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Uncomplicated treatments for pulpitis and periodontitis continues to be challenging and regenerative approaches could meet this contingency. Dental pulp stem cells (DPSCs) represent a good candidate for oral recovering therapies. Here, we investigated changes in morphology, proliferation, and in vitro differentiation toward mesenchymal and neuronal phenotypes of human DPSCs harvested from differently aged donors. Aging is a physiologic phenomenon occurring with time that hamper body's capability to maintain homeostasis also affecting the functional reserve. Cytofluorimetric, immunohistochemical, quantitative reverse-transcription polymerase chain reaction (qRT-PCR), and western blot analyses were performed to gain insight for successful regenerative strategies in elderly. We observed a decline in DPSCs proliferation and differentiation potential with age. Interestingly, these cells behaved differently under osteogenic or odontogenic stimuli, showing different age-related mineralization capabilities. Similarly, neurogenic differentiation decreased with age. In conclusion, our observations represent a valid tool for the development of tailored regenerative strategies in an aging society.
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Proliferación Celular , Senescencia Celular , Pulpa Dental/citología , Células Madre Mesenquimatosas/fisiología , Células-Madre Neurales/fisiología , Osteoblastos/fisiología , Regeneración , Adulto , Factores de Edad , Anciano , Proliferación Celular/genética , Forma de la Célula , Células Cultivadas , Senescencia Celular/genética , Regulación de la Expresión Génica , Humanos , Células Madre Mesenquimatosas/metabolismo , Persona de Mediana Edad , Células-Madre Neurales/metabolismo , Neurogénesis , Osteoblastos/metabolismo , Osteogénesis , Fenotipo , Regeneración/genética , Medicina Regenerativa/métodos , Ingeniería de Tejidos , Adulto JovenRESUMEN
Purpose: To shed light on the idea that mesenchymal stem/stromal cells (MSCs) recruited in synovium (SM) (i.e. Synovium-Derived Stromal Cells, SDSCs) could be involved in Osteoarthritis (OA) pathophysiology. Attention was also paid to a further stromal cell type with a peculiar ultrastructure called telocytes (TCs), whose role is far from clarified. Methods: In the present in vitro study, we compared SDSCs isolated from healthy and OA subjects in terms of phenotype, morphology and differentiation potential as well as in their capability to activate normal Peripheral Blood Mononuclear Cells (PBMCs). Histological, immunohistochemical and ultrastructural analyses were integrated by qRT-PCR and functional resorbing assays. Results: Our data demonstrated that both SDSC populations stimulated the formation of osteoclasts from PBMCs: the osteoclast-like cells generated by healthy-SDSCs via transwell co-cultures were inactive, while OA-derived SDSCs have a much greater effectiveness. Moreover, the presence of TCs was more evident in cultures obtained from OA subjects and suggests a possible involvement of these cells in OA. Conclusions: Osteoclastogenic differentiation capability of PBMCs from OA subjects, also induced by B synoviocytes has been already documented. Here we hypothesized that SDSCs, generally considered for their regenerative potential in cartilage lesions, have also a role in the onset/maintenance of OA. Clinical relevance: Our observations may represent an interesting opportunity for the development of a holistic approach for OA treatment, that considers the multifaceted capability of MSCs in relation to the environment.
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Osteoartritis/etiología , Osteogénesis , Células del Estroma/fisiología , Membrana Sinovial/citología , Anciano de 80 o más Años , Diferenciación Celular , Femenino , Humanos , Masculino , Microscopía Electrónica de Rastreo , Microscopía Electrónica de Transmisión , Osteoartritis/metabolismo , Osteoartritis/fisiopatología , Osteogénesis/fisiología , Reacción en Cadena en Tiempo Real de la Polimerasa , Células del Estroma/ultraestructura , Membrana Sinovial/fisiopatología , Telocitos/fisiologíaRESUMEN
Senescence can impair the therapeutic potential of stem cells. In this study, senescence-associated morphofunctional changes in periosteum-derived progenitor cells (PDPCs) from old and young individuals were investigated by combining cytofluorimetry, immunohistochemistry, and transmission electron microscopy. Cell cycle analysis demonstrated a large number of G0/G1 phase cells in PDPCs from old subjects and a progressive accumulation of G0/G1 cells during passaging in cultures from young subjects. Cytofluorimetry documented significant changes in light scattering parameters and closely correlated with the ultrastructural features, especially changes in mitochondrial shape and autophagy, which are consistent with the mitochondrial-lysosomal axis theory of ageing. The combined morphological, biofunctional, and ultrastructural approach enhanced the flow cytometric study of PDPC ageing. We speculate that impaired autophagy, documented in replicative senescent and old PDPCs, reflect a switch from quiescence to senescence. Its demonstration in a tissue with limited turnover-like the cambium layer of the periosteum, where reversible quiescence is the normal stem cell state throughout life-adds a new piece to the regenerative medicine jigsaw in an ageing society.
