Publications by authors named "Hélène Rouard"

57 Publications

Progressive and Coordinated Mobilization of the Skeletal Muscle Niche throughout Tissue Repair Revealed by Single-Cell Proteomic Analysis.

Cells 2021 Mar 28;10(4). Epub 2021 Mar 28.

Université Paris Est Creteil, INSERM, IMRB, F-94010 Creteil, France.

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. 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. 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. : 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.
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http://dx.doi.org/10.3390/cells10040744DOI Listing
March 2021

Osteonecrosis of the Femoral Head Safely Healed with Autologous, Expanded, Bone Marrow-Derived Mesenchymal Stromal Cells in a Multicentric Trial with Minimum 5 Years Follow-Up.

J Clin Med 2021 Feb 1;10(3). Epub 2021 Feb 1.

INSERM U957, Lab. Pathophysiology of Bone Resorption, Faculty of Medicine, University of Nantes, 44035 Nantes, France.

: 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). : 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. : 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. : 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.
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http://dx.doi.org/10.3390/jcm10030508DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7867148PMC
February 2021

Early injection of autologous bone marrow concentrates decreases infection risk and improves healing of acute severe open tibial fractures.

Injury 2020 Dec 31. Epub 2020 Dec 31.

University Paris East Hôpital Henri Mondor, 94010 Creteil, France.

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.
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http://dx.doi.org/10.1016/j.injury.2020.12.007DOI Listing
December 2020

Distinct Phases of Postnatal Skeletal Muscle Growth Govern the Progressive Establishment of Muscle Stem Cell Quiescence.

Stem Cell Reports 2020 09 6;15(3):597-611. Epub 2020 Aug 6.

Univ Paris Est Creteil, INSERM, EFS, IMRB, Creteil 94010, France. Electronic address:

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.
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http://dx.doi.org/10.1016/j.stemcr.2020.07.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7486220PMC
September 2020

Subchondral bone or intra-articular injection of bone marrow concentrate mesenchymal stem cells in bilateral knee osteoarthritis: what better postpone knee arthroplasty at fifteen years? A randomized study.

Int Orthop 2021 Feb 2;45(2):391-399. Epub 2020 Jul 2.

Orthopedic Department Henri Mondor Hospital, University Paris East, Creteil, France.

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.
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http://dx.doi.org/10.1007/s00264-020-04687-7DOI Listing
February 2021

Early efficacy evaluation of mesenchymal stromal cells (MSC) combined to biomaterials to treat long bone non-unions.

Injury 2020 Apr 26;51 Suppl 1:S63-S73. Epub 2020 Feb 26.

INSERM U957, Lab. Pathophysiology of bone resorption, Faculty of Medicine, University of Nantes, Nantes, France.

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.
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http://dx.doi.org/10.1016/j.injury.2020.02.070DOI Listing
April 2020

Human Apoptotic Cells, Generated by Extracorporeal Photopheresis, Modulate Allogeneic Immune Response.

Front Immunol 2019 18;10:2908. Epub 2019 Dec 18.

Assistance Publique-Hôpitaux de Paris (AP-HP), Groupe Hospitalo-Universitaire Chenevier Mondor, Centre d'Investigation Clinique Biothérapie, Créteil, France.

The induction of specific and sustainable tolerance is a challenging issue in organ transplantation. The discovery of the immunosuppressive properties of apoptotic cells in animal models has paved the way for their use in human transplantation. In this work, we aimed to define a stable, reproducible, and clinically compatible production procedure of human apoptotic cells (Apo-cells). Using a clinically approved extracorporeal photopheresis technique, we have produced and characterized phenotypically and functionally human apoptotic cells. These Apo-cells have immunosuppressive properties proved and in NOD/SCID/γC mice by their capacity to modulate an allogeneic response following both a direct and an indirect antigen presentation. These results brought the rationale for the use of Apo-cells in tolerance induction protocol for organ transplantation.
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http://dx.doi.org/10.3389/fimmu.2019.02908DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6930166PMC
November 2020

Long-Term Results of Cultured Limbal Stem Cell Versus Limbal Tissue Transplantation in Stage III Limbal Deficiency.

Stem Cells Transl Med 2019 12 5;8(12):1230-1241. Epub 2019 Sep 5.

Banque de tissus, Etablissement Français du Sang-Ile-de-France, Paris, France.

We aimed to evaluate efficiency and safety of transplantation of limbal stem cells (LSC) cultured on human amniotic membrane with no feeders and to compare cultured LSC with limbal tissue transplantation. Thirty eyes with stage III LSC deficiency were treated with autologous (autoLSC) or allogeneic (alloLSC) cultured LSC transplantation (prospective phase II clinical trial; average follow-up time, 72 months) or autologous (autoLT) or allogeneic (alloLT) limbal tissue transplantation (retrospective control group; average follow-up time, 132 months) between 1993 and 2014. The 5-year graft survival defined by absence of recurrence of the clinical signs of limbal deficiency was 71% for autoLSC, 0% for alloLSC, 75% for autoLT, and 33% for alloLT. Visual acuity improved by 9.2 lines for autoLSC and 3.3 lines for autoLT. It decreased by 0.7 lines for alloLSC and 1.9 lines for alloLT. Adverse events were recorded in 1/7 autoLSC, 7/7 alloLSC, 6/8 autoLT, and 8/8 alloLT patients. Corneal epithelial defect was the only adverse event recorded after autoLSC, whereas severe sight-threatening adverse events were recorded in the remaining three groups. Compared with failed grafts, successful grafts featured greater decrease in fluorescein staining, greater superficial vascularization-free corneal area, lower variability of the corneal epithelial thickness, and higher corneal epithelial basal cell density. Autologous cultured LSC transplantation was associated with high long-term survival and dramatic improvement in vision and was very safe. Autologous limbal tissue transplantation resulted in similar efficiency but was less safe. Cadaver allogeneic grafts resulted in low long-term success rate and high prevalence of serious adverse events. Stem Cells Translational Medicine 2019;8:1230&1241.
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http://dx.doi.org/10.1002/sctm.19-0021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6877763PMC
December 2019

Design and Validation of an Automated Process for the Expansion of Peripheral Blood-Derived CD34 Cells for Clinical Use After Myocardial Infarction.

Stem Cells Transl Med 2019 08 29;8(8):822-832. Epub 2019 Apr 29.

CellProthera and IRHT, Mulhouse, France.

