Dynamic Survival Risk Prognostic Model and Genomic Landscape for Atypical Teratoid/Rhabdoid Tumors: A Population-Based, Real-World Study.

BACKGROUND: An atypical teratoid/rhabdoid tumor (AT/RT) is an uncommon and aggressive pediatric central nervous system neoplasm. However, a universal clinical consensus or reliable prognostic evaluation system for this malignancy is lacking. Our study aimed to develop a risk model based on comprehensive clinical data to assist in clinical decision-making. METHODS: We conducted a retrospective study by examining data from the Surveillance, Epidemiology, and End Results (SEER) repository, spanning 2000 to 2019. The external validation cohort was sourced from the Children's Hospital Affiliated to Chongqing Medical University, China. To discern independent factors affecting overall survival (OS) and cancer-specific survival (CSS), we applied Least Absolute Shrinkage and Selection Operator (LASSO) and Random Forest (RF) regression analyses. Based on these factors, we structured nomogram survival predictions and initiated a dynamic online risk-evaluation system. To contrast survival outcomes among diverse treatments, we used propensity score matching (PSM) methodology. Molecular data with the most common mutations in AT/RT were extracted from the Catalogue of Somatic Mutations in Cancer (COSMIC) database. RESULTS: The annual incidence of AT/RT showed an increasing trend (APC, 2.86%; 95% CI:0.75-5.01). Our prognostic study included 316 SEER database participants and 27 external validation patients. The entire group had a median OS of 18 months (range 11.5 to 24 months) and median CSS of 21 months (range 11.7 to 29.2). Evaluations involving C-statistics, DCA, and ROC analysis underscored the distinctive capabilities of our prediction model. An analysis via PSM highlighted that individuals undergoing triple therapy (integrating surgery, radiotherapy, and chemotherapy) had discernibly enhanced OS and CSS. The most common mutations of AT/RT identified in the COSMIC database were SMARCB1, BRAF, SMARCA4, NF2, and NRAS. CONCLUSIONS: In this study, we devised a predictive model that effectively gauges the prognosis of AT/RT and briefly analyzed its genomic features, which might offer a valuable tool to address existing clinical challenges.

NSUN5/TET2-directed chromatin-associated RNA modification of 5-methylcytosine to 5-hydroxymethylcytosine governs glioma immune evasion.

Malignant glioma exhibits immune evasion characterized by highly expressing the immune checkpoint CD47. RNA 5-methylcytosine(m5C) modification plays a pivotal role in tumor pathogenesis. However, the mechanism underlying m5C-modified RNA metabolism remains unclear, as does the contribution of m5C-modified RNA to the glioma immune microenvironment. In this study, we demonstrate that the canonical 28SrRNA methyltransferase NSUN5 down-regulates ß-catenin by promoting the degradation of its mRNA, leading to enhanced phagocytosis of tumor-associated macrophages (TAMs). Specifically, the NSUN5-induced suppression of ß-catenin relies on its methyltransferase activity mediated by cysteine 359 (C359) and is not influenced by its localization in the nucleolus. Intriguingly, NSUN5 directly interacts with and deposits m5C on CTNNB1 caRNA (chromatin-associated RNA). NSUN5-induced recruitment of TET2 to chromatin is independent of its methyltransferase activity. The m5C modification on caRNA is subsequently oxidized into 5-hydroxymethylcytosine (5hmC) by TET2, which is dependent on its binding affinity for Fe2+ and α-KG. Furthermore, NSUN5 enhances the chromatin recruitment of RBFOX2 which acts as a 5hmC-specific reader to recognize and facilitate the degradation of 5hmC caRNA. Notably, hmeRIP-seq analysis reveals numerous mRNA substrates of NSUN5 that potentially undergo this mode of metabolism. In addition, NSUN5 is epigenetically suppressed by DNA methylation and is negatively correlated with IDH1-R132H mutation in glioma patients. Importantly, pharmacological blockage of DNA methylation or IDH1-R132H mutant and CD47/SIRPα signaling synergistically enhances TAM-based phagocytosis and glioma elimination in vivo. Our findings unveil a general mechanism by which NSUN5/TET2/RBFOX2 signaling regulates RNA metabolism and highlight NSUN5 targeting as a potential strategy for glioma immune therapy.

Integrated analysis of single-cell and bulk RNA sequencing data reveals the association between hypoxic tumor cells and exhausted T cells in predicting immune therapy response.

