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Hypoxia Maintains the Connective Tissue Microenvironment
Rowena McBeath, MD, PhD; A. Lee Osterman, MD
The Philadelphia Hand Center, Thomas Jefferson University, Philadelphia, PA

Purpose: The role of oxygen in connective tissue regeneration is poorly understood. We have recently discovered that human tenocytes cultured in a low oxygen environment transdifferentiate to a fibrochondrocyte phenotype. The purpose of this study was to evaluate the effect of normoxia (22%02) or hypoxia (1%02) on human tenocyte differentiation in a three dimensional microenvironment.

Methods: Human tenocytes were isolated from patients undergoing revision amputation from traumatic hand injury ('normal' tenocytes) or delayed tendon reconstruction ('adherent' tenocytes) using IRB-approved protocols. Cells were cultured in normoxia until passage four then plated onto PLA electrospun nanofiber scaffolds. Scaffolds were cultured in normoxia or hypoxia for 1,2, 4 or 8 weeks and then harvested for immunohistochemical stains for Collagen I , Aggrecan or Collagen II as well as qRT- PCR of tenocyte and fibrochondrocyte molecular markers.

Results: Normal and adherent human tenocyte scaffolds develop dense cellular microarchitecture when cultured in hypoxia, but fail to develop consistent tissue in normoxia. Tissue quality is maximal at week 8 in hypoxia. Interestingly, immunohistochemical staining for collagen I was increased in normal tenocyte scaffolds cultured in normoxia while decreased in hypoxia, while the converse was true of aggrecan staining: aggrecan staining was increased in normal tenocyte scaffolds cultured in hypoxia. Furthermore, adherent tenocyte scaffolds demonstrated increased aggrecan expression in hypoxia compared to normal tenocyte scaffolds. These findings were confirmed by qRT-PCR.

Conclusions: In transitioning from the two dimensional to three dimensional environment it is clear that hypoxia is key to maintaining the tendon cellular microenvironment as well as fibrochondrocyte differentiation, in particular:

  1. Tendon tissue maintains its cellular microarchitecture in the hypoxic environment more than the normoxic environment
  2. Adherent tendon tissue produces more proteoglycans in the hypoxic environment than in the normoxic environment
  3. Adherent tendon tissue produces more proteoglycans in the hypoxic environment than normal tendon tissue
  4. In the absence of mechanical forces, tendon tissue transdifferentiates to a fibrochondrocyte phenotype in the hypoxic environment
  5. These findings may explain the tendency for tendon adhesion formation in the hypoxic environment -- as demonstrated by increased proteoglycan formation and decreased collagen I formation -- and provide insight into tendon tissue regeneration and repair.
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