The Synergistic Effect of Human Platelet Lysate and Macromolecular Crowding Regulate in Vitro Extracellular Matrix Deposition for the Development of Tendon Tissue Equivalents

Tendon injuries constitute a significant portion of musculoskeletal diseases, pose a heavy burden on healthcare and reduce patients' quality of life. Current treatments fall short of fully restoring tendon function. Tissue-engineered therapies using autologous tenocytes (hTCs) offer a promising solution for tendon regeneration; however, they are not commercially available due to the long culture time required to obtain sufficient tendon extracellular matrix (ECM) for implantation. Macromolecular crowding (MMC) is a promising tool capable of dramatically increase ECM deposition rate by hTCs. Moreover, hTCs have been traditionally cultured using foetal bovine serum (FBS), which is associated with rapid phenotypic drift. Allogeneic alternatives to FBS like human platelet lysate (hPL) have the potential to overcome this issue. Herein we explore the combined effect of MMC and hPL for hTCs culture to maintain cell function and develop tissue equivalents for tendon cell therapy. hTCs were isolated from tendons using culture medium supplemented with FBS or hPL. hTCs were then cultured with two MMC agents, κλ carrageenan (κλ CR) and λ carrageenan (λ CR). Cell function was assessed by AlamarBlue®, Live/Dead™ and nuclei counting. Collagen type I (COL I) deposition was assessed by SDS-PAGE and immunocytochemistry. Cell layers cultured with hPL for 8 days were then detached, sectioned and subjected to immunofluorescence analysis to determine the deposited COL I layer thickness. hTCs cultured with hPL showed a significant increase in proliferation and metabolic activity compared to FBS for every MMC condition, at every time point. SDS-PAGE and immunocytochemistry revealed that κλ CR supplementation significantly increased COL I deposition in hTCs cultured either with FBS or hPL. Cell layer sections revealed that hPL +κλ CR allowed the deposition of a significantly thicker COL I bed (69.8 ± 16.0 μm) compared to hPL -MMC (12.8 ± 1.3 μm). This work demonstrates the efficacy of MMC combined with hPL to enhance the deposition of tendon ECM in vitro from hTCs. In particular, hTCs supplemented with hPL and κλ CR maximized COL I deposition and allowed the formation of ECM-rich cell layers. The optimal cell culture conditions herein obtained pave the way to the development of in vitro tendon tissue substitute in a timely manner while eliminating the use of xenogeneic media supplements.