Maria Eugenia Leite Duarte

Characterization of a bovine collagen–hydroxyapatite composite scaffold for bone tissue engineering

Different biomaterials have been used as scaffolds for bone tissue engineering. Here we characterize a biomaterial composed of sintered (1100 degrees C) and powdered hydroxyapatite (HA) and type I collagen (Coll), both of bovine origin, designed for osteoconductive and osteoinductive scaffolds. Coll/HA proportions were 1/2.6 and 1/1 (wet weight), and particles sizes varied from 200 to 400 microm. Vv (volume density) and Sv (surface to volume density) for the HA particles in the composite ranged from 0.48 +/- 0.06 to 0.55 +/- 0.02 and 5.090 +/- 0.545 to 6.366 +/- 0.289 microm(-1), respectively. Due to the relatively small changes in Vv and Sv, a macroporosity could be characterized for the biocomposite. X-ray diffraction and infrared spectroscopy showed that the sintered bone was composed essentially of HA with minimum additional groups such as surface calcium hydroxide, surface and crystal water, free carbon dioxide and possibly brushite. Mass spectrometry detected carbonates at A and B sites of HA, and weakly bound to the structure. Human osteoblasts adhered and spread on both the HA particle surface and the collagen fibers, which seemed to guide cells between adjacent particles. The biocomposite studied has several characteristics considered as ideal for its use as a scaffold for osteoconduction and osteoinduction.

Synovial fluid and synovial membrane mesenchymal stem cells: latest discoveries and therapeutic perspectives

Mesenchymal stem cells (MSCs) have the ability to differentiate into osteoblasts, chondroblasts, adipocytes, and even myoblasts. Most studies have focused on finding MSCs in different parts of the body for medical treatment. Every joint structure, including bone, joint fat, articular cartilage, and synovium, potentially contains resident MSCs. Recently, a progenitor cell population has been found in synovial fluid and showed similarities with both bone marrow and synovial membrane MSCs. Synovial fluid MSCs have been studied in healthy persons and osteoarthritic patients in order to explore its potential for treatment of some orthopedic disorders. Here, we briefly review the current knowledge on synovial fluid MSCs, their origin, relation to some orthopedic diseases, and future applications.

Identification of periodontal pathogens in healthy periimplant sites.

Purpose:The aim of this study was to evaluate the presence of periodontopathogens in subgingival periimplant sites in partially edentulous patients using polymerase chain reaction procedures, with regard to areas with clinical and radiographic signs of health and areas presenting periimplant disease. Materials and Methods:Thirty nonsmoking, partially edentulous patients, aged 30 to 76 years, were included in this study and divided in 3 groups according their clinical and radiographic characteristics. Group A (n = 10) presented periimplant health, group B (n = 10) presented periimplant mucositis, and group C (n = 10) were patients with periimplantitis. Periimplant tissues were clinically examined as regards the color of mucosae, presence of bacterial plaque, depth and bleeding on probing, and local suppuration. History of periodontal disease was also considered. Radiographic analysis evaluated the presence of bone loss around the implant. Samples of periimplant crevicular fluid were collected to analyze the presence of periodontal pathogens, Actinobacillus actinomycetemcomitans (Aa), Porphyromonas gingivalis (Pg), Prevotella intermedia (Pi), Tannerella forsythensis (Tf), and Treponema denticola (Td). Results:The results showed that the history of periodontal disease is associated with periimplant disease. The bacteria Aa, Pg, Pi, Td, and Tf were present in periimplant sites clinically and radiographically characterized, as healthy periimplant tissues, mucositis, and periimplantitis. Conclusions:We concluded that Aa, Pg, Pi, Td, and Tf are present in healthy and diseased conditions. Therefore, these periodontal pathogens are not strictly related to periimplant disease sites.

Polymorphisms in BMP4 and FGFR1 genes are associated with fracture non-union

Fracture healing is a complex process influenced by a multitude of factors and expression of several thousand genes. Polymorphisms in these genes can lead to an extended healing process and explain why certain patients are more susceptible to develop non‐union. A total of 16 SNPs within five genes involved in bone repair pathogenesis (FAM5C, BMP4, FGF3, FGF10, and FGFR1) were investigated in 167 patients with long bone fractures, 101 with uneventful healing, and 66 presenting aseptic non‐unions. Exclusion criteria were patients presenting pathological fractures, osteoporosis, hypertrophic and infected non‐unions, pregnancy, and children. All genetic markers were genotyped using TaqMan real‐time PCR. Chi‐square test was used to compare genotypes, allele frequencies, and haplotype differences between groups. Binary logistic regression analyzed the significance of many covariates and the incidence of non‐union. Statistical analysis revealed open fracture to be a risk factor for non‐union development (p < 0.001, OR 3.6 [1.70–7.67]). A significant association of haplotype GTAA in BMP4 (p = 0.01) and FGFR1 rs13317 (p = 0.005) with NU could be observed. Also, uneventful healing showed association with FAM5C rs1342913 (p = 0.04). Our work supported the role of BMP4 and FGFR1 in NU fracture independently of the presence of previously described risk factors.

