Setting a Successful Sorting for Extracellular Vesicle Isolation.

Extracellular vesicles (EVs) released by mesenchymal stromal cells (MSCs) contain a set of microRNAs with regenerative and anti-inflammatory roles. Therefore, purified MSC-EVs are envisioned as a next-generation therapeutic option for a wide array of diseases. In this protocol, we report the strategy for successfully sorting EVs from the supernatant of adipose-derived MSCs (ASCs), often used in orthopedics regenerative medicine applications. First, we described the sample preparation, focusing on EV isolation and labeling steps with carboxyfluorescein succinimidyl ester (CFSE) for fluorescence detection; subsequently, we detailed the sorting process, which constitutes the main part of the protocol. In addition to the rules defined by MISEV 2023 and MIFlowCyt EV guidelines, we applied specific experimental conditions concerning nozzle size, frequency, and sheath pressure. Morphological parameters are established using beads of diameters selected to cover the theoretical range of EV size. After ASC-EVs sorting, we performed a purity check of the sorted fraction by re-analyzing it with the sorter and verifying the EV size distribution with the nanoparticle tracking analysis technique. Due to the increasing importance of EVs, having a pure population to study and characterize is becoming crucial. Here, we demonstrate a winner strategy to set up sorting to achieve this goal.

Cell-based therapies have disease-modifying effects on osteoarthritis in animal models: A systematic review by the ESSKA Orthobiologic Initiative. Part 3: Umbilical cord, placenta, and other sources for cell-based injectable therapies.

PURPOSE: This systematic review aimed to investigate in animal models the presence of disease-modifying effects driven by non-bone marrow-derived and non-adipose-derived products, with a particular focus on umbilical cord and placenta-derived cell-based therapies for the intra-articular injective treatment of osteoarthritis (OA). METHODS: A systematic review was performed on three electronic databases (PubMed, Web of Science and Embase) according to PRISMA guidelines. The results were synthesised to investigate disease-modifying effects in preclinical animal studies comparing injectable umbilical cord, placenta, and other sources-derived products with OA controls. The risk of bias was assessed using the SYRCLE tool. RESULTS: A total of 80 studies were included (2314 animals). Cell therapies were most commonly obtained from the umbilical cord in 33 studies and placenta/amniotic tissue in 18. Cell products were xenogeneic in 61 studies and allogeneic in the remaining 19 studies. Overall, 25/27 (92.6%) of studies on umbilical cord-derived products documented better results compared to OA controls in at least one of the following outcomes: macroscopic, histological and/or immunohistochemical findings, with 19/22 of studies (83.4%) show positive results at the cartilage level and 4/6 of studies (66.7%) at the synovial level. Placenta-derived injectable products documented positive results in 13/16 (81.3%) of the studies, 12/15 (80.0%) at the cartilage level, and 2/4 (50.0%) at the synovial level, but 2/16 studies (12.5%) found overall worse results than OA controls. Other sources (embryonic, synovial, peripheral blood, dental pulp, cartilage, meniscus and muscle-derived products) were investigated in fewer preclinical studies. The risk of bias was low in 42% of items, unclear in 49%, and high in 9% of items. CONCLUSION: Interest in cell-based injectable therapies for OA treatment is soaring, particularly for alternatives to bone marrow and adipose tissue. While expanded umbilical cord mesenchymal stem cells reported auspicious disease-modifying effects in preventing OA progression in animal models, placenta/amniotic tissue also reported deleterious effects on OA joints. Lower evidence has been found for other cellular sources such as embryonic, synovial, peripheral blood, dental-pulp, cartilage, meniscus, and muscle-derived products. LEVEL OF EVIDENCE: Level II.

Starvation and Inflammation Modulate Adipose Mesenchymal Stromal Cells' Molecular Signature.

