Zn‐Doped Hydroxyapatite Nanorods With Dual Antibacterial‐Osteogenic Functions for Periodontal Regeneration

Residents' journal review

Objectives The use of Sclerostin Antibody(Scl-Ab) as a bone anabolic agent has shown significant benefit in bone disorders in preclinical animal models and human clinical trials. Currently available evidence on the use of Scl-Ab in alveolar bone regeneration is limited to animal studies and hence this scoping review encompasses the animal studies conducted to ascertain the effectiveness of Scl-Ab on alveolar bone regeneration. Materials and methods The search strategy was aimed to locate published animal studies in which the treatment arm includes Sclerostin antibody administration for alveolar bone preservation or regeneration. The search terms used were (((Animal model) OR Rodent) AND Alveolar bone defect) AND Anti sclerostin antibody) OR Sclerostin antibody) AND Alveolar bone regeneration) OR Bone regeneration) AND Bone fill. Results Of the 559 results from Medline/PubMed, Scopus, Web of Science, Google scholar and additional articles from the references, six were included in the review. Scl-Ab was found to be effective in improving the bone quality and quantity. It was also observed that Scl-Ab was useful in reduced bone density associated with diseases and conditiona affecting osteoblast activity. Conclusion The review concluded that Scl-Ab promotes alveolar bone augmentation and improves bone quality without surgical interventions.

Bone tissue regeneration is orchestrated by the surrounding supporting tissues and involves the build-up of osteogenic cells, which orchestrate remodeling/healing through the expression of numerous mediators and signaling molecules. Periodontal regeneration models have proven useful for studying the interaction and communication between alveolar bone and supporting soft tissue. We applied a quantitative proteomic approach to analyze and compare proteins with altered expression in gingival soft tissue and alveolar bone following tooth extraction. For target identification and validation, hard and soft tissue were extracted from mini-pigs at the indicated times after tooth extraction. From triplicate experiments, 56 proteins in soft tissue and 27 proteins in alveolar bone were found to be differentially expressed before and after tooth extraction. The expression of 21 of those proteins was altered in both soft tissue and bone. Comparison of the activated networks in soft tissue and alveolar bone highlighted their distinct responsibilities in bone and tissue healing. Moreover, we found that there is crosstalk between identified proteins in soft tissue and alveolar bone with respect to cellular assembly, organization, and communication. Among these proteins, we examined in detail the expression patterns and associated networks of ATP5B and fibronectin 1. ATP5B is involved in nucleic acid metabolism, small molecule biochemistry, and neurological disease, and fibronectin 1 is involved in cellular assembly, organization, and maintenance. Collectively, our findings indicate that bone regeneration is accompanied by a profound interaction among networks regulating cellular resources, and they provide novel insight into the molecular mechanisms involved in the healing of periodontal tissue after tooth extraction.

