Hydrogels in the treatment of rheumatoid arthritis: drug delivery systems and artificial matrices for dynamic in vitro models.

Rheumatoid arthritis (RA) is an autoimmune and chronic inflammatory disorder that mostly affects the synovial joints and can promote both cartilage and bone tissue destruction. Several conservative treatments are available to relieve pain and control the inflammation; however, traditional drugs administration are not fully effective and present severe undesired side effects. Hydrogels are a very attractive platform as a drug delivery system to guarantee these handicaps are reduced, and the therapeutic effect from the drugs is maximized. Furthermore, hydrogels can mimic the physiological microenvironment and have the mechanical behavior needed for use as cartilage in vitro model. The testing of these advanced delivery systems is still bound to animal disease models that have shown low predictability. Alternatively, hydrogel-based human dynamic in vitro systems can be used to model diseases, bypassing some of the animal testing problems. RA dynamic disease models are still in an embryonary stage since advances regarding healthy and inflamed cartilage models are currently giving the first steps regarding complexity increase. Herein, recent studies using hydrogels in the treatment of RA, featuring different hydrogel formulations are discussed. Besides, their use as artificial extracellular matrices in dynamic in vitro articular cartilage is also reviewed.

Microfluidics for Angiogenesis Research.

Angiogenesis is a natural and vital phenomenon of neovascularization that occurs from pre-existing vasculature, being present in many physiological processes, namely in development, reproduction and regeneration. Being a highly dynamic and tightly regulated process, its abnormal expression can be on the basis of several pathologies. For that reason, angiogenesis has been a subject of major interest among the scientific community, being transverse to different areas and founding particular attention in tissue engineering and cancer research fields. Microfluidics has emerged as a powerful tool for modelling this phenomenon, thereby surpassing the limitations associated to conventional angiogenic models. Holding a tremendous flexibility in terms of experimental design towards a specific goal, microfluidic systems can offer an unlimited number of opportunities for investigating angiogenesis in many relevant scenarios, namely from its fundamental comprehension in normal physiological processes to the identification and testing of new therapeutic targets involved on pathological angiogenesis. Additionally, microvascular 3D in vitro models are now opening up new prospects in different fields, being used for investigating and establishing guidelines for the development of next generation of 3D functional vascularized grafts. The promising applications of this emerging technology in angiogenesis studies are herein overviewed, encompassing fundamental and applied research.

Biomaterials and Microfluidics for Drug Discovery and Development.

Microfluidic devices are now one of the most promising tools to mimic in vivo like conditions, either in normal or disease scenarios, such as tumorigenesis or pathogenesis. Together with the potential of biomaterials, its combination with microfluidics represents the ability to more closely mimic cells' natural microenvironment concerning its three-dimensional (3D) nature and continuous perfusion with nutrients and cells' crosstalk. Due to miniaturization and increased experimental throughput, microfluidics have generated significant interest in the drug discovery and development domain. Herein, the most recent advances in the field of microfluidics for drug discovery are overviewed, and the role of biomaterials in 3D in vitro models and the contribution of organ-on-a-chip technologies highlighted.

Scaffolds and coatings for bone regeneration.

Bone tissue has an astonishing self-healing capacity yet only for non-critical size defects (<6 mm) and clinical intervention is needed for critical-size defects and beyond that along with non-union bone fractures and bone defects larger than critical size represent a major healthcare problem. Autografts are, still, being used as preferred to treat large bone defects. Mostly, due to the presence of living differentiated and progenitor cells, its osteogenic, osteoinductive and osteoconductive properties that allow osteogenesis, vascularization, and provide structural support. Bone tissue engineering strategies have been proposed to overcome the limited supply of grafts. Complete and successful bone regeneration can be influenced by several factors namely: the age of the patient, health, gender and is expected that the ideal scaffold for bone regeneration combines factors such as bioactivity and osteoinductivity. The commercially available products have as their main function the replacement of bone. Moreover, scaffolds still present limitations including poor osteointegration and limited vascularization. The introduction of pores in scaffolds are being used to promote the osteointegration as it allows cell and vessel infiltration. Moreover, combinations with growth factors or coatings have been explored as they can improve the osteoconductive and osteoinductive properties of the scaffold. This review focuses on the bone defects treatments and on the research of scaffolds for bone regeneration. Moreover, it summarizes the latest progress in the development of coatings used in bone tissue engineering. Despite the interesting advances which include the development of hybrid scaffolds, there are still important challenges that need to be addressed in order to fasten translation of scaffolds into the clinical scenario. Finally, we must reflect on the main challenges for bone tissue regeneration. There is a need to achieve a proper mechanical properties to bear the load of movements; have a scaffolds with a structure that fit the bone anatomy.

