Effects of Coculture Fibroblasts and Vascular Endothelial Cells on Proliferation and Osteogenesis of Adipose Stem Cells

Background. The development of tissue engineering provides a new method for the clinical treatment of bone defects, but the problems of slow formation and slow vascularization of tissue engineered bone have always existed. Studies have shown that the combined culture system of vascular endothelial cells and adipose stem cells is superior to single cell in repairing bone defects. With the excellent proliferation ability, secretion of synthetic collagen and a variety of regulatory factors and fibroblasts can differentiate into osteoblasts and have the potential to be excellent seed cells involved in tissue engineering bone construction. Objective. To investigate the effects of combined culture of fibroblasts, vascular endothelial cells, and adipose stem cells on proliferation and osteogenic differentiation of adipose stem cells. Methods. The cells were divided into 4 groups: adipose stem cell group, adipose stem cell+vascular endothelial cell coculture group, adipose stem cell+fibroblast coculture group, and adipose stem cell+vascular endothelial cell+fibroblast coculture group. The morphological changes of the cells were observed under an inverted microscope. After 1, 3, 5, 7, and 9 days of coculture, the proliferation of adipose stem cells in each group was detected by a CCK-8 method and the growth curve was plotted. Adipose stem cells in each group were stained with alizarin red and alkaline phosphatase at days 7, 14, 21, and 28. At the third week of coculture, Western blot was used to detect the expression level of bone morphogenetic protein 2 of adipose stem cells in each group. Results and Conclusions. (1) After 14 days of culture, some cells in the adipose stem cell+vascular endothelial cell+fibroblast coculture group fused into clumps and distributed in nests, while the adipose stem cells in the adipose stem cell group had a single cell morphology and no cell clusters were observed. (2) The cell growth curves were basically the same in each group, and the absorbance value increased gradually. The absorbance value of the adipocyte+vascular endothelial cell+fibroblast coculture group was the highest, followed by the adipocyte+fibroblast coculture group and then the adipocyte+fibroblast coculture group. (3) Alizarin red staining showed negative reaction in each group on the 7th day, and a small number of red positive cells gradually appeared in each group as time went on. On the 28th day, red positive cells were found in all groups, and most of them were in the coculture group of adipose stem cells+vascular endothelial cells+fibroblasts, showing red focal. The coculture group of adipose stem cells+vascular endothelial cells and adipose stem cells+fibroblasts was less, and the adipose stem cell group was the least. On day 28 of alkaline phosphatase staining, cells in each group had red positive particles, and the adipose stem cell+vascular endothelial cell+fibroblast coculture group and adipose stem cell+fibroblast coculture group had the most, followed by the adipose stem cell+vascular endothelial cell coculture group and then the adipose stem cell group. (4) Bone morphogenetic protein 2 was expressed in all groups, especially in adipose stem cell+fibroblast coculture group and adipose stem cell+vascular endothelial cell+ fibroblast coculture group. (5) Fibroblast could promote adipose stem cell osteogenic differentiation better than vascular endothelial cells, but the proliferation effect was not as good as vascular endothelial cells. The coculture system of fibroblast combined with vascular endothelial cells and adipose stem cells promoted the proliferation of adipose stem cells and the rapid and efficient differentiation of adipose stem cells into osteoblasts.

Adipose Stem Cell-Derived Exosomes As a Macrophage-Based Treatment for Chronic Rotator Cuff Tendinopathy: From a Mouse Model to a Study in Human Tissue

