Optimisation of primary osteoblast cell culture from suckling mouse / 基础医学与临床

Objective To develop a suitable medium and optimize culture time for the primary osteoblast culture from suckling mouse,so to provide an improved experimental protocol for primary osteoblast culture in vitro.Meth-ods Primary osteoblasts were collected from skull of CD1 suckling mouse by interrupted enzyme digestion.The pu-rified osteoblasts were harvested by differential centrifugation.The incubation time,concentration of fetal bovine se-rum(FBS),β-glycerophosphate sodium and dexamethasone were tested and optimized.The change of osteoblast maturation marker was examined by Western blot(WB)and immunofluorescence staining(IF).The osteogenic ac-tivity was determined by alkaline phosphatase staining,alizarin red staining and ultrastructure.Results Primary osteoblast were obtained from sucleling mouse skull bone by interrupted enzyme digestion for proliferation and trans-generational expansion.The expression of osteoblast maturation markers was parallel to the time of induction culture and the concentration of FBS.Mature osteoblasts were obtained by culturing the cells with 10％ FBS for 14 days.The differentiation of primary osteoblasts was induced by different concentrations of β-glycerophosphate and dexam-ethasone.The results showed that the expression of osteoblast maturation markers was higher under the culture con-ditions of 10 mmol/L β-glycerophosphate and 5 nmol/L dexamethasone(P<0.01),and the staining of alkaline phosphatase and alizarin red was obvious,and the osteogenic activity was better too.Conclusions Primary osteo-blasts isolated from the skull of suckling CD1 mice cultured in induction medium containing 10％fetal bovine ser-um,10 mmol/L β-glycerophosphate sodium and 5 nmol/L dexamethasone for 14 days show good osteogenic activity and are suitable for in vitro experimental studies.

PD98059 Inhibits the Proliferation and Differentiation of Osteoblasts in Rat Otocyst / 听力学及言语疾病杂志

Objective To investigate the effect of ERK inhibitor PD98059 on the proliferation and differentia-tion of rat otocyst osteoblasts.Methods SD neonatal rat osteoblasts were extracted by two-step digestion with 0.25％pancreatin and type Ⅰ collagenase,and co-cultured with ERK inhibitor PD98059 at concentrations of 0 μmol/L,10 μmol/L,25 μmol/L and 50 μmol/L,respectively.Then,the osteoblasts proliferation of the four groups were assessed by EDU method for 4 consecutive days.The proliferation trend of each group was compared and analyzed.Osteoblasts were differentiated by β-sodium glycerophosphate,L-vitamin C and dexamethasone at concentrations of 10 mmol/L,50 ug/ml and 10-7 mol/L.After 24 h,the mRNA expression levels of osteogenic fac-tors which include Ocn,Bsp,Runx2,Bmp2,OPG and RANKL in each group were detected by RT-qPCR,and the differences of the results were analyzed.Results All the concentrations of ERK inhibitor PD98059 could inhibit the proliferation of osteoblasts in SD neonatal rat,and the inhibitory effect of PD98059 at concentrations of 10 μmol/L was significantly greater than that of the other three groups(P＜0.05).In addition,all the concentrations of PD98059 could inhibit the expressions of Ocn,Bsp,Runx2,Bmp2 and OPG mRNA.The mRNA expressions of Ocn,Bsp,Runx2 and Bmp2 in 10 μmol/L PD98059 group were significantly lower than those in 0 μmol/L,25μmol/L and 50 μmol/L PD98059 groups(P＜0.05).The mRNA expressions of OPG in 10 and 25 μmol/L PD98059 groups were significantly lower than those in 0 and 50 μmol/L PD98059 groups(P＜0.05),and there was no significant difference between the first two groups(P＞0.05).The CT value of RANKL mRNA was not detec-ted in all groups.Conclusion ERK pathway inhibitor PD98059 can both inhibit the proliferation and differentiation of osteoblasts in rat otocyst.Therefore,we speculate that ERK1/2-MAPK pathway may mediate the formation of tympanosclerosis by affecting the proliferation and differentiation of rat otocyst osteoblasts.

Corylin in Treatment of Metabolic Bone Diseases： A Review / 中国实验方剂学杂志

Bone is a dynamic tissue undergoing continuous regeneration and reconstruction， and its metabolic activities are mainly regulated by bone formation mediated by osteoblasts and bone resorption mediated by osteoclasts. In addition， a variety of cells such as adipocytes， inflammatory cells， endothelial cells， and nerve cells can affect bone metabolism by changing the bone marrow microenvironment. The incidence of bone metabolic diseases caused by bone metabolism disorders is increasing with aging of the population. At present， the clinical treatment of bone metabolic diseases has the disadvantages of long cycle， high cost and many side effects. Therefore， there is an urgent need for safe and effective prevention and treatment drugs. Corylin is an isoflavonoid extracted from Psoraleae Fructus， which has a variety of pharmacological effects such as anti-inflammatory， anti-oxidation， anti-tumor， anti-atherosclerosis， attenuating obesity and improving insulin resistance. Studies have shown that corylin not only exerts osteoprotective effects by promoting osteoblast differentiation and inhibiting osteoclast differentiation， but also plays a positive role in bone metabolism by regulating lipid metabolism， inflammatory response， angiogenesis and anti-aging. The current review overviews the effects and mechanisms of corylin on regulating bone metabolism directly or indirectly， hoping to open up a new perspective for the prevention and treatment of osteoporosis， fracture， osteoarthritis and other related diseases.

Molecular mechanism of sulforaphane promoting the differentiation of bone marrow stem cells into osteoblasts / 局解手术学杂志

