Biomaterials science and surface engineering strategies for dental peri-implantitis management.

Peri-implantitis is a bacterial infection that causes soft tissue inflammatory lesions and alveolar bone resorption, ultimately resulting in implant failure. Dental implants for clinical use barely have antibacterial properties, and bacterial colonization and biofilm formation on the dental implants are major causes of peri-implantitis. Treatment strategies such as mechanical debridement and antibiotic therapy have been used to remove dental plaque. However, it is particularly important to prevent the occurrence of peri-implantitis rather than treatment. Therefore, the current research spot has focused on improving the antibacterial properties of dental implants, such as the construction of specific micro-nano surface texture, the introduction of diverse functional coatings, or the application of materials with intrinsic antibacterial properties. The aforementioned antibacterial surfaces can be incorporated with bioactive molecules, metallic nanoparticles, or other functional components to further enhance the osteogenic properties and accelerate the healing process. In this review, we summarize the recent developments in biomaterial science and the modification strategies applied to dental implants to inhibit biofilm formation and facilitate bone-implant integration. Furthermore, we summarized the obstacles existing in the process of laboratory research to reach the clinic products, and propose corresponding directions for future developments and research perspectives, so that to provide insights into the rational design and construction of dental implants with the aim to balance antibacterial efficacy, biological safety, and osteogenic property.

MRI-based vertebral bone quality score as a novel bone status marker of patients with adolescent idiopathic scoliosis.

To investigate the application of MRI-based vertebral bone quality (VBQ) score in assessing bone mineral density (BMD) for patients with adolescent idiopathic scoliosis (AIS). We reviewed the data of AIS patients between January 2021 and October 2023 with MRI, whole-spine plain radiographs, quantitative computed tomography (QCT) and general information. VBQ score was calculated using T1-weighted MRI. Univariate analysis was applied to present the differences between variables of patients with normal BMD group (QCT Z-score > - 2.0) and low BMD group (QCT Z-score ≤ - 2.0). The correlation between VBQ score and QCT Z-score was analyzed with Pearson correlation test. A multivariate logistic regression model was used to determine the independent factors related to low BMD. Receiver operating characteristic curve (ROC) was drawn to analyze the diagnostic performance of VBQ score in distinguishing low BMD. A total of 136 AIS patients (mean age was 14.84 ± 2.10 years) were included, of which 41 had low BMD. The low BMD group had a significantly higher VBQ score than that in normal group (3.48 ± 0.85 vs. 2.62 ± 0.62, P < 0.001). The VBQ score was significantly negative correlated with QCT Z score (r = - 0.454, P < 0.001). On multivariate analysis, VBQ score was independently associated with low BMD (OR: 4.134, 95% CI 2.136-8.000, P < 0.001). The area under the ROC curve indicated that the diagnostic accuracy of the VBQ score for predicting low BMD was 81%. A sensitivity of 65.9% with a specificity of 88.4% could be achieved for distinguishing low BMD by setting the VBQ score cutoff as 3.18. The novel VBQ score was a promising tool in distinguishing low BMD in patients with AIS and could be useful as opportunistic assessment for screening and complementary evaluation to QCT before surgery.

TRIB3 Promotes Osteogenic Differentiation of Human Adipose-derived Mesenchymal Stem Cells Levelled by Post-transcriptional Regulation of miR-24-3p.

