Study on 3D printed MXene-berberine-integrated scaffold for photo-activated antibacterial activity and bone regeneration.

The repair of mandibular defects is a challenging clinical problem, and associated infections often hinder the treatment, leading to failure in bone regeneration. Herein, a multifunctional platform is designed against the shortages of existing therapies for infected bone deficiency. 2D Ti3C2 MXene and berberine (BBR) are effectively loaded into 3D printing biphasic calcium phosphate (BCP) scaffolds. The prepared composite scaffolds take the feature of the excellent photothermal capacity of Ti3C2 as an antibacterial, mediating NIR-responsive BBR release under laser stimuli. Meanwhile, the sustained release of BBR enhances its antibacterial effect and further accelerates the bone healing process. Importantly, the integration of Ti3C2 improves the mechanical properties of the 3D scaffolds, which are beneficial for new bone formation. Their remarkable biomedical performances in vitro and in vivo present the outstanding antibacterial and osteogenic properties of the Ti3C2-BBR functionalized BCP scaffolds. The synergistic therapy makes it highly promising for repairing infected bone defects and provides insights into a wide range of applications of 2D nanosheets in biomedicine.

Recent advances in 2D material-based phototherapy.

Phototherapy, which generally refers to photothermal therapy (PTT) and photodynamic therapy (PDT), has received significant attention over the past few years since it is non-invasive, has effective selectivity, and has few side effects. As a result, it has become a promising alternative to traditional clinical treatments. At present, two-dimensional materials (2D materials) have proven to be at the forefront of the development of advanced nanomaterials due to their ultrathin structures and fascinating optical properties. As a result, much work has been put into developing phototherapy platforms based on 2D materials. This review summarizes the current developments in 2D materials beyond graphene for phototherapy, focusing on the novel approaches of PTT and PDT. New methods are being developed to go above and beyond conventional treatment to fully use the potential of 2D materials. Additionally, the efficacy of cutting-edge phototherapy is assessed, and the existing difficulties and future prospects of 2D materials for phototherapy are covered.

Berberine regulates bone metabolism in apical periodontitis by remodelling the extracellular matrix.

OBJECTIVES: The objectives of this study were to explore the role and related mechanism of berberine in repairing bone destruction in apical periodontics (AP). MATERIALS AND METHODS: AP was established in 14 of 21 male Wistar rats (four weeks of age; 70-80 g) for 3 weeks. The canals were cleaned and administered berberine (2 mg/ml; n = 7) or calcium hydroxide (100 mg/ml; control; n = 7), followed by glass ionomer cement sealing. After 3 weeks, specimen collection followed by micro-computed tomography (µ-CT) and histological staining was performed, including haematoxylin and eosin staining, Masson's trichrome staining, tartrate-resistant acid phosphatase staining, immunohistochemistry and immunofluorescence histochemistry. RESULTS: µ-CT showed that AP lesion volume reduced in the berberine group. Histopathology showed that berberine decreased the activity and number of osteoclasts but increased the expression of proteins related to osteoblast differentiation, including alkaline phosphatase and osterix. The immune cell, T cell, dendritic cell and macrophage counts were significantly decreased in the berberine group. In the berberine group, the expression of extracellular matrix-degraded proteases, metalloproteinases, was decreased; however, that of extracellular matrix-stable proteases, lysyl oxidases, was increased. CONCLUSIONS: Berberine controlled the inflammatory response and regulated bone metabolism in AP by reducing metalloproteinase expression and increasing lysyl oxidases expression.

Role of Slit/Robo Signaling pathway in Bone Metabolism.

Slit/Robo signals were initially found to play an essential role in nerve development as axonal guidance molecules. In recent years, with in-depth study, the role of Slit/Robo in other life activities, such as tumor development, angiogenesis, cell migration, and bone homeostasis, has gradually been revealed. Bone is an organ with an active metabolism. Bone resorption and bone formation are closely related through precise spatiotemporal coordination. There is much evidence that slit, as a new bone coupling factor, can regulate bone formation and resorption. For example, Slit3 can promote bone formation and inhibit bone resorption through Robo receptors, which has excellent therapeutic potential in metabolic bone diseases. Although the conclusions of some studies are contradictory, they all affirm the vital role of Slit/Robo signaling in regulating bone metabolism. This paper reviews the research progress of Slit/Robo signaling in bone metabolism, briefly discusses the contradictions in the existing research, and puts forward the research direction of Slit/Robo in the field of bone metabolism in the future.