Mesenchymal stromal cells (MSCs) and their released factors (secretome) are intriguing options for regenerative medicine approaches based on the management of inflammation and tissue restoration, as in joint disorders like osteoarthritis (OA). Production strategy may modulate cells and secretome fingerprints, and for the latter, the effect of serum removal by starvation used in clinical-grade protocols has been underestimated. In this work, the effect of starvation on the molecular profile of interleukin 1 beta (IL1ß)-primed adipose-derived MSCs (ASCs) was tested by assessing the expression level of 84 genes related to secreted factors and 84 genes involved in defining stemness potential. After validation at the protein level, the effect of starvation modulation in the secretomes was tested in a model of OA chondrocytes. IL1ß priming in vitro led to an increase in inflammatory mediators' release and reduced anti-inflammatory potential on chondrocytes, features reversed by subsequent starvation. Therefore, when applying serum removal-based clinical-grade protocols for ASCs' secretome production, the effects of starvation must be carefully considered and investigated.

Effects of Pulsed Electromagnetic Field Treatment on Skeletal Muscle Tissue Recovery in a Rat Model of Collagenase-Induced Tendinopathy: Results from a Proteome Analysis.

Tendon disorders often result in decreased muscle function and atrophy. Pulsed Electromagnetic Fields (PEMFs) have shown potential in improving tendon fiber structure and muscle recovery. However, the molecular effects of PEMF therapy on skeletal muscle, beyond conventional metrics like MRI or markers of muscle decline, remain largely unexplored. This study investigates the metabolic and structural changes in PEMF-treated muscle tissue using proteomics in a rat model of Achilles tendinopathy induced by collagenase. Sprague Dawley rats were unilaterally induced for tendinopathy with type I collagenase injection and exposed to PEMFs for 8 h/day. Gastrocnemius extracts from untreated or PEMF-treated rats were analyzed with LC-MS/MS, and proteomics differential analysis was conducted through label-free quantitation. PEMF-treated animals exhibited decreased glycolysis and increased LDHB expression, enhancing NAD signaling and ATP production, which boosted respiratory chain activity and fatty acid beta-oxidation. Antioxidant protein levels increased, controlling ROS production. PEMF therapy restored PGC1alpha and YAP levels, decreased by tendinopathy. Additionally, myosins regulating slow-twitch fibers and proteins involved in fiber alignment and force transmission increased, supporting muscle recovery and contractile function. Our findings show that PEMF treatment modulates NAD signaling and oxidative phosphorylation, aiding muscle recovery through the upregulation of YAP and PGC1alpha and increasing slow myosin isoforms, thus speeding up physiological recovery.

Platelet-rich plasma injections for the management of knee osteoarthritis: The ESSKA-ICRS consensus. Recommendations using the RAND/UCLA appropriateness method for different clinical scenarios.

PURPOSE: The aim of this consensus was to develop evidence- and expert-based patient-focused recommendations on the appropriateness of intra-articular platelet-rich plasma (PRP) injections in different clinical scenarios of patients with knee osteoarthritis (OA). METHODS: The RAND/UCLA Appropriateness Method was used by the European Society of Sports Traumatology, Knee Surgery, and Arthroscopy (ESSKA), as well as the International Cartilage Regeneration and Joint Preservation Society (ICRS) to reach a consensus and produce recommendations for specific patient categories combining best available scientific evidence with the collective judgement of a panel of experts. RESULTS: Scenarios were defined based on first treatment vs first injective treatment vs second injective treatment, age (<50/50-65/66-80/>80), tibiofemoral vs patellofemoral involvement, OA level (Kellgren-Lawrence/KL 0-I/II-III/IV), and joint effusion (dry knee, minor-mild or major effusion). Out of 216 scenarios, in 84 (38.9%) the indication was considered appropriate, in 9 (4.2%) inappropriate and in 123 (56.9%) uncertain. The parameters associated with the highest consensus were PRP use after failed injective treatments (62.5%), followed by PRP after failed conservative treatments and KL 0-III scenarios (58.3%), while the highest uncertainty was found for PRP use as first treatment and KL IV OA (91.7% and 87.5% of uncertain scenarios, respectively). CONCLUSION: This ESSKA-ICRS consensus established recommendations on the appropriateness or inappropriateness of PRP injections for the treatment of knee OA, providing a useful reference for clinical practice. PRP injections are considered appropriate in patients aged ≤80 years with knee KL 0-III OA grade after failed conservative non-injective or injective treatments, while they are not considered appropriate as first treatment nor in KL IV OA grade. LEVEL OF EVIDENCE: Level I.

