Cellular and Structural Changes in Achilles and Patellar Tendinopathies: A Pilot In Vivo Study.

Tendinopathies continue to be a challenge for both patients and the medical teams providing care as no universal clinical practice guidelines have been established. In general, tendinopathies are typically characterized by prolonged, localized, activity-related pain with abnormalities in tissue composition, cellularity, and microstructure that may be observed on imaging or histology. In the lower limb, tendinopathies affecting the Achilles and the patellar tendons are the most common, showing a high incidence in athletic populations. Consistent diagnosis and management have been challenged by a lack of universal consensus on the pathophysiology and clinical presentation. Current management is primarily based on symptom relief and often consists of medications such as non-steroidal anti-inflammatories, injectable therapies, and exercise regimens that typically emphasize progressive eccentric loading of the affected structures. Implementing the knowledge of tendon stem/progenitor cells (TSPCs) and assessing their potential in enhancing tendon repair could fill an important gap in this regard. In the present pilot in vivo study, we have characterized the structural and cellular alterations that occur soon after tendon insult in models of both Achilles and patellar tendinopathy. Upon injury, CD146+ TSPCs are recruited from the interfascicular tendon matrix to the vicinity of the paratenon, whereas the observed reduction in M1 macrophage polarization is related to a greater abundance of reparative CD146+ TSPCs in situ. The robust TSPCs' immunomodulatory effects on macrophages were also demonstrated in in vitro settings where TSPCs can effectively polarize M1 macrophages towards an anti-inflammatory therapeutic M2 phenotype. Although preliminary, our findings suggest CD146+ TSPCs as a key phenotype that could be explored in the development of targeted regenerative therapies for tendinopathies.

Modification of Mesenchymal Stem/Stromal Cell-Derived Small Extracellular Vesicles by Calcitonin Gene Related Peptide (CGRP) Antagonist: Potential Implications for Inflammation and Pain Reversal.

During the progression of knee osteoarthritis (OA), the synovium and infrapatellar fat pad (IFP) can serve as source for Substance P (SP) and calcitonin gene-related peptide (CGRP), two important pain-transmitting, immune, and inflammation modulating neuropeptides. Our previous studies showed that infrapatellar fat pad-derived mesenchymal stem/stromal cells (MSC) acquire a potent immunomodulatory phenotype and actively degrade Substance P via CD10 both in vitro and in vivo. On this basis, our hypothesis is that CD10-bound IFP-MSC sEVs can be engineered to target CGRP while retaining their anti-inflammatory phenotype. Herein, human IFP-MSC cultures were transduced with an adeno-associated virus (AAV) vector carrying a GFP-labelled gene for a CGRP antagonist peptide (aCGRP). The GFP positive aCGRP IFP-MSC were isolated and their sEVs' miRNA and protein cargos were assessed using multiplex methods. Our results showed that purified aCGRP IFP-MSC cultures yielded sEVs with cargo of 147 distinct MSC-related miRNAs. Reactome analysis of miRNAs detected in these sEVs revealed strong involvement in the regulation of target genes involved in pathways that control pain, inflammation and cartilage homeostasis. Protein array of the sEVs cargo demonstrated high presence of key immunomodulatory and reparative proteins. Stimulated macrophages exposed to aCGRP IFP-MSC sEVs demonstrated a switch towards an alternate M2 status. Also, stimulated cortical neurons exposed to aCGRP IFP-MSC sEVs modulate their molecular pain signaling profile. Collectively, our data suggest that yielded sEVs can putatively target CGRP in vivo, while containing potent anti-inflammatory and analgesic cargo, suggesting the promise for novel sEVs-based therapeutic approaches to diseases such as OA.

Aseptic Meningitis Linked to Borrelia afzelii Seroconversion in Northeastern Greece: An Emerging Infectious Disease Contested in the Region.

