Biology subjects

Yang, T.

Publications and source records attributed to Yang, T..

At least 109 records · Page 6Linked to original sources

bioRxiv2024

Genomic evolution reshapes cell type diversification in the amniote brain

Over 320 million years of evolution, amniotes have developed complex brains and cognition through largely unexplored genetic and gene expression mechanisms. We created a comprehensive single-cell atlas of over 1.3 million cells from the telencephalon and cerebellum of turtles, zebra finches, pigeons, mice, and macaques, employing single-cell resolution spatial transcriptomics to validate gene expression patterns across species. Our study revealed significant species-specific variations in cell types, highlighting their conservation and diversification in evolution. We found pronounced differences in telencephalon excitatory neurons (EX) and cerebellar cell types between birds and mammals. Birds predominantly express SLC17A6 in EX, whereas mammals expressed SLC17A7 in neocortex and SLC17A6 elsewhere, possibly due to loss of SLC17A7 function loss in birds. Additionally, we identified a novel bird-specific Purkinje cell subtype (SVIL+), implicating the LSD11/KDM1A pathway in learning and circadian rhythms, and related numerous positively selected genes in birds, suggesting an evolutionary optimization of cerebellar functions for ecological and behavioral adaptation. Our findings elucidate the complex interplay between genetic evolution and environmental adaptation, underscoring the role of genetic diversification in the development of specialized cell types across amniotes.

evolutionary biology↗

bioRxiv2024

Spatiotemporal transcriptomic maps of mouse intracerebral hemorrhage at single-cell resolution

Intracerebral hemorrhage (ICH) is a severe and widespread disease that results in high mortality and morbidity. Despite significant advances made in clinical and preclinical research, the prognosis of ICH remains poor due to an incomplete understanding of the complex pathological responses. To address this challenge, we generated single-cell resolution spatiotemporal transcriptomic maps of mouse brain 1, 3, 7, 14, and 28 days after ICH. Our analysis revealed the cellular constituents and their dynamic responses following ICH. We found changes in gene expression that are indicative of active phagocytosis and lipid processing in the lesion core and identified genes associated with brain repair at the lesion border. Persistent lipid accumulation culminated in the phenotypic transition of macrophages into foam cells. Furthermore, our results demonstrate that ICH could stimulate neural stem cells in the subventricular zone, thereby enhancing neurogenesis in this niche. This study provides a spatiotemporal molecular atlas of mouse ICH that advances the understanding of both local and global responses of brain cells to ICH and offers a valuable resource that can aid the development of novel therapies for this devastating condition.

neuroscience↗

bioRxiv2024

Protein S-glutathionylation confers cell resistance to ferroptosis

Ferroptosis is a type of cell death that is strongly associated with the cellular redox state. Glutathione is the key to buffering lipid peroxidation in ferroptosis and can also modify proteins by S-glutathionylation under oxidative stress. Here, we showed that the strong associations among glutathione pools, protein S-glutathionylation, and susceptibility to ferroptosis existed broadly in ferroptosis induced by erastin or acetaminophen. Deficiency of CHAC1, a glutathione-degrading enzyme, led to decreased glutathione pools and reduced protein S-glutathionylation, improved liver function and attenuated hepatocyte ferroptosis upon acetaminophen challenge, which could be retarded by CHAC1 overexpression. We conducted quantitative redox proteomics in primary mouse hepatocytes to identify glutathione pool-sensitive S-glutathionylated proteins and found that S-glutathionylation is required to maintain the function of ADP-ribosylation factor 6 (ARF6). Our data suggest that aberrant ARF6 S-glutathionylation increases the labile iron pool by delaying the recycling of transferrin receptors, thereby promoting ferroptosis. Our study reveals the importance of protein S-glutathionylation in conferring cell resistance to ferroptosis. O_FIG O_LINKSMALLFIG WIDTH=186 HEIGHT=200 SRC="FIGDIR/small/592374v1_ufig1.gif" ALT="Figure 1"> View larger version (42K): org.highwire.dtl.DTLVardef@82dbaeorg.highwire.dtl.DTLVardef@1247cc5org.highwire.dtl.DTLVardef@7b7147org.highwire.dtl.DTLVardef@49ff07_HPS_FORMAT_FIGEXP M_FIG C_FIG HIGHLIGHTSO_LIHighly upregulated CHAC1 decreases glutathione pools and protein S-glutathionylation. C_LIO_LIReduced protein S-glutathionylation associated with decreased glutathione pools promotes ferroptosis. C_LIO_LIS-glutathionylation of ARF6 at Cys90 promotes ARF6 activation. C_LIO_LIReduced S-glutathionylation of ARF6 provides a labile iron pool to drive ferroptosis. C_LI

