BACKGROUND: It is still disputed whether several growth factors of platelet-rich plasma and the fascia with blood vessels can promote bone regeneration and vascularization. OBJECTIVE: To evaluate the effects of platclet-rich plasma and the fascia with blood vessels on the vascularization of tissue-engineered bone composited by marrow stromal stem cells (MSCs) and decalcified bone matrix (DBM). DESIGN, TIME AND SETTING: The present single-sample observation, self-control animal experiment was performed at the Central Laboratory Center, Affiliated Hospital of Qingdao University Medical College between October 2004 and November 2007. MATERIALS: Twelve healthy hybrid dogs, aged 11-12 months, weighing 20-25 kg, equal number of males and females, were included for this study. METHODS: ①MSCs were isolated from dog bone marrow by density gradient centrifugation, followed by in vitro adherent culture and ontogenesis culture.Dog femur was taken for preparation of DBM and composited with MSCs.②The back of each dog was divided into 4 regions (A,B,C,and D).In the regions A and B, DBM/MSCs/platelet-rich plasma composites were transplanted. In the regions C and D, DBM/MSCs composites were transplanted. The implants for the regions A and C were wrapped with latissimus dorsi fascia with blood vessels. The implants for the regions B and D were wrapped with blood vessels-free superficial fascia from back. At weeks 4,8,and 12,4 dogs comprising 2 males and 2 females, were sacrificed following anesthesia for specimen harvesting. MAIN OUTCOME MEASURES:①MSC morphology was observed utilizing a phase contrast microscope, and cell growth curves were portrayed by methyl thiazolyl tetrazolium colorimetric assay.②Morphology of osteoblasts induced was observed by modified alkaline phospharase (ALP) staining (Ca-Co method), Von Kossa method, alizarin red method and calcium node staining.③Composite structure was observed through the use of inverted phase contrast microscope and scanning electron microscope.④Gross, radiological, and histological observations. RESULTS: ①Both induced osteoblasts and MSCs exhibited ALP and calcium node stainings positive.②The growth curves of induced osteoblasts and MSCs presented with typical "S" shape.③Following composite culture of allogenic DBM and MSCs, MSCs grew well, rapidly proliferated and could secrete a great amount of extraccllular matrix.④At each time point, osteogenic bone mass, bone absorbance, and vascularization were better in regions A,B,and C than in region D (P＜0.05). CONCLUSION: Histological and radiological observations have confirmed that both platelet-rich plasma and the fascia with blood vessels can synergistically promote the formation and vascularization of tissue-engineered bone.

Objective: To study the effects of tissue engineered bone in the repair of mandibular defect.Methods:The experiments were conducted in 12 dogs.Bone marrow stromal stem cells(BMSCs) of dog were cultured in DMEM containing 100 ml/L fetal bovine serum and induced to differentiate towards osteoblasts.Then the cells were seeded onto absorbable polylactic acid(PLA) compounded with rhBMP-2(0.16 mg for each implant), the composite was implanted into the oval-shaped mandibular bone defect in the size of 30 mm?12 mm on one side,another side was used as blank control.The dogs were divided into 4 grups with 3 in each group.PLA/rhBMP/BMSCs,PLA/rhBMP,PLA/BMSCs and PLA were used as the implants in group A,B,C and D respectively. 2, 4 and 8 weeks after implantation,the effectiveness of bone formation was evaluated by means of gross observation,histological and scanning electronic microscope ( SEM) examination. Results: In group A new bone formation in the implanted defects was observed 4 weeks after operation, the defects were replaced by muture bone tissue 8 weeks after operation. In group B a little new bone was found in the implanted area 4 weeks after operation and fibrous bone was observed 8 weeks after operation. In group C chondral ossification was found and in group D fibrous tissue was found in the bone defects 8 weeks after operation.Conclusion:PLA/rhBMP/BMSCs may be feasible in the repair of bone defect.

The electromyography of the superior and inferior heads of lateral pterygoid muscle of 15 human subjects were recorded. It has been observed that marked active potential occurred in the superior head of the ipsilateral pterygoid muscle and the inferior head of the contralateral muscle during a one-side molar bite. Marked active Potential appeared in the superior head of the lateral pterygoid muscle during the closing movement of the jaw, whereas it appeared in the inferior head during the opening movement.It has not been confirmed that the lateral pterygoid muscle is an important protractor of the mandible, as only relatively weak active potential was recorded in the two heads of the muscles on both sides during the protraction of the jaw. Very marked active potential has been observed in the ipsilateral prenygoid muscle during the lateral movement of the mandible, while in the contralatenal superior head it occurred only slightly. The. above finding does not correspond to the descriptions found in current text-books.The two heads of the lateral pterygoid can therefonose considered as two functionally and structurally distinct muscles.

