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Lycorine-nanoparticles (LYC-NPs) were successfully synthesized using anti-solvent precipitation-freeze drying method, and characterized using transmission electron microscopy (TEM), particle size analysis and Fourier transform infrared spectroscopy (FTIR). Then, the antitumor effects of LYC-NPs against HepG2 cells were investigated, and the underlying molecular mechanisms were explored. Our results showed that LYC-NPs displayed potent antiproliferative against HepG2 cells concentration dependently. Flow cytometry analysis exhibited that LYC-NPs triggered apoptosis and impeded cell cycle in G0/G1 phase. Moreover, the up-regulated expression of cleaved caspases-3 and Bax, and decrease of mitochondrial membrane potential and the Bcl-2 expression were involved in LYC-NPs apoptosis, implying that LYC-NPs induced apoptosis via the mitochondrial-mediated apoptosis pathway. Furthermore, LYC-NPs distinctly impaired HepG2 cells migration and invasion with down-regulation of matrix metalloproteinase-2 (MMP-2) and MMP-9 expression. These results indicated that LYC-NPs could be an favorable agent for restraining the growth and metastasis of HepG2 cells.

Background: Primary synovial chondromatosis (PSC) is a rare idiopathic pathology characterized by the formation of osseocartilaginous nodules within synovial joints, tendons, or bursae. The mineralization pattern of PSC nodules is poorly understood and has yet to be investigated using elemental analysis. Mapping this pattern could elucidate the progression of the disease. Materials and methods: PSC nodules discovered during dissection of a formalin fixed donor were analyzed. Scanning electron microscopy paired with energy dispersive X-ray spectroscopy (SEM-EDS) was used to quantify calcium and phosphorus levels to distinguish mineralized components from cartilage, indicated by increased carbon and oxygen concentrations. Results: Nine nodules with average dimensions 1.76cm x 1.25cm were identified in the semimembranosus bursa. SEM-EDS demonstrated increased calcium phosphate levels in nodular cores, while outer margins contained primarily carbon and oxygen. Quantification of these elements revealed nodular peripheries to contain 68.0% carbon, 30.2% oxygen, 0.8% calcium, and 1.0% phosphate, while cores were comprised of 38.1% carbon, 42.1% oxygen, 14.1% calcium, and 5.7% phosphate. Conclusions: Nodules were found to have mineralized cores embedded within a cartilaginous matrix. This pattern suggests disease progression is facilitated by endochondral ossification, opening the potential for new therapeutic techniques.

The recent discovery of mesenchymal stem cells within periapical lesions (PL-MSC) has presented novel opportunities for managing periradicular diseases in adult teeth by way of enhancing tissue regeneration. This discovery coincides with the current paradigm shift toward biologically driven treatment strategies in endodontics, which have typically been reserved for non-vital immature permanent teeth. One such approach that shows promise is utilizing local endogenous non-collagenous dentine extracellular matrix components (dECM) to recruit and upregulate the intrinsic regenerative capacity of PL-MSCs

As human stem cells with the special pluripotency play important roles in the innovative drug discovery and regenerative medicine, development of extracellular matrix (ECM) mimetics or functional materials that can support stem cell growth and propagation is of high significance. Despite numerous efforts spent, one major limitation restricting the wide applications of stem cells to the clinical translation is the lack of efficient strategies for low cost and large-scale stem cell production under xeno-free culture conditions. Herein, we reported a new strategy with peptides-modified polystyrene-based polymers coated onto the surface of coverslips for the growth and reproduction of human embryonic stem cells (hESCs). The modified peptides are the active parts of proteins which has been shown to contribute to the pluripotent stem cell attachment or proliferation. The peptides were linked to the glass coverslips coated by the polymer materials via chemical crosslinking, and the composite substrates successfully maintain the long-term growth of HUES-7, H7 and DF699. Our study shows that the coating of polystyrene-derived polymer modified by our developed peptides is a good matrix for long-term growth and reproduction of stem cells. This polystyrene-derived polymer substrate can be produced in large scale and stored for a long time. The most important thing is that it can support the growth of undifferentiated human pluripotent stem cells (hPSCs) for more than ten passages, which could provide a new and relatively easy way to amplify hESCs in vitro.

The repair of bone defects, especially for the large segment of bone defects, has always been an urgent problem in orthopedic clinic and attracted researchers’ attention. Nowadays, the application of tissue engineering bone in the repair of bone defects has become the research hotspot. With the rapid development of tissue engineering, the novel and functional scaffold materials for bone repair have emerged. In this review, we have summarized the multi-functional roles of osteoclasts in bone remodeling. The development of matrix-based tissue engineering bone has laid a theoretical foundation for further investigation about the novel bone regeneration materials which could perform high bioactivity. From the point of view on preserving pre-osteoclasts and targeting mature osteoclasts, this review introduced the novel matrix-based tissue engineering bone based on osteoclasts in the field of bone tissue engineering, which provides a potential direction for the development of novel scaffold materials for the treatment of bone defects.

Fetal bovine serum (FBS) is a widely used supplement in cell culture medium, despite its known variability in composition which greatly affects cellular function and consequently the outcome of studies. In bone tissue engineering, the deposited mineralized matrix is one of the main outcome parameters, but using different brands of FBS can result in large variations. Alkaline phosphatase (ALP) is present in FBS. Not only is ALP used to judge the osteogenic differentiation of bone cells, it may affect deposition of mineralized matrix. The present study focused on the enzymatic activity of ALP in FBS of different suppliers and its contribution to mineralization in osteogenic differentiation cultures. It was hypothesized that culturing cells in a medium with high intrinsic ALP activity of FBS will lead to higher mineral deposition compared to media with lower ALP activity. The used FBS types were shown to have significant differences in enzymatic ALP activity. Our results indicate that the ALP activity of the medium not only affected the deposited mineralized matrix but also the osteogenic differentiation of cells as measured by a changed cellular ALP activity of human bone marrow derived mesenchymal stromal cells (hBMSC). In media with low inherent ALP activity, the cellular ALP activity was increased and played the major role in the mineralization process; while, in media with high intrinsic ALP activity contribution from the serum, less cellular ALP activity was measured and the ALP activity of the medium also contributed to mineral formation substantially. Our results highlight the diverse effects of ALP activity intrinsic to FBS on osteogenic differentiation and matrix mineralization and how FBS can determine the experimental outcomes, in particular for studies investigating matrix mineralization. Once again, the need to replace FBS with more controlled and known additives is highlighted.Graphical abstract

It is well known that the biomechanical and tribological performance of articular cartilage is inextricably linked to its extracellular matrix (ECM) structure and zonal heterogeneity. Furthermore, it is understood that the presence of native ECM components, such as collagen II and aggrecan, promote healthy homeostasis in the resident chondrocytes. What is less frequently discussed is how chondrocyte metabolism is related to the extracellular mechanical environment, at both the macro and microscale. The chondrocyte is in immediate contact with the pericellular matrix of the chondron, which acts as a mechanocoupler, transmitting external applied loads from the ECM to the chondrocyte. Therefore, components of the pericellular matrix also play essential roles in chondrocyte mechanotransduction and metabolism. Recreating the biomechanical environment through tuning material properties of a scaffold and/or the use of external cyclic loading can induce biosynthetic responses in chondrocytes. Decellularized scaffolds, which retain the native tissue macro- and microstructure also represent an effective means of recapitulating such an environment. The use of such techniques in tissue engineering applications can ensure the regeneration of skeletally mature articular cartilage with appropriate biomechanical and tribological properties to restore joint function. Despite the pivotal role in graft maturation and performance, biomechanical and tribological properties of such interventions is often underrepresented. This review outlines the role of biomechanics in relation to native cartilage performance and chondrocyte metabolism, and how application of this theory can enhance the future development and successful translation of biomechanically relevant tissue engineering interventions. Impact statement Physiological cartilage function is a key criterion in the success of a cartilage tissue engineering solution. The

Glioblastoma (GBM) displays diffusive invasion throughout the brain microenvironment that is partially responsible for its short median survival rate (