cell biology↗

bioRxiv2024

Post-mortem evidence for a reciprocal relationship between genomic DNA damage and alpha-synuclein pathology in dementia with Lewy bodies.

DNA damage and DNA damage repair (DDR) dysfunction are insults with broad implications on cellular physiology, including in proteostasis, and have been recently implicated in many neurodegenerative diseases. Alpha-synuclein (aSyn), a pre-synaptic and nuclear protein associated with neurodegenerative disorders known as synucleinopathies, has been implicated in DNA double strand break (DSB) repair function. Consistently, DSB induction has been demonstrated in cell and animal models of synucleinopathy. Nevertheless, the types of DNA damage and the contribution of DNA damage towards Lewy body (LB) formation in synucleinopathies are unknown. Here, we demonstrate the increase of DSB in neuronal and non-neuronal cellular populations of post-mortem temporal cortex tissue from dementia with Lewy body (DLB) patients and demonstrate increases in DSBs early at a presymptomatic age of aSyn transgenic mice. Strikingly, in postmortem DLB tissue, DNA damage-derived ectopic cytoplasmic genomic material (eCGM) was evident within the majority of LBs examined. The observed cellular pathology was consistent with nucleoproteasomal upregulation of associated DNA damage repair proteins, particularly in base excision repair and DSB repair pathways. Collectively our study demonstrates the early occurrence of DNA damage and associated nucleoproteasomal changes in response to nuclear aSyn pathology. Furthermore, the data suggests a potential involvement for DNA damage derived eCGM for the facilitation of cytoplasmic aSyn aggregates. Ultimately, uncovering pathological mechanisms underlying DNA damage in DLB sheds light into novel disease mechanisms and opens novel possibilities for diagnosing and treating synucleinopathies.

neuroscience↗

bioRxiv2024

Metabolic engineering of narrow-leafed lupin for the production of enantiomerically pure (-)-sparteine

Sparteine is a plant-derived alkaloid widely known for its utility as chiral ligand in asymmetric synthesis1-3. However, its variable market price and availability have failed to meet the demand for a cheap and reliable product4-6. Sparteine is naturally synthesized by a sub-group of legume plants, which typically accumulate complex mixtures of closely related alkaloids. Here, we identified two enzymes from narrow-leafed lupin (NLL, L. angustifolius) that can sequentially oxidize (-)-sparteine to (+)-lupanine. The first enzyme is a cytochrome P450 monooxygenase belonging to family 71 (CYP71D189) and the second one is a short-chain dehydrogenase/reductase (SDR1). We also screened a non-GMO NLL mutant library and isolated a knockout in CYP71D189. The knockout displayed an altered metabolic profile where (-)-sparteine accounted for 96% of the alkaloid content in the seeds (GC-MS basis). The (-)-sparteine isolated from the mutant seeds was enantiomerically pure (99% ee). Apart from the altered alkaloid profile, the mutant did not have any noticeable phenotype. Our work demonstrates that (-)-sparteine is the precursor of most QAs in NLL and provides a convenient source of this valuable compound for academia and industry.