Silk Fibroin-Based Scaffold for Bone Tissue Engineering.

Regeneration of diseased or damaged skeletal tissues is one of the challenge that needs to be solved. Although there have been many bone tissue engineering developed, scaffold-based tissue engineering complement the conventional treatment for large bone by completing biological and functional environment. Among many materials, silk fibroin (SF) is one of the favorable material for applications in bone tissue engineering scaffolding. SF is a fibrous protein mainly extracted from Bombyx mori. and spiders. SF has been used as a biomaterial for bone graft by its unique mechanical properties, controllable biodegradation rate and high biocompatibility. Moreover, SF can be processed using conventional and advanced biofabrication methods to form various scaffold types such as sponges, mats, hydrogels and films. This review discusses about recent application and advancement of SF as a biomaterial.

Seaweed polysaccharide-based hydrogels used for the regeneration of articular cartilage.

This manuscript provides an overview of the in vitro and in vivo studies reported in the literature focusing on seaweed polysaccharides based hydrogels that have been proposed for applications in regenerative medicine, particularly, in the field of cartilage tissue engineering. For a better understanding of the main requisites for these specific applications, the main aspects of the native cartilage structure, as well as recognized diseases that affect this tissue are briefly described. Current available treatments are also presented to emphasize the need for alternative techniques. The following part of this review is centered on the description of the general characteristics of algae polysaccharides, as well as relevant properties required for designing hydrogels for cartilage tissue engineering purposes. An in-depth overview of the most well known seaweed polysaccharide, namely agarose, alginate, carrageenan and ulvan biopolymeric gels, that have been proposed for engineering cartilage is also provided. Finally, this review describes and summarizes the translational aspect for the clinical application of alternative systems emphasizing the importance of cryopreservation and the commercial products currently available for cartilage treatment.

In vivo biofunctional evaluation of hydrogels for disc regeneration.

PURPOSE: Regenerative strategies aim to restore the original biofunctionality of the intervertebral disc. Different biomaterials are available, which might support disc regeneration. In the present study, the prospects of success of two hydrogels functionalized with anti-angiogenic peptides and seeded with bone marrow derived mononuclear cells (BMC), respectively, were investigated in an ovine nucleotomy model. METHODS: In a one-step procedure iliac crest aspirates were harvested and, subsequently, separated BMC were seeded on hydrogels and implanted into the ovine disc. For the cell-seeded approach a hyaluronic acid-based hydrogel was used. The anti-angiogenic potential of newly developed VEGF-blockers was investigated on ionically crosslinked metacrylated gellan gum hydrogels. Untreated discs served as nucleotomy controls. 24 adult merino sheep were used. After 6 weeks histological, after 12 weeks histological and biomechanical analyses were conducted. RESULTS: Biomechanical tests revealed no differences between any of the implanted and nucleotomized discs. All implanted discs significantly degenerated compared to intact discs. In contrast, there was no marked difference between implanted and nucleotomized discs. In tendency, albeit not significant, degeneration score and disc height index deteriorated for all but not for the cell-seeded hydrogels from 6 to 12 weeks. Cell-seeded hydrogels slightly decelerated degeneration. CONCLUSIONS: None of the hydrogel configurations was able to regenerate biofunctionality of the intervertebral disc. This might presumably be caused by hydrogel extrusion. Great importance should be given to the development of annulus sealants, which effectively exploit the potential of (cell-seeded) hydrogels for biological disc regeneration and restoration of intervertebral disc functioning.

Hydroxyapatite/alginate/gellan gum inks with osteoconduction and osteogenic potential for bioprinting bone tissue analogues.

