Deep learning for automated segmentation and counting of hypocotyl and cotyledon regions in mature Pinus radiata D. Don. somatic embryo images.

In commercial forestry and large-scale plant propagation, the utilization of artificial intelligence techniques for automated somatic embryo analysis has emerged as a highly valuable tool. Notably, image segmentation plays a key role in the automated assessment of mature somatic embryos. However, to date, the application of Convolutional Neural Networks (CNNs) for segmentation of mature somatic embryos remains unexplored. In this study, we present a novel application of CNNs for delineating mature somatic conifer embryos from background and residual proliferating embryogenic tissue and differentiating various morphological regions within the embryos. A semantic segmentation CNN was trained to assign pixels to cotyledon, hypocotyl, and background regions, while an instance segmentation network was trained to detect individual cotyledons for automated counting. The main dataset comprised 275 high-resolution microscopic images of mature Pinus radiata somatic embryos, with 42 images reserved for testing and validation sets. The evaluation of different segmentation methods revealed that semantic segmentation achieved the highest performance averaged across classes, achieving F1 scores of 0.929 and 0.932, with IoU scores of 0.867 and 0.872 for the cotyledon and hypocotyl regions respectively. The instance segmentation approach demonstrated proficiency in accurate detection and counting of the number of cotyledons, as indicated by a mean squared error (MSE) of 0.79 and mean absolute error (MAE) of 0.60. The findings highlight the efficacy of neural network-based methods in accurately segmenting somatic embryos and delineating individual morphological parts, providing additional information compared to previous segmentation techniques. This opens avenues for further analysis, including quantification of morphological characteristics in each region, enabling the identification of features of desirable embryos in large-scale production systems. These advancements contribute to the improvement of automated somatic embryogenesis systems, facilitating efficient and reliable plant propagation for commercial forestry applications.

Environmentally Benign Fast-Degrading Conductive Composites.

An environmentally benign conductive composite that rapidly degrades in the presence of warm water via enzyme-mediated hydrolysis is described. This represents the first time that hydrolytic enzymes have been immobilized onto eco-friendly conductive carbon sources with the express purpose of degrading the encapsulating biodegradable plastic. Amano Lipase (AL)-functionalized carbon nanofibers (CNF) were compounded with polycaprolactone (PCL) to produce the composite film CNFAL-PCL (thickness ∼ 600 µm; CNFAL = 20.0 wt %). To serve as controls, films of the same thickness were also produced, including CNF-AL5-PCL (CNF mixed with AL and PCL; CNF = 19.2 wt % and AL = 5.00 wt %), CNF-PCL (CNF = 19.2 wt %), ALx-PCL (AL = x = 1.00 or 5.00 wt %), and PCL. The electrical performance of the CNF-containing composites was measured, and conductivities of 14.0 ± 2, 22.0 ± 5, and 31.0 ± 6 S/m were observed for CNFAL-PCL, CNF-AL5-PCL, and CNF-PCL, respectively. CNFAL-PCL and control films were degraded in phosphate buffer (2.00 mg/mL film/buffer) at 50 °C, and their average percent weight loss (Wtavg%) was recorded over time. After 3 h CNFAL-PCL degraded to a Wtavg% of 90.0% and had completely degraded after 8 h. This was considerably faster than CNF-AL5-PCL, which achieved a total Wtavg% of 34.0% after 16 days, and CNF-PCL, which was with a Wtavg% of 7.00% after 16 days. Scanning electron microscopy experiments (SEM) found that CNFAL-PCL has more open pores on its surface and that it fractures faster during degradation experiments which exposes the interior enzyme to water. An electrode made from CNFAL-PCL was fabricated and attached to an AL5-PCL support to form a fast-degrading thermal sensor. The resistance was measured over five cycles where the temperature was varied between 15.0-50.0 °C. The sensor was then degraded fully in buffer at 50 °C over a 48 h period.

Interleukin-1ß More Than Mechanical Loading Induces a Degenerative Phenotype in Human Annulus Fibrosus Cells, Partially Impaired by Anti-Proteolytic Activity of Mesenchymal Stem Cell Secretome.

