Freeze‐dry processing of three‐dimensional cell constructs for bone graft materials

Abstract

Freeze‐dry processing improves the operability and stability of cell‐based biomaterials and facilitates sterilization for clinical application. However, there is no established freeze‐drying protocol for engineered tissues. Recently, we reported that biomimetic bone tissues can be fabricated using scaffold‐free three‐dimensional (3D) cell constructs with potential applications as bone graft materials. The purpose of this study was to assess the influence of freeze drying on the morphology and components of 3D cell constructs. Cell constructs freeze dried in phosphate buffered saline (PBS) maintained organic and inorganic components; whereas sodium citrate buffer (SCB)‐treated constructs had significantly lower amounts of calcium and bone‐related proteins. Alkaline phosphatase (ALP) activity in cell constructs was maintained by freeze drying in 10% sucrose‐containing PBS, whereas cell constructs treated with PBS without sucrose or with sucrose‐containing SCB showed significant reductions of ALP activity. In this study, we found that sucrose‐containing phosphate buffer was suitable for freeze drying to maintain minerals and protein functions within 3D cell constructs, whereas citrate buffer was inappropriate. The insights gained by this study may facilitate the development of novel cell‐based biomaterials fabricated by tissue engineering approaches and bone graft biomaterials.

Three Potential Elements of Developing Nerve Guidance Conduit for Peripheral Nerve Regeneration

“…[ 237 ] Sucrose is a protein protectant to stabilize the protein conformation widely used in freeze/dry processing. The suppression of protein denaturation [ 238 ] by a high concentration of sucrose makes it possible to allow the bioactive molecule loading in vivo to engage in the regenerative process. Furthermore, superfluous sucrose involves glycometabolism for synthesizing myelin lipids; [ 239 ] thus, it is unnecessary to worry about spillover sucrose affecting NGC in vivo.…”

“…However, some drawbacks of freeze‐dry processing should be of concern as well. The freeze‐dried cell body produces irregular pores [ 211 ] and inevitably changes the volume, which may influence the inorganic or organic constituent for regeneration [ 238 ] when processed with cells or growth factors.…”

Large three-dimensional cell constructs for tissue engineering

“…To establish the freeze-dry protocol for 3D cell constructs as a biomimetic bone tissue, the BMSC constructs were pretreated with or without sucrose [ 83 ]. The experiments showed that the size of the BMSC construct decreased and the morphology was maintained ( Figure 8 ).…”

“…In contrast, 10 wt% sucrose in PBS maintained the ALP activity in the constructs. These results indicated that the freeze-dry processing of 3D cell constructs with suitable protein protectants enabled the fabrication of a novel bone substitute comprising only cells [ 83 ].

Figure 8.

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“…Scale bar: 500 μm. Reproduced with permission from Sasaki et al [ 83 ]. Copyright 2019, Wiley Periodicals, Inc…”