Borreliosis (Lyme disease) is a zoonosis, mediated to humans and small mammals through specific vectors (ticks), with increasing global incidence. It is associated with a variety of clinical manifestations and can, if not promptly recognized and left untreated, lead to significant disability. In Europe, the main Borrelia species causing disease in humans are Borrelia burgdorferi s.s., Borrelia afzelii, Borrelia garinii, and Borrelia spielmanii. The Ixodes ricinus tick is their principal vector. Although Lyme disease is considered endemic in the Balkan region and Turkey, and all three main Lyme pathogens have been detected in ticks collected in these countries, autochthonous Lyme disease remains controversial in Greece. We report a case of aseptic meningitis associated with antibody seroconversion against Borrelia afzelii in a young female patient from the prefecture of Thasos without any relevant travel history. The patient presented with fever and severe headache, and the cerebrospinal fluid examination showed lymphocytic pleocytosis. Serum analysis was positive for specific IgG antibodies against Borrelia afzelii. In the absence of typical erythema migrans, serological evidence of infection is required for diagnosis. Although atypical in terms of clinical presentation, the seasonality and geographical location of potential disease transmission in the reported patient should raise awareness among clinicians for a still controversial and potentially underreported emerging infectious disease in Greece.

Mesenchymal Stem/Stromal Cell-Derived Small Extracellular Vesicles (MSC-sEVs): A Promising Treatment Modality for Diabetic Foot Ulcer.

Diabetic foot ulcer (DFU) is associated with neuropathy and/or peripheral artery disease of the lower limb in diabetic patients, affecting quality of life and leading to repeated hospitalizations and infections [...].

Mesenchymal Stem/Stromal Cells: Immunomodulatory and Bone Regeneration Potential after Tumor Excision in Osteosarcoma Patients.

Osteosarcoma (OS) is a type of bone cancer that is derived from primitive mesenchymal cells typically affecting children and young adults. The current standard of treatment is a combination of neoadjuvant chemotherapy and surgical resection of the cancerous bone. Post-resection challenges in bone regeneration arise. To determine the appropriate amount of bone to be removed, preoperative imaging techniques such as bone and CT scans are employed. To prevent local recurrence, the current standard of care suggests maintaining bony and soft tissue margins from 3 to 7 cm beyond the tumor. The amount of bone removed in an OS patient leaves too large of a deficit for bone to form on its own and requires reconstruction with metal implants or allografts. Both methods require the bone to heal, either to the implant or across the allograft junction, often in the setting of marrow-killing chemotherapy. Therefore, the issue of bone regeneration within the surgically resected margins remains an important challenge for the patient, family, and treating providers. Mesenchymal stem/stromal cells (MSCs) are potential agents for enhancing bone regeneration post tumor resection. MSCs, used with scaffolds and growth factors, show promise in fostering bone regeneration in OS cases. We spotlight two MSC types-bone marrow-derived (BM-MSCs) and adipose tissue-derived (ASCs)-highlighting their bone regrowth facilitation and immunomodulatory effects on immune cells like macrophages and T cells, enhancing therapeutic outcomes. The objective of this review is two-fold: review work demonstrating any ability of MSCs to target the deranged immune system in the OS microenvironment, and synthesize the available literature on the use of MSCs as a therapeutic option for stimulating bone regrowth in OS patients post bone resection. When it comes to repairing bone defects, both MB-MSCs and ASCs hold great potential for stimulating bone regeneration. Research has showcased their effectiveness in reconstructing bone defects while maintaining a non-tumorigenic role following wide resection of bone tumors, underscoring their capability to enhance bone healing and regeneration following tumor excisions.

The Ongoing Epidemic of West Nile Virus in Greece: The Contribution of Biological Vectors and Reservoirs and the Importance of Climate and Socioeconomic Factors Revisited.

Emerging infectious diseases have inflicted a significant health and socioeconomic burden upon the global population and governments worldwide. West Nile virus, a zoonotic, mosquito-borne flavivirus, was originally isolated in 1937 from a febrile patient in the West Nile Province of Uganda. It remained confined mainly to Africa, the Middle East, and parts of Europe and Australia until 1999, circulating in an enzootic mosquito-bird transmission cycle. Since the beginning of the 21st century, a new, neurotropic, more virulent strain was isolated from human outbreaks initially occurring in North America and later expanding to South and South-eastern Europe. Since 2010, when the first epidemic was recorded in Greece, annual incidence has fluctuated significantly. A variety of environmental, biological and socioeconomic factors have been globally addressed as potential regulators of the anticipated intensity of the annual incidence rate; circulation within the zoonotic reservoirs, recruitment and adaptation of new potent arthropod vectors, average winter and summer temperatures, precipitation during the early summer months, and socioeconomic factors, such as the emergence and progression of urbanization and the development of densely populated areas in association with insufficient health policy measures. This paper presents a review of the biological and socioenvironmental factors influencing the dynamics of the epidemics of West Nile virus (WNV) cases in Greece, one of the highest-ranked European countries in terms of annual incidence. To date, WNV remains an unpredictable opponent as is also the case with other emerging infectious diseases, forcing the National Health systems to develop response strategies, control the number of infections, and shorten the duration of the epidemics, thus minimizing the impact on human and material resources.