OBJECTIVE: To explore the effect of TRIB3 on the osteogenic differentiation of human adipose-derived mesenchymal stem cells (hASCs) and reveal the potential role of TRIB3 in bone regeneration. METHODS: TRIB3-knockdown and TRIB3-overexpression hASCs were used to explore the effect of TRIB3 on osteogenic differentiation by alkaline phosphatase (ALP) staining, alizarin red S (ARS) staining, quantitative real-time polymerase chain reaction (qRT-PCR) and heterotopic bone formation. The regulation of miR-24-3p on TRIB3 was detected by qRT-PCR and western blot. Ribonucleic acid (RNA) sequencing was performed to investigate the downstream regulatory network of TRIB3. RESULTS: TRIB3 promoted the osteogenic differentiation of hASCs both in vitro and in vivo. This process was regulated epigenetically by the post-transcriptional regulation of miR-24-3p, which could bind directly to the three prime untranslated region (3'UTR) of TRIB3 and inhibit TRIB3 expression. The downstream regulatory network of TRIB3-mediated osteogenic differentiation was related to calcium ion binding and cell metabolism, extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) and nuclear factor-κB (NF-κB) signalling pathways. CONCLUSION: TRIB3 is a promising therapeutic target for hASC-based bone tissue engineering and the epigenetic regulation of TRIB3 through miR-24-3p permits regulatory controllability, thus promoting osteogenesis through an important metabolic target while obtaining a safe and controllable effect via post-transcriptional epigenetic regulation.

Origin and Clinical Applications of Neural Crest-Derived Dental Stem Cells.

Over the past few decades, scientific research into neural crest-derived stem cells has progressed rapidly. The migration and differentiation of neural crest-derived stem cells has been an interesting area of research. Stem cells within teeth originating from the embryonic neural crest have attracted increasing attention in clinical and scientific research because they are easy to obtain and have superb stemness. The stem cells within the teeth include dental pulp stem cells (DPSCs), dental follicle stem cells (DFSCs), stem cells from apical papilla (SCAPs), stem cells from human exfoliated deciduous teeth (SHEDs), and periodontal ligament stem cells (PDLSCs). To date, there have been several interesting studies focusing on dental pulp regeneration, neural regeneration and the revascularization for therapeutic applications.

Transcriptomics and Functional Analysis of Graphene-Guided Osteogenic Differentiation of Mesenchymal Stem Cells.

OBJECTIVE: To explore graphene's effects on the gene expression profile of mesenchymal stem cells, and to reveal the mechanisms of graphene-guided osteogenic differentiation. METHODS: Human adipose-derived mesenchymal stem cells (hASCs) were cultured on single-layer graphene-coated titanium disks or titanium disks in proliferation medium (control) or osteoinduction medium for 7 days before RNA extraction. After library construction and RNA sequencing, identification of differentially expressed genes was performed through Limma package of R platform, with a cut-off value of log fold change (logFC) > = |1|. Pathway and Gene ontology (GO) analyses were conducted on DAVID Bioinformatics Resources 6.8 (NIAID/NIH). Network analyses were performed by the Ingenuity Pathways Analysis (IPA). RESULTS: Signalling pathway analysis revealed the top five pathways - cytokine-cytokine receptor interaction, neuroactive-ligand receptor interaction, calcium signalling pathway, PI3K-Akt signalling pathway and cell adhesion molecules. GO analyses demonstrated significant changes on cell adhesion, calcium signalling, and epigenetic regulation. IPA network analyses demonstrated that inflammation-related pathways were influenced by graphene, while the downstream factors of histone H3 and H4 were also altered especially under the existence of osteoinduction medium. CONCLUSION: Graphene promotes osteogenic differentiation of hASCs mainly by influencing cell adhesion, cytokine-cytokine receptor interactions, inflammatory responses, and potentially influences histone H3 and H4 through epigenetic regulation.

Differences in carbon source usage by dental plaque in children with and without early childhood caries.

Early childhood caries (ECC) is a considerable pediatric and public health problem worldwide. Preceding studies have focused primarily on bacterial diversity at the taxonomic level. Although these studies have provided significant information regarding the connection between dental caries and oral microbiomes, further comprehension of this microbial community's ecological relevance is limited. This study identified the carbon source metabolic differences in dental plaque between children with and without ECC. We compared the microbial community functional diversity in 18 caries-free subjects with 18 severe ECC patients based on sole carbon source usage using a Biolog assay. The anaerobic microbial community in the ECC patients displayed greater metabolic activity than that of the control group. Specific carbon source metabolism differed significantly between the two groups. Subjects from the two groups were well distinguished by cluster and principal component analyses based on discriminative carbon sources. Our results implied that the microbial functional diversity between the ECC patients and healthy subjects differed significantly. In addition, the Biolog assay furthered our understanding of oral microbiomes as a composite of functional abilities, thus enabling us to identify the ecologically relevant functional differences among oral microbial communities.

