Aims. The value of core decompression (CD) in the treatment of osteonecrosis of the femoral head (ONFH) remains controversial. We conducted a systematic review and meta-analysis to evaluate whether CD combined with other treatments could improve the clinical and radiological outcomes of ONFH patients compared with CD alone. Methods. We searched the PubMed, Embase, Web of Science, and Cochrane Library databases until June 2020. All randomized controlled trials (RCTs) and clinical controlled trials (CCTs) comparing CD alone and CD combined with other measures (CD + cell therapy, CD + bone grafting, CD + porous tantalum rod, etc.) for the treatment of ONFH were considered eligible for inclusion. The primary outcomes of interest were Harris Hip Score (HHS), ONFH stage progression, structural failure (collapse) of the femoral head, and conversion to total hip arthroplasty (THA). The pooled data were analyzed using Review Manager 5.3 software. Results. A total of 20 studies with 2,123 hips were included (CD alone = 768, CD combined with other treatments = 1,355). The combination of CD with other therapeutic interventions resulted in a higher HHS (mean difference (MD) = 6.46, 95% confidence interval (CI) = 2.10 to 10.83, p = 0.004) and Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) score (MD = −10.92, 95% CI = -21.41 to -4.03, p = 0.040) and a lower visual analogue scale (VAS) score (MD = −0.99, 95% CI = -1.56 to -0.42, p < 0.001) than CD alone. For the rates of disease stage progression, 91 (20%) progressed in the intervention group compared to 146 (36%) in the control group (odds ratio (OR) = 0.32, 95% CI = 0.16 to 0.64, p = 0.001). In addition, the intervention group had a more significant advantage in delaying femoral head progression to the collapsed stage (OR = 0.32, 95% CI = 0.17 to 0.61, p < 0.001) and reducing the odds of conversion to THA (OR = 0.35, 95% CI = 0.23 to 0.55, p < 0.001) compared to the control group. There were no serious adverse events in either group. Subgroup analysis showed that the addition of cell therapy significantly improved clinical and radiological outcomes compared to CD alone, and this approach appeared to be more effective than other therapies, particularly in precollapse (stage I to II) ONFH patients. Conclusion. There was marked heterogeneity in the studies. There is a trend towards improved clinical outcomes with the addition of stem cell therapy to CD. Cite this article: Bone Joint Res 2021;10(7):445–458

Aims. The aim of the HIPGEN consortium is to develop the first cell therapy product for hip fracture patients using PLacental-eXpanded (PLX-PAD) stromal cells. Methods. HIPGEN is a multicentre, multinational, randomized, double-blind, placebo-controlled trial. A total of 240 patients aged 60 to 90 years with low-energy femoral neck fractures (FNF) will be allocated to two arms and receive an intramuscular injection of either 150 × 10. 6. PLX-PAD cells or placebo into the medial gluteal muscle after direct lateral implantation of total or hemi hip arthroplasty. Patients will be followed for two years. The primary endpoint is the Short Physical Performance Battery (SPPB) at week 26. Secondary and exploratory endpoints include morphological parameters (lean body mass), functional parameters (abduction and handgrip strength, symmetry in gait, weightbearing), all-cause mortality rate and patient-reported outcome measures (Lower Limb Measure, EuroQol five-dimension questionnaire). Immunological biomarker and in vitro studies will be performed to analyze the PLX-PAD mechanism of action. A sample size of 240 subjects was calculated providing 88% power for the detection of a 1 SPPB point treatment effect for a two-sided test with an α level of 5%. Conclusion. The HIPGEN study assesses the efficacy, safety, and tolerability of intramuscular PLX-PAD administration for the treatment of muscle injury following arthroplasty for hip fracture. It is the first phase III study to investigate the effect of an allogeneic cell therapy on improved mobilization after hip fracture, an aspect which is in sore need of addressing for the improvement in standard of care treatment for patients with FNF. Cite this article: Bone Jt Open 2022;3(4):340–347

