Lianxu Chen

Effects of transforming growth factor-β1 and vascular endothelial growth factor 165 gene transfer on Achilles tendon healing

Repaired Achilles tendons typically take weeks before they are strong enough to handle physiological loads. Gene therapy is a promising treatment for Achilles tendon defects. The aim of the present study was to evaluate the histological/biomechanical effects of Transforming growth factor-beta1 (TGF-beta1) and vascular endothelial growth factor 165 (VEGF(165)) gene transfer on Achilles tendon healing in rabbits. Bone Marrow-Derived Mesenchymal Stem Cells (BMSCs) were transduced with adenovirus carrying human TGF-beta1 cDNA (Ad-TGF-beta1), human VEGF(165) cDNA (Ad-VEGF(165)), or both (PIRES-TGF-beta1/VEGF(165)) Viruses, no cDNA (Ad-GFP), and the BMSCs without gene transfer and the intact tendon were used as control. BMSCs were surgically implanted into the experimentally injured Achilles tendons. TGF-beta1 distribution, cellularity, nuclear aspect ratio, nuclear orientation angle, vascular number, collagen synthesis, and biomechanical features were measured at 1, 2, 4, and 8 weeks after surgery. The TGF-beta1 and TGF beta 1/VEGF(165) co-expression groups exhibited improved parameters compared with other groups, while the VEGF(165) expression group had a negative impact. In the co-expression group, the angiogenesis effects of VEGF(165) were diminished by TGF-beta1, while the collagen synthesis effects of TGF-beta1 were unaltered by VEGF(165). Thus treatment with TGF-beta1 cDNA-transduced BMSCs grafts is a promising therapy for acceleration and improvement of tendon healing, leading to quicker recovery and improved biomechanical properties of Achilles tendons.

The roles of TGF-β1 gene transfer on collagen formation during Achilles tendon healing

Collagen content and cross-linking are believed to be major determinants of tendon structural integrity and function. The current study aimed to investigate the effects of transforming growth factor (TGF)-beta1 on the collagen content and cross-linking of Achilles tendons, and on the histological and biomechanical changes occurring during Achilles tendon healing in rabbits. Bone marrow-derived mesenchymal stem cells (BMSCs) transfected with the TGF-beta1 gene were surgically implanted into experimentally injured Achilles tendons. Collagen proteins were identified by immunohistochemical staining and fiber bundle accumulation was revealed by Sirius red staining. Achilles tendons treated with TGF-beta1-transfected BMSCs showed higher concentrations of collagen I protein, more rapid matrix remodeling, and larger fiber bundles. Thus TGF-beta1 can promote mechanical strength in healing Achilles tendons by regulating collagen synthesis, cross-link formation, and matrix remodeling.

Rat adipose-derived stromal cells expressing BMP4 induce ectopic bone formation in vitro and in vivo.

AbstractAim:Bone morphogenetic protein 4 (BMP4) is one of the main local contributing factors in callus formation in the early phase of fracture healing. Adipose-derived stromal cells (ADSC) are multipotent cells. The present study was conducted to investigate the osteogenic potential of ADSC when exposed to adenovirus containing BMP4 cDNA (Ad-BMP4).Methods:ADSC were harvested from Sprague-Dawley rats. After exposure to Ad-BMP4, ADSC were assessed by alkaline phosphatase activity (ALP) assay, RT-PCR and von Kossa staining. BMP4 expression was assessed by RT-PCR, immunofluorescence and Western blot analysis. ADSC transduced with Ad-BMP4 were directly injected into the hind limb muscles of athymic mice. ADSC Ad-EGFP(enhanced green fluorescence protein) served as controls. All animals were examined by X-ray film and histological analysis.Results:The expression of BMP4 was confirmed at both mRNA and protein levels. The expression of the osteoblastic gene, ALP activity and von Kossa staining confirmed that ADSC transduced with Ad-BMP4 underwent rapid and marked osteoblast differentiation, whereas ADSC transduced with Ad-EGFP and cells left alone displayed no osteogenic differentiation. X-ray and histological examination confirmed new bone formation in athymic mice transplanted with ADSC transduced with Ad-BMP4.Conclusion:Our data demonstrated successful osteogenic differentiation of ADSC transduced with Ad-BMP4 in vitro and in vivo. ADSC maybe an ideal source of mesenchyme lineage stem cells for gene therapy and tissue engineering.

