Delineation of in vitro chondrogenesis of human synovial stem cells following preconditioning using decellularized matrix
Graphical abstract
Introduction
Articular cartilage has a limited capacity for self-repair. Once damaged, the injured cartilage will develop defects. Despite promising results from autologous chondrocyte implantation [1], donor tissue availability is a challenge. Recent advances make adult stem cells an attractive cell source for cartilage regeneration, especially tissue-specific stem cells such as synovium-derived stem cells (SDSCs) [2], [3]. Since in vitro expansion is a necessary step before in vivo application, accompanying cell senescence and dedifferentiation represent a formidable challenge for stem cell-based cartilage repair [4].
We found that decellularized extracellular matrix (dECM) deposited by mesenchymal stem cells could rejuvenate stem cells [5], [6], [7], [8], [9], [10], [11] and primary cells [12], [13], [14] in both proliferation and differentiation capacity. For instance, dECM deposited by SDSCs significantly promoted expanded porcine SDSCs (pSDSCs) in both proliferation and chondrogenic potential [5]. In vivo transplantation of dECM expanded pSDSCs demonstrated efficacy in promoting cartilage regeneration in a partial thickness cartilage defect porcine model [15].
Our recent reports suggested that this in vitro cell expansion system also benefits human SDSC (hSDSC) expansion and rejuvenation of chondrogenic potential [16], [17], which brings hope for the potential use of this approach in clinical treatment [18], [19]. However, a concomitant up-regulation of type X collagen (COL10A1) was also observed in chondrogenically differentiated hSDSCs that have undergone dECM expansion, indicating a tendency toward chondrogenic hypertrophy.
In this study, we fully characterized cell morphology, volume, elasticity, and surface phenotypes in hSDSCs following dECM expansion. We not only defined proliferation and chondrogenic potential in dECM expanded hSDSCs but also examined whether increased expression of COL10A1 could be a sign of endochondral bone formation. Since both the mitogen-activated protein kinase (MAPK) and Wnt signals are critical pathways for chondrogenesis and have crosstalk in stem cell mediated cartilage regeneration [20], these two signals were evaluated for their changes in both cell expansion and chondrogenic induction of hSDSCs after preconditioning using dECM and conventional plastic flasks, which might provide evidence for further investigation of potential mechanisms underlying the rejuvenation of hSDSCs by dECM expansion.
Section snippets
SDSC culture
Adult human synovial fibroblasts (4 donors, two male and two female, average 43 years old, all had no known joint disease), referred to as hSDSCs [16], [17], were obtained from Asterand (North America Laboratories, Detroit, MI). Human SDSCs were plated and cultured in a growth medium [alpha minimum essential medium (αMEM) containing 10% fetal bovine serum (FBS), 100 U/mL penicillin, 100 μg/mL streptomycin, and 0.25 μg/mL fungizone (Invitrogen, Carlsbad, CA)] at 37 °C in a humidified 5% CO2 and 21% O2
Expansion on dECM changed cell morphology and increased cell proliferation
Before plating cells, SEM data showed that, compared with a flat surface on Plastic substrate, dECM coated flasks exhibited a three-dimensional structure with interconnected nanofibers (Fig. 1(A)) that was corroborated by morphological data from AFM (Fig. 1(B)), in which the diameter of matrix fibers was 109.5 ± 39.6 nm (n = 82). SEM data also showed that hSDSCs displayed a well-organized fibroblast-like cell shape when expanded on dECM rather than a broad and flat cell shape when grown on Plastic
Discussion
Our previous work found that dECM could rejuvenate pSDSCs for both proliferation and chondrogenic potential. We recently found this in vitro cell expansion system also applied to hSDSCs. In order to make this approach applicable to future clinical treatment, in this study, we characterized dECM and expanded cells using both SEM and AFM and compared them to the Plastic control; we also characterized proliferation and chondrogenic capacity of adult hSDSCs after expansion on dECM and determined
Conclusion and perspectives
Our study demonstrated that dECM deposited by hSDSCs formed a three-dimensional nanostructured matrix; expansion on dECM yielded a large quantity of hSDSCs with enhanced chondrogenic potential; despite an increase of type X collagen in chondrogenically induced cells, dECM expanded cells had significantly lower potential for endochondral bone formation. Even though the composition of dECM has been characterized in a recent report [16], research on the crosstalk between expanded SDSCs and dECMs
Competing Interest Statement
The authors do not have any conflicts of interest.
Acknowledgments
The authors thank Suzanne Danley for help in editing the manuscript. We also thank Ting Zheng and Dr. Cerasela Zoica Dinu for their help with the AFM instrument. This project was partially supported by research grants from the AO Foundation (S-12-19P) and the National Institutes of Health (NIH) (1 R03 AR062763-01A1 and P20 GM103434).
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