Cell migration and molecular mechanisms during healing of damaged vascular or muscle tissues are emerging fields of interest worldwide. The study herein focuses on evaluating the role of allogenic adipose-derived mesenchymal stem cells (ADMSCs) in restoring damaged tissues. Using a hindlimb ischemic mouse model, ADMSC-mediated induction of cell migration and gene expression related to myocyte regeneration and angiogenesis were evaluated. ADMSCs were labeled with GFP (ADMSC-GFP). The proximal end of the femoral blood vessel of mice (over 6 months of age) are ligated at two positions then cut between the two ties. Hindlimb ischemic mice were randomly divided into two groups: Group I (n = 30) which was injected with PBS (100 μL) and Group II (n = 30) which was transplanted with ADMSC-GFP (106 cells/100 μL PBS) at the rectus femoris muscle. The migration of ADMSC-GFP in hindlimb was analyzed by UV-Vis system. The expression of genes related to angiogenesis and muscle tissue repair was quantified by real-time RT-PCR. The results showed that ADMSCs existed in the grafted hindlimb for 7 days. Grafted cells migrated to other damaged areas such as thigh and heel. In both groups the ischemic hindlimb showed an increased expression of several angiogenic genes, including Flt-1, Flk-1, and Ang-2. In particular, the expression of Ang-2 and myogenic-related gene MyoD was significantly increased in the ADMSC-treated group compared to the PBS-treated (control) group; the expression increased at day 28 compared to day 3. The other factors, such as VE-Cadherin, HGF, CD31, Myf5, and TGF-β, were also more highly expressed in the ADMSC-treated group than in the control group. Thus, grafted ADMSCs were able to migrate to other areas in the injured hindlimb, persist for approximately 7 days, and have a significantly positive impact on stimulating expression of myogenic- and angiogenesis-related genes.

Ischemia are common conditions related to lack of blood supply to tissues. Depending on the ischemic sites, ischemia can cause different diseases, such as hindlimb ischemia, heart infarction and stroke. This study aims to evaluate how extracellular vesicles (EVs) derived from ETV2 transfected fibroblasts affect endothelial cell proliferation and neovascularization in a murine model of hindlimb ischemia. Human fibroblasts were isolated and cultured under standard conditions and expanded to the 3th passage before use in experiments. Human fibroblasts were transduced with a viral vector containing the ETV2 gene. Transduced cells were selected by puromycin treatment. These cells were further cultured for collection of EVs, which were isolated from culture supernatant. Following co-culture with endothelial cells, EVs were evaluated for their effect on endothelial cell proliferation and were directly injected into ischemic tissues of a murine model of hindlimb ischemia. The results showed that EVs could induce endothelial cell proliferation in vitro and improved neovascularization in a murine model of hindlimb ischemia. Our results suggest that EVs derived from ETV2-transfected fibroblasts can be promising non-cellular products for the regeneration of blood vessels.

In vitro production of tissues or tissue engineering is a promising approach to produce artificial tissues for regenerative medicine. There are at least three important components of tissue engineering, including stem cells, scaffolds and growth factors. This study aimed to produce cartilage tissues in vitro from culture and chondrogenic differentiation of rabbit bone marrow-derived mesenchymal stem cells (BMMSCs), induced by chondrogenesis medium, on biodegradable polycaprolactone (PCL) scaffolds. BMMSCs were isolated from rabbit bone marrow according to the standard protocol. The adherence, proliferation and differentiation of BMMSCs on scaffolds were investigated using two scaffold systems: PCL scaffolds and collagen-coated PCL (PCL/col) scaffolds. The results showed that BMMSCs could attach and grow on both PCL and PCL/col scaffolds. However, the adhesion efficacy of BMMSCs on the PCL/col scaffolds was significantly better than on PCL scaffolds. Under induced conditions, BMMSCs on PLC/col scaffolds showed increased aggrecan accumulation and upregulated expression of chondrogenesis-associated genes (e.g. collagen type II, collagen type I, aggrecan and collagen type X) after 3, 7, 21 and 28 days of induction. These in vitro cartilage tissues could form mature chondrocyte-like cells after they were grafted into rabbits. The results suggest that use of BMMSCs in combination with polycaprolactone scaffolds and chondrogenesis medium can be a way to form in vitro cartilage tissue.