biochemistry↗

bioRxiv2024

Top-down Mass Spectrometric Analysis of a Lipooligosaccharide from the Human Commensal Bacteroides fragilis

Lipopolysaccharides (LPS) and lipooligosaccharides (LOS) are ubiquitous structures found on the outer membrane of gram-negative bacteria. Bacteroides fragilis, is a gram-negative anaerobe commonly inhabiting the human colon. The LOS of this organism is known to trigger a type I interferon response in dendritic cells. However, detailed structural analysis of this LOS has been largely elusive. Using top-down mass spectrometry, we have unraveled the comprehensive fine structure of B. fragilis LOS. Our analysis reveals that this LOS has a poly-galactose-rhamnose-KDO-lipid A architecture, which can be modified by hexuronic acid and ethanolamine via phosphodiester linkages. The lipid moiety typically includes three to five acyl chains of varying length on a glucosamine disaccharide. This investigation lays the groundwork for deeper immunological exploration of B. fragilis LOS and underscores the efficacy of top-down mass spectrometry in characterizing intact LOS/LPS structures and their modifications.

biochemistry↗

bioRxiv2024

Problem-Solving as a Language: A Computational Lens into Human and Monkey Intelligence

1Human intelligence is characterized by our remarkable ability to solve complex problems. This involves planning a sequence of actions that leads us from an initial state to a desired goal state. Quantifying and comparing problem-solving capabilities across species and finding its evolutional roots is a fundamental challenge in cognitive science, and is critical for understanding how the brain carries out this intricate process. In this study, we introduce the Language of Problem-Solving (LoPS) model as a novel quantitative framework that investigates the structure of problem-solving behavior through a language model. We adapted the classic Pac-Man game as a cross-species behavioral paradigm to test both humans and macaque monkeys. Using the LoPS model, we extracted the latent structure -- or grammar -- embedded in the agents gameplay, revealing the non-Markovian temporal structure of their problem-solving behavior. The LoPS model captured fine-grained individual differences among the players and revealed the striking differences in the complexity and hierarchical organization of problem-solving behavior between humans and monkeys, reflecting the distinct cognitive capabilities of each species. Furthermore, both humans and monkeys evolved their LoPS grammars during learning, progressing from simpler to more complex ones, suggesting that the language of problem-solving is not fixed, but rather evolves to support more sophisticated and efficient problem-solving. Through the lens of a language model, our study provides insights into how humans and monkeys break down problem-solving into compositional units and navigate complex tasks. This framework deepens our understanding of human intelligence and its evolution, and establishes a foundation for future investigations of the neural mechanisms of problem-solving.

neuroscience↗

bioRxiv2024

Srs2 binding to PCNA and its sumoylation contribute to RPA antagonism during the DNA damage response

Activation of the DNA damage checkpoint upon genotoxin treatment induces a multitude of cellular changes to cope with genome stress. After prolonged genotoxin treatment, the checkpoint can be downregulated to allow cell cycle and growth resumption. In yeast, downregulation of the DNA damage checkpoint requires the Srs2 DNA helicase, which removes the ssDNA binding complex RPA and the associated Mec1 checkpoint kinase from DNA, thus dampening Mec1-mediated checkpoint. However, it is unclear whether the anti-checkpoint role of Srs2 is temporally and spatially regulated to allow timely checkpoint termination while preventing superfluous RPA removal. Here we address this question by examining regulatory elements of Srs2, such as its phosphorylation, sumoylation, and protein-interaction sites. Our genetic analyses and checkpoint level assessment suggest that the RPA countering role of Srs2 is promoted by Srs2 binding to PCNA, which recruits Srs2 to a subset of ssDNA containing regions. RPA antagonism is further fostered by Srs2 sumoylation, which we found depending on the Srs2-PCNA interaction and Mec1, and peaking after Mec1 activity reaches maximal levels. These data suggest that Srs2 recruitment to PCNA proximal ssDNA-RPA filaments followed by its sumoylation can promote checkpoint recovery, whereas Srs2 action is minimized at regions with no proximal PCNA to permit RPA-mediated ssDNA protection.

