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Strontium (Sr) is known to stimulate osteogenesis, while inhibiting osteoclastogenesis, thus encouraging research on its application as a therapeutic agent for bone repair/regeneration. It has been suggested that it may possess immunomodulatory properties, which might act synergistically in bone repair/regeneration processes. To further explore this hypothesis we have designed a Sr-hybrid system composed of an in situ forming Sr-crosslinked RGD-alginate hydrogel reinforced with Sr-doped hydroxyapatite (HAp) microspheres and studied its in vitro osteoinductive behaviour and in vivo inflammatory response. The Sr-hybrid scaffold acts as a dual Sr 2+ delivery system, showing a cumulative Sr 2+ release of ca. 0.3 mM after 15 days. In vitro studies using Sr 2+ concentrations within this range (0 to 3 mM Sr 2+ ) confirmed its ability to induce osteogenic differentiation of mesenchymal stem/stromal cells (MSC), as well as to reduce osteoclastogenesis and osteoclasts (OC) functionality. In comparison with a similar Sr-free system, the Sr-hybrid system stimulated osteogenic differentiation of MSC, while inhibiting the formation of OC. Implantation in an in vivo model of inflammation, revealed an increase in F4/80 + /CD206 + cells, highlighting its ability to modulate the inflammatory response as a pro-resolution mediator, through M2 macrophage polarization. Therefore, the Sr-hybrid system is potentially an appealing biomaterial for future clinical applications., (Copyright © 2019. Published by Elsevier B.V.)

Strontium (Sr) has been described as having beneficial influence in bone strength and architecture. However, negative systemic effects have been reported on oral administration of Sr ranelate, leading to strict restrictions in clinical application. We hypothesized that local delivery of Sr improves osteogenesis without eliciting detrimental side effects. Therefore, the in vivo response to an injectable Sr-hybrid system composed of RGD-alginate hydrogel cross-linked in situ with Sr and reinforced with Sr-doped hydroxyapatite microspheres, was investigated. The system was injected in a critical-sized bone defect model and compared to a similar Sr-free material. Micro-CT results show a trend towards higher new bone formed in Sr-hybrid group and major histological differences were observed between groups. Higher cell invasion was detected at the center of the defect of Sr-hybrid group after 15 days with earlier bone formation. Higher material degradation with increase of collagen fibers and bone formation in the center of the defect after 60 days was observed as opposed to bone formation restricted to the periphery of the defect in the control. These histological findings support the evidence of an improved response with the Sr enriched material. Importantly, no alterations were observed in the Sr levels in systemic organs or serum.

The hypothesis behind this work is that fibrinogen (Fg), classically considered a pro-inflammatory protein, can promote bone repair/regeneration. Injury and biomaterial implantation naturally lead to an inflammatory response, which should be under control, but not necessarily minimized. Herein, porous scaffolds entirely constituted of Fg (Fg-3D) were implanted in a femoral rat bone defect and investigated at two important time points, addressing the bone regenerative process and the local and systemic immune responses, both crucial to elucidate the mechanisms of tissue remodelling. Fg-3D led to early infiltration of granulation tissue (6 days post-implantation), followed by bone defect closure, including periosteum repair (8 weeks post-injury). In the acute inflammatory phase (6 days) local gene expression analysis revealed significant increases of pro-inflammatory cytokines IL-6 and IL-8, when compared with non-operated animals. This correlated with modified proportions of systemic immune cell populations, namely increased T cells and decreased B, NK and NKT lymphocytes and myeloid cell, including the Mac-1+ (CD18+/CD11b+) subpopulation. At 8 weeks, Fg-3D led to decreased plasma levels of IL-1β and increased TGF-β1. Thus, our data supports the hypothesis, establishing a link between bone repair induced by Fg-3D and the immune response. In this sense, Fg-3D scaffolds may be considered immunomodulatory biomaterials., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Current challenges in the development of scaffolds for bone regeneration include the engineering of materials that can withstand normal dynamic physiological mechanical stresses exerted on the bone and provide a matrix capable of supporting cell migration and tissue ingrowth. The objective of the present work was to develop and characterize a hybrid polymer–ceramic injectable system that consists of an alginate matrix crosslinked in situ in the presence of strontium(Sr), incorporating a ceramic reinforcement in the form of Sr-rich microspheres. The incorporation of Sr in the microspheres and in the vehicle relies on the growing evidence that Sr has beneficial effects in bone remodeling and in the treatment of osteopenic disorders and osteoporosis. Sr-rich porous hydroxyapatite microspheres with a uniform size and a mean diameter of 555 μm were prepared, and their compression strength and friability tested. A 3.5% (w/v) ultrapure sodium alginate solution was used as the vehicle and its in situ gelation was promoted by the addition of calcium (Ca) or Sr carbonate and Glucone-δ-lactone. Gelation times varied with temperature and crosslinking agent, being slower for Sr than for Ca, but adequate for injection in both cases. Injectability was evaluated using a device employed in vertebroplasty surgical procedures, coupled to a texture analyzer in compression mode. Compositions with 35%w of microspheres presented the best compromise between injectability and compression strength of the system, the force required to extrude it being lower than 100 N.Micro CT analysis revealed a homogeneous distribution of the microspheres inside the vehicle, and a mean inter-microspheres space of 220 μm. DMA results showed that elastic behavior of the hybrid is over the viscous one and that the higher storage modulus was obtained for the 3.5%Alg–35%Sr-HAp-Sr formulation.

