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BACKGROUND: Inflammation affects the osteogenic differentiation of periodontal ligament stem cells, and the osteogenic ability and autophagy level of periodontal ligament stem cells are closely related. However, there are no relevant reports on whether inflammation affects the osteogenic ability and autophagy level of periodontal ligament stem cells at different stages of osteogenic differentiation. OBJECTIVE: To explore alkaline phosphatase expression and autophagy periodontal ligament stem cells levels in periodontitis and normal conditions. METHODS: Periodontal ligament stem cells from normal and periodontitis patients were isolated and cultured, and underwent Vimentin, pan-CK, and Stro1 fluorescence staining. At 3, 7, and 14 days of osteogenic differentiation, western blot assay was used to detect the protein expression levels of alkaline phosphatase, LC3B, Beclin1, and ATG5 in normal and inflammatory periodontal ligament stem cells. The mRNA expression levels of alkaline phosphatase, bone sialoprotein, osteocalcin, Runx2, LC3B, Beclin1, and ATG5 were detected by real-time PCR. RESULTS AND CONCLUSION: (1) Stro-1 was positive, Vimentin was positive, and pan CK was negative in periodontal ligament stem cells. (2) At 3, 7, and 14 days after osteogenic differentiation, compared with normal periodontal ligament stem cells, the mineralization nodules formed by periodontal ligament stem cells from inflammatory sources were significantly reduced (P < 0.01); the expression of alkaline phosphatase protein and mRNA was significantly lower (P < 0.05); the mRNA expression levels of bone sialoprotein, osteocalcin, and Runx2 were significantly decreased (P < 0.05). (3) At 7 and 14 days after osteogenic differentiation, compared with normal periodontal ligament stem cells, the expression levels of ATG5, LC3B, and Beclin1 proteins and mRNA of periodontal ligament stem cells were downregulated (P < 0.05). These findings suggest that inflammation reduces the activity of periodontal ligament stem cells in mineralizing nodule formation and the expression of alkaline phosphatase and weakens the autophagy potential of periodontal ligament stem cells at 7 and 14 days after osteogenic differentiation. [ABSTRACT FROM AUTHOR]

BACKGROUND: Psoralen has a strong anti-osteoporotic activity and may have a restorative effect on chemotherapy-induced osteoporosis. OBJECTIVE: To explore the restorative effect of psoralen on the osteogenic differentiation of bone marrow mesenchymal stem cells in mice inhibited by cyclophosphamide and its mechanism. METHODS: C57BL/6 mouse bone marrow mesenchymal stem cells were isolated and cultured. Effect of psoralen on viability of bone marrow mesenchymal stem cells was detected by MTT assay. Osteogenic induction combined with alkaline phosphatase staining was used to determine the optimal dose of psoralen to restore the osteogenic differentiation of bone marrow mesenchymal stem cells inhibited by cyclophosphamide. The mRNA expression levels of Runx2, alkaline phosphatase, Osteocalcin, osteoprotegerin, and Wnt/β-catenin signaling pathway-related genes Wnt1, Wnt4, Wnt10b, β-catenin, and c-MYC were measured by RT-qPCR at different time points under the intervention with psoralen. The protein expression of osteogenic specific transcription factor Runx2 and Wnt/β-catenin signaling pathway related genes Active β-catenin, DKK1, c-MYC, and Cyclin D1 was determined by western blot assay at different time points under the intervention with psoralen. RESULTS AND CONCLUSION: (1) There was no significant effect of different concentrations of psoralen on the viability of bone marrow mesenchymal stem cells. The best recovery of the inhibition of osteogenic differentiation of bone marrow mesenchymal stem cells caused by cyclophosphamide was under the intervention of psoralen at a concentration of 200 μmol/L. (2) Psoralen reversed the reduction in osteogenic differentiation marker genes Runx2, alkaline phosphatase, Osteocalcin and osteoprotegerin mRNA expression and Runx2 protein expression in bone marrow mesenchymal stem cells caused by cyclophosphamide conditioned medium. (3) Psoralen reversed the decrease in Wnt/β-catenin pathway-related genes Wnt4, β-catenin, c-MYC mRNA and Active β-catenin, c-MYC, and Cyclin D1 protein expression and the increase in DKK1 protein expression in bone marrow mesenchymal stem cells caused by cyclophosphamide conditioned medium. (4) The results showed that cyclophosphamide inhibited osteogenic differentiation of bone marrow mesenchymal stem cells in mice, and psoralen had a restorative effect on it. The best intervention effect was achieved at a concentration of 200 μmol/L psoralen, and this protective effect might be related to the activation of Wnt4/β-catenin signaling pathway by psoralen. [ABSTRACT FROM AUTHOR]

