Regeneration of Mechanically Enhanced Tissue-Engineered Cartilage Based on the Decalcified Bone Matrix Framework.

Human decalcified bone matrix (HDBM) is a framework with a porous structure and good biocompatibility. Nevertheless, its oversized pores lead to massive cell loss when seeding chondrocytes directly over it. Gelatin (GT) is a type of protein obtained by partial hydrolysis of collagen. The GT scaffold can be prepared from the GT solution through freeze-drying. More importantly, the pore size of the GT scaffold can be controlled by optimizing the concentration of the GT solution. Similarly, when different concentrations of gelatin are combined with HDBM and then freeze-dried, the pore size of the HDBM can be modified to different degrees. In this study, the HDBM framework was modified with 0.3, 0.6, and 0.9%GT, resulting in an improved pore size and adhesion rate. Results showed that the HDBM framework with 0.6%GT (HDBM-0.6%GT) had an average pore size of 200 µm, which was more suitable for chondrocyte seeding. Additionally, our study validated that porcine decalcified bone matrix (PDBM) had a proper pore structure. Chondrocytes were in vitro seeded on the three frameworks for 4 weeks and then implanted in nude mice and autologous goats, respectively. The in vivo cartilage regeneration results showed that HDBM-0.6%GT and PDBM frameworks compensated for the oversized pores of the HDBM framework. Moreover, they showed successfully regenerated more mature cartilage tissue with a certain shape in animals.

Regeneration of articular cartilage defects: Therapeutic strategies and perspectives.

Articular cartilage (AC), a bone-to-bone protective device made of up to 80% water and populated by only one cell type (i.e. chondrocyte), has limited capacity for regeneration and self-repair after being damaged because of its low cell density, alymphatic and avascular nature. Resulting repair of cartilage defects, such as osteoarthritis (OA), is highly challenging in clinical treatment. Fortunately, the development of tissue engineering provides a promising method for growing cells in cartilage regeneration and repair by using hydrogels or the porous scaffolds. In this paper, we review the therapeutic strategies for AC defects, including current treatment methods, engineering/regenerative strategies, recent advances in biomaterials, and present emphasize on the perspectives of gene regulation and therapy of noncoding RNAs (ncRNAs), such as circular RNA (circRNA) and microRNA (miRNA).

Targeting macrophage polarization as a promising therapeutic strategy for the treatment of osteoarthritis.

Osteoarthritis (OA) is a chronic osteoarthropathy characterized by the progressive degeneration of articular cartilage and synovial inflammation. Early OA clinical treatments involve intra-articular injection of glucocorticoids, oral acetaminophen and non-steroidal anti-inflammatory drugs (NSAIDs), which are used for anti-inflammation and pain relief. However, long-term use of these agents will lead to inevitable side effects, even aggravate cartilage loss. At present, there are no disease-modifying OA drugs (DMOADs) yet approved by regulatory agencies. Polarization regulation of synovial macrophages is a new target for OA treatment. Inhibiting M1 polarization and promoting M2 polarization of synovial macrophages can alleviate synovial inflammation, relieve joint pain and inhibit articular cartilage degradation, which is a promising strategy for OA treatment. In this study, we describe the molecular mechanisms of macrophage polarization and its key role in the development of OA. Subsequently, we summarize the latest progress of strategies for OA treatment through macrophage reprogramming, including small molecule compounds (conventional western medicine and synthetic compounds, monomer compounds of traditional Chinese medicine), biomacromolecules, metal/metal oxides, cells, and cell derivatives, and interprets the molecular mechanisms, hoping to provide some information for DMOADs development.

Assessment of mandibular retromolar space in adults with regard to third molar eruption status.