Mesenchymal stem/stromal cell (MSC)-based therapies for low back pain and intervertebral disc (IVD) degeneration have been emerging, despite the poor knowledge of their full mechanism of action. As failure of the annulus fibrosus (AF) is often associated with IVD herniation and inflammation, the objective of the present study was to investigate the impact of the MSC secretome on human AF cells exposed to mechanical loading and a pro-inflammatory environment. Human AF cells isolated from IVD biopsies from patients with adolescent idiopathic scoliosis (AIS) or disc degeneration (DD) were exposed to physiological cyclic tensile strain (CTS) for 72 h in a custom-made device, with or without interleukin (IL)-1ß medium supplementation. AF cells stimulated with CTS + IL-1ß were then treated with secretome from IL-1ß-preconditioned MSCs for 48 h. AF cell metabolic activity, gene expression, protein secretion, matrix metalloproteinase (MMP) activity, and tissue inhibitor of MMPs (TIMP) concentration were evaluated. Expanded AF cells from AIS and DD patients revealed similar metabolic activity and gene expression profiles. CTS stimulation upregulated collagen type I (COL1A1) expression, while IL-1ß significantly stimulated IL-6, IL-8, MMP-1, and MMP-3 gene expression and prostaglandin E2 production by AF cells but downregulated COL1A1. The combination of CTS + IL-1ß had a similar outcome as IL-1ß alone, accompanied by a significant upregulation of elastin. The MSC secretome did not show any immunomodulatory effect on CTS + IL-1ß-stimulated AF cells but significantly decreased MMP-1, MMP-2, MMP-3, and MMP-9, while increasing the production of TIMP-1. The obtained results demonstrate a stronger impact of the inflammatory milieu on human AF cells than upper physiologic mechanical stress. In addition, a new MSC mechanism of action in degenerated IVD consisting of the modulation of AF MMP activity was also evidenced, contributing to the advancement of knowledge in AF tissue metabolism.

Differential Response of Bovine Mature Nucleus Pulposus and Notochordal Cells to Hydrostatic Pressure and Glucose Restriction.

OBJECTIVE: The nucleus pulposus of the human intervertebral disc contains 2 cell types: notochordal (NC) and mature nucleus pulposus (MNP) cells. NC cell loss is associated with disc degeneration and this process may be initiated by mechanical stress and/or nutrient deprivation. This study aimed to investigate the functional responses of NC and MNP cells to hydrostatic pressures and glucose restriction. DESIGN: Bovine MNP and NC cells were cultured in 3-dimensional alginate beads under low (0.4-0.8 MPa) and high (1.6-2.4 MPa) dynamic pressure for 24 hours. Cells were cultured in either physiological (5.5 mM) glucose media or glucose-restriction (0.55 mM) media. Finally, the combined effect of glucose restriction and high pressure was examined. RESULTS: Cell viability and notochordal phenotypic markers were not significantly altered in response to pressure or glucose restriction. MNP cells responded to low pressure with an increase in glycosaminoglycan (GAG) production while high pressure significantly decreased ACAN gene expression compared with atmospheric controls. NC cells showed no response in matrix gene expression or GAG production with either loading regime. Glucose restriction decreased NC cell TIMP-1 expression but had no effect on MNP cells. The combination of glucose restriction and high pressure only affected MNP cell gene expression, with decreased ACAN, Col2α1, and ADAMTS-5 expression. CONCLUSION: This study shows that NC cells are more resistant to acute mechanical stresses than MNP cells and provides a strong rationale for future studies to further our understanding the role of NC cells within the disc, and the effects of long-term exposure to physical stresses.

GEORG SCHMORL PRIZE OF THE GERMAN SPINE SOCIETY (DWG) 2018: combined inflammatory and mechanical stress weakens the annulus fibrosus: evidences from a loaded bovine AF organ culture.