Objective To investigate the molecular mechanism of sulforaphane(Sul)promoting bone marrow stem cells(BMSCs)differentiating into osteoblasts.Methods BMSCs were divided into the control group(without any treatment),induction group(induction of osteogenic differentiation),and induction+Sul group(induction of osteogenic differentiation with the addition of 40 μmol/L of Sul).The adenovirus-shRNA-Mock,-shRNA-TET1,-shRNA-TET2,and-shRNA-TET3 were transfected into BMSCs as the shRNA-Mock group,shRNA-TET1 group,shRNA-TET2 group,and shRNA-TET3 group.BMSCs were cultured in cell culture medium containing osteogenic differentiation induction medium and 40 μmol/L of Sul,and then transfected with adenovirus-shRNA-TET1,-shRNA-TET2,-shRNA-TET3,and-shRNA-Mock as the induction+Sul+shRNA-TET1 group,induction+Sul+shRNA-TET2 group,induction+Sul+shRNA-TET3 group,and induction +Sul+shRNA-Mock group.The mRNA and protein expression levels of Runx2 after BMSCs differentiated into osteoblasts were determined by qPCR and Western blot.The DNA content of Runx2 promoter region bound to Histone H3 after BMSCs differentiated into osteoblasts was determined by chromatin immunocoprecipitation(ChIP).The methylation level of Runx2 promoter region of BMSCs differentiated into osteoblasts was determined by HpaⅡenzyme and MspⅠenzyme digestion combined with qPCR.The degree of BMSCs differentiated into osteoblasts was determined by alizarin red staining.Results Compared with the induction group,the mRNA and protein expression levels of Runx2 in the induction+Sul group were significantly increased(P<0.05);the content of DNA in the Runx2 promoter region bound to Histone H3 was increased(P<0.05),the methylation level of Runx2 promoter region was reduced(P<0.05),and the alizarin red staining score was elevated(P<0.05).Compared with the induction+Sul group,the content of DNA in the Runx2 promoter region bound to Histone H3 in the induction+Sul+shRNA-TET1 group was decreased(P<0.05),the methylation level of Runx2 promoter region was increased(P<0.05),and the alizarin red staining score was decreased(P<0.05).While there was no significant change among the induction+Sul+shRNA-TET2 group,induction+Sul+shRNA-TET3 group,induction+Sul+shRNA-Mock group(P>0.05).Conclusion Sul can promote the differentiation of BMSCs into osteoblasts through promoting DNA demethylation of Runx2 promoter region by TET1.

Construction of double-layer bone-on-a-chip containing bone matrix / 中华创伤杂志

Objective:To construct a double-layer bone-on-a-chip containing bone matrix, with which the process of osteoblast and osteoclast differentiation in vitro is stimulated, aiming to provide a new platform for the development of osteoporosis medications. Methods:Software WorkSoild was used to design the double-layer and double-channel bone-on-a-chip and the template was fabricated by photolithography. With polydimethylsiloxane (PDMS) as the raw material, the main body of the chip was prepared by mold fabrication. The inlets and outlets of the four channels of the culture room were separated with bovine cortex bones and sealed with liquid storage columns. In the chip verification experiment, chips were divided into osteogenic and osteoclastic induction groups and osteogenic and osteoclastic control groups. In the osteogenic and osteoclastic induction groups, precursor cells of mouse embryonic osteoblast, MC3T3-E1 and mouse macrophage RAW264.7 were inoculated on the chip separately. Osteogenic induction lasted 14 days and osteoclastic induction 7 days. MC3T3-E1 cells and RAW264.7 cells were not induced in the osteogenic and osteoclastic control groups. The following indicators were observed: (1) Appearance and sealing performance of the chip: After the chip was prepared, photos were taken to observe its appearance and sealing tests were conducted to observe its sealing performance. (2) Biocompatibility: At 3 days after MC3T3-E1 cells were inoculated onto the chip and cultured and at 1, 3 and 5 days after RAW264.7 cells were inoculated onto the chip and cultured, the cell survival was observed with calcein acetoxymethyl ester/propidium iodide (AM/PI) staining and Cell Counting Kit 8 (CCK-8). (3) Osteogenic differentiation: Alkaline phosphatase (ALP) staining and alizarin red staining were performed on the cells in the osteogenic induction group to observe the osteogenic induction. RNA was collected from the osteogenic induction group and the osteogenic control group, the expression of osteoblast marker Runt-related transcription factor 2 (RUNX2), osteocalcin (OCN) and type I collagen (COL1A1) was detected by real-time florescent quantitative PCR (qPCR), and the differentiation degree and osteogenic ability of osteoblasts were observed. (4) Osteoclast differentiation: tartrate-resistant acid phosphatase (TRAP) staining was performed on cells in the osteoclastic induction group to observe osteoclast differentiation. RNA was extracted from the osteoclastic induction group and the osteoclastic control group for qPCR of osteoclast differentiation-related genes, and the expression levels of the osteoclast marker gene TRAP, cathepsin K (CTSK) and dendritic cell specific transmembrane protein (DC-STAMP) were detected.Results:The double-layer bone-on-a-chip containing bone matrix was 3 cm×3 cm in size and transparent as a whole. The structure of the system on the chip system was compact and had no seepage. It was shown by calcein AM/PI staining that at 3 days after MC3T3-E1 cells and RAW264.7 cells were cultured, very few red fluorescent dead cells were found. CCK-8 test showed that within 5 days after being cultured, the cell viability was all above 90%, indicating that the biocompatibility of the chip was good and the cells could survive and proliferate normally. The results of ALP and alizarin red staining showed that MC3T3-E1 cells successfully differentiated into osteoblasts and produced calcified nodules in the osteogenic induction group at 14 days after the induction. The qPCR results showed that the relative expression level of RUNX2 in MC3T3-E1 cells in the osteogenic induction group was 4.98±0.74, which was significantly higher than that of the control group (0.99±0.03) ( P<0.01). The relative expression level of OCN in MC3T3-E1 cells was 7.98±0.76, which was significantly higher than that of the control group (1.00±0.06) ( P<0.01). The relative expression level of COL1A1 in MC3T3-E1 cells was 7.07±0.56, which was significantly higher than that of the control group (0.97±0.03) ( P<0.01). The TRAP staining results showed that the RAW264.7 cells in the osteoclastic induction group differentiated to giant multinucleated osteoclasts, and TRAP protein was expressed in large quantity in the osteoclasts. The results of qPCR showed that the relative expression level of TRAP in RAW264.7 cells in the osteoclastic induction group was 3.35±0.37, which was significantly higher than that of the control group (1.01±0.06) ( P<0.01). The relative expression level of CTSK in RAW264.7 cells was 3.46±0.79, which was significantly higher than that of the control group (1.01±0.05) ( P<0.01). The relative expression level of DC-STAMP in RAW264.7 cells was 1.92±0.12, which was significantly higher than that of the control group (0.98±0.08) ( P<0.01). Conclusions:The double-layer bone-on-a-chip containing bone matrix is compact in structure, can be cultured in vitro for a long time, has good biocompatibility and can be used for inducing osteogenic and osteoclast differentiation. Therefore, it is expected to provide a new research platform for exploring the mechanism of osteoporosis and medication screening.