Ischemia is the reduction of blood flow to tissues by injury of blood vessels. Depending on the sites of tissues and grade of ischemia, ischemia can cause many serious complications. This study aimed to evaluate the effects of the E-twenty six (ETS) factor Ets variant 2 (ETV2) gene expression in angiogenesis and the effect of ETV2 gene therapy in a mouse model of hindlimb ischemia. The role of ETV2 on endothelial cell proliferation was evaluated in vitro. Knockdown of ETV2 expression was done using short hairpin RNA (shRNA) lentiviral viral particles. The ETV2 viral vector was injected into the skeletal muscles at the ligated and burned sites of the hindlimb and evaluated for its efficacy as a gene therapy modality for ischemia. Vascular regeneration in mice was indirectly evaluated by changes in mouse survival, necrotic grades of the leg, normal blood oxygen saturation level (SpO(2)), and blood flow by trypan blue injection assay. Preliminary data showed that ETV2 expression played a role in angiogenesis of endothelial cells. ETV2 overexpression could trigger and stimulate proliferation of skeletal endothelial cells. In vivo knockdown of ETV2 expression inhibited the auto-recovery of ischemic hindlimb, while overexpression of ETV2 helped to rescue leg loss and reduce necrosis, significantly improving angiogenesis in hindlimb ischemia. Our findings demonstrate that ETV2 gene therapy is a potentially effective modality for vascular regeneration.

Introduction: Pathological animal models provide the foundation for developing new methods for disease treatment. This research aims to establish a rabbit model of femoral head necrosis. Methods: Osteonecrosis of the femoral head (ONFH) was induced in rabbits by using methylprednisolone (MPS) combined with Complete Freund's Adjuvant (CFA). New Zealand White rabbits were divided into two groups. ONFH group (n=10) was given an intramuscular injection of 0.5 mg/kg CFA and 40 mg/kg methylprednisolone. Normal group (n=6) received normal saline at the same location and same volume as those in ONFH group. The efficiency of the ONFH rabbit model was assessed at week 7 after the last injection. Body weight was detected, and the histological structure of head femoral and bone were assessed by H&E staining. The empty lacunae were counted. Cartilage degeneration was evaluated using image analysis software. Blood vessel density was assessed after ink artery infusion. The cell cycle of bone marrow-derived mononuclear cells was analyzed by flow cytometry. Results: The results showed that there was no difference in body weight changes of rabbits between the two groups. However, the bone morphology and cartilage surface of the femoral head showed abnormalities in the ONFH group. The percentage of empty osteocyte lacunae was significantly higher in ONFH group than normal group. Chondrocyte degeneration and fibrocartilage expression were observed in the ONFH group. Compared to the normal group, the ONFH group had less ink-stained blood vessels. However, the fraction of bone marrow-derived mononuclear cells in S phase and G2/M phase of the cell cycle was significantly increased in the ONFH group. Conclusion: Thus, CFA combined with MPS for 7 weeks can be used to establish an early-stage femoral head necrosis model in rabbits.

Introduction: Bone marrow derived MSCs (BM-MSCs) and adipose derived MSCs (AD-MSCs) are among the types of stem cells most commonly studied. Our study aims to compare the therapeutic efficacy of allograft AD-MSCs versus BM-MSCs in a mouse model of hindlimb ischemia. Methods: AD-MSCs were isolated from belly fat and BM-MSCs were isolated from femur bone marrow. They were used to treat mice with acute hindlimb ischemia. Treatment efficacy was compared among 4 groups: injected with BM-MSCs, injected with AD-MSCs, non-treated and injected with phosphate buffered saline. Mice in the groups were evaluated for the following: necrosis grade of leg, leg edema, blood flow, muscle cell restructure and new blood vessel formation. Results: Results showed that AD-MSC transplantation significantly recovered acute limb ischemia, with 76.5% of mice fully recovered, while the ratio was only 48.5% in BM-MSC transplanted group, and 0% in the non-treated and PBS groups. Evaluation of leg edema, blood flow, muscle cell restructure and new blood vessel formation also supported the observation that AD-MSC transplantation was superior over BM-MSC transplantation. Conclusion: Therefore, AD-MSCs may serve as the more suitable MSC for hindlimb ischemia treatment and angiogenesis therapy.

