scholarly journals FGF-2-Induced Human Amniotic Mesenchymal Stem Cells Seeded on a Human Acellular Amniotic Membrane Scaffold Accelerated Tendon-to-Bone Healing in a Rabbit Extra-Articular Model

2020 ◽  
Vol 2020 ◽  
pp. 1-14
Author(s):  
Jun Zhang ◽  
Ziming Liu ◽  
Yuwan Li ◽  
Qi You ◽  
Jibin Yang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Amniotic Membrane ◽  
Bone Healing ◽  
Chondrogenic Differentiation ◽  
Biomechanical Analysis ◽  
Specific Marker ◽  
Amniotic Mesenchymal Stem Cells

Background. FGF-2 (basic fibroblast growth factor) has a positive effect on the proliferation and differentiation of many kinds of MSCs. Therefore, it represents an ideal molecule to facilitate tendon-to-bone healing. Nonetheless, no studies have investigated the application of FGF-2-induced human amniotic mesenchymal stem cells (hAMSCs) to accelerate tendon-to-bone healing in vivo. Objective. The purpose of this study was to explore the effect of FGF-2 on chondrogenic differentiation of hAMSCs in vitro and the effect of FGF-2-induced hAMSCs combined with a human acellular amniotic membrane (HAAM) scaffold on tendon-to-bone healing in vivo. Methods. In vitro, hAMSCs were transfected with a lentivirus carrying the FGF-2 gene, and the potential for chondrogenic differentiation of hAMSCs induced by the FGF-2 gene was assessed using immunofluorescence and toluidine blue (TB) staining. HAAM scaffold was prepared, and hematoxylin and eosin (HE) staining and scanning electron microscopy (SEM) were used to observe the microstructure of the HAAM scaffold. hAMSCs transfected with and without FGF-2 were seeded on the HAAM scaffold at a density of 3×105 cells/well. Immunofluorescence staining of vimentin and phalloidin staining were used to confirm cell adherence and growth on the HAAM scaffold. In vivo, the rabbit extra-articular tendon-to-bone healing model was created using the right hind limb of 40 New Zealand White rabbits. Grafts mimicking tendon-to-bone interface (TBI) injury were created and subjected to treatment with the HAAM scaffold loaded with FGF-2-induced hAMSCs, HAAM scaffold loaded with hAMSCs only, HAAM scaffold, and no special treatment. Macroscopic observation, imageological analysis, histological assessment, and biomechanical analysis were conducted to evaluate tendon-to-bone healing after 3 months. Results. In vitro, cartilage-specific marker staining was positive for the FGF-2 overexpression group. The HAAM scaffold displayed a netted structure and mass extracellular matrix structure. hAMSCs or hAMSCs transfected with FGF-2 survived on the HAAM scaffold and grew well. In vivo, the group treated with HAAM scaffold loaded with FGF-2-induced hAMSCs had the narrowest bone tunnel after three months as compared with other groups. In addition, macroscopic and histological scores were higher for this group than for the other groups, along with the best mechanical strength. Conclusion. hAMSCs transfected with FGF-2 combined with the HAAM scaffold could accelerate tendon-to-bone healing in a rabbit extra-articular model.

The negatively charged microenvironment of collagen hydrogels regulates the chondrogenic differentiation of bone marrow mesenchymal stem cells in vitro and in vivo

2020 ◽  
Vol 8 (21) ◽  
pp. 4680-4693
Author(s):  
Jirong Yang ◽  
Yumei Xiao ◽  
Zizhao Tang ◽  
Zhaocong Luo ◽  
Dongxiao Li ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Protein Adsorption ◽  
Cell Morphology ◽  
Chondrogenic Differentiation ◽  
Collagen Hydrogels ◽  
Marrow Mesenchymal Stem Cells

The different negatively charged microenvironments of collagen hydrogels affect the protein adsorption, cell morphology, and chondrogenic differentiation of BMSCs in vitro and in vivo.

scholarly journals Different Phenotypes and Chondrogenic Responses of Human Menstrual Blood and Bone Marrow Mesenchymal Stem Cells to activin A and TGF-β3