Continuous stimulation of tumor neoantigens and various cytokines in the tumor microenvironment leads to T cell dysfunction, but the specific mechanisms by which these key factors are distributed among different cell subpopulations and how they affect patient outcomes and treatment response are incompletely characterized. By integrating single-cell and bulk sequencing data of non-small cell lung cancer patients, we constructed a clinical outcome-associated T cell exhaustion signature. We discovered a significant association between the T cell exhaustion state and tumor cell hypoxia. Hypoxic malignant cells were significantly correlated with the proportion of exhausted T cells, and they co-occurred in patients at advanced stage. By analyzing the ligand-receptor interactions between these two cell states, we observed that T cells were recruited towards tumor cells through production of chemokines such as CXCL16-CXCR6 axis and CCL3/CCL4/CCL5-CCR5 axis. Based on 15 immune checkpoint blockade (ICB)-treatment cohorts, we constructed an interaction signature that can be used to predict the response to immune checkpoint blockade therapy. Among genes composed of the signature, CXCR6 alone has similarly high prediction efficacy (Area Under Curve (AUC) = 1, 0.89 and 0.73 for GSE126044, GSE135222 and GSE93157, respectively) with the signature and thus could serve as a potential biomarker for predicting immunotherapy response. Together, we have discovered and validated a significant association between exhausted T cells and hypoxic malignant cells, elucidating key interaction factors that significantly associated with response to immunotherapy.

High expression of VTA1 is an adverse prognostic factor in lung adenocarcinoma.

Lung adenocarcinoma (LUAD) is a common pathological type of non-small cell lung cancer; identifying preferable biomarkers has become one of the current challenges. Given that VTA1 has been reported associated with tumor progression in various human solid cancers but rarely reported in LUAD, herein, RNA sequencing data from TCGA and GTEx were obtained for analysis of VTA1 expression and differentially expressed gene (DEG). Furthermore, functional enrichment analysis of VTA1-related DEGs was performed by GO/KEGG, GSEA, immune cell infiltration analysis, and protein-protein interaction (PPI) network. In addition, the clinical significance of VTA1 in LUAD was figured out by Kaplan-Meier Cox regression and prognostic nomogram model. R package was used to analyze incorporated studies. As a result, VTA1 was highly expressed in various malignancies, including LUAD, compared with normal samples. Moreover, high expression of VTA1 was associated with poor prognosis in 533 LUAD samples, as well as T stage T2&T3&T4, N stage N1&N2&N3, M stage M1, pathologic stage II&III&IV, and residual tumor R1&R2, et al. (P < 0.05). High VTA1 was an independent prognostic factor in Cox regression analysis; Age and cytogenetics risk were included in the nomogram prognostic model. Furthermore, a total of 4232 DEGs were identified between the high- and the low-expression group, of which 736 genes were up-regulated and 3496 genes were down-regulated. Collectively, high expression of VTA1 is a potential biomarker for adverse outcomes in LUAD. The DEGs and pathways recognized in the study provide a preliminary grasp of the underlying molecular mechanisms of LUAD carcinogenesis and progression.

Pharmacogenomic Analysis of Combined Therapies against Glioblastoma Based on Cell Markers from Single-Cell Sequencing.

Glioblastoma is the most common and aggressive form of primary brain cancer and the lack of viable treatment options has created an urgency to develop novel treatments. Personalized or predictive medicine is still in its infancy stage at present. This research aimed to discover biomarkers to inform disease progression and to develop personalized prophylactic and therapeutic strategies by combining state-of-the-art technologies such as single-cell RNA sequencing, systems pharmacology, and a polypharmacological approach. As predicted in the pyroptosis-related gene (PRG) transcription factor (TF) microRNA (miRNA) regulatory network, TP53 was the hub gene in the pyroptosis process in glioblastoma (GBM). A LASSO Cox regression model of pyroptosis-related genes was built to accurately and conveniently predict the one-, two-, and three-year overall survival rates of GBM patients. The top-scoring five natural compounds were parthenolide, rutin, baeomycesic acid, luteolin, and kaempferol, which have NFKB inhibition, antioxidant, lipoxygenase inhibition, glucosidase inhibition, and estrogen receptor agonism properties, respectively. In contrast, the analysis of the cell-type-specific differential expression-related targets of natural compounds showed that the top five subtype cells targeted by natural compounds were endothelial cells, microglia/macrophages, oligodendrocytes, dendritic cells, and neutrophil cells. The current approach-using the pharmacogenomic analysis of combined therapies-serves as a model for novel personalized therapeutic strategies for GBM treatment.