CD10-Bound Human Mesenchymal Stem/Stromal Cell-Derived Small Extracellular Vesicles Possess Immunomodulatory Cargo and Maintain Cartilage Homeostasis under Inflammatory Conditions.

The onset and progression of human inflammatory joint diseases are strongly associated with the activation of resident synovium/infrapatellar fat pad (IFP) pro-inflammatory and pain-transmitting signaling. We recently reported that intra-articularly injected IFP-derived mesenchymal stem/stromal cells (IFP-MSC) acquire a potent immunomodulatory phenotype and actively degrade substance P (SP) via neutral endopeptidase CD10 (neprilysin). Our hypothesis is that IFP-MSC robust immunomodulatory therapeutic effects are largely exerted via their CD10-bound small extracellular vesicles (IFP-MSC sEVs) by attenuating synoviocyte pro-inflammatory activation and articular cartilage degradation. Herein, IFP-MSC sEVs were isolated from CD10High- and CD10Low-expressing IFP-MSC cultures and their sEV miRNA cargo was assessed using multiplex methods. Functionally, we interrogated the effect of CD10High and CD10Low sEVs on stimulated by inflammatory/fibrotic cues synoviocyte monocultures and cocultures with IFP-MSC-derived chondropellets. Finally, CD10High sEVs were tested in vivo for their therapeutic capacity in an animal model of acute synovitis/fat pad fibrosis. Our results showed that CD10High and CD10Low sEVs possess distinct miRNA profiles. Reactome analysis of miRNAs highly present in sEVs showed their involvement in the regulation of six gene groups, particularly those involving the immune system. Stimulated synoviocytes exposed to IFP-MSC sEVs demonstrated significantly reduced proliferation and altered inflammation-related molecular profiles compared to control stimulated synoviocytes. Importantly, CD10High sEV treatment of stimulated chondropellets/synoviocyte cocultures indicated significant chondroprotective effects. Therapeutically, CD10High sEV treatment resulted in robust chondroprotective effects by retaining articular cartilage structure/composition and PRG4 (lubricin)-expressing cartilage cells in the animal model of acute synovitis/IFP fibrosis. Our study suggests that CD10High sEVs possess immunomodulatory miRNA attributes with strong chondroprotective/anabolic effects for articular cartilage in vivo. The results could serve as a foundation for sEV-based therapeutics for the resolution of detrimental aspects of immune-mediated inflammatory joint changes associated with conditions such as osteoarthritis (OA).

Therapeutic Perspectives for Inflammation and Senescence in Osteoarthritis Using Mesenchymal Stem Cells, Mesenchymal Stem Cell-Derived Extracellular Vesicles and Senolytic Agents.

Osteoarthritis (OA) is the most common cause of disability worldwide among the elderly. Alarmingly, the incidence of OA in individuals less than 40 years of age is rising, likely due to the increase in obesity and post-traumatic osteoarthritis (PTOA). In recent years, due to a better understanding of the underlying pathophysiology of OA, several potential therapeutic approaches targeting specific molecular pathways have been identified. In particular, the role of inflammation and the immune system has been increasingly recognized as important in a variety of musculoskeletal diseases, including OA. Similarly, higher levels of host cellular senescence, characterized by cessation of cell division and the secretion of a senescence-associated secretory phenotype (SASP) within the local tissue microenvironments, have also been linked to OA and its progression. New advances in the field, including stem cell therapies and senolytics, are emerging with the goal of slowing disease progression. Mesenchymal stem/stromal cells (MSCs) are a subset of multipotent adult stem cells that have demonstrated the potential to modulate unchecked inflammation, reverse fibrosis, attenuate pain, and potentially treat patients with OA. Numerous studies have demonstrated the potential of MSC extracellular vesicles (EVs) as cell-free treatments that comply with FDA regulations. EVs, including exosomes and microvesicles, are released by numerous cell types and are increasingly recognized as playing a critical role in cell-cell communication in age-related diseases, including OA. Treatment strategies for OA are being developed that target senescent cells and the paracrine and autocrine secretions of SASP. This article highlights the encouraging potential for MSC or MSC-derived products alone or in combination with senolytics to control patient symptoms and potentially mitigate the progression of OA. We will also explore the application of genomic principles to the study of OA and the potential for the discovery of OA phenotypes that can motivate more precise patient-driven treatments.

