Genetic correction of adipose tissue-derived mesenchymal stem cells mediated by TALEN targeting the GDF5 gene.

Stem cells and other associated cell types may be a potential alternative to treat various genetic disorders that currently do not benefit from traditional approaches. Functional recovery of cells could be induced via directional differentiation or genetic manipulation. In this study, mesenchymal stem cells (MSCs) were obtained from a patient with osteoarthritis (OA) carrying a functional single­nucleotide polymorphism (SNP rs143383, C/T transition) within the 5'­UTR of growth and differentiation factor 5 (GDF5) gene. The SNP causes GDF5 expression to be reduced and thus increases OA susceptibility. Aiming to correct the dysfunctional gene, a pair of transcription activator­like effector nucleases (TALENs) were designed to cleave the DNA around the mutated locus, coupled with a short single stranded DNA complementary to the cleavage site. Following in vitro cell colony formation and selection, two genetically corrected MSC colonies were identified out of a total of 142. These MSCs were induced and differentiated into chondrocytes. As a result, genetically corrected chondrocytes exhibited normal morphology and lower levels of apoptosis compared with cells carrying the SNP. In cultured cells, the secretion of matrix metalloproteinases was suppressed and TIMP metallopeptidase inhibitor 1 was increased by correction of the mutation. Furthermore, the expression of GDF5 target genes, cell vitality­associated genes and extracellular matrix degrading genes were returned to normal levels in corrected cells compared with mutation­carrying cells, indicating the functional recovery of these corrected chondrocytes. The present study demonstrated that TALEN­mediated genetic correction can be used to edit genes in adipose­derived MSCs from patients with OA and may have clinical potential.

Hsa_circ_0045714 regulates chondrocyte proliferation, apoptosis and extracellular matrix synthesis by promoting the expression of miR-193b target gene IGF1R.

In recent years, some studies have been made on the effects of circular RNA (circRNA) in osteoarthritis (OA) and so on; however, its mechanisms remain to be further explored. Studies have shown that tumor necrosis factor-alpha can inhibit hsa_circ_0045714 expression in chondrocytes so as to upregulate miR-193b expression. Dual-luciferase reporter assay showed that insulin-like growth factor 1 receptor (IGF1R) is a key target gene of miR-193b. Hsa_circ_0045714 over-expression does not influence miR-193b expression, but can inhibit its transcriptional activity, thereby upregulating IGF1R expression. Hsa_circ_0045714 can promote the expression of type II collagen and aggrecan, and upregulate chondrocyte proliferation, while its linear sequences cannot. IGF1R has similar function, while miR-193b can inhibit the expression of type II collagen and aggrecan, and downregulate chondrocyte proliferation but enhance their apoptosis. IGF1R overexpression can reverse the effect of miR-193b, while miR-193b mimics or IGF1R siRNA can inhibit the function of hsa_circ_0045714. Therefore, hsa_circ_0045714 can regulate extracellular matrix synthesis as well as proliferation and apoptosis of chondrocytes by promoting the expression of miR-193b target gene IGF1R. The findings will provide new proofs for studies on the applications of circRNA in OA and other orthopedic diseases.

[Application of 3D digital orthopedic techniques in treatment of acetabular fracture].

OBJECTIVE: To evaluate the therapeutic effect of three-dimensional digital orthopedic techniques in treatment of acetabular fractures. METHODS: We retrospectively analyzed 50 cases of acetabular fracture treated between March, 2007 and December, 2013. The lamellar CT scanning data were imported into Mimics software, and 3D anatomical models of the pelvic and proximal femur were reconstructed. Computer-assisted analysis was carried out to understand the condition of fractures and simulate fracture reduction. The pelvic models were manufactured by rapid prototyping technique for definite diagnosis and typing of acetabular fractures and subsequent surgical treatment. RESULTS: Three-dimensional reconstruction images and rapid prototyping pelvic models faithfully represented the findings in operations. Preoperative simulation of the operation shortened the time of operation and reduced the volume of bleeding in the operation. All the patients were followed up for 6 to 24 months. According to Matta imaging score, anatomical reduction was achieved in 41 cases and satisfactory reduction in 9 cases. According to the Harris functional criteria, 32 patients had excellent, 12 had good and 6 had acceptable outcomes with a rate of excellent and good outcomes of 88%. CONCLUSION: Three-dimensional digital orthopedic techniques allow accurate display of the acetabulum and the spatial relation of the anatomic structures to assist in fracture diagnosis, typing and treatment.

[ECTOPIC OSTEOGENESIS EVALUATION OF RECOMBINANT HUMAN BONE MORPHOGENETIC PROTEIN 2 LOADED CHITOSAN/DEXTRAN SULFATE BY MICRO-CT].