We previously demonstrated that intracardiac delivery of autologous peripheral blood-derived CD34 stem cells (SCs), mobilized by granulocyte-colony stimulating factor (G-CSF) and collected by leukapheresis after myocardial infarction, structurally and functionally repaired the damaged myocardial area. When used for cardiac indication, CD34 cells are now considered as Advanced Therapy Medicinal Products (ATMPs). We have industrialized their production by developing an automated device for ex vivo CD34 -SC expansion, starting from a whole blood (WB) sample. Blood samples were collected from healthy donors after G-CSF mobilization. Manufacturing procedures included: (a) isolation of total nuclear cells, (b) CD34 immunoselection, (c) expansion and cell culture recovery in the device, and (d) expanded CD34 cell immunoselection and formulation. The assessment of CD34 cell counts, viability, and immunophenotype and sterility tests were performed as quality tests. We established graft acceptance criteria and performed validation processes in three cell therapy centers. 59.4 × 10  ± 36.8 × 10 viable CD34 cells were reproducibly generated as the final product from 220 ml WB containing 17.1 × 10  ± 8.1 × 10 viable CD34 cells. CD34 identity, genetic stability, and telomere length were consistent with those of basal CD34 cells. Gram staining and mycoplasma and endotoxin analyses were negative in all cases. We confirmed the therapeutic efficacy of both CD34 -cell categories in experimental acute myocardial infarct (AMI) in immunodeficient rats during preclinical studies. This reproducible, automated, and standardized expansion process produces high numbers of CD34 cells corresponding to the approved ATMP and paves the way for a phase I/IIb study in AMI, which is currently recruiting patients. Stem Cells Translational Medicine 2019;8:822&832.
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http://dx.doi.org/10.1002/sctm.17-0277DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6646685PMC
August 2019

Feasibility of Bioengineered Tracheal and Bronchial Reconstruction Using Stented Aortic Matrices.

JAMA 2018 06;319(21):2212-2222

Assistance Publique-Hôpitaux de Paris, Unité de Recherche Clinique, Hôpitaux Saint Louis-Lariboisière-Fernand Widal, Université Paris Diderot, Paris, France.

Importance: Airway transplantation could be an option for patients with proximal lung tumor or with end-stage tracheobronchial disease. New methods for airway transplantation remain highly controversial.

Objective: To establish the feasibility of airway bioengineering using a technique based on the implantation of stented aortic matrices.

Design, Setting, And Participants: Uncontrolled single-center cohort study including 20 patients with end-stage tracheal lesions or with proximal lung tumors requiring a pneumonectomy. The study was conducted in Paris, France, from October 2009 through February 2017; final follow-up for all patients occurred on November 2, 2017.

Exposures: Radical resection of the lesions was performed using standard surgical techniques. After resection, airway reconstruction was performed using a human cryopreserved (-80°C) aortic allograft, which was not matched by the ABO and leukocyte antigen systems. To prevent airway collapse, a custom-made stent was inserted into the allograft. In patients with proximal lung tumors, the lung-sparing intervention of bronchial transplantation was used.

Main Outcomes And Measures: The primary outcome was 90-day mortality. The secondary outcome was 90-day morbidity.

Results: Twenty patients were included in the study (mean age, 54.9 years; age range, 24-79 years; 13 men [65%]). Thirteen patients underwent tracheal (n = 5), bronchial (n = 7), or carinal (n = 1) transplantation. Airway transplantation was not performed in 7 patients for the following reasons: medical contraindication (n = 1), unavoidable pneumonectomy (n = 1), exploratory thoracotomy only (n = 2), and a lobectomy or bilobectomy was possible (n = 3). Among the 20 patients initially included, the overall 90-day mortality rate was 5% (1 patient underwent a carinal transplantation and died). No mortality at 90 days was observed among patients who underwent tracheal or bronchial reconstruction. Among the 13 patients who underwent airway transplantation, major 90-day morbidity events occurred in 4 (30.8%) and included laryngeal edema, acute lung edema, acute respiratory distress syndrome, and atrial fibrillation. There was no adverse event directly related to the surgical technique. Stent removal was performed at a postoperative mean of 18.2 months. At a median follow-up of 3 years 11 months, 10 of the 13 patients (76.9%) were alive. Of these 10 patients, 8 (80%) breathed normally through newly formed airways after stent removal. Regeneration of epithelium and de novo generation of cartilage were observed within aortic matrices from recipient cells.

Conclusions And Relevance: In this uncontrolled study, airway bioengineering using stented aortic matrices demonstrated feasibility for complex tracheal and bronchial reconstruction. Further research is needed to assess efficacy and safety.

Trial Registration: clinicaltrials.gov Identifier: NCT01331863.
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http://dx.doi.org/10.1001/jama.2018.4653DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6134437PMC
June 2018

History of concentrated or expanded mesenchymal stem cells for hip osteonecrosis: is there a target number for osteonecrosis repair?

Int Orthop 2018 07 24;42(7):1739-1745. Epub 2018 May 24.

Hôpital Henri Mondor, 94010, Creteil, France.

Purpose: Despite multiple possible treatments, the risk of collapse remains the main problem of osteonecrosis. Heart failure (HF). In an effort to address the reverse this issue, curative strategies with regenerative medicine are increasingly being considered. The aim of this technology is to halt or reverse progression of the disease to collapse.

Material And Methods: The pioneering report by Hernigou published in 2002 was the first pilot study suggesting that injection of bone marrow stem cells was a safe approach able to improve osteonecrosis in patients with early stages. Since then, an impressive number of studies and trials employing unselected BM-derived cells (1000 the last 2 years) showed that delivery of those cells to the site of osteonecrosis during core decompression was somehow able to ameliorate the patient with osteonecrosis. In order to translate the promise of this cell therapy into better clinical benefit, many questions need to be addressed. In this review, we therefore analyzed current clinical experience of the literature and our experience of 4000 cases to address these questions and particularly the number of cells that should be injected.

Results: After almost 20 years of clinical research in this field, we are still far from having drawn conclusions on the number of cells we should inject in regenerating hip osteonecrosis. Findings are difficult to interpret due to heterogeneity of causes of osteonecrosis, as well as differences in the cells count, sample quality, and stages of osteonecrosis. The authors address specific issues, as cell quality, cell numbers, volume of osteonecrosis, concentration of cells, and ex vivo expansion. Bone marrow mesenchymal stem cells are supposed to be "functionally competent," but are collected from the bon, marrow of patients with diseases and risk factors of osteonecrosis. The recipient organ (bone osteonecrosis) is a tissue where several alterations have already occurred. These questions are addressed in this review.

