Enhanced Extracellular Matrix Research Articles

Injectable and Microporous Microgel Assembly with Sequential Bioactive Factor Release for the Endogenous Repair of Nucleus Pulposus

AbstractHarnessing endogenous stem/progenitor cells to enhance extracellular matrix (ECM) deposition in the nucleus pulposus (NP) is considered as a potential treatment for intervertebral disc degeneration (IVDD), but is limited by the lack of suitable scaffolds for cell infiltration and growth, as well as the tailored release of bioactive factors. Herein, an injectable cell‐free microgel assembly (MA‐TNS) with interconnected microporous structure is developed to recruit stem/progenitor cells and induce the differentiation of the recruited cells by releasing stromal cell‐derived factor‐1α (SDF‐1α) and transforming growth factor‐β1 (TGF‐β1) sequentially. TGF‐β1 is initially loaded by the chondroitin sulfate/gelatin nanoparticles. Together with SDF‐1α, these nanoparticles are co‐encapsulated into the photo‐cross‐linked microgels, which are then assembled into a microporous scaffold via dynamic boronate ester bonds. An initial rapid release of SDF‐1α followed by a sustained release of TGF‐β1 lasting over 28 days is achieved. MA‐TNS significantly promotes the recruitment, infiltration, and chondrocyte‐like differentiation of marrow mesenchymal stem cells in vitro. Furthermore, in vivo experiments on rats and cynomolgus monkeys prove that MA‐TNS can inhibit IVDD and recover ECM deposition markedly, thereby enabling long‐term NP reconstruction. The resulting efficacy in rats and nonhuman primates supports that MA‐TNS is a promising scaffold for intervertebral disc repair and regeneration.

Effectiveness of the production of tissue-engineered living bone graft: a comparative study using perfusion and rotating bioreactor systems

Bioreactor systems are very precious tools to generate living bone grafts in vitro. The aim of this study was to compare the effectiveness of rotating and perfusion bioreactor in the production of a living bone construct. Human bone marrow-derived mesenchymal stem cells (BMDSCs) were seeded on the surfaces of hydroxyapatite-based scaffolds and cultured for 21 days in three different conditions: (1) static 3D culture, (2) 3D culture in a perfusion bioreactor, and (3) dynamic 3D culture in a rotating bioreactor. Quantitative evaluation of cell number showed that cultivation in the perfusion bioreactor significantly reduced cell proliferation compared to the rotating bioreactor and static culture. Osteogenic differentiation test demonstrated that BMDSCs cultured in the rotating bioreactor produced significantly greater amount of osteopontin compared to the cells cultured in the perfusion bioreactor. Moreover, Raman spectroscopy showed that cultivation of BMDSCs in the rotating bioreactor enhanced extracellular matrix (ECM) mineralization that was characterized by B-type carbonated substitution of hydroxyapatite (associated with PO43− groups) and higher mineral-to-matrix ratio compared to the ECM of cells cultured in the perfusion system. Thus, it was concluded that the rotating bioreactor was much more effective than the perfusion one in the generation of bone tissue construct in vitro.

The addition of zinc ions to polymer-ceramic composites accelerated osteogenic differentiation of human mesenchymal stromal cells

Critical-sized bone defects, caused by congenital disorders or trauma, are defects that will not heal spontaneously and require surgical intervention. Recent advances in biomaterial design for the treatment of such defects focus on improving their osteoinductive properties. Here, we propose a bioactive composite with high ceramic content composed of poly(ethyleneoxide terephthalate)/poly(butylene terephthalate) (1000PEOT70PBT30, PolyActive, PA) and 50 % beta-tricalcium phosphate (β-TCP) with the addition of zinc in a form of a coating on the TCP particles. Due to its essential role in bone homeostasis, we hypothesised that the addition of zinc to the polymer-ceramic composite will further enhance its osteogenic properties. β-TCP particles were immersed in a zinc solution with a concentration of 15 or 45 mM. The addition of zinc did not alter the β-TCP composition or the release of calcium or phosphate ions. 3D porous 1000PEOT70PBT30 - β-TCP scaffolds were additively manufactured by “3D fibre deposition” and their ability to support the osteogenic differentiation was assessed by culturing clinically relevant human mesenchymal stromal cells (hMSCs) on the scaffolds for 3, 7, 14 and 28 days. The expression of osteogenic gene markers was increased in the presence of both zinc concentrations. Remarkably, upregulation of osteocalcin (OCN), a late osteogenic marker, was observed after three days of culture. Furthermore, enhanced extracellular matrix (ECM) production and mineralization was observed. These findings support the existing evidence on the osteogenic properties of zinc and further demonstrate that the incorporation of zinc into a polymer-ceramic composite could be a promising strategy in the field of regeneration of critical-sized bone defects.

