Low-density cultured cartilage cells expanded in platelet lysate present distinct features to develop an innovative clinical treatment for diffuse cartilage lesions.

PURPOSE: Chondrocyte-based cell therapies are effective for the treatment of chondral lesions, but remain poorly indicated for diffuse lesions in the context of early osteoarthritis (OA). The aim of this study was to develop a protocol to obtain chondroprogenitor cells suitable for the treatment of diffuse chondral lesions within early OA. METHODS: Cartilage cells were expanded at low density in human platelet lysate (hPL). A test was performed to exclude senescence. The expression of surface cluster of differentiation 146, cluster of differentiation 166, major histocompatibility complex (MHC)-I and MHC-II and of genes of interest were evaluated, as well as the trophic potential of these cells, by the assessment of lubricin and matrix production. The immunomodulatory potential was assessed through their co-culture with macrophages. RESULTS: Cartilage cells expanded at low density in hPL showed higher proliferation rate than standard-density cells, no replicative senescence, low immunogenicity and expression of lubricin. Moreover, they presented an increased expression of chondrogenic and antihypertrophic markers, as well as a superior matrix deposition if compared to cells cultured at standard density. Cartilage cells induced on macrophages an upregulation of CD206, although a higher increase of CD163 expression was observed in the presence of low-density cells. CONCLUSIONS: These findings lay the grounds to explore the clinical usefulness of low-density cultured cartilage cells to treat diffuse lesions in early OA joints for both autologous and allogenic use. LEVEL OF EVIDENCE: Not applicable.

A personalized osteoarthritic joint-on-a-chip as a screening platform for biological treatments.

Osteoarthritis (OA) is a highly disabling pathology, characterized by synovial inflammation and cartilage degeneration. Orthobiologics have shown promising results in OA treatment thanks to their ability to influence articular cells and modulate the inflammatory OA environment. Considering their complex mechanism of action, the development of reliable and relevant joint models appears as crucial to select the best orthobiologics for each patient. The aim of this study was to establish a microfluidic OA model to test therapies in a personalized human setting. The joint-on-a-chip model included cartilage and synovial compartments, containing hydrogel-embedded chondrocytes and synovial fibroblasts, separated by a channel for synovial fluid. For the cartilage compartment, a Hyaluronic Acid-based matrix was selected to preserve chondrocyte phenotype. Adding OA synovial fluid induced the production of inflammatory cytokines and degradative enzymes, generating an OA microenvironment. Personalized models were generated using patient-matched cells and synovial fluid to test the efficacy of mesenchymal stem cells on OA signatures. The patient-specific models allowed monitoring changes induced by cell injection, highlighting different individual responses to the treatment. Altogether, these results support the use of this joint-on-a-chip model as a prognostic tool to screen the patient-specific efficacy of orthobiologics.

Using Macrophage Polarization in Human Platelet Lysate to Test the Immunomodulatory Potential of Cells for Clinical Use.

Macrophage-based co-cultures are used to test the immunomodulatory function of candidate cells for clinical use. This study aimed to characterize a macrophage polarization model using human platelet lysate (hPL) as a GMP-compliant alternative to Fetal Bovine Serum (FBS). Primary human monocytes were differentiated into unpolarized (M0) or polarized (M1, M2a, and M2c) macrophages in an hPL- or FBS-based medium. The protein secretion profiles and expression of phenotypic markers (CD80 for M1, CD206 for M2a, and CD163 for M2c) were analyzed. Subsequently, chondrocytes were tested in an hPL-based co-culture model to assess their immunomodulatory function in view of their possible use in patients with osteoarthritis. The results showed similar marker regulation between hPL and FBS cultures, but lower basal levels of CD206 and CD163 in hPL-cultured macrophages. Functional co-culture experiments with chondrocytes revealed increased CD206 expression both in hPL and in FBS, indicating an interaction between macrophages and chondrocytes. While markers in FBS-cultured macrophages were confirmed in hPL-cultured cells, the interpretation of marker modulation in immunomodulatory assays with hPL-based cultures should be carried out cautiously due to the observed differences in the basal marker levels for CD206 and CD163. This research underscores the utility of hPL as a GMP-compliant alternative to FBS for macrophage-based co-cultures and highlights the importance of understanding marker expressions in different culture conditions.