2020 ◽  
Author(s):  
Ilona Uzieliene ◽  
Edvardas Bagdonas ◽  
Kazuto Hoshi ◽  
Tomoaki Sakamoto ◽  
Atsuhiko Hikita ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Chondrogenic Differentiation ◽  
Collagen Type ◽  
Activin A ◽  
Cell Surface Markers ◽  
Type Ii ◽  
Marrow Mesenchymal Stem Cells

Abstract Background: Due to its low capacity for self-repair, articular cartilage is highly susceptible to damage and deterioration, which leads to the development of degenerative joint diseases such as osteoarthritis. Menstrual blood-derived mesenchymal stem cells (MenSCs) are much less characterized compared to bone marrow mesenchymal stem cells (BMMSCs). However, MenSCs seem an attractive alternative to classical BMMSCs due to ease of access and broader differentiation capacity. The aim of this study was to evaluate chondrogenic differentiation potential of MenSCs and BMMSCs stimulated with transforming growth factor β (TGF-β3) and activin A, member of the TGF-β superfamily of proteins.Methods: MenSCs (n=6) and BMMSCs (n=5) were isolated from different healthy donors. Expression of cell surface markers CD90, CD73, CD105, CD44, CD45, CD14, CD36, CD55, CD54, CD63, CD106, CD34, CD10, Notch1 was analysed by flow cytometry. Cell proliferation capacity was determined using CCK-8 proliferation kit. Adipogenic differentiation capacity was evaluated according to Oil-Red staining, osteogenic differentiation - Alizarin Red staining. Chondrogenic differentiation (Activin A and TGF-β3 stimulation) was induced in vitro and in vivo (subcutaneous scaffolds in nude BALB/c mice) and investigated by histologically and by expression of chondrogenic genes (collagen type II, aggrecan). Activin A protein production was evaluated by ELISA.Results: MenSCs exhibited a higher proliferation rate, as compared to BMMSCs, and a different expression profile of several cell surface markers. Activin A stimulated collagen type II gene expression and glycosaminoglycan synthesis in TGF-β3 treated MenSCs but not in BMMSCs, both in vitro and in vivo, although the effects of TGF-β3 alone were more pronounced in BMMSCs in vitro. Conclusion: These data suggest that activin A exerts differential effects on the induction of chondrogenic differentiation in MenSCs vs. BMMSCs, which implies that different mechanisms of chondrogenic regulation are activated in these cells. Following further optimisation of differentiation protocols and the choice of growth factors, potentially including activin A, MenSCs may turn out to be a promising population of stem cells for the development of cell-based therapies with the capacity to stimulate cartilage repair and regeneration.Trial registration: Not applicable.

scholarly journals Sodium hyaluronate supplemented culture medium combined with joint-simulating mechanical loading improves chondrogenic differentiation of human mesenchymal stem cells

2021 ◽  
Vol 41 ◽  
pp. 616-632
Author(s):  
G Monaco ◽  
◽  
AJ El Haj ◽  
M Alini ◽  
MJ Stoddart
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Mechanical Loading ◽  
Chondrogenic Differentiation ◽  
Culture Medium ◽  
In Vitro Models ◽  
Matrix Deposition ◽  
Sulphated Glycosaminoglycan

In vitro models aim to recapitulate the in vivo situation. To more closely mimic the knee joint environment, current in vitro models need improvements to reflect the complexity of the native tissue. High molecular weight hyaluronan (hMwt HA) is one of the most abundant bioactive macromolecules in healthy synovial fluid, while shear and dynamic compression are two joint-relevant mechanical forces. The present study aimed at investigating the concomitant effect of joint-simulating mechanical loading (JSML) and hMwt HA-supplemented culture medium on the chondrogenic differentiation of primary human bone-marrow-derived mesenchymal stem cells (hBM-MSCs). hBM-MSC chondrogenesis was investigated over 28 d at the gene expression level and total DNA, sulphated glycosaminoglycan, TGF-β1 production and safranin O staining were evaluated. The concomitant effect of hMwt HA culture medium and JSML significantly increased cartilage-like matrix deposition and sulphated glycosaminoglycan synthesis, especially during early chondrogenesis. A stabilisation of the hBM-MSC-derived chondrocyte phenotype was observed through the reduced upregulation of the hypertrophic marker collagen X and an increase in the chondrogenic collagen type II/X ratio. A combination of JSML and hMwt HA medium better reflects the complexity of the in vivo synovial joint environment. Thus, JSML and hMwt HA medium will be two important features for joint-related culture models to more accurately predict the in vivo outcome, therefore reducing the need for animal studies. Reducing in vitro artefacts would enable a more reliable prescreening of potential cartilage repair therapies.