Bioorthogonal DOPA-NGF activated tissue engineering microunits for recovery from traumatic brain injury by microenvironment regulation

Stem cell treatment is vital for recovery from traumatic brain injury (TBI). However, severe TBI usually leads to excessive inflammation and neuroinhibitory factors in the injured brain, resulting in poor neural cell survival and uncontrolled formation of glial scars. In this study, a bioorthogonal microenvironment was constructed on biodegradable poly(lactide-co-glycolide) (PLGA) microcarriers through immobilization of mussel-inspired bioorthogonal 3,4-dihydroxyphenylalanine-containing recombinant nerve growth factor (DOPA-NGF) and human umbilical cord mesenchymal stem cells (hUMSCs) for minimally invasive therapy of TBI. Cell culture and RNA-seq analysis revealed enhanced extracellular matrix (ECM) secretion and viability of hUMSCs on PLGA microcarriers compared to 2D culture. Immobilized DOPA-NGF further promoted adhesion, proliferation, and gene expression in RSC96 neurotrophic cells and hUMSCs. Specifically, the neurotrophin receptor of NT-3 (NTRK3) in hUMSCs was activated by DOPA-NGF, leading to MYC transcription and paracrine enhancement to build an adjustable biomimetic microenvironment. After transplantation of microunits in animal models, the motor and learning-memory ability of TBI mice were improved through rollbacks of overactivated inflammatory reaction regulation, neuronal death, and glial scar formation after injury. This was attributed to the paracrine enhancement of hUMSCs activated by the DOPA-NGF. Our study provides a neural regenerative microenvironment-based therapeutic strategy to advance the effects of transplanted hUMSCs in cell-based regenerative medicine for TBI therapy. Statement of significanceExtensive studies have demonstrated the importance of the microenvironment for posttraumatic brain injury recovery. However, an efficient method that can mimic the neural regenerative microenvironment to strengthen stem cell therapy and brain injury recovery is still absent. In this study, the minimally invasive transplantation of DOPA-NGF immobilized biodegradable microcarriers with mesenchymal stem cells was found to be an effective method for regeneration of injured brain. Moreover, transcriptome analysis revealed that neurotrophin receptor of NT-3 (NTRK3) was activated by DOPA-NGF for MYC transcription and paracrine enhancement to build a kind of adjustable biomimetic microenvironment for brain injury therapy. This study provides a neural regenerative microenvironment-based therapeutic strategy to advance the transplanted hUMSCs in cell-based regenerative medicine for neural recovery.

Carbon dots enhance extracellular matrix secretion for dentin-pulp complex regeneration through PI3K/Akt/mTOR pathway-mediated activation of autophagy.

Pulp injury is one of the most common clinical diseases, and severe cases are usually associated with the functional loss of the tooth, while the current clinical treatment modality is only a cavity filling procedure without the regeneration of the dentin-pulp complex, thus leading to a devitalized and brittle tooth. In this study, carbon dots (CDots) with excellent biocompatibility are prepared from ascorbic acid and polyethyleneimine via a hydrothermal method. The as-prepared CDots can enhance extracellular matrix (ECM) secretion of human dental pulp stem cells (DPSCs), giving rise to increased cell adhesion on ECM and a stronger osteogenic/odontogenic differentiation capacity of DPSCs. Further, the mechanism underlying CDots-enhanced ECM secretion is revealed by the transcriptome analysis, Western blot assay and molecular dynamics simulation, identifying that the pharmacological activities of CDots are originated from a reasonable activation of the autophagy, which is mediated by regulating phosphoinositide 3-kinase/protein kinase B/mammalian target of rapamycin signaling pathway. Based on the abundant CDots-induced ECM and thereby the reinforcement of the cell-ECM adhesion, an intact dental pulp stem cell sheet can be achieved, which in return promote in vivo the efficient regeneration of dentin-pulp complex as well as blood vessels.