Conclusion: In this review, we analyzed current clinical experience regarding cell therapy and address issues that should be a guide for future cell-based therapeutic application in osteonecrosis.
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http://dx.doi.org/10.1007/s00264-018-4000-1DOI Listing
July 2018

A novel in vivo porcine model of intervertebral disc degeneration induced by cryoinjury.

Int Orthop 2018 09 10;42(9):2263-2272. Epub 2018 May 10.

Université Paris-Est-Créteil (UPEC), Laboratoire de bio-ingénierie cellulaire, tissulaire et sanguine à visée thérapeutique (BCTSVT), INSERM/IMRB-U955, équipe n°10, groupe 5, Créteil, France.

Purpose: Degenerative disc disease involves sequential events that lead to the loss of cells, a decrease in disc matrix production, disc dehydration, and alteration of its biomechanical properties. The aim of this study was to determine whether cryoinjury of the nucleus pulposus performed through endplate perforation contributes to disc degeneration and to compare this technique with standard methods.

Method: Under general anesthesia, the lumbar discs of six pigs were exposed and randomly submitted to needle puncture of the annulus fibrosus (NeP), isolated endplate injury (EP), or cryoinjury using a 2.5-J Thompson cryoprobe applied through a single endplate perforation (EP+cryo). The remaining discs served as controls. Animals were sacrificed at two months and the harvested lumbar spines were submitted to CT scan and MRI investigations. Histologic analysis was performed to assess the degree of disc degeneration.

Results: CT scan showed that decrease in average disc height was more important after cryoinjury (49.3%) than after endplate perforation (16.9%) (P < 0.0001) or needle puncture (19.4%) (P < 0.0001). On MRI, the dehydration ratio was significantly more important after EP+cryo (60%) than after NP (40%) or EP (30%) (P < 0.0001). After cryoinjury, the histologic score developed for this study was significantly higher than after needle puncture or endplate perforation (P < 0.0001).

Conclusions: Imaging and histological analysis showed that disc cryoinjury applied through endplate perforation was superior to the classical NeP and EP models to induce experimental disc degeneration. This model appears suitable for testing safety and efficacy of novel treatments of intervertebral disc degeneration.
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http://dx.doi.org/10.1007/s00264-018-3971-2DOI Listing
September 2018

Cell therapy versus simultaneous contralateral decompression in symptomatic corticosteroid osteonecrosis: a thirty year follow-up prospective randomized study of one hundred and twenty five adult patients.

Int Orthop 2018 07 9;42(7):1639-1649. Epub 2018 May 9.

Hôpital Henri Mondor, 94010, Creteil, France.

Purpose: Symptomatic osteonecrosis related to corticosteroids has a high risk of progression to collapse in absence of treatment. The purposes of this study were to evaluate the results of autologous bone marrow grafting of the symptomatic hip in adult patients with osteonecrosis and to compare the results with core decompression alone in the contralateral symptomatic hip.

Materials And Methods: A total of 125 consecutive patients (78 males and 47 females) with bilateral osteonecrosis (ON) and who had both hips symptomatic and at the same stage on each side (stage I or II) were included in this study from 1988 to 1998. The volume of osteonecrosis was measured with MRI in both hips; the smaller size ON was treated with core decompression, and the contralateral hip with the larger ON was treated with percutaneous mesenchymal cell (MSC) injection obtained from bone marrow concentration. The average total number of MSCs (counted as number of colony forming units-fibroblast) injected in each hip was 90,000 ± 25,000 cells (range 45,000 to 180,000 cells).

Results: At the most recent FU (average 25 years after the first surgery, range 20 to 30 years), among the 250 hips included in the study, 35 hips (28%) had collapsed at the most recent follow-up after bone marrow grafting, and 90 (72%) after core decompression (CD). Ninety-five hips (76%) in the CD group underwent total hip replacement and 30 hips (24%) in the bone marrow graft group (p < 0.0001). Hips undergoing only CD were approximately three times more likely to undergo a primary THA (odds ratio: 10.0278; 95% CI: 5.6117 to 17.9190; p < 0.0001) as compared with hips undergoing an initial bone marrow grafting. For the 90 hips treated with bone marrow injection and without collapse, the mean volume of repair evaluated by MRI at the most recent follow-up was 16.4 cm (range 12 to 21 cm) corresponding to a decrease of the pre-operative average volume from 22.4 cm (range 35-15 cm) to 6 cm (range 12-0 cm); as percentage of the volume of the femoral head, the decrease moved from 44.8 to 12%.

Conclusion: Core decompression with bone marrow injection improved the outcome of the disease as compared with core decompression alone in the same patient.
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http://dx.doi.org/10.1007/s00264-018-3941-8DOI Listing
July 2018

Feasibility and safety of treating non-unions in tibia, femur and humerus with autologous, expanded, bone marrow-derived mesenchymal stromal cells associated with biphasic calcium phosphate biomaterials in a multicentric, non-comparative trial.

Biomaterials 2019 03 19;196:100-108. Epub 2018 Mar 19.

INSERM U957, Lab. Pathophysiology of Bone Resorption, Faculty of Medicine, University of Nantes, Nantes, France.

Background: ORTHO-1 is a European, multicentric, first in human clinical trial to prove safety and feasibility after surgical implantation of commercially available biphasic calcium phosphate bioceramic granules associated during surgery with autologous mesenchymal stromal cells expanded from bone marrow (BM-hMSC) under good manufacturing practices, in patients with long bone pseudarthrosis.

Methods: Twenty-eight patients with femur, tibia or humerus diaphyseal or metaphyso-diaphyseal non-unions were recruited and surgically treated in France, Germany, Italy and Spain with 100 or 200 million BM-hMSC/mL associated with 5-10 cc of bioceramic granules. Patients were followed up during one year. The investigational advanced therapy medicinal product (ATMP) was expanded under the same protocol in all four countries, and approved by each National Competent Authority.

Findings: With safety as primary end-point, no severe adverse event was reported as related to the BM-hMSC. With feasibility as secondary end-point, the participating production centres manufactured the BM-hMSC as planned. The ATMP combined to the bioceramic was surgically delivered to the non-unions, and 26/28 treated patients were found radiologically healed at one year (3 out of 4 cortices with bone bridging).

Interpretation: Safety and feasibility were clinically proven for surgical implantation of expanded autologous BM-hMSC with bioceramic.