Constructing an APOBEC-related gene signature with predictive value in the overall survival and therapeutic sensitivity in lung adenocarcinoma.

Background: APOBEC family play an important role in cancer mutagenesis and tumor development. The role of APOBEC family in lung adenocarcinoma (LUAD) has not been studied comprehensively. Materials and methods: The expression data of pan-cancer as well as LUAD was obtained from public databases. The expression level of APOBEC family genes was analyzed in different normal and cancer tissues. APOBEC mutagenesis enrichment score (AMES) was utilized to evaluate the APOBEC-induced mutations and the relation of APOBEC with genomic instability. Gene set enrichment analysis was used to identify differentially enriched pathways. Univariate Cox regression and Lasso regression were applied to screen key prognostic genes. The immune cell infiltration was estimated by CIBERSORT. RT-qPCR assay, CCK-8 and Transwell assay were conducted to explore gene expression and lung cancer cell invasion. Results: Cancer tissues had significantly altered expression of APOBEC family genes and the expression patterns of APOBEC family were different in different cancer types. APOBEC3B was the most aberrantly expressed in most cancer types. In LUAD, we observed a significantly positive correlation of AMES with intratumor heterogeneity (ITH), tumor neoantigen burden (TNB), and tumor mutation burden (TMB). High AMES group had high mutation counts of DNA damage repair pathways, and high enrichment of cell cycle and DNA repair pathways. We identified four prognostic genes (LYPD3, ANLN, MUC5B, and FOSL1) based on AMES, and constructed an AMES-related gene signature. The expressions of four genes were enhanced and accelerated the invasion ability and viability of lung cancer cells. Furthermore, we found that high group increased oxidative stress level. Conclusions: APOBEC family was associated with genomic instability, DNA damage-related pathways, and cell cycle in LUAD. The AMES-related gene signature had a great potential to indicate the prognosis and guide immunotherapy/chemotherapy for patients suffering from LUAD.

Long non-coding RNA AC018926.2 regulates palmitic acid exposure-compromised osteogenic potential of periodontal ligament stem cells via the ITGA2/FAK/AKT pathway.

Although obesity has been proposed as a risk factor for periodontitis, the influence of excessive fat accumulation on the development of periodontitis and periodontal recovery from disease remains largely unknown. This study investigated the cellular response of periodontal ligament stem cells (PDLSCs) to elevated levels of a specific fatty acid, namely, palmitic acid (PA). The mechanism by which PA exposure compromises the osteogenic potential of cells was also explored. It was found that exposure of PDLSCs to abundant PA led to decreased cell osteogenic differentiation. Given that long non-coding RNAs (lncRNAs) play a key role in the stem cell response to adverse environmental stimuli, we screened the lncRNAs that were differentially expressed in PDLSCs following PA exposure using lncRNA microarray analysis, and AC018926.2 was identified as the lncRNA that was most sensitive to PA. Next, gain/loss-of-function studies illustrated that AC018926.2 was an important regulator in PA-mediated osteogenic differentiation of PDLSCs. Mechanistically, AC018926.2 upregulated integrin α2 (ITGA2) expression and therefore activated ITGA2/FAK/AKT signalling. Further functional studies revealed that inactivation of ITGA2/FAK/AKT signalling by silencing ITGA2 counteracted the pro-osteogenic effect induced by AC018926.2 overexpression. Moreover, the results of bioinformatics analysis and RNA immunoprecipitation assay suggested that AC018926.2 might transcriptionally regulate ITGA2 expression by binding to PARP1 protein. Our data suggest that AC018926.2 may serve as a therapeutic target for the management of periodontitis in obese patients.

Therapeutic targeting m6A-guided miR-146a-5p signaling contributes to the melittin-induced selective suppression of bladder cancer.