Assessing the response of human primary macrophages to defined fibrous architectures fabricated by melt electrowriting.

The dual role of macrophages in the healing process depends on macrophage ability to polarize into phenotypes that can propagate inflammation or exert anti-inflammatory and tissue-remodeling functions. Controlling scaffold geometry has been proposed as a strategy to influence macrophage behavior and favor the positive host response to implants. Here, we fabricated Polycaprolactone (PCL) scaffolds by Melt Electrowriting (MEW) to investigate the ability of scaffold architecture to modulate macrophage polarization. Primary human macrophages unpolarized (M0) or polarized into M1, M2a, and M2c phenotypes were cultured on PCL films and MEW scaffolds with pore geometries (square, triangle, and rhombus grid) characterized by different angles. M0, M2a, and M2c macrophages wrapped along the fibers, while M1 macrophages formed clusters with rounded cells. Cell bridges were formed only for angles up to 90°. No relevant differences were found among PCL films and 3D scaffolds in terms of surface markers. CD206 and CD163 were highly expressed by M2a and M2c macrophages, with M2a macrophages presenting also high levels of CD86. M1 macrophages expressed moderate levels of all markers. The rhombus architecture promoted an increased release by M2a macrophages of IL10, IL13, and sCD163 compared to PCL films. The proangiogenic factor IL18 was also upregulated by the rhombus configuration in M0 and M2a macrophages compared to PCL films. The interesting findings obtained for the rhombus architecture represent a starting point for the design of scaffolds able to modulate macrophage phenotype, prompting investigations addressed to verify their ability to facilitate the healing process in vivo.

Advances in Musculoskeletal Cell Therapy: Basic Science and Translational Approaches.

Nowadays, the real need in orthopedic research is to strictly validate advanced regenerative medicine approaches in preclinical models, with the hope that this unique and straightforward approach can facilitate a safe and effective translation into everyday clinical practice [...].

Inflammatory priming with IL-1ß promotes the immunomodulatory behavior of adipose derived stem cells.

Inflammatory processes contribute to osteoarthritis (OA) severity and progression. Mesenchymal stem cells, particularly those derived from adipose tissue (ASCs), are able to sense and control the inflammatory environment. This immunomodulatory potential can be boosted by different priming strategies based on inflammatory stimulation. The aim of the present study is to investigate the transcriptional modulation of a huge panel of genes and functionally verify the predicted immunomodulatory ability of ASCs after interleukin one beta (IL-1ß) priming. ASCs were isolated from adipose tissue obtained from three donors and expanded. After stimulation with 1 ng/ml of IL-1ß for 48 h, cells were collected for gene array and functional tests. Pooled cells from three donors were used for RNA extraction and gene array analysis. Gene Ontology (GO) enrichment analysis and Gene Set Enrichment Analysis (GSEA) were performed to assess the involvement of the modulated genes after priming in specific biological processes and pathways. Functional co-culture tests of ASCs with T cells and macrophages were performed to assess the ability of primed ASCs to modulate immune cell phenotype. Among the overall genes analyzed in the gene array, about the 18% were up- or down-regulated in ASCs after IL-1ß priming. GO enrichment analysis of up- or down-regulated genes in ASCs after IL-1ß priming allowed identifying specific pathways involved in the modulation of inflammation and extracellular matrix remodeling. The main processes enriched according to the GSEA are related to the inflammatory response and cell proliferative processes. Functional tests on immune cells showed that primed and non-primed ASCs induced a decrease in the CD3+ T lymphocytes survival rate and an anti-inflammatory macrophage polarization. In conclusion, IL-1ß priming represents a tailored strategy to enhance the ability of ASCs to direct macrophages towards an anti-inflammatory phenotype and, consequently, improve the efficacy of ASCs in counteracting the OA inflammatory component.

Immunomodulatory potential of secretome from cartilage cells and mesenchymal stromal cells in an arthritic context: From predictive fiction toward reality.