Introduction: Both ETV-2 and HGF factors are known to be important factors which trigger neo-angiogenesis both in vitro and in vivo models. This study aimed to treat hindlimb ischemia in mice by co-injection of ETV-2 and HGF viral vectors. Methods: ETV-2 and HGF vectors were prepared per previous publications. The mouse ischemic hindlimb model was performed by ligating and burning the artery at the hindlimb. ETV-2 and HGF viral vectors were co-injected into the ligated and burnt sites. Results: The results showed that co-injection of the vectors significantly improved angiogenesis as well as reduced leg loss in mice compared to placebo treatment. The percentage of mice who succumbed to ischemia was also significantly reduced compared to control. Conclusion: Overall, this study suggests a potential impact of combining ETV-2 and HGF to treat angiogenesis. Use of ETV-2 and HGF viral vectors can be a promising therapy for ischemia treatment in the future.

Nowaday, the regenerative medicine based on the use of biomaterial scaffold combine to stem cells was considered as a necessary treatment strategy for injury or diseases involving cartilage. In this study, we investigated the adherence, proliferation and chondrogenic differentiation of rabbit bone marrow-derived mesenchymal stem cells (rBM-MSCs) on biodegradable polycaprolactone scaffold (PCL). MSCs were isolated from bone marrow of rabbit. rBM-MSC (2x105cell/scaffold) were seeded on PCL and collagen-coated PCL (PCL/col) scaffolds. The cell adhesion and proliferation abilities on the PCL and PCL/col scaffolds were compared to each other to find the better using MTT assay. Then 3D cultured-cells were induced in chondrogenic differentiation medium. The change of scaffold surface structure was evaluated by scanning electron microscope (SEM). The accumulation of aggrecan protein was detected by Alcian Blue staining. The chondrogenic-related gene expression such as sox9, collagen type 1, collagen type 2, collagen type 10, aggrecan, runx2 was assessed by RT-PCR. The existence of artificial cartilage after transplantation of chondrocyte-seeded scaffold under rabbit skin was evaluated by staining histological sections with Alcian Blue. The results showed that rBM-MSCs attached and grown on PCL/col and PCL scaffolds. However, the adhesion efficient of rBM-MSC on PCL/col scaffold was better than PCL scaffolds. Therefore, PCL/col scaffold was used for next experiments. The morphology of rBM-MSCs on PLC/col scaffold were changed and the aggrecan accumulation increased after cultured in chondrogenic differentiation medium. The scaffold surface modification was detected with the presentation of collagen-like fiber structures. The expression of chondrogenesis-associated genes increased after 3, 7, 21, 28 days of chondrogenic induction, especially for collagen type II, collagen type I, aggrecan and collagen type X. Transplanted artificial cartilage was existed in rabbit for six weeks. The mature chondrocyte-like cells were detected and cartilage specific-proteoglycan accumulated more in differentiated cell-scaffold than scaffold seeded undifferentiated-cells. Thus artificial cartilage could be created when differentiating rBM-MSC into chondrocyte on collagen-coated PCL scaffold.