Abnormal RNA methylation and dysregulation of miRNA are frequently occurred in bladder cancer. Melittin is a potential drug candidate for intravesical chemotherapy against bladder cancer. However, the underlying epigenetic mechanism by which melittin-induced anti-tumor effect remains unclear. Here, we showed that melittin selectively induced apoptosis of bladder cancer cells in a METTL3-dependent manner. Ectopic expression of METTL3 significantly blocked melittin-induced apoptosis in vitro and in vivo. MicroRNA-sequence analysis identified miR-146a-5p suppression contributed to the melittin-induced selective antitumor effect. Further investigation revealed that METTL3-guided m6A modification methylated pri-miR-146 at the flanking sequence, which was responsible for the pri-miR-146 maturation. Moreover, NUMB/NOTCH2 axis was identified as a downstream target signal that mediated the pro-survival role of miR-146a-5p in bladder cancer cells. Importantly, METTL3 and miR-146a-5p were positively correlated with recurrence and poor prognosis of patients with bladder cancer. Our study indicates that METTL3 acts as a fate determinant that controls the sensitivity of bladder cancer cells to melittin treatment. Moreover, METTL3/miR-146a-5p/NUMB/NOTCH2 axis plays an oncogenic role in bladder cancer pathogenesis and could be a potential therapeutic target for recurrent bladder cancer treatment.

Analysis of correlation factors influencing the outcome of initial 131I remnant ablative therapy in intermediate- to high-risk patients with papillary thyroid microcarcinoma.

OBJECTIVE: To investigate the factors influencing the outcome of initial 131I remnant ablative therapy in intermediate- to high-risk patients with papillary thyroid microcarcinoma (PTMC). METHODS: We divided 99 patients with PTMC who underwent total thyroidectomy into two groups according to their response to initial 131I remnant ablative therapy: excellent response (ER) and non-ER groups. Clinical and laboratory characteristics were collected and retrospectively analyzed using univariate and multivariate binary logistic regression. Receiver operator characteristic (ROC) curves and diagnostic cutoff values were analyzed to evaluate the predictive value of significant quantitative influencing factors for 131I treatment outcomes. A prognostic nomogram model based on the above independent risk factors was established. RESULTS: Of the 99 eligible patients who accepted the initial 131I treatment following total thyroidectomy, 76 (76.7%) were classified into the ER group and 23 (23.3%) into the non-ER group. The univariate and multivariate analyses showed that extrathyroidal extension [ETE; odds ratio (OR) = 4.769; P = 0.041], preablative thyrotropin (TSH; OR = 0.972; P = 0.017), and stimulated thyroglobulin (sTg; OR = 1.614; P = 0.040) were independent predictors for the therapeutic effect of 131I treatment. Patients with higher sTg (>1.37 ng/ml) and lower TSH (<67.97 mU/l) and ETE tended to have a poor response to initial 131I treatment. The quantification of the therapeutic effect of initial 131I therapy in patients with PTMC using our newly constructed nomogram showed that ETE, preablative sTg, and TSH were contributors to non-ER. CONCLUSION: Intermediate- to high-risk patients with PTMC after total thyroidectomy who had low pretreatment sTg and high preablative TSH levels and negative ETE were more likely to achieve satisfactory response to initial 131I remnant ablative therapy. Our prognostic nomogram is a valuable tool to enable patients and clinical professionals to be better informed about patients' therapeutic response to initial 131I remnant ablative therapy.

Bufalin targets the SRC-3/MIF pathway in chemoresistant cells to regulate M2 macrophage polarization in colorectal cancer.

M2-polarized macrophages are one of critical factors in tumour chemoresistance. An increasing number of studies have shown that M2 macrophage polarization can be promoted by chemoresistance. A large number of evidences indicate that Bufalin has significant antitumour effect, previous studies have found that Bufalin can reduce the polarization of M2 macrophages to play an anti-tumour effect in vivo, but the mechanism remains unclear. In our study, we found that Bufalin reduced the polarization of M2 macrophages induced by chemoresistant cells both in vivo and in vitro; however, Bufalin had no obvious direct effect on M2 macrophage polarization. Furthermore, we demonstrated that Bufalin targeted the SRC-3 protein to reduce MIF release in chemoresistant cells in order to regulate the polarization of M2 macrophages. More interestingly, we also found that Cinobufacini, Bufalin is its main active monomer, which its could regulate the polarization of M2 macrophages to enhance the anti-tumour effect of oxaliplatin in vivo and in the clinic. Overall, this study provides a theoretical basis for the clinical application of drugs containing Bufalin as the main active ingredient in combination with established chemotherapy for the treatment of colorectal cancer.

Periodontitis-compromised dental pulp stem cells secrete extracellular vesicles carrying miRNA-378a promote local angiogenesis by targeting Sufu to activate the Hedgehog/Gli1 signalling.

