3D-printed near-infrared-light-responsive on-demand drug-delivery scaffold for bone regeneration.

Although several bioactive 3D-printed bone scaffolds loaded with multiple kinds of biomolecules for enhanced bone regeneration have been recently developed, the manipulation of on-demand release profiles of different biomolecules during bone regeneration remains challenging. Herein, a 3D-printed dual-drug-loaded biomimetic scaffold to regulate the host stem cell recruitment and osteogenic differentiation in a two-stage process for bone regeneration was successfully fabricated. First, a chemotactic small-molecule drug, namely, simvastatin (SIM) was directly incorporated into the hydroxyapatite/collagen bioink for printing and could be rapidly released during the early stage of bone regeneration. Further, near-infrared (NIR)-light-responsive polydopamine-coated hydroxyapatite nanoparticles were designed to deliver the osteogenic drug, i.e., pargyline (PGL) in a controllable manner. Together, our scaffold displayed an on-demand sequential release of those two drugs and could optimize their therapeutic effects to align with the stem cell recruitment and osteoblastic differentiation, thereby promoting bone regeneration. The results confirmed the suitable mechanical strength, high photothermal conversion efficiency, good biocompatibility of our scaffold. The scaffold loaded with SIM could efficiently accelerate the migration of stem cells. In addition, the scaffold with on-demand sequential release promoted alkaline phosphatase (ALP) activity, significantly upregulated gene expression levels of osteogenesis-related markers, and enhanced new-bone-formation capabilities in rabbit cranial defect models. Altogether, this scaffold not only offers a promising strategy to control the behavior of stem cells during bone regeneration but also provides an efficient strategy for controllable sequential release of different biomolecule in bone tissue engineering.

The design and processing of a 3D-printed high-performance biological fixation plate.

In order to generate a high-performance personalized biological fixation plate with matching mechanical properties and biocompatibility, reverse reconstruction and fracture reduction of a femur were performed by combining reverse and forward approaches, and the surface was extracted according to the installation position of the plate to complete plate modeling by shifting, thickening, and performing other operations. Subsequently, topology optimization and three-dimensional (3D) printing were performed, and the properties of the manufactured plate were probed. The results showed that the maximum displacement of the plate was 4.13 mm near the femoral head, the maximum stress was 5.15e2 MPa on both sides of the plate across its entire length, and the stress concentration decreased following topology optimization. The plate with optimized topology and filled with porous structure has a good filling effect. The final mass of the H-shaped plate was 12.05 g, while that of the B-shaped plate was 11.05 g, which dropped by 20.93% and 27.49%, respectively, compared with the original plate. The surface of the 3D-printed plate was bright and new, with a clear pore structure and good lap joint. The B-shaped and H-shaped plates were closely dovetailed with the host bone, which met the assembly requirements. This lays a foundation for the direct application of a high-performance personalized biological fixation plate.

Establishment and evaluation of risk prediction model for the esophageal cancer via whole transcriptome analysis / 中国胸心血管外科临床杂志

@#Objective    To establish the gene-based esophageal cancer (ESCA) risk score prediction models via whole transcriptome analysis to provide ideas and basis for improving ESCA treatment strategies and patient prognosis. Methods    RNA sequencing data of esophageal squamous cell carcinoma (ESCC), esophageal adenocarcinoma (EAC) and adjacent tissues were obtained from The Cancer Genome Atlas database. The edgeR method was used to screen out the differential genes between ESCA tissue and normal tissue, and the key genes affecting the survival status of ESCC and EAC patients were initially identified through univariate Cox regression analysis. The least absolute shrinkage and selection operator regression analysis and multivariate Cox regression analysis were used to further screen genes and establish ESCC and EAC risk score prediction models. Results    The risk score prediction models were the independent prognostic factors for ESCA, and the risk score was significantly related to the survival status of patients. In ESCC, the risk score was related to T stage. In EAC, the risk score was related to lymph node metastasis, distant metastasis and clinical stage. The constructed nomogram based on risk score showed good predictive ability. In ESCC, the risk score was related to tumor immune cell infiltration and the expression of immune checkpoint genes. However, this feature was not obvious in EAC. Conclusion 聽 聽The ESCC and EAC risk score prediction models have shown good predictive capabilities, which provide certain inspiration and basis for optimizing the management of ESCA and improving the prognosis of patients.