The chimera of reaching a universal consensus on platelet-rich plasma treatment for knee osteoarthritis: a review of recent consensus statements and expert opinion.

INTRODUCTION: Osteoarthritis (OA) is a prevalent cause of disability worldwide, affecting millions and posing significant socioeconomic burdens. Various conservative measures like hyaluronic acid (HA) and platelet-rich plasma (PRP) injections aim to manage OA symptoms and delay surgical interventions. Despite the increasing utilization of PRP, consensus on its efficacy remains elusive, reflecting the evolving landscape of OA management. AREAS COVERED: This study reviews guidelines and recommendations on intra-articular PRP injections for OA globally, highlighting divergent perspectives among different medical societies. A comprehensive literature search identified 19 relevant guidelines, indicating a temporal and geographic evolution in attitudes toward PRP use. While some guidelines endorse PRP for mild-to-moderate OA, others express caution due to concerns about product standardization and clinical evidence heterogeneity. EXPERT OPINION: The lack of universal consensus on PRP for OA underscores the complex interplay between clinical evidence, practice patterns, and evolving perspectives. Recent shifts toward endorsing PRP may reflect advancements in preparation techniques and personalized medicine approaches. However, challenges persist, including patient selection and product standardization. Efforts to develop consensus and refine PRP classification systems are essential for guiding clinical practice and advancing OA management.

Exploration of effects of galvanic vestibular stimulation on circadian rhythms and its associations with sleep and spatial memory in patients with breast cancer: The ICANSLEEP-2 protocol.

BACKGROUND: Patients with breast cancer (BC) exhibit circadian rhythm disruptions, mainly of rest-activity rhythm (RAR), of which sleep is an essential component, and cortisol rhythm. Sleep complaints such as insomnia and cognitive impairments are prevalent in BC. In general population, sleep is known to contribute greatly to cognition. Thus, improving RAR (and particularly sleep) could help limiting cognitive impairments in BC patients. It has recently been suggested that, in addition to its essential role in spatial memory, the vestibular system contributes to RAR synchronization. Its stimulation could therefore limit both sleep disturbances and spatial memory deficits in BC. OBJECTIVES: The main aim of the ICANSLEEP-2 study is to assess the effects of galvanic vestibular stimulation (GVS) on circadian rhythms. The secondary aim is to assess whether GVS improves sleep and spatial memory in BC patients. METHODS: Two groups with insomnia complaints (Insomnia Severity Index > 7) will be included: a patients' group with BC (n = 50) and a healthy control group without history of cancer (n = 25). There will be two assessment sessions, before and after 2 weeks of GVS. Patients will be randomly assigned to either a GVS group or a sham group (noneffective stimulation). Controls will receive GVS. GVS effects will be quantified and compared between groups. Assessments will include actigraphy, salivary cortisol, polysomnography, a cognitive test battery (including a computer-based task for spatial memory) and validated questionnaires (for psychological functioning and sleep complaints). DISCUSSION: Current methods for improving sleep in BC have had controversial outcomes regarding sleep structure. We expect GVS to offer a new mean of directly targeting RAR disruptions in BC patients, with beneficial effects on sleep structure. Given the crucial impact of sleep on cognitive functioning, notably spatial memory, improving sleep of BC patients should enhance their cognitive functioning. ETHICS AND DISSEMINATION: This study received ethical approval from the Ile de France IV institutional review board on 19 April 2022 (no. ID-RCB: 2022-A00437-36). The findings yielded by this protocol will be presented at various conferences and in peer-reviewed journals. CLINICALTRIALS.GOV REGISTRATION NUMBER: NCT05414357.