Background: Low-intensity pulsed ultrasound (LIPUS) has been used as an effective noninvasive method for treating fractures and osteoarthrosis, but the application in the field of oral implantation is in its infancy. This study aimed to clarify the effect and mechanism of LIPUS on the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) and implant osseointegration, and to provide an experimental basis for future clinical applications. Methods: Dental implants were inserted into Wistar rat femurs, and LIPUS was performed for 4 weeks. Micro-CT and toluidine blue staining were used to assess implant osseointegration. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were used to identify enriched functional terms and signalling pathways for differentially expressed genes from LIPUS-treated rat BMSC RNAseq data obtained from the GEO database. The random forest method was used to identify key risk genes according to the mean decrease Gini (MDG) coefficient. Then, LIPUS was applied to treat rat BMSCs, and alkaline phosphatase (ALP) staining, alizarin red staining, RT-PCR and western blotting were used to determine whether LIPUS could promote BMSC osteogenic differentiation via integrin α11 (ITGA11) and the focal adhesion pathway. Results: Our in vivo experimentations verified that LIPUS significantly increased new bone formation and osseointegration around the implant in rats. Bioinformatics analysis of RNA-seq data revealed that the upregulated genes in BMSCs after LIPUS treatment were significantly enriched in osteoblast differentiation-related functions and focal adhesion-related pathways. Random forest analysis revealed that ITGA11 was the most significant factor affecting BMSC osteogenic differentiation among the differentially expressed genes. In addition, LIPUS significantly increased ALP expression and mineralized nodule formation in rat BMSCs by upregulating ITGA11 and increasing the activity of FAK/PI3K/AKT/GSK3β/β-catenin pathway. Conclusions: LIPUS can effectively promote implant osseointegration in rats and improve rat BMSC osteogenic differentiation by upregulating ITGA11 and increasing the activity of the downstream focal adhesion pathway. [ABSTRACT FROM AUTHOR]

The multiscale hierarchical porous titanium scaffolds significantly improve biomechanical adaptation and biological activity by simulating bone structure, presenting attractive application prospects. To achieve the controllable preparation of multiscale hierarchical porous Ti-3Zr-2Sn-3Mo-25Nb (TLM) scaffolds, this study innovatively proposed the combination of additive manufacturing and electrochemical dealloying. The macro-scale pores of TLM were fabricated using selective laser melting (SLM), with a pore size of 640 μm and a porosity of 70.92 %. Then, a biphasic microstructure was obtained by heat treatment. Finally, the micro- and nano-scale structure was prepared via electrochemical dealloying. The elastic modulus of the multiscale hierarchical porous TLM scaffolds was 1.43 GPa, which closely matched the human bone tissue, indicating excellent biomechanical adaptation. Meanwhile, in vitro experiments further demonstrated that these scaffolds significantly enhanced BMSCs adhesion. The alkaline phosphatase (ALP) activity, calcium deposition, and collagen secretion were up-regulated, while lipid differentiation was down-regulated, indicating the scaffolds exhibited the potential for osseointegration. This research provides a valuable method for the preparation of multiscale hierarchical porous titanium scaffolds and offers innovative insights for the development of customized hard-tissue implants.

