Adipose Tissue-Derived Mesenchymal Stromal Cells from Ex-Morbidly Obese Individuals Instruct Macrophages towards a M2-Like Profile In Vitro.

Obesity, which continues to increase worldwide, was shown to irreversibly impair the differentiation potential and angiogenic properties of adipose tissue mesenchymal stromal cells (ADSCs). Because these cells are intended for regenerative medicine, especially for the treatment of inflammatory conditions, and the effects of obesity on the immunomodulatory properties of ADSCs are not yet clear, here we investigated how ADSCs isolated from former obese subjects (Ex-Ob) would influence macrophage differentiation and polarization, since these cells are the main instructors of inflammatory responses. Analysis of the subcutaneous adipose tissue (SAT) of overweight (OW) and Ex-Ob subjects showed the maintenance of approximately twice as many macrophages in Ex-Ob SAT, contained within the CD68＋/FXIII-A- inflammatory pool. Despite it, in vitro, coculture experiments revealed that Ex-Ob ADSCs instructed monocyte differentiation into a M2-like profile, and under inflammatory conditions induced by LPS treatment, inhibited HLA-DR upregulation by resting M0 macrophages, originated a similar percentage of TNF-α＋ cells, and inhibited IL-10 secretion, similar to OW-ADSCs and BMSCs, which were used for comparison, as these are the main alternative cell types available for therapeutic purposes. Our results showed that Ex-Ob ADSCs mirrored OW-ADSCs in macrophage education, favoring the M2 immunophenotype and a mixed (M1/M2) secretory response. These results have translational potential, since they provide evidence that ADSCs from both Ex-Ob and OW subjects can be used in regenerative medicine in eligible therapies. Further in vivo studies will be fundamental to validate these observations.

The Effects of Titanium Dioxide Nanoparticles on Osteoblasts Mineralization: A Comparison between 2D and 3D Cell Culture Models.

Although several studies assess the biological effects of micro and titanium dioxide nanoparticles (TiO2 NPs), the literature shows controversial results regarding their effect on bone cell behavior. Studies on the effects of nanoparticles on mammalian cells on two-dimensional (2D) cell cultures display several disadvantages, such as changes in cell morphology, function, and metabolism and fewer cell-cell contacts. This highlights the need to explore the effects of TiO2 NPs in more complex 3D environments, to better mimic the bone microenvironment. This study aims to compare the differentiation and mineralized matrix production of human osteoblasts SAOS-2 in a monolayer or 3D models after exposure to different concentrations of TiO2 NPs. Nanoparticles were characterized, and their internalization and effects on the SAOS-2 monolayer and 3D spheroid cells were evaluated with morphological analysis. The mineralization of human osteoblasts upon exposure to TiO2 NPs was evaluated by alizarin red staining, demonstrating a dose-dependent increase in mineralized matrix in human primary osteoblasts and SAOS-2 both in the monolayer and 3D models. Furthermore, our results reveal that, after high exposure to TiO2 NPs, the dose-dependent increase in the bone mineralized matrix in the 3D cells model is higher than in the 2D culture, showing a promising model to test the effect on bone osteointegration.

The role of macrophages in fracture healing: a narrative review of the recent updates and therapeutic perspectives.

Objective: This review addresses the latest advances in research on the role of macrophages in fracture healing, exploring their relationship with failures in bone consolidation and the perspectives for the development of advanced and innovative therapies to promote bone regeneration. Background: The bone can fully restore its form and function after a fracture. However, the regenerative process of fracture healing is complex and is influenced by several factors, including macrophage activity. These cells have been found in the fracture site at all stages of bone regeneration, and their general depletion or the knockdown of receptors that mediate their differentiation, polarization, and/or function result in impaired fracture healing. Methods: The literature search was carried out in the PubMed database, using combinations of the keywords "macrophage", "fracture healing, "bone regeneration", and "bone repair". Articles published within the last years (2017-2022) reporting evidence from in vivo long bone fracture healing experiments were included. Conclusions: Studies published in the last five years on the role of macrophages in fracture healing strengthened the idea that what appears to be essential when it comes to a successful consolidation is the right balance between the M1/M2 populations, which have different but complementary roles in the process. These findings opened promising new avenues for the development of several macrophage-targeted therapies, including the administration of molecules and/or biomaterials intended to regulate macrophage differentiation and polarization, the local transplantation of macrophage precursors, and the use of exosomes to deliver signaling molecules that influence macrophage activities. However, more research is still warranted to better understand the diversity of macrophage phenotypes and their specific roles in each step of fracture healing and to decipher the key molecular mechanisms involved in the in vivo crosstalk between macrophages and other microenvironmental cell types, such as endothelial and skeletal stem/progenitor cells.