OBJECTIVES: Previously, our investigations demonstrated robust pro-angiogenic potentials of extracellular vesicles secreted by periodontitis-compromised dental pulp stem cells (P-EVs) when compared to those from healthy DPSCs (H-EVs), but the underlying mechanism remains unknown. MATERIALS AND METHODS: Here, circulating microRNAs (miRNAs) specifically found in P-EVs (compared with H-EVs) were identified by Agilent miRNA microarray analysis, and the roles of the candidate miRNA in P-EV-enhanced cell angiogenesis were confirmed by cell transfection and RNA interference methods. Next, the direct binding affinity between the candidate miRNA and its target gene was evaluated by luciferase reporter assay. CCK-8, transwell/scratch wound healing and tube formation assays were established to investigate the proliferation, migration, and tube formation abilities of endothelial cells (ECs). Western blot was employed to measure the protein levels of Hedgehog/Gli1 signalling pathway components and angiogenesis-related factors. RESULTS: The angiogenesis-related miRNA miR-378a was found to be enriched in P-EVs, and its role in P-EV-enhanced cell angiogenesis was confirmed, wherein Sufu was identified as a downstream target gene of miR-378a. Functionally, silencing of Sufu stimulated EC proliferation, migration and tube formation by activating Hedgehog/Gli1 signalling. Further, we found that incubation with P-EVs enabled the transmission of P-EV-contained miR-378a to ECs. Subsequently, the expressions of Sufu, Gli1 and vascular endothelial growth factor in ECs were significantly influenced by P-EV-mediated miR-378a transmission. CONCLUSIONS: These data suggest that P-EVs carrying miR-378a promote EC angiogenesis by downregulating Sufu to activate the Hedgehog/Gli1 signalling pathway. Our findings reveal a crucial role for EV-derived miR-378a in cell angiogenesis and hence offer a new target for modifying stem cells and their secreted EVs to enhance vessel regenerative potential.

An unusual case of reactivated latent pulmonary cryptococcal infection in a patient after short-term steroid and azathioprine therapy: a case report.

BACKGROUND: Cryptococcus is one of the major fungal pathogens infecting the lungs. Pulmonary cryptococcal infection is generally considered a community-acquired condition caused by inhalation of dust contaminated with fungal cells from the environment. Here, we report a case developing pulmonary cryptococcosis 3 months after hospital admission, which has rarely been reported before. CASE PRESENTATION: A 73-year-old female patient who was previously immunocompetent experienced persistent dry cough for 2 weeks, 3 months after admission. Chest computed tomography (CT) showed a new solitary pulmonary nodule developed in the upper lobe of the left lung. Staining and culture of expectorated sputum smears were negative for bacteria, acid-fast bacilli, or fungus. The patient then underwent biopsy of the lesion. Histopathology findings and a positive serum cryptococcal antigen titer (1:8) indicated pulmonary cryptococcosis. Daily intravenous 400 mg fluconazole was administered initially followed by oral fluconazole therapy. Follow-up chest CT after 3 months of antifungal therapy showed complete disappearance of the pulmonary nodule. Respiratory symptoms of the patient also resolved. A complete investigation excluded the possibility of a patient-to-patient transmission or primarily acquiring the infection from the hospital environment. Based on the patient's history of exposure to pigeons before admission and recent steroid and azathioprine use after admission for the treatment of myasthenic crisis, reactivation of a latent pulmonary cryptococcal infection acquired before admission, in this case, is impressed. CONCLUSIONS: Although rarely reported, pulmonary cryptococcal infection should be included in the differential diagnosis of hospitalized patients with respiratory symptoms, especially in those with predisposing risk factors. Chest image studies and further surgical biopsy are needed for confirmation.

Effect of ¹³¹I Therapy on Complete Blood Count in Patients with Differentiated Thyroid Cancer.

BACKGROUND The aim of this study was to investigate the effects of ¹³¹I therapy on complete blood count (CBC) in patients with differentiated thyroid cancer (DTC). MATERIAL AND METHODS We analyzed CBC in 542 patients with DTC who were grouped according to treatment cycles and cumulative dose and then subdivided by sex and age. The effects of ¹³¹I therapy among the different groups and subgroups were analyzed. RESULTS After sorting patients by treatment cycles and doses, ¹³¹I therapy was found to have different effects on CBC depending on patient sex and age. The effect on white blood cell (WBC) counts persisted longer in women, while increases in hemoglobin (Hb) were more significant in men. The influence on red blood cell (RBC) counts was short-lived in patients aged 45 to 54 years. Monocyte counts were significantly decreased only in patients aged 55 years and older who had undergone 3 or 4 treatment cycles. In men, CBC was more affected by cumulative dose. ¹³¹I therapy only influenced platelet and monocyte counts in patients aged 55 years or older. Hb was significantly decreased and increased in the high- and low-dose groups, respectively. No significant complications were observed during follow-up. CONCLUSIONS ¹³¹I therapy had a greater impact on WBC counts in women, while changes in RBC counts and Hb were more obvious in men. During ¹³¹I therapy, clinicians should pay attention to different CBC indicators based on a patient's sex and age, but risks associated with an altered CBC are unlikely to outweigh the benefits of 131I. The results of the present study may help alleviate the concerns of a large proportion of patients with DTC and their families about the effects of ¹³¹I therapy on CBC.

