Characterization of Human Chondrocytes Exposed to Simulated Microgravity

Abstract

Background: Tissue engineering is a strategy of cartilage regeneration, but scaffolds, required for 3D growth of chondrocytes, are still a problem. Methods: Searching for possibilities to improve scaffold-free engineering of cartilage, we characterized human chondrocytes incubated on a random positioning machine (RPM) to simulate microgravity (µg). Results: When cultured in simulated µg, human chondrocytes start forming 3D cell assemblies within 5 days. After 24h, we could not detect caspase-3, Fas, p53 or Bcl-2 proteins in these cells, Annexin V flow cytometry, however, revealed 18% of apoptotic chondrocytes in 1g cultures but only 10% on the RPM. Both rates of apoptosis were not changed, when vascular endothelial growth factor (VEGF) or basic fibroblast growth factor (bFGF) was added. 24 h, simulated microgravity also had significantly decreased collagen type I and X, but did not change collagen type IV and laminin, while collagen type II, chondroitin sulfate and aggrecan were elevated as compared with 1g controls. The production of collagen type II/X, chondroitin sulfate and aggrecan was modified, when external bFGF or VEGF had been applied. Conclusion: Chondrocytes exposed to simulated µg seem to change their extracellular matrix production behavior, while they rearrange their cytoskeletal proteins prior to forming 3D aggregates.

Growing Tissues in Real and Simulated Microgravity: New Methods for Tissue Engineering

“…2,11,[49][50][51] Only a part of the cells of a monolayer change their growth behavior, when exposed to microgravity, while the other part continues to grow adherently. [52][53][54] However, when exposed to microgravity, both the adherently and the 3D growing cells alter their gene expression patterns. 55 Very often the decrease or increase of the protein content found in a cell population under microgravity is accompanied by a change in the number of cells bearing the respective antigen.…”

“…In addition, the signals delivered by removal or randomization of gravity forces cause the formation of different types of aggregates depending on the kind of cells cultured. While cancer cells mainly assemble to spheres, 52 for example, ECs form tubes 8 and chondrocytes form pieces of cartilage, 54 in vitro like they naturally do in vivo. Although all these cells sense the lack of gravity within seconds, 20,59 it takes at least 12 h until 3D spheroids are observed 52,53 and up to 7 days until constructs like tubes resembling an intima can be recognized floating in a culture flask.…”

“…116 RPM-cultivated chondrocytes created tissue with matrix composition characteristics in between the control 1 g and the ISS-produced neocartilages, but produced neocartilage with more evenly dispersed and further spaced apart cells. 54 The number of cells that can be obtained from a clinically relevant-sized biopsy is not sufficient for cell-based therapies such as autologous cartilage implantation or to produce a functional neocartilage implant. Thus, the classic expansion phase in monolayer is needed to increase cell numbers, adding to the probability of reduced chondrogenic capacities.…”

The Impact of Vascular Endothelial Growth Factor and Basic Fibroblast Growth Factor on Cardiac Fibroblasts Grown under Altered Gravity Conditions

“…The so-called 'Random Positioning Machine' is a well established method to culture cells in altered gravity conditions [32][33][34]. A principal aim of this study is to answer the question how these conditions influence CF.…”

Tissue-Engineered Cartilaginous Constructs for the Treatment of Caprine Cartilage Defects, Including Distribution of Laminin and Type IV Collagen

“…This is in agreement with previous work showing that chondrocytes can synthesize the two molecules in vitro. 18 It has also been shown that chondrocytes can synthesize the molecules in vivo 7 ; however, the source of the laminin and type IV collagen in the reparative tissue in this study was not investigated.…”