Stimuli-Responsive Codelivery System-Embedded Polymeric Nanofibers with Synergistic Effects of Growth Factors and Low-Intensity Pulsed Ultrasound to Enhance Osteogenesis Properties.

The present work aims to develop optimized scaffolds for bone repair by incorporating mesoporous nanoparticles into them, thereby combining bioactive factors for cell growth and preventing rapid release or loss of effectiveness. We synthesized biocompatible and biodegradable scaffolds designed for the controlled codelivery of curcumin (CUR) and recombinant human bone morphogenic protein-2 (rhBMP-2). Active agents in dendritic silica/titania mesoporous nanoparticles (DSTNs) were incorporated at different weight percentages (0, 2, 5, 7, 9, and 10 wt %) into a matrix of polycaprolactone (PCL) and polyethylene glycol (PEG) nanofibers, forming the CUR-BMP-2@DSTNs/PCL-PEG delivery system (S0, S2, S5, S7, S9, and S10, respectively, with the number showing the weight percentage). To enhance the formation process, the system was treated using low-intensity pulsed ultrasound (LIPUS). Different advanced methods were employed to assess the physical, chemical, and mechanical characteristics of the fabricated scaffolds, all confirming that incorporating the nanoparticles improves their mechanical and structural properties. Their hydrophilicity increased by approximately 25%, leading to ca. 53% enhancement in their water absorption capacity. Furthermore, we observed a sustained release of approximately 97% for CUR and 70% for BMP-2 for the S7 (scaffold with 7 wt % DSTNs) over 28 days, which was further enhanced using ultrasound. In vitro studies demonstrated accelerated scaffold biodegradation, with the highest level observed in S7 scaffolds, approximately three times higher than the control group. Moreover, the cell viability and proliferation on DSTNs-containing scaffolds increased when compared to the control group. Overall, our study presents a promising nanocomposite scaffold design with notable improvements in structural, mechanical, and biological properties compared to the control group, along with controlled and sustained drug release capabilities. This makes the scaffold a compelling candidate for advanced bone tissue engineering and regenerative therapies.

A Multifunctional Scaffold for Bone Infection Treatment by Delivery of microRNA Therapeutics Combined With Antimicrobial Nanoparticles.

Treating bone infections and ensuring bone repair is one of the greatest global challenges of modern orthopedics, made complex by antimicrobial resistance (AMR) risks due to long-term antibiotic treatment and debilitating large bone defects following infected tissue removal. An ideal multi-faceted solution would will eradicate bacterial infection without long-term antibiotic use, simultaneously stimulating osteogenesis and angiogenesis. Here, a multifunctional collagen-based scaffold that addresses these needs by leveraging the potential of antibiotic-free antimicrobial nanoparticles (copper-doped bioactive glass, CuBG) to combat infection without contributing to AMR in conjunction with microRNA-based gene therapy (utilizing an inhibitor of microRNA-138) to stimulate both osteogenesis and angiogenesis, is developed. CuBG scaffolds reduce the attachment of gram-positive bacteria by over 80%, showcasing antimicrobial functionality. The antagomiR-138 nanoparticles induce osteogenesis of human mesenchymal stem cells in vitro and heal a large load-bearing defect in a rat femur when delivered on the scaffold. Combining both promising technologies results in a multifunctional antagomiR-138-activated CuBG scaffold inducing hMSC-mediated osteogenesis and stimulating vasculogenesis in an in vivo chick chorioallantoic membrane model. Overall, this multifunctional scaffold catalyzes killing mechanisms in bacteria while inducing bone repair through osteogenic and angiogenic coupling, making this platform a promising multi-functional strategy for treating and repairing complex bone infections.

Development of miR-26a-activated scaffold to promote healing of critical-sized bone defects through angiogenic and osteogenic mechanisms.

