Extracellular Matrix Mineralization Research Articles

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.

Cell osteogenic bioactivity mediated precisely by varying scaled micro-pits on ordered micro/nano hierarchical structures of titanium.

Hierarchical surface structures with micro–nano scale play a crucial role in regulation of cell proliferation and osteogenic differentiation. It has been proven that cells are extremely sensitive to the nanoscaled structure and show multifarious phenotypes. Though a vital function of microstructure on osseointegration has been confirmed, the cell performances response to different microscaled structure is needed to be further dissected and in depth understood. In this work, the ordered micro–nano hierarchical structures with varying micro-scaled pits were precisely fabricated on titanium successfully by the combination of electrochemical, chemical etching and anodization as well. In vitro systematical assessments indicated that the micro–nano multilevel structures on titanium exhibited excellent cells adhesion and spreading ability, as well as steerable proliferation and osteogenic differentiation behaviors. It is shown that smaller micro-pits and lower roughness of the hierarchical structures enabled faster cell propagation. Despite cell growth was delayed on micro–nano titanium with relatively larger cell-match-size micro-pits and roughness, osteogenic-specific genes were significantly elevated. Furthermore, the alkaline phosphatase activity, collagen secretion and extracellular matrix mineralization of MC3T3-E1 on multi-scaled titanium were suppressed by a large margin after adding IWP-2 (an inhibitor of Wnt/β-catenin signal pathway), indicating this pathway played a crucial part in cell osteogenic differentiation modulated by micro–nano structures.

Li-Doped Ti Surface for the Improvement of Osteointegration.

Aseptic loosening is the main factor that leads to the failure of orthopedic implants. Enhancing the early osteointegration of a bone implant can lower the risk of aseptic loosening. Here, a Li-doped surface was constructed on a Ti surface via plasma electrolytic oxidation (PEO) to improve osteointegration. The prepared Li-doped PEO coating showed a porous morphology and the sustained release of Li ions. In vitro results of rat bone marrow mesenchymal stem cell (rBMSC) culture studies suggested that the Li-doped Ti surface significantly favored cell adhesion. Moreover, it was found that the Li-doped surface enhanced alkaline phosphatase activity and extracellular matrix mineralization of rBMSCs. In addition, the surface improved the expression of osteogenesis-related genes. Furthermore, a bone implantation model indicated that the Li-doped Ti surface showed improved osteointegration. The incorporation of Li into a Ti surface is a promising method for orthopedic applications.

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.Graphical abstract

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.

ZIF-8 modified multifunctional injectable photopolymerizable GelMA hydrogel for the treatment of periodontitis

Periodontitis is a chronic inflammatory disease caused by plaque that leads to alveolar bone resorption. In the treatment of periodontitis, it is necessary to reduce the bacterial load and promote alveolar bone regeneration. In this study, zeolitic imidazolate framework-8 (ZIF-8) is used in the treatment of periodontitis, and an injectable photopolymerizable ZIF-8/gelatin methacryloyl (GelMA) composite hydrogel (GelMA-Z) is constructed. We confirm that ZIF-8 nanoparticles are successfully loaded into GelMA, which demonstrates fluidity and photopolymerizability. GelMA-Z continuously releases Zn2+ and shows good cytocompatibility. In vitro, GelMA-Z can effectively upregulate the expression of osteogenesis-related genes and proteins, increase alkaline phosphatase activity, promote extracellular matrix mineralization by rat bone mesenchymal stem cells, and exert an obvious antibacterial effect against Porphyromonas gingivalis. In vivo, GelMA-Z reduces the bacterial load, relieves inflammation and promotes alveolar bone regeneration in a rat model. The above results show that GelMA-Z has potential prospects in the treatment of periodontitis. Statement of significanceVarious methods have been explored for the treatment of periodontitis. However, current regiments have difficulty achieving ideal alveolar bone regeneration. In this study, we constructed a zeolitic imidazolate framework-8 (ZIF-8)/gelatin methacryloyl (GelMA) composite hydrogel (GelMA-Z). (1) The injectable and photopolymerizable GelMA-Z showed biocompatibility in vitro and in vivo. (2) GelMA-Z continually released zinc ions to promote the osteogenic differentiation of bone mesenchymal stem cells and kill bacteria in vitro. (3) In a rat model, the GelMA-Z pregel solution was used to fill the periodontal pocket and then crosslinked by UV exposure. GelMA-Z can stably remain in the periodontal pocket to reduce the bacterial load, relieve inflammation and promote alveolar bone regeneration. In conclusion, GelMA-Z has great potential for use in the treatment of periodontitis, especially in promoting alveolar bone regeneration.

