 overexpression; this was accompanied by increased osteogenic differentiation of MSCs. Moreover, PCK2 regulated osteogenic differentiation of MSCs via AMP-activated protein kinase (AMPK)/unc-51 like autophagy activating kinase 1(ULK1)-dependent autophagy. Collectively, our present study unveiled a novel role for PCK2 in integrating autophagy and bone formation, providing a potential target for stem cell-based bone tissue engineering that may lead to improved therapies for metabolic bone diseases. Stem Cells 2019;37:1542-1555.

Extracellular vesicles as a novel therapeutic tool for cell-free regenerative medicine in oral rehabilitation.

Oral maxillofacial defects may always lead to complicated hard and soft tissue loss, including bone, nerve, blood vessels, teeth and skin, which are difficult to restore and severely influence the life quality of patients. Extracellular vesicles (EVs), including exosomes, microvesicles and apoptotic bodies, are emerging as potential solutions for complex tissue regeneration through cell-free therapies. In this review, we highlight the functional roles of EVs in the regenerative medicine for oral maxillofacial rehabilitation, specifically bone, skin, blood vessels, peripheral nerve and tooth-related tissue regeneration. Publications were reviewed by two researchers independently basing on three databases (PubMed, MEDLINE and Web of Science), until 31 December 2018. Basing on current researches, we classified the origin of EVs for regenerative medicine into four categories: related cells in the regenerative niche, mesenchymal stem cells, immune cells and body fluids. The secretome of different cells are distinct, while the same cells secrete different EVs under varied conditions; therefore, the content profiles of EVs and regulatory mechanisms on target cells are compared and emphasised. By unravelling the regulatory mechanisms of EVs in tissue regeneration, modified cells and tailored EVs with specific target may be produced for precision medicine with high efficacy.

UNC-5 netrin receptor B regulates adipogenesis of human adipose-derived stem cells through JNK pathway.

BACKGROUND: There is a balance between adipogenic differentiation and osteogenic differentiation of human adipose-derived stem cells (hASCs). It is essential to explore the mechanism of hASCs lineage commitment. In our previous study, UNC-5 netrin receptor B (UNC5B) was identified as a positive regulator for osteogenesis. OBJECTIVE: To further explore the potential roles and mechanisms of UNC5B during adipogenic differentiation and to provide a new method to regulate adipogenesis and osteogenesis of hASCs. METHODS: Lentivirus containing UNC5B shRNA was used for UNC5B knockdown. Plasmids overexpressing UNC5B gene were used for UNC5B upregulation. To investigate the role of UNC5B in adipogenesis in vitro and in vivo, Oil Red O staining, RT-qPCR and transplantation into nude mice were performed. Western blotting analyses were performed to explore the mechanisms of UNC5B in adipogenic differentiation. RESULTS: UNC5B expression in hASCs was significantly increased during adipogenic differentiation. Knockdown of UNC5B enhanced adipogenic differentiation in vitro. Both H&E staining and Oil Red O staining showed more adipose tissue-like constructs in UNC5B-knockdown cells in vivo. Upregulation of UNC5B significantly impaired adipogenic differentiation in vitro. Downregulation of UNC5B could increase phosphorylation of JNK in hASCs. JNK inhibitors reduced adipogenic differentiation of hASCs. CONCLUSION: Our findings showed that UNC5B inhibited adipogenesis of hASCs through JNK signalling. As a whole, UNC5B regulates both adipogenesis and osteogenesis of hASCs.

UNC-5 netrin receptor B mediates osteogenic differentiation by modulating bone morphogenetic protein signaling in human adipose-derived stem cells.

UNC-5 netrin receptor B (UNC5B) is a dependence receptor of netrin-1 that plays an essential role in mediating angiogenesis and tumorigenesis. Despite its significant roles, there is limited knowledge about the role played by UNC5B in osteogenesis. In the present study, we first demonstrated that UNC5B was required for osteogenic differentiation of human adipose-derived stem cells (hASCs), both in vitro and in vivo. We also found that mechanistically, UNC5B promotes osteogenic differentiation by activating bone morphogenetic protein signaling. These findings point to a new important function of UNC5B and provide a potential basis for hASCs-mediated bone regeneration.