Ridge bone maintenance in human after extraction.

Purpose:The aim of this study was to evaluate, clinically and histologically, the tissues formed in human alveolar sockets filled with bovine morphogenetic protein/bovine organic matrix (BOM) and absorbable membrane (AM) immediately after extraction. Materials:Forty-six human alveolar sockets, exhibiting buccal bone defects were selected for this study. Group 1 received no biomaterial to serve as control. Sockets from group 2 were filled with bovine bone morphogenetic protein (bBMP) associated with bOM. The association of bBMP/bOM/AM filled the alveolar defects from group 3. AM was placed over the defects from group 4. Clinical evaluation analyzed ridge width before biomaterial filling and 4 months after filling. Fifteen specimens were collected from groups 2, 3, and 4 for histologic analyses. Results:Clinical results showed no significant augmentation on the control group (−0.16 ± 0.28 mm). All test sites, groups 2, 3, and 4, showed relevant ridge width augmentation (3.0 ± 0.5 mm, 2.4 ± 0.3 mm, and 2.9 ± 0.6 mm, respectively) and no resorption. Histologically, all experimental alveolar sockets showed active bone formation with osteoid, osteoblasts, and cell differentiation. Conclusion:On the basis of this study, we concluded that bBMP/bOM with or without AM could preserve the ridge showing viable bone formation for future implant placement.

Fine structure and molecular content of human chondrocytes encapsulated in alginate beads

Preservation of the chondrocytic phenotype in vitro requires a 3D (three‐dimensional) culture model. Diverse biomaterials have been tested as scaffolds for culture of animal chondrocytes; however, to date, none is considered a gold standard in regenerative medicine. Here, we studied the fine structure and the GAGs (glycosaminoglycans) content of human chondrocytes encapsulated in alginate beads by using electron microscopy and radioactive sulfate [35S] incorporation, respectively. Cells were obtained from human cartilage, encapsulated in alginate beads and cultured for 28 days. [35S]Na2SO4 was added to the culture media and later isolated for quantification of the sulfated GAGs found in three compartments: IC (intracellular), IB (intra‐bead) and EB (extra‐bead). Round cells were seen isolated or forming small groups throughout the alginate. Human chondrocytes presented the features of active cells such as euchromatic nuclei, abundant RER (rough endoplasmic reticulum) and many transport vesicles. We observed an extracellular matrix rich in collagen fibres and electrondense material adjacent to the cells. Most of the GAGs produced (74%) were found in the culture medium (EB), indicating that alginate has a limited capacity to retain the GAGs. CS (chondroitin sulfate), the major component of aggrecan, was the most prominent GAG produced by the encapsulated cells. Human chondrocytes cultured in alginate can sustain their phenotype, confirming the potential application of this biomaterial for cartilage engineering.

Fc receptor-like 3 ( −169T>C ) polymorphism increases the risk of tendinopathy in volleyball athletes: a case control study

BackgroundTendinopathy pathogenesis is associated with inflammation. Regulatory T (Treg) cells contribute to early tissue repair through an anti-inflammatory action, with the forkhead box P3 (FOXP3) transcription factor being essential for Treg function, and the FC-receptor-like 3 (FCRL3) possibly negatively regulating Treg function. FCRL3 –169T>C and FOXP3 –2383C>T polymorphisms are located near elements that regulate respective genes expression, thus it was deemed relevant to evaluate these polymorphisms as risk factors for tendinopathy development in athletes.MethodsThis case-control study included 271 volleyball athletes (146 tendinopathy cases and 125 controls) recruited from the Brazilian Volleyball Federation. Genotyping analyses were performed using TaqMan assays, and the association of the polymorphisms with tendinopathy evaluated by multivariate logistic regression.ResultsTendinopathy frequency was 63% patellar, 22% rotator cuff and 15% Achilles tendons respectively. Tendinopathy was more common in men (OR = 2.87; 95% CI = 1.67–4.93). Higher age (OR = 8.75; 95% CI = 4.33–17.69) and more years of volleyball practice (OR = 8.38; 95% CI = 3.56–19.73) were risk factors for tendinopathy. The FCRL3 –169T>C frequency was significantly different between cases and controls. After adjustment for potential confounding factors, the FCRL3 –169C polymorphism was associated with increased tendinopathy risk (OR = 1.44; 95% CI = 1.02–2.04), either considering athletes playing with tendon pain (OR = 1.98; 95% CI = 1.30–3.01) or unable to train due to pain (OR = 1.89; 95% CI = 1.01–3.53). The combined variant genotypes, FCRL3 –169TC or –169CC and FOXP3 –2383CT or –2383TT, were associated with an increased risk of tendinopathy among athletes with tendon pain (OR = 2.24; 95% CI: 1.14–4.40 and OR = 2.60; 95% CI: 1.11–6.10). The combined analysis of FCRL3 –169T>C and FOXP3 –2383C>T suggests a gene-gene interaction in the susceptibility to tendinopathy.ConclusionsFCRL3 –169C allele may increase the risk of developing tendinopathy, and together with knowledge of potential risk factors (age, gender and years playing) could be used to personalize elite athletes’ training or treatment in combination with other approaches, with the aim of minimizing pathology development risk.

