Objective To investigate the diagnostic efficacy of silkworm larvae plasma (SLP) colorimetry in the accurate diagnosis of periprosthetic joint infection (PJI). Methods Ninety healthy male New Zealand white rabbits were used for knee arthroplasty with Swanson prosthesis. Then they were randomly divided into 3 groups according to different pathogenic bacteria: group A (Staphylococcus aureus group), group B (Staphylococcus epidermidis group) and group C (Escherichia coli group), with 30 rats in each group. The PJI model was prepared by knee injection with 1 mL of pathogenic bacteria of different concentrations. Samples were taken before inoculation and at 7, 14, and 21 days after inoculation, and based on the 2018 PJI Philadelphia International Consensus diagnostic criteria, the success rate of modeling among 3 groups of experimental animals was determined. The sensitivity, specificity, positive predictive value, negative predictive value, and diagnostic efficiency of SLP colorimetry were calculated. Results At 21 days after inoculation, 26, 18, and 23 rabbits in groups A, B, and C were diagnosed as infection, respectively. The success rates of modeling were 86.7%, 60.0%, and 76.7%, respectively, showing no significant difference among the 3 groups (χ2=5.724, P=0.073). The results of PJI colorimetry showed that 1 false-positive animal (specificity 75.0%) appeared in group A at 7 days, and the specificity of SLP increased to 100.0% over time (on 14 and 21 days); on 14 and 21 days, another animal appeared false-negative results (sensitivity decreased from 100.0% to 96.2%). One false-positive animal appeared in group B at 7 days (specificity 91.7%), the specificity returned to 100.0% over time; 1 and 4 false-negative animals appeared at 14 and 21 days, respectively (sensitivity 94.4% and 83.3%, respectively). In group C, two false-positive animals (specificity 71.4%) were found at 7 days, and then returned to 100.0%. The diagnostic efficiency of groups A and C was very high at 21 days (96.7% and 100.0%), even for the low virulence Staphylococcus epidermidis in group B, the diagnostic efficiency could be maintained at 90.0% (21 days), and the overall diagnostic efficiency was very good (95.6%). Conclusion SLP colorimetry has high sensitivity, specificity, and diagnostic efficiency in the diagnosis of PJI, which is a potential diagnostic method.
Objective To summarize operative procedure and the effectiveness of open reduction with internal fixation or radial head replacement for the treatment of Essex-Lopresti injury. Methods Between November 2002 and October 2010, 10 patients with Essex-Lopresti injury were treated. There were 8 males and 2 females with a mean age of 36 years (range, 20-56 years). Eight cases were fresh closed fracture within 2 days. According to Mason classification, 5 fracture were typeII, 3 were type III. The other 2 cases were old fracture within 3 months. Wrist joint X-ray revealed that all the patients had distal radioulnar joint dislocation. Open reduction with internal mini-plate or absorbable screw fixation was performed in 5 cases, and radial head replacement in 5 cases; meanwhile, the distal radioulnar joint was reducted and fixed. Results All incisions healed by first intention without infection or bone nonunion. The patients were followed up 7 to 24 months with an average of 14.7 months. The X-ray films showed fracture heal ing at 9-20 weeks (mean, 16.3 weeks); distal radioulnar joint was stable without shortening or shift of proximal radius. According to elbow cl inical evaluation system, the results were excellent in 5 cases, good in 3 cases, and fair in 2 cases. According to wrist cl inical evaluation, the results were excellent in 7 cases, good in 2 cases, and fair in 1 case. All patients had good elbow stabil ity, and recovered quickly. Conclusion Early diagnosis, operation, and functional exercises are important to obtain an excellent result in treating Essex-Lopresti injury.
