Telocytes: current methods of research, challenges and future perspectives.

Telocytes (TCs) are CD34-positive interstitial cells that have long cytoplasmic projections, called telopodes; they have been identified in several organs and in various species. These cells establish a complex communication network between different stromal and epithelial cell types, and there is growing evidence that they play a key role in physiology and pathology. In many tissues, TC network impairment has been implicated in the onset and progression of pathological conditions, which makes the study of TCs of great interest for the development of novel therapies. In this review, we summarise the main methods involved in the characterisation of these cells as well as their inherent difficulties and then discuss the functional assays that are used to uncover the role of TCs in normal and pathological conditions, from the most traditional to the most recent. Furthermore, we provide future perspectives in the study of TCs, especially regarding the establishment of more precise markers, commercial lineages and means for drug delivery and genetic editing that directly target TCs.

Bioengineering extracellular vesicles: smart nanomaterials for bone regeneration.

In the past decade, extracellular vesicles (EVs) have emerged as key regulators of bone development, homeostasis and repair. EV-based therapies have the potential to circumnavigate key issues hindering the translation of cell-based therapies including functional tissue engraftment, uncontrolled differentiation and immunogenicity issues. Due to EVs' innate biocompatibility, low immunogenicity, and high physiochemical stability, these naturally-derived nanoparticles have garnered growing interest as potential acellular nanoscale therapeutics for a variety of diseases. Our increasing knowledge of the roles these cell-derived nanoparticles play, has made them an exciting focus in the development of novel pro-regenerative therapies for bone repair. Although these nano-sized vesicles have shown promise, their clinical translation is hindered due to several challenges in the EV supply chain, ultimately impacting therapeutic efficacy and yield. From the biochemical and biophysical stimulation of parental cells to the transition to scalable manufacture or maximising vesicles therapeutic response in vivo, a multitude of techniques have been employed to improve the clinical efficacy of EVs. This review explores state of the art bioengineering strategies to promote the therapeutic utility of vesicles beyond their native capacity, thus maximising the clinical potential of these pro-regenerative nanoscale therapeutics for bone repair.

Epigenetic Reprogramming via Synergistic Hypomethylation and Hypoxia Enhances the Therapeutic Efficacy of Mesenchymal Stem Cell Extracellular Vesicles for Bone Repair.

Mesenchymal stem cells (MSCs) are a promising cell population for regenerative medicine applications, where paracrine signalling through the extracellular vesicles (EVs) regulates bone tissue homeostasis and development. MSCs are known to reside in low oxygen tension, which promotes osteogenic differentiation via hypoxia-inducible factor-1α activation. Epigenetic reprogramming has emerged as a promising bioengineering strategy to enhance MSC differentiation. Particularly, the process of hypomethylation may enhance osteogenesis through gene activation. Therefore, this study aimed to investigate the synergistic effects of inducing hypomethylation and hypoxia on improving the therapeutic efficacy of EVs derived from human bone marrow MSCs (hBMSCs). The effects of the hypoxia mimetic agent deferoxamine (DFO) and the DNA methyltransferase inhibitor 5-azacytidine (AZT) on hBMSC viability was assessed by quantifying the DNA content. The epigenetic functionality was evaluated by assessing histone acetylation and histone methylation. hBMSC mineralisation was determined by quantifying alkaline phosphate activity, collagen production and calcium deposition. EVs were procured from AZT, DFO or AZT/DFO-treated hBMSCs over a two-week period, with EV size and concentration defined using transmission electron microscopy, nanoflow cytometry and dynamic light scattering. The effects of AZT-EVs, DFO-EVs or AZT/DFO-EVs on the epigenetic functionality and mineralisation of hBMSCs were evaluated. Moreover, the effects of hBMSC-EVs on human umbilical cord vein endothelial cells (HUVECs) angiogenesis was assessed by quantifying pro-angiogenic cytokine release. DFO and AZT caused a time-dose dependent reduction in hBMSC viability. Pre-treatment with AZT, DFO or AZT/DFO augmented the epigenetic functionality of the MSCs through increases in histone acetylation and hypomethylation. AZT, DFO and AZT/DFO pre-treatment significantly enhanced extracellular matrix collagen production and mineralisation in hBMSCs. EVs derived from AZT/DFO-preconditioned hBMSCs (AZT/DFO-EVs) enhanced the hBMSC proliferation, histone acetylation and hypomethylation when compared to EVs derived from AZT-treated, DFO-treated and untreated hBMSCs. Importantly, AZT/DFO-EVs significantly increased osteogenic differentiation and mineralisation of a secondary hBMSC population. Furthermore, AZT/DFO-EVs enhanced the pro-angiogenic cytokine release of HUVECs. Taken together, our findings demonstrate the considerable utility of synergistically inducing hypomethylation and hypoxia to improve the therapeutic efficacy of the MSC-EVs as a cell-free approach for bone regeneration.

