Background:
Mature osteocytes embedded in the three-dimensional lacuna-canalicular network (3D-LCN) structure are known to sense local compressive strain and initiate strain-dependent new bone formation by osteoblasts, utilizing cytokines such as sclerostin and Dkk1 expressed by the osteocytes as major signaling molecules. Studying these osteocytes wholly is a challenge, as deeply embedded osteocytes in bone tissue are difficult to access. As of now, no laboratory model is capable of reproducing physiological phenotype and sensing and responding to mechanical stimuli functions of osteocytes for routine use in biomedical research and preclinical drug evaluation. The reasons for these problems are: (1) The observable characteristics derived from observable osteocytes from their interaction towards their environment cannot be maintained in current 2-D culture; (2) Commonly used osteocyte-like cell lines such as MLO-Y4 are sufficiently altered so that they do not express sclerostin at detectable levels; and (3)In lab-based differentiation of osteoblasts into 3D mature osteocytes with network formation and sclerostin expression has not been realized. A novel in lab-based bone tissue model with a reconstructed 3D osteocyte network could be extremely useful for studying fundamental biological mechanisms associated with osteocytes. The model can be preliminarily validated by reconstructing the bone-like tissue with a 3D mouse osteocyte network and comparing it to the in a vivo mouse data. This can ghelp in developing primary human osteocytes. Operated at a microfluidic scale, such a human 3D bone tissue model may possibly replace animal testing in preclinical evaluation of authentic human tissue response to drugs.
Summary:
The present invention comprises a method of culturing osteocytes in a microfluidic chamber. A suspension of pre-osteocytes is mixed with microbeads, and the mixture is deposited in a microfluidic chamber to form a bed of closely packed microbeads with pre-osteocytes distributed among the microbeads. The bed is then perfused with a culture medium such that the pre-osteocytes differentiate and develop into osteocytes. The cultured osteocytes along with the close-packed microbeads form a bone-like tissue. These microbeads have diameters such that interstitial spaces between adjacent microbeads may be occupied by no more than one of the cells. The embedded cells form 3D network emulating the LCN structure in native bone tissues. A drug or other biologically active substance is included in the culture medium, and the effect of the drug or other biologically-active on the development of the bone-like tissue is assessed. The microbeads include biphasic calcium phosphate. This invention comprises a microfluidic device having a chamber defining an opening and having an inlet spaced away from the opening, a semipermeable barrier closing said opening, an impermeable substrate across the semipermeable barrier, and an outlet for collecting liquid passing through the semipermeable barrier and conveying the liquid away from the microfluidic device. The microfluidic device includes a liquid-permeable bed of microbeads and cells within the chamber.
Full Patent: Microfluidic three-dimensional osteocyte network reconstructed with microbeads as scaffold
Image Source: https://www.nature.com/articles/boneres201526/figures/1