Osteogenic Differentiation of Three-Dimensional Bioprinted Constructs Consisting of Human Adipose-Derived Stem Cells In Vitro and In Vivo.

Here, we aimed to investigate osteogenic differentiation of human adipose-derived stem cells (hASCs) in three-dimensional (3D) bioprinted tissue constructs in vitro and in vivo. A 3D Bio-plotter dispensing system was used for building 3D constructs. Cell viability was determined using live/dead cell staining. After 7 and 14 days of culture, real-time quantitative polymerase chain reaction (PCR) was performed to analyze the expression of osteogenesis-related genes (RUNX2, OSX, and OCN). Western blotting for RUNX2 and immunofluorescent staining for OCN and RUNX2 were also performed. At 8 weeks after surgery, osteoids secreted by osteogenically differentiated cells were assessed by hematoxylin-eosin (H&E) staining, Masson trichrome staining, and OCN immunohistochemical staining. Results from live/dead cell staining showed that most of the cells remained alive, with a cell viability of 89%, on day 1 after printing. In vitro osteogenic induction of the 3D construct showed that the expression levels of RUNX2, OSX, and OCN were significantly increased on days 7 and 14 after printing in cells cultured in osteogenic medium (OM) compared with that in normal proliferation medium (PM). Fluorescence microscopy and western blotting showed that the expression of osteogenesis-related proteins was significantly higher in cells cultured in OM than in cells cultured in PM. In vivo studies demonstrated obvious bone matrix formation in the 3D bioprinted constructs. These results indicated that 3D bioprinted constructs consisting of hASCs had the ability to promote mineralized matrix formation and that hASCs could be used in 3D bioprinted constructs for the repair of large bone tissue defects.

[Promoted role of bone morphogenetic protein 2/7 heterodimer in the osteogenic differentiation of human adipose-derived stem cells].

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 150 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 14 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) staining and quantification. The expressions of the osteoblast-related genes were tested on days 1, 4, 7 and 14. 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) ß-TCP scaffold only (scaffold control group); (2) ß-TCP scaffold with hASCs cultured by PM in vitro for 1 week (PM control group); (3) ß-TCP scaffold with hASCs cultured by OM in vitro for 1 week (OM control group); (4) ß-TCP scaffold with hASCs cultured by OM with 150 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 content (P>0.05). After 4 days, the cellular DNA content increased under the stimulation of BMP-2/7 (P<0.05). On day 7, there was no significant difference among the three groups (P>0.05). ALP activity was higher by the induction of BMP-2/7 than in OM alone and PM (P<0.05). More mineralization deposition and more expressions of osteoblast-related genes such as Runx2, ALP, COL-1A1 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 ECM with 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 osteogenesis of hASCs and is able to enhance the osteogenic differentiation of hASCs in vitro and in vivo.

[Constructing 3-dimensional colorized digital dental model assisted by digital photography].