Mineralized Peyronie's plaque (MPP) impairs penile function. The association, colocalization, and dynamic interplay between organic and inorganic constituents can provide insights into biomineralization of Peyronie's plaque. Human MPPs (n = 11) were surgically excised, and the organic and inorganic constituents were spatially mapped using multiple high-resolution imaging techniques. Multiscale image analyses resulted in spatial colocalization of elements within a highly porous material with heterogenous composition, lamellae, and osteocytic lacuna-like features with a morphological resemblance to bone. The lower (520 ±179 mg/cc) and higher (1024 ± 155 mg/cc) mineral density regions were associated with higher (11%) and lower (7%) porosities in MPP. Energy dispersive X-ray and micro-X-ray fluorescent spectroscopic maps in the higher mineral density regions of MPP revealed higher counts of calcium (Ca) and phosphorus (P), and a Ca/P ratio of 1.48 ± 0.06 similar to bone. More importantly, higher counts of zinc (Zn) were localized at the interface between softer (more organic to inorganic ratio) and harder (less organic to inorganic ratio) tissue regions of MPP and adjacent softer matrix, indicating the involvement of Zn-related proteins and/or pathways in the formation of MPP. In particular, dentin matrix protein-1 (DMP-1) was colocalized in a matrix rich in proteoglycans and collagen that contained osteocytic lacuna-like features. This combined materials science and biochemical with correlative microspectroscopic approach provided insights into the plausible cellular and biochemical pathways that incite mineralization of an existing fibrous Peyronie's plaque. Statement of Significance Aberrant human penile mineralization is known as mineralized Peyronie's plaque (MPP) and often results in a loss of form and function. This study focuses on investigating the spatial association of matrix proteins and elemental composition of MPP by colocalizing calcium, phosphorus, and trace metal zinc with dentin matrix protein 1 (DMP-1), acidic proteoglycans, and fibrillar collagen along with the cellular components using high resolution correlative microspectroscopic techniques. Spatial maps provided insights into cellular and biochemical pathways that incite mineralization of fibrous Peyronie's plaque in humans.

Intestinal organoids are emerging tools in intestinal disease modeling and regeneration. Matrigel, a basement membrane matrix extracted from mouse sarcoma, is the gold standard material for intestinal organoid culture. Yet, the tumor origin of this matrix does not effectively provide organoids with native intestine-like microenvironments. To overcome this limitation, we demonstrate a decellularized small intestine-derived hydrogel reinforced with three extracellular matrix (ECM) proteins that are essential for intestinal development. To identify optimal conditions for intestinal organoid culture, we optimized the concentrations of the ECM hydrogel derived from decellularized intestine and three ECM proteins. The intestinal organoids grown in the established ECM hydrogels have shown similarity to those grown in Matrigel in terms of organoid forming efficiency, size, and gene expression patterns of intestine-specific markers. Our intestine-mimetic hydrogel could increase the feasibility of intestinal organoids for more clinically relevant applications in disease modeling and regenerative medicine.

Background: The healing of diabetic wounds is poor due to a collagen deposition disorder. Matrix metalloproteinase-9 (MMP-9) is closely related to collagen deposition in the process of tissue repair. Many studies have demonstrated that extracellular vesicles derived from adipose-derived stem cells (ADSC-EVs) promote diabetic wound healing by enhancing collagen deposition. Objective: In this study, we explored whether ADSC-EVs could downregulate the expression of MMP-9 in diabetic wounds and promote wound healing by improving collagen deposition. The potential effects of ADSC-EVs on MMP-9 and diabetic wound healing were tested both in vitro and in vivo. Methods: We first evaluated the effect of ADSC-EVs on the proliferation and MMP-9 secretion of HaCaT cells treated with advanced glycation end product-bovine serum albumin (AGE-BSA) using CCK-8, western blot and MMP-9 enzyme-linked immunosorbent assay(ELISA). Next, the effects of ADSC-EVs on healing, re-epithelialisation, collagen deposition, and MMP-9 concentration in diabetic wound fluids were evaluated in an immunodeficient mouse model via MMP-9 ELISA and haematoxylin and eosin, Masson’s trichrome, and immunofluorescence staining for MMP-9. Results: In vitro, ADSC-EVs promoted the proliferation and MMP-9 secretion of HaCaT cells. In vivo, ADSC-EVs accelerated diabetic wound healing by improving re-epithelialisation and collagen deposition and by inhibiting the expression of MMP-9. Conclusion: ADSC-EVs possess the potential of healing of diabetic wounds in a mouse model by inhibiting downregulating MMP-9 and improving collagen deposition. Thus, ADSC-EVs are a promising candidate for the treatment of diabetic wounds.

Bone and bone marrow are vital to mammalian structure, movement, and immunity. These tissues are also commonly subjected to molecular alterations giving rise to debilitating diseases like rheumatoid arthritis, osteoporosis, osteomyelitis, and cancer. Technologies such as matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) enable the discovery of spatially resolved chemical information in biological tissue samples to help elucidate the complex molecular processes underlying pathology. Traditionally, preparation of native osseous tissue for MALDI IMS has been difficult due to the mineralized composition and heterogenous morphology of the tissue, and compensation for these challenges with decalcification and fixation protocols can remove or delocalize molecular species. Here, sample preparation methods were advanced to enable multimodal MALDI IMS of undecalcified, fresh-frozen murine femurs allowing the distribution of endogenous lipids to be linked to specific tissue structures and cell types. Adhesive-bound bone sections were mounted onto conductive glass slides with a microscopy-compatible glue and freeze-dried to minimize artificial bone marrow damage. Subliming matrix does not induce further bone marrow cracking, and recrystallizing the deposited matrix improves lipid signal. High spatial resolution (10 μm) MALDI IMS was employed to characterize lipid distributions in fresh-frozen bone, and use of complementary microscopy modalities aided tissue and cell assignments. For example, various phosphatidylcholines localize to bone marrow, adipose tissue, marrow adipose tissue, and muscle. Further, sphingomyelin(42:1) was abundant in megakaryocytes, whereas sphingomyelin(42:2) was diminished in this cell type. These data reflect the vast molecular and cellular heterogeneity indicative of the bone marrow and the soft tissue surrounding the femur. Multimodal MALDI IMS has the potential to advance bone-related biomedical research by offering deep molecular coverage with spatial relevance in a preserved native bone microenvironment.

Collagen XII is a regulator of corneal stroma structure and function. The current study examined the roles of collagen XII in regulating corneal stromal transforming growth factor (TGF)-β activation and latency. Specifically, with the use of conventional collagen XII null mouse model, the roles of collagen XII in the regulation of TGF-β latency and activity in vivo were investigated. Functional quantification of latent TGF-β in stromal matrix was performed by using transformed mink lung reporter cells that produce luciferase as a function of active TGF-β. Col12a1 knock-down with short hairpin RNA (shRNA) was used to test the role of collagen XII in TGF-β activation. We found Col12a1–/– hypertrophic stromas with keratocyte hyperplasia. Increased collagen fibril forward signal was found by second harmonic generation (SHG) microscopy in the absence of collagen XII. Collagen XII regulated mRNA synthesis of Serpine1, Col1a1, and Col5a1 and deposition of collagens in the extracellular matrix. A functional plasminogen activator inhibitor (PAI-1) luciferase assay showed that collagen XII is necessary for latent TGF-β storage in the extracellular matrix and that collagen XII downregulates active TGF-β. Collagen XII dictates stromal structure and function by regulating TGF-β activity. A hypertrophic phenotype in Col12a1–/– corneal tissue can be explained by abnormal upregulation of TGF-β activation and decreased latent storage.