Insulin-like growth factor 1 promotes neural differentiation of human stem cells from the apical papilla.

OBJECTIVE: Insulin-like growth factor 1 (IGF1) is one of the vital factors in regenerative endodontics. Previous studies have focused on the role of IGF1 in the mineralization of dental tissues. However, the role of IGF1 in the neural differentiation of dental stem cells was little discussed. DESIGN: IGF1 was overexpressed in human stem cells from the apical papilla (hSCAPs) by lentivirus and knocked down in hSCAPs by small interfering RNA. The neural differentiation level of hSCAPs was investigated histologically by HE staining and Nissl staining after neural induction for 3 days. The expression of proteins was examined by western blot and immunofluorescence. RESULTS: IGF1 promoted neural differentiation of hSCAPs, more cell processes and Nissl-positive body stained cells. IGF1 overexpression could both promote glial differentiation in hSCAPs, characterized by the increase of S100ß and GFAP proteins, and neuronal differentiation, characterized by the increase of ßIII-tubulin and functional GAD67/vGLUT1 proteins. Conversely, IGF1 knockdown suppressed both glial and neuronal differentiation. IGF1 activated AKT to regulate the early neural differentiation of hSCAPs. CONCLUSIONS: The results indicate IGF1 could promote neural differentiation of hSCAPs by activating AKT signaling and provide a cue for the candidate of induced neural seeding cells in regenerative endodontics.

The Expression of Semaphorin 7A in Human Periapical Lesions.

INTRODUCTION: Semaphorin 7A (SEMA7A) is a membrane-bound or secretory protein exerting multiple functions in the regulation of inflammation, neural degradation, and cancer progression. Human periapical lesions are chronic and infectious diseases mainly caused by bacteria. However, the involvement of SEMA7A in human periapical lesions is still unclear. This study aimed to explore the expression of SEMA7A in human periapical lesions accompanied by the potential association of SEMA7A with matrix metalloproteinase (MMP)-1 and MMP-3 during the progression of apical periodontitis. METHODS: Samples of periapical lesions and healthy controls were collected. Total RNA and protein were extracted respectively for quantitative real-time polymerase chain reaction and Western blot analysis. Additionally, 6 healthy samples and 27 periapical lesion samples were fixed, dehydrated, and embedded for further histologic and immunochemical analysis. The expression of SEMA7A was quantified by average integrated optical density. Immunofluorescence analysis was conducted to explore the colocalization of SEMA7A/MMP-1 and SEMA7A/MMP-3. RESULTS: Compared with healthy controls, the messenger RNA and protein expression of SEMA7A was markedly up-regulated in periapical lesions. A stronger expression of MMP-1, MMP-3, and inflammatory cytokines was exhibited in periapical lesions than in healthy groups. An increasing expression of SEMA7A can be observed in both the periapical granuloma group and the radicular cyst group compared with the normal group (P < .01). Immunofluorescence results showed the colocalization of SEMA7A with both MMP-1 and MMP-3 in vascular vessels and extracellular matrix. CONCLUSIONS: SEMA7A was up-regulated in periapical periodontitis and might be involved in the tissue destruction and infiltration of immune cells in periapical lesions.

[The Roles of RUNX1 in the Proliferation and Osteogenic and Adipogenic Differentiation of Dental Pulp Stem Cells].

OBJECTIVE: To investigate the influence of Runt-related transcription factor 1 (RUNX1) on the proliferation, osteogenic differentiation and adipogenic differentiation of dental pulp stem cells (DPSC) in vitro. METHODS: DPSCs were transfected through lentiviral vector carrying the target gene RUNX1 and green fluorescent protein (GFP). After 48 h, transfection efficiency was determined with the fluorescent marking of GFP and Western blot. The effect of the overexpression of RUNX1 on DPSC proliferation and colony formation was determined with CCK-8 and colony formation assay; cell cycle of DPSC was detected by flow cytometry. RUNX1 siRNA was transfected into the DPSCs. After mineralized induction, the effect of RUNX1 overexpression/silencing on the osteogenetic differentiation of DPSC was tested by alkaline phosphatase (ALP) staining and alizarin red staining. After adipogenic induction, oil red O staining was done in order to observe the effect of overexpression/silencing of RUNX1 on the adipogenic differentiation of DPSC. RESULTS: RUNX1 protein was overexpressed in DPSC after lentiviral transfection. Fluorescent test showed successful transfection of lentiviral transfection and over 70% of the cells showed stable expression of GFP protein. The proliferation and colony-formation efficiency of DPSC was enhanced significantly and the proportion of DPSCs in the S phase was significantly increased in the RUNX1-overexpessed group ( P<0.05). ALP activity and mineralized nodule formation ability increased, while lipid droplets decreased in the RUNX1-overexpessed group ( P<0.05). ALP activity and mineralized nodule formation ability decreased, while lipid droplets increased in the RUNX1 knockdown group ( P<0.05) . CONCLUSION: RUNX1 promotes DPSC proliferation and osteogenic differentiation while it inhibits DPSC adipogenic differentiation.

