Effect of (, TLZT) gel preparation on p53 /miR-502-5p /NF-κBp65 in synovial tissue of knee osteoarthritis / 中国骨伤

OBJECTIVE@#To observe effects of (, TLZT) gel preparation on p53, miR-502-5p, NF-κBp65 in synovial tissue of knee osteoarthritis (KOA), and to explore mechanism of TLZT gel preparation in treating KOA.@*METHODS@#Thirthy-six Wistar rats aged 8 weeks and weighed 200 to 220 g (meaned 208 g) were randomly divided into normal group, model group and traditional Chinese medicine (TCM) group, 12 rats in each group. KOA model was established by modified Hulth method. After 4 weeks of modeling, TCM group treated with TLZT gel preparation for external use, 3 times daily for 2 weeks;normal group and model group were fed normally without intervention. After treatment, morphological changes of specimens in each group were observed, changes of miR-502-5p in synovial tissue were detected by qPCR, and contents of p53, NF-κBp65, IL-1β, TNF-α, MMP-13 in synovial tissue were detected by qPCR and Western Blot respectively.@*RESULTS@#(1)Morphological observation of specimens showed that the articular cartilage in model group was hyaline and uneven, the synovial membranes were hypertrophic and proliferative with a large number of inflammatory cells infiltrating, the joint fluid was thicker in texture;the articular cartilage in TCM group was more transparent and smooth, synovial hyperplasia was mild with a small amount of inflammatory cell infiltration, the texture of articular fluid was clear and sparse. (2) Compared with normal group, content of miR-502-5p of synovial tissue in model and TCM group were increased, mRNA and expression of p53 decreased, expression of NF-κBp65, IL-1β, TNF-α, MMP-13 increased. (3)Compared with model group, content of miR-502-5p in synovial tissue of TCM group decreased (<0.05), mRNA and protein expression of p53 increased (<0.05), mRNA and protein expression of NF-κBp65, IL-1β, TNF-α, MMP-13 decreased (<0.05).@*CONCLUSION@#Expression of p53, miR-502 -5p, NF -κBp65 in synovial tissue is closely related to synovial hyperplasia and inflammatory reaction, TLZT gel preparation may reduce proliferation and inflammatory reaction of KOA synovium by regulating the expression of p53, miR- 502-5p, NF-κBp65 in synovial tissues.

Expression and significance of β-catenin and NF-κB signaling pathway in knee osteoarthritis synovial inflammation / 中国骨伤

OBJECTIVE@#To explore expression of β-catenin and NF-κB signaling pathway in synovial tissue of rats with different degrees of knee osteoarthritis (KOA).@*METHODS@#Forty-eight SPF male rats weighed (200±20) g were randomly divided into three groups, namely model group (32 rats), sham operation group (8 rats) and control group (8 rats). KOA model rats were established by Hulth method, and 8 rats were killed at 2, 4, 8, 12 weeks respectively after modeling, in order to establish KOA model rats with moderate, early, mild and severe degree. Sham operation group was only cut off capsule of knee joint and suture to exclude interference factor, control group was untreated. Behavior, synovial hyperplasia and cartilage degeneration of rats among each group were observed. Expression of NF-κB and signaling pathway and β-catenin in synovial tissue of rats were detected by real-time PCR.@*RESULTS@#KOA rat model was successfully established, and synovial hyperplasia was observed in KOA model at mild and early degree, and then gradually decreased; while cartilage degeneration in the early moderate and severe KOA model was significantly expressed, and gradually aggravated with time. The results of PCR showed that expression of β-catenin in 4-week group (8.57±0.46) and 8-week group (4.23±0.09) were higher than those in control group (<0.05); expression of TLR-2 in 2-week group (12.04±4.02) and 4-week group (8.54±2.13) were higher than those in control group(<0.05), and TLR-4 in 2-week group(5.04±0.93), 4-week group (3.29±0.58) and 8-week group (1.63±0.12) were higher than those in control group; expression of NF-κB was significantly higher in 2-week group (10.15±2.04), 4-week group (15.97±4.17), 8-week group (7.69±1.48) and 12-week group (6.70±1.58) than that in control group (<0.05), and expression of IL-1β was significantly higher in 4-week group (2.79±0.25) and 8-week group (2.46±0.32) than that of control group (<0.05).@*CONCLUSIONS@#On the RNA expression level, both of β-catenin and NF-κB signaling pathways are involved in synovial inflammation in KOA model rats, and they play a regulatory role in expression of IL-1β, degeneration of KOA.