The roles of Bajitianwan formula on Aβ-injured osteoblasts and the mechanism based on network pharmacology / 药学实践与服务

Objective To explore the effect of Bajitianwan（BJTW）formula on bone formation of Aβ-injured osteoblasts and its mechanism. Methods Osteoblasts isolated from neonatal 24-hour Wistar rats were used for the study, and osteoblasts were subjected to damage with Aβ1-42 oligomers, and pharmacological intervention was performed with the aqueous extract of BJTW formula. The MTT assay, alkaline phosphatase（ALP）activity assay, catalase（CAT）activity assay, superoxide dismutase（SOD）activity assay, glutathione（GSH）activity assay and malondialdehyde（MDA）activity assay were carried out respectively. The expression levels of bone morphogenetic protein 2（BMP2）, osteogenic specific transcription factor（RUNX-2）and osteoprotective protein（OPG）were detected by Western blotting. After confirming the effect of BJTW formula on Aβ-injured osteoblasts, the network pharmacology method was used to predict the potential pathways. Results The BJTW formula significantly promoted the proliferation of Aβ-injured osteoblasts, increased ALP, SOD and GSH activity, inhibited MDA activity, and promoted the expression of bone formation-related proteins BMP2, RUNX-2 and OPG. Network pharmacological analysis showed that the effect of ameliorating of Aβ-injured osteoblasts by BJTW formula was mainly mediated by AGE-RAGE, PI3K-Akt, MAPK and neuroactive ligand-receptor interaction signaling pathways. Conclusion In this study, the effect of BJTW formula on improving the osteoblasts damaged by Aβ was confirmed for the first time, and its related mechanism was explored based on network pharmacology method. The results lay a strong foundation for the clinical application of traditional formula BJTW against osteoporosis.

Effect of energy level of photobiomodulation therapy on bone repair in rats / Efecto del nivel de energía de la terapia de fotobiomodulación sobre la reparación ósea en ratas

Abstract The aim of this experimental study was to determine the effect of photobiomodulation therapy on bone repair in a rat tibia osteotomy model at 15 and 30 days. The sample consisted of 36 male Holtzman rats that were randomized into 6 equal groups. Groups A1 and A2: osteotomy + 1 J laser energy. Groups B1 and B2: osteotomy + 3 J laser energy. Groups C1 and C2 (controls): osteotomy only. The bone repair was analyzed by histological evaluation of osteoblasts and osteocytes both at 15 days (groups A1, B1, and C1) and at 30 days (groups A2, B2, and C2). Within the results, in all groups a greater number of osteoblasts was found at 15 days vs 30 days (p<0.05), and a greater number of osteocytes in B1 and C2 vs B2 and C1, respectively (p<0.05). When evaluating the 3 groups worked up to 15 days, more osteoblasts were found in A1 and C1 vs B1 (p<0.001); and osteocytes predominated in A1 and B1 vs C1 (p<0.001). At 30 days there was a greater quantity of osteoblasts in C2 vs A2 and B2 (p<0.05) and of osteocytes in C2 vs B2 (p<0.05). It is concluded that 1 J photobiomodulation therapy improved bone repair at 15 days; however, this improvement was not observed at 30 days because there were no differences between the irradiated groups and the control.

Resumen El objetivo de este estudio experimental fue determinar el efecto de terapia de fotobiomodulación sobre la reparación ósea en un modelo de osteotomía de tibia de rata a los 15 y 30 días. La muestra estuvo compuesta por 36 ratas Holtzman macho que se aleatorizaron en 6 grupos iguales. Grupos A1 y A2: osteotomía + energía láser de 1 Joule. Grupos B1 y B2: osteotomía + energía láser 3 Joule. Grupos C1 y C2 (controles): solo osteotomía. La reparación ósea fue analizada por evaluación histológica de osteoblastos y osteocitos tanto a los 15 días (grupos A1, B1 y C1) como a los 30 días (grupos A2, B2 y C2). Como resultados se encontró que en todos los grupos hubo mayor número de osteoblastos a los 15 días vs. 30 días (p<0,05), y mayor número de osteocitos en B1 y C2 vs B2 y C1, respectivamente (p<0,05). Al evaluar a los animales a los 15 días, se observó mayor número de osteoblastos en A1 y C1 vs B1 (p<0.001); y mayor número de osteocitos en A1 y B1 vs C1 (p<0,001). Al evaluar a los ratones a los 30 días hubo mayor cantidad de osteoblastos en C2 vs A2 y B2 (p<0,05) y de osteocitos en C2 vs B2 (p<0,05). Se concluye que la terapia de fotobiomodulación con 1 Joule mejoró la reparación ósea a los 15 días; sin embargo, dicha mejora no se observó a los 30 días porque no hubo diferencias entre los grupos irradiados y el control.

Physical-chemical influences and cell behavior of natural compounds on titanium dental surfaces

Abstract The present study evaluated the influence of carvacrol, terpinene-4-ol, and chlorhexidine on the physical-chemical properties of titanium surfaces, cell viability, proliferation, adhesion, and spreading of fibroblasts and osteoblasts in vitro. Titanium surfaces (Ti) were treated with Carvacrol (Cvc), Terpinen-4-ol (T4ol), Chlorhexidine (CHX), DMSO, and ultrapure water (Control group). Physical-chemical modifications were evaluated by surface wettability, the surface free energy (SFE) calculated from the contact angle values using the Owens-Wendt-Rabel-Kaeble (OWRK) equation, scanning electron microscopy (SEM) and energy dispersive spectrometry probe (EDS) system. Cells were seeded onto Ti-treated surfaces and incubated for 24 h and 72 h, then evaluated by Alamar blue assay and fluorescence microscopy. Surfaces treated with Cvc and T4ol showed the presence of Na, O, and Cl. All surfaces showed hydrophilic characteristics and SFE values between 5.5 mN/m and 3.4 mN/m. On the other hand, EDS peaks demonstrated the presence of O and Cl after CHX treatment. A reduction of cell viability and adhesion was noted on titanium surfaces treated with CHX after 24 and 72h. In conclusion, the results indicate that the decontamination with Cvc and T4ol on Ti surfaces does not alter the surface proprieties and allows an adequate interaction with cells involved in the re-osseointegration process such as fibroblasts and osteoblasts.

Resumo O presente estudo avaliou a influência do carvacrol, terpineno-4-ol e clorexidina nas propriedades físico-químicas de superfícies de titânio, viabilidade celular, proliferação, adesão e esplhamento de fibroblastos e osteoblastos in vitro. Superfícies de titânio (Ti) foram tratadas com Carvacrol (Cvc), Terpinen-4-ol (T4ol), Clorexidina (CHX), DMSO e água ultrapura (Grupo Controle). As modificações físico-químicas foram avaliadas pela molhabilidade da superfície, a energia livre de superfície (ELS) calculada a partir dos valores do ângulo de contato usando a equação de Owens-Wendt-Rabel-Kaeble (OWRK), microscopia eletrônica de varredura (MEV) e espectroscopia de raios X por energia dispersiva (EDS). As células foram semeadas em superfícies tratadas com Ti e incubadas por 24 h e 72 h, e avaliadas pelo ensaio Alamar blue e microscopia de fluorescência. As superfícies tratadas com Cvc e T4ol mostraram a presença de Na, O e Cl. Todas as superfícies apresentaram características hidrofílicas e valores de ELS entre 5,5 mN/m e 3,4 mN/m. Por outro lado, os picos de EDS demonstraram a presença de O e Cl após o tratamento com CHX. Uma redução da viabilidade celular e adesão foi observada em superfícies de titânio tratadas com CHX após 24 e 72h. Em conclusão, os resultados indicam que a descontaminação com Cvc e T4ol em superfícies de Ti não altera as propriedades da superfície e permite uma interação adequada com células envolvidas no processo de reosseointegração como fibroblastos e osteoblastos.