Bone regeneration seems to be dependant on cell communication between osteogenic and endothelial cells arising from surrounding blood vessels. This study aims to determine whether endothelial cells can regulate the osteogenic potential of osteoprogenitor cells in vitro and in vivo, in a long bone defect, when co-immobilized in alginate microspheres. Alginate is a natural polymer widely used as a biomaterial for cell encapsulation. Human osteoprogenitors (HOP) from bone marrow mesenchymal stem cells were immobilized alone or together with human umbilical vein endothelial cells (HUVEC) inside irradiated, oxidized and RGD-grafted alginate microspheres. Immobilized cells were cultured in dynamic conditions and cell metabolic activity increased during three weeks. The gene expression of alkaline phosphatase and osteocalcin, both specific markers of the osteoblastic phenotype, and mineralization deposits were upregulated in co-immobilized HOPs and HUVECs, comparing to the immobilization of monocultures. VEGF secretion was also increased when HOPs were co-immobilized with HUVECs. Microspheres containing co-cultures were further implanted in a bone defect and bone formation was analysed by muCT and histology at 3 and 6 weeks post-implantation. Mineralization was observed inside and around the implanted microspheres containing the immobilized cells. However, when HOPs were co-immobilized with HUVECs, mineralization significantly increased. These findings demonstrate that co-immobilization of osteogenic and endothelial cells within RGD-grafted alginate microspheres provides a promising strategy for bone tissue engineering.

Background: The vast and promising class of long non-coding RNAs (lncRNAs) has been under investigation for distinct therapeutic applications. Nevertheless, their role as molecular drivers of bone regeneration remains poorly studied. The lncRNA H19 mediates osteogenic differentiation of Mesenchymal Stem/Stromal Cells (MSCs) through the control of intracellular pathways. However, the effect of H19 on the extracellular matrix (ECM) components is still largely unknown. This research study was designed to decode the H19-mediated ECM regulatory network, and to reveal how the decellularized siH19-engineered matrices influence MSC proliferation and fate. This is particularly relevant for diseases in which the ECM regulation and remodeling processes are disrupted, such as osteoporosis. Methods: Mass spectrometry-based quantitative proteomics analysis was used to identify ECM components, after oligonucleotides delivery to osteoporosis-derived hMSCs. Moreover, qRT-PCR, immunofluorescence and proliferation, differentiation and apoptosis assays were performed. Engineered matrices were decellularized, characterized by atomic force microscopy and repopulated with hMSC and pre-adipocytes. Clinical bone samples were characterized by histomorphometry analysis. Results: Our study provides an in-depth proteome-wide and matrisome-specific analysis of the ECM proteins controlled by the lncRNA H19. Using bone marrow-isolated MSC from patients with osteoporosis, we identified fibrillin-1 (FBN1), vitronectin (VTN) and collagen triple helix repeat containing 1 (CTHRC1), among others, as having different pattern levels following H19 silencing. Decellularized siH19-engineered matrices are less dense and have a decreased collagen content compared with control matrices. Repopulation with naïve MSCs promotes a shift towards the adipogenic lineage in detriment of the osteogenic lineage and inhibits proliferation. In pre-adipocytes, these siH19-matrices enhance lipid droplets formation. Mechanistically, H19 is targeted by miR-29c, whose expression is decreased in osteoporotic bone clinical samples. Accordingly, miR-29c impacts MSC proliferation and collagen production, but does not influence ALP staining or mineralization, revealing that H19 silencing and miR-29c mimics have complementary but not overlapping functions. Conclusion: Our data suggest H19 as a therapeutic target to engineer the bone ECM and to control cell behavior. [ABSTRACT FROM AUTHOR]

