Objective:To prepare the hydrogel scaffolds with different concentrations of laponite and compare their osteogenic properties.Methods:The scaffolds of gelatin/sodium alginate hydrogel into which laponite was added according to the mass ratios of 0%, 1%, 2%, and 3% were assigned into groups T0, T1, T2, and T3. In each group, the compressive modulus was measured and the leaching solution for 24 h extracted to measure the ion release. Bone marrow mesenchymal stem cells (BMSCs) were cultured in the extract medium from each group and common medium (blank group) ( n=3) in the in vitro experiments to determine the expression of osteogenic genes Runt-related transcription factor 2 (Runx2), alkaline phosphatase (ALP) and type I collagen after 7 days of culture. In the in vivo experiments, the scaffolds were implanted into the femoral condyle defects in rats, and a blank group with no scaffolds was set. The bone repair in each group was evaluated by hematoxylin-eosin(HE) staining and immunohistochemical staining. Results:The compressive modulus in group T2 [(139.05±6.43) kPa] was significantly higher than that in groups T0, T1 and T3 [(68.83±3.76) kPa, (101.18±3.68) kPa and (125.40±3.28) kPa] ( P<0.05). The ion contents of lithium, magnesium and silicon released from the 24 h leaching solution in group T2 were (0.031±0.005) μg/mL, (3.047±0.551) μg/mL and (5.243±0.785) μg/mL, insignificantly different from those in group T3 ( P> 0.05) but significantly larger than those in group T1 ( P>0.05). The in vitro experiments showed that the expression levels of Runx2, ALP and type I collagen in group T2 were 1.59±0.11, 2.02±0.08 and 1.06±0.17, significantly higher than those in the other groups ( P<0.05). HE staining showed that the implanted hydrogel was tightly bound to the bone tissue. Immunohistochemical staining showed that the numbers of Runx2 and osteocalcin positive cells in group T2 were significantly higher than those in the other groups. Conclusions:With ideal biocompatibility, hydrogel scaffolds with different concentrations of laponite can slowly release the decomposed ions of lithium, magnesium and silicon to promote the osteogenic differentiation of BMSCs and the repair of bone defects in vivo. A 2% concentration of laponite in the hydrogel scaffolds may result in the best results.

Objective:To characterize the biological activity of fibroblasts on the surface of titanium alloy sheets with different ridge widths by investigating the effects of ridge widths on the adhesion, proliferation and differentiation of fibroblasts.Methods:Five groups of titanium sheets with ridge widths of 50 μm, 80 μm, 100 μm, 150 μm and 200 μm were prepared, with all the groove depths being 10 μm. The titanium sheets with no ridges were taken as a control group. After fibroblasts were incubated on the sheets, states of their adhesion were observed by scanning electron microscopy (SEM) at different time points. CCK-8 cell proliferation test and immunofluorescence staining were used to observe proliferation and shape of the cells. The effects of ridge widths on adhesion of fibroblasts were evaluated by Vinculin immunofluorescence staining, and the effects of ridge widths on expression of α-smooth muscle actin ( α-SMA) by immunofluorescence. Results:SEM showed that the cells adhered to the ridges on the titanium sheets 48 hours after inoculation. In the groups with smaller ridge widths (from 50 μm to 150 μm), the cells were slender in shape and grew along the ridge direction. CCK-8 indicated that different ridge widths had no significant effect on the proliferation of fibroblasts between the 6 groups ( P>0.05). Immunofluorescence staining showed that the cells arranged in an orderly direction along the ridges; the long axis of the cells in the 50 μm group showed the best consistency with the extending direction of the ridge, with significant differences among the 6 groups ( P<0.05). The Vinculin test found that the secretion of cell adhesion protein was concentrated in the ridge and semi-quantitative analysis showed that the 50 μm group had the most Vinculin secretion, with significant differences among the 6 groups ( P<0.05). The α-SMA test showed that the ridge width had a regulatory effect on the myogenic differentiation of fibroblasts, and the 50 μm group had the strongest expression of α-SMA, with significant differences among the 6 groups ( P<0.05). Conclusions:Modification of ridges on the surface of titanium sheets may affect arrangement, adhesion and myogenic differentiation of fibroblasts. The ridges of 50 μm in width may lead to stronger polarized arrangement of fibroblasts, more secretion of adhesion-related protein and more pronounced myogenic differentiation of fibroblasts.