Unveiling the role of osteoblastic micro RNAs in the skeleton: from biological functions to therapeutic potential / Rol de los micro-ARN osteoblásticos en el esqueleto: desde las funciones biológicas al potencial terapéutico

MicroRNAs (miRNAs) are small non-coding RNA molecules that play critical roles in post-transcriptional gene regulation. They function by binding to target messenger RNA (mRNA) molecules, leading to their degradation or inhibiting their translation into proteins. In the context of skeletal diseases, such as osteoporosis, osteoarthritis, and bone metastasis, there is growing evidence osteoblastic miRNAs, are involved in the regulation of bone formation and maintenance.Osteoblasts are bone-forming cells responsible for synthesizing and depositing the extracellular matrix, which ultimately mineralizes to form bone tissue. Osteoblastic miRNAs modulate various aspects of osteoblast function, including proliferation, differentiation, mineralization, and apoptosis. Dysregulation of these miRNAs can disrupt the balance between bone formation and resorption, leading to skeletal diseases.The therapeutic implications of targeting osteoblastic miRNAs in skeletal diseases are significant. Modulating the expression levels of specific miRNAs holds promise for developing novel therapeutic strategies to enhance bone formation, prevent bone loss, and promote bone regeneration. Potential therapeutic approaches include the use of synthetic miRNA mimics to restore miRNA expression in diseases associated with miRNA downregulation or the use of anti-miRNA oligonucleotides to inhibit miRNA function in diseases associated with miRNA upregulation.miRNA-based therapies are still in the early stages of development, and further research is needed to fully understand the complexity of miRNA networks. Additionally, the delivery of miRNAs to specific target tissues and cells remains a challenge that needs to be addressed for effective clinical translation. Nonetheless, targeting osteoblastic miRNAs represents a promising avenue for future therapeutic interventions in skeletal diseases. (AU)

Los micro-ARNs (miARNss) son pequeños ARN no codificantes que desempeñan un papel fundamental en la regulación génica postranscripcional. Ejercen su función al unir-se a moléculas de ARN mensajero (ARNm), promoviendo su degradación e inhibiendo su traducción en proteínas. En el contexto de las enfermedades esqueléticas, como la osteoporosis, la osteoartritis y la metástasis ósea existe evidencia de que los miARNs osteoblásticos están involucrados en la regulación de la formación y del mantenimiento óseo. Los osteoblastos son células formadoras de hueso responsables de sintetizar y depositar la matriz extracelular, que finalmente se mineraliza para formar el hueso. Los miARNs derivados de osteoblastos modulan varios aspectos de la función de estas células, incluida la proliferación, diferenciación, mineralización y la apoptosis. La desregulación de estos miARNs puede alterar el equilibrio entre la formación y la resorción ósea, lo que lleva a enfermedades óseas. Las implicaciones terapéuticas de los miARNs osteoblásticos en enfermedades esqueléticas son significativas. La modulación de los niveles de expresión de miARNs específicos es prometedora para desarrollar nuevas estrate-gias terapéuticas a fin de mejorar la formación, prevenir la pérdida y promover la regeneración ósea. Los enfoques terapéuticos potenciales incluyen el uso de miméticos de miARNs para restaurar la expresión de miARNs o el uso de oligonucleótidos anti-miARNs para inhibir su función. Las terapias basadas en miARNs aún se encuentran en las primeras etapas de desarrollo. La administración de miARNs a las células y los tejidos específicos sigue siendo un desafío para lograr una aplicación clínica eficaz. (AU)

Low concentrations of grape seed extract maintain osteoblast morphology, cell adhesion, and mineralization

Abstract The increase in life expectancy has led to a higher incidence of osteoporosis, characterized by an imbalance in bone remodeling. Several drugs are used for its treatment, but most promote undesirable side effects. The present investigation evaluated the effects of two low concentrations of grape seed extract (GSE) rich in proanthocyanidins on MC3T3-E1 osteoblastic cells. The cells were cultured in an osteogenic medium and divided into control (C), 0.1 µg/mL GSE (GSE0.1), and 1.0 µg/mL GSE (GSE1.0) groups to evaluate cell morphology, adhesion, and proliferation, in situ alkaline phosphatase (ALP) detection, mineralization and immunolocalization of osteopontin (OPN). The data obtained were analyzed by statistical tests for a significance of 5%. Cell morphology was maintained with both GSE concentrations, whereas cell adhesion significantly increased within three days in all groups. Cell proliferation increased significantly at seven days of culture, followed by a significant decrease in all experimental periods, with no statistical difference among them. In situ detection of ALP and mineralization increased with time, but within each period, no statistical differences among groups were observed. The expression of osteopontin was distributed regularly with more intensity after 24 hours in the GSE0.1 group. After three days, OPN expression was more intense in the control group, followed by GSE0.1 and GSE1.0 groups. Data obtained suggest that low concentrations of GSE do not affect the morphology and may stimulate the functional activity of osteoblastic cells.

Resumo O aumento da expectativa de vida tem levado a uma maior incidência de osteoporose, caracterizada por um desequilíbrio na remodelação óssea. Vários medicamentos são utilizados para o seu tratamento, contudo, a maioria promove efeitos colaterais indesejáveis. A presente investigação avaliou os efeitos de duas baixas concentrações de extrato de semente de uva (GSE) rico em proantocianidinas em células osteoblásticas MC3T3-E1. As células foram cultivadas em meio osteogênico e divididas em grupos controle (C), 0,1 µg/mL de GSE (GSE0.1) e 1,0 µg/mL de GSE (GSE1.0) para avaliar morfologia, adesão e proliferação celular, detecção in situ de fosfatase alcalina (ALP), mineralização e imunolocalização da proteína osteopontina (OPN). Os dados obtidos foram analisados por testes estatísticos para um nível de significância de 5%. A proliferação celular aumentou significativamente aos sete dias de cultura, seguido de uma diminuição significativa em todos os períodos experimentais, sem diferença estatística entre eles. A detecção in situ de ALP e mineralização aumentou com o tempo, mas dentro de cada período não foram observadas diferenças estatísticas entre os grupos. A morfologia celular foi mantida com ambas as concentrações de GSE, enquanto a adesão celular aumentou significativamente aos três dias em todos os grupos. A expressão de osteopontina distribuiu-se regularmente com maior intensidade após 24 horas no grupo GSE0.1. Após três dias, a expressão de OPN foi mais intensa no grupo controle, seguida pelos grupos GSE0.1 e GSE1.0. Os dados obtidos sugerem que baixas concentrações de GSE não afetam a morfologia e podem estimular a atividade funcional das células osteoblásticas.