The purpose of the present study is to predict by bioinformatics the activity of the extracellular vesicle (EV)-embedded micro RNA (miRNAs) secreted by cartilage cells (CCs), adipose tissue-derived- (ASCs), and bone marrow-derived stem cells (BMSCs) and verify their immunomodulatory potential supporting our bioinformatics findings to optimize the autologous cell-based therapeutic strategies for osteoarthritis (OA) management. Cells were isolated from surgical waste tissues of three patients who underwent total hip replacement, expanded and the EVs were collected. The expression of EV-embedded miRNA was evaluated with the QuantStudio 12 K Flex OpenArray® platform. Mientournet and ingenuity pathway analysis (IPA) were used for validated target prediction analysis and to identify miRNAs involved in OA and inflammation. Cells shared the expression of 325 miRNAs embedded in EVs and differed for the expression of a small number of them. Mienturnet revealed no results for miRNAs selectively expressed by ASCs, whereas miRNA expressed by CCs and BMSCs were putatively involved in the modulation of cell cycle, senescence, apoptosis, Wingless and Int-1 (Wnt), transforming growth factor beta (TGFß), vascular endothelial growth factor (VEGF), Notch, Hippo, tumor necrosis factor alpha (TNFα), interleukin 1 beta (IL-1ß), insulin like growth factor 1 (IGF-1), RUNX family transcription factor 2 (RUNX2), and endochondral ossification pathways. Cartilage homeostasis, macrophages and T cells activity and inflammatory mediators were identified by IPA as targets of the miRNAs found in all the cell populations. Co-culture tests on macrophages and T cells confirmed the immuno-modulatory ability of CCs, ASCs, and BMSCs. The study findings support the rationale behind the use of cell-based therapy for the treatment of OA.

Systematic review and meta-analysis on the use of human platelet lysate for mesenchymal stem cell cultures: comparison with fetal bovine serum and considerations on the production protocol.

Mesenchymal stem cell (MSC) culturing for cell therapies needs a step forward to be routinely used in clinical settings. Main concerns regard the use of animal origin reagents, in particular supplementing the culture medium with FBS. Lately, Human Platelet Lysate (HPL) has been proposed as animal-free alternative, described as an excellent supplement for culturing MSCs. The aim of this systematic review was to analyze the current literature on the effect of HPL and FBS on ASCs and BMSCs. The primary outcome was the proliferation rate of cells cultured with FBS and HPL. Differences in terms of doubling time (DT) and population doubling (PD) were evaluated by meta-analysis, subgrouping data according to the cell type. A total of 35 articles were included. BMSCs and ASCs were used in 65.7% (23) and 28.6% (10) studies, respectively. Only two studies included both cell types. Overall, 22 studies were eligible for the meta-analysis. Among them, 9 articles described ASCs and 13 BMSCs. The results showed that BMSCs and ASCs cultured with 10% HPL and 5% HPL have lower DT and higher PD compared to cells cultured with 10% FBS. A possible correlation between the DT decrease and the application of at least 3 freeze/thaw cycles to induce platelet lysis was found. Additionally, HPL increased VEGF secretion and maintained the immuno-modulatory abilities for both cell types. The clarification reported here of the higher efficiency of HPL compared to FBS can help the transition of the scientific community towards clinical-related procedures. 1. The meta-analysis shows that HPL induces a population doubling increase and a doubling time decrease of both ASCs and BMSCs compared to FBS. 2. When at least 3 freeze/thaw cycles are applied to induce platelet lysis, the doubling time of HPL-cultured cells is lower than FBS-cultured cells (Created with BioRender.com).

Detailed Methodology to Establish a Synovium/Cartilage-on-a-Chip Model to Investigate the Process of Monocyte Extravasation.

Microfluidics allows for recapitulating organotypic environments in miniaturized cell culture platforms. This ability paves the way to the investigation of complex biological processes in a relevant milieu. Here we describe the protocols to generate an organotypic model including a vascularized compartment mimicking the synovial membrane and designed for the study of monocyte extravasation during osteoarthritis.

Vancomycin-nanofunctionalized peptide-enriched silk fibroin to prevent methicillin-resistant Staphylococcus epidermidis-induced femoral nonunions in rats.

