The Role of Epigenetic Functionalization of Implants and Biomaterials in Osseointegration and Bone Regeneration—A Review

The contribution of epigenetic mechanisms as a potential treatment model has been observed in cancer and autoimmune/inflammatory diseases. This review aims to put forward the epigenetic mechanisms as a promising strategy in implant surface functionalization and modification of biomaterials, to promote better osseointegration and bone regeneration, and could be applicable for alveolar bone regeneration and osseointegration in the future. Materials and Methods: Electronic and manual searches of the literature in PubMed, MEDLINE, and EMBASE were conducted, using a specific search strategy limited to publications in the last 5 years to identify preclinical studies in order to address the following focused questions: (i) Which, if any, are the epigenetic mechanisms used to functionalize implant surfaces to achieve better osseointegration? (ii) Which, if any, are the epigenetic mechanisms used to functionalize biomaterials to achieve better bone regeneration? Results: Findings from several studies have emphasized the role of miRNAs in functionalizing implants surfaces and biomaterials to promote osseointegration and bone regeneration, respectively. However, there are scarce data on the role of DNA methylation and histone modifications for these specific applications, despite being commonly applied in cancer research. Conclusions: Studies over the past few years have demonstrated that biomaterials are immunomodulatory rather than inert materials. In this context, epigenetics can act as next generation of advanced treatment tools for future regenerative techniques. Yet, there is a need to evaluate the efficacy/cost effectiveness of these techniques in comparison to current standards of care.     

L-Arginine/nanofish bone nanocomplex enhances bone regeneration via antioxidant activities and osteoimmunomodulatory properties

In this study,a promising strategy has been developed to promote bone regeneration by combining antioxidant activities and osteoimmunomodulatory properties.Herein,an L-arginine/nanofish bone(Arg/NFB) nanocomplex has been prepared and evaluated in vitro and in vivo.The Arg/NFB nanocomplex possesses good antioxidant activities and could modulate the polarization of non-activated macrophage into different types and induce the secretion of pre-inflammato ry,anti-inflammatory,osteogenic as well as angiogenic cytokines.Additionally,the regulated immune microenvironment can enhance the osteogenic differentiation of mouse embryo osteoblast precursor cells(MC3 T3-E1) and angiogenic capacity of human umbilical vein endothelial cells(HUVECs),leading to the improved formation of mineralized nodules,alkaline phosphatase activity and angiogenic effects.In vivo results with cranial defect models reveal that the treatment of Arg/NFB nanocomplex exhibited significant improvement of new bone formation and angiogenesis.All the results demonstrate Arg/NFB nanocomplex with antioxidant activities and osteoimmunomodulatory properties could be a new idea for developing the next generation of bone regeneration biomaterials.     

Bone‐guided regeneration: from inert biomaterials to bioactive polymer (nano)composites

Natural bone is a unique nanostructured material made of collagen fibre matrix and hydroxyapatite (HA) nanocrystals, providing mechanical support and protection from the vertebrate skeleton. However, in severe cases like bone‐deficiencies, bone needs to be “externally” repaired. Initially, different biological solutions were developed in bone‐guided regeneration. However, due to the limitations with the existing biological grafts, a lot of researches have been devoted toward biomaterials including metals, ceramics, and polymers. On the basis of the interface reactions between the implant and the surrounding tissues, these biomaterials may be classified as “nearly inert” or bioactive. Interestingly, the bioactive materials exhibit a specific biological response, leading to the formation of a natural bonding junction between the bone and the implant during bone regeneration. Recently, a special attention has been paid to a new generation of bioactive materials, i.e. (nano)structured biomaterials composed of a bioresorbable polymer matrix reinforced with bioactive inorganic compounds. While (bio)ceramic component provides the bioactivity, these materials can be readily engineered in such a way that their resorption rate in the body match the formation rate of the new tissue. This review hence reports the different biological and non‐biological solutions developed in bone‐guided regeneration, with a special emphasis on polymer‐based materials, and our recent results obtained in osseointegration The bone physiology, and its natural regeneration are also described. Copyright © 2011 John Wiley & Sons, Ltd.     

