title: Manual cryopreservation of human alveolar periosteal tissue segments: Effects of pre-culture on recovery rate.
abstract: Cultured human periosteal sheets constitute a promising grafting material for periodontal tissue regenerative therapy. However, preparation of these sheets usually requires six weeks or longer, and this lengthy commitment and delay limits both clinical applicability and availability. The aim of this study is to develop an efficient, practical, cost-effective cryopreservation method for periosteal tissue segments (PTSs). Human PTSs were aseptically excised from alveolar bone and pre-cultured in Medium 199+10% fetal bovine serum (FBS) for the indicated number of days before they were slowly frozen down to -75°C in a commercial freezing vessel using medium containing 10% dimethyl sulfoxide (Me(2)SO) and various concentrations of FBS. After fast-thawing at 37°C, PTSs were again cultured, and their growth and responses to standard osteogenic induction were evaluated (vs. freshly excised PTSs). Proliferating cells were obtained at the highest levels from cryopreserved PTSs that were pre-cultured for 14 days before freezing. When a concentration of 50% or more FBS was included in the cryopreservation solution, cells migrated out more actively and grew faster. Importantly, osteoinduction up-regulated alkaline phosphatase (ALP) activity and osteoblastic marker mRNAs in cryopreserved PTS-derived sheets just as effectively as it did in native PTS-derived ones. These data suggest that pre-conditioned PTSs can be efficiently cryopreserved in a freezing solution containing high FBS by traditional manual cryopreservation methods without aid of a program freezer or more elaborate equipment.
authors.affiliation: Division of Oral Bioengineering, Department of Tissue Regeneration and Reconstitution, Institute of Medicine and Dentistry, Niigata University, Niigata 951-8514, Japan. kawase@dent.niigata-u.ac.jp
authors.forename: Tomoyuki
authors.initials: T
authors.lastname: Kawase
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Kogami
authors.forename: Masaki
authors.initials: M
authors.lastname: Nagata
authors.forename: Kohya
authors.initials: K
authors.lastname: Uematsu
authors.forename: Kazuhiro
authors.initials: K
authors.lastname: Okuda
authors.forename: Douglas M
authors.initials: DM
authors.lastname: Burns
authors.forename: Hiromasa
authors.initials: H
authors.lastname: Yoshie
mesh_terms: D000328:Adult; D000469:Alkaline Phosphatase / Q000378:metabolism; D000539:Alveolar Process / Q000166:cytology*; D049109:Cell Proliferation; D002478:Cells, Cultured; D024042:Collagen Type I / Q000378:metabolism; D050659:Core Binding Factor Alpha 1 Subunit / Q000378:metabolism; D015925:Cryopreservation / Q000295:instrumentation / Q000379:methods*; D003451:Cryoprotective Agents / Q000494:pharmacology; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D005260:Female; D006801:Humans; D008297:Male; D010006:Osteoblasts / Q000378:metabolism; D010521:Periosteum / Q000166:cytology* / Q000378:metabolism*; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism; D044967:Serum; D013696:Temperature
chemical_list: D024042:Collagen Type I; D050659:Core Binding Factor Alpha 1 Subunit; D003451:Cryoprotective Agents; D018809:Proliferating Cell Nuclear Antigen; C493709:RUNX2 protein, human; D000469:Alkaline Phosphatase; D004121:Dimethyl Sulfoxide