This work describes the preparation and characterization of biomimetic chitosan/multiwall carbon nanotubes/nano-hydroxyapatite (CTS/MWCNT/nHAp) scaffolds and their viability for bone tissue engineering applications. The cryogenic process ice segregation-induced self-assembly (ISISA) was used to fabricate 3D biomimetic CTS scaffolds. Proper combination of cryogenics, freeze-drying, nature and molecular ratio of solutes give rise to 3D porous interconnected scaffolds with clusters of nHAp distributed along the scaffold surface. The effect of doping in CNT (e.g. with oxygen and nitrogen atoms) on cell viability was tested. Under the same processing conditions, pore size was in the range of 20-150 μm and irrespective on the type of CNT. Studies on cell viability with scaffolds were carried out using human cells from periosteum biopsy. Prior to cell seeding, the immunophenotype of mesenchymal periosteum or periosteum-derived stem cells (MSCs-PCs) was characterized by flow cytometric analysis using fluorescence-activated and characteristic cell surface markers for MSCs-PCs. The characterized MSCs-PCs maintained their periosteal potential in cell cultures until the 2nd passage from primary cell culture. Thus, the biomimetic CTS/MWCNT/nHAp scaffolds demonstrated good biocompatibility and cell viability in all cases such that it can be considered as promising biomaterials for bone tissue engineering.

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Several ocular diseases affect the corneal surface; the development of effective technologies for the treatment of corneal lesions has brought about an improvement in the quality of life of affected patients. The aim of this study is to culture and characterize limbal stem cells cultured on gamma ((60)Co) radiosterilized human amnion (RHA). Limbal stem cells were isolated from ten preserved samples of corneal transplant. The cells were cultured since primary culture until expanded cells on RHA and stained with monoclonal antibodies to establish their immunophenotype, after which cytokeratin 12 and Vimentin were positive by immunohistochemistry. The immunophenotype remained constant since primary culture until expanded cells in RHA. The RHA and cells construct were structurally integrated. Immunohistochemistry was cytokeratin 12, Vimentin positive, and cytokeratin 19 negative. In vitro limbal cells maintain a constant epithelial transition immunophenotype in culture up to primary culture until expanded cells on RHA.

SUMMARY:The aim of our research was to create an osteogenic unit in the skulls of athymic mice; however, the first challenge we faced was to find sufficient and adequate data that would allow us to determine the morphological, immunohistochemical and microtopographical characteristics that could be used as normality standards in athymic mice skulls and, hence, a reference in the event of achieving the formation of de novo bone using the osteogenic unit we proposed. Knowing the normal bone morphology in the skull of athymic mice was a necessary precondition to develop subsequently an osteogenic unit possessing the Osteogenesis, Osteoinduction and Osteoconductivity that could be compared versus those in the normal bone during its formations and remodeling processes. Therefore, we conducted a pilot study to determine bone morphological characteristics in the skull of athymic mice by means of specific histological staining: hematoxylin-eosin and Von Kossa, for osteoid tissue and mineralized bone, and Masson Tri-chrome for ossified areas. We also use immunohistochemistry to detect bone formation markers: alkaline phosphatase resulting from osteoblastic activity stimulation, type 1 collagen a bonematrix structural protein; Osteopontine, a protein specifically synthesized by osteoblasts that favors cell proliferation and remodeling in bone defects; Osteocalcine, a peptide hormone produced by osteoblasts during bone formation; and, Runx 2, a transcription factor expressed by stem cells which stimulates bone differentiation. Likewise, we used electron microscopy on the newly formed tissue to determine the presence of organic deposits, such as calcium, phosphate and magnesium in bone tissue.