BACKGROUND Superficial thrombophlebitis (ST) is a frequent pathology, but its exact incidence remains to be determined. This study tested the hypothesis whether relationships exist among smooth muscle cells (SMCs) derived from ST, varicose great saphenous veins (VGSVs), and normal great saphenous veins (GSVs). METHODS Forty-one samples of ST, VGSVs, and GSVs were collected. SMCs were isolated and cultured. Proliferation, migration, adhesion, and senescence in SMCs from the three vein walls were compared by various methods. Bax, Bcl-2, caspase-3, matrix metalloproteinase-2 (MMP-2), MMP-9, tissue inhibitor of metalloproteinase-1 (TIMP-1), and TIMP-2 messenger RNA (mRNA) and protein expressions were detected by fluorescence quantitative PCR and Western blot. RESULTS An obvious decrease in cytoskeletal filaments was observed in thrombophlebitic vascular smooth muscle cells (TVSMCs). The quantity of proliferation, migration, adhesion, and senescence in TVSMCs was significantly higher than in varicose vascular smooth muscle cells and normal vascular smooth muscle cells (NVSMCs) (all P < 0.05). Bax and caspase-3 mRNA and protein expression were decreased, while Bcl-2 mRNA and protein expression were increased in the TVSMCs compared with the varicose vascular smooth muscle cells and the NVSMCs (all P < 0.05). MMP-2, MMP-9, TIMP-1, and TIMP-2 mRNA and protein expression were significantly increased in the TVSMCs compared with the VVGSVs and the NVSMCs (all P < 0.05). CONCLUSION SMCs derived from ST are more dedifferentiated and demonstrate increased cell proliferation, migration, adhesion, and senescence, as well as obviously decreased cytoskeletal filaments. These results suggest that the phenotypic and functional differences could be related to the presence of atrophic and hypertrophic vein segments during the disease course among SMCs derived from ST, VGSVs, and GSVs.

Modification of the extracellular matrix (ECM) is a critical aspect of developing a metastasis-supportive organ niche. Recent work investigating ECM changes that facilitate metastasis has revealed ways in which different metastatic organ niches are similar as well as the distinct characteristics that make them unique. In this review, we present recent findings regarding how ECM modifications support metastasis in four frequent metastatic sites: the lung, liver, bone, and brain. We discuss ways in which these modifications are shared between metastatic organs as well as features specific to each location. We also discuss areas of technical innovation that could be advantageous to future research and areas of inquiry that merit further investigation.

There is increasing evidence that cells cultured in three-dimensional (3D) settings have superior performance compared to their traditional counterparts in monolayers. This has been attributed to cell-cell and cell-extracellular matrix interactions that more closely resemble the in vivo tissue architecture. The rapid adoption of 3D cell culture systems as experimental tools for diverse applications has not always been matched by an improved understanding of cell behavior in different 3D environments. Here, we studied human mesenchymal stem/stromal cells (hMSCs) as scaffold-free self-assembled aggregates of low and high cell number and compared them to cell-laden alginate hydrogels with and without arginine-glycine-aspartic acid peptides. We observed a significant decrease in the size of cell-only aggregates over 14 days in culture compared to the cells encapsulated in alginate hydrogels. Alginate hydrogels had persistently more living cells for a longer period (14 days) in culture as measured by total DNA content. Proliferation studies revealed that a weeklong culture of hMSCs in 3D culture, whether as aggregates or cell-laden alginate hydrogels, reduced their proliferation over time. Cell cycle analysis found no significant differences between days 1 and 7 for the different culture systems. The findings of this study improve our understanding of how aggregate cultures differ with or without a hydrogel carrier, and whether aggregation itself is important when it comes to the 3D culture of hMSCs.

In this study, a hybrid of TiO2@LDH was applied to fabricate a bone scaffold to take the advantages of both nanostructures. In addition to the role of LDH in the biomineralization process, it was used to provide a suitable bed for the better dispersion of TiO2 nanoparticles and prevent them from aggregations. The main limitation in the usage of these nanoceramics in the preparation of bone scaffolds is their powder state, which prevents their fixation in the desired place. So, alginate (Alg) was selected as the matrix to embed this nanohybrid and support its stability and fixation. Bioactivity of the prepared Alg/TiO2@LDH scaffolds was assessed by incubation in the simulated body fluid at 37 °C for 28 days. Field emission scanning electron microscopy as well as energy dispersive X-ray were used to prove the biomineralization on the scaffolds. MTT test showed that the scaffold containing 6 wt% of the TiO2@LDH do not have any toxicity effects on the MG-63 cell line. Also, this scaffold presented outstanding antibacterial ability against Staphylococcus aureus, which is known as an important infecting agent in the bone scaffolds. The proposed Alg/TiO2@LDH scaffold in this study could be a proper candidate to be used in bone tissue engineering.

Bone is a hard-connective tissue composed of matrix, cells and bioactive factors with a hierarchical structure, where the matrix is mainly composed of type I collagen and hydroxyapatite. Collagen fibers assembled by collagen are the template for mineralization and make an important contribution to bone formation and the bone remodeling process. Therefore, collagen has been widely clinically used for bone/cartilage defect regeneration. However, pure collagen implants, such as collagen scaffolds or sponges, have limitations in the bone/cartilage regeneration process due to their poor mechanical properties and osteoinductivity. Different forms of collagen-based composites prepared by incorporating natural/artificial polymers or bioactive inorganic substances are characterized by their interconnected porous structure and promoting cell adhesion, while they improve the mechanical strength, structural stability and osteogenic activities of the collagen matrix. In this review, various forms of collagen-based biocomposites, such as scaffolds, sponges, microspheres/nanoparticles, films and microfibers/nanofibers prepared by natural/synthetic polymers, bioactive ceramics and carbon-based materials compounded with collagen are reviewed. In addition, the application of collagen-based biocomposites as cytokine, cell or drug (genes, proteins, peptides and chemosynthetic) delivery platforms for proangiogenesis and bone/cartilage tissue regeneration is also discussed. Finally, the potential application, research and development direction of collagen-based biocomposites in future bone/cartilage tissue regeneration are discussed.

3D printed bioactive glass or bioceramic particle reinforced composite scaffolds for bone tissue engineering currently suffer from low particle concentration (100% breaking strain) by adding poly(ethylene glycol) which is biocompatible and FDA approved. The scaffolds require no post-printing washing to remove hazardous components. More exposure of HA microparticles on strut surfaces is enabled by incorporating higher HA concentrations. Compared to scaffolds with 72 wt% HA, scaffolds with higher HA content (90 wt%) enhance matrix formation but not new bone volume after 12 weeks implantation in rat calvarial defects. Histological analyses demonstrate that bone regeneration within the 3D printed scaffolds is via intramembranous ossification and starts in the central region of pores. Fibrous tissue that resembles non-union tissue within bone fractures is formed within pores that do not have new bone. The amount of blood vessels is similar between scaffolds with mainly fibrous tissue and those with more bone tissue, suggesting vascularization is not a deciding factor for determining the type of tissues regenerated within the pores of 3D printed scaffolds. Multinucleated immune cells are commonly present in all scaffolds surrounding the struts, suggesting a role of managing inflammation in bone regeneration within 3D printed scaffolds.

Aging is accompanied by declining lung function and increasing susceptibility to lung diseases. The role of endothelial dysfunction and vascular remodeling in these changes is supported by growing evidence, but underlying mechanisms remain elusive. In this review we summarize functional, structural, and molecular changes in the aging pulmonary vasculature and explore how interacting aging and mechanobiological cues may drive progressive vascular remodeling in the lungs.