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Autofagia , Senescencia Celular/fisiología , Células Madre Mesenquimatosas , Periostio/patología , Adulto , Anciano de 80 o más Años , Células Cultivadas , Femenino , Citometría de Flujo , Humanos , Inmunohistoquímica , Masculino , Células Madre Mesenquimatosas/fisiología , Células Madre Mesenquimatosas/ultraestructura , Microscopía Electrónica de TransmisiónRESUMEN
Osteoarthritis (OA) is a joint degenerative pathology characterized by mechanical and inflammatory damages affecting synovium, articular cartilage (AC), and subchondral bone (SB). Several in vitro, in vivo, and ex vivo models are developed to study OA, but to date the identification of specific pharmacological targets seems to be hindered by the lack of models with predictive capabilities. This study reports the development of a biomimetic in vitro model of AC and SB interface. Gellan gum methacrylated and chondroitin sulfate/dopamine hydrogels are used for the AC portion, whereas polylactic acid functionalized with gelatin and nanohydroxyapatite for the SB. The physiological behavior of immortalized stem cells (Y201s) and Y201s differentiated in chondrocytes (Y201-Cs), respectively, for the SB and AC, is demonstrated over 21 days of culture in vitro in healthy and pathological conditions, whilst modeling the onset of cytokines-induced OA. The key metrics are: lower glycosaminoglycans production and increased calcification given by a higher Collagen X content, in the AC deep layer; higher expression of pro-angiogenic factor (vegf) and decreased expression of osteogenic markers (coll1, spp1, runx2) in the SB. This novel approach provides a new tool for studying the development and progression of OA.
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Cartílago Articular , Osteoartritis , Humanos , Huesos/metabolismo , Cartílago Articular/metabolismo , Cartílago Articular/patología , Condrocitos , Osteoartritis/metabolismo , Osteoartritis/patología , Osteogénesis , Ingeniería de Tejidos/métodosRESUMEN
Graded lattice scaffolds based on rhombic dodecahedral (RD) elementary unit cell geometry were manufactured in 316L stainless steel (SS) by laser powder bed fusion (LPBF). Two different strategies based on varying strut thickness layer-by-layer in the building direction were adopted to obtain the graded scaffolds: a) decreasing strut size from core to edge to produce the dense-in (DI) structure and b) increasing strut size in the same direction to produce the dense-out (DO) structure. Both graded structures (DI and DO) were constructed with specular symmetry with respect to the central horizontal axis. Structural, mechanical, and biological characterizations were carried out to evaluate feasibility of designing appropriate biomechanical performances of graded scaffolds in the perspective of bone tissue regeneration. Results showed that mechanical behavior is governed by graded geometry, while printing parameters influence structural properties of the material such as density, textures, and crystallographic phases. The predominant failure mechanism in graded structures initiates in correspondence of thinner struts, due to high stress concentrations on strut junctions. Biological tests evidenced better proliferation of cells in the DO graded scaffold, which in turn exhibits mechanical properties close to cortical bone. The combined control of grading strategy, printing parameters and elementary unit cell geometry can enable implementing scaffolds with improved biomechanical performances for bone tissue regeneration.
