Decellularized tissue exhibits large differences of extracellular matrix properties dependent on decellularization method: novel insights from a standardized characterization on skeletal muscle.

Decellularized matrices are an attractive choice of scaffold in regenerative medicine as they can provide the necessary extracellular matrix (ECM) components, signals and mechanical properties. Various detergent-based protocols have already been proposed for decellularization of skeletal muscle tissue. However, a proper comparison is difficult due to differences in species, muscle origin and sample sizes. Moreover, a thorough evaluation of the remaining acellular matrix is often lacking. We compared an in-house developed decellularization protocol to four previously published methods in a standardized manner. Porcine skeletal muscle samples with uniform thickness were subjected to in-depth histological, ultrastructural, biochemical and biomechanical analysis. In addition, 2D and three-dimensional cytocompatibility experiments were performed. We found that the decellularization methods had a differential effect on the properties of the resulting acellular matrices. Sodium deoxycholate combined with deoxyribonuclease I was not an effective method for decellularizing thick skeletal muscle tissue. Triton X-100 in combination with trypsin, on the other hand, removed nuclear material but not cytoplasmic proteins at low concentrations. Moreover, it led to significant alterations in the biomechanical properties. Finally, sodium dodecyl sulphate (SDS) seemed most promising, resulting in a drastic decrease in DNA content without major effects on the ECM composition and biomechanical properties. Moreover, cell attachment and metabolic activity were also found to be the highest on samples decellularized with SDS. Through a newly proposed standardized analysis, we provide a comprehensive understanding of the impact of different decellularizing agents on the structure and composition of skeletal muscle. Evaluation of nuclear content as well as ECM composition, biomechanical properties and cell growth are important parameters to assess. SDS comes forward as a detergent with the best balance between all measured parameters and holds the most promise for decellularization of skeletal muscle tissue.

The Immunoarchitecture of Human Extraocular Muscles.

Purpose: The purpose of this study was to describe the immunoarchitecture of normal extraocular muscles (EOMs) in terms of presence, distribution, and organization of various immune cells. Methods: We performed unilateral orbital exenterations in six fresh human cadavers from elderly patients, followed by dissection of the medial, lateral, superior and inferior rectus, superior and inferior oblique, and superior palpebral levator muscle in their entirety. We further cross sectioned each EOM in an anterior, central, and posterior third. After immunohistochemical staining for CD3, CD8, CD20, CD138, CD68, and podoplanin, quantitative analysis was performed. Results: We found all EOMs (rectus, oblique, and levator muscles) to harbor both T- and B-lymphocytes, with a B-lymphocyte dominance and an absence of plasma cells. The highest prevalence of immune cells was seen in the muscle bellies, with, on average, 488 ± 63 CD3+ T-lymphocytes and 44 ± 110 CD20+ B-lymphocytes per mm2, and significant differences from the anterior (T-lymphocytes) and posterior (T- and B-lymphocytes) thirds. T- and B-lymphocytes were primarily organized in hotspots in the vicinity of blood vessels. In addition, a small resident population of macrophages scattered throughout the specimens was detected. No lymphatic vessels were found in any of the EOMs. Conclusions: These findings can serve as a reference dataset in the assessment of EOM biopsies in the diagnostic process of inflammatory orbital and systemic disorders. Moreover, from a regenerative perspective, our results highlight the importance of taking into account the presence of a resident immune cell population when studying the host immune response on transplanted tissues or engineered constructs.

Decellularized skeletal muscle: A versatile biomaterial in tissue engineering and regenerative medicine.

