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[This corrects the article DOI: 10.1016/j.mbplus.2024.100158.].
Platelet concentrates and enamel matrix derivatives (EMD) have been used extensively for periodontal regenerative procedures. Modifications in processing protocols have resulted in a variety of concentrated platelet formulations. The present study evaluated the combination of a modified platelet concentrate, advanced platelet-rich fibrin plus (A-PRF+), with Emdogain® on the proliferation and migration of human periodontal ligament fibroblasts. The study utilized an in vitro scratch and MTT assay for the stated purpose. We prepared A-PRF+ from healthy volunteer blood samples and procured Emdogain® from Straumann India Pvt. Ltd. We compared A-PRF+, EMD, and the combination with distilled water for cell migration, proliferation, and wound closure at 0, 24, and 48 h. Increased cell migration and proliferation were observed from 24 to 48 h in all the groups with significant wound closure. After 48 h, the A-PRF+ and EMD group showed complete wound closure and the highest proliferation rate compared to either EMD or A-PRF+ alone, and distilled water. The combined application of A-PRF+ and EMD promoted enhanced wound healing, surpassing the effects of EMD and A-PRF+ used individually. The study also demonstrated the superior ability of EMD alone in periodontal fibroblast healing. Cell migration and proliferation propensity of A-PRF+ endorse it as an option for regenerative therapy.
[This corrects the article DOI: 10.1016/j.mbplus.2023.100130.].
At least 10% of the global population is impacted by chronic kidney disease (CKD) and ageing is a key risk factor. CKD is characterised by the build-up of extracellular matrix and a loss of functional nephrons. However, the mechanisms that maintain matrix homeostasis across the physiological lifespan remain elusive. Using 13C-lysine metabolic labelling, we quantified kidney matrix protein turnover in healthy mice at four timepoints (8, 22, 52, and 78 weeks). We found that basement membrane components, including collagen IV, laminin-521, nidogens and perlecan, were more long-lived over age, with collagen IV half-lives extending from weeks in young kidneys to years in aged kidneys, suggesting a reduced capacity for basement membrane renewal. The half-lives of fibrillar collagens I and III also increased over age up to forty-fold, which is consistent with minimal degradation. In contrast, collagen XV retained rapid turnover despite increased abundance, indicating a persistent role in tissue remodelling. Using peptide location fingerprinting to predict structural alterations and proteolytic processing we identified age-dependent meprin oligomerisation and altered nidogen-laminin interaction states. We predicted structural alterations within assembly domains of collagen VI and reduced accessibility of integrin-binding regions, suggesting altered microfibril organisation and cell-surface binding. Collagen XV had predicted structural changes across the NC1 domain encoding the matrikine restin, indicative of altered protease accessibility and matrikine release during ageing. These findings are consistent with age-related kidney fibrosis being driven by impaired matrix degradation rather than increased synthesis, with protease accessibility and altered matrix interactions likely contributing to this remodelling process.
Older adults are the primary population for cell-based therapies for age-related diseases, but the efficacy of administering autologous mesenchymal stem cells (MSCs) is impaired due to biological aging. In the present study, we cultured aging adipose (AD)-derived MSCs from > 65-year-old donors on extracellular matrix (ECM) synthesized by human amniotic fluid-derived pluripotent stem cells (ECM Plus) versus tissue culture plastic (TCP) and hypothesized that ECM Plus provided an ideal "young" microenvironment for reactivating and preserving early-stage progenitor cells within aging AD-MSCs. To test our hypothesis, we serially sub-cultured aging AD-MSCs on ECM Plus or TCP and characterized the cells both phenotypically and functionally, and then analyzed the cells at the single-cell transcriptomic level for the mechanisms that control cell fate. The results showed that the maintenance of aging AD-MSCs on ECM Plus significantly restored their quantity and quality. The mechanisms responsible for these effects were associated with a remarkable up-regulation of intracellular CD74 when cells were maintained on ECM Plus compared to TCP, which triggered activation of the phosphoinositide-3-kinase (PI3K) pathway as a key modulator of cell survival (anti-apoptosis) and suppression of cellular senescence. Moreover, AD-MSCs maintained on ECM Plus increased their expression of HLA-DR and stimulated T cell activity. These findings challenge the "immune privilege" of allogeneic MSCs as a universal source for MSC-based therapies. The present study leads to a new paradigm for treating age-related diseases: serial administration of rejuvenated autologous MSCs, which may not only replace aged MSCs but also gradually reverse the aged microenvironment.
[This corrects the article DOI: 10.1016/j.mbplus.2024.100155.].