Stem cells are defined by their potential for self-renewal and the ability to differentiate into numerous cell types, including cartilage and bone cells. Although basic laboratory studies demonstrate that cell therapies have strong potential for improvement in tissue healing and regeneration, there is little evidence in the scientific literature for many of the available cell formulations that are currently offered to patients. Numerous commercial entities and ‘regenerative medicine centres’ have aggressively marketed unproven cell therapies for a wide range of medical conditions, leading to sometimes indiscriminate use of these treatments, which has added to the confusion and unpredictable outcomes. The significant variability and heterogeneity in cell formulations between different individuals makes it difficult to draw conclusions about efficacy. The ‘minimally manipulated’ preparations derived from bone marrow and adipose tissue that are currently used differ substantially from cells that are processed and prepared under defined laboratory protocols. The term ‘stem cells’ should be reserved for laboratory-purified, culture-expanded cells. The number of cells in uncultured preparations that meet these defined criteria is estimated to be approximately one in 10 000 to 20 000 (0.005% to 0.01%) in native bone marrow and 1 in 2000 in adipose tissue. It is clear that more refined definitions of stem cells are required, as the lumping together of widely diverse progenitor cell types under the umbrella term ‘mesenchymal stem cells’ has created confusion among scientists, clinicians, regulators, and our patients. Validated methods need to be developed to measure and characterize the ‘critical quality attributes’ and biological activity of a specific cell formulation. It is certain that ‘one size does not fit all’ – different cell formulations, dosing schedules, and culturing parameters will likely be required based on the tissue being treated and the desired biological target. As an alternative to the use of exogenous cells, in the future we may be able to stimulate the intrinsic vascular stem cell niche that is known to exist in many tissues. The tremendous potential of cell therapy will only be realized with further basic, translational, and clinical research. Cite this article: Bone Joint J 2019;101-B:361–364

Symptomatic and non-symptomatic hip osteonecrosis related to sickle cell disease (SCD) has a high risk of progression to collapse and total hip arthroplasty (THA) in this disease has a high rate of complications. We asked question about the benefit of performing an IRM to detect and treat with cell therapy an early (stage I or II) contralateral osteonecrosis. 430 consecutive SCD adult (32 years, 18 to 51) patients (225 males) with bilateral osteonecrosis (diagnosed with MRI) were included in this study from 1990 to 2010. One side with collapse was treated with THA and the contralateral without collapse (stage I or II) treated with cell therapy. The volume of osteonecrosis was measured with MRI. For cell therapy, the average total number of mesenchymal stem cells (MSCs) counted as number of colony forming units-fibroblast injected in each hip was 160,000 ± 45,000 cells (range 75,000 to 210,000 cells). At the most recent FU (20 years, range 10 to 30), among the 430 hips treated with cell therapy, 45 hips (10.5%) had collapsed and had required THA at 10 years (range 5 to 14 years) and 380 hips (88%) were without collapse and asymptomatic (or with few symptoms) with a decrease percentage of necrosis on MRI from 45% to 11%. Among the 430 contralateral THA, 96 (22.3%) had required one revision, 28 had a re-revision, and 12 a third re-revision with aseptic loosening (85% of revisions) and/or infection (6% of revisions). Hips undergoing cell therapy were approximately three times less likely to undergo revision or re-revision surgery (p < 0.01) as compared with hips undergoing a primary THA. THA is the usual treatment of collapsed ON in patients with SCD. In this population, it is worth looking with MRI for an early stage on the contralateral hip and performing (when necessary) bone marrow cell implantation during the same anesthesia as for arthroplasty

Autologous cell therapy using stem cells and progenitor cells is considered to be a popular approach in regenerative medicine for the repair and regeneration of tissue and organs. In orthopaedic practice, autologous cell therapy has become a major focus, particularly, as a feasible treatment for tendon injury. Tendons are dense connective tissue that bridge bone to muscle and transmit forces between muscle and bone to maintain mechanical movement. Tendons are poorly vascularised and have very little capacity to self-regenerate. Degeneration of tendon is often caused by injury. The pathogenesis of tendon injury, commonly known as tendinosis, is not an inflammatory condition but is secondary to degenerative changes, including disruption of the collagen matrix, calcification, vascularisation and adipogenesis. The aetiology of tendinosis is considered to be multifactorial and the pathogenesis is still unclear. Intrinsic factors such as a lack of blood and nutrition supply and extrinsic factors such as acute trauma and overuse injury caused by repetitive strain, have been implicated as contributors to the pathogenesis of tendinosis. More recent studies suggest that programmed tendon cell death (tenocyte apoptosis) may play a major role in the development of tendinosis. Such cellular abnormalities may influence the capacity of tendon to maintain its integrity. Traditional treatments such as anti-inflammatory drugs, steroid injections and physiotherapy are aimed at symptom relief and do not address the underlying pathological changes of degeneration. Here, we propose that autologous cell therapy may be an innovative and promising treatment for tendon injury. We will present evidence that suggest that autologous tendon cell therapy may be feasible to repair and regenerate tendon. We will also present data summarising the preclinical evaluation of autologous tendon cell therapy in animal models and the safety and tolerability of autologous tendon cell therapy in humans in studies, which are currently conducted at the Centre for Orthopaedic Research at the University of Western Australia