High efficiency protocol for platelet derived fibrin gel loaded with mesenchymal stromal cells extracellular vesicles.

Introduction: Extracellular vesicles from mesenchymal stromal cells (MSC-EVs) are potent stimulators of naïve cartilage and their injection is studied in clinical trials for cartilage lesions, since often cartilage repaired with conventional approaches is incomplete or less performant leading to joint degeneration. The main pitfall of these innovative approaches is the high EVs dispersion into the joint cavity and consequent low concentration at lesion site. Thus, biological scaffolds for concentration of EVs where needed might be a promising option. This work aimed at producing an enhanced platelet-derived fibrin gel loaded with adipose-derived MSCs (ASCs)-EVs. Methods: EVs' embedment efficiency in platelet gel, their release and incorporation in OA chondrocytes and cartilage explants were monitored by flow cytometry, microfluidic approaches, scansion electron microscopy and real-time quantitative multimodal nonlinear optics imaging. The effect of released EVs was tested in OA chondrocytes by gene expression studies. Results: A protocol ensuring high incorporation EVs efficiency in platelet gels was defined, relying on a one-step modification of the standard procedure used in current clinical practice. Trapped EVs were released continuously for up to 4 weeks and uptaken in pathologic chondrocytes and cartilage explants. The release of the EVs-loaded platelet gel had stronger and synergic anti-inflammatory/matrix remodelling effects with respect to both EVs per se and unloaded gel released products. Conclusions: These results suggest the feasibility of producing a platelet gel loaded with MSC-EVs at high efficiency that can be used as an enhanced tool to foster chondrocyte homeostasis, a key requisite for proper cartilage healing.

Donor Sites and Harvesting Techniques Affect miRNA Cargos of Extracellular Vesicles Released by Human Adipose-Derived Mesenchymal Stromal Cells.

Osteoarthritis (OA) is a degenerative joint disorder characterized by the progressive deterioration of articular cartilage driven and sustained by catabolic and inflammatory processes that lead to pain and functional impairment. Adipose-derived stem cells (ASCs) have emerged as a promising therapeutic strategy for OA due to their regenerative potential, which mainly relies on the adaptive release of paracrine molecules that are soluble or encapsulated in extracellular vesicles (EVs). The biological effects of EVs specifically depend on their cargo; in particular, microRNAs (miRNAs) can specifically modulate target cell function through gene expression regulation. This study aimed to investigate the impact of collection site (abdominal vs. peri-trochanteric adipose tissue) and collection method (surgical excision vs. lipoaspiration) on the miRNAs profile in ASC-derived EVs and their potential implications for OA therapy. EV-miRNA cargo profiles from ASCs of different origins were compared. An extensive bioinformatics search through experimentally validated and OA-related targets, pathways, and tissues was conducted. Several miRNAs involved in the restoration of cartilage homeostasis and in immunomodulation were identified in all ASC types. However, EV-miRNA expression profiles were affected by both the tissue-harvesting site and procedure, leading to peculiar characteristics for each type. Our results suggest that adipose-tissue-harvesting techniques and the anatomical site of origin influence the therapeutic efficacy of ASC-EVs for tissue-specific regenerative therapies in OA, which warrants further investigation.

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.

Corticosteroid injections for the treatment of osteoarthritis present a wide spectrum of effects ranging from detrimental to disease-modifying: A systematic review of preclinical evidence by the ESSKA Orthobiologic Initiative.