M2 Macrophages Enhance the Cementoblastic Differentiation of Periodontal Ligament Stem Cells via the Akt and JNK Pathways.

Although macrophage (Mφ) polarization has been demonstrated to play crucial roles in cellular osteogenesis across the cascade of events in periodontal regeneration, how polarized Mφ phenotypes influence the cementoblastic differentiation of periodontal ligament stem cells (PDLSCs) remains unknown. In the present study, human monocyte leukemic cells (THP-1) were induced into M0, M1, and M2 subsets, and the influences of these polarized Mφs on the cementoblastic differentiation of PDLSCs were assessed in both conditioned medium-based and Transwell-based coculture systems. Furthermore, the potential pathways and cyto-/chemokines involved in Mφ-mediated cementoblastic differentiation were screened and identified. In both systems, M2 subsets increased cementoblastic differentiation-related gene/protein expression levels in cocultured PDLSCs, induced more PDLSCs to differentiate into polygonal and square cells, and enhanced alkaline phosphatase activity in PDLSCs. Furthermore, Akt and c-Jun N-terminal Kinase (JNK) signaling was identified as a potential pathway involved in M2 Mφ-enhanced PDLSC cementoblastic differentiation, and cyto-/chemokines (interleukin (IL)-10 and vascular endothelial growth factor [VEGF]) secreted by M2 Mφs were found to be key players that promoted cell cementoblastic differentiation by activating Akt signaling. Our data indicate for the first time that Mφs are key modulators during PDLSC cementoblastic differentiation and are hence very important for the regeneration of multiple periodontal tissues, including the cementum. Although the Akt and JNK pathways are involved in M2 Mφ-enhanced cementoblastic differentiation, only the Akt pathway can be activated via a cyto-/chemokine-associated mechanism, suggesting that players other than cyto-/chemokines also participate in the M2-mediated cementoblastic differentiation of PDLSCs. Stem Cells 2019;37:1567-1580.

Modulating macrophage responses to promote tissue regeneration by changing the formulation of bone extracellular matrix from filler particles to gel bioscaffolds.

Extracellular matrices (ECMs) derived from native tissues/organs have been used as biomaterials for tissue engineering and regenerative medicine in a wide range of preclinical and clinical settings. The success or failure of these applications is largely contingent on the host responses to the matrices in vivo. Despite retaining their native structural and functional proteins, bone ECM-based transplants have been reported to evoke adverse immune responses in many cases; thus, optimizing the immunomodulatory properties of bone ECMs is critical for ensuring downstream regenerative outcomes. Using a simple digestion-neutralization protocol, we transformed the commonly used bone-derived filler particles into gel bioscaffolds. Instead of inducing macrophages toward proinflammatory (M1) polarization, as reported in the literature and confirmed in the present study for ECM particles, the ECM gels were found to be more likely to polarize macrophages toward regulatory/anti-inflammatory (M2) phenotypes, leading to enhanced tissue regeneration in a rat periodontal defect model. The present work demonstrates a simple, practical and economical strategy to modify the immunomodulatory properties of bone ECMs before their in vivo transplantation and hence has important implications that may facilitate the use of ECM-based bioscaffolds derived from diverse sources of tissues for regenerative purposes.

Building capacity for macrophage modulation and stem cell recruitment in high-stiffness hydrogels for complex periodontal regeneration: Experimental studies in vitro and in rats.