Oxidative Epigallocatechin Gallate Coating on Polymeric Substrates for Bone Tissue Regeneration

Plant derived flavonoids have not been well explored in tissue engineering applications due to difficulties in efficient formulations with biomaterials for controlled presentation. Here, the authors report that surface coating of epigallocatechin gallate (EGCG) on polymeric substrates including poly (L‐lactic acid) (PLLA) nanofibers can be performed via oxidative polymerization of EGCG in the presence of cations, enabling regulation of biological functions of multiple cell types implicated in bone regeneration. EGCG coating on the PLLA nanofiber promotes osteogenic differentiation of adipose‐derived stem cells (ADSCs) and is potent to suppress adipogenesis of ADSCs while significantly reduces osteoclastic maturation of murine macrophages. Moreover, EGCG coating serves as a protective layer for ADSCs against oxidative stress caused by hydrogen peroxide. Finally, the in vivo implantation of EGCG‐coated nanofibers into a mouse calvarial defect model significantly promotes the bone regeneration (61.52 ± 28.10%) as compared to defect (17.48 ± 11.07%). Collectively, the results suggest that EGCG coating is a simple bioinspired surface modification of polymeric biomaterials and importantly can thus serve as a promising interface for tuning activities of multiple cell types associated with bone fracture healing.     

Mesenchymal stem cells reciprocally regulate the M1/M2 balance in mouse bone marrow-derived macrophages

Mesenchymal stem cells (MSCs) have been widely studied for their applications in stem cell-based regeneration. During myocardial infarction (MI), infiltrated macrophages have pivotal roles in inflammation, angiogenesis and cardiac remodeling. We hypothesized that MSCs may modulate the immunologic environment to accelerate regeneration. This study was designed to assess the functional relationship between the macrophage phenotype and MSCs. MSCs isolated from bone marrow and bone marrow-derived macrophages (BMDMs) underwent differentiation induced by macrophage colony-stimulating factor. To determine the macrophage phenotype, classical M1 markers and alternative M2 markers were analyzed with or without co-culturing with MSCs in a transwell system. For animal studies, MI was induced by the ligation of the rat coronary artery. MSCs were injected within the infarct myocardium, and we analyzed the phenotype of the infiltrated macrophages by immunostaining. In the MSC-injected myocardium, the macrophages adjacent to the MSCs showed strong expression of arginase-1 (Arg1), an M2 marker. In BMDMs co-cultured with MSCs, the M1 markers such as interleukin-6 (IL-6), IL-1β, monocyte chemoattractant protein-1 and inducible nitric oxide synthase (iNOS) were significantly reduced. In contrast, the M2 markers such as IL-10, IL-4, CD206 and Arg1 were markedly increased by co-culturing with MSCs. Specifically, the ratio of iNOS to Arg1 in BMDMs was notably downregulated by co-culturing with MSCs. These results suggest that the preferential shift of the macrophage phenotype from M1 to M2 may be related to the immune-modulating characteristics of MSCs that contribute to cardiac repair.     

Laponite–Gelatin Nanofibrous Microsphere Promoting Human Dental Follicle Stem Cells Attachment and Osteogenic Differentiation for Noninvasive Stem Cell Transplantation

Nanofibrous microspheres (NFM) are emerging as prominent next-generation biomimetic injectable scaffold system for stem cell delivery and different tissue regeneration where nanofibrous topography facilitates ECM-like stem cells niches. Addition of osteogenic bioactive nanosilicate platelets within NFM can provide osteoconductive cues to facilitate matrix mediated osteogenic differentiation of stem cells and enhance the efficiency of bone tissue regeneration. In this study, gelatin nanofibrous microspheres are prepared containing fluoride-doped laponite XL21 (LP) using the emulsion mediated thermal induce phase separation (TIPS) technique. Systematic studies are performed to understand the effect of physicochemical properties of biomimicking NFM alone and with different concentrations of LP on human dental follicle stem cells (hDFSCs), their cellular attachment, proliferation, and osteogenic differentiation. The study highlights the effect of LP nanosilicate with biomimicking nanofibrous injectable scaffold system aiding in enhancing stem cell differentiation under normal physiological conditions compared to NFM without LP. The laponite–NFM shows suitability as excellent injectable biomaterials system for stem cell attachment, proliferation and osteogenic differentiation for stem cell transplantation and bone tissue regeneration.     