Introduction: Implant-related infections and infected fractures are significant burdens in orthopedics. Staphylococcus epidermidis is one of the main causes of bone infections related to biofilm formation upon implants. Current antibiotic prophylaxis/therapy is often inadequate to prevent biofilm formation and results in antibiotic resistance. The development of bioactive materials combining antimicrobial and osteoconductive properties offers great potential for the eradication of microorganisms and for the enhancement of bone deposition in the presence of infections. The purpose of this study is to prevent the development of methicillin-resistant S. epidermidis (MRSE)-infected nonunion in a rat model. Methods: To this end, a recently developed in our laboratories bioactive material consisting of antibiotic-loaded nanoparticles based on carboxylic acid functionalized hyperbranched aliphatic polyester (CHAP) that are integrated into peptide-enriched silk fibroin sponges with osteoconductive properties (AFN-PSF) was employed, whose biocompatibility and microbiological tests provided proof of its potential for the treatment of both orthopedic and dental infections. In particular, non-critical femoral fractures fixed with plates and screws were performed in Wistar rats, which were then randomly divided into three groups: 1) the sham control (no infection, no treatment); 2) the control group, infected with MRSE and treated with peptide-enriched silk fibroin sponges incorporating non-drug-loaded functionalized nanoparticles (PSF); 3) the treated group, infected with MRSE and treated with peptide-enriched silk fibroin sponges incorporating vancomycin-loaded functionalized nanoparticles (AFN-PSF). After 8 weeks, bone healing and osteomyelitis were clinically assessed and evaluated by micro-CT, microbiological and histological analyses. Results: The sham group showed no signs of infection and complete bone healing. The PSF group failed to repair the infected fracture, displaying 75% of altered bone healing and severe signs of osteomyelitis. The AFN-PSF treated group reached 70% of fracture healing in the absence of signs of osteomyelitis, such as abscesses in the cortical and intraosseous compartments and bone necrosis with sequestra. Discussion: AFN-PSF sponges have proven effective in preventing the development of infected nonunion in vivo. The proposed nanotechnology for local administration of antibiotics can have a significant impact on patient health in the case of orthopedic infections.

Recapitulating monocyte extravasation to the synovium in an organotypic microfluidic model of the articular joint.

The synovium of osteoarthritis (OA) patients can be characterized by an abnormal accumulation of macrophages originating from extravasated monocytes. Since targeting monocyte extravasation may represent a promising therapeutic strategy, our aim was to develop an organotypic microfluidic model recapitulating this process. Synovium and cartilage were modeled by hydrogel-embedded OA synovial fibroblasts and articular chondrocytes separated by a synovial fluid channel. The synovium compartment included a perfusable endothelialized channel dedicated to monocyte injection. Monocyte extravasation in response to chemokines and OA synovial fluid was quantified. The efficacy of chemokine receptor antagonists, RS-504393 (CCR2 antagonist) and Cenicriviroc (CCR2/CCR5 antagonist) in inhibiting extravasation was tested pre-incubating monocytes with the antagonists before injection. After designing and fabricating the chip, culture conditions were optimized to achieve an organotypic model including synovial fibroblasts, articular chondrocytes, and a continuous endothelial monolayer expressing intercellular adhesion molecule-1 and vascular cell adhesion molecule-1. A significantly higher number of monocytes extravasated in response to the chemokine mix (p< 0.01) and OA synovial fluid (p< 0.01), compared to a control condition. In both cases, endothelium pre-activation enhanced monocyte extravasation. The simultaneous blocking of CCR2 and CCR5 proved to be more effective (p< 0.001) in inhibiting monocyte extravasation in response to OA synovial fluid than blocking of CCR2 only (p< 0.01). The study of extravasation in the model provided direct evidence that OA synovial fluid induces monocytes to cross the endothelium and invade the synovial compartment. The model can be exploited either to test molecules antagonizing this process or to investigate the effect of extravasated monocytes on synovium and cartilage cells.

Peptide-Enriched Silk Fibroin Sponge and Trabecular Titanium Composites to Enhance Bone Ingrowth of Prosthetic Implants in an Ovine Model of Bone Gaps.