The alteration of A disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) in the knee joints of osteoarthritis mice.

The A disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) family is gradually being recognized as an important family of mediators that, along with the matrix metalloproteinases (MMPs), control the degradation process in osteoarthritis (OA). The objective of this study was to uncover the detailed alterations of ADAMTS1, ADAMTS2, and ADAMTS5 in the knee joint of OA mice. The OA model was established by anterior cruciate ligament transection (ACLT) on the knee joints of C57BL/6 J mice. The mice showed representative phenotypes of ACLT-induced OA, including obvious deterioration of the cartilage, reductions in the collagen and proteoglycan components in the cartilage matrix of OA mice, and increased inflammation and osteoclast activity. By qPCR, the gene expression levels of Adamts1, -2, and -5 were the top-ranked among Adamts1-5 in cartilage/chondrocytes, osteogenic tissue/osteoblasts, and cortical bone/osteocytes. Moreover, the protein expression levels of ADAMTS1, -2, and -5 were all increased in articular cartilage, the growth plate, and subchondral bone of the knee joint. The results suggest the important roles of ADAMTS1, -2, and -5 in OA disease, which will be helpful in further research on degenerative changes in OA.

Preparation and application of chitosan biomaterials in dentistry.

Chitosan is a biodegradable and biocompatible natural polysaccharide that has a wide range of applications in the field of dentistry due to its functional versatility and ease of access. Recent studies find that chitosan and its derivatives can be embedded in materials for dental adhesives, barrier membranes, bone replacement, tissue regeneration, and antimicrobial agent to better manage oral diseases. In this paper, we provide a comprehensive overview on the preparation, applications, and major breakthroughs of chitosan biomaterials. Furthermore, incorporation of chitosan additives for the modification and improvement of dental materials has been discussed in depth to promote more advanced chitosan-related research in the future.

Oral Microbiome and SARS-CoV-2: Beware of Lung Co-infection.

The new coronavirus SARS-CoV-2, the cause of COVID-19, has become a public health emergency of global concern. Like the SARS and influenza pandemics, there have been a large number of cases coinfected with other viruses, fungi, and bacteria, some of which originate from the oral cavity. Capnocytophaga, Veillonella, and other oral opportunistic pathogens were found in the BALF of the COVID-19 patients by mNGS. Risk factors such as poor oral hygiene, cough, increased inhalation under normal or abnormal conditions, and mechanical ventilation provide a pathway for oral microorganisms to enter the lower respiratory tract and thus cause respiratory disease. Lung hypoxia, typical symptoms of COVID-19, would favor the growth of anaerobes and facultative anaerobes originating from the oral microbiota. SARS-CoV-2 may aggravate lung disease by interacting with the lung or oral microbiota via mechanisms involving changes in cytokines, T cell responses, and the effects of host conditions such as aging and the oral microbiome changes due to systemic diseases. Because the oral microbiome is closely associated with SARS-CoV-2 co-infections in the lungs, effective oral health care measures are necessary to reduce these infections, especially in severe COVID-19 patients. We hope this review will draw attention from both the scientific and clinical communities on the role of the oral microbiome in the current global pandemic.

Berberine mediates root remodeling in an immature tooth with apical periodontitis by regulating stem cells from apical papilla differentiation.