Design of Silica‐Oligourethane‐Collagen Membranes for Inflammatory Response Modulation: Characterization and Polarization of a Macrophage Cell Line

The polarization of macrophages M0 to M1 or M2 using molecules embedded in matrices and hydrogels is an active field of study. The design of biomaterials capable of promoting polarization has become a paramount need nowadays, since in the healing process macrophages M1 and M2 modulate the inflammatory response. In this work, several immunocytochemistry and ELISA tests strongly suggest the achievement of polarization using collagen‐based membranes crosslinked with tri‐functionalized oligourethanes and coated with silica. Measuring the amount of TGF‐β1 secreted to culture media by macrophages growth on these materials, and quantifying the macrophage morphology, it is proved that it is possible to stimulate the anti‐inflammatory pathway toward M2, having measurements with p ≤ 0.05 of statistical significance between the control and the collagen‐based membranes. Furthermore, some physicochemical characteristics of the hybrid materials are tested envisaging future applications: collagenase degradation resistance, water uptake, collagen fiber diameter, and deformation resistance are increased for all the crosslinked biomaterials. It is considered that the biological and physicochemical properties make the material suitable for the modulation of the inflammatory response in the chronic wounds and promising for in vivo studies.     

An Injectable,Bifunctional Hydrogel with Photothermal Effects for Tumor Therapy and Bone Regeneration

Significant attention has been focused on bone tumor therapy recently. At present, the treatment in clinic typically requires surgical intervention. However, a few tumor cells remain around bone defects after surgery and subsequently proliferate within several days. Thus, fabrication of biomaterials with dual functions of tumor therapy and bone regeneration is significant. Herein, the injectable hydrogel containing cisplatin (DDP) and polydopamine‐decorated nano‐hydroxyapatite is prepared via Schiff base reaction between the aldehyde groups on oxidized sodium alginate and amino groups on chitosan. The hydrogel exhibits sustained release properties for DDP due to the immobilization of DDP via abundant functional groups on polydopamine (PDA). Additionally, given the intense absorption of PDA in the near‐infrared region, the hydrogel exhibits excellent photothermal effects when exposed to the NIR laser (808 nm). Based on the properties, the hydrogel effectively ablates tumor cells (4T1 cells) in vitro and suppresses tumor growth in vivo. Furthermore, the hydrogel promotes the adhesion and proliferation of bone mesenchymal stem cells in vitro due to the abundant functional groups on PDA and further induces bone regeneration in vivo. Therefore, the study extends research on novel biomaterials with dual functions of tumor therapy and bone regeneration.     

Application of carbon fibers to biomaterials: a new era of nano-level control of carbon fibers after 30-years of development

Carbon fibers are state-of-the-art materials with properties that include being light weight, high strength, and chemically stable, and are applied in various fields including aeronautical science and space science. Investigation of applications of carbon fibers to biomaterials was started 30 or more years ago, and various products have been developed. Because the latest technological progress has realized nano-level control of carbon fibers, applications to biomaterials have also progressed to the age of nano-size. Carbon fibers with diameters in the nano-scale (carbon nanofibers) dramatically improve the functions of conventional biomaterials and make the development of new composite materials possible. Carbon nanofibers also open possibilities for new applications in regenerative medicine and cancer treatment. The first three-dimensional constructions with carbon nanofibers have been realized, and it has been found that the materials could be used as excellent scaffolding for bone tissue regeneration. In this critical review, we summarize the history of carbon fiber application to the biomaterials and describe future perspectives in the new age of nano-level control of carbon fibers (122 references).     

