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@#Objective To observe the efficacy of a kind of complex composed of biphasic ceramic biologic bone (BCBB), bone morphogenetic protein (BMP) and basic fibroblast growth factor (bFGF) on the repair of necrotic areas of the femoral head. Methods The femoral head necrosis model of 64 femoral heads in 32 rabbits induced with microwave heating were randomly divided into four groups, which implanted with nothing (group A), BCBB/BMP (group B), BCBB/BMP/bFGF (group C) and with cancellous bone autograft (group D). The specimens were harvested separately at the end of 2, 4, 8 and 12 weeks after operation. 4 femoral heads were taken off at each interval in every group. A series of examinations were carried out including of naked eyes and gross anatomic observation, X-ray, histology, and blood vessel immunohistochemical staining. Results In group A, 1 femoral head collapsed by the end of 12 weeks, and there was only a little osteoid tissue formed. At the same time, a lot of new bone formed in group B and group C, and the boundary between the bone grafting area and the post bone still existed, but the boundary was unclear in group D, with the density consistent to the post bone. Under X-ray, the defect could be found and one femoral head collapsed in group A by the end of 12 weeks. The density of bone grafting area was high and the boundary to the post bone was unclear in group B and in group C. The density of bone grafting area was the same as the post bone and the boundary between them was unclear in group D. There was only a little osteoid tissue formed in group A by the end of 4 weeks. At the same time, there was a little new bone formed in group B, and BCBB was partly degraded. There was a lot of new bone formed in group C and group D, and BCBB was partly degraded in group C, but cancellous bone autograft was almost absorbed in group D. The new bone area by the end of 4, 8 and 12 weeks from more to less were: group C and group D (P>0.05), group B, and group A (P<0.05). At the end of 2, 4 and 8 weeks, the blood vessel area of group C was more than that of group A, group B, and group D (P<0.05). Conclusion The BCBB/BMP/bFGF complex can induced osteoinduction and revascularization, to repair rabbit femoral head necrosis as effective as cancellous bone autograft.
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Objective To compare the curative effect of anatomical plate and locking plate in treatment of calcaneous intraarticular fracture. Methods 67 petiants with calcaneous intraarticular fracture were randomly divided into anatomical plate group (n=33) and locking plate group (n=34) . The Bo..hler angle, the Gissane angle, the length of calcaneal axis, the width and height of calcaneous and the Maryland grade were compared at 1 week and 6 month after operation. Results (1) week after operation, the Bo..hler angle, the Gissane angle, the height and width of calcaneous, the length of calcaneal axis, the Maryland grade had no significant difference between 2 groups . 6 months after operation, the Bo..hler angle, the Gissane angle, the height of calcaneous had significant differe nce between 2 groups. There was no significant difference in the length of calcaneal axis and the grade of Maryland between 2 groups. Conclusions The locking plate group is better than anatomical plate group in major anatomical measure indicators in 6 months follow up. The therapy of locking plate is worth of clinical promotion.
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BACKGROUND: Small intestinal submucosa (SIS) has good compatibility with cells and tissues, and has good degradabUity. It is an ideal scaffold for tissue engineering. Inducing adipose derived mesenchymal stem cells (ADSCs) seeded on SIS can construct target tissues, which has the potential to be used in clinical treatment.OBJECTIVE: To prepare decellularized porcina SIS matrix, and testify its biocompatibility with rabbit ADSCs cultured in vitro. METHODS: SIS was processed by enzyme digestion-hypertonic saline decellularization, lyophilized at low temperature, and sterilized by gamma radiation. Paraffin sections were used to observe the effect of decellularization of SIS, and the surface structures of SIS were observed by scanning electron microscope (SEM). Rabbit ADSCs were isolated and cultured, and passage 3 ADSCs were seeded onto one side or both sides of SIS. After one weak of co-culture, the cell-scaffold composites were observed.RESULTS AND CONCLUSION: SIS was white and semi-transparent film. Paraffin sections showed no cells on SIS matrix; electron microscopy showed loose weave structure of serosal surface and dense packing structure of mucosal surface. After one week of co-cultivation, plenty of ADSCs were observed on the surface of SIS. In ADSCs seeded onto one side of SIS group, a large number of cells grew on the superior surface, and few even no cells were observed on inferior surface of SIS. When ADSCs were seeded onto both sides of SIS, cells adhered to SIS in paraffin sections. Results show that enzyme digestion-hypartonic saline decellulariation can decellularize SIS completely, and SIS can support ADSCs growth.
