Generation of bone grafts using cryopreserved mesenchymal stromal cells and macroporous collagen‐nanohydroxyapatite cryogels

Abstract

Bone tissue engineering strategy involves the 3D scaffolds and appropriate cell types promoting the replacement of the damaged area. In this work, we aimed to develop a fast and reliable clinically relevant protocol for engineering viable bone grafts, using cryopreserved adipose tissue‐derived mesenchymal stromal cells (MSCs) and composite 3D collagen‐nano‐hydroxyapatite (nanoHA) scaffolds. Xeno‐ and DMSO‐free cryopreserved MSCs were perfusion‐seeded into the biomimetic collagen/nanoHA scaffolds manufactured by cryotropic gelation and their osteoregenerative potential was assessed in vitro and in vivo. Cryopreserved MSCs retained the ability to homogenously repopulate the whole volume of the scaffolds during 7 days of post‐thaw culture. Moreover, the scaffold provided a suitable microenvironment for induced osteogenic differentiation of cells, confirmed by alkaline phosphatase activity and mineralization. Implantation of collagen‐nanoHA cryogels with cryopreserved MSCs accelerated woven bone tissue formation, maturation of bone trabeculae, and vascularization of femur defects in immunosuppressed rats compared to cell‐free collagen‐nanoHA scaffolds. The established combination of xeno‐free cell culture and cryopreservation techniques together with an appropriate scaffold design and cell repopulation approach accelerated the generation of viable bone grafts.

Nature-Based Biomaterials and Their Application in Biomedicine

“…Natural ECM can act as a porous 3D microenvironment "scaffold", with its multitude of growth factors, effector molecules, enzymes and cellular adhesion motifs influencing cell proliferation, gene expression and intracellular signalling [19][20][21]. Due to the complexity and variability of ECM, however, scaffolds used in tissue engineering are typically more tailored to particular functions, such as promoting cell adhesion or differentiation [22,23]. Regardless of the clinical objective or environment, all biomaterial scaffolds share the following essential properties.…”

Modulation of mesenchymal stromal cells properties by the microenvironment in 3D culture

“…Samples were poured in the plastic Petri dishes (35 mm in diameter; the layer thickness was 2 mm) and frozen at -15 °C for 3 h. The obtained disks were stored in the 70 % aqueous ethanol prior to use. 5 mm diameter disks of blood plasma-based macroporous scaffolds were thoroughly washed 5 times with Hanks solution and then twice with a culture medium and seeded with cells as we described early [9].…”

“…Earlier, we demonstrated that encapsulation of MSCs in alginate microspheres led to a cessation of division while retaining the cell ability to differentiation into osteogenic, adipogenic, and chondrogenic lineages [7,8]. In another alginatebased 3D system, namely macroporous sponge-like carriers (scaffolds), MSCs retained their proliferation ability and similar to microspheres were capable to multilineage differentiation in the presence of inductors [9]. In the first system, cells are surrounded by a homogeneous hydrogel, while in the second system, they are situated at the interface between phasesthe solid surface of the pores and the liquid culture medium.…”

Cryogel Scaffolds for Tissue-Engineering: Advances and Challenges for Effective Bone and Cartilage Regeneration

“…As an alternative, the same polymers can be chemically cross-linked using DVS in alkaline ambient. This cross-linker has been used to form stable polysaccharidic networks [46], as the hydroxyl groups of polysaccharides can react with the two electronic-deficient double bonds of DVS, leading to polymer cross-linking.…”

“…These reagents are used to couple carboxylic groups of one polymer with primary amino groups or Other cross-linking protocols, largely used for the fabrication of cryogel scaffolds suitable for bone or cartilage regeneration, are based on coupling reactions with bifunctional agents, such as glutaraldehyde (GA) and divinyl sulfone (DVS) [15,44,45], reported, respectively, in scheme 2 and 3 of Figure 5. A GA coupling reaction can been used for the cross-linking of polymers bearing nucleophiles, such as primary amino groups of gelatin [15], collagen [46], and chitosan [19]. As an alternative, the same polymers can be chemically cross-linked using DVS in alkaline ambient.…”