molecular biology↗

bioRxiv2024

Pathway-enhanced Transformer-based model for robust enumeration of cell types from the cell-free transcriptome

Analyzing cell types of origin of cell-free RNA can enhance the resolution of liquid biopsies, thereby deepening the understanding of molecular and cellular changes in development and disease processes. Existing deconvolution methods typically rely on meticulously curated gene expression profiles or employ deep neural network with vast and complex solution spaces that are difficult to interpret. These approaches overlook the synergistic and co-expression effects among genes in biological signaling pathways, compromising their generalizability and robustness. we developed Deconformer, a Transformer-based deconvolution model that integrates biological signaling pathways at the embedding stage, to address these issues. Compared to popular methods on multiple datasets, Deconformer demonstrates superior performance and robustness, and is capable of tracking the developmental process of the fetal and placenta. Additionally, pathway-level interpretability of Deconformer offers new insights into crosstalk, dependencies, and other interactions within cell-free RNA pathways, supporting further biological discoveries. We posit that Deconformer represents a significant advancement in the precise analysis of the cell-free transcriptome. It holds the promise of describing disease progression and severity with a new level of accuracy, focusing on the contributions of originating cell types and their pathway dependencies. This model has the potential to catalyze the development of non-invasive diagnostic tools and enhance our understanding of the underlying biology of diseases.

bioinformatics↗

bioRxiv2024

Multifocal Ectopic Purkinje Premature Contractions due to neutralization of an SCN5A negative charge: structural insights into the gating pore hypothesis

BackgroundWe identified a novel SCN5A variant, E171Q, in a neonate with very frequent ectopy and reduced ejection fraction which normalized after arrhythmia suppression by flecainide. This clinical picture is consistent with multifocal ectopic Purkinje-related premature contractions (MEPPC). Most previous reports of MEPPC have implicated SCN5A variants such as R222Q that neutralize positive charges in the S4 voltage sensor helix of the channel protein NaV1.5 and generate a gating pore current. Methods and ResultsE171 is a highly conserved negatively-charged residue located in the S2 transmembrane helix of NaV1.5 domain I. E171 is a key component of the Gating Charge Transfer Center, a region thought to be critical for normal movement of the S4 voltage sensor helix. We used heterologous expression, CRISPR-edited induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs), and molecular dynamics simulations to demonstrate that E171Q generates a gating pore current, which was suppressed by a low concentration of flecainide (IC50 = 0.71{+/-}0.07 {micro}M). R222Q shifts voltage dependence of activation and inactivation in a negative direction but we observed positive shifts with E171Q. E171Q iPSC-CMs demonstrated abnormal spontaneous activity and prolonged action potentials. Molecular dynamics simulations revealed that both R222Q and E171Q proteins generate a water-filled permeation pathway that underlies generation of the gating pore current. ConclusionPreviously identified MEPPC-associated variants that create gating pore currents are located in positively-charged residues in the S4 voltage sensor and generate negative shifts in the voltage dependence of activation and inactivation. We demonstrate that neutralizing a negatively charged S2 helix residue in the Gating Charge Transfer Center generates positive shifts but also create a gating pore pathway. These findings implicate the gating pore pathway as the primary functional and structural determinant of MEPPC and widen the spectrum of variants that are associated with gating pore-related disease in voltage-gated ion channels.

genetics↗

bioRxiv2024

A single-cell 3D spatiotemporal multi-omics atlas from Drosophila embryogenesis to metamorphosis