Article Figures and data Abstract Editor's evaluation Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Oral inflammatory diseases such as apical periodontitis are common bacterial infectious diseases that may affect the periapical alveolar bone tissues. A protective process occurs simultaneously with the inflammatory tissue destruction, in which mesenchymal stem cells (MSCs) play a primary role. However, a systematic and precise description of the cellular and molecular composition of the microenvironment of bone affected by inflammation is lacking. In this study, we created a single-cell atlas of cell populations that compose alveolar bone in healthy and inflammatory disease states. We investigated changes in expression frequency and patterns related to apical periodontitis, as well as the interactions between MSCs and immunocytes. Our results highlight an enhanced self-supporting network and osteogenic potential within MSCs during apical periodontitis-associated inflammation. MSCs not only differentiated toward osteoblast lineage cells but also expressed higher levels of osteogenic-related markers, including Sparc and Col1a1. This was confirmed by lineage tracing in transgenic mouse models and human samples from oral inflammatory-related alveolar bone lesions. In summary, the current study provides an in-depth description of the microenvironment of MSCs and immunocytes in both healthy and disease states. We also identified key apical periodontitis-associated MSC subclusters and their biomarkers, which could further our understanding of the protective process and the underlying mechanisms of oral inflammatory-related bone disease. Taken together, these results enhance our understanding of heterogeneity and cellular interactions of alveolar bone cells under pathogenic and inflammatory conditions. We provide these data as a tool for investigators not only to better appreciate the repertoire of progenitors that are stress responsive but importantly to help design new therapeutic targets to restore bone lesions caused by apical periodontitis and other inflammatory-related bone diseases. Editor's evaluation Data from scRNA-Seq analysis demonstrated that acute inflammation stimulates periodontal stem cells to differentiate into osteoblast lineage cells to protect the alveolar bone. In murine models and patients with apical periodontitis, the genes and proteins associated with osteogenesis were enriched. The studies help us understand how MSCs respond to inflammation during apical periodontitis disease progression. https://doi.org/10.7554/eLife.82537.sa0 Decision letter Reviews on Sciety eLife's review process Introduction Oral diseases, particularly dental caries and periodontal diseases, affect 3.5 billion people worldwide (Disease et al., 2018). Untreated dental caries can directly lead to pulp necrosis and periapical lesions, resulting in apical periodontitis (AP). Individuals with at least one tooth affected by AP comprise up to 52% of cases, indicating that AP is a highly prevalent disease (Tibúrcio-Machado et al., 2021). Inflammation in the oral cavity can lead to destruction of surrounding periapical tissues and resorption of hard tissues, a consequence of the unbalanced interaction between infection and the immune response (Gazivoda et al., 2009; Márton and Kiss, 2014). Restoring and regenerating the destroyed periapical alveolar bone structures have always been a challenging task in clinical practice. Active inflammation, the tissue injury and the protective process all occur simultaneously in the setting of chronic AP (Márton and Kiss, 2014). Importantly, there is a complex assemblage of immune cell types involved in the pathogenesis, highlighting the importance of polymorphonuclear leukocytes, lymphocytes, and monocyte/macrophages in periapical defense (Braz-Silva et al., 2019; Nair, 2004). Notably, an increasing number of studies report the involvement of mesenchymal stem cells (MSCs) in the protective action that occurs during oral inflammatory diseases, whereby MSCs exert immunomodulatory effects and have regenerative potential (Li et al., 2014; Márton and Kiss, 2000; Nair, 2004). MSC markers such as CD44, CD73, CD90, CD106, and STRO-1 have been observed in human periapical inflammatory tissues (Estrela et al., 2019; Liao et al., 2011). Cells isolated from the inflamed periapical region were able to produce colony-forming unit-fibroblasts (CFU-Fs) with high-osteogenic capacity. It is also reported that interference with MSC mobilization toward the periapex region in an AP mouse model led to enlargement of lesions, accompanied by decreased wound healing markers and increased inflammatory cytokines (Araujo-Pires et al., 2014). These findings indicate the involvement of MSCs in the repair and regeneration of oral inflammatory-related bone lesions. They also suggest that MSCs present promising targets for treating bone lesions, with great potential for modulating inflammation and promoting tissue regeneration. However, most studies have surveyed whole tissues to understand the transcriptomic and cellular profile of these diseases. Specific cell populations and their regulatory molecules, as well as the interaction among different cell populations, remain far from clear. The advances in single-cell technologies offer an unbiased approach for identifying heterogeneous cell subsets, tracking the trajectories of distinct cell clusters and uncovering regulatory relationships between genes (Hwang et al., 2018; Tang et al., 2009). In this study, we collected mandibular alveolar bone samples from control and AP in mice and subjected them to single-cell RNA sequencing (scRNA-seq). The atlas of the mandibular alveolar bone explored the distinct cell subsets and their expression profiles relevant to AP. We also investigated the relationship between MSCs and immune cell subsets. The results reveal the role of a subset of MSCs in inflammation, which showed increased frequency and which formed a self-supporting network. Moreover, MSCs exhibited upregulated osteogenic potential, which was confirmed in transgenic mouse models and human patients with chronic AP. These results advance our understanding of heterogeneity and interactions of alveolar bone cells in the pathogenesis of inflammatory-related bone diseases. Defining key cellular subsets such as MSCs and their biomarkers in inflamed tissue will be important for identifying new therapeutic targets for oral inflammatory-related bone diseases. Results Single-cell transcriptional profiling identified 15 discrete populations in homeostasis and chronic AP samples Individual cells were isolated from alveolar bone of healthy mice and mice with AP. We modeled AP using a well-established AP mouse model in which the mandibular first molar pulp was exposed and subsequently developed chronic AP over a 3 wk period (Taira et al., 2019). Bar-coded cDNA libraries from individual cells were obtained using the 10× Genomics Chromium Controller platform (Zheng et al., 2017; Figure 1A). The combined libraries from healthy and AP alveolar bone contained 15,148 individual cells. The median value of feature_RNA was between 1000 and 2000 (Figure 1—figure supplement 1B). After quality control filtering and removal of the batch effect between batches, the t-stochastic neighbor embedding (t-SNE) method was applied to reduce the dimensionality. Seurat's unbiased cluster detection algorithm identified 15 distinct cell populations (Figure 1B and C). Cluster-specific transcripts were utilized to annotate cell types with classic markers as described in a previous study (Lin et al., 2021). These included B cell (Cd79a), hematopoietic stem cell (HSC) (Cd34), MSC (Col1a1), natural killer (NK) cell (Klrd1), T cell (Cd3g), dendritic cell (Siglech), epithelial cell (Epcam), erythrocyte (Hbb-bt), macrophage (Adgre1), mast cell (Fcer1a), megakaryocyte (Gp1bb), monocyte (Ly6c2), myeloid progenitor (Mpo), neutrophil (S100a8), and pre-B cell (Vpreb1; Figure 1E and F). The top 20 enriched genes in each defined cluster were identified and compared (Figure 1G). Figure 1 with 1 supplement see all Download asset Open asset Identification of the single-cell atlas of alveolar bone using scRNA-sequencing (scRNA-seq) and unbiased clustering. (A) Schematic diagram of the experimental design. (B–C) t-Stochastic neighbor embedding (t-SNE) representation of aligned gene expression data in single cells extracted from mandibles of control mice (n=8340) and apical periodontitis (AP) mice (n=6808) showing 15 distinct clusters and cellular origin. (D) Relative abundance of 15 cell populations composing alveolar bone under healthy and AP conditions. (E) Expression of gene markers in distinct cell types. (F) Gene expression patterns projected onto t-SNE plots of marker genes. Scale: log-transformed gene expression. (G) Heatmap showing the 20 most upregulated genes (ordered by decreasing Padj value) in each cluster defined in B. Scale: log2 fold change. AP led to significant changes in frequency and transcriptional expression of cell populations All the identified cell clusters were present in both AP and control samples, but there were significant differences in the cellular compositions of particular clusters. T cell, B cell, NK cell, macrophage, epithelial cell, and MSC had significantly increased frequency in AP samples. Neutrophil, myeloid progenitor, monocyte, megakaryocyte, mast cell, HSC, and dendritic cell were markedly decreased (Figures 2A and 1D). Figure 2 Download asset Open asset Changes in frequency and transcriptional expression pattern in each cell population from control and apical periodontitis (AP) groups. (A) Bar plot of cells per cluster (AP versus control). Normalization to overall number of inputs per condition. Fisher's exact test with Bonferroni correction was used. *p<0.05, **p<0.01, and ****p<0.0001. All data were exhibited as mean ± SEM. (B) Violin plots of cluster-specific expression of representative genes. (C–G) Violin plots showing genes that significantly changed in each cluster from control and AP. AP is a complex inflammatory process involving innate and adaptive immune responses (Cotti et al., 2014). A variety of inflammatory cells such as neutrophils, mast cells, monocytes, macrophages, and lymphocytes are involved in periapical lesions, highlighting the direct involvement of the immune response in the pathogenesis of AP (Nair, 2004). Neutrophils are important components in the acute phase of AP as a first line of defense. But they are also important in the progression of AP by interacting with microorganisms, leading to tissue damage and chemotaxis (Braz-Silva et al., 2019). Single-cell differential expression analysis revealed that the most significantly enriched genes in neutrophils were various proinflammatory chemokines and cytokines. These included C-X-C motif chemokine ligand 2 (Cxcl2), C-C motif chemokine ligand 6 (Ccl6), NLR family pyrin domain containing 3 (Nlrp3), and Interleukin-1β (Il1b). Notably, we found that C-C motif chemokine receptor like 2 (Ccrl2) was upregulated in neutrophils during AP (Figure 2D). It is responsible for the innate defense against pathogens and is also involved in the regulation of neutrophil migration (Del Prete et al., 2017; Kolaczkowska and Kubes, 2013; Mantovani et al., 2011). Mast cells, monocytes, and macrophages have critical roles in the inflammatory infiltrate during chronic AP (Braz-Silva et al., 2019). The production of Interleukin-6 (Il6) was present in these cell populations with the highest expression level in mast cells. The pro-inflammatory cytokine IL-1β is a key mediator of host response to microbial infection and is associated with the persistence of AP (Morsani et al., 2011; Ng et al., 2008). We found Il1b transcripts in a series of cell types, such as monocyte, macrophage, mast cell, and neutrophil. Of these, macrophages had the highest Il1b expression. Another major cytokine, tumor necrosis factor (Tnf; Cotti et al., 2014), was detected in immunoresponsive cell clusters, such as monocyte, macrophage, mast cell, myeloid progenitor, neutrophil, and HSC, with the highest expression observed in the monocyte population (Figure 2B). Furthermore, gene signatures from monocytes showed that the interferon-induced transmembranes (IFITMs) protein 1 and 2 (Ifitm1 and Ifitm2; Figure 2E) were upregulated the most during AP. These factors have been associated with signal transduction of anti-inflammation activity in the immune system (Yánez et al., 2020). We also detected upregulated expression levels of Ccl9 in the monocyte population from AP. Ccl9 is an important cytokine and is involved in the survival of osteoclasts during the destruction of the periapical bone (Silva et al., 2007). Also, genes coding for pro-inflammatory calcium-binding S100 family proteins such as S100a9 and S100a11 had increased expression in the AP monocyte cluster (Figure 2E). Previous reports demonstrated that macrophages are capable of secreting pro- and anti-inflammatory substances which act on the development and repair of the AP lesions (Italiani and Boraschi, 2014; Shapouri-Moghaddam et al., 2018). Indeed, several genes encoding pro-inflammatory mediators, including Cxcl2, Cxcl16, Il1a, and Ptgs2, were upregulated in macrophages