UNLABELLED: OBJECTIVE To evaluate the ectopic osteogenesis of recombinant human bone morphogenetic protein 2 (rhBMP-2) loaded chitosan (CS)/dextran sulfate (DS) by micro-CT. METHODS: rhBMP-2/CS/DS microspheres were prepared by the ionic crosslinking and its shape was observed under the scanning electron microscope. The release of rhBMP-2 was determined from resultant microspheres by ELISA assay. Forty-eight Sprague Dawley male rats were randomly divided into 4 groups (n = 12), quadriceps muscle bag model was made, gelatin sponge (group A), CS/DS microspheres (group B), rhBMP-2 (group C), and CS/DS/rhBMP-2 microspheres (group D) were implanted into the bags respectively. The tissue samples with heterotopic ossification were harvested for micro-CT scanning at 4, 8, 12, and 16 weeks. The tissue mineral density (TMD), bone volume fraction (BVF), trabecular thickness (Tb.Th), trabecular number (Tb.N), bone mineral density (BMD), and tissue mineral content (TMC) were measured. RESULTS: The prepared rhBMP-2/CS/DS microspheres with smooth surfaces were spherical and evenly disperses without obvious agglomeration. At 2 hours, microsphere started a sudden release period in vitro; the release reached a peak at 2 days; and the release cycle lasted about 20 days. The rats survived to the end of the experiment. At each time point after operation, no radiation developed and no osteogenesis was observed by three dimensional reconstruction in groups A and B. However, radioactive strength and reconstructed bone tissue gradually increased in groups C and D, and group D had more radioautography and more bone tissues than group C. At each time point, TMD, BVF, Tb.Th, Tb.N, BMD, and TMC of groups A and B were zero. Ectopic bone formed with time, the other parameters showed an increasing trend except Tb.N in groups C and D, showing significant difference when compared with groups A and B at each time point (P < 0.05). There was no significant difference between groups C and D at 4 weeks (P > 0.05); the parameters of group D were significantly higher than those of group C at 8-16 weeks (P < 0.05). CONCLUSION: rhBMP-2/CS/DS microspheres have stronger ability of ectopic bone formation than single rhBMP-2.

[DESIGN AND CLINICAL APPLICATION OF LESSER TROCHANTERIC REDUCTION FIXATION SYSTEM].

OBJECTIVE: To design and produce a lesser trochanteric reduction fixation system and verify its value and effectiveness. METHODS: A lesser trochanteric reduction fixation system was designed and produced according to the anatomical features of the lesser trochanteric fractures. Sixty-six patients with intertrochanteric fractures of Evans type III were included between January 2010 and July 2012. Of 66 patients, 32 were treated with dynamic hip screw (DHS) assisted with the lesser trochanteric reduction fixation system (study group), and 34 cases were treated with DHS only (control group). The 2 groups were comparable with no significant difference in gender, age, the reasons, and the types of the fractures (P > 0.05). The operation time, intraoperative blood loss, neck-shaft angle, bone healing time, ratio of successful fixations, and the functional evaluation of the hip joint after operation were compared between 2 groups. RESULTS: The study group had shorter operation time [(58.4 ± 5.3) minutes] and less intraoperative blood loss [(186.3 ± 6.6) mL than the control group [(78.5 ± 6.2)minutes and (246.2 ± 8.7) mL], showing significant differences (t = -14.040, P = 0.000; t = -31.145, P = 0.000). There was no significant difference in neck-shaft angle between study group [(138.6 ± 3.0)] and control group [(139.4 ± 2.9) degrees] (t = -1.044, P = 0.301). The wounds healed by first intention in both groups. The 30 and 31 patients were followed up 12 to 24 months (mean, 15 months) in the study group, and 13 to 25 months (mean, 16 months) in the control group, respectively. All fractures healed well in 2 groups. The study group had significantly shorter healing time [(8.8 ± 2.0) weeks] than the control group [(10.7 ± 3.4) weeks] (t = -2.871, P = 0.006). At 12 months after operation, coxa vara happened in 2 cases of the study group with a successful fixation ratio of 93.3% and in 10 cases of the control group with a successful fixation ratio of 67.7%, showing significant difference (Χ2 = 6.319, P = 0.022). According to Harris hip score, the excellent and good rate was 83.3% in the study group (25/30) and was 58.1% in the control group (18/31), showing significant difference (Χ2 = 4.680, P = 0.049). CONCLUSION: The application of the lesser trochanteric reduction fixation system can reduce stripping of the soft tissue around the fracture fragments, shorten the operation time and the healing time, and preserve the function of the hip joint maximumly.

[Computer aid designed operation of complicated calcaneal fracture by 3D simulation].

OBJECTIVE: To precisely treat complicated calcaneal fracture by 3D simulation through computer aid designed operation. METHODS: From November 2007 to March 2008, 38 patients of calcaneal fracture were treated. There were 29 males and 9 females aged 14-69 years old (average 29.8 years old). According to Sanders classification, there were 4 patients of type I, 14 of type II, 12 of type III, and 8 of type IV. The time between injury and surgery was 3 hours to 5 days. The CT images of calcaneal fracture of 38 patients were put into computer for 3D reconstruction, then the Böhler angles were measured and bone grafting angles were designed. According to the angle surveyed by the computer, the individual-oriented operation program was made, and then the operation was done under C-arm X-ray machine. RESULTS: The preoperative Böhler angel was (34.58 +/- 4.38) degrees in the normal side and (8.33 +/- 12.62) degrees in the injured side, indicating there was significant difference (P < 0.05). During the process of the poking reduction by 3D simulation, when the bone rotating angle was (28.84 +/- 6.51) degrees, the Böhler angel was restored to (32.86 +/- 1.72) degrees, indicating there was no significant difference when compared with the normal side before operation (P > 0.05), and significant difference compared with the injured side before operation (P < 0.05). Twenty-eight patients were followed up for 12-22 months (average 18 months). The Böhler angel was restored to (32.41 +/- 1.42) degrees 1 year after operation. According to the foot function scoring system made by American Ankle Surgery Association, 16 cases were graded as excellent, 10 as good, 1 as fair, 1 as poor, and the excellent and good rate was 92.9%. CONCLUSION: Computer aid designed operation of complicated calcaneal fracture by 3D simulation technique can restore the Böhler angel and subtalar joint precisely. It is an effective supplementary treatment method for calcaneal fracture.