Objective To screen an optimum method for in vitro culture of Helicobacter pylori-spe-cific CD4+T cells and apply it to immunodominant Th epitopes screening .Methods PBMCs were isolated from subjects positive for Helicobacter pylori infection and were stimulated with HpaA recombinant protein . Various induction conditions including serum containing mediums , concentrations of antigen and time were screened to obtain an optimum method for in vitro culture of Helicobacter pylori-specific CD4+T cells.The cells were harvested and stimulated using HpaA synthesized overlapping peptide pool .The percentage of an-tigen-specific CD4+T cells was evaluated by intercellular cytokine staining of interferon-γand the results were compared under different conditions .The possible immunodominant Th epitopes were screened by using synthetic overlapping peptides .Results Antigen-specific CD4+T cells were well cultured in RPMI 1640 culture medium containing human AB serum in comparison with those cultured in fetal bovine serum based medium.The highest percentage of antigen-specific CD4+T cells was achieved when stimulated with HpaA recombinant protein at the concentration of 0.2 μmol/L.CD4+T cells in response to the stimulation of 0.2μmol/L of HpaA recombinant protein was observed on the ninth day after culture and its peak was reached on the fifteenth day .A possible immunodominant Th epitope ( HpaA220-237 ) was screened in subjects with He-licobacter pylori-infection by using synthetic overlapping peptides .Conclusion Helicobacter pylori-specific CD4+T cells were successfully cultured in vitro by using RPMI 1640 culture medium containing human AB serum and stimulated with 0.2 μmol/L of HpaA recombinant protein for fifteen consecutive days .This cul-ture method could be applied to immunodominant Th epitopes screening and provide evidences for further in -vestigation on the development of Helicobacter pylori epitope-based vaccine .

The characterization and management of oral and maxillofacial spaces infections in diabetic patients were studied in order to determine the pattern of this clinical condition and formulate a management plan.There were 31 cases with average age of 61 years(s=9);the mean hospitalization time was 14 days(s=6);the average fasting blood glucose level on admission was 10.4 mmol/L.Of the 31 patients 20 were multiple-space infections and 11 were single-space infections.13 patients had major complications during admission.Odontogenic infection was the most common cause of the space infections.Streptococcus viridians and staphylococcus aureus were common organisms(5/19,4/19)identified through pus and/or blood cultures.Early surgical incision and drainage,perfect blood glucose control,intravenous antimicrobial therapy,preventing asphyxia and managing major complications are necessary and effective approaches for the management plan.

Objective To study the antileukemia immune response of IL-18 gene transfected dendritic cells(DCs) of chronic myelogeous leukemia(CML).Methods DCs were transfected with IL-18 gene by liposomes in CML.The expression of IL-18 in IL-18 transfected DCs was detected.The percentages of CD80+ and CD86+ cells in IL-18 tranfected DCs were determined by FCM.The proliferation of T cell,NK and specific CTL kill activity induced by IL-18 gene transfected DCs were detected.Results The quantity of IL-18 in IL-18 tranfected DCs was(596?34.1)pg/2?106cells/48 h,while the culture medium of mock-transduced DCs and DCs did not secrete detectable levels of IL-18.The percentages of CD80+ and CD86+ cells in IL-18 tranfected DCs were higher than that in mock-transfected DCs(P

BACKGROUND: Odontogenic infection factor has been given much importance in the study of the etiology of secondary trigeminal neuralgia,and the theory of jaw bone cavities is proposed. OBJECTIVE: To study the relationship of the jaw bone cavities and the etiology of trigeminal neuralgia.DESIGN: A self-controlled trial.SETTING: Department of oral and maxillofacial surgery of a university hospital.PARTICIPANTS: The patients with the trigeminal neuralgia were treated in Department of Oral and Maxillofacial Surgery, Affiliated Hospital of the Medical College of Qingdao University from February 1994 to December 2003, from whom 45 were selected for this study, including 15 males and 30 females with altogether 74 jaw bone cavities.METHODS: Curettage of the jaw bone cavities was performed in these cases, and visual analogue scale(VAS) was adopted for evaluation of the postoperative pain.MAIN OUTCOME MEASURES: ① VAS; ② Pathological examination and bacteria culture of the specimens.RESULTS: Pain relief was achieved in 33 cases(73.3% ) after the first surgery and in 10 cases(22.2% ) after a second or third surgery. In 2 cases (4.5%), the pain was alleviated but medication was still needed for pain control. Pathological examinations in most cases identified predominantly in flammatory and granulation tissues.CONCLUSION: Jaw bone cavities may be one of the major etiologic factors of trigeminal neuragia.