Bone tissue engineering using 3D silk scaffolds and human dental pulp stromal cells epigenetic reprogrammed with the selective histone deacetylase inhibitor MI192.

Epigenetics plays a critical role in regulating mesenchymal stem cells' (MSCs) fate for tissue repair and regeneration. There is increasing evidence that the inhibition of histone deacetylase (HDAC) isoform 3 can enhance MSC osteogenesis. This study investigated the potential of using a selective HDAC2 and 3 inhibitor, MI192, to promote human dental pulp stromal cells (hDPSCs) bone-like tissue formation in vitro and in vivo within porous Bombyx Mori silk scaffolds. Both 2 and 5 wt% silk scaffolds were fabricated and characterised. The 5 wt% scaffolds possess thicker internal lamellae, reduced scaffold swelling and degradation rates, whilst increased compressive modulus in comparison to the 2 wt% silk scaffold. MI192 pre-treatment of hDPSCs on 5 wt% silk scaffold significantly enhanced hDPSCs alkaline phosphatase activity (ALP). The expression of osteoblast-related genes (RUNX2, ALP, Col1a, OCN) was significantly upregulated in the MI192 pre-treated cells. Histological analysis confirmed that the MI192 pre-treated hDPSCs-silk scaffold constructs promoted bone extracellular matrix (ALP, Col1a, OCN) deposition and mineralisation compared to the untreated group. Following 6 weeks of subcutaneous implantation in nude mice, the MI192 pre-treated hDPSCs-silk scaffold constructs enhanced the vascularisation and extracellular matrix mineralisation compared to untreated control. In conclusion, these findings demonstrate the potential of using epigenetic reprogramming and silk scaffolds to promote hDPSCs bone formation efficacy, which provides evidence for clinical translation of this technology for bone augmentation.

Controlled Release of Epigenetically-Enhanced Extracellular Vesicles from a GelMA/Nanoclay Composite Hydrogel to Promote Bone Repair.

Extracellular vesicles (EVs) have garnered growing attention as promising acellular tools for bone repair. Although EVs' potential for bone regeneration has been shown, issues associated with their therapeutic potency and short half-life in vivo hinders their clinical utility. Epigenetic reprogramming with the histone deacetylase inhibitor Trichostatin A (TSA) has been reported to promote the osteoinductive potency of osteoblast-derived EVs. Gelatin methacryloyl (GelMA) hydrogels functionalised with the synthetic nanoclay laponite (LAP) have been shown to effectively bind, stabilise, and improve the retention of bioactive factors. This study investigated the potential of utilising a GelMA-LAP hydrogel to improve local retention and control delivery of epigenetically enhanced osteoblast-derived EVs as a novel bone repair strategy. LAP was found to elicit a dose-dependent increase in GelMA compressive modulus and shear-thinning properties. Incorporation of the nanoclay was also found to enhance shape fidelity when 3D printed compared to LAP-free gels. Interestingly, GelMA hydrogels containing LAP displayed increased mineralisation capacity (1.41-fold) (p ≤ 0.01) over 14 days. EV release kinetics from these nanocomposite systems were also strongly influenced by LAP concentration with significantly more vesicles being released from GelMA constructs as detected by a CD63 ELISA (p ≤ 0.001). EVs derived from TSA-treated osteoblasts (TSA-EVs) enhanced proliferation (1.09-fold), migration (1.83-fold), histone acetylation (1.32-fold) and mineralisation (1.87-fold) of human bone marrow stromal cells (hBMSCs) when released from the GelMA-LAP hydrogel compared to the untreated EV gels (p ≤ 0.01). Importantly, the TSA-EV functionalised GelMA-LAP hydrogel significantly promoted encapsulated hBMSCs extracellular matrix collagen production (≥1.3-fold) and mineralisation (≥1.78-fold) in a dose-dependent manner compared to untreated EV constructs (p ≤ 0.001). Taken together, these findings demonstrate the potential of combining epigenetically enhanced osteoblast-derived EVs with a nanocomposite photocurable hydrogel to promote the therapeutic efficacy of acellular vesicle approaches for bone regeneration.