Despite osteoarthritis (OA) representing a large burden for healthcare systems, there remains no effective intervention capable of regenerating the damaged cartilage in OA. Mesenchymal stromal cells (MSCs) are adult-derived, multipotent cells which are a candidate for musculoskeletal cell therapy. However, their precise mechanism of action remains poorly understood. The effects of an intra-articular injection of human bone-marrow derived MSCs into a knee osteochondral injury model were investigated in C57Bl/6 mice. The cell therapy was retrieved at different time points and single cell RNA sequencing was performed to elucidate the transcriptomic changes relevant to driving tissue repair. Mass cytometry was also used to study changes in the mouse immune cell populations during repair. Histological assessment reveals that MSC treatment is associated with improved tissue repair in C57Bl/6 mice. Single cell analysis of retrieved human MSCs showed spatial and temporal transcriptional heterogeneity between the repair tissue (in the epiphysis) and synovial tissue. A transcriptomic map has emerged of some of the distinct genes and pathways enriched in human MSCs isolated from different tissues following osteochondral injury. Several MSC subpopulations have been identified, including proliferative and reparative subpopulations at both 7 days and 28 days after injury. Supported by the mass cytometry results, the immunomodulatory role of MSCs was further emphasised, as MSC therapy was associated with the induction of increased numbers of regulatory T cells correlating with enhanced repair in the mouse knee. The transcriptomes of a retrieved MSC therapy were studied for the first time. An important barrier to the translation of MSC therapies is a lack of understanding of their heterogeneity, and the consequent lack of precision in its use. MSC subpopulations with different functional roles may be implicated in the different phases of tissue repair and this work offers further insights into repair process

Aims An estimated 5–10% of fractures fail to heal adequately. Novel therapies in the treatment of problem fractures include the use of culture expanded cells. An animal model of delayed fracture union is required to parallel the clinical scenario so that variations in cell therapy techniques can be rapidly assessed. Material and Methods A simple unilateral external fixator was designed for use in the rat. The fixator was applied following open osteotomy of the femur and a reproducible externally fixated femoral fracture model was established (n=41). Fracture union was assessed by digital radiography, histology and biomechanical strength testing (four point bending) at weeks 4, 6 and 8. Histological examination was also undertaken at day 4 and weeks 1 and 2. A delayed union in the fracture model was created by periosteal and endosteal stripping (n=14). Radiography and biomechanical strength testing were performed at week 8. The use of cell therapy was tested in the delayed union model. Osteogenic cells were culture expanded for 6 weeks before re-implantation. Reimplantation was facilitated by the use of a drill hole through the fracture site . Animals were randomized to one of three groups – i) drill hole & cells in a carrier ii) drill hole & carrier only iii) no drill hole, cells or carrier. Results In the fracture model radiological and histological evidence of fracture union was apparent at week 6. Biomechanical testing showed a significant difference in load to failure and stiffness of the fracture between weeks 4 and 8 (p=0.009 and 0.008 respectively). There was also a significant difference in biomechanical properties between the fracture model and the delayed union model at week 8. Drilling with the injection of a carrier significantly improved the biomechanical properties (p=0.03) of a delayed union at week 14. Surprisingly this effect was negated by the introduction of cells. Conclusion A fracture and delayed union model in the rat has been established for the testing of cell therapy. The application of cell therapy to a delayed union has been less advantageous in improving union than expected. This prompts the need for further work required in optimising cell culture techniques and cell delivery

The HIPGEN study funded under EU Horizon 2020 (Grant 7792939) has the aim to investigate the potential of the first regenerative cell therapy for the improvement of recovery after muscle injury in hip fracture patients. For this aim we intramuscularly injected placental derived mesenchymal stromal cells during hip fracture arthroplasty. Despite not having reached the primary endpoint, which was the Short Physical Performance Battery, we could observe an increase in abductor muscle strength and a faster return to balance looking at symmetry in insole measurements during follow up