There is a growing demand for engineered bone tissues custom-designed to match the patient-specific defect size and in vitro models for studying bone diseases and/or drug screening. Herein, we propose a bioprinted bone tissue construct using SaOs-2 cells within alginate/gellan gum/hydroxyapatite inks. Different ink formulations were developed with varying hydroxyapatite content and then evaluated for viscoelasticity, printability, biomineralization properties, post-printing viability, proliferation, metabolic activity, and osteogenic phenotype of SaOs-2-encapsulated cells. Results indicate that ink formulations exhibit non-Newtonian shear-thinning behaviour, maintaining shape integrity and structural stability post-printing. Ink mineralization rates increase with the hydroxyapatite content, rendering them suitable for bone defect strategies. Post-printed cells in the developed constructs remain live, spreading, and metabolically active but do not proliferate. Osteogenic gene and protein expression, both early and late, show upregulation at day 7 relative to day 1, followed by downregulation at day 14. Lower hydroxyapatite content inks demonstrate up to fourfold upregulation in genes and proteins at most time points. Additionally, these constructs release calcium and phosphate at levels conducive to mineralization. Overall, the tissue-engineered miniaturized constructs not only meet the criteria for early-stage bone defect/fracture regeneration but also serve as a promising platform for drug screening and evaluating potential therapeutic treatments.

Innovative nanotheranostics: Smart nanoparticles based approach to overcome breast cancer stem cells mediated chemo- and radioresistances.

The alarming increase in the number of breast cancer patients worldwide and the increasing death rate indicate that the traditional and current medicines are insufficient to fight against it. The onset of chemo- and radioresistances and cancer stem cell-based recurrence make this problem harder, and this hour needs a novel treatment approach. Competent nanoparticle-based accurate drug delivery and cancer nanotheranostics like photothermal therapy, photodynamic therapy, chemodynamic therapy, and sonodynamic therapy can be the key to solving this problem due to their unique characteristics. These innovative formulations can be a better cargo with fewer side effects than the standard chemotherapy and can eliminate the stability problems associated with cancer immunotherapy. The nanotheranostic systems can kill the tumor cells and the resistant breast cancer stem cells by novel mechanisms like local hyperthermia and reactive oxygen species and prevent tumor recurrence. These theranostic systems can also combine with chemotherapy or immunotherapy approaches. These combining approaches can be the future of anticancer therapy, especially to overcome the breast cancer stem cells mediated chemo- and radioresistances. This review paper discusses several novel theranostic systems and smart nanoparticles, their mechanism of action, and their modifications with time. It explains their relevance and market scope in the current era. This article is categorized under: Therapeutic Approaches and Drug Discovery > Nanomedicine for Oncologic Disease.

Mn-Based Methacrylated Gellan Gum Hydrogels for MRI-Guided Cell Delivery and Imaging.

This work aims to engineer a new stable injectable Mn-based methacrylated gellan gum (Mn/GG-MA) hydrogel for real-time monitored cell delivery into the central nervous system. To enable the hydrogel visualization under Magnetic Resonance Imaging (MRI), GG-MA solutions were supplemented with paramagnetic Mn2+ ions before its ionic crosslink with artificial cerebrospinal fluid (aCSF). The resulting formulations were stable, detectable by T1-weighted MRI scans and also injectable. Cell-laden hydrogels were prepared using the Mn/GG-MA formulations, extruded into aCSF for crosslink, and after 7 days of culture, the encapsulated human adipose-derived stem cells remained viable, as assessed by Live/Dead assay. In vivo tests, using double mutant MBPshi/shi/rag2 immunocompromised mice, showed that the injection of Mn/GG-MA solutions resulted in a continuous and traceable hydrogel, visible on MRI scans. Summing up, the developed formulations are suitable for both non-invasive cell delivery techniques and image-guided neurointerventions, paving the way for new therapeutic procedures.

Biocomposite Macrospheres Based on Strontium-Bioactive Glass for Application as Bone Fillers.