The development of a multicellular organism is a highly intricate process tightly regulated by numerous genes and pathways in both spatial and temporal manners. Here, we present Flysta3D, a comprehensive multi-omics atlas of the model organism Drosophila, spanning its developmental lifespan from embryo to pupa. Our datasets encompass 3D single-cell spatial transcriptomic, single-cell transcriptomic, and single-cell chromatin accessibility information. By integrating these multi-dimensional data, we constructed cell state trajectories that uncover the detailed profiles of tissue development. With a focus on the central nervous system (CNS) and midgut, we dissected the spatiotemporal dynamics of gene regulatory networks, cell type diversity, and morphological changes from a multi-omics perspective. This extensive atlas provides an unprecedentedly rich resource and serves as a systematic platform for studying Drosophila development with integrated single-cell data at an ultra-high spatiotemporal resolution.

developmental biology↗

bioRxiv2024

Cryo-EM structures of human cone visual pigments

Trichromatic color vision in humans constitutes a pivotal evolutionary adaptation, endowing individuals with the capacity to discern and discriminate a diverse spectrum of colors. This unique visual capability confers a selective advantage crucial for successful adaptation, survival, and reproductive success in the natural environment. Color vision in humans is facilitated by the red, green, and blue cone visual pigments within cone photoreceptor cells. These pigments consist of a G-protein-coupled receptor opsin apoprotein and a chromophore covalently linked to opsins. Despite the elucidated structure of rhodopsin, the structures of cone visual pigments have yet to be determined. Here, we present the cryo-EM structures of three human cone visual pigments in complex with G proteins. Our structural analysis reveals detailed interactions between cone opsins, all-trans-retinal, and G proteins, indicating their active state. We also provide a concise summary and analysis of mutations in human cone opsins, elucidating potential relationships between residue substitutions and spectral tuning. Notably, S1162.67Y, A2335.52S, Y2776.44F were found to induce a blue shift in the absorption spectrum of the red-pigment, while the substitutions W2816.48Y and K3127.43A resulted in the absence of the absorption spectrum. The structural elucidation of human cone visual pigments significantly contributes to our understanding of how distinct types of cone cells perceive light across varying wavelengths. Furthermore, it provides a deeper insight into the functioning of the human trichromatic vision system, probing the mechanisms enabling humans to perceive a broad spectrum of colors.

biophysics↗

bioRxiv2024

YamOmics: A comprehensive data resource on yam multi-omics

Yams (Dioscorea spp.) are a highly important class of horticultural crops, serving as a staple food for millions of people in Africa and contributing significantly to food security. They are also widely cultivated in East Asia as medicinal herbs, bringing substantial economic incomes. Diverse omics data play a pivotal role in advancing yam research and breeding. However, these data are often scattered, lacking in systematic organization and analysis, which underscores the need for centralized and comprehensive data management. In view of this, we gathered extensive omics data and developed the Yam Omics Database (YamOmics; https://biotec.njau.edu.cn/yamdb). The database currently offers a vast and diverse range of omics data, covering genomic, transcriptomic and plastomic data from 41 distinct yam species, along with detailed records of genomic variants from [~]1000 germplasms, and gene expression profiles from [~]200 samples. Additionally, the database features thorough annotations, encompassing aspects like genome synteny, ortholog groups, signaling pathways, gene families and protein interactions. To support yam basic biology and breeding research, it is also equipped with a suite of user-friendly online tools, including PCR primer design, CRISPR design, expression analysis, enrichment analysis, and kinship analysis tools.

plant biology↗

bioRxiv2024

Root-specific secondary metabolism at the single-cell level: a case study of theanine metabolism and regulation in the roots of tea plants (Camellia sinensis)

Root-synthesized secondary metabolites are critical quality-conferring compounds of foods, plant-derived medicines, and beverages. However, information at a single-cell level on root-specific secondary metabolism remains largely unexplored. L-theanine, an important quality component of tea, is primarily synthesized in roots, from which it is then transported to new shoots of tea plant. In this study, we present a single-cell RNA sequencing (scRNA-seq)-derived map for the tea plant root, which enabled cell-type-specific analysis of glutamate and ethylamine (two precursors of theanine biosynthesis) metabolism, and theanine biosynthesis, storage, and transport. Our findings support a model in which the theanine biosynthesis pathway occurs via multicellular compartmentation and does not require high co-expression levels of transcription factors and their target genes within the same cell cluster. This study provides novel insights into theanine metabolism and regulation, at the single-cell level, and offers an example for studying root-specific secondary metabolism in other plant systems.