from AP samples (Figure 2C). Expression of anti-inflammatory-associated genes such as Ifitm1 and Ifitm2 was significantly increased in the AP macrophage cluster compared to control cells. Furthermore, Fcγ receptor IIB (Fcgr2b) was markedly upregulated in macrophages from AP samples. Fcgr2b is expressed in most tissue-resident macrophages (Gautier et al., 2012) and functions to inhibit Fcγ-dependent phagocytosis. It also inhibits release of cytokines such as IL-6, TNF-α, IL-1α, as well as neutrophil chemotactants (Clatworthy and Smith, 2004; Espéli et al., 2016). In addition, expression of Apolipoprotein E (Apoe), which can suppress the pro-inflammatory response (Jofre-Monseny et al., 2007), was significantly increased in the macrophage population (Figure 2C). These data indicated the activation of anti-inflammatory factors by macrophages during local inflammation by AP. The major classes of lymphocytes are T lymphocytes, B lymphocytes, and the NK cells. T and B lymphocytes comprise the majority of the inflammatory infiltrate in AP (Graunaite et al., 2012). A significant increase in the expression of inflammatory-associated genes, such as Srgn, Emb, Ctla4, and Il7r could be observed in the AP T cell population (Figure 2F). In the AP B lymphocytes cluster, inflammation-responsive genes (Fth1, Ftl1, Ebf1, and mt-Nd5) were upregulated (Figure 2G). Interestingly, Lars2, the gene encoding a mitochondrial leucyl tRNA synthase (Carminho-Rodrigues et al., 2020; 't Hart et al., 2005), was significantly upregulated in T and B lymphocytes, indicating changes in mitochondrial metabolism in both clusters. Inflammation induces osteoclasts differentiation leading to periapical alveolar bone destruction Bone destruction is a major pathological factor in chronic inflammatory diseases such as AP. Various cytokines including TNF-α, IL-1α, and IL-6 were released by immunocytes to recruit the osteoclast precursors and induce the maturation of osteoclasts (Lyu et al., 2022). We have detected osteoclast markers including Ctsk, Acp5, Mmp9, and Nfatc1 by scRNA-seq. Moreover, Csfr1, Cx3cr1, Itgam, and Tnfrs11a were used to identify osteoclast precursors. Markers of osteoclast and osteoclast precursors were highly expressed in the clusters of monocyte and macrophage (Figure 3A and B). Gene Ontology (GO) analysis showed that inflammation related immune reactions and bone resorption activity were significantly enriched in macrophage cluster (Figure 3C). To further study the differential trajectory of osteoclasts, pseudotime analysis was performed for the clusters of macrophage and monocyte. Two independent branch points were determined, and five monocyte/macrophage subclusters were scattered at different branches in the developmental tree (Figure 3D and G). The results showed that the monocyte cluster differentiated into the macrophage cluster (Figure 3E). During this trajectory, the gene expression pattern across pseudotime showed that osteoclastic genes, such as Ctsk, Acp5, Mmp9, Atp6v0d2, and Dcstamp, were progressively elevated (Figure 3F). Of note, we have observed a branch which was highly positive for Ctsk and Acp5 (Figure 3H), indicating the mature osteoclasts were differentiated from monocyte/macrophage lineage and contributed to inflammatory bone resorption during AP. We have also analyzed the expression of osteoclast related genes using the bulk RNA-seq library built on mandibular samples extracted from mice with AP. Markers of osteoclast and osteoclast precursors were significantly upregulated, confirming the osteoclasts activity in the inflammatory-related bone lesion (Figure 3I). Figure 3 with 1 supplement see all Download asset Open asset Inflammatory-related bone resorption under apical periodontitis (AP) situation. (A) The expression levels of markers of osteoclasts and osteoclast precursors. (B) Violin plots of the expression of osteoclastogenesis genes. (C) Gene Ontology (GO) enrichment analysis of the biological functions of macrophage cluster. (D) Trajectory order of the monocyte/macrophage populations by pseudotime value. (E) The differentiation trajectory of monocyte and macrophage clusters presented on a t-stochastic neighbor embedding (t-SNE) visualization. (F) The expression patterns of osteoclast markers during the trajectory of monocyte/macrophage populations. (G) Distribution of monocytes/macrophages on the developmental tree by clusters. (H) Heatmap of differential genes of three states. (I) Heatmap of genes associated with osteoclastogenesis in bulk RNA-seq analysis. AP leads to reduced transcriptionally inferred cellular interactions with an increased self-supporting network in MSCs We next sought to characterize the cell-cell communication related to the perturbation of signaling pathways detected in the AP samples by employing CellphoneDB (Efremova et al., 2020; Nagai et al., 2021). We identified a close interaction among MSC, macrophage, and dendritic cells under homeostasis conditions (Figure 4A). A similarly close communication was found among MSC, macrophage, and dendritic cells under inflammatory conditions (Figure 4B). Next, we compared the differential cell-cell interaction (CCI) network between AP and control samples using CrossTalkeR (Nagai et al., 2021). The results suggested that AP is associated with an overall decrease in cellular interactions. However, MSC intercellular communication with mast cells and monocytes is upregulated and accompanied by the highest number of interactions within MSCs themselves (Figure 4C). These results indicate that, although cell populations lose their normal physiological interactions, MSCs were able to establish a self-interacting network and coordinate with certain types of cells during chronic AP. Next, we ranked the individual ligands by the number of their interactions. Inflammatory-related proteins (Lgals9, Tnf, and Ccl4), extracellular matrix protein (Fn1), and protein involved in biomineralization (Spp1) were among the highest interactions. Also, Tgfb1, Vegfb, and Vegfa were in the top 10 most abundant ligands (Figure 4D). Bar plots were also generated to display the top 10 upregulated gene/cell pairs, showing that inflammation associated genes (Tnf/neutrophil, Ccl3/mast cell, Ccl3/monocyte, and Il1b/macrophage) and matrix related genes (Sele/MSC, Fn1/MSC, and Fn1/monocyte) were the most influential ligands during AP when compared to control state (Figure 4E). We used a Sankey plot to further focus on MSC-related interactions (Figure 4F). The results indicated that Sele was primarily directed by MSC toward the MSC cluster via multiple receptors including Glg1, Selplg, and Cd44. Moreover, Fn1 was secreted by MSC, monocyte, macrophage, and mast cells toward MSCs (Figure 4G). Of note, Sele and Fn1 are important cell adhesion molecules that mediate cell homing and migration (Frenette et al., 1998; To and Midwood, 2011). This was in accordance with the increased frequency of the MSC cluster (Figure 2A) as well as the upregulated cellular interaction among MSCs and other cell populations. Figure 4 Download asset Open asset Apical periodontitis (AP) suppressed transcriptionally inferred cellular interactions and increased a self-supporting network within the mesenchymal stem cell (MSC) cluster. (A–C) Network plot of ligand-receptor activity in control (A), AP (B), and AP versus control (C). (D) Bar plot of top 10 most abundant ligands in all inferred ligand-receptor interactions. (E) Ranking of ligand/source regarding communication gains in AP state. (F and G) Sankey plot listing all predicted source, receptor, and receiver interactions associated with Fn1 and Sele. scRNA-seq based identification of AP-associated MSC population MSCs represented the non-immune cell population in alveolar bone, constituting 1.76% of total identified cells. This cell population could be decomposed into four subclusters (Figure 5A). The most dominant subcluster was characterized by high expression of Prrx1, platelet-derived growth factor receptor β (Pdgfrb), and hematopoiesis supporting factors such as C-X-C motif chemokine 12 (Cxcl12) and angiopoietin (Angpt1). It was also characterized by osteogenic-related markers, such as Runt-related transcription factor 2 (Runx2), Sp7, and was thus classified as the MSC_osteolineage cells (OLCs). The other subclusters were identified as MSC_endothelial and Figure and B). The cell composition of the MSC cluster indicated an of subcluster during the subcluster was reduced (Figure Of note, we performed lineage tracing using and transgenic mouse models to identify stem cells in alveolar bone and the surrounding periodontal tissues et al., 2020; et al., et al., 2020; et al., 2020). The results suggested increased of and as well as upregulated and periodontal stem cells in AP lesions (Figure supplement 1A). We compared the MSC marker genes, identified among the four enrichment analysis confirmed that there are four MSC populations. were enriched for tissue system and osteoblast MSC_endothelial cells enrichment for and cells were enriched for of and suggested highly of cells, including regulation of protein from mitochondrial and (Figure supplement Figure with 3 see all Download asset Open asset Identification and of apical periodontitis mesenchymal stem cell (MSC) population and (A) t-Stochastic neighbor embedding (t-SNE) representation an of single cells within the MSC cluster. (B) Violin plots of MSC expression of representative genes. (C) The of four subclusters of MSC population were in control and AP groups. (D) Violin plots the changes in the expression of top upregulated genes in the MSC cluster. and of Sparc (E) and (F) in (G) of in periodontal stem cells (H) tracing analysis of and AP stimulates MSC differentiation toward osteoblast lineage cells We next compared the data from the MSC cluster between AP and control groups. the top upregulated genes, we observed a significant in the single cell expression levels of such as secreted protein that is and in and accompanied by a toward increased expression (Figure We their by and bulk RNA analysis. The results revealed that and were significantly upregulated, and to increase during AP (Figure supplement B). In confirmed the upregulated expression of osteogenic-related markers, such as Sparc and in the AP were increased of cells in the bone the protective of MSC to differentiate into and under AP conditions (Figure and F). tracing analysis further revealed that to AP lesions and differentiated into (Figure Moreover, using mouse we identified cells in the alveolar bone, confirming that inflammation could the progenitor cells in the bone and toward thus to the protective during AP (Figure We investigated the heterogeneity of MSCs by branch expression analysis and pseudotime analysis in 2 et al., The results revealed three distinct states. Of these, state 2 and state 3 represented differentiated cell populations Figure We the of the MSCs with to their states. We the pseudotime of each cluster cell in the state and the results indicate that with MSC_endothelial cells, MSCs were able to differentiate into and cells. cells may to an state (Figure Interestingly, in state as osteogenic exhibited the highest toward differentiation with upregulated of and (Figure Notably, this MSC population increased significantly in the AP (Figure A of the of the gene expression pattern across pseudotime revealed of osteogenic genes such as and during inflammation when compared to the state (Figure the MSC subcluster exhibited gene expression levels of osteogenic markers that increased during AP. These results were in accordance with our previous single-cell analysis of gene expression in the AP (Figure Figure 6 Download asset Open asset Apical periodontitis (AP) stimulates mesenchymal stem cell (MSC) differentiation toward osteoblast lineage cells. (A) analysis of the MSC (B) cell differentiation trajectory of MSC populations in control and AP groups. (C) Heatmap of differential genes of three states. (D) Bar plot of changes between AP and control in as identified in pseudotime analysis. Fisher's exact test with Bonferroni correction was used. ****p<0.0001. All data were exhibited as mean ± SEM. (E) between the pseudotime gene trajectories of MSC showed of and line and indicate marker levels were identified in alveolar bone from patients with AP We next osteogenesis within the MSC population was in human patients with AP. The of and were significantly enhanced in alveolar bone from patients with accompanied by in and (Figure Moreover, detected a of and in AP alveolar bone confirming higher osteogenic potential under AP conditions (Figure of markers in AP lesions from patients is with our previous an of osteogenic within the MSC subcluster in alveolar bone under AP conditions. Figure Download asset Open asset alveolar bone in apical periodontitis (AP) is associated with higher subcluster (A) Gene expression of and in samples of healthy and AP patients showed an upregulated expression in AP in healthy and in AP All data are as the mean ± SEM. (B–C) of and in bone of human alveolar bone exhibited high osteogenic protein levels in AP. 10 In summary, the current study the of MSCs and immunoresponsive cells under healthy and chronic AP including heterogeneity in their of We that this analysis a