Recently, we found that although high-stiffness matrices stimulated osteogenic differentiation of bone marrow-derived stromal cells (BMSCs), the macrophages (Mφs) in high-stiffness transglutaminase crosslinked gelatins (TG-gels) tended to undergo M1 polarization and hence compromised cell osteogenesis. In this study, we hypothesized that the copresentation of interleukin (IL)-4 and stromal cell-derived factor (SDF)-1α in high-stiffness TG-gels may enhance periodontal regeneration by modulating Mφ polarization and promoting endogenous stem cell recruitment. We found that Mφs were more likely to polarize toward an immunomodulatory M2 state in the presence of IL-4 and hence positively influence the osteogenic differentiation of BMSCs when these cells coexisted in either indirect or direct co-culture systems. In cell migration assays, BMSCs exhibited an enhanced capability to move toward gels containing SDF-1α, and more cells could be recruited into the three-dimensional matrix of TG-gels. When TG-gels containing IL-4 and/or SDF-1α were used to repair periodontal defects, more new bone (MicroCT) was formed in animals that received the dual cytokine-loaded transplants at 4 weeks postsurgery. Mφs were recruited to all the transplanted gels, and after one week, more M1-phenotype cells were found in the groups without IL-4, while the presence of IL-4 was more likely to result in M2 polarization (immunofluorescence staining). When the tissue biopsies were histologically examined, the TG-gels containing both IL-4 and SDF-1α led to a generally satisfactory regeneration with respect to attachment recovery (epithelial and connective tissue) and hybrid tissue regeneration (bone, periodontal ligament and cementum). Our data suggest that the incorporation of IL-4 into high-stiffness TG-gels may promote the M2 polarization of Mφs and that SDF-1α can be applied to guide endogenous cell homing. Overall, building capacity for Mφ modulation and cell recruitment in high-stiffness hydrogels represents a simple and effective strategy that can support high levels of periodontal tissue regeneration. STATEMENT OF SIGNIFICANCE: The development of hydrogel-based regenerative therapies centered on the mobilization and stimulation of native cells for therapeutics opens a window toward realizing periodontal endogenous regeneration. In the present study, the parallel use of immunomodulatory and homing factors in high-stiffness hydrogel materials is shown to induce stem cell homing, modulate cell differentiation and indeed induce regrowth of the periodontium. We found that incorporation of interleukin (IL)-4 in high-stiffness TG-gels coaxed macrophages to polarize into M2 phenotypes, and stromal cell-derived factor (SDF)-1α could be applied to direct endogenous cell homing. Hence, we present for the first time a clinically relevant strategy based on macrophage modulation and host cell recruitment that can support high levels of periodontal tissue regeneration.

Macrophage involvement affects matrix stiffness-related influences on cell osteogenesis under three-dimensional culture conditions.

Accumulating evidence indicates that the physicochemical properties of biomaterials exert profound influences on stem cell fate decisions. However, matrix-based regulation selected through in vitro analyses based on a given cell population do not genuinely reflect the in vivo conditions, in which multiple cell types are involved and interact dynamically. This study constitutes the first investigation of how macrophages (Mφs) in stiffness-tunable transglutaminase cross-linked gelatin (TG-gel) affect the osteogenesis of bone marrow-derived mesenchymal stem cells (BMMSCs). When a single cell type was cultured, low-stiffness TG-gels promoted BMMSC proliferation, whereas high-stiffness TG-gels supported cell osteogenic differentiation. However, Mφs in high-stiffness TG-gels were more likely to polarize toward the pro-inflammatory M1 phenotype. Using either conditioned medium (CM)-based incubation or Transwell-based co-culture, we found that Mφs encapsulated in the low-stiffness matrix exerted a positive effect on the osteogenesis of co-cultured BMMSCs. Conversely, Mφs in high-stiffness TG-gels negatively affected cell osteogenic differentiation. When both cell types were cultured in the same TG-gel type and placed into the Transwell system, the stiffness-related influences of Mφs on BMMSCs were significantly altered; both the low- and high-stiffness matrix induced similar levels of BMMSC osteogenesis. Although the best material parameter for synergistically affecting Mφs and BMMSCs remains unknown, our data suggest that Mφ involvement in the co-culture system alters previously identified material-related influences on BMMSCs, such as matrix stiffness-related effects, which were identified based on a culture system involving a single cell type. Such Mφ-stem cell interactions should be considered when establishing proper matrix parameter-associated cell regulation in the development of biomimetic biomaterials for regenerative applications. STATEMENT OF SIGNIFICANCE: The substrate stiffness of a scaffold plays critical roles in modulating both reparative cells, such as mesenchymal stem cells (MSCs), and immune cells, such as macrophages (Mφs). Although the influences of material stiffness on either Mφs or MSCs, have been extensively described, how the two cell types respond to matrix cues to dynamically affect each other in a three-dimensional (3D) biosystem remains largely unknown. Here, we report our findings that, in a platform wherein Mφs and bone marrow-derived MSCs coexist, matrix stiffness can influence stem cell fate through both direct matrix-associated regulation and indirect Mφ-based modulation. Our data support future studies of the MSC-Mφ-matrix interplay in the 3D context to optimize matrix parameters for the development of the next biomaterial.