Recent Advances in Biomaterials-Based Therapies for Alleviation and Regeneration of Traumatic Brain Injury

Traumatic brain injury (TBI), a major public health problem accompanied with numerous complications, usually leads to serve disability and huge financial burden. The adverse and unfavorable pathological environment triggers a series of secondary injuries, resulting in serious loss of nerve function and huge obstacle of endogenous nerve regeneration. With the advances in adaptive tissue regeneration biomaterials, regulation of detrimental microenvironment to reduce the secondary injury and to promote the neurogenesis becomes possible. The adaptive biomaterials could respond and regulate biochemical, cellular, and physiological events in the secondary injury, including excitotoxicity, oxidative stress, and neuroinflammation, to rebuild circumstances suitable for regeneration. In this review, the development of pathology after TBI is discussed, followed by the introduction of adaptive biomaterials based on various pathological characteristics. The adaptive biomaterials carried with neurotrophic factors and stem cells for TBI treatment are then summarized. Finally, the current drawbacks and future perspective of biomaterials for TBI treatment are suggested.     

Metabolomics Insights of the Immunomodulatory Activities of Phlorizin and Phloretin on Human THP-1 Macrophages

Dihydrochalcones, phlorizin (PZ) and its aglycone phloretin (PT), have evidenced immunomodulatory effects through several mechanisms. However, the differential metabolic signatures that lead to these properties are largely unknown. Since macrophages play an important role in the immune response, our study aimed to characterise human THP-1 macrophages under PZ and PT exposure. A multiplatform-based untargeted metabolomics approach was used to reveal metabolites associated with the anti-inflammatory mechanisms triggered by the dihydrochalcones in LPS-stimulated macrophages, for the first time. Results showed differential phenotypic response in macrophages for all treatments. Dihydrochalcone treatment in LPS-stimulated macrophages mimics the response under normal conditions, suggesting inhibition of LPS response. Antagonistic effects of dihydrochalcones against LPS was mainly observed in glycerophospholipid and sphingolipid metabolism besides promoting amino acid biosynthesis. Moreover, PT showed greater metabolic activity than PZ. Overall, the findings of this study yielded knowledge about the mechanisms of action PZ and PT at metabolic level in modulating inflammatory response in human cells.     

Ordered mesoporous materials in the context of drug delivery systems and bone tissue engineering

Chemistry, materials science and medicine are research areas that converge in the field of drug delivery systems and tissue engineering. This paper tries to introduce an example of such an interaction, aimed at solving health issues within the world of biomaterials. Ordered mesoporous materials can be loaded with different organic molecules that would be released afterwards, in a controlled fashion, inside a living body. These materials can also react with the body fluids giving rise to carbonated nanoapatite particles as the products of such a chemical interaction; these particles, equivalent to biological apatites, enable the regeneration of bone tissue.     

Protein and Polysaccharide-Based Electroactive and Conductive Materials for Biomedical Applications

Electrically responsive biomaterials are an important and emerging technology in the fields of biomedical and material sciences. A great deal of research explores the integral role of electrical conduction in normal and diseased cell biology, and material scientists are focusing an even greater amount of attention on natural and hybrid materials as sources of biomaterials which can mimic the properties of cells. This review establishes a summary of those efforts for the latter group, detailing the current materials, theories, methods, and applications of electrically conductive biomaterials fabricated from protein polymers and polysaccharides. These materials can be used to improve human life through novel drug delivery, tissue regeneration, and biosensing technologies. The immediate goal of this review is to establish fabrication methods for protein and polysaccharide-based materials that are biocompatible and feature modular electrical properties. Ideally, these materials will be inexpensive to make with salable production strategies, in addition to being both renewable and biocompatible.     

