Chondrogenically differentiated mesenchymal stromal cell pellets stimulate endochondral bone regeneration in critical-sized bone defects

Abstract

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.

MiR‐455‐3p regulates early chondrogenic differentiation via inhibiting Runx2

“…As we know, chondrogenic cells commonly begin hypertrophic differentiation after 21 d [28][29][30][31], and we also showed that the hypertrophic marker Col10a1 was significantly increased after 21 d of chondrogenic differentiation; thus, miR-455-3p might be involved in the acceleration of early chondrogenesis, but not hypertrophic differentiation.…”

Osteogenic Differentiation of Mesenchymal Stem Cells by Mimicking the Cellular Niche of the Endochondral Template

“…Another limitation of this study is the use of ascorbic acid and dexamethasone in the chondrogenic medium, which might be expected to cause MSCs to differentiate toward the osteogenic lineage. However, the use of dexamethasone and ascorbic acid in the chondrogenic medium is well documented 53,60,66,78,[91][92][93][94][95][96][97][98][99][100][101][102] and has not been shown to cause mineralization. Moreover, alizarin red staining of the aggregates before the coculture showed no positive staining and the control groups were all exposed to the same chondrogenic medium, so any differences seen between the groups were not because of exposure to these growth factors.…”

Endochondral Ossification for Enhancing Bone Regeneration: Converging Native Extracellular Matrix Biomaterials and Developmental Engineering In Vivo

“…61,67,70,71,75 Most of these studies utilized a heterotopic bone formation model in immunocompromised animals to provide evidence of osteo-and chondro-conductivity and inductivity. However, more recent studies by Bahney et al, 110 van der Stok et al, 106 Harada et al, 105 and Shoji et al 108 (Table 2) showed critical-size defect regeneration in rat femurs and mice tibias via EC ossification. Likewise, studies by Montufar-Solis et al 27 and Doan et al 62 (Table 3) showed critical-sized defect regeneration in murine calvaria.…”

“…[1][2][3][4][5][6][7][8][9] An emerging group of successful research efforts have focused on exploiting this condition over the past decade. These recent publications (Tables 2-4) have demonstrated the capacity of cartilage tissue constructs to promote EC ossification in vivo after in vitro priming with seeded BMSCs, 60,63,64,66,67,[72][73][74][75][76][103][104][105][106][107] ESCs, 27,62,67,76 ADSCs, 108 iPSCs, 109 and articular chondrocytes (ACs). 61,67,70,71,75 Most of these studies utilized a heterotopic bone formation model in immunocompromised animals to provide evidence of osteo-and chondro-conductivity and inductivity.…”