Objective:To prepare biomimetic tissue engineering scaffolds of gelatin/sodium alginate/laponite composite hydrogel loaded with BMSCs by 3D biological printing technique，and explore the osteogenic effect of 3D printing on hydrogel scaffolds containing bone marrow mesenchymal stem cells（BMSCs）.Methods:BMSCs were routinely extracted and identified by flow cytometry. Gelatin，sodium alginate and laponite were mixed and then BMSCs were added to prepare cell-containing composite hydrogel scaffolds using 3D bioprinting. Non-printed scaffolds containing cells were prepared by injection molding method. In vitro，the prepared scaffolds were divided into the printing group with cells and non-printing group with cells according to whether they were printed，with 12 samples per group. Another simple cell culture group was set as control. Then，the internal structure of the composite hydrogel was observed by scanning electron microscope，and the expansion rate and water content of the scaffolds were measured by freeze-drying method. At day 3 after culture，the growth status of BMSCs was observed by phalloidine staining. cell counting kit（CCK）-8 assay was used to detect cell activity in scaffolds at days 1，3，and 7 after culture and RT-PCR to detect the expression of osteogenesis related genes Osterix，osteocalcin（OCN）and collagen I at days 7 and 14 ofter culture. In vivo，four groups were set according to printing or not and whether containing cells or not：printing implant group with cells，non-printing implant group with cells，printing implant group without cells and non-printing implant group without cells，with 9 samples per group. Scaffolds in four groups were implanted to the posterior gluteal muscle pouches（random on left or right）of 36 8-week-old SD rats，respectively. The samples were taken X-ray images at 2，4 and 8 weeks after operation，respectively. The osteogenic differentiation of tissues at 8 weeks was observed by HE and Masson staining. Results:The flow cytometry showed that the cells were BMSCs. Internal pores of hydrogels were obvious，and cells stretched freely in the pores. Differences of the swelling rate and water content were not statistically significant between printing group with cells［（1，039.37±30.66）%，（91.21±0.26）%］and non-printing group with cells［（1，032.38±35.05）%，（91.16±0.28）%］（ P>0.05）. At day 3 after culture in vitro，the cells grew well in the hydrogel. After culturing for 1 day in vitro，there was no significant difference in absorbance between printing group with cells and non-printing group with cells（ P>0.05）. At day 3 after culture，there was no significant difference in absorbance between printing group with cells and non-printing group with cells，but both groups showed a higher level than simple cell culture group（ P<0.05）. At day 7 after culture，the absorbance in printing group with cells（2.72±0.17）was higher than that in non-printing group with cells（2.35±0.11），and both of which were higher than that in simple cell culture group（1.95±0.12）（ P<0.05）. At day 7 after culture in vitro，there was no statistically significant difference in the expression of osteogenic differentiation-related genes between printing group with cells and the non-printing group with cells（ P>0.05），but they were all higher than those in simple cell culture group（ P<0.05）. At day 14 after culture in vitro，the expression of osteogenesis-related genes Osterix（1.650±0.095），OCN（2.725±0.091），collagen I（2.024±0.091）in printing group with cells were higher than those in non-printing group with cells（1.369±0.114，2.174±0.198，1.617±0.082，respectively）and those in simple cell culture group（1.031±0.094，1.116±0.092，0.736±0.140，respectively）（ P<0.05）. After implantation for 2 weeks in vivo，with no statistically significant difference in the gray values of X-ray films in each group（ P>0.05）. At weeks 4 and 8 after implantation，the gray values of X-ray films in printing implant group with cells and non-printing implant group with cells were higher than those in printing implant group without cells and non-printing implant group without cells（ P<0.01）. At 8 weeks after implantation，HE staining showed that the scaffolds were degraded in different degrees and immersed with cells，with collagen production seen in Masson staining as well. Conclusions:Composite hydrogel scaffolds can provide a good three-dimensional environment for BMSCs growth. 3D bioprinting can promote the proliferation and osteogenic differentiation of BMSCs in hydrogel scaffolds. In addition，BMSCs-loaded scaffolds can be degraded slowly in vivo with good ectopic osteogenic ability.

