Differentiation and Characterization of Cystic Fibrosis Transmembrane Conductance Regulator Knockout Human Pluripotent Stem Cells into Salivary Gland Epithelial Progenitors

The differentiation of pluripotent stem cells has been used to study disease mechanisms and development. We previously described a method for differentiating human pluripotent stem cells (hPSCs) into salivary gland epithelial progenitors (SGEPs). Here, cystic fibrosis transmembrane conductance regulator (CFTR) knockout hPSCs were differentiated into SGEPs derived from CFTR knockout hESCs (CF-SGEPs) using the same protocol to investigate whether the hPSC-derived SGEPs can model the characteristics of CF. CF—a disease that affects salivary gland (SG) function—is caused by mutations of the CFTR gene. Firstly, we successfully generated CFTR knockout hPSCs with reduced CFTR protein expression using the CRISPR-Cas9 system. After 16 days of differentiation, the protein expression of CFTR decreased in SGEPs derived from CFTR knockout hESCs (CF-SGEPs). RNA-Seq revealed that multiple genes modulating SG development and function were down-regulated, and positive regulators of inflammation were up-regulated in CF-SGEPs, correlating with the salivary phenotype of CF patients. These results demonstrated that CFTR suppression disrupted the differentiation of hPSC-derived SGEPs, which modeled the SG development of CF patients. In summary, this study not only proved that the hPSC-derived SGEPs could serve as manipulable and readily accessible cell models for the study of SG developmental diseases but also opened up new avenues for the study of the CF mechanism.

Determining Osteogenic Differentiation Efficacy of Pluripotent Stem Cells by Telomerase Activity

BACKGROUND: Bone tissue engineering based on pluripotent stem cells (PSCs) is a new approach to deal with bone defects. Protocols have been developed to generate osteoblasts from PSCs. However, the low efficiency of this process is still an important issue that needs to be resolved. Many studies have aimed to improve efficiency, but developing accurate methods to determine efficacy is also critical. Studies using pluripotency to estimate efficacy are rare. Telomerase is highly associated with pluripotency. METHODS: We have described a quantitative method to measure telomerase activity, telomeric repeat elongation assay based on quartz crystal microbalance (QCM). To investigate whether this method could be used to determine the efficiency of in vitro osteogenic differentiation based on pluripotency, we measured the pluripotency pattern of cultures through stemness gene expression, proliferation ability and telomerase activity, measured by QCM. RESULTS: We showed that the pluripotency pattern determined by QCM was similar to the patterns of proliferation ability and gene expression, which showed a slight upregulation at the late stages, within the context of the general downregulation tendency during differentiation. Additionally, a comprehensive gene expression pattern covering nearly every stage of differentiation was identified. CONCLUSION: Therefore, this assay may be powerful tools for determining the efficiency of differentiation systems based on pluripotency. In this study, we not only introduce a new method for determining efficiency based on pluripotency, but also provide more information about the characteristics of osteogenic differentiation which help facilitate future development of more efficient protocols.

Study of oral microbial adhesion and biofilm formation on the surface of nano-fluorohydroxyapatite/polyetheretherketone composite / 中华口腔医学杂志

<p><b>OBJECTIVE</b>To develop novel polyetheretherketone (PEEK) based nanocomposites which possess the favorable antibacterial property, and to investigate the oral microbial adhesion and biofilm formation on the surfaces of PEEK, nano-fluorohydroxyapatite (n-FHA)-PEEK and nano-hydroxyaptite (n-HA)-PEEK.</p><p><b>METHODS</b>The bacterial adhesion and biofilm formation on the surfaces of n-FHA-PEEK, n-HA-PEEK were investigated via microbial viability assay kit and laser scanning confocal microscope (LSCM), respectively, with pure PEEK as control group.</p><p><b>RESULTS</b>No significantly statistical difference were found in the bacterial adhesion amounts on the surfaces of n-FHA-PEEK, n-HA-PEEK and PEEK at 1 h and 4 h. However, the number of bacteria on the n-FHA-PEEK surface decreased dramatically at 2 h (0.496 ± 0.008) compared with n-HA-PEEK groups (0.543 ± 0.015, P < 0.01). Although the biofilms formation on surfaces observed by LSCM had similar morphology and thickness at 3, 7, 14 d, that on the n-FHA-PEEK surface showed the highest dead-to-live bacteria ratio among the three materials at 14 d.</p><p><b>CONCLUSIONS</b>The combination of n-HA, especially for the n-FHA could inhibit the bacteria adhesion and accelerate the bacterial death, eventually may have an influence on the structure of biofilms and reduce the risk of peri-implantitis. Therefore, n-FHA-PEEK nanocomposites presented a good prospect for clinical applications as dental implant materials.</p>