Coating Ti6Al4V implants with nanocrystalline diamond functionalized with BMP-7 promotes extracellular matrix mineralization in vitro and faster osseointegration in vivo

The present study investigates the effect of an oxidized nanocrystalline diamond (O-NCD) coating functionalized with bone morphogenetic protein 7 (BMP-7) on human osteoblast maturation and extracellular matrix mineralization in vitro and on new bone formation in vivo. The chemical structure and the morphology of the NCD coating and the adhesion, thickness and morphology of the superimposed BMP-7 layer have also been assessed. The material analysis proved synthesis of a conformal diamond coating with a fine nanostructured morphology on the Ti6Al4V samples. The homogeneous nanostructured layer of BMP-7 on the NCD coating created by a physisorption method was confirmed by AFM. The osteogenic maturation of hFOB 1.19 cells in vitro was only slightly enhanced by the O-NCD coating alone without any increase in the mineralization of the matrix. Functionalization of the coating with BMP-7 resulted in more pronounced cell osteogenic maturation and increased extracellular matrix mineralization. Similar results were obtained in vivo from micro-CT and histological analyses of rabbit distal femurs with screws implanted for 4 or 12 weeks. While the O-NCD-coated implants alone promoted greater thickness of newly-formed bone in direct contact with the implant surface than the bare material, a further increase was induced by BMP-7. It can be therefore concluded that O-NCD coating functionalized with BMP-7 is a promising surface modification of metallic bone implants in order to improve their osseointegration.

Fabrication of an ordered micro-/nanotextured titanium surface to improve osseointegration

Ordered microscale titanium (Ti) surface topography with homogeneous cell-sized microholes (20 µm in diameter) was fabricated using simple electrochemical etching. The as-prepared surface imposed with uniform titania nanotubes (TNTs, 70 nm in diameter) through electrochemical anodization showed no considerable change in the initial microscale morphology. Bone marrow mesenchymal stem cells (BMSCs) were used in evaluating the bioactivity. Compared with polished Ti and unordered microtextured Ti, the ordered microtextured Ti formed by electrochemical etching remarkably promoted cell attachment, alkaline phosphatase activity, collagen secretion, extracellular matrix mineralization, and osteogenesis-related gene expression but considerably inhibited cell proliferation. After TNTs were introduced to the ordered microtextured Ti, cell attachment and osteogenic differentiation indexes were further enhanced, and cell proliferation recovered over time. The ordered micro-/nanotextured Ti surface was more conducive to the cell attachment, proliferation, and osteogenesis of BMSCs than polished Ti with and without TNTs, unordered microtextured Ti with and without TNTs, and unitary ordered microtextured Ti. Thus, the novel ordered bio-inspired micro-/nanotextured structure composed of cell-sized microholes and TNTs on the Ti surface possessed a favorable interfacial environment that improved osseointegration, potentially optimizing Ti implant surface topography.

Cerium Oxide Nanoparticles Promote Osteoplastic Precursor Differentiation by Activating the Wnt Pathway

Osteoplastic precursors are critical for fracture repair and bone homeostasis maintenance. Cerium oxide nanoparticles (CeO2 NPs) can promote the osteogenic differentiation of mesenchymal stem cells and secrete vascular endothelial growth factors. However, little is known about its role in precursor osteoblasts; therefore, we further investigated the effect and mechanism of CeO2 NPs in precursor osteoblasts. Cell counting kit-8 analysis was utilized to detect the toxicity of CeO2 NPs on MC3T3-E1 mouse osteogenic precursor cells. Then, alizarin red S staining was employed to assess the degree of extracellular matrix mineralization, and quantitative real-time polymerase chain reaction analysis was performed to measure the levels of osteogenesis-related genes. To identify differentially expressed genes, mRNA-sequencing was performed. Subsequently, GO and KEGG analyses were deployed to identify the major downstream pathways, whereas Western blot was used for verification. CeO2 NPs significantly enhanced the ability of MC3T3-E1 precursor osteoblasts to enhance matrix mineralization and increased the expression of osteogenic genes such as runt-related transcription factor 2, collagen Iα1, and osteocalcin. Pathway analysis revealed that CeO2 NPs enhanced the nuclear translocation of β-catenin and activated the Wnt pathway by promoting family with sequence similarity 53 member B/simplet expression, while Western blot analysis indicated the same results. After using a Wnt pathway inhibitor (KYA1797K), the simulative effect of CeO2 NPs was abolished. This study revealed that CeO2 NPs promoted MC3T3-E1 precursor osteoblast differentiation by activating the Wnt pathway.

