Induction of Tendon-Specific Markers in Adipose-Derived Stem Cells in Serum-Free Culture Conditions

Abstract

Differentiation of stem cells as a cell-based therapy for repairing, replacing or restoring damaged tissues such as bone, cartilage, and tendon is becoming increasingly attractive within the field of musculoskeletal tissue engineering. Towards this end, there are numerous published and well-defined protocols to differentiate stem cells towards cartilage and bone tissues, but the protocols towards tendon tissue are still emerging and thus less developed. Recent studies focused on the induction of tendon-specific markers in cultured stem cells using different Growth Factors (GFs) including Bone Morphogenetic Proteins (BMPs) and Transforming Growth Factor (TGF) isoforms. However, the inclusion of serum in relatively high concentration across these studies is less favorable, since the components within serum may interfere with the induction of the markers. Alternatively, in vitro studies with low concentration or absence of serum would be ideal. In this study, we assessed the induction effect of BMP-12 and TGF-β1 on tendon-specific markers in Adipose-Derived Stem cells (ADSCs), in serum-free conditions. Specifically, we investigated the temporal and dosing effects of both GFs on several markers. Our results demonstrate that BMP-12 induces late expression of the transcription factors Scleraxis (SCX) and Mohawk (MKX), whereas TGF-β1 induced their earlier expression. Moreover, BMP-12 induced Decorin (DCN) but was inhibited by TGF-β1. Other markers such as Collagen Iα1 (COL1A1) likewise showed this pattern. Importantly, the protein analysis generally supported the gene expression data. Interestingly, differences were observed in the cellular localisation of SCX between BMP-12 and TGF-β1 stimulations. Furthermore, the addition of Ascorbic Acid (AA) with either BMP-12 or TGF-β1 resulted in increased deposition of Collagen I. Our results enhance the existing protocols for the differentiation of ADSCs towards the tenogenic lineage in serum-free conditions and contribute to the understanding and the development of tenogenic induction protocols. Statement of significance Herein we describe the tenogenic effect of BMP-12 and TGF-β1 in cultured ADSCs in serum-free conditions. This culture system provides an insight into serum-free culture conditions in stem cell differentiation protocols. A positive response of the ADSCs to the tenogenic induction was observed. In particular, the different Growth Factors used in this study displayed notable differences both on the gene and on the protein expression of the tendon-specific markers. The results underline the positive outcome of the serum removal in tenogenic differentiation protocols, contributing to the development of future cell-based therapies for tendon regeneration and repair.

Supporting Cell-Based Tendon Therapy: Effect of PDGF-BB and Ascorbic Acid on Rabbit Achilles Tenocytes In Vitro

“…Small bioactive molecules like ascorbic acid (AA), i.e., Vitamin C [29][30][31] or glutamine [12] have also been investigated. Interestingly, bioactive molecules are often investigated concerning their effect on stem cell tenogenic differentiation under serum+ conditions [18,[32][33][34] and serum-free conditions [35,36], while effects on tenocytes have not been investigated in-depth so far, and only few reports for human or animal tenocytes exist [37][38][39]. Additionally, it is beneficial to determine the effects of biomolecules on tenocyte culture not only for pre-treatment for cell-based therapy but also since tenocytes are the main cell population affected by the delivery of drugs explored for aiding tendon tissue healing and repair.…”

Directing iPSC Differentiation into iTenocytes using Combined Scleraxis Overexpression and Cyclic Loading

“…Cells were grown for two weeks in tenogenic media (low glucose DMEM supplemented with 2mM glutamine, 10% FBS, 1% AAS, and 50µg/ml ascorbic acid). 35,36 After 2 weeks, cells were lysed with RLT plus lysis buffer (RLT plus buffer, Qiagen, Valencia, CA containing β-mercaptoethanol, M3148, Sigma, St. Louis, MO), and stored at -80 o C until processing for gene expression analysis.…”

Engineering the Tenogenic Niche In Vitro with Microenvironmental Tools

“…Ascorbic acid, for example, is a vitamin that prevents free radical formation [95] and protects tenocytes from oxidative stress. [96] It has been used along with various tenogenic inductive treatments, [97][98][99][100] as it facilitates enhanced collagen type I synthesis in vitro. [101,102] Low glucose dose has also been shown [102] Glucose Low doses upregulated expression of scleraxis, SOX9, COL1A1, and COL1A2, but not COL2A [103] TSPCs Tendon-derived matrix Stimulated proliferation and maintained the stemness [216] Collagen type I/fibronectin Demonstrated greater attachment on fibronectin than on collagen [217] Exhibited spindle-shaped morphology with long processes on collagen, but not on fibronectin [217] BMSC Collagen type I Tenogenic differentiation was improved when PDMS replicas of Achilles tendon sections were coated with collagen type I, however this was shown to be concentration-dependent [218] Collagen type I/fibronectin Collagen promoted scleraxis, tenomodulin and tenascin C expression, and inhibited osteogenic differentiation, while fibronectin upregulated RUNX-2 and ALP [91] ADSCs Tendon-derived matrix/fibronectin Deposited more collagen type I and exhibited greater collagen type I to collagen type III ratio on tendon-derived matrix [219] Tendon-derived matrix hydrogel/Collagen type I hydrogel Tendon-derived matrix demonstrated higher proliferation; upregulation of scleraxis, tenascin C, and tenomodulin; enhanced mechanical properties; lower osteogenic potential [220] Collagen type V Reduction of osteogenic differentiation markers, even in the presence of osteogenic media [221] Gelatin Did not show any additional benefits beyond improved adhesion [222] Did not affect proliferation and decreased protein and collagen synthesis [223] 2.7 Tenocyte proliferation was increased at early passage cells [207,212] TSPCs 0.5 Inhibited self-renewal capacity and reduced adipogenic, chondrogenic, and osteogenic potential in differentiation media [224] 2 Increased proliferation, retained their tenogenic differentiation potential, and had a lower osteogenic, chondrogenic, and adipogenic differentiation capacity [212] Reduced Sox-9 and RUNX-2 expression level, did not affect PPAR expression and culture in differentiation media led to enhanced adipogenesis, chondrogenesis, and osteogenesis…”

“…Ascorbic acid and BMP-12 or TGF-1 promoted expression of scleraxis and Mohawk and increased collagen type I synthesis and deposition [97] 3D architecture Growth factors…”

“…Ascorbic acid, for example, is a vitamin that prevents free radical formation and protects tenocytes from oxidative stress . It has been used along with various tenogenic inductive treatments, as it facilitates enhanced collagen type I synthesis in vitro . Low glucose dose has also been shown to promote tenogenic phenotype, as evidenced by upregulated tenogenic marker expression and enhanced collagen expression in tenocyte culture .…”