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BACKGROUND: Studies demonstrated that small intestinal submucosa (SIS) had no immunogenicity, which can not lead to rejection following transplantation, thus, this is an ideal skin substitutes for natural skin.OBJECTIVE: Basic fibroblast growth factor (bFGF) gene was transfected into bone marrow mesenchymal stem cells (BMSCs),and combined the cells with SIS to construct tissue-engineered skin.METHODS: BMSCs were obtained from Japanese big-earad rabbits, and in vitro cultured. Then the subculturad BMSCs were transfected by pCDNA3.1 plasmid, followed by incubation on swine SIS to construct the tissue-engineered skin. The growth of cells and phenotype of BMSCs were detected by flow cytometry. In addition, the result of transfecting BMSCs with pCDNA-bFGF vector was measured by Western blot, and the structure of tissue-engineered skin was observed.RESULTS AND CONCLUSION: After passaged, BMSCs were grown quickly with Iong-fusiform shape. The cells were positive expressed CD90 and CD44, but negative expressed CD45. bFGF had been transfected into BMSCs, and stable expressed. The transfected BMSCs grew well in SIS. By this method, tissue-engineered skin can be constructed in vitro.
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BACKGROUND:Simple medication therapy can not promote the healing of articular cartilage defects effectively;the insufficient sources of autogenous cartilage also limited cartilage transDlantation.OBJECTIVE:The types of articular cartilage injury and local micro-environment changes were analyzed to summarize the progress of tissue engineered seed cell transplantation and cell transplantation therapy for repairing articular cartilage injury.METHODS:The databases of PubMed database(http://www.ncbi.nlm.nih.gov/sites/entrez/)and CNKI(http://www.cnki.net/)were retrieved with the key words of"Tissue engineering,cell transplantation,articular cartilage defects",and the literature was limited to English and Chinese languages.Studies concerning articular cartilage injury or was closely related to tissue engineered seed cells transplantatiOn were included.Repetitive studies were excluded Survival and migration of seed cells,as well as the recovery of joint function and adverse reactions were served as evaluation index.RESULTS AND CONCLUSION:A total of 201 literatures were seized by computers,according to the inclusion criteria,papers concerning tissue engineering cells for repairing articular cartilage injury were analyzed.Clinically,articular cartilage damage was commonly resulted by traumatic arthritis,which is difficult to repair due to the poor self-repair capacity of articular cartilage.The emergence of tissue engineered cell transplantation brought new hope for articular cartilage injury healing Cell transplantation has obtained good effects in articular cartilage repair,but the technology is still room for improvement.How to technically improve the tissue-engineered 3 elements,namely,cells,scaffold materials,and biological activity factors were the research focuses.Autologous cartilage regeneration was the theoretical support for articular cartilage defects repairing,which needs to optimize its regeneration and to maintain a more stable chondrocyte phenotype in further research The tissue-engineered cells had received good effects in repairing articular cartilage injury,and it is expected to be a new clinical treatment for articular cartilage injury with the in-depth study of tissue engineering.
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BACKGROUND:Subcutaneous fat of human body is a rich reservoir of adipose derived stromal stem cells(ADSCs).ADSCs can proliferate rapidly when being cultured in vitro,and has the capacity of multi-directional differentiation.ADSCs attracted much attention in research of tissue engineered seed cells.OBJECTIVE:To isolate and culture stromal vascular fraction(SVF)cells from rabbit subcutaneous fat in vitro,and to testify whether it has multiple differentiation capacity.METHODS:SVF cells were isolated in vitro from rabbits,and cultured under standard condition.Cellular surface antigens CD44,CD45 and CD29 of passage 3 SVF cells were examined using flow cytometry.Passage 3 SVF calls were induced to differentiate into osteoblasts,chondrocytes,and lipocytes.Oil red staining was used to examine lipocyte induction.Alkaline phosphatase(ALP)staining,alizarin red staining and von Kossa staining were used to examine osteoblast induction.Type Ⅱ collagen immunohistochemical staining and type Ⅱ collagen mRNA RT-PCR were used to examine chondrocyte differentiation.RESULTS AND CONCLUSION:Primary SVF cells were multi-angular or short spindle-shaped.Passage 3 SVF calls were long spindle-shaped.Flow cytometry showed CD44+,CD29+,CD45.Oil red staining exhibited positive reaction in lipocyte induction group.ALP staining,alizarin red staining and Von kossa staining demonstrated positive reactions in osteoblast induction group.Type Ⅱ Collagen immunohistochemical staining and alcian blue staining have suggested positive reactions at 14 days of chondrogenic induction group.RT-PCR of type Ⅱ collagen mRNA test showed that the product band had strong signal at 14 days of chondrogenic induction group compared with that before induction.Above mentioned results have indicated that SVF cells isolated from rabbit subcutaneous fat have identical surface makers of stem cells,and have the ability to differentiate into lipocytes,ostsoblasts and chondrocytes in vitro by induction,and it could be concluded that the SVF cells were ADSCs.
