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Background: Adult skeletal muscle contains resident muscle stem cells (MuSC) with high myogenic and engraftment potentials, making them suitable for cell therapy and regenerative medicine approaches. However, purification process of MuSC remains a major hurdle to their use in the clinic. Indeed, muscle tissue enzymatic dissociation triggers a massive activation of stress signaling pathways, among which P38 and JNK MAPK, associated with a premature loss of MuSC quiescence. While the role of these pathways in the myogenic progression of MuSC is well established, the extent to which their dissociation-induced activation affects the functionality of these cells remains unexplored., Methods: We assessed the effect of P38 and JNK MAPK induction on stemness marker expression and MuSC activation state during isolation by pharmacological approaches. MuSC functionality was evaluated by in vitro assays and in vivo transplantation experiments. We performed a comparative analysis of the transcriptome of human MuSC purified with pharmacological inhibitors of P38 and JNK MAPK (SB202190 and SP600125, respectively) versus available RNAseq resources., Results: We monitored PAX7 protein levels in murine MuSC during muscle dissociation and demonstrated a two-step decline partly dependent on P38 and JNK MAPK activities. We showed that simultaneous inhibition of these pathways throughout the MuSC isolation process preserves the expression of stemness markers and limits their premature activation, leading to improved survival and amplification in vitro as well as increased engraftment in vivo. Through a comparative RNAseq analysis of freshly isolated human MuSC, we provide evidence that our findings in murine MuSC could be relevant to human MuSC. Based on these findings, we implemented a purification strategy, significantly improving the recovery yields of human MuSC., Conclusion: Our study highlights the pharmacological limitation of P38 and JNK MAPK activities as a suitable strategy to qualitatively and quantitatively ameliorate human MuSC purification process, which could be of great interest for cell-based therapies., (© 2024. The Author(s).)

We investigated the impact of mesenchymal stem cell (MSC) therapy on treating bilateral human hip osteonecrosis, analyzing 908 cases. This study assesses factors such as tissue source and cell count, comparing core decompression with various cell therapies. This research emphasizes bone repair according to pre-treatment conditions and the specificities of cell therapy in osteonecrosis repair, indicating a potential for improved bone repair strategies in hips without femoral head collapse. This study utilized a single-center retrospective analysis to investigate the efficacy of cellular approaches in the bone repair of osteonecrosis. It examined the impact on bone repair of tissue source (autologous bone marrow concentrate, allogeneic expanded, autologous expanded), cell quantity (from none in core decompression alone to millions in cell therapy), and osteonecrosis stage and volume. Excluding hips with femoral head collapse, it focused on patients who had bilateral hip osteonecrosis, both pre-operative and post-operative MRIs, and a follow-up of over five years. The analysis divided these patients into seven groups based on match control treatment variations in bilateral hip osteonecrosis, primarily investigating the outcomes between core decompression, washing effect, and different tissue sources of MSCs. Younger patients (<30 years) demonstrated significantly better repair volumes, particularly in stage II lesions, than older counterparts. Additionally, bone repair volume increased with the number of implanted MSCs up to 1,000,000, beyond which no additional benefits were observed. No significant difference was observed in repair outcomes between different sources of MSCs (BMAC, allogenic, or expanded cells). The study also highlighted that a 'washing effect' was beneficial, particularly for larger-volume osteonecrosis when combined with core decompression. Partial bone repair was the more frequent event observed, while total bone repair of osteonecrosis was rare. The volume and stage of osteonecrosis, alongside the number of injected cells, significantly affected treatment outcomes. In summary, this study provides comprehensive insights into the effectiveness and variables influencing the use of mesenchymal stem cells in treating human hip osteonecrosis. It emphasizes the potential of cell therapy while acknowledging the complexity and variability of results based on factors such as age, cell count, and disease stage.

Competing Interests: The authors declare no conflict of interest.