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Huesos , Prótesis e Implantes , Polvos , Rayos Láser , Andamios del Tejido/químicaRESUMEN
This experimental study aims to extend the know-how on biomechanical performances of duplex stainless steel (DSS) for tissue engineering applications to a graded lattice geometry scaffold based on the F53 DSS (UNS S32750 according to ASTM A182) produced by laser powder bed fusion (LPBF). The same dense-out graded geometry based on rhombic dodecahedral elementary unit cells investigated in previous work on 316L stainless steel (SS) was adopted here for the manufacturing of the F53 DSS scaffold (SF53). Microstructural characterization and mechanical and biological tests were carried out on the SF53 scaffold, using the in vitro behavior of the 316L stainless steel scaffold (S316L) as a control. Results show that microstructure developed as a consequence of different volume energy density (VED) values is mainly responsible for the different mechanical behaviors of SF53 and S316L, both fabricated using the same LPBF manufacturing system. Specifically, the ultimate compressive strength (σUC) and elastic moduli (E) of SF53 are three times and seven times higher than S316L, respectively. Moreover, preliminary biological tests evidenced better cell viability in SF53 than in S316L already after seven days of culture, suggesting SF53 with dense-out graded geometry as a viable alternative to 316L SS for bone tissue engineering applications.
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Alzheimer's disease (AD) is the most prevalent form of dementia and is characterized by progressive neurodegeneration leading to severe cognitive, memory, and behavioral impairments. The onset of AD involves a complex interplay among various factors, including age, genetics, chronic inflammation, and impaired energy metabolism. Despite significant efforts, there are currently no effective therapies capable of modifying the course of AD, likely owing to an excessive focus on the amyloid hypothesis and a limited consideration of other intracellular pathways. In the present review, we emphasize the emerging concept of AD as a metabolic disease, where alterations in energy metabolism play a critical role in its development and progression. Notably, glucose metabolism impairment is associated with mitochondrial dysfunction, oxidative stress, Ca2+ dyshomeostasis, and protein misfolding, forming interconnected processes that perpetuate a detrimental self-feeding loop sustaining AD progression. Advanced glycation end products (AGEs), neurotoxic compounds that accumulate in AD, are considered an important consequence of glucose metabolism disruption, and glyceraldehyde (GA), a glycolytic intermediate, is a key contributor to AGEs formation in both neurons and astrocytes. Exploring the impact of GA-induced glucose metabolism impairment opens up exciting possibilities for creating an easy-to-handle in vitro model that recapitulates the early stage of the disease. This model holds great potential for advancing the development of novel therapeutics targeting various intracellular pathways implicated in AD pathogenesis. In conclusion, looking beyond the conventional amyloid hypothesis could lead researchers to discover promising targets for intervention, offering the possibility of addressing the existing medical gaps in AD treatment.
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Enfermedad de Alzheimer , Humanos , Enfermedad de Alzheimer/metabolismo , Gliceraldehído/metabolismo , Estrés Oxidativo , Productos Finales de Glicación Avanzada/metabolismo , Glucosa/metabolismo , Péptidos beta-Amiloides/metabolismoRESUMEN
Alzheimer's disease (AD) is a widespread neurodegenerative disorder, affecting a large number of elderly individuals worldwide. Mitochondrial dysfunction, metabolic alterations, and oxidative stress are regarded as cooperating drivers of the progression of AD. In particular, metabolic impairment amplifies the production of reactive oxygen species (ROS), resulting in detrimental alterations to intracellular Ca2+ regulatory processes. The Na+/Ca2+ exchanger (NCX) proteins are key pathophysiological determinants of Ca2+ and Na+ homeostasis, operating at both the plasma membrane and mitochondria levels. Our study aimed to explore the role of NCX1 and NCX3 in retinoic acid (RA) differentiated SH-SY5Y cells treated with glyceraldehyde (GA), to induce impairment of the default glucose metabolism that typically precedes Aß deposition or Tau protein phosphorylation in AD. By using an RNA interference-mediated approach to silence either NCX1 or NCX3 expression, we found that, in GA-treated cells, the knocking-down of NCX3 ameliorated cell viability, increased the intracellular ATP production, and reduced the oxidative damage. Remarkably, NCX3 silencing also prevented the enhancement of Aß and pTau levels and normalized the GA-induced decrease in NCX reverse-mode activity. By contrast, the knocking-down of NCX1 was totally ineffective in preventing GA-induced cytotoxicity except for the increase in ATP synthesis. These findings indicate that NCX3 and NCX1 may differently influence the evolution of AD pathology fostered by glucose metabolic dysfunction, thus providing a potential target for preventing AD.
