Enhancing Tendon Regeneration: Investigating the Impact of Topography on the Secretome of Adipose-Derived Stem Cells

Abstract

Tendons are vital for maintaining integrity and movement, but current treatment options are insufficient for their regeneration after injuries. Previous studies have shown that the secretome from mesenchymal stem cells (MSCs) promoted tendon regeneration. However, limited studies have explored the impact of the physical microenvironment on the secretome's efficacy of MSCs. In this study, it is shown that the topographic orientation regulates the secretome of human adipose-derived stem cells (ADSCs) and promotes tendon regeneration. Conditioned medium (CM) is collected from ADSCs cultured on the scaffolds with different topography. The results show that CM generated from aligned structure group has a potent effect in promoting cell migration and proliferation, tenogenic differentiation, macrophage polarization toward M2 phenotype, tendon structure and mechanical function recovery. Proteomic analysis revealed that the aligned structure can up-regulate the secretion of Extracellular matrix (ECM) proteins while down-regulate proinflammatory factors. This modulation activates the MAPK, GPCR and Integrin signaling pathways which may account for the enhanced effect on tendon regeneration. This study offers a promising and safer non-cell-based treatment option for tendon repair.

Keywords: ADSCs; paracrine; proteomics; tendon regeneration; topology.

Figure 1

Preparation of aligned and random silk fibroin scaffolds and collection of ADSCs‐derived CM. CM, conditioned medium; RCM, ADSCs derived CM from random oriented silk fibroin scaffolds; ACM, ADSCs derived CM from aligned oriented silk fibroin scaffolds.

Figure 2

Characterization of ADSCs and scaffolds with different topographies. A) Cell morphology of ADSCs. B–D) Multi‐lineage differentiation properties of ADSCs were evaluated. ADSCs were cultured in osteogenic differentiation media for 1 week and evaluated by ALP staining (B). ADSCs were cultured in adipogenic differentiation media for 2 weeks and evaluated by staining lipid droplets with oil red O (C). ADSCs were cultured in chondrogenic differentiation media for 2 weeks and evaluated by Alician Blue staining (D). E–L) The topographic orientation of silk fibrin scaffolds and ADSCs cultured on them were visualized under SEM. E–H) Random scaffold. I–L) Aligned scaffold. M) Overlapping pore orientation color map showing degree of anisotropy in random and aligned scaffolds. N–P) Representative histogram curve in random and aligned scaffolds of the orientation shown in the color map images where the plugin from Fiji, Directionality, was used to calculate the number of fibers (Fiji count) per direction (degree).

Figure 3

Effects of RCM and ACM on TSPCs migration and cell proliferation. A) Cell migration of TSPCs treated with RCM or ACM using scratch assay at 0, 12, and 24 h. TSPCs were pre‐stained using Dil. Scale bars = 500 µm. B) The relative migration rate was quantified and compared, n = 4 technically independent samples for each group, *p < 0.05, ***p < 0.001. C,D) Live/dead staining of TSPCs after 1 day and 3 days of culture in RCM and ACM (live cells in green and dead cells in red). Scale bar = 200 µm. The cell viability was calculated by ImageJ, n = 4 randomly‐selected microscopic images per group. E) Proliferation of TSPCs in RCM and ACM for 1, 3, 5, and 7 days measured by CCK‐8, n = 5 technically independent samples for each group, ***p < 0.001 ****p < 0.0001. F) Gene expression of PCNA in RCM and ACM at day 1. The level at the RCM group was set as 1. Data were shown as Mean ± SEM, n = 3 technically independent samples for each group, *p < 0.05. G) The expression of Ki67 was evaluated by immunofluorescence staining. Scale bar = 100 µm. H) Ki67+ cells/total cells were quantified and compared, n = 3 randomly‐selected microscopic images per group, ***p < 0.001.

Figure 4

Effect of RCM and ACM on tendinous differentiation. A–C) TSPCs were cultured in RCM and ACM for 3 days. Gene expression of tendon markers was compared on days 1, 2, and 3. Levels of the RCM group were set as 1. Data are shown as Mean ± SEM, n = 3 technically independent samples for each group. D,F) The expression of SCX and TNMD were evaluated by immunofluorescence staining. Scale bar = 100 µm. E,G) Relative intensity of SCX and TNMD were quantified, n = 3 randomly‐selected microscopic images per group, *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 5

The effects of RCM and ACM on the polarization of macrophages. A–C) The expression of M2‐related anti‐inflammatory genes in RAW264.7 cultured in RCM and ACM for 2 days. Levels at the RCM group were set as 1, n = 3 technically independent samples for each group. D–G) The expression of ARG‐1 and CD206 were evaluated by immunofluorescence staining after 2 days, and the fluorescence positive area were quantified by ImageJ. Scale bar = 100 µm. n = 3 randomly‐selected microscopic images per group. H–J) The expression of M1‐related inflammatory genes. Levels at the RCM group were set as 1, n = 3 technically independent samples for each group. K,L) The expression of iNOS were evaluated by immunofluorescence staining after 2 days, and fluorescence positive area were quantified by ImageJ. Scale bar = 100 µm. n = 3 randomly‐selected microscopic images per group, *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 6

Histological examination and immunohistochemical staining of macrophage polarization markers in early stage of repaired tendons. A) Schematic diagram of in vivo experimental operation. The tendon defect is performed bilaterally. The figure was generated with BioRender (https://biorender.com/). B) H&E staining of patellar tendons from RCM and ACM groups at one week postoperatively. Red arrows indicate immune cells. Scale bar = 200 µm, 50 µm. C) The proportion rates of immune cells, fibroblast‐like cells, and other cells were quantified and compared. n = 9 randomly‐selected microscopic images per group, *p < 0.05, **p < 0.01. D) Immunohistochemical staining of macrophage polarization markers in repaired tendons. ARG‐1 and CD206 (M2, anti‐inflammatory), iNOS (M1, pro‐inflammatory). Scale bar = 100 µm. E) Quantifications of immunohistochemical stainings. n = 6 randomly‐selected microscopic images per group, **p < 0.01, ****p < 0.0001.