Purpose: Several reports have identified prognostic factors for hip osteonecrosis treated with cell therapy, but no study investigated the accuracy of artificial intelligence method such as machine learning and artificial neural network (ANN) to predict the efficiency of the treatment. We determined the benefit of cell therapy compared with core decompression or natural evolution, and developed machine-learning algorithms for predicting ten year collapse-free survival in hip osteonecrosis treated with cell therapy. Using the best algorithm, we propose a calculator for "prognosis hip osteonecrosis cell therapy (PHOCT)" accessible for clinical use., Methods: A total of 3145 patients with 5261 osteonecroses without collapses were included in this study, comprising 1321 (42%) men and 1824 (58%) women, with a median age of 34 (12-62) years. Cell therapy was the treatment for 3021 hips, core decompression alone for 1374 hips, while absence of treatment was the control group of 764 hips. First, logistic regression and binary logistic regression analysis were performed to compare results of the three groups at ten years. Then an artificial neural network model was developed for ten year collapse-free survival after cell therapy. The models' performances were compared. The algorithms were assessed by calibration, and performance, and with c-statistic as measure of discrimination. It ranges from 0.5 to 1.0, with 1.0 being perfect discrimination and 0.5 poor (no better than chance at making a prediction)., Results: Among the 3021 hips with cell therapy, 1964 hips (65%) were collapse-free survival at ten years, versus 453 (33%) among those 1374 treated with core decompression alone, and versus 115 (15%) among 764 hips with natural evolution. We analyzed factors influencing the prediction of collapse-free period with classical statistics and artificial intelligence among hips with cell therapy. After selecting variables, a machine learning algorithm created a prognosis osteonecrosis cell therapy calculator (POCT). This calculator proved to have good accuracy on validation in these series of 3021 hip osteonecroses treated with cell therapy. The algorithm had a c-statistic of 0.871 suggesting good-to-excellent discrimination when all the osteonecroses were mixed. The c-statistics were calculated separately for subpopulations of categorical osteonecroses. It retained good accuracy, but underestimated ten year survival in some subgroups, suggesting that specific calculators could be useful for some subgroups. This study highlights the importance of multimodal evaluation of patient parameters and shows the degree to which the outcome is modified by some decisions that are within a surgeon's control, as the number of cells to aspirate, the choice of injecting in both the osteonecrosis and the healthy bone, the choice between unilateral or bilateral injection, and the possibility to do a repeat injection., Conclusion: Many disease conditions and the heterogeneities of patients are causes of variation of outcome after cell therapy for osteonecrosis. Predicting therapeutic effectiveness with a calculator allows a good discrimination to target patients who are most likely to benefit from this intervention., (© 2023. The Author(s) under exclusive licence to SICOT aisbl.)

Over the past 25 years, we have demonstrated the feasibility of airway bioengineering using stented aortic matrices experimentally then in a first-in-human trial (n = 13). The present TRITON-01 study analyzed all the patients who had airway replacement at our center to confirm that this innovative approach can be now used as usual care. For each patient, the following data were prospectively collected: postoperative mortality and morbidity, late airway complications, stent removal and status at last follow-up on November 2, 2021. From October 2009 to October 2021, 35 patients had airway replacement for malignant (n = 29) or benign (n = 6) lesions. The 30-day postoperative mortality and morbidity rates were 2.9% (n = 1/35) and 22.9% (n = 8/35) respectively. At a median follow-up of 29.5 months (range 1-133 months), 27 patients were alive. There have been no deaths directly related to the implanted bioprosthesis. Eighteen patients (52.9%) had stent-related granulomas requiring a bronchoscopic treatment. Ten among 35 patients (28.6%) achieved a stent free survival. The actuarial 2- and 5-year survival rates (Kaplan-Meier estimates) were respectively 88% and 75%. The TRITON-01 study confirmed that airway replacement using stented aortic matrices can be proposed as usual care at our center. Clinicaltrials.gov Identifier: NCT04263129., (© 2022 The American Society of Transplantation and the American Society of Transplant Surgeons.)

