Bioinspired Silk Fibroin Mineralization for Advanced In Vitro Bone Remodeling Models

Abstract

Human in vitro bone models can create the possibility for investigation of physiological bone remodeling while addressing the principle of replacement, reduction and refinement of animal experiments (3R). Current in vitro models lack cell-matrix interactions and their spatiotemporal complexity. To facilitate these analyses, a bone-mimetic template is developed in this study, inspired by bone's extracellular matrix composition and organization. Silk fibroin (SF) is used as an organic matrix, poly-aspartic acid (pAsp) is used to mimic the functionality of noncollagenous proteins, and 10× simulated body fluid serves as mineralization solution. By using pAsp in the mineralization solution, minerals are guided toward the SF material resulting in mineralization inside and as a coating on top of the SF. After cytocompatibility testing, remodeling experiments are performed in which mineralized scaffold remodeling by osteoclasts and osteoblasts is tracked with nondestructive microcomputed tomography and medium analyses over a period of 42 d. The mineralized scaffolds support osteoclastic resorption and osteoblastic mineralization, in the physiological bone remodeling specific sequence. This model could therefore facilitate the investigation of cell-matrix interactions and may thus reduce animal experiments and advance in vitro drug testing for bone remodeling pathologies like osteoporosis, where cell-matrix interactions need to be targeted.

A 3D cell-free bone model shows collagen mineralization is driven and controlled by the matrix

“…[10c, 34] The absence of the pAsp specific 1789 cm −1 peak in the mineralized matrix shows – in contrast to what is often assumed [35] – that the pAsp did not enter the osteoid, but only acts as carrier for the mineral that prevents its precipitation outside collagen matrix. [36]…”

“…[10c, 34]] The absence of the pAsp specific 1789 cm -1 peak in the mineralized matrix shows -in contrast to what is often assumed [35] -that the pAsp did not enter the osteoid, but only acts as carrier for the mineral that prevents its precipitation outside collagen matrix. [36] Comparison of averaged spectra from the control bone, the unmineralized collagen and the partially mineralized collagen around the osteon (after normalization to the amide III vibration at 1255 cm -1) ) (Figure 4h right inset), revealed no changes in the matrix related to the subsequent demineralization and remineralization steps, apart from the intensity of the orientation-dependent amide I vibration (1660 cm -1 ) that decreased upon demineralization and increased again during the remineralization process. This indicates changes in the collagen orientation, and imply thereby that mineral-matrix interactions are also a determinant factor in the molecular scale organization of the organic matrix.…”

A dialysis medium refreshment cell culture set-up for an osteoblast-osteoclast coculture

“…Thus, the supplements either stayed active or stimulated the coculture enough in the beginning. The possible cells' secretome consists of high MW molecules that are assumed not to be able to pass the membrane such as ALP (86 kDa), RUNX-2 (57 kDa), osteopontin (55 kDa), TRAP (30)(31)(32)(33)(34)(35) and extracellular vesicles (large range of MW).…”

“…Small molecules such as glucose (180 Da) and lactate (90 Da) were able to pass the membrane continuously over the whole culture time. Large molecules such as TRAP (30)(31)(32)(33)(34)(35) were maintained in the cell culture compartment. Moreover, the cells remained The dialysis culture system was used to study osteogenic differentiation of MSCs and MCs into osteoblasts and osteoclasts.…”

“…MCs need a mineralized surface to attach to and to become osteoclasts. Recently, de Wildt et al reported a pre-mineralized SF scaffold that acts as a bone-mimetic template [31]. They used poly aspartic acid and simulated body fluid to premineralize silk fibroin scaffolds before cell seeding.…”