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Combinatorial Cassettes for Higher-Throughput Screening of Osteogenesis in Mice


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.

combi-cassette schematic
Left: Photo of a combi-cassette that is loaded with bone tissue engineering constructs. Middle: Schematic of a 19-hole combi-cassette. Right: Dimensions of a hole in a 19-hole combi-cassette.
Credit: Subhadip Bodhak


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


combi-cassette histology
Cell-Based Formulation: Primary human bone marrow stromal cells (hBMSCs) loaded onto hydroxyapatite/tricalcium phosphate particles (HA/TCP) were placed into 19-hole combi-cassettes and implanted subcutaneously into the backs of mice for 8 weeks. Controls with HA/TCP only (no hBMSCs) were also tested.  Images in the same row are of the same field of view (b = bone, ft = fibrous tissue, s = HA/TCP scaffold). New bone that was autofluorescent was formed in the osteogenic constructs (hBMSCs + HA/TCP). No new bone was formed in the non-osteogenic constructs (HA/TCP alone).
Credit: Subhadip Bodhak


combi-cassette X-ray uCT
Growth Factor-Based Formulation: Constructs of bone morphogenetic protein 2 (BMP2) loaded into gelatin sponges were placed into 19-hole combi-cassettes and implanted subcutaneously into the backs of mice for 8 weeks. Non-osteogenic controls were included that had gelatin sponge only (no BMP2).  Left: Key to the placement of the formulations into the 19-hole combi-cassette.  Middle: After 8 weeks of implantation, the combi-cassettes were retrieved from the mice and imaged by X-ray microcomputed tomography.  A 3D reconstruction of one of the cassettes is shown and it is in the same orientation as the key on the left. Note that new bone formation occurs primarily in the holes loaded with osteogenic formulations. Right: A cross-section of the same combi-cassette that is shown in “middle."
Credit: Subhadip Bodhak

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.

Slides: Combi_Cassettes.pdf

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

pile of cassettes
Please contact Carl Simon for some samples for testing

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



Created November 22, 2019, Updated July 9, 2021