Grafting bone defects or atrophic non-unions with mesenchymal stromal cells (MSCs)-based grafts is not yet successful. MSC-based grafts typically use undifferentiated or osteogenically differentiated MSCs and regenerate bone through intramembranous ossification. Endochondral ossification might be more potent but requires chondrogenic differentiation of MSCs. Here, we determined if chondrogenically differentiated MSC (ch-MSC) pellets could induce bone regeneration in an orthotopic environment through endochondral ossification. Undifferentiated MSC pellets (ud-MSC) and ch-MSC pellets were generated from MSCs of human donors cultured on chondrogenic medium for respectively 3 (ud-MSC) and 21 (ch-MSC) days. A 6 mm femoral bone defect was made and stabilised with an internal plate in 27 athymic rats. Defects were left empty for 6 weeks to develop an atrophic non-union before they were grafted with ch-MSC pellets or ud-MSC pellets. Micro-CT scans made 4 and 8 weeks after grafting showed that ch-MSC pellets resulted in significantly more bone than ud-MSC pellets. This regenerated bone could completely bridge the defect, but the amount of bone regeneration was donordependent. Histology after 7 and 14 days showed slowly mineralising pellets containing hypertrophic chondrocytes, as well as TRAP-positive and CD34-positive cells around the ch-MSC pellets, indicating osteoclastic resorption and vascularisation typical for endochondral ossification. In conclusion, grafting critical femoral bone defects with chondrogenically differentiated MSC pellets led to rapid and pronounced bone regeneration through endochondral ossification and may therefore be a more successful MSCbased graft to repair large bone defects or atrophic nonunions. But, since bone regeneration was donor-depend, the generation of potent chondrogenically differentiated MSC pellets for each single donor needs to be established first.

Regeneration of load-bearing segmental bone defects is a major challenge in trauma and orthopaedic surgery. The ideal bone graft substitute is a biomaterial that provides immediate mechanical stability, while stimulating bone regeneration to completely bridge defects over a short period. Therefore, selective laser melted porous titanium, designed and fine-tuned to tolerate full load-bearing, was filled with a physiologically concentrated fibrin gel loaded with bone morphogenetic protein-2 (BMP-2). This biomaterial was used to graft critical-sized segmental femoral bone defects in rats. As a control, porous titanium implants were either left empty or filled with a fibrin gels without BMP-2. We evaluated bone regeneration, bone quality and mechanical strength of grafted femora using in vivo and ex vivo µCT scanning, histology, and torsion testing. This biomaterial completely regenerated and bridged the critical-sized bone defects within eight weeks. After twelve weeks, femora were anatomically re-shaped and revealed open medullary cavities. More importantly, new bone was formed throughout the entire porous titanium implants and grafted femora regained more than their innate mechanical stability: torsional strength exceeded twice their original strength. In conclusion, combining porous titanium implants with a physiologically concentrated fibrin gels loaded with BMP-2 improved bone regeneration in load-bearing segmental defects. This material combination now awaits its evaluation in larger animal models to show its suitability for grafting loadbearing defects in trauma and orthopaedic surgery.

This study aimed at investigating in vitro and in vivo the efficiency of commercially available fibrin as a carrier for controlled and sustained bone morphogenetic protein-2 (BMP-2) release to induce bone formation and reduce the side effects of its use. In vitro release and activity of low-dose recombinant human BMP-2 (rhBMP-2) (37.5 µg/mL) embedded in commercially available fibrin were evaluated and, subsequently, criticalsize femur defects in rats were grafted to study bone regeneration and vascularisation by micro-computed tomography (µCT) and histology. In vitro experiments showed a sustained BMP-2 release with a high BMP activity remaining after 28 d. In vivo, fibrin loaded with BMP-2 showed an extremely fast bone healing, with a large amount of new bone formation throughout the entire defect in the first 4 weeks and complete cortical repair and fusion after 8 weeks, with no ectopic bone formation. In contrast, the control fibrin group did not fuse after 12 weeks. Vascularisation was similar in both groups at 4 and 12 weeks after implantation. In conclusion, commercially available fibrin is a very efficient carrier for rhBMP-2 to graft critical-size cortical bone defects and might be a more optimal delivery vehicle for BMP-2-induced bone regeneration than currently available collagen sponges.

The aim of the current study is to assess the biological performance of self‐healing hydrogels based on calcium phosphate (CaP) nanoparticles and bisphosphonate (BP) conjugated hyaluronan (HA) in a critical size segmental femoral bone defect model in rats. Additionally, these hydrogels are loaded with bone morphogenetic protein 2 (BMP‐2) and their performance is compared in healthy and osteoporotic bone conditions. Treatment groups comprise internal plate fixation and placement of a PTFE tube containing hydrogel (HABP‐CaP) or hydrogel loaded with BMP‐2 in two dosages (HABP‐CaP‐lowBMP2 or HABP‐CaP‐highBMP2). Twelve weeks after bone defect surgery, bone formation is analyzed by X‐ray examination, micro‐CT analysis, and histomorphometry. The data show that critical size, segmental femoral bone defects cannot be healed with HABP‐CaP gel alone. Loading of the HABP‐CaP gel with low dose BMP‐2 significantly improve bone formation and resulted in defect bridging in 100% of the defects. Alternatively, high dose BMP‐2 loading of the HABP‐CaP gel does not improve bone formation within the defect area, but leads to excessive bone formation outside the defect area. Bone defect healing is not affected by osteoporotic bone conditions.