Pneumocephalus; a rare cause of coma.

We report the case of an 84-years old female patient who developed cerebral air embolism in association with the indwelling hemodialysis central venous catheter. Pneumocephalus, even though rare, should be included in the differential diagnosis of acute manifestation of neurologic deficits, especially in association with central venous access, surgical interventions or trauma, and requests prompt management. Brain computed tomography scanning remains the investigation of choice.

The roles and therapeutic potentialof mesenchymal stem/stromal cells and their extracellular vesicles in tendinopathies.

Tendinopathies encompass a highly prevalent, multi-faceted spectrum of disorders, characterized by activity-related pain, compromised function, and propensity for an extended absence from sport and the workplace. The pathophysiology of tendinopathy continues to evolve. For decades, it has been related primarily to repetitive overload trauma but more recently, the onset of tendinopathy has been attributed to the tissue's failed attempt to heal after subclinical inflammatory and immune challenges (failed healing model). Conventional tendinopathy management produces only short-term symptomatic relief and often results in incomplete repair or healing leading to compromised tendon function. For this reason, there has been increased effort to develop therapeutics to overcome the tissue's failed healing response by targeting the cellular metaplasia and pro-inflammatory extra-cellular environment. On this basis, stem cell-based therapies have been proposed as an alternative therapeutic approach designed to modify the course of the various tendon pathologies. Mesenchymal stem/stromal cells (MSCs) are multipotent stem cells often referred to as "medicinal signaling cells" due to their immunomodulatory and anti-inflammatory properties that can produce a pro-regenerative microenvironment in pathological tendons. However, the adoption of MSCs into clinical practice has been limited by FDA regulations and perceived risk of adverse events upon infusion in vivo. The introduction of cell-free approaches, such as the extracellular vesicles of MSCs, has encouraged new perspectives for the treatment of tendinopathies, showing promising short-term results. In this article, we review the most recent advances in MSC-based and MSC-derived therapies for tendinopathies. Preclinical and clinical studies are included with comment on future directions of this rapidly developing therapeutic modality, including the importance of understanding tissue loading and its relationship to any treatment regimen.

Anaerobic Spondylodiscitis caused by Parvimonas Micra in a Rheumatoid Arthritis Patient: Case Report and Review of the Literature.

We report the rare case of Parvimonas micra bacteraemia and secondary spondylodiscitis probably triggered by tooth injury in a rheumatoid arthritis patient. Anaerobic bacteria associated spondylodiscitis may evade diagnosis due to atypical clinical presentation usually lacking fever, and the difficulties related to microbiological characterisation of the pathogen. Even though anaerobic spinal infections may constitute <3% of the total, clinical suspicion should remain high, especially in the case of positive history for pre-existing oral cavity or gastrointestinal/gynaecological tract infections.

CD146+ Endometrial-Derived Mesenchymal Stem/Stromal Cell Subpopulation Possesses Exosomal Secretomes with Strong Immunomodulatory miRNA Attributes.

