Biotech

A tissue bandage for pelvic ganglia injury – Nature Communications

Time Stamp: October 16, 2024 8:00 PM
Source Node: 695762

TB simulates mini-tissue and can potentially promote angiogenesis

The scaffold comprises a bilayer structure, with the bottom layer designed as a square grid featuring diagonal lines extending in both directions, creating a “✳” shape node between fibers. The top layer consists of strength fibers arranged in a single direction (Fig. 2a). The basal mini fibers have an average diameter of 27.90 ± 2.49 μm, with the 45° gap measuring 186.66 ± 8.60 μm and the 90° gap measuring 279.06 ± 11.08 μm. The strength fibers have 167.99 ± 7.39 μm diameter and a gap of 814.45 ± 41.89 μm (Fig. 2b). It provides the ability for the bandage to roll on one axis, similar to bandages used in clinical settings that can easily wrap around the ganglia (Fig. 2c). However, the pore size is uneven within one square, due to the limitation of printing accuracy and the addition of strength fibers (Fig. 2b). The frequency of pore size has distinct peaks at 2-3k and 7-8k (Fig. 2d). The cumulative frequency of pore size could be effectively fitted by the Sigmoidal-Boltzmann model (adjusted R² exceeding 99.77%), leading to the derivation of formula (1):

$${{y}}={{A}}2+frac{{{A}}1-{{{rm{A}}}}2}{1+{e}^{(x-x0)/{dx}}}$$

(1)

where A1 is the initial value, A2 is the final value, x0 is the center, and dx is the time constant. Specifically, A1 = −12.78, A2 = 100.41, x0 = 7.42, dx = 3.12.

**Fig. 2: Mini-tissue-like properties of TB.**

The cumulative frequencies of 10% (d10), 50% (d50), and 90% (d90) illustrate that the primary size distribution falls in the range of 3.11k–24.97k.

The scaffold provides excellent resistance to stretching, strength fibers exhibiting a tensile modulus 3.77 times higher than non-strength fibers due to their larger diameter. These added strength fibers also limit rolling in one direction, similar to clinical bandages, enhancing user-friendliness (Fig. 2e, f and Supplementary Movie 1). Supplementary Movie 2 showcases the TB fabrication process, which is simple and replicable, akin to standard cell culture practices (Fig. S1a–d). After analyzing different cell seeding densities, we found that using 300k cells minimized cell loss compared to 100k and 500k (Fig. S2b, c). This approach maintained over 90% cell fusion and reached 500k cells per unit within 5 days (Figs. 2g and S2a, d). In contrast, there is a decrease in both fusion efficiency and cell count over time in the 500k cell group. Therefore, 300k cells was employed for TB construction by the evidence of cell loss and fusion rates. The mature TB can accommodate over 5.97 × 10⁵ cells/cm², approximately 9.01 times more than petri dish (2D) can hold (Fig. 2h). Subsequent evaluation using an EdU kit showed that cells within TB maintained a high proportion in the G1 phase and a low proportion in the S and G2 phase (Fig. 2i–l). Furthermore, CCK8 analysis confirmed low proliferation rates in TB (Fig. 2m). These findings underscore the presence of contact inhibition in TB, a crucial factor for cell integration and assessing TB’s safety to prevent tumor formation in vivo²².

After 5 days of cultivation, the cells were firmly attached and well-integrated with each other. To verify its maturity, we evaluated the expression of vascular endothelial-related proteins in the TB. As depicted in Fig. 2n, cells were evenly spread on the bandage and showed endothelial markers like CD31, ZO-1, and Vinculin, indicating a mature and functional HUVECs phenotype on the scaffold^23,24,25. CD31 is crucial for identifying endothelial cells and maintaining cell-cell junction integrity on TB²⁴. Vinculin plays a pivotal role in cell-cell connections and ECM stability². ZO-1 is a marker for barrier function in forming TJs²⁶. Endothelial solid cell-cell junctions, cytoskeletal organization, and barrier function are essential for endothelial cell function in mimicking vascular tissue and enhancing TB survival in challenging in vivo environments²³. Additionally, transmission electron microscope confirmed the presence of TJs in TB (Fig. 2o)²⁷. These results provide initial evidence of successful mini-tissue construction.