Macrophage behavior upon biomaterial implantation conditions the inflammatory response and subsequent tissue repair. The hypothesis behind this work was that fibrinogen (Fg) and magnesium (Mg) biomaterials, used in combination (FgMg) could act synergistically to modulate macrophage activation, promoting a pro-regenerative phenotype. Materials were characterized by scanning electron microscopy, Fg and Mg degradation products were quantified by atomic absorption spectroscopy and ELISA. Whole blood immune cells and primary human monocyte-derived macrophages were exposed to the biomaterials extracts in unstimulated (M0) or pro-inflammatory LPS or LPS-IFNγ (M1) conditions. Macrophage phenotype was evaluated by flow cytometry, cytokines secreted by whole blood cells and macrophages were measured by ELISA, and signaling pathways were probed by Western blotting. The secretomes of macrophages preconditioned with biomaterials extracts were incubated with human mesenchymal stem/stromal cells (MSC) and their effect on osteogenic differentiation was evaluated via Alkaline Phosphatase (ALP) activity and alizarin red staining. Scaffolds of Fg, alone or in the FgMg combination, presented similar 3D porous architectures. Extracts from FgMg materials reduced LPS-induced TNF-α secretion by innate immune cells, and macrophage M1 polarization upon LPS-IFNγ stimulation, resulting in lower cell surface CD86 expression, lower NFκB p65 phosphorylation and reduced TNF-α secretion. Moreover, while biomaterial extracts per se did not enhance MSC osteogenic differentiation, macrophage secretome, particularly from cells exposed to FgMg extracts, increased MSC ALP activity and alizarin red staining, compared with extracts alone. These findings suggest that the combination of Fg and Mg synergistically influences macrophage pro-inflammatory activation and crosstalk with MSC. Modulating macrophage phenotype by degradable and bioactive biomaterials is an increasingly explored strategy to promote tissue repair/regeneration. Fibrinogen (Fg) and magnesium (Mg)-based materials have been explored in this context. Previous work from our group showed that monocytes interact with fibrinogen adsorbed onto chitosan surfaces through TLR4 and that fibrinogen scaffolds promote in vivo bone regeneration. Also, magnesium ions have been reported to modulate macrophage pro-inflammatory M1 stimulation and to promote bone repair. Here we report, for the first time, the combination of Fg and Mg materials, hypothesizing that it could act synergistically on macrophages, directing them towards a pro-regenerative phenotype. As a first step towards proving/disproving our hypothesis we used extracts obtained from Fg, Mg and FgMg multilayer constructs. We observed that FgMg extracts led to a reduction in the polarization of macrophages towards a pro-inflammatory phenotype. Also, the secretome of macrophages exposed to extracts of the combination material promoted the expression of osteogenic markers by MSCs. Image, graphical abstract [ABSTRACT FROM AUTHOR]

Fibrinogen (Fg) is a pro‐inflammatory protein with pro‐healing properties. Previous work showed that fibrinogen 3D scaffolds (Fg‐3D) promote bone regeneration, but the cellular players were not identified. Osteoclasts are bone resorbing cells that promote bone remodeling in close crosstalk with osteoblasts. Herein, the capacity of osteoclasts differentiated on Fg‐3D to degrade the scaffolds and promote osteoblast differentiation was evaluated in vitro. Fg‐3D scaffolds were prepared by freeze‐drying and osteoclasts were differentiated from primary human peripheral blood monocytes. Results obtained showed osteoclasts expressing the enzymes cathepsin K and tartrate resistant acid phosphatase colonizing Fg‐3D scaffolds. Osteoclasts were able to significantly degrade Fg‐3D, reducing the scaffold's area, and increasing D‐dimer concentration, a Fg degradation product, in their culture media. Osteoclast conditioned media from the first week of differentiation promoted significantly stronger human primary mesenchymal stem/stromal cell (MSC) osteogenic differentiation, evaluated by alkaline phosphatase activity. Moreover, week 1 osteoclast conditioned media promoted earlier MSC osteogenic differentiation, than chemical osteogenesis inductors. TGF‐β1 was found increased in osteoclast conditioned media from week 1, when compared to week 3 of differentiation. Taken together, our results suggest that osteoclasts are able to differentiate and degrade Fg‐3D, producing factors like TGF‐β1 that promote MSC osteogenic differentiation. [ABSTRACT FROM AUTHOR]