Multifunctional hydrogel/platelet-rich fibrin/nanofibers scaffolds with cell barrier and osteogenesis for guided tissue regeneration/guided bone regeneration applications

Craniofacial bone defects caused by tumors, trauma, long-term tooth loss, or periodontal disease are a major challenge in the field of tissue engineering. In periodontitis and peri-implantitis, reconstructive therapy is also a major challenge for the dental surgeon. Lipoxins, resolvins, protectins, and maresins, known as specialized pro-resolving lipid mediators (SPMs), have been widely studied in the field of dental, oral, and craniofacial research for bone regeneration for their actions in restoring tissue homeostasis and promoting tissue healing and regeneration. Therefore, this study focuses on a survey of the use of SPMs for craniofacial and alveolar bone regeneration. Thus, electronic searches of five databases were performed to identify pre-clinical studies that evaluated the actions of SMPs on craniofacial and alveolar bone regeneration. Of the 523 articles retrieved from the electronic databases, 19 were included in the analysis. Resolvin (Rv) E1 was the mostly assessed SPM (n=8), followed by maresins (Ma) R1 (n=3), lipoxins (Lx) A4 (n=3), RvD1 (n=3), RvD2 (n=1), LxB4 (n=1), and maresin (M)-CTR3 (n=1). Meta-analysis showed that SPMs increased the newly formed bone by 14.85% compared to the control group (p<0.00001), decreased the area of the remaining defect by 0.35 mm2 (p<0.00001), and decreased the linear distance between the defect to the bone crest by 0.53 mm (p<0.00001). RvE1 reduced inflammatory bone resorption in periodontal defects and calvarial osteolysis and enhanced bone regeneration when RvE1 was combined with a bovine bone graft. RvD2 induced active resolution of inflammation and tissue regeneration in periapical lesions, while RvD1 controlled the inflammatory microenvironment in calvarial defects in rats, promoting bone healing and angiogenesis. MaR1 induced the proliferation and migration of mesenchymal stem cells, osteogenesis, and angiogenesis in calvarial defects, and benzo (b)-LxA4 and LxA4 promoted bone regeneration calvarial and alveolar bone defects in rats, inducing regeneration under inflammatory conditions. In summary, SPMs have emerged as pivotal contributors to the resolution of inflammation and the facilitation of bone neoformation within craniofacial and alveolar bone defects. These results are based on pre-clinical studies, in vivo and in vitro, and provide an updated review regarding the impact of SPMs in tissue engineering.