The bottom layer provides both mechanical strength and anchors for cell adhesion, growth, and migration, forming a holistic cell bandage (Fig. 2p). A micro 3D structure within a scaffold effectively houses many cells arranged in layers (Fig. S3a). Vesicular structures on TB measuring approximately 346.17 ± 37.78 nm in diameter, aligning with the characteristic size of exosomes (Fig. S3b)²⁸. Cell migration with pseudopodia on the bandage is similar to vehicles navigating a highway (Supplementary Movie 2 and Fig. S3c). Additionally, flow cytometry reveals intercellular connections and angiogenesis-promoting functions in TB, with ZO-1, Vinculin, VE (an endothelial barrier protein controlling endothelial permeability and leukocyte transmigration), and VEGFA all exceeding 97% (Fig. 2q–s)²⁹. These findings bolster the concept that TB exhibits characteristics akin to mini-tissue.

Cryopreservation is a crucial process for TB applications. We found that a commercial SFCM is superior to the conventional 10% v/v DMSO method, resulting in significantly lower cell shedding and a reduced ratio of dead cells, preserving TB viability for up to 5 months (Figs. 2t and S4a, b). This method meets the clinical storage needs for TB products. After TB implantation, PCL triggers a foreign body reaction, recruiting macrophages for digestion and absorption. Initially, PCL degrades via ester bond hydrolysis into fragments encapsulated in 1k–6k nm² vesicles (Fig. S5a, b). Evaluation of fiber area shows a decrease at the early stage, followed by an increase after 6 weeks as neighboring fibers fuse with the integration of foreign-body giant cells (Fig. S5c, d). PCL degrades into ε-caprolactone and hydroxyhexanoic acid, which are metabolized into CO₂ and water without significant toxicity, confirming PCL’s safety for in vivo use²¹. These features make TB suitable for clinical use as a cell delivery product.

RNA-sequencing (RNA-seq) analysis demonstrated a distinctive variation in gene expression patterns between cells on TB and 2D (Fig. 3a). A total of 999 significant differentially expressed genes were obtained, including 548 up-regulated genes and 451 down-regulated genes (Fig. 3b). The Gene Ontology (GO) analysis suggested that these genes mainly enriched in angiogenesis, hypoxia, and ECM remodeling (Fig. 3c). The Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment results showed that the HIF-1 signaling pathway was promoted, which indicates a hypoxia status in TB (Fig. 3d). Since a high cell density within TB induces a hypoxic microenvironment that triggers elevated expression of VEGFA. VEGFA upregulation enhances the angiogenic capabilities of HUVECs to improve oxygen acquisition³⁰. Then, we extracted genes related to angiogenesis function. The comparison showed that these genes are mostly positively correlated (Fig. 3e). We found TB exhibited a substantial increase in VEGFA expression, exceeding that of the 2D culture by more than 4-fold. Moreover, the upregulation of proliferation and development genes (FOS, JUNB, and WNT5A) alongside neurotrophic and neuroprotective genes (F3, ENO2, and DNER) in TB further supports its potential for neuronal repair (Fig. 3f)^{31,32,33,34,35}. Accordingly, it seems that TB could be a potential strategy for therapy of impaired MPG.

**Fig. 3: RNA-seq analysis between TB and 2D culture.**

TB regulates voiding behavior and bladder function after MPG injury

The essential surgical procedure involved exposure, separation, crushing, insertion, and bandaging. Post-crushing, a noticeable impression was observed on MPG (Fig. 4a). Therapeutic efficacy was evaluated after 2 weeks. The injury and scaffold groups showed signs of urinary retention, such as significant bladder enlargement and increased residual urine. Additionally, the injury group showed diverticula. In contrast, the TB group displayed moderate bladder size and residual urine levels (Fig. 4b, c).