Histone H3K9 Acetyltransferase PCAF Is Essential for Osteogenic Differentiation Through Bone Morphogenetic Protein Signaling and May Be Involved in Osteoporosis.

Human mesenchymal stem cells (MSCs) are multipotent progenitor cells that can differentiate into osteoblasts, chondrocytes, and adipocytes. The importance of epigenetic regulation for osteogenic differentiation of MSCs is widely accepted. However, the molecular mechanisms are poorly understood. Here, we show that histone H3K9 acetyltransferase PCAF plays a critical role in osteogenic differentiation of MSCs. Knockdown of PCAF significantly reduced the bone formation both in vitro and in vivo. Mechanistically, PCAF controls BMP signaling genes expression by increasing H3K9 acetylation. Most importantly, PCAF expression is significantly decreased in bone sections of ovariectomized or aged mice. Histone modification enzyme is chemically modifiable; therefore, PCAF may represent a novel therapeutic target for stem cell-mediated regenerative medicine and the treatment of osteoporosis. Stem Cells 2016;34:2332-2341.

[Promoted role of bone morphogenetic protein 2/7 heterodimer in the osteogenic differentiation of human adipose-derived stem cells].

OBJECTIVE: To investigate the role of bone morphogenetic protein 2/7 heterodimer (BMP-2/7) in the osteogenesis of human adipose-derived stem cells (hASCs). METHODS: hASCs were exposed to three different treatments in vitro: osteogenic medium with 150 µg/L BMP-2/7 (experimental group), osteogenic medium alone (OM group) and proliferation medium (PM group). After 1, 4 and 7 days of osteogenic induction, the amount of cellular DNA was measured to investigate the cytotoxicity. After 7 and 14 days, alkaline phosphatase (ALP) staining and quantification were performed to test the activity of ALP. After 21 and 28 days, the calcification deposition was determined by Alizarin Red S (ARS) staining and quantification. The expressions of the osteoblast-related genes were tested on days 1, 4, 7 and 14. In the in vivo study, 6 nude mice were used and 4 groups were set and implanted subcutaneously into the back of nude mice: (1) ß-TCP scaffold only (scaffold control group); (2) ß-TCP scaffold with hASCs cultured by PM in vitro for 1 week (PM control group); (3) ß-TCP scaffold with hASCs cultured by OM in vitro for 1 week (OM control group); (4) ß-TCP scaffold with hASCs cultured by OM with 150 µg/L BMP-2/7 in vitro for 1 week (test group). After 4 weeks of implantation, histological staining was performed to evaluate the in vivo osteogenesis of hASCs. RESULTS: After induction for 1 day, there was no significant difference between the experimental group and the PM group on the cellular DNA content (P>0.05). After 4 days, the cellular DNA content increased under the stimulation of BMP-2/7 (P<0.05). On day 7, there was no significant difference among the three groups (P>0.05). ALP activity was higher by the induction of BMP-2/7 than in OM alone and PM (P<0.05). More mineralization deposition and more expressions of osteoblast-related genes such as Runx2, ALP, COL-1A1 and OC were determined in the experimental group at different time points (P<0.05). HE staining showed that, in the test group and OM control group, the extracellular matrix (ECM) with eosinophilic staining were observed around hASCs, and newly-formed bone-like tissues could be found in ECM around the scaffold materials. Moreover, compared with the OM control group, more bone-like tissues could be observed in ECM with typical structure of bone tissue in the test group. Masson's trichrome staining showed that more expression of collagen could be observed in ECM in the test group compared with the other groups. There was small amount of expression of collagen in the OM and PM control groups. No obvious positive results were found in the scaffold group. CONCLUSION: BMP-2/7 heterodimer plays a significant role in the osteogenesis of hASCs and is able to enhance the osteogenic differentiation of hASCs in vitro and in vivo.

[Application of patient-participated digital design in esthetic rehabilitation of anterior teeth].