The balance between bone resorption and bone formation is vital for maintenance and regeneration of alveolar bone and supporting structures around teeth and dental implants. Tissue regeneration in the oral cavity is regulated by multiple cell types, signaling mechanisms, and matrix interactions. A goal for periodontal tissue engineering/regenerative medicine is to restore oral soft and hard tissues through cell, scaffold, and/or signaling approaches to functional and aesthetic oral tissues. Bony defects in the oral cavity can vary significantly, ranging from smaller intrabony lesions resulting from periodontal or peri-implant diseases to large osseous defects that extend through the jaws as a result of trauma, tumor resection, or congenital defects. The disparity in size and location of these alveolar defects is compounded further by patient-specific and environmental factors that contribute to the challenges in periodontal regeneration, peri-implant tissue regeneration, and alveolar ridge reconstruction. Efforts have been made over the last few decades to produce reliable and predictable methods to stimulate bone regeneration in alveolar bone defects. Tissue engineering/regenerative medicine provide new avenues to enhance tissue regeneration by introducing bioactive models or constructing patient-specific substitutes. This review presents an overview of therapies (e.g., protein, gene, and cell based) and biomaterials (e.g., resorbable, nonresorbable, and 3-dimensionally printed) used for alveolar bone engineering around teeth and implants and for implant site development, with emphasis on most recent findings and future directions.

