In recent years, tissue-engineered microtissue strategy has made significant research progress in many research fields. In bone tissue engineering, Totaro et al.  mixed human mesenchymal stem cells (hMSCs) and polycaprolactone (PCL) - hydroxyapatite (HA) microscaffolds and placed them in a rotating flask for dynamic culture to construct bone microtissue. Compared with 2D culture, hMSCs in the microtissue have robust osteogenic differentiation ability. In cartilage tissue engineering, Wang et al.  cultured nanofiber microcarrier mimicking ECM and bone marrow-derived mesenchymal stem cells (BMSCs) into functional cartilage microtissues under microgravity condition, and implanted the microtissues directly into the knee cartilage defects of Sprague-Dawley rats, achieving promising repair effects. In myocardial tissue engineering, the researchers encapsulated induced pluripotent stem cells (iPSCs) into the omental ECM hydrogel in microfluidic system to make myocardial microtissues. The injection of microtissue into the gastrocnemius muscle did not damage the cellular activity within the microtissue . Similarly, the application of microtissue strategy in adipose tissue engineering [22, 28], vascular tissue engineering [41, 42], oncology research [43, 44] and other fields has also achieved extensive achievements. However, the application of microtissue strategy in neural tissue engineering is rare, we used microtissue in the current study to conduct research on nerve injury repair.
At present, there are four main approaches of microtissue construction, including hanging-drop cell culture , spheroid-based cell culture , microcarrier or microscaffold-based cell culture [19, 24] and microgel or microsphere-based cell culture . The first two kinds of microtissue are formed by cell self-aggregation with microgravity or low adherent cell culture materials, and the latter two kinds of microtissue are formed using microcarriers or microgel loaded cells. In our study, to explore the direct interaction between microtissue and SCs or DRG in vitro, we chose the hanging-drop method and spheroid method to construct microtissue. However, we found that the microtissue constructed by the hanging-drop method was loose, and easy to disintegrate with the culture time, so we chose spheroid method to construct the microtissue for subsequent experiments.
An important component of tissue-engineered microtissue is the seed cell. MSCs, derived from pluripotent adult precursors with self-renewal ability in the germ layer of the mesenchymal layer, can promote nerve regeneration by inducing the secretion of various neurotrophic factors and are promising seed cells for tissue engineering [45, 54]. Although BMSCs have been used to repair tissue damage, including that of the peripheral nerve, the low yield and invasiveness in the extraction process seriously limit the clinical application of BMSCs . Compared with BMSCs, ASCs with lower invasiveness in the extraction process, higher yield and faster proliferation rate in vitro are a better choice for tissue engineering . Moreover, the application of ASCs has achieved a promising repair effect in PNI [57–60]. Taken together, these advantages indicated that ASCs are a good choice for the construction of microtissues.
Regarding the preparation of microtissues, the number of cells contained in each microtissue varies greatly, from 1440 to 40,000 cells per microtissue [22, 61, 62]. Paradoxically, the function of microtissue is poor when there are too few cells, and cell necrosis will appear in the depths of microtissue when there are too many cells. To explore the appropriate number of cells in microtissue, we constructed microtissues using 5000, 7500, and 10,000 cells, respectively. After three days of culture, Live/Dead staining was performed on the microtissues, showing that most of the cells in the three types of microtissues remained viable. To maximize the function of the microtissues, we selected microtissues containing 10,000 cells for subsequent experiments. With the culture time, the diameter of microtissues decreased gradually and presented a compacted state, which was consistent with the research results of Colle et al. .
ECM is a 3D network without cells. Its main components are collagen, proteoglycan/glycosaminoglycan, elastin, fibronectin, laminin and other glycoproteins . Fibronectin can be expressed in the ECMs of various important functional cells in vertebrates . Fibronectin molecules secreted by cells are assembled into supramolecular fibers, connected to form a fibrous network, and cells adhere to the fibrous network . Laminin is a large heterotrimeric glycoprotein. Laminin molecules participate in the composition of ECM and cell adhesion through the interaction with other ECM components and resident cells [66, 67]. In the current study, immunofluorescence analysis showed that red-stained fibronectin and green-stained laminin were all over the microtissue, indicating that there was ECM in the microtissue, and it was because of the existence of ECM that the cells could aggregate into microtissue.
qRT-PCR and ELISA results indicated that BDNF expression at transcriptional and translational levels in the Microtissue group was significantly higher than the 2D group. BDNF can enhance the intrinsic capacity of axonal regeneration. On the one hand, BDNF can activate the BDNF/TrkB pathway to form the actin waves; on the other hand, BDNF can improve cAMP levels and upregulates the CREB-cjun-STAT3-Gap-43 pathway to improve the ability of axon growth [68, 69]. The neural electrophysiological evaluation showed that the peak amplitude of CAMPs in the Microtissue group with a higher expression level of BDNF was significantly higher than the 2D group and the Hollow group and similar to that in the ANG group (Fig. 7c). In addition to promoting axonal growth, BDNF also plays an important role in the proliferation of Schwann cells. BDNF can regulate the proliferation of SCs to promote remyelination . A previous study indicated that long-chain non-coding RNA MALAT1 was enhanced after peripheral nerve injury, which increased the expression and secretion of BDNF through sponging miR-129-5p, thus promoting proliferation and migration of Schwann cells . Under the effect of BDNF, the proliferation rate of SCs in the Microtissue group was significantly higher than the 2D group in the transwell system.