To investigate the preventive effect of TGF-β1 neutral izing antibody on collagen production and adhesion formation of flexor tendon. Methods Tendon fibroblasts, epitenon tenocytes, and endotenon tenocytes were obtained from 6 New Zealand rabbit flexor tendons. Each cell culture was supplemented with 1 ng/mL of TGF-β along with increasing dose of TGF-β1 neutral izing antibody. Col I production was measured by enzyme-l inked immunoabsorbent assay after 3 days. Eighty-four adult New Zealand White rabbits forepaws underwent sharp transection of middle digit flexor digitorumprofundus and immediate repair. Then the rabbits were divided into three groups: the normal saline (NS group, n=36), 1.0 µg/ mL TGF-β1neutral izing antibody (1.0 µg/mL TGF-β1group, n=36) and 2.0 µg/mL TGF-β1 neutral izing antibody (2.0 µg/mL TGF-β1 group, n=12) were injected in tendon sheath respectively. Tendons were harvested at 4 and 8 weeks for biomechanics testing, histological evaluation and scanning electron microscope observation. Tendons were harvested at 1, 2, 4 and 8 weeks to determine the mRNA expression of TGF-β1 and Col I by in situ hybridization. Results ELISA exhibed that TGF-β1 enhanced Col I production and the neutral izing antibody significantly inhibited TGF-β1-induced Col I production in all 3 cell culture with a dose-dependent. At 4 and 8 weeks after operation the gl iding excursion of the tendon and the simulated active flexion in NS group were less than that of 1.0 µg/mL TGF-β1 group and 2.0 µ g/mL TGF-β1 group. There was significant difference between NS group and 1.0 µ g/mL TGF-β1 group, 2.0 µ g/mL TGF-β1 group (P lt; 0.05). The tendon anastomosis breaking strength showed no significant differences among three groups (P gt; 0.05). Scanning electron microscope and histological observation showed that collagen fibers arranged irregularly in NS group, but arranged regularly in 1.0 µ g/mL TGF-β1 group and 2.0 µ g/mL TGF-β1group at 4 and 8 weeks after operation. The in situ hybridization results revealed that TGF-β1 and Col I mRNA expression in 1.0 µ g/mL TGF-β1 group was lower than that in NS group at each time. There was significant difference between two groups (P lt; 0.05). Conclusion TGF-β1neutral izing antibody can inhibit the function of the TGF-β1 effectively and prevent adhesion formation after the flexor tendon injured and repaired.
Objective To summarize the selection criteria and clinical application of surgical methods for hip fractures (femoral neck fracture and intertrochanteric fracture) in the elderly. Methods The related literature concerning the surgical methods for femoral neck fracture and intertrochanteric fracture in the elderly at home and abroad was extensively reviewed and summarized. Results Among the elderly patients with femoral neck fracture, the closed reduction and internal fixation or dynamic hip screw (DHS), and total hip arthroplasty are recommended for patients under 65 years old and 65–80 years old respectively and without special surgical contraindication; whereas hemiarthroplasty is recommended for patients with poor physical conditions. Among the patients with intertrochanteric fracture, DHS or the 3rd generation of Gamma nails is recommended for patients with stable fracture while the intramedullary fixation systems (e.g., proximal femoral nail anti-rotation, intertrochanteric antegrade nail) and the extramedullary fixation systems (e.g., proximal femoral locking compression plate and less invasive stabilization system) can be applied to the patients with unstable fracture according to the fracture type and bone conditions. Notably, hip arthroplasty is recommended for elderly patients with comminuted fracture. Conclusion The surgical method and internal fixator should be chosen according to the fracture type and bone condition in the elderly in order to improve the effectiveness and reduce the complication.
Objective To explore the biomechanic effects of multi ple freeze-thaw on human allograft tendons. Methods Thirty tendons (24 flexor digitorum superficial is tendons and 6 flexor poll icis longus tendons) were harvested from 3 fresh cadaver donors and were divided into 6 groups randomly (fresh group; 1 cycle, 2 cycle, 3 cycle, 5 cycle, and 10 cycle freeze-thaw groups). There was 4 flexor digitorum superficial is tendons and 1 flexor poll icis longus tendon in each group. The structural and mechanical properties as well as viscoelastic change were estimated. Results The results of the structural and mechanical properties in 1 cycle, 2 cycle, and 3 cycle freeze-thaw groups were similar to that of the fresh group (P gt;0.05). The tendons in 5 cycle and 10 cycle freeze-thaw groups showed a significantly lower ultimate load and maximum stress when compared with those of fresh group (P lt; 0.05), but there was no significant difference in maximum tensile or maximum strain (P gt; 0.05). Moreover, the tendons in 5 cycle and 10 cycle freeze-thaw groups had a significant increase in viscoelastic properties when compared with fresh group (P lt; 0.05). Conclusion In the cryopreservation of tendon allografts, the cycle of freeze-thaw should not exceed 3 times. Multiple cycle freeze-thaw will weaken the biomechanical properties of tendon allografts, which make grafts easier to fatigue or even rupture.