Aging is a physiological process with profound impact on the biology and function of biosystems, including the human dentition. While resilient, human teeth undergo wear and disease, affecting overall physical, psychological, and social human health. However, the underlying mechanisms of tooth aging remain largely unknown. Root dentin is integral to tooth function in that it anchors and dissipates mechanical load stresses of the tooth-bone system. Here, we assess the viscoelastic behavior, composition, and ultrastructure of young and old root dentin using nano-dynamic mechanical analysis, micro-Raman spectroscopy, small angle X-ray scattering, atomic force and transmission electron microscopies. We find that the root dentin overall stiffness increases with age. Unlike other mineralized tissues and even coronal dentin, however, the ability of root dentin to dissipate energy during deformation does not decay with age. Using a deconstruction method to dissect the contribution of mineral and organic matrix, we find that the damping factor of the organic matrix does deteriorate. Compositional and ultrastructural analyses revealed higher mineral-to-matrix ratio, altered enzymatic and non-enzymatic collagen cross-linking, increased collagen d-spacing and fibril diameter, and decreased abundance of proteoglycans and sulfation pattern of glycosaminoglycans . Therefore, even in the absence of remodeling, the extracellular matrix of root dentin shares traits of aging with other tissues. To explain this discrepancy, we propose that altered matrix-mineral interactions, possibly mediated by carbonate ions sequestered at the mineral interface and/or altered glycosaminoglycans counteract the deleterious effects of aging on the structural components of the extracellular matrix. STATEMENT OF SIGNIFICANCE: Globally, a quarter of the population will be over 65 years old by 2050. Because many will retain their dentition, it will become increasingly important to understand and manage how aging affects teeth. Dentin is integral to the protective, biomechanical, and regenerative features of teeth. Here, we demonstrate that older root dentin not only has altered mechanical properties, but shows characteristic shifts in mineralization, composition, and post-translational modifications of the matrix. This strongly suggests that there is a mechanistic link between mineral and matrix components to the biomechanical performance of aging dentin with implications for efforts to slow or even reverse the aging process.

The self-healing of chemotherapy-induced oral mucositis was difficultly practiced because of both local bacterial infection and severe inflammation. Herein, in situ mucoadhesive hydrogels (PPP_E) was successfully prepared by using temperature-sensitive PLGA-PEG-PLGA (PPP) as matrix and EGCG as adhesion enhancer. Series of PPP_E precursor solution with various EGCG concentration (1%, 2% and 5%) were prepared by fixing the PPP concentration at 25%. EGCG slightly decreased the sol-gel transition temperature and shorten sol-gel transition time of PPP hydrogel. Moreover, the incorporation of EGCG could significantly increase the tissue adhesion properties of PPP hydrogel at 37℃. PPP_2%E displayed the suitable gelation temperature (36.2℃), gelation time (100s) and storage moduli (48Pa). Tripeptide KPV as model drug was easily dissolved in cold PPP_2%E precursor solution to prepare KPV@PPP_2%E hydrogel. The anti-inflammatory activity and promoting cell migration potential of KPV in PPP-2% E hydrogel were well maintained. Moreover, KPV@PPP_2%E exhibited the strong antibacterial efficacy against S. aureus. PPP_2%E precursor solution rapidly transited to hydrogel and adhered to the wound surface for 7 hours when administrated to the gingival mucosa of rats. Treatment with KPV@PPP_2%E hydrogel greatly improved the food intake and body weight recovery of chemotherapy-induced rats with oral mucositis. Moreover, the tissue morphology of the ulcerated gingival after KPV@PPP_E hydrogel was also well repaired via promoting CK10 and PCNA expression. Besides, the inflammatory cytokines including IL-1β and TNF-α were significantly inhibited by KPV@PPP_2%E hydrogel while IL-10 was up-regulated. KPV@PPP_2%E hydrogel also had the anti-bacteria effect on MRSA-infected gingival ulcer wound, which resulted in the obvious inhibition of inflammatory cells infiltration in submucosal tissues. Conclusively, KPV@PPP_E may be a promising practical application for cancer people with chemotherapy-induced oral mucositis.

The gap between demand and supply for the required bone graft and donor is widening day by day. Tissue engineering approach is used to overcome the limitations of bone graft substitutes. In Tissue Engineering, a biocompatible scaffold is placed inside the body, which over a period of time gets converted into the bone Extra-Cellular Matrix (ECM). Hydroxyapatite (HA) and β-Tricalcium Phosphate (β-TCP) are extensively studied to fabricate bone scaffolds for tissue engineering applications by conventional methods. However, the scaffold needs to be produced with controlled pore size, porosity, and pore interconnectivity for homing of the cells which can be achieved by additive manufacturing. Also, the scaffolds produced with conventional methods lack mechanical properties which limit the use of hydroxyapatite (HA) and β-tricalcium phosphate (β-TCP) at the recipient site. In this paper, scaffolds with different materials' compositions, different layers orientations, and pore sizes are designed as an input parameter for different scaffold architecture which has not been previously studied considering extrusion-based additive manufacturing. These parameters are considered to predict the mechanical properties of the scaffold architecture. It has been found that in all combinations the scaffold with 0°- 90°- 0°- 90° orientation layer gives Young’s modulus that is comparable to natural human bone. However, the scaffolds with 0°- 90° -0°- 90° orientation layer and 350 µm pore size gives comparatively higher effective Young’s modulus of about 30.948 GPa for 5% HA composition. In the future 0°- 90°- 0°- 90° orientation can be considered to fabricate 3D scaffold architecture using extrusion-based additive manufacturing method.

Studies on animal models with mutations in ameloblastin gene have suggested that the extracellular matrix protein ameloblastin (AMBN) plays important roles in controlling cell-matrix adhesion and ameloblast polarization during amelogenesis. In order to examine the function of AMBN in cell polarization and morphology, we developed an in vitro 3D cell culture model to examine the effect of AMBN and amelogenin (AMEL) addition on ameloblast cell line ALC. We further used high resolution confocal microscopy to detect expression of polarization markers in response to AMBN addition. Addition of AMBN to the 3D culture matrix resulted in the clustering and elongation (higher aspect ratio) of ALC in a dose dependent manner. The molar concentration of AMEL required to exact this response from ALC was 2.75- times greater than that of AMBN. This polarization effect of ameloblastin was attributable directly to an evolutionary conserved domain within its exon 5-encoded region. The exon 6-encoded region also influenced AMBN-cell interactions but to a lesser extent. The clusters formed with AMBN were polarized with expression of E-cadherin, Par3 and Cldn1 assembly at the nascent cell-cell junctions. The elongation effect was specific to epithelial cells of ameloblastic lineage ALC and LS8 cells. Our data suggest that AMBN may play critical signaling roles in the initiation of cell polarity by acting as a communicator between cell-cell and cell-matrix interactions. Our investigation has important implications for understanding the function of ameloblastin in enamel-cell matrix adhesion and the outcomes may contribute to efforts to develop strategies for enamel tissue regeneration.

Hydrogels are often used to study the impact of biomechanical and topographical cues on cell behavior. Conventional hydrogels are designed a priori, with characteristics that cannot be dynamically changed in an externally controlled, user-defined manner. We developed a composite hydrogel, termed an acoustically-responsive scaffold (ARS), that enables non-invasive, spatiotemporally controlled modulation of mechanical and morphological properties using focused ultrasound. An ARS consists of a phase-shift emulsion distributed in a fibrin matrix. Ultrasound non-thermally vaporizes the emulsion into bubbles, which induces localized, radial compaction and stiffening of the fibrin matrix. In this in vitro study, we investigate how this mechanism can control the differentiation of fibroblasts into myofibroblasts, a transition correlated with substrate stiffness on 2D substrates. Matrix compaction and stiffening was shown to be highly localized using confocal and atomic force microscopies, respectively. Myofibroblast phenotype, evaluated by α-smooth muscle actin (α-SMA) immunocytochemistry, significantly increased in matrix regions proximal to bubbles compared to distal regions, irrespective of the addition of exogenous transforming growth factor-β1 (TGF-β1). Introduction of the TGF-β1 receptor inhibitor SB431542 abrogated the proximal enhancement. This approach providing spatiotemporal control over biophysical signals and resulting cell behavior could aid in better understanding fibrotic disease progression and the development of therapeutic interventions for chronic wounds. STATEMENT OF SIGNIFICANCE: Hydrogels are used in cell culture to recapitulate both biochemical and biophysical aspects of the native extracellular matrix. Biophysical cues like stiffness can impact cell behavior. However, with conventional hydrogels, there is a limited ability to actively modulate stiffness after polymerization. We have developed an ultrasound-based method of spatiotemporally-controlling mechanical and morphological properties within a composite hydrogel, termed an acoustically-responsive scaffold (ARS). Upon exposure to ultrasound, bubbles are non-thermally generated within the fibrin matrix of an ARS, thereby locally compacting and stiffening the matrix. We demonstrate how ARSs control the differentiation of fibroblasts into myofibroblasts in 2D. This approach could assist with the study of fibrosis and the development of therapies for chronic wounds.