Traditional bioactive glass powders are typically composed of irregular particles that can be packed into dense configurations presenting low interconnectivity, which can limit bone ingrowth. The use of novel biocomposite sphere formulations comprising bioactive factors as bone fillers are most advantageous, as it simultaneously allows for packing the particles in a 3-dimensional manner to achieve an adequate interconnected porosity, enhanced biological performance, and ultimately a superior new bone formation. In this work, we develop and characterize novel biocomposite macrospheres of Sr-bioactive glass using sodium alginate, polylactic acid (PLA), and chitosan (CH) as encapsulating materials for finding applications as bone fillers. The biocomposite macrospheres that were obtained using PLA have a larger size distribution and higher porosity and an interconnectivity of 99.7%. Loose apatite particles were observed on the surface of macrospheres prepared with alginate and CH by means of soaking into a simulated body fluid (SBF) for 7 days. A dense apatite layer was formed on the biocomposite macrospheres' surface produced with PLA, which served to protect PLA from degradation. In vitro investigations demonstrated that biocomposite macrospheres had minimal cytotoxic effects on a human osteosarcoma cell line (SaOS-2 cells). However, the accelerated degradation of PLA due to the degradation of bioactive glass may account for the observed decrease in SaOS-2 cells viability. Among the biocomposite macrospheres, those composed of PLA exhibited the most promising characteristics for their potential use as fillers in bone tissue repair applications.

Fibroblasts regulate osteoblasts through gap junctional communication.

BACKGROUND AIMS: Fibroblasts are present in most tissues of the body, playing an active role in the regulation of homeostasis in such tissues. While fibroblast heterotypic interactions are acknowledged in the regeneration of tissues such as skin and periodontal ligament, their role in bone regeneration is far from being understood. We hypothesized that fibroblasts could influence osteoblasts, and as connexin 43 is the predominant connexin in both cell types, we speculated that those heterotypic interactions could occur through gap junctional communication (GjC). METHODS: Direct co-cultures of human mesenchymal stromal cell (hMSC)-derived osteoblasts and human dermal fibroblasts (hDFb) were established in the presence and absence of the GjC inhibitor α-glycyrrhetinic acid. Communication between osteoblasts and hDFb via GjC was verified by transference of the gap junction-permeable dye calcein-AM. Cell proliferation was assessed by dsDNA quantification, while osteogenic differentiation was evaluated by measuring alkaline phosphatase (ALP) activity and the expression of osteogenic markers by real-time polymerase chain reaction (PCR). RESULTS: The amount of calcein-AM transferred between the different cell types decreased when α-glycyrrhetinic acid was used. While the proliferation of the hMSC-derived osteoblasts was not affected by the presence of the hDFb, the level of osteogenic markers such as ALP activity and osteocalcin in transcripts in osteoblasts was severely diminished. This effect was partially reversed by adding α-glycyrrhetinic acid to the co-cultures. CONCLUSIONS: The results strongly suggest that fibroblasts regulate osteoblast behavior partially through GjC. This information could be critical for predicting the outcome of strategies aimed at promoting bone regeneration as, for example, in bone tissue-engineering approaches.

Nanoparticles for neurotrophic factor delivery in nerve guidance conduits for peripheral nerve repair.

Peripheral nerve injuries are a major source of disabilities, and treatment of long nerve gap autografts is the gold standard. However, due to poor availability and donor-site morbidity, research is directed towards the development of regenerative strategies based on the use of artificial nerve guidance conduits (NGCs). Several properties and characteristics of the NGCs can be fine-tuned, such as the architecture of the conduit, the surface topography and the addition of bioactive molecules and cells to speed up nerve regeneration. In this review, US FDA-approved NGCs are described. The recent works, in which polymeric, magnetic, silica-based and lipidic NPs are employed to introduce growth factors (GFs) to NGCs, are overviewed and discussed in depth herein.

Nerves present in the extremities of the body are often injured, and this can lead to disabilities. To treat this problem, nerve sections from other body parts can be used, but the main disadvantage of this technique is poor availability and donor-site morbidity. To tackle these difficulties, research is focused on the development of artificial nerves, which are known as nerve guidance conduits (NGCs). This review article focuses on advances in this field, which is mainly related to the optimization of the material for conduit synthesis, on architecture and topography, and on how the functionalization of the NGCs with bioactive molecules can support nerve regeneration at the injured site. Currently commercialized NGCs are presented, and an in-depth discussion on strategies comprising neurotrophic factors administered alone, or included in the NGCs using nanoparticles, is also provided.

Tissue engineering and regenerative medicine strategies in meniscus lesions.