Objective By culturing tendon sheath fibroblasts, epitenon tenocytes and endotenon tenocytes of rabbits’ tendon in vitro, to study the effects of mannose-6-phosphate on transforming growth factor β (TGF-β) peptide and receptor expression, and to provide the experimental basis for preventing the tendon heal ing adhesion by mannose- 6-phosphate. Methods Eight adult New Zealand white rabbits, regardless of their gender and weighing 4.0-4.5 kg, were selected. Tendon sheath fibroblasts, epitenon tenocytes, and endotenon tenocytes were isolated from rabbit flexor tendon and cultured separately. All 3 cells were divided into 2 groups at random after cells were adjusted to a concentration of 4 × 104 per well and 1 × 104/mL. The first was the control group without supplementation. The experimental group was supplemented withmannose-6-phosphate. The expressions of TGF-β and TGF-β receptor were quantified with enzyme-l inked immunosorbent assay. The expression of TGF-β1 mRNA was also assessed with in situ hybridization and the expression of TGF-β1 was assessed with immunohistochemistry. Results The expressions of TGF-β and TGF-β receptor in experimental group were significantly lower than that in control group (P lt; 0.05). The expression levels of TGF-β1 and TGF-β2 decreased in descending order of tendon sheath fibroblasts (36.1%, 37.9%), epitenon tenocytes (31.0%, 32.1%), and endotenon tenocytes (31.2%, 27.0%). The expression levels of TGF-β3 decreased in descending order of endotenon tenocytes (42.5%), tendon sheath fibroblasts (41.2%), and epitenon tenocytes (33.3%). The expression levels of TGF-β receptor 1 and TGF-β receptor 2 decreased in descending order of epitenon tenocytes (29.9%, 26.2%), endotenon tenocytes (27.8%, 23.5%), and tendon sheath fibroblasts (23.1%, 20.0%). The expression levels of TGF-β receptor 3 decreased in descending order of endotenon tenocytes (26.1%), epitenon tenocytes (19.2%), and tendon sheath fibroblasts (15.8%). In experimental group, the positive expression of TGF-β1 mRNA and the expression level of intracellular TGF-β1 mRNA in all 3 tendon cells were significantly lower than those in the control group (P lt; 0.05). Immunohistochemical staining showed the expressions of TGF-β1 in all 3 tendon cells were significantly lower in theexperimental group than in the control group. Conclusion Mannose-6-phosphate can significantly decrease the expressions of TGF-β peptide, TGF-β receptor, and TGF-β1 mRNA. Modulation of mannose-6-phosphate levels may provide a mean of modulating the effects of TGF-β on adhesion formation in flexor tendon wound heal ing.