Optimizing Barium Titanate Nanocomposite Bone Scaffolds for Biomineralization in Dynamic Compression Bioreactors Using Time-Lapsed Microstructural Imaging and Smart Thresholding

Bone scaffolds made of calcium phosphate polymer nanocomposites have limited osteoinductive properties. Piezoelectric materials have attracted considerable interest in bone tissue engineering due to their potential to promote osteogenesis through additional electrical stimulation. Time-lapsed micro-CT imaging is a time-effective tool for in vitro optimization of such scaffolds but is challenged by nanocomposites with a high attenuation coefficient, such as one containing high amounts of piezoelectric barium titanate. We used high-resolution end-point micro-CT scans combined with histology and Raman spectroscopy to screen polydopamine functionalized nanocomposites containing 3–27 vol% barium titanate for collagenous extracellular matrix formation and mineralization. All compositions showed well-connected extracellular matrix and birefringent matured collagen after seven weeks of static human mesenchymal stem cell cultures. Nevertheless, high-resolution micro-CT analysis combined with smart thresholding during image processing enabled us to observe modest differences in ECM mineralization between groups suggesting that a volume fraction of 9–21% barium titanate facilitated the formation of dense mineral clusters in the pores even in the absence of mechanical stimuli, further corroborated by Raman spectroscopy. The same image processing approach facilitated the analysis of time-lapsed micro-CT images of scaffold cultures in dynamic compression bioreactors where 9 vol% barium titanate was the best nanocomposite composition, resulting in a significant twofold increased maturation rate under dynamic conditions. On the other hand, barium titanate content of ≥15 vol% did not improve mineralization. At 27 vol%, the biomineralization of the collagenous extracellular matrix was even impeded in the nanocomposite scaffolds, as evidenced by histology stainings. Overall, our approach enables time-lapsed quantitative assessment of high X-ray absorbing nanocomposite scaffolds for biomineralization under dynamic compression, facilitating the optimization of such mechanically responsive scaffolds.

Optimizing the strontium content to achieve an ideal osseointegration through balancing apatite-forming ability and osteogenic activity

Implant failure caused by unsatisfying osseointegration is still a noteworthy clinical problem. Strontium (Sr) has been confirmed to be a bioactive element that facilitates bone growth. In this study, Sr was surface incorporated in titanium (Ti) implant with different contents. The XRD results demonstrated that Sr existed mainly in the form of SrTiO3. All Sr-contained implants showed sustainable Sr2+ release behavior. Meanwhile, the Sr2+ release rate was proportional to the Sr content. The in vitro immersing test showed that the apatite-forming ability on the implant surface was decreased with the increase of Sr content. Conversely, the cell experiments manifested that implants with high content of Sr were more favorable to cell spreading, proliferation, osteogenic differentiation, and extracellular matrix mineralization. The in vivo implant experiment revealed that Sr-incorporation could improve osseointegration, new bone formation and mineralization, and bone-implant bonding strength. In addition, Ti5Sr, which possessed a combined good osteogenic activity and apatite-forming ability, exhibited the best in vivo overall performance. In summary, we first put forward the competitive effect of osteogenic activity and apatite-forming ability on bone-implant osseointegration, which would provide a new strategy for implant design.

Physical characterization and biological tests of bioactive titanium surfaces prepared by short-time micro-arc oxidation in green electrolyte

