RESUMO
In vitro-expanded bone marrow stromal cells (BMSCs) have long been proposed for the treatment of complex bone-related injuries because of their inherent potential to differentiate into multiple skeletal cell types, modulate inflammatory responses, and support angiogenesis. Although a wide variety of methods have been used to expand BMSCs on a large scale by using good manufacturing practice (GMP), little attention has been paid to whether the expansion procedures indeed allow the maintenance of critical cell characteristics and potency, which are crucial for therapeutic effectiveness. Here, we described standard procedures adopted in our facility for the manufacture of clinical-grade BMSC products with a preserved capacity to generate bone in vivo in compliance with the Brazilian regulatory guidelines for cells intended for use in humans. Bone marrow samples were obtained from trabecular bone. After cell isolation in standard monolayer flasks, BMSC expansion was subsequently performed in two cycles, in 2- and 10-layer cell factories, respectively. The average cell yield per cell factory at passage 1 was of 21.93 ± 12.81 × 106 cells, while at passage 2, it was of 83.05 ± 114.72 × 106 cells. All final cellular products were free from contamination with aerobic/anaerobic pathogens, mycoplasma, and bacterial endotoxins. The expanded BMSCs expressed CD73, CD90, CD105, and CD146 and were able to differentiate into osteogenic, chondrogenic, and adipogenic lineages in vitro. Most importantly, nine out of 10 of the cell products formed bone when transplanted in vivo. These validated procedures will serve as the basis for in-house BMSC manufacturing for use in clinical applications in our center.
RESUMO
INTRODUCTION: Femoral shaft fractures generally occur in young adults following a high-energy trauma and are prone to delayed union/non-union. Novel therapies to stimulate bone regeneration will have to mimic some of the aspects of the biology of fracture healing; however, which are these aspects is unclear. Locked intramedullary nailing is the current treatment of choice for the stabilisation of femur shaft fractures, and it is associated with accelerated healing and increased union rates. These benefits were partially attributed to the reaming procedure, which, regardless of significantly destroying the haematoma, stimulates the healing response. To better understand how reaming influences healing, we evaluated the viability of the nucleated cell fraction and the frequency of CD146+ skeletal progenitors, which contain multipotent cells, in the post-reaming haematoma. We also screened the concentrations of inflammatory mediators and growth factors in the fracture site after reaming compared with those in the original haematoma. METHODS: Pre- and post-reaming haematomas were percutaneously aspirated from the fracture site of 15 patients with closed femoral shaft fractures. Cellular viability and the percentage of CD146+ progenitors were analysed by flow cytometry. The concentrations of cytokines and growth factors were determined by ELISA. RESULTS: AnnexinV/Pi analysis showed that the viability of the total nucleated cell fraction was decreased in the post-reaming haematoma. However, the procedure increased the percentage of CD146+ skeletal progenitors in the fracture site. Analysis of cytokines and growth factors in supernatants showed a decreased concentration of the inflammatory mediators IL-6, CCL-4, and MCP-1, along with an increase of anti-inflammatory IL-10, and the growth factors bFGF and PDGF-AB. CONCLUSION: These findings support the view that the positive effects of reaming on fracture healing might result from mechanically grafting the fracture site with a population of skeletal progenitors that contain multipotent cells; transitioning the signalling environment to a less inflammatory state, and enhancing the availability of specific osteogenic and angiogenic factors. A better understanding of the requisite stimuli for optimal bone repair, considering the disturbances made by orthopaedic treatments, will be determinant for the development of innovative treatments for bone repair.
Assuntos
Basigina/metabolismo , Fraturas do Fêmur/cirurgia , Fixação Intramedular de Fraturas , Consolidação da Fratura/fisiologia , Hematoma/patologia , Mediadores da Inflamação/metabolismo , Osteogênese/fisiologia , Adulto , Pinos Ortopédicos , Ensaio de Imunoadsorção Enzimática , Feminino , Fraturas do Fêmur/imunologia , Citometria de Fluxo , Consolidação da Fratura/imunologia , Hematoma/etiologia , Humanos , Masculino , Pessoa de Meia-Idade , Adulto JovemRESUMO
OBJECTIVE: To present the results and conclusions of our study on surgical treatment for unstable pelvic fractures in children subjected to surgical reduction and stabilisation. METHODS: We analysed the cases of fourteen skeletally immature patients with unstable pelvic fractures who underwent surgery for this condition between March 2004 and January 2011. The surgical technique used was based on the principle of surgical reduction and stabilisation of anterior and posterior lesions of the pelvic ring. This was a retrospective study, based on clinical assessment and X-ray analyses. RESULTS: The mean age of patients at the time of the condition was 9.4 years (range 2-13 years). Eight patients were female and six were male. The cause of the trauma was being hit by a car in ten cases, falls in three cases and an accident involving a motorcycle in one case. Five patients presented with other associated injuries, including fracture of the clavicle, femur shaft, proximal humerus, tibial shaft or olecranon, and bladder damage. All the patients assessed showed excellent clinical progress. Pelvic asymmetry prior to surgery varied from 1.1 to 2.9 cm (mean 1.5 cm) and dropped to a range of 0.2 to 0.9 cm (mean 0.4 cm) after reduction. In none of the cases was there a change between the pelvic asymmetry measured immediately after surgery and at the end of the follow-up period. CONCLUSION: Pelvic fracture in skeletally immature patients is rare and surgery is not normally indicated. Various authors have questioned this conservative type of treatment due to complications encountered. Bone remodelling does not seem to be sufficient to ensure an improvement in pelvic asymmetry, which justifies opting for surgery to reduce and correct deformities in the pelvic ring.