Role of dendritic cells in MYD88-mediated immune recognition and osteoinduction initiated by the implantation of biomaterials / 国际口腔科学杂志·英文版

Bone substitute material implantation has become an important treatment strategy for the repair of oral and maxillofacial bone defects. Recent studies have shown that appropriate inflammatory and immune cells are essential factors in the process of osteoinduction of bone substitute materials. Previous studies have mainly focused on innate immune cells such as macrophages. In our previous work, we found that T lymphocytes, as adaptive immune cells, are also essential in the osteoinduction procedure. As the most important antigen-presenting cell, whether dendritic cells (DCs) can recognize non-antigen biomaterials and participate in osteoinduction was still unclear. In this study, we found that surgical trauma associated with materials implantation induces necrocytosis, and this causes the release of high mobility group protein-1 (HMGB1), which is adsorbed on the surface of bone substitute materials. Subsequently, HMGB1-adsorbed materials were recognized by the TLR4-MYD88-NFκB signal axis of dendritic cells, and the inflammatory response was activated. Finally, activated DCs release regeneration-related chemokines, recruit mesenchymal stem cells, and initiate the osteoinduction process. This study sheds light on the immune-regeneration process after bone substitute materials implantation, points out a potential direction for the development of bone substitute materials, and provides guidance for the development of clinical surgical methods.

Optimized design and key performance factors of a gas circulation filtration system in a metal 3D printer.

To further improve the quality of parts in metal 3D printers, it is necessary to optimize the structure and study the performance of their gas circulation filtration systems. First, we used the parametric modeling method to complete the formed cavity modeling. We then optimized the design of the air inlet structure of the formed cavity using the moldflow simulation method, and finally, we evaluated the optimized design results through assembly experiments and measurements of the molded parts' components. The combination of parametric modeling and moldflow simulation methods produced a high modeling efficiency and had a good effect on the optimized design of the gas circulation filtration systems. After optimizing the design, the turbulence intensities and distribution areas of the formed cavities were reduced. During the 3D printing of the curved guide plate, the plane of the guide plate holder was inclined 55° relative to the machining datum plane, which improved the form quality. The 3D printed curved guide plate closely matched the inlet end of the printer's air duct, and the upper guide plate was fixed at a suitable position using screws. The niobium contents of the parts formed by the guide plate in Design 2 were low, which lays a foundation for the 3D printing of high-performance metal parts.

Biometry of width between labial transitional line angles in anterior teeth:an observational study

PURPOSE@#The maximum width between the mesial and distal labial transitional line angles, described as “esthetic width” herein, could significantly influence the visual perception of the teeth and smile. This study aimed to conduct biometric research on esthetic width and to explore whether regular distribution exists in the esthetic width of human teeth. @*MATERIALS AND METHODS@#A total of 4,264 maxillary and mandibular anterior teeth were measured using the Geomagic studio software program. The proportions of maxillary to mandibular homonymous teeth and proportions between the adjacent teeth were calculated. Bilateral symmetry and the correlation between the esthetic and mesiodistal widths were both accounted for during the measurement procedures. @*RESULTS@#The mean esthetic widths were 6.773 ± 0.518 mm and 4.329 ± 0.331 mm for maxillary and mandibular central incisors, respectively, 5.451 ± 0.487 mm and 5.008 ± 0.351 mm for maxillary and mandibular lateral incisors, respectively, and 3.340 ± 0.353 mm and 5.958 ± 0.415 mm for maxillary and mandibular canines, respectively. Except for the mandibular canines, no significant difference in esthetic width was found among homonymous teeth from the same jaw. A high linear correlation was found between the esthetic and mesiodistal widths of the same tooth, except for the maxillary canines. Esthetic width proportions among different tooth categories showed some regular patterns, which were similar to those of the mesiodistal width. @*CONCLUSION@#Esthetic width is regularly distributed among the teeth in the Chinese population. This could provide an important reference for anterior dental restorations and dimension recovery in esthetic reconstruction of anterior teeth.