[Display omitted] • SMF facilitates osteogenic differentiation of hPDLSCs. • SMF contributes to H19 expression in hPDLSCs. • SMF enhances osteogenic differentiation of hPDLSCs by up-regulating H19. • The Wnt/β-catenin pathway is downstream of H19. Static magnetic field (SMF) as an effective physical stimulus is capable of osteogenic differentiation for multiple mesenchymal stem cells, including human periodontal ligament stem cells (hPDLSCs). However, the exact molecular mechanism is still unknown. Therefore, this study intends to excavate molecular mechanisms related to SMF in hPDLSCs using functional experiments. hPDLSCs were treated with different intensities of SMF, H19 lentivirus, and Wnt/β-catenin pathway inhibitor (XAV939). Changes in osteogenic markers (Runx2, Col Ⅰ, and BMP2), Wnt/β-catenin markers (β-catenin and GSK-3β), and calcified nodules were examined using RT-qPCR, western blotting, and alizarin red staining in hPDLSCs. SMF upregulated the expression of H19, and SMF and overexpressing H19 facilitated the expression of osteogenic markers (Runx2, Col Ⅰ, and BMP2), activation of the Wnt/β-catenin pathway, and mineralized sediment in hPDLSCs. Knockdown of H19 alleviated SMF function, and treatment with XAV939 limited SMF- and H19-mediated osteogenic differentiation of hPDLSCs. Notably, the expression of hsa-miR-532-3p, hsa-miR-370-3p, hsa-miR-18a-5p, and hsa-miR-483-3p in hPDLSCs was regulated by SMF, and may form an endogenous competitive RNA mechanism with H19 and β-catenin. SMF contributes to the osteogenic differentiation of hPDLSCs by mediating the H19/Wnt/β-catenin pathway, and hsa-miR-532-3p, hsa-miR-370-3p, hsa-miR-18a-5p, and hsa-miR-483-3p may be the key factors in it. [ABSTRACT FROM AUTHOR]

The study aims to elucidate the mechanism through which 17β-estradiol facilitates osteogenic differentiation in bone marrow mesenchymal stem cells (BMSCs). In our study, lentiviral transfection was employed to establish apoptosis repressor with caspase recruitment domain (ARC) knockdown or overexpression in BMSCs. The impact of 17β-estradiol on ARC expression was assessed using western blot, RT-PCR and immunofluorescence. Techniques such as ALP staining, ALP activity assay, western blot, RT-PCR and immunofluorescence staining were utilized to examine the influence of ARC expression levels on the osteogenic differentiation of BMSCs and the osteoclastic differentiation of Raw264.7 cell lines. Mitophagy flux levels in BMSCs were detected using the mitophagy detection kit. RNA sequencing and bioinformatics analyses were conducted to explore potential mechanisms of ARC regulation in BMSCs osteogenic differentiation. To sum up, 17β-estradiol can modulate bone homeostasis by adjusting ARC expression. ARC stimulates mitophagy in BMSCs via MAPK/Akt pathway, identifying ARC as a promising therapeutic target for postmenopausal osteoporosis (PMOP) treatment., Competing Interests: Declarations. Ethics approval and consent to participate: Not applicable. Consent for publication: Not applicable. Competing interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024. The Author(s).)

Bioprinting of nanohydroxyapatite (nHA)-based bioinks has attracted considerable interest in bone tissue engineering. However, the role and relevance of the physicochemical properties of nHA incorporated in a bioink, particularly in terms of its printability and the biological behavior of bioprinted cells, remain largely unexplored. In this study, two bioinspired nHAs with different chemical compositions, crystallinity, and morphologies were synthesized and characterized: a more crystalline, needle-like Mg 2+ -doped nHA (N-HA) and a more amorphous, rounded Mg 2+ - and CO 3 -doped nHA (R-HA). To investigate the effects of the different compositions and morphologies of these nanoparticles on the bioprinting of human bone marrow stromal cells (hBMSCs), gelatin and gelatin methacryloyl (GelMA) were selected as the bioink backbone. The addition of 1% (w/w) of these bioceramic nanoparticles significantly improved the printability of GelMA in terms of extrudability, buildability, and filament spreading. The biological potential of the bioinks was evaluated by examining the hBMSC viability, metabolic activity, and osteogenic differentiation over 21 days. Both nHAs showed high cell viability, with N-HA showing a significant increase in metabolic activity under nonosteogenic conditions and R-HA showing a notable increase with osteogenic stimulation. These results suggest that the two nHAs interact differently with their environment, highlighting the importance of both the chemistry and morphology in bioink performance. In addition, osteogenic differentiation further highlighted how the physicochemical properties of nHAs influence osteogenic markers at both the RNA and protein levels. Clearly, tailoring the physicochemical properties of hydroxyapatite nanoparticles is critical to developing more biomimetic bioinks with great potential for advancing bone bioprinting applications.2- -doped nHA (R-HA). To investigate the effects of the different compositions and morphologies of these nanoparticles on the bioprinting of human bone marrow stromal cells (hBMSCs), gelatin and gelatin methacryloyl (GelMA) were selected as the bioink backbone. The addition of 1% (w/w) of these bioceramic nanoparticles significantly improved the printability of GelMA in terms of extrudability, buildability, and filament spreading. The biological potential of the bioinks was evaluated by examining the hBMSC viability, metabolic activity, and osteogenic differentiation over 21 days. Both nHAs showed high cell viability, with N-HA showing a significant increase in metabolic activity under nonosteogenic conditions and R-HA showing a notable increase with osteogenic stimulation. These results suggest that the two nHAs interact differently with their environment, highlighting the importance of both the chemistry and morphology in bioink performance. In addition, osteogenic differentiation further highlighted how the physicochemical properties of nHAs influence osteogenic markers at both the RNA and protein levels. Clearly, tailoring the physicochemical properties of hydroxyapatite nanoparticles is critical to developing more biomimetic bioinks with great potential for advancing bone bioprinting applications.