Poor antioxidant and thermal-shock capacities of C/C composites thermal barrier coating (TBC) caused by cracking and shedding of coatings has been a major obstacle blocking the development of C/C composites. Herein, in-situ growth of whisker reinforced silicon carbide transition layer and inter-embedding mechanism of multi-gradient coatings were brought into the design of TBC to enhance the antioxidant and thermal-shock capacities. A three-layer gradient coating SiC-SiCw/ZrB2-SiC/ZrSiO4-aluminosilicate glass (ZAG) from inside to outside, in which ZrB2-SiC/ZAG serve as oxygen barrier layers with self-healing ability and SiC-SiCw provides thermal stress buffering and bonding against cracking and shedding of coatings, is designed. The ZAG mainly forms a dense oxygen blocking frontier with self-healing ability through fluidized glass, while the ZrB2-SiC can react actively with infiltrated oxygen in a way of self-sacrifice, preventing oxygen erosion to C/C matrix and SiC-SiCw transition layer. As a result, the collaborative work among layers endows this coating with excellent high temperature service performance. This work provides a new insight for the design of excellent TBC.

Objectives:\ud Antibiotic resistance is increasingly a growing global threat. This study aimed to investigate the potential use of newly developed scandium-doped phosphate-based glasses (Sc-PBGs) as an antibacterial and anticariogenic agent through controlled release of Sc3+ ions.\ud \ud Methods:\ud Sc-PBGs with various calcium and sodium oxide contents were produced and characterised using thermal and spectroscopic analysis. Degradation behaviour, ion release, antibacterial action against Streptococcus mutans, anti-matrix metalloproteinase-2 (MMP-2) activity, remineralisation potential and in vivo biocompatibility were also investigated.\ud \ud \ud Results:\ud The developed glass system showed linear Sc3+ ions release over time. The released Sc3+ shows statistically significant inhibition of S. mutans biofilm (1.2 log10 CFU reduction at 6 h) and matrix metalloproteinase-2 (MMP-2) activity, compared with Sc-free glass and positive control. When Sc-PBGs were mounted alongside enamel sections, subjected to acidic challenges, alternating hyper- and hypomineralisation layers consistent with periods of re- and demineralisation were observed demonstrating their potential remineralising action. Furthermore, Sc-PBGs produced a non-toxic response when implanted subcutaneously for 2 weeks in Sprague Dawley rats.\ud \ud Significance:\ud Since Sc3+ ions might act on various enzymes essential to the biological mechanisms underlying caries, Sc-PBGs could be a promising therapeutic agent against cariogenic bacteria.

Endothelial tip cell outgrowth of blood-vessel sprouts marks the initiation of angiogenesis which is critical in physiological and pathophysiological procedures. However, how mechanical characteristics of extracellular matrix (ECM) modulates tip cell formation has been largely neglected. In this study, we found enhanced CD31 expression in the stiffening outer layer of hepatocellular carcinoma than in surrounding soft tissues. Stiffened matrix promoted sprouting from endothelial cell (EC) spheroids and upregulated expressions of tip cell-enriched genes in vitro. Moreover, tip cells showed increased cellular stiffness, more actin cytoskeleton organization and enhanced YAP nuclear transfer than stalk and phalanx ECs. We further uncovered that substrate stiffness regulates FAK and Paxillin phosphorylation in focal adhesion of ECs promoting Rac1 transition from inactive to active state. YAP is subsequently activated and translocated into nucleus, leading to increased tip cell specification. p-Paxillin can also loosen the intercellular connection which also facilitates tip cell specification. Collectively our present study shows that matrix stiffness modulates tip cell formation through p-PXN-Rac1-YAP signaling axis, shedding light on the role of mechanotransduction in tip cell formation. This is of special significance in biomaterial design and treatment of some pathological situations., Graphical abstract Image 1, Highlights • Mechanotransduction is implicated in angiogenesis and tip cell formation. • Tip cells showed different mechanical properties from stalk and phalanx ECs. • Paxillin, Rac1 and YAP might be novel treatment targets for some diseases. • Material stiffness affects tip cell specification.

Made available in DSpace on 2022-04-28T17:30:34Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-12-28 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Introduction: Gingival recession (GR) is a relevant clinical condition due to its high prevalence worldwide, which leads to aesthetic demands and dentin hypersensitivity. Collagen matrices have been associated with different designs of coronally advanced flaps (CAFs) to treat GR defects. However, the literature lacks long-term follow-up of this treatment option. The aim of this study is to present the long-term follow-up (up to 7 years) of two GR defects (associated or not with noncarious cervical lesion) treated with a CAF and a collagen matrix (CM). Case presentation Case 1 underwent a CAF associated with a CM to treat a single GR defect. After 7 years, Case 1 presented with 3 mm of recession reduction, which corresponds with the 85.7% of root coverage. Case 2 presented two GR defects associated with noncarious cervical lesions (NCCLs). The NCCLs were partially restored with resin composite and then underwent a modified CAF for multiple defects and a CM. After 5 years of follow-up, Case 2 presented with 1.5 and 2.5 mm of recession reduction, which corresponds with the average 83.3% defect coverage. Conclusion These two cases may show that CMs can provide long-term stable outcomes in the treatment of GR defects. Fed Univ Pernambuco UFPE, Dept Prosthesis & Surg Orofacial, Recife, PE, Brazil Sao Paulo State Univ, Inst Sci & Technol, Div Periodont, UNESP, Av Engn Francisco Jose Longo 777, BR-12245000 Sao Jose Dos Campos, SP, Brazil Private Educ Inst, Sao Paulo, Brazil Sao Paulo State Univ, Inst Sci & Technol, Div Periodont, UNESP, Av Engn Francisco Jose Longo 777, BR-12245000 Sao Jose Dos Campos, SP, Brazil CAPES: 001 CNPq: 304269/2019-0

The zona pellucida (ZP) is an extracellular matrix surrounding mammalian oocytes. It is composed of three to four glycoproteins, ZP1-ZP4. ZP3 is essential for sperm binding and zona matrix formation. Here, we identified a novel heterozygous mutation (NM_001110354.2:c.502_504delGAG) of ZP3, occurring in a pair of sisters with empty follicle syndrome (EFS). A mouse model with the same mutation was established using the CRISPR/Cas9 gene-editing system. As in the above family, F0 -, F1 -, and F2 -generation female mice with the mutation were all infertile. Further analysis using the Chinese hamster ovary cells (CHO-K1) also showed that this mutation weakens the strength of binding between ZP3 and ZP2, which hinders the assembly of ZP and results in unstable ZP formation. Immunohistochemical analysis using ovarian serial sections in both humans and mice demonstrated that the ZP of preantral follicles was thinner than normal control, or even absent. Our study presents a new gene mutation that leads to EFS, providing new evidence and support for the genetic diagnosis of infertile individuals with similar phenotypes. Our results also show that the loop of ZP3 is not only a linker between two amphiphilic helices but may play a critical role in specifying the correct heterodimerization partner. This article is protected by copyright. All rights reserved.

BACKGROUND Keratinized gingival insufficiency is a disease attributed to long-term tooth loss, can severely jeopardizes the long-term health of implants. A simple and effective augmentation surgery method should be urgently developed. CASE SUMMARY A healthy female patient, 45-year-old, requested implant restoration of the her left mandibular first molar and second molar. Before considering a stage II, as suggested from the probing depth measurements, the widths of the mesial, medial, and distal buccal keratinized gingiva of second molar (tooth #37) were measured and found to be 0.5 mm, 0.5 mm, and 0 mm, respectively. This suggested that the gingiva was insufficient to resist damage from bacterial and mechanical stimulation. Accordingly, modified apically repositioned flap (ARF) surgery combined with xenogeneic collagen matrix (XCM) and platelet-rich fibrin (PRF) was employed to increase the width of gingival tissue. After 1 mo of healing, the widths of mesial, medial, and distal buccal keratinized gingiva reached 4 mm, 4 mm, and 3 mm, respectively, and the thickness of the augmented mucosa was 4.5 mm. Subsequently, through the second-stage operation, the patient obtained an ideal soft tissue shape around the implant. CONCLUSION For cases with keratinized gingiva widths around implants less than 2mm，the soft tissue width and thickness could be increased by modified ARF surgery combined with XCM and PRF. Moreover, this surgery significantly alleviated patients’ pain and ameliorated oral functional comfort.