Funding: EU-FP7-HEALTH-2009, REBORNE Project (GA: 241876).
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http://dx.doi.org/10.1016/j.biomaterials.2018.03.033DOI Listing
March 2019

Subchondral stem cell therapy versus contralateral total knee arthroplasty for osteoarthritis following secondary osteonecrosis of the knee.

Int Orthop 2018 11 27;42(11):2563-2571. Epub 2018 Mar 27.

Hôpital Henri Mondor, 94010, Creteil, France.

Purpose: Total knee arthroplasty (TKA) implanted in patients with secondary osteonecrosis (ON) related to corticosteroids have relatively poor outcome (20% revision rate) at a mean follow-up of only eight years. With the hypothesis that subchondral bone marrow injection might improve knees in these patients, we evaluated 30 patients who had bilateral knee osteoarthritis with severe joint space narrowing and received TKA in one knee and subchondral bone marrow concentrate injection in the contralateral knee.

Material And Methods: A prospective randomized controlled clinical trial was carried out in 60 knees of 30 patients (mean age 28 years, 18-41) who presented bilateral osteoarthritis secondary to knee ON related to corticosteroids in relation with different severe medical conditions. During the same anesthesia, one knee received TKA; for the other knee, a bone marrow graft containing an average of 6500 MSCs/mL (counted as CFU-F, range 3420 to 9830) was delivered to the subchondral bone of the femur and tibia. The length of anesthesia related to each procedure (bone marrow aspiration and subchondral injection of concentrated bone marrow versus total knee arthroplasty) was measured. Peri-operative outcomes, morbidity, complications, and safety of the two procedures were compared. Subsequent admissions for revision surgery were identified. At the most recent follow-up (average of 12 years, range 8 to 16 years), clinical outcomes of the patient (Knee Society score) were obtained along with radiological imaging outcomes (MRIs for knees with subchondral bone marrow injection).

Results: Anesthesia related to the TKA side was longer than for the cell therapy group. Medical and surgical complications were more frequent after TKA. A higher number of thrombophlebitis was observed on the side with TKA (15%) versus none on the side with cell therapy (0%). At the most recent follow-up (average of 12 years, range 8 to 16 years), six (out of 30) TKA knees needed subsequent surgery versus only one with cell therapy. The Knee Score had improved and remained similar in the TKA and cell therapy groups (respectively 80.3 points ± 11 versus 78.3 ± 23); 21 patients preferred the knee with cell therapy and 9 preferred the knee with TKA. Knees with cell therapy had improvement on cartilage and bone marrow lesions observed at the site of bone marrow subchondral injection.

Conclusions: Subchondral autologous bone marrow concentrate was an effective procedure for treating young patients with knee osteoarthritis following secondary ON of the knee related to corticosteroids with a lower complication rate and a quicker recovery as compared with TKA.
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http://dx.doi.org/10.1007/s00264-018-3916-9DOI Listing
November 2018

Stem cell therapy in early post-traumatic talus osteonecrosis.

Int Orthop 2018 12 5;42(12):2949-2956. Epub 2018 Jan 5.

Department of Orthopaedic Surgery, University Paris East (UPEC), Hôpital Henri Mondor, 94010, Creteil, France.

Purpose: Avascular necrosis of the talus is one of the most notable complications associated with talar neck fractures with frequent evolution of the osteonecrosis into a difficult arthrodesis. We tested whether the injection of bone marrow mesenchymal stem cells (MSCs) could improve the repair process of the osteonecrosis.

Material And Methods: Forty-five early (without collapse) post-traumatic talus osteonecroses (group 1; study group) were treated between 1995 and 2012 with percutaneous injection of progenitor cells (autologous bone marrow concentrate from the iliac crest). The number of MSCs transplanted in each ankle of group 1 was 124 × 103 cells (range 101 × 10 to 164 × 10 cells). The evolution of these osteonecroses treated with autologous bone marrow implantation was compared with the evolution of a control group of 34 talar osteonecroses without collapse and treated with only core decompression (group 2; control group) between 1985 and 1995. The outcome was determined by progression in radiographic stages to collapse, by the need of arthrodesis, and by the time to successfully achieve fusion for patients who needed arthrodesis.

Results: For the 45 ankles with autologous concentrate bone marrow grafting, collapse frequency was lower (27%, 12 among 45 versus 71%, 24 among 34; odds ratio 0.1515, 95% CI 0.0563-0.4079; P = 0.0002) and follow-up showed longer duration of survival before collapse or arthrodesis, compared to 34 ankles of the control patients with core decompression alone. Furthermore, the time to successfully achieve fusion after arthrodesis was significantly shorter in patients treated with bone marrow progenitors as compared with the other ankles, which had core decompression alone.

Conclusion: In our study the early conservative surgical treatment with autologous bone marrow grafting improved the natural course of the disease as compared with core decompression alone.
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http://dx.doi.org/10.1007/s00264-017-3716-7DOI Listing
December 2018

Single-stage treatment of infected tibial non-unions and osteomyelitis with bone marrow granulocytes precursors protecting bone graft.

Int Orthop 2018 10 13;42(10):2443-2450. Epub 2017 Nov 13.

Department of Orthopaedic Surgery, University Paris East (UPEC), Hôpital Henri Mondor, 94010, Creteil, France.

Purpose: Infected non-unions present a clinical challenge, especially with risk of recurrent infection. Bone marrow contains granulocyte precursors identified in vitro as colony forming units-granulocyte macrophage (CFU-GM) have a prophylactic action against infection. We therefore tested the hypothesis that bone marrow concentrated granulocytes precursors added to a standard bone graft could decrease the risk of recurrence of infection when single-stage treatment of infected tibial non-unions is performed with bone graft.

Methods: During a single-stage procedure 40 patients with infected tibial non-union received a spongious bone graft supercharged with granulocytes precursors after debridement (study group). A control group (40 patients) was treated in a single stage with local debridement and standard bone graft obtained from the iliac crest. The antibiotic therapy protocol was the same (60 days) in the two groups. CFU-GM progenitors were harvested from bone marrow aspirated on the opposite iliac crest of the site where the cancellous bone was obtained. Union (radiographs and CT scan), a recurrence of clinical infection, and need for subsequent surgery were evaluated.

Results: Thirty-eight (95%) patients who received graft supercharged with granulocytes precursors achieved successful union without recurrence of infection during the seven-year follow-up versus 28 (70%) control patients; for the control group the mean graft resorption volume was 40%, while no bone graft resorption was found for the study group.