PURPOSE: Aim of this systematic review of preclinical evidence was to determine the effects of intra-articular corticosteroid (CS) injections in joints affected by osteoarthritis (OA). METHODS: A systematic review was performed on animal studies evaluating intra-articular CS injections for OA joints. The search was performed on PubMed, Cochrane, and Web of Science databases. A synthesis of the results was performed investigating CS effects by evaluating studies comparing CS with control groups. Morphological, histological, immunohistochemistry evaluations, clinical outcomes, biomarkers and imaging results were evaluated. The risk of bias was assessed according to the Systematic Review Centre for Laboratory Animal Experimentation's tool. RESULTS: Thirty-two articles analysing CS effects in OA animal models were included (1079 joints), 18 studies on small and 14 on large animals. CS injections showed overall positive effects in at least one of the outcomes in 68% of the studies, while 16% reported a deleterious effect. CS improved cartilage and synovial outcomes in 68% and 60% of the studies, but detrimental effects were documented in 11% and 20% of the studies, respectively. Clinical parameters evaluated in terms of pain, lameness or joint swelling improved in 63% of the studies but deteriorated in 13%. Evidence is limited on imaging and biomarkers results, as well as on the best CS type, dose, formulation and injection protocol. The risk of bias assessment revealed a 28% low and an 18% high risk of bias. CONCLUSION: Intra-articular CS injections induced a wide range of results on OA joints in experimental animal models, from disease-modifying and positive effects on pain and joint function at short-term evaluation to the lack of benefit or even negative effects. This underlines the need to identify more specific indications and treatment modalities to avoid possible detrimental effects while maximising the anti-inflammatory properties and the benefits of intra-articular CS in OA joints. LEVEL OF EVIDENCE: Level II.

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.

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.

The use of injectable orthobiologics for knee osteoarthritis: A European ESSKA-ORBIT consensus. Part 1-Blood-derived products (platelet-rich plasma).

PURPOSE: The aim of this European Society of Sports Traumatology, Knee Surgery and Arthroscopy (ESSKA) consensus is to provide recommendations based on evidence and expert opinion to improve indications, decision-making and administration-related aspects when using blood-derived orthobiologics (for simplicity indicated as PRP-platelet-rich plasma-with PRP being the most common product) for the management of knee osteoarthritis (OA). METHODS: Leading European expert clinicians and scientists were divided into a steering group, a rating group and a peer review group. The steering group prepared 28 question-statement sets divided into three sections: PRP rationale and indications, PRP preparation and characterisation and PRP protocol. The quality of the statements received grades of recommendation ranging from A (high-level scientific support) to B (scientific presumption), C (low-level scientific support) or D (expert opinion). The question-statement sets were then evaluated by the rating group, and the statements scored from 1 to 9 based on their degree of agreement with the statements produced by the steering group. Once a general consensus was reached between the steering and rating groups, the document was submitted to the peer review group who evaluated the geographic adaptability and approved the document. A final combined meeting of all the members of the consensus was held to produce the official document. RESULTS: The literature review on the use of blood-derived products for knee OA revealed that 9 of 28 questions/statements had the support of high-level scientific literature, while the other 19 were supported by a medium-low scientific quality. Three of the 28 recommendations were grade A recommendations: (1) There is enough preclinical and clinical evidence to support the use of PRP in knee OA. This recommendation was considered appropriate with a strong agreement (mean: 8). (2) Clinical evidence has shown the effectiveness of PRP in patients for mild to moderate degrees of knee OA (KL ≤ 3). This recommendation was considered appropriate with a strong agreement (mean: 8.1). (3) PRP injections have been shown to provide a longer effect in comparison to the short-term effect of CS injections. They also seem to provide a safer use profile with less potential related complications. This recommendation was considered appropriate with a very strong agreement (mean: 8.7). Six statements were grade B recommendations, 7 were grade C and 12 were grade D. The mean rating score was 8.2 ± 0.3. CONCLUSIONS: The consensus group reached a high level of agreement on all the questions/statements despite the lack of clear evidence for some questions. According to the results from this consensus group, given the large body of existing literature and expert opinions, PRP was regarded as a valid treatment option for knee OA and as a possible first-line injectable treatment option for nonoperative management of knee OA, mainly for KL grades 1-3. LEVEL OF EVIDENCE: Level II.

Men and women undergoing total hip arthroplasty have different clinical presentations before surgery and different outcomes at 1-year follow-up.