The perivascular localization of endometrial mesenchymal stem/stromal cells (eMSC) allows them to sense local and distant tissue damage, promoting tissue repair and healing. Our hypothesis is that eMSC therapeutic effects are largely exerted via their exosomal secretome (eMSC EXOs) by targeting the immune system and angiogenic modulation. For this purpose, EXOs isolated from Crude and CD146+ eMSC populations were compared for their miRNA therapeutic signatures and immunomodulatory functionality under inflammatory conditions. eMSC EXOs profiling revealed 121 in Crude and 88 in CD146+ miRNAs, with 82 commonly present in both populations. Reactome and KEGG analysis of miRNAs highly present in eMSC EXOs indicated their involvement among others in immune system regulation. From the commonly present miRNAs, four miRNAs (hsa-miR-320e, hsa-miR-182-3p, hsa-miR-378g, hsa-let-7e-5p) were more enriched in CD146+ eMSC EXOs. These miRNAs are involved in macrophage polarization, T cell activation, and regulation of inflammatory cytokine transcription (i.e., TNF-α, IL-1ß, and IL-6). Functionally, stimulated macrophages exposed to eMSC EXOs demonstrated a switch towards an alternate M2 status and reduced phagocytic capacity compared to stimulated alone. However, eMSC EXOs did not suppress stimulated human peripheral blood mononuclear cell proliferation, but significantly reduced secretion of 13 pro-inflammatory molecules compared to stimulated alone. In parallel, two anti-inflammatory proteins, IL-10 and IL-13, showed higher secretion, especially upon CD146+ eMSC EXO exposure. Our study suggests that eMSC, and even more, the CD146+ subpopulation, possess exosomal secretomes with strong immunomodulatory miRNA attributes. The resulting evidence could serve as a foundation for eMSC EXO-based therapeutics for the resolution of detrimental aspects of tissue inflammation.

Long noncoding RNAs in mesenchymal stromal/stem cells osteogenic differentiation: Implications in osteoarthritis pathogenesis.

This letter focuses on a recently published article that provided an exceptional description of the effect of epigenetic modifications on gene expression patterns related to skeletal system remodeling. Specifically, it discusses a novel modality of epigenetic regulation, the long noncoding RNAs (lncRNAs), and provides evidence of their involvement in mesenchymal stromal/stem cells osteo-/adipo-genic differentiation balance. Despite focus on lncRNAs, there is an emerging cross talk between lncRNAs and miRNAs interaction as a novel mechanism in the regulation of the function of the musculoskeletal system, by controlling bone homeostasis and bone regeneration, as well as the osteogenic differentiation of stem cells. Thus, we touched on some examples to demonstrate this interaction. In addition, we believe there is still much to discover from the effects of lncRNAs on progenitor and non-progenitor cell differentiation. We incorporated data from other published articles to review lncRNAs in normal progenitor cell osteogenic differentiation, determined lncRNAs involved in osteoarthritis pathogenesis in progenitor cells, and provided a review of lncRNAs in non-progenitor cells that are differentially regulated in osteoarthritis. In conclusion, we really enjoyed reading this article and with this information we hope to further our under standing of lncRNAs and mesenchymal stromal/stem cells regulation.

Human infrapatellar fat pad mesenchymal stem cells show immunomodulatory exosomal signatures.

Within the human knee infrapatellar fat pad (IFP) and synovium, resident synoviocytes and macrophages contribute to the onset and progression of inflammatory joint diseases. Our hypothesis is that IFP-derived mesenchymal stem cells (IFP-MSC) robust immunomodulatory therapeutic effects are largely exerted via their exosomal (IFP-MSC EXOs) secretome by attenuating synoviocytes and macrophages pro-inflammatory activation. IFP-MSC EXOs showed distinct miRNA and protein immunomodulatory profiles. Reactome analysis of 24 miRNAs highly present in exosomes showed their involvement in the regulation of six gene groups, including immune system. Exosomes were enriched for immunomodulatory and reparative proteins that are involved in positive regulation of cell proliferation, response to stimulus, signal transduction, signal receptor activity, and protein phosphorylation. Stimulated synoviocytes or macrophages exposed to IFP-MSC EXOs demonstrated significantly reduced proliferation, altered inflammation-related molecular profiles, and reduced secretion of pro-inflammatory molecules compared to stimulated alone. In an acute synovial/IFP inflammation rat model, IFP-MSC EXOs therapeutic treatment resulted in robust macrophage polarization towards an anti-inflammatory therapeutic M2 phenotype within the synovium/IFP tissues. Based on these findings, we propose a viable cell-free alternative to MSC-based therapeutics as an alternative approach to treating synovitis and IFP fibrosis.

Characterization and response to inflammatory stimulation of human endometrial-derived mesenchymal stem/stromal cells.

