Serum deprivation limits loss and promotes recovery of tenogenic phenotype in tendon cell culture systems

Abstract

Current knowledge gaps on tendon tissue healing can partly be ascribed to the limited availability of physiologically relevant culture models. An unnatural extracellular matrix, high serum levels and random cell morphology in vitro mimic strong vascularization and lost cell elongation in pathology, and discord with a healthy, in vivo cell microenvironment. The thereby induced phenotypic drift in tendon‐derived cells (TDCs) compromises the validity of the research model. Therefore, this research quantified the extracellular matrix (ECM)‐, serum‐, and cell morphology‐guided phenotypic changes in tendon cells of whole tendon fascicle explants with intact ECM and TDCs cultured in a controlled microenvironmental niche. Explanted murine tail tendon fascicles were cultured in serum‐rich or serum‐free medium and phenotype was assessed using transcriptome analysis. Next, phenotypic marker gene expression was measured in in vitro expanded murine tail TDCs upon culture in serum‐rich or serum‐free medium on aligned or random collagen I patterns. Freshly isolated fascicles or TDCs served as native controls. In both systems, the majority of tendon‐specific genes were similarly attenuated in serum‐rich culture. Strikingly, 1‐week serum‐deprived culture—independent of cell morphology—converged TDC gene expression toward native levels. This study reveals a dynamic serum‐responsive tendon cell phenotype. Extracting fascicles or TDCs from their native environment causes large changes in cellular phenotype, which can be limited and even reversed by serum deprivation. We conclude that serum‐derived factors override matrix‐integrity and cell morphology cues and that serum‐deprivation stimulates a more physiological microenvironment for in vitro studies.

Tendon-Derived Biomimetic Surface Topographies Induce Phenotypic Maintenance of Tenocytes In Vitro

“…Additionally, serum-deprivation, i.e. depletion of medium-associated growth factors rescued the lost tenocyte phenotype [21]. In addition, the small molecule tazaratone, which targets the retinoic acid receptor, preserves tenocyte phenotype via scleraxis [22].…”

Dynamic Load Model Systems of Tendon Inflammation and Mechanobiology

“…This indicates that the muscle and bone likely play a role in the inflammatory signaling and ultimate degeneration of tendon after injury. Culture conditions, specifically serumsupplemented media, likely play a role in directing tenocytes to a pro-inflammatory phenotype as well in both whole tendon explants and 2D culture (van Vijven et al, 2021). Underlying presence of pro-inflammatory factors is generally unknown or varies in serum batches, which may contribute to the proinflammatory phenotypes.…”

Impact of isolation method on cellular activation and presence of specific tendon cell subpopulations during in vitro culture

“…It has long been understood that once isolated, tendon cells from many different species do not retain their in vivo characteristics, a phenomenon referred to as “phenotypic drift” 15 . This process is typically described as a loss of tenogenic markers, including scleraxis (Scx), tenomodulin, Mohawk (Mkx), and collagen type I (Col1), increases in the expression of injury‐associated collagens including collagen type III (Col3), alterations in cell morphology away from the typical spindle shape, and changes in proliferative capacity over time in culture 15‐19 . This has traditionally been viewed as a negative outcome, and investigators have been advised to use isolated tendon cells at early passages in an attempt to preserve the tendon cell phenotype seen in vivo 17 …”

“…15 This process is typically described as a loss of tenogenic markers, including scleraxis (Scx), tenomodulin, Mohawk (Mkx), and collagen type I (Col1), increases in the expression of injury-associated collagens including collagen type III (Col3), alterations in cell morphology away from the typical spindle shape, and changes in proliferative capacity over time in culture. [15][16][17][18][19] This has traditionally been viewed as a negative outcome, and investigators have been advised to use isolated tendon cells at early passages in an attempt to preserve the tendon cell phenotype seen in vivo. 17 Though they are referred to by many different names, the majority of the cells that reside in tendons are fibroblasts.…”