Optimization of Non-Cross-Linked Hyaluronic Acid and Adipose-Derived Mesenchymal Stem Cells for Regenerative Applications: An in Vitro Study Publisher



Kafaei E ; Forozesh L ; Barghian E ; Barghian B ; Malekan M ; Gholizadeh F ; Khani E ; Abdollahiasl A ; Nilforoushzadeh MA ; Sisakht MM
Source: Cell Biochemistry and Biophysics Published:2026

Abstract

Hyaluronic acid (HA) and adipose-derived mesenchymal stem cells (AD-MSCs) are both promising agents for skin rejuvenation. Combining non-cross-linked HA with AD-MSCs may enhance regenerative outcomes; however, the optimal HA concentration and its compatibility with AD-MSCs remain unclear. This study evaluated AD-MSCs encapsulated in varying concentrations of non-cross-linked HA, including a Proage® HA formulation, to identify the most biocompatible scaffold for regenerative applications. AD-MSCs were isolated from human adipose tissue and characterized using flow cytometry and tri-lineage differentiation. Cells were encapsulated in HA matrices ranging from 2% to 10%, including a Proage® 2% HA formulation. Metabolic activity was assessed using the Alamar Blue assay on Days 1, 3, 5, and 7. Gene expression of COL1A1, COL3A1, LMNA, IL10, TIMP1, MMP1, CD49f, and CD146 was analyzed via qRT-PCR to evaluate extracellular matrix remodeling and inflammatory responses. AD-MSCs maintained high metabolic activity across HA concentrations, with 4% HA demonstrating the highest metabolic activity by Day 5, whereas higher concentrations (>5%) reduced activity. Gene expression analysis showed significant upregulation of COL1A1, COL3A1, LMNA, TIMP1, CD49f, and CD146 in 3D HA conditions compared with 2D culture. Specifically, 4% HA increased COL1A1, LMNA, CD146, and TIMP1 expression, while the Proage® HA formulation significantly upregulated COL3A1, LMNA, CD49f, CD146, and TIMP1. No significant changes were observed for IL1B or MMP1, and IL10 showed a non-significant upward trend under 3D conditions. Overall, non-cross-linked 4% HA provided an optimal microenvironment for AD-MSC metabolic activity and regenerative gene expression. The Proage® 2% HA formulation also demonstrated strong biocompatibility and regenerative potential, particularly through upregulation of collagen and stemness markers. These findings indicate that AD-MSCs tolerate the 4% non-cross-linked HA scaffold well, maintaining high metabolic activity and growth potential, thereby supporting its potential use to enhance regenerative outcomes in skin rejuvenation therapies. © The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature 2026.