DR7dA, a Novel Antioxidant Peptide Analog, Demonstrates Antifibrotic Activity in Pulmonary Fibrosis In Vivo and In Vitro.

Pulmonary fibrosis (PF), which is characterized by enhanced extracellular matrix (ECM) deposition, is an interstitial lung disease that lacks an ideal clinical treatment strategy. It has an extremely poor prognosis, with an average survival of 3-5 years after diagnosis. Our previous studies have shown that the antioxidant peptide DR8 (DHNNPQIR-NH2), which is extracted and purified from rapeseed, can alleviate PF and renal fibrosis. However, natural peptides are easily degraded by proteases in vivo, which limits their potency. We have since synthesized a series of DR8 analogs based on amino acid scanning substitution. DR7dA [DHNNPQ (D-alanine) R-NH2] is an analog of DR8 in which L-isoleucine (L-Ile) is replaced with D-alanine (D-Ala), and its half-life is better than that of DR8. In the current study, we verified that DR7dA ameliorated tumor growth factor (TGF)-β1-induced fibrogenesis and bleomycin-induced PF. The results indicated that DR7dA reduced the protein and mRNA levels of TGF-β1 target genes in TGF-β1-induced models. Surprisingly, DR7dA blocked fibrosis in a lower concentration range than DR8 in cells. In addition, DR7dA ameliorated tissue pathologic changes and ECM accumulation in mice. BLM caused severe oxidative damage, but administration of DR7dA reduced oxidative stress and restored antioxidant defense. Mechanistic studies suggested that DR7dA inhibits ERK, P38, and JNK phosphorylation in vivo and in vitro All results indicated that DR7dA attenuated PF by inhibiting ECM deposition and oxidative stress via blockade of the mitogen-activated protein kinase (MAPK) pathway. Hence, compared with its parent peptide, DR7dA has higher druggability and could be a candidate compound for PF treatment in the future. SIGNIFICANCE STATEMENT: In order to improve druggability of DR8, we investigated the structure-activity relationship of it and replaced the L-isoleucine with D-alanine. We found that the stability and antifibrotic activity of DR7dA were significantly improved than DR8, as well as DR7dA significantly attenuated tumor growth factor (TGF)-β1-induced fibrogenesis and ameliorated bleomycin-induced fibrosis by inhibiting extracellular matrix deposition and oxidative stress via blockade of the MAPK pathway, suggesting DR7dA may be a promising candidate compound for the treatment of PF.

The RNA-binding protein SND1 promotes the degradation of GPX4 by destabilizing the HSPA5 mRNA and suppressing HSPA5 expression, promoting ferroptosis in osteoarthritis chondrocytes.

Heat shock protein family A member 5 (HSPA5), a recently identified suppressor of ferroptosis, was reported to potentially regulating osteoarthritis. However, the exact role of HSPA5 and how its expression was regulated in osteoarthritis are largely unclear. Rat primary chondrocytes were treated with 10ng/mL IL-1β for 24h and incubated with ferrostatin-1 (a ferroptosis inhibitor). Cell viability, production of TNF-α, ROS and MDA, expression levels of collagen II, MMP13, GPX4, and SND1, and Fe2+ concentration were detected. Gain- and loss-of-function manipulations were performed to investigate the effect of HSPA5 on chondrocyte functions, and SND1 shRNA (sh-SND1) was transfected into IL-1β-treated primary chondrocytes alone or together with sh-HSPA5. Furthermore, the interaction between HSPA5 and GPX4 and the regulation of HSPA5 on GPX4 were explored. Finally, SND1 was knocked down in the rats with osteoarthritis, and the histopathology, expression of HSPA5-GPX4 axis, and levels of oxidative stress markers were evaluated. IL-1β treatment could enhance extracellular matrix (ECM) degradation (collagen II reduced and MMP13 increased), promote ferroptosis, manifested by decreased cell viability, increased levels of TNF-α, ROS, MDA, and Fe2+ concentrations, and decreased level of GPX4 protein, and increase SND1 expression in chondrocytes, which could be reversed by ferrostatin-1. Knockdown of SND1 enhanced ECM degradation and suppressed ferroptosis IL-1β-treated chondrocytes, which could be eliminated by knockdown of HSPA5. SND1 bound with HSPA5 at the 3'UTR and destabilized the HSPA5 mRNA. HSPA5 protein directly bound with GPX4 protein and positively regulate its expression. HSPA5 overexpression suppressed IL-1β-induced chondrocyte ferroptosis, while this effect was counteracted by GPX4 silencing. Knockdown of SND1 upregulated HSPA5 and GPX4 in rat cartilage, inhibited inflammatory damage and ferroptosis, and alleviated OA progression. The RNA-binding protein SND1 promotes the degradation of GPX4 by destabilizing the HSPA5 mRNA and suppressing HSPA5 expression, promoting ferroptosis in osteoarthritis chondrocytes.