Introduction: Open fractures are at risk of nonunion; surgeons are reluctant to propose early standard bone grafting after open fractures, preferring to wait in order to adequately assess the facture status of infection. Bone marrow contains mesenchymal stem cells (MSCs) and granulocyte and macrophage precursors identified in vitro as colony forming units-granulocyte macrophage (CFU-GM), both of which have a prophylactic action against infection. We therefore tested the hypothesis that early injection of bone marrow concentrate would be useful in these fractures., Methods: We evaluated a series of 231 patients who had received early percutaneous implantation of bone marrow concentrate (BMC) to treat open fractures (with gap less than 10 mm) that were Gustilo-Anderson Type II or III. The results were compared with those of 67 control (no early graft) patients and with those of 76 patients treated with an early, standard of care, iliac bone graft. All patients were treated with external fixation and were considered to have an aseptic fracture at the time of early grafting, but the actual status of infection was re-assessed at the time of grafting by histology and/or analysis of the aspirate. The bone marrow graft contained after concentration 49,758 ± 21,642 CFU-GM-derived colonies/cc and 9400 ± 1435 MSCs/cc which represents an important increase compared to the level of CFU-GM cells and MSCs present in a standard auto-graft. Healing was evaluated at 9 months., Results: The rate of unsuspected infections was higher than 15% in the 3 groups. Bone union and removal of external fixation was achieved at 9 months by 50.7% of patients in the Control Group, by 86.8% of patients in the group with a standard bone graft, and by 87.4% of patients in the bone marrow group. A 90% risk reduction (p = 0.005) in the need for an invasive standard bone graft to treat a nonunion and in the risk of infection was observed when bone marrow was proposed as early injection to the treatment of type II or type-III tibial fractures., Conclusion: Bone marrow concentrate for early grafting in open fractures with limited gap was efficient for healing while decreasing infection., (Copyright © 2020. Published by Elsevier Ltd.)

Purpose: Corticoid treatment associated with haematologic treatments can lead to ankle osteonecrosis in children's survivors of acute leukemia (ALL). Based on the efficiency of mesenchymal stem cells (MSCs) in hip osteonecrosis, we performed an evaluation of this treatment in 51 children and adolescents who had symptomatic ankle osteonecrosis after therapy for haematologic cancer., Material and Methods: The 51 patients had a total of 79 osteonecrosis sites on MRI, with 29 talus sites, 18 metaphyseal tibia sites, 12 epiphyseal tibia sites, eight calcaneus sites, six fibula sites, four navicular sites, and two cuboid sites. In this prospective randomized trial, 37 ankles were addressed for cell therapy, 37 others for core decompression alone, and 20 were considered as a control group without treatment. We analyzed the outcome of this treatment osteonecrosis, the number and characteristics of bone marrow mesenchymal cells (MSCs) that could be transplanted, and the risks of tumorigenesis in these patients with haematologic cancers. The patients were operated on over a period of ten years from 2000 to 2010 and were monitored through December 31, 2019., Results: Despite a normal systemic blood cells count, MSCs in the iliac crest (counted as CFU-F) were in low number (1021 MSCs/mL; range 314-3015) and were of host origin after even allogeneic bone marrow transplantation. Better clinical outcomes (pain, foot and ankle deformity) and osteonecrosis repair on MRI with absence of collapse were obtained in ankles that received cell therapy as compared with those with core decompression alone or those without initial surgery. No tumour was found on MRI at the sites of injection and this study found no increased risk of recurrence or of new cancer in this population after an average follow-up of 15 years., Conclusions: These results suggest that autologous MSCs can improve the quality of life of leukemia survivors with ankle osteonecrosis., (© 2021. SICOT aisbl.)

