COVID-19 and rheumatic diseases: A mini-review.

Joint pain and arthralgia can be manifestations of COVID-19, and studies evaluating long COVID symptoms identified the persistence of these disorders. Moreover, some case reports highlighted the development of new inflammatory arthritis in patients with COVID-19, suggesting a possible relation. Viral infections and rheumatic diseases share a documented relationship; they have been associated with genetic and environmental risk factors responsible for some of them. There is crosstalk between viruses and the immune system during the development of several rheumatic diseases. Moreover, infections may participate in the pathogenesis of autoimmune rheumatic diseases and contribute to patient mortality. Therefore, it is crucial to provide a clearer insight into the interaction between viral infections and rheumatic diseases. Here, we provide a mini-review of the current literature with the aim of shedding light on the relationship between COVID-19 and rheumatic or musculoskeletal diseases, which is still unclear. Specifically, we examined several aspects: risk for the rheumatic population of acquiring the virus or developing severe symptoms, similarities of COVID-19 and arthritis, the possible rheumatic consequence of COVID-19, of rheumatic drugs and vaccines, and COVID-19 prevention in rheumatic patients through vaccination.

Current concepts and perspectives for articular cartilage regeneration.

Articular cartilage injuries are common in the population. The increment in the elderly people and active life results in an increasing demand for new technologies and good outcomes to satisfy longer and healthier life expectancies. However, because of cartilage's low regenerative capacity, finding an efficacious treatment is still challenging for orthopedics.Since the pioneering studies based on autologous cell transplantation, regenerative medicine has opened new approaches for cartilage lesion treatment.Tissue engineering combines cells, biomaterials, and biological factors to regenerate damaged tissues, overcoming conventional therapeutic strategies. Cells synthesize matrix structural components, maintain tissue homeostasis by modulating metabolic, inflammatory, and immunologic pathways. Scaffolds are well acknowledged by clinicians in regenerative applications since they provide the appropriate environment for cells, can be easily implanted, reduce surgical morbidity, allow enhanced cell proliferation, maturation, and an efficient and complete integration with surrounding articular cartilage. Growth factors are molecules that facilitate tissue healing and regeneration by stimulating cell signal pathways.To date, different cell sources and a wide range of natural and synthetic scaffolds have been used both in pre-clinical and clinical studies with the aim to find the suitable solution for recapitulating cartilage microenvironment and inducing the formation of a new tissue with the biochemical and mechanical properties of the native one. Here, we describe the current concepts for articular cartilage regeneration, highlighting the key actors of this process trying to identify the best perspectives.

COVID-19 Impact on Musculoskeletal Regenerative Medicine Research: Maintaining Lab Continuity.

BACKGROUND: Research in the fields of musculoskeletal tissue engineering and regenerative medicine may suffer a slowdown during the ongoing COVID-19 pandemic emergency. This is likely to harm the development of new therapeutic strategies and their translation into the clinic in the long term. Recently, the need to maintain continuity in research activities in those fields has assumed even greater importance due to the accumulation of data concerning the effects of SARS-CoV-2 on the musculoskeletal system. This study is aimed at the identification of a series of safe handling practices against COVID-19 diffusion to apply in a research environment, thus allowing the maintenance of research lab activities. METHODS: The control measures to apply to mitigate the COVID-19 risk were identified and categorized utilizing the Hierarchy of Controls. We also compared our analysis with that assessed before the pandemic to consider the additional risk of COVID-19. RESULTS: Results highlighted that the most relevant implemented measures to control SARS-CoV-2 were based on protecting people through engineering (e.g., ventilation and social distancing), and administrative (e.g., hand sanitization, work shifts) measures or Personnel Protective Equipment, rather than eliminating hazards at the source (e.g., smart working). CONCLUSIONS: Work continuity in research labs during the COVID-19 emergency should be guaranteed by ensuring the protection of researchers in the workplace and considering the physical environment, the type of operators and work activity, and the proven ability of workers to face biological risks. The increased knowledge and awareness on lab' risks should be useful to prevent and mitigate future viral outbreaks.

Cartilage Tissue Engineering by Extrusion Bioprinting: Process Analysis, Risk Evaluation, and Mitigation Strategies.