Osteoarthritis frequently requires arthroplasty. Cementless implants are widely used in clinics to replace damaged cartilage or missing bone tissue. In cementless arthroplasty, the risk of aseptic loosening strictly depends on implant stability and bone-implant interface, which are fundamental to guarantee the long-term success of the implant. Ameliorating the features of prosthetic materials, including their porosity and/or geometry, and identifying osteoconductive and/or osteoinductive coatings of implant surfaces are the main strategies to enhance the bone-implant contact surface area. Herein, the development of a novel composite consisting in the association of macro-porous trabecular titanium with silk fibroin (SF) sponges enriched with anionic fibroin-derived polypeptides is described. This composite is applied to improve early bone ingrowth into the implant mesh in a sheep model of bone defects. The composite enables to nucleate carbonated hydroxyapatite and accelerates the osteoblastic differentiation of resident cells, inducing an outward bone growth, a feature that can be particularly relevant when applying these implants in the case of poor osseointegration. Moreover, the osteoconductive properties of peptide-enriched SF sponges support an inward bone deposition from the native bone towards the implants. This technology can be exploited to improve the biological functionality of various prosthetic materials in terms of early bone fixation and prevention of aseptic loosening in prosthetic surgery.

Advanced Microfluidic Models of Cancer and Immune Cell Extravasation: A Systematic Review of the Literature.

Extravasation is a multi-step process implicated in many physiological and pathological events. This process is essential to get leukocytes to the site of injury or infection but is also one of the main steps in the metastatic cascade in which cancer cells leave the primary tumor and migrate to target sites through the vascular route. In this perspective, extravasation is a double-edged sword. This systematic review analyzes microfluidic 3D models that have been designed to investigate the extravasation of cancer and immune cells. The purpose of this systematic review is to provide an exhaustive summary of the advanced microfluidic 3D models that have been designed to study the extravasation of cancer and immune cells, offering a perspective on the current state-of-the-art. To this end, we set the literature search cross-examining PUBMED and EMBASE databases up to January 2020 and further included non-indexed references reported in relevant reviews. The inclusion criteria were defined in agreement between all the investigators, aimed at identifying studies which investigate the extravasation process of cancer cells and/or leukocytes in microfluidic platforms. Twenty seven studies among 174 examined each step of the extravasation process exploiting 3D microfluidic devices and hence were included in our review. The analysis of the results obtained with the use of microfluidic models allowed highlighting shared features and differences in the extravasation of immune and cancer cells, in view of the setup of a common framework, that could be beneficial for the development of therapeutic approaches fostering or hindering the extravasation process.

Cellulose nanocrystals: a multimodal tool to enhance the targeted drug delivery against bone disorders.

Aim: We investigated the use of cellulose nanocrystals (CNCs) as drug nanocarriers combining an anti-osteoporotic agent, alendronate (ALN), and an anti-cancer drug, doxorubicin (DOX). Materials & methods: CNC physicochemical characterization, in vivo imaging coupled with histology and in vitro uptake and toxicity assays were carried out. Results:In vivo CNC-ALN did not modify bone tropism and lung penetration, whereas its liver and kidney accumulation was slightly higher compared with CNCs alone. In vitro studies showed that CNC-ALN did not impair ALN's effect on osteoclasts, whereas CNC-DOX confirmed the therapeutic potential against bone metastatic cancer cells. Conclusions: This study provides robust proof of the potential of CNCs as easy, flexible and specific carriers to deliver compounds to the bone.

Innovative Visualization and Quantification of Extracellular Vesicles Interaction with and Incorporation in Target Cells in 3D Microenvironments.