Granulatosides D,E and other polar steroid compounds from the starfish Choriaster granulatus. Their immunomodulatory activity and cytotoxicity

Two new steroid glycosides, granulatosides D (1) and E (2), belonging to the group of bi- and monoglycosides of polyhydroxysteroids, respectively, were isolated from the ethanolic extract of the starfish Choriaster granulatus along with thirteen previously known glycosides of polyhydroxysteroids (3–15) and one steroid heptaol (16). The structures of the new compounds were elucidated by extensive NMR and ESIMS techniques. Cytotoxic and immunomodulatory activities of compounds 1, 3–8, and 10–16 using murine splenocytes and peritoneal macrophages were studied. At a dose of 0.1 μM new glycoside 1 showed immunomodulatory properties, increasing the intracellular ROS (reactive oxygen species) level in peritoneal murine macrophages by 20% and decreasing intracellular ROS level by 21% in pre-treated with endotoxic lipopolysaccharide from E. coli (LPS) peritoneal macrophages.     

Dielectrophoretic characterization of macrophage phenotypes

Different macrophage phenotypes play important roles in diverse biological processes and diseases. In this study, we have characterized the dielectrophoretic responses of human monocytes and macrophage phenotypes: nonactivated (M0), pro-inflammatory (M1), and pro-healing (M2a). Dielectrophoretic responses of cells change as a function of frequency of the applied electric field. We measured the crossover frequency at which cells transition from negative to positive dielectrophoresis (DEP) or vice versa using interdigitated electrodes. For these characterization experiments, we also developed a new low-conductivity media formulation that retained 100% of the initial viability for 1 h. Human THP1 monocytes showed a distinguishable DEP response from mature macrophages. M1 macrophages also showed a distinct DEP response compared to M0 and M2a macrophages. No clear distinction could be drawn between M0 and M2a. The median values of the crossover frequencies of monocytes, M0, M1, and M2a were 38, 21, 11, and 23 kHz, respectively. Membrane capacitances of these cells were calculated consequently, and the values were 0.0111, 0.0128, 0.0244, and 0.0117 F/m² for monocytes, M0, M1, and M2a, respectively. These results show how bioelectric properties are influenced by changes in macrophage phenotype.     

Revisiting ceramics for medical applications

The most significant demand for biomaterials has emerged as a consequence of the need to provide clinical treatment to a large number of patients. The search for potential solutions produces a large demand for materials suitable for bone repair or replacement. Calcium phosphates, bio-glasses, bio-glass ceramics and ordered silica mesoporous materials, among other types of materials, will be reviewed and studied from the point of view of their potential applications as replacement materials in bone repair and regeneration, as potential substrates in tissue engineering, and also as drug delivery systems. An overview on the present achievements, but also on the "missing links" will be presented.     

α‐Mannosyl‐Functionalized Cationic Nanohydrogel Particles for Targeted Gene Knockdown in Immunosuppressive Macrophages

Immunosuppressive M2 macrophages govern the immunophathogenic micromilieu in many severe diseases including cancer or fibrosis, thus, their re‐polarization through RNA interference is a promising concept to support combinatorial therapies. For targeted siRNA delivery, however, safe and stable carriers are required that manage cell specific transport to M2 macrophages. Here, siRNA‐loaded cationic nanogels are reported with α‐mannosyl decorated surfaces that target and modify M2 macrophages selectively. Via amphiphilic precursor block copolymers bearing one single α‐mannosyl moiety at their chain end mannosylated cationic nanohydrogel particles (ManNP) were obtained of 20 nm diameter determined by dynamic light scattering and cryogenic electron transmission microscopy. α‐Mannosyl surface modification is confirmed by agglutination with concanavalin A. SiRNA‐loaded ManNP preferentially targets the overexpressed mannose receptor CD206 on M2 macrophages, as shown by in vitro cell uptake studies in M2 polarized primary macrophages. This specificity is confirmed, since ManNP uptake could be reduced by blocking of CD206 with mannan. Effective ManNP‐guided siRNA delivery is confirmed by sequence‐specific gene knockdown of CSF‐1R in M2‐type macrophages exclusively, while the expression levels in M1‐polarized macrophages is not affected. In conclusion, α‐mannosyl‐functionalized ManNPs are promising universal siRNA carriers for targeted immunomodulatory treatment of immunosuppressive macrophages.     