Cytotoxicity and genotoxicity of Bio-C Repair, Endosequence BC Root Repair, MTA Angelus and MTA Repair HP

Abstract The aim was to evaluate in vitro cytotoxicity and genotoxicity of Bio-C Repair (BCR), compared to Endosequence BC Root Repair (ERRM), MTA Angelus (MTA-Ang), and MTA Repair HP (MTA-HP). MC3T3 osteoblastic cells were exposed to extracts of the repairing bioceramic cements. After 1, 3, and 7 days, cytotoxicity and genotoxicity were evaluated by MTT and Micronucleus tests, respectively. Cells not exposed to biomaterials were used as a negative control. Data were compared using ANOVA two-way, followed by the Tukey Test (α=5%). MTA-Ang and MTA-HP showed no difference in relation to control regarding cytotoxicity in any experimental times. BCR and ERRM reduced cell viability after 3 and 7 days (p<0.05); however, the reduction caused by BCR was less than that caused by ERRM. Considering the micronucleus formation, all biomaterials caused an increase after 3 and 7 days (p<0.05), being greater for the BCR and ERRM groups. It can be concluded that BCR is non-cytotoxic in osteoblastic cells, as well as MTA-Ang e MTA Repair HP. BCR and ERRM showed greater genotoxicity than others tested biomaterials.

Resumo O objetivo foi avaliar in vitro a citotoxicidade e genotoxicidade do Bio-C Repair (BCR), em comparação com o Endosequence BC Root Repair (ERRM), MTA Angelus (MTA-Ang) e MTA Repair HP (MTA-HP). As células osteoblásticas MC3T3 foram expostas aos extratos dos cimentos biocerâmicos reparadores. Após 1, 3 e 7 dias, a citotoxicidade e a genotoxicidade foram avaliadas pelos testes MTT e Micronúcleo, respectivamente. Células não expostas aos biomateriais foram utilizadas como controle negativo. Os dados foram comparados por ANOVA de dois fatores, seguido do Teste de Tukey (p = 5 %). MTA-Ang e MTA-HP não apresentaram diferença em relação ao controle quanto à citotoxicidade em nenhum dos tempos experimentais. BCR e ERRM reduziram a viabilidade celular após 3 e 7 dias (p < 0,05); no entanto, a redução causada pelo BCR foi menor que aquela causada pelo ERRM. Todos os biomateriais causaram aumento na formação de micronúcleos após 3 e 7 dias (p < 0,05), sendo maior para os grupos BCR e ERRM. O BCR não é citotóxico em células osteoblásticas, assim como cimentos MTA-Ang e MTA Repair HP. BCR e ERRM apresentaram maior genotoxicidade do que outros biomateriais testados.

Expression Changes of Hypoxia-Inducible Factor-1α in G-CSF Induced Hematopoietic Stem Cell Mobilization / 中国实验血液学杂志

OBJECTIVE@#To investigate the expression and its relative mechanism of hypoxia-inducible factor-1α(HIF-1α) in bone marrow(BM) of mice during G-CSF mobilization of hematopoietic stem cells (HSC) .@*METHODS@#Flow cytometry was used to detect the proportion of Lin-Sca-1+ c-kit+ (LSK) cells in peripheral blood of C57BL/6J mice before and after G-CSF mobilization. And the expression of HIF-1α and osteocalcin (OCN) mRNA and protein were detected by RQ-PCR and immunohistochemistry. The number of osteoblasts in bone marrow specimens of mice was counted under the microscope.@*RESULTS@#The proportion of LSK cells in peripheral blood began to increase at day 4 of G-CSF mobilization, and reached the peak at day 5, which was significantly higher than that of control group (P<0.05). There was no distinct difference in the expression of HIF-1α mRNA between bone marrow nucleated cells and osteoblasts of steady-state mice (P=0.073), while OCN mRNA was mainly expressed in osteoblasts, which was higher than that in bone marrow nucleated cells (P=0.034). After mobilization, the expression level of HIF-1α increased, but OCN decreased, and the number of endosteum osteoblasts decreased. The change of HIF-1α expression was later than that of OCN and was consistent with the proportion of LSK cells in peripheral blood.@*CONCLUSION@#The expression of HIF-1α in bone marrow was increased during the mobilization of HSC mediated by G-CSF, and one of the mechanisms may be related to the peripheral migration of HSC induced by osteoblasts inhibition.

Teriparatide regulates osteoblast differentiation in high-glucose microenvironment through the cAMP/PKA/CREB signaling pathway / 南方医科大学学报

OBJECTIVE@#To investigate the effect of teriparatide on the differentiation of MC3T3-E1 cells in high-glucose microenvironment and explore the possible mechanism.@*METHODS@#MC3T3-E1 cells cultured in normal glucose or high-glucose (25 mmol/L) medium were treated with 10 nmol/L teriparatide with or without co-treatment with H-89 (a PKA inhibitor). CCK-8 assay was used to detect the changes in cell proliferation, and cAMP content in the cells was determined with ELISA. Alkaline phosphatase (ALP) activity and mineralized nodules in the cells were detected using ALP kit and Alizarin red staining, respectively. The changes in cell morphology were detected by cytoskeleton staining. Real-time PCR was used to detect the mRNA expressions of PKA, CREB, RUNX2 and Osx in the treated cells.@*RESULTS@#The treatments did not result in significant changes in proliferation of MC3T3-E1 cells (P > 0.05). Compared with the cells in routine culture, the cells treated with teriparatide showed significantly increased cAMP levels (P < 0.05) with enhanced ALP activity and increased area of mineralized nodules (P < 0.05). Teriparatide treatment also resulted in more distinct visualization of the cytoskeleton in the cells and obviously up-regulated the mRNA expressions of PKA, CREB, RUNX2 and Osx (P < 0.05). The opposite changes were observed in cells cultured in high glucose. In cells exposed to high glucose, treatment with teriparatide significantly increased cAMP levels (P < 0.05), ALP activity and the area of mineralized nodules (P < 0.05) and enhanced the clarity of the cytoskeleton and mRNA expressions of PKA, CREB, RUNX2 and Osx; the effects of teriparatide was strongly antagonized by co-treatment with H-89 (P < 0.05).@*CONCLUSION@#Teriparatide can promote osteoblast differentiation of MC3T3-E1 cells in high-glucose microenvironment possibly by activating the cAMP/PKA/CREB signaling pathway.