Evaluation of functional suitable digital complete denture system based on 3D printing technology

PURPOSE@#To improve the clinical effects of complete denture use and simplify its clinical application, a digital complete denture restoration workflow (Functional Suitable Digital Complete Denture System, FSD) was proposed and preliminary clinical evaluation was done. MATERIALS AND METHODS Forty edentulous patients were enrolled, of which half were treated by a prosthodontic chief physician, and the others were treated by a postgraduate student. Based on the primary impression and jaw relation obtained at the first visit, diagnostic denture was designed and printed to create a definitive impression, jaw relation, and esthetic confirmation at the second visit. A redesigned complete denture was printed as a mold to fabricate final denture that was delivered at the third visit.To evaluate accuracy of impression made by diagnostic denture, the final denture was used as a tray to make impression, and 3D comparison was used to analyze their difference. To evaluate the clinical effect of FSD, visual analogue scores (VAS) were determined by both dentists and patients. @*RESULTS@#Two visits were reduced before denture delivery. The RMS values of 3D comparison between the impression made via diagnostic dentures and the final dentures were 0.165 ± 0.033 mm in the upper jaw and 0.139 ± 0.031 mm in the lower jaw. VAS ratings were between 8.5 and 9.6 in the chief physician group, while 7.7 and 9.5 in the student group; there was no statistical difference between the two groups. @*CONCLUSION@#FSD can simplify the complete denture restoration process and reduce the number of visits. The accuracy of impressions made by diagnostic dentures was acceptable in clinic. The VASs of both dentists and patients were satisfied.

Asymmetrical methyltransferase PRMT3 regulates human mesenchymal stem cell osteogenesis via miR-3648.

Histone arginine methylation, which is catalyzed by protein arginine methyltransferases (PRMTs), plays a key regulatory role in various biological processes. Several PRMTs are involved in skeletal development; however, their role in the osteogenic differentiation of mesenchymal stem cells (MSCs) is not completely clear. In this study, we aimed to elucidate the function of PRMT3, a type-I PRMT that catalyzes the formation of ω-mono- or asymmetric dimethyl arginine, in MSCs osteogenesis. We found that PRMT3 promoted MSCs osteogenic commitment and bone remodeling. PRMT3 activated the expression of miR-3648 by enhancing histone H4 arginine 3 asymmetric dimethylation (H4R3me2a) levels at promoter region of the gene. Overexpression of miR-3648 rescued impaired osteogenesis in PRMT3-deficient cells. Moreover, administration of Prmt3 shRNA or a chemical inhibitor of PRMT3 (SGC707) caused an osteopenia phenotype in mice. These results indicate that PRMT3 is a potential therapeutic target for the treatment of bone regeneration and osteopenia disorders.

Effects of Suspension Training and Chiropractic on Degenerative Lumbar Spondylolisthesis / 中国康复理论与实践

@#Objective To observe the effects of suspension training along with chiropractic on degenerative lumbar spondylolisthesis. Methods From June to December, 2016, 64 patients with degenerative lumbar spondylolisthesis were randomly divided into control group (n=32) and treatment group (n=32). The control group accepted McKenzie approach, lumbar traction and functional training, while the treatment group accepted suspension training and chiropractic, for 45 days. They were evaluated with Meyerding Rating, Visual Analogue Scale (VAS) of pain and Oswestry Disability Index (ODI) before and after treatment. Results The scores of Meyerding Rating, VAS and ODI improved in both groups after treatment (t>9.157, P<0.001), and improved more in the treatment group than in the control group (t>2.069, P<0.05). Conclusion Suspension training combining with chiropractic is safe and effective for degenerative lumbar spondylolisthesis.

Research progress on highly active anti-retroviral therapy induced secondary osteoporosis / 中国实验动物学报

Highly active anti-retroviral therapy(HAART)is a principal therapy method for AIDS. However, HAART is also one of crucial factors inducing bone mass loss and osteoporosis but its related mechanisms are obscure. Of note,nucleotide reverse transcriptase inhibitor(NRTI)and protease inhibitors(PI)may play key roles in inducing osteoporosis. This review summarizes the research progress in epidemiology and associated mechanisms on osteoporosis induced by NRTI and PI.

Research and development of digital design and fabrication of complete denture / 中华口腔医学杂志

Removable complete denture are still the primary prosthetic solution for edentulous patients. Functional pressure impression obtaining, jaw relation recording, personalized balance occlusion and highly precise fabrication of denture are difficult. The digital restoration technique represented by intraoral three-dimensional scanning and three-dimensional (3D) printing compensates for the shortages of the manual techniques, but there are still many limitations in the application of complete dentures. At present, a few computer aided design and computer aided manufacture (CAD/CAM) complete denture systems have been developed both domestically and abroad, and these system are mainly focused on the digital design and manufacture of denture, and are seldom used for the recording of impression and jaw relation. This review is based on the main clinical procedures of the traditional complete denture restoration, elaborating the research and application status of digital technique in each steps, in order to provide reference for clinical application.