Effective repair of large bone defects through bone tissue engineering (BTE) remains an unmet clinical challenge. Successful BTE requires optimal and synergistic interactions among biocompatible scaffolds, osteogenic factors, and osteoprogenitors to form a highly vascularized microenvironment for bone regeneration and osseointegration. We sought to develop a highly effective BTE system by using 3D printed citrate-based mPOC/hydroxyapatite (HA) composites laden with BMP9-stimulated human urine stem cells (USCs). Specifically, we synthesized and characterized methacrylate poly(1,8 octamethylene citrate) (mPOC), mixed it with 0%, 40% or 60% HA (i.e., mPOC-0HA, mPOC-40HA, or mPOC-60HA), and fabricated composite scaffold via micro-continuous liquid interface production (μCLIP). The 3D-printed mPOC-HA composite scaffolds were compatible with human USCs that exhibited high osteogenic activity in vitro upon BMP9 stimulation. Subcutaneous implantation of mPOC-HA scaffolds laden with BMP9-stimulated USCs revealed effective bone formation in all three types of mPOC-HA composite scaffolds. Histologic evaluation revealed that the mPOC-60HA composite scaffold yielded the most mature bone, resembling native bone tissue with extensive scaffold-osteointegration. Collectively, these findings demonstrate that the citrate-based mPOC-60HA composite, human urine stem cells, and the potent osteogenic factor BMP9 constitute a desirable triad for effective bone tissue engineering.

The selective androgen receptor (AR) modulator YK11 promotes the anabolism of muscle cells and osteoblastic precursor cells. However, its effects on bone marrow-derived mesenchymal stem cells (BMSCs) and the repair of cranial bone defects are unknown. Here, the effects of different concentrations of YK11 on the osteogenic differentiation of BMSCs were determined. Subsequently, the AR was inhibited to investigate whether the effect of YK11 on the osteogenic differentiation of BMSCs was affected. A model of cranial defects was constructed to investigate the effects of the YK11-equipped hydrogel on cranial defect repair as well as the effects of YK11 on cranial defect repair after inhibiting the AR. Finally, the possible pathway of YK11 regulating the osteogenic differentiation of BMSCs was explored. Our results show 2 μM YK11 promoted the proliferation of BMSCs. A dose of 0.25-4 μM YK11 could promote osteogenesis of BMSCs, and the promoting effect was gradually enhanced with increasing concentration. In vivo, 0.5 and 1 mg/mL YK11 could promote the repair of cranial bone defects. After inhibiting the AR, the effects of YK11 on promoting both the osteogenic differentiation of BMSCs and repair of cranial defects were suppressed. YK11 may regulate the osteogenic differentiation of BMSCs through the BMP2 (bone morphogenetic protein 2)/Smad signaling pathway. In conclusion, YK11 promoted the osteogenic differentiation of BMSCs by activation on the AR. Meanwhile, YK11 promoted the repair of cranial bone defects in rats in vivo. The BMP2/Smad signaling pathway may be involved in the regulation of the osteogenic differentiation of BMSCs by YK11.