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In a scaffold-based approach for bone tissue regeneration, the control over morphometry allows for balancing scaffold biomechanical performances. In this experimental work, trabecular geometry was obtained by a generative design process, and scaffolds were manufactured by vat photopolymerization with 60% (P60), 70% (P70) and 80% (P80) total porosity. The mechanical and biological performances of the produced scaffolds were investigated, and the results were correlated with morphometric parameters, aiming to investigate the influence of trabecular geometry on the elastic modulus, the ultimate compressive strength of scaffolds and MG-63 human osteosarcoma cell viability. The results showed that P60 trabecular geometry allows for matching the mechanical requirements of human mandibular trabecular bone. From the statistical analysis, a general trend can be inferred, suggesting strut thickness, the degree of anisotropy, connectivity density and specific surface as the main morphometric parameters influencing the biomechanical behavior of trabecular scaffolds, in the perspective of tissue engineering applications.
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Given the lack of in vitro models faithfully reproducing the osteoarthritis (OA) disease on-set, this work aimed at manufacturing a reliable and predictive in vitro cytokine-based Articular Cartilage (AC) model to study OA progression. Cell spheroids of primary human fetal chondrocytes (FCs) and h-TERT mesenchymal stem cells differentiated chondrocytes (Y201-C) were analysed in terms of growth kinetics, cells proliferation and apoptosis over 10 days of culture, in healthy condition or in presence of cytokines (interleukin-1ß, -6 and TNF-α). Then, the spheroids were assembled into chondrospheres using a bottom-up strategy, to obtain an in vitro cytokines-induced OA model. The resulting chondrospheres were evaluated for gene expression and anabolic ECM proteins. Compared to the healthy environment, the simulated OA environment induced chondrocyte hyperproliferation and apoptotic pathway, decreased expression of anabolic ECM proteins, and diminished biosynthetic activity, resembling features of early-stage OA. These characteristics were observed for both Y201-C and HC at high and low concentrations of cytokines. Both HC and Y201-C demonstrated the suitability for the manufacturing of a scaffold-free in vitro OA model to facilitate studies into OA pathogenesis and therapeutic strategies. Our approach provides a faithful reproduction of early-stage osteoarthritis, demonstrating the ability of obtaining different disease severity by tuning the concentration of OA-related cytokines. Given the advantages in easy access and more reproducible performance, Y201-C may represent a more favourable source of chondrocytes for establishing more standardized protocols to obtain OA models.
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Reactive oxygen species (ROS) are versatile molecules that, even if produced in the background of many biological processes and responses, possess pleiotropic roles categorized in two interactive yet opposite domains. In particular, ROS can either function as signaling molecules that shape physiological cell functions, or act as deleterious end products of unbalanced redox reactions. Indeed, cellular redox status needs to be tightly regulated to ensure proper cellular functioning, and either excessive ROS accumulation or the dysfunction of antioxidant systems can perturb the redox homeostasis, leading to supraphysiological concentrations of ROS and potentially harmful outcomes. Therefore, whether ROS would act as signaling molecules or as detrimental factors strictly relies on a dynamic equilibrium between free radical production and scavenging resources. Of notice, the mammalian brain is particularly vulnerable to ROS-mediated toxicity, because it possesses relatively poor antioxidant defenses to cope with the redox burden imposed by the elevated oxygen consumption rate and metabolic activity. Many features of neurodegenerative diseases can in fact be traced back to causes of oxidative stress, which may influence both the onset and progression of brain demise. This review focuses on the description of the dual roles of ROS as double-edge sword in both physiological and pathological settings, with reference to Alzheimer's and Parkinson's diseases.
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Bone homeostasis is the equilibrium between organic and inorganic components of the extracellular matrix (ECM) and cells. Alteration of this balance has consequences on bone mass and architecture, resulting in conditions such as osteoporosis (OP). Given ECM protein mutual regulation and their effects on bone structure and mineralization, further insight into their expression is crucial to understanding bone biology under normal and pathological conditions. This study focused on Type I Collagen, which is mainly responsible for structural properties and mineralization of bone, and selected proteins implicated in matrix composition, mineral deposition, and cell-matrix interaction such as Decorin, Osteocalcin, Osteopontin, Bone Sialoprotein 2, Osteonectin and Transforming Growth Factor beta. We developed a novel multidisciplinary approach in order to assess bone matrix in healthy and OP conditions more comprehensively by exploiting the Fourier Transform Infrared Imaging (FTIRI) technique combined with histomorphometry, Sirius Red staining, immunohistochemistry, and Western Blotting. This innovatory procedure allowed for the analysis of superimposed tissue sections and revealed that the alterations in OP bone tissue architecture were associated with warped Type I Collagen structure and deposition but not with changes in the total protein amount. The detected changes in the expression and/or cooperative or antagonist role of Decorin, Osteocalcin, Osteopontin, and Bone Sialoprotein-2 indicate the deep impact of these NCPs on collagen features of OP bone. Overall, our strategy may represent a starting point for designing targeted clinical strategies aimed at bone mass preservation and sustain the FTIRI translational capability as upcoming support for traditional diagnostic methods.