PURPOSE: The purpose of this study was to investigate the influence of sex on patients undergoing total hip arthroplasty (THA) for hip osteoarthritis (HOA), aiming to assess the clinical and functional outcomes using patient-reported outcome measures (PROMs). METHODS: A retrospective analysis of patients undergoing THA at Ospedale Galeazzi-Sant'Ambrogio between 2016 and 2022 was conducted. Inclusion criteria encompassed Kellgren-Lawrence grade III or IV HOA, with preoperative and 12-month postoperative PROMs. Enroled patients have been selected from a larger cohort without matching design for confounders. The analyses were performed using R software v4.0.3 (R Core Team) and data distributions were assessed using the Shapiro-Wilk normality test. RESULTS: One hundred ninety patients (72 male and 118 female) who had both preoperative and postoperative PROMs have been analysed from our institutional prosthesis registry (Datareg). Baseline and 12-month post-THA PROMs showed significant improvements overall. VAS score dropped notably from baseline to 3 months postsurgery (7.1 ± 2.1 vs. 0.9 ± 1.7). Functional and mental PROMs, including Harris Hip Score-functional (HHS-F), Harris Hip Score-total (HHS-t), SF-12PS and SF-12MS, exhibited substantial improvements post-THA. Stratifying by sex, males had lower baseline VAS, higher HHS-F, SF-12MS and hip disability and osteoarthritis outcome score-physical function short form (HOOS-PS). At 12 months, males displayed significantly better VAS, HHS-F, SF-12PS and HOOS-PS scores. Complication rates were minimal (1.5%), with stable rates across genders, mostly involving dislocation and periprosthetic fractures. Implant survival at 12 months reached an impressive 99%. CONCLUSION: THA remains an effective treatment for severe HOA. However, females presented with worse baseline conditions and showed relatively less improvement at 1-year postsurgery compared to males. This difference could be attributed to physiological and psychosocial factors associated with sex, including hormonal changes, muscle mass decline and perception of pain. Longer follow-ups and prospective studies are necessary to validate these findings and facilitate personalised approaches in HOA treatment, emphasising the need for careful consideration of sex-related variables in clinical decision-making for THA patients. LEVEL OF EVIDENCE: Level III.

An Updated Overview of Risk Factors for Shoulder Stiffness. / Risikofaktoren für die Entstehung der primären Schultersteife: ein aktueller Überblick.

A painful reduction in shoulder mobility, known as "shoulder stiffness", can occur both as a primary idiopathic condition and as a secondary condition, for example, following surgical procedures. Various factors can contribute to the development of primary shoulder stiffness. In this review we summarize the pathophysiological mechanisms, genetic influences, endocrine disorders, metabolic conditions, as well as other diseases and medical-therapeutic approaches that might have an impact on the development of primary shoulder stiffness.

Stable Housekeeping Genes in Bone Marrow, Adipose Tissue, and Amniotic Membrane-Derived Mesenchymal Stromal Cells for Orthopedic Regenerative Medicine Approaches.

The therapeutic effect of mesenchymal stromal cells (MSCs) has been described for a variety of disorders, including those affecting musculoskeletal tissues. In this context, the literature reports several data about the regenerative effectiveness of MSCs derived from bone marrow, adipose tissue, and an amniotic membrane (BMSCs, ASCs, and hAMSCs, respectively), either when expanded or when acting as clinical-grade biologic pillars of products used at the point of care. To date, there is no evidence about the superiority of one source over the others from a clinical perspective. Therefore, a reliable characterization of the tissue-specific MSC types is mandatory to identify the most effective treatment, especially when tailored to the target disease. Because molecular characterization is a crucial parameter for cell definition, the need for reliable normalizers as housekeeping genes (HKGs) is essential. In this report, the stability levels of five commonly used HKGs (ACTB, EF1A, GAPDH, RPLP0, and TBP) were sifted into BMSCs, ASCs, and hAMSCs. Adult and fetal/neonatal MSCs showed opposite HKG stability rankings. Moreover, by analyzing MSC types side-by-side, comparison-specific HKGs emerged. The effect of less performant HKG normalization was also demonstrated in genes coding for factors potentially involved in and predicting MSC therapeutic activity for osteoarthritis as a model musculoskeletal disorder, where the choice of the most appropriate normalizer had a higher impact on the donors rather than cell populations when compared side-by-side. In conclusion, this work confirms HKG source-specificity for MSCs and suggests the need for cell-type specific normalizers for cell source or condition-tailored gene expression studies.