Establishing a Diaphyseal Femur Fracture Model in Mice.

Bones have a significant regenerative capacity. However, fracture healing is a complex process, and depending on the severity of the lesions and the age and overall health status of the patient, failures can occur, leading to delayed union or nonunion. Due to the increasing number of fractures resulting from high-energy trauma and aging, the development of innovative therapeutic strategies to improve bone repair based on the combination of skeletal/mesenchymal stem/stromal cells and biomimetic biomaterials is urgently needed. To this end, the use of reliable animal models is fundamental to better understanding the key cellular and molecular mechanisms that determine the healing outcomes. Of all the models, the mouse is the preferred research model because it offers a wide variety of transgenic strains and reagents for experimental analysis. However, the establishment of fractures in mice may be technically challenging due to their small size. Therefore, this article aims to demonstrate the procedures for the surgical establishment of a diaphyseal femur fracture in mice, which is stabilized with an intramedullary wire and resembles the most common bone repair process, through cartilaginous callus formation.

The Isolation and Manufacture of GMP-Grade Bone Marrow Stromal Cells from Bone Specimens.

Bone marrow stromal cells (BMSCs, also known as bone marrow mesenchymal stem cells) are a plastic-adherent heterogeneous cell population that contain inherent skeletal progenitors and a subset of multipotential skeletal stem cells (SSCs). Application of BMSCs in therapeutic protocols implies its isolation and expansion under good manufacturing practices (GMP). Here we describe the procedures we have found to successfully generate practical BMSCs numbers, with preserved biological potency.

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.

Combinatorial cassettes to systematically evaluate tissue-engineered constructs in recipient mice.

Ectopic bone formation in mice is the gold standard for evaluation of osteogenic constructs. By regular procedures, usually only 4 constructs can be accommodated per mouse, limiting screening power. Combinatorial cassettes (combi-cassettes) hold up to 19 small, uniform constructs from the time of surgery, through time in vivo, and subsequent evaluation. Two types of bone tissue engineering constructs were tested in the combi-cassettes: i) a cell-scaffold construct containing primary human bone marrow stromal cells with hydroxyapatite/tricalcium phosphate particles (hBMSCs + HA/TCP) and ii) a growth factor-scaffold construct containing bone morphogenetic protein 2 in a gelatin sponge (BMP2+GS). Measurements of bone formation by histology, bone formation by X-ray microcomputed tomography (µCT) and gene expression by quantitative polymerase chain reaction (qPCR) showed that constructs in combi-cassettes were similar to those created by regular procedures. Combi-cassettes afford placement of multiple replicates of multiple formulations into the same animal, which enables, for the first time, rigorous statistical assessment of: 1) the variability for a given formulation within an animal (intra-animal variability), 2) differences between different tissue-engineered formulations within the same animal and 3) the variability for a given formulation in different animals (inter-animal variability). Combi-cassettes enable a more high-throughput, systematic approach to in vivo studies of tissue engineering constructs.

Atrophic nonunion stromal cells form bone and recreate the bone marrow environment in vivo.

