Functional Scaffold-Free Bone Equivalents Induce Osteogenic and Angiogenic Processes in a Human In Vitro Fracture Hematoma Model

Abstract

After trauma, the formed fracture hematoma within the fracture gap contains all the important components (immune/stem cells, mediators) to initiate bone regeneration immediately. Thus, it is of great importance but also the most susceptible to negative influences. To study the interaction between bone and immune cells within the fracture gap, up-to-date in vitro systems should be capable of recapitulating cellular and humoral interactions and the physicochemical microenvironment (eg, hypoxia). Here, we first developed and characterized scaffold-free bone-like constructs (SFBCs), which were produced from bone marrow-derived mesenchymal stromal cells (MSCs) using a macroscale mesenchymal condensation approach. SFBCs revealed permeating mineralization characterized by increased bone volume (μCT, histology) and expression of osteogenic markers (RUNX2, SPP1, RANKL). Fracture hematoma (FH) models, consisting of human peripheral blood (immune cells) mixed with MSCs, were co-cultivated with SFBCs under hypoxic conditions. As a result, FH models revealed an increased expression of osteogenic (RUNX2, SPP1), angiogenic (MMP2, VEGF), HIF-related (LDHA, PGK1), and inflammatory (IL6, IL8) markers after 12 and 48 hours cocultivation. Osteogenic and angiogenic gene expression of the FH indicate the osteoinductive potential and, thus, the biological functionality of the SFBCs. IL-6, IL-8, GM-CSF, and MIP-1β were detectable within the supernatant after 24 and 48 hours of co-cultivation. To confirm the responsiveness of our model to modifying substances (eg, therapeutics), we used deferoxamine (DFO), which is well known to induce a cellular hypoxic adaptation response. Indeed, DFO particularly increased hypoxia-adaptive, osteogenic, and angiogenic processes within the FH models but had little effect on the SFBCs, indicating different response dynamics within the co-cultivation system. Therefore, based on our data, we have successfully modeled processes within the initial fracture healing phase in vitro and concluded that the cross-talk between bone and immune cells in the initial fracture healing phase is of particular importance for preclinical studies.

Enhancement of Immunosuppressive Activity of Mesenchymal Stromal Cells by Platelet-Derived Factors is Accompanied by Apoptotic Priming

“…Blood- [ 7 ] or MSC-based [ 8 ] scaffolds/hydrogels have become attractive tools for investigating physiological or delayed bone healing processes as well as developing functional bone substitutes. This is particularly important since bone substitutes often require additional biological supplementation to support bone repair.…”

Temporal dynamics of immune-stromal cell interactions in fracture healing

“…While the gold standard of preclinical studies is animal models, these models may have limited transferability due to differences in timeline, physiologic structure, pharmacologic response, and variation in specific gene pathways across species, supporting the need for in vitro models using human cells and tissues to complement animal work ( 4 , 127 , 128 ). In vitro models have been developed over the past decade to create a more physiologically-relevant system for studying human fracture.…”

Strategies for Enhancing Vascularization of Biomaterial‐Based Scaffold in Bone Regeneration

“…[169] Reports have also suggested that co-administration of deferoxamine with other angiogenic signaling cues promotes hypoxic stimulation of the vascular intermediaries as compared to the control group. [170,171] Collagen type 1 is the major protein-polymer found in ECM, contributing to the strength and stiffness which is laid down by the osteoblasts and fibroblasts. Its properties can be modified to fabricate bone scaffolds and vascularized grafts to aid neovascularization.…”

“…Simvastatin doping onto mesoporous HA microsphere‐collagen composite causes VEGF, HIF‐1α expression from stem cells with the interdependent intensification of tubule formation, cell migration illustrating pro‐angiogenesis [169] . Reports have also suggested that co‐administration of deferoxamine with other angiogenic signaling cues promotes hypoxic stimulation of the vascular intermediaries as compared to the control group [170,171] …”