Very large bone defects significantly diminish the vascular, blood, and nutrient supply to the injured site, reducing the bone's ability to self-regenerate and complicating treatment. Delivering nanomedicines from biomaterial scaffolds that induce host cells to produce bone-healing proteins is emerging as an appealing solution for treating these challenging defects. In this context, microRNA-26a mimics (miR-26a) are particularly interesting as they target the two most relevant processes in bone regeneration-angiogenesis and osteogenesis. However, the main limitation of microRNAs is their poor stability and issues with cytosolic delivery. Thus, utilising a collagen-nanohydroxyapatite (coll-nHA) scaffold in combination with cell-penetrating peptide (RALA) nanoparticles, we aimed to develop an effective system to deliver miR-26a nanoparticles to regenerate bone defects in vivo. The microRNA-26a complexed RALA nanoparticles, which showed the highest transfection efficiency, were incorporated into collagen-nanohydroxyapatite scaffolds and in vitro assessment demonstrated the miR-26a-activated scaffolds effectively transfected human mesenchymal stem cells (hMSCs) resulting in enhanced production of vascular endothelial growth factor, increased alkaline phosphatase activity, and greater mineralisation. After implantation in critical-sized rat calvarial defects, micro CT and histomorphological analysis revealed that the miR-26a-activated scaffolds improved bone repair in vivo, producing new bone of superior quality, which was highly mineralised and vascularised compared to a miR-free scaffold. This innovative combination of osteogenic collagen-nanohydroxyapatite scaffolds with multifunctional microRNA-26a complexed nanoparticles provides an effective carrier delivering nanoparticles locally with high efficacy and minimal off-target effects and demonstrates the potential of targeting osteogenic-angiogenic coupling using scaffold-based nanomedicine delivery as a new "off-the-shelf" product capable of healing complex bone injuries.

3D bioprinting of cartilaginous templates for large bone defect healing.

Damaged or diseased bone can be treated using autografts or a range of different bone grafting biomaterials, however limitations with such approaches has motivated increased interest in developmentally inspired bone tissue engineering (BTE) strategies that seek to recapitulate the process of endochondral ossification (EO) as a means of regenerating critically sized defects. The clinical translation of such strategies will require the engineering of scaled-up, geometrically defined hypertrophic cartilage grafts that can be rapidly vascularised and remodelled into bone in mechanically challenging defect environments. The goal of this study was to 3D bioprint mechanically reinforced cartilaginous templates and to assess their capacity to regenerate critically sized femoral bone defects. Human mesenchymal stem/stromal cells (hMSCs) were incorporated into fibrin based bioinks and bioprinted into polycaprolactone (PCL) frameworks to produce mechanically reinforced constructs. Chondrogenic priming of such hMSC laden constructs was required to support robust vascularisation and graft mineralisation in vivo following their subcutaneous implantation into nude mice. With a view towards maximising their potential to support endochondral bone regeneration, we next explored different in vitro culture regimes to produce chondrogenic and early hypertrophic engineered grafts. Following their implantation into femoral bone defects within transiently immunosuppressed rats, such bioprinted constructs were rapidly remodelled into bone in vivo, with early hypertrophic constructs supporting higher levels of vascularisation and bone formation compared to the chondrogenic constructs. Such early hypertrophic bioprinted constructs also supported higher levels of vascularisation and spatially distinct patterns of new formation compared to BMP-2 loaded collagen scaffolds (here used as a positive control). In conclusion, this study demonstrates that fibrin based bioinks support chondrogenesis of hMSCs in vitro, which enables the bioprinting of mechanically reinforced hypertrophic cartilaginous templates capable of supporting large bone defect regeneration. These results support the use of 3D bioprinting as a strategy to scale-up the engineering of developmentally inspired templates for BTE. STATEMENT OF SIGNIFICANCE: Despite the promise of developmentally inspired tissue engineering strategies for bone regeneration, there are still challenges that need to be addressed to enable clinical translation. This work reports the development and assessment (in vitro and in vivo) of a 3D bioprinting strategy to engineer mechanically-reinforced cartilaginous templates for large bone defect regeneration using human MSCs. Using distinct in vitro priming protocols, it was possible to generate cartilage grafts with altered phenotypes. More hypertrophic grafts, engineered in vitro using TGF-ß3 and BMP-2, supported higher levels of blood vessel infiltration and accelerated bone regeneration in vivo. This study also identifies some of the advantages and disadvantages of such endochondral bone TE strategies over the direct delivery of BMP-2 from collagen-based scaffolds.

Development of collagen-poly(caprolactone)-based core-shell scaffolds supplemented with proteoglycans and glycosaminoglycans for ligament repair.