OBJECTIVE: To explore a method of constructing universal 3-dimensional (3D) colorized digital dental model which can be displayed and edited in common 3D software (such as Geomagic series), in order to improve the visual effect of digital dental model in 3D software. METHODS: The morphological data of teeth and gingivae were obtained by intra-oral scanning system (3Shape TRIOS), constructing 3D digital dental models. The 3D digital dental models were exported as STL files. Meanwhile, referring to the accredited photography guide of American Academy of Cosmetic Dentistry (AACD), five selected digital photographs of patients'teeth and gingivae were taken by digital single lens reflex camera (DSLR) with the same exposure parameters (except occlusal views) to capture the color data. In Geomagic Studio 2013, after STL file of 3D digital dental model being imported, digital photographs were projected on 3D digital dental model with corresponding position and angle. The junctions of different photos were carefully trimmed to get continuous and natural color transitions. Then the 3D colorized digital dental model was constructed, which was exported as OBJ file or WRP file which was a special file for software of Geomagic series. For the purpose of evaluating the visual effect of the 3D colorized digital model, a rating scale on color simulation effect in views of patients'evaluation was used. Sixteen patients were recruited and their scores on colored and non-colored digital dental models were recorded. The data were analyzed using McNemar-Bowker test in SPSS 20. RESULTS: Universal 3D colorized digital dental model with better color simulation was constructed based on intra-oral scanning and digital photography. For clinical application, the 3D colorized digital dental models, combined with 3D face images, were introduced into 3D smile design of aesthetic rehabilitation, which could improve the patients' cognition for the esthetic digital design and virtual prosthetic effect. CONCLUSION: Universal 3D colorized digital dental model with better color simulation can be constructed assisted by 3D dental scanning system and digital photography. In clinical practice, the communication between dentist and patients could be improved assisted by the better visual perception since the colorized 3D digital dental models with better color simulation effect.

[Establishing a luciferase reporter system to evaluate osteogenic differentiation potential of human adipose-derived stem cells].

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 vivo. 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 OC(pro)-Luc-Puro construct. Then, the OC(pro)-Luc-Puro construct together with three assistant vectors: pMDLg/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 reporter gene driven by osteocalcin promotor were created with the lentivirus carrying OC(pro)-Luc-Puro cassette under puromycin selection. The OC(pro)-Luc-hASCs were then cultured in the absence or presence of osteogenic differentiation medium. On the 7th and 14th 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 osteocalcin (OC), runt-related transcription factor 2 (Runx2) and alkaline phosphatase (ALP) were used to assess the osteogenic differentiation ability of OC(pro)-Luc-hASCs. RESULTS: OC(pro)-Luc-Puro plasmid and OCpro-Luc-hASCs were successfully generated. On the 7th and 14th days after osteogenic induction, the luciferase activity of the cellular extracts from OC(pro)-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 OC(pro)-Luc-hASCs. CONCLUSION: We established a luciferase reporter system that could be used to rapidly, quantitatively and specifically determine osteogenic 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 differentiation of hASCs in a high throughput manner, but also provides a way to monitor ostogenic differentiation of cells in vivo.

[Different multidisciplinary approaches of two traumatic teeth fractures in the esthetic zone: a case report].

In this article, different methods to deal with teeth fractures were discussed by presenting a case of traumatic crown-root fracture in the anterior esthetic zone. The traumatic crown-root fracture is a common problem in clinic. When a fracture line locates in close proximity to or below the alveolar bone crest, the fracture most likely involve the junctional epithelium and the connective tissue attachment. This type of fracture becomes a challenge for restorative dentists because it involves biologic, functional, and esthetic considerations, especially when the fracture occurs in an esthetic area. In this case, a young patient presented with two fractured upper anterior teeth to the Department of Periodontics, Peking University School and Hospital of Stomatology. After the comprehensive clinical evaluation, the right central incisor was decided to extract for implant therapy and the right lateral incisor was decided to retain by one modified crown lengthening surgery. The most common technique applied to save a retained root is a clinical crown lengthening procedure. However, the aggressive alveolar bone resection of both target and adjacent teeth to reestablish the bone width and periodontal health may compromise functional and esthetic outcomes. To reduce loss of excessive osseous tissue during osteotomy procedure, the modified crown lengthening of the right lateral incisor was performed, including minor bone resection and root reshaping. Regarding the right central incisor, the retained root was all located below the alveolar bone crest. The extraction and implant procedure, combined with guided bone graft were performed to avoid the damage to neighbor teeth during traditional restorative therapy and to reshape a preferable buccal contour. At the last visit, the patient was recalled with healthy periodontium, normal tooth function and favorable esthetic results.