Tissue and organ regeneration is the dynamic process by which a population of cells rearranges into a specific form with specific functions. Traditional tissue regeneration utilizes tissue grafting, cell implantation, and structured scaffolds to achieve clinical efficacy. However, tissue grafting methods face a shortage of donor tissue, while cell implantation may involve leakage of the implanted cells without a supportive 3D matrix. Cell migration, proliferation, and differentiation in structured scaffolds may disorganize and frustrate the artificially pre-designed structures, and sometimes involve immunogenic reactions. To overcome this limitation, the self-organizing properties and innate regenerative capability of tissue/organism formation in the absence of guidance by structured scaffolds has been investigated. This review emphasizes the growing subfield of the regulated self-organizing approach for neotissue formation and describes advances in the subfield using diverse, cutting-edge, inter-disciplinarity technologies. We cohesively summarize the directed self-organization of cells in the micro-engineered cell-ECM system and 3D/4D cell printing. Mathematical modeling of cellular self-organization is also discussed for providing rational guidance to intractable problems in tissue regeneration. It is envisioned that future self-organization approaches integrating biomathematics, micro-nano engineering, and gene circuits developed from synthetic biology will continue to work in concert with self-organizing morphogenesis to enhance rational control during self-organizing in tissue and organ regeneration.

Soft tissue remodeling is a sophisticated process that sequentially provides dynamic biological signals to guide cell behavior. However, capturing these signals within hydrogel and directing over time has still been unrealized owing to the poor comprehension of physiological processes. Here, a bio-mimicking hydrogel is designed via thiol-ene click reaction to capture the early physical signal triggered by inflammation, and the chemical signals provided with chemokine and natural adhesion sites, which guaranteed the precise soft tissue remodeling. This bio-mimicking hydrogel efficiently facilitated cell anchoring, migration, and invasion in the 3D matrix due to the permissive space and the interaction with integrin receptors. Besides, the covalently grafted chemokine-like peptide is optimal for colonization and functional differentiation of endothelial cells through a HIF-1α dependent signal pathway. Furthermore, the early polarization of macrophages, collagen deposition and angiogenesis in rat acute wound model, and the increased blood perfusion in mouse skin flap model have confirmed that the bio-mimicking hydrogel realized precise soft tissue remodeling and opens new avenues for the phased repair of different tissues such as nerve, myocardium, and even bone.

The gold standard in cryopreservation is still conventional slow freezing of single cells or small aggregates in suspension, although major cell loss and limitation to non-specialised cell types in stem cell technology are known drawbacks. The requirement for rapidly available therapeutic and diagnostic cell types is increasing constantly. In the case of human induced pluripotent stem cells (hiPSCs) or their derivates, more sophisticated cryopreservation protocols are needed to address this demand. These should allow a preservation in their physiological, adherent state, an efficient re-cultivation and upscaling upon thawing towards high-throughput applications in cell therapies or disease modelling in drug discovery. Here, we present a novel vitrification-based method for adherent hiPSCs, designed for automated handling by microfluidic approaches and with ready-to-use potential e.g. in suspension-based bioreactors after thawing. Modifiable alginate microcarriers serve as a growth surface for adherent hiPSCs that were cultured in a suspension-based bioreactor and subsequently cryopreserved via droplet-based vitrification in comparison to conventional slow freezing. Soft (0.35%) versus stiff (0.65%) alginate microcarriers in concert with adhesion time variation have been examined. Findings revealed specific optimal conditions leading to an adhesion time and growth surface (matrix) elasticity dependent hypothesis on cryo-induced damaging regimes for adherent cell types. Deviations from the found optimum parameters give rise to membrane ruptures assessed via SEM and major cell loss after adherent vitrification. Applying the optimal conditions, droplet-based vitrification was superior to conventional slow freezing. A decreased microcarrier stiffness was found to outperform stiffer material regarding cell recovery, whereas the stemness characteristics of rewarmed hiPSCs were preserved.

The new method of the formation of nanocomposite materials based on carbon nanotubes for the regeneration of connective tissues has been developed.Aim. Study of the structure, mechanical characteristics and biocompatibility of the obtained materials.Materials and methods. The experimental samples of nanocomposite materials were based on multi-walled and singlewalled carbon nanotubes, the matrix was bovine serum albumin. A layer of liquid dispersion of the components on a silicon substrate or in a container was irradiated with laser radiation to form the solid nanocomposite material. The microstructure of the obtained samples was analyzed with X-ray microtomography, the tensile strength was investigated using a testing machine. Fibroblast cells were incubated with experimental samples for 3, 24, 48, and 72 h and then fixed with glutaraldehyde. Cell growth during incubation with samples was studied using optical and atomic force microscopy.Results. It was found that a slight decrease in tensile strength and increase in the degree of deformation were observed with an increase in the concentration of carbon nanotubes. At the same time, the mechanical parameters of the samples corresponded to the requirements for materials for the restoration of connective tissue defects. Microscopic studies indicate good adhesion of cells to the nanocomposite material, no toxic effect of the samples on the cells was found. After 3 hours of incubation, the cells had their original rounded shape, after 24 hours of incubation cells began to proliferate on the sample's surface and were spindle-shaped. After 48 and 72 hours, the cells practically formed a monolayer on the surface of the samples.Conclusion. The results of the study show that the structural and mechanical parameters of the developed nanocomposite materials meet the requirements of biomedicine. It was also shown that nanocomposite materials do not suppress cell growth and can serve as a scaffold for the regeneration of damaged tissues.

The angle-ply multilaminate structure of the annulus fibrosus is not reestablished following discectomy which leads to reherniation of the intervertebral disc (IVD). Biomimetic scaffolds developed to repair these defects should be evaluated for their ability to support tissue regeneration by endogenous and exogenous cells. Herein a collagen-based, angle-ply multilaminate patch designed to repair the outer annulus fibrosus was assessed for its ability to support mesenchymal stromal and annulus fibrosus cell viability, elongation, alignment, extracellular matrix gene expression, and scaffold remodeling. Results demonstrated that the cells remained viable, elongated, and aligned along the collagen fiber preferred direction of the scaffold, upregulated genes associated with annulus fibrosus matrix and produced collagen on the scaffold yielding biaxial mechanical properties that resembled native annulus fibrosus tissue. In conclusion, these scaffolds have demonstrated their potential to promote a living repair of defects in the annulus fibrosus and thus may be used to prevent recurrent IVD herniations.

BACKGROUND: The treatment of pancreatic cancer (PDAC) remains clinically challenging, and neoadjuvant therapy (NAT) offers down staging and improved surgical resectability. Abundant fibrous stroma is involved in malignant characteristic of PDAC. We aimed to investigate tissue remodelling, particularly the alteration of the collagen architecture of the PDAC microenvironment by NAT. METHODS: We analysed the alteration of collagen and gene expression profiles in PDAC tissues after NAT. Additionally, we examined the biological role of Ephrin-A5 using primary cultured cancer-associated fibroblasts (CAFs). RESULTS: The expression of type I, III, IV, and V collagen was reduced in PDAC tissues after effective NAT. The bioinformatics approach provided comprehensive insights into NAT-induced matrix remodelling, which showed Ephrin-A signalling as a likely pathway and Ephrin-A5 (encoded by EFNA5) as a crucial ligand. Effective NAT reduced the number of Ephrin-A5(+) cells, which were mainly CAFs; this inversely correlated with the clinical tumour shrinkage rate. Experimental exposure to radiation and chemotherapeutic agents suppressed proliferation, EFNA5 expression, and collagen synthesis in CAFs. Forced EFNA5 expression altered CAF collagen gene profiles similar to those found in PDAC tissues after NAT. CONCLUSION: These results suggest that effective NAT changes the extracellular matrix with collagen profiles through CAFs and their Ephrin-A5 expression.