A wide range of synthetic and natural biomaterials is available for skeletal muscle tissue engineering. One class of natural biomaterials consists of the extracellular matrix (ECM) from donor skeletal muscle. To obtain this ECM, the cellular compartment must be completely removed while retaining the native composition and ultrastructure of the tissue as much as possible. In this review, the progress and challenges in the field of skeletal muscle decellularization are discussed by reviewing the different decellularization methods available and by highlighting the different applications of the scaffolds. Decellularized skeletal muscle has mainly been studied in the context of regeneration with a focus on its tissue-specific morphological features as well as biochemical cues to stimulate muscle regeneration. However, in this review, the potential applications of decellularized skeletal muscle are expanded beyond the regenerative setting to demonstrate its versatility as a biomaterial. Acellular matrices are discussed as a platform to study cell-matrix interactions and drug screening. Decellularized skeletal muscle ECM can also be further processed to re-engineer its structure. An overview is presented of materials processed from decellularized skeletal muscle, ranging from injectable hydrogels, bioinks for 3D bioprinting, electrospun nanofibers to coatings for cell culture.

Detergent-based decellularized peripheral nerve allografts: An in vivo preclinical study in the rat sciatic nerve injury model.

Nerve autograft is the gold standard technique to repair critical nerve defects, but efficient alternatives are needed. The present study evaluated the suitability of our novel Roosens-based (RSN) decellularized peripheral nerve allografts (DPNAs) in the repair of 10-mm sciatic nerve defect in rats at the functional and histological levels after 12 weeks. These DPNAs were compared with the autograft technique (AUTO) and Sondell (SD) or Hudson (HD) based DPNAs. Clinical and functional assessments demonstrated a partial regeneration in all operated animals. RSN-based DPNAs results were comparable with SD and HD groups and closely comparable with the AUTO group without significant differences (p > .05). Overall hematological studies confirmed the biocompatibility of grafted DPNAs. In addition, biochemistry revealed some signs of muscle affection in all operated animals. These results were confirmed by the loss of weight and volume of the muscle and by muscle histology, especially in DPNAs. Histology of repaired nerves confirmed an active nerve tissue regeneration and partial myelination along with the implanted grafts, being the results obtained with HD and RSN-based DPNAs comparable with the AUTO group. Finally, this in vivo study suggests that our novel RSN-based DPNAs supported a comparable tissue regeneration, along the 10-mm nerve gap, after 12-week follow-up to HD DPNAs, and both were superior to SD group and closely comparable with autograft technique. However, further improvements are needed to overcome the efficacy of the nerve autograft technique.

Qualitative and Quantitative Evaluation of a Novel Detergent-Based Method for Decellularization of Peripheral Nerves.

Tissue engineering is an emerging strategy for the development of nerve substitutes for peripheral nerve repair. Especially decellularized peripheral nerve allografts are interesting alternatives to replace the gold standard autografts. In this study, a novel decellularization protocol was qualitatively and quantitatively evaluated by histological, biochemical, ultrastructural and mechanical methods and compared to the protocol described by Sondell et al. and a modified version of the protocol described by Hudson et al. Decellularization by the method described by Sondell et al. resulted in a reduction of the cell content, but was accompanied by a loss of essential extracellular matrix (ECM) molecules such as laminin and glycosaminoglycans. This decellularization also caused disruption of the endoneurial tubes and an increased stiffness of the nerves. Decellularization by the adapted method of Hudson et al. did not alter the ECM composition of the nerves, but an efficient cell removal could not be obtained. Finally, decellularization by the method developed in our lab by Roosens et al. led to a successful removal of nuclear material, while maintaining the nerve ultrastructure and ECM composition. In addition, the resulting ECM scaffold was found to be cytocompatible, allowing attachment and proliferation of adipose-derived stem cells. These results show that our decellularization combining Triton X-100, DNase, RNase and trypsin created a promising scaffold for peripheral nerve regeneration.

Evaluation methods as quality control in the generation of decellularized peripheral nerve allografts.

