Healing of a Large Long-Bone Defect through Serum-Free In Vitro Priming of Human Periosteum-Derived Cells

Abstract

SummaryClinical translation of cell-based strategies for regenerative medicine demands predictable in vivo performance where the use of sera during in vitro preparation inherently limits the efficacy and reproducibility. Here, we present a bioinspired approach by serum-free pre-conditioning of human periosteum-derived cells, followed by their assembly into microaggregates simultaneously primed with bone morphogenetic protein 2 (BMP-2). Pre-conditioning resulted in a more potent progenitor cell population, while aggregation induced osteochondrogenic differentiation, further enhanced by BMP-2 stimulation. Ectopic implantation displayed a cascade of events that closely resembled the natural endochondral process resulting in bone ossicle formation. Assessment in a critical size long-bone defect in immunodeficient mice demonstrated successful bridging of the defect within 4 weeks, with active contribution of the implanted cells. In short, the presented serum-free process represents a biomimetic strategy, resulting in a cartilage tissue intermediate that, upon implantation, robustly leads to the healing of a large long-bone defect.

Single-cell characterization and metabolic profiling of in vitro cultured human skeletal progenitors with enhanced in vivo bone forming capacity

“…The herein presented in vitro priming of progenitor cells generates a clear enrichment in specified skeletal progenitors. This leads to secretion of crucial signaling molecules that upon implantation can function in a paracrine signaling manner with both graft‐ and host‐derived progenitors, allowing for full integration and further remodeling at the fracture site …”

“…Recruitment of osteochondrogenic precursor cells to the fracture site relies on a strictly controlled temporal and spatial chemotactic navigation of signals. In this setting, secreted growth and differentiation factors play a crucial role . Commonly, these soluble factors signal through cell surface receptors.…”

“…This leads to secretion of crucial signaling molecules that upon implantation can function in a paracrine signaling manner with both graftand host-derived progenitors, allowing for full integration and further remodeling at the fracture site. 12 Recruitment of osteochondrogenic precursor cells to the fracture site relies on a strictly controlled temporal and spatial chemotactic navigation of signals. In this setting, secreted growth and differentiation factors play a crucial role.…”

“…vitro expanded hPDCs could be associated with elevated mRNA transcripts of the BMP type 1 and type 2 receptors. 12 This is of particular interest since BMP-ligands are indispensable stimulatory factors in the periosteum during fracture healing 13 and signal through a complex of type 1 (ALK1/ACVRL1, ALK2/ACVR1, ALK3/BMPR1A, and ALK6/ BMPR1B) and type 2 (BMPR2, ACVR2A, and ACVR2B) transmembrane serine/threonine kinase receptors. As such, the expression level and availability of the BMP-receptors on a progenitor cell is crucial for their ability to respond and undergo subsequent differentiation upon BMP-stimulation.…”

“…Notably, exogenous BMP‐stimulation could further enhance the bone forming capacity . Moreover, it was reported that the osteochondrogenic potential of in vitro expanded hPDCs could be associated with elevated mRNA transcripts of the BMP type 1 and type 2 receptors . This is of particular interest since BMP‐ligands are indispensable stimulatory factors in the periosteum during fracture healing and signal through a complex of type 1 (ALK1/ACVRL1, ALK2/ACVR1, ALK3/BMPR1A, and ALK6/BMPR1B) and type 2 (BMPR2, ACVR2A, and ACVR2B) transmembrane serine/threonine kinase receptors.…”