Once pulp necrosis or apical periodontitis occurs on immature teeth, the weak root and open root apex are challenging to clinicians. Berberine (BBR) is a potential medicine for bone disorders, therefore, we proposed to apply BBR in root canals to enhance root repair in immature teeth. An in vivo model of immature teeth with apical periodontitis was established in rats, and root canals were filled with BBR, calcium hydroxide or sterilized saline for 3 weeks. The shape of the roots was analyzed by micro-computed tomography and histological staining. In vitro, BBR was introduced into stem cells from apical papilla (SCAPs). Osteogenic differentiation of stem cells from apical papilla was investigated by alkaline phosphatase activity, mineralization ability, and gene expression of osteogenic makers. The signaling pathway, which regulated the osteogenesis of SCAPs was evaluated by quantitative real time PCR, Western blot analysis, and immunofluorescence. In rats treated with BBR, more tissue was formed, with longer roots, thicker root walls, and smaller apex diameters. In addition, we found that BBR promoted SCAPs osteogenesis in a time-dependent and concentration-dependent manner. BBR induced the expression of ß-catenin and enhanced ß-catenin entering into the nucleus, to up-regulate more runt-related nuclear factor 2 downstream. BBR enhanced root repair in immature teeth with apical periodontitis by activating the canonical Wnt/ß-catenin pathway in SCAPs.

Gap junction-mediated cell-to-cell communication in oral development and oral diseases: a concise review of research progress.

Homoeostasis depends on the close connection and intimate molecular exchange between extracellular, intracellular and intercellular networks. Intercellular communication is largely mediated by gap junctions (GJs), a type of specialized membrane contact composed of variable number of channels that enable direct communication between cells by allowing small molecules to pass directly into the cytoplasm of neighbouring cells. Although considerable evidence indicates that gap junctions contribute to the functions of many organs, such as the bone, intestine, kidney, heart, brain and nerve, less is known about their role in oral development and disease. In this review, the current progress in understanding the background of connexins and the functions of gap junctions in oral development and diseases is discussed. The homoeostasis of tooth and periodontal tissues, normal tooth and maxillofacial development, saliva secretion and the integrity of the oral mucosa depend on the proper function of gap junctions. Knowledge of this pattern of cell-cell communication is required for a better understanding of oral diseases. With the ever-increasing understanding of connexins in oral diseases, therapeutic strategies could be developed to target these membrane channels in various oral diseases and maxillofacial dysplasia.

TGF-ß1 facilitates cell-cell communication in osteocytes via connexin43- and pannexin1-dependent gap junctions.

Connexins and pannexins are two families of channel forming proteins that are able to pass small molecules to achieve communication between cells. While connexins have been recognized to mediate gap junctional intercellular communication (GJIC), pannexins are far less known. Our previous study reported the potential role of TGF-ß1 in mediating of connexins in osteocytes in vitro. Herein, we aimed to elucidate the influence of TGF-ß1 on cell-cell communication based on gap junctions assembled by connexins and pannexins in vitro and ex vivo. We first showed that TGF-ß1 positively affected the elongation of dendritic processes of osteocytes. Our data indicated that TGF-ß1 increased expressions of connexin43 (Cx43) and pannexin1 (panx1), which are indispensable for hemichannel formation in gap junctions, in osteocytes in vitro and ex vivo. TGF-ß1 enhanced gap junction formation and impacted cell-cell communication in living osteocytes, as indicated by the scrape loading and Lucifer yellow transfer assays. TGF-ß1 enhanced the expressions of Cx43 and panx1 via activation of ERK1/2 and Smad3/4 signalling. The TGF-ß1-restored expressions of Cx43 and panx1 in osteocytes in the presence of an ERK inhibitor, U0126, further demonstrated the direct participation of Smad3/4 signalling. TGF-ß1 increased the accumulation of Smad3 in the nuclear region (immunofluorescence assay) and promoted the enrichment of Smad3 at the binding sites of the promoters of Gja1 (Cx43) and Panx1 (ChIP assay), thereby initiating the enhanced gene expression. These results provide a deep understanding of the molecular mechanisms involved in the modulation of cell-cell communication in osteocytes induced by TGF-ß1.

Disturbed bone remodelling activity varies in different stages of experimental, gradually progressive apical periodontitis in rats.