Core-shell scaffolds offer a promising regenerative solution to debilitating injuries to anterior cruciate ligament (ACL) thanks to a unique biphasic structure. Nevertheless, current core-shell designs are impaired by an imbalance between permeability, biochemical and mechanical cues. This study aimed to address this issue by creating a porous core-shell construct which favors cell infiltration and matrix production, while providing mechanical stability at the site of injury. The developed core-shell scaffold combines an outer shell of electrospun poly(caprolactone) fibers with a freeze-dried core of type I collagen doped with proteoglycans (biglycan, decorin) or glycosaminoglycans (chondroitin sulphate, dermatan sulphate). The aligned fibrous shell achieved an elastic modulus akin of the human ACL, while the porous collagen core is permeable to human mesenchymal stem cell (hMSC). Doping of the core with the aforementioned biomolecules led to structural and mechanical changes in the pore network. Assessment of cellular metabolic activity and scaffold contraction shows that hMSCs actively remodel the matrix at different degrees, depending on the core's doping formulation. Additionally, immunohistochemical staining and mRNA transcript levels show that the collagen-chondroitin sulphate formulation has the highest matrix production activity, while the collagen-decorin formulation featured a matrix production profile more characteristic of the undamaged tissue. Together, this demonstrates that scaffold doping with target biomolecules leads to distinct levels of cell-mediated matrix remodeling. Overall, this work resulted in the development of a versatile and robust platform with a combination of mechanical and biochemical features that have a significant potential in promoting the repair process of ACL tissue.

Inflammation and biomaterials: role of the immune response in bone regeneration by inorganic scaffolds.

The regulatory role of the immune system in maintaining bone homeostasis and restoring its functionality, when disturbed due to trauma or injury, has become evident in recent years. The polarization of macrophages, one of the main constituents of the immune system, into the pro-inflammatory or anti-inflammatory phenotype has great repercussions for cellular crosstalk and the subsequent processes needed for proper bone regeneration such as angiogenesis and osteogenesis. In certain scenarios, the damaged osseous tissue requires the placement of synthetic bone grafts to facilitate the healing process. Inorganic biomaterials such as bioceramics or bioactive glasses are the most widely used due to their resemblance to the mineral phase of bone and superior osteogenic properties. The immune response of the host to the inorganic biomaterial, which is of an exogenous nature, might determine its fate, leading either to active bone regeneration or its failure. Therefore, various strategies have been employed, like the modification of structural/chemical features or the incorporation of bioactive molecules, to tune the interplay with the immune cells. Understanding how these particular modifications impact the polarization of macrophages and further osteogenic and osteoclastogenic events is of great interest in view of designing a new generation of osteoimmunomodulatory materials that support the regeneration of osseous tissue during all stages of bone healing.

The effect of biomimetic calcium deficient hydroxyapatite and sintered ß-tricalcium phosphate on osteoimmune reaction and osteogenesis.

Biomaterial implantation triggers inflammatory reactions. Understanding the effect of physicochemical features of biomaterials on the release of inflammatory cytokines from immune cells would be of great interest in view of designing bone graft materials to enhance the healing of bone defects. The present work investigated the interactions of two chemically and texturally different calcium phosphate (CaPs) substrates with macrophages, one of the main innate immune cells, and its further impact on osteogenic differentiation of bone forming cells. The behaviour of macrophages seeded on biomimetic calcium deficient hydroxyapatite (CDHA) and sintered ß-tricalcium phosphate (ß-TCP) was assessed in terms of the release of inflammatory cytokines and osteoclastogenic factors. The osteogenic differentiation of bone progenitor cells (bone marrow stromal cells (BMSCs) and osteoblastic cell line (SaOS-2)) were subsequently studied by incubating with the conditioned medium induced by macrophage-CaPs interaction in order to reveal the effect of immune cell reaction to CaPs on osteogenic differentiation. It was found that the incubation of macrophages with CaPs substrates caused a decrease of pro-inflammatory cytokines, more pronounced for ß-TCP compared with CDHA showing significantly decreased IL-6, TNF-a, and iNOS. However, the macrophage-CDHA interaction resulted in a more favourable environment for osteogenic differentiation of osteoblasts with more collagen type I production and osteogenic genes (Runx2, BSP) expression, suggesting that osteogenic differentiation of bone cells is not only determined by the nature of biomaterials, but also significantly influenced by the inflammatory environment generated by the interaction of immune cells and biomaterials. STATEMENT OF SIGNIFICANCE: The field of osteoimmunology highlights the importance of the cross-talk between immune and bone cells for effective bone regeneration. This tight interaction opens the door to new strategies that encompass the development of smart cell-instructive biomaterials which performance covers the events from early inflammation to osteogenesis. The present work links the anti-inflammatory and osteoimmunomodulatory features of synthetic bone grafts to their chemistry and texture, focussing on the cross-talk between macrophages and two major orchestrators of bone healing, namely primary mesenchymal stem cells and osteoblasts. The results emphasize the importance of the microenvironment created through the interaction between the substrate and the immune cells as it can stimulate osteogenic events and subsequently foster bone healing.

