Hypoxia and Tension Maintain Human Tenocyte Tissue Constructs in the 3D Microenvironment

Abstract

Grant received from: NIA AG048118 (2014) and Jahnigen foundation/AGS GEMSSTAR (2014) There is no financial information to disclose. Tendon tissue constructs are urgently needed in surgical treatment of patients with traumatic tendon loss. Previous studies have elucidated the effects of time, cell density and oxygen levels on maintenance of the human tenocyte phenotype in vitro; we hypothesized that these conditions could be translated to a 3D construct fit for future in vivo clinical applications. Human tenocytes were isolated from patients (ages 20-35) undergoing revision amputation for traumatic hand injury using IRB-exempt protocols (#14E.621). To form the tenocyte tissue constructs, electrospun nanofiber scaffolds were fabricated using collagen I solution expressed through a spinneret along 15kV electric field over a defined air gap, collected onto a rotating mandrel, and sterilized under UV prior to cell seeding according to established protocols (1). Human tenocytes (p4) were seeded onto scaffolds and cultured in normal oxygen (21%02, normoxic) and low oxygen (1%02, hypoxic) conditions, and harvested after 2, 4, and 8 weeks in static or dynamic (bioreactor, (2)) culture for immunohistochemical, qRT-PCR and western blot analysis of tenocyte (collagen I, scleraxis, tenomodulin), fibrocartilage (collagen III) and chondrocyte (collagen II, sox9) markers. Human tenocytes engrafted onto electrospun nanofiber scaffolds and maintained a tissue-like phenotype over time in hypoxic culture. When cultured statically -- in the absence of tension or compression -- culture of tenocyte tissue constructs in hypoxic conditions resulted in more robust tenocyte tissue appearance, increased collagen I and decreased aggrecan expression compared to normoxic conditions at 8 weeks. When cultured dynamically under bioreactor tensile conditions, tenocyte tissue constructs collagen III expression levels were inhibited to a greater extent than those in static conditions. •3-dimensional tenocyte tissue constructs can be created using human tenocytes engrafted onto electrospun nanofiber scaffolds, and maintained in low and normal oxygen conditions over time.•Human tenocyte tissue constructs achieve a tendon-like phenotype to a greater extent in hypoxic conditions, under tensile stress, as compared to static normoxic conditions.•Knowledge of the molecular mechanism underlying maintenance of the human tenocyte phenotype in a 3-dimensional environment over time will enhance construct survival and success in the in vivo environment.