Study on musculuskeletal ultrasound features and correlation of knee osteoarthritis / 中国骨伤

OBJECTIVE@#To compare the correlation between musculoskeletal ultrasound features, dysfunction and X-ray findings in patients with knee osteoarthritis, and to analyze the pathological mechanism of soft tissue inflammation in knee osteoarthritis.@*METHODS@#Cross-sectional method was performed in this research (Evidence level: III). The patients with knee osteoarthritis were collected according to the screening criteria from September 2016 to January 2017 in Orthopedic clinic in our hospital. Musculoskeletal ultrasound and X-ray images were obtained and measured, knee function was measured by Lysholm scale. Pearson coefficient, t test and Wilcoxon were applied to analyze the correlation between soft tissue inflammation, knee dysfunction and X-ray features.@*RESULTS@#Total 123 patients with knee osteoarthritis were recruited in this research. Soft tissue inflammation around knee had a high incidence in patients with knee osteoarthritis (infrapatellar fat pad inflammation 81%), and the synovial membrane thickness, joint effusion depth and meniscus bulging were beyond the normal range. Correlation analysis showed that the about Lysholm score and joint effusion depth had negative correlations with "Squat" score(=-0.21, =0.02). and Medial meniscus bulging had negative correlations with "Sustain" score(=-0.26, <0.01) and Lysholm total score (=-0.19, =0.04). Lateral meniscus bulging had a negative correlation with "Unstable" score (=-0.22, =0.02). The X-ray features, and medial joint space narrow had negative correlations with joint effusion depth(=-0.27, <0.01) and synovial membrane thickness(=-0.17, =0.007), and had a positive correlation with medial meniscus bulging. Medial joint space narrow was significantly correlated with patellar ligament inflammation and fat pad inflammation(<0.05). Lateral joint space narrow was significantly correlated with patellar ligament inflammation(=0.02).@*CONCLUSIONS@#Soft tissue inflammation around the knee-a major pathological manifestation of knee osteoarthritis, has significant correlations with knee dysfunction and bony structure lesions, and affects the progression of knee osteoarthritis by damaging knee joint function and promoting the destruction of articular cartilage.

Analysis of the relationship between living habit and cervical instability in adolescent patients with neck pain / 中国骨伤

<p><b>OBJECTIVE</b>To investigate the relationship between living habit and cervical instability in adolescent patients with neck pain.</p><p><b>METHODS</b>Fifty-nine adolescent patients with neck pain(neck pain group) and seventeen healthy teenagers (control group) were recruited and divided into two groups, and clinical information, living habit were collected. In addition, all people were taken lateral, hyperextension and hyperflexion radiography to analyze relationship between living habit and cervical instability.</p><p><b>RESULTS</b>There was no obvious difference in age, height, weight and body mass index between two groups. The neck pain group using cellphone time per day is longer than control group, while control group had more exercise time than neck pain group(<0.01). The incidence of instability in neck pain group was significantly higher than that in control group(<0.01). In hyperflexion, angular displacement(AD) of neck pain group in vertebral body between C₃-C₄, C₄-C₅ and C₅-C₆ was significantly higher than that of control group. In neck pain group, AD of hyperflexion was higher than that of hyperextension on C₄-C₅(<0.01), and AD of hyperextension is higher than that of hyperflexion on C₆-C₇(<0.05). In neck pain group, AD of hyperextension on C₄-C₅ was positively correlated with time of using cellphone every day(=0.275, =0.035). And AD of hyperflexion was significantly positive correlated with time of using cellphone(=0.577, <0.001), but was negatively correlated with exercise time(=-0.279, =0.032). The AD of hyperflexion on C₅-C₆ was negatively correlated with exercise time every day(=-0.292, =0.025), AD of hyperextension was negatively correlated with time of using computer every day(=-0.262, =0.045).</p><p><b>CONCLUSIONS</b>Adolescent neck pain patients had more time to use cellphone than normal teens every day, and exercise time is less than healthy teenagers, and occurrence rate of cervical instability is higher on C₃-C₄, C₄-C₅, C₅-C₆ segment. The longer daily exercise time, the smaller C₄-C₅ and C₅-C₆ AD values; the longer cellphone usage every day, the greater C4-C5 AD values.</p>