Effect of nano-structural properties of biomimetic hydroxyapatite on osteoimmunomodulation.

Immune cells are sensitive to the microstructural and textural properties of materials. Tuning the structural features of synthetic bone grafts could be a valuable strategy to regulate the specific response of the immune system, which in turn modulates the activity of bone cells. The aim of this study was to analyse the effect of the structural characteristics of biomimetic calcium deficient hydroxyapatite (CDHA) on the innate immune response of macrophages and the subsequent impact on osteogenesis and osteoclastogenesis. Murine RAW 264.7 cells were cultured, under standard and inflammatory conditions, on chemically identical CDHA substrates that varied in microstructure and porosity. The impact on osteogenesis was evaluated by incubating osteoblastic cells (SaOS-2) with RAW-CDHA conditioned extracts. The results showed that macrophages were sensitive to different textural and structural properties of CDHA. Under standard conditions, the impact of inflammatory cytokine production by RAW cells cultured on CDHA played a significant role in the degradation of substrates, suggesting the impact of resorptive behaviour of RAW cells on biomimetic surfaces. Osteoblast differentiation was stimulated by the conditioned media collected from RAW cells cultured on needle-like nanostructured CDHA. The results demonstrated that needle-like nanostructured CDHA was able to generate a favourable osteoimmune environment to regulate osteoblast differentiation and osteogenesis. Under inflammatory conditions, the incubation of RAW cells with less porous CDHA resulted in a decreased gene expression and release of pro-inflammatory cytokines.

Osteoinduction by Foamed and 3D-Printed Calcium Phosphate Scaffolds: Effect of Nanostructure and Pore Architecture.

Some biomaterials are osteoinductive, that is, they are able to trigger the osteogenic process by inducing the differentiation of mesenchymal stem cells to the osteogenic lineage. Although the underlying mechanism is still unclear, microporosity and specific surface area (SSA) have been identified as critical factors in material-associated osteoinduction. However, only sintered ceramics, which have a limited range of porosities and SSA, have been analyzed so far. In this work, we were able to extend these ranges to the nanoscale, through the foaming and 3D-printing of biomimetic calcium phosphates, thereby obtaining scaffolds with controlled micro- and nanoporosity and with tailored macropore architectures. Calcium-deficient hydroxyapatite (CDHA) scaffolds were evaluated after 6 and 12 weeks in an ectopic-implantation canine model and compared with two sintered ceramics, biphasic calcium phosphate and ß-tricalcium phosphate. Only foams with spherical, concave macropores and not 3D-printed scaffolds with convex, prismatic macropores induced significant ectopic bone formation. Among them, biomimetic nanostructured CDHA produced the highest incidence of ectopic bone and accelerated bone formation when compared with conventional microstructured sintered calcium phosphates with the same macropore architecture. Moreover, they exhibited different bone formation patterns; in CDHA foams, the new ectopic bone progressively replaced the scaffold, whereas in sintered biphasic calcium phosphate scaffolds, bone was deposited on the surface of the material, progressively filling the pore space. In conclusion, this study demonstrates that the high reactivity of nanostructured biomimetic CDHA combined with a spherical, concave macroporosity allows the pushing of the osteoinduction potential beyond the limits of microstructured calcium phosphate ceramics.

Clustering of PK-trisaccharides on amphiphilic cyclodextrin reveals unprecedented affinity for the Shiga-like toxin Stx2.