Objective To analyze the stabil ity and cl inical outcomes of arthroscopic anterior cruciate l igament (ACL) reconstruction with γ irradiated patellar tendon allograft compared with autograft. Methods From January 2004 to October 2007, 69 patients undergoing arthroscopic ACL reconstruction were prospectively randomized consecutively into two groups: group A (autograft, n=36) and group B (γ irradiated allograft, n=33). In group A, there were 30 males and 6 females with an average age of 30.1 years, including 30 cases of simple ACL rupture and 6 cases of ACL rupture with medial accessory l igament injury; ACL rupture was caused by sports in 28 cases, by traffic accident in 5 cases, and by others in 3 cases; and the time from injury to operation was 1.4 months on average. In group B, there were 26 males and 7 femaleswith an average age of 32.5 years, including 27 cases of simple ACL rupture and 6 cases of ACL rupture with medial accessory l igament injury; ACL rupture was caused by sports in 27 cases, by traffic accident in 4 cases, and by others in 2 cases; and the time from injury to operation was 1.5 months on average. There were no significant differences in general data between two groups (P gt; 0.05). The same arthroscopic technique was used in all ACL reconstructions done by the same surgeon. The cl inical outcome was evaluated and compared by general conditions, pivot shift test, Lachman test, KT-2000 arthrometer testing, Daniel’s one-leg hop test, International Knee Documental Committee (IKDC) scoring, Lysholm knee scoring scale, and Tegner activity score. Results All patients were followed up for 39.5 months (group A) and 37.6 months (group B). In group A, patella fracture occurred in 1 case and anterior knee pain in 2 cases postoperatively. No compl ication occurred in group B. The hospital ization times in groups A and B were (15.6 ± 2.4) days and (15.5 ± 1.5) days, respectively, showing no significant difference (P gt; 0.05). The operation time of group A was longer than that of group B and the fever time of group A was shorter than that of group B, showing significant differences (P lt; 0.05). At the final follow-up, there were significant differences (P lt; 0.05) in Lachman test and the pivot shift test between two groups, between pre- and post-operation; there were no significant differences (P gt; 0.05) in Daniel’s one-leg hop test, the IKDC, Lysholm, and Tegner activity scores between two groups, however, there was a decreased trend in the functional and activity levels in group B. And there was significant difference between pre- and post-operation (P lt; 0.05). At the final follow-up, the differences between normal side and affected side were (2.4 ± 0.6) mm in group A and (5.5 ± 3.6) mm in group B, showing significant difference (P lt; 0.05). There was significant difference in tibial advancement between pre- and post-operation (P lt; 0.05). Conclusion The functional and activity level of the knee after ACL reconstruction with autograft and γ irradiated patellar tendon allograft were similar, but anterior and rotational stabil ity of the involved knee decreases significantly in the group with γ irradiated patellar tendon allograft.
Objective To explore effect of platelet-rich plasma (PRP) on rabbit BMSCs differentiation into SC in vitro and to detect secretory function of the differentiated cells. Methods BMSCs isolated from 5 mL bone marrow of 2-montholdNew Zealand white rabbit were cultured using density gradient centrifugation and adherence screening methods. A total of 5 mL femoral vein blood was obtained from rabbits to prepare PRP using modified Appel method. The BMSCs at passage 3 were divided into three groups: the combined induction group, in which the cells were cultured with complete medium containing PRP after β-mercaptoethanol and retinoic acid inductions; the simple induction group, in which the cells were cultured with L-DMEM complete medium without PRP afterβ-mercaptoethanol and retinoic acid induction; the control group, in which the cells were cultured with L-DMEM complete medium. Growth condition of the cells in each group was observed using inverted microscope. cell identification was conducted at 4, 7, 9, and 11 days after culture using immunofluorescence staining method, and NGF content was detected by ELISA method. NGF mRNA expression was assayed by RT-PCR 11 days after culture. Results Most cells in the combined induction and the simple induction group were out of BMSCs typical cell morphology 4 days after culture; cells in the combined induction group were out of BMSCs typical cell morphology and changed into cells resembl ing SC in terms of morphology and contour 9 days after culture. The cells in the control group showed no obvious morphological changes. S-100 protein expression in the cells was evident in the combined induction and the simple induction group at each time point after induced culture; the positive expression rate of cell in each group was increased over time, and significant differences were evident between the combined induction group and the simple induction group 7, 9, and 11 days after culture (P lt; 0.05). Control groupwas negative for the expression. There were significant differences when comparing the control group with the combined induction group or the simple induction group in terms of NGF content at each time point (P lt; 0.01). Significant difference was evident between the combined induction group and the simple induction group 7, 9, and 11 days after culture (P lt; 0.05), and no significant difference was noted 4 days after culture (P gt; 0.05). Relative intensity of NGF mRNA expression in the combined induction group was greater than that of the simple induction group 11 days after culture (P lt; 0.05). Conclusion Rabbit BMSCs can differentiate into SC excreting NGF under certain induction condition in vitro. PRP can remarkably promote BMSCs differentiation into SC.