Objectives: Chronic rotator cuff tendinopathy is one of the most prevalent causes of shoulder pain. Growing evidence suggests that macrophages play a significant role in the pathogenesis of tendinopathy. Therefore, a treatment that targets macrophages would be useful for patients with this common musculoskeletal disorder. To investigate whether adipose stem cell-derived exosomes (ASC-Exos) mediate polarization of macrophages and contribute to the healing of chronic rotator cuff tendinopathy. Methods: First, we compared the effects of ASC-Exos on polarization of mouse bone marrow-derived macrophages between a classically activated phenotype (M1φ) and an alternatively activated phenotype (M2φ) in vitro. Seventy-two C57Bl/6 mice were assigned to normal cage activity (n=24) or 5 weeks of treadmill overuse (n=48). The enthesis of supraspinatus in each mouse was then injected with ASC-Exos (ASC-Exos group, n=24) or saline (saline group, n=24). Histological and biomechanical outcomes were assessed 4 weeks after treatment. Finally, tissue samples from human patients with rotator cuff tendinopathy were obtained to determine the effect of ASC-Exos on the M1φ/M2φ balance in tissue-resident macrophages. Results: ASC-Exos inhibited M1φ polarization and augmented M2φ polarization both in mouse bone marrow-derived macrophages, mouse in vivo model and human tissue-resident macrophages. Mice in the ASC-Exos group showed less severe pathological changes than the saline group, including less cellular infiltration, disorganization of collagen, and ground substance deposition. The mean modified Bonar score was significantly lower in the ASC-Exos group than in the saline group (9.81 ± 0.96 vs 7.69 ± 1.03; P<.05). Furthermore, the maximum failure load was significantly higher in the ASC-Exos group than in the saline group (4.23 ± 0.66 N vs 3.86 ± 0.65 N; P<.05) as was stiffness (3.38 ± 0.34 N/m vs. 2.68 ± 0.49 N/m; P<.05). Conclusions: ASC-Exos-mediated balance of M1φ/M2φ contributes to healing of tendinopathy. Regulation of the M1φ/M2φ balance could be a new target for treatment of chronic rotator cuff tendinopathy.

Adipose Stem Cell–Derived Exosomes Ameliorate Chronic Rotator Cuff Tendinopathy by Regulating Macrophage Polarization: From a Mouse Model to a Study in Human Tissue

Background: Chronic rotator cuff (RC) tendinopathy is one of the most prevalent causes of shoulder pain. Growing evidence suggests that macrophages play a significant role in the proinflammatory response, resolution of inflammation, and tissue healing of tendinopathy. In particular, enhancement of M2 macrophage (M2φ) activity contributes to the accelerated healing of tendinopathy. Therefore, a treatment that enhances M2φ polarization would be useful for patients with this common musculoskeletal disorder. Purpose: To investigate whether adipose stem cell–derived exosomes (ASC-Exos) enhance M2φ polarization and ameliorate chronic RC tendinopathy. Study Design: Controlled laboratory study. Methods: First, we compared the effects of ASC-Exos on polarization of mouse bone marrow–derived macrophages between a classically activated phenotype (M1φ) and an alternatively activated phenotype (M2φ) in vitro. In total, 72 C57BL/6 mice were assigned to normal cage activity (n = 24) or 5 weeks of treadmill overuse (n = 48). The supraspinatus tendon of each treadmill overuse mouse was treated with ASC-Exos (n = 24) or saline (n = 24). Histological and biomechanical outcomes were assessed 4 weeks after treatment. Finally, tissue samples from human patients with RC tendinopathy were obtained to assay the effect of ASC-Exos on the M1φ/M2φ balance in tissue-resident macrophages. Results: ASC-Exos inhibited M1φ polarization and augmented M2φ polarization in vitro and in vivo. Mice in the ASC-Exos group showed less severe pathological changes than those in the saline group, including less cellular infiltration, disorganization of collagen, and ground substance deposition. The modified Bonar score of the ASC-Exos group (mean ± SD, 7.68 ± 1.03) was significantly lower than that of the saline group (9.81 ± 0.96; P < .05). Furthermore, the maximum failure load was significantly higher in the ASC-Exos group than in the saline group (4.23 ± 0.66 N vs 3.86 ± 0.65 N; P < .05), as was stiffness (3.38 ± 0.34 N/m vs 2.68 ± 0.49 N/m; P < .05). Conclusion: ASC-Exos–mediated polarization balance of M1φ/M2φ contributes to the amelioration of chronic RC tendinopathy. Regulation of the M1φ/M2φ balance could be a new target for the treatment of chronic RC tendinopathy. Clinical Relevance: Administration of ASC-Exos is a cell-free approach that may become a novel treatment option for chronic RC tendinopathy and should be explored further.