Using amphiphilic cyclodextrin as a scaffold, the first class of PK-glycoconjugates capable of high avidity binding to both Stx1 and Stx2 toxins in solid-phase assay formats is reported. The generated glycomicroarray effectively mimics the plasma membrane surface while discriminating binding of the two Stx toxins, with unprecedented affinity to Stx2.

Synthetic glycoconjugates characterize the fine specificity of Brucella A and M monoclonal antibodies.

The dominant cell wall antigen of Brucella bacteria is the O-polysaccharide component of the smooth lipopolysaccharide. Infection by various Brucella biovars causes abortions and infertility in a wide range of domestic and wild animals and debilitating disease in humans. Diagnosis relies on the detection of antibodies to the A and M antigens expressed in the O-polysaccharide. This molecule is a homopolymer of the rare monosaccharide, 4-formamido-4,6-dideoxy-d-mannopyranose (Rha4NFo). The A epitope is created by a uniform α1,2 linked internal polymeric sequence capped by a distinct tetrasaccharide sequence defining the M antigen. Unique oligosaccharides only available by chemical synthesis and conjugated via reducing and non-reducing residues to bovine serum albumin have revealed the structural basis of the fine specificity that allows the discrimination of these closely related A and M epitopes. All three M specific monoclonal antibodies (mAbs) are inferred to possess groove type binding sites open at each end, and recognize an α1,3 linked Rha4NFo disaccharide as a part of a trisaccharide epitope, which in two mAbs includes the terminal Rha4NFo residue. The binding site of one of these antibodies is sufficiently large to engage up to six Rha4NFo residues and involves weak recognition of α1,2 linked Rha4NFo residues. The third mAb binds an internal trisaccharide epitope of the M tetrasaccharide. Two A specific mAbs also possess groove type binding sites that accommodate six and four α1,2 linked Rha4NFo residues.

Molecular recognition of Brucella A and M antigens dissected by synthetic oligosaccharide glycoconjugates leads to a disaccharide diagnostic for brucellosis.

The cell wall O-polysaccharides of pathogenic Brucella species are homopolymers of the rare sugar 4,6-dideoxy-4-formamido-α-D-mannopyranose. Despite the apparent simplicity of the polysaccharide it appears to be a "block copolymer" composed of A and M polysaccharide sequences expressed as a single molecule. The simultaneous presence of both in the cell wall has complicated the understanding of the molecular recognition of these antigens by antibodies present in the serum of infected animals and humans and by monoclonal antibodies. Since presumptive diagnosis of brucellosis, a serious disease in domestic livestock, wild animals, and humans, is based on detection of these antibodies it is important to separate the two antigenic epitopes, one of which is also found in other bacteria. Chemical synthesis provides the only means to achieve this outcome. A series of six oligosaccharides from di to hexasaccharides 1-6 were synthesized and conjugated to proteins to provide glycoconjugate antigens and conjugate vaccines. These chemically defined antigens identified the M antigenic determinant and provided a structural basis for understanding the fine specificity of monoclonal and polyclonal antibodies that bind the M antigen. This resulted in the discovery of a disaccharide that shows considerable potential as an unambiguous diagnostic antigen for detecting brucellosis in humans and animals and two hexasaccharide conjugate vaccine candidates that produce high levels of O-polysaccharide specific antibodies in mice.

Oligosaccharides and peptide displayed on an amphiphilic polymer enable solid phase assay of hapten specific antibodies.

Copovidone, a copolymer of vinyl acetate and N-vinyl-2-pyrrolidone, was synthesized via reversible addition-fragmentation chain transfer (RAFT) polymerization, and after deacetylation the polymer was functionalized by introduction of amino, azide, and alkyne pendant groups to allow attachment of glycans and peptide. Candida albicans ß-mannan trisaccharides 1 and 2 and M. tuberculosis arabinan hexasaccharide 3 with appropriate tethers were conjugated to the polymers by squarate or click chemistry. C. albicans T-cell peptide 4 bearing a C-terminal ε-azidolysine was also conjugated to copovidone by click chemistry. The resulting conjugates provide convenient non-protein-based antigens that are readily adsorbed on ELISA plates, and display excellent characteristics for assay of antibody binding to the haptenic group of interest. Copovidone and BSA glycoconjugates exhibited similar adsorption characteristics when used to coat ELISA plates, and both conjugates were optimal when used as coating solutions at low nanogram/mL concentrations. Provided that the copovidone conjugated glycan is stable to acid, assay plates can be easily processed for reuse at least three times without detectable variation or degradation in ELISA readout.