Bioceramics and pharmaceuticals: A remarkable synergy

The research on controlled drug delivery systems using bioceramics as host matrices presents two distinct sides; one route aims at embedding pharmaceuticals in biomaterials designed for the reconstruction or regeneration of living tissues, in order to counteract inflammatory responses, infections, bone carcinomas and so forth, while the other route deals with the more traditional drug introduction systems, i.e. oral administration.The incorporation of pharmaceuticals to bioceramic matrices could be very interesting in clinical practice. It is rather common in these days for an orthopedic surgeon working in bone reconstruction to use bioceramics. An added value to the production of these ceramics would be the optional addition of pharmaceuticals such as antibiotics, anti-inflammatories, anti-carcinogens, etc. In this sense, if we take into account the infections statistics at hip joint prostheses, the incidence varies between 2 and 4%, reaching up to a 45% in bolts used as external fixation. One of the main problems in these situations is the access to the infected area of the bone, in order to deliver the adequate antibiotic. If the pharmaceutical could be included within the implant itself, the added value would be straightforward.And if the bioceramic is bioactive, and therefore precursor of new bone tissue, the capability to introduce peptides, proteins or growth factors at its pores could accelerate the bone regeneration processes. We are facing a fine example of multidisciplinary research, where the so-called transversal supply of knowledge from and between the domains of materials science, biology and medicine will empower the know-how and applications that shall, undoubtedly, give rise to new advances in science and technology.     

Cycloolefin-Copolymer/Polyethylene (COC/PE) Blend Assists with the Creation of New Articular Cartilage

It is shown that the initial biomechanical stability of the applied COC/PE blend in the treated tissue locality, the initial integrity of biomaterials substituting the subchondral bone by polymer implant, the initial bearing capacity and the vertical position of these biomaterials have a mayor influence on the regeneration of new articular cartilage and subchondral bone. These aspects are essential for a prosperous treatment of osteochondral defects. Results reveal that the initial biomechanical stiffness of materials (substituting the subchondral bone) has a fundamental influence on both the quality and the quantity of new articular cartilage and subchondral bone. Research is also aimed at the application of suitable biologically tolerated polymer material, its surface modifications, verifications of the vertical position of implants in relation to the articular cartilage surface and ensuring the initial biomechanical stability of the polymer implant.     

Molecular Cloning,Expression and Macrophage Activation of an Immunoregulatory Protein from Cordyceps militaris

Protein components of C. militaris have been reported to possess various biological activities. In our previous research, a Cordyceps militaris-derived immunoregulatory protein (CMIP) was naturally isolated and showed the activity of inhibiting the metastasis of breast cancer cells. This study aimed to obtain recombinant CMIP (rCMIP) using recombinant expression and elucidate its ability to activate macrophages. Recombinant CMIP showed one band at approximately 15 kDa or 30 kDa, or two bands at 15 kDa and 30 kDa, under different denaturation conditions of electrophoresis. The cell binding assay showed that rCMIP selectively binds to the surface of macrophages. After adhesion, it did not induce the apoptosis of RAW 264.7 cells, but promoted their proliferation. Moreover, rCMIP significantly induced the expression of M1 macrophage polarization-related molecules. The mean fluorescence intensity (MFI) of CD 86 was enhanced by 2.1-fold and 3.2-fold under 0.64 μM and 1.6 μM of rCMIP treatment, respectively. Cytokines typically expressed in M1 macrophages, such as TNF-α, iNOS, IL-6, CCL 4, CCL 5 and CXCL 10, were also considerably induced by rCMIP, while the expression of cytokines in typical M2 macrophages, like Arg-1, CCL17 and CCL22, were not changed or slightly decreased. Under rCMIP treatment, the release of NO was also appreciably induced. In the present study, we reported cloning, expression and functional characterization of rCMIP, which was naturally isolated from the fruiting body of C. militaris in our previous study. The data imply that rCMIP possesses immunomodulatory activity in macrophages.