INTRODUCTION: Nonunion is a challenging condition in orthopaedics as its etiology is not fully understood. Clinical interventions currently aim to stimulate both the biological and mechanical aspects of the bone healing process by using bone autografts and surgical fixation. However, recent observations showed that atrophic nonunion tissues contain putative osteoprogenitors, raising the hypothesis that its reactivation could be explored to achieve bone repair. METHODS: Here we characterized atrophic nonunion stromal cells (NUSC) in vitro, using bone marrow stromal cells (BMSC) and osteoblasts as controls cells of the osteoblastic lineage, and evaluated its ability to form bone in vivo. RESULTS: NUSC had proliferative and senescence rates comparable to BMSC and osteoblasts, and homogeneously expressed the osteolineage markers CD90 and CD73. Regarding CD105 and CD146 expression, NUSC were closely related to osteoblasts, both with an inferior percentage of CD105+/CD146+ cells as compared to BMSC. Despite this, NUSC differentiated along the osteogenic and adipogenic lineages in vitro; and when transplanted subcutaneously into immunocompromised mice, new bone formation and hematopoietic marrow were established. CONCLUSIONS: This study demonstrates that NUSC are osteogenically competent, supporting the hypothesis that their endogenous reactivation could be a strategy to stimulate the bone formation while reducing the amount of bone autograft requirements.

Vertical femoral neck fractures in young adults: a closed fixation strategy using a transverse cancellous lag screw.

Vertical femoral neck fractures (Pauwels type III classification) in young adults generally occur as a consequence of high-energy trauma and are frequently seen in association with multiple injuries. Considering the controversies regarding the optimal fixation for this fracture, our aim was to evaluate the clinical outcome of a closed fixation strategy for vertical femoral neck fractures in young adults using two parallel and one transverse cancellous lag screws. This was a single-surgeon, prospective study including 20 young adults with average age of 38.75 years (range 18-59 years) with a high-energy Pauwels III femoral neck fracture. Closed reduction and internal fixation with three cancellous lag screws were performed. The first screw was inserted crosswise to avoid further shear forces. Second and third parallel screws were placed above the lesser trochanter and centrally on the greater trochanter, respectively. Clinical outcomes were assessed by comparing postoperative and final follow-up radiographs 24 months post-injury. Eleven patients had an isolated vertical femoral neck fracture. Of these, five had further femoral neck comminution. Nine patients had an associated ipsilateral femoral shaft fracture. All fractures were displaced at the time of the first radiological evaluation. Closed reduction quality was considered excellent or good in 15 patients. After 24 months, bone union was achieved in 16 cases. Osteonecrosis of the femoral head developed in association with two fractures, and a nonunion developed in association with two fractures. We conclude that vertical high-energy femoral neck fractures can be treated successfully with internal fixation with two parallel cancellous lag screws positioned above the lesser trochanter and a third screw inserted centrally on the greater trochanter at an angle perpendicular to the fracture line.

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.

Mesenchymal stromal cells from bone marrow treated with bovine tendon extract acquire the phenotype of mature tenocytes.

OBJECTIVE: This study evaluated in vitro differentiation of mesenchymal stromal cells isolated from bone marrow, in tenocytes after treatment with bovine tendon extract. METHODS: Bovine tendons were used for preparation of the extract and were stored at -80 °C. Mesenchymal stromal cells from the bone marrow of three donors were used for cytotoxicity tests by means of MTT and cell differentiation by means of qPCR. RESULTS: The data showed that mesenchymal stromal cells from bone marrow treated for up to 21 days in the presence of bovine tendon extract diluted at diminishing concentrations (1:10, 1:50 and 1:250) promoted activation of biglycan, collagen type I and fibromodulin expression. CONCLUSION: Our results show that bovine tendon extract is capable of promoting differentiation of bone marrow stromal cells in tenocytes.

OBJETIVO: O estudo avalia a diferenciação in vitro das células mesenquimais isoladas do estroma da medula óssea em tenócitos após tratamento com extrato de tendão bovino. MÉTODOS: Tendões bovinos foram usados para confecção do extrato e estocados a −80 °C. Células mesenquimais do estroma da medula óssea (BMSCs) de três doadores foram usadas para os testes de citotoxicidade por MTT e diferenciação celular por qPCR. RESULTADOS: Os dados mostram que células mesenquimais do estroma da medula óssea tratadas por até 21 dias em presença do extrato de tendão bovino diluído em concentrações crescentes (1:10, 1:50 e 1:250) promovem a ativação da expressão de biglican, colágeno tipo I e fibromodulina. CONCLUSÃO: Nossos resultados mostram que o extrato de tendão bovino é capaz de promover a diferenciação das BMSCs em tenócitos.