Local administration of TGFβ-1/VEGF165 gene-transduced bone mesenchymal stem cells for Achilles allograft replacement of the anterior cruciate ligament in rabbits.

Graft remodeling following anterior cruciate ligament (ACL) reconstruction requires a long period of recovery before it is capable of withstanding physiological loads. Graft revascularization is extremely important in the remodeling process. In ACL reconstruction, the local administration of vascular endothelial growth factor (VEGF) significantly increased revascularization of the graft, but did not significantly affect the mechanical properties of the graft after implantation (Ju et al., 2006; Yoshikawa, et al., 2006). Our previous studies showed that transforming growth factor-β1 (TGFβ1) could promote improvements in mechanical strength in Achilles tendon regeneration, by regulating collagen type I and type III synthesis, cross-link formation, and matrix-remodeling (Hou et al., 2009). The current study aims to investigate whether the co-expression of TGFβ1/VEGF(165) could beneficially affect the remodeling of ACL grafts. Bone marrow-derived mesenchymal stem cells (BMSCs), transfected with an adenoviral vector encoding TGFβ1, VEGF(165) or TGFβ1/VEGF(165), were surgically implanted into experimental ACL grafts, with non-transfected cells as a control. HE and toluidine blue staining, vascular number, and biomechanical features were analyzed at 3, 6, 12, and 24 weeks after surgery. The results suggest that TGFβ1 expression, in the TGFβ1/VEGF(165)-transfected BMSCs, could accelerate the remodeling of the reconstructed ligament. The cross-talk between TGFβ1 and VEGF(165) has positive consequences, as TGFβ1/VEGF(165)-transfected BMSCs significantly promoted angiogenesis of the reconstructed ligament at 3, 6, 12 weeks, with the best mechanical properties being achieved at 24 weeks. Furthermore, co-expression of these genes is more powerful and efficient than single gene therapy.

Assessment of the profiling microRNA expression of differentiated and dedifferentiated human adult articular chondrocytes.

MicroRNA has an important role in regulating gene expression during cell differentiation. In this study we identified the expression pattern of microRNA in the differentiated and dedifferentiated chondrocytes. Adult human articular chondrocytes were cultured in monolayer. RNA was isolated from the differentiated chondrocytes (collected after isolation) and the fifth‐passage (dedifferentiated) chondrocytes, and subjected to gene expression analysis using microRNA and cDNA microarray analysis. Real‐time RT‐PCR was also performed to confirm the differentially expressed genes. Furthermore, we integrated microRNA and cDNA microarray data together with computational approaches, such as microRNA gene target prediction algorithms, to reveal the role of microRNAs involved in chondrocyte homeostasis. The results showed a dramatic change in expression of microRNA between the two cell types. Thirteen up‐regulated and 12 down‐regulated microRNAs were detected in differentiated chondroctes. We also revealed microRNA–gene target pairs potentially involved in dedifferentiation process. Our results revealed novel findings of differential expression of microRNA in dedifferentiation, and microRNA could have an important role in the maintenance of chondrocytes homeostasis. MicroRNA may be a target for cartilage tissue engineering and regenerative medicine.