Background: Pathological animal models provide the foundation for developing new methods for treating. This research aims to establish a rabbit model of femoral head necrosis. Osteonecrosis of the femoral head (ONFH) was induced in rabbits by using methylpresnisolone combined with Complete Freund's Adjuvant (CFA). New Zealand White rabbits were divided into two groups. Group A (n=10) was given an intramuscular injection of 0.5 mg/kg (CFA) and 40mg/kg methylprednisolone (MPS). Group B (n=6) was received normal saline at the same location and same volume as those in Group A. The efficiency of ONFH rabbit model was assessed at 7 weeks after the last injection. Body weight was weighed. The histological structure of head femoral and bone were deteded by H&E staining. The empty lacuna was counted. Cartilage degeneration was evaluated using image analysis software. Blood vessel density was assessed after ink artery infusion. The cell cycle of bone marrow-derived mononuclear cells was analyzed by flow cytometry. The results showed that there was no difference in body weight change of rabbits between two groups. However, the bone morphology and cartilage surface of femoral head were abnormalities at group A. The percentage of empty osteocyte lacunae were significantly higher in Group A than Group B. Chondrocyte degeneration and fibrocartilage expression were observed at Group A. Compare to group B, Group A had less ink-stained blood vessels. Moreover, the fraction of bone marrow-derived mononuclear at S phase and G2/M phase of the cell cycle was significantly decreased in group A. Thus, CFA combined with MPS can be used to establishing an early-stage femoral head necrosis model of rabbit.

Endothelial progenitor cells (EPCs) play an important role in angiogenesis. However, they exist in limited numbers in the human body. This study was aimed to produce EPCs, for autologous transplantation, using direct reprogramming of skin fibroblasts under GMP-compliant conditions. Fibroblasts were collected and cultured from the skin in DMEM/F12 medium supplemented with 5% activated platelet-rich plasma and 1% antibiotic-antimycotic solution. They were then transfected with mRNA ETV2 and incubated in culture medium under hypoxia (5% oxygen) for 14 d. Phenotype analysis of transfected cells confirmed that single-factor ETV2 transfection successfully reprogrammed dermal fibroblasts into functional EPCs. Our results showed that ETV2 mRNA combined with hypoxia can give rise to functional EPCs. The cells exhibited functional phenotypes similar to endothelial cells derived from umbilical cord vein; they expressed CD31 and VEGFR2, and formed capillary-like structures in vitro. Moreover, these EPCs could significantly improve hindlimb ischemia in mouse models. Although the direct conversion efficacy was low (3.12 ± 0.98%), altogether our study demonstrates that functional EPCs can be produced from fibroblasts and can be used in clinical applications.

The title of the article “A mouse model of osteonecrotic femoral head induced by methylprednisolone and liposaccharide” published in 25 March 2016, in 2016 at Vol 3, Issue 3, from pages: 548-556 (DOI: 10.7603/s40730-016-0012-x) should be read as: “A mouse model of osteonecrotic femoral head induced by methylprednisolone and lipopolysaccharide”.

Introductions: Joint replacements have considerably improved the quality of life of patients with damaged joints. Over the past 30 years, there has been much effort and investigations in ways to repair damages in joints, including knee and hip joints. Materials for joint production have also been developed. Many improvements have been made in the joint replacement materials to increase their biocompatibility and longevity. This study is aimed at evaluating the in vitro and in vivo biocompatibility of Ti-6Al-4V titanium alloy and UHMWPE polymer used in total hip replacements. Methods: Ti-6Al-4V titanium alloy and UHMWPE polymer were carefully washed with sterile distilled water then autoclaved. The materials were used directly or indirectly to evaluate pyrogens, endotoxins, animal cell cytotoxicity, gene mutation, animal cell transformation, DNA synthesis, immunogenicity, histology reactions, and immune response. All assays were performed according to ISO10993 guidelines. Results: The results showed that Ti-6Al-4V titanium alloy and Chirulen 1020 UHMWPE polymer satisfied all criteria for implantable materials.

