Efficient decellularization for tissue engineering of the tendon-bone interface with preservation of biomechanics

Abstract

Interfaces between tendon/ligament and bone (“entheses”) are highly specialized tissues that allow for stress transfer between mechanically dissimilar materials. Entheses show very low regenerative capacity resulting in high incidences of failure after surgical repair. Tissue engineering is a promising approach to recover functionality of entheses. Here, we established a protocol to decellularize porcine entheses as scaffolds for enthesis tissue engineering. Chemical detergents as well as physical treatments were investigated with regard to their efficiency to decellularize 2 mm thick porcine Achilles tendon entheses. A two-phase approach was employed: study 1 investigated the effect of various concentrations of sodium dodecyl sulfate (SDS) and t-octylphenoxypolyethoxy-ethanol (Triton X-100) as decellularization agents. The most efficient combination of SDS and Triton was then carried forward into study 2, where different physical methods, including freeze-thaw cycles, ultrasound, perfusion, and hydrostatic washing were used to enhance the decellularization effect. Cell counts, DNA quantification, and histology showed that washing with 0.5% SDS + 1% Triton X-100 for 72 h at room temperature could remove ~ 98% cells from the interface. Further investigation of physical methods proved that washing under 200 mmHg hydrostatic pressure shortened the detergent exposing time from 72 h to 48 h. Biomechanical tensile testing showed that the biomechanical features of treated samples were preserved. Washing under 200 mmHg hydrostatic pressure with 0.5% SDS + 1% Triton X-100 for 48 h efficiently decellularized entheses with preservation of matrix structure and biomechanical features. This protocol can be used to efficiently decellularize entheses as scaffolds for tissue engineering.

Residual sodium dodecyl sulfate in decellularized muscle matrices leads to fibroblast activation in vitro and foreign body response in vivo

“…This result is consistent with recently published work that showed intact tissue morphology in trachea decellularized under 1 Torr negative pressure, a stronger vacuum than used in our study (Butler et al, ). Athough we did not specifically test the effects of negative pressure on increasing efficiency of decellularization, a recent study using an SDS‐based method to decellularize trachea under 200 mmHg pressure compared to atmospheric pressure suggested they could reduce the treatment time from 72 to 48 hr while obtaining high decellularization efficiencies, which corroborates our anecdotal observation that negative pressure speeds decellularization (Xu et al, ).…”

Decellularization methods for developing porcine corneal xenografts and future perspectives

“…The efficacy of decellularization agents greatly fluctuates with the tissue being treated. For instance, while TX is able to decellularize cartilage, corneal decellularization studies have shown great variability . Du et al found that decellularization of pig corneas using TX was unachievable, regardless of high concentrations (5% w/v) and prolonged time (96 hours).…”

Next generation tissue engineering of orthopedic soft tissue-to-bone interfaces

“…[47,56–61] Decellularized or demineralized native matrices have also been used to engineer the enthesis. [48,59,60,62,63] …”

“…[150] Decellularized and demineralized bone matrix applied to the tendon-to-bone surgical suture site improved tendon-to-bone healing, demonstrated through increased amounts of fibrocartilage and mineralized fibrocartilage in the repair site enthesis and reduced rates of tendon failure. [63] Recent work has successfully decellularized the entire tendon-to-bone insertion, [62] with significant improvements in pullout strength using the decellularized tendon-to-bone insertion over the direct suture technique. [151] Cells were able to repopulate the graft and exhibited a transition from a cartilage-like to tenocyte-like morphology across the interface.…”