Inflammation has been associated with early degradative changes in articular cartilage and immune responses are key factor influencing normal tissue regeneration and repair. With synovitis a prominent feature in osteoarthritis (OA) and associated with the progressive degradation of articular cartilage, immune factors need to be factored into efforts to achieve efficient cartilage repair/regeneration. Recent efforts have focused on the use of autologous or allogeneic mesenchymal stem/stromal cells (MSCs) to modulate the inflammatory environment in the injured or osteoarthritic joint. Intraarticular injection of MSCS has modulated cartilage degradation in a variety of pre-clinical OA models. Results from early clinical trials have also shown effects on pain and function-associated outcome measures. Other cell types may also have some capacity for use as a therapy for OA. For example, primary allogeneic chondrocytes also seem to have some immune-privilege in the synovial joint and are immunomodulatory in a rat model. Although MSCs isolated from bone marrow that are induced to undergo chondrogenic differentiation do not retain these properties, MSCs isolated from the synovium or chondroprogenitors generated from cartilage itself may represent the future of cell therapy for OA

Abstract. Objectives. Bone marrow aspirate concentrate (BMAC), together with fibrin glue (Tisseel, Baxter, UK) and Hyaluronic acid (HA) were used as a one-step cell therapy treating patients with ankle cartilage defects in our hospital. This therapy was proven to be safe, with patients demonstrating a significant improvement 12 months post-treatment. Enriched mesenchymal stem cells (MSCs) in BMAC are suggested inducers of cartilage regeneration, however, currently there is no point-of-care assessment for BMAC quality; especially regarding the proportion of MSCs within. This study aims to characterise the cellular component of CCR-generated BMAC using a point-of-care device, and to investigate if the total nucleated cell (TNC) count and patient age are predictive of MSC concentration. Methods. During surgery, 35ml of bone marrow aspirate (BMA) was collected from each patients’ iliac crest under anaesthesia, and BMAC was obtained via a commercial kit (Cartilage Regeneration kit, CCR, Innotec. ®. , UK). BMAC was then mixed with thrombin (B+T) for injection with HA and fibrinogen. In our study, donor-matched BMA, BMAC and B+T were obtained from consented patients (n=12, age 41 ± 16years) undergoing surgery with BMAC therapy. TNC, red blood cell (RBC) and platelet (PLT) counts were measured via a haematology analyser (ABX Micros ES 60, Horiba, UK), and the proportion of MSCs in BMA, BMAC and B+T were assessed via colony forming unit-fibroblast (CFU-F) assays. Significant differences data in matched donors were tested using Friedman test. All data were shown as mean ± SD. Results. Mean TNC counts in BMA and BMAC were not significantly different (14.0 ± 4.4 million/ml and 19.4 ± 32.9 million/ml, respectively, P>0.9999). However, TNC counts were significantly lower in B+T compared to BMAC (9.7 ± 24.5 million/ml and 19.4 ± 32.9 million/ml, respectively, P=0.0167). Similarly, PLT counts were decreased in B+T compared to BMAC (40.7 ± 30.7 million/ml and 417.5 ± 365.5 million/ml, respectively, P<0.0001), however, PLTs were significantly concentrated in BMAC compared to BMA (417.5 ± 365.5 million/ml and 114.8 ± 61.6 million/ml, respectively, P=0.0429). RBC counts were significantly decreased in BMAC and B+T compared to BMA (P=0.0322 and P<0.0001, respectively). Higher concentration of MSCs were observed in BMAC compared to BMA (0.006% ± 0.01% and 0.00007% ± 0.0001%, respectively, P=0.0176). Similar to TNCs and PLTs, the proportion of MSCs significantly decreased in B+T compared to BMAC (0.0004% ± 0.001% and 0.006% ± 0.01%, respectively, P=0.0023). Furthermore, patient age and TNC counts did not correlate with MSC concentration (Spearman's Rank test, P=0.3266 and P=0.4880, respectively). Conclusions. BMAC successfully concentrated PLTs, but BMAC preparations were highly variable. Mixing BMAC and thrombin however, as described in the CCR protocol, resulted in a dramatic reduction in TNCs, PLTs and MSCs. TNC counts and patient age could not be used to predict the MSC proportion in the BMAC based on current data. Future work aims to look at the biomolecule profile of BMAC plasma, and to correlate them to patient clinical outcomes. Declaration of Interest. (a) fully declare any financial or other potential conflict of interest