Notoginsenoside R1 Facilitated Wound Healing in High-Fat Diet/Streptozotocin-Induced Diabetic Rats.

Diabetic ulcers bring about high morbidity and mortality in patients and cause a great economic burden to society as a whole. Since existing treatments cannot fulfil patient requirements, it is urgent to find effective therapies. In this study, the wound healing effect of topical notoginsenoside R1 (NR1) treatment on diabetic full-thickness wounds in type II diabetes mellitus (T2DM) was induced by the combination of a high-fat diet and streptozotocin (STZ) injection. NR1 significantly increased the wound closure rate, enhanced extracellular matrix (ECM) secretion, promoted collagen growth, increased platelet endothelial cell adhesion molecule-1 (CD31) expression, and decreased cleaved caspase-3 expression. RNA-Seq analysis identified ECM remodeling and inflammation as critical biological processes and Timp1 and Mmp3 as important targets in NR1-mediated wound healing. Further experiments showed that NR1-treated wounds demonstrated higher expression of tissue inhibitor of metalloproteinase 1 (TIMP1) and transforming growth factor-β1 (TGFβ1) and lower expression of matrix metallopeptidase 9 (MMP9), matrix metallopeptidase 3 (MMP3), interleukin-1β (IL-1β), and interleukin-6 (IL-6) than diabetic wounds. These investigations promote the understanding of the mechanism of NR1-mediated diabetic wound healing and provide a promising therapeutic drug to enhance diabetic wound healing.

A review on 3D printing in tissue engineering applications

Abstract In tissue engineering, 3D printing is an important tool that uses biocompatible materials, cells, and supporting components to fabricate complex 3D printed constructs. This review focuses on the cytocompatibility characteristics of 3D printed constructs, made from different synthetic and natural materials. From the overview of this article, inkjet and extrusion-based 3D printing are widely used methods for fabricating 3D printed scaffolds for tissue engineering. This review highlights that scaffold prepared by both inkjet and extrusion-based 3D printing techniques showed significant impact on cell adherence, proliferation, and differentiation as evidenced by in vitro and in vivo studies. 3D printed constructs with growth factors (FGF-2, TGF-β1, or FGF-2/TGF-β1) enhance extracellular matrix (ECM), collagen I content, and high glycosaminoglycan (GAG) content for cell growth and bone formation. Similarly, the utilization of 3D printing in other tissue engineering applications cannot be belittled. In conclusion, it would be interesting to combine different 3D printing techniques to fabricate future 3D printed constructs for several tissue engineering applications.

Freestanding Magnetic Microtissues for Tissue Engineering Applications.

A long-sought goal in tissue engineering (TE) is the development of tissues able to recapitulate the complex architecture of the native counterpart. Microtissues, by resembling the functional units of living structures, can be used to recreate tissues' architecture. Howbeit, microfabrication methodologies fail to reproduce cell-based tissues with uniform shape. At the macroscale, complex tissues are already produced by magnetic-TE using solely magnetized cells as building materials. The enhanced extracellular matrix (ECM) deposition guaranties the conservation of tissues' architecture, leading to a successful cellular engraftment. Following the same rational, now the combination of a versatile microfabrication-platform is proposed with magnetic-TE to generate robust micro-tissues with complex architecture for TE purposes. Small tissue units with circle, square, and fiber-like shapes are designed with high fidelity acting as building blocks for engineering complex tissues. Notably, freestanding microtissues maintain their geometry after 7 days post-culturing, overcoming the challenges of microtissues fabrication. Lastly, the ability of microtissues in invading distinct tissue models while releasing trophic factors is substantiated in methacryloyl laminarin (LAM) and platelet lysates (PLMA) hydrogels. By simply using cells as building units and such microfabrication-platform, the fabrication of complex multiscale and multifunctional tissues with clinical relevance is envisaged, including for therapies or disease models.