OBJECTIVE: The aim of this study was to compare the difference in length and width of the mandibular retromolar space (RMS) stratified by the different eruption and impaction statuses of the third molars in patients with skeletal Class I malocclusion. MATERIALS AND METHODS: The right mandibular RMS in 186 adult patients categorized according to the different statuses of the third molar was analyzed by using cone-beam computed tomography (CBCT). The shortest distances between the inner lingual cortex of the mandibular body and second molar root were measured parallel to the posterior occlusal line (POL) at depths of 2, 4, 6, 8, and 10 mm (mandibular retromolar space length in root level, RLin2,4,6,8,10) on the axial slices with the cementoenamel junction (CEJ) as the reference level. The width of the RMS and second molar root was measured vertical to the POL at the terminal point of the molar distalization at depths of 2, 4, 6, 8, and 10 mm (width of the mandibular retromolar space, BW2,4,6,8,10/ width of the second molar distal root, TW2,4,6,8,10) from the CEJ. RESULTS: RL in different measurement planes was 2.72 ± 2.22 ~ 3.74 ± 2.26 for Group A, 5.27 ± 1.68 ~ 9.10 ± 2.04 for Group B, 1.94 ± 2.34 ~ 5.71 ± 4.37 for Group C, 1.83 ± 2.95 ~ 5.05 ± 4.24 for Group D, and 5.93 ± 3.97 ~ 10.52 ± 2.16 for Group E. The BW measurement results for A ~ E group were 9.71 ± 1.41 ~ 10.51 ± 1.81, 9.83 ± 1.39 ~ 12.55 ± 2.11, 9.96 ± 1.21 ~ 12.17 ± 1.62, 9.82 ± 1.47 ~ 12.28 ± 2.77, and 10.02 ± 1.20 ~ 12.75 ± 0.82, respectively. There was no significant difference between men and women in any measurements (P > 0.05). Patients with normal third molars erupted and those vertically impacted possessed larger RMS lengths than those in which the third molars were missing, horizontally impacted or mesially impacted (P < 0.05). In each measurement plane, TW was significantly smaller than BW (P < 0.05). CONCLUSIONS: Sex had no effect on the length or width of the mandibular RMS. Different statuses of third molars can also differentially affect the mandibular RMS. The mandibular RMS width is not a limit for mandibular molar distalization. CLINICAL RELEVANCE: When considering the distalization of mandibular molars, more attention should be directed to the lingual cortex of the mandible, and CBCT scans are recommended for patients who require significant mandibular molar distalization. The mandible buccal shelf and retromolar area maybe a safe zone to insert the miniscrew for molar distalization.

Analysis of regional vegetable circulation efficiency and its spatial effect considering carbon emission.

In order to explore the evolution process of regional vegetable circulation efficiency and its influencing factors, this paper uses Super-SBM model considering unexpected output and GML index to calculate the vegetable circulation efficiency of 13 provinces (cities) in Central China and its surrounding areas from 2015 to 2019, then conducts spatial autocorrelation analysis on the vegetable circulation efficiency in this region through Moran index. Finally, SPDM model is constructed to explore the spatial effect of the influencing factors on the vegetable circulation efficiency in this region. The results show that (1) the vegetable circulation efficiency of most provinces (cities) in this region is low. (2) The Global Moran's I of the vegetable circulation efficiency in this region is positive, that is, the vegetable circulation efficiency in this region shows a certain degree of spatial agglomeration effect. (3) The level of scientific and technological innovation and the degree of government support have significant positive direct and indirect effects on the efficiency of vegetable circulation in the region, the quality of workers has significant positive indirect effects, and the level of economic development and industrial structure have significant negative indirect effects.

Surgery-first treatment of a skeletal Class III malocclusion with a completely digital workflow.

Completely digital design/completely digital manufacturing (CDD/CDM) workflows have been widely used in orthodontic and orthognathic treatments. This case report introduces a CDD/CDM workflow consisting of clear aligners and virtual planning for a surgery-first approach (SFA) in a patient with a skeletal Class III malocclusion. Following a shortened treatment time of 5 months, the patient's facial appearance improved significantly, and well-balanced occlusion was obtained. SFAs with clear aligners can enable patients to achieve complete esthetic satisfaction during the therapeutic period. The CDD/CDM workflow provided accurate results, improved the clinical outcome, and reduced treatment time.

The orphan GPR50 receptor interacting with TßRI induces G1/S-phase cell cycle arrest via Smad3-p27/p21 in BRL-3A cells.