Human platelet lysate supports the formation of robust human periodontal ligament cell sheets.

The use of stem cell-derived sheets has become increasingly common in a wide variety of biomedical applications. Although substantial evidence has demonstrated that human platelet lysate (PL) can be used for therapeutic cell expansion, either as a substitute for or as a supplement to xenogeneic fetal bovine serum (FBS), its impact on cell sheet production remains largely unexplored. In this study, we manufactured periodontal ligament stem cell (PDLSC) sheets in vitro by incubating PDLSCs in sheet-induction media supplemented with various ratios of PL and FBS, i.e. 10% PL without FBS, 7.5% PL + 2.5% FBS, 5% PL + 5% FBS, 2.5% PL + 7.5% FBS or 10% FBS without PL. Cultures with the addition of all the designed supplements led to successful cell sheet production. In addition, all the resultant cellular materials exhibited similar expression profiles of matrix-related genes and proteins, such as collagen I, fibronectin and integrin ß1. Interestingly, the cell components within sheets generated by media containing both PL and FBS exhibited improved osteogenic potential. Following in vivo transplantation, all sheets supported significant new bone formation. Our data suggest that robust PDLSC sheets can be produced by applying PL as either an alternative or an adjuvant to FBS. Further examination of the relevant influences of human PL that benefit cell behaviour and matrix production will pave the way towards optimized and standardized conditions for cell sheet production.

The effects of conditioned media generated by polarized macrophages on the cellular behaviours of bone marrow mesenchymal stem cells.

Macrophages (Mφs) are involved in a variety of physiological and pathological events including wound healing and tissue regeneration, in which they play both positive and negative roles depending on their polarization state. In this study, we investigated the cellular behaviours of bone marrow mesenchymal stem cells (BMMSCs) after incubation in different conditioned media (CMs) generated by unpolarized Mφs (M0) or polarized Mφs (M1 and M2). Mφ polarization was induced by stimulation with various cytokines, and CMs were obtained from in vitro Mφ cultures termed CM0, CM1 and CM2 based on each Mφ phenotype. We found that CM1 supported the proliferation and adipogenic differentiation of BMMSCs, whereas CM0 had a remarkable effect on cell osteogenic differentiation. To a certain degree, CM2 also facilitated BMMSC osteogenesis; in particular, cells incubated with CM2 exhibited an enhanced capacity to form robust stem cell sheets. Although incubation with CM1 also increased production of extracellular matrix components, such as fibronectin, COL-1 and integrin ß1during sheet induction, the sheets generated by CM2-incubated cells were thicker than those generated by CM1-incubated cells (P < 0.001). Our data suggest that each Mφ phenotype has a unique effect on BMMSCs. Fine-tuning Mφ polarization following transplantation may serve as an effective method to modulate the therapeutic potential of BMMSCs.

Administration of signalling molecules dictates stem cell homing for in situ regeneration.

Ex vivo-expanded stem cells have long been a cornerstone of biotherapeutics and have attracted increasing attention for treating intractable diseases and improving tissue regeneration. However, using exogenous cellular materials to develop restorative treatments for large numbers of patients has become a major concern for both economic and safety reasons. Advances in cell biological research over the past two decades have expanded the potential for using endogenous stem cells during wound healing processes, and in particular, recent insight into stem cell movement and homing has prompted regenerative research and therapy based on recruiting endogenous cells. Inspired by the natural healing process, artificial administration of specific chemokines as signals systemically or at the injury site, typically using biomaterials as vehicles, is a state-of-the-art strategy that potentiates stem cell homing and recreates an anti-inflammatory and immunomodulatory microenvironment to enhance in situ tissue regeneration. However, pharmacologically coaxing endogenous stem cells to act as therapeutics in the field of biomedicine remains in the early stages; its efficacy is limited by the lack of innovative methodologies for chemokine presentation and release. This review describes how to direct the homing of endogenous stem cells via the administration of specific signals, with a particular emphasis on targeted signalling molecules that regulate this homing process, to enhance in situ tissue regeneration. We also provide an outlook on and critical considerations for future investigations to enhance stem cell recruitment and harness the reparative potential of these recruited cells as a clinically relevant cell therapy.