Discovery of inhibitors of Shiga toxin type 2 by on-plate generation and screening of a focused compound library.

A new microtiter-plate-based method for the rapid generation and evaluation of focused compound libraries was developed and applied to screening ligand analogues for the E. coli Shiga-like toxin Stx2a. The method is general, it mitigates the masking of intrinsic affinity gains by multivalency and enables the discovery of potential hits when starting from ligands that exhibit extremely low affinity with proteins that depend on multivalency for their function.

Impact of the nature and size of the polymeric backbone on the ability of heterobifunctional ligands to mediate shiga toxin and serum amyloid p component ternary complex formation.

Inhibition of AB(5)-type bacterial toxins can be achieved by heterobifunctional ligands (BAITs) that mediate assembly of supramolecular complexes involving the toxin's pentameric cell membrane-binding subunit and an endogenous protein, serum amyloid P component, of the innate immune system. Effective in vivo protection from Shiga toxin Type 1 (Stx1) is achieved by polymer-bound, heterobifunctional inhibitors-adaptors (PolyBAITs), which exhibit prolonged half-life in circulation and by mediating formation of face-to-face SAP-AB(5) complexes, block receptor recognition sites and redirect toxins to the spleen and liver for degradation. Direct correlation between solid-phase activity and protective dose of PolyBAITs both in the cytotoxicity assay and in vivo indicate that the mechanism of protection from intoxication is inhibition of toxin binding to the host cell membrane. The polymeric scaffold influences the activity not only by clustering active binding fragments but also by sterically interfering with the supramolecular complex assembly. Thus, inhibitors based on N-(2-hydroxypropyl) methacrylamide (HPMA) show significantly lower activity than polyacrylamide-based analogs. The detrimental steric effect can partially be alleviated by extending the length of the spacer, which separates pendant ligand from the backbone, as well as extending the spacer, which spans the distance between binding moieties within each heterobifunctional ligand. Herein we report that polymer size and payload of the active ligand had moderate effects on the inhibitor's activity.

Multifunctional multivalency: a focused library of polymeric cholera toxin antagonists.

Structural pre-organization of the multivalent ligands is important for successful interaction with multimeric proteins. Polymer-based heterobifunctional ligands that contain pendant groups prearranged into heterodimers can be used to probe the active site and surrounding area of the receptor. Here we describe the synthesis and activities of a series of galactose conjugates on polyacrylamide and dextran. Conjugation of a second fragment resulted in nanomolar inhibitors of cholera toxin, while the galactose-only progenitors showed no detectable activity.

In vivo supramolecular templating enhances the activity of multivalent ligands: a potential therapeutic against the Escherichia coli O157 AB5 toxins.

We demonstrate that interactions between multimeric receptors and multivalent ligands are dramatically enhanced by recruiting a complementary templating receptor such as an endogenous multimeric protein but only when individual ligands are attached to a polymer as preorganized, covalent, heterobifunctional pairs. This effect cannot be replicated by a multivalent ligand if the same recognition elements are independently arrayed on the scaffold. Application of this principle offers an approach to create high-avidity inhibitors for multimeric receptors. Judicious selection of the ligand that engages the templating protein allows appropriate effector function to be incorporated in the polymeric construct, thereby providing an opportunity for therapeutic applications. The power of this approach is exemplified by the design of exceptionally potent Escherichia coli Shiga toxin antagonists that protect transgenic mice that constitutively express a human pentraxin, serum amyloid P component.

Heterobifunctional multivalent inhibitor-adaptor mediates specific aggregation between Shiga toxin and a pentraxin.

[reaction: see text] The first example of a multivalent heterofunctional inhibitor-adaptor, called "BAIT", is described. This multivalent inhibitor-adaptor is able to capture a "target" receptor (Shiga toxin) through its recognition of one ligand of a heterobivalent headgroup while the other ligand binds to an endogenous "trap" protein (serum amyloid P component, SAP). BAIT showed markedly enhanced inhibition of toxin activity. An efficient synthesis of this multivalent cluster containing heterobifunctional ligands was accomplished by chemical and chemoenzymatic approaches.