A systematic investigation of the effects of TGF-ß3 and mechanical stimulation on tenogenic differentiation of mesenchymal stromal cells in a poly(ethylene glycol)/gelatin-based hydrogel.

Background: High post-surgical failure rates following tendon injury generate high medical costs and poor patient recovery. Cell-based tendon tissue engineering has the potential to produce fully functional replacement tissue and provide new strategies to restore tendon function and healing. In this endeavour, the application of mesenchymal stromal cells (MSCs) encapsulated in biomaterial scaffolds has shown great promise. However, a consensus on optimal promotion of tenogenic differentiation of MSCs has yet to be reached, although growth factors and mechanical cues are generally acknowledged as important factors. Methods: In this study, we prepared a hydrogel cell culture system consisting of methacrylated poly(d,l-lactic acid-ethylene glycol-d,l-lactic acid) (P(LA-EG-LA)) and gelatin methacrylate (GelMA) to encapsulate human bone marrow-derived MSCs (hBMSCs). We further systematically investigated the influence of static and intermittent cyclic uniaxial strain mechanical stimulation, in combination with transforming growth factor-ß3 (TGF-ß3) supplementation, on tenogenic differentiation of hBMSCs. Results: Increased TGF-ß3 concentration upregulated the tenogenic genes Scleraxis (SCX) and collagen type I (COL1A1) but showed no effects on tenascin-c (TNC) and collagen type III (COL3A1) expression. Mechanical stimulation had no observable effect on gene expression, but intermittent cyclic uniaxial strain stimulation improved matrix deposition. Together, these data provide new insights into how TGF-ß3 and mechanical stimulation regulate MSC tenogenesis, with TGF-ß3 promoting the expression of key tenogenic genes whilst mechanical stimulation aided matrix deposition in the engineered tissue. Furthermore, intermittent cyclic uniaxial strain at 3% elongation and 0.33 â€‹Hz for 1 â€‹h/day showed improved matrix effects compared to static strain. Conclusion: Together, the most promising result for tenogenic differentiation of hBMSCs was identified as treatment with 5 â€‹ng/ml TGF-ß3 under intermittent cyclic uniaxial strain (3% strain; 0.33 â€‹Hz; 1 â€‹h/day). This knowledge is of importance for the development of an improved protocol for tenogenic differentiation of MSCs and thereby for tendon tissue engineering. The translational potential of this article: Tissue-engineered strategies for tendon repair require a consensus on the differentiation of mesenchymal stromal cells to tenocytes, which is currently lacking. This article provides a systematic investigation of two main tenogenic differentiation conditions to further development of a tenogenic differentiation protocol.

Substrate mechanical properties bias MSC paracrine activity and therapeutic potential.

Mesenchymal stromal cells (MSCs) have significant therapeutic potential due to their ability to differentiate into musculoskeletal lineages suitable for tissue-engineering, as well as the immunomodulatory and pro-regenerative effects of the paracrine factors that these cells secrete. Cues from the extracellular environment, including physical stimuli such as substrate stiffness, are strong drivers of MSC differentiation, but their effects upon MSC paracrine activity are not well understood. This study, therefore sought to determine the impact of substrate stiffness on the paracrine activity of MSCs, analysing both effects on MSC fate and their effect on T-cell and macrophage activity and angiogenesis. The data show that conditioned medium (CM) from MSCs cultured on 0.2 kPa (soft) and 100 kPa (stiff) polyacrylamide hydrogels have differing effects on MSC proliferation and differentiation, with stiff CM promoting proliferation whilst soft CM promoted differentiation. There were also differences in the effects upon macrophage phagocytosis and angiogenesis, with the most beneficial effects from soft CM. Analysis of the media composition identified differences in the levels of proteins including IL-6, OPG, and TIMP-2. Using recombinant proteins and blocking antibodies, we confirmed a role for OPG in modulating MSC proliferation with a complex combination of factors involved in the regulation of MSC differentiation. Together the data confirm that the physical microenvironment has an important influence on the MSC secretome and that this can alter the differentiation and regenerative potential of the cells. These findings can be used to tailor the culture environment for manufacturing potent MSCs for specific clinical applications or to inform the design of biomaterials that enable the retention of MSC activity after delivery into the body. STATEMENT OF SIGNIFICANCE: • MSCs cultured on 100 kPa matrices produce a secretome that boosts MSC proliferation • MSCs cultured on 0.2 kPa matrices produce a secretome that promotes MSC osteogenesis and adipogenesis, as well as angiogenesis and macrophage phagocytosis • IL-6 secretion is elevated in MSCs on 0.2 kPa substrates • OPG, TIMP-2, MCP-1, and sTNFR1 secretion are elevated in MSCs on 100 kPa substrates.

Tendon tissue engineering: Current progress towards an optimized tenogenic differentiation protocol for human stem cells.

Tendons are integral to our daily lives by allowing movement and locomotion but are frequently injured, leading to patient discomfort and impaired mobility. Current clinical procedures are unable to fully restore the native structure of the tendon, resulting in loss of full functionality, and the weakened tissue following repair often re-ruptures. Tendon tissue engineering, involving the combination of cells with biomaterial scaffolds to form new tendon tissue, holds promise to improve patient outcomes. A key requirement for efficacy in promoting tendon tissue formation is the optimal differentiation of the starting cell populations, most commonly adult tissue-derived mesenchymal stem/stromal cells (MSCs), into tenocytes, the predominant cellular component of tendon tissue. Currently, a lack of consensus on the protocols for effective tenogenic differentiation is hampering progress in tendon tissue engineering. In this review, we discuss the current state of knowledge regarding human stem cell differentiation towards tenocytes and tendon tissue formation. Tendon development and healing mechanisms are described, followed by a comprehensive overview of the current protocols for tenogenic differentiation, including the effects of biochemical and biophysical cues, and their combination, on tenogenesis. Lastly, a synthesis of the key features of these protocols is used to design future approaches. The holistic evaluation of current knowledge should facilitate and expedite the development of efficacious stem cell tenogenic differentiation protocols with future impact in tendon tissue engineering. STATEMENT OF SIGNIFICANCE: The lack of a widely-adopted tenogenic differentiation protocol has been a major hurdle in the tendon tissue engineering field. Building on current knowledge on tendon development and tendon healing, this review surveys peer-reviewed protocols to present a holistic evaluation and propose a pathway to facilitate and expedite the development of a consensus protocol for stem cell tenogenic differentiation and tendon tissue engineering.