**Fig. 4: The voiding behavior and urodynamic analysis 2 weeks later.**

Behaviorally, void spots in the sham group were concentrated in two corners with larger spot areas, indicating effective bladder voiding and emptying capacity. The void spots in the scaffold and injury groups were randomly distributed, with smaller spot sizes and higher counts, indicating filling incontinence and loss of voluntary voiding (Fig. 4d–f). Similar to the sham group, the TB group had void spots mainly in corners but smaller and scattered, suggesting partial recovery of impaired voiding function. There was no significant difference in total spot area (Fig. 4e).

Urodynamic tests revealed distinct urinary patterns among the different groups. The sham group exhibited standard urination patterns characterized by effective contraction peaks (ECP) during urination and a well-regulated urine output cycle. In contrast, the TB group demonstrated prolonged voiding intervals and reduced frequency despite showing ECP, signifying delayed nerve conduction. Meanwhile, the scaffold and injury groups showed reduced urine volumes, with stable bladder pressure during perfusion and no noticeable ECP (Fig. 4g). However, instances of irregular urine overflows hinted at urine retention and incontinence, early signs of NB after MPG injury. There were no significant variances in baseline pressure across all groups. The scaffold and injury groups exhibited decreased ECP compared to the sham and TB groups (Fig. 4h). The ECP variation is significantly different. The total voiding times in these groups was higher than TB group, primarily due to urinary leakage (Fig. 4i). The TB group, experiencing delayed nerve signal conduction, displayed increased effectual urine output (EUO) compared to other groups, including max, mean, and total EUO (Fig. 4j). The cryopreservation TB also showed effective repairment (Fig. S6a–c). In summary, the TB group maintained the ability of voluntary voiding, while the scaffold and injury groups lost it.

TB mitigates histomorphology changes in the bladder

Bladder morphology analysis revealed significant inflation following MPG injury in all groups except the sham group. The inflation observed in the TB group was relatively minor. Continuous fullness following MPG injury led to increased bladder compliance, accompanied by thinning and remodeling of the bladder wall, and shallow mucosal folds (Fig. 5a). The scaffold and injury groups exhibited prominently damaged and denuded urothelium, compromising barrier function and potentially triggering bladder inflammation (Fig. 5b, c)^36,37. A notable reduction in mucosal thickness and transitional columnar epithelium of pan Cytokeratin (CKpan) expression was observed (Fig. 5d, e). In contrast, the TB group maintained higher mucosal thickness and an intact epithelial layer. The increased bladder pressure from urinary retention causes damage and atrophy of the bladder muscle, resulting in decreased muscle content within the bladder muscle layer. This effect intensifies at 4 weeks, manifested as widening of muscle space and fibrous tissue deposition. However, collagen deposition slightly increases in the bladder of scaffold and injury groups at 4 weeks, suggesting a longer time may be needed for full fibrosis development (Fig. S7a–d). As expected, vessel distribution also varied, with the TB group showing increase in expression compared to the sham and injury groups (Fig. 5f, g). The cryopreserved TB demonstrated the ability to protect bladder structure (Fig. S6d, e). In conclusion, MPG injury had a detrimental impact on bladder structure, but TB therapy mitigated these effects, suggesting its potential in preserving it.

**Fig. 5: Histomorphology change of bladder 2 weeks later.**

TB protects the MPG and supports its recovery

MPG serves as a vital neural structure, acting as a relay station for signal transmission^38,39. Its health is crucial for downstream organ function. Luciferase imaging revealed that TB can survive for approximately 5 days in vivo, indicating its involvement in early repairment stage (Fig. S8a, b). In Fig. 6a, b, the sham group exhibited well-organized MPG morphology with neatly distributed neural bodies and randomly scattered small vessels. In contrast, the TB, scaffold, and injury groups showed disruptions in morphology, decreased neuron numbers, inflammatory infiltration, and hemorrhage. Even in the injury group, necrosis and fibrosis were observed, while the scaffold group displayed neutrophil infiltration. Nissl bodies play a crucial role in protein synthesis within neurons. Toluidine blue staining revealed neuronal damage in all groups, except for the sham group. The scaffold and injury groups exhibited more severe conditions, including degradation, deformation, vacuolization, and decreased cellular contents (Figs. 6c and S9a). This is further supported by Fig. 6f, which shows reduction in Nissl bodies in the scaffold and injury groups compared to the sham and TB groups. In Fig. 6d, compared to the typical arterial and venous concomitant flow in sham group, the other groups exhibited smaller vessels and higher vessel density. Additionally, the TB group displayed constriction in small arteries. Further analysis of CD31 expression revealed upregulation of blood vessels in the TB group, validating the RNA-seq results indicating TB’s promotion of local neovascularization (Figs. 6g and S9b, c). The thawed TB can also protect MPG (Fig. S6f, g).