BackgroundDental follicle stem cells (DFSCs) show mesenchymal stem cell properties with the potential for alveolar bone regeneration. Stem cell properties can be impaired by reactive oxygen species (ROS), prompting us to examine the importance of scavenging ROS for stem cell-based tissue regeneration. This study aimed to investigate the effect and mechanism of N-acetylcysteine (NAC), a promising antioxidant, on the properties of DFSCs and DFSC-based alveolar bone regeneration.MethodsDFSCs were cultured in media supplemented with different concentrations of NAC (0–10 mM). Cytologic experiments, RNA-sequencing and antioxidant assays were performed in vitro in human DFSCs (hDFSCs). Rat maxillary first molar extraction models were constructed, histological and radiological examinations were performed at day 7 post-surgery to investigate alveolar bone regeneration in tooth extraction sockets after local transplantation of NAC, rat DFSCs (rDFSCs) or NAC-treated rDFSCs.Results5 mM NAC-treated hDFSCs exhibited better proliferation, less senescent rate, higher stem cell-specific marker and immune-related factor expression with the strongest osteogenic differentiation; other concentrations were also beneficial for maintaining stem cell properties. RNA-sequencing identified 803 differentially expressed genes between hDFSCs with and without 5 mM NAC. “Developmental process (GO:0032502)” was prominent, bioinformatic analysis of 394 involved genes revealed functional and pathway enrichment of ossification and PI3K/AKT pathway, respectively. Furthermore, after NAC treatment, the reduction of ROS levels (ROS, superoxide, hydrogen peroxide), the induction of antioxidant levels (glutathione, catalase, superoxide dismutase), the upregulation of PI3K/AKT signaling (PI3K-p110, PI3K-p85, AKT, phosphorylated-PI3K-p85, phosphorylated-AKT) and the rebound of ROS level upon PI3K/AKT inhibition were showed. Local transplantation of NAC, rDFSCs or NAC-treated rDFSCs was safe and promoted oral socket bone formation after tooth extraction, with application of NAC-treated rDFSCs possessing the best effect.ConclusionsThe proper concentration of NAC enhances DFSC properties, especially osteogenesis, via PI3K/AKT/ROS signaling, and offers clinical potential for stem cell-based alveolar bone regeneration.

Three-Year Follow-Up of a Single Immediate Implant Placed in an Infected Area: A New Approach for Harvesting Autogenous Symphysis Graft

Bone tissue regeneration is a critical aspect of dental surgery, given the common occurrence of bone resorption leading to alveolar bone defects. The aim of this paper was to conduct a systematic review to provide a comprehensive summary of the evidence regarding the regenerative properties of dentin biomaterial. This systematic review was conducted through comprehensive searches in the PubMed, Scopus, and Web of Science databases, as well as an extensive exploration of the gray literature sources, including WorldCat, The New York Academy of Medicine Library, and Trip Database, following the established PRISMA protocol. Keywords such as tooth, dentin, grinder, and autograft guided the search, with a focus on a standardized procedure involving dentin grinders within laboratory, experimental, and clinical settings. Initially, a pool of 1942 articles was identified with 452 duplicates removed. An additional 1474 articles were excluded for not aligning with the predefined topics, and three more were excluded due to the unavailability of the full text. Ultimately, 13 articles met the strict inclusion criteria and were included in the review. The chemical composition of the dentin particles was similar to natural bone in terms of oxygen, carbon, calcium, phosphorus, sodium, and magnesium content, as well as in terms of the Ca/P ratio. In addition, the dentin also contained amide I and amide II structures, as well as aliphatic and hydroxyl functional groups. The chemically treated dentin was free of microorganisms. The dentin had characteristic tubules that opened after chemical treatment. At the cellular level, dentin released bone morphogenetic protein 2, induced significant cell growth, and stimulated the reorganization of the fibroblast cytoskeleton. Most clinical studies have focused on alveolar bone regeneration. After the transplantation of demineralized dentin particles, studies have observed new bone formation, a reduction in residual bone, and an increase in connective tissue. Clinical reports consistently indicate uncomplicated healing and recovery post-transplantation. However, there is a notable gap in the evidence concerning complication rates, patient-reported outcomes, and the presence of pro-inflammatory factors. In conclusion, dentin biomaterial emerges as a versatile bone substitute, demonstrating high biocompatibility and ease of acquisition. The preservation of its internal structure containing organic matter and growth factors enhances its potential for effective bone regeneration. Particularly, in dental surgery, dentin-derived materials present a promising alternative to traditional autologous bone autografts, offering the potential to reduce patient morbidity and treatment costs.

