The role of the PCM in reducing oxidative stress induced by radical initiated photoencapsulation of chondrocytes in poly(ethylene glycol) hydrogels.
OBJECTIVE
The objectives for this study were to determine whether radical initiated photopolymerizations typically employed for cell encapsulations lead to oxidative stress incurred by chondrocytes and whether the development of a pericellular matrix (PCM) decreases this oxidative stress and has longer-term benefits on chondrocyte function.


METHODS
Freshly isolated bovine chondrocytes were encapsulated in poly(ethylene glycol) (PEG) hydrogels devoid of a PCM or with a PCM, confirmed by immunocytochemistry (IC), and cultured for up to 2 weeks. Reactive oxygen species (ROS) production and damage to cell membrane by lipid peroxidation were accomplished using carboxy-2,7-difluorodihydrofluorescein diacetate (carboxy-H(2)DFFDA) and by malondialdehyde (MDA) content, respectively. Gene expression and proteoglycan synthesis were analyzed using reverse transcription (RT)-quantitative PCR (qPCR) and (35)SO(4) incorporation, respectively.


RESULTS
The photopolymerization reaction, which alone generates radicals and extracellular ROS, led to oxidative stress in chondrocytes evidenced by increased intracellular ROS and lipid peroxidation. The presence of a PCM decreased intracellular ROS and abrogated membrane lipid peroxidation, improved aggrecan, collagen II and collagen VI expression, and enhanced proteoglycan synthesis.


CONCLUSIONS
The development of the PCM prior to photoencapsulation in PEG hydrogels reduces oxidative stress and improves chondrocyte anabolic activity. Our data suggest this reduction occurs by decreased ROS diffusion into the cell and decreased membrane damage. Our findings suggest that minimizing oxidative stress, such as through the presence of a PCM, may have long-term beneficial effects on tissue elaboration when employing photopolymerizations to encapsulate chondrocytes for cartilage tissue engineering applications.
Department of Chemical and Biological Engineering, University of Colorado Boulder, Boulder, CO 80309, USA.
N
N
Farnsworth
C
C
Bensard
S J
SJ
Bryant
D053674:Aggrecans / Q000096:biosynthesis / Q000235:genetics; D000818:Animals; D002417:Cattle; D002478:Cells, Cultured; D019902:Chondrocytes / Q000166:cytology* / Q000378:metabolism / Q000528:radiation effects; D003094:Collagen / Q000235:genetics / Q000378:metabolism; D005109:Extracellular Matrix / Q000378:metabolism* / Q000528:radiation effects; D005609:Free Radicals / Q000737:chemistry* / Q000528:radiation effects; D015870:Gene Expression / Q000528:radiation effects; D020100:Hydrogels / Q000737:chemistry / Q000528:radiation effects; D053509:Matrix Metalloproteinase 13 / Q000235:genetics / Q000378:metabolism; D018384:Oxidative Stress / Q000502:physiology* / Q000528:radiation effects; D055668:Photochemical Processes / Q000528:radiation effects; D011092:Polyethylene Glycols / Q000737:chemistry; D058105:Polymerization / Q000528:radiation effects; D017382:Reactive Oxygen Species / Q000378:metabolism / Q000528:radiation effects; D023822:Tissue Engineering / Q000379:methods; D014466:Ultraviolet Rays
D053674:Aggrecans; D005609:Free Radicals; D020100:Hydrogels; D017382:Reactive Oxygen Species; D011092:Polyethylene Glycols; D003094:Collagen; D053509:Matrix Metalloproteinase 13