Background : Skeletal muscle is one of the only mammalian tissues capable of rapid and efficient regeneration after trauma or in pathological conditions. Skeletal muscle regeneration is driven by the muscle satellite cells, the stem cell population in interaction with their niche. Upon injury, muscle fibers undergo necrosis and muscle stem cells activate, proliferate and fuse to form new myofibers. In addition to myogenic cell populations, interaction with other cell types such as inflammatory cells, mesenchymal (fibroadipogenic progenitors-FAPs, pericytes) and vascular (endothelial) lineages are important for efficient muscle repair. While the role of the distinct populations involved in skeletal muscle regeneration is well characterized, the quantitative changes in the muscle stem cell and niche during the regeneration process remain poorly characterized. Methods : We have used mass cytometry to follow the main muscle cell types (muscle stem cells, vascular, mesenchymal and immune cell lineages) during early activation and over the course of muscle regeneration at D0, D2, D5 and D7 compared with uninjured muscles. Results : Early activation induces a number of rapid changes in the proteome of multiple cell types. Following the induction of damage, we observe a drastic loss of myogenic, vascular and mesenchymal cell lineages while immune cells invade the damaged tissue to clear debris and promote muscle repair. Immune cells constitute up to 80% of the mononuclear cells 5 days post-injury. We show that muscle stem cells are quickly activated in order to form new myofibers and reconstitute the quiescent muscle stem cell pool. In addition, our study provides a quantitative analysis of the various myogenic populations during muscle repair. Conclusions : We have developed a mass cytometry panel to investigate the dynamic nature of muscle regeneration at a single-cell level. Using our panel, we have identified early changes in the proteome of stressed satellite and niche cells. We have also quantified changes in the major cell types of skeletal muscle during regeneration and analyzed myogenic transcription factor expression in satellite cells throughout this process. Our results highlight the progressive dynamic shifts in cell populations and the distinct states of muscle stem cells adopted during skeletal muscle regeneration. Our findings give a deeper understanding of the cellular and molecular aspects of muscle regeneration.

Background : Osteonecrosis (ON) of the femoral head represents a potentially severe disease of the hip where the lack of bone regeneration may lead to femoral head collapse and secondary osteoarthritis, with serious pain and disability. The aim of this European, multicentric clinical trial was to prove safety and early efficacy to heal early femoral head ON in patients through minimally invasive surgical implantation of autologous mesenchymal stromal cells (MSC) expanded from bone marrow (BM) under good manufacturing practices (GMP). Methods : Twenty-two patients with femoral head ON (up to ARCO 2C) were recruited and surgically treated in France, Germany, Italy and Spain with BM-derived, expanded autologous MSC (total dose 140 million MSC in 7 mL). The investigational advanced therapy medicinal product (ATMP) was expanded from BM under the same protocol in all four countries and approved by each National Competent Authority. Patients were followed during two years for safety, based on adverse events, and for efficacy, based on clinical assessment (pain and hip score) and imaging (X-rays and MRIs). Patients were also reviewed after 5 to 6 years at latest follow-up for final outcome. Results : No severe adverse event was recalled as related to the ATMP. At 12 months, 16/20 per protocol and 16/22 under intention-to-treat (2 drop-out at 3 and 5 months) maintained head sphericity and showed bone regeneration. Of the 4 hips with ON progression, 3 required total hip replacement (THR). At 5 years, one patient (healed at 2 years visit) was not located, and 16/21 showed no progression or THR, 4/21 had received THR (all in the first year) and 1 had progressed one stage without THR. Conclusions : Expanded MSCs implantation was safe. Early efficacy was confirmed in 80% of cases under protocol at 2 years. At 5 years, the overall results were maintained and 19% converted to THR, all in the first year.

Purpose: There is an increasing number of reports on the treatment of knee osteoarthritis (OA) using mesenchymal stem cells (MSCs). However, it is not known what would better drive osteoarthritis stabilization to postpone total knee arthroplasty (TKA): targeting the synovial fluid by injection or targeting on the subchondral bone with MSCs implantation., Methods: A prospective randomized controlled clinical trial was carried out between 2000 and 2005 in 120 knees of 60 patients with painful bilateral knee osteoarthritis with a similar osteoarthritis grade. During the same anaesthesia, a bone marrow concentrate of 40 mL containing an average 5727 MSCs/mL (range 2740 to 7540) was divided in two equal parts: after randomization, one part (20 mL) was delivered to the subchondral bone of femur and tibia of one knee (subchondral group) and the other part was injected in the joint for the contralateral knee (intra-articular group). MSCs were counted as CFU-F (colony fibroblastic unit forming). Clinical outcomes of the patient (Knee Society score) were obtained along with radiological imaging outcomes (including MRIs) at two year follow-up. Subsequent revision surgeries were identified until the most recent follow-up (average of 15 years, range 13 to 18 years)., Results: At two year follow-up, clinical and imaging (MRI) improvement was higher on the side that received cells in the subchondral bone. At the most recent follow-up (15 years), among the 60 knees treated with subchondral cell therapy, the yearly arthroplasty incidence was 1.3% per knee-year; for the 60 knees with intra-articular cell therapy, the yearly arthroplasty incidence was higher (p = 0.01) with an incidence of 4.6% per knee-year. For the side with subchondral cell therapy, 12 (20%) of 60 knees underwent TKA, while 42 (70%) of 60 knees underwent TKA on the side with intra-articular cell therapy. Among the 18 patients who had no subsequent surgery on both sides, all preferred the knee with subchondral cell therapy., Conclusions: Implantation of MSCs in the subchondral bone of an osteoarthritic knee is more effective to postpone TKA than injection of the same intra-articular dose in the contralateral knee with the same grade of osteoarthritis.