Mesenchymal stem cells (MSCs) transplantation for the treatment of acute hindlimb ischemia is recently attracting the attention of many scientists. Identifying the role of donor cells in the host is a crucial factor for improving the efficiency of treatment. This study evaluated the injury repair role of xenogeneic adipose-derived stem cell (ADSC) transplantation in acute hindlimb ischemia mouse model. Human ADSCs were transplanted into the limb of ischemic mouse. The survival rate of grafted cells and expression of human VEGF-R2 and CD31 positive cells were assessed in the mouse. In addition, the morphological and functional recovery of ischemic hindlimb was also assessed. The results showed that one-day post cell transplantation, the survival percentage of grafted cells was 3.62% ± 2.06% at the injection site and 15.71% ± 12.29% around the injection site. The rate of VEGFR2-positive cells had highest expression at 4 days post transplantation, 5.46% ± 2.13% at the injection site; 9.12% ± 7.17% at the opposite of injection site, and 7.22% ± 4.59% at the lateral gastrocnemius. The percentage of CD31 positive cells increased on day 4 at the injection site to 0.8% ± 1.60%, and further increased on day 8 at the lateral gastrocnemius site and the opposite injection site to 1.56% ± 0.44% and 1.17% ± 1.69%, respectively. After 14 days, the cell presentation and the angiogenesis marker expression were decreased to zero, except for CD31 expression at the opposite of injection site (0.72% ± 1.03%). Histological structure of the cell-injected muscle tissue remained stable as that of the normal muscle. New small blood vessels were found growing in hindlimb. On the other hand, approximately 66.67% of mice were fully recovered from ischemic hindlimb at grade 0 and I after cell injection. Thus, xenotransplantation of human ADSCs might play a significant role in the formation of new blood vessel and can assist in the treatment of mouse with acute hindlimb ischemia.

The adipose tissue contains a large numbers of stem cells; adipose-derived stem cells (ADSCs) can be em- ployed in regenerative medicine. This study was aimed at isolating ADSCs and evaluating the safety of ADSCs in mouse models. Stromal vascular fraction (SVF) was collected from the adipose tissue using collagenase. ADSCs were then isolated from SVFs by in vitro culture. The stemness of the ADSCs was evaluated in vitro based on their self-renewal potential, po- tential to differentiate into osteoblasts, and adipocytes, and the expression of specific markers. Finally, the tumor forma- tion ability of ADSCs was evaluated in vivo in athymic mice. Results showed that 100% of the ADSC samples developed well and maintained homogeneity up to passage 10. The ADSCs were completely sterilized and could not form tumors in athymic mice. These initial results showed that ADSCs were safe for use in stem cell therapy.

Acute lower limb ischemia is a common peripheral artery disease whose treatment presents many difficulties. Stem cell transplantation is considered a novel and promising method of treating this disease. Umbilical cord blood (UCB) is rich in stem cells, including hematopoietic stem cells (HSCs), mesenchymal stem cells (MSCs) and endothelial progenitor cells (EPCs). However, historically, banked umbilical cord blood has been used mainly to treat blood-related diseases. Therefore, this study compared the efficacy of umbilical cord bloodderived mononuclear cells (UCB-MNCs) with EPC transplantation for the treatment of acute hindlimb ischemia (ALI) in mouse models. MNCs were isolated from UCB by Ficoll gradient centrifugation, after which the EPCs were sorted based on CD34+ and CD133+ markers and cultured according to a previously published protocol. To induce ALI, mice were immuno-suppressed using busulfan (BU) and cyclophosphamide (CY), after which the femoral arteries were burned. Induction of ALI in the immune suppressed mice was confirmed by the grade of tissue damage, pedal frequency in water, tissue edema, changes in histology, total white blood cell count, and white blood cell composition. Model mice were injected with a dose of MNCs or EPCs and un-treated control mice were injected with phosphate buffered saline. The efficiency of treatment was evaluated by comparing the grade of tissue damage between the three groups of mice. Mice aged 6–12 months were suitable for ALI, with 100% of mice exhibiting ischemia from grade I 10%, grade III 50%, grade IV 40%. For all ALI mice, a gradual increase in pedal frequency in water, increased tissue edema, necrosis of muscle tissue, and loss of hindlimb function were observed after 20 days. Transplanted MNCs and EPCs significantly improved hindlimb ischemia compared with control treatment. Moreover, EPC transplantation significantly improved hindlimb ischemia compared with MNC transplantation. Following EPC and MNC transplantation, 44.44% and 33.33% of the mice recovered fully (grade 0), respectively. Specifically, all recovered mice exhibited hindlimb activities similar to those of normal mice. Transplantation of UCB-derived MNCs and EPCs are promising therapies for hindlimb ischemia.