Application of proteomic methods in biomaterials biological evaluation / 国际生物医学工程杂志

As one of the most important strategies of no drug therapy,biomaterials have drawn extensive application in clinical use.Current animal tests and assays of cell level haven't reached the satisfying depth on the mechanism of biocompatibility and material-host interaction.As the typical method of post-genome era,proteomics contributed to investigation in the biomarker of diseases and the global theory of physiological function and drug effect.Along with the increase of the inquiry of biomaterial,high quality evaluation of bio-effects and the reliability is required.Utilizing proteomic method to study the dynamic variation of the whole proteins after contacting with certain biomaterial is an effective means to explore the direction of its further improvement.This review summarizes most representative studies on function and biocompatibility of biomaterials and focus on the application in dental implant materials,which indicate the significance of the combination of the biomaterials development and the progressive biology assay technique.

Progress of seed cells and scaffolds for skin tissue engineering / 中国组织工程研究

OBJECTIVE: Based on the overview of progress regarding seed cells and scaffolds for skin tissue engineering, to introduce the research of three dimensional nanofiber scaffolds prepared by electrospinning technique, and its application prospect in tissue engineered skin.DATA SOURCES: The databases of CNKI, Sciencedirect, and I.S.I were retrieved by the first author with key words of "tissue-engineering, skin, wound healing, seed cell, scaffold, electrospunning" in both Chinese and English from 1992 to 2009.DATA SELECTION: Major accomplishments of research on skin tissue engineering published in recent years.MAIN OUTCOME MEASURES: ①Papers related to treating skin destruction using tissue engineered materials. ②Articles correlative to seed cells and scaffold materials. ③Papers regarding electrospinning technique.RESULTS: The preparation of artificial skins is the research direction of tissue-engineered skin, in particular, studies regarding epidermal stem cells, bone marrow mesenchymal stem cells, hair follicle stem cells, and adipose-derived stem cells are attracted more attention. As tissue engineering, it is a key problems to prepare a scaffold to meet the needs of mechanical property,physical composition and biocompatibility. Scaffold materials comprise micrometer porous scaffold and nano-fiber scaffolds. The electrospinning technique is newly developed method for preparing nano-fiber scaffolds with the advantage of fast and convenience, and the scaffolds possess greater porosity, which not only benefit for the blood circulation and oxygen exchange,but also prevent the loss of water content and protein from wound surface.CONCLUSIONS: Tissue engineered skin is an important ingredient of regenerative medicine, seed cells and scaffold matrixes are two core problems that call for long term investigation. With the incessant development and integration among life sciences,nanotechnology and computer technology, marvelous progress has been achieved in the perspective of comprehending the interaction between seed cells, the mutual regulation mechanism and how the architecture and properties of scaffold materials affecting the regenerative procedures.

Preparation, Crystallization and Preliminary X-ray Crystallographic Analysis of Smu.776 From Caries Pathogen Streptococcus mutans / 生物化学与生物物理进展

The gene smu. 776 encodes a possible S-adenosylmethionine-dependent methyltransferase of 385 residues in Streptococcus mutans, a primary pathogen for human dental caries. The DNA fragment of smu.776 was cloned into pET28a and expressed in good amount from the E. coli strain BL21 (DE3). Smu.776 protein was purified to homogeneity in a two-step procedure ofNi2+ chelating and size exclusion chromatography. Crystals were obtained by hanging-drop vapor diffusion method and diffracted to 2.0 (A) resolution.The crystal belongs to orthorhombic space group C2 with cell dimension of a=168.47 (A), b= 50.66 (A), c=53.96 (A), β=104.22°. The asymmetric unit is expected to contain one molecule with solvent content of 51.3%.