Dynamic Pressure Stimulation Upregulates Collagen II and Aggrecan in Nucleus Pulposus Cells Through Calcium Signaling.

An in vitro study to investigate the effect of pressure stimulation on nucleus pulposus (NP) cells. The aim of this study was to investigate the question whether physical stimulation can be leveraged to enhance extracellular matrix (ECM) synthesis as a preventive measure for intervertebral disc (IVD) degeneration. ECM plays an important role in regulating hydration and pressure balance of the IVD. Cellular stimulation devices with different pressurizing protocols were used to create a pressurized environment to cells cultures. The setup was used to mimic the pressurized conditions within IVD to investigate the effect of pressure stimulation on NP cells. Pressure stimulation at 300 kPa can enhance the synthesis of ECM proteins Collagen II and aggrecan in NP cells and the effect of dynamic pressure stimulation outperformed the static one. The difference between static and dynamic pressure stimulation was due primarily to calcium signaling activated by pressure fluctuation. The superior effect of dynamic pressure holds for a wide range of stimulation durations, relating to the range of spontaneous calcium oscillations in NP cells. The results link mechanotransduction to the downstream ECM protein synthesis and suggest slow exercises that correspond with spontaneous calcium oscillations in NP cells can be effective to stimulate ECM synthesis in IVD.

Lysosomal Acid Lipase, a Novel Biomarker for Hepatic Fibrosis

Background: Hepatic fibrosis (HF) can be easily developed irreversible liver cirrhosis or even liver cancer. Lysosomal acid lipase (LAL), encoded by the lipase A (Lipa) gene, is a critical enzyme involved in cholesterol ester metabolism and is considered a biomarker for liver cirrhosis. However, the association between LAL and HF, as well as the role of LAL in HF development are poorly understood. In this study, we aimed to determine whether LAL served a novel biomarker for HF development and reveal its underlying mechanism. Methods: The expression of LAL was surveyed in the liver tissues of CCl 4 and high-fat diet (HFD)-induced HF animals. Alpha smooth muscle actin level, collagen and Gata family expression was analyzed by Real-time PCR and Western blot. The identification of putative transcription factor binding sites in the DNA sequences of Lipa was located by PROMO-ALGGEN v8.3 online software and ENCODE ChIP-Seq Significance Tool. MD simulation was performed using Desmond to explore the protein-ligand interaction. Findings: Our results showed that LAL expression was reduced in TGF-β1-induced HSC-T6 cells and liver tissues. Gata 3, a member in the Gata family that transcribes Lipa, was involved in the pathological process of HF. More importantly, morroniside, an active compound of Cornus officinalis Sieb. et Zucc., regulated the GATA3/Lipa axis, inhibited HSCs activation and enhanced extracellular matrix (ECM) degradation. These findings, for the first time, suggest a novel biomarker for HF, which is also a molecular target of morroniside for its anti-HF effects. Interpretation: This study provides strong scientific evidence to support the development of morroniside as novel alternative or complementary therapeutics for HF prevention and treatment. Funding: This work was supported by the Basic and Applied Basic Research Fund Project of Guangdong Province (NO.2019A1515110202), the China Postdoctoral Science Foundation (NO.2020M672604), The National Natural Science Foundation of China (82074019 and 81930114), Characteristic innovation projects of universities in Guangdong Province (2020KTSCX030), Guangdong Key Laboratory for Translational Cancer research of Chinese Medicine (2018B030322011), Key-Area Research and Development Program of Guangdong Province (No. 2020B1111100004). Research Grant Council of HKSAR (HKBU-22103017-ECS), Innovation & Technology Commission (PRP/015/19FX), National Natural Science Foundation of China (SCM-2016-NSFC-003) and Natural Science Foundation of Guangdong Province (2018A0303130122). Declaration of Interest: None to declare. Ethical Approval: All animals received humane care, and the experimental protocol was approved by the Committee of Laboratory Animals and was performed according to institutional guidelines.