Mesenchymal stromal cells from bone marrow treated with bovine tendon extract acquire the phenotype of mature tenocytes / Células mesenquimais do estroma da medula óssea tratadas com extrato de tendão bovino adquirem o fenótipo de tenócitos maduros

This study evaluated in vitro differentiation of mesenchymal stromal cells isolated from bone marrow, in tenocytes after treatment with bovine tendon extract. METHODS: Bovine tendons were used for preparation of the extract and were stored at -80 °C. Mesenchymal stromal cells from the bone marrow of three donors were used for cytotoxicity tests by means of MTT and cell differentiation by means of qPCR. RESULTS: The data showed that mesenchymal stromal cells from bone marrow treated for up to 21 days in the presence of bovine tendon extract diluted at diminishing concentrations (1:10, 1:50 and 1:250) promoted activation of biglycan, collagen type I and fibromodulin expression. CONCLUSION: Our results show that bovine tendon extract is capable of promoting differentiation of bone marrow stromal cells in tenocytes.

O estudo avalia a diferenciação in vitro das células mesenquimais isoladas do estroma da medula óssea em tenócitos após tratamento com extrato de tendão bovino. MÉTODOS: Tendões bovinos foram usados para confecção do extrato e estocados a -80 °C. Células mesenquimais do estroma da medula óssea (BMSCs) de três doadores foram usadas para os testes de citotoxicidade por MTT e diferenciação celular por qPCR. RESULTADOS: Os dados mostram que células mesenquimais do estroma da medula óssea tratadas por até 21 dias em presença do extrato de tendão bovino diluído em concentrações crescentes (1:10, 1:50 e 1:250) promovem a ativação da expressão de biglican, colágeno tipo I e fibromodulina. CONCLUSÃO: Nossos resultados mostram que o extrato de tendão bovino é capaz de promover a diferenciação das BMSCs em tenócitos.

PS1/γ-Secretase-Mediated Cadherin Cleavage Induces ß-Catenin Nuclear Translocation and Osteogenic Differentiation of Human Bone Marrow Stromal Cells.

Bone marrow stromal cells (BMSCs) are considered a promising tool for bone bioengineering. However, the mechanisms controlling osteoblastic commitment are still unclear. Osteogenic differentiation of BMSCs requires the activation of ß-catenin signaling, classically known to be regulated by the canonical Wnt pathway. However, BMSCs treatment with canonical Wnts in vitro does not always result in osteogenic differentiation and evidence indicates that a more complex signaling pathway, involving cadherins, would be required to induce ß-catenin signaling in these cells. Here we showed that Wnt3a alone did not induce TCF activation in BMSCs, maintaining the cells at a proliferative state. On the other hand, we verified that, upon BMSCs osteoinduction with dexamethasone, cadherins were cleaved by the PS1/γ-secretase complex at the plasma membrane, and this event was associated with an enhanced ß-catenin translocation to the nucleus and signaling. When PS1/γ-secretase activity was inhibited, the osteogenic process was impaired. Altogether, we provide evidence that PS1/γ-secretase-mediated cadherin cleavage has as an important role in controlling ß-catenin signaling during the onset of BMSCs osteogenic differentiation, as part of a complex signaling pathway responsible for cell fate decision. A comprehensive map of these pathways might contribute to the development of strategies to improve bone repair.

The effect of autologous concentrated bone-marrow grafting on the healing of femoral shaft non-unions after locked intramedullary nailing.

