Abstract

Introduction: Stem cells hold promise for tissue repair due to their secretome, which is influenced by the microenvironment. To improve cartilage regeneration, a three-dimensional (3D) natural cartilage scaffold system has been developed to create a more chondrogenic secretome. As human cartilage is avascular, a hypoxic environment may better mimic in vivo conditions. This study investigates if oxygen levels (normoxic vs. hypoxic) affect MSC chondrogenic potential and secretome composition in this 3D scaffold.
Methods: This study used a randomized time series design to investigate how oxygen levels affect the transformation of MSCs into cartilage cells within a 3D cartilage scaffold. A control group (C) was grown in a standard medium. Two experimental groups, P1 and P2, were cultured n a 3D cartilage scaffold under normal (21%) and hypoxic (5%) oxygen concentration. respectively. The hypothesis was that a hypoxic environment would promote superior  chondrogenic  differentiation. Key  markers  were  evaluated  at  specific  time  points  using immunohistochemistry and enzyme-linked assay.
Results: In the 3D cultures, the cells exposed to low oxygen (hypoxic) generally showed higher expression of markers related to cartilage formation (SOX-9, RUNX-2, Coll-II, and aggrecan) compared to those in normal oxygen levels (normoxic). Additionally, the hypoxic group had consistently higher levels of several growth factors important for tissue development and repair (BMP-2, BMP-7, TGF-β3, IGF-1, and FGF-2) throughout the study.
Conclusion: Hypoxic conditions in 3D culture led to increased expression of all analyzed chondrogenic markers in mesenchymal stem cells, suggesting that a low-oxygen environment effectively promotes chondrocyte proliferation and differentiation.
Keywords: Stem Cells, Three-Dimensional, Culture Technique, Secretome, Cartilage. 

INTRODUCTION

Cartilage damage and loss, whether due to injury, disease, or aging, trigger subchondral bone remodeling and lead to osteoarthritis (OA).1 As cartilage has limited regenerative capacity and current treatments offer limited success, new therapies are needed to slow OA progression2–4. Mesenchymal stem cells (MSCs) hold promises for cartilage repair due to their ability to self-renew and differentiate into chondrocytes5. However, challenges like immune rejection, tumor risk, cost, and infection transmission hinder their clinical application6,7. Recent research suggests that MSCs' therapeutic effects primarily stem from secreted paracrine factors, collectively known as the secretome, rather than direct cell engraftment. This discovery opens new avenues for OA treatment by focusing on isolating and utilizing individual secretome components, thus bypassing the limitations of direct stem cell transplantation6,8. To maximize cartilage regeneration, the secretome composition should be optimized to be more chondrogenic, enriched with proteins crucial for cartilage formation like aggrecan, type II collagen, TGF-β, IGF-1, FGF-2, and BMP-2,7.  The  microenvironment,  including  chemical  and  physical  stimuli,  significantly  influences  the secretome's properties. In particular, the culture medium and oxygen concentration that mimic the natural microenvironment might play pivotal roles in producing a secretome with desired characteristics9,10. Three-dimensional (3D) culture systems offer advantages over traditional 2D cultures by mimicking the natural tissue environment, enhancing cell growth, proliferation, and self-renewal, while preventing premature differentiation. This  approach  facilitates  better  molecular  exchange,  nutrient  uptake,  gas  exchange,  and  waste  removal, mirroring in vivo conditions9,11. To replicate the in vivo environment more accurately, a hypoxic (low oxygen) condition is preferred, as it aligns with the avascular nature of human cartilage. Hypoxia can induce essential transcription  factors  for  chondrogenesis,  enhance  stem  cell  proliferation  and  multipotency,  and  promote chondrocyte marker expression (e.g., collagen type II, aggrecan, SOX-9), ultimately leading to increased extracellular matrix synthesis. Both in vitro and in vivo studies have demonstrated improved chondrocyte proliferation and differentiation under hypoxic conditions in 3D scaffolds. However, the specific effects of oxygen concentration on the chondrogenic potential of 3D cultures remain unexplored12,13.  By optimizing both culture media and oxygen concentration, we aim to create an ideal environment for chondrogenic  differentiation.  This  study  investigates  whether  difference  in  oxygen  levels  (normoxic  vs. hypoxic) influence MSC chondrogenic potential and the composition of their secreted secretomes within a 3D cartilage scaffold.