Effect of endocannabinoid system on senile osteoporosis / 中华骨科杂志

Osteoporosis is a common disease of old age that affects millions of people worldwide. Besides, it has been a chronic disease difficult to treat in the elderly, so it is of great significance to develop new drugs for the treatment of senile osteoporosis. The endocannabinoid system contains cannabinoid ligands, endocannabinoid receptors, and enzymes required for the synthesis and degradation of endocannabinoids, which play an important role in bone metabolism. Preclinical studies using endocannabinoid system-based therapies in animal models and in vitro have shown that endocannabinoid systems can prevent senile osteoporosis and highlight their therapeutic potential for senile osteoporosis. In this paper, PubMed, ScienceDirect, CNKY, and Wanfang databases were searched for articles related to the endocannabinoid system and osteoporosis. This paper analyzed the pathogenesis of senile osteoporosis (such as calcium, active vitamin D3 deficiency or insufficiency, sex hormone deficiency, cell function decline and secondary to chronic diseases, etc.), and reviewed the various components of the endocannabinoid system and their application in osteoporosis by regulating bone homeostasis in recent years, providing a new direction for the clinical treatment of senile osteoporosis.

Research progress on an autophagy-mediating AMPK pathway involving bone metabolism-related cells that regulate bone homeostasis in periodontitis / 口腔疾病防治

@#Osteoclasts are the only cells responsible for bone resorption in the body, and osteoblasts are the main cells responsible for bone regeneration in the body. Under physiological conditions, these cells maintain a dynamic balance to maintain bone homeostasis. It was widely believed that the imbalance of bone metabolism is mainly affected by the expression of related inflammatory factors. However, with the gradual expansion of related studies in recent years, autophagy has been shown to be closely related to the differentiation, apoptosis and functions of osteoclasts and osteoblasts. AMP-activated protein kinase (AMPK) is an important regulator of energy metabolism in vivo and is involved in the regulation of autophagy and bone homeostasis in bone metabolism-related cells. Periodontitis is a chronic infectious disease, and its typical symptoms are alveolar bone resorption. At present, controlling the level of periodontal inflammation and alveolar bone resorption more effectively in clinical practice remains a challenge. The detection of AMPK and autophagy levels in bone metabolism-related cells shows certain prospects for the clinical prevention and treatment of periodontitis in the future. Therefore, this article reviews the regulation of periodontal inflammation levels and bone homeostasis through cell autophagy related to AMPK-mediated bone metabolism.

Efeito biológico do laser de baixa intensidade em osteoblastos cultivados na superfície de arcabouços poliméricos tridimensionais / Biological effect of low-intensity laser on osteoblasts cultured on the surface of three-dimensional polymeric scaffolds

A primeira parte do trabalho avaliou, através de uma revisão sistemática de estudos in vitro, a aplicabilidade da fotobiomodulação como uma ferramenta auxiliar na engenharia de tecidos. De 8373 estudos inicialmente identificados a partir das estratégias de busca, dez artigos atingiram os critérios de inclusão para análise. Os dados obtidos na maioria dos estudos revisados indicaram que a laserterapia de baixa intensidade (LBI) pode aumentar a proliferação e diferenciação de células cultivadas na superfície dos biomateriais. Na segunda parte do trabalho foi avaliado o efeito da LBI na dose de 4 J/cm2 na proliferação de osteoblastos (OFCOL II) cultivados na superfície de arcabouços poliméricos tridimensionais (3D) de ácido polilático (PLA) e de PLA associado a quitosana (PLA/Q) produzidos pela técnica de fiação por sopro em solução. O ensaio do Alamar Blue demonstrou que as células OFCOL II cultivadas sobre os arcabouços 3D de PLA e irradiadas apresentaram uma maior atividade proliferativa quando comparadas aos grupos não irradiados no intervalo de 72 h. Além disso, as células OFCOL II cultivadas sobre arcabouços de PLA/Q também apresentaram uma maior atividade proliferativa em 24 h. A análise pela microscopia eletrônica de varredura (MEV) mostrou que os osteoblastos se encontravam ancorados em concavidades das fibras nos arcabouços examinados. Concluiu-se que o modelo proposto apresentou um potencial para estudos na área da engenharia tecidual óssea. Na terceira parte do trabalho foi avaliada a influência da LBI infravermelha (IV) e vermelha (V) em diferentes dosagens (1 J/cm², 4 J/cm² e 6 J/cm²) na proliferação e viabilidade das células OFCOL II. O ensaio do Alamar Blue mostrou diferenças significativas (p<0,05) na atividade mitocondrial do grupo IV utilizando a dose de 1 J/cm2 e 4 J/cm2, nos intervalos de 24 e 48 h. Já o ensaio do Live/Dead evidenciou que a LBI induziu aumento da viabilidade celular no grupo IV na dose de 4 J/cm2, quando comparada com os demais grupos. Em conjunto, os resultados sugerem que a LBI pode promover bioestimulação in vitro de osteoblastos, inclusive quando cultivados na superfície de arcabouços poliméricos 3D, representando assim uma ferramenta promissora nas técnicas de engenharia tecidual óssea (AU).

The first part of the work evaluated, through a systematic review of in vitro studies, the applicability of photobiomodulation as an auxiliary tool in tissue engineering. Of 8373 studies initially identified from the search strategies, ten articles met the inclusion criteria for analysis. Data obtained from most of the reviewed studies indicated that low-intensity laser therapy (LLLT) could increase the proliferation and differentiation of cells cultured on the surface of biomaterials. The second part of the work evaluated the effect of LLLT at a dose of 4 J/cm² on the proliferation of osteoblasts (OFCOL II) cultivated on the surface of threedimensional (3D) polymer scaffolds of polylactic acid (PLA) and PLA associated with chitosan (PLA/Q) produced by the solution blow spinning technique. The Alamar Blue assay demonstrated that OFCOL II cells cultured on 3D PLA scaffolds and irradiated showed more significant proliferative activity when compared to non-irradiated groups within 72 h. Furthermore, OFCOL II cells cultured on PLA/Q scaffolds showed higher proliferative activity at 24 h. Analysis by scanning electron microscopy (SEM) showed that the osteoblasts were anchored in the concavities of the fibers of the examined scaffolds. It was concluded that the proposed model showed potential for studies in the field of bone tissue engineering. The third part of the work evaluated the influence of infrared (IR) and red (R) laser therapy at different dosages (1 J/cm², 4 J/cm², and 6 J/cm²) on the proliferation and viability of OFCOL II cells. The Alamar Blue assay showed significant differences (p<0.05) in the mitochondrial activity of group IR using the dose of 1 J/cm² and 4 J/cm² at 24 and 48 h. The Live/Dead assay showed that LLLT induced an increase in cell viability in the IR group at a dose of 4 J/cm² compared to the other groups. Taken together, the results suggest that LLLT can promote in vitro biostimulation of osteoblasts, even when cultivated on the surface of 3D polymeric scaffolds, thus representing a promising tool in bone tissue engineering techniques (AU).

