Abstract
BackgroundCryopreservation has been extensively applied to the long-term storage of a diverse range of biological materials. However, no comprehensive study is currently available on the cryopreservation of periodontal ligament stem cell (PDLSC) sheets which have been suggested as excellent transplant materials for periodontal tissue regeneration. The aim of this study is to investigate the effect of cryopreservation on the structural integrity and functional viability of PDLSC sheets.MethodsPDLSC sheets prepared from extracted human molars were divided into two groups: the cryopreservation group (cPDLSC sheets) and the freshly prepared control group (fPDLSC sheets). The cPDLSC sheets were cryopreserved in a solution consisting of 90% fetal bovine serum and 10% dimethyl sulfoxide for 3 months. Cell viability and cell proliferation rates of PDLSCs in both groups were evaluated by cell viability assay and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, respectively. The multilineage differentiation potentials of the cells were assessed by von Kossa staining and Oil Red O staining. The chromosomal stability was examined by karyotype analysis. Moreover, the cell sheets in each group were transplanted subcutaneously into the dorsal site of nude mice, after which Sirius Red staining was performed to analyze the efficiency of tissue regeneration.ResultsThe PDLSCs derived from both groups of cell sheets showed no significant difference in their viability, proliferative capacities, and multilineage differentiation potentials, as well as chromosomal stability. Furthermore, transplantation experiments based on a mouse model demonstrated that the cPDLSC sheets were equally effective in generating viable osteoid tissues in vivo as their freshly prepared counterparts. In both cases, the regenerated tissues showed similar network patterns of bone-like matrix.ConclusionsOur results offer convincing evidence that cryopreservation does not alter the biological properties of PDLSC sheets and could enhance their clinical utility in tissue regeneration.
Highlights
Cryopreservation has been extensively applied to the long-term storage of a diverse range of biological materials
The Extracellular matrix (ECM) in Cryopreservation periodontal ligament stem cell (cPDLSC) sheets was not disrupted by freezing or thawing We began our study by investigating whether the cryopreservation method that we used had any detrimental effects on the vital structural organization of periodontal ligament stem cell (PDLSC) sheets. Both cPDLSC and Freshly prepared periodontal ligament stem cell (fPDLSC) sheets, which were induced only by Alpha-modified Eagle’s medium (α-MEM) supplemented with 15% fetal bovine serum (FBS) and 20 μg/mL vitamin C (Vc), retained their “sheet-like” structure (Fig. 1a and f ) with the ECM remaining largely intact (Fig. 1c–e and h–j)
The results showed that all three types of matrix proteins were abundantly distributed around the PDLSCs in both the cryopreserved sheets (Fig. 1c–e) and in their freshly prepared counterparts (Fig. 1h–j), implying that the ECM structure remained intact and undisrupted despite cryopreservation
Summary
Cryopreservation has been extensively applied to the long-term storage of a diverse range of biological materials. No comprehensive study is currently available on the cryopreservation of periodontal ligament stem cell (PDLSC) sheets which have been suggested as excellent transplant materials for periodontal tissue regeneration. The aim of this study is to investigate the effect of cryopreservation on the structural integrity and functional viability of PDLSC sheets. We reported the development of an effective and reliable method based on vitamin C (Vc) treatment that facilitated the construction of highly viable and functional periodontal ligament stem cell (PDLSC) sheets and the subsequent regeneration of periodontal tissues [9]. Clinical application of PDLSC sheets was limited by the fact that their laboratory preparation was very time consuming. Using conventional methods, it would require at least 10 days to grow the cell sheets in our laboratory which precluded any therapeutic usage in the event of medical emergency
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