Protein Preparation, Crystallization and Preliminary X-ray Crystallographic Analysis of Smu.260 From Streptococcus mutans--a Cariogenic Dental Pathogen / 生物化学与生物物理进展

Smu. 260 encodes a putative protein of 200 residues in Streptococcus mutans, a primary pathogen for human dental caries. Smu. 260 was cloned into expression vector pET28a and expressed in good amount trom the E. coli strain BL21 (DE3). Smu.260 protein was purified to homogeneity in a two-step procedure of Ni2+ chelating and size exclusion chromatography. The purified protein exists in two forms, a dimer form about 46 ku with yellow color and a tetramer form without apparent color. Crystals were obtained from the dimer protein by hanging-drop vapor-diffusion method. The crystals diffracted to about 2.3 A resolution and belong to orthorhombic space group P212121 with cell dimensions of a = 89.88A, b = 90.91 A, c = 105.17 A. The asymmetric unit is expected to contain two dimers with solvent content of 53%.

Study on injectable bioactive bone repairing material of nano-hydroxyapatite and polyamide-66 composite / 生物医学工程学杂志

The aim of this study was to evaluate the injectability, histocompatibility, function and other properties of the injectable bioactive bone repairing material of nano-hydroxyapatite and polyamide-66 (n-HA/PA66) composite. The XRD pattern, the relationship between the injectability and liquid-powder ratio, setting time and liquid-powder ratio, compressive strength and liquid-powder ratio were assessed. The size of the composite was determined to be 70 nm in length and 30 to 50 nm in width, and the molecular weight of polyamides-66 was 18000. The diameter of pores of the composite was about 200 to 400 micrometer. To evaluate the histocompatibility and function, 8 male dogs were studied with the injectable n-HA/PA66 composite implanted in the artificial defected alveolus of mandible on only one side to be compared with the intact alveolus on the other side. The specimens were taken at 4, 8, 12, 16 months after the implantation and the results were evaluated. The XRD pattern of the solidificated n-HA/PA66 composite was the same as the powdered n-HA/PA66 composite. The injectable n-HA/PA66 composite had a good injectability, 25 to 30 minutes setting time and about 37 MPa compressive strength when the liquid-powder ratio was 0.50. The healing of the gingiva was well at the implanted areas in all animals. The height of the repaired alveolar bone was obvious higher than that of the blank control. The earlier sign of ossification was histologically observed at 16 weeks after implantation. The injectable n-HA/PA66 composite has good biocompatibility and osteoconductive property. As an injectable material, with good maneuverability, it is useful for repairing irregular bone defects, especially in oral and maxillofacial surgery.

The experimental study of PDLLA/rhBMP-2 compound screws for internal fixation of mandibular fracture / 华西口腔医学杂志

<p><b>OBJECTIVE</b>To study the effect of poly D,L-lactic acid (PDLLA) screws with PDLLA-rhBMP compound on bone regeneration in the screw holes and fracture ends of dog mandibles.</p><p><b>METHODS</b>A self-control study was carried out in 4 dogs. PDLLA/rhBMP-2 compound screws were implanted to fix the mental fractures and PDLLA screws were used as control. The samples from mandibles were collected at 4, 8, 12, 16 weeks after implantation and observed by radiography and histology.</p><p><b>RESULTS</b>All dogs showed a greater degree of bone regeneration around PDLLA/rhBMP-2 screws than PDLLA ones and all fractures were fixed and healed well.</p><p><b>CONCLUSION</b>The PDLLA-rhBMP screw has a better effect of inducing osteogenesis than PDLLA screw, and is able to exert a good fixation to fracture.</p>

Simultaneous repair of cleft lip and closure of cleft hard palate with vomer flaps in patients with unilateral complete cleft lip and palate / 华西口腔医学杂志

<p><b>OBJECTIVE</b>The purpose of this study was to retrospect the prognosis of simultaneous repair of cleft lip and closure of cleft hard palate with vomer flaps in patients with unilateral complete cleft lip and palate.</p><p><b>METHODS</b>A retrospective study was carried out in 47 patients with unilateral complete cleft lip and palate and, simultaneously received repair of cleft lip and closure of cleft hard palate with vomer flaps. The duration of operation, as well as the blood loss during the operation was recorded, and compared with those patients who only received cleft lip repair.</p><p><b>RESULTS</b>All the operations were successful, and the wound healed well. The procedure of simultaneous repair of cleft lip and closure of cleft hard palate with vomer flaps did not prolong the operating time, compared with simple cleft lip repair. No blood transfusion was needed due to closure of cleft hard palates with vomer flaps.</p><p><b>CONCLUSION</b>Simultaneous repairs of cleft lip and closure of cleft hard palate with vomer flaps are safe for patients with unilateral complete cleft lip and palate.</p>