Enhancing osteogenic potential of hDPSCs by resveratrol through reducing oxidative stress via the Sirt1/Nrf2 pathway.

CONTEXT: The osteogenic potential of the human dental pulp stromal cells (hDPSCs) was reduced in the state of oxidative stress. Resveratrol (RSV) possesses numerous biological properties, including osteogenic potential, growth-promoting and antioxidant activities. OBJECTIVE: This study investigates the osteogenic potential of RSV by activating the Sirt1/Nrf2 pathway on oxidatively stressed hDPSCs and old mice. MATERIALS AND METHODS: The hDPSCs were subjected to reactive oxygen species (ROS) fluorescence staining, cell proliferation assay, ROS activity assay, superoxide dismutase (SOD) enzyme activity, the glutathione (GSH) concentration assay, alkaline phosphatase staining, real-time polymerase chain reaction (RT-PCR) and Sirt1 immunofluorescence labelling to assess the antioxidant stress and osteogenic ability of RSV. Forty female Kunming mice were divided into Old, Old-RSV, Young and Young-RSV groups to assess the repair of calvarial defects of 0.2 mL RSV of 20 mg/kg/d for seven days by injecting intraperitoneally at 4 weeks after surgery using micro-computed tomography, nonlinear optical microscope and immunohistochemical analysis. RESULTS: RSV abates oxidative stress by alleviating the proliferation, mitigating the ROS activity, increasing the SOD enzyme activity and ameliorating the GSH concentration (RSV IC50 in hDPSCs is 67.65 ± 9.86). The antioxidative stress and osteogenic capabilities of RSV were confirmed by the up-regulated gene expression of SOD1, xCT, RUNX2 and OCN, as well as Sirt1/Nrf2. The collagen, bone matrix formation and Sirt1 expression, are significantly increased after RSV treatment in mice. DISCUSSION AND CONCLUSIONS: For elderly or patients with oxidative stress physiological states such as hypertension, heart disease, diabetes, etc., RSV may potentially improve bone augmentation surgery in regenerative medicine.

The Selective Histone Deacetylase Inhibitor MI192 Enhances the Osteogenic Differentiation Efficacy of Human Dental Pulp Stromal Cells.

The use of human dental pulp stromal cells (hDPSCs) has gained increasing attention as an alternative stem cell source for bone tissue engineering. The modification of the cells' epigenetics has been found to play an important role in regulating differentiation, with the inhibition of histone deacetylases 3 (HDAC3) being linked to increased osteogenic differentiation. This study aimed to induce epigenetic reprogramming using the HDAC2 and 3 selective inhibitor, MI192 to promote hDPSCs osteogenic capacity for bone regeneration. MI192 treatment caused a time-dose-dependent change in hDPSC morphology and reduction in viability. Additionally, MI192 successfully augmented hDPSC epigenetic functionality, which resulted in increased histone acetylation and cell cycle arrest at the G2/M phase. MI192 pre-treatment exhibited a dose-dependent effect on hDPSCs alkaline phosphatase activity. Quantitative PCR and In-Cell Western further demonstrated that MI192 pre-treatment significantly upregulated hDPSCs osteoblast-related gene and protein expression (alkaline phosphatase, bone morphogenic protein 2, type I collagen and osteocalcin) during osteogenic differentiation. Importantly, MI192 pre-treatment significantly increased hDPSCs extracellular matrix collagen production and mineralisation. As such, for the first time, our findings show that epigenetic reprogramming with the HDAC2 and 3 selective inhibitor MI192 accelerates the osteogenic differentiation of hDPSCs, demonstrating the considerable utility of this MSCs engineering approach for bone augmentation strategies.

CircRNA expression profiles in human dental pulp stromal cells undergoing oxidative stress.