scholarly journals Amniotic Mesenchymal Stem Cells Can Enhance Angiogenic Capacity via MMPsIn VitroandIn Vivo

2015 ◽  
Vol 2015 ◽  
pp. 1-15 ◽  
Author(s):  
Fei Jiang ◽  
Jie Ma ◽  
Yi Liang ◽  
Yuming Niu ◽  
Ning Chen ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Umbilical Vein ◽  
3D Culture ◽  
Positive Staining ◽  
Vascular Networks ◽  
Amniotic Mesenchymal Stem Cells ◽  
3D Culture Model

The aim of this study was to evaluate the angiogenic capacity and proteolytic mechanism of coculture using human amniotic mesenchymal stem cells (hAMSCs) with human umbilical vein endothelial cells (HUVECs)in vivoandin vitroby comparing to those of coculture using bone marrow mesenchymal stem cells with HUVEC. For thein vivoexperiment, cells (HUVEC-monoculture, HUVEC-hAMSC coculture, and HUVEC-BMMSC coculture) were seeded in fibrin gels and injected subcutaneously in nude mice. The samples were collected on days 7 and 14 and histologically analyzed by H&E and CD31 staining. CD31-positive staining percentage and vessel-like structure (VLS) density were evaluated as quantitative parameters for angiogenesis. The increases of CD31-positive staining area and VLS density in both HUVEC-hAMSC group and HUVEC-BMMSC group were found between two time points, while obvious decline of those was observed in HUVEC-only group. For thein vitroexperiment, we utilized the same 3D culture model to investigate the proteolytic mechanism related to capillary formation. Intensive vascular networks formed by HUVECs were associated with hAMSCs or BMMSCs and related to MMP2 and MMP9. In conclusion, hAMSCs shared similar capacity and proteolytic mechanism with BMMSCs on neovascularization.

The dependence of in vivo stable ectopic chondrogenesis by human mesenchymal stem cells on chondrogenic differentiation in vitro

Biomaterials ◽  
2008 ◽  
Vol 29 (14) ◽  
pp. 2183-2192 ◽  
Author(s):  
Kai Liu ◽  
Guang Dong Zhou ◽  
Wei Liu ◽  
Wen Jie Zhang ◽  
Lei Cui ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Chondrogenic Differentiation ◽  
Human Mesenchymal Stem Cells

scholarly journals Exosomes derived from M0, M1 and M2 macrophages exert distinct influences on the proliferation and differentiation of mesenchymal stem cells

PeerJ ◽  
2020 ◽  
Vol 8 ◽  
pp. e8970 ◽  
Author(s):  
Yu Xia ◽  
Xiao-Tao He ◽  
Xin-Yue Xu ◽  
Bei-Min Tian ◽  
Ying An ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Chondrogenic Differentiation ◽  
Adipogenic Differentiation ◽  
Underlying Mechanism ◽  
Suppressive Effect ◽  
Complete Medium ◽  
Marrow Mesenchymal Stem Cells