The aim of this study was to assess the union rates in a series of patients with failed femoral shaft aseptic non-union who were treated with percutaneous concentrated autologous bone marrow grafting. Bone marrow harvesting and cell injection were performed under general anaesthesia in a single surgical procedure. Radiographic union was diagnosed in fractures with a score ≥ 10 according to the radiographic union scale in tibial fractures (RUST) and confirmed by clinical examination. Eight out of 16 patients progressed to consolidation (RUST score ≥ 10). Radiographic evidence of fracture union was observed at an average of 4.75 ± 1.75 months (range 3 to 8 months). All eight patients who did not progress to union within 12 months following the cell grafting procedure had a RUST score ≤ 10 (range 4 to 9). There were no differences in age, number of previous surgeries, duration of nonunion and preoperative RUST score between the patients that developed solid union and those with failed consolidation. However, a relationship between the number of osteoprogenitors injected and the rate of union was noted, 20.2 ± 8.6 × 10(8) versus 9.8 ± 4.3 × 10(8), p<0.005, between the patients with and without union, respectively. The efficacy of percutaneous autologous concentrated bone marrow grafting seems to be related to the number of osteoprogenitors available in the aspirates. Optimisation of the aspiration technique and concentration process is of paramount importance to increase the incidence of a successful outcome.

Late adherent human bone marrow stromal cells form bone and restore the hematopoietic microenvironment in vivo.

Bone marrow stromal cells (BMSCs) are a valuable resource for skeletal regenerative medicine because of their osteogenic potential. In spite of the very general term "stem cell," this population of cells is far from homogeneous, and different BMSCs clones have greatly different phenotypic properties and, therefore, potentially different therapeutic potential. Adherence to a culture flask surface is a primary defining characteristic of BMSCs. We hypothesized that based on the adherence time we could obtain an enriched population of cells with a greater therapeutic potential. We characterized two populations of bone marrow-derived cells, those that adhered by three days (R-cells) and those that did not adhere by three days but did by six days (L-cells). Clones derived from L-cells could be induced into adipogenic, chondrogenic, and osteogenic differentiation in vitro. L-cells appeared to have greater proliferative capacity, as manifested by larger colony diameter and clones with higher CD146 expression. Only clones from L-cells developed bone marrow stroma in vivo. We conclude that the use of late adherence of BMSCs is one parameter that can be used to enrich for cells that will constitute a superior final product for cell therapy in orthopedics.

Heterologous mesenchymal stem cells successfully treat femoral pseudarthrosis in rats.

BACKGROUND: This study evaluated the effectiveness of treating pseudarthrosis in rats by using bone marrow cell suspensions or cultures of bone marrow mesenchymal stromal cells METHODS: Thirty-eight specific pathogen-free (SPF) animals were randomly assigned to four groups: Group 1, Control, without surgical intervention; Group 2 (Placebo), experimental model of femoral pseudarthrosis treated only with saline solution; Group 3, experimental model of femoral pseudarthrosis treated with heterologous bone marrow cells suspension; Group 4, experimental model of femoral pseudarthrosis treated with cultures of heterologous mesenchymal stromal cells from bone marrow. When pseudarthrosis was confirmed by simple radiological studies, digital radiography and histopathology after a 120-day postoperative period, Groups 2, 3 and 4 were treated as above. At 30, 60 and 90 days after the treatment, all animals were evaluated by simple radiological studies, and at the end of the experiment, the animals were assessed by computed axial tomography and anatomopathological and histomorphometric examinations. RESULTS: Injected cells were detected in the areas affected by pseudarthrosis using scintigraphy within the first 24 hours after their administration. After 60 days, the animals of Group 3 showed callus formation while the animals of Group 4 presented periosteal reaction and had some consolidated areas. In contrast, Group 2 showed a predominance of fibro-osteoid tissue. After 90 days, bone consolidation and remodeling was observed in all animals from Group 3 whereas animals from Group 4 exhibited partial consolidation and those ones from Group 2 persisted with pseudarthrosis. CONCLUSION: The treatment with heterologous bone marrow cells suspension proved to be effective in the treatment of pseudarthrosis whereas cultures of heterologous bone marrow mesenchymal stromal cells did not show the same potential to aid bone healing.