Extracellular vesicles (EVs) showed therapeutic properties in several applications, many in regenerative medicine. A clear example is in the treatment of osteoarthritis (OA), where adipose-derived mesenchymal stem cells (ASCs)-EVs were able to promote regeneration and reduce inflammation in both synovia and cartilage. A still obscure issue is the effective ability of EVs to be internalized by target cells, rather than simply bound to the extracellular matrix (ECM) or plasma membrane, since the current detection or imaging technologies cannot fully decipher it due to technical limitations. In the present study, human articular chondrocytes (ACHs) and fibroblast-like synoviocytes (FLSs) isolated from the same OA patients were cocultured in 2D as well as in 3D conditions with fluorescently labeled ASC-EVs, and analyzed by flow cytometry or confocal microscopy, respectively. In contrast with conventional 2D, in 3D cultures, confocal microscopy allowed a clear detection of the tridimensional morphology of the cells and thus an accurate discrimination of EV interaction with the external and/or internal cell environment. In both 2D and 3D conditions, FLSs were more efficient in interacting with ASC-EVs and 3D imaging demonstrated a faster uptake process. The removal of the hyaluronic acid component from the ECM of both cell types reduced their interaction with ASC-EVs only in the 2D system, showing that 2D and 3D conditions can yield different outcomes when investigating events where ECM plays a key role. These results indicate that studying EVs binding and uptake both in 2D and 3D guarantees a more precise and complementary characterization of the molecular mechanisms involved in the process. The implementation of this strategy can become a valuable tool not only for basic research, but also for release assays and potency prediction for clinical EV batches.

Microfluidic Biofabrication of 3D Multicellular Spheroids by Modulation of Non-geometrical Parameters.

Three-dimensional (3D) cell spheroids are being increasingly applied in many research fields due to their enhanced biological functions as compared to conventional two-dimensional (2D) cultures. 3D cell spheroids can replicate tissue functions, which enables their use both as in vitro models and as building blocks in tissue biofabrication approaches. In this study, we developed a perfusable microfluidic platform suitable for robust and reproducible 3D cell spheroid formation and tissue maturation. The geometry of the device was optimized through computational fluid dynamic (CFD) simulations to improve cell trapping. Experimental data were used in turn to generate a model able to predict the number of trapped cells as a function of cell concentration, flow rate, and seeding time. We demonstrated that tuning non-geometrical parameters it is possible to control the size and shape of 3D cell spheroids generated using articular chondrocytes (ACs) as cellular model. After seeding, cells were cultured under perfusion at different flow rates (20, 100, and 500 µl/min), which induced the formation of conical and spherical spheroids. Wall shear stress values on cell spheroids, computed by CFD simulations, increased accordingly to the flow rate while remaining under the chondroprotective threshold in all configurations. The effect of flow rate on cell number, metabolic activity, and tissue-specific matrix deposition was evaluated and correlated with fluid velocity and shear stress distribution. The obtained results demonstrated that our device represents a helpful tool to generate stable 3D cell spheroids which can find application both to develop advanced in vitro models for the study of physio-pathological tissue maturation mechanisms and to obtain building blocks for the biofabrication of macrotissues.

Organs-on-a-chip as model systems for multifactorial musculoskeletal diseases.

Multifactorial diseases affecting musculoskeletal tissues are characterized by the interactions between multiple tissues, such as muscle and nerves in neuromuscular diseases, or multiple cellular components in a tissue, as in the case of bone tumors, interacting with bone cells. For these diseases also the influence of different biophysical and biochemical stimuli, such as mechanical overload and inflammatory molecules in osteoarthritis, play a key role. To investigate these complex phenomena, organ-on-a-chip systems have been developed, taking into account specific disease characteristics such as being directly derived from patients, the presence of specifically mutated cells, or a combination of relevant biophysical and/or biochemical stimuli. Depending on the envisaged application, different issues remain to be addressed. In particular, improving automation and output sensors are key for drug screening applications, while refining model microarchitecture to enhance physiological fidelity is needed for more basic science studies.

Bone Marrow-Derived Cell Therapies to Heal Long-Bone Nonunions: A Systematic Review and Meta-Analysis-Which Is the Best Available Treatment?