BACKGROUND: Oxidative stress has a determinantal effect on human dental pulp stromal cells (hDPSCs), including affecting their longevity and functionality. Circular RNAs (circRNAs) play an essential role in stromal cell behavior; however, the exact mechanism in which circRNAs functions within hDPSCs were undergoing oxidative stress remains unclear. The purpose of this study is to assess the global changes and characteristics of circRNAs in hDPSCs undergoing oxidative stress. METHODS: Using an oxidative stress model of hDPSCs, we applied microarray analysis to examine the circRNAs profiles. We confirmed the changes in circRNAs by quantitative Real-Time PCR (qRT-PCR). Furthermore, bioinformatics tools, including a miRcode map, TargetScan, gene ontology (GO) analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, were reconstructed for further assessment. SIRT1 gene and protein expression were tested by qRT-PCR and In Cell-Western analysis. RESULTS: We revealed 330 upregulated, and 533 downregulated circRNAs undergoing oxidative stress in hDPSCs and confirmed three circRNAs distinct expressions (hsa_circ_0000257, hsa_circ_0087354, and hsa_circ_0001946) in hDPSCs undergoing oxidative stress by qRT-PCR. GO, and KEGG pathway enrichment revealed the differentially expressed circRNAs might participate in p53 and cell cycle signaling networks associated with oxidative stress. SIRT1 gene and protein expression was reduced in the oxidatively stressed cells (OSC) group compared to untreated cells (UC). CONCLUSIONS: The findings of this study has provided new insights into circRNAs and a basis for further studies assessing the potential functions of hsa_circ_0000257, hsa_circ_0087354, and hsa_circ_0001946 in oxidatively stressed hDPSCs.

Study on the effect of protein lysine lactylation modification in macrophages on inhibiting periodontitis in rats.

BACKGROUND: Protein lysine lactylation (Kla) has been proved to be closely related to inflammatory diseases, but its role in periodontitis (PD) is unclear. Therefore, this study aimed to establish the global profiling of Kla in PD models in rats. METHODS: Clinical periodontal samples were collected, the inflammatory state of tissues was verified by H&E staining, and lactate content was detected by a lactic acid kit. Kla levels were detected by immunohistochemistry (IHC) and Western blot. Subsequently, the rat model of PD was developed and its reliability verified by micro-CT and H&E staining. Mass spectrometry analysis was conducted to explore the expression profile of proteins and Kla in periodontal tissues. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis were performed, and a protein-protein interaction (PPI) network was constructed. The lactylation in RAW264.7 cells was confirmed by IHC, immunofluorescence and Western blot. The relative expression levels of inflammatory factors IL-1ß, IL-6, TNF-α, macrophage polarization-related factors CD86, iNOS, Arg1, and CD206 in RAW264.7 cells were detected by real time-quantitative polymerase chain reaction (RT-qPCR). RESULTS: We observed substantial inflammatory cell infiltration in the PD tissues, and the lactate content and lactylation levels were significantly increased. The expression profiles of protein and Kla were obtained by mass spectrometry based on the established rat model of PD. Kla was confirmed in vitro and in vivo. After inhibiting the "writer" of lactylation P300 in RAW264.7 cells, the lactylation levels decreased, and the expression of inflammatory factors IL-1ß, IL-6, and TNF-α increased. Meanwhile, the levels of CD86 and iNOS increased, and Arg1 and CD206 decreased. CONCLUSIONS: Kla may play an important role in PD, regulating the release of inflammatory factors and polarization of macrophages.

Additively manufactured bioceramic scaffolds based on triply periodic minimal surfaces for bone regeneration.

The study focused on the effects of a triply periodic minimal surface (TPMS) scaffolds, varying in porosity, on the repair of mandibular defects in New Zealand white rabbits. Four TPMS configurations (40%, 50%, 60%, and 70% porosity) were fabricated with ß-tricalcium phosphate bioceramic via additive manufacturing. Scaffold properties were assessed through scanning electron microscopy and mechanical testing. For proliferation and adhesion assays, mouse bone marrow stem cells (BMSCs) were cultured on these scaffolds. In vivo, the scaffolds were implanted into rabbit mandibular defects for 2 months. Histological staining evaluated osteogenic potential. Moreover, RNA-sequencing analysis and RT-qPCR revealed the significant involvement of angiogenesis-related factors and Hippo signaling pathway in influencing BMSCs behavior. Notably, the 70% porosity TPMS scaffold exhibited optimal compressive strength, superior cell proliferation, adhesion, and significantly enhanced osteogenesis and angiogenesis. These findings underscore the substantial potential of 70% porosity TPMS scaffolds in effectively promoting bone regeneration within mandibular defects.