Figure 7

Histological examination and immunohistochemical staining of different markers in repaired tendons. H&E (A) and Masson's trichrome (B) staining of patellar tendons from RCM and ACM groups at 4 weeks postoperatively. Scale bar = 200 µm, 50 µm. C) Histological scoring of regenerated patellar tendon at 4 weeks postoperation. n = 5 randomly‐selected microscopic images per group, ***p < 0.001. D) Immunohistochemical staining of different markers in repaired tendons. Tendon‐related markers: SCX, TNMD, COL I; Macrophage polarization markers: ARG‐1 (M2, anti‐inflammatory), iNOS (M1, pro‐inflammatory). Scale bar = 100 µm. E) Quantifications of immunohistochemical stainings. n = 6 randomly‐selected microscopic images per group, **p < 0.01, ***p < 0.001, ****p < 0.0001. F) Picrosirius red‐staining of patellar tendons from RCM and ACM groups with polarized light. Scale bar = 200 µm, 50 µm. The proportion rates of collagen I (red or light orange area), collagen III fibers (green area), and dark area were quantified. n = 6 randomly‐selected microscopic images per group. G) TEM images of the collagen ultrastructure of RCM and ACM group. Scale bar = 250 nm. The collagen fibrils’ diameters were quantified. n = 100 randomly‐selected collagen fibrils per group, ****p < 0.0001.

Figure 8

Gait analysis and biomechanical evaluation for the rats after being administered with RCM and ACM. A) Schematic diagram of gait analysis and representative gait screen shot, gait pattern, gait intensity, and 3D footprint intensity. The schematic diagram of gait analysis was generated with BioRender (https://biorender.com/). B) Quantification of hind stand in 2 weeks and 4 weeks. C) Quantification of hind print area in 2 weeks and 4 weeks. D) Quantification of hind stand/swing time ratio in 2 weeks and 4 weeks. E) Quantification of hind stand/step cycle time in 2 weeks and 4 weeks. F) Quantification of hind mean intensity in 2 weeks and 4 weeks. The data were analyzed by paired t‐test. RF right front, LF left front, RH right hind, LH left hind. n = 16 randomly‐selected results from 8 biological samples, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 G) Comparison of max force between groups. n = 8 biologically independent samples, *p < 0.05. H) Comparison of mechanical tensile modulus between groups. n = 8 biologically independent samples, *p < 0.05.

Figure 9

Proteomic analysis of ACM and RCM. A) Schematic diagram of the proteomic profiling of ACM and RCM (n = 3 independent experimental units (EUs)). The figure was generated with BioRender (https://biorender.com/). B) PCA plot of proteomic data in ACM and RCM groups. C) Differentially‐expression analysis of secretome between ACM and RCM. Significantly up‐ or down‐regulated proteins are in red or blue. D) Heatmap profiling the hierarchical cluster analysis of secretome of ACM and RCM. E) Heatmaps of the all up‐regulated and the top 23 down‐regulated proteins in ACM. F,G) Gene ontology (GO) analysis of the differentially‐expressed proteins. H) Gene set enrichment analysis (GSEA) of differentially‐expressed proteins.

Figure 10

ACM improves cell proliferation and tendon differentiation of TSPCs by activating MAPK and Integrin pathways. A) Western blot of p‐MEK and MEK in TSPCs after treatment of RCM, RCM+U0126, ACM, or ACM+U0126 for 18 h. B) Western blot of p‐ERK, ERK in TSPCs after treatment of RCM, RCM+U0126, ACM, or ACM+U0126 treatment for 18 h. C) Gene expression of PCNA in different groups at day 3. D) Proliferation of TSPCs measured by CCK‐8 at 1, 3, and 5 days, n = 5 technically independent samples for each group, **p < 0.01 ****p < 0.0001. E) Gene expression of SCX, TNMD, and MKX in different groups at day 3. Levels at the RCM group were set as 1. Data were shown as Mean ± SD, n = 3 technically independent samples for each group, *p < 0.05, **p < 0.01. F) Gene expression of PCNA in different groups at day 3, ***p < 0.001. G) Proliferation of TSPCs measured by CCK‐8 at 1, 3, and 5 days, n = 5 technically independent samples for each group, ****p < 0.0001. H) Gene expression of SCX, TNMD, and MKX in different groups at day 3. Levels at the RCM group were set as 1. Data were shown as Mean ± SD, n = 3 technically independent samples for each group, *p < 0.05.

Figure 11

Schematic illustration of topology structure in promoting tendon regeneration by potentiating the paracrine effects of ADSC secretome. The figure was generated with BioRender (https://biorender.com/).