Bone remodelling keeps going through the lifespan of human by bone formation and bone resorption. In the craniofacial region, mandibles act as the main force for biting and chewing, and also become susceptible to a common bone-loss disease, namely, apical periodontitis, once infected dental pulp is not treated timely, during which bone resorption occurs from the apical foramen to the apical bone area. Although conventional root canal treatment (RCT) can remove the most of the infection, chronical apical periodontitis due to incomplete removal of dental pulp and subsequent microleakage will become refractory and more challenging, and this process has scarcely been specifically studied as a bone remodelling issue in rat models. Therefore, to study chronical and refractory apical periodontitis owing to incomplete cleaning of infected dental pulp and microleackage in vivo, we establish a modified rat model of gradually progressive apical periodontitis by sealing residual necrotic dental pulp and introducing limited saliva, which simulates gradually progressive apical periodontitis, as observed in the clinical treatment of chronical and refractory apical periodontitis. We show that bone-loss is inevitable and progressive in this case of apical periodontitis, which confirms again that complete and sound root canal treatment is crucial to halt the progression of chronical and refractory apical periodontitis and promote bone formation. Interestingly, bone remodelling was enhanced at the initial stage of apical periodontitis in this model while reduced with a high osteoblast number afterwards, as shown by the time course study of the modified model. Suggesting that the pathological apical microenvironment reserve its hard tissue formation ability to some degree but in a disturbed manner. Hopefully, our findings can provide insights for future bone regenerative treatment for apical periodontitis-associated bone loss.

Dual modulation of crystallinity and macro-/microstructures of 3D printed porous titanium implants to enhance stability and osseointegration.

The macro architecture and micro surface topological morphology of implants play essential roles in bone tissue regeneration. 3D printing technology provides enormous advantages for the rapid fabrication of personalized bone tissue repair implants. This study presents a demonstration of dual-modulation (DM) 3D printed porous titanium implants to enhance stability and osseointegration. Titanium implants with the first level of modulation of macro porous architecture and mechanical properties are obtained using macro architecture design and 3D printing fabrication. The first level of modulation achieved scaffolds with a wide range of compressive strengths (36.76-139.97 MPa) when varying the scaffold macro architectures. In the second level of modulation of surface topological morphology, alkali treatment, heat treatments and electrochemical deposition of hydroxyapatite coating were conducted for further regulating the biological function of implants. DM 3D printed scaffolds significantly promoted bone marrow mesenchyme stem cell adhesion and proliferation, indicating good cytocompatibility. In addition, in vivo osseointegration experiments indicated that the DM scaffolds formed better tissue-materials interfaces. New bone formation rates in DM scaffolds are higher than those in conventional 3D printed scaffolds after 6 months of implantation (58.1% versus 36.1%). These results demonstrate that DM scaffolds could enhance early stability and osseointegration. This study may provide new insights into the design, fabrication and post-processing of 3D printed porous titanium implants for various applications in personalized bone tissue regeneration.

Transforming growth factor-ß1 up-regulates connexin43 expression in osteocytes via canonical Smad-dependent signaling pathway.

Connexin 43 (Cx43)-mediated gap junctional intercellular communication (GJIC) has been shown to be important in regulating multiple functions of bone cells. Transforming growth factor-ß1 (TGF-ß1) exhibited controversial effects on the expression of Cx43 in different cell types. To date, the effect of TGF-ß1 on the Cx43 expression of osteocytes is still unknown. In the present study, we detected the expression of TGF-ß1 in osteocytes and bone tissue, and then used recombinant mouse TGF-ß1 to elucidate its effect on gap junctions (GJs) of osteocytes. Our data indicated that TGF-ß1 up-regulated both mRNA and protein expression of Cx43 in osteocytes. Together with down-regulation of Cx43 expression after being treated with TGF-ß type I receptor inhibitor Repsox, we deduced that TGF-ß1 can positively regulate Cx43 expression in osteocytes. Thus we next focussed on the downstream signals of TGF-ß and found that TGF-ß1-mediated smads, Smad3 and Smad4, to translocate into nucleus. These translocated signal proteins bind to the promoter of Gja1 which was responsible for the changed expression of Cx43. The present study provides evidence that TGF-ß1 can enhance GJIC between osteocytes through up-regulating Cx43 expression and the underlying mechanism involved in the activation of Smad-dependent pathway.

Aberrant activation of latent transforming growth factor-ß initiates the onset of temporomandibular joint osteoarthritis.