A novel tissue-engineered bone constructed by using human adipose-derived stem cells and biomimetic calcium phosphate scaffold coprecipitated with bone morphogenetic protein-2 / 北京大学学报(医学版)

Objective:To construct a novel biomimetic calcium phosphate (BioCaP) scaffold loaded with bone morphogenetic protein-2 (BMP-2),and to investigate its role in the osteogenesis of human adipose-derived stem cells (hASCs) in vitro and in vivo.Methods:The BioCaP scaffold coprecipitated with BMP-2 (BMP-2-BioCaP) was constructed in this study.Field emission scanning electron microscopy (SEM) was used to analyze the morphology of the surfaces.The release kinetics was measured to evaluate the slow-release characteristics in vitro.BMP-2-BioCaP was immersed in proliferation medium (PM) or osteogenic medium (OM),respectively.The supernatants were collected and used to culture hASCs in vitro.Cell numbers were determined using the cell-counting kit-8 (CCK-8) to assess the cell proliferation.After 7 and 14 days,alkaline phosphatase (ALP) staining and quantification were performed to test the activity of ALP.After 14 and 21 days,the calcification deposition was determined by alizarin red S (ARS) staining and quantification.The expressions of the osteoblast-related genes were tested on day 4 and day 14.In the in vivo study,6 nude mice were used and implanted subcutaneously into the back of the nude mice for 4 groups:(1) BioCaP scaffold only,(2) BioCaP scaffold + hASCs,(3) BMP-2-BioCaP scaffold,(4) BMP-2-BioCaP scaffold + hASCs (test group).After 4 weeks of implantation,hematoxylin-eosin (HE) staining was performed to evaluate the in vivo osteogenesis of hASCs.Results:SEM observations showed that BioCaP and BMP-2-BioCaP scaffold were entirely composed of straight,plate-like and sharp-edged crystal units,and the length of the crystal units varied between 5 and 10 μm.Release kinetics analysis demonstrated that BMP-2 incorporated with BioCaP could be released at certain concentration and last for more than 21 days,and the accumulative protein release could reach 20％.CCK-8 assays showed that cell proliferation was not significantly affected by BMP-2BioCaP.ALP activity was higher by the induction of OM + BMP-2-BioCaP than of the other groups (P ＜0.01).More mineralization deposition and more expressions of osteoblast-related genes such as Runt-related transcription factor 2 (RUNX2),ALP,osteopontin (OPN) and osteocalcin (OC) were determined in the OM + BMP-2-BioCaP group at different time points (P ＜0.01).HE staining showed that,in the test group and BMP-2-BioCaP scaffold group,the extracellular matrix (ECM) with eosinophilic staining were observed around hASCs,and newly-formed bone-like tissues could be found in ECM around the scaffold materials.Moreover,compared with the BMP-2-BioCaP scaffold group,more bone-like tissues could be observed in ECM with typical structure of bone tissue in the test groups.No obvious positive results were found in the other groups.Conclusion:BMP-2-BioCaP scaffold could achieve slow-release of BMP-2 and promote the osteogenic differentiation of hASCs in vitro and in vivo.The novel tissue-engineered bone composed of hASCs and BMP-2-BioCaPis promising for the repair of bone defect.

A study on establishment of examination criteria for the clinical practice of dental students in the department of prosthodontics by Delphi method / 中华医学教育探索杂志