To analyze the clinical characteristics and genetic data of a child with type Ⅱ Bruce syndrome (type Ⅱ BS) admitted to the Department of Orthopedics, Shanxi Children′s Hospital at May 2020.A 3-day-old boy was admitted due to the pain and swelling of the right lower limb 3 days after birth.Due to the patient had multiple fractures, callus formation after clavicle and rib fracture, greendstick fracture of the humerus, right femur fracture, left tibia and fibula fracture, congenital clubfoot, and congenital contracture of wrist, elbow, hip and knee joint, and therefore, chromosome diseases were considered.Gene sequencing data showed gene mutation in PLOD2 with compound heterozygosity deletion of the child (proband), and mutation and heterozygosity deletion came from their parents, respectively.The patient was diagnosed as type Ⅱ BS.This case report alarms clinicians to identify a missense mutation of PLOD2 and loss of heterozygosity, so as to reduce the misdiagnosis rate and achieve early diagnosis and treatment.

Objective: To establish an efficient classification and treatment system for limb long bone defects. Methods: Based on the length of bone defect, soft tissue injury and wound infection, a new classification and treatment system was proposed with reference to Gustilo-Anderson classification for open fractures and Orthopedic Trauma Association (OTA) classification. Results: We divided the limb long bone defects into 3 types, each of which was subdivided into 4 subtypes depending on concomitant soft tissue defect and/or infection. Type Ⅰ are bone defects less than 4 cm in length, including type Ⅰa (simple bone defects with a limited extent), type Ⅰb (bone and soft tissue defects), type Ⅰc (bone defects with infection) and type Ⅰd (bone defects with infection and soft tissue defects). Type Ⅱ are bone defects ranging from 4 to 10 cm in length, including type Ⅱa (simple bone defects with a large extent), type Ⅱb (bone and soft tissue defects), type Ⅱc (bone defects with infection) and type Ⅱd (bone defects with infection and soft tissue defects). Type Ⅲ are bone defects larger than 10 cm in length, including type Ⅲa (simple bone defects with a very large extent), type Ⅲb (bone and soft tissue defects), type Ⅲc (bone defects with infection) and type Ⅲd (bone defects with infection and soft tissue defects). Conclusion Our new classification and treatment system for long limb bone defects is more efficient and intuitive, facilitating clinical diagnosis and treatment of limb long bone defects.

Objective: To evaluate the effect of 3DBody software assisted problem-based learning (PBL) teaching in orthopedic teaching. Methods: Undergraduates of clinical medicine from grade 2013 who had internship in our hospital were divided into experimental group and control group. Undergraduates in the experimental group were taught by 3DBody software assisted PBL teaching, with designed questions and the method of using 3DBody software handing to students before class. While undergraduates in the control group were taught by traditional teaching, with the use of textbooks and multimedia courseware. Examination scores of theory and probation were compared between the two groups. Questionnaires were used to evaluate the subjective perception of different teaching method among the participants in each group. Results: Scores of theoretical examination in the experimental group (84.6±5.9) were higher than those in the control group (73.2±6.1); scores of probation examination in the experimental group (17.7±2.1) were significantly higher than those in the control group (12.7±1.9); the degree of satisfaction in the experimental group (9.2±0.8) was significantly higher than that in the control group (7.2±1.3); all differences were statistically significant (P<0.05). Conclusion 3DBody software assisted PBL teaching can significantly improve the effectiveness of orthopedic teaching and enhance students' learning initiative and interest, which is worth promoting.