Conclusion: Supercharging the cancellous bone graft with bone marrow granulocytes precursors protect the site of infected non-union from recurrence of infection and bone resorption of the graft.
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http://dx.doi.org/10.1007/s00264-017-3687-8DOI Listing
October 2018

Intracavernous Injections of Bone Marrow Mononucleated Cells for Postradical Prostatectomy Erectile Dysfunction: Final Results of the INSTIN Clinical Trial.

Eur Urol Focus 2017 12 24;3(6):643-645. Epub 2017 Jun 24.

Etablissement Français du Sang, Unité d'Ingénierie et de thérapie cellulaire, Créteil, France.

We recently reported stage I of a phase 1/2 clinical trial of cell therapy to treat postradical prostatectomy erectile dysfunction (INSTIN, INtra-cavernous STem-cell INjection clinical trial, NCT01089387). In this first stage, four doses of intracavernous autologous bone marrow mononuclear cells (BM-MNCs) were tested in 12 patients. Here, we report the results of stage II, in which six additional patients received the optimal dose identified in stage I (10 BM-MNCs), and the long-term results in the 12 patients included in stage I. The objectives were to assess the safety and efficacy of this new treatment. In stage II, no patients had side effects, and the erectile function improvements were similar to those seen in stage I: after 6 months, significant improvements versus baseline were noted in International Index of Erectile Function-15 intercourse satisfaction (7.8±3.1 vs 2.2±3.4, p=0.033) and erectile function (18±8.3 vs 3.7±4.1, p=0.035) domains. In stage I patients, after a mean follow-up of 62.1±11.7 mo, there were no prostate cancer recurrences, and erectile function scores were somewhat lower compared with the 1-yr time point. These findings suggest that intracavernous BM-MNC injections are safe and improve erectile function. The decline in erectile function over time suggests a need for assessing repeated injections.

Patient Summary: We report a phase 1/2 pilot clinical trial of cell therapy consisting in intracavernous injection of bone marrow mononuclear cells to treat postradical prostatectomy erectile dysfunction. Erectile function was improved after 6 mo in the patients given 1×10 cells. No serious side effects (life threatening or requiring hospitalisation) occurred after a mean follow-up of 62.1 mo in the first 12 patients.
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http://dx.doi.org/10.1016/j.euf.2017.06.009DOI Listing
December 2017

Enhanced human bone marrow mesenchymal stromal cell adhesion on scaffolds promotes cell survival and bone formation.

Acta Biomater 2017 09 19;59:94-107. Epub 2017 Jun 19.

IMRB U955-E10, INSERM, Creteil, France; Faculty of Medicine, Paris Est University, Creteil, France; Engineering and Cellular Therapy Unit, Etablissement Français du Sang, Créteil, France. Electronic address:

In order to induce an efficient bone formation with human bone marrow mesenchymal stromal cells (hBMSC) associated to a scaffold, it is crucial to determine the key points of the hBMSC action after in vivo transplantation as well as the appropriate features of a scaffold. To this aim we compared the hBMSC behavior when grafted onto two biomaterials allowing different bone potential in vivo. The cancellous devitalized Tutoplast®-processed bone (TPB) and the synthetic hydroxyapatite/β-tricalcium-phosphate (HA/βTCP) which give at 6weeks 100% and 50% of bone formation respectively. We first showed that hBMSC adhesion is two times favored on TPB in vitro and in vivo compared to HA/βTCP. Biomaterial structure analysis indicated that the better cell adhesion on TPB is associated to its higher and smooth open pore architecture as well as its content in collagen. Our 6week time course analysis, showed using qPCR that only adherent cells are able to survive in vivo giving thus an advantage in term of cell number on TPB during the first 4weeks after graft. We then showed that grafted hBMSC survival is crucial as cells participate directly to bone formation and play a paracrine action via the secretion of hIGF1 and hRANKL which are known to regulate the bone formation and resorption pathways respectively. Altogether our results point out the importance of developing a smooth and open pore scaffold to optimize hBMSC adhesion and ensure cell survival in vivo as it is a prerequisite to potentiate their direct and paracrine functions.

Statement Of Significance: Around 10% of skeletal fractures do not heal correctly causing nonunion. An approach involving mesenchymal stromal cells (MSC) associated with biomaterials emerges as an innovative strategy for bone repair. The diversity of scaffolds is a source of heterogeneity for bone formation efficiency. In order to better determine the characteristics of a powerful scaffold it is crucial to understand their relationship with cells after graft. Our results highlight that a biomaterial architecture similar to cancellous bone is important to promote MSC adhesion and ensure cell survival in vivo. Additionally, we demonstrated that the grafted MSC play a direct role coupled to a paracrine effect to enhance bone formation and that both of those roles are governed by the used scaffold.
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http://dx.doi.org/10.1016/j.actbio.2017.06.018DOI Listing
September 2017

Family cord blood banking for sickle cell disease: a twenty-year experience in two dedicated public cord blood banks.

Haematologica 2017 06 16;102(6):976-983. Epub 2017 Mar 16.

Eurocord, Paris-Diderot University EA 3518, Saint-Louis Hospital, Assistance Publique-Hôpitaux de Paris, France.

Efforts to implement family cord blood banking have been developed in the past decades for siblings requiring stem cell transplantation for conditions such as sickle cell disease. However, public banks are faced with challenging decisions about the units to be stored, discarded, or used for other endeavors. We report here 20 years of experience in family cord blood banking for sickle cell disease in two dedicated public banks. Participants were pregnant women who had a previous child diagnosed with homozygous sickle cell disease. Participation was voluntary and free of charge. All mothers underwent mandatory serological screening. Cord blood units were collected in different hospitals, but processed and stored in two public banks. A total of 338 units were stored for 302 families. Median recipient age was six years (11 months-15 years). Median collected volume and total nucleated cell count were 91 mL (range 23-230) and 8.6×10 (range 0.7-75×10), respectively. Microbial contamination was observed in 3.5% (n=12), positive hepatitis B serology in 25% (n=84), and homozygous sickle cell disease in 11% (n=37) of the collections. Forty-four units were HLA-identical to the intended recipient, and 28 units were released for transplantation either alone (n=23) or in combination with the bone marrow from the same donor (n=5), reflecting a utilization rate of 8%. Engraftment rate was 96% with 100% survival. Family cord blood banking yields good quality units for sibling transplantation. More comprehensive banking based on close collaboration among banks, clinical and transplant teams is recommended to optimize the use of these units.
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http://dx.doi.org/10.3324/haematol.2016.163055DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5451329PMC
June 2017

Local transplantation of bone marrow concentrated granulocytes precursors can cure without antibiotics infected nonunion of polytraumatic patients in absence of bone defect.