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Osteopontina , Osteoporosis , Colágeno , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Decorina/metabolismo , Cabeza Femoral/química , Cabeza Femoral/metabolismo , Cabeza Femoral/patología , Análisis de Fourier , Humanos , Sialoproteína de Unión a Integrina/genética , Sialoproteína de Unión a Integrina/metabolismo , Osteocalcina/análisis , Osteocalcina/genética , Osteocalcina/metabolismo , Osteonectina , Osteopontina/genética , Osteopontina/metabolismo , Osteoporosis/diagnóstico por imagen , Osteoporosis/patología , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
Hydrogel-based bioinks are the main formulations used for Articular Cartilage (AC) regeneration due to their similarity to chondral tissue in terms of morphological and mechanical properties. However, the main challenge is to design and formulate bioinks able to allow reproducible additive manufacturing and fulfil the biological needs for the required tissue. In our work, we investigated an innovative Manuka honey (MH)-loaded photocurable gellan gum methacrylated (GGMA) bioink, encapsulating mesenchymal stem cells differentiated in chondrocytes (MSCs-C), to generate 3D bioprinted construct for AC studies. We demonstrated the beneficial effect of MH incorporation on the bioink printability, leading to the obtainment of a more homogenous filament extrusion and therefore a better printing resolution. Also, GGMA-MH formulation showed higher viscoelastic properties, presenting complex modulus G∗ values of â¼1042 âPa, compared to â¼730 âPa of GGMA. Finally, MH-enriched bioink induced a higher expression of chondrogenic markers col2a1 (14-fold), sox9 (3-fold) and acan (4-fold) and AC ECM main element production (proteoglycans and collagen).
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The presence of Reactive Oxygen Species (ROS) in bone can influence resident cells behaviour as well as the extra-cellular matrix composition and the tissue architecture. Aging, in addition to excessive overloads, unbalanced diet, smoking, predisposing genetic factors, lead to an increase of ROS and, if it is accompanied with an inappropriate production of scavengers, promotes the generation of oxidative stress that encourages bone catabolism. Furthermore, bone injuries can be triggered by numerous events such as road and sports accidents or tumour resection. Although bone tissue possesses a well-known repair and regeneration capacity, these mechanisms are inefficient in repairing large size defects and bone grafts are often necessary. ROS play a fundamental role in response after the implant introduction and can influence its success. This review provides insights on the mechanisms of oxidative stress generated by an implant in vivo and suitable ways for its modulation. The local delivery of active molecules, such as polyphenols, enhanced bone biomaterial integration evidencing that the management of the oxidative stress is a target for the effectiveness of an implant. Polyphenols have been widely used in medicine for cardiovascular, neurodegenerative, bone disorders and cancer, thanks to their antioxidant and anti-inflammatory properties. In addition, the perspective of new smart biomaterials and molecular medicine for the oxidative stress modulation in a programmable way, by the use of ROS responsive materials or by the targeting of selective molecular pathways involved in ROS generation, will be analysed and discussed critically.