The liver has the powerful capacity to regenerate after injury or resection. In one of our previous studies, GPR50 was observed to be significantly upregulated at 6 h, following a partial hepatectomy (PH) in rat liver regeneration (LR) via gene expression profile. However, little research has been done on the regulation and mechanism of GPR50 in the liver. Herein, we observed that the overexpression of GPR50 inhibited the proliferation of BRL-3A cells. To further explore the molecular mechanisms of GPR50 in the regulation of BRL-3A cell proliferation, interaction between GPR50 and transforming growth factor-beta I (TßRI) and iTRAQTM differential proteomic analysis were elucidated, which suggested that GPR50 may interact with TßRI to activate the TGF-ß signaling pathway and arrest BRL-3A cell cycle G1/S transition. Subsequently, the potential mechanism underlying the role of GPR50 in hepatocyte growth was also explored through the addition of a signaling pathway inhibitor. These data suggested that interaction between the orphan GPR50 receptor and TßRI induced the G1/S-phase cell cycle arrest of BRL-3A cells via the Smad3-p27/p21 pathway.

AMPK-mTOR-ULK1-mediated autophagy protects carbon tetrachloride-induced acute hepatic failure by inhibiting p21 in rats.

Autophagy is a lysosomal-dependent degradation pathway in eukaryotic cells. Recent studies have reported that autophagy can facilitate the activation of hepatic stellate cells (HSCs) and fibrogenesis of the liver during long-term carbon tetrachloride (CCl4) exposure. However, little is known about the role of autophagy in CCl4-induced acute hepatic failure (AHF). This study aimed to identify whether modulation of autophagy can affect CCl4-induced AHF and evaluate the upstream signaling pathways mediated by CCl4-induced autophagy in rats. The accumulation of specific punctate distribution of endogenous LC3-II, increased expression of LC3-II, Atg5, and Atg7 genes/proteins, and decreased expression of p62 gene were observed after acute liver injury was induced by CCl4 in rats, indicating that CCl4 resulted in a high level of autophagy. Moreover, loss of autophagic function by using chloroquine (CQ, an autophagic inhibitor) aggravated liver function, leading to increased expression of p21 (a cyclin-dependent kinase inhibitor) in CCl4-treated rats. Furthermore, the AMPK-mTORC1-ULK1 axis was found to serve a function in CCl4-induced autophagy. These results reveal that AMPK-mTORC1-ULK1 signaling-induced autophagy has a protective role in CCl4-induced hepatotoxicity by inhibiting the p21 pathway. This study suggests a useful strategy aimed at ameliorating CCl4-induced acute hepatotoxicity by autophagy.

A combined proteomic and metabolomic analyses of the priming phase during rat liver regeneration.

By comparing differentially abundant proteins and metabolites, the protein expression, metabolic changes and metabolic regulation mechanisms during the priming phase of liver regeneration (LR) were investigated. We combined proteomic analysis via isobaric tags for relative and absolute quantification (iTRAQ) with metabolomic analysis via nontargeted liquid chromatography-mass spectrometry (LC-MS). LC-MS was used to examine 29 energy metabolites expression alterations in targeted metabolomics. A total number of 441 differentially expressed proteins and 65 metabolites were identified. PSMB10, PSMB5, RCG_63409, PSME4 and PSMB7 were key node proteins, these proteins are involved in the proteasome pathway. The most strongly enriched transcription factor motif was TP63. These results point out a critical role of the proteasome pathway (defense mechanisms) and of TP63 (metabolic regulator) as the key transcription factor during the priming phase of LR. Metabolomic and metabolite analysis showed that profiling indicates upregulation of arginine biosynthesis and glycolysis as the main ATP-delivering pathway. Integrative proteomic and metabolomic analysis showed that biomolecular changes were primarily related to the neurological disease, cell death and survival and cell morphology. What's more, neurotransmitters may play an important role in the regulation of LR.

circRNA-14723 promotes hepatocytes proliferation in rat liver regeneration by sponging rno-miR-16-5p.