Correlation of genes expression on MyD 88-dependent signaling pathway in progression of knee osteoarthritis / 中国骨伤

<p><b>OBJECTIVE</b>To investigate expression features and correlation of genes expression on MyD88-dependent signaling pathway in synovial membrane (SM) of progression of knee osteoarthritis (OA).</p><p><b>METHODS</b>Sixty Wistar rats were randomly divided into 6 groups, including blank group (N), false surgical group, model groups[2 weeks (2W), 4 weeks (4W), 8 weeks (8W) and 12 weeks (12W)], with 10 rats in each group. The models were established by using Hulth method. Control group was experienced no surgery, while false surgical group was only opened joint cavity and sutured. The SM samples was collected according to the time designed above. The relative expression quantity of MyD88, TLR4 and NF-κB was detected by Real-time PCR after the extraction of the total RNA and reverse transcription. The correlation analysis was obtained by SPSS.</p><p><b>RESULTS</b>There was no significant difference in each gene mRNA expression between false surgical and blank group(> 0.05), while enhanced expression was found in the model groups(<0.05). The correlation index among MyD88, TLR4 and NF-κB was 0.91 and 0.86 respectively, and had significant difference among them.</p><p><b>CONCLUSIONS</b>Positively relative among MyD88, TLR4 and NF-κB played main role in TLR4/NF-κB signal passway, and could predicate the expression of other genes in the passway. It also could further provide the basis for clarify the pathologic mechanism of knee OA.</p>

Observation of curative effects of musculoskeletal ultrasound-guided needle-knife on the degenerative meniscus disease / 中国骨伤

<p><b>OBJECTIVE</b>To observe the curative effect of musculoskeletal ultrasound-guided needle-knife on the degenerative meniscus disease, and to provide a new method in the treatment of degenerative meniscus disease.</p><p><b>METHODS</b>Seventy-seven patients with degenerative meniscus disease treated in the Third Affiliated Hospital of Beijing University of Chinese Medicine from January 2015 to September 2015 were selected, including 30 males and 47 females, aged from 44 to 66 years old, with an average of 57.5 years old. VAS scores, Lysholm scores and distance of meniscal protrusion were analyzed and compared before treatment, 2 weeks and 1 month after treatment. The curative effect was summarized at last.</p><p><b>RESULTS</b>The mean Lysholm scores were 51.63±15.26(before treatment), 77.13±11.82(2 weeks after treatment) and 87.56±8.65(1 month after treatment). The mean VAS scores were 7.080±1.574 (before treatment), 2.630±0.310(2 weeks after treatment) and 0.850±0.177(1 month after treatment). The mean of the distance of meniscal protrusion scores were 0.400±0.156 (before treatment), 0.298±0.140 (2 weeks after treatment) and 0.240±0.110 (1 month after treatment). VAS scores and Lysholm scores were improved significantly compared with preoperative results. The distance of meniscal protrusion showed an obvious improvement after treatment.</p><p><b>CONCLUSIONS</b>The treatment of musculoskeletal ultrasound-guided needle-knife has advantages of high accuracy position and excellent effectiveness for degenerative meniscus disease. The treatment provides safety operation and significantly improves quality of life in patients without any complications.</p>

Analysis and comparison about musculoskeletal ultrasonoLranhv and x-rav of knee osteoarthritis / 中国骨伤