Muscle stem cells (or muscle satellite cells [MuSCs]) are required for postnatal growth. Yet, the detailed characterization of myogenic progression and establishment of quiescence during this process remains poorly documented. Here, we provide an overview of myogenic cells heterogeneity and dynamic from birth to adulthood using flow cytometry. We demonstrated that PAX7+ cells acquire an increasing ability to progress in the myogenic program from birth to adulthood. We then simultaneously analyzed the cycling state (KI67 expression) of the MuSCs and progenitors (PAX7+) and their progression into myogenic precursors (PAX7-MYOD+) and differentiating cells (MYOG+) in vivo. We identified two distinct peaks of myogenic differentiation between P7-P10 and P21-P28, and showed that the quiescent MuSC pool is established between 7 and 8 weeks of age. Overall our study provides a comprehensive in vivo characterization of myogenic heterogeneity and demonstrates the highly dynamic nature of skeletal muscle postnatal growth process., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Background and Study Aim: Advanced therapy medicinal products (ATMP) frequently lack of clinical data on efficacy to substantiate a future clinical use. This study aims to evaluate the efficacy to heal long bone delayed unions and non-unions, as secondary objective of the EudraCT 2011-005441-13 clinical trial, through clinical and radiological bone consolidation at 3, 6 and 12 months of follow-up, with subgroup analysis of affected bone, gender, tobacco use, and time since the original fracture., Patients and Methods: Twenty-eight patients were recruited and surgically treated with autologous bone marrow derived mesenchymal stromal cells expanded under Good Manufacturing Practices, combined to bioceramics in the surgical room before implantation. Mean age was 39 ± 13 years, 57% were males, and mean Body Mass Index 27 ± 7. Thirteen (46%) were active smokers. There were 11 femoral, 4 humeral, and 13 tibial non-unions. Initial fracture occurred at a mean ± SD of 27.9 ± 31.2 months before recruitment. Efficacy results were expressed by clinical consolidation (no or mild pain if values under 30 in VAS scale), and by radiological consolidation with a REBORNE score over 11/16 points (value of or above 0.6875). Means were statistically compared and mixed models for repeated measurements estimated the mean and confidence intervals (95%) of the REBORNE Bone Healing scale. Clinical and radiological consolidation were analyzed in the subgroups with Spearman correlation tests (adjusted by Bonferroni)., Results: Clinical consolidation was earlier confirmed, while radiological consolidation at 3 months was 25.0% (7/28 cases), at 6 months 67.8% (19/28 cases), and at 12 months, 92.8% (26/28 cases including the drop-out extrapolation of two failures). Bone biopsies confirmed bone formation surrounding the bioceramic granules. All locations showed similar consolidation, although this was delayed in tibial non-unions. No significant gender difference was found in 12-month consolidation (95% confidence). Higher consolidation scale values were seen in non-smoking patients at 6 (p = 0.012, t-test) and 12 months (p = 0.011, t-test). Longer time elapsed after the initial fracture did not preclude the occurrence of consolidation., Conclusion: Bone consolidation was efficaciously obtained with the studied expanded hBM-MSCs combined to biomaterials, by clinical and radiological evaluation, and confirmed by bone biopsies, with lower consolidation scores in smokers., Competing Interests: Declaration of Competing Interest The authors confirm that there are no known conflicts of interest associated with this publication and there has been no financial support for this work that could have influenced its outcome. The authors do not communicate any conflict of interest about this work and manuscript., (Copyright © 2020. Published by Elsevier Ltd.)