Circular RNA (circRNA) is a subclass of noncoding RNA (ncRNA) detected within mammalian tissues and cells. However, its regulatory role during the proliferation phase of rat liver regeneration (LR) remains unreported. This study was designed to explore their regulatory mechanisms in cell proliferation of LR. The circRNA expression profile was detected by high-throughput sequencing. It was indicated that 260 circRNAs were differentially expressed during the proliferation phase of rat LR. Among them, circ-14723 displayed a significantly differential expression. We further explored its regulatory mechanism in rat hepatocytes (BRL-3A cells). First, EdU, flow cytometry and western blot (WB) indicated that knocking down circ-14723 inhibited BRL-3A cells proliferation. Second, RNA-Pulldown and dual-luciferase report assay showed that circ-14723 could sponge rno-miR-16-5p. At last, WB showed that the reported target genes of rno-miR-16-5p, CCND1, and CCNE1 were downregulated after knocking down circ-14723. In conclusion, we found that circ-14723 exerted a critical role in G1/S arrest to promote cell proliferation via rno-miR-16-5p/CCND1 and CCNE1 axis in rat LR. This finding further revealed the regulatory mechanisms of circRNA on cell proliferation of LR, and might provide a potential target for clinical problems.

lncRNA Expression Reveals the Potential Regulatory Roles in Hepatocyte Proliferation during Rat Liver Regeneration.

Liver regeneration is a tissue growth process after loss or injury of liver tissue, which is a compensatory hyperplasia rather than true regeneration, mainly depending on hepatocyte proliferation. Currently, a large number of studies on hepatocyte proliferation have been conducted. However, studies on the regulation of long noncoding RNA (lncRNA) on hepatocyte proliferation are still limited. To identify specially expressed lncRNA during rat liver regeneration, high-throughput sequencing technology was performed, and a total of 2446 lncRNAs and 4091 mRNAs were identified as significantly differentially expressed. Gene ontology (GO) enrichment analysis was performed to analyze the role of differentially expressed mRNAs, and 695 mRNAs were identified to be related to cell proliferation. Then, an lncRNA-mRNA coexpression network based on the differentially expressed lncRNAs and proliferation-related genes was constructed to analyze the potential function of lncRNAs on hepatocyte proliferation, and ten lncRNAs, NONRATT003557.2, NONRATT005357.2, NONRATT003292.2, NONRATT001466.2, NONRATT003289.2, NONRATT001047.2, NONRATT005180.2, NONRATT004419.2, NONRATT005336.2, and NONRATT005335.2, were selected as key regulatory factors, which may play crucial roles in hepatocyte proliferation during rat liver regeneration. Finally, a protein-protein interaction (PPI) network was established to illuminate the interaction between proliferation-related genes, and ten hub genes (Aurkb, Cdk1, Cdc20, Bub1b, Mad2l1, Kif11, Prc1, Ccna2, Top2a, and Ccnb1) were screened with the MCC method in the PPI network, which may be important biomarkers involved in the hepatocyte proliferation during rat liver regeneration. These results may provide clues for a more comprehensive understanding of the molecular mechanism of hepatocyte proliferation during rat liver regeneration.

An integrated analysis of the circRNA-miRNA-mRNA network reveals novel insights into potential mechanisms of cell proliferation during liver regeneration.

Cell proliferation constitutes the fundamental process and driving force behind regrowth during liver regeneration (LR). However, it remains unclear how competing endogenous RNA (ceRNA) networks affect hepatocyte proliferation and liver regeneration. Therefore, this study was designed to explore an LR-specific ceRNA network, which regulates cell proliferation. Based on the microarray data of mRNAs, and high-throughput sequencing data of miRNAs and circRNAs from regenerating livers, this study initially applied known 1484 LR associated mRNAs to perform GO analysis, and then selected 169 LR associated mRNAs involved in cell proliferation and the cell cycle. Subsequently, 188 interactive miRNA-mRNA pairs and 5206 circRNA-miRNA pairs, respectively, were predicted using bioinformatics methods. Next, in view of the differential expressions of these ceRNAs during LR, 26 miRNA-mRNA pairs and 71 circRNA-miRNA pairs were applied to generate a circRNA-miRNA-mRNA regulatory network, and only 14 triple interactive groups were obtained based on the predicted inverse interactions among ceRNAs. Finally, circ_19698/miR-423-5p axis was demonstrated to promote cell proliferation by modulating the expression of MYC, CCNA2, and CCND1 in rat BRL-3A cells. This study suggests a potential regulatory mechanism of cell proliferation in regenerating livers, as well as a novel pathway for modulating ceRNA networks to promote liver regeneration.

Large-scale quantitative genomics analyzes the circRNA expression profile and identifies the key circRNA in regulating cell proliferation during the proliferation phase of rat LR.