<p><b>OBJECTIVE</b>To analyze and compare the characteristics of musculoskeletal ultrasonography and X-ray of knee osteoarthritis, and to investigate the advantages of them.</p><p><b>METHODS</b>According to the inclusion and exclusion criteria, 57 cases (66 knees) were collected from February 2015 to May 2015. Among them, there were 48 females and 9 males with an average age of (58.9 +/- 9.8) years old (ranged, 41 to 78 years old). The main symptoms included unilateral or bilateral knee pain and locked joints explicit areas of tender points. The mean course of disease was (13.6 +/- 3.0) months. The results of musculoskeletal ultrasound and X-ray examinations were analyzed.</p><p><b>RESULTS</b>According to Kellgren-Lawrence classification of knee joint on the X-ray: the musculoskeletal ultrasound results of patients with I degree synovial hyperplasia in 9 cases, joint effusion in 20 cases, meniscal disease in 13 cases, patellar pad inflammation in 5 cases, and patellar lesion in 8 cases. The musculoskeletal ultrasound results of patients with III degree: synovial hyperplasia in 20 cases,joint effusion in 31 cases, meniscal disease in 22 cases, patellar pad inflammation in 16 cases and patellar lesion in 17 cases. The musculoskeletal ultrasound results of patients with III degree: synovial hyperplasia in 6 cases,joint effusion in 6 cases, meniscal disease in 7 cases, patellar pad inflammation in 7 cases and patellar lesion in 5 cases.</p><p><b>CONCLUSION</b>The musculoskeletal ultrasound can detect the pathological changes of knee soft tissue sensitively, provide an accurate location of lesions,and find lesions early. The musculoskeletal ultrasound should be applicated in the diagnosis of knee osteoarthritis.</p>

Study about Traditional Chinese Medicine syndrome of adolescent neck pain / 中国骨伤

<p><b>OBJECTIVE</b>To preliminarily research the formular about the Traditional Chinese Medicine (TCM) syndrome of adolescent neck pain.</p><p><b>METHODS</b>An observation table of adolescent neck pain syndromes was formulated,and 1 397 patients with adolescent neck pain were investigated to establish a database of adolescent neck pain. The Descriptive Statistical Analysis and Hierarchical Cluster Analysis were performed by statistical software.</p><p><b>RESULTS</b>Totally 60 TCM symptoms was clustered into 4 TCM syndromes by Hierarchical Cluster Analysis. The expert panel of TCM syndromes preliminarily formulate 4 TCM syndromes of adolescent neck pain by analyzing the result of Cluster Analysis and discussing their clinical experience.</p><p><b>CONCLUSION</b>Adolescent neck pain is a category of Tendon Trauma's Bi-syndrome of TCM. Ying, Wei, Qi and blood block caused by exopathy, strains, and internal injury is considered as the main pathogenesis of adolescent neck pain. Base on statistical result and expert's opinions, 4 TCM syndromes about adolescent neck pain were formulated: cold-dampness syndrome, dampness-heat blockage syndrome, liver-stagnation and spleen-deficiency syndrome, Qi and Yin deficiency of both heart and kidney syndrome.</p>

Progress on establishment of animal model of osteoarthritis by intra-articular injection / 中国骨伤

Osteoarthritis (Osteoarthritis, OA) is a common clinical degenerative joint disease with increased incidence rate in recent years. Animal experiment is one of the important ways to explore pathogenesis and treatment of OA, while induced animal model is the most important part in animal experiment. Intra-articular injection of drugs is a classical method for establishing animal model of OA. Choose of animal should follows the principle of correlation, appropriateness and practicability, injections should perform in accordance with experimental purposes and subject, detections means and evaluation methods also should corresponding to experimental reality. The gold standard of OA animal model and intra-articular injections has not build, need further study.

Soft tissue changes observed through musculoskeletal ultrasound in the treatment of knee osteoarthritis with needle-knife / 中国骨伤

<p><b>OBJECTIVE</b>To study soft tissue changes observed through musculoskeletal ultrasound (MSUS) in the treatment of knee osteoarthritis with needle-knife, so as to provide MSUS basis for needle-knife in the treatment of knee osteoarthritis.</p><p><b>METHODS</b>Forty patients with knee osteoarthritis treated in the Third Affiliated Hospital of Beijing University of Chinese Medicine from December 2011 to December 2012 were selected according to inclusion and exclusion criteria. All the patients were treated with needle-knife release method. The VAS scores and knee joint circumference were recorded before treatment and 2 weeks after treatment. The changes of knee joint hydrops articuli and joint synovial thickness were measured through MSUS.</p><p><b>RESULTS</b>The knee pain index was 6.850 +/- 1.417 before treatment and 2.790 +/- 1.299 after treatment;the index after treatment was lower than that of before treatment. The knee joint circumference was 407.320 +/- 45.151 mm before treatment and 391.240 +/- 41.129 mm after treatment; the knee joint circumference decreased after treatment. The amount of hydrops articuli observed by musculoskeletal ultrasound showed that 47 knees were cured, 19 knees improved and 2 knees failed. The synovial membrane thickness: 43 knees cured, 17 knees improved and 8 knees failed.</p><p><b>CONCLUSION</b>The hydrops articuli and synovial thickness of knee joint of patients with knee osteoarthritis observed under the MSUS is consistent with the main symptoms and signs, which suggests that MSUS observation on soft tissue changes before and after needle knife in the treatment of knee osteoarthritis with high reliability.</p>

