Regenerative Features of Adipose Tissue for Osteoarthritis Treatment in a Rabbit Model: Enzymatic Digestion Versus Mechanical Disruption

Evaluating cell migration after cell-based treatment is important for several disorders, including osteoarthritis (OA), as it might influence the clinical outcome. This research explores migrating expanded-adipose stromal cells (ASCs) and adipose niches after enzymatic and mechanical processes. Bilateral anterior cruciate ligament transection induced a mild grade of OA at eight weeks in adult male New Zealand rabbits. ASCs, enzymatic stromal vascular fraction (SVF), and micro fragmented adipose tissue (MFAT) were intra-articularly injected in the knee joint. Assessments of cell viability and expression of specific markers, including CD-163 wound-healing macrophages, were done. Cell migration was explored through labelling with PKH26 dye at 7 and 30 days alongside co-localization analyses for CD-146. All cells showed good viability and high percentages of CD-90 and CD-146. CD-163 was significantly higher in MFAT compared to SVF. Distinct migratory potential and time-dependent effects were observed among cell-based treatments. At day 7, both ASCs and SVF migrated towards synovium, whereas for MFAT versus cartilage, a different migration pattern was noticed at day 30. The long-term distinct cell migration of ASCs, SVF, and MFAT open interesting clinical insights on their potential use for OA treatment. Moreover, the highest expression of CD-163 in MFAT, rather than SVF, might have an important role in directly mediating cartilage tissue repair responses

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

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

Hyaluronan scaffold supports osteogenic differentiation of bone marrow concentrate cells

Osteochondral lesions are considered a challenge for orthopedic surgeons. Currently, the treatments available are often unsatisfactory and unable to stimulate tissue regeneration. Tissue engineering offers a new therapeutic strategy, taking into account the role exerted by cells, biomaterial and growth factors in restoring tissue damage. In this light, Mesenchymal Stem Cells (MSCs) have been indicated as a fascinating tool for regenerative medicine thanks to their ability to differentiate into bone, cartilage and adipose tissue. However, in vitro-cultivation of MSCs could be associated with some risks such as de-differentiation/reprogramming, infection and contaminations of the cells. To overcome these shortcomings, a new approach is represented by the use of Bone Marrow Concentrate (BMC), that could allow the delivery of cells surrounded by their microenvironment in injured tissue. For this purpose, cells require a tridimensional scaffold that can support their adhesion, proliferation and differentiation. This study is focused on the potentiality of BMC seeded onto a hyaluronan-based scaffold (Hyaff-11) to differentiate into osteogenic lineage. This process depends on the specific interaction between cells derived from bone marrow (surrounded by their niche) and scaffold, that create an environment able to support the regeneration of damaged tissue. The data obtained from the present study demonstrate that BMC grown onto Hyaff-11 are able to differentiate toward osteogenic sense, producing specific osteogenic genes and matrix proteins

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”

Overview of Anti-Inflammatory and Anti-Nociceptive Effects of Polyphenols to Halt Osteoarthritis: From Preclinical Studies to New Clinical Insights

Knee osteoarthritis (OA) is one of the most multifactorial joint disorders in adults. It is characterized by degenerative and inflammatory processes that are responsible for joint destruction, pain and stiffness. Despite therapeutic advances, the search for alternative strategies to target inflammation and pain is still very challenging. In this regard, there is a growing body of evidence for the role of several bioactive dietary molecules (BDMs) in targeting inflammation and pain, with promising clinical results. BDMs may be valuable non-pharmaceutical solutions to treat and prevent the evolution of early OA to more severe phenotypes, overcoming the side effects of anti-inflammatory drugs. Among BDMs, polyphenols (PPs) are widely studied due to their abundance in several plants, together with their benefits in halting inflammation and pain. Despite their biological relevance, there are still many questionable aspects (biosafety, bioavailability, etc.) that hinder their clinical application. This review highlights the mechanisms of action and biological targets modulated by PPs, summarizes the data on their anti-inflammatory and anti-nociceptive effects in different preclinical in vitro and in vivo models of OA and underlines the gaps in the knowledge. Furthermore, this work reports the preliminary promising results of clinical studies on OA patients treated with PPs and discusses new perspectives to accelerate the translation of PPs treatment into the clinics

Modeling and fabrication of silk fibroin-gelatin-based constructs using extrusion-based three-dimensional bioprinting