Objective To prepare biphasic calcium phosphate/polyvinyl alcohol scaffolds by 3D printing at room temperature and explore the effect of 3D scaffolds on in vitro osteogenic differentiation of the bone marrow mesenchymal stem cells (BMSCs).Methods After biphasic calcium phosphate and polyvinyl alcohol solutions were mixed,the biphasic calcium phosphate/polyvinyl alcohol composite scaffolds were prepared by room temperature 3D printing combined with freeze drying technique.Non-printing scaffolds were prepared by injection molding.The surface microstructure,porosity,elastic modulus and hydrophilicity of the 2 sorts of scaffolds were measured.The cytological experiments were carried out in 3 groups (n =3):printed scaffold group,non-printed scaffold group and blank control group (no scaffold).After the BMSCs were seeded onto the scaffolds for 7 and 14 days,the 3 groups were compared in terms of cellular proliferation,alkaline phosphatase activity and expression levels of osteogenesis-related genes.Results 3D composite scaffolds with controllable pore size and porosity were prepared successfully,with an average porosity of 59.6％ ± 3.6％ and an average elastic modulus of 429.3 ± 54.3 kPa.After culture for 7 and 14 days,the cellular absorbance values in the printed scaffold group (0.987 ± 0.047 and 1.497 ± 0.076) were significantly higher than those in the non-printed scaffold group (0.767 ±0.063 and 1.181 ±0.098) (P ＜ 0.05) which were in turn significantly higher than those in the blank control group (0.532 ±0.046 and 0.895 ± 0.062) (P ＜ 0.05).After culture for 7 and 14 days,the ALP activity and expression levels of osteogenesis-related genes in the printed and non-printed scaffold groups showed no significant between-group differences (P ＞ 0.05),but were significantly higher than those in the blank control group (P ＜ 0.05).Conclusions Tissue-engineered composite biphasic calcium phosphate/polyvinyl alcohol scaffolds with controllable pore size and good connectivity can be prepared by freeze-drying and room temperature 3D printing techniques.Co-culture of the scaffolds and BMSCs in vitro promotes adhesion,proliferation and osteogenic differentiation of the cells.

Objective To compare the effectiveness of expandable intramedullary nail(EIMN) versus locked compression plate (LCP) in the treatment of humeral shaft fractures of AO types A and B.Methods The clinical data were retrospectively analyzed and compared of the 44 humeral shaft fractures which had been treated between May 2012 and February 2016.There were 26 men and 18 women,from 23 to 66 years of age (average,41.5 years).By AO classification,22 cases were type 12-A and 22 type 12-B.EINM was used in 22 patients with an average age of 41.3 ± 1 1.7 years and LCP in 22 ones with an average age of 41.6 ± 10.3 years.The 2 groups were compared in terms of intraoperative blood loss,operative time,hospital stay,union time,union rate,Constant and Mayo scores at the final follow-ups,and complications as well.Results The 2 groups were compatible without significant differences in the preoperative demographic data (P ＞ 0.05).All the 44 patients were followed up for 10 to 18 months (average,12 months).The intraoperative blood loss (76.4 ± 18.66 mL),operative time (69.1 ± 13.2 min),incision length (5.8 ± 1.5 cm) and union time (13.2 ± 8.4 w) in the EIMN group were significantly better than those in the LCP group (138.6 ± 39.4 mL,96.4 ± 14.2 min,8.5 ± 1.4 cm and 18.4 ± 6.6 w,respectively) (P ＜ 0.05).There was also a significant difference between the 2 groups in the total complication rate[18.2％ (4/22) versus 50.0％ (11/22)] (P ＜0.05).No deep infection or should pain was observed in either group.Conclusions Inthe treatment of humeral shaft fractures of AO types 42-A and 42-B,compared with LCP,EIMN may have advantages of less intraoperative blood loss,operative time,union time and complications.The 2 methods are similar in hospital stay and final functional recovery of the should joint.

