Biology subjects

Awad, H. A.

Publications and source records attributed to Awad, H. A..

9 recordsLinked to original sources

bioRxiv2026

A human synovial tendon-on-a-chip models key features of peritendinous adhesions and offers a new approach methodology for testing anti-fibrotic drugs

Peritendinous adhesions are a debilitating complication of tendon injury characterized by excessive matrix deposition and chronic inflammation. Due to limitations of current preclinical models, the underlying mechanisms of adhesion pathogenesis remain poorly defined, and there are no approved drugs to prevent or resolve adhesions. Here, we develop a human synovial tendon-on-a-chip (synToC) that integrates synovial fibroblasts, tendon-resident fibroblasts, immune cells, and vascular endothelium to reconstruct the intrasynovial tendon microenvironment. We show that synovial fibroblast activation promoted tendon contraction and inflammatory cytokine secretion dominated by IL-6, leading to monocyte infiltration and formation of fibronectin- and collagen III-rich matrix bridges between tendon and synovial compartments resembling nascent peritendinous adhesions. These phenotypes emerged even in the absence of exogenous TGF-{beta}1, indicating that synovial fibroblast-mediated crosstalk is sufficient to initiate adhesion-like pathology. Importantly, pharmacological inhibition of the IL-6/JAK/STAT pathway suppressed synovial activation, blunted inflammatory cytokine signaling, and attenuated fibrotic matrix deposition and interfacial adhesion formation. These findings establish the synToC as a human-relevant new approach methodology (NAM) to interrogate the multicellular drivers of tendon adhesions and to accelerate the development of anti-fibrotic therapies.

bioengineering↗

bioRxiv2025

Taurine Transporter SLC6A6 Expression Promotes Mesenchymal Stromal Cell Function

Mesenchymal stromal cell (MSC) differentiation is critical for the development, maintenance, and repair of bone tissue. This occurs within the bone marrow microenvironment, which consists of various stromal populations and structural components that support skeletal growth, repair and homeostasis. MSCs also promote self-renewal of hematopoietic stem cells and regulate their differentiation. Analysis of our own and publicly available single-cell RNA sequencing datasets of the murine non-immune bone and bone marrow indicates that Slc6a6 expression is enriched in MSCs. Slc6a6 encodes for an ion-dependent transporter of taurine (TauT). Although taurine supplements have been shown to mitigate the onset of bone defects in aged populations, the absence of TauT expression in osteolineage cells suggests that taurines effect on bone may be secondary to its function in other populations, such as MSCs. Using young TauT genetic loss-of-function murine models we find that TauT loss impacts MSC populations in vivo and impairs MSC osteogenic differentiation in vitro. This correlates with decreased bone mineral density and bone strength in young TauT knockout mice. Importantly, shRNA-based knockdown of SLC6A6 expression in primary human MSCs reduces osteogenic differentiation, indicating a key role of taurine uptake in human MSC function. Consistent with a decline in MSC function with TauT loss, we find that TauT null MSCs are unable to support self-renewal and expansion of co-cultured hematopoietic stem/progenitor populations. Mechanistically, our RNA-sequencing analysis identifies downregulation of Wnt/beta-catenin signaling in MSCs in the absence of TauT. These cells also show reduced oxidative phosphorylation, and increased ROS levels, indicating that impaired Wnt signaling and elevated oxidative stress may contribute to the observed defects in osteogenic differentiation capacity. Collectively, our data identify taurine uptake as a key regulator of mesenchymal stromal cell maintenance and osteogenic fate determination.