**Fig. 6: Histomorphology changes and axon variations in MPG at 2 weeks.**

The severity of peripheral nerve injuries hinges on demyelination and the extent of axonal and connective tissue damage⁴⁰. In Fig. 6e, the sham group showed normal myelinated axons, mitochondria (mit), and compact myelin sheath (MS). The injury and scaffold groups showed severe axonal degeneration, containing loss of axons, an irregular laminated structure of MS, reduced volume, myelin debris, and absence of mit. While the TB group maintained a certain number of axons, there were irregularities in the MS and decreased volume and mit number. We speculate that TB facilitates microvasculature involvement with myelinating glial cells regeneration, protects axon and MS through neurotrophic assistance⁴¹.

The G-ratio is a critical measure associated with nerve impulse conduction velocity, showing a positive correlation⁴². In the sham group, the average G-ratio was 0.74 ± 0.02. The injury and scaffold groups had lower G-ratios of 0.42 ± 0.02 and 0.51 ± 0.03, leading to a disorder of signal conduction. However, TB treatment maintained the G-ratio at 0.72 ± 0.02, mitigating the negative effects (Fig. 6h). Analysis of the G-ratio perimeter and area indicated that the injury and scaffold groups displayed abnormal thickening of the MS compared to the sham and TB groups, due to the relaxation of the sheath structure caused by axonal degeneration and clearance of myelin debris (Fig. 6i).

MPG is a complex ganglia that innervates the pelvic organs in rats. It consists of sympathetic and parasympathetic neurons, sensory neurons, interneurons, and possibly enteric neurons, all working in concert to regulate pelvic organ functions⁴³. Tuj1 is a specific marker to identify neuron presence and distribution. Our observations revealed a patchy pattern of Tuj1-marked neurons within the MPG. This pattern was consistent across the sham, TB, and scaffold groups, while the injury group significantly reduced expression levels (Fig. 7a).

**Fig. 7: Intertwined neural structures in MPG.**

Calcitonin Gene-Related Peptide (CGRP) is a marker for specific sensory neurons⁴⁴. Tyrosine Hydroxylase (TH) is an enzyme critical for catecholamine synthesis to identify sympathetic neurons⁴⁵. S100β is widely recognized as a marker for Schwann cells, reflecting neuron survival and health status⁴⁶. Choline Acetyltransferase (ChAT) is essential for acetylcholine synthesis and specifically marks cholinergic neurons, including those of the parasympathetic system⁴⁷. Enteric cholinergic neurons also express ChAT⁴⁸. Nestin is an intermediate filament found in neuroepithelial stem cells⁴⁹. NF200 is used to identify mature neurons. Neuronal nitric oxide synthase (nNOS) is a crucial enzyme for nitric oxide production and a specific marker for interneurons⁵⁰. Additionally, c-Fos is employed as an indicator of neuronal activity⁵¹. These markers have been instrumental in identifying and visualizing various neuronal populations within the MPG.

Figure 7b–d depicts intertwined neurofilaments collectively enveloping the body of neuron within the MPG. Tuj1, S100 β, and NF200 markers exhibited higher expression levels across all groups than other markers. Activation of neural stem cells was observed in all groups, as evidenced by the expression of Nestin. The functional neuron markers, including CGRP, TH, ChAT, and nNOS, were positively expressed in both the sham and TB groups; however, their expression levels were reduced in the TB group. In contrast, the expression levels of functional neurons and neurofilaments in the injury and scaffold groups were significantly diminished or absent, indicating severe damage to the MPG. The MPG in the TB group maintained a favorable condition, with functional neurons present at appropriate levels, thereby ensuring proper functionality.