[Ectopic osteogenesis of stromal cell-derived factor 1 combined with simvastatin-loaded collagen scaffold in vivo].

OBJECTIVE: To construct and evaluate a novel tissue-engineered bone composed of murine stromal cell-derived factor 1(mSDF-1), simvastatin (SIM) and collagen scaffold (Bio-Oss®), serving as a cell-homing approach for bone formation. METHODS: In the study, 32 ICR mice were randomly divided into 4 groups,each group including 8 mice. The drug-loaded collagen scaffolds were implanted subcutaneously onto the cranium of each mouse according to the groups: (1) 1:50 (volume ratio) dimethyl sulfoxide (DMSO)/phosphate-buffered saline (PBS) solution + collagen scaffold (blank control group); (2) 10⁻³ mol/L SIM solution + collagen scaffold (SIM group); (3) 200 mg/L mSDF-1 solution + collagen scaffold (mSDF-1 group); and (4) 10® mol/L SIM +200 mg/L mSDF-1 solution + collagen scaffold (SIM + mSDF-1 group). One week after implantation, the mice were treated by injecting the same drug solution mentioned above around the scaffold once a day for two days. The specimens were harvested 6 weeks after implantation and the bone formation was evaluated by soft X-ray analysis, HE staining and immunohistochemical staining. Angiogenesis of each group was checked by calculation of vessels in each tissue section. RESULTS: Six weeks after implantation, the collagen scaffolds were retrieved. The value of gray scale for the SIM+mSDF-1 group [(421 836.5 ± 65 425.7)pixels] was significantly higher than that of the blank control group[(153 345.6 ± 45 222.2) pixels, P<0.01], the SIM group [(158 119.2 ± 100 284.2)pixels, P<0.01], and the mSDF-1 group[(255 529.5 ± 152 142.4)pixels, P<0.05]; HE staining analysis revealed that significant bone formation was achieved in the SIM + mSDF-1 group; The immunohistochemical staining showed the existence of osteopontin and osteocalcin in the SIM + mSDF-1 group; There were more vessels in the SIM+mSDF-1 group[(46 ± 8)vessels/mm²] than in the blank control group [(23 ± 7) vessels/mm2, P<0.01], and the SIM group[(24 ± 6) vessels/mm2, P<0.01]. CONCLUSION: The novel tissue-engineered bone composed of mSDF-1, SIM and collagen scaffolds has the potential to form bone subcutaneously in vivo. It represents a novel method of in vivo bone re-generation without seed cell delivery.

The effect of simvastatin on chemotactic capability of SDF-1α and the promotion of bone regeneration.

The purpose of this study was to investigate the cooperative effects of simvastatin (SIM) and stromal cell-derived factor-1α (SDF-1α) on the osteogenic and migration capabilities of mesenchymal stem cells (MSCs), and construct a cell-free bone tissue engineering system comprising SIM, SDF-1α and scaffold. We found that 0.2 µm SIM significantly increased alkaline phosphatase activity (P < 0.05) of mouse bone marrow MSCs with no inhibition of cell proliferation, and enhanced the chemotactic capability of SDF-1α (P < 0.05). Next, we constructed a novel cell-free bone tissue engineering system using PLGA loaded with SIM and SDF-1α, and applied it in critical-sized calvarial defects in mice. New bone formation in the defect was evaluated by micro-CT, HE staining and immunohistochemistry. The results showed that PLGA loaded with SIM and SDF-1α promoted bone regeneration significantly more than controls. We investigated possible mechanisms, and showed that SDF-1α combined with SIM increased MSC migration and homing in vivo, promoted angiogenesis and enhanced the expression of BMP-2 in newly-formed bone tissue. In conclusion, SIM enhanced the chemotactic capability of SDF-1α and the cell-free bone tissue engineering system composed of SIM, SDF-1α and scaffold promoted bone regeneration in mouse critical-sized calvarial defects.