Analysis of X-rays of cervical imbalances syndrome before and after the treatment of osteopathy and traction intervention in 187 youth cases / 中国骨伤

<p><b>OBJECTIVE</b>To observe X-ray features of before and after treatment of cervical imbalance syndrome with osteopathy and traction intervention in youth patients, then to investigate the clinical effect of the treatment of the cervical imbalances syndrome in youth by osteopathy.</p><p><b>METHODS</b>From September 2007 to December 2010, one hundred and eighty-seven students of 19 to 22 years (means 21 years) with neck pain as the main symptom in Beijing university of Chinese medicine were selected and divide them into osteopathy group and traction groups randomly. In osteopathy group, there were 94 patients including 40 males and 54 females; in traction group, there were 93 patients including 42 males and 51 females. The treatment of osteopathy group lasted for 3 weeks,three times per week; traction group by traction treatment three weeks,three times a week, too. X-ray before treatment and 3 weeks after treatment were collected.</p><p><b>RESULTS</b>The osteopathy group:94 cases, before treatment, abnormal curvature in 57 cases, spinous position change in 45 cases and angular displacement in 44 cases, vertebral sliding in 15 cases, Ruth Jackson line intersect proneness change in 70 cases,stretch change in 47 cases; after treatment, abnormal curvature in 35 cases, spinous position change in 24 cases and angular displacement in 18 cases, vertebral sliding in 3 cases, Ruth Jackson line intersect proneness change in 41 cases, extension change in 33 cases; X-Ray measurement results: cervical curvature improved from (7.070 +/- 4.629) degrees before treatment to (7.660 +/- 4.156) degrees after treatment, angular displacement improved from (13.790 +/- 2.590) degrees before treatment to (11.050 +/- 2.560) degrees after treatment; vertebral sliding improved from (3.770 +/- 0.350) mm before treatment to (3.160 +/- 0.485) mm after treatment. The change of angular displacement and vertebral sliding were so significant before and after treatment in this group, there were statistical defferences between before and after the treatment (P < 0.01) in osteopathy group. The traction group: 93 cases, before treatment,abnormal curvature in 60 cases, spinous position change in 39 cases and angular displacement in 39 cases, vertebral sliding in 15 cases, Ruth Jackson line intersect proneness change in 70 cases, stretch in 47 cases; after treatment,abnormal curvature in 50 cases, spinous position change in 29 cases and angular displacement in 17 cases; vertebral sliding in 3 cases, Ruth Jackson line intersect proneness change in 41 cases, stretch in 33 cases; X-Ray measurement results: cervical curvature improved from (5.590 +/- 4.639) degrees before treatment to (5.990 +/- 4.330) degrees after treatment, angular displacement improved from (13.360 +/- 2.064) degrees before treatment to (11.210 +/- 1.872) degrees after treatment; vertebral sliding improved from (3.790 +/- 0.339) mm before treatment to (3.480 +/- 0.332) mm after treatment. The change of angular displacement and vertebral sliding were also so significant in traction group before and after treatment, there were statistical defferences between before and after the treatment (P < 0.01).</p><p><b>CONCLUSION</b>(1) Pathological changes can be called youth cervical imbalance syndrome. (2) Stress points, angular displacement, cervical vertebral slip, curvature straightened, spinous position change are main X-ray performances. (3) Both osteopathy and traction intervention on are efficient in youth neck pain. (4) Youths cervical vertebra imbalance is early but reversible performance.</p>

The clinical application of infrared imaging technology in teenagers with cervicodynia / 中国骨伤