The potential mechanism of MPG injuring and repairing

Cell therapies have great potential for treating diseases, but the need to understand their biological mechanisms hampers product approval and slows progress in the field⁵². Proteomics is a crucial strategy for learning disease progression. Based on our results, the scaffold acts as a delivery tool for HUVECs transplantation without repairing MPG. Therefore, we conducted Tandem Mass Tags (TMT) Proteomics analysis on sham, TB, and injury groups. The data showed good repeatability and stability (Fig. S10a–d). A total of 329 significant differential expression proteins (DEPs) were identified. From the subcellular localization, they are clustered with 24.96% nucleus protein, 22.99% cytoplasm protein, and so on (Fig. S10e).

By comparing the sham group and the injury group, a total of 144 DEPs were found, including 83 up-regulated and 61 down-regulated (Figs. 8a and S10f). GO enrichment indicated that 74 of 144 DEPs were enriched in 27 GO terms (Fig. 8b). We have identified several possible causes of damage, including ion transport disorder, immune response, cellular structural damage, lipid metabolism disorders, ischemia, and oxidative stress. KEGG analysis mainly focused on immune response (Fig. 8c). The Protein-Protein Interaction (PPI) network of DEPs was constructed using the STRING database. Its protein topology further categorized the causes of damage into four categories: neural cell structural and functional disorder (most proteins downregulated), immune response (most proteins upregulated), fatty acid metabolism disorder (downregulated proteins), and others (Fig. 8d). We provided related heatmaps for each PPI network to visualize the related protein interactions (Fig. 8e). Overall, crush to MPG is mechanical damage that triggers many adverse effects, including severe cytoskeletal changes, a robust immune response, homeostasis disruptions, a lipid metabolism disorder, and oxidative stress.

**Fig. 8: TMT analysis uncovers the causes of crush injury to MPG between the injury and sham groups.**

Comparing the MPG from the TB group to the injury group, we identified 79 DEPs, with 58 up-regulated and 21 down-regulated (Figs. 9a and S10g). GO analysis revealed that the DEPs were mainly associated with the cytoskeleton, structural molecule activity, and cell redox homeostasis (Fig. 9b). KEGG pathway analysis focused on muscle contraction (Fig. 9c). PPI network and related heatmap were categorized into cytoskeleton reorganization, keratins, and other classifications (Fig. 9d, e). To assess the immune status in the TB group, we implanted TB in MPG and compared it with sham group. As shown in Fig. S11a, there was an increase in macrophage markers (CD86 for pro-inflammatory and CD206 for anti-inflammatory), indicating initiation of the immune response⁵³. While, the significantly higher expression of CD206 suggested an anti-inflammatory reaction in the MPG of the TB group (Fig. S11b, c). These findings suggest that TB has a propensity to trigger protective responses in MPG. By responsing to mechanical stimulation, TB enhances the synthesis of cytoskeletal proteins to improve structural stability and mechanical support for cells, aiding in preserving cellular homeostasis, mitigating the impact of external forces, and preventing further damage. Additionally, keratins, which are the primary intermediate filament proteins, enhance cellular mechanical strength and support neuron regeneration and recovery⁵⁴. These findings underscore the effectiveness of TB therapy in promoting cytoskeletal reorganization for MPG repair.

**Fig. 9: The repair reason for TB on MPG injury between the TB and injury groups.**

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Source: https://www.nature.com/articles/s41467-024-53302-5

A tissue bandage for pelvic ganglia injury – Nature Communications

TB simulates mini-tissue and can potentially promote angiogenesis

TB regulates voiding behavior and bladder function after MPG injury

TB mitigates histomorphology changes in the bladder

TB protects the MPG and supports its recovery

The potential mechanism of MPG injuring and repairing

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