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BACKGROUND:The existed femoral head necrosis(FHN)model can not reflex the clinical necrosis of femoral head correctly.Microwave heating provides a new approach for model preparation.but the concrete standard remains uncertain.OBJECTIVE:To study the optimal temperature and time jn preparing a FHN model induced by microwave heating.METHODS:Totally 48 rabbits were randomly divided into 4 groups on the basis of the microwave temperature(50,55,60℃)and heating time(10,20 minutes).The microwave antenne was inserted into the rabbit femoral head.The rabbits were sacrificed immediately and at 1,2,4,8 and 1 2 weeks after operation.A series of examinations were performed including gross observation,X-ray.histology and MRI to observe the femoral head necrosis and repair status.RESULTS AND CONCLUSION:Marrow partially solidified in the group(50℃,10 minutes)at 1 week,and the osteonecrosis returned to normaI at 8 weeks after operation.1n the group(55℃,10 minutes),marrow was completely coagulated at 1 week and low signal on T1 weighted images and increased signal on T2 images were identified at 2 weeks.Osteonecrosis and repair occurred at the same time at 4 weeks.At 12 weeks,the osteonecrosis continued and the repair stopped,and the femorel heads started to collapse.All femoral heads collapsed at 8 weeks in group(50℃,20 minutes)and group(60℃,10 minutes).Accordingly.microwave heating is a good method in developing FHN model.55℃ and 10 minutes are the optimal temperature and time for the development of FHN model of rabbits induced by microwave heating.
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BACKGROUND: Basic fibroblast growth factor (bFGF) has significant promotion effects on repair in trauma, but local application cannot play a role for a long time.OBJECTIVE: To observe expression of bFGF gene in rabbit bone marrow mesenchymai stem cells (BMSCs) following transfection.DESIGN, TIME AND SETTING: The cytogene in vitro observation was performed at the College of Life Science, Yunnan University from March to August 2009.MATERIALS: One Japan flap-eared rabbit was purchased from the Department of Animal, Kunming Medical College. pCDNA3.1plasmid (Invitrogen, USA) was used in this study.METHODS: Bone marrow was extracted from rabbit anterior superior iliac spine. BMSCs were harvested by the adherent method.Cells were digested and subcultured when 80% confluent. According to GeneBank bFGF cDNA sequence, gene was designed and synthesized. Following electropherosis, the gel was retrieved using xhol I, BamH I enzyme digestion. Restriction enzyme was used to perform enzyme digestion, electropherosis and gene recovery in PcDNA Vector plasmid. bFGF DNA was connected with PcDNA Vector plasmid. PcDNA-bFGF eukaryotic expression vector was constructed. Recombinant was transfected into rabbit BMSCs using liposome infection protocol, and stable transfected line was screened.MAIN OUTCOME MEASURES: BMSC surface antigen expression was measured. Western blot was utilized to determine the expression of target protein.RESULTS: Results of flow cytometry showed that cultured cells were positive for CD90 and CD44, but negative for CD45.Results of immunohistochemistry demonstrated that vessel-derived BMSCs were negative for CD34, but positive for CD44. In cell disruption liquid of bFGF-transfected BMSCs, a significant positive zone of hybridization was visible at M, 23 000. However, no positive band was found in protein from pCDNA3.1(-) blank vector-transfected BMSCs.CONCLUSION: The bFGF gene was successfully transfected into BMSCs, and this target gene can stably express.