The effects of cardiac stretch on atrial fibroblasts: analysis of the evidence and potential role in atrial fibrillation.

Atrial fibrillation (AF) is an important clinical problem. Chronic pressure/volume overload of the atria promotes AF, particularly via enhanced extracellular matrix (ECM) accumulation manifested as tissue fibrosis. Loading of cardiac cells causes cell stretch that is generally considered to promote fibrosis by directly activating fibroblasts, the key cell type responsible for ECM production. The primary purpose of this article is to review the evidence regarding direct effects of stretch on cardiac fibroblasts, specifically: (i) the similarities and differences among studies in observed effects of stretch on cardiac fibroblast function; (ii) the signalling pathways implicated; and (iii) the factors that affect stretch-related phenotypes. Our review summarizes the most important findings and limitations in this area and gives an overview of clinical data and animal models related to cardiac stretch, with particular emphasis on the atria. We suggest that the evidence regarding direct fibroblast activation by stretch is weak and inconsistent, in part because of variability among studies in key experimental conditions that govern the results. Further work is needed to clarify whether, in fact, stretch induces direct activation of cardiac fibroblasts and if so, to elucidate the determining factors to ensure reproducible results. If mechanical load on fibroblasts proves not to be clearly profibrotic by direct actions, other mechanisms like paracrine influences, the effects of systemic mediators and/or the direct consequences of myocardial injury or death, might account for the link between cardiac stretch and fibrosis. Clarity in this area is needed to improve our understanding of AF pathophysiology and assist in therapeutic development.

Berberine accelerated wound healing by restoring TrxR1/JNK in diabetes.

The high disability, mortality and morbidity of diabetic ulcers make it urgent to explore effective strategies for diabetic wound repair. TrxR1 plays a vital role in regulating redox homeostasis in various pathologies. In the present study, the effect of berberine (BBR) on diabetic wounds was investigated in streptozotocin (STZ)-induced diabetic rats and a high glucose (HG)-induced cell model, and the mechanism of BBR on TrxR1 was elucidated. BBR treatment remarkably accelerated wound healing and enhanced extracellular matrix (ECM) synthesis and significantly inhibited HG-induced HaCaT cell damage. Further analysis indicated that BBR activated TrxR1, suppressed its downstream JNK signaling, thereby inhibiting oxidative stress and apoptosis, promoted cell proliferation, down-regulated matrix metalloproteinase (MMP) 9 (MMP9) and up-regulated transforming growth factor-β1 (TGF-β1) and tissue inhibitors of MMP 1 (TIMP1), resulting in accelerated wound healing. Importantly, the enhancement of BBR on wound repair was further abolished by TrxR1 inhibitor. Moreover, in diabetic wounds induced by a combination of STZ injection and high-fat diet, BBR significantly increased wound closure rate and TrxR1 expression, and this was reversed by TrxR1 inhibitor. These data indicated that topical BBR treatment accelerated diabetic wound healing by activating TrxR1. Targeting TrxR1 may be a novel, effective strategy for restoring redox homeostasis and promoting diabetic wound healing.

Demineralized bone matrix scaffold modified with mRNA derived from osteogenically pre-differentiated MSCs improves bone repair

Gene therapy based on mRNA provides a promising approach for bone regeneration. Quick mRNA translation and controlled protein production could be earned by implantation of mRNA-activated scaffold in bone remodeling region. Furthermore, the expression levels of osteogenic-related mRNA in the cytoplasm of osteogenically pre-differentiated mesenchymal stem cells (MSCs) were high and the expression levels were different at different stages of osteogenically differentiated MSCs. This study intended to investigate the effect of osteoinductive-mRNAs (Oi-mRNAs), derived from osteogenically pre-differentiated MSCs at various stages (Day 1 (Oi1-mRNA), Day 3 (Oi3-mRNA), Day 7 (Oi7-mRNA), Day 14 (Oi14-mRNA) and Day 21 (Oi21-mRNA), respectively), on the osteogenic differentiation of MSCs. Further, the Oi-mRNAs combined with cationic polymer polyethylenimine (PEI) were loaded onto demineralized bone matrix (DBM) scaffold (Oi-mRNA/DBM). The results revealed that the Oi1-mRNA, Oi3-mRNA and Oi21-mRNA had no obvious effect on the osteogenic differentiation of MSCs, while the Oi7-mRNA increased the expression of alkaline phosphatase (ALP) and the Oi14-mRNA significantly promoted the expression of osteocalcin (OC) and osteopontin (OPN), and calcium deposition. In addition, the Oi14-mRNA/DBM scaffold could significantly enhance extracellular matrix (ECM) secretion and new collagen formation of MSCs. After being implanted into rat critical-sized cranium defect model, the Oi14-mRNA/DBM scaffold could promote the infiltration of cells and repair of bone defect in vivo. The DBM scaffold loaded with mRNA encoding osteoinductive protein may provide a powerful tool for bone defect repair.