Extrusion bioprinting is considered promising in cartilage tissue engineering since it allows the fabrication of complex, customized, and living constructs potentially suitable for clinical applications. However, clinical translation is often complicated by the variability and unknown/unsolved issues related to this technology. The aim of this study was to perform a risk analysis on a research process, consisting in the bioprinting of a stem cell-laden collagen bioink to fabricate constructs with cartilage-like properties. The method utilized was the Failure Mode and Effect Analysis/Failure Mode and Effect Criticality Analysis (FMEA/FMECA) which foresees a mapping of the process to proactively identify related risks and the mitigation actions. This proactive risk analysis allowed the identification of forty-seven possible failure modes, deriving from seventy-one potential causes. Twenty-four failure modes displayed a high-risk level according to the selected evaluation criteria and threshold (RPN > 100). The results highlighted that the main process risks are a relatively low fidelity of the fabricated structures, unsuitable parameters/material properties, the death of encapsulated cells due to the shear stress generated along the nozzle by mechanical extrusion, and possible biological contamination phenomena. The main mitigation actions involved personnel training and the implementation of dedicated procedures, system calibration, printing conditions check, and, most importantly, a thorough knowledge of selected biomaterial and cell properties that could be built either through the provided data/scientific literature or their preliminary assessment through dedicated experimental optimization phase. To conclude, highlighting issues in the early research phase and putting in place all the required actions to mitigate risks will make easier to develop a standardized process to be quickly translated to clinical use.

A Roadmap of In Vitro Models in Osteoarthritis: A Focus on Their Biological Relevance in Regenerative Medicine.

Osteoarthritis (OA) is a multifaceted musculoskeletal disorder, with a high prevalence worldwide. Articular cartilage and synovial membrane are among the main biological targets in the OA microenvironment. Gaining more knowledge on the accuracy of preclinical in vitro OA models could open innovative avenues in regenerative medicine to bridge major gaps, especially in translation from animals to humans. Our methodological approach entailed searches on Scopus, the Web of Science Core Collection, and EMBASE databases to select the most relevant preclinical in vitro models for studying OA. Predicting the biological response of regenerative strategies requires developing relevant preclinical models able to mimic the OA milieu influencing tissue responses and organ complexity. In this light, standard 2D culture models lack critical properties beyond cell biology, while animal models suffer from several limitations due to species differences. In the literature, most of the in vitro models only recapitulate a tissue compartment, by providing fragmented results. Biotechnological advances may enable scientists to generate new in vitro models that combine easy manipulation and organ complexity. Here, we review the state-of-the-art of preclinical in vitro models in OA and outline how the different preclinical systems (inflammatory/biomechanical/microfluidic models) may be valid tools in regenerative medicine, describing their pros and cons. We then discuss the prospects of specific and combinatorial models to predict biological responses following regenerative approaches focusing on mesenchymal stromal cells (MSCs)-based therapies to reduce animal testing.

Learning from Monocyte-Macrophage Fusion and Multinucleation: Potential Therapeutic Targets for Osteoporosis and Rheumatoid Arthritis.

Excessive bone resorption by osteoclasts (OCs) covers an essential role in developing bone diseases, such as osteoporosis (OP) and rheumatoid arthritis (RA). Monocytes or macrophages fusion and multinucleation (M-FM) are key processes for generating multinucleated mature cells with essential roles in bone remodelling. Depending on the phenotypic heterogeneity of monocyte/macrophage precursors and the extracellular milieu, two distinct morphological and functional cell types can arise mature OCs and giant cells (GCs). Despite their biological relevance in several physiological and pathological responses, many gaps exist in our understanding of their formation and role in bone, including the molecular determinants of cell fusion and multinucleation. Here, we outline fusogenic molecules during M-FM involved in OCs and GCs formation in healthy conditions and during OP and RA. Moreover, we discuss the impact of the inflammatory milieu on modulating macrophages phenotype and their differentiation towards mature cells. Methodological approach envisaged searches on Scopus, Web of Science Core Collection, and EMBASE databases to select relevant studies on M-FM, osteoclastogenesis, inflammation, OP, and RA. This review intends to give a state-of-the-art description of mechanisms beyond osteoclastogenesis and M-FM, with a focus on OP and RA, and to highlight potential biological therapeutic targets to prevent extreme bone loss.

Articular Cartilage Regeneration in Osteoarthritis.