Influence of melatonin associated with the Bio-Gide® membrane on osteoblast activity: an in vitro Study / Influência da melatonina associada à membrana bio gide® na atividade de osteoblastos: um estudo in vitro

ABSTRACT Melatonin (MLT) is a hormone responsible for regulating several physiological processes. It has been shown that MLT can be an important mediator in bone formation and stimulation, promoting osteoblast differentiation. In clinical practice, in tissue regeneration procedures, it is necessary to use membranes or barriers, associated with biomaterials, or not. The aim of this in vitro study was to assess the effect of melatonin on the activity of osteoblastic cells, associated, or not, with a resorbable collagen membrane (Bio-Gideä). For this, mice-derived pre-osteoblastic cells MC3T3 obtained from the ATCC (American Type Culture Collection) were used. Cultured cells were subject to the following treatments: MLT with a concentration of 1mM, a Bio-Gideä membrane and a membrane associated with MLT (Bio-Gideä + MLT). Proliferation and cell viability assays and protein lysate (ELISA test) quantification for the BMP-2 protein were carried out, in periods of 72 hours, 7 days and 10 days. After analyzing the data (one-way ANOVA, alpha=5%) it was observed that when MLT was used in isolation, there was an increase in cell proliferation and viability in osteoblastic cells (p<0.05). But, when MLT was associated with resorbable membranes, there was an inverse behavior, both in terms of proliferation and viability (p<0.05). In the case of the ELISA test, no secretion of BMP-2 was detected in any of the analyzed groups. It is concluded that MLT has a stimulatory effect on osteoblasts, but, when associated with Bio-Gideä resorbable membranes, it does not show any viable action in osteoblastic cell stimulation.

RESUMO A melatonina (MLT) é um hormônio responsável pela regulação de diversos processos fisiológicos no nosso organismo. Tem sido demonstrado que a melatonina possa ser um importante mediador na formação e estimulação óssea, promovendo a diferenciação dos osteoblastos. Clinicamente, para o procedimento de regeneração tecidual, faz-se necessário a utilização de membranas ou barreiras, associadas ou não a biomateriais. Assim, o objetivo deste estudo in vitro foi avaliar o efeito da melatonina na atividade de células osteoblásticas, associada ou não a uma membrana de colágeno reabsorvível (Bio-Gide®). Para isto foram utilizadas células pré-osteoblásticas MC3T3 do ATCC (American Type Culture Collection), de camundongos. As células em cultura foram submetidas aos seguintes tratamentos: MLT na concentração de 1mM, membrana Bio Gide® e membrana associada à MLT (Bio-Gide® + MLT). Foram realizados os ensaios de proliferação e viabilidade celular e quantificação do lisado proteico (teste ELISA), para a proteína BMP-2, nos períodos de 72 horas, 7 e 10 dias. Após a análise dos dados (ANOVA um critério, alfa=5%) pode-se observar que a MLT quando utilizada sozinha, resultou em um aumento na proliferação e viabilidade celular nas células osteoblásticas (p<0,05). Entretanto, quando a MLT foi associada à membrana reabsorvível foi observado um comportamento inverso, tanto na proliferação quanto na viabilidade (p<0,05). Para o teste ELISA realizado, não houve secreção detectável de BMP-2 para nenhum grupo analisado. Conclui-se que a melatonina possui uma ação estimuladora nos osteoblastos, mas quando associada à membrana reabsorvível Bio-Gide®, não demonstra uma ação viável na estimulação de células osteoblásticas.

Experimental study on necroptosis of osteoblasts induced by fluoride / 中华地方病学杂志

Objective:To investigate whether the necroptosis pathway receptor interacting protein 1-receptor interacting protein 3-mixed lineage kinase domain-like protein (RIP1-RIP3-MLKL) is involved in fluoride-induced osteoblastic death.Methods:Human osteosarcoma cell line (Saos-2 cells) were cultured in vitro and divided into NC group, sodium fluoride (NaF) groups (5.0, 10.0, 20.0 and 40.0 mg/L NaF), necroptosis inhibitor Necrostatin-1 (Nec-1) group (50.0 μmol/L Nec-1) and NaF + Nec-1 groups (5.0, 10.0, 20.0 and 40.0 mg/L NaF + 50.0 μmol/L Nec-1). After cultured for 24 and 48 h, respectively, cell proliferation was determined via the CCK-8 method, and the activity of lactate dehydrogenase (LDH) was determined by chemical colorimetry. To further analyze the influence of NaF on RIP1-RIP3-MLKL pathway, Saos-2 cells were divided into NCⅡgroup and NaFⅡgroups (2.5, 5.0, 10.0, 20.0 and 40.0 mg/L NaF). After cultured for 24 and 48 h, respectively, the protein expression levels of RIP1, RIP3 and MLKL were determined by Western blotting. Results:The cell proliferation rates (%: 100.00 ± 0.59, 104.90 ± 0.44, 104.16 ± 0.41, 82.45 ± 1.91, 64.59 ± 1.83, 103.56 ± 0.41, 107.18 ± 0.87, 105.35 ± 1.28, 89.63 ± 1.20, 77.51 ± 1.30; 100.00 ± 0.33, 107.92 ± 0.44, 101.78 ± 1.06, 75.45 ± 0.39, 57.94 ± 1.17, 106.74 ± 0.21, 111.85 ± 0.21, 107.82 ± 0.68, 82.34 ± 0.56, 70.19 ± 0.99) among all groups were significantly different at both 24 and 48 h ( F = 77.13, 2 313.43, P < 0.05). Except the cell proliferation rate of the 10.0 mg/L NaF + Nec-1 group that was not significantly different with that of the 10.0 mg/L NaF group at 24 h ( P > 0.05), the cell proliferation rates of other NaF + Nec-1 groups were significantly higher than those of corresponding NaF groups at both 24 and 48 h ( P < 0.05). The proliferation rate was negatively correlated with fluoride concentration ( r24 h = - 0.976, r48 h = - 0.969, P < 0.001). The LDH activity in all concentrations of NaF groups was significantly higher than that in NC group and corresponding NaF + Nec-1 groups at both 24 and 48 h ( P < 0.05). The LDH activity was positively correlated with fluoride concentration ( r24 h = 0.985, r48 h = 0.988, P < 0.001). The protein expression levels of RIP1, RIP3 and MLKL of 5.0 mg/L NaFⅡ group at 24 h, RIP3 of 5.0 mg/L NaFⅡ group at 48 h, and RIP1, RIP3 and MLKL of 10.0, 20.0 and 40.0 mg/L NaFⅡ groups at both 24 and 48 h were higher than that in NCⅡ group ( P < 0.05). The protein expression levels of RIP1, RIP3 and MLKL were positively correlated with fluoride concentration ( r24 h-RIP1 = 0.881, r48 h-RIP1 = 0.952, r24 h-RIP3 = 0.867, r48 h-RIP3 = 0.938, r24 h-MLKL = 0.758, r48 h-MLKL = 0.907, P < 0.001). Conclusion:Fluoride can directly cause necroptosis of osteoblasts through the RIP1-RIP3-MLKL pathway, and the severity of cell damage is closely related to fluoride concentration, Nec-1 has partially reversed the effects of fluoride.

