The Manufacture of GMP-Grade Bone Marrow Stromal Cells with Validated In Vivo Bone-Forming Potential in an Orthopedic Clinical Center in Brazil.

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.

Bone intramedullary reaming grafts the fracture site with CD146+ skeletal progenitors and downmodulates the inflammatory environment.

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.

Immediate dental implant failure associated with nasopalatine duct cyst.

This case report presents an analysis of the clinical, radiographic, and histological features of a peri-implant lesion around an implant placed immediately after extraction of a tooth with a periapical lesion. A 52-year-old man received an immediate implant (3.75 x 11.5 mm2) placed in the anterior region of the maxilla. Three years after implant placement, the patient presented with swelling in the anterior portion of the maxilla. Radiographic examination showed a well-circumscribed radiolucency around the implant. The implant and the lesion were removed and fixed in 10% buffered formalin and processed. Histological analysis showed 3 types of epithelium: respiratory, cuboidal, and non-keratinized stratified squamous. In the cyst wall peripheral nerves, arteries, veins, and chronic inflammation were present. The diagnosis was nasopalatine duct cyst. We concluded that the nasopalatine duct cyst can develop in association with dental implants. Clinically, the lesion is similar to the classical nasopalatine duct cyst. Histological analysis should be mandatory in all cases of peri-implant lesions and in all dental periapical lesions before immediate implant placement.

Efeito anti-tumoral dos bisfosfonatos: uma nova perspectiva terapêutica / Anti-tumor effect of bisphosphonates: a new therapeutic perpective

Os bisfosfonatos (BFs) são potentes inibidores da reabsorção óssea mediada por osteoclastos. Essas drogas sãoefetivas na redução do cálcio sérico em pacientes com hipercalcemia maligna, assim como também no tratamentoda dor óssea, osteoporose e metástases ósseas.Diversos estudos demonstram que os BFs possuem efeito em outras células além dos osteoclastos. Em célulastumorais podem agir induzindo a apoptose, inibindo a proliferação celular, inibindo a adesão e a invasividadecelular ou as metástases ósseas. O mecanismo molecular subjacente a estes efeitos parece ser a inibição de enzimasda via do mevalonato, o que leva a um impedimento da prenilação de GTPases como Ras e Rho, importantes paraa manutenção da integridade do citoesqueleto e tráfego de vesículas nas células.As evidências dos recentes estudos laboratoriais e clínicos sugerem que os BFs têm um papel importante comotratamento suplementar e possivelmente complementar na terapia do câncer. Um entendimento mais profundo e completo sobre o efeito anti-tumoral destas drogas pode sugerir possibilidades terapêuticas novas e seletivas.

Bisphosphonates are potent osteoclast-mediated bone reabsorption inhibitors. These drugs are effective in reducingserum calcium levels in patients with malignant hypercalcaemia, as well as in bone pain, osteoporosis and bonemetastasis treatment. Several studies have demonstrated that bisphosphonates are effective in other cell types than osteoclasts. In tumourcells they can act inducing apoptosis, inhibiting cell proliferation, inhibiting cell adhesion and invasion or bonemetastasis. The underlying molecular mechanism to these effects seems to be the inhibition of mevalonate pathwayenzymes, which leads to a prenilation impairment of GTPases like Ras and Rho, important in maintaining celularcytoskeleton integrity and vesicles trafficking. Evidences from recent laboratorial and clinical studies suggest that bisphosphonates have an important role as a supplemental, and possibly as complementary treatment in cancer therapy. A more complete understanding concerning the anti-tumor effect of these drugs may suggest new and selective therapeutical possibilities.