cell biology↗

bioRxiv2025

Automated Joint Space Detection Improves Bone Segmentation Accuracy

Quantitative description of complex anatomical structures remains challenging due to the expertise necessary for manual segmentation, labor, and interobserver variability. To overcome this, automated detection of specific landmarks can be provided by digital image analysis techniques including deep learning (DL) models. To this end, we undertook supervised automated analysis of micro-computed tomography (micro-CT) datasets of murine hindpaws and forepaws (30-33 bones). Advancing beyond previously published semi-automated (SA) marker-based watershed algorithms, we added structure enhancement, tensor voting, and output dilation to identify joint spaces. Segmentation was enhanced by the use of a DL joint space prediction model (3D U-Net architecture, ResNet-18 backbone) using wild-type (WT) hindpaw labels as ground truth. Prediction was then extended to hindpaws (52.4% in test group) and forepaws from WT and tumor necrosis factor transgenic (TNF-Tg) mice with inflammatory-erosive arthritis of both sexes across age. Segmentation accuracy improved dramatically using the DL methodology (WT male: SA 79.39{+/-}5.73% vs DL 98.16{+/-}1.47%, p<0.0001; WT female: SA 79.16{+/-}4.84% vs DL 99.19{+/-}1.63%, p<0.0001). Accuracy declined with increased disease severity and age in TNF-Tg mice (TNF-Tg male 93.54{+/-}4.73%, female 91.81{+/-}2.80%, p[≤]0.01). Subsequent testing in forepaws also displayed progressive reduction in accuracy with increasing arthritic severity (i.e., WT 87.29{+/-}2.07%, TNF-Tg male 72.65{+/-}11.70%, p<0.0001). Overall, this supervised automated model outperforms recent SA approaches in healthy joints to enhance investigation of complex bone anatomy. Although flexible application to novel and disease-modified datasets demonstrates deprecated performance, utilization may nonetheless catalyze structure-specific segmentation model development.

pathology↗

bioRxiv2024

Fluid Flow Impacts Endothelial-Monocyte Interactions in a Model of Vascular Inflammatory Fibrosis

The aberrant vascular response associated with tendon injury results in circulating immune cell infiltration and a chronic inflammatory feedback loop leading to poor healing outcomes. Studying this dysregulated tendon repair response in human pathophysiology has been historically challenging due to the reliance on animal models. To address this, our group developed the human tendon-on-a-chip (hToC) to model cellular interactions in the injured tendon microenvironment; however, this model lacked the key element of physiological flow in the vascular compartment. Here, we leveraged the modularity of our platform to create a fluidic hToC that enables the study of circulating immune cell and vascular crosstalk in a tendon injury model. Under physiological shear stress consistent with postcapillary venules, we found a significant increase in the endothelial leukocyte activation marker intercellular adhesion molecule 1 (ICAM-1), as well as enhanced adhesion and transmigration of circulating monocytes across the endothelial barrier. The addition of tissue macrophages to the tendon compartment further increased the degree of circulating monocyte infiltration into the tissue matrix. Our findings demonstrate the importance of adding physiological flow to the human tendon-on-a-chip, and more generally, the significance of flow for modeling immune cell interactions in tissue inflammation and disease.

bioengineering↗

bioRxiv2021

Transcriptional regulation of cyclophilin D by BMP/SMAD signaling and its role in osteogenic differentiation

Cyclophilin D (CypD) promotes opening of the mitochondrial permeability transition pore (MPTP) which plays a key role in both cell physiology and pathology. It is, therefore beneficial for cells to tightly regulate CypD and MPTP but little is known about such regulation. We have reported before that CypD is downregulated and MPTP deactivated during differentiation in various tissues. Herein, we identify BMP/Smad signaling, a major driver of differentiation, as a transcriptional repressor of the CypD gene, Ppif. Using osteogenic induction of mesenchymal lineage cells as a model of BMP/Smad-dependent differentiation, we show that CypD is in fact transcriptionally repressed during this process. The importance of such CypD downregulation is evidenced by the negative effect of CypD rescue via gain-of-function on osteogenesis both in vitro and in vivo. In sum, we characterized BMP/Smad signaling as a regulator of CypD expression and elucidated the role of CypD downregulation during cell differentiation.

cell biology↗

bioRxiv2021

Determination of best Raman spectroscopy spatial offsets for transcutaneous bone quality assessments in human hands

Spatially offset Raman spectroscopy (SORS) is able to detect bone signal transcutaneously and could assist in predicting bone fracture risk. Criteria for optimal source-detector offsets for transcutaneous human measurements, however, are not well-established. Although larger offsets yield a higher percentage of bone signal, the absolute amount of bone signal decreases. Spectral unmixing into bone, adipose, and non-adipose components was employed to quantify changes in bone signal to noise ratio across a range of offsets, and optimal offsets for phalanx and metacarpal measurements were determined. The bone signal to noise ratio was maximized at offsets ranging from 4-6 mm.

bioengineering↗

bioRxiv2021

Altered TGFB1 regulated pathways promote accelerated tendon healing in the superhealer MRL/MpJ mouse

