Development of a Refined Tenocyte Differentiation Culture Technique for Tendon Tissue Engineering

Abstract

We have established that human tenocytes can differentiate in the absence of exogenous fetal bovine serum (FBS) but in the presence of insulin-like growth factor-1 (IGF-1) and transforming growth factor-β₃ (TGF-β₃). The extent of tenocyte differentiation was assessed by examining cell survival, collagen synthesis, cell morphology and expression of tenocyte differentiation markers such as scleraxis (Scx), tenomodulin (Tnmd), collagen type I (Col-I) and decorin (Dcn). Our results indicate that 50 ng/ml IGF-1 and 10 ng/ml TGF-β₃ (in the absence of FBS) were capable of maintaining in vitro human tenocyte survival in 14-day cultures. The extent of collagen synthesis and messenger ribonucleic acid expression of Scx, Tnmd, Col-I and Dcn were significantly upregulated in response to IGF-1 and TGF-β₃. These findings have shown for the first time that human tenocytes can be maintained in long-term culture, in serum-free conditions, making this approach a suitable one for the purpose of tendon tissue engineering.

Three-Dimensional Platelet-Rich Plasma Hydrogel Model to Study Early Tendon Healing

“…SCX, TNMD, and DCN, has been reported in the presence of IGF-1 and TGF-β 3 in 2D cultures [Qiu et al, 2013]. Interestingly, in our experimental conditions, expression of SCX and TNMD could be regained not only at low passages (e.g.…”

Use of biomimetic microtissue spheroids and specific growth factor supplementation to improve tenocyte differentiation and adaptation to a collagen-based scaffold in vitro

“…Caliari et al [22] demonstrated that IGF-1 at concentrations ranging from 10 to 200 ng/ml was capable of significantly enhancing COL1A2, COL3A1 and decorin (DCN) gene expression in equine tenocytes cultured in collagen scaffolds in the absence of serum for 7 days. By contrast, a study performed by Qiu et al [24] demonstrated that IGF-I (50 ng/ml) alone was unable to significantly influence COL1, SCX or TNMD expression in human tenocytes. However, incubation of cells with TGF-b3 (10 ng/ml), or TGF-b3 and IGF-1 together, significantly enhanced gene expression above that of control cultures incubated in the presence of serumsupplemented growth medium.…”

“…This has been highlighted in several studies in which the expression of tendon-associated markers, including COL1, COL3, SCX and TNMD, were shown to be downregulated in tenocytes cultured for extended periods [19,20]. These limitations however, could be overcome to some degree by incorporation of tenocytes into 3D culture systems [25,27,41] or through the addition of specific growth factors [22,24,25]. In the current report, we initially investigated the effectiveness of culturing equine tenocytes as hanging drops as a means by which to generate scaffold-free 3D microtissue spheroids.…”

Lactoferrin and parathyroid hormone are not harmful to primary tenocytes in vitro, but PDGF may be

“…Clinically, PDGF receptor expression is increased in diseased tendons, and is associated with hypercellularity 32 , and taken into context with our findings on the poor effects of PDGF on tendon-related genes, this suggests that PDGF may not be the most suitable factor for improving tendon healing outcomes. IGF-1 and TGF-β are both present within the tendon matrix 18 , and in vitro have been shown to be involved in tendon cell growth, collagen production and matrix remodelling [19][20][21][22][23][24][25] . Developmental studies suggest a key role for TGFβ1 in tendon development 33 , and inhibiting TGFβ1 has generally resulted in poor healing outcomes in in vivo tendon defect models 34 .…”

“…These were chosen as PDGF is among the most widely studied tendon factors, and IGF-1 and TGF-β are both present within the tendon matrix. Furthermore, all are upregulated during tendon healing, and are known to enhance tendon cell growth, collagen production and matrix remodelling, in vitro [18][19][20][21][22][23][24][25][26] . Furthermore, we have focussed on not only classical tendon-related outputs, such as cell proliferation and collagen production, we have also assessed the tenocyte gene expression profile, assessing genes important in tenocyte, chondrocyte and osteoblast biology.…”