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Materiales Biocompatibles , Estrés Oxidativo , Antioxidantes/farmacología , Materiales Biocompatibles/farmacología , Huesos , Especies Reactivas de OxígenoRESUMEN
Ageing is among the main risk factors for human disease onset and the identification of the hallmarks of senescence remains a challenge for the development of appropriate therapeutic target in the elderly. Here, we compare senescence-related changes in two cell populations of mesenchymal stromal cells by analysing their miRNA profiling: Human Dental Pulp Stromal Cells (hDPSCs) and human Periosteum-Derived Progenitor Cells (hPDPCs). After these cells were harvested, total RNA extraction and whole genome miRNA profiling was performed, and DIANA-miRPath analysis was applied to find the target/pathways. Only 69 microRNAs showed a significant differential expression between dental pulp and periosteum progenitor cells. Among these, 24 were up regulated, and 45 were downregulated in hDPSCs compared to hPDPCs. Our attention was centered on miRNAs (22 upregulated and 34 downregulated) involved in common pathways for cell senescence (i.e. p53, mTOR pathways), autophagy (i.e. mTOR and MAPK pathways) and cell cycle (i.e. MAPK pathway). The p53, mTOR and MAPK signaling pathways comprised 43, 37 and 112 genes targeted by all selected miRNAs, respectively. Our finding is consistent with the idea that the embryological origin influences cell behavior and the ageing process. Our study strengthens the hypothesis that ageing is driven by numerous mediators interacting through an intricate molecular network, which affects adult stem cells self-renewal capability. Graphical abstract.
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Células Madre Mesenquimatosas , Anciano , Envejecimiento/genética , Humanos , MicroARNs/genética , Serina-Treonina Quinasas TOR , Proteína p53 Supresora de Tumor/genéticaRESUMEN
Developing biomaterial formulations with specific biochemical characteristics and physical properties suitable for bioprinting of 3D scaffolds is a pivotal challenge in tissue engineering. Therefore, the design of novel bioprintable formulations is a continuously evolving research field. In this work, the authors aim at expanding the library of biomaterial inks by blending two natural biopolymers: pectin and gelatin. Cytocompatible formulations are obtained by combining pectin and gelatin at different ratios and using (3-glycidyloxypropyl)trimethoxysilane (GPTMS) as single crosslinking agent. It is shown that the developed formulations are all suitable for extrusion-based 3D bioprinting. Self-supporting scaffolds with a designed macroporosity and micropores in the bioprinted struts are successfully obtained by combining extrusion-based bioprinting and freeze-drying. The presence of gelatin in these formulations allows for the modulation of porosity, of water uptake and of scaffold stiffness in respect to pure pectin scaffolds. Results demonstrate that these new biomaterial formulations, processed with this specific approach, are promising candidates for the fabrication of tissue-like scaffolds for tissue regeneration.
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Bioimpresión , Materiales Biocompatibles/química , Gelatina/química , Hidrogeles/química , Pectinas , Impresión Tridimensional , Ingeniería de Tejidos/métodos , Andamios del Tejido/químicaRESUMEN
Gelatin is a natural biopolymer extensively used for tissue engineering applications due to its similarities to the native extracellular matrix. However, the rheological properties of gelatin formulations are not ideal for extrusion-based bioprinting. In this work, we present an approach to improve gelatin bioprinting performances by using pectin as a rheology modifier of gelatin and (3-glycidyloxypropyl)trimethoxysilane (GPTMS) as a gelatin-pectin crosslinking agent. The preparation of gelatin-pectin formulations is initially optimized to obtain homogenous gelatin-pectin gels. Since the use of GPTMS requires a drying step to induce the completion of the crosslinking reaction, microporous gelatin-pectin-GPTMS sponges are produced through freeze-drying, and the intrinsic properties of gelatin-pectin-GPTMS networks (e.g., porosity, pore size, degree of swelling, compressive modulus, and cell adhesion) are investigated. Subsequently, rheological investigations together with bioprinting assessments demonstrate the key role of pectin in increasing the viscosity and the yield stress of low viscous gelatin solutions. Water stable, three-dimensional, and self-supporting gelatin-pectin-GPTMS scaffolds with interconnected micro- and macroporosity are successfully obtained by combining extrusion-based bioprinting and freeze-drying. The proposed biofabrication approach does not require any additional temperature controller to further modulate the rheological properties of gelatin solutions and it could furthermore be extended to improve the bioprintability of other biopolymers.