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BACKGROUND: An ideal model of osteonecrosis of the femoral head is beneficial to the study on the cause of disease, pathogenesy and treatment. So far there has not been a coherent method to prepare this model. OBJECTIVE: To establish a rabbit model of osteonecrosis of femoral head induced by microwave heating, and to decide optimum microwave temperature and heating time.DESIGN: Randomized controlled animal trial.SETTING: Animal Experimental Center of Kunming Medical College.MATERIALS: The experiment was performed at the Animal Experimental Center of Kunming Medical College between September 2004 and November 2005. Forty-eight healthy adult New Zealand rabbits, either male or female, were provided by the Animal Experimental Center of Kunming Medical College. The animal procedure was accorded with the ethical standards. GW-92C multi-functional microwave therapy apparatus was the product of Grand World Medical Apparatus (Tianjin) Co., Ltd. METHODS: The microwave antenna was inserted into the rabbit femoral head. Ninety-six femoral heads in forty-eight rabbits were randomly divided into four groups (n =24) according to the microwave temperature and heating time: microwave heating at 50 ℃ for 10 minutes group; 55 ℃ for 10 minutes group; 50 ℃ for 20 minutes group and 60 ℃ for 10 minutes group. The models of osteonecrosis of femoral head were induced by microwave heating using multi-functional microwave therapy apparatus according to the temperature and heating time of grouping. MAIN OUTCOME MEASURES: In each group, two rabbits (four femoral heads) were killed immediately, one, two, four, eight and twelve weeks after operation, respectively. A series of examinations were carried out, including gross observation, X-ray to observe bone trabecular arrangement, cystis degeneration, head collapse or hip joint destruction, MRI to observe the necrotic area, and HE staining to observe the osteonecrosis and bone repair. RESULTS: Marrow tissues partially coagulated in the microwave heating at 50 ℃ for 10 minutes group at the end of the 1st week, and the osteonecrosis returned to normality at the end of the 8th week. In 55 ℃ for 10 minutes group, marrow tissues were completely coagulated at the end of the 1st week, and decreased signal on T1 weighted images and increased signal on T2 images were identified at the end of the 2nd week. In the 4th week, bone repair was found simultaneously when osteonecrosis occurred. At the end of the 12th week, the osteonecrosis continued and the repair stopped, and the femoral head collapse occurred. All femoral heads collapsed at the end of the 8th week in 50 ℃ for 20 minutes group and 60 ℃ for 10 minutes group. CONCLUSION: Microwave heating at 55 ℃ for 10 minutes is the optimal choice to develop a rabbit model of osteonecrosis of the femoral head.
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Objective To induce bone marrow mesenchymal stem cells(BMSCs) or bone marrow stroma stem cell(MSCs) to differentiate directionally towards chondrocytes in vitro and then identify the differentiated cells.Methods Bone marrow was harvested from the iliac bone of 16-week-old Japanese white rabbits.After gradient centrifugation,cultivation,amplification,the 3rd-passaged BMSC were implanted in six-hole-plate according to a certain proportion and induced by chondrogenic inducers including transforming growth factor-?1,dexamethasone and vitamin C.Chondrocytes were selected and fixed at different time.The features of chondrocytes were identified by toluidine blue staining and collagen types II immunohistochemical assay.Results The structure of cellular cartilage from BMSCs was uniformly positive of toluidine blue staining and collagen types II immunohistochemical staining.Conclusion Rabbit BMSC,obtained under the experimental conditions,develops stably,proliferates rapidly and is differentiated successfully into chondrocytes by induction in vitro.
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[Objective]To study the dynamic changes of the VEGF and BMP-2 genes expressions inside the necrotic femoral head of rabbits.[Method]Made rabbit model of femoral head necrosis(FHN) with the method of twice intravenous injection with low dosage lipo polysaccharide(LPS)(10 ?g/kg body weight,at a time interval of 24 hours.)and 3 times intramuscular injection with methylprednisolone(MPS)(20mg/kg body weight,at a time interval of 24 hours.().)Immunohistochemistry,in situ hybridization and extract total RNA for real-time quantitative PCR were performed and successfully designed the suitable probes and primerswas used to detect the dynamic changes of the VEGF and BMP-2 genes expressions inside the femoral head of rabbits.[Result]The expressions of the VEGF and BMP-2 genes in group B(model group)at the 4,8,12 th and 16 th week were negative and real-time quantitative PCR showed that the level of the VEGF and BMP-2 mRNA genes expressions were obviously lower than that of control group.[Conclusion]The VEGF and BMP-2 genes expressions are inhibited in FNH model induced by LPS and MPS.