[This corrects the article DOI: 10.1016/j.mbplus.2024.100162.].
[This retracts the article DOI: 10.1016/j.mbplus.2020.100029.].
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Osteogenesis imperfecta (OI) encompasses a genetically diverse spectrum of skeletal dysplasias, commonly linked to pathogenic variants in COL1A1 and COL1A2. Emerging data identify terminal nucleotidyltransferase 5A (TENT5A)-a noncanonical cytoplasmic poly(A) polymerase essential for mRNA stability of extracellular matrix (ECM) proteins-as a novel OI gene. However, human phenotypes associated with TENT5A impairment and its molecular underpinnings remain poorly characterized. We performed detailed clinical, biochemical, and transcriptomic characterization of a Palestinian male with homozygous TENT5A variant (NM_001370434: c.972C>G, p. Ile324Met). Functional studies were conducted on patient-derived fibroblasts subjected to osteogenic induction, assessing matrix mineralization, mRNA expression, poly(A) tail dynamics (via Nanopore direct RNA sequencing), and stress response. RNA-seq and qPCR were used to profile osteogenic and epigenetic gene expression. Despite normal stature and preserved long bone architecture, the proband exhibited recurrent small bone fractures and persistently elevated alkaline phosphatase. Fibroblasts from the proband demonstrated reduced mineralization and significantly shortened poly(A) tails in COL1A1 and COL1A2 transcripts, corresponding to decreased expression. Transcriptomic analysis revealed downregulation of key ECM, osteogenic, and chromatin-regulatory genes, alongside increased ribosomal transcripts and translational machinery. Poly(A) tail shortening was selective, affecting ECM and regulatory genes but sparing housekeeping genes. Under metabolic stress, TENT5A-impaired fibroblasts showed markedly reduced viability, indicating broader cellular vulnerability. Despite these molecular disruptions, clinical severity remained mild, possibly modulated by early bisphosphonate therapy. This case defines a distinct form of OI arising from post-transcriptional dysregulation in matrix-producing cells. TENT5A alteration compromises ECM production by destabilizing collagen mRNAs, disrupting osteogenic and epigenetic programs, and impairing cellular resilience. Our findings identify a novel homozygous TENT5A variant and demonstrate its pathogenic effect on osteoblast function and matrix mineralization, supporting the inclusion of TENT5A in diagnostic panels for unexplained skeletal fragility.
Immune checkpoint inhibitors (ICI) have demonstrated clinical benefit in head and neck squamous cell carcinoma (HNSCC); however, single-agent efficacy is limited, leaving significant unmet needs. Metformin may synergize with ICIs, offering promise to improve response rates. We leveraged multiomic data from a randomized, presurgical neoadjuvant trial (NCT03618654) evaluating a single infusion of the anti-PD-L1 ICI durvalumab with or without daily, standard dose metformin in previously untreated, nondiabetic patients with HNSCC to understand predictors of response and the effect of combination therapy. Clinical, pathologic, and correlative data were analyzed to investigate response and resistance mechanisms. We present an in-depth multiomic analysis of primary tumor specimens to study treatment response/resistance in human papillomavirus-positive HNSCC. Baseline samples revealed that myofibroblastic cancer-associated fibroblast and extracellular matrix signatures were enriched in durvalumab plus metformin nonresponders, which were localized to the leading tumor edge on spatial transcriptomics. In contrast, baseline responder samples were enriched for the Langerhans-like dendritic cell (DC) state and IFN signatures. Treatment increased intratumoral CD8+ T-cell and IFN signatures and peripheral blood CCL2 levels. Responders demonstrated macrophage and DC enrichment and antigen processing and presentation upregulation. Enrichment of cell cycle-related gene sets, specifically the MYC targets V1 hallmark gene set, correlated with nonresponse. Early response and resistance dynamics for durvalumab plus metformin in human papillomavirus-positive HNSCC reveal baseline extracellular matrix-myofibroblastic cancer-associated fibroblast as predictive of nonresponse. In contrast, responders were distinguished by baseline enrichment in the Langerhans-like DC state and posttreatment antigen-presenting gene sets.