There has been considerable advancement over the last few years in the treatment of osteoarthritis, common chronic disease and a major cause of disability in older adults. In this pathology, the entire joint is involved and the regeneration of articular cartilage still remains one of the main challenges, particularly in an actively inflammatory environment. The recent strategies for osteoarthritis treatment are based on the use of different therapeutic solutions such as cell and gene therapies and tissue engineering. In this review, we provide an overview of current regenerative strategies highlighting the pros and cons, challenges and opportunities, and we try to identify areas where future work should be focused in order to advance this field.

3D printing of musculoskeletal tissues: impact on safety and health at work.

Additive manufacturing (commonly referred to as 3D printing) created an attractive approach for regenerative medicine research in musculoskeletal tissue engineering. Given the high number of fabrication technologies available, characterized by different working and physical principles, there are several related risks that need to be managed to protect operators. Recently, an increasing number of studies demonstrated that several types of 3D printers are emitters of ultrafine particles and volatile organic compounds whose harmful effects through inhalation, ingestion and skin uptake are known. Confirmation of danger of these products is not yet final, but this provides a basis to adopt preventive measures in agreement with the precautionary principle. The purpose of this investigation was to provide a useful tool to the researcher for managing the risks related to the use of different kinds of three-dimensional printers (3D printers) in the lab, especiallyconcerning orthopedic applications, and to define appropriate control measures. Particular attention was given to new emerging risks and to developing response strategies for a comprehensive coverage of the health and safety of operators.

Three-Dimensional Bioprinting of Cartilage by the Use of Stem Cells: A Strategy to Improve Regeneration.

Cartilage lesions fail to heal spontaneously, leading to the development of chronic conditions which worsen the life quality of patients. Three-dimensional scaffold-based bioprinting holds the potential of tissue regeneration through the creation of organized, living constructs via a "layer-by-layer" deposition of small units of biomaterials and cells. This technique displays important advantages to mimic natural cartilage over traditional methods by allowing a fine control of cell distribution, and the modulation of mechanical and chemical properties. This opens up a number of new perspectives including personalized medicine through the development of complex structures (the osteochondral compartment), different types of cartilage (hyaline, fibrous), and constructs according to a specific patient's needs. However, the choice of the ideal combination of biomaterials and cells for cartilage bioprinting is still a challenge. Stem cells may improve material mimicry ability thanks to their unique properties: the immune-privileged status and the paracrine activity. Here, we review the recent advances in cartilage three-dimensional, scaffold-based bioprinting using stem cells and identify future developments for clinical translation. Database search terms used to write this review were: "articular cartilage", "menisci", "3D bioprinting", "bioinks", "stem cells", and "cartilage tissue engineering".

Scaffolds for Bone Tissue Engineering: State of the art and new perspectives.

This review is intended to give a state of the art description of scaffold-based strategies utilized in Bone Tissue Engineering. Numerous scaffolds have been tested in the orthopedic field with the aim of improving cell viability, attachment, proliferation and homing, osteogenic differentiation, vascularization, host integration and load bearing. The main traits that characterize a scaffold suitable for bone regeneration concerning its biological requirements, structural features, composition, and types of fabrication are described in detail. Attention is then focused on conventional and Rapid Prototyping scaffold manufacturing techniques. Conventional manufacturing approaches are subtractive methods where parts of the material are removed from an initial block to achieve the desired shape. Rapid Prototyping techniques, introduced to overcome standard techniques limitations, are additive fabrication processes that manufacture the final three-dimensional object via deposition of overlying layers. An important improvement is the possibility to create custom-made products by means of computer assisted technologies, starting from patient's medical images. As a conclusion, it is highlighted that, despite its encouraging results, the clinical approach of Bone Tissue Engineering has not taken place on a large scale yet, due to the need of more in depth studies, its high manufacturing costs and the difficulty to obtain regulatory approval. PUBMED search terms utilized to write this review were: "Bone Tissue Engineering", "regenerative medicine", "bioactive scaffolds", "biomimetic scaffolds", "3D printing", "3D bioprinting", "vascularization" and "dentistry".

Standard operating procedure for the good manufacturing practice-compliant production of human bone marrow mesenchymal stem cells.