PURPOSE: The aim of this systematic review was to address tissue engineering and regenerative medicine (TERM) strategies applied to the meniscus, specifically (1) clinical applications, indications, results, and pitfalls and (2) the main trends in research assessed by evaluation of preclinical (in vivo) studies. METHODS: Three independent reviewers performed a search on PubMed, from 2006 to March 31, 2011, using the term "meniscus" with all of the following terms: "scaffolds," "constructs," "cells," "growth factors," "implant," "tissue engineering," and "regenerative medicine." Inclusion criteria were English language-written, original clinical research (Level of Evidence I to IV) and preclinical studies of TERM application in knee meniscal lesions. Reference lists and related articles on journal Web sites of selected articles were checked until prepublication for potential studies that could not be identified eventually by our original search. The modified Coleman Methodology score was used for study quality analysis of clinical trials. RESULTS: The PubMed search identified 286 articles (a similar search from 2000 to 2005 identified 161 articles). Non-English-language articles (n = 9), Level V publications (n = 19), in vitro studies (n = 118), and 102 studies not related to the topic were excluded. One reference was identified outside of PubMed. Thirty-eight references that met the inclusion criteria were identified from the original search. On the basis of our prepublication search, 2 other references were included. A total of 9 clinical and 31 preclinical studies were selected for further analysis. Of the clinical trials, 1 was classified as Level I, 2 as Level II, and 6 as Level IV. Eight referred to acellular scaffold implantation for partial meniscal replacement, and one comprised fibrin clot application. The mean modified Coleman Methodology score was 48.0 (SD, 15.7). Of the preclinical studies, 11 original works reported on studies using large animal models whereas 20 research studies used small animals. In these studies the experimental design favored cell-seeded scaffolds or scaffolds enhanced with growth factors (GFs) in attempts to improve tissue healing, as opposed to the plain acellular scaffolds that were predominant in clinical trials. Injection of mesenchymal stem cells and gene therapy are also presented as alternative strategies. CONCLUSIONS: Partial meniscal substitution using acellular scaffolds in selected patients with irreparable loss of tissue may be a safe and promising procedure. However, there is only 1 randomized controlled study supporting its application, and globally, many methodologic issues of published trials limit further conclusions. We registered a different trend in preclinical trials, with most considering augmentation of scaffolds by cells and/or GFs, as opposed to the predominantly acellular approach in clinical trials. Different TERM approaches to enhance meniscal repair or regeneration are in preclinical analysis, such as the use of mesenchymal stem cells, gene therapy, and GFs alone or in combination, and thus could be considered in the design of subsequent trials. LEVEL OF EVIDENCE: Level IV, systematic review of Level I to IV studies.

Influence of gellan gum-hydroxyapatite spongy-like hydrogels on human osteoblasts under long-term osteogenic differentiation conditions.

The scientific community has been doing significant efforts towards engineering new 3D bone models in recent years. Osteocytes are mechanosensitive cells that play significant roles in the maintenance of bone homeostasis. Currently, as far as we know, there are no 3D models that faithfully recapitulate a bone microenvironment capable of promoting the differentiation of osteoblasts towards osteocytes. Besides, in the existing models, the use of human cells does not prevail over the animal cell lines. For so, we propose a 3D model that may have important implications for ongoing efforts towards a better understanding of bone physiology and disease. The main aim of the current work was the promotion of an effective differentiation of osteoblasts into osteocytes by mean of using a 3D model composed of primary human osteoblasts (hOBs) cultured on Gellan Gum-Hydroxyapatite (GG-HAp) matrix under a long-term osteogenic culture. The results revealed that GG-HAp matrix stimulated a fast cell migration/entrapment, attachment, spreading, and mineralization. Moreover, the transition process from osteoblasts to osteocytes was confirmed by the expression of the osteogenic-related (ALP, Runx2, COL I, OC, OPN and OSX) and osteocyte-related (hPDPN) marker throughout the culture time. Overall, the developed 3D model holds a great promise for the treatment of various bone diseases, namely on diagnostic applications and for bone regeneration purposes.

Bioengineered Nanoparticles Loaded-Hydrogels to Target TNF Alpha in Inflammatory Diseases.