Laser texturing of additively manufactured implants: A tool to programme biological response.

The advent of additive manufacturing (AM) is rapidly shaping healthcare technologies pushing forward personalisation and enhanced implant functionalisation to improve clinical outcomes. AM techniques such as powder bed fusion (PBF) have been adopted despite the need to modify the as-built surface post manufacture. Medical device manufacturers have focused their efforts on refining various physical and chemical surface finishing approaches, however there is little consensus and some methods risk geometry alteration or contamination. This has led to a growing interest in laser texturing technologies to engineer the device surface. Herein, several bioinspired micro and nano textures were applied to laser PBF Ti-6Al-V4 substrates to alter physicochemical properties and in-turn we sought to understand what influences these alterations had on a human osteosarcoma cell line (MG63). Significant variations in roughness and time dependent contact angles were revealed between different patterns provide a tool to elicit desired biological responses. All surface treatments effectively enhanced early cell behaviour and in particular coverage was increased for the micro-textures. Influence of the patterns on cell differentiation was less consistent with alkaline phosphatase content increased only for the channel, grid and dual textures. While long term (21 days) mineralisation was found to be significantly enhanced in grids, dual, triangles and shark skin textures. Further regression analysis of all physicochemical and biological variables indicated that several properties should be used to strongly correlate cell behaviour, resulting in 82 % of the 21 day mineralisation dataset explained through a combination of roughness kurtosis and glycerol contact angle. Overall, this manuscript demonstrates the ability of laser texturing to offer tailored cell-surface interactions, which can be tuned to offer a tool to drive functional customisation of anatomically customised medical devices.

An ECM-Mimetic Hydrogel to Promote the Therapeutic Efficacy of Osteoblast-Derived Extracellular Vesicles for Bone Regeneration.

The use of extracellular vesicles (EVs) is emerging as a promising acellular approach for bone regeneration, overcoming translational hurdles associated with cell-based therapies. Despite their potential, EVs short half-life following systemic administration hinders their therapeutic efficacy. EVs have been reported to bind to extracellular matrix (ECM) proteins and play an essential role in matrix mineralisation. Chitosan and collagen type I are naturally-derived pro-osteogenic biomaterials, which have been demonstrated to control EV release kinetics. Therefore, this study aimed to develop an injectable ECM-mimetic hydrogel capable of controlling the release of osteoblast-derived EVs to promote bone repair. Pure chitosan hydrogels significantly enhanced compressive modulus (2.48-fold) and osteogenic differentiation (3.07-fold), whilst reducing gelation times (2.09-fold) and proliferation (2.7-fold) compared to pure collagen gels (p ≤ 0.001). EV release was strongly associated with collagen concentration (R2 > 0.94), where a significantly increased EV release profile was observed from chitosan containing gels using the CD63 ELISA (p ≤ 0.001). Hydrogel-released EVs enhanced human bone marrow stromal cells (hBMSCs) proliferation (1.12-fold), migration (2.55-fold), and mineralisation (3.25-fold) compared to untreated cells (p ≤ 0.001). Importantly, EV-functionalised chitosan-collagen composites significantly promoted hBMSCs extracellular matrix mineralisation when compared to the EV-free gels in a dose-dependent manner (p ≤ 0.001). Taken together, these findings demonstrate the development of a pro-osteogenic thermosensitive chitosan-collagen hydrogel capable of enhancing the therapeutic efficacy of osteoblast-derived EVs as a novel acellular tool for bone augmentation strategy.

Photocurable GelMA Adhesives for Corneal Perforations.