Introduction. The natural history of osteonecrosis of the femoral head (ONFH) is not cleanly understood, but most of them progresse to the joint destruction and requires total hip replacement arthroplasty. There are several head preserving procedure, but no single therapeutic method proved to be effective in preventing progression of the disease. The possibility has been raised that implantation of bone marrow containing osteogenic precursors may be effective in the treatment of this disease. However, there are no long-term follow-up results of cell therapy for ONFH. AS far as we know, there are no reports about bone graft and cell therapy for ONFH. Therefore, we performed a prospective clinical and radiological evaluation on ONFH treated with core decompression combined with autoiliac bone graft and an implantation of autologous bone marrow cells as a therapeutic method of ONFH. Materials and Methods. Sixty-one hips in 52 patients with ONFH were included in this study. The average follow-up of the patients was 68 (60∼88) months. The necrotic lesions were classified according to their size and location, and we compared the results. Results. At the last follow-up, the rate of excellent or good results was 80% (12/15 hips) in the small lesion group, 65% (17/26 hips) in the medium size group, and 28% (6/20 hips) in the large size group. The procedures were a clinical success in 4 of 5 hips(80%) with stage I, 23 of 35 hips (65.7%) with Stage II and 7 hips of 18 hips(38.9%) with stage III and 1 of 3 hips(33.3%) with stage IV. Among the 20 cases with large sized necrotic lesions, 17 cases were laterally located and this group showed the worst outcome with 13 hips (76.5%) having bad or failed clinical results. Conclusions. The outcome of cancellous bone grafting combined with implantation of autologous bone marrow cells differed depending on the size and location of the lesion. The patients who have a large sized lesion or medium sized lateral located lesion would not be considered as a good candidate for head preserving procedure. However for the medium sized lesions, this procedure showed a competent clinical result against other head preserving procedures

Introduction: Eighty percent of individuals experience low back pain in their lifetime. This is often due to disc injury or degeneration. Conservative treatment of discogenic pain is often unsuccessful whilst surgery with the use of spacers or fusion is non-physiological. Aim: To develop an animal model to assess the viability of autologous disc cell therapy. Methods: The fat sand rat (Psammomys obesus obesus) was chosen because of its predisposition to the early development of spondylosis. Using microsurgical techniques fragments of annulus and nucleus were harvested from a single disc in 50 sand rats. Vascular clips were placed on the adjacent psoas muscle to mark the harvested level. Disc material was initially cultured in a monolayer then transferred into a three-dimensional culture medium of agarose. This technique yields greater cellular proliferation and the development of cell growth in colonies. Cells were labelled with bromodeoxyuridine for later immunohistochemical identification. Twenty thousand cells in a carrier medium were then reimplanted at a second operation at an adjacent disc level in the same animal. The rat was subsequently sacrificed and the histology of the disc space was reviewed. Results: To date, 50 primary disc harvests and 30 reimplantations have been performed. Two rats died prior to reimplantation. All histological specimens confirmed the presence of viable transplanted disc cells. Conclusions: Autologous disc cell transplantation can be performed in the rat. Further modification of these techniques may lead to the development of autologous disc cell therapy comparable to that currently successfully used in hyaline cartilage defects of synovial joints in humans