Objective To establish the examination criteria for the prosthodontic clinical practice using Delphi method,to improve the evaluation level of clinical practice of dental students.Method 15 clinical experts in the department of prosthodontics,Peking University School of Stomatology were selected in this study.The evaluation indicators system of prosthodontic clinical practice was established by two rounds of investigations by Delphi method.Initiative ratio and authority coefficient (Cr) of the experts were used to evaluate the reliability of the new examination criteria.Next,the eight-year program students in class of 2007 (38 students) and 2008 (34 students) were examined by old and new examination criteria respectively and the feedback of the students were collected by the method of questionnaire.SPSS 16.0 was used for data processing.Results In this study,the initiative and the authority of the experts was very high,and the initiative ratio and authority coefficient were 100％ and 0.860 respectively.A new examination criterion of prosthodontic clinical practice was established,including 3 first-level indicators,9 second-level indicators and 19 third-level indicators.Questionnaire analysis revealed that only 12％ (n=4) and 9％ (n=3)of the students in class of 2008 considered the new examination criteria was not comprehensive enough or objective enough,which was obviously less than the students in class of 2007 [45％ (n=17) and 36％ (n=14)].Conclusion A new examination criterion of prosthodontic clinical practice was successfully established by Delphi method.The new examination criterion is more comprehensive and objective than the old one and can promote the educational quality of clinical practice of dental students.The method of this study also provides reference for the educational reform of clinical practice in other fields.

Diagnossis and treatment of complicated anterior teeth esthetic defects by combination of whole-process digital esthetic rehabilitation with periodontic surgery / 北京大学学报(医学版)

Objective:To explore a new method of whole-process digital esthetic prosthodontic rehabilitation combined with periodontic surgery for complicated anterior teeth esthetic defects accompanied by soft tissue morphology,to provide an alternative choice for solving this problem under the guidance of three-dimensional (3 D) printing digital dental model and surgical guide,thus completing periodontic surgery and digital esthetic rehabilitation of anterior teeth.Methods:In this study,12 patients with complicated esthetic problems accompanied by soft tissue morphology in their anterior teeth were included.The dentition and facial images were obtained by intra-oral scanning and three-dimensional (3D) facial scanning and then calibrated.Two esthetic designs and prosthodontic outcome predictions were created by computer aided design/computer aided manufacturing (CAD/CAM) software combined with digital photography,including consideration of white esthetics and comprehensive consideration of pink-white esthetics.The predictive design of prostheses and the facial appearances of the two designs were evaluated by the patients.If the patients chose the design of comprehensive consideration of pink-white esthetics,they would choose whether they would receive periodontic surgery before esthetic rehabilitation.The dentition design cast of those who chose periodontic surgery would be 3D printed for the guide of periodontic surgery accordingly.Results:In light of the two digital designs based on intra-oral scanning,facing scanning and digital photography,the satisfaction rate of the patients was significantly higher for the comprehensive consideration of pink-white esthetic design (P ＜ 0.05) and more patients tended to choose priodontic surgery before esthetic rehabilitation.The 3 D printed digital dental model and surgical guide provided significant instructions for periodontic surgery,and achieved success transfer from digital design to clinical application.The prostheses were fabricated by CAD/CAM,thus realizing the whole-process digital esthetic rehabilitation.Conclusion:The new method for esthetic rehabilitation of complicated anterior teeth esthetic defects accompanied by soft tissue morphology,including patient-involved digital esthetic analysis,design,esthetic outcome prediction,3D printing surgical guide for periodontic surgery and digital fabrication is a practical technology.This method is useful for improvement of clinical communication efficiency between doctor-patient,doctor-technician and doctors from different departments,and is conducive to multidisciplinary treatment of this complicated anterior teeth esthetic problem.

Application of three-point double eyelid blepharoplasty combined with correcting epicanthal folds by using asymmetric Z-plasty with a two curve design / 中华医学美学美容杂志

Objective To explore the clinical effect of three-point double eyelid blepharoplasty combined with correcting epicanthal folds by using asymmetric Z-plasty with a two curve design.Methods We retrospectively reviewed the application and effect of three-point double eyelid blepharoplasty combined with correcting epicanthal folds by using asymmetric Z-plasty with a two curve design between January 2015 and June 2016.There were 36 cases of severe epicanthus in this group,including 20 cases of thin eyelid,13 cases of mild swollen eye and 3 cases of moderate swollen eye.Results Follow-up by telephone or WeChat,satisfactory for the 26 cases,the basic satisfaction of 9 cases,1 case of dissatisfaction,moderate swollen eye and severe epicanthus.Conclusions In the case of severe epicanthus with a single eyelid and a mild swollen eyelid without thinning of the eyelid,the use of three-point double eyelid blepharoplasty combined with correcting epicanthal folds by using asymmet ric Z-plasty with a two curve design is one of good choices of methods for the correction of epicanthus.