Matrix metalloproteinases (MMPs) have been implicated not only in the regulation of developmental processes but also in the release of biologically active molecules and in the modulation of repair during tertiary dentine formation. Although efforts to preserve dentine have focused on inhibiting the activity of these proteases, their function is much more complex and necessary for dentine repair than expected. The present review explores the role of MMPs as bioactive components of the dentine matrix involved in dentine formation, repair and regeneration. Special consideration is given to the mechanical properties of dentine, including those of reactionary and reparative dentine, and the known roles of MMPs in their formation. MMPs are critical components of the dentine matrix and should be considered as important candidates in dentine regeneration.

Purpose The stiffness of the pericellular matrix (PCM) decreases in the most common degenerative joint disease, osteoarthritis (OA). This study was undertaken to explore the potential functional role of transient receptor potential vanilloid 4 (TRPV4), Piezo1, and Piezo2 in transducing different PCM stiffness in chondrocytes. Methods and results Polydimethylsiloxane (PDMS) substrates with different stiffness (designated 197 kPa, 78 kPa, 54 kPa, or 2 kPa, respectively) were first prepared to simulate the decrease in stiffness of the PCM that chondrocytes encounter in osteoarthritic cartilage. Next, the TRPV4-, Piezo1-, or Piezo2-knockdown primary chondrocytes (designated TRPV4-KD, Piezo1-KD, or Piezo2-KD cells) were seeded onto these different PDMS substrates. Then, using a Ca2+-imaging system, substrate stiffness-regulated intracellular Ca2+ influx ([Ca2+]i) in chondrocytes was examined to investigate the role of TRPV4, Piezo1, and Piezo2 in Ca2+ signaling in response to different stiffness. Results showed that the characteristics of intracellular [Ca2+]i in chondrocytes regulated by PDMS substrate exhibited stiffness-dependent differences. Additionally, stiffness-evoked [Ca2+]i changes were suppressed in TRPV4-KD, Piezo1-KD, or Piezo2-KD cells compared with control siRNA-treated cells, implying that any channel is fundamental for Ca2+ signaling induced by substrate stiffness. Furthermore, TRPV4-mediated Ca2+ signaling played a central role in the response of chondrocytes to 197 kPa and 78 kPa substrate, while Piezo1/2-mediated Ca2+ signaling played a central role in the response of chondrocytes to 54 kPa and 2 kPa substrate. Conclusions Collectively, these findings indicate that chondrocytes might perceive and distinguish the different PCM stiffness by using different mechanosensitive ion channels.

Current challenges in the biofabrication of stable, cell-seeded fibrin gels are faced with a lack of microstructural and mechanical properties, limiting their use as bone extracellular matrix-mimicking constructs. While current approaches focus on generating fibrin gels of various fibrinogen and thrombin concentrations, i.e. 5–50 mg mL−1 and 1–250 units mL−1, respectively, processes that modulate gel fibrillar alignment and mechanical properties have yet to be explored. Herein, it was demonstrated that the automated gel aspiration-ejection (GAE) of precursor fibrin hydrogels of low concentration (i.e. 1 mg mL−1 fibrinogen and 0.5 units mL−1 thrombin) resulted in their immediate compaction while tailoring their fibrillar alignment and mechanical properties for potential target applications. Furthermore, this gel microenvironment accelerated the osteoblastic differentiation of seeded mesenchymal stem cells and their matrix mineralization, in vitro. Therefore, the ability to modulate the properties of dense fibrin constructs through automated GAE may be a novel biofabrication approach for cell delivery in bone regeneration.

Made available in DSpace on 2022-05-01T10:35:01Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-01-01 Fluconazole-sensitive (CaS) and -resistant (CaR) C. albicans were grown as single-species and dual-species biofilms with Lactobacillus casei (Lc) and Lactobacillus rhamnosus (Lr). Single-species Lc and Lr were also evaluated. Biofilm analysis included viable plate counts, the extracellular matrix components, biomass, and structural organization. Lc reduced the viability of CaS, water-soluble polysaccharides, and eDNA in CaS + Lc biofilm. Lc biofilm presented more eDNA than CaS. The total biomass of CaS + Lc biofilm was higher than the single-species biofilms. The viability of Lc and Lr was reduced by CaR dual-species biofilms. The total and insoluble biomass in CaS + Lr was higher than in single-species CaS biofilms. Lc hindered the growth of CaS, and their association hampered matrix components linked to the structural integrity of the biofilm. These findings allow understanding of how the implementation of probiotics influences the growth of C. albicans biofilms and thereby helps with the development of novel approaches to control these biofilms. Department of Dental Materials and Prosthodontics São Paulo State University (UNESP) School of Dentistry Department of Cariology Operative Dentistry & Dental Public Health Indiana University School of Dentistry Department of Surgery and Diagnosis São Paulo State University (Unesp) School of Dentistry Department of Dental Materials and Prosthodontics São Paulo State University (UNESP) School of Dentistry Department of Surgery and Diagnosis São Paulo State University (Unesp) School of Dentistry

Osteoarthritis is a prevalent degenerative joint disease characterized by progressive articular cartilage loss and destruction. The resultant increase in friction causes severe pain. The collagen I matrix (COL I) has been used clinically for cartilage repair; however, how COL I acts at cartilage surfaces is unclear. Here, we studied adsorption and lubrication of synovial fluid components, albumin, γ-globulin, and the phospholipid DPPC, on COL I under physiological conditions using surface plasmon resonance and an in situ sensing surface force apparatus. Our results revealed COL I had poor lubrication ability, a fairly high coefficient of friction (COF, μ = 0.651 ± 0.013), and surface damage under a 7 mN load. DPPC formed an improved lubricating layer on COL I (μ = 0.072 ± 0.016). In sharp contrast, albumin and γ-globulin exhibited poor lubrication with an order of magnitude higher COF but still provided benefits by protecting COL I from wear. Hence, DPPC on COL I may help optimize COL I implantation design.

The skeletal muscle tissue comprises a hierarchical fibrous structure with fully aligned myofibers. To obtain a unique aligned engineering construct for regenerating muscle tissue, we adopted a submerged bioprinting process. Here, 3 wt % collagen and 6 wt % alginate solutions were used as a matrix cell-encapsulating bioink and supporting solution in the printing bath, respectively. By manipulating the processing parameters (various alginate weight fractions in the bath, nozzle moving speed, and hydrostatic pressure), cell-laden filaments (∼50 μm in diameter) were successfully fabricated. They presented a high degree of alignment of the fibrillated collagen and meaningful initial viability (∼90%) of the C2C12 myoblasts. In vitro cellular responses indicated that fully aligned F-actin filaments of myoblasts were developed, resulting in a high degree of alignment/formation of myotubes, compared to that in the controls (>100 μm diameter of cell-laden filaments). Furthermore, the expression levels of various myogenic genes (Myod1, Myh2, and Myog) were measured using a reverse transcription polymerase chain reaction on day 21 of the cell culture, and the results showed that the cell-laden filaments with a small diameter had considerably greater gene expression levels (2.2-8-fold) than those with a relatively large diameter. Thus, the printing process described herein can provide a new potential biofabricating platform to obtain cell-laden engineering constructs for various tissues.