[Application of patient-participated digital design in esthetic rehabilitation of anterior teeth].

OBJECTIVE: To explore a new method of patient-involved digital design, esthetic outcome prediction and fabrication for the esthetic rehabilitation of anterior teeth, and to provide an alternative choice for the restoration of anterior teeth. METHODS: In this study, 32 patients with esthetic problems in their anterior teeth were included and divided into two groups randomly: the experimental group (16 patients) and control group (16 patients). In the experimental group, the dentition and facial images were obtained by intra-oral scanning and Three-dimensional (3D) facial scanning and then calibrated. The design of the rehabilitation and the esthetic outcome prediction were created by computer-aided design (CAD) software. After morphologic modification according to the patients' opinions, prostheses were fabricated according to the final design by computer-aided manufacturing (CAM) equipment. As for the control group, the regular design method was applied to restore their anterior teeth. The time consuming in the first insertion of each restoration in both groups was recorded. The quality of the prostheses was assessed by another prosthedontist. The satisfaction to prostheses and the facial appearance were evaluated by the patients. RESULTS: The process of the patient-involved digital design and outcome anticipation was successfully established. The patients were satisfied with the esthetic effects of the anterior restoration made by the digital technique. The acceptance rate of the patients on the digital rehabilitation in the experimental group was 100%. There was no significant difference of the quality of the prostheses between the two groups. The satisfaction rate of the patients on prostheses and facial appearance was significantly higher in the experimental group than in the control group (P < 0.05). In addition, the time consuming in the first insertion of the experimental group was much shorter than that in the control group (P < 0.01). CONCLUSION: The new method of the patient-involved digital design, esthetic outcome prediction and fabrication for the esthetic rehabilitation of anterior teeth is a practical technique. This method is useful in shortening the time consuming of the restoration of anterior teeth and improving the patient satisfaction with the esthetic outcome.

[Effect of human adipose-derived stromal cells on osteogenesis in vivo].

OBJECTIVE: To explore the effect of human adipose-derived stromal cells (hASCs) on the osteogenesis during the process of bone formation in vivo, and to lay the foundation of further investigations on the mechanism of in vivo osteogenesis of hASCs. METHODS: hASCs were isolated from adipose tissue by the method of collagenase digestion, and were routinely proliferated and passaged. In the in vivo study 16 nude mice were used and 4 groups were set and implanted subcutaneously into the back of nude mice: (1) blank; (2) ß-tricalcium phosphate (ß-TCP) scaffold only (scaffold control group); (3) ß-TCP scaffold with human fibroblasts (negative cell control group); (4) ß-TCP scaffold with hASCs (test group). After 1 week, 2 weeks, 4 weeks and 6 weeks of implantation, samples from the 4 nude mice were collected at each time point. Scanning electron microscope (SEM) observation and histological staining were performed to evaluate the in vivo osteogenesis of hASCs. RESULTS: SEM images showed that large amount of extracellular matrix (ECM) could be observed around hASCs in test group after 2 weeks of implantation. At the time point of 4 weeks, mineral deposit was found in ECM. At the time point of 6 weeks, the mineral deposit was observed to increase significantly. HE staining showed that the ECM with eosinophilic staining could be observed around hASCs after 2 weeks of implantation. At the time point of 4 weeks, newly-formed bone-like tissue could be found in ECM around the scaffold materials. At the time point of 6 weeks, more bone-like tissues were observed in ECM with typical structure of bone tissue. In comparison, no obvious mineralization and bone-like tissue were found in other groups. CONCLUSION: hASCs play important roles in the process of osteogenesis in vivo, including secretion of large amount of ECM, acceleration of the mineralization of ECM and guidance for the formation of bone-like tissues.

[Recombinant human tumor necrosis factor-alpha promotes human adipose-derived stromal cells transforming into osteoblast in vitro].