The current treatments for the management of corneal and scleral perforations include sutures and adhesives. While sutures are invasive, induce astigmatism and carry a risk of infection, cyanoacrylate glues are toxic, proinflammatory and form an opaque and rough surface that precludes vision. Consequently, the clinical need for a fast curing and strong tissue adhesive with minimised cytotoxicity and host inflammation remains unmet. In this paper, we engineer a gelatine methacryloyl (GelMA) adhesive that can be crosslinked in situ within 2 min using UV or visible light and a riboflavin (RF)/sodium persulfate (SPS) system. Optical coherence tomography (OCT) images demonstrated that the flowable GelMA adhesive could completely fill corneal wounds and restore the ocular curvature by forming a smooth contour on the ocular surface. Further, ex vivo studies in porcine eyes showed that GelMA bioadhesives exhibited burst pressures that were comparable to cyanoacrylates (49 ± 9 kPa), with the hydrogels exhibiting a transmittance (90%), water content (85%) and storage modulus (5 kPa) similar to the human cornea. Finally, using human dermal fibroblasts, we showed that our GelMA adhesive was non-toxic and could effectively support cell adhesion and proliferation. Taken together, the adhesive's performance, injectability and ease of administration, together with gelatin's availability and cost-effectiveness, make it a potential stromal filler or sealant for corneal and conjunctival applications.

SDF-1 Functionalized Hydrogel Microcarriers for Skin Flap Repair.

Critically sized skin flaps used to treat skin defects often suffer from necrosis due to insufficient blood supply. Hence there is an urgent need to improve the survival rate of skin flaps by promoting local angiogenesis. The delivery of growth factor loaded microcarriers have shown promise in enhancing defect repair, however, their rapid clearance from the defect site limits their regenerative potential. Thus, it is critical to develop microcarriers which can promote the sustained release of bioactive factors to effectively stimulate tissue repair. This study aimed to develop a stromal cell-derived factor 1 (SDF-1) loaded microcarrier coated with Matrigel (MC@SDF-1@Mat) to promote skin flap repair. SEM imaging showed that the surface of the microcarrier was coated by a porous Matrigel film. The drug release experiment showed that the Matrigel-coated microcarriers enhanced the sustained release of the model drug methylene blue when compared to uncoated group. MC@SDF-1@Mat significantly promoted the proliferation, migration, and angiogenesis of HUVECs via CCK-8, wound healing assay, and tube formation assay, respectively. Moreover, the murine random skin flap model was further established and treated. It was found that the flap necrosis area in the MC@SDF-1@Mat treated group was significantly reduced. H&E and Masson staining showed the histological structure and collagen organization exhibited a normal phenotype in the MC@SDF-1@Mat treated group. Additionally, CD31 immunohistochemical analysis showed that the MC@SDF-1@Mat treated group exhibited the greatest degree of neovascularization. In conclusion, our SDF-1 functionalized gelatin-based hydrogel microcarrier has potential clinical applications in promoting skin flap repair and drug delivery.

GelMA Hydrogel Reinforced with 3D Printed PEGT/PBT Scaffolds for Supporting Epigenetically-Activated Human Bone Marrow Stromal Cells for Bone Repair.

Epigenetic approaches using the histone deacetylase 2 and 3 inhibitor-MI192 have been reported to accelerate stem cells to form mineralised tissues. Gelatine methacryloyl (GelMA) hydrogels provide a favourable microenvironment to facilitate cell delivery and support tissue formation. However, their application for bone repair is limited due to their low mechanical strength. This study aimed to investigate a GelMA hydrogel reinforced with a 3D printed scaffold to support MI192-induced human bone marrow stromal cells (hBMSCs) for bone formation. Cell culture: The GelMA (5 wt%) hydrogel supported the proliferation of MI192-pre-treated hBMSCs. MI192-pre-treated hBMSCs within the GelMA in osteogenic culture significantly increased alkaline phosphatase activity (p ≤ 0.001) compared to control. Histology: The MI192-pre-treated group enhanced osteoblast-related extracellular matrix deposition and mineralisation (p ≤ 0.001) compared to control. Mechanical testing: GelMA hydrogels reinforced with 3D printed poly(ethylene glycol)-terephthalate/poly(butylene terephthalate) (PEGT/PBT) scaffolds exhibited a 1000-fold increase in the compressive modulus compared to the GelMA alone. MI192-pre-treated hBMSCs within the GelMA-PEGT/PBT constructs significantly enhanced extracellular matrix collagen production and mineralisation compared to control (p ≤ 0.001). These findings demonstrate that the GelMA-PEGT/PBT construct provides enhanced mechanical strength and facilitates the delivery of epigenetically-activated MSCs for bone augmentation strategies.