The most implanted tissue after blood is a bone, which consists of collagen and hydroxyapatite as the major components. Collagen is a nature-based polymer that is available in skin and tendons, whereas hydroxyapatite is a natural ceramic that is available abundantly in teeth. The composites of hydroxyapatite/collagen (HA/Col) have been reported to be the most encouraging bone graft because of the likenesses with the natural bones. Furthermore, the integration of the nano silvers in the composites’ matrix has been predicted to lead to antimicrobial reactions. This study combined the natural fish scales hydroxyapatite (FsHAp), fish scale collagen (FsCol), and nanosilver (Ag) to develop the composites of FsHAp/FsCol/nAg with various ratios of content. FTIR, XRD, EDX, and SEM were employed to identify the chemical structure and morphology of the tested composites. Similar processes were conducted to study the swelling ratio, biostability, and antibacterial features of the composites. The cytotoxicity of the FsHAp/FsCol/AgNPs composites was evaluated using Alamar blue assay. The outcome revealed that 80:20 of the HA/Col composite showed higher stability than other ratio compositions. The morphology of the composites demonstrated a homogeneous surface with nanosilvers scattered evenly in the matrix. Additionally, the activities of the antimicrobial in the composites were found against Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus). For the cytotoxicity study, the composites showed non-toxic effects on MG-63 human cells at high concentrations.

Cell adhesion on biomaterial surfaces has been extensively studied from the perspective of the adsorption properties of adhesive ligands, while recent research on mechanobiology has been revealing a critical role of the mechanical properties of the extracellular milieu in the control of cell adhesion, such as the stiffness and viscoelasticity of the matrix. Although the effects of the lateral mobility of an adhesive ligand have been intensively investigated in a model substrate with water-soluble polymer layers, less is known about those in the setting of lateral deformability of hydrophobic condensed polymer layers. In this study, to help clarify this issue, we used PNIPAAm-grafted substrates with a well-controlled degree of graft-polymerization (DGP) as a typical hydrophobic condensed polymer surface at a cell culture temperature of 37 °C. We observed a clear negative correlation between cell spreading and DGP of PNIPAAm regardless of the amount of fibronectin adsorbed on the substrates, which was found to be attributable to the lateral deformability of a condensed PNIPAAm layer based on lateral force microscopic analysis. The surface-lateral-deformation-induced modulation in stability and maturation of focal adhesion of the cells is discussed in relation to the matrix-strain-induced alteration of the density distribution of adsorbed adhesive ligands. To clarify the role of lateral deformation of condensed polymer surface on cell adhesion, the responses of cell spreading were characterized at a cell culture temperature on the poly(N-isopropylacrylamide)-grafted surfaces with different degree of graft-polymerization (DGP). A clear negative correlation between cell spreading and DGP of PNIPAAm was found regardless of the amount of fibronectin adsorbed on the substrates. The microscopic local strain of the condensed polymers by cellular traction forces was considered to modulate the density distribution of adsorbed adhesive ligands beneath the focal adhesions and the cell spreading.

Three types of resorbable composite materials containing the protein phase were examined in the work. They were: 1 – PLA matrix/PLA fibers/albumin, 2 – PLA matrix/calcium alginate fibers/albumin, 3...

Decellularized extracellular matrix (dECM)–based biomaterials are of great clinical utility in soft tissue repair applications due to their regenerative properties. Multi-layered dECM devices have been developed for clinical indications where additional thickness and biomechanical performance are required. However, traditional approaches to the fabrication of multi-layered dECM devices introduce additional laminating materials or chemical modifications of the dECM that may impair the biological functionality of the material. Using an established dECM biomaterial, ovine forestomach matrix, a novel method for the fabrication of multi-layered dECM constructs has been developed, where layers are bonded via a physical interlocking process without the need for additional bonding materials or detrimental chemical modification of the dECM. The versatility of the interlocking process has been demonstrated by incorporating a layer of hyaluronic acid to create a composite material with additional biological functionality. Interlocked composite devices including hyaluronic acid showed improved in vitro bioactivity and moisture retention properties.

Leukocyte extravasation into inflamed tissue is a complex process that is difficult to capture as a whole in vitro. We employed a blood-vessel-on-a-chip model in which endothelial cells were cultured in a tube-like lumen in a collagen-1 matrix. The vessels are leak-tight, creating a barrier for molecules and leukocytes. Addition of inflammatory cytokine TNF-α caused vasoconstriction, actin remodelling and upregulation of ICAM-1. Introducing leukocytes into the vessels allowed real-time visualisation of all different steps of the leukocyte transmigration cascade including migration into the extracellular matrix. Individual cell tracking over time distinguished striking differences in migratory behaviour between T-cells and neutrophils. Neutrophils cross the endothelial layer more efficiently than T-cells, but upon entering the matrix, neutrophils display high speed but low persistence, whereas T-cells migrate with low speed and rather linear migration. In conclusion, 3D imaging in real-time of leukocyte extravasation in a vessel-on-a-chip enables detailed qualitative and quantitative analysis of different stages of the full leukocyte extravasation process in a single assay.SummaryA functional hydrogel-based blood-vessel-on-a-chip model is used to study the complete leukocyte transendothelial migration process in real time. T-lymphocytes and neutrophils exhibit distinct migration dynamics in the extravascular matrix after transendothelial migration, which can be altered using a chemotactic gradient.

Background We have developed a novel imaging analysis procedure that is highly predictive of local failure after chemoradiation in head and neck cancer. In this study we investigated whether any pretreatment biomarkers correlated with key imaging parameters. Methods Pretreatment biopsy material was available for 28 patients entered into an institutional trial of adaptive radiotherapy in which FDG-PET images were collected weekly during treatment. The biopsies were immunohistochemically stained for CD44, EGFR, GLUT1, ALDH1, Ki-67 and p53 and quantified using image analysis. Expression levels were correlated with previously derived imaging parameters, the pretreatment SUVmax and the dose response matrix (DRM). Results The different parameters of the SUVmax and DRM did not correlate with each other. We observed a positive and highly significant (p = 0.0088) correlation between CD44 expression and volume of tumor with a DRM greater than 0.8. We found no correlation between any DRM parameter and GLUT1, p53, Ki-67 and EGFR or ALDH1. GLUT1 expression did correlate with the maximum SUV0 and the volume of tumor with an SUV0 greater than 20. Conclusions The pretreatment SUVmax and DRM are independent imaging parameters that combine to predict local recurrence. The significant correlation between CD44 expression, a known cancer stem cell (CSC) marker, and volume of tumor with a DRM greater than 0.8 is consistent with concept that specific foci of cells are responsible for tumor recurrence and that CSCs may be randomly distributed in tumors in specific niches. Dose painting these small areas may lead to improved tumor control.

Osteoblasts in vivo form an epithelial-like layer with tight junctions between cells. Bone formation involves mineral transport into the matrix and acid transport to balance pH levels. To study the importance of the pH gradient in vitro, we used Transwell inserts composed of polyethylene terephthalate (PET) membranes with 0.4 μm pores at a density of (2 ± 0.4) × 10(6) pores per cm(2). Mesenchymal stem cells (MSCs) prepared from murine bone marrow were used to investigate alternative conditions whereby osteoblast differentiation would better emulate in vivo bone development. MSCs were characterized by flow cytometry with more than 90% CD44 and 75% Sca-1 labeling. Mineralization was validated with paracellular alkaline phosphatase activity, collagen birefringence, and mineral deposition confirming MSCs identity. We demonstrate that MSCs cultured and differentiated on PET inserts form an epithelial-like layer while mineralizing. Measurement of the transepithelial resistance was ~ 1,400 ohms•cm(2) at three weeks of differentiation. The pH value of the media above and under the cells were measured while cells were in proliferation and differentiation. In mineralizing cells, a difference of 0.145 pH unit was observed between the medium above and under the cells indicating a transepithelial gradient. A significant difference in pH units was observed between the medium above and below the cells in proliferation compared to differentiation. Data on pH below membranes were confirmed by pH-dependent SNARF1 fluorescence. Control cells in proliferative medium did not form an epithelial-like layer, displayed low transepithelial resistance, and there was no significant pH gradient. By transmission electron microscopy, membrane attached osteoblasts in vitro had abundant mitochondria consistent with active transport that occurs in vivo by surface osteoblasts. In keeping with osteoblastic differentiation, scanning electron microscopy identified deposition of extracellular collagen surrounded by hydroxyapatite. This in vitro model is a major advancement in modeling bone in vivo for understanding of osteoblast bone matrix production.