Abstract
BackgroundPeripheral nerve injury (PNI) is one of the essential causes of physical disability with a high incidence rate. The traditional tissue engineering strategy, Top-Down strategy, has some limitations. A new tissue-engineered strategy, Bottom-Up strategy (tissue-engineered microtissue strategy), has emerged and made significant research progress in recent years. However, to the best of our knowledge, microtissues are rarely used in neural tissue engineering; thus, we intended to use microtissues to repair PNI.MethodsWe used a low-adhesion cell culture plate to construct adipose-derived mesenchymal stem cells (ASCs) into microtissues in vitro, explored the physicochemical properties and microtissues components, compared the expression of cytokines related to nerve regeneration between microtissues and the same amount of two-dimension (2D)-cultured cells, co-cultured directly microtissues with dorsal root ganglion (DRG) or Schwann cells (SCs) to observe the interaction between them using immunocytochemistry, quantitative reverse transcription polymerase chain reaction (qRT-PCR), enzyme-linked immunosorbent assay (ELISA). We used grafts constructed by microtissues and polycaprolactone (PCL) nerve conduit to repair sciatic nerve defects in rats.ResultsThe present study results indicated that compared with the same number of 2D-cultured cells, microtissue could secrete more nerve regeneration related cytokines to promote SCs proliferation and axons growth. Moreover, in the direct co-culture system of microtissue and DRG or SCs, axons of DRG grown in the direction of microtissue, and there seems to be a cytoplasmic exchange between SCs and ASCs around microtissue. Furthermore, microtissues could repair sciatic nerve defects in rat models more effectively than traditional 2D-cultured ASCs.ConclusionTissue-engineered microtissue is an effective strategy for stem cell culture and therapy in nerve tissue engineering.
Highlights
Peripheral nerve injury (PNI) is one of the essential causes of physical disability with a high incidence rate
Characterization of adipose-derived mesenchymal stem cells (ASCs) Flow cytometry showed that the stem cell markers CD73 (87.35%), CD90 (99.96%), and CD105 (95.14%) were generally highly expressed in ASCs, and less than 2% of ASCs were immunopositive for the hematopoietic stem cell markers CD34, CD45 (Additional file 2: Figure S2)
Cell viability was evaluated by live/ dead staining, which showed that the majority of ASCs remained viable (Fig. 1c)
Summary
Peripheral nerve injury (PNI) is one of the essential causes of physical disability with a high incidence rate. Traditional tissue engineering adopts the Top-Down strategy: the graft skeleton matching the size and shape of the recipient region is constructed through three-dimension (3D) printing and other methods, and high-density seed cells with or without bioactive molecules are added to scaffold to construct the tissue engineered graft [10,11,12]. Traditional two-dimension (2D)-cultured cells require trypsin digestion before use, which can destroy the extracellular matrix (ECM) and the microenvironment for cell growth and reduce cell activity [13, 14]; cells were unevenly distributed in the scaffold, and cells in the central region were necrotic due to hypoxia and difficult excrement of metabolic waste [15, 16]; after transplantation to the recipient region, single cells are vulnerable to the interference of various factors, such as ischemia and inflammation, leading to cell loss or death [16]
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