Tenocyte cell density, migration, and extracellular matrix deposition with amniotic suspension allograft

Abstract

Amniotic suspension allografts (ASA), derived from placental tissues, contain particulated amniotic membrane and amniotic fluid cells. Recently, ASA and other placental-derived allografts have been used in orthopaedic applications, including tendinopathies and tendon injuries. The purpose of this study was to determine the potential effects of ASA on tenocyte cell density, migration, and responses to inflammatory stimuli. Tenocyte cell density was measured using AlamarBlue over multiple time points, while migration was determined using a Boyden chamber assay. Deposition of ECM markers were measured using BioColor kits. Gene expression and protein production of cytokines and growth factors following stimulus with pro-inflammatory IL-1β and TNF-α was measured using qPCR and ELISAs. Conditioned media (CM) was made from ASA and used for all assays in this study. In vitro, ASA CM treatment significantly promoted tenocyte increases in cell density and migration compared to assay media controls. ASA CM also increased the deposition of extracellular matrix (ECM) proteins, including collagen, elastin, and sGAG. Following inflammatory stimulation and treatment with ASA CM, tenocytes downregulated IL-8 gene expression, a pro-inflammatory cytokine normally elevated during the inflammatory phase of tendon healing. Additionally, tenocytes treated with ASA CM had significantly lower protein levels of TGF-β1 compared to controls. This study evaluated ASA and its effect on tenocytes; specifically, treatment with ASA resulted in increased cell density, more robust migration and matrix deposition, and some alteration of inflammatory targets. © 2018 The Authors. Journal of Orthopaedic Research® Published by Wiley Periodicals, Inc. on behalf of Orthopaedic Research Society. J Orthop Res 37:412-420, 2019.

Keywords: amniotic suspension allograft; regenerative; repair; tendon; tenocytes.

Figure 1

Evaluation of effects of amniotic suspension allograft (ASA) conditioned media (CM) on tenocyte cell density over 14 days. (A) Cell density was measured for cells at days 3, 7, 10, and 14. Mean ± standard deviation reported; n = 12 per group. All statistical comparisons are within the particular day and compared to assay media. *p < 0.05, ***p < 0.001, and §p < 0.0001. (B) At 14 days, cell monolayers were stained with 5‐chloromethylfluorescein diacetate (CMFDA). Representative images demonstrating cell density for each group at 4× objective are shown.

Figure 2

Evaluation of amniotic suspension allograft (ASA) conditioned media (CM) effects on tenocyte migration. (A) Quantitative analysis of tenocyte migration was evaluated over 24 hours. Mean ± standard deviation reported; n = 24 per group. §p < 0.0001 compared to assay media. (B) Qualitative images of cells stained with crystal violet for each group are shown at 4× objective.

Figure 3

Impact of amniotic suspension allograft (ASA) conditioned media (CM) on extracellular matrix (ECM) deposition of tenocytes. Deposition of extracellular matrix molecules was evaluated including (A) collagen, (B) elastin, and (C) soluble glycosaminoglycans (sGAG). For all groups, data are presented as mean ± standard deviation; n = 9 per group. §p < 0.0001 compared to assay media at the same time point.

Figure 4

Evaluation of amniotic suspension allograft (ASA) conditioned media (CM) effect on tenocyte gene expression in pro‐inflammatory environment. Data is presented as fold change of the target of interest normalized to glyceraldehyde 3‐phosphate dehydrogenase (GAPDH). (A) interleukin‐1 beta (IL‐1β), (B) tumor necrosis factor alpha (TNF‐α), (C) interleukin‐6 (IL‐6), (D) interleukin‐8 (IL‐8), (E) matrix metalloproteinase 1 (MMP‐1), and (F) transforming growth factor beta 1 (TGF‐β1). Mean ± standard deviation reported; n = 12 per group. *p < 0.05 compared to untreated condition.

Figure 5

Evaluation of effect of amniotic suspension allograft (ASA) conditioned media (CM) on protein production of tenocytes in a pro‐inflammatory environment. Concentrations of (A) matrix metalloproteinase 1 (MMP‐1) and (B) transforming growth factor beta 1 (TGF‐β1) were measured. Mean ± standard deviation reported; n = 9 per group. *p < 0.05, **p < 0.01, and §p < 0.0001 compared to untreated condition.