The purpose of this study was to compare the modulation effects of Microcurrent Therapy (MT) and Low-Level Laser Therapy (LLLT) on Matrix Metalloproteinases (MMPs) and tissue inhibitors of Metalloproteinases (TIMPs) expressions during healing of surgical wounds using appendectomy wound as a model. Ninety patients who recently underwent appendectomy were randomly divided into 3 main groups of equal numbers. All cases in the three groups received ordinary medical therapy. Moreover, group A (MT group) received Microcurrent Therapy for 20 min. In addition to a designed physical therapy treatment protocol for 20 min. Group B (LLLT group) received Low-Level Laser Therapy for 20 min., plus the same designed physical therapy treatment protocol for 20 min. Group C (placebo group) received placebo shame LLLT for 20 min. plus the same designed physical therapy treatment protocol for 20 min. Enzyme-linked immunosorbent assay (ELISA) and Western Blot Technique (WBT) were used to determine expression levels of MMP-8, MMP-9, and TIMP-1 at the beginning of treatment and after the end of twelve successive sessions. Following therapies, results showed a statistically significant decrease in the MMP-8 and MMP-9 expressions with significantly increased expression levels of TIMP-1 in each group separately (P < 0.05). These changes in the expression levels towards proper healing of surgical wounds were more obvious in MT and LLLT groups compared to the placebo group, with significantly better effect in the LLLT group compared to the MT group . Microcurrent therapy and low-level laser therapy have a notable impact in improving wound healing process as they can significantly affect the expression levels of matrix metalloproteinases and tissue inhibitors of metalloproteinases towards good prognosis of healing process and decreasing possible wound healing complication, with superior effect of low-level laser therapy.
Calcific aortic valve disease (CAVD) is characterized by progressive extracellular matrix (ECM) remodeling that promotes valve fibrosis and calcification. However, its molecular and structural basis remains unclear. In this study, we comprehensively analyzed ECM remodeling in human CAVD valves, focusing on collagen dynamics and key ECM-associated regulatory components. Histopathological analysis revealed fibrous layer thickening, collagen disorganization, and focal loss of the spongiosa in the CAVD group. Polarized picrosirius red staining demonstrated increased yellow-orange birefringence in the fibrotic and calcified regions, indicating altered collagen organization. Quantitative liquid chromatography-mass spectrometry analysis showed region-specific shifts toward an increased type III collagen proportion in fibrotic and calcific regions despite the reduced total collagen content in calcified areas. Collagen with improper triple-helical structure primarily accumulated around the calcified nodules, suggesting abnormal collagen turnover. Transmission electron microscopy revealed thinner and more heterogeneous collagen fibrils in lesioned regions than that in pre-lesional region. In normal valves, immunohistochemistry suggested that the hyaluronan-versican-fibrillin complex contributes to local regulation of Transforming growth factor-beta 1 (TGF-β1) activity via latent TGF-β binding proteins (LTBP); however, this regulatory structure was disrupted in CAVD. Notably, LTBP-4 showed strong, regionally restricted localization in the fibrotic and calcific regions and was positively correlated with collagen yellow-orange birefringence. Collectively, these findings indicate that CAVD is associated with a localized shift toward a structurally heterogeneous, type III collagen-enriched matrix, accompanied by collagen denaturation and abnormal accumulation of LTBP-4, highlighting ECM dysregulation as a key feature of disease progression.
The process of aging is an integral but complex component of life that has been intensely studied for decades, from the molecular level to whole organisms. At the tissue level, bone is one of the most difficult to study due to its composite nature of inorganic and organic phases, but advancements in proteomics are enhancing our understanding of the latter, improving our understanding of skeletal aging through identifying temporal changes across the bone proteome. The relative longevity of extracellular matrix (ECM) proteins can make them more susceptible to accumulating damage modifications over time. In addition, their informational density, including their 2D and 3D structure, protein folding, post translational modifications and proteomic composition, as well as their functional importance, has made them a target of interest in the study of aging and medical conditions such as osteoporosis and arthritis. ECM proteins are also increasingly utilised in forensic science for determining biological sex/age because of their longevity. Following recent developments in peptide location fingerprinting methods that improve capabilities of identifying regional changes in protein structure, this study aimed to identify age-associated regional changes along protein structures in Rattus norvegicus from whole limb LC-MC/MS data. Regional changes in protein structure were identified in a variety of collagenous and non-collagenous ECM proteins, providing evidence for increased remodeling in juvenile rats and a reduced ability in adult rats, alongside damage accumulation in the adult ECM. This research highlights the importance of fibrillar collagen remodeling but is also indicative of potential new roles for osteopontin, thrombin, apolipoproteins and wider ECM regulators such as cartilage oligomeric matrix protein. This demonstrates, in this case, the utility of peptide location fingerprinting as a screening tool to identify biomarker candidates of bone aging between juvenile and adult rats.