This study investigates the mechanism of PHF20 in osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs). BMSCs from Balb/c mouse were cultured and identified through osteogenesis, adipogenesis, and flow cytometry. After osteogenic induction, the levels of OPN and OCN in BMSCs were detected by RT-qPCR. Alizarin red staining and alkaline phosphatase (ALP) staining were used to evaluate the osteogenic differentiation ability of BMSCs. PHF20, METTL14, and HOXA13 expressions were detected by RT-qPCR and Western blot. After quantitative analysis of m6A level, RNA immunoprecipitation (RIP) was performed to measure the enrichment of IGF2BP3 or m6A on HOXA13 mRNA. HOXA13 mRNA stability was assessed after actinomycin D treatment. PHF20, METT14, and HOXA13 expressions gradually increased during osteogenic differentiation of BMSCs. Suppression of PHF20 expression repressed the osteogenic differentiation of BMSCs, mainly resulted in a decrease in OPN and OCN levels, reduced mineralization, and weakened ALP activity. Mechanistically, PHF20 elevated METTL14 expression by enhancing the enrichment of H3K4me3 on its promoter, and METTL14 strengthened HOXA13 m6A methylation to maintain HOXA13 mRNA stability through IGF2BP3. In conclusion, PHF20 elevates METTL14 expression by enhancing H3K4me3 enrichment on its promoter and enhances HOXA13 mRNA stability via IGF2BP3-mediated m6A modification, thus facilitating HOXA13 expression and eventually inducing osteogenic differentiation of BMSCs., Competing Interests: Declarations. Competing interests: The authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Aims: Human periodontal ligament stem cells (hPDLSCs) exhibit an enormous potential to regenerate periodontal tissue. However, their translatability to the clinical setting is constrained by technical difficulties in standardizing culture conditions. The aim was to assess complex culture conditions using a proteomic-based protocol to standardize multi-layer hPDLSC cultivation methodology., Materials and Methods: hPDLSC-derived constructs were created with varying biological complexity. The simplest constructs were monolayer sheets of hPDLSCs cultured with fetal bovine serum (FBS) or Plasma Rich in Growth Factors supernatant (PRGFsn). The most complex constructs were triple-layered cell structures cultured with PRGFsn, with or without PRGF fibrin membrane (mPRGF). Ultrastructure and proteomic analyses were performed on these constructs., Results: PRGF supernatant improved protein expression related to extracellular matrix, adhesion, proliferation, and migration in hPDLSCs. PRGF fibrin scaffold upregulates proteins for cell activation, respiration, and electron transport. hPDLSCs on fibrin membrane show robust osteogenic potential through differential protein expression (ossification, tissue remodeling, morphogenesis, or cell migration) and overall homeostasis relative to less complex structures., Conclusion: Our data reveal the far-reaching potential of 3-dimensional constructs in combination with PRGF technology in periodontal regenerative applications.

Mechanical loading plays a pivotal role in regulating bone anabolic processes. Understanding the optimal mechanical loading parameters for cellular responses is critical for advancing strategies in orthopedic bioreactor-based bone tissue engineering. This study developed a poly (sorbitol sebacate) (PSS) filmscaffold with a sorbitol-to-sebacic acid molar ratio of 1:4. The scaffold underwent extensive characterization, including physical and mechanical property evaluations, biocompatibility assessments, and cell adhesion analysis. The Young's modulus of the PSS polymer was determined to be 6.81 ± 0.44 MPa under dry conditions, 6.37 ± 1.09 MPa in a wet state, and 6.38 ± 0.71 MPa after ethanol washing (70 %). The average contact angle of the PSS film was measured at 88.806 ± 1.644°, indicating moderate hydrophilicity. To investigate the osteogenic potential, a fluid flow inducing a shear stress of 1 Pa at a frequency of 1 Hz was applied to mesenchymal stem cells (MSCs) cultured on the PSS scaffold. Cells were exposed to dynamic fluid flow for one hour daily on days 4, 5, 6, and 7 of culture, followed by a static culture period of 14 days. The expression of osteogenic differentiation markers, including osteopontin (OPN), osteocalcin (OCN), type I collagen, and calcium deposition, was significantly elevated under dynamic conditions compared to static culture. This study highlights the importance of mechanical stimulation in enhancing MSC osteogenesis and underscores the osteoconductive properties of the PSS scaffold. These findings provide valuable insights into scaffold design and mechanical loading strategies for laboratory-based bone tissue engineering applications., (Copyright © 2025 Elsevier Ltd. All rights reserved.)