According to the European Regulation (EC 1394/2007), Mesenchymal Stem Cells expanded in culture for clinical use are considered as Advanced Therapy Medicinal Products. As a consequence, they must be produced in compliance with Good Manufacturing Practice in order to ensure safety, reproducibility, and efficacy. Here, we report a Standard Operating Procedure describing the Good Manufacturing Practice-compliant production of Bone Marrow-derived Mesenchymal Stem Cells suitable for autologous implantation in humans. This procedure can be considered as a template for the development of investigational medicinal Mesenchymal Stem Cells-based product protocols to be enclosed in the dossier required for a clinical trial approval. Possible clinical applications concern local uses in the regeneration of bone tissue in nonunion fractures or in orthopedic and maxillofacial diseases characterized by a bone loss.

Media fill for validation of a good manufacturing practice-compliant cell production process.

According to the European Regulation EC 1394/2007, the clinical use of Advanced Therapy Medicinal Products, such as Human Bone Marrow Mesenchymal Stem Cells expanded for the regeneration of bone tissue or Chondrocytes for Autologous Implantation, requires the development of a process in compliance with the Good Manufacturing Practices. The Media Fill test, consisting of a simulation of the expansion process by using a microbial growth medium instead of the cells, is considered one of the most effective ways to validate a cell production process. Such simulation, in fact, allows to identify any weakness in production that can lead to microbiological contamination of the final cell product as well as qualifying operators. Here, we report the critical aspects concerning the design of a Media Fill test to be used as a tool for the further validation of the sterility of a cell-based Good Manufacturing Practice-compliant production process.

Cell manipulation in autologous chondrocyte implantation: from research to cleanroom.

In the field of orthopaedics, autologous chondrocyte implantation is a technique currently used for the regeneration of damaged articular cartilage. There is evidence of the neo-formation of tissue displaying characteristics similar to hyaline cartilage. In vitro chondrocyte manipulation is a crucial phase of this therapeutic treatment consisting of different steps: cell isolation from a cartilage biopsy, expansion in monolayer culture and growth onto a three-dimensional biomaterial to implant in the damaged area. To minimise the risk of in vitro cell contamination, the manipulation must be performed in a controlled environment such as a cleanroom. Moreover, the choice of reagents and raw material suitable for clinical use in humans and the translation of research protocols into standardised production processes are important. In this study we describe the preliminary results obtained by the development of chondrocyte manipulation protocols (isolation and monolayer expansion) in cleanrooms for the application of autologous implantation.

Induction of original phenotype of human immortalized chondrocytes: a quantitative gene expression analysis.

We previously established a line of immortalized normal human articular chondrocytes, lbpva55, expressing the E6 and E7 transforming genes of the human papilloma virus type 16. With this study we investigated the phenotypic modulation ability of this cell line, cultured in different conditions, with the aim of validating its use for studies on cartilage metabolism and physiology. To this end, we performed a quantitative analysis, using real-time PCR technology, of the expression of the main structural components of the cartilage matrix (collagens I, II and aggrecan), of two transcription factors regulating chondrocyte differentiation (Sox-9 and Egr-1) and of some enzymes involved in matrix turnover (cathepsin B, MMP-1 and MMP-13). Results showed that, under defined conditions, lbpva55 cells were able to re-express the chondrocyte phenotype that was lost in a conventional monolayer condition, as demonstrated by an up-regulation of collagen II, the main marker of hyaline cartilage and Sox-9, a master gene regulator of chondrocytic differentiation. The gene expression profile of our immortalized cells compared with that of normal articular chondrocytes showed that this line could be used as a valid in vitro model for a better understanding of cell molecular mechanisms relevant for the development of new therapeutic approaches in rheumatic diseases and for the cartilage engineering field.

Human chondrocytes and mesenchymal stem cells grown onto engineered scaffold.