Intraoperative anthropometric measurements of tibial morphology: comparisons with the dimensions of current tibial implants

PurposesThis study analyzed morphological differences in the resected proximal tibial surfaces of Chinese males and females undergoing total knee arthroplasty (TKA) and compared the measurements with the dimensions of five currently used tibial implants.MethodsThe mediolateral (ML), middle anteroposterior (AP), medial anteroposterior (MAP), and lateral anteroposterior (LAP) dimensions of the resected tibial surfaces of 976 Chinese TKA knees (177 male, 799 female) were measured. The ML/AP ratio of every knee was calculated. These morphological data were compared with the dimensions of five currently used tibial implants.ResultsThe ML, AP, MAP, and LAP dimensions of the resected proximal tibias showed significant differences according to gender. Compared with currently used tibial implants, the smaller implants showed tibial ML undersizing and the larger implants showed tibial ML overhang. The ML/AP aspect ratio progressively decreased with increasing AP dimension in the resected proximal tibias, which contrasts with the relatively constant or increased (NexGen) aspect ratio in currently used tibial implants. Males showed a higher ML/AP aspect ratio than females for a given AP dimension. This indicates that for an implant with a given AP dimension, the tibial ML dimension tends to be undersized in males and to overhang in females.ConclusionThe results of this study may provide fundamental data for designing suitable tibial implants for use in the Chinese population, especially for design of gender-specific prostheses.Level of evidenceII.

Double-Bundle Anterior Cruciate Ligament Reconstruction Using Bone–Patellar Tendon–Bone Allograft Technique and 2- to 5-Year Follow-up

Background: Nonanatomic transtibial single-bundle anterior cruciate ligament reconstruction (SB-ACLR) with a bone–patellar tendon–bone (BPTB) allograft has been used for a long time and has shown the same satisfactory clinical results as an autograft; however, it has not been reported if a double-bundle ACLR (DB-ACLR) could be performed with a BPTB allograft and achieve even better results. Hypothesis: The DB-ACLR with a BPTB allograft is technically feasible and will be superior to the SB technique in restoring better anterior and rotating stability. Study Design: Cohort study; Level of evidence, 2. Methods: The study was performed with 56 patients, and 52 (25 in the DB group and 27 in the SB group) of them were followed up at 2 to 5 years. With an irradiated deep-frozen BPTB allograft, a standard single-incision arthroscopic technique was used, and the graft was fixed with bioabsorbable interference screws on both the femoral and tibial sides. Outcome assessment at final follow-up included International Knee Documentation Committee (IKDC), Tegner, and Lysholm scores; side-to-side difference by conventional KT-2000 arthrometer; total anteroposterior (AP) laxity by the back-pushing KT-2000 arthrometer; pivot shift (0, +, ++); range of motion (ROM); and isokinetic muscle strength evaluation. Results: Mean follow-up was 47.3 ± 11.5 and 58.2 ± 6.6 months for the DB group and SB group, respectively. A statistically significant difference in favor of the DB group was found with the total AP laxity at 30° (P < .05). The overall incidence of pivot shift in the DB group (4% ++) was significantly lower than that in the SB group (26%: 19% + and 7% ++; P = .029). No significant differences were found between the 2 groups in terms of IKDC score, Lysholm score, Tegner score, conventional KT-2000 arthrometer anterior laxity, ROM, and muscle strength. Conclusion: A DB-ACLR with a BPTB allograft is feasible and achieved more satisfactory results than the transtibial SB technique in terms of total AP stability and rotational stability in spite of no significant differences among other clinical parameters.

Computed tomographic measurement of gender differences in bowing of the sagittal femoral shaft in persons older than 50 years.

Surgeons do not give enough weight to the effects of bowing of the sagittal femoral shaft in total knee arthroplasty (TKA), which can result in damage to the cortex, fractures, or malalignment of the femoral component. To determine gender differences in bowing, we used spiral computed tomography to scan the femurs of 26 men and 47 women older than 50 years who required TKA. Skeletal extraction of the total sagittal femoral shaft from computed tomographic images was done by a matrix laboratory. The extracted curves were evenly divided into 3 sections. Comparison of the curvature on different sections of the same side of the femur showed that the distal third was significantly bowed. In addition, the curvature of the distal third was significantly larger in women than in men. Such morphological characteristics put forward new requirements in how intramedullary guide rods are used in TKA.