Titanium (Ti) and its alloys are the most used biomaterials in dental and orthopedic implant applications. However, despite the good performance of these materials, implants may fail; therefore, several surface modification methodologies have been developed to increase the bioactivity of the metal surface, accelerating the osseointegration process while promoting improved corrosion performance. In this work, the production of a TiO2 coating on titanium through a short-time micro-arc oxidation (MAO) in a green electrolyte (obtained by a mixed solution of K3PO4 and Ca(CH3COO)2.H2O) is proposed, aiming at obtaining a porous oxide layer with Ca and P incorporation through an environmentally friendly experimental condition. The morphology, chemical composition, crystalline structure, wettability, hardness and bioactivity of the modified Ti surfaces were characterized. The MAO process at 250 V for 1 min in the green electrolyte solution allowed the production of a highly porous oxide surface in the anatase crystalline phase, with effective Ca and P incorporation. Pre-osteoblastic cells were used in in-vitro assays to analyze viability, adhesion, proliferation and ability to perform extracellular matrix mineralization on the Ti surfaces (polished and MAO-treated Ti). The MAO-treated Ti surface exhibited better results in the bioactivity tests, presenting more calcium phosphate precipitates. This surface also presented higher hardness, lower hydrophilicity and better performance in biological tests than the polished surface. The here-reported MAO-treated Ti surface is promising for dental implants, especially in patients having poor bone quality that requires greater stimulation for osteogenesis.

Matrix Vesicles as a Therapeutic Target for Vascular Calcification.

Vascular calcification (VC) is linked to an increased risk of heart disease, stroke, and atherosclerotic plaque rupture. It is a cell-active process regulated by vascular cells rather than pure passive calcium (Ca) deposition. In recent years, extracellular vesicles (EVs) have attracted extensive attention because of their essential role in the process of VC. Matrix vesicles (MVs), one type of EVs, are especially critical in extracellular matrix mineralization and the early stages of the development of VC. Vascular smooth muscle cells (VSMCs) have the potential to undergo phenotypic transformation and to serve as a nucleation site for hydroxyapatite crystals upon extracellular stimulation. However, it is not clear what underlying mechanism that MVs drive the VSMCs phenotype switching and to result in calcification. This article aims to review the detailed role of MVs in the progression of VC and compare the difference with other major drivers of calcification, including aging, uremia, mechanical stress, oxidative stress, and inflammation. We will also bring attention to the novel findings in the isolation and characterization of MVs, and the therapeutic application of MVs in VC.

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.

In vitro evaluation of freeze-drying chitosan-mineralized collagen/Mg-Ca alloy composites for osteogenesis.

Magnesium (Mg) alloy with good mechanical properties and biodegradability is considered as one of the ideal bone repair materials. However, the rapid corrosion of Mg-based metals can pose harm to the function of an implant in clinical applications. In this study, micro-arc oxidation coating was prepared on the surface of the Mg-Ca matrix, then the chitosan and mineralized collagen (nano-hydroxyapatite/collagen; nHAC) were immobilized on the surface of the MAO/Mg-Ca matrix to construct the CS-nHAC/Mg-Ca composites of different component proportions (the ratio of CS to nHAC is 2:1, 1:1, and 1:2, respectively). The corrosion resistance, osteogenic activity, and angiogenic ability were extensively investigated. The results indicated that the CS-nHAC reinforcement materials can improve the corrosion resistance of the Mg matrix significantly and promote the proliferation and adhesion of mouse embryo osteoblast precursor cells (MC3T3-E1) and human umbilical vein endothelial cells (HUVECs). In addition, the CS-nHAC/Mg-Ca composites can not only promote the alkaline phosphatase (ALP) activity and extracellular matrix mineralization of MC3T3-E1 cells but also enhance the migration motility and vascular endothelial growth factor (VEGF) expression of HUVECs. Meanwhile, the 2CS-1nHAC/Mg-Ca composite exhibited the optimum function characteristics compared with other samples. Therefore, considering the improvement of corrosion resistance and biocompatibility, the CS-nHAC/Mg-Ca composites are expected to be a promising orthopedic implant.

Corrosion behavior, antibacterial ability, and osteogenic activity of Zn-incorporated Ni-Ti-O nanopore layers on NiTi alloy