OBJECTIVE: To explore a new method of patient-involved digital design, esthetic outcome prediction and fabrication for the esthetic rehabilitation of anterior teeth, and to provide an alternative choice for the restoration of anterior teeth. METHODS: In this study, 32 patients with esthetic problems in their anterior teeth were included and divided into two groups randomly: the experimental group (16 patients) and control group (16 patients). In the experimental group, the dentition and facial images were obtained by intra-oral scanning and Three-dimensional (3D) facial scanning and then calibrated. The design of the rehabilitation and the esthetic outcome prediction were created by computer-aided design (CAD) software. After morphologic modification according to the patients' opinions, prostheses were fabricated according to the final design by computer-aided manufacturing (CAM) equipment. As for the control group, the regular design method was applied to restore their anterior teeth. The time consuming in the first insertion of each restoration in both groups was recorded. The quality of the prostheses was assessed by another prosthedontist. The satisfaction to prostheses and the facial appearance were evaluated by the patients. RESULTS: The process of the patient-involved digital design and outcome anticipation was successfully established. The patients were satisfied with the esthetic effects of the anterior restoration made by the digital technique. The acceptance rate of the patients on the digital rehabilitation in the experimental group was 100%. There was no significant difference of the quality of the prostheses between the two groups. The satisfaction rate of the patients on prostheses and facial appearance was significantly higher in the experimental group than in the control group (P < 0.05). In addition, the time consuming in the first insertion of the experimental group was much shorter than that in the control group (P < 0.01). CONCLUSION: The new method of the patient-involved digital design, esthetic outcome prediction and fabrication for the esthetic rehabilitation of anterior teeth is a practical technique. This method is useful in shortening the time consuming of the restoration of anterior teeth and improving the patient satisfaction with the esthetic outcome.

Apoptotic vesicles derived from human red blood cells promote bone regeneration via carbonic anhydrase 1.

Apoptotic vesicles (apoVs) are nanoscale vesicles derived from billions of apoptotic cells involved in the maintenance of the human body's homeostasis. Previous researches have shown that some apoVs, such as those derived from mesenchymal stem cells, contribute to bone formation. However, those apoVs cannot be extracted from patients in large quantities, and cell expansion is needed before apoV isolation, which limits their clinical translation. Mature RBCs, which have no nuclei or genetic material, are easy to obtain, showing high biological safety as a source of extracellular vesicles (EVs). Previous studies have demonstrated that RBC-derived EVs have multiple biological functions, but it is unknown whether RBCs produce apoVs and what effect these apoVs have on bone regeneration. In this study, we isolated and characterized RBC-derived apoVs (RBC-apoVs) from human venous blood and investigated their role in the osteogenesis of human bone mesenchymal stem cells (hBMSCs). We showed that RBCs could produce RBC-apoVs that expressed both general apoVs markers and RBC markers. RBC-apoVs significantly promoted osteogenesis of hBMSCs and enhanced bone regeneration in rat calvarial defects. Mechanistically, RBC-apoVs regulated osteogenesis by transferring carbonic anhydrase 1 (CA1) into hBMSCs and activating the P38 MAPK pathway. Our results indicated that RBC-apoVs could deliver functional molecules from RBCs to hBMSCs and promote bone regeneration, pointing to possible therapeutic use in bone tissue engineering.

A balance of biocompatibility and antibacterial capability of 3D printed PEEK implants with natural totarol coating.

OBJECTIVE: Polyetheretherketone (PEEK), a biomaterial with appropriate bone-like mechanical properties and excellent biocompatibility, is widely applied in cranio-maxillofacial and dental applications. However, the lack of antibacterial effect is an essential drawback of PEEK material and might lead to infection and osseointegration issues. This study aims to apply a natural antibacterial agent, totarol coating onto the 3D printed PEEK surface and find an optimized concentration with balanced cytocompatibility, osteogenesis, and antibacterial capability. METHODS: In this study, a natural antibacterial agent, totarol, was applied as a coating to fused filament fabrication (FFF) 3D printed PEEK surfaces at a series of increasing concentrations (1 mg/ml, 5 mg/ml, 10 mg/ml, 15 mg/ml, and 20 mg/ml). The samples were then evaluated for cytocompatibility with L929 fibroblast and SAOS-2 osteoblast using live/dead staining and CCK-8 assay. The antibacterial capability was assessed by crystal violet staining, live/dead staining, and scanning electron microscopy (SEM) utilizing the oral primary colonizer S. gordonii and isolates of mixed oral bacteria in a stirring system simulating the oral environment. The appropriate safe working concentration for totarol coating is selected based on the results of the cytocompatibility and antibacterial test. Subsequently, the influence on osteogenic differentiation was evaluated by alkaline phosphatase (ALP) and alizarin red staining (ARS) analysis of pre-osteoblasts. RESULTS: Our results showed that the optimal concentration of totarol solution for promising antibacterial coating was approximately 10 mg/ml. Such surfaces could play an excellent antibacterial role by inducing a contact-killing effect with an inhibitory effect against biofilm development without affecting the healing of soft and hard tissues around FFF 3D printed PEEK implants or abutments. SIGNIFICANCE: This study indicates that the totarol coated PEEK has an improved antibacterial effect with excellent biocompatibility providing great clinical potential as an orthopedic/dental implant/abutment material.