Similarly, the expression of VEGF in both transcriptional and translational levels in the Microtissue group was higher than in the 2D group. Although VEGF can promote vasculogenesis and angiogenesis, increasing evidence suggests that VEGF plays a key role in the nervous system, such as promoting axon growth in various neurons . VEGF-A-165 is the dominant isoform in most mammalian tissues, two isoform families are produced by alternative splicing: VEGF-A-165a and VEGF-A-165b, and VEGF-A-165a have nerve regeneration potential [73, 74]. Previous studies have shown that VEGF has a significant effect on the axonal growth of DRG . Moreover, VEGF attracts and influences the speed and size of the growth cone during nerve regeneration . Ruiz de Almodovar et al. showed that VEGF expressed and secreted by floor plate and VEGF receptor Flk1 expressed and secreted by commissural neurons jointly guided axonal growth in a spinal cord ventral midline model system . Taken together, VEGF can promote axon growth and guide axon growth direction with its receptor Flk1. In the direct co-culture system of microtissue and DRG, DRG axons are much longer and grow towards microtissue, which may be due to the large amount of VEGF secreted by microtissue (Fig. 5).
qRT-PCR results indicated that anti-inflammatory cytokines (IL-4, IL-10, and IL-13) expression at the transcriptional level in the Microtissue group was significantly higher than the 2D group. We performed ELISA for IL-13, and the results showed that the secretion level of IL-13 in the Microtissue group was significantly higher than the 2D group. In the early stage of injury, the inflammatory reaction can better limit the necrotic and apoptotic cells or foreign bodies in a certain area to prevent the expansion of lesions. However, in the later stage, inflammatory cells often overreact, infiltrate a large number of local areas, release a large number of pro-inflammatory factors, but further aggravate tissue damage . Compared with 2D ASCs, microtissues can secrete various anti-inflammatory factors and better inhibit the inflammatory response, which may reduce tissue damage and achieve the purpose of repair. In addition, IL-4 has been reported to regulate cell survival, proliferation, and branching in the nervous system, and promote peripheral axonal regeneration .
ELISA results indicated that NGF expression at translational level in the Microtissue group was significantly higher than those in the 2D group. NGF is widely distributed in various tissues and organs of the body, the first isolated neurotrophic factor that promotes axon growth and elongation . A previous study indicated that NGF could promote axon growth by abolishing neuronal growth cone-collapsing factor semaphorin3A (Sema3A)-induced axon growth inhibition . Moreover, exogenous NGF can activate the autophagy of dedifferentiated SCs, accelerate the clearance and phagocytosis of myelin fragments, promoting axon and myelin regeneration . In addition to promoting axon growth, NGF may also be involved in myelin formation. Previous studies reported that NGF may induce remyelination through its activity on congenital oligodendrocyte precursors, and can act directly on various cells involved in myelination (such as Schwann cells) [82, 83]. This explains why the density of myelinated nerve fibers and the thickness of myelin sheath in the Microtissue group were significantly higher than those in the Hollow and 2D groups. In addition, better myelin regeneration leads to a shorter latency of CAMPs in the Microtissue group.
In vitro, we used the transwell system to indirectly co-culture DRG with microtissues or monolayer cells. The upper insert and lower chamber were separated by a 0.4µm polyester membrane, allowing the upper and lower chambers to exchange cytokines and block cell contact. Therefore, we used the transwell system to study the difference between the two groups of paracrine effects on promoting DRG axon growth. Compared with the traditional single-layer cultured cells, the microtissues can secrete more BDNF, NGF, VEGF and IL-13, all of them can promote the growth of axons, so the length of axons in the Microtissue group is significantly longer than the 2D group. The axonal growth-promoting effects of above neurotrophic factors, angiogenic factors and anti-inflammatory factors have been verified in vivo. In the early stage of transplantation (4 weeks), axons in the Microtissue group penetrated the whole nerve graft, whereas the ratio of axon length in the 2D group and the Hollow group was only 66% and 47%. In the later stage of transplantation (12 weeks), although the axons of all four groups penetrated the nerve grafts, immunofluorescence analysis showed that the axon density of the Microtissue group was significantly higher than the 2D group and the Hollow group. In addition, higher SFI and better gastrocnemius recovery also indicated that the Microtissue group had achieved better nerve repair effect than the 2D group and Hollow group.
Microtissue can secrete many neurotrophic factors, angiogenesis factors and anti-inflammatory factors to promote nerve regeneration. In addition to the paracrine effect, whether microtissue can directly act on DRG or Schwann cells is also worthy to explore. To solve this problem, we conducted a direct contact co-culture of microtissues with DRG or Schwann cells to explore cell-to-cell interaction between them. After 7 days of direct co-culture between microtissues and DRG, the axon length of DRG was much longer than the indirect co-culture system, and the axon grew in the direction of microtissues. As discussed earlier, VEGF secreted by microtissues can promote and guide the growth of axons. In the indirect co-culture system, VEGF was evenly dispersed in the medium, while in the direct co-culture system, the instantaneous concentration of VEGF around microtissues was higher than that in other areas, so axons grew in the microtissues direction. After 3 days of direct co-culture between microtissues and SCs, we found an interesting phenomenon. Red and green double-stained cells appeared around the red-stained microtissues and green-stained Schwann cells. These cells were spindle-like in morphology, which is similar to SCs, and the two kinds of cells appeared to have undergone cytoplasmic exchange. This phenomenon may have implications for the research of the outcome of stem cells implantation in vivo, and the causes and mechanisms of direct cellular interactions need to be explored further.