BACKGROUND:Conventional methods,including trypsin digestion and cells transfer using single call suspension,have many drawbacks,which limit the development of bone tissue engineering.OBJECTIVE:To culture bone marrow stromal stem calls,induce osteoblastic differentiation,and prepare cell sheets.METHODS:Canine bone marrow stromal calls were isolated by density gradient centrifugation technique,inoculated into DMEM medium,and induced to differentiate into osteoblasts.Complete call sheets were harvested by call sheet engineering based on the temperature change of temperature-responsive medium.RESULTS AND CONCLUSION:Immediately after inoculation,primary calls were scattered on the bottom of culture flask,presenting a transparent spherical body with a good refractive capacity.At 12 hours,calls exhibited a long shuttle shape,reached complete confluency,and grew in a whirlpool-like fashion.After osteoblastic induction,the majority of bone marrow stromal stem calls appeared tetragonal,polygonal,and squamose.At 21-28 days,round or oval-shaped calcified nodules formed.When the bone marrow stromal stem calls in the temperature-responsive culture dishes were cooled below the critical temperature 32℃,cells were gradually detached from the bottom of culture flask and formed complete bone marrow stromal stem call sheets.These findings indicate that density gradient cantrifugation technique can be used to successfully isolate and culture canine bone marrow stromal stem cells to differentiate into osteoblasts and call sheet engineering enables to harvest complete bone marrow stromal stem call sheets.

BACKGROUND:There are some disadvantages in harvesting and transferring cells in the traditional tissue engineering technique,and it is difficult to form dense tissues,which significantly limits the development of tissue engineering.OBJECTIVE:To explore the culture and fabrication of dog bone marrow mesenchymal stem cell(BMSC)sheet in vitro.METHODS:Bone marrow was extracted from dogs following anesthesia.BMSCs were isolated with the method of density gradient centrifugation in vitro.BMSCs at passage 4 at a density of 1×10~9/L were incubated in the temperature-responsive culture dishes with a diameter of 3.5 cm,and cultured in an incubator at 37 ℃,5% CO_2 and saturated humidity.The temperature of the incubator was changed from to 37 V to 20 ℃ to prepare BMSCs cell sheet for 20 minutes.Cell morphological changes and cell sheet formation were observed under an inverted microscope.RESULTS AND CONCLUSION:Dog BMSCs following 24 hours of primary culture presented ellipse or polygonal shape.Most cells adhered at hour 72,and cell colonies were visible at day 7.Cells showed long spindle and completely confluence at day 12,with unclear boundary.BMSCs in the temperature-responsive culture dishes presented short spindle shape,and gradually separated from the dish bottom,forming entire cell sheet containing extracellular matrix at 20 V.These verified that dog BMSCs can be effectively obtained with method of density gradient centrifugation.Complete cell sheet layer can be fabricated with temperature-responsive culture dishes.

BACKGROUND: How to reconstruct tissue-engineered bone with structure similar to natural bone iS a problem in the development of tissue engineering. Cell sheet engineering technology enables novel approaches to construction of tissue-engineered bone. OBJECTIVE: To observe the biocompatibility of call sheets to decalcified bone matrix (DBM) and their growth on DBM. DESIGN, TIME AND SETTING: An in vitro observation was performed at the Central Laboratory, Affiliated Hospital, Qingdao University Medical College between June and September 2009.MATERIALS: Dog bone marrow stromal cell sheets were prepared using temperatura-responsive medium. Dog DBM was prepared by defatting, decalcification, and noncotlagen protein removal procedures. METHODS: DBM surface was covered by call sheets prepared by temperature-responsive technology and cultured with DMEM containing 10% fetal bovine serum and osteoinductive agent.MAIN OUTCOME MEASURES: Under scanning electron microscope, DBM structure, as well as the attachment and growth of cell sheets on DBM surface, was observed. Porosity and aperture size of DBM were calculated. RESULTS: DBM exhibited a three-dimensional latticed structure, with a porosity of approximately 75%. The mean aperture size was (250.11±98.89) μm, exhibiting a normal distribution. Cell sheets well attached to and grew on DBM surface, and rapidly proliferated.CONCLUSION: Cell sheets show good biocompatibility to DBM. DBM/cell sheets complex can be applied in tissue-engineered bones, which promotes the construction of tissue-engineered bone with structure similar to natural bone.