• Zn-incorporated nanopores layers with different lengths were prepared on the NiTi substrate through anodization and hydrothermal treatment. • The loaded amounts and release kinetics of Zn ions was controlled by the Ni-Ti-O nanopores. • Zn incorporation enhanced the corrosion resistance. • Zn-incorporated nanopores layers enhanced the antibacterial property and exhibited long-lasting antibacterial ability. • Zn incorporation promoted the generation of alkaline phosphatase products and significantly promotesd type I collagen synthesis and extracellular matrix mineralization. Development of bone fixation devices with excellent corrosion resistance, antibacterial ability, and osteogenic activity is critical for promoting fracture healing. In this study, Zn-incorporated nanopore (NP) layers were prepared on the NiTi alloy through anodization and hydrothermal treatment. Results show that Zn can be evenly incorporated into the NP layers in the form of ZnTiO 3 . The Zn-incorporated samples exhibit good corrosion resistance and significantly reduce Ni 2+ release. Meanwhile, the samples can continuously release Zn 2+ , which is responsible for excellent long-term antibacterial ability. Furthermore, the synergetic effect of Zn 2+ release and nanoporous structure of the NP layers endues the NiTi alloy excellent osteogenic activity, as verified by upregulated alkaline phosphatase activity, secretion of type I collagen, and extracellular matrix mineralization. Therefore, Zn-incorporated Ni-Ti-O NP layers have great potential as biomedical coatings of NiTi-based implant materials.

GENE-ACTIVATED MATRIСES IMPREGNATED WITH POLYPLEXES WITH BMP-2 GENE FOR BONE REGENERATION

Gene-activated matrices based on chitosan hydrogel and polylactide particles impreg-nated with polyplexes with BMP-2 gene have been developed for bone regeneration. The obtained matrices have a significant osteoinductive effect on MSCs: extracellular matrix mineralization, an increase in gene expression and BMP-2 and ALPL proteins production are observed.

A nano-structured TiO2/CuO/Cu2O coating on Ti-Cu alloy with dual function of antibacterial ability and osteogenic activity

• A TiO 2 /CuO/Cu 2 O composite coating with nanostructure was deposited on Ti-Cu alloy by alkali-heat treatment. • TiO 2 /CuO/Cu 2 O coating exhibited strong antibacterial ability against S. aureus . • TiO 2 /CuO/Cu 2 O coating promoted initial adhesion and spreading of MC3T3-E1 cells, as well as osteogenic differentiation. Bacteria-associated infection and poor osseointegration are two main reasons for orthopedic implant failure. Ti-Cu alloy exhibited excellent antibacterial property, but still presented unsatisfied osteogenic activities. Therefore, Ti-Cu alloy was surface modified by an alkali-heat treatment in this paper to improve the osteogenic ability without reduction in antibacterial ability. A TiO 2 /CuO/Cu 2 O composite coating with nanostructure was deposited on Ti-Cu alloy. The coating showed increased roughness and great hydrophilicity. Antibacterial tests indicated that the modified Ti-Cu alloy exhibited stronger antibacterial ability against Staphylococcus aureus ( S. aureus ) than Ti-Cu alloy. Meanwhile, cell experiments demonstrated that the composite coating promoted initial adhesion and spreading of MC3T3-E1 cells, enhanced alkaline phosphatase (ALP) activities as well as extracellular matrix (ECM) mineralization, and significantly upregulated osteogenesis-related gene expressions. It was suggested that the nano-structured TiO 2 /CuO/Cu 2 O coating on Ti-Cu alloy might provide a potential strategy for orthopedic implant failure.

Effects of a novel self-assembling peptide scaffold on bone regeneration and controlled release of two growth factors.

RADA16 is a self-assembling peptide material with good bioactivity. To improve the bioactivity of a material, some specific functional motifs can be added to its peptide sequence. Here, we report a self-assembling peptide nanogel, RADA16-RGD, that has better bioactivity than RADA16 and can simultaneously carry and control the release of two growth factors, VEGF and BMP-2, which have synergistic effects on bone formation. The peptide materials were characterized by transmission electron microscopy and scanning electron microscopy. The mechanical properties of the peptides were evaluated by the rheology test. The biocompatibility of the materials was evaluated via the use of the CCK-8 test, live/dead staining and confocal laser scanning microscopy. Osteogenesis capability in vitro was evaluated by means of ALP staining, extracellular matrix mineralization and detection of osteogenic markers. The controlled release of growth factors was examined by ELISA. The results showed that RADA16-RGD exhibited a better ability than RADA16 to promote cell proliferation, adhesion and bone formation. In addition, RADA16-RGD had good biocompatibility and exhibited effective controlled release of VEGF and BMP-2. More importantly, compared with RADA16-RGD loaded with single growth factor or without growth factors, RADA16-RGD loaded with two growth factors exhibited a stronger ability to promote cell proliferation and osteogenesis. This study provides a promising strategy for the application of self-assembling peptides to promote osteogenesis and controlled release of proteins.

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 (50μM) for 48h 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.