UBE2C orchestrates bone formation through stabilization of SMAD1/5.

While previous studies have demonstrated the role of ubiquitin-conjugating enzyme 2C (UBE2C) in promoting ß-cell proliferation and cancer cell lineage expansion, its specific function and mechanism in bone marrow mesenchymal stem/stromal cells (BMSCs) growth and differentiation remain poorly understood. Our findings indicate that mice with conditional Ube2c deletions in BMSCs and osteoblasts exhibit reduced skeletal bone mass and impaired bone repair. A significant reduction in the proliferative capacity of BMSCs was observed in conditional Ube2c knockout mice, with no effect on apoptosis. Additionally, conditional Ube2c knockout mice exhibited enhanced osteoclastic activity and reduced osteogenic differentiation. Furthermore, human BMSCs with stable UBE2C knockdown exhibited diminished capacity for osteogenic differentiation. Mechanistically, we discovered that UBE2C binds to and stabilizes SMAD1/5 protein expression levels. Interestingly, UBE2C's role in regulating osteogenic differentiation and SMAD1/5 expression levels appears to be independent of its enzymatic activity. Notably, UBE2C regulates osteogenic differentiation through SMAD1/5 signaling. In conclusion, our findings underscore the pivotal role of UBE2C in bone formation, emphasizing its contribution to enhanced osteogenic differentiation through the stabilization of SMAD1/5. These results propose UBE2C as a promising target for BMSC-based bone regeneration.

Mgp High-Expressing MSCs Orchestrate the Osteoimmune Microenvironment of Collagen/Nanohydroxyapatite-Mediated Bone Regeneration.

Activating autologous stem cells after the implantation of biomaterials is an important process to initiate bone regeneration. Although several studies have demonstrated the mechanism of biomaterial-mediated bone regeneration, a comprehensive single-cell level transcriptomic map revealing the influence of biomaterials on regulating the temporal and spatial expression patterns of mesenchymal stem cells (MSCs) is still lacking. Herein, the osteoimmune microenvironment is depicted around the classical collagen/nanohydroxyapatite-based bone repair materials via combining analysis of single-cell RNA sequencing and spatial transcriptomics. A group of functional MSCs with high expression of matrix Gla protein (Mgp) is identified, which may serve as a pioneer subpopulation involved in bone repair. Remarkably, these Mgp high-expressing MSCs (MgphiMSCs) exhibit efficient osteogenic differentiation potential and orchestrate the osteoimmune microenvironment around implanted biomaterials, rewiring the polarization and osteoclastic differentiation of macrophages through the Mdk/Lrp1 ligand-receptor pair. The inhibition of Mdk/Lrp1 activates the pro-inflammatory programs of macrophages and osteoclastogenesis. Meanwhile, multiple immune-cell subsets also exhibit close crosstalk between MgphiMSCs via the secreted phosphoprotein 1 (SPP1) signaling pathway. These cellular profiles and interactions characterized in this study can broaden the understanding of the functional MSC subpopulations at the early stage of biomaterial-mediated bone regeneration and provide the basis for materials-designed strategies that target osteoimmune modulation.

BHLHE40 Maintains the Stemness of PαS Cells In Vitro by Targeting Zbp1 through the Wnt/ß-Catenin Signaling Pathway.