Objective To study the effect of platelet lysate (PL) on chondrogenic differentiation of human umbil ical cord derived mesenchymal stem cells (hUCMSCs) in vitro. Methods Umbil ical cords were voluntarily donated by healthy mothers. The hUCMSCs were isolated by collagenase digestion and cultured in vitro. The surface markers of the cells were detected by flow cytometer. According to different components of inductive medium, the cultured hUCMSCs were divided into 3 groups: group A [H-DMEM medium, 10% fetal bovine serum (FBS), and 10%PL]; group B [H-DMEM medium, 10%FBS,10 ng/mL transforming growth factor β1 (TGF-β1), 1 × 10-7 mol/L dexamethasone, 50 μg/mL Vitamin C, and 1% insul intransferrin- selenium (ITS)]; and group C (H-DMEM medium, 10%FBS, 10 ng/mL TGF-β1, 1 × 10-7mol/L dexamethasone, 50 μg/ mL vitamin C, 1%ITS, and 10%PL). The hUCMSCs were induced in the mediums for 2 weeks. Toluidine blue staining was used to detect the secretion of chondrocyte matrix. Immunofluorescence method was used to identify the existence of collagen trpe II. The expressions of Aggrecan and collagen type II were detected by semiquantitative RT-PCR. Results Flow cytometer results showed that the hUCMSCs did not express the surface markers of hematopoietic cell CD34, CD45, and human leukocyte antigen DR, but expressed the surface markers of adhesion molecule and mesenchymal stem cells CD44, CD105, and CD146. Toluidine blue staining and immunofluorescence showed positive results in group C, weak positive results in group B, and negative results in group A. Semiquantitative RT-PCR showed the expressions of Aggrecan and collagen type II at mRNA level in groups B and C, but no expression in group A. The mRNA expressions of Aggrecan and collagen type II were higher in group C than in group B (P lt; 0.05). Conclusion Only 10%PL can not induce differentiation of hUCMSCs into chondrocytes, but it can be a supplement to the induced mediums. PL can improve hUCMSCs differentiating into chondrocytes obviously in vitro. This study provides new available conditions for constructing tissue engineered cartilage.
Objective To construct inducible lentiviral vector containing human bone morphogenetic protein 2 (hBMP-2) gene and to study its expression in human umbil ical cord blood mesenchymal stem cells (HUMSCs). Methods hBMP-2 gene was ampl ified by PCR from a plasmid and was cloned into pDown by BP reaction. pLV/EXPN2-Neo-TRE-hBMP-2 and pLV/EXPN2-Puro-EF1A-reverse transactivator (rtTA) were obtained with GATEWAY technology, and then were sequenced and analyzed by PCR. The recombinant vectors were transfected into 293FT cells respectively through l ipofectamine, and the lentiviral viruses were harvested from 293FT cells, then the titer was determined. Viruses were used to infect HUMSCs in tandem. In order to research the influence of induction time and concentration, one group of HUMSCs was induced by different doxycl ine concentrations (0, 10, 100 ng/mL, and 1, 10, 100 μg/mL) in the same induction time (48 hours), and the other by the same concentration (10 μg/mL) in different time points (12, 24, 48, and 72 hours). The expression of target gene hBMP-2 was indentified by ELISA method. After 2-week osteogenic induction of transfected HUMSCs, the mineral ization nodes were detected with Al izarin bordeaux staining method. Results Therecombinant inducible lentiviral vectors (pLV/EXPN2-Neo-TRE-hBMP-2 and pLV/EXPN2-Puro-EF1A-rtTA) were successfully constructed. The lentiviruses were also obtained and mediated by 293FT cells, and the virus titers were 3.5 × 108 TU/mL and 9.5 × 107 TU/mL respectively. HUMSCs could expression hBMP-2 by induction of doxycycl ine. The expression of hBMP-2 reached the peak at 10 μg/mL doxycl ine at 48 hours of induction. After 2-week osteogenic induction, a lot of mineral ization nodes were observed. Conclusion The recombinant inducible lentiviral vectors containing hBMP-2 gene can be successfully constructed, which provide an effective and simple method for the further study of stem cells and animal experiment in vivo.