The development of improved methods for treatment of chondral defects using autologous cells in combination with biomaterials leads to a new generation of implantable devices. Their association gives rise to a hybrid construct combining biological and material components that can be specifically committed. The comprehension of cellular and molecular mechanisms of cartilage repair and the use of biomaterials in combination with chondrocytes or mesenchymal stem cells in the treatment of cartilage defects has opened a new era of therapeutical strategies. Recently, their applicability in the treatment of early lesions in osteoarthritis is under investigation. To obtain new information on the behaviour of chondrocytes and mesenchymal stem cells grown on a hyaluronan derivative scaffold (Hyaff-11) already used in cartilage repair, we analysed a series of molecules expressed by these cells by Real-Time RT-PCR and immunohistochemical analyses. The data obtained with this work showed that this biomaterial is able to reduce the expression of some catabolic molecules by human chondrocytes and provide a good environment to support the differentiation of mesenchymal stem cells in chondrogenic sense. These observations confirm Hyaff-11 as a suitable scaffold both for chondrocytes and mesenchymal stem cells for the treatment of articular cartilage defects.

Transplantation of chondrocytes seeded on collagen-based scaffold in cartilage defects in rabbits.

Recent success in tissue engineering by restoring cartilage defects by transplanting autologous chondrocyte cells on a three-dimensional scaffold has prompted the improvement of this therapeutic strategy. Here we describe a new approach investigating the healing of rabbit cartilage by means of autologous chondrocytes seeded on a biomaterial made of an equine collagen type I-based scaffold. Full-thickness defects were created bilaterally in the weight-bearing surface of the medial femoral condyle of both femora of New Zealand male rabbits. The wounds were then repaired by using both chondrocytes seeded on the biomaterial and biomaterial alone. Controls were similarly treated but received either no treatment or implants of the delivery substance. Histological examination of the reconstructed tissues at 1, 3, 6, and 12 months after transplantation showed that at 1 and 3 months there was no formation of reconstructed tissue in any of the groups evaluated; after 6 months there was evidence of a newly regenerated tissue with some fibrocartilaginous features only in the group treated with biomaterial-seeded cells, and at 12 months a more organized tissue was evident in the same group. With regards to the group transplanted with biomaterial alone and the untreated control group, there was no evidence of new tissue production. These results advocate the use of this collagen-based scaffold for further in vivo studies on large size animals and, finally, in human clinical trials for the treatment of knee cartilage defects.

Down regulation of degenerative cartilage molecules in chondrocytes grown on a hyaluronan-based scaffold.

Hyaluronic-acid-based biomaterials used for cartilage repair allow the expression of specific extracellular matrix molecules by human chondrocytes grown onto them. We investigated whether these biomaterials could also create an environment in which the cells downregulate the expression of some catabolic factors. Chondrocytes were isolated from human articular cartilage obtained from the knees of patients with a history of trauma. First, the cells were expanded in monolayers and then they were seeded on a hyaluronic-acid derivative scaffold. Constructs and surnatants were collected and analysed at 1, 3, 7, 14 and 21 days after seeding. Immunohistochemical analysis for CD44 and caspase was carried out on paraffin-embedded sections. The Tunel method was used to identify chondrocyte apoptosis status. Secretion of MMP-1 and MMP-13 in the surnatants of the cells grown onto the biomaterial was measured by enzyme-linked immunosorbent assay. Nitric oxide (NO) production was evaluated by estimating the stable NO metabolite nitrite by the Griess method. A real-time RT-PCR analysis was performed on the constructs to evaluate the expression of type I and II collagens, aggrecan, Sox-9, MMP-1 and MMP-13 mRNAs at the different experimental times evaluated. Decreased levels of metalloproteinases and nitric oxide were observed in the surnatants of chondrocytes grown onto the hyaluronan-based scaffold. This was also confirmed by real-time PCR analysis which showed that the cells expressed the specific differentiated phenotype downregulating the expression of some catabolic molecules. Cells apoptosis decreased during the culture period, which further supported the biochemical data. The ability of the hyaluronan scaffold to reduce the expression and production of molecules involved in cartilage degenerative diseases indicates its use to treat early lesions of osteoarthritic patients.

Molecular and immunohistological characterization of human cartilage two years following autologous cell transplantation.