Modulation of inflammatory responses to implanted biomaterials towards tissue regeneration has gained prominence as an innovative tissue engineering strategy. Recent in vitro and in vivo studies showed that Fibrinogen (Fg) adsorbed to Chitosan (Ch) substrates modulates immune cell responses, enhances the production of osteogenic factors by monocytes/macrophages and promotes bone regeneration, but the mechanisms involved remain poorly understood. Thus, the present work was conducted to clarify the molecular mechanisms of interaction between primary human monocytes and the above substrates. Cell surface expression of TLR-4 was significantly downregulated in the presence of pre-adsorbed Fg, when compared to Ch control, indicating an interaction via this receptor. The same substrate triggered MAPK activation, specifically the ERK 1/2 and JNK pathways. Importantly, both ERK 1/2 and JNK phosphorylation were reduced when TLR-4 signalling was blocked using a specific pharmacological inhibitor. Functionally, adsorbed Fg induced production of the potent osteogenic mediator BMP-2 by monocytes, while TLR-4 inhibition resulted in a significant decrease of BMP-2 mRNA and protein levels, in response to Fg stimulation. Overall, our data reveals that adsorbed Fg exerts a pro-osteogenic effect on human monocytes through its interaction with TLR-4 and subsequent production of BMP-2, elucidating two key aspects of the immunomodulatory action of adsorbed Fg in bone regeneration. Statement of Significance Recent studies showed that when Fibrinogen (Fg) is used to modify Chitosan (Ch) substrates, it modulates the immune response, enhances production of osteogenic factors by monocytes/macrophages, and promotes bone regeneration. However, the mechanisms involved in monocyte-Fg interaction, were only partially known. Current work addresses the interaction between primary human monocytes and Ch surfaces modified by Fg adsorption (Ch-Fg) at the molecular level. Results show that monocytes interact specifically with Ch-Fg via TLR-4, triggering particular intracellular signalling pathways (ERK and JNK, but not p38), downstream of TLR-4. Functionally, Ch-Fg induced monocytes to produce the osteogenic mediator BMP-2. Thus, we clarify herein two essential aspects of the interaction between adsorbed Fg and monocytes, with impact on immunomodulation and regeneration, upon biomaterial implantation. [ABSTRACT FROM AUTHOR]

The normal bone regeneration process is a complex and coordinated series of events involving different cell types and molecules. However, this process is impaired in critical-size/large bone defects, with non-unions or delayed unions remaining a major clinical problem. Novel strategies are needed to aid the current therapeutic approaches. Mesenchymal stem/stromal cells (MSCs) are able to promote bone regeneration. Their beneficial effects can be improved by modulating the expression levels of specific genes with the purpose of stimulating MSC proliferation, osteogenic differentiation or their immunomodulatory capacity. In this context, the genetic engineering of MSCs is expected to further enhance their pro-regenerative properties and accelerate bone healing. Herein, we review the most promising molecular candidates (protein-coding and non-coding transcripts) and discuss the different methodologies to engineer and deliver MSCs, mainly focusing on in vivo animal studies. Considering the potential of the MSC secretome for bone repair, this topic has also been addressed. Furthermore, the promising results of clinical studies using MSC for bone regeneration are discussed. Finally, we debate the advantages and limitations of using MSCs, or genetically-engineered MSCs, and their potential as promoters of bone fracture regeneration/repair. [ABSTRACT FROM AUTHOR]

Abstract: There is a need for new therapeutic strategies to treat bone defects caused by trauma, disease or tissue loss. Injectable systems for cell transplantation have the advantage of allowing the use of minimally invasive surgical procedures, and thus for less discomfort to patients. In the present study, it is hypothesized that Arg–Gly–Asp (RGD)-coupled in a binary (low and high molecular weight) injectable alginate composition is able to influence bone cell differentiation in a three-dimensional (3D) structure. Viability, metabolic activity, cytoskeleton organization, ultrastructure and differentiation (alkaline phosphatase (ALP), von Kossa, alizarin red stainings and osteocalcin quantification) of immobilized cells were assessed. Cells within RGD-modified alginate microspheres were able to establish more interactions with the synthetic extracellular matrix as visualized by confocal laser scanning microscope and transmission electron microscopy imaging, and presented a much higher level of differentiation (more intense ALP and mineralization stainings and higher levels of osteocalcin secretion) when compared to cells immobilized within unmodified alginate microspheres. These findings demonstrate that peptides covalently coupled to alginate were efficient in influencing cell behavior within this 3D system, and may provide adequate preparation of osteoblasts for cell transplantation. [Copyright &y& Elsevier]