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BACKGROUND:The treatment of avascular necrosis of femoral head remains a clinical challenge. However, recent study shows that basic fibroblast growth factor (Bfgf) is an effective angiogenic growth factor. We suppose that Bfgf has the potential in treating avascular necrosis of femoral head through revascularizing effect and steogenesis.OBJECTIVE: To develop a rabbit model that simulates the clinical situation with trapdoor bone rafting and evaluate the revascularizing effect in the repair of femoral head defect with Bfgf/partially deproteinised bone (PDPB).DESIGN: A completely randomized controlled trial.SETTING: Animal Experimental Center of Kunming Medical College.MATERIALS: The experiment was conducted in the Animal Experimental Center of Kunming Medical College between July 2002 and July 2003.Totally 27 adult male healthy New Zealand rabbits, weighing 2.2 to 2.8 kg,were recruited and randomly divided into Bfgf/composite PDPB group,single PDPB group, and blank control group with 9 rabbits in each group.INTERVENTIONS: ① Preparation of Bfgf/PDPB: containing Bfgf 10 ng/mm3. ② Establishment and repair of femoral head defect model:Totally 27 rabbits were chosen. Bone defect model was stablished by opening a trapdoor between femoral head and femoral neck. Bfgf/PDPB was implanted in composite group; single PDPB was implanted in PDPB group; no implants were given in blank control group. The rabbits were killed after injection through blood vessels with prepared Chinese ink 2, 4and 8 weeks after operation, and then the femoral head of each rabbit was taken out as specimen.MAIN OUTCOME MEASURES: ① Histological examination and blood vessel counting. ② Image analysis of microvessel area.RESULTS: Twenty-seven rabbits all entered the result analysis. ① Histological examination and vessel count of femoral head specimens in rabbits:8 weeks after operation, composite bone group: implants were replaced with bone tissues and medullary cavity of bone formed. A lot of medullary vessels were present. DPB group: implants were enwrapped by bonelike tissues and most of them were absorbed. Blank control group: the area of femoral head defect was filled with fibrous tissues. New bonelike tissues and scattered chondrocyte island appeared in the adjacent connective tissues of defect area, with a small number of blood vessels. Microvessel count at week 2 in composite group was significantly higher than that in PDPB group and blank control group [(31.833±7.914) vs (22.917±2.079)and (11.250±4.220) (number of blood vessel/field), P ＜ 0.01, P ＜ 0.05].The number of microvessels at weeks 4 and 8 in composite bone group and DPB group was significantly greater than that in blank control group. ②Image analysis of microvessel area: The transparent samples of 20 μm thick were observed under the optical microscope: 2, 4 and 8 weeks after operation, there were many vessels woven into nets. Many vessels woven into nets were also found in PDPB group; there were scattered vessels in blank control group.CONCLUSION: Bfgf has the revascularizing effect on the repair of femoral head defect, and has the potential and advantages in treating avascular necrosis of femoral head.