Nonunions represent one of the major indications for clinical settings with stem cell-based therapies. The objective of this research was to systematically assess the current evidence for the efficacy of bone marrow-derived cell-based approaches associated or not with bone scaffolds for the treatment of nonunions. We searched MEDLINE (PubMed) and CENTRAL up to July 2019 for clinical studies focused on the use of cell-based therapies and bone marrow derivatives to treat bone nonunions. Three investigators independently extracted the data and appraised the risk of bias. We analysed 27 studies including a total number of 347 participants exposed to four interventions: bone marrow concentrate (BMAC), BMAC combined with scaffold (BMAC/Scaffold), bone marrow-derived mesenchymal stromal cells (BMSCs), and BMSC combined with scaffold (BMSC/Scaffold). Two controlled studies showed a positive trend in bone healing in favour of BMAC/Scaffold or BMSC/Scaffold treatment against bone autograft, although the difference was not statistically significant (RR 0.11, 95% CI -0.05; 0.28). Among single cohort studies, the highest mean pooled proportion of healing rate was reported for BMAC (77%; 95% CI 63%-89%; 107 cases, n = 8) and BMAC/Scaffold treatments with (71%; 95% CI 50%-89%; 117 cases, n = 8) at 6 months of follow-up. At 12 months of follow-up, an increasing proportion of bone healing was observed in all the treatment groups, ranging from 81% to 100%. These results indicate that BMAC or BMAC/Scaffold might be considered as the primary choice to treat nonunions with a successful healing rate at a midterm follow-up. Moreover, this meta-analysis highlighted that the presence of a scaffold positively influences the healing rate at a long-term follow-up. More case-control studies are still needed to support the clinical improvement of cell-based therapies against autografts, up to now considered as the gold standard for the treatment of nonunions.

Injective mesenchymal stem cell-based treatments for knee osteoarthritis: from mechanisms of action to current clinical evidences.

PURPOSE: Osteoarthritis (OA) represents a relevant social and economic burden worldwide. "Mesenchymal stem cells" or, as recently proposed, "medicinal signaling cells" (MSCs) have been recently introduced as injective treatments for OA with the aim of restoring joint homeostasis. The aim of this review is to provide the reader with the tools necessary to interpret the currently available clinical data, focusing on the MSC mechanisms of action which might help to clarify what we should expect from this treatment. METHODS: Clinical studies reporting MSC injections for the treatment of knee OA, either freshly isolated or culture-expanded cells, have been included and commented in relation to the supposed therapeutic effect that MSCs might exert giving their supposed mode of actions. RESULTS: The majority of the studies reports significant improvements in terms of pain and knee function compared to baseline values, up to 24 months of follow-up. Although these data support the expected therapeutic effect of this therapy giving the features of these cells, only 14% of the studies present a control group and more than one-third of them report the results on less than ten patients. CONCLUSIONS: Despite the constant presence of positive and satisfactory results in the studies analyzed, the complexity of MSC metabolism and related therapeutic effects as well as the weakness of most of the studies do not allow withdrawing definitive conclusions about the superiority of one tissue source over another, as well as about the best cell dose and the long-term durability of the effects of these procedures. Given the high potential value of these therapies in the treatment of OA, further studies accurately designed, carefully defining the type of patients to be included and pursuing minimal standard requirements in terms of follow-up, number of patients, and types of measurements should be conducted to finally assess the efficacy of MSC-based injective treatments.

Enhancing all-in-one bioreactors by combining interstitial perfusion, electrical stimulation, on-line monitoring and testing within a single chamber for cardiac constructs.

Tissue engineering strategies have been extensively exploited to generate functional cardiac patches. To maintain cardiac functionality in vitro, bioreactors have been designed to provide perfusion and electrical stimulation, alone or combined. However, due to several design limitations the integration of optical systems to assess cardiac maturation level is still missing within these platforms. Here we present a bioreactor culture chamber that provides 3D cardiac constructs with a bidirectional interstitial perfusion and biomimetic electrical stimulation, allowing direct cellular optical monitoring and contractility test. The chamber design was optimized through finite element models to house an innovative scaffold anchoring system to hold and to release it for the evaluation of tissue maturation and functionality by contractility tests. Neonatal rat cardiac fibroblasts subjected to a combined perfusion and electrical stimulation showed positive cell viability over time. Neonatal rat cardiomyocytes were successfully monitored for the entire culture period to assess their functionality. The combination of perfusion and electrical stimulation enhanced patch maturation, as evidenced by the higher contractility, the enhanced beating properties and the increased level of cardiac protein expression. This new multifunctional bioreactor provides a relevant biomimetic environment allowing for independently culturing, real-time monitoring and testing up to 18 separated patches.