<p><b>OBJECTIVE</b>To compare the temperature distribution of both sides of shoulder, provide objective reference for the application thermoview in the diagnosis of cervicodynia in teenagers.</p><p><b>METHODS</b>Forty-five adolescents with cervicodynias from March 2009 to December 2009 were collected. There were 23 males and 22 females, with an average age of 21 years (ranged from 19 to 22 years). The course of disease ranged from 2 to 20 weeks (averaged 13 weeks). C7 horizontal line were used to divide the back into the neck area and shoulders area, and the midline to subdivide the chosen area into left and right area. Thermal infrared imaging was used to observe the temperature both sides of neck and shoulders, the data was analyzed by the computer.</p><p><b>RESULTS</b>The temperature of shoulder was higher than neck. There were significant differences in the highest and average temperature both of the left and right side of neck and shoulder (P < 0.05), but no difference in the lowest temperature both of left and right side of neck.</p><p><b>CONCLUSION</b>The thermal infrared imaging is important for diagnosing cervical imbalance syndrome, finding the sign of abnormal muscle metabolism of shoulder and providing the basis for prevention.</p>

Case-control study on therapeutic effects between bone-setting and herbal fumigation for the treatment of the obsolete malleolus joint sprains / 中国骨伤

<p><b>OBJECTIVE</b>To compare the clinical effects of bone-setting technique and herbal fumigation for the treatment of the obsolete malleolus joint sprains.</p><p><b>METHODS</b>From March 2008 to May 2011, 76 patients were divided into treatment group (39 cases) and control group (37 cases). In the treatment group: 15 males and 24 females; the age ranged from 20 to 59 years with an average of (42.97 +/- 9.21) years; the course of disease ranged from 1 to 60 months; the average score of ankle joint function was (71.27 +/- 4.50). In the control group: 11 males and 26 females; the age ranged from 25 to 57 years with an average of (41.29 +/- 8.77) years; the course of disease ranged from 1 to 36 months with an average of (8.47 +/- 7.37) months; the average score of ankle joint function was (71.45 +/- 4.61). The patients in the treatment group were treated with bone-setting technique two times a week, and the patients in the control group were treated with herbal fumigation once a day. The ankle joint function scores and treatment effects of the two groups were compared after 3 weeks by using Baird-Jackson ankle function score.</p><p><b>RESULTS</b>After 3 weeks of the treatment, the average score of ankle joint function of the treatment group was (93.44 +/- 4.91), and in the control group was (85.8 +/- 16.57), the difference has statistical significance. The treatment group score was better than that of the control group. Before and after treatment,the average ankle score of the treatment group was (71.27 +/- 4.50) and (93.44 +/- 4.91), the difference has statistical significance. Before and after treatment, the average ankle score of the control group was (71.45 +/- 4.61) and (85.81 +/- 6.57), the difference has statistical significance. In the treatment group, 16 cases got an excellent result, 18 good, 3 fair, 2 poor; in the control group, 9 cases got an excellent result, 14 good, 5 fair,9 poor. The difference has statistical significance.</p><p><b>CONCLUSION</b>The bone-setting techniques and herbal fumigation treatment of obsolete malleolus joint sprains both have a certain effect, and the former is better than the latter.</p>

Multiple linear regression analysis of X-ray measurement and WOMAC scores of knee osteoarthritis / 中国骨伤

<p><b>OBJECTIVE</b>To perform Multiple Linear Regression analysis of X-ray measurement and WOMAC scores of knee osteoarthritis, and to analyze their relationship with clinical and biomechanical concepts.</p><p><b>METHODS</b>From March 2011 to July 2011, 140 patients (250 knees) were reviewed, including 132 knees in the left and 118 knees in the right; ranging in age from 40 to 71 years, with an average of 54.68 years. The MB-RULER measurement software was applied to measure femoral angle, tibial angle, femorotibial angle, joint gap angle from antero-posterir and lateral position of X-rays. The WOMAC scores were also collected. Then multiple regression equations was applied for the linear regression analysis of correlation between the X-ray measurement and WOMAC scores.</p><p><b>RESULTS</b>There was statistical significance in the regression equation of AP X-rays value and WOMAC scores (P<0.05), while there was no statistical significance in the regression equation of lateral X-ray value and WOMAC scores (P>0.05).</p><p><b>CONCLUSION</b>1) X-ray measurement of knee joint can reflect the WOMAC scores to a certain extent. 2) It is necessary to measure the X-ray mechanical axis of knee, which is important for diagnosis and treatment of osteoarthritis. 3) The correlation between tibial angle,joint gap angle on antero-posterior X-ray and WOMAC scores is significant, which can be used to assess the functional recovery of patients before and after treatment.</p>