Researchers have been exploring the genetic mechanisms underlying the control of liver regeneration (LR). However, an integrated analysis of circRNAs expression of rat regenerating livers during the proliferation phase has not been performed yet. For this purpose, circRNAs expression profile was globally analyzed by high-throughput sequencing. It showed that 10,003 circRNAs were detected, and 164 circRNAs were differentially expressed. Subsequently, 27 circRNAs were predicted to bind to 58 candidate miRNAs and compete for miRNA-binding sites with 2195 mRNAs. By applying GO and KEGG analysis, it was predicted that these circRNAs significantly participated in tissue regeneration, regulation of cell proliferation and Ras, p53, Wnt, Jak-STAT, MAPK signalling pathways. Based on the number of the corresponding miRNAs and their role enriched and reported in cell proliferation of LR or hepatocellular carcinoma, four kinds of circRNAs (circ_03848, circ_08236, circ_13398 and circ_15013) were considered as the key circRNAs. The predicted competing endogenous RNA networks and bioinformatics analysis revealed the potential role of these circRNAs in LR, which would provide useful information for understanding the mechanism of LR.

Comprehensive analysis of lncRNA-miRNA-mRNA during proliferative phase of rat liver regeneration.

This study aims to reveal the regulatory mechanism of lncRNAs-miRNAs-mRNAs network during the proliferative phase of liver regeneration (LR). High-throughput sequencing technology was performed, and a total of 1,738 differentially expressed lncRNAs (DE lncRNAs), 167 known differentially expressed miRNAs (DE miRNAs), and 2,727 differentially expressed mRNAs were identified. Then, the target DE lncRNAs and DE mRNAs regulated by the same miRNAs were screened and a ceRNA regulatory network containing 32 miRNAs, 107 lncRNAs, and 270 mRNAs was constructed. Insulin signaling pathway, pyrimidine metabolism, axon guidance, carbohydrate digestion and absorption, and pyruvate metabolism were significantly enriched in the network. Through literature review and the regulatory relationship between lncRNAs and miRNAs, nine core lncRNAs were identified, which might play important roles during the proliferative phase of rat LR. This study analyzed lncRNA-miRNA-mRNA regulatory network for the first time during the proliferative phase of rat LR, providing clues for exploring the mechanism of LR and the treatment of liver diseases.

Comparison of gene expression in liver regeneration and hepatocellular carcinoma formation.

BACKGROUND: Liver -cell proliferation occurs in hepatocellular carcinoma (HCC) and liver regeneration (LR). The development and progression of HCC and LR have many similar molecular pathways with very different results. In simple terms, LR is a controllable process of organ recovery and function reconstruction, whereas liver cancer is uncontrollable. Do they share common key pathways and genes? METHODS: In this study, the dynamic transcriptome profile at ten time points (0, 2, 6, 12, 24, 30, 36, 72, 120, and 168 hours) during LR in rats after two-thirds hepatectomy and eight stages (normal, cirrhosis without HCC, cirrhosis, low-grade dysplastic, high-grade dysplastic, and very early, early advanced, and very advanced HCC) representing a stepwise carcinogenic process from preneoplastic lesions to end-stage HCC were analyzed in detail. A variety of bioinformatic methods, including MaSigPro, weighted gene-coexpression network analysis, and spatial analysis of functional enrichment, were used to analyze, elucidate, and compare similarities and differences between LR and HCC formation. RESULTS: Key biological processes and genes were identified. From the comparison, we found that cell proliferation and angiogenesis were the most significantly dysregulated processes shared by LR and HCC. The pattern of cell-proliferation-related gene expression in progression stage during LR is similar to the transition process from dysplasia to early-stage HCC. LR and HCC showed different expression patterns as a whole. Some key genes, including FYN, XPO1, FOXM1, EZH2, and NRF1, were identified as playing critical roles in both LR and HCC. CONCLUSION: These findings could contribute to revealing the molecular mechanism of development and regulation mechanism of normal and abnormal proliferation, which could provide new ideas and treatment methods for regenerative medicine, oncological drug development, and oncological treatment.

In silico analysis of expression data during the early priming stage of liver regeneration after partial hepatectomy in rat.