Patient-derived osteosarcoma cells are resistant to methotrexate

Osteosarcoma is the most common primary bone tumor in children and young adults. The median survival of osteosarcoma patients has not significantly improved since 1990, despite administration of different classes of chemotherapy agents, such as methotrexate, cisplatin and doxorubicin. Cancer stem cells (CSCs) are responsible for the resistance of osteosarcoma to chemotherapy and OCT4, SOX2 and SSEA4 have been used to identify CSCs in osteosarcoma. Here, we used low-passage patient-derived osteosarcoma cells and osteosarcoma cells directly isolated from patients before and after chemotherapy treatments to evaluate the effects of chemotherapy on stem cell markers expression. We demonstrate that primary osteosarcoma cells are resistant to methotrexate treatment and sensitive to cisplatin and doxorubicin in vitro. We also verified that cisplatin and doxorubicin reduce the expression of SOX2 and OCT4 in primary osteosarcoma cells whereas methotrexate does not alter SOX2 and OCT4 expression, however it increases SSEA4 expression in primary osteosarcoma cells. Finally, we found that, although the combination treatment cisplatin plus doxorubicin inhibited the in vivo growth of osteosarcoma cells in NOD-SCID gamma mice subcutaneously injected with SaOs2, the combination treatment cisplatin plus doxorubicin plus methotrexate did not inhibit the in vivo growth of these cells. These observations may provide an explanation for the poor response of osteosarcomas to chemotherapy and point to the need of reevaluating the therapeutic strategies for human osteosarcomas.

Skeletal Resident Stem Cells

Bone is a specialized type of connective tissue with a specialized extracellular matrix impregnated with hydroxyapatite crystal, which gives bone the characteristic of a hard tissue, with limited flexibility. The three main cell types that participate in bone formation and maintenance are the osteoblasts, osteocytes, and osteoclasts. Osteocytes are considered the mature end point cell of the osteogenic lineage cascade. Osteoblasts are bone-producing cells, which synthesize and secrete collagen fibers and other noncollagen proteins and begin the process of mineralization of bone matrix. Osteoclasts are the third specialized cell type found in bone tissue and are responsible for bone resorption. Osteoblasts and osteocytes derive from a common precursor, identified as a mesenchymal stem cell, also known as stromal stem cell or skeletal stem cell. Under proper conditions, these cells give rise to colonies of fibroblastoid cells, each colony derived from a single cell clone, a single precursor, termed a colony forming unit–fibroblast (CFU-F). These stem cells are also recognized by their differentiation potential. Because of their ability to be grown in vitro without losing their multipotency, mesenchymal stem cells have been explored in the repair or regeneration of injured mesenchymal tissues such as fracture repair, tendinous lesions, and cartilage. In the bone marrow microenvironment, skeletal stem cells are not randomly distributed. They are located mainly around the precapillary arteriole, a site identified as a perivascular niche. This intimate functional and morphologic relationship led to the observation of a widespread distribution of mesenchymal progenitors in the whole organism. Both endothelial and mesenchymal cells have been shown to be essential in the bone treatment. Although the main focus of using mononuclear cells in the regeneration and reconstruction of bone tissue is the combined action of osteoprogenitor cells and angioblasts, several groups described a likely paracrine activity by lymphocytes and monocytes present in this population. These cells secrete cytokines such as interleukin-6 and TGFb, which stimulate the proliferation and differentiation of cells from the bone and the vascular system. However, these studies are very recent and real paracrine effects still need to be clarified.