Yantian Chen

Characterization and Optimization of Cell Seeding in Scaffolds by Factorial Design: Quality by Design Approach for Skeletal Tissue Engineering

Cell seeding into scaffolds plays a crucial role in the development of efficient bone tissue engineering constructs. Hence, it becomes imperative to identify the key factors that quantitatively predict reproducible and efficient seeding protocols. In this study, the optimization of a cell seeding process was investigated using design of experiments (DOE) statistical methods. Five seeding factors (cell type, scaffold type, seeding volume, seeding density, and seeding time) were selected and investigated by means of two response parameters, critically related to the cell seeding process: cell seeding efficiency (CSE) and cell-specific viability (CSV). In addition, cell spatial distribution (CSD) was analyzed by Live/Dead staining assays. Analysis identified a number of statistically significant main factor effects and interactions. Among the five seeding factors, only seeding volume and seeding time significantly affected CSE and CSV. Also, cell and scaffold type were involved in the interactions with other seeding factors. Within the investigated ranges, optimal conditions in terms of CSV and CSD were obtained when seeding cells in a regular scaffold with an excess of medium. The results of this case study contribute to a better understanding and definition of optimal process parameters for cell seeding. A DOE strategy can identify and optimize critical process variables to reduce the variability and assists in determining which variables should be carefully controlled during good manufacturing practice production to enable a clinically relevant implant.

Enhancement of osteogenic gene expression for the differentiation of human periosteal derived cells

The osteogenic differentiation of progenitor populations allows analysis of cell functionality as well as creating a platform for investigating stem cells for bone tissue engineering. Protocols used for osteogenic differentiation of progenitor cells are often identical to those detailed for bone marrow mesenchymal stem cells, however this may be flawed due to cell populations residing in different niches and being in distinct stages of differentiation. We herein describe the individual and combined effects of known osteo-inductive agents; dexamethasone (Dex), 1,25-dihydroxyvitamin D3 (VitD3), all trans-retinoic acid (atRA), cyclic AMP (cAMP) and bone morphogenic protein 2 (BMP2) in combination with fetal bovine serum (FBS) on osteogenesis of human periosteal derived cells (hPDCs). The addition of Dex&FBS was essential for the transition of hPDCs to an ALP positive cell population. Subsequently, atRA, Dex&FBS and BMP2 were required for the expression of transcription factors governing osteogenesis and hence differentiation towards a mature osteoblast. It is also hypothesized that Dex has no direct effect on the differentiation of hPDCs, instead its effect is to augment differentiation in combination with other factors. These data provide a comprehensive assessment of known osteogenic factors, in a novel multiplex system, to evaluate their effect on progenitor cell differentiation.