There is currently no effective medical treatment for temporomandibular joint osteoarthritis (TMJ-OA) due to a limited understanding of its pathogenesis. This study was undertaken to investigate the key role of transforming growth factor-ß (TGF-ß) signalling in the cartilage and subchondral bone of the TMJ using a temporomandibular joint disorder (TMD) rat model, an ageing mouse model and a Camurati-Engelmann disease (CED) mouse model. In the three animal models, the subchondral bone phenotypes in the mandibular condyles were evaluated by µCT, and changes in TMJ condyles were examined by TRAP staining and immunohistochemical analysis of Osterix and p-Smad2/3. Condyle degradation was confirmed by Safranin O staining, the Mankin and OARSI scoring systems and type X collagen (Col X), p-Smad2/3a and Osterix immunohistochemical analyses. We found apparent histological phenotypes of TMJ-OA in the TMD, ageing and CED animal models, with abnormal activation of TGF-ß signalling in the condylar cartilage and subchondral bone. Moreover, inhibition of TGF-ß receptor I attenuated TMJ-OA progression in the TMD models. Therefore, aberrant activation of TGF-ß signalling could be a key player in TMJ-OA development.

Epigenetics in Odontogenesis and its Influences.

BACKGROUND: Odontogenesis is fundamentally controlled by the genome. However, epigenetic factors have indispensable effects during odontogenesis. Previous studies have shown that exogenous factors, such as the environment, that cause hypomethylation and hypermethylation in DNA may lead to dental differences in monozygotic twin pairs. In addition, abnormalities in epigenetic regulation could induce disruptions in odontogenesis, thereby causing tooth malformation or agenesis. OBJECTIVE: This review overviews the epigenetic mechanisms involved in odontogenesis with the aim of establishing a fundamental vision of tooth development, which might be useful in further research in odontogenesis and therapy for dental diseases. RESULTS: We summarized articles about epigenetics in odontogenesis. Here, we present concrete epigenetic regulation mechanisms in odontogenesis that have been reported previously, following the order of microRNA, DNA methylation and histone modification. CONCLUSION: Epigenetic factors influence the proliferation, differentiation or apoptosis of cells that play indispensable roles during the process of odontogenesis which have the ability to exquisitely regulate the tooth number, size and shape.

Creating hierarchical porosity hydroxyapatite scaffolds with osteoinduction by three-dimensional printing and microwave sintering.

Hierarchical porosity, which includes micropores and macropores in scaffolds, contributes to important multiple biological functions for tissue regeneration. This paper introduces a two-step method of combining three-dimensional printing (3DP) and microwave sintering to fabricate two-level hierarchical porous scaffolds. The results showed that 3D printing made the macroporous structure well-controlled and microwave sintering generated micropores on the macropore surface. The resulting hierarchical macro/microporous hydroxyapatite scaffold induced bone formation following intramuscular implantation. Moreover, when comparing the hierarchical macro/microporous hydroxyapatite scaffold to the non-osteoinductive hydroxyapatite scaffolds (either 3D printed or H2O2 foamed) subjected to muffle sintering which do not have micropores, the critical role of micropores in material-driven bone formation was shown. The findings presented herein could be useful for the further optimization of bone grafting materials for bone regeneration.

MicroRNA 224 Regulates Ion Transporter Expression in Ameloblasts To Coordinate Enamel Mineralization.

Enamel mineralization is accompanied by the release of protons into the extracellular matrix, which is buffered to regulate the pH value in the local microenvironment. The present study aimed to investigate the role of microRNA 224 (miR-224) as a regulator of SLC4A4 and CFTR, encoding the key buffering ion transporters, in modulating enamel mineralization. miR-224 was significantly downregulated as ameloblasts differentiated, in parallel with upregulation of SLC4A4 and CFTR. Overexpression of miR-224 downregulated SLC4A4 and CFTR expression in cultured human epithelial cells. A microRNA luciferase assay confirmed the specific binding of miR-224 to the 3' untranslated regions (UTRs) of SLC4A4 and CFTR mRNAs, thereby inhibiting protein translation. miR-224 agomir injection in mouse neonatal incisors resulted in normal enamel length and thickness, but with disturbed organization of the prism structure and deficient crystal growth. Moreover, the enamel Ca/P ratio and microhardness were markedly reduced after miR-224 agomir administration. These results demonstrate that miR-224 plays a pivotal role in fine tuning enamel mineralization by modulating SLC4A4 and CFTR to maintain pH homeostasis and support enamel mineralization.