Background Different phenotypes of macrophages (M0, M1 and M2 Mφs) have been demonstrated to play distinct roles in regulating mesenchymal stem cells in various in vitro and in vivo systems. Our previous study also found that cell-conditioned medium (CM) derived from M1 Mφs supported the proliferation and adipogenic differentiation of bone marrow mesenchymal stem cells (BMMSCs), whereas CM derived from either M0 or M2 Mφs showed an enhanced effect on cell osteogenic differentiation. However, the underlying mechanism remains incompletely elucidated. Exosomes, as key components of Mφ-derived CM, have received increasing attention. Therefore, it is possible that exosomes may modulate the effect of Mφ-derived CM on the property of BMMSCs. This hypothesis was tested in the present study. Methods In this study, RAW264.7 cells were induced toward M1 or M2 polarization with different cytokines, and exosomes were isolated from the unpolarized (M0) and polarized (M1 and M2) Mφs. Mouse BMMSCs were then cultured with normal complete medium or inductive medium supplemented with M0-Exos, M1-Exos or M2-Exos. Finally, the proliferation ability and the osteogenic, adipogenic and chondrogenic differentiation capacity of the BMMSCs were measured and analyzed. Results We found that only the medium containing M1-Exos, rather than M0-Exos or M2-Exos, supported cell proliferation and osteogenic and adipogenic differentiation. This was inconsistent with CM-based incubation. In addition, all three types of exosomes had a suppressive effect on chondrogenic differentiation. Conclusion Although our data demonstrated that exosomes and CM derived from the same phenotype of Mφs didn’t exert exactly the same cellular influences on the cocultured stem cells, it still confirmed the hypothesis that exosomes are key regulators during the modulation effect of Mφ-derived CM on BMMSC property.

EFFECTS OF SHORT-TERM CYCLIC HYDROSTATIC PRESSURE ON INITIATING AND ENHANCING THE EXPRESSION OF CHONDROGENIC GENES IN HUMAN ADIPOSE-DERIVED MESENCHYMAL STEM CELLS

2014 ◽  
Vol 14 (04) ◽  
pp. 1450054 ◽  
Author(s):  
FARZANEH SAFSHEKAN ◽  
MOHAMMAD TAFAZZOLI SHADPOUR ◽  
MOHAMMAD ALI SHOKRGOZAR ◽  
NOOSHIN HAGHIGHIPOUR ◽  
SEYED HAMED ALAVI
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Growth Factor ◽  
Hydrostatic Pressure ◽  
Chondrogenic Differentiation ◽  
Negative Control ◽  
Cartilage Tissue ◽  
Control Group

Cartilage tissue engineering is a promising treatment for damaged or diseased cartilage that requires thorough understanding of influential parameters involved in chondrogenic differentiation. This study examined how 4-h application of cyclic hydrostatic pressure (CHP) of 5 MPa at 0.5 Hz could modulate chondroinduction of human adipose-derived mesenchymal stem cells (hAMSCs) in vitro. Four groups were examined including a negative control group, a chemical group treated by growth factor for 10 days, a mechanical group exposed to 4-h loading on the 10th day of pellet culture without any chondrogenic stimulator, and finally a chemical-mechanical group subjected to both growth factor and loading. Application of cyclic hydrostatic pressure increased the expression of chondrogenic genes, including sox9 and aggrecan to higher levels than those of the chemical group. This study indicates that cyclic hydrostatic pressure initiates and enhances the chondrogenic differentiation of mesenchymal stem cells with or without growth factors in vitro and confirms the important role of hydrostatic pressure during chondrogenesis in vivo.

scholarly journals Sox9-Increased miR-322-5p Facilitates BMP2-Induced Chondrogenic Differentiation by Targeting Smad7 in Mesenchymal Stem Cells

2021 ◽  
Vol 2021 ◽  
pp. 1-11
Author(s):  
Yongsheng Zeng ◽  
Chengcheng Du ◽  
Pengcheng Xiao ◽  
Yiting Lei ◽  
Piao Zhao ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Chondrogenic Differentiation ◽  
In Vitro Study ◽  
Underlying Mechanism ◽  
Bone Morphogenetic Protein 2 ◽  
Luciferase Reporter ◽  
Smad7 Expression