Bone marrow is the tissue where hemopoiesis occurs in close contact with the stromal microenvironment which support hemopoietic stem cell growth and differentiation. The bone marrow stroma is composed of a variety of different cell types providing structural and functional support for hemopoiesis: endothelial cells, adipocytes, smooth muscle cells, reticular cells, osteoblasts and stromal fibroblasts. Among these cell types, stromal fibroblasts have a peculiar biologic relevance. They are in fact able to support hemopoiesis, to differentiate towards osteogenic, chondrogenic and adipogenic lineage and to form a bone structure complete of hemopoietic marrow in in vivo assays. Their in vitro clonogenic counterpart is represented by Colony Forming Units-fibroblasts (CFU-f), which in turn give rise to Bone Marrow Stromal Cells (BMSC). In vivo bone formation by BMSC has been strikingly demonstrated and therefore these cells are considered a progenitor compartment for osteoblasts, responsible for the maintenance of bone turnover throughout life. BMSC can be easily isolated from bone marrow aspirates. Nevertheless, given the low frequency of BMSC in a marrow sample, a step of extensive in vitro expansion is required to obtain a consistent number of cells available for both reconstruction and repair of mesodermally derived tissues. Moreover, their use for gene and cell therapy of skeletal diseases requires the long-lasting engraftment of BMSC endowed with a residual proliferation potential sufficient to sustain the low, but continuous, bone turnover in adulthood. The maintenance of BMSC stemness and the possibility to reprogram their commitment is therefore a field of primary interest given their potential use in regenerative medicine. Cell therapy of bone lesions by ex vivo expanded BMSC is passing from the phase of experimental animal model to the phase of clinical trials. Bone is repaired via local delivery of cells within a scaffold. Extremely appealing is the possibility of using mesenchymal progenitors in the therapy of genetic bone diseases via systemic infusion. Under some conditions where the local microenvironment is either altered (i.e. injury) or under important remodelling processes (i.e. fetal growth), engraftment of stem and progenitor cells seems to be enhanced. A better understanding of the mechanisms controlling BMSC differentiation and engraftment is required for their exploitation in therapy of human diseases. Furthermore, a better understanding of the interactions occurring between BMSC and biomaterials used to deliver cells in vivo will hopefully extend the field of therapeutic applications of mesenchymal progenitors. In this talk we will go through our experimental evidences on: a) influence of signaling molecule; b) transplantation route and engraftment; c) biomaterials. Growth factors are essential for a number of cellular functions. Our results show that FGF-2 supplemented BMSC primary cultures display better differentiation potential, a higher degree of osteogenicity and undergo an early increase in telomere size followed by a gradual decrease, whereas in control cultures telomere length decreases with increasing population doublings. In conjunction with clonogenic culture conditions, FGF-2 supplementation extends the life-span of BMSC to over 70 doublings and preserves their differentiation potential up to 50 doublings. All together, these data suggest that FGF-2 supplementation in vitro selects for the survival of a particular subset of cells enriched in pluripotent mesenchymal precursors and may be useful to obtain a large number of cells for mesenchymal tissue repair. BMSC intravenous infusion has been proposed as a means to support the hematopoiesis in Bone Marrow Transplants or as a vehicle for gene therapy. However, it seems that this route of injection leads to engraftment of a small proportion of BMSC. We have transplanted human BMSC transduced with the human erythropoietin gene, either intravenously or subcutaneously in NOD/SCID mice. Efficiency of engraftment was evaluated monitoring the hematocrit levels. Systemic infusion never increased hematocrit levels, whereas subcutaneous transplantation of the same number of cells induced an important increase of the hematocrit for at least two months. To determine whether the transient effect was due to cell loss or to reduction in expression, we recovered the cells implanted into a tridimensional scaffold, after the normalization of the hematocrit, expanded them in vitro, and re-implanted them in a new group of mice. Again the hematocrit levels rose one week after the transplantation. These results demonstrate that ex-vivo expanded human BMSC are not transplantable by systemic infusion, whereas the local implantation into a 3D scaffold allows their long term engraftment. Biomaterials for bone regeneration should have a suitable structure to allow cell adhesion and an ideal level of vascularisation, a key factor to achieve new bone formation. Furthermore, they have to be informative, driving the cells towards osteogenesis and allowing the deposition of bone extracellular matrix. Our results indicate that BMSC need a mineralized scaffold to initiate bone formation which will occur with an extent proportional to the availability of biomaterial surface

Purpose/introduction: 80% of individuals experience low back pain in their lifetime. This is often due to disc injury or degeneration. Conservative treatment of discogenic pain is often unsuccessful whilst surgery with the use of spacers of fusion is non-physiological. The aim of this study was to develop an animal model to assess the viability of autologous disc cell therapy. Method: The Fat Sand Rat (Psammomys obesus obesus) was chosen due to its predisposition to the early development of spondylosis. Using microsurgical techniques fragments of annulus and nucleus were harvested from a single disc in 52 sand rats. Vascular clips were placed on the adjacent psoas muscle to mark the harvested level. Disc material was initially cultured in monolayer then transferred into a three dimensional culture media of agarose. This technique yields greater cellular proliferation and the development of cell growth in colonies. Cells were labelled with Bromodeoxyuridine for later immunohistochemical identification. 20 000 cells in a carrier media were then re-implanted at a second operation at an adjacent disc level in the same animal. The rat was subsequently euthanised and the histology of the disc space reviewed. Results: To date 52 primary disc harvests and 20 reimplantations have been performed. 15 rats have been euthanised and sectioned. Average age at primary surgery was 6.8 months reimplantation eight months and euthanisation 11.2 months. Cell colony viability was inversely related to rat age at harvest. Immunohistochemical analysis of colony extracellular matrix revealed production of type 1 and 2 collagen, chondroitin and keratin sulphate Two rats died prior to reimplantation. All histological specimens confirm the presence of viable transplanted disc cells. Transplanted cells did not alter the progression of degenerative changes on x-ray. Conclusion: Autologous disc cell transplantation can be performed in the rat. Further modification of these techniques may lead to the development of autologous disc cell therapy comparable to that currently successfully used in hyaline cartilage defects of synovial joints in humans