The study of biocompatibility of super high molecular weight poly D,L-lactic acid implant / 中华口腔医学杂志

<p><b>OBJECTIVE</b>To evaluate the biocompatibility of the super high molecular weight poly D,L-lactic acid (SHMW-PDLLA) implant.</p><p><b>METHODS</b>The SHMW-PDLLA plates were implanted into the SD-rats between the masseter and ramus of the mandible. The blood specimens were gained at 3, 6, 9, 12 months after the operation. The proteins, electrolyte, enzyme and other indices were tested by use of Beckman automatic biochemical analysis device. The soft tissue specimens around the SHMW-PDLLA plates were gained at 3, 6, 9, 12 months after the operation and the tissue reaction was observed with the pathological and haematological methods.</p><p><b>RESULTS</b>There were not any abnormal findings in the blood after the SHMW-PDLLA plates implanted in the body of SD-rats. The implanted SHMW-PDLLA plates were degraded gradually in 6 to 12 months after the operation. There was not any abnormal tissue reaction found to the soft tissue around the SHMW-PDLLA plates by histological and pathological observations.</p><p><b>CONCLUSIONS</b>The SHMW-PDLLA implant has a good biocompatibility to SD-rats.</p>

Experimental research on degradation and biocompatibility of super-high-molecular-weight poly-DL-lactic acid / 华西口腔医学杂志

<p><b>OBJECTIVE</b>The super-high-molecular-weight poly-DL-lactic acid (PDLLA), with the molecular weight of 900 kD, is a newly emerging biomaterial and potentially used in the therapy of bone fracture because of its excellent mechanical property. However the biocompatibility of this material has not been reported so far, therefore this experiment was designed to examine whether the super-high-molecular-weight PDLLA was harmful to creatures, when it was implanted in the body of animals for a long period.</p><p><b>METHODS</b>The material was prepared in small cuboids, with the size of 1.0 mm x 1.5 mm x 2.0 mm, and these blocks were implanted into the masseteric space of SD rats and, the activity of the SD-rats was monitored continuously. The animals were sacrificed in the 3rd, 6th, 9th, 12th months after the operation and, the specimens were taken out from the animals. The examination included anatomical, pathological and haematological methods. The data were analyzed with SPSS 8.0.</p><p><b>RESULTS</b>The wound healed well after the operation. Super-high-molecular-weight PDLLA degraded 6 months after the implantation. In the 3rd month after the operation, a thin fiber membrane around the materials was formed. In the 6th month, the membrane was much thinner than that in the 3rd month and completely disappeared in the 9th month. The pathological examination showed that slightly inflammatory reaction appeared in the tissue around these blocks in the 3rd month, but the inflammatory reactions were gradually remitted in the following 6th, 9th and 12th months. Further, the haematological examination did not show any abnormity during the 12-month observation period.</p><p><b>CONCLUSION</b>The super-high-molecular-weight PDLLA can be degrade when it is implanted into the body of creatures, which proves its good biocompatibility.</p>

Repair of cleft hard palate with absorbable membrane of poly-DL-lactic acid: A clinical report / 实用口腔医学杂志

Objective:To study the effects of poly-DL-lactic acid(PDLLA) biomembrane in the repair of cleft hard palate. Methods:Thirty-two cleft palate patients, age 2.67 to 12.83 years old, were treated. The traditional surgical method was used to close the cleft soft palate, and the PDLLA biomembrane was implanted into the surgical gap between the periosteum and bone at the hard palate and fixed with suture to close the cleft hard palate. Clinical follow-up was conducted for 6 months.Results:Operations on all 32 patients were completed successfully.The average surgical time was not prolonged, and post-operative complication was not increased. Wound healing of soft palate, uvula and hard palate was uneventful with no incidence of fistula or dehiscence. Conclusion: PDLLA absorbable biomembrane can be used to repair cleft hard palate.