Osteogenic potential of murine periosteum for critical-size cranial defects.

Tissue engineering of bone has combined bespoke scaffolds and osteoinductive factors to maintain functional osteoprogenitor cells, and the periosteum has been confirmed as a satisfactory source of osteoblasts. Suitable matrices have been identified that support cell proliferation and differentiation, including demineralised bone matrix (both compatible and osteoinductive) and acellular human dermis. We have evaluated the osteogenic potential of an osteogenic unit, developed by combining periosteum, demineralised bone matrix, and acellular human dermis, in rodents with critical-size cranial defects. Briefly, remnants from the superior maxillary periosteum were used to harvest cells, which were characterised by flow cytometry and reverse retrotranscriptase-polymerase chain reaction (RT-PCR). Cells were cultured into the osteogenic unit and assessed for viability before being implanted into 3 rodents, These were compared with the control group (n=3) after three months. Histological analyses were made after staining with haematoxylin and eosin and Von Kossa, and immunostaining, and confirmed viable cells that stained for CD90, CD73, CD166, runt-related transcription factor, osteopontin, and collagen type I in the experimental group, while in the control group there was only connective tissue on the edges of the bone in the injury zone. We conclude that osteogenic unit constructs have the osteogenic and regenerative potential for use in engineering bone tissue.

Trypsin as enhancement in cyclical tracheal decellularization: Morphological and biophysical characterization.

There are different types of tracheal disorders (e.g. cancer, stenosis and fractures). These can cause respiratory failure and lead to death of patients. Several attempts have been made for trachea replacement in order to restore the airway, including anastomosis and implants made from synthetic or natural materials. Tracheal allotransplantation has shown high rejection rates, and decellularization has emerged as a possible solution. Decellularization involves the removal of antigens from cells in the organ or tissue, leaving a matrix that can be used as 3D cell-scaffold. Although this process has been used for tracheal replacement, it usually takes at least two months and time is critical for patients with tracheal disorders. Therefore, there is necessary to develop a tracheal replacement process, which is not only effective, but also quick to prepare. The aim of this research was to develop a faster trachea decellularization protocol using Trypsin enzyme and Ethylenediaminetetraacetic acid (EDTA) as decellularization agents. Three protocols of cyclic trachea decellularization (Protocols A, B, and C) were compared. Following Protocol A (previously described in the literature), 15 consecutive cycles were performed over 32 days. Protocol B (a variation of Protocol A) ­ EDTA being added ­ with 15 consecutive cycles performed over 60 days. Finally, Protocol C, with the addition of Trypsin as a decellularization agent, 5 consecutive cycles being performed over 10 days. For the three protocols, hematoxylin­eosin (H&E) staining and DNA residual content quantification were performed to establish the effectiveness of the decellularization process. Scanning Electron Microscopy (SEM) was used to observe the changes in porosity and microarrays. To evaluate the structural matrices integrity, Thermogravimetric Analysis (TGA) and biomechanical test were used. None of the protocols showed significant alteration or degradation in the components of the extracellular matrix (ECM). However, in Protocol C, more cellular components were removed in less time, making it the most efficient process. In addition, the cell tracking and viability was evaluated with chondrocytes seeding on the scaffold obtained by Protocol C, which showed an adequate cell scaffolding ability of this matrix.

Association of Metastasis with Clinicopathological Data in Mexican Patients with Osteosarcoma, Giant Cell Tumor of Bone and Chondrosarcoma.

BACKGROUND: Bone tumors are neoplasias with a high overall mortality; one of the main factors that reduce survival is their high capacity to develop metastases. It has been reported that finding lung metastases at diagnosis of osteosarcoma (OS), chondrosarcoma (CS) and giant cell tumor of bone (GCTb) is quite common. In this study, we inquire the relationship of metastases caused by these tumors with different clinical and pathological aspects, in order to guide medical personnel in the diagnosis and opportune treatment of metastases or micro metastases. MATERIALS AND METHODS: We collected data of 384 patients with clinical, radiological and histopathological diagnosis of OS, GCTb and CS that attended the National Rehabilitation Institute (INR) during 2006 to 2014. Chi-square and Fisher's exact tests were performed for data analysis. RESULTS: In the three tumor types, the presence of metastases at diagnosis was variable (p=0.0001). Frequency of metastases was 36.7%, 31.7% and 13.2% for OS, CS and GCTb respectively. The average age had no significant difference (p>0.05) in relation to metastases, even so, patients with OS and GCTb and metastases, were older while patients with CS and metastases were younger, in comparison to patients without metastases. Males had a higher frequency of metastases (68.2%, p = 0.09) in contrast to CS and GCTb, in which the metastases was more frequent in women with 51.9% (p = 0.44) and 57.9% (p = 0.56) respectively. Broadly, metastasis was associated with primary tumors located in the femur (44.4%), followed by the tibia (15.6%); metastases was more frequent when primary tumor of GCTb and OS were in the same bones, but were located in the hip (26.3%) for CS. CONCLUSIONS: The frequency of metastases in OS, GCTb and CS is high in our population and is determined by different clinicopathological variables related to the kind of tumor. Further studies are needed in order to evaluate metastases subsequent to diagnosis and associations with survival and clinicopathological factors , as well as to determine the sensitivity and specificity of current methods of detection.