The destruction of periodontal alveolar bone (AB) caused by periodontitis is regarded as one of the major reasons for tooth loss. The inhibition of bone resorption and regeneration of lost AB are the desirable outcomes in clinical practice but remain in challenge. The use of mesenchymal stem cells (MSCs) is one current approach for achieving true restoration of AB defects (ABD). Antler stem cells (AnSC) are capable of renewing a huge mammalian bony appendage, the deer antler, suggesting an unparalleled potential for bone regeneration. Herein, we investigated the effectiveness of deer AnSCs conditioned medium (CM, AnSC‐CM) for repair of surgically‐created ABD using a rat model and sought to define the underlying mechanisms. The results showed that AnSC‐CM effectively induced regeneration of AB tissue; the outcome was significantly better than human bone marrow mesenchymal stem cell conditioned medium (hBMSC‐CM). AnSC‐CM treatment upregulated osteogenic factors and downregulated osteoclastic differentiation factors; stimulated proliferation, migration and differentiation of resident MSCs toward osteogenic lineage cells; modulated macrophage polarization toward the M2 phenotype and suppressed osteoclastogenesis. That AnSC‐CM resulted in better outcomes than hBMSC‐CM in treating ABD was attributed to the cell compatibility as both AnSCs and AB tissue are neural crest‐derived. In conclusion, the effects of AnSC‐CM on AB tissue regeneration were achieved through both promotion of osteogenesis and inhibition of osteoclastogenesis. We believe that AnSC‐CM is a candidate for effective treatment of ABD in dental clinical practice but will require investment in further development.

Most common techniques for alveolar bone augmentation are guided bone regeneration (GBR) and autologous bone grafting. GBR studies demonstrated long-term reabsorption using heterologous bone graft. A general consensus has been achieved in implant surgery for a minimal amount of 2 mm of healthy bone around the implant.A current height loss of about 3-4 mm will result in proper deeper implant insertion when alveolar bone expansion is not planned because of the dome shape of the alveolar crest. To manage this situation a split crest technique has been proposed for alveolar bone expansion and the implants’ insertion in one stage surgery. Platelet-rich fibrin (PRF) is a healing biomaterial with a great potential for bone and soft tissue regeneration without inflammatory reactions, and may be used alone or in combination with bone grafts, promoting hemostasis, bone growth, and maturation.AimThe aim of this study was to demonstrate the clinical effectiveness of PRF combined with a new split crest flapless modified technique in 5 patients vs. 5 control patients.Materials and methodsTen patients with horizontal alveolar crests deficiency were treated in this study, divided into 2 groups: Group 1 (test) of 5 patients treated by the flapless split crest new procedure; Group 2 (control) of 5 patients treated by traditional technique with deeper insertion of smaller implants without split crest. The follow-up was performed with x-ray orthopantomography and intraoral radiographs at T0 (before surgery), T1 (operation time), T2 (3 months) and T3 (6 months) post-operation.ResultsAll cases were successful; there were no problems at surgery and post-operative times. All implants succeeded osteointegration and all patients underwent uneventful prosthetic rehabilitation. Mean height bone loss was 1 mm, measured as bone-implant most coronal contact (Δ-BIC), and occurred at immediate T2 post-operative time (3 months). No alveolar bone height loss was detected at implant insertion time, which was instead identified in the control group because of deeper implant insertion.ConclusionThis modified split crest technique combined with PRF appears to be reliable, safe, and to improve the clinical outcome of patients with horizontal alveolar crests deficiency compared to traditional implanting techniques by avoiding alveolar height-loss related to deeper insertion of smaller implants.