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The punctual analysis of bone Extracellular Matrix (ECM) proteins represents a pivotal point for medical research in bone diseases like osteoporosis. Studies in this field, historically done to appreciate bone biology, were mainly conducted on animal samples and, up to today, only a few studies on protein detection in human bone are present. The challenges in bone ECM protein extraction and quantitation protocols are related to both the separation of proteins from the mineral content (i.e. hydroxyapatite) and the difficulty of avoiding protein denaturation during the extraction processes. The aim of the present work was to define appropriate protocol(s) for bone ECM protein extraction that could be applied to investigate both normal and pathological conditions. We compared and optimised some of the most used protocols present in the literature, modifying the protein precipitation method, the buffer used for resuspension and/or the volume of reagent used. Bradford and BCA assays and Western Blotting were used to evaluate the variations in the total protein recovery and the amount of selected proteins (Type I Collagen, TGF-ß, IGF-1, Decorin, Osteopontin, Bone Sialoprotein-2 and Osteocalcin). Collectively, we were capable to draw-up two single-extract protocols with optimal recovery and ideal protein content, that can be used for a detailed analysis of ECM proteins in pathological bone samples. Time-consuming multi-extract procedures, optimised in their precipitation methods, are however crucial for a precise detection of specific proteins, like osteocalcin. As the matter of fact, also the demineralization processes, commonly suggested and performed in several protocols, could hinder an accurate protein detection, thus inherently affecting the study of a pathological bone ECM. This study represents a starting point for the definition of appropriate strategies in the study of bone extracellular matrix proteins involved in the onset and maintenance of bone diseases, as well as a tool for the development of customized scaffolds capable to modulate a proper feedback loop in bone remodelling, altered in case of diseases like osteoporosis.
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Proteínas de la Matriz Extracelular , Osteoporosis , Animales , Huesos/metabolismo , Matriz Extracelular/metabolismo , Proteínas de la Matriz Extracelular/metabolismo , Humanos , OsteocalcinaRESUMEN
Bone is an extremely dynamic tissue, undergoing continuous remodeling for its whole lifetime, but its regeneration or augmentation due to bone loss or defects are not always easy to obtain. Bone tissue engineering (BTE) is a promising approach, and its success often relies on a "smart" scaffold, as a support to host and guide bone formation through bone cell precursors. Bone homeostasis is maintained by osteoblasts (OBs) and osteoclasts (OCs) within the basic multicellular unit, in a consecutive cycle of resorption and formation. Therefore, a functional scaffold should allow the best possible OB/OC cooperation for bone remodeling, as happens within the bone extracellular matrix in the body. In the present work OB/OC co-culture models, with and without scaffolds, are reviewed. These experimental systems are intended for different targets, including bone remodeling simulation, drug testing and the assessment of biomaterials and 3D scaffolds for BTE. As a consequence, several parameters, such as cell type, cell ratio, culture medium and inducers, culture times and setpoints, assay methods, etc. vary greatly. This review identifies and systematically reports the in vitro methods explored up to now, which, as they allow cellular communication, more closely resemble bone remodeling and/or the regeneration process in the framework of BTE. STATEMENT OF SIGNIFICANCE: Bone is a dynamic tissue under continuous remodeling, but spontaneous healing may fail in the case of excessive bone loss which often requires valid alternatives to conventional treatments to restore bone integrity, like bone tissue engineering (BTE). Pre-clinical evaluation of scaffolds for BTE requires in vitro testing where co-cultures combining innovative materials with osteoblasts (OBs) and osteoclasts (OCs) closely mimic the in vivo repair process. This review considers the direct and indirect OB/OC co-cultures relevant to BTE, from the early mouse-cell models to the recent bone regenerative systems. The co-culture modeling of bone microenvironment provides reliable information on bone cell cross-talk. Starting from improved knowledge on bone remodeling, bone disease mechanisms may be understood and new BTE solutions are designed.
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Osteoblastos , Osteoclastos , Animales , Remodelación Ósea , Huesos , Diferenciación Celular , Técnicas de Cocultivo , RatonesRESUMEN
A high incidence of heterotopic ossification (HO) has been reported in patients with diffuse idiopathic skeletal hyperostosis (DISH), a metabolic disease characterized by calcifications of entheses at spine and peripheral sites. We performed histological and immunohistochemical analyses in five different HO sites in a patient with DISH to study a possible mutual interaction of bone morphogenetic protein 2 (BMP-2), transforming growth factor beta (TGF-ß), and decorin, crucial for bone mass increasing, matrix calcification, and endochondral bone formation. We speculated that the surgical trauma triggered HO, inducing TGF-ß release at the lesion site. TGF-ß recruits osteoblast precursor cells and determines the overexpression of BMP-2 in the surrounding skeletal muscle, inducing a further osteogenic differentiation, contributing to HO onset.