The priming stage is the first step of liver regeneration (LR). This stage is characterized by the transition from G0 to cell cycle for 4 hours in rat. In this study, individual gene level and gene set level (GSEA) was performed to identify the candidate genes and significantly changed biological processes at 2 h after partial hepatectomy (PH). The leading edge analysis is performed to identify the key genes and iRegulon was employed for transcription factor (TF) analysis. A total of 53 differentially expressed genes were identified using RMA package based on R language at 2 h after PH, including the transcription factor, enzyme and cytokine. As the most important genes in our analysis, Socs3 was selected with a special analysis so as to find the pathways correlate to the expression of it. The changed significantly pathways in LR involved response to stress, ATP metabolism, and regulation of cell cycle mainly. Several transcription factors were identified including Stat5a, Cnot3 and zfp384. Taken together, at the early priming stage of LR in rat, the liver is experiencing some changes including response to stress, activated ATP metabolism and inhibition of cell cycle. Our analysis provided a detailed and comprehensive map for further research of the early priming stage of LR in rat.

Expression analysis on 14-3-3 proteins in regenerative liver following partial hepatectomy

Abstract 14-3-3 proteins play a vital part in the regulation of cell cycle and apoptosis as signaling integration points. During liver regeneration, the quiescent hepatocytes go through hypertrophy and proliferation to restore liver weight. Therefore, we speculated that 14-3-3 proteins regulate the progression of liver regeneration. In this study, we analyzed the expression patterns of 14-3-3 proteins during liver regeneration of rat to provide an insight into the regenerative mechanism using western blotting. Only four isoforms (γ, ε, σ and τ/θ) of the 14-3-3 proteins were expressed in regenerative liver after partial hepatectomy (PH). The dual effects, the significant down-regulation of 14-3-3ε and the significant up-regulation of 14-3-3τ/θ at 2 h after PH, might play particularly important roles in S-phase entry. The significant peaks of 14-3-3σ at 30 h and of ε and τ/θ at 24 h might be closely related not only to the G2/M transition but also to the size of hepatocytes. Possibly, the peak of 14-3-3ε expression seen at 168 h plays critical roles in the termination of liver regeneration by inhibiting cellular proliferation.

Expression analysis on 14-3-3 proteins in regenerative liver following partial hepatectomy.

14-3-3 proteins play a vital part in the regulation of cell cycle and apoptosis as signaling integration points. During liver regeneration, the quiescent hepatocytes go through hypertrophy and proliferation to restore liver weight. Therefore, we speculated that 14-3-3 proteins regulate the progression of liver regeneration. In this study, we analyzed the expression patterns of 14-3-3 proteins during liver regeneration of rat to provide an insight into the regenerative mechanism using western blotting. Only four isoforms (γ, ε, σ and τ/θ) of the 14-3-3 proteins were expressed in regenerative liver after partial hepatectomy (PH). The dual effects, the significant down-regulation of 14-3-3ε and the significant up-regulation of 14-3-3τ/θ at 2 h after PH, might play particularly important roles in S-phase entry. The significant peaks of 14-3-3σ at 30 h and of ε and τ/θ at 24 h might be closely related not only to the G2/M transition but also to the size of hepatocytes. Possibly, the peak of 14-3-3ε expression seen at 168 h plays critical roles in the termination of liver regeneration by inhibiting cellular proliferation.

[Three novel genes BM390716, BI274487 and AA963863 involed in extracellular matrix metabolism of eight rat regenerating liver cell types].

To explore the roles of three novel genes BM390716, BI274487 and AA963863 and the correlation between them during liver regeneration of rats, eight kinds of liver cells were isolated using the combined percoll density gradient centrifugation and immunomagnetic bead method. Rat genome 230 2.0 array was used to detect the changes in expression of genes involved in metabolism of extracellular matrix and the novel genes in rat genome. Correlation between sequence homology, co-expression of the above genes and the physiological activities they involed in were analyzed using Microsoft Excel and BLAST software. The results showed that BM390716 was homologous to and co-expressed with pparα, BI274487 was homologous to and co-expressed with timp2, and AA963863 was homologous to and co-expressed with csgalnact1. It is predicted that BM390716, BI274487, and AA963863 were involved in extracellular matrix metabolism in eight types of rat regenerating liver cells.