Int Orthop 2016 Nov 1;40(11):2331-2338. Epub 2016 Mar 1.

Orthopaedic Surgeon, University Paris East (UPEC), Hôpital Henri Mondor, 94010, Creteil, France.

Purpose: Infected, long bone non-unions present a significant clinical challenge. New and alternative therapies are needed to address this problem. The purposes of this study were to compare the number of circulating granulocyte-macrophage colony-forming units (CFU-GM) in the peripheral blood of polytraumatic patients with infected tibial non-unions and in the peripheral blood of control patients with the hypothesis that their number was decreased in polytraumatic patients; and to treat their infection without antibiotics and with local transplantation of bone marrow concentrated granulocytes precursors.

Methods: Thirty (18 atrophic and 12 hyperthrophic ) infected tibial non-unions (without bone defect) that occurred after open fractures in polytraumatic patients were treated without antibiotics and with percutaneous injection of autologous bone marrow concentrate (BMC) containing granulocytes precursors (CFU-GM). CFU-GM progenitors were assessed in the bone marrow aspirate, peripheral blood, and fracture site of these patients. The number of these progenitors was compared with the CFU-GM progenitors of control patient samples (healthy donors matched for age and gender). Outcome measures were: timing of union, callus formation (radiographs and CT scan), and recurrence of clinical infection.

Results: As compared to control patients, the number of CFU GM derived colonies was lower at peripheral blood in patients with infected nonunions. The bone marrow graft injected in nonunions contained after concentration 42 621 ± 20 350 CFU-GM-derived colonies/cc. Healing and cure of infection was observed at six months for 25 patients and at one year follow up for 30 patients. At the median ten year follow-up (range: 5 to 15), only one patient had clinical recurrent infection after healing (between 6 months and last follow-up).

Conclusion: The peripheral blood of these polytraumatic patients with infected nonunions had a remarkable decrease in CFU-GM-derived colonies as compared with normal controls. Local transplantation of concentrated CFU-GM-derived colonies aspirated from bone marrow allowed cure of infection and healing without antibiotics.
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http://dx.doi.org/10.1007/s00264-016-3147-xDOI Listing
November 2016

Allografts supercharged with bone-marrow-derived mesenchymal stem cells possess equivalent osteogenic capacity to that of autograft: a study with long-term follow-ups of human biopsies.

Int Orthop 2017 Jan 24;41(1):127-132. Epub 2016 Aug 24.

EFS Cell Therapy Facility University Paris East (UPEC), Hôpital Henri Mondor, 94010, Creteil, France.

Purpose: Bone-marrow-derived mesenchymal stem cells (BM-MSCs) have been proposed to enhance bone formation in allografts. However, it is not known whether a combination of MSCs, contained in bone marrow concentrate (BMC) and structural allograft could be better than an allograft without MSCs and equivalent to a femoral head autograft in terms of histologic bone formation and long-term cellularity in the graft. After ten years of follow-up, three types of grafts: those initially loaded with BM-MSCs; dead, irradiated allografts; autografts.

Materials And Methods: Twenty patients received acetabular grafting during hip surgery and subsequently underwent femoral hip revision eight to 13 years later (average 10 years). Revision surgery was for reasons other than graft failure. These 20 patients had received eight allografts initially loaded with BM-MSCs: six dead irradiated allografts and six autografts. The number of MSCs present in the three types of graft were evaluated at the time of initial surgery and at revision. New bone formation associated in the acetabular graft was assessed by histology and calculated as a percentage of total available bony area.

Results: At the most recent follow-ups (average 10 years), concentration of MSCs in allografts previously loaded with BM-MSCs was higher than that found in autografts. There were low or no MSCs found in uncharged allografts. New-bone-formation analysis showed that allografts loaded with BM-MSCs produced more new bone (35 %; range 20-50 %) compared with either uncharged allografts (9 %; range 2-15 %) or autografts (24 %; range 12-32 %).

Conclusions: Our observations with allografts charged with BM-MSCs provides evidence in support of a long-term benefit of supercharging bone allografts with autologous BM-MSCs.
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http://dx.doi.org/10.1007/s00264-016-3263-7DOI Listing
January 2017

Stem Cell Therapy for the Treatment of Hip Osteonecrosis: A 30-Year Review of Progress.

Clin Orthop Surg 2016 Mar 13;8(1):1-8. Epub 2016 Feb 13.

Department of Orthopaedic Surgery, University Paris East (UPEC), Hôpital Henri Mondor, Creteil, France.

Avascular necrosis of the femoral head is caused by a multitude of etiologic factors and is associated with collapse with a risk of hip arthroplasty in younger populations. A focus on early disease management with the use of stem cells was proposed as early as 1985 by the senior author (PH). We undertook a systematic review of the medical literature to examine the progress in cell therapy during the last 30 years for the treatment of early stage osteonecrosis.
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http://dx.doi.org/10.4055/cios.2016.8.1.1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4761591PMC
March 2016

Osteogenic progenitors in bone marrow aspirates have clinical potential for tibial non-unions healing in diabetic patients.

Int Orthop 2016 Jul 17;40(7):1375-9. Epub 2015 Nov 17.

Orthopaedic Surgery, Hôpital Henri Mondor, University Paris East (UPEC), 94010, Creteil, France.

Purpose: There is a significantly higher incidence of delayed unions, non-unions, and increased healing time in diabetic patients compared with non-diabetic patients. Studies suggest that diabetics suffer from deficiencies of pancreatic stem/progenitor cells, and a clinically relevant question arises concerning the availability and functionality of progenitor cells obtained from bone marrow of diabetics for applications in bone repair.

Methods: We have evaluated the cellularity and frequency of osteogenic mesenchymal stem cells (MSCs) in bone marrow from 54 diabetic patients (12 with type 1 and 42 with type 2) with tibial non-unions. These patients were treated with bone marrow MSCs (BM-MSCs) delivered in an autologous bone marrow concentrate (BMC). Clinical outcomes and marrow cellularity were compared to 54 non-diabetic, matched patients with tibial non-unions also treated with BMC.