Robotic dispensing-based 3D bioprinting represents one of the most powerful technologies to develop hydrogel-based 3D constructs with enormous potential in the field of regenerative medicine. The optimization of hydrogel printing parameters, proper geometry and internal architecture of the constructs, and good cell viability during the bioprinting process are the essential requirements. In this paper, an analytical model based on the hydrogel rheological properties was developed to predict the extruded filament width in order to maximize the printed structure's fidelity to the design. Viscosity data of two natural hydrogels were imputed to a power-law model to extrapolate the filament width. Further, the model data were validated by monitoring the obtained filament width as the output. Shear stress values occurring during the bioprinting process were also estimated. Human mesenchymal stromal cells (hMSCs) were encapsulated in the silk fibroin-gelatin (G)-based hydrogel, and a 3D bioprinting process was performed to produce cell-laden constructs. Live and dead assay allowed estimating the impact of needle shear stress on cell viability after the bioprinting process. Finally, we tested the potential of hMSCs to undergo chondrogenic differentiation by evaluating the cartilaginous extracellular matrix production through immunohistochemical analyses. Overall, the use of the proposed analytical model enables defining the optimal printing parameters to maximize the fabricated constructs' fidelity to design parameters before the process execution, enabling to achieve more controlled and standardized products than classical trial-and-error approaches in the biofabrication of engineered constructs. Employing modeling systems exploiting the rheological properties of the hydrogels might be a valid tool in the future for guaranteeing high cell viability and for optimizing tissue engineering approaches in regenerative medicine applications

A 3D Collagen-Based Bioprinted Model to Study Osteosarcoma Invasiveness and Drug Response

The biological and therapeutic limits of traditional 2D culture models, which only partially mimic the complexity of cancer, have recently emerged. In this study, we used a 3D bioprinting platform to process a collagen-based hydrogel with embedded osteosarcoma (OS) cells. The human OS U-2 OS cell line and its resistant variant (U-2OS/CDDP 1 μg) were considered. The fabrication parameters were optimized to obtain 3D printed constructs with overall morphology and internal microarchitecture that accurately match the theoretical design, in a reproducible and stable process. The biocompatibility of the 3D bioprinting process and the chosen collagen bioink in supporting OS cell viability and metabolism was confirmed through multiple assays at short- (day 3) and long- (day 10) term follow-ups. In addition, we tested how the 3D collagen-based bioink affects the tumor cell invasive capabilities and chemosensitivity to cisplatin (CDDP). Overall, we developed a new 3D culture model of OS cells that is easy to set up, allows reproducible results, and better mirrors malignant features of OS than flat conditions, thus representing a promising tool for drug screening and OS cell biology research

Repair Potential of Matrix-Induced Bone Marrow Aspirate Concentrate and Matrix-Induced Autologous Chondrocyte Implantation for Talar Osteochondral Repair: Patterns of Some Catabolic, Inflammatory, and Pain Mediators

Objective: The low regenerative potential of cartilage contributed to the development of different cell therapies aimed to improve the clinical outcome in young patients with Osteochondral Lesions of the Talus (OLT). This study is designed to assess the regenerative potential of autologous matrix-induced Bone Marrow Aspirate Concentrate (mBMAC) and matrix-induced Autologous Chondrocyte Implantation (mACI) evaluating, on a small number of osteochondral biopsies, the expression of some catabolic, inflammatory, and pain mediators. Design: Twenty-two patients with OLT were analyzed in this study; 7 were treated with mACI and 15 with mBMAC. Informed consent was obtained from all the patients. Clinical assessments were performed pre-operatively and at 12, 24, and 36 months after surgery using the American Orthopedic Foot and Ankle Society (AOFAS). Histology and immunohistochemistry were used to assess cartilage repair at 24 months. Data were analyzed using non-parametric Wilcoxon-Mann-Whitney and Spearman tests. Results: A remarkable improvement in AOFAS score was noticed for both treatments up to 36 months; however, patients treated with mACI reported the best AOFAS score. Various degrees of tissue remodeling were observed by histological analysis for both cell strategies. However, mBMAC treatment showed a higher expression of some fibrous and hypertrophic markers compared to mACI group. A mild positivity for nerve growth factor, as pain mediator, was noticed for both treatments.M Conclusions: Our findings demonstrated the best histological and clinical results following mACI treatment since different fibrotic and hypertrophic features were evident in the mBMAC group at 24-month follow-up