plant biology↗

bioRxiv2024

Feedback loop regulation between viperin and viral hemorrhagic septicemia virus through competing protein degradation pathways

Viperin is an antiviral protein that exhibits a remarkably broad spectrum of antiviral activity. Viperin-like proteins are found all kingdoms of life, suggesting it is an ancient component of the innate immune system. However, viruses have developed strategies to counteract viperins effects. Here, we describe a feedback loop between viperin and viral hemorrhagic septicemia virus (VHSV), a common fish pathogen. We show that Lateolabrax japonicus viperin (Ljviperin) is induced by both IFN-independent and IFN-dependent pathways, with the C-terminal domain of Ljviperin being important for its anti-VHSV activity. Ljviperin exerts an antiviral effect by binding both the nucleoprotein (N) and phosphoprotein (P) of VHSV and induces their degradation through the autophagy pathway, which is an evolutionarily conserved antiviral mechanism. However, counteracting viperins activity, N protein targets and degrades transcription factors that up-regulate Ljviperin expression, interferon regulatory factor (IRF) 1 and IRF9, through ubiquitin-proteasome pathway. Together, our results reveal a previously unknown feedback loop between viperin and virus, providing potential therapeutic targets for VHSV prevention. ImportanceViral hemorrhagic septicaemia (VHS) is a contagious disease caused by the viral hemorrhagic septicaemia virus (VHSV), which poses a threat to over 80 species of marine and freshwater fish. Currently, there are no effective treatments available for this disease. Understanding the mechanisms of VHSV-host interaction is crucial for preventing viral infections. Here, we found that, as an ancient antiviral protein, viperin degrades the N and P proteins of VHSV through the autophagy pathway. Additionally, the N protein also impacts the biological functions of IRF1 and IRF9 through the ubiquitin-proteasome pathway, leading to the suppression of viperin expression. Therefore, the N protein may serve as a potential virulence factor for the development of VHSV vaccines and screening of antiviral drugs. Our research will serve as a valuable reference for the development of strategies to prevent VHSV infections.

immunology↗

bioRxiv2024

Cryo-EM structure and biochemical analysis of human chemokine receptor CCR8

The C-C motif chemokine receptor 8 (CCR8) is a class A G-protein coupled receptor that has emerged as a promising therapeutic target in cancer and autoimmune diseases. Although the structures of human CCR8 in complex an antagonist antibody Fab1 and an endogenous agonist ligand CCL1 have been solved, the structure of ligand-free CCR8 remains to be determined. Here, we solved the cryo-electron microscopy (cryo-EM) structure of the human CCR8-Gi complex in the absence of a ligand. Structural analysis and comparison revealed that CCR8 in our apo structure undergoes some conformational change and is similar to that in the CCL1-CCR8 complex structure, indicating an active state. In addition, the key residues of CCR8 involved in the recognition of LMD-009, a potent nonpeptidic agonist, were investigated by mutating CCR8 and testing the calcium flux induced by LMD-009-CCR8 interaction. Two mutants of CCR8, Y1133.32A, and E2867.39A, showed a dramatically decreased ability in mediating calcium mobilization, indicating their key interaction with LMD-009. These structural and biochemical analyses provided molecular insights into the agonism and activation of CCR8 and will also facilitate CCR8-targeted therapy.

biophysics↗

bioRxiv2023

Identification of a short, single site matriglycan that maintains neuromuscular function in the mouse