Primary bone mesenchymal stem cells (BMSCs) gradually lose stemness during in vitro expansion, which significantly affects the cell therapeutic effects. Here, we chose murine PαS (SCA-1+PDGFRα+CD45-TER119-) cells as representative of BMSCs and aimed to explore the premium culture conditions for PαS cells. Freshly isolated (fresh) PαS cells were obtained from the limbs of C57/6N mice by fluorescence-activated cell sorting (FACS). We investigated the differences in the stemness of PαS cells by proliferation, differentiation, and stemness markers in vitro and by ectopic osteogenesis and chondrogenesis ability in vivo, as well as the changes in the stemness of PαS cells during expansion in vitro. Gain- and loss-of-function experiments were applied to investigate the critical role and underlying mechanism of the basic helix-loop-helix family member E40 (BHLHE40) in maintaining the stemness of PαS cells. The stemness of fresh PαS cells representative in vivo was superior to that of passage 0 (P0) PαS cells in vitro. The stemness of PαS cells in vitro decreased gradually from P0 to passage 4 (P4). Moreover, BHLHE40 plays a critical role in regulating the stemness of PαS cells during in vitro expansion. Mechanically, BHLHE40 regulates the stemness of PαS cells by targeting Zbp1 through the Wnt/ß-catenin signaling pathway. This work confirms that BHLHE40 is a critical factor for regulating the stemness of PαS cells during expansion in vitro and may provide significant indications in the exploration of premium culture conditions for PαS cells.

Characterization of mesenchymal stem cells in human fetal bone marrow by single-cell transcriptomic and functional analysis.

Bone marrow mesenchymal stromal/stem cells (MSCs) are a heterogeneous population that can self-renew and generate stroma, cartilage, fat, and bone. Although a significant progress has been made toward recognizing about the phenotypic characteristics of MSCs, the true identity and properties of MSCs in bone marrow remain unclear. Here, we report the expression landscape of human fetal BM nucleated cells (BMNCs) based on the single-cell transcriptomic analysis. Unexpectedly, while the common cell surface markers such as CD146, CD271, and PDGFRa used for isolating MSCs were not detected, LIFR+PDGFRB+ were identified to be specific markers of MSCs as the early progenitors. In vivo transplantation demonstrated that LIFR+PDGFRB+CD45-CD31-CD235a- MSCs could form bone tissues and reconstitute the hematopoietic microenvironment (HME) effectively in vivo. Interestingly, we also identified a subpopulation of bone unipotent progenitor expressing TM4SF1+CD44+CD73+CD45-CD31-CD235a-, which had osteogenic potentials, but could not reconstitute HME. MSCs expressed a set of different transcription factors at the different stages of human fetal bone marrow, indicating that the stemness properties of MSCs might change during development. Moreover, transcriptional characteristics of cultured MSCs were significantly changed compared with freshly isolated primary MSCs. Our cellular profiling provides a general landscape of heterogeneity, development, hierarchy, microenvironment of the human fetal BM-derived stem cells at single-cell resolution.

Alkaline shear-thinning micro-nanocomposite hydrogels initiate endogenous TGFß signaling for in situ bone regeneration.

Recruiting endogenous stem cells to bone defects without stem cell transplantation and exogenous factor delivery represents a promising strategy for bone regeneration. Herein, we develop an alkaline shear-thinning micro-nanocomposite hydrogel (10-MmN), aiming to alkaline-activate endogenous TGFß1 and achieve in situ bone regeneration. It contains polyethyleneimine (PEI)-modified gelatin, laponite nanoplatelets (LAP), a bicarbonate buffer with a pH of 10, and gelatin microspheres (MSs). PEI-modified gelatin plays a pivotal role in hydrogel fabrication. It endows the system with sufficient positive charges, and forms a shear-thinning nanocomposite matrix in the pH 10 buffer (10-mN) with negatively charged LAP via electrostatic gelation. For biological functions, the pH 10 buffer dominates alkaline activation of endogenous serum TGFß1 to recruit rat bone marrow stem cells through the Smad pathway, followed by improved osteogenic differentiation. In addition, MSs are incorporated into 10-mN to form 10-MmN, and function as substrates to provide good attachment sites for the recruited stem cells and facilitate further their osteogenic differentiation. In a rat critical-sized calvarial defect model, 10-MmN exhibits excellent biocompatibility, biodegradability, hydrogel infusion and retention in bone defects with flexible shapes and active bleeding. Importantly, it repairs ~95% of the defect areas in 3 months by recruiting TGFßR2+ and CD90+CD146+ stem cells, and promoting cell proliferation, osteogenic differentiation and bone formation. The present study provides a biomaterial-based strategy to regulate alkalinity in bone defects for the initiation of endogenous TGFß signaling, which can be extended to treat other diseases.