Results: After adjusting for age and sex, no differences were identified with respect to bone marrow cellularity and MSC number among the diabetic and non-diabetic groups and both groups received approximately the same number of MSCs on average. BMC treatment promoted non-union healing in 41 diabetic patients (76 %) and 49 non-diabetic patients (91 %), but the non-diabetic patients healed more quickly and produced a larger volume of callus.

Conclusion: We recommend that diabetic patients be treated with an increased number of progenitor cells by increasing the bone marrow aspiration volume. We also anticipate a need to extend the time of casting and non-weight bearing for diabetic patients as compared with non-diabetic patients.
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http://dx.doi.org/10.1007/s00264-015-3046-6DOI Listing
July 2016

Autologous bone marrow stromal cells are promising candidates for cell therapy approaches to treat bone degeneration in sickle cell disease.

Stem Cell Res 2015 Nov 8;15(3):584-594. Epub 2015 Oct 8.

Université Paris-Est, Faculté de médecine, Laboratoire de "Bioingénierie cellulaire, tissulaire et sanguine", EA3952, Créteil, France; Etablissement Français du Sang d'Ile-de-France, Unité d'Ingénierie et de Thérapie Cellulaire, Créteil, France. Electronic address:

Osteonecrosis of the femoral head is a frequent complication in adult patients with sickle cell disease (SCD). To delay hip arthroplasty, core decompression combined with concentrated total bone marrow (BM) treatment is currently performed in the early stages of the osteonecrosis. Cell therapy efficacy depends on the quantity of implanted BM stromal cells. For this reason, expanded bone marrow stromal cells (BMSCs, also known as bone marrow derived mesenchymal stem cells) can be used to improve osteonecrosis treatment in SCD patients. In this study, we quantitatively and qualitatively evaluated the function of BMSCs isolated from a large number of SCD patients with osteonecrosis (SCD-ON) compared with control groups (patients with osteonecrosis not related to SCD (ON) and normal donors (N)). BM total nuclear cells and colony-forming efficiency values (CFE) were significantly higher in SCD-ON patients than in age and sex-matched controls. The BMSCs from SCD-ON patients were similar to BMSCs from the control groups in terms of their phenotypic and functional properties. SCD-ON patients have a higher frequency of BMSCs that retain their bone regeneration potential. Our findings suggest that BMSCs isolated from SCD-ON patients can be used clinically in cell therapy approaches. This work provides important preclinical data that is necessary for the clinical application of expanded BMSCs in advanced therapies and medical products.
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http://dx.doi.org/10.1016/j.scr.2015.09.016DOI Listing
November 2015

Safety of Intracavernous Bone Marrow-Mononuclear Cells for Postradical Prostatectomy Erectile Dysfunction: An Open Dose-Escalation Pilot Study.

Eur Urol 2016 06 4;69(6):988-91. Epub 2015 Oct 4.

Etablissement Français du Sang, Unité d'Ingénierie et de thérapie cellulaire, Créteil, France.

Unlabelled: Evidence from animal models replicating postradical prostatectomy erectile dysfunction (pRP-ED) suggests intracavernous injection of bone marrow-mononuclear cells (BM-MNCs) as a promising treatment approach for pRP-ED. We conducted a phase 1/2 pilot clinical trial of intracavernous autologous BM-MNC injection to treat pRP-ED (NCT01089387). Twelve patients with localized prostate cancer and vasculogenic pRP-ED refractory to maximal medical treatment were divided into four equal groups treated with escalating BM-MNC doses (2×10(7), 2×10(8), 1×10(9), 2×10(9)). Tolerance was the primary endpoint. Secondary endpoints were the effects on erectile function and penile vascularization at 6 mo, as assessed using the International Index of Erectile Function-15 and Erection Hardness Scale questionnaires, and color duplex Doppler ultrasound. We measured the peak systolic velocity in cavernous arteries and assessed endothelial function using the penile nitric oxide release test. No serious side effects occurred. At 6 mo versus baseline, significant improvements of intercourse satisfaction (6.8±3.6, 3.9±2.5, p=0.044) and erectile function (17.4±8.9, 7.3±4.5, p=0.006) domains of the International Index of Erectile Function-15 and Erection Hardness Scale (2.6±1.1, 1.3±0.8, p=0.008) were observed in the total population. Spontaneous erections showed significantly greater improvement with the higher doses. Clinical benefits were associated with improvement of peak systolic velocity and of % penile nitric oxide release test and sustained after 1 yr. Our results need to be confirmed by phase 2 clinical trials.

Patient Summary: We report a phase 1/2 pilot clinical trial investigating cell therapy with injection of bone marrow mononucleated cells to treat postradical prostatectomy erectile dysfunction. No serious side effects occurred. Improvements of erectile function and penile vascularization were noted. Further studies are required to confirm these preliminary results.
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http://dx.doi.org/10.1016/j.eururo.2015.09.026DOI Listing
June 2016

Development of a simple procedure for the treatment of femoral head osteonecrosis with intra-osseous injection of bone marrow mesenchymal stromal cells: study of their biodistribution in the early time points after injection.

Stem Cell Res Ther 2015 Apr 13;6:68. Epub 2015 Apr 13.

Université Paris-Est, Faculté de médecine, Laboratoire de "Bioingénierie cellulaire, tissulaire et sanguine", EA3952, 5 rue Gustave Eiffel, 94000, Créteil, France.

Introduction: Osteonecrosis of the femoral head (ONFH) is a degenerative disease progressing to a femoral head (FH) collapse. Injection of osteoprogenitor cells like bone marrow mesenchymal stromal cells (BMSCs) into the FH appears to be a good therapeutic treatment. However, safety and efficacy of BMSCs to treat bone defect are the main preclinical data required for clinical application. Efficacy and the lack of risk of cell transformation after amplification of BMSCs have been extensively described. The main objectives of this study were to develop a simple and usable procedure for clinicians and control its feasibility by evaluating the biodistribution of BMSCs after injection into the FH in a large animal model. The impact of this approach was evaluated on one natural pig ONFH.

Methods: BMSCs were directly injected in the pig FH, and then the biodistribution of grafted cells was detected by quantitative real-time polymerase chain reaction, cytometry, or a combination of classic histology analysis and in situ hybridization (ISH). BMSC efficacy on bone regeneration was evaluated by magnetic resonance imaging (MRI) and histology.