OBJECTIVE: To investigate the effect of recombinant human tumor necrosis factor alpha (rhTNF-α) on the osteogenesis potential of the osteo-induced human adipose-derived stromal cells (hASCs) in vitro. METHODS: hASCs at passage 4 were divided into four groups according to culturing conditions: basal medium [BM, DMEM + 10% FBS + antibiotics], BM with 10 µg/L rhTNF-α, osteogenic medium (OM, BM + dexamethasone + L-ascorbate + ß-glycerophosphate) and OM with 10 µg/L rhTNF-α. On days 3, 7, 14 and 21, alkaline phosphatase (ALP) activities were examined. On days 14 and 21, the staining and quantitation of calcium deposition were performed. For the cells under osteogenic induction, osteoblast-related genes, such as core-binding factor α1 (Cbfa1), Osterix (Osx) and osteocalcin (OC) were analyzed with reverse transcription PCR on days 3, 7, 14, and 21, and real time PCR was performed to confirm the effect of rhTNF-α on genes expression on day 3 . RESULTS: rhTNF-α promoted ALP activities of induced hASCs on day 14 (3.527 ± 0.415 vs. 2.345 ± 0.354,P<0.01) and on day 21 (3.106 ± 0.105 vs. 2.442 ± 0.163,P<0.01) and promoted calcium deposition of induced hASCs on day 14 (2.896 ± 0.173 vs. 0.679 ± 0.173,P<0.01) and on day 21 (2.231 ± 0.233 vs. 1.729 ± 0.229, P<0.01). RT-PCR and Real-time PCR assays showed that rhTNF-α augmented the expression of Cbfa1, Osx and OC of these cells. CONCLUSION: The findings indicate that 10 µg/L rhTNF-α can promote the osteogenic potential of osteogenetically induced hASCs in vitro.

[Osteogenic capability of primary human adipose-derived stromal cells in vivo].

OBJECTIVE: To investigate the osteogenic capability of primary human adipose-derived stromal cells (hASCs) in vivo. METHODS: hASCs were isolated from adipose tissue by the method of collagenase digestion. After 7 and 14 days of osteogenic induction, alkaline phosphatase (ALP) staining and Alizarin Red staining were performed to test the osteogenic potential of hASCs in vitro. After 14 days of adipogenic induction, the adipogenic potential of hASCs was assayed by Oil Red O staining.In the in vivo part, 12 nude mice were used. Test group (scaffold with hASCs) and control group (scaffold only) were symmetrically implanted into the back of nude mice. After 4 weeks and 8 weeks of implantation, samples were collected. Histological and immunohistochemical staining were performed to investigate the osteogenic capability of hASCs. RESULTS: Approximately 6×10(7) hASCs could be isolated from 300 mL adipose tissue. ALP, Alizarin Red and Oil Red O staining of hASCs showed positive results after specific inductions. These results demonstrated the osteogenic and adipogenic potentials of hASCs in vitro. Bone-like tissue could be observed in the test group at 4 weeks and 8 weeks after the implantation. Immunohistochemical staining showed that there were positive results of osteocalcin, ALP and anti-human nuclei in the bone-like tissue areas. CONCLUSION: A large number of primary hASCs can be isolated from human adipose tissue; hASCs combined with scaffold show osteogenic capability in vivo.

[Application of human adipose-derived stromal cells in bone tissue engineering].

Human adipose-derived stromal cells (hASCs) can be obtained from adipose tissues that offer an abundant and easily accessible pool of stem cells. Thus, hASCs have become a highly attractive source of seed cells in bone tissue engineering and have promising prospects in bone regeneration. Since 2002, our research group has performed a series of experiments on hASCs and its application in bone tissue engineering, including: to substitute dexamethasone by 1,25 (OH)2 vitamin D3 to induce osteogenic differentiation of hASCs; to explore the effect of epigenetic regulation and to inflammation on the osteogenic differentiation of hASCs; to construct a novel and simple tissue engineered bone system by hASCs and human platelet-rich plasma (hPRP) and to investigate the bone formation capability of this tissue engineered bone and the stimulatory effect of simvastatin. Our results suggested that 1,25 (OH)2 vitamin D3 could replace dexamethasone to induce the osteogenic differentiation of hASCs; retinoblastoma binding protein 2 (RBP2), as one of histone demethylases, could regulate the osteogenic differentiation of hASCs epigenetically while tumor necrosis factor α (TNFα), as a inflammatory factor, could also influence the osteogenic differentiation of hASCs. Moreover, we found that in vivo bone formation could be detected by our novel tissue engineered bone composed with hASCs and hPRP; simvastatin could enhance the bone formation capability of this tissue-engineered structure.

