Combinatorial Cassettes for Higher-Throughput Screening of Osteogenesis in Mice

Summary

Ectopic bone formation in mice is the gold standard for evaluation of osteogenic constructs. By regular procedures, usually only 4 constructs can be accommodated per mouse, limiting screening power. Combinatorial cassettes (combi-cassettes) hold up to 19 small, uniform constructs from the time of surgery, through time in vivo, and subsequent evaluation. Two types of bone tissue engineering constructs were tested in the combi-cassettes: i) a cell-scaffold construct containing primary human bone marrow stromal cells with hydroxyapatite/ tricalcium phosphate particles (hBMSCs + HA/TCP) and ii) a growth factor-scaffold construct containing bone morphogenetic protein 2 in a gelatin sponge (BMP2+GS). Measurements of bone formation by histology, bone formation by X-ray microcomputed tomography (μCT) and gene expression by quantitative polymerase chain reaction (qPCR) showed that constructs in combi-cassettes were similar to those created by regular procedures. Combi-cassettes afford placement of multiple replicates of multiple formulations into the same animal, which enables, for the first time, rigorous statistical assessment of: 1) the variability for a given formulation within an animal (intra-animal variability), 2) differences between different tissue-engineered formulations within the same animal and 3) the variability for a given formulation in different animals (inter-animal variability). In summary, combi-cassettes enable a more high-throughput, systematic approach to in vivo studies of tissue engineering constructs.

Description

Combinatorial cassettes (combi-cassettes) are planar, hexagonal structures, made from polytetrafluoroethylene (PTFE) and have small holes. Tissue engineering formulations are loaded into the holes, and the loaded combi-cassettes are placed subcutaneously into mice. Combi-cassettes enable 19 tissue-engineered constructs to be placed into one mouse, improving the quantitative rigor of animal tests.

The reliability of combi-cassettes for screening tissue engineering constructs was tested by using 2 types of known osteogenic formulations.

1. Cell-based formulation: primary human bone marrow stromal cells (hBMSCs) loaded onto hydroxyapatite/tricalcium phosphate particles (HA/TCP)
2. Growth factor-based formulation: bone morphogenetic protein 2 (BMP2) loaded into gelatin sponges

Paper: Bodhak S, Fernandez de Castro Diaz L, Kuznetsov SA, Maeda A, Bonfim D, Robey PG, Simon Jr CG (2018) Combinatorial cassettes to systematically evaluate tissue-engineered constructs in recipient mice. Biomaterials 186, 31-43.  https://doi.org/10.1016/j.biomaterials.2018.09.035

Slides: Combi_Cassettes.pdf

Sample: Please contact Carl Simon (carl.simon [at] nist.gov (carl[dot]simon[at]nist[dot]gov)) if you would like a few combi-cassettes to test in your system.

Contributors: Subhadip Bodhak, Pamela Robey, Luis de Castro, Sergei Kuznetsov, Carl Simon