A high level of scientific evidence is available to guide treatment of primary shoulder stiffness: The SIAGASCOT consensus.

PURPOSE: Shoulder stiffness (SS) is a condition characterised by active and passive restricted glenohumeral range of motion, which can occur spontaneously in an idiopathic manner or be associated with a known underlying aetiology. Several treatment options are available and currently no consensus has been obtained on which treatment algorithm represents the best choice for the patient. Herein we present the results of a national consensus on the treatment of primary SS. METHODS: The project followed the modified Delphi consensus process, involving a steering, a rating and a peer-review group. Sixteen questions were generated and subsequently answered by the steering group after a thorough literature search. A rating group composed by professionals specialised in the diagnosis and treatment of shoulder pathologies rated the question-answer sets according to the scientific evidence and their clinical experience. RESULTS: Recommendations were rated with an average of 8.4 points out of maximum 9 points. None of the 16 answers received a rating of less than 8 and all the answers were considered as appropriate. The majority of responses were assessed as Grade A, signifying a substantial availability of scientific evidence to guide treatment and support recommendations encompassing diagnostics, physiotherapy, electrophysical agents, oral and injective medical therapies, as well as surgical interventions for primary SS. CONCLUSIONS: A consensus regarding the conservative and surgical treatment of primary SS could be achieved at a national level. This consensus sets basis for evidence-based clinical practice in the management of primary SS and can serve as a model for similar initiatives and adaptable guidelines in other European countries and potentially on a global scale. LEVEL OF EVIDENCE: Level I.

Housekeeping Gene Stability in Adipose Mesenchymal Stromal Cells Cultivated in Serum/Xeno-Free Media for Osteoarthritis.

Among the available therapeutics for the conservative treatment of osteoarthritis (OA), mesenchymal stromal cells (MSCs)-based products appear to be the most promising. Alongside minimally manipulated cell-based orthobiologics, where MSCs are the engine of the bioactive properties, cell expansion under good manufacturing practice (GMP) settings is actively studied to obtain clinical-grade pure populations able to concentrate the biological activity. One of the main characteristics of GMP protocols is the use of clinical-grade reagents, including the recently released serum-free/xeno-free (SFM/XFM) synthetic media, which differ significantly from the traditional reagents like those based on fetal bovine serum (FBS). As SFM/XFM are still poorly characterized, a main lack is the notion of reliable housekeeping genes (HKGs) for molecular studies, either standalone or in combination with standard conditions. Indeed, the aim of this work was to test the stability of five commonly used HKGs (ACTB, EF1A, GAPDH, RPLP0, and TBP) in adipose-derived MSCs (ASCs) cultivated in two commercially available SFM/XFM and to compare outcomes with those obtained in FBS. Four different applets widely recognized by the scientific community (NormFinder, geNorm, comparative ΔCt method, and BestKeeper) were used and data were merged to obtain a final stability order. The analysis showed that cells cultured in both synthetic media had a similar ranking for HKGs stability (GAPDH being best), albeit divergent from FBS expanded products (EF1A at top). Moreover, it was possible to identify specific HKGs for side by side studies, with EF1A/TBP being the most reliable normalizers for single SFM/XFM vs. FBS cultured cells and TBP the best one for a comprehensive analysis of all samples. In addition, stability of HKGs was donor-dependent. The normalization effect on selected genes coding for factors known to be involved in OA pathology, and whose amount should be carefully considered for the selection of the most appropriate MSC-based treatment, showed how HKGs choice might affect the perceived amount for the different media or donor. Overall, this work confirms the impact of SFM/XFM conditions on HKGs stability performance, which resulted similarly for both synthetic media analyzed in the study.