Nowadays, the high incidence of peripheral nerve injuries and the low success ratio of surgical treatments are driving research to the generation of novel alternatives to repair critical nerve defects. In this sense, tissue engineering has emerged as a possible alternative with special attention to decellularization techniques. Tissue decellularization offers the possibility to obtain a cell-free, natural extracellular matrix (ECM), characterized by an adequate 3D organization and proper molecular composition to repair different tissues or organs, including peripheral nerves. One major problem, however, is that there are no standard quality control methods to evaluate decellularized tissues. Therefore, in this review, a brief description of current strategies for peripheral nerve repair is given, followed by an overview of different decellularization methods used for peripheral nerves. Furthermore, we extensively discuss the available and currently used methods to demonstrate the success of tissue decellularization in terms of the cell removal, preservation of essential ECM molecules and maintenance or modification of biomechanical properties. Finally, orientative guidelines for the evaluation of decellularized peripheral nerve allografts are proposed.

Effects of different sterilization methods on the physico-chemical and bioresponsive properties of plasma-treated polycaprolactone films.

For most tissue engineering applications, surface modification and sterilization of polymers are critical aspects determining the implant success. The first part of this study is thus dedicated to modifying polycaprolactone (PCL) surfaces via plasma treatment using a medium pressure dielectric barrier discharge, while the second part focuses on the sterilization of plasma-modified PCL. Chemical and physical surface changes are examined making use of water contact angle goniometry (WCA), x-ray photoelectron spectroscopy and atomic force microscopy. Bioresponsive properties are evaluated by performing cell culture tests. The results show that air and argon plasmas decrease the WCA significantly due to the incorporation of oxygen-containing functionalities onto the PCL surface, without modifying its morphology. Extended treatment times lead to PCL degradation, especially in the case of air plasma. In addition to surface modification, the plasma potential to sterilize PCL is studied with appropriate treatment times, but sterility has not been achieved so far. Therefore, plasma-modified films are subjected to UV, H2O2 plasma (HP) and ethylene oxide (EtO) sterilizations. UV exposure of 3 h does not alter the PCL physico-chemical properties. A decreased wettability is observed after EtO sterilization, attributable to the modification of PCL chain ends reacting with EtO molecules. HP sterilization increases the WCA of the plasma-treated samples, presumably due to the scission of the hydrophilic bonds generated during the prior plasma treatments. Moreover, HP modifies the PCL surface morphology. For all the sterilizations, an improved cell adhesion and proliferation is observed on plasma-treated films compared to untreated ones. EtO shows the lowest proliferation rate compared to HP and UV. Overall, of the three sterilizations, UV is the most effective, since the physical alterations provoked by HP might interfere with the structural integrity when it comes to 3D scaffolds, and the chemical modifications caused by EtO, in addition to its toxicity, interfere with PCL bioactivity.

Redifferentiation of High-Throughput Generated Fibrochondrocyte Micro-Aggregates: Impact of Low Oxygen Tension.

In meniscus tissue engineering strategies, enhancing the matrix quality of the neomeniscal tissue is important. When the differentiated phenotype of fibrochondrocytes is lost, the quality of the matrix becomes compromised. The objective of this study was to produce uniform fibrochondrocyte micro-aggregates with desirable phenotype and tissue homogeneity in large quantities using a simple and reproducible method. Furthermore, we investigated if hypoxia could enhance the matrix quality. Porcine fibrochondrocytes were expanded at 21% oxygen until passage 3 (P3) and a gene expression profile was determined. P3 fibrochondrocytes were cultivated in chondrogenic medium at 5 and 21% oxygen in high-throughput agarose chips containing 2,865 microwells 200 µm in diameter. Evaluation included live/dead staining, histological examination, immunohistochemistry, dimethylmethylene blue assay and real-time reverse transcriptase quantitative polymerase chain reaction of the micro-aggregates. Gene expression analysis showed a drastic decline in collagen II and high expression of collagen I during monolayer culture. After 4 days, uniform and stable micro-aggregates could be produced. The redifferentiation and matrix quality of the hypoxic cultured micro-aggregates were enhanced relative to the normoxic cultures. Sulfated glycosaminoglycan synthesis was significantly higher, and collagen II expression and the collagen II/collagen I ratio were significantly upregulated in the hypoxic cultures. High-throughput production of uniform microtissues holds promise for the generation of larger-scale tissue engineering constructs or optimization of redifferentiation mechanisms for clinical applications.