Effects of hydrogen peroxide on biological characteristics and osteoinductivity of decellularized and demineralized bone matrices.

Due to the similar collagen composition and closely physiological relationship with soft connective tissues, demineralized bone matrices (DBMs) were used to repair the injured tendon or ligament. However, the osteoinductivity of DBMs would be a huge barrier of these applications. Hydrogen peroxide (H2 O2 ) has been proved to reduce the osteoinductivity of DBMs. Nevertheless, the biological properties of H2 O2 -treated DBMs have not been evaluated completely, while the potential mechanism of H2 O2 compromising osteoinductivity is also unclear. Hence, the purpose of this study was to characterize the biological properties of H2 O2 -treated DBMs and search for the proof that H2 O2 could compromise osteoinductivity of DBMs. Decellularized and demineralized bone matrices (DCDBMs) were washed by 3% H2 O2 for 12 h to fabricate the H2 O2 -treated DCDBMs (HPTBMs). Similar biological properties including collagen, biomechanics, and biocompatibility were observed between DCDBMs and HPTBMs. The immunohistochemistry staining of bone morphogenetic protein 2 (BMP-2) was negative in HPTBMs. Furthermore, HPTBMs exhibited significantly reduced osteoinductivity both in vitro and in vivo. Taken together, these findings suggest that the BMP-2 in DCDBMs could be the target of H2 O2 . HPTBMs could be expected to be used as a promising scaffold for tissue engineering. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2019.

An engineered tendon/ligament bioscaffold derived from decellularized and demineralized cortical bone matrix.

Demineralized bone matrix (DBM), as an extracellular matrix (ECM), has had limited use as a medical replacement although studies have reported a possibility for its use in tendon or ligament tissue engineering. To be an acid-extracted organic matrix, DBM contains much of bone protein, with a small amount of inorganic solids and some cell debris. However, cell debris is a critical factor that triggers inflammatory reaction in clinical reconstructions using ECM. In this study, we used a protocol incorporating the use of detergent with nuclease treatment to prepare decellularized DBM (DCDBM). DNA quantification analysis and histological observation confirmed that cells were completely removed from DBM. The inherent ultrastructure of DBM was well preserved after decellularization as observed through scanning electron microscopy. Additionally, calcium and phosphorus were absent and the specific functional groups of collagen remained after decellularization. Moreover, 79.71% of the tensile strength of DBM was retained and the viscoelastic properties were similar to the ligament. Furthermore, DCDBM promoted the adhesion and proliferation of NIH-3T3 fibroblasts in vitro and triggered less inflammation response at 12 weeks subcutaneous implantation in a rat model. These results demonstrate that the DCDBM has the potential to be used for tendon and ligament replacement. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 468-478, 2018.

Fabrication and characterization of a decellularized bovine tendon sheet for tendon reconstruction.

Obtaining a performing decellularized tendon scaffold with proper dimensions and adequate availability is highly desirable. However, the combined study of complete decellularization and detailed characterization of native tendon extracellular matrix (ECM) from large animals is still lacking. In the present study, we developed a new decellularization protocol, including physical methods and enzymatic solutions for processing bovine Achilles tendons, and produced a decellularized bovine tendon sheet (DBTS) scaffold for tendon reconstruction. The decellularization effectiveness was demonstrated by DNA quantification and histological qualification. The removal of the alpha-gal epitopes was confirmed by ELISA analysis and immunohistochemical staining. After decellularization, there were no significant alterations of the native tendon extracellular matrix (ECM) properties, including the internal ultrastructure, biochemical compositions such as collagen, glycosaminoglycans (GAGs), basic fibroblast growth factor (bFGF) and transforming growth factor-ß1 (TGF-ß1), fibronectin and decorin, as well as substantial mechanical strength. Furthermore, the DBTS scaffold showed no cytotoxic and promoted the proliferation of NIH-3T3 fibroblasts in vitro. When implanted into rat subcutaneous tissue, the DBTS scaffold displayed excellent histocompatibility in vivo. Our results, while offering a new decellularization protocol for large tendons, can provide a promising biologic scaffold with a combination of mechanical strength and tendon ECM bioactive factors that may have many potential applications in tendon reconstruction. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 2299-2311, 2017.

The utilization of decellularized tendon slices to provide an inductive microenvironment for the proliferation and tenogenic differentiation of stem cells.

The extracellular matrix (ECM) microenvironment for the stem cell niches, including but not limited to the biochemical composition, matrix topography, and stiffness, is crucial to stem cell proliferation and differentiation. The purpose of this study was to explore the capacity of the decellularized tendon slices (DTSs) to induce stem cell proliferation and tenogenic differentiation. Rat adult stem cells, including tendon-derived stem cells (TDSCs) and bone marrow-derived stem cells (BMSCs), were identified to have universal stem cell characteristics. The DTSs were found to retain the native tendon ECM microenvironment cues, including the inherent surface topography, well-preserved tendon ECM biochemical composition and similar stiffness to native tendon. When the TDSCs and BMSCs were cultured on the DTSs respectively, the LIVE/DEAD assay, alamarBlue® assay, scanning electron microscopy examination and qRT-PCR analysis demonstrated that the DTSs have the capacity to support these stem cells homogeneous distribution, alignment, significant proliferation and tenogenic differentiation. Taken together, the findings of this study indicate that the DTSs can provide a naturally inductive microenvironment for the proliferation and tenogenic differentiation of TDSCs and BMSCs, supporting the use of decellularized tendon ECM as a promising and valuable approach for tendon repair/reconstruction.