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BACKGROUND: Proper physiochemical method can alleviate or eliminate the antigenicity of natural xenogenic bone and preserve its natural porous reticular system so that its structure and components are in conformity with physiological requirements. It is expected that xenogenic bone becomes a kind of substitutive material in bone transplantation.OBJECTIVE: It was to adopt generative culture of marrow stromal cell and complex culture of ceramic xenogenic bone in vitro to understand the influence of ceramic xenogenic bone on morphology, growth, adhesion and proliferation of marrow stromal cell in rabbits.DESIGN: Blank, control experiment was designed.SETTING: Center Laboratory of First Hospital Affiliated to Kunming Medical College.MATERIALS: Five Japanese big ear white rabbits of 3-month old were employed, body mass varied from 1.5 to 2.0 kg, of either sex.INTERVENTIONS: The experiment was performed in Center Laboratory of First Hospital Affiliated to Kunming Medical College from June to October 2003. With physiochemical management, the fresh pig rib bought from market was prepared into ceramic xenogenic bone and was verified with Xray diffractometry to be mainly composed of hydroxyapatite. It was verified with scanning electron microscopic observation that such material presented porous meshwork system as osteogenie tissue. Five Japanese big ear white rabbits were employed to collect 1mL marrow for each. PRMI 1640culture solution was used to dilute it into suspension of marrow stromal cell and the cells of upper layer were collected for culture. With 1×106 cells/bottle, they were vaccinated to 8 culture bottles of 25 mL and generated at the ratio of 1:1. The prepared ceramic xenogenic bone was placed into culture bottles, 5 pieces in each bottle, totally for 5 bottles, taken as experimental group and the other 3 bottles (without xenogenic bone ) were taken as the control. The culture ended in 2 weeks and a part of cultured material was used for observation with phase microscope and scanning electron microscope.MAIN OUTCOME MEASURES: Influence of ceramic xenogenie bone on morphology, growth, adhesion and proliferation of marrow stromal cell in rabbits were observed with phase microscope and scanning electron microscope.to 14 days after vaccination, cell proliferation was connected to be monolayer meshwork. Generative culture: in 24 hours, adhesion was accomplished and the cells grew well in both experimental group and the cortrol.In the experimental group, fibroblastic cells were arranged more closely rabbits with scanning electron microscope: in experimental group, it was visible in 2 weeks of generative culture that some counected reticular fibroblastic cells adhered with the inner wall of reticular pores and porous base of ceramic xenogenic bone as well as the surface of bone trabecula.The cells were mostly fusiform, triangular or multiangular.CONCLUSION: Ceramic xenogenic bone is mainly composed of hydroxyapatite and presents reticular porous structural system of osteogenic tissue, which will not produce harmful effects on morphology, growth, adhesion and proliferation of marrow stromal cell and it is expected to be used in complex graft for repair of bone defects with bone marrow.
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There are many factors which affect the effects of acetabular fracture treatment. According to the literature, they can be concluded as follows: First,factors before the injury, such as quality of bone, age, other diseases of the patient and the expectation of the patient for functional recovery; Secondly, injury related factors, such as type of fracture, presence or absence of complicated nerve injury, dislocation of the femoral head, soft tissue damage and other complications, and the degree of cartilage damage of the femoral head; Thirdly, operational factors, such as the time gap between trauma and surgery, accuracy of reduction, stability of the coxa after the operation, experience of the surgeon, selection of operative approach, conditions of the hospital and operative complications.
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Objective To explore the causes and management of delayed un ion and non-union of long bone fracture of the leg. Methods 13 cases of delayed union and non-union of long bone fracture of the leg were treated with interlo cking intramedullary nails. 2 cases were given close implanting, and 11 cases we re managed with open reduction, of whom 9 were also grafted with autogenous bone . All patients were given early static interlocking, and 4 cases were given late dynamic interlocking. Results X-rays and follow-up showed that all patients h ealed up after an average of 14.5 months.Conclusions Interlocking intramedullary nails are a satisfactory method to treat delayed union and non-union of long b one fracture of the leg. The key points of the operation are reaming, selecting suitable nails and dynamic interlocking when necessary.
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Acetabulum fracture, the inner fracture of the joint, has be en divided into 10 kinds. Because acetabulum is an irregular body, no single ope ration approach can solve all kinds of acetabular fracture. Appropriate selectio n of the operative approach according to the type of fracture is very important. Kocher-Langenbeck approach, extended-iliofemoral approach and ilioinguinal ap proach are often used in clinic now. Because of its complicated procedures and m any complications resulting from the approach, the application of extended-ilio femoral approach is decreasing. Because of its predominant advantage and improve ment of the operation techniques, the use of ilioinguinal approach is obviously increasing in proportion. Since the united approach can reveal clearly so that i t facilitates reduction and internal fixation, it is recommended by the surgeons at home and abroad.
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Sacroiliac Syndrome is a common cause of low back pain.With reporting 24 cases we reviewed literatures related to sacroiliac joint diseases and made a brief discussion. The mechanism and the definition of this syndrome were proposed, as well as the clinic types and the methods of physical examination. It is emphasized that sacroiliac pain associated with reffered pain in the lower extremity be Signified clinical importance. The role of radiological per- spectives and the effective methods of treatment were described.
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0.05)but there was a correlation of IL-1?/IL-10 ratio with thrombus mass (Pearson r=0.87, P=0.01