Human Macrophage- and Osteoclast-Based Constructs Do Not Induce Ectopic Bone Formation

Abstract

PurposeAn increasing body of evidence suggests that bone resorbing osteoclasts are important—but as yet underrated—cellular initiators of bone formation. Furthermore, macrophages also have shown stimulatory effects on the osteogenic differentiation of mesenchymal stromal cells (MSCs). Consequently, we here investigated whether human macrophage- and osteoclast-laden carrier materials can induce ectopic bone formation upon subcutaneous implantation in nude mice.MethodsHuman osteoclast precursors were isolated and differentiated toward macrophages. Subsequently, these macrophages were seeded onto two types of cell carrier materials (i.e., electrospun polymeric scaffolds and devitalized bovine bone granules) and differentiated for 14 days toward osteoclasts. DNA assay and fluorescent nuclei staining were performed. Osteoclast differentiation was assessed by a tartrate-resistant acid phosphatase (TRAP)-activity assay, TRAP, and immunocytochemical staining for β3 integrin. After 60 days of implantation into nude mice, specimens were retrieved, histologically processed, and stained with hematoxylin and eosin (HE) as well as for TRAP to study ectopic bone formation and osteoclast activity, respectively.ResultsOsteoclast precursors limitedly adhered to both material types. Osteoclast-laden samples showed increased intracellular gross TRAP-activity on both cell carrier types, TRAP staining on polymeric electrospun scaffolds, and positive β3 integrin staining on decellularized bovine bone granules compared to the macrophage-laden materials. We observed that only the positive control samples loaded with bone morphogenetic protein-2 (BMP-2) induced ectopic bone formation and TRAP signal.ConclusionWe conclude that neither human macrophage- nor osteoclast-laden constructs are capable to induce ectopic bone formation under the current experimental set-up.Lay summaryInterestingly, increasing amounts of evidence suggest that osteoclasts—the cells responsible for breaking down bone tissue—can trigger bone formation. Therefore, we here aimed to study whether blood-derived macrophages and osteoclasts can induce bone formation in vivo. Consequently, we generated human macrophage- and osteoclast-laden constructs using two types of scaffold materials and implanted them underneath the skin of nude mice. Although we confirmed the presence of macrophages and osteoclasts on the materials, we found no signs of bone formation.