Quantitative evaluation of printing accuracy and tissue surface adaptation of mandibular complete denture polylactic acid pattern fabricated by fused deposition modeling technology / 中华口腔医学杂志

Objective@#To quantitatively evaluate the adaptation of polylactic acid (PLA) pattern of mandibular complete denture fabricated by fused deposition modeling (FDM) technology.@*Methods@#A mandibular complete denture digital model was designed through a complete denture design software based on a pair of standard maxillomandibular edentulous plaster model and their occlusion bases. Ten PLA mandibular complete dentures were printed with a FDM machine. The dentures were scanned with and without the plaster model using a three-dimensional (3D) scanner. In Geomagic software, the scanning data of printed dentures were registered to its computer aided design (CAD) data, and the printing error was analyzed using the multipoint registration command. For quantitatively evaluating the adaptation of the denture, the data of plaster model and PLA denture were registered to the whole data of denture located in the plaster model using the best-fit alignment command, the 3D deviation of the plaster model and tissue surface of the denture represent the space between them. The overall area was separated into three parts: primary stress-bearing area, secondary stress-bearing area and border seal area, and the average deviations of these three parts were measured. The values were analyzed using analysis of variance.@*Results@#Compared with the CAD data, the printing error was (0.013±0.004) mm. The overall 3D deviation between PLA denture and plaster model was (0.164±0.033) mm, in which the primary stress-bearing area was (0.165± 0.045) mm, the secondary stress-bearing area was (0.153 ± 0.027) mm, the border seal area was (0.186 ± 0.043) mm. These showed a good fit in the majority parts of the FDM denture to the plaster model. No statistically significant difference was observed between the three areas (F=1.857, P=0.175>0.05).@*Conclusions@#Combined with the 3D scanning, CAD and FDM technology, a FDM 3D printing process of complete denture for injection moulding can be established. As a result, high efficiency and low cost can be used to print out the complete denture, to lay the basis for further clinical applications.

Application and outlook of three-dimensional printing in prosthetic dentistry / 中华口腔医学杂志

At present, three-dimensional (3D) printing has been applied in many aspects in the field of prosthodontics, such as dental models, wax patterns, guide plates, dental restoration and customized implants. The common forming principles include light curing, sintering and melting-condensation, the materials include pure wax, resin, metal and ceramics. However, the printing precision and the strength of multi-material integrated forming, remains to be improved. In addition, as a technology by which the internal structure of a material can be customized manufacturing, further advantage of 3D printing used in the manufacture of dental restoration lies in the customization functional bionic micro-structures, but the related research is still in its infancy. The review briefly summarizes the commonly used 3D printing crafts in prosthetic dentistry, and details clinical applications and evaluations, provides references for clinical decision and further research.

Topographies of biomaterials regulate cell functions under epigenetic mechanisms / 中华口腔医学杂志

Topographies of biomaterials can act as potent regulators of cell functions, including proliferation, migration, differentiation, and reprograming. The mechanisms involve not only signaling pathways, but also epigenetic regulations. A clearer picture of how topographies of biomaterials alter epigenetic states facilitates the design of highly-functionalized and individualized biomaterials, and provides novel insights into epigenetic manipulation in controlling cell fates in regenerative medicine and disease treatment.

Establishing a luciferase reporter system to evaluate osteogenic differentiation poten-tial of human adipose-derived stem cells / 北京大学学报(医学版)