To better understand the molecular mechanisms of tendon healing, we investigated the Murphy Roths Large (MRL) mouse, which is considered a model of mammalian tissue regeneration. We show that compared to C57Bl/6J (C57) mice, injured MRL tendons have reduced fibrotic adhesions and cellular proliferation, with accelerated improvements in biomechanical properties. Transcriptional analysis of biological drivers showed positive enrichment of TGFB1 in both C57 and MRL healing tendons. However, only MRL tendons exhibited downstream transcriptional effects of cell cycle regulatory genes, with negative enrichment of the cell senescence-related regulators, compared to the positively-enriched inflammatory and ECM organization pathways in the C57 tendons. Serum cytokine analysis revealed decreased levels of circulating senescence-associated circulatory proteins (SASP) in response to injury in the MRL mice compared to the C57 mice. These data collectively demonstrate altered TGFB1 regulated inflammatory, fibrosis, and cell cycle pathways in flexor tendon repair.

cell biology↗

bioRxiv2020

Improved prediction of femoral fracture toughness in mice by combining standard medical imaging with Raman spectroscopy

Bone fragility and fracture risk are assessed by measuring the areal bone mineral density (aBMD) using dual-energy X-ray absorptiometry (DXA). While aBMD correlates with bone strength, it is a poor predictor of fragility fracture risk. Alternatively, fracture toughness assesses the bones resistance to crack propagation and fracture, making it a suitable bone quality metric. Here, we explored how femoral midshaft measurements from DXA, micro-computed tomography (CT), and Raman spectroscopy could predict fracture toughness. We hypothesized that ovariectomy (OVX) decreases aBMD and fracture toughness compared to controls and we can optimize a multivariate assessment of bone quality by combining results from X-ray and Raman spectroscopy. Female mice underwent an OVX (n=5) or sham (n=5) surgery at 3 months of age. Femurs were excised 3 months after ovariectomy and assessed with Raman spectroscopy, CT, and DXA. Subsequently, a notch was created on the anterior side of the mid-diaphysis of the femurs. Three-point bending induced a controlled fracture that initiated at the notch. The OVX mice had a significantly lower aBMD, cortical thickness, and fracture toughness when compared to controls (p<0.05). A leave one out cross-validated (LOOCV) partial least squares regression (PLSR) model based only on the combination of aBMD and cortical thickness showed no significant predictive correlations with fracture toughness, whereas a PLSR model based on principal components derived from the full Raman spectra yielded significant prediction (r2=0.71, p<0.05). Further, the PLSR model was improved by incorporating aBMD, cortical thickness, and principal components from Raman spectra (r2=0.92, p<0.001). This exploratory study demonstrates combining X-ray with Raman spectroscopy leads to a more accurate assessment of bone fracture toughness and could be a useful diagnostic tool for the assessment of fragility fracture risk.

bioengineering↗

bioRxiv2020

Tendon Cell Deletion of IKKβ/NF-κB Drives Functionally Deficient Tendon Healing and Altered Cell Survival Signaling In Vivo

Acute tendon injuries are characterized by excessive matrix deposition that impedes regeneration and disrupts functional improvements. Inflammation is postulated to drive pathologic scar tissue formation, with nuclear factor kappa B (NF-{kappa}B) signaling emerging as a candidate pathway in this process. However, characterization of the spatial and temporal activation of canonical NF-{kappa}B signaling during tendon healing in vivo, including identification of the cell populations activating NF-{kappa}B, is currently unexplored. Therefore, we aimed to determine which cell populations activate canonical NF-{kappa}B signaling following flexor tendon repair with the goal of delineating cell-specific functions of NF-{kappa}B signaling during scar mediated tendon healing. Immunofluorescence revealed that both tendon cells and myofibroblasts exhibit prolonged activation of canonical NF-{kappa}B signaling into the remodeling phase of healing. Using cre-mediated knockout of the canonical NF-{kappa}B kinase (IKK{beta}), we discovered that suppression of canonical NF-{kappa}B signaling in Scleraxis-lineage cells increased myofibroblast content and scar tissue formation. Interestingly, Scleraxis-lineage specific knockout of IKK{beta} increased the incidence of apoptosis, suggesting that canonical NF-{kappa}B signaling may be mediating cell survival during tendon healing. These findings suggest indispensable roles for canonical NF-{kappa}B signaling during flexor tendon healing. One Sentence SummaryScleraxis-lineage specific knockdown of persistent canonical IKK{beta}/NF-{kappa}B drives scar formation and apoptotic signaling during flexor tendon healing.

molecular biology↗