BACKGROUND: There are only a few studies concerning the cellular, biochemical, and genetic processes that occur during the remodeling of graft tissue after autologous chondrocyte transplantation. The purpose of the present study was to quantify the expression of genes encoding extracellular matrix proteins and regulatory factors that are essential for cell differentiation in cartilage biopsy specimens from patients who had this treatment two years previously. METHODS: Two cartilage biopsy specimens from each of four patients who had been treated with autologous chondrocyte transplantation and from two multiorgan donors were used. Real-time reverse transcriptase-polymerase chain reaction analysis was performed to evaluate the expression of types I, II, and X collagen; aggrecan; cathepsin B; and early growth response protein-1 (Egr-1) and Sry-type high-mobility-group box transcription factor-9 (Sox-9) mRNAs. Immunohistochemical analysis for matrix proteins and regulatory proteins was carried out on paraffin-embedded sections. RESULTS: Type-I collagen mRNA was expressed in all of the samples evaluated. Type-II collagen was present in autologous chondrocyte transplantation samples but at lower levels than in the controls. Type-X collagen messenger was undetectable. Aggrecan mRNA was present in all of the samples at lower levels than in the controls, while cathepsin-B messenger levels were higher and Egr-1 and Sox-9 mRNAs were expressed at lower levels. The immunohistochemical analysis showed slight positivity for type-I collagen in all of the sections. Type-II collagen was found in all of the samples with positivity confined inside the cells, while the controls displayed a positivity that was diffuse in the extracellular matrix. Cathepsin B was slightly positive in all of the samples, while the controls were negative. Egr-1 protein was particularly evident in the areas negative for type-II collagen. Sox-9 was positive in all samples, with evident localization in the superficial and middle layers. CONCLUSIONS: In biopsy specimens from autologous chondrocyte transplantation tissue at two years, there is evidence of the formation of new tissue, which displays varying degrees of organization with some fibrous and fibrocartilaginous features. Long-term follow-up investigations are needed to verify whether, once all of the remodeling processes are completed, the newly formed tissue will acquire the more typical features of articular cartilage.

Enhanced lipid peroxidation in synoviocytes from patients with osteoarthritis.

OBJECTIVE: To evaluate the degree of lipid peroxidation of synoviocytes from patients with rheumatoid arthritis (RA), osteoarthitis (OA), and controls and to look at the production of nitric oxide (NO) and its involvement in this process. METHODS: Human synoviocytes were isolated from synovial tissues from patients with RA, OA, and from healthy controls. Cells were maintained in culture for up to 3 culture passages. Lipid peroxidation, verified by the production of malonaldehyde (MDA) and 4-hydroxy-2(E)-nonenal (4-HNE), was determined by colorimetric assay. NO was evaluated by estimating the stable NO metabolite nitrite by the Griess method in the supernatants of unstimulated and interleukin (IL)-1beta and tumor necrosis factor (TNF)-a stimulated cells. RESULTS: Increased levels of lipid peroxidation were observed for OA-derived synoviocytes compared to RA and controls. The cells in each experimental group produced low amounts of NO both in basal and in stimulated conditions. CONCLUSION: In OA, synovial cells underwent a lipid peroxidation process that did not occur in synoviocytes from RA or controls even in the absence of a detectable production of the reactive nitrogen intermediate NO. We can postulate that this peroxidation process might be due to the action of NO secreted by chondrocytes that are known to produce higher levels of this radical in OA compared to RA.

Cathepsin B as a soluble marker to monitor the phenotypic stability of engineered cartilage.

The clinical need for improved human autologous chondrocyte transplantation has motivated the use of different biomaterials, which are aimed at fixing the cells in the defect area and permit their proliferation and differentiation. The maintenance of the original phenotype by isolated chondrocytes grown in vitro is an important requisite for their use in repairing damaged articular cartilage. The methods to verify the expression of cartilage-specific molecules usually involve destructive procedures to recover the cells from the scaffolds for tests. The aim of our study was to find a soluble marker able to attest the occurrence of a differentiation process by chondrocytes grown onto a biomaterial used for cell transplantation. We turned our attention to cathepsin B which is known to be abnormally synthesized in de-differentiated chondrocytes and scarcely produced in the differentiated ones. The production of cathepsin B by human articular chondrocytes expanded in vitro and then grown onto a hyaluronan-based polymer derivative (Hyaff-11) three-dimensional scaffold was evaluated with a specific enzyme-immunoassay at different experimental times together with the expression of mRNA by real-time PCR. We showed that cathepsin B, which is abundantly produced by chondrocytes grown in a monolayer culture, decreases significantly after the cells are seeded onto the scaffold, giving further evidence of a re-differentiation process. This result suggests cathepsin B a practical soluble marker to evaluate the "good" quality of transplantable constructs.