Serum procalcitonin predicting mortality in exertional heatstroke.

BACKGROUND: The aim of this study was to test if Procalcitonin PCT value at the time of admission is a predictor of mortality and/or a diagnostic marker of concomitant infection in exertional heatstroke. METHODS: 68 patients with exertional heatstroke admitted to the multidisciplinary intensive care unit were studied. Serum PCT was detected by means of a specific and ultrasensitive immunoluminometric assay within 2 h of admission. The Acute Physiology and Chronic Health Evaluation (APACHE) II score was evaluated within 24 h of admission. RESULTS: There was no significant difference in PCT levels between concomitant infection and non-infection patients (p=0.712). Elevated PCT level in exertional heatstroke patients was associated with a more critical pathological state. PCT values in patients with multiple organ dysfunction syndrome (MODS) were significantly higher than those without MODS (p=0.007.). PCT values were also positively correlated with APACHE II scores (r=0.588, p=0.016). PCT values in non-survivors were higher than in survivors at univariate regression analysis (p=0.017). After adjusting for confounders, PCT concentration also remained an independent determinant of mortality (OR 2.98; 95% CI 1.02 to 4.41; p=0.039). Receiver operating characteristic curve for PCT concentration was located above the reference line, which shows an association with mortality. The area under the curve for PCT concentration (0.705; 95% CI 0.547 to 0.862) was statistical significantly (p=0.019). As a predictor of mortality, PCT value was inferior to APACHE II score. CONCLUSIONS: PCT value at the time of admission is an independent predictor of mortality, but maybe not a good indicator of concomitant infection in exertional heatstroke.

[Inhibition of retinoblastoma binding protein 2 promotes osteogenic differentiation of human adipose-derived stromal cells].

OBJECTIVE: To explore the effect of retinoblastoma binding protein 2 (RBP-2), a histone H3K4 demethylase, on osteogenic differentiation of human adipose-derived stromal cell (hASC). METHODS: According to the GenBank sequence information of RBP-2, four different small interfering RNAs (siRNA) targeting RBP-2 gene were designed and the corresponding short hairpin RNAs (shRNA) were cloned into pLL 3.7 lentivirus RNA interference vector. The lentivirus with RBP-2-siRNA was packaged in 293T cells. The effective sequence was examined and selected by Western blotting and real-time PCR. The lentiviruses with efficient knockdown effects were used to infect hASC. On the 14th day after osteogenic differentiation, alkaline phosphatase (ALP) activities of hASC were quantitatively tested and at the same time, ALP staining and alizarin red staining were performed to assess the difference of osteogenic differentiation between the knockdown group and the control group. RESULTS: The recombinant lentivirus siRNA targeting RBP-2 was successfully constructed and the expression of RBP-2 mRNA and protein were dramatically suppressed by infection with RBP-2-siRNA lentivirus. On the 14th day after osteogenic induction, ALP activity of hASC in the knockdown group [(299.2 ± 22.7), (224.3 ± 17.7) U/g] was much stronger than that in the control group [(129.9 ± 12.9) U/g, P < 0.05] and the same result was achieved for the ALP staining and alizarin red staining. CONCLUSIONS: The constructed RBP-2-siRNA lentivirus could markedly decrease the expression of RBP-2 and promote osteogenic differentiation of hASC. It indicated that RBP-2 can repress the osteogenic differentiation of hASC.