Novel nano-microspheres containing chitosan, hyaluronic acid, and chondroitin sulfate deliver growth and differentiation factor-5 plasmid for osteoarthritis gene therapy / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

OBJECTIVE@#To construct a novel non-viral vector loaded with growth and differentiation factor-5 (GDF-5) plasmid using chitosan, hyaluronic acid, and chondroitin sulfate for osteoarthritis (OA) gene therapy.@*METHODS@#Nano-microspheres (NMPs) were prepared by mixing chitosan, hyaluronic acid, and chondroitin sulfate. GDF-5 plasmid was encapsulated in the NMPs through electrostatic adsorption. The basic characteristics of the NMPs were observed, and then they were co-cultured with chondrocytes to observe their effects on extracellular matrix (ECM) protein expression. Finally, NMPs loaded with GDF-5 were injected into the articular cavities of rabbits to observe their therapeutic effects on OA in vivo.@*RESULTS@#NMPs exhibited good physicochemical properties and low cytotoxicity. Their average diameter was (0.61±0.20) μm, and encapsulation efficiency was (38.19±0.36)%. According to Cell Counting Kit-8 (CCK-8) assay, relative cell viability was 75%-99% when the total weight of NMPs was less than 560 μg. Transfection efficiency was (62.0±2.1)% in a liposome group, and (60.0±1.8)% in the NMP group. There was no significant difference between the two groups (P>0.05). Immunohistochemical staining results suggested that NMPs can successfully transfect chondrocytes and stimulate ECM protein expression in vitro. Compared with the control groups, the NMP group significantly promoted the expression of chondrocyte ECM in vivo (P<0.05), as shown by analysis of the biochemical composition of chondrocyte ECM. When NMPs were injected into OA model rabbits, the expression of ECM proteins in chondrocytes was significantly promoted and the progression of OA was slowed down.@*CONCLUSIONS@#Based on these data, we think that these NMPs with excellent physicochemical and biological properties could be promising non-viral vectors for OA gene therapy.

Icaritin promotes chondrogenic differentiation of BMSCs by Wnt/β-catenin signaling pathway / 中国中药杂志

To investigate the effect of icaritin (ICT) combined with GDF-5 on chondrogenic differentiation of bone marrow stromal cells (BMSCs), and discuss the action of Wnt signaling pathway, full bone marrow adherent method was used to isolate and culture SD rats BMSCs, and the cells at P3 generation were taken and divided into 6 groups： BMSCs group, ICT group, GDF-5 group, GDF-5+ICT group, GDF-5+ICT+SB216763 group, and GDF-5+ICT+ XAV-939 group. The cells were induced and cultured for 14 days. The morphology change was observed by inverted microscope. Alcian blue staining method was used to detect the changes of proteoglycans. RT-PCR was used to detect the mRNA expressions of aggrecan, Col2, Sox9, Dvl1, Gsk3β, and β-catenin. The protein expressions of collagen 2 (COL2) and β-catenin were detected by Western blot. The results indicated that, compared with the BMSCs group, gradual increase was present in proteoglycan Alcian blue staining; mRNA expressions of cartilage differentiation marker genes aggrecan, COL2, Sox9 and the protein expression of COL2, as well as mRNA and protein expressions of Wnt signaling pathway-related gene β-catenin, but with gradual decrease in Gsk3β mRNA expressions in GDF-5 group, GDF-5+ICT group and GDF-5+ICT+SB216763 group. On the contrary, compared with GDF-5+ICT group, there was a decrease in expressions of Dvl1, and β-catenin related to chondrogenic differentiation and Wnt signaling pathway, a increase in Gsk3β mRNA expression, and also a decrease in protein expressions of COL2 and β-catenin in GDF-5+ICT+XAV-939 group, with statistically significant difference between two groups. GDF-5 in combination with icaritin can induce chondrogenic differentiation of BMSCs in rats, and icaritin (ICT) can promote the chondrogenic differentiation. ICT can promote the chondrogenic differentiation of BMSCs in vitro probably by activating the Wnt/β-catenin signaling pathway.

MicroRNA-21 controls the development of osteoarthritis by targeting GDF-5 in chondrocytes

Osteoarthritis is a common cause of functional deterioration in older adults and is an immense burden on the aging population. Altered chondrogenesis is the most important pathophysiological process involved in the development of osteoarthritis. However, the molecular mechanism underlying the regulation of chondrogenesis in patients with osteoarthritis requires further elucidation, particularly with respect to the role of microRNAs. MiR-21 expression in cartilage specimens was examined in 10 patients with knee osteoarthritis and 10 traumatic amputees. The effect of miR-21 on chondrogenesis was also investigated in a chondrocyte cell line. The effect of miR-21 on the expression of growth differentiation factor 5 (GDF-5) was further assessed by luciferase reporter assay and western blot. We found that endogenous miR-21 is upregulated in osteoarthritis patients, and overexpression of miR-21 could attenuate the process of chondrogenesis. Furthermore, we identified GDF-5 as the direct target of miR-21 during the regulation of chondrogenesis. Our data suggest that miR-21 has an important role in the pathogenesis of osteoarthritis and is a potential therapeutic target.