BACKGROUND AIMS: The human endometrium has emerged as an attractive source of endometrial-derived mesenchymal stem/stromal cells (eMSCs) that can be easily isolated by non-invasive procedures. The prominent capacity of the endometrium for efficient and scarless regeneration each menstrual cycle indicates the increased eMSC immunomodulatory and pro-angiogenic properties. Herein the authors investigated the molecular responses of eMSCs to an inflammatory environment and whether those intrinsic responses affected their functional attributes. METHODS: Human eMSCs immunophenotypic, transcriptional and secretory profiles were evaluated at passage three (P3) and passage eight (P8) to determine culture effects. Functionally, P3 and P8 non-induced and TNF-α/IFN-γ-induced eMSCs were interrogated for their capacity to suppress stimulated peripheral blood mononuclear cell (PBMC) proliferation, whereas non-induced eMSCs were assessed for their support to vascular network formation in co-cultures with human umbilical vein endothelial cells in vitro. RESULTS: Non-induced P3 and P8 eMSCs exhibited similar spindle-shaped morphology and clonogenic capacity. Nevertheless, P8 eMSCs showed reduced growth rate capacity and telomere length. The eMSCs displayed the typical MSC-related immunophenotypic profile, with P3 and P8 eMSCs expressing high levels (>98%) of CD140ß, intermediate levels (35-60%) of CD146 and SUSD2 and low levels (∼8%) of NG2 pericytic markers. Non-induced P3 and P8 showed similar transcriptional and secretory profiles, though the expression of immunomodulatory HLA-G and IL-8 genes was significantly downregulated in P8 compared with P3 eMSCs. Upon TNF-α/IFN-γ induction, eMSCs showed an immunophenotypic profile similar to that of non-induced eMSCs, except for significant upregulation of HLA-DR protein expression in both induced P3 and P8 eMSCs. However, induced P3 and P8 eMSCs showed significant upregulation of CD10, HLA-G, IDO, IL-6, IL-8, LIF and TSG gene expression compared with non-induced cultures. TNF-α/IFN-γ induction strongly increased the secretion of inflammatory-/angiogenesis-related molecules, whereas growth factor secretion was similar to the non-induced eMSCs. Functionally, P3 and P8 eMSCs showed a strong inhibitory effect on stimulated PBMC proliferation and the capacity to support neovascularization in vitro. CONCLUSIONS: The authors' study suggests that serial expansion does not affect eMSC immunophenotypic, transcriptional and secretory profiles. This is directly reflected by the functional immunomodulatory and pro-angiogenic properties of eMSCs, which remain unaltered until P8 in vitro. However, exposure of eMSCs to inflammatory environments enhances their immunomodulatory transcriptional and inflammatory-/angiogenesis-related secretory profiles. Therefore, the resulting evidence of eMSCs serial expansion and exposure to inflammation could serve as a foundation for improved eMSCs manufacturing and potential clinical translation efforts.

Single-Cell RNA-Sequencing Identifies Infrapatellar Fat Pad Macrophage Polarization in Acute Synovitis/Fat Pad Fibrosis and Cell Therapy.

The pathogenesis and progression of knee inflammatory pathologies is modulated partly by residing macrophages in the infrapatellar fat pad (IFP), thus, macrophage polarization towards pro-inflammatory (M1) or anti-inflammatory (M2) phenotypes is important in joint disease pathologies. Alteration of M1/M2 balance contributes to the initiation and progression of joint inflammation and can be potentially altered with mesenchymal stem cell (MSC) therapy. In an acute synovial/IFP inflammation rat model a single intra-articular injection of IFP-MSC was performed, having as controls (1) diseased rats not receiving IFP-MSC and (2) non-diseased rats. After 4 days, cell specific transcriptional profiling via single-cell RNA-sequencing was performed on isolated IFP tissue from each group. Eight transcriptomically distinct cell populations were identified within the IFP across all three treatment groups with a noted difference in the proportion of myeloid cells across the groups. Largely myeloid cells consisted of macrophages (>90%); one M1 sub-cluster highly expressing pro-inflammatory markers and two M2 sub-clusters with one of them expressing higher levels of canonical M2 markers. Notably, the diseased samples (11.9%) had the lowest proportion of cells expressing M2 markers relative to healthy (14.8%) and MSC treated (19.4%) samples. These results suggest a phenotypic polarization of IFP macrophages towards the pro-inflammatory M1 phenotype in an acute model of inflammation, which are alleviated by IFP-MSC therapy inducing a switch towards an alternate M2 status. Understanding the IFP cellular heterogeneity and associated transcriptional programs may offer insights into novel therapeutic strategies for disabling joint disease pathologies.