Objective:Human adipose-derived stem cells (hASCs)are a highly attractive source in bone tissue engineering.To generate a luciferase reporter system that could be used to quantitatively and rapidly examine osteogenic differentiation potential of human adipose-derived stem cells (hASCs ) in vitro,and eventually make it possible to monitor the osteogenic differentiation of transplanted cells in vi-vo.Methods:The genomic DNA harboring promotor regions of osteocalcin and DNA sequences encoding luciferase genes were amplified by PCR and cloned into the pLVX-pTRE-puro vector to generate the OCpro-Luc-Puro construct.Then,the OCpro-Luc-Puro construct together with three assistant vectors:pM-DLg/pRRE,pRSV-REV,and pVSVG,were transiently transfected into HEK293T cells followed by viral supernatants collection,filtration and concentration.Next,the hASCs stably expressing luciferase repor-ter gene driven by osteocalcin promotor were created with the lentivirus carrying OCpro-Luc-Puro cassette under puromycin selection.The OCpro-Luc-hASCs were then cultured in the absence or presence of osteo-genic differentiation medium.On the 7th and 1 4th days,after osteogenic induction,cellular extracts were collected and analyzed by luciferase reporter assay.Meanwhile,alizarin red staining and quantification as well as quantitative reverse transcription PCR (qRT-PCR)analysis of osteogenic associated genes osteo-calcin (OC),runt-related transcription factor 2 (Runx2)and alkaline phosphatase (ALP)were used to assess the osteogenic differentiation ability of OCpro-Luc-hASCs.Results:OCpro-Luc-Puro plasmid and OCpro-Luc-hASCs were successfully generated.On the 7th and 1 4th days after osteogenic induction,the luciferase activity of the cellular extracts from OCpro-Luc-hASCs was dramatically increased.Consistently, the extracellular matrix mineralization,as shown by Alizarin red S (ARS)staining and quantification was also markedly intensified and a marked increase of the mRNA expression levels of OC,Runx2 and ALP, although to variable extent,was observed upon osteogenic differentiation.These results indicated that the observations from traditional experiments examining hASCs osteogenic differentiation were largely in agreement with that of our luciferase reporter assay in OCpro-Luc-hASCs.Conclusion:We established a luciferase reporter system that could be used to rapidly,quantitatively and specifically determine osteo-genic differentiation ability of hASCs.Comparing with the traditional time-consuming methods,the system we generated here was highly effective.This system not only can be used to examine ostogenic differentia-tion of hASCs in a high throughput manner,but also provides a way to monitor ostogenic differentiation of cells in vivo.

Promoted role of bone morphogenetic protein 2/7 heterodimer in the osteogenic differentiation of human adipose-derived stem cells / 北京大学学报(医学版)

Objective:To investigate the role of bone morphogenetic protein 2/7 heterodimer (BMP-2/7)in the osteogenesis of human adipose-derived stem cells (hASCs).Methods:hASCs were exposed to three different treatments in vitro:osteogenic medium with 1 50 μg/L BMP-2/7 (experimental group), osteogenic medium alone (OM group)and proliferation medium (PM group).After 1 ,4 and 7 days of osteogenic induction,the amount of cellular DNA was measured to investigate the cytotoxicity.After 7 and 1 4 days,alkaline phosphatase (ALP)staining and quantification were performed to test the activity of ALP.After 21 and 28 days,the calcification deposition was determined by Alizarin Red S (ARS)stai-ning and quantification.The expressions of the osteoblast-related genes were tested on days 1 ,4,7 and 1 4.In the in vivo study,6 nude mice were used and 4 groups were set and implanted subcutaneously into the back of nude mice:(1 )β-TCP scaffold only (scaffold control group );(2 )β-TCP scaffold with hASCs cultured by PMin vitro for 1 week (PMcontrol group);(3)β-TCP scaffold with hASCs cultured by OM in vitro for 1 week (OM control group);(4)β-TCP scaffold with hASCs cultured by OM with 1 50 μg/L BMP-2/7 in vitro for 1 week (test group).After 4 weeks of implantation,histological staining was performed to evaluate the in vivo osteogenesis of hASCs.Results:After induction for 1 day,there was no significant difference between the experimental group and the PM group on the cellular DNA con-tent (P>0.05 ).After 4 days,the cellular DNA content increased under the stimulation of BMP-2/7 (P0.05).ALP ac-tivity was higher by the induction of BMP-2/7 than in OMalone and PM(P<0.05).More mineraliza-tion deposition and more expressions of osteoblast-related genes such as Runx2,ALP,COL-1 A1 and OC were determined in the experimental group at different time points (P<0.05).HE staining showed that, in the test group and OM control group,the extracellular matrix (ECM)with eosinophilic staining were observed around hASCs,and newly-formed bone-like tissues could be found in ECM around the scaffold materials.Moreover,compared with the OM control group,more bone-like tissues could be observed in ECMwith typical structure of bone tissue in the test group.Masson’s trichrome staining showed that more expression of collagen could be observed in ECM in the test group compared with the other groups.There was small amount of expression of collagen in the OM and PM control groups.No obvious positive results were found in the scaffold group.Conclusion:BMP-2/7 heterodimer plays a significant role in the osteo-genesis of hASCs and is able to enhance the osteogenic differentiation of hASCs in vitro and in vivo.