Preparation and characterization of decellularized tendon slices for tendon tissue engineering

Abstract

To develop a naturally derived tendon tissue engineering scaffold with the preservation of the native ultrastructure, tensile strength, and biochemical composition of the tendon extracellular matrix (ECM), decellularized tendon slices (DTSs) were prepared using repetitive freeze/thaw of the intact Achilles tendons, frozen section, and nuclease treatment. The DTSs were characterized in the native ultrastructure, mechanical properties, biochemical composition, and cytocompatibility. Histological examination and DNA quantification analysis confirmed that cells were completely removed from tendon tissue by repetitive freeze/thaw in combination with nuclease treatment 12 h. The intrinsic ultrastructure of tendon tissue was well preserved based on scanning electron microscopy examination. The tensile strength of the DTSs was retained 85.62% of native tendon slice. More than 93% of proteoglycans (fibromodulin, biglycan) and growth factors (TGF-β1, IGF-1, VEGF, and CTGF) inherent in tendon ECM were preserved in the DTSs according to ELISA analysis. Furthermore, the DTSs facilitated attachment and repopulation of NIH-3T3 fibroblasts in vitro. Overall, the DTSs are sheet scaffolds with a combination of elemental mechanical strength and tendon ECM bioactive factors that may have many potential applications in tendon tissue engineering.

Freeze-Thaw Cycles Enhance Decellularization of Large Tendons

“…10 The ideal scaffold should not only be biocompatible and match the natural biomechanical properties, but should also comprise naturally structured, extracellular matrix proteins that can interact with the repopulating cells and direct them toward tenogenic differentiation and matrix remodeling. 10,11 Decellularization of tendon tissue offers the unique opportunity of obtaining a scaffold with a natural extracellular matrix structure that is hypoimmunogenic 13,14 and displays biomechanical properties very similar to the original tissue, [15][16][17][18] providing great advantages for potential clinical application and research use.…”

“…[15][16][17][18][19][20][21] Results obtained after applying different detergent-based protocols remain conflicting. While t-octyl-phenoxypolyethoxyethanol (Triton X-100) did not lead to satisfactory results in an early study, 15 it was found to be most effective by others.…”

“…25 However, our own unpublished data showed that this procedure, although reducing the content of vital cells, did not remove DNA, which was recently supported by others. 18 Freeze-thaw cycles with subsequent nuclease treatment led to nearly complete removal of cells and DNA, 18 but nuclease treatment was performed on sliced tendon only, 18,21 thus, this protocol does not appear applicable for decellularization of whole, large tendon constructs.…”

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.…”