Objective To prepare a bionic artificial bone scaffold using a room temperature three dimensional (3D) printing technique and evaluate its biocompatibility and bioactivity in vitro.Methods A room temperature 3D printing technique was applied to fabricate 3D bionic artificial bone scaffolds using collagen/hydroxyapatite.The physico-chemical structure,porosity and mechanical strength of the scaffolds were assessed.The extract liquid of scaffolds was cocultured with bone mesenchymal stem cells (BMSCs) to evaluate the toxicity of scaffolds.There were 3 experimental groups:blank control with no scaffolds,printed scaffolds group and non-printed scaffolds group.The condition of BMSCs on the scaffolds was observed via scanning electron microscopy(SEM) and immunostaining.3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay and SEM were applied to monitor the proliferation of BMSCs on the scaffolds.At last,alkaline phosphatase (ALP) activity and mRNA expression levels of osteogenesis-related genes were detected to assess the osteoinductive property of the scaffolds.Results The 3D printed scaffolds fabricated in the present study were characterized by highly interconnected pores which were controllable and even in size.The cross section of the scaffolds presented an irregular honeycomb-like microstructure.The porosity of printed 3D scaffolds (71.14％ ± 2.24％) was significantly higher than that of non-printed scaffolds (59.04％ ±2.98％) (P ＜ 0.05).The physico-chemical structures of the materials were preserved after printing without additional cytotoxicity.The MTT results at 7 and 14 days revealed that the printed scaffolds had a significantly more cell numbers than the non-printed scaffolds(P ＜ 0.05).SEM showed that the BMSCs adhered well onto the printed scaffolds and proliferated and migrated through the pores.Compared with the blank control,the printed scaffolds showed obviously better osteogenic outcomes.Conclusion The 3D bionic artificial bone scaffolds of collagen/hydroxyapatite manufactured by a room temperature 3D printing technique can provide a good extracellular matrix for BMSCs to proliferate and differentiate.

Objective To study the feasibility of microsurgical technique to denervate sympathetic of femoral artery in rabbit, providing a reliable animal experimental model for further study of the mechanism of neuralization in bone tissue engineering.Methods From July, 2014 to July, 2015, 21 New Zealand white rabbits were divided into 4 groups randomly: the control group (n =3), the 4 weeks group (n =6), the 8 weeks group (n =6) and the 12 weeks group (n =6).Bilateral femoral arteries of the 21 rabbits were exposed.Adventitia of femoral arteries in 3 test groups were removed for about 2cm by microsurgical technique, whereas adventitia of the control group remained intact without any treatment.The arteries samples were collected at 4 weeks, 8 weeks and 12 weeks after treatment.The structure of vascular were indicated by hematoxylin-eosin (HE) staining, and the distribution and volume of the sympathetic fibers were evidenced by glyoxylic acid staining and the expression of tyrosine hydroxylase (TH), the marked protein of sympathetic.Results The adventitia of 3 test groups were invisible or lost most of it while the control group remained intact shown by HE staining.For glyoxylic acid staining, the fluorescence intensity value of the control group, 4 weeks group, 8 weeks and 12 weeks were 0.08124 ± 0.00260, 0.02920 ± 0.00206, 0.02661 ± 0.00233, 0.03094 ± 0.00211, respectively (n =6).The distribution and fluorescence intensity of sympathetic nerve were both significantly reduced in test groups compared to the control group (P ＜ 0.05).And there was no statistical difference among the 3 test groups (P ＞ 0.05).Semi-quantitative analysis of the expression of TH was 0.8626 ± 0.03519, 0.3631 ± 0.03019, 0.3964 ± 0.02239, 0.3487 ± 0.02356 respectively, which showed the same tendency as glyoxylic acid staining test.Conclusion Microsurgical technique is promising as an ideal method for the local denervation of sympathetic nerve from artery system as it can significantly reduce sympathetic fibers on adventitia without regeneration during the experimental period.