Matriglycan (-1,3-{beta}-glucuronic acid-1,3--xylose-) is a polysaccharide that is synthesized on -dystroglycan, where it functions as a high-affinity glycan receptor for extracellular proteins, such as laminin, perlecan and agrin, thus anchoring the plasma membrane to the extracellular matrix. This biological activity is closely associated with the size of matriglycan. Using high-resolution mass spectrometry and site-specific mutant mice, we show for the first time that matriglycan on the T317/T319 and T379 sites of -dystroglycan are not identical. T379-linked matriglycan is shorter than the previously characterized T317/T319-linked matriglycan, although it maintains its laminin binding capacity. Transgenic mice with only the shorter T379-linked matriglycan exhibited mild embryonic lethality, but those that survived were healthy. The shorter T379-linked matriglycan exists in multiple tissues and maintains neuromuscular function in adult mice. In addition, the genetic transfer of -dystroglycan carrying just the short matriglycan restored grip strength and protected skeletal muscle from eccentric contraction-induced damage in muscle-specific dystroglycan knock-out mice. Due to the effects that matriglycan imparts on the extracellular proteome and its ability to modulate cell-matrix interactions, our work suggests that differential regulation of matriglycan length in various tissues optimizes the extracellular environment for unique cell types.

biochemistry↗

bioRxiv2023

Bioactive coatings on 3D printed polycaprolactone scaffolds for bone regeneration: a novel murine femur defect model for examination of the biomaterial capacity for repair.

Bone tissue engineering is a rapidly advancing field that seeks to develop efficacious approaches for treating non-healing fractures and large bone defects. Healing complications arise due to trauma, disease, infection, aseptic loosening of orthopaedic implants or iatrogenic causes. An ideal biodegradable scaffold would induce and support bone formation until the bone matrix is sufficiently stable to facilitate healing. The current study has examined bone augmentation, using functionalised coated scaffolds, with the hypothesised potential to induce skeletal cell differentiation for the repair of critical-sized bone defects. However, challenges in clinical translation arise from the alterations in cellular microenvironment that are present in the translation from in vitro to in vivo with the application of animal models of progressively increasing size and complexity of the implantation site. 3D printed, porous poly(caprolactone) trimethacrylate (denoted PCL-TMA900) scaffolds were applied within a murine femur defect, stabilised by a polyimide intramedullary pin, to assess the efficacy of select coatings in inducing bone formation. The PCL-TMA900 scaffolds were coated with i) elastin-like polypeptide (ELP), ii) poly(ethyl acrylate)/fibronectin/bone morphogenetic protein-2 (PEA/FN/BMP-2), iii) both ELP and PEA/FN/BMP-2 concurrently, or iv) Laponite nanoclay binding BMP-2, as bioactive coatings. The murine femur defect model was refined to assess the coated PCL-TMA900 scaffolds in an osseous defect, with sequential microcomputed tomography ({micro}CT) and histological analysis of the new bone tissue. Overall, PCL-TMA900 was found to be an optimal robust, biocompatible, 3D printable scaffold material. All PCL-TMA900 scaffolds, uncoated and coated, showed integration with the femur. The PCL-TMA900 scaffold coated with the nanoclay material Laponite and BMP-2 induced consistent, significant bone formation compared to the uncoated PCL-TMA900 scaffold. Bone formation was observed within the pores of the Laponite/BMP-2 coated scaffold. Critically, no heterotopic bone formation was observed as the BMP-2 was retained around the scaffold and not released into the tissues, producing bone around the scaffold in the desired shape and volume, compared to bone formation observed with the positive control (collagen sponge/BMP-2 construct). In comparison, the ELP coated and PEA/FN/BMP-2 scaffolds did not demonstrate significant or consistent bone formation compared to uncoated PCL-TMA900 control scaffolds. In summary, nanoclay Laponite/BMP-2 coated PCL-TMA900 scaffolds offer a biodegradable, osteogenic construct for bone augmentation with potential for development into a large scale polymer scaffold for translation to the clinic.

bioengineering↗