Photocurable antimicrobial silk-based hydrogels for corneal repair.

Corneal transplantation is the current gold standard treatment to restore visual acuity to patients with severe corneal diseases and injuries. Due to severe donor tissue shortage, efforts to develop a corneal equivalent have been made but the challenge remains unmet. Another issue of concern in ocular surgery is the difficult instillation and fast drainage of antibiotic ocular eye drops as bacterial infections can jeopardize implant success by delaying or impairing tissue healing. In this study, we developed antimicrobial silk-based hydrogels that have the potential to be photoactivated in situ, fully adapting to the corneal injury shape. Gentamicin-loaded methacrylated-silk (SilkMA) hydrogels were prepared within minutes using low UV intensity (3 mW/cm2 ). SilkMA gels provided a Young's modulus between 21 and 79 kPa together with a light transmittance spectrum and water content (83%-90%) similar to the human cornea. Polymer concentration (15%-25%) was found to offer a tool for tailoring the physical properties of the hydrogels. We confirmed that the methacrylation did not affect the material's in vitro degradation and biocompatibility by observing fibroblast adhesion and proliferation. Importantly, agar diffusion tests showed that the synthesized hydrogels were able to inhibit Staphylococcus aureus and Pseudomonas aeruginosa growth for 72 h. These characteristics along with their injectability and viscoelasticity demonstrate the potential of SilkMA hydrogels to be applied in several soft tissue engineering fields. As such, for the first time we demonstrate the potential of photocurable antimicrobial SilkMA hydrogels as a novel biomaterial to facilitate corneal regeneration.

A long-lasting guided bone regeneration membrane from sequentially functionalised photoactive atelocollagen.

The fast degradation of collagen-based membranes in the biological environment remains a critical challenge, resulting in underperforming Guided Bone Regeneration (GBR) therapy leading to compromised clinical results. Photoactive atelocollagen (AC) systems functionalised with ethylenically unsaturated monomers, such as 4-vinylbenzyl chloride (4VBC), have been shown to generate mechanically competent materials for wound healing, inflammation control and drug delivery, whereby control of the molecular architecture of the AC network is key. Building on this platform, the sequential functionalisation with 4VBC and methacrylic anhydride (MA) was hypothesised to generate UV-cured AC hydrogels with reduced swelling ratio, increased proteolytic stability and barrier functionality for GBR therapy. The sequentially functionalised atelocollagen precursor (SAP) was characterised via TNBS and ninhydrin colourimetric assays, circular dichroism and UV-curing rheometry, which confirmed nearly complete consumption of collagen's primary amino groups, preserved triple helices and fast (< 180 s) gelation kinetics, respectively. Hydrogel's swelling ratio and compression modulus were adjusted depending on the aqueous environment used for UV-curing, whilst the sequential functionalisation of AC successfully generated hydrogels with superior proteolytic stability in vitro compared to both 4VBC-functionalised control and the commercial dental membrane Bio-Gide®. These in vitro results were confirmed in vivo via both subcutaneous implantation and a proof-of-concept study in a GBR calvarial model, indicating integrity of the hydrogel and barrier defect, as well as tissue formation following 1-month implantation in rats. STATEMENT OF SIGNIFICANCE: Collagen-based membranes remain a key component in Guided Bone Regeneration (GBR) therapy, but their properties, e.g. proteolytic stability and soft tissue barrier functionality, are still far from optimal. This is largely attributed to the complex molecular configuration of collagen, which makes chemical accessibility and structure-function relations challenging. Here, we fabricated a UV-cured hydrogel network of atelocollagen, whereby triple helices were sequentially functionalised with two distinct ethylenically unsaturated monomers. The effects of the sequential functionalisation and UV-curing on the macroscopic properties, degradation behaviour and GBR capability were investigated in vitro and in vivo. The results highlight the key role of the sequential functionalisation and provide important insights for the design of future, longer-lasting resorbable membranes for GBR therapy.

Hydrostatic pressure promotes chondrogenic differentiation and microvesicle release from human embryonic and bone marrow stem cells.