Periodontitis is a highly prevalent chronic inflammatory disease affecting more than 60% of the population, which leads to destruction of the tooth-supporting tissues. The periodontium is composed of both hard (bone and cementum) and soft tissues (periodontal ligament and gingiva), requiring a tissue-engineering approach to allow a precisely coordinated and compartmentalised healing response for subsequent structural and functional regeneration. The present study investigated the functionalisation of highly porous scaffolds with decellularised cell-laid extracellular matrix for periodontal regeneration. This novel technique allowed the combination of a three-dimensional scaffold providing mechanical support with a native ECM providing tissue specific biological activity. The decellularisation of such constructs allows the maintenance of an intact ECM structure and composition while removing the immunogenic cellular component, thus generating an acellular implant. By combining a bone-like ECM-decorated scaffold (bone compartment) with periodontal ligament cell-sheets (PDLcs), the aim was to achieve specific bone and periodontal ligament regeneration. The first part of this study (Chapter 2) focused on the optimisation of cell seeding on highly porous scaffolds. Indeed, cell seeding on such structures is challenging, resulting in both poor and heterogeneous cellular attachment, impeding in vitro characterisation of the constructs and hence their clinical translation. Several parameters affecting the quality of cell seeding were investigated, and we successfully identified pre-incubation of the scaffolds in FBS as a reproducible and repeatable protocol, which significantly improved cell seeding efficiency and subsequent scaffold maturation. The second part of the study (Chapter 3) investigated the effect of culture time on ECM deposition and its composition. To this end, human osteoblasts were seeded on 250 μm pore size polycaprolactone melt electrowritten scaffolds and cultured in osteogenic medium for 1, 2 or 4 weeks, allowing cell proliferation, differentiation and ECM deposition. The constructs were subsequently decellularised, using an in-house optimised protocol for PDLcs decellularisation. Cellularised and decellularised constructs were then extensively characterised in vitro to assess cellular and extracellular composition. The decellularised constructs were recellularised with osteoblasts to study their biological activity in vitro. In vivo performance of the different groups for bone regeneration was assessed in vivo in a rodent calvarial defect model. The various culture periods demonstrated a significant difference in ECM morphology and quantity between 1, 2 and 4 weeks. At the early time points, the fibres were decorated with collagen which mineralised over time and gradually obstructed the pores of the PCL scaffold. Although longer culture times resulted in higher osteogenic activity of reseeded cells, the more mature matrix impeded in vivo bone regeneration. Scaffold porosity is crucial for host cell colonisation and vascularisation, which are indispensable for tissue regeneration. The decoration of the 250 μm pore size construct in the previous study altered its porosity and subsequent regeneration. In the third study (Chapter 4) scaffolds with different pore sizes (250, 500 and 750 μm) were cultured for 1, 2 and 4 weeks. The scaffolds with 750 μm pore sizes did not exhibit appropriate mechanical properties and were not further characterised. 250 and 500 μm scaffolds cultured for 1, 2 and 4 weeks were decellularised, characterised and recellularised with osteoblasts or macrophages. All decellularised constructs were implanted in a rodent calvarial defect and evaluated for bone regeneration. Although 500 μm pores enabled maintenance of the porosity even after 4 weeks of in vitro maturation, both pore sizes performed similarly in vivo. Again, shorter in vitro maturation was more beneficial for bone regeneration and more mature ECM impaired bone regeneration as observed 6 weeks post-implantation. In the last part of this study (Chapter 5), the best performing bone compartment (250 μm pore scaffold maturated for 1 week) was combined with a PDLcs prior to decellularisation, in order to fabricate a biphasic scaffold for periodontal regeneration. Cell removal and ECM preservation were confirmed in vitro before implanting in a periodontal defect. Freshly decellularised constructs were compared before and after freeze drying and long-term storage. Freeze drying allows stabilisation of biological components, potentially increasing products stability, shelf life and therefore clinical translation. Although our biphasic construct did not induce bone regeneration in vivo, fresh and freeze dried constructs displayed a higher potential in periodontal regeneration. Both groups displayed enhanced cementum formation and periodontal attachment, and prevented the formation of ankylosis, as opposed to the control groups. In conclusion, the ECM-decorated melt electrowritten scaffolds were shown to support bone and periodontal regeneration. Optimisation of the cell culture time was shown to be essential for efficient in vivo regeneration. Longer maturation time did not automatically increase scaffold performance, and indeed the more mature matrix appeared to inhibit in vivo bone regeneration. The combination of our optimised bone compartment with a mature periodontal ligament cell-sheet before decellularisation successfully generated a construct capable of promoting compartmentalised periodontal regeneration.

Post-extraction bleeding and alveolar bone resorption are the two frequently encountered complications after tooth extraction that result in poor healing and rehabilitation difficulties. The present study covalently bonded polyphosphate onto a collagen scaffold (P-CS) by crosslinking. The P-CS demonstrated improved hemostatic property in a healthy rat model and an anticoagulant-treated rat model. This improvement is attributed to the increase in hydrophilicity, increased thrombin generation, platelet activation and stimulation of the intrinsic coagulation pathway. In addition, the P-CS promoted the in-situ bone regeneration and alveolar ridge preservation in a rat alveolar bone defect model. The promotion is attributed to enhanced osteogenic differentiation of bone marrow stromal cells. Osteogenesis was improved by both polyphosphate and blood clots. Taken together, P-CS possesses favorable hemostasis and alveolar ridge preservation capability. It may be used as an effective treatment option for post-extraction bleeding and alveolar bone loss.Statement of significanceCollagen scaffold is commonly used for the treatment of post-extraction bleeding and alveolar bone loss after tooth extraction. However, its application is hampered by insufficient hemostatic and osteoinductive property. Crosslinking polyphosphate with collagen produces a modified collagen scaffold that possesses improved hemostatic performance and augmented bone regeneration potential.

Mesenchymal stem cells (MSCs) have shown significant potential in bone regeneration and regenerative medicine in recent years. With the advancement of tissue engineering, MSCs have been increasingly applied in bone repair and regeneration, and their clinical application potential has grown through interdisciplinary approaches involving biomaterials and genetic engineering. However, there is a lack of systematic reviews summarizing their applications in bone regeneration. To address this gap, we analyzed the latest research on MSCs for bone regeneration published from 2013 to 2023. Using the Web of Science Core Collection, we conducted a literature search in December 2024 and employed bibliometric tools like CiteSpace and VOSviewer for a comprehensive analysis of the key research trends. Our findings focus on the development of cell engineering, highlighting the advantages, limitations, and future prospects of MSC applications in bone regeneration. These insights aim to enhance understanding of MSC-based bone regeneration, inspire new research directions, and facilitate the clinical translation of MSC research.