Bone morphogenetic protein 2 (BMP2) induces effective chondrogenesis of mesenchymal stem cells (MSCs) by promoting Sox9 expression. However, BMP2 also induces chondrocyte hypertrophy and endochondral ossification by upregulating Smad7 expression, which leads to the disruption of chondrogenesis. In addition, Smad7 can be inhibited by Sox9. Therefore, the underlying mechanism is not clear. Currently, an increasing number of studies have shown that microRNAs play a pivotal role in chondrogenic and pathophysiological processes of cartilage. The purpose of this study was to determine which microRNA is increased by Sox9 and targets Smad7, thus assisting BMP2 in maintaining stable chondrogenesis. We found that miR-322-5p meets the requirement through next-generation sequencing (NGS) and bioinformatic analysis. The targeting relationship between miR-322-5p and Smad7 was confirmed by dual-luciferase reporter assays, qPCR, and western blotting (WB). The in vitro study indicated that overexpression of miR-322-5p significantly inhibited Smad7 expression, thus causing increased chondrogenic differentiation and decreased hypertrophic differentiation, while silencing of miR-322-5p led to the opposite results. Flow cytometry (FCM) analysis indicated that overexpression of miR-322-5p significantly decreased the rate of early apoptosis in BMP2-stimulated MSCs, while silencing of miR-322-5p increased the rate. A mouse limb explant assay revealed that the expression of miR-322-5p was negatively correlated with the length of the BMP2-stimulated hypertrophic zone of the growth plate. An in vivo study also confirmed that miR-322-5p assisted BMP2 in chondrogenic differentiation. Taken together, our results suggested that Sox9-increased miR-322-5p expression can promote BMP2-induced chondrogenesis by targeting Smad7, which can be exploited for effective tissue engineering of cartilage.

295 EQUINE AMNION-DERIVED MESENCHYMAL STEM CELLS: POSSIBLE IMPLICATION IN ENDOMETRIAL REGENERATION

2013 ◽  
Vol 25 (1) ◽  
pp. 295
Author(s):  
B. Corradetti ◽  
R. Perego ◽  
A. Meucci ◽  
D. Bizzaro ◽  
F. Cremonesi ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Stem Cell ◽  
Amniotic Membrane ◽  
Female Reproductive Tract ◽  
Endometrial Tissue ◽  
Expression Of Genes ◽  
Endometrial Regeneration

In veterinary medicine, as in human, amnion is an attractive source of mesenchymal stem cells (MSC), which poses no ethical dilemmas, allows highly efficient recovery of cells without the requirement of invasive procedures and shows immunomodulatory properties. We previously demonstrated that equine amniotic membrane-derived cells (AMC) not only exhibit specific stem cell properties with respect to expression of pluripotent (OCT-4, TRA-1-60, and SSEA-4) and adult (CD44, CD105, CD29, and CD166) stem cell markers but also possess differentiation potential in vitro and the capability to regenerate tendons in vivo after spontaneous lesions when allogeneically transplanted. Moreover, we reported evidence that at the first passages (P) in culture (until P5), AMC express MHC-class I but not MHC-class II and are well tolerated in vivo. In the present study, we further characterised AMC in vitro in order to evaluate their potential application in the treatment of endometritis in vivo. In particular, the amniotic membrane in toto and AMC have been compared to the endometrial tissue in toto and to cells isolated from endometrium for the expression of genes involved in the proliferation and differentiation of uterine MSC during early pregnancy (AbdB-like Hoxa genes) and those influencing preimplantation conceptus development (progesterone receptor, PR, and oestrogen receptors ERα and ERβ). The AMC were isolated as recently reported by Lange-Consiglio et al. (2011 Open J. Tissue Eng. Regen. Med. 4), and endometrial cells were obtained according to the protocol described by Donofrio et al. (2008 Reprod. Biol. Endocrinol. 6, 65) for bovine cells, and slightly modified for equine cells. Total RNA was extracted from both tissues and from AMC and endometrium-derived cells immediately after isolation (P0). Reverse transcription-PCR was performed according to the standard procedures, using GAPDH and HPRT1 as reference genes. Expression of HOXA9 and PR was confirmed in all samples examined, whereas mRNA for ERβ was only detected in endometrial tissue and in cells derived from it. Expression of ERα was observed only in endometrial tissue. The expression of genes crucially involved in patterning of the female reproductive tract (HOXA9 and PR) in amnion and cells derived from it suggests that this source shares similar molecular properties with endometrium. Further studies are required to explore uterine mesenchymal-like features shared by AMC (i.e. verifying the expression of Wnt7α, Wnt5α, and Wnt4α, or the presence of the more recently characterised membrane-bound intracellular progesterone receptors PGRMC1 and mPR). These preliminary results provide an intriguing indication of the possible implication of amnion-derived cells in endometrial regeneration, in particular when poor endometrial proliferation is associated with infertility or poor pregnancy outcome.

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