The effects of Connexin-43 on osteoblasts cells proliferation and osteogenic differentiation / 中华骨科杂志

Objective:To investigate the effects of Connexin-43 (Cx43) on osteoblasts proliferation and osteogenic differentiation and its regulatory mechanism.Methods:Osteoblasts were isolated and cultured in vitro. The osteogenic activity of osteoblasts was detected by alizarin red staining and alkaline phosphatase (ALP) staining after dexamethasone treatment. The expression of Cx43, Runt-related transcription factor 2 (Runx2), ALP, collagen I type (COL-I) and proliferation-related proteins PCNA and CDK4 in osteoblasts were detected by Western-blot. The expressions of osteoblast proteins were detected by immunofluorescence staining. The proliferation of osteoblasts was detected by CCK8 assay. The lentivirus-mediated Cx43 gene overexpression plasmid (Lv-Cx43) was constructed and transfected into osteoblasts. The osteogenic activity and proliferation ability of osteoblasts were further detected by the above methods. Cx43 in osteoblasts was overexpressed by pretreating PD98059. The osteogenic activity and proliferation of Cx43 in overexpressed osteoblasts was detected by CCK8 and alizarin red staining.Results:The isolated osteoblasts have osteogenic differentiation ability. Compared with the control group, 1×10 -6 mol/L dexamethasone treatment could reduce the formation of calcium nodules in osteoblasts. With the increase of dexamethasone treatment duration, the protein expression of Cx43, Runx2, ALP and COL-I in osteoblasts decreased gradually, while the expression of PCNA, CDK4 and p-ERK1/2 decreased. The OD values of normal osteoblasts at 0, 1, 2, 3 and 4 d were 0.316±0.043, 0.891±0.623, 1.683±0.154, 2.315±0.721 and 2.891±0.323, respectively. However, The OD values of osteoblasts treated with dexamethasone were 0.376±0.021, 0.657±0.121, 1.124±0.285, 1.521±0.272, 1.987±0.584, respectively. OD values of dexamethasone treated osteoblasts were lower than those of normal group at 2, 3 and 4 days ( P<0.05). The relative expression levels of Cx43 mRNA in control group, Lv-NC group and Lv-Cx43 group were 0.541±0.086, 0.598±0.018 and 1.000±0.082, respectively. The mRNA expression level of Cx43 in Lv-Cx43 group was higher than that in control group and Lv-NC group ( P<0.05). The ratio of Cx43 protein band to the gray value of GAPDH band in control group, Lv-NC group and Lv-Cx43 group were 0.816±0.737, 0.738±0.643 and 1.145±1.101, respectively. The expression level of Lv-Cx43 was higher than that in control group and Lv-NC group ( P<0.05). The expressions of Runx2, ALP, COL-I mRNA and related marker proteins in Lv-Cx43 group were higher than those in control group and Lv-NC group ( P<0.05). The number of calcium nodules in the Lv-Cx43 group was significantly higher than that in the control group and Lv-NC group. The OD value of osteoblasts and the number of calcium nodules in Lv-Cx43+PD98059 group were significantly lower than those in Lv-Cx43 group ( P<0.05). Conclusion:The proliferation and differentiation ability of osteoblasts is significantly decreased after the treatment of dexamethasone with decreased expression of Cx43. Overexpression of Cx43 can promote the proliferation and osteogenic differentiation of osteoblasts, which may be regulated through the ERK1/2 pathway.

Effect of porous zirconia ceramics on proliferation and differentiation of osteoblasts / 北京大学学报(医学版)

OBJECTIVE@#To investigate the effect of porous surface morphology of zirconia on the proliferation and differentiation of osteoblasts.@*METHODS@#According to different manufacturing and pore-forming methods, the zirconia specimens were divided into 4 groups, including milled sintering group (M-Ctrl), milled porous group (M-Porous), 3D printed sintering group (3D-Ctrl) and 3D printed porous group (3D-Porous). The surface micromorphology, surface roughness, contact angle and surface elements of specimens in each group were detected by scanning electron microscope (SEM), 3D laser microscope, contact angle measuring device and energy-dispersion X-ray analysis, respectively. MC3T3-E1 cells were cultured on 4 groups of zirconia discs. The cell morphology of MC3T3-E1 cells on zirconia discs was eva-luated on 1 and 7 days by SEM. The cell proliferation was detected on 1, 3 and 5 days by cell counting kit-8 (CCK-8). After osteogenic induction for 14 days, the relative mRNA expression of alkaline phosphatase (ALP), type Ⅰ collagen (Colla1), Runt-related transcription factor-2 (Runx2) and osteocalcin (OCN) in MC3T3-E1 cells were detected by real-time quantitative polymerase chain reaction.@*RESULTS@#The pore size [(419.72±6.99) μm] and pore depth [(560.38±8.55) μm] of 3D-Porous group were significantly larger than the pore size [(300.55±155.65) μm] and pore depth [(69.97±31.38) μm] of M-Porous group (P < 0.05). The surface of 3D-Porous group appeared with more regular round pores than that of M-Porous group. The contact angles of all the groups were less than 90°. The contact angles of 3D-Ctrl (73.83°±5.34°) and M-Porous group (72.7°±2.72°) were the largest, with no significant difference between them (P>0.05). Cells adhered inside the pores in M-Porous and 3D-Porous groups, and the proliferation activities of them were significantly higher than those of M-Ctrl and 3D-Ctrl groups after 3 and 5 days' culture (P < 0.05). After 14 days' incubation, ALP, Colla1, Runx2 and OCN mRNA expression in 3D-Porous groups were significantly lower than those of M-Ctrl and 3D-Ctrl groups (P < 0.05). Colla1, Runx2 and OCN mRNA expressions in M-Porous group were higher than those of 3D-Porous group (P < 0.05).@*CONCLUSION@#The porous surface morphology of zirconia can promote the proliferation and adhesion but inhibit the differentiation of MC3T3-E1 cells.