Epidemiological Aspects of Osteosarcoma, Giant Cell Tumor and Chondrosarcoma Musculoskeletal Tumors--Experience of the National Rehabilitation Institute, Mexico City.

BACKGROUND: Primary bone neoplasms are rare, contributing only 0.2% of the global burden of all human malignancies. Osteosarcoma (OS) and chondrosarcoma (CS) are the most common malignancies of bone. The giant cell tumor of bone (GCTb) is a benign tumor with behavior characterized by osteolytic bone destruction. The OS, CS and GCTb affect both sexes, all races and generally have incidence peaks regarding the age of the patient which vary according to the tumor type. We analyzed the incidences of OS, CS and GCTb and their relations with gender and age in patients treated in the National Rehabilitation Institute (INR, for its acronym in Spanish) over a period of nine years. MATERIALS AND METHODS: In the study period, clinic pathological data for 384 patients were obtained with clinical, radiological and histopathological diagnosis for OS, GCTb and CS. Data analysis was performed using the chi-square and Fisher's exact tests. RESULTS: From 2006 to 2014 were recorded 384 cases of bone malignancies in the database of INR. The GCTb had the highest incidence (53.1%), followed by OS (31.3%) and finally the CS (15.6%). The overall average age was 33.6±15.8 years and the overall frequency of gender had a ratio of 1/1.03 male/female. The states with the highest incidence were Distrito Federal and Estado de Mexico with 29.2% and 25.3% respectively. Malignant neoplasms of bone assessed in the course of nine years show three significant increases in 2008, 2011 and 2014 (p=0.14). We found association between sex and tumor type (p=0.03), GCTb and CS predominated in females (54.9% and 56.6% respectively), while for the OS males were most affected (59.1%). Age was different in relation with tumor type (p=0.0001), average age was 24.3±11.2 years for OS, 34.5±13 years for GCTb and 49.2±18.5 years for CS. Furthermore, associations of tumor type with topographic location of the primary tumor (P=0.0001) were found. CONCLUSIONS: In this study we can see that incidence of musculoskeletal tumor in our population is continuously increasing and in nine years an approximately 200% increase of musculoskeletal tumor cases was observed.

Chondrocyte differentiation for auricular cartilage reconstruction using a chitosan based hydrogel.

Tissue engineering with the use of biodegradable and biocompatible scaffolds is an interesting option for ear repair. Chitosan-Polyvinyl alcohol-Epichlorohydrine hydrogel (CS-PVA-ECH) is biocompatible and displays appropriate mechanical properties to be used as a scaffold. The present work, studies the potential of CS-PVA-ECH scaffolds seeded with chondrocytes to develop elastic cartilage engineered-neotissues. Chondrocytes isolated from rabbit and swine elastic cartilage were independently cultured onto CS-PVA-ECH scaffolds for 20 days to form the appropriate constructs. Then, in vitro cell viability and morphology were evaluated by calcein AM and EthD-1 assays and Scanning Electron Microscopy (SEM) respectively, and the constructs were implanted in nu/nu mice for four months, in order to evaluate the neotissue formation. Histological analysis of the formed neotissues was performed by Safranin O, Toluidine blue (GAG's), Verhoeff-Van Gieson (elastic fibers), Masson's trichrome (collagen) and Von Kossa (Calcium salts) stains and SEM. Results indicate appropriate cell viability, seeded with rabbit or swine chondrocyte constructs; nevertheless, upon implantation the constructs developed neotissues with different characteristics depending on the animal species from which the seeded chondrocytes came from. Neotissues developed from swine chondrocytes were similar to auricular cartilage, while neotissues from rabbit chondrocytes were similar to hyaline cartilage and eventually they differentiate to bone. This result suggests that neotissue characteristics may be influenced by the animal species source of the chondrocytes isolated.