Circular RNA derived from TIMP2 functions as a competitive endogenous RNA and regulates intervertebral disc degeneration by targeting miR‑185‑5p and matrix metalloproteinase 2.

Intervertebral disc degeneration (IDD) is an important cause of lower back pain, although the underlying mechanisms remain poorly understood. The present study aimed to examine the role of a circular RNA derived from tissue inhibitor of metallopeptidases 2 (circ-TIMP2) in degenerative nucleus pulposus (NP) tissues, and to validate its function in cultured human NP cells. Overexpression of miR-185-5p in NP cells markedly inhibited the enhanced extracellular matrix (ECM) catabolism induced by tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) treatment. Bioinformatics analysis demonstrated that matrix metalloproteinase 2 (MMP2) was a potential target of miR-185-5p. MMP2 protein expression levels were increased following treatment with TNF-α and IL-1β in NP cells compared with those in untreated cells, and this effect was attenuated by transfection with miR-185-5p. Compared with normal NP tissues, IDD samples exhibited higher circ-TIMP2 expression levels. In addition, overexpres-sion of circ-TIMP2 promoted ECM catabolism and suppressed ECM anabolism. Furthermore, circ-TIMP2 sequestered miR-185-5p, which may potentially upregulate the target genes associated with ECM degradation. In conclusion, the results of the present study revealed that circ-TIMP2 promoted TNF-α- and IL-1β-induced NP cell imbalance between ECM anabolism and catabolism via miR-185-5p-MMP2 signaling. These findings provide a potential therapeutic option for the treatment of IDD.

The circular RNA FAM169A functions as a competitive endogenous RNA and regulates intervertebral disc degeneration by targeting miR-583 and BTRC

Intervertebral disc degeneration (IDD) is an important factor leading to low back pain, although the underlying mechanisms remain poorly understood. In this study we examined the role of circular RNA FAM169A (circ-FAM169A) in degenerative nucleus pulposus (NP) tissues, and validated its function in cultured human NP cells. Overexpression of circ-FAM169A in NP cells markedly enhanced extracellular matrix (ECM) catabolism and suppressed ECM anabolism in NP cells. Furthermore, circ-FAM169A sequestered miR-583, which could potentially upregulate BTRC, an inducer of the NF-κB signaling pathway. In conclusion, the present study revealed that circ-FAM169A promotes IDD development via miR-583/BTRC signaling. These findings provide a potential therapeutic option for the treatment of IDD.

3D-Printed Bioreactor Enhances Potential for Tendon Tissue Engineering

Bioreactors have immense value within tissue engineering by enabling important control over design inputs within a microenvironment; however, they are often complex and expensive. Herein, we demonstrate a strategy where a bioreactor was 3D-printed as a means to provide an accessible route to culture scaffolds under physiological inputs and to determine an ideal tendon scaffold structure based on remodeling and degradation events. Furthermore, real-time monitoring of tissue remodeling is introduced through the bioreactor design. Following in vitro uniaxial stretch conditioning, scaffolds were positively altered to reflect improved biomechanical function, enhanced extracellular matrix (ECM) content, and increased mechanical properties of elastic moduli. A bioreactor was constructed to meet the following criteria: real-time readout of resistance, ability to be easily cleaned and sterilized, control of strain, speed, and time parameters, and capability to withstand 3 weeks of continuous load with minimal maintenance. The scaffolds cultured in a dynamic setting showed improved mechanics and promising ECM and collagen content generated. Overall, the bioreactor was effective as a tool to provide mechanical culture conditions that promote the tendon cell niche. The bioreactor supports the scaffolds as a possible therapeutic venture in tendon tissue engineering. Additionally, the 3D-printed bioreactor is a significant contribution to the tissue engineering field by making dynamic culturing more accessible for researchers. Bioreactors present physiological and dynamic conditions for tissue testing and are crucial in tissue engineering for product development, standardization, quality control, and large-scale synthesis. The low-cost, 3D-printed, programmable bioreactor provides cyclic stretch to scaffolds with real-time resistance readout and demonstrates tissue changes over time. The bioreactor enhances scaffold biomechanics and biofunctionality towards natural tendon by promoting cell alignment through mechanical force. The design components are culture chamber for tendon constructs, motor control of uniaxial stretching, and a base for stabilization. The results from conditioning scaffolds in the bioreactor lend to a better understanding of how specific inputs affect tissue outcomes.