Sulfate plays a critical role in bone health and development. More than 90 sulfate-related genes are highly conserved across mammalian species, but very few of these genes have been linked to adverse bone phenotypes in humans. To extend our knowledge of sulfate-related gene expression dynamics during mineralization, this study leveraged 6 publicly available transcriptomic datasets, covering human osteosarcoma cell line Saos-2 mineralization, 2 mouse calvarial osteoblast mineralization models, vascular smooth muscle cell (VSMC) calcification, and 2 neurogenic heterotopic ossification datasets. We focused on a total of 12 sulfate-related genes that were upregulated during mineralization of Saos-2 cells. Six of these genes (Slc26a11, Sgsh, Sqor, Sult1a1, Tpst1, and Ust) were also consistently upregulated during mouse osteoblast and VSMC mineralization. Additionally, 3 genes (Cth, Got1, and Sulf1) were upregulated in Saos-2 mineralization but downregulated in mouse primary osteoblasts. Cbs, Chst3, and Chst13 were unchanged in the mouse primary cell models. Cbs, Chst13, Sgsh, Sulf1, and Ust also increased in models of heterotopic ossification. We have now identified several genes (CHST13, TPST1, UST, SULF1, GOT1, SLC26A11, and SULT1A1) that have not previously been considered for adverse bone conditions in humans, suggesting that additional sulfate biology genes may be linked with human skeletal conditions. Network analysis showed large co-expression clusters of genes, including sulfate biology and bone genes, that were upregulated across the calcification time courses. Gene ontology term enrichment analysis demonstrated significant enrichment in terms associated with mineralization, including ossification, bone mineralization, cartilage development, and extracellular matrix organization for these clusters of genes. This study provides a collated list of sulfate-related genes and networks that are associated with mineralization, which will facilitate future functional studies of sulfation pathways associated with bone pathology.
Elastin is the extracellular matrix protein responsible for properties of extension and energy-efficient elastic recoil in large blood vessels, lung parenchyma and other vertebrate tissues. Monomeric tropoelastin assembles by phase separation into an extended polymeric matrix covalently cross-linked through lysine residues, producing a robust biomaterial able to withstand hundreds of millions of cycles of extension and recoil. Elastin functions as an entropic elastomer, whose properties are the direct result of the inability of the protein to fold into a fixed, stable structure. Most investigations of how the unusual properties of polymeric elastin arise from the sequence of tropoelastin have utilized molecular biological/biophysical methodologies. This study takes an alternative approach, using a comprehensive, well-curated database of Amniote tropoelastin sequences to identify characteristics conserved through >300 million years of evolution. Conserved characteristics included preservation of not only regions of positional sequence but also collective or compositional characteristics derived from but not strictly dependent on positional sequence. A plausible overall consensus sequence for Amniote tropoelastins allowed quantification of residue-by-residue, domain-by-domain and region-by-region levels of sequence conservation. Regions of low positional sequence conservation nevertheless maintained a recognizable sequence style characterized by tandem repeats and partial repeats of short, non-polar motifs. Motif analysis suggested hPGhGG, with numerous insertions and deletions, as the underlying repeating unit in all Amniote tropoelastins. The data identify significant evolutionary constraints dictated by fundamental requirements for formation and functionality of the extracellular elastin matrix, and suggest a rich source of evolutionarily permitted opportunities for modulating properties to meet specific species requirements.
Genome-scale metabolic models (GEMs) are indispensable for studying cellular metabolism. We present iCHO3K, a community-consensus, manually curated reconstruction of the Chinese hamster metabolic network. Spanning 11,004 reactions linked to 3,597 genes, iCHO3K augments the network with 3,489 protein structures and physicochemical descriptors for >70% of 7,377 metabolites, enabling structure-aware analyses. We applied iCHO3K to contextualize transcriptomics and metabolomics from a fed-batch Chinese hamster ovary (CHO) cell line engineered to abolish lactate secretion. The model indicated reduced glycolytic flux with enhanced tricarboxylic acid (TCA) activity and elevated intracellular NADH and phosphoenolpyruvate (PEP), consistent with experimental measurements. Leveraging iCHO3K's structural annotations, we evaluated potential off-target binding of NADH and PEP across early glycolytic enzymes and identified a putative allosteric PEP interaction with phosphofructokinase, suggesting a structural mechanism underlying reduced glucose uptake and glycolytic flux. Overall, iCHO3K provides a framework for systematic multi-omics integration, improved flux prediction, and structure-guided mechanistic insight, advancing CHO cell engineering and biomanufacturing. A record of this paper's transparent peer review process is included in the supplemental information.