Human Tendon Stem/Progenitor Cell Features and Functionality Are Highly Influenced by in vitro Culture Conditions.

Our understanding of tendon biology continues to evolve, thus leading to opportunities for developing novel, evidence-based effective therapies for the treatment of tendon disorders. Implementing the knowledge of tendon stem/progenitor cells (TSPCs) and assessing their potential in enhancing tendon repair could fill an important gap in this regard. We described different molecular and phenotypic profiles of TSPCs modulated by culture density, as well as their multipotency and secretory activities. Moreover, in the same experimental setting, we evaluated for different responses to inflammatory stimuli mediated by TNFα and IFNγ. We also preliminarily investigated their immunomodulatory activity and their role in regulating degradation of substance P. Our findings indicated that TSPCs cultured at low density (LD) exhibited cobblestone morphology and a reduced propensity to differentiate. A distinctive immunophenotypic profile was also observed with high secretory and promising immunomodulatory responses when primed with TNFα and IFNγ. In contrast, TSPCs cultured at high density (HD) showed a more elongated fibroblast-like morphology, a greater adipogenic differentiation potential, and a higher expression of tendon-related genes with respect to LD. Finally, HD TSPCs showed immunomodulatory potential when primed with TNFα and IFNγ, which was slightly lower than that shown by LD. A shift from low to high culture density during TSPC expansion demonstrated intermediate features confirming the cellular adaptability of TSPCs. Taken together, these experiments allowed us to identify relevant differences in TSPCs based on culture conditions. This ability of TSPCs to acquire distinguished morphology, phenotype, gene expression profile, and functional response advances our current understanding of tendons at a cellular level and suggests responsivity to cues in their in situ microenvironment.

Increased Mesenchymal Stem Cell Functionalization in Three-Dimensional Manufacturing Settings for Enhanced Therapeutic Applications.

Mesenchymal stem/stromal cell (MSC) exist within their in vivo niches as part of heterogeneous cell populations, exhibiting variable stemness potential and supportive functionalities. Conventional extensive 2D in vitro MSC expansion, aimed at obtaining clinically relevant therapeutic cell numbers, results in detrimental effects on both cellular characteristics (e.g., phenotypic changes and senescence) and functions (e.g., differentiation capacity and immunomodulatory effects). These deleterious effects, added to the inherent inter-donor variability, negatively affect the standardization and reproducibility of MSC therapeutic potential. The resulting manufacturing challenges that drive the qualitative variability of MSC-based products is evident in various clinical trials where MSC therapeutic efficacy is moderate or, in some cases, totally insufficient. To circumvent these limitations, various in vitro/ex vivo techniques have been applied to manufacturing protocols to induce specific features, attributes, and functions in expanding cells. Exposure to inflammatory cues (cell priming) is one of them, however, with untoward effects such as transient expression of HLA-DR preventing allogeneic therapeutic schemes. MSC functionalization can be also achieved by in vitro 3D culturing techniques, in an effort to more closely recapitulate the in vivo MSC niche. The resulting spheroid structures provide spatial cell organization with increased cell-cell interactions, stable, or even enhanced phenotypic profiles, and increased trophic and immunomodulatory functionalities. In that context, MSC 3D spheroids have shown enhanced "medicinal signaling" activities and increased homing and survival capacities upon transplantation in vivo. Importantly, MSC spheroids have been applied in various preclinical animal models including wound healing, bone and osteochondral defects, and cardiovascular diseases showing safety and efficacy in vivo. Therefore, the incorporation of 3D MSC culturing approach into cell-based therapy would significantly impact the field, as more reproducible clinical outcomes may be achieved without requiring ex vivo stimulatory regimes. In the present review, we discuss the MSC functionalization in 3D settings and how this strategy can contribute to an improved MSC-based product for safer and more effective therapeutic applications.