PURPOSE: The pathomechanism of annulus fibrosus (AF) failure is still unknown. We hypothesise that mechanical overload and an inflammatory microenvironment contribute to AF structural weakening. Therefore, the objective of this study was to investigate the influence of these factors on the AF, particularly the translamellar bridging network (TLBN) which connects the AF lamellae. METHODS: A bovine AF organ culture (AF-OC) model of standardised AF rings was used to study the individual and combined effects of cyclic tensile strain (CTS) and IL-1ß (1 ng/mL) culture medium supplementation. AF-OCs were analysed for PGE2 production (ELISA) and deposition of IL-6, COX-2, fibrillin, and MMP3 in the tissue (immunohistochemistry, IHC). The mechanical strength of the TLBN was evaluated using a peel test to measure the strength required to separate an AF segment along a lamellar bound. RESULTS: The combination of CTS + IL-1ß led to a significant increase in PGE2 production compared to Control (p < 0.01). IHC evaluations showed that the CTS + IL-1ß group exhibited higher production of COX-2 and MMP3 within the TLBN regions compared to the adjacent lamellae and a significant increase in IL-6 ratio compared to Control (p < 0.05). A significant decrease in the annular peel strength was observed in the CTS + IL1ß group compared to Control (p < 0.05). CONCLUSION: Our findings suggest that CTS and IL-1ß act synergistically to increase pro-inflammatory and catabolic molecules within the AF, particularly the TLBN, leading to a weakening of the tissue. This standardised model enables the investigation of AF/TLBN structure-function relationship and is a platform to test AF-focused therapeutics. These slides can be retrieved under Electronic Supplementary Material.

Same-species phenotypic comparison of notochordal and mature nucleus pulposus cells.

PURPOSE: The ratio of notochordal (NC) cells to mature nucleus pulposus (MNP) cells in the nucleus pulposus varies with species, age and health. Studies suggest that loss of NC cells is a key component of intervertebral disc degeneration. However, few studies have examined the phenotypes of these two cell populations. Therefore, this study aimed to isolate NC and MNP cells from the same intervertebral disc and study phenotypic differences in extracellular matrix production and cell morphology in 3D culture over 7 days. METHODS: Sequential mechanical dissociation and enzymatic digestion were used to isolate NC cell clusters and single MNP cells from bovine caudal discs. Cells were cultured in alginate beads and subsequently analysed for viability, cytokeratin-8 expression, GAG production and extracellular matrix gene expression. RESULTS: Mechanical dissociation allowed NC cells to be extracted as intact cell clusters. NC cells represented 8% of the NP cell population and expressed both cytokeratin-8 and vimentin. MNP cells expressed vimentin, only. Both cells types were viable for 7 days. In addition to morphological differences, NC cells produced up to 30 times more total proteoglycan than MNP cells. NC cells had significantly higher aggrecan and brachyury expression. CONCLUSIONS: NC and MNP cells can be isolated from the same bovine disc and maintain their distinct phenotypes in 3D culture.

Development of a method for the measurement of primary cilia length in 3D.

BACKGROUND: Primary cilia length is an important measure of cell and tissue function. While accurate length measurements can be calculated from cells in 2D culture, measurements in tissue or 3D culture are inherently difficult due to optical distortions. This study uses a novel combination of image processing techniques to rectify optical distortions and accurately measure cilia length from 3D images. METHODS: Point spread functions and experimental resolutions were calculated from subresolution microspheres embedded in 3D agarose gels for both wide-field fluorescence and confocal laser scanning microscopes. The degree of axial smearing and spherical aberration was calculated from xy:xz diameter ratios of 3D image data sets of 4 µm microspheres that had undergone deconvolution and/or Gaussian blurring. Custom-made 18 and 50 µm fluorescent microfibers were also used as calibration objects to test the suitability of processed image sets for 3D skeletonization. Microfiber length in 2D was first measured to establish an original population mean. Fibers were then embedded in 3D agarose gels to act as ciliary models. 3D image sets of microfibers underwent deconvolution and Gaussian blurring. Length measurements within 1 standard deviation of the original 2D population mean were deemed accurate. Finally, the combined method of deconvolution, Gaussian blurring and skeletonization was compared to previously published methods using images of immunofluorescently labeled renal and chondrocyte primary cilia. RESULTS: Deconvolution significantly improved contrast and resolution but did not restore the xy:xz diameter ratio (0.80). Only the additional step of Gaussian blurring equalized xy and xz resolutions and yielded a diameter ratio of 1.02. Following image processing, skeletonization successfully estimated microfiber boundaries and allowed reliable and repeatable measurement of fiber lengths in 3D. We also found that the previously published method of calculating length from 2D maximum projection images significantly underestimated ciliary length. CONCLUSIONS: This study used commercial and public domain image processing software to rectify a long-standing problem of 3D microscopy. We have shown that a combination of deconvolution and Gaussian blurring rectifies optical distortions inherent in 3D images and allows accurate skeletonization and length measurement of microfibers and primary cilia that are bent or curved in 3D space.