Root canal sealers play a crucial role in the success of endodontic treatment, facilitating healing and regeneration of the periapical region. This study aimed to evaluate the biological, physicochemical and structural properties of two sealers, AH Plus and ROEKO GuttaFlow 2. Scanning electron microscopy (SEM) analysis reveals polyhedral particles uniformly distributed within the porous organic matrix of AH Plus, whereas ROEKO GuttaFlow 2 exhibits a heterogeneous structure, with particles distributed evenly. Fourier-Transform Infrared Spectroscopy (FT-IR) analysis confirmed the characteristic chemical bonds associated with both the organic and inorganic phases of each material, while X-Ray diffraction analysis identified the main crystalline phases (CaWO4 and ZrO2 for AH Plus and ZrSiO4 and ZrO2 for ROEKO GuttaFlow 2). The biocompatibility tests were performed on human osteosarcoma cells (ATCC-G 292 CRL-1423). An in vitro metabolic activity and viability test (MTT) showed a significant decrease by ~92% (* p < 0.05) and ~87% after 24 and 48 h for samples incubated with AH Plus versus the control. Regarding ROEKO GuttaFlow 2, MTT levels increased by ~8% in the first 24 h, while after 48 h they decreased by ~11% versus control. Lactate dehydrogenase levels significantly increased at 24 and 48 h for cells incubated with AH Plus (*** p < 0.001, ** p < 0.01). ROEKO GuttaFlow 2 significantly decreased the LDH level at 24 h (** p < 0.01), while at 48 h a rise was observed. The significantly increased levels of nitric oxide observed in cells incubated with the materials at 24 and 48 h (** p < 0.01, *** p < 0.001) suggest a cellular adaptation to our experimental environment. Overall, ROEKO GuttaFlow 2 exhibited a more favorable profile under our testing conditions.
Transforming growth factor-beta (TGF-β), a cytokine embedded in the bone matrix, is released during bone resorption, influencing osteoclast differentiation and coupling factor production, which affect osteoblasts and osteocytes. This study investigates the role of TGF-β in bone remodeling using an in vitro model with calcium phosphate-coated plates covalently bonded to latent TGF-β (LTGF-β(+)-CaP plates). This model replicates the natural release of TGF-β and its effects on RAW264 macrophage-like cells, which differentiate into osteoclasts upon stimulation of RANKL. Cells cultured on LTGF-β(+)-CaP plates formed resorption pits and released TGF-β, upregulating osteoclast differentiation- and resorption-related genes during early differentiation. During the resorption phase, TGF-β-enhanced osteoblast activation and coupling factor expression supporting bone formation in surrounding cells. In osteocytes, it differentially regulated gene expression by upregulating osteoprotegerin and downregulating sclerostin, suggesting a dual role in remodeling. Our findings demonstrate that TGF-β plays a critical role in bone homeostasis by directly promoting osteoclast differentiation and resorption while indirectly facilitating osteoblast differentiation through coupling factors. These results provide insights into the dynamic interactions between osteoclasts, osteoblasts, and osteocytes, emphasizing TGF-β's role in linking bone resorption and formation. This study establishes a novel in vitro platform to examine TGF-β-mediated bone remodeling and its underlying molecular mechanisms. Furthermore, our model can be used to explore how TGF-β signaling affects cellular communication in the bone and may contribute to identifying new therapeutic targets for osteoporosis and other bone-resorptive disorders.
Healthy tendon is associated with organized and aligned extracellular matrix (ECM) that becomes disorganized with disease, yet the mechanisms and pathogenesis of tendon disease remain poorly understood. Tendons that "wrap-around" joints, such as the flexor digitorum longus (FDL) tendon, contain a unique ECM that shares similarities with diseased tendons, which can be leveraged to study tendon cell biology across distinct loading environments. The pericellular matrix (PCM) is a critical matrix structure that is understudied in tendon and is likely involved in tendon mechanosensation and homeostasis. Two components of the tendon PCM, biglycan and collagen VI, are known regulators of tendon function and are implicated in tendon disease. Given the implication of PCM molecules in regulating tendon properties, this work sought to define the regional development of a murine wrap-around tendon, how biglycan influences these regional properties, and the extent to which these mechanisms involve collagen VI. Gene expression and histological analyses demonstrated regional divergence in FDL tendon properties by P14. While biglycan knockout did not result in broad disruptions to gene expression or the behavior of Scx+ or Col6a1+ cells, several genes were dysregulated with biglycan deficiency. These changes corresponded with inferior mechanical properties in biglycan-deficient tendons. This work defines the development of regional FDL tendon properties and demonstrates that biglycan knockout impacts these regional properties through a collagen VI-independent mechanism. Results from this work provide understanding of biglycan regulation in tendon and lay the foundation for future work researching tendon mechanosensitive mechanisms.