The resection of bone tumors results in large bone defects with some residual tumor cells, and the treatment of this type of bone defect area often faces a dilemma, namely, the trade-off between bone repair and antitumor after the resection of bone tumors. In order to promote local bone repair, and at the same time inhibit tumor recurrence by continuous and controlled drug administration, we developed a multifunctional NIR-responsive scaffold, whose main components are polylactic acid and MXene, and loaded with PLGA/DOX microspheres, and we hope that the scaffold can take into account both antitumor and bone repair in the bidirectional modulation effect of NIR. The results showed that the scaffold with 1% MXene content had relatively good performance in photothermal therapy (PT) and other aspects, and it could be smoothly increased to 50 °C within 2 min under NIR illumination, and the drug release of microspheres was increased by 10% after illumination compared with that at body temperature. In vivo experiments in animals showed that this scaffold effectively limited the in situ recurrence of tumor cells and lung metastasis and was able to promote osteogenic differentiation under NIR irradiation. Therefore, this scaffold can not only control the release of antitumor drugs but also enhance the antitumor effect through the bidirectional modulation effect of PT and at the same time promote bone formation, which provides a good application solution for the integrated treatment of the bone defect area after bone tumor surgery.

Spinal fusion is the ultimate choice for most patients with severe disc degeneration, and bone tissue engineering offers novel strategies to improve intervertebral bone growth and fusion. In this study, we utilized graphene oxide (GO) and methacrylated gelatin (GelMA) to prepare GelMA/GO composite hydrogel scaffolds with different GO concentrations. By characterizing the various properties of the scaffolds, it was learned that the composite scaffold containing 1.2 mg/mL GO possessed the best overall performance, and we used it for subsequent experiments. GelMA/GO composite scaffolds containing different bone-forming peptide-1 (BFP-1) concentrations were constructed and cocultured with bone marrow mesenchymal stem cells (BMSCs), and the results showed that GelMA/GO composite scaffolds containing 0.4 mg/mL BFP-1 induced the cells to produce more ALP and mineralized matrix. The above scaffold was further investigated as a GelMA/GO@BFP-1 composite, and the results showed that it promoted the production of ALP and mineralized matrix in BMSCs, and significantly enhanced the expression of osteogenesis-related genes (ALP, Runx-2, OCN, OPN) and proteins (Runx-2, OCN). It suggests that the GelMA/GO@BFP-1 complex promotes osteogenic differentiation of BMSCs and has the potential tobe used as a bone implant for improving intervertebral bone fusion., (© 2024 The Author(s). Journal of Biomedical Materials Research Part A published by Wiley Periodicals LLC.)

This study demonstrates that dissolution products of inorganic/organic SiO 2 -CaO CME /PTHF/PCL-diCOOH hybrid (70S30C CME -CL) drive human bone marrow stromal cells (h-BMSCs) down an osteogenic pathway with the production of mineralised matrix. We investigated osteogenesis through combined analyses of mRNA dynamics for key markers and targeted staining of mineralised matrix. We demonstrate that h-BMSCs undergo accelerated differentiation in vitro in response to the 70S30C CME -CL ionic milieu, as compared to incubation with osteogenic media. Extracts from 70S30C CME -CL promote osteogenesis by inducing changes in cellular metabolic activity, promoting changes in cell morphology consistent with the osteogenic lineage, and by enhancing mineralisation of hydroxyapatite in the extracellular matrix. Additionally, our results show that 70S30C CME -CL hybrids prove sustained functional resilience by maintaining osteostimulatory effects despite cumulated dissolution cycles. In co-differentiation medium, 70S30C CME -CL ionic release can modulate signalling pathways associated with non-osteogenic functions, further supporting their potential for bone regeneration applications. Overall, our study provides compelling experimental evidence that the 70S30C CME -CL hybrid is a promising biomaterial for bone tissue regeneration applications., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this study. All data presented in the manuscript was collected by the authors and the authors take complete responsibility for the integrity of the data and the accuracy of the data analyses., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)