Bioactivity Evaluation of Biphasic Hydroxyapatite Bone Substitutes Immersed and Grown with Supersaturated Calcium Phosphate Solution.

Recently, the frequency of use of bone substitute materials for the purpose of bone augmentation has increased in implant treatment, but bone formation with bone substitute materials alone is limited. Calcification of bone in the body progresses as Ca2+, H2PO4-, and HPO42- in the body form hydroxyapatite (HA) crystals. In this study, therefore, we prepared a biphasic bone substitute with biological activity to promote bone formation by inducing precipitation and growth of HA crystals on the surface of a bone substitute and evaluated it. Biphasic bone substitute granules were prepared by immersing HA granules in a supersaturated calcium phosphate solution prepared by mixing five medical infusion solutions, the precipitate was analyzed, and the biological activities of biphasic HA granules were evaluated in vitro and in vivo. As a result, the precipitated calcium phosphate crystals were identified as low crystalline HA. On the surface of the HA granules, low-crystalline HA grew markedly as needle-shaped crystals and significantly promoted cell proliferation and bone differentiation. In animal experiments, biphasic HA granules had a significantly higher bone mineral density, new bone volume ratio, and new bone area ratio. Therefore, it suggests that biphasic hydroxyapatite is a useful bone substitute for bone augmentation in dental implant treatment.

Production of progenitor cells from primary human epithelial cell monolayer cultures.

Primary keratinocytes derived from human epidermis are widely used in tissue engineering and regenerative medicine. An important aspect in clinical applications is the preservation of human skin keratinocyte stem cells. However, it is difficult to expand the number of human skin keratinocyte stem cells, which are undifferentiated and highly proliferative in culture by using standard cell culture methods. It is even more difficult to identify them, since universal specific markers for human skin keratinocyte stem cells have not been identified. In this paper, we show a method to produce a large number of primary progenitor human skin keratinocytes by using our novel culture techniques. Primary human skin keratinocyte monolayers are cultured using twice the volume of medium without serum and lacking essential fatty acids. Once the cells reach 70-80% confluence, they begin to float up into the overlying medium and are called "epithelial pop-up keratinocytes (ePUKs)" allowing the cells to be passaged without the use of trypsin. We analyzed the properties of ePUKs by cell size, cell viability, immunocytofluorescence biomarker staining, and cell cycle phase distribution by fluorescence-activated cell sorting (FACS). Our results showed that these ePUKs appear to be progenitor epithelial cells, which are small in size, undifferentiated, and have a high proliferative capacity. We believe that ePUKs are suitable for use in medical applications requiring a large number of primary human progenitor skin keratinocytes.

Utilisation of a Bioreactor for Culture and Expansion of Epithelial Cells without the use of Trypsin or Enzymes.

OBJECTIVE: To develop a bioreactor for automated culture, maintenance, and collection of normal human keratinocytes using an enzyme-free propagation method. METHODS: The culture of normal human epithelial keratinocytes was compared in two culture methods - a study team-developed automated bioreactor utilising an enzyme-free passage method, and a manual culture method. Cell size, glucose utilisation, and the proliferative capacity of the two cultures were evaluated. RESULTS: An automated bioreactor, not using enzymes for passage, but instead using the novel Epithelial Pop Up Keratinocytes (ePUK)1 culture technique, resulted in an extended culture longevity and proliferative capacity in normal primary human keratinocytes. Daughter cells were collected up to three times per day utilising the bioreactor. The daughter cells produced by the bioreactor were smaller than daughter cells produced by the manual culture method. The proliferative capacity and health of the parent monolayer within both the bioreactor and the manual culture flask was dependent upon sufficient glucose availability. Due to the contact inhibition nature of epithelial keratinocytes, the bioreactor enabled the study of an adherent cell type soon after cytokinesis and before the cell has integrated as part of an adherent matrix. CONCLUSION: The study demonstrates that increasing the number of media changes per day as necessary, based on glucose utilisation, is necessary for prolonged longevity and functional productivity of ePUK cultures.

Comparison of two decellularized dermal equivalents.

Immunologically inert allogeneic acellular dermal scaffolds provide a matrix with molecular architecture close to native tissues, which synthetic scaffolds cannot. Not all nature-derived scaffolds possess the same biological and physical properties. The different properties of scaffolds supporting cellular growth used for manufacturing tissue engineered grafts could lead to different implantation results. The scaffold properties should be carefully considered in order to meet the expected outcomes of tissue engineered grafts. In this report, we evaluated the cellular growth on AlloDerm® and Allopatch, 2 acellular scaffolds derived from human cadaver skin, using a fabricated 3D organotypic culture with primary human oral keratinocytes to produce an ex vivo produced oral mucosa equivalent (EVPOME). A well stratified epithelium could be constructed on both scaffolds. AlloDerm® and Allopatch EVPOMEs were also implanted into severe combined immunodeficiency mice to compare the ingrowth of blood vessels into the dermal component of the two EVPOMEs. Blood vessel counts were 3.3 times higher (p = .01) within Allopatch EVPOMEs than within AlloDerm® EVPOMEs. An oral and skin keratinocyte co-culture, separated by a physical barrier to create a cell-free zone, was used to evaluate cell migration on AlloDerm® and Allopatch. Slower cell migration was observed on Allopatch than on AlloDerm®.

Controlled release of simvastatin from biodegradable hydrogels promotes odontoblastic differentiation.

The objective of this study was to investigate the odontoblastic differentiation of dental pulp stem cells (DPSC) by biodegradable hydrogels incorporating simvastatin micelles, both in vitro and in vivo. Simvastatin (ST) was incorporated into the micelles of gelatin grafted with L-lactic acid oligomers (LAo) to allow water-solubilization. The simvastatin-LAo-grafted gelatin (LAo-g-gelatin) micelles were mixed with gelatin, followed by chemical crosslinking to form gelatin hydrogels (ST Mi/GH). The ST Mi were released from the gelatin hydrogel granules (GH) through enzymatic degradation. The ST Mi enhanced alkaline phosphatase activity, calcium deposition, and bone morphogenic protein-2 secretion of DPSC. When implanted subcutaneously into mice, the ST Mi/GH treated group exhibited increased dentin sialoprotein and calcium deposition, compared with those treated with GH plus free ST. It is possible to achieve odontoblastic differentiation of DPSC through the controlled release of ST from GH.

Electrodeposition of pronectin for titanium to augment gingival epithelium adhesion.

This paper is one trial of surface modification of titanium with pronectin F+ (PN) of an artificial protein to enhance gingival adhesion. Titanium plates were electrodeposited in the PN solution to prepare PN-electrodeposited titanium plates. When PN detachment from the PN-electrodeposited titanium plates was investigated, no detachment was observed, in contrast to the case of titanium plates simply coated with PN. A cell culture experiment demonstrated that electrodeposited PN had an inherent ability to enhance the initial attachment of gingival epithelial cells. The PN-electrodeposited titanium plates were implanted between the gingival epithelium and the underlying bone tissue of rabbits to evaluate epithelial growth on the plates and their gingival adhesion. Non-treated and PN-coated titanium plates were used as controls. PN electrodeposition enhanced epithelial growth and adhesion of titanium plates to a significantly great extent compared with PN-coated plates. These findings demonstrate that PN electrodeposition is a promising method to enhance epithelium adhesion onto a titanium surface.