Results: After 30-minute and 24-hour follow-up, grafted cells were detected at the injection site and no BMSCs were detected in filter organs or body fluids. The combination of classic histology analysis and ISH showed a good homogeneity of cell distribution in FH. Local delivery of BMSCs onto a bone scaffold associated with bone formation in vivo confirmed the preferential tropism of BMSCs to the bone tissue as well as their efficacy to form bone. Treatment of a natural pig ONFH by autologous BMSCs indicated a beginning of bone healing as early as 2 weeks with a complete healing after 9 weeks. At this stage, MRI and histological analysis were similar to those of a normal FH.

Conclusions: Intra-osseous injection of BMSCs in FH seems to be a good strategy for ONFH treatment as the safety concerning the biodistribution of BMSCs is ensured. Moreover, the efficacy of BMSCs in natural ONFH seems to indicate that this is a promising approach. Altogether, these results constitute the preclinical data necessary for the setup of a clinical application with expanded BMSCs in the context of advanced therapy medicinal products.
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http://dx.doi.org/10.1186/s13287-015-0036-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4448288PMC
April 2015

Percutaneous injection of bone marrow mesenchymal stem cells for ankle non-unions decreases complications in patients with diabetes.

Int Orthop 2015 Aug 22;39(8):1639-43. Epub 2015 Mar 22.

Department of Orthopaedic Surgery, Henri-Mondor Hospital, University of Paris, Créteil Cedex, France,

Purpose: Clinical studies in diabetic patients have demonstrated that there is a high incidence of complications in distal tibia and ankle fracture treatments. One strategy to mitigate issues with wound healing and infection in diabetic patients is to use a percutaneous technique in which autologous, bone marrow-derived, concentrated cells are injected at the site of non-unions.

Methods: Eighty-six ankle non-union in diabetic patients were treated with bone marrow mesenchymal stem cells (BM-MSCs) delivered in an autologous bone marrow concentrate (BMC). Clinical outcomes of the 86 diabetic non-union patients treated with BMC were compared with 86 diabetic matched non-unions treated with a standard bone iliac crest autograft.

Results: Treatment with BMC promoted non-union healing in 70 among 86 diabetic patients (82.1 %) with a low number of complications. Of the 86 diabetic patients treated with iliac bone graft, 53 (62.3 %) had healing; major complications were observed: 5 amputations, 11 osteonecroses of the fracture wound edge and 17 infections.

Conclusions: In diabetic patients with ankle non-unions, treatment with BM-MSCs from bone marrow concentrate may be preferable in view of the high risks of major complications after open surgery and iliac bone grafting, and improved healing rates compared with standard iliac bone autograft treatment.
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http://dx.doi.org/10.1007/s00264-015-2738-2DOI Listing
August 2015

Reduced levels of mesenchymal stem cells at the tendon-bone interface tuberosity in patients with symptomatic rotator cuff tear.

Int Orthop 2015 Jun 12;39(6):1219-25. Epub 2015 Mar 12.

Orthopaedic Surgery, Hospital Henri Mondor, Créteil, France,

Purpose: While the use of bone marrow concentrate (BMC) has been described in the treatment of rotator cuff tears, the impact of a rotator cuff injury on the mesenchymal stem cells (MSCs) content present in the human shoulder has not been determined, especially with regard to changes in the levels of MSCs at the tendon-bone interface. With the hypothesis that there was a decreased level of MSCs at the tendon-bone interface tuberosity in patients with rotator cuff tear, we assessed the level of MSCs in the tuberosity of the shoulder of patients undergoing a rotator cuff repair.

Methods: We analysed the data of 125 patients with symptomatic rotator cuff tears and of 75 control patients without rotator cuff injury. We recorded the following data: size of tear, number of torn tendons, aetiology of the tear, lag time between onset of shoulder symptoms/injury and repair, and also fatty infiltration of muscles. Mesenchymal stem cell content at the tendon-bone interface tuberosity was evaluated by bone marrow aspiration collected in the humeral tuberosities of patients at the beginning of surgery.

Results: A significant reduction in MSC content (from moderate, 30-50 %, to severe >70 %) at the tendon-bone interface tuberosity relative to the MSC content of the control was observed in all rotator cuff repair study patients. Severity of the decrease was statistically correlated to a number of factors, including the delay between onset of symptoms and surgery, number of involved tendons, fatty infiltration stage and increasing patient age.

Conclusion: This study demonstrates that the level of MSCs present in the greater tuberosity of patients with a rotator cuff tear decreases as a function of a number of clinical factors, including lag time from tear onset to treatment, tear size, number of tears and stage of fatty infiltration, among others. This information may help the practices in using biologic augmentation of a rotator cuff repair.
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http://dx.doi.org/10.1007/s00264-015-2724-8DOI Listing
June 2015

Bone-Forming Capacity and Biodistribution of Bone Marrow-Derived Stromal Cells Directly Loaded Into Scaffolds: A Novel and Easy Approach for Clinical Application of Bone Regeneration.

Cell Transplant 2015 28;24(10):1945-55. Epub 2014 Oct 28.

Université Paris-Est Créteil, Faculté de médecine, Laboratoire de "Bioingénierie Cellulaire, Tissulaire et Sanguine," Créteil, France.

In the context of clinical applications of bone regeneration, cell seeding into scaffolds needs to be safe and easy. Moreover, cell density also plays a crucial role in the development of efficient bone tissue engineering constructs. The aim of this study was to develop and evaluate a simple and rapid cell seeding procedure on hydroxyapatite/β-tricalcium phosphate (HA/βTCP), as well as define optimal cell density and control the biodistribution of grafted cells. To this end, human bone marrow-derived stromal cells (hBMSCs) were seeded on HA/βTCP scaffolds, and we have compared bone formation using an ectopic model. Our results demonstrated a significantly higher bone-forming capacity of hBMSCs directly loaded on HA/βTCP during surgery compared to hBMSCs preseeded for 7 days in vitro on HA/βTCP before ectopic implantation. The extent of new bone formation increases with increasing hBMSC densities quantitatively, qualitatively, and in frequency. Also, this study showed that grafted hBMSCs remained confined to the implantation site and did not spread toward other tissues, such as liver, spleen, lungs, heart, and kidneys. In conclusion, direct cell loading into a scaffold during surgery is more efficient for bone regeneration, as well as quick and safe. Therefore direct cell loading is suitable for clinical requirements and cell production control, making it a promising approach for orthopedic applications. Moreover, our results have provided evidence that the formation of a mature bone organ containing hematopoietic islets needs a sufficiently high local density of grafted hBMSCs, which should guide the optimal dose of cells for clinical use.
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http://dx.doi.org/10.3727/096368914X685276DOI Listing
August 2016