Rheumatoid Arthritis (RA) is an incurable autoimmune disease that promotes the chronic impairment of patients' mobility. For this reason, it is vital to develop therapies that target early inflammatory symptoms and act before permanent articular damage. The present study offers two novel therapies based in advanced drug delivery systems for RA treatment: encapsulated chondroitin sulfate modified poly(amidoamine) dendrimer nanoparticles (NPs) covalently bonded to monoclonal anti-TNF α antibody in both Tyramine-Gellan Gum and Tyramine-Gellan Gum/Silk Fibroin hydrogels. Using pro-inflammatory THP-1 (i.e., human monocytic cell line), the therapy was tested in an inflammation in vitro model under both static and dynamic conditions. Firstly, we demonstrated effective NP-antibody functionalization and TNF-α capture. Upon encapsulation, the NPs were released steadily over 21 days. Moreover, in static conditions, the approaches presented good anti-inflammatory activity over time, enabling the retainment of a high percentage of TNF α. To mimic the physiological conditions of the human body, the hydrogels were evaluated in a dual-chamber bioreactor. Dynamic in vitro studies showed absent cytotoxicity in THP-1 cells and a significant reduction of TNF-α in suspension over 14 days for both hydrogels. Thus, the developed approach showed potential for use as personalized medicine to obtain better therapeutic outcomes and decreased adverse effects.

Correction: Strategies for the hypothermic preservation of cell sheets of human adipose stem cells.

[This corrects the article DOI: 10.1371/journal.pone.0222597.].

PAMAM dendrimers functionalised with an anti-TNF α antibody and chondroitin sulphate for treatment of rheumatoid arthritis.

Rheumatoid arthritis is a chronic autoimmune disease characterised by joint synovial inflammation, along with cartilage and bone tissue destruction. Dendrimers can offer new opportunities as drug delivery systems of molecules of interest. Herein we aimed to develop poly(amidoamine) dendrimers (PAMAM), functionalised with chondroitin sulphate (CS), lined with anti-TNF α antibodies (Abs) to provide anti-inflammatory properties. Physicochemical characterisation demonstrated that anti-TNFα Abs-CS/PAMAM dendrimer NPs were successfully produced. The in vitro studies revealed that CS/PAMAM dendrimer NPs did not affect the ATDC5 and THP-1 cell lines' metabolic activity and proliferation, presenting good cytocompatibility and hemocompatibility. Moreover, anti-TNFα Abs-CS/PAMAM dendrimer NPs showed suitable TNF α capture capacity, making them appealing for new immunotherapies in RA patients.

Decellularized hASCs-derived matrices as biomaterials for 3D in vitro approaches.

Tissue regeneration strategies have been greatly evolving in the last years due to the use of more realistic approaches. These approaches rely in the use of biomaterials for the development of three dimensional (3D) structures that emulate the in vivo microenvironment of different tissues. Recently, extracellular matrices (ECM) secreted by cells have been caught a great deal of attention as an attractive biomaterial for the development 3D structures. In fact, different cells and/or different cellular culture conditions gave rise to different ECM's compositions, which can be used for the development of more physiologically relevant 3D structures. Nevertheless, the recovery of cell-derived ECM requires the use of a proper decellularization protocol. Herein, we report a decellularization protocol to recover the ECM produced by human adipose derived stem cells. This protocol comprises multiple steps (chemical, physical or enzymatic) which are described here in more detail. Furthermore, it is describe the methods that have been used to evaluate the effectiveness of this decellularization protocol. Overall, this protocol enables the production of hASCs-derived matrices that can be further used for the production of more physiologically relevant 3D in vitro models for tissue regeneration strategies.

Human Microcirculation-on-Chip Models in Cancer Research: Key Integration of Lymphatic and Blood Vasculatures.

Cancer metastasis is a highly complex and multistep process, which is initiated by the invasion of tumor cells into the microcirculation system. A diverse variety of organ-on-chip models are described investigating this critical event. However, most of these models solely integrate the blood vasculature and overlook the fundamental role of the lymphatic system despite the solid evidence showing that cancer cells mainly use this vascular network to initiate metastasis. Herein, the latest advances in the field of organ-on-chip models of the human microcirculation system in cancer research are reviewed. The reported models are employed to investigate the mechanistic determinants of tumor physiopathology and for the screening of new anticancer drugs under the scope of the microcirculation bed. Overall, the development of complete microcirculation-on-chip models integrating the blood and lymphatic vasculatures is expected to provide key insights into the drug delivery process, the screening of novel therapeutic compounds, or the mechanism of tumor invasion and metastasis, among others.