Mechanical stimulation plays in an important role in regulating stem cell differentiation and their release of extracellular vesicles (EVs). In this study, effects of low magnitude hydrostatic pressure (HP) on the chondrogenic differentiation and microvesicle release from human embryonic stem cells (hESCs) and human bone marrow stem cells (hBMSCs) are examined. hESCs were differentiated into chondroprogenitors and then embedded in fibrin gels and subjected to HP (270 kPa, 1 Hz, 5 days per week). hBMSC pellets were differentiated in chondrogenic media and subjected to the same regime. HP significantly enhanced ACAN expression in hESCs. It also led to a significant increase in DNA content, sGAG content and total sGAG/DNA level in hBMSCs. Furthermore, HP significantly increased microvesicle protein content released from both cell types. These results highlight the benefit of HP bioreactor in promoting chondrogenesis and EV production for cartilage tissue engineering.

Methacrylated Silk Fibroin Hydrogels: pH as a Tool to Control Functionality.

The last decade has witnessed significant progress in the development of photosensitive polymers for in situ polymerization and 3D printing applications. Light-mediated sol-gel transitions have immense potential for tissue engineering applications as cell-laden materials can be crosslinked within minutes under mild environmental conditions. Silk fibroin (SF) is extensively explored in regenerative medicine applications due to its ease of modification and exceptional mechanical properties along with cytocompatibility. To efficiently design SF materials, the in vivo assembly of SF proteins must be considered. During SF biosynthesis, changes in pH, water content, and metal ion concentrations throughout the silkworm gland divisions drive the transition from liquid silk to its fiber form. Herein, we study the effect of the glycidyl-methacrylate-modified SF (SilkMA) solution pH on the properties and secondary structure of SilkMA hydrogels by testing formulations prepared at pH 5, 7, and 8. Our results demonstrate an influence of the prepolymer solution pH on the hydrogel rheological properties, compressive modulus, optical transmittance, and network swellability. The hydrogel pH did not affect the in vitro viability and morphology of human dermal fibroblasts. This work demonstrates the utility of the solution pH to tailor the SilkMA conformational structure development toward utility and function and shows the need to strictly control the pH to reduce batch-to-batch variability and ensure reproducibility.

MI192 induced epigenetic reprogramming enhances the therapeutic efficacy of human bone marrows stromal cells for bone regeneration.

Human bone marrow stromal cells (hBMSCs) have been extensively utilised for bone tissue engineering applications. However, they are associated with limitations that hinder their clinical utility for bone regeneration. Cell fate can be modulated via altering their epigenetic functionality. Inhibiting histone deacetylase (HDAC) enzymes have been reported to promote osteogenic differentiation, with HDAC3 activity shown to be causatively associated with osteogenesis. Therefore, this study aimed to investigate the potential of using an HDAC2 & 3 selective inhibitor - MI192 to induce epigenetic reprogramming of hBMSCs and enhance its therapeutic efficacy for bone formation. Treatment with MI192 caused a time-dose dependant reduction in hBMSCs viability. MI192 was also found to substantially alter hBMSCs epigenetic function through reduced HDAC activity and increased histone acetylation. hBMSCs were pre-treated with MI192 (50 µM) for 48 h prior to osteogenic induction. MI192 pre-treatment significantly upregulated osteoblast-related gene/protein expression (Runx2, ALP, Col1a and OCN) and enhanced alkaline phosphatase specific activity (ALPSA) (1.43-fold) (P ≤ 0.001). Moreover, MI192 substantially increased hBMSCs extracellular matrix calcium deposition (1.4-fold) (P ≤ 0.001) and mineralisation when compared to the untreated control. In 3D microtissue culture, MI192 significantly promoted hBMSCs osteoblast-related gene expression and ALPSA (> 2.41-fold) (P ≤ 0.001). Importantly, MI192 substantially enhanced extracellular matrix deposition (ALP, Col1a, OCN) and mineralisation (1.67-fold) (P ≤ 0.001) within the bioassembled-microtissue (BMT) construct. Following 8-week intraperitoneal implantation within nude mice, MI192 treated hBMSCs exhibited enhanced extracellular matrix deposition and mineralisation (2.39-fold) (P ≤ 0.001) within the BMT when compared to the untreated BMT construct. Taken together, these results demonstrate that MI192 effectively altered hBMSCs epigenetic functionality and is capable of promoting hBMSCs osteogenic differentiation in vitro and in vivo, indicating the potential of using epigenetic reprogramming to enhance the therapeutic efficacy of hBMSCs for bone augmentation strategies.