A familial case with brachydactyly type C with a GDF5 mutation

Brachydactyly type C is a limb malformation characterized by shortening of the second, third, and fifth middle and/or proximal phalanges, but it has variable phenotypic expressivity. Mutations in the growth differentiation factor-5 (GDF5) gene cause isolated brachydactyly C. Herein, we report a familial case with isolated brachydactyly type C characterized by brachymesophalangy of both second and third digits, with a GDF5 missense mutation, and discuss the phenotypic variability of the condition. Identifying more cases with genetic confirmation will help elucidate the clinical and genetic characteristics of this condition in the Korean population.

Identification of a GDF5 Mutation in a Korean Patient with Brachydactyly Type C without Foot Involvement

Brachydactyly type C (BDC) is characterized by shortening of the middle phalanges of the index, middle, and little fingers. Hyperphalangy of the index and middle finger and shortening of the first metacarpal can also be observed. BDC is a rare genetic condition associated with the GDF5 gene, and this condition has not been confirmed by genetic analysis so far in the Korean population. Herein, we present a case of a 6-yr-old girl diagnosed with BDC confirmed by molecular genetic analysis. The patient presented with shortening of the second and third digits of both hands. Sequence analysis of the GDF5 gene was performed and the pathogenic mutation, c.1312C>T (p.Arg438Cys), was identified. Interestingly, this mutation was previously described in a patient who presented with the absence of the middle phalanges in the second through fifth toes. However, our patient showed no involvement of the feet. Considering intrafamilial and interfamilial variability, molecular analysis of isolated brachydactyly is warranted to elucidate the genetic origin and establish a diagnosis.

Effect of growth differentiation factor 5 on connexin 43 expression during chondrogenic differentiation of mouse bone marrow sternal cells in vitro / 中华创伤骨科杂志

Objective To investigate the effect of growth differentiation factor 5(GDF-5)on expression of gap junctional protein,connexin 43,during ehondrogenic differentiation of bone marrow sternal cells(BMSCs)in vitro.Methods BMSCs were isolated from mouse bone marrow and cultured in vitro.The cells in passage 3 were chosen to be induced into chondrogenic differentiation.After induction for 72 hours,TypeⅡcollagen protein was examined by immunocytochemistry and the sulfate glycosaminoglycan was measured by Alcian blue staining.With induction for 24,48 and 72 hours,the proliferation effects of BMSCs were investigated by MTT assay;connexin 43 mRNA and protein were examined by RT-PCR,western blotting and immunocytochemistry respectively at different time points during induction.Results According to MTT assay,GDF-5 had no effect on the proliferation of BMSCs at different time points of induction;RT-PCR,western blotting and immunocytochemistry showed that GDF-5 could promote expressions of connexin 43 mRNA and protein at different times during induction.After 72 hours of induction,immunocytochemistry showed expression of TypeⅡcollagen protein,and AIcian blue staining of proteo- glycan revealed deposition of typical cartilage extracellular matrix.Conclusion GDF-5 can enhance chondrogenic differentiation of mouse BMSCs in vitro by up-regulating the expression of gap junctional protein,connexin 43.

Effects of human growth/differentiation factor 5 gene transfer on the proliferation and differentiation of rabbit bone marrow mesenchymal stem cells in vitro / 西安交通大学学报(医学版)

Objective To investigate the effects of gene transfection with human growth/differentiation factor 5(hGDF5)on the differentiation and proliferation of rabbit bone marrow mesenchymal stem cells (BMSCs).Methods BMSCs were obtained from adult New zealand rabbits and purified by gradient centrifuge.Exogenous recombinant human GDF5 was transfected into BMSCs with liposome method.Then hGDF5 expression at mRNA and protein level was measured separately by reverse transcription-polymerase reaction and indirect immunofluorescence method.Activity of alkaline phosphates(ALP),expression of TypeⅡcollagen(ColⅡ),proteoglycan and growth of the cells were all measured by biological methods to evaluate the effects of hGDF5 gene transfer on the differentiation and proliferation of rabbit BMSCs.Results After hGDF5 gene transfection,BMSCs expressed hGDF5 mRNA and protein,and compared with the control groups,expression of proteoglycan and ColⅡ increased significantly,but no significant difference appeared in ALP activity and cell proliferation.Conclusion Gene transfer with hGDF5 is an effective way to enhance the expression of GDF5 at mRNA and protein.The expression of heterogenetic hGDF5 gene can induce BMSCs' differentiation to chondrogenic cells.But the gene transfection has no obvious effects on the proliferation and ALP activity of BMSCs.