Purpose: Prolonged denervation resulting from deferred nerve repair or long distance between the muscle and the repaired nerve, leads to major alterations concerning muscle fibre degeneration and their replacement by fibrous or fatty tissue. These structural modifications of the muscle are unfavourable for reinnervation and consequently affect the final functional outcome after peripheral nerve repair with its corollary of reduced muscle force. The purpose of this work was to assess the potential for regeneration of denervated-reinnervated muscles and their improvement with adjuvant cell therapy using in situ transfer of cultured autologus satellite cells. Material and methods: This work was conducted with the tibialis anterior muscle in different groups of New Zealand rabbits. The experimental model was a sectioned common fibular nerve and immediate or deferred (two months) microsurgical nerve suture. In vivo functional measurements and histomorphological analyses were performed four months after nerve repair. Results: Reinervation led to loss of mucle weight and maximal force (Fmax) which were greater with longer deferral of repair. Transfer of satellite cells performed immediately after reinervation did not improve muscle properties. Conversely, transfer of satellite cells two months after nerve suture increased Fmax 25% (p < 0.01) and muscle weight 28% (p = 0.005) in comparison with control muscles undergoing reinervation without cell transfer. Furthermore, the morphology of the muscle was improved as demonstrated by anti-myosine labelling studies. Discussion: Adjuvant cell therapy allows, in certain conditions, an improvement in functional recovery after peripheral nerve injury. Its clinical application still raises a certain number of ethical issues but taking into consideration data currently available, it would be reasonable to propose this therapeutic approach in humans to reduce involution of the denervated muscle and improve its receptivity for regenerating axons after peripheral nerve repair. Better post-operative results could be expected

Due to the presence of megakaryocytes, platelets and clotting factors, bone marrow aspirate (BMA) tends to coagulate. For the first time, starting from our previous studies on mesenchymal vertebral stem cells, it has been hypothesized that coagulated BMA represents a safe and effective autologous biological scaffold for bone regeneration in spinal surgery. The present research involved advanced preclinical in vitro models and the execution of a pilot clinical study.

Evaluation of cell morphology, growth kinetics, immunophenotyping, clonogenicity, trilineage-differentiation, growth-factors and HOX and TALE gene expression were analyzed on clotted- and un-clotted human V-BMA. In parallel, a pilot clinical study on ten patients with degenerative spine diseases submitted to instrumented posterior arthrodesis, is ongoing to assess the ability of clotted-V-BMA to improve spinal fusion at 6- and 12-months follow-up.

Results demonstrated that clotted-V-BMA have significantly higher growth-factor expression and mesenchymal stem cell (MSCs) viability, homogeneity, clonogenicity, and ability to differentiate towards the osteogenic phenotype than un-clotted-V-BMA. Clotted-V-BMA also highlighted significant reduced expression of PBX1 and of MEIS3 genes negatively involved in osteoblast maturation and differentiation. From December 2020, eight patients have already been enrolled with first promising results that will be finally evaluated in the next two months.

The application of V-BMA-clot as carrier of progenitors and cytokines and as natural scaffold with a structural texture represents a point-of-care orthobiologic product to improve spinal fusion. Clinical application seems to be efficacy, and we will confirm and strengthen these data with the final results of the pilot clinical study.

Purpose of the study: Irrespective of the technique used, the average rate of bone-tendon healing after rotator cuff repair is about 50% One of the reasons is the poor vitality of the tissues implicated in repair, particularly progressive destruction of the enthesis. Using the rat Achilles tendon, we destroyed the enthesis mechanically then repaired it with and without local injection of chondrocytes in order to study the effect of cell therapy on healing phenomena. Material and method: Sixty 3-month-old Wistar rats were operated on under general anaesthesia to detach the Achilles tendon and destroy the enthesis. In the first group (RI), the tendon was reinserted via a transosseous tunnel using a 4/0 non absorbable knitted thread. In the second group (RIC), joint chondrocytes, harvested from 4-day-old rats were injected locally during the same repair procedure. Animals were sacrificed every 15 days (n=15 per group) for a biomechanical and histology study. Results: In group RI, the non-healing rate was 50% versus 33% in group RIC; the difference was not significant (n=0.3). Tear resistance was increased significantly at 45 days in the RC group (p=0.04). The histology study showed a statistically significant development of a neoenthesis in the RIC group (p< 0.05), which was not observed in the RI group. Discussion: This animal model is valid for exploring rotator cuff healing with a spontaneous rate of healing to the order of 50%. Addition of chondrocytes during the surgical repair induces the production of an enthesis and increases the healing rate 50% and the value of the different biomechanical parameters at 30 days, with a statistically significant difference at 45 days