PO-229 Transient tissue ‘priming’ via FAK inhibition to impair pancreatic ductal adenocarcinoma (PDAC) progression to improve sensitivity to gemcitabine/abraxane

IntroductionThe extensive stromal deposition and remodelling of pancreatic ductal adenocarcinoma (PDAC) alters mechanical tumour-stroma integrations, promoting tumour development and metastatic spread. Few effective therapies mean that PDAC is predicted to be the second leading cause of cancer mortality by 2030. In highly metastatic mouse models of PDAC, we observed enhanced extracellular matrix (ECM) deposition and remodelling throughout disease progression. This was paralleled by an increased focal adhesion kinase (FAK) expression and activity, suggesting a role for FAK in the increased desmoplastic reaction that is typical of PDAC. Consequently, fine tuned manipulation of the dense stroma by streamlined FAK inhibition (FAKi) presents a novel opportunity for PDAC management and improved response to chemotherapy.Material and methodsIntravital imaging of the FUCCI cell cycle reporter was used to dynamically monitor tumour cell response to combined FAKi and standard-of-care therapy with gemcitabine/Abraxane. This was overlaid with second harmonic generation (SHG) imaging of collagen fibres, to assess the efficacy of FAKi to disrupt the dense PDAC ECM. To complement our in vivo metastatic studies, we used sophisticated 3D in vitro models of invasion, anchorage-independent growth and shear-stress, in both primary and patient-derived PDAC cell lines.Results and discussionsWe systematically demonstrated that using FAKi to modulate ECM prior to standard-of-care therapy enhanced treatment efficacy whilst also reducing metastatic spread in vivo. Further analysis revealed that FAKi sensitised cells to shear stress, impairing metastatic colonisation and the establishment of fibrotic niches in the liver. Stratified patient samples revealed a subset of patients likely to respond to FAK priming regimes, where fine-tuned ECM manipulation prior to chemotherapy may offer a novel opportunity in metastatic PDAC.ConclusionThis subtype-specific fine-tuned stromal manipulation may allow us to maximise gemcitabine/Abraxane therapy whilst reducing drug toxicity and potentially reducing metastatic spread in a preclinical setting.

Establishment of an in vitro organoid model of dermal papilla of human hair follicle.

Human hair dermal papilla (DP) cells are specialized mesenchymal cells that play a pivotal role in hair regeneration and hair cycle activation. The current study aimed to first develop three-dimensional (3D) DP spheroids (DPS)with or without a silk-gelatin (SG) microenvironment, which showed enhanced DP-specific gene expression, resulting in enhanced extracellular matrix (ECM) production compared with a monolayer culture. We tested the feasibility of using this DPS model for drug screening by using minoxidil, which is a standard drug for androgenic alopecia. Minoxidil-treated DPS showed enhanced expression of growth factors and ECM proteins. Further, an attempt has been made to establish an in vitro 3D organoid model consisting of DPS encapsulated by SG hydrogel and hair follicle (HF) keratinocytes and stem cells. This HF organoid model showed the importance of structural features, cell-cell interaction, and hypoxia akin to in vivo HF. The study helped to elucidate the molecular mechanisms to stimulate cell proliferation, cell viability, and elevated expression of HF markers as well as epithelial-mesenchymal crosstalks, demonstrating high relevance to human HF biology. This simple in vitro DP organoid model system has the potential to provide significant insights into the underlying mechanisms of HF morphogenesis, distinct molecular signals relevant to different stages of the hair cycle, and hence can be used for controlled evaluation of the efficacy of new drug molecules.