Purpose of the study: Monolayer cultures of chondrocytes multiply and rapidly lose their chondrocyte phenotype, limiting their potential for tissue engineering. Mesenchymatous stem cells can preserve their phenotypic characteristics after several monolayer passages, offering a promising alternative for cartilage repair. The purpose of this work was to study the influence of transforming growth factor beta-1 (TGF-beta1) and bone morphogenic protein-2 (BMP2) and/or culture supplements (hyaluronic acid) on matrix synthesis and chondrocyte differentiation of human mesenchymatous stem cells (MSC) cultured on collagen sponges. Material and methods: MSC were isolated from bone marrow harvested during hip arthroplsty. At the third passage in monolayer culture, the MSC were reseeded on collagen sponges and cultured in vitro for 28 days under seven differ conditions: insulin transferrin selenium (ITS), foetal calf serum (FCS), ITS+TGFbeta1, ITS+ hyaluronate, ITS+TGFbeta1+hyaluronate, ITS+TGFbeta1+BMP2, ITS +TGFbeta1+BMP2+hyaluronate. The phenotypic evolution was followed using the expression of different genes of interest with PCRq (collagen2, collagen1, collagen3, collagen10, agrecanne, versicanne, COMP, Sox9). Synthesis of matrix material was assessed histologically and immunohistochemically. Results: Used alone, hyaluronic acid did not trigger chondrocyte differentiation of MSC. For the additives FCS, ITS, or hyaluronate, the synthesis of matrix material in the sponge was weak and poor in major constituents of cartilage. Conversely, the other conditions in presence of TGFbeta1±BMP2 induced important expression of collagen2, agrecanne and COMP as well as increased matrix synthesis with a strong content in proteoglycans and collagen. Discussion: The usefulness of MSC is growing due to their pluripotent characteristics. The conditions leading to their differentiation into the chondrocyte phenotype remains a subject of discussion. Our results show the particular importance of TGFbeta1 in the process of differentiation. Conclusion: Chondrogenic differentiation of MSC cultured in collagen sponges as well as the synthesis of the cartilaginous matrix requires the presence of TGFbeta1 in the culture medium and to a lesser extent BMP2. These results suggest the perspective of using MSC for guided cell therapy targeting cartilage

Long bone fractures in patients with diabetes mellitus (DM) are slow to heal, often resulting in delayed reunion or non-union. It is reasonable to postulate that the underlying cause of these DM-associated complications is a reduced population of bone marrow progenitor cells and/or their dysfunction. With the hypothesis that the administration of healthy, allogeneic adult bone marrow-derived mesenchymal stromal cells (MSCs) can enhance DM fracture healing, the aim of this endeavour was to assess the efficacy of MSC administration to support fracture repair using two doses. Here 250,000 or 500,000 human bone marrow-derived MSCs were locally introduced to femoral fractures in diabetic mice, and the quality of de novo bone assessed 8 weeks later. Preliminary bone bridging was evident in all animals; however, a large circumferential reparative callus was consistently retained indicating non-union. Micro-CT analysis elucidated consistent callus dimensions, bone mineral density, bone volume/total volume in all groups, but an increase in bone surface area/bone volume in cell-treated fractures. Moreover, greater amounts of mature bone were identified in fractures treated with a low dose of MSCs. Four-point bending evaluation of the mechanical integrity of the repairing fracture indicated a statistically significant improvement in flexure strength and flexure modulus in DM fractures treated with 250,000 MSCs as compared to controls. An improvement in total energy required for failure was observed in both groups that received MSCs. Therefore, the administration of non-DM bone marrow-derived MSCs supported the development of more mature bone in the reparative callus, resulting in greater mechanical integrity.

Background

Delayed bone healing and nonunion are complications of long bone fractures, with prolonged pain and disability. Regenerative therapies employing mesenchymal stromal cells (MSC) and/or bone substitutes are increasingly applied to enhance bone consolidation. Within the REBORNE project, a multi-center orthopaedic clinical trial was focused on the evaluation of efficacy of expanded autologous bone marrow (BM) derived MSC combined with a CaP-biomaterial to enhance bone healing in patients with nonunion of diaphyseal fractures. To complement the clinical and radiological examination of patients, bone turnover markers (BTM) were assayed as potential predictors of bone healing or non-union.