Enhanced bone formation in large segmental radial defects by combining adipose-derived stem cells expressing bone morphogenetic protein 2 with nHA/RHLC/PLA scaffold

Abstract

In this study, rabbit adipose-derived stem cells (rASCs) were isolated, cultured in vitro, and transfected with recombinant adenovirus vector containing human bone morphogenetic protein 2 (Ad-hBMP2). These cells were combined with a nano-hydroxyapatite/recombinant human-like collagen/poly(lactic acid) scaffold (nHA/RHLC/PLA) to fabricate a new biocomposite (hBMP2/rASCs-nHA/RHLC/PLA, group 1) and cultured in osteogenic medium. Non-transfected rASCs mixed with nHA/RHLC/PLA (rASCs-nHA/RHLC/PLA, group 2) and nHA/RHLC/PLA scaffold alone (group 3) served as controls. Scanning electron microscope (SEM) demonstrated integration of rASCs with the nHA/RHLC/PLA scaffold. Quantitative real-time RT-PCR analyses of collagen I, osteonectin, and osteopontin cDNA expression indicated that the osteogenic potency of rASCs was enhanced by transfection with Ad-hBMP2. After in vitro culture for seven days, three groups were implanted into 15-mm length critical-sized segmental radial defects in rabbits. After 12 weeks, radiographic and histological analyses were performed. In group 1, the medullary cavity was recanalised, bone was rebuilt and moulding was finished, the bone contour had begun to remodel and scaffold was degraded completely. In contrast, bone defects were not repaired in groups 2 or 3. Furthermore, the scaffold degradation rate in group 1 was significantly higher than in groups 2 or 3. In summary, after transduction with Ad-hBMP2, the osteogenesis of rASCs was enhanced; a new biocomposite created with these cells induced repair of a critical bone defect in vivo in a relatively short time.

Fig. 1

Representative fluorescence of rabbit adipose-derived stem cells (rASCs) transfected with adenovirus containing enhanced green fluorescence protein (Ad-EGFP) at a multiplicity of infection (MOI) rate of 400. Scale bar 50 µm. a No obvious cell toxicity was observed. b >90% rASCs expressed EGFP

Fig. 2

Relative expression levels of COL I (a), ON (b) and OP (c). *p < 0.05 statistically significant compared with rASCs-nHA/RHLC/PLA (group 2)

Fig. 3

SEM observations. Scale bar 100 µm. a nHA/RHLC/PLA porous scaffold. b Magnified view of the white rectangle frame from (a). c Co-culturing of rASCs combined with nHA/RHLC/PLA porous scaffold under osteogenic medium after seven days. d Magnified view of the white rectangle frame from (a)

Fig. 4

Radiographic results after implantation of different groups into radial bone defect including hBMP2/rASCs-nHA/RHLC/PLA (group 1) at six weeks (a) and 12 weeks (b), rASCs-nHA/RHLC/PLA (group 2) at six weeks (c) and 12 weeks (d), nHA/RHLC/PLA (group 3) at 12 weeks (e) and blank control (group 4) (f) at 12 weeks. Scale bar 0.5 cm

Fig. 5

Histological analyses of hBMP2/rASCs-nHA/RHLC/PLA (group 1) and rASCs-nHA/RHLC/PLA (group 2) at six weeks (a, c) and 12 weeks (b, d). Scale bar 500 µm. b new bone tissue, s residual scaffold (grey color), ct connective tissue, bc bone cortex* Recanalised medullary cavity

Fig. 6

Proportion of new bone area of hBMP2/rASCs-nHA/RHLC/PLA (group 1) and rASCs-nHA/RHLC/PLA (group 2) at six weeks and 12 weeks. *p < 0.05 was statistically significant compared with rASCs-nHA/RHLC/PLA (group 2)

Fig. 7

Proportion of residual scaffold area of hBMP2/rASCs-nHA/RHLC/PLA (group 1), rASCs-nHA/RHLC/PLA (group 2) and nHA/RHLC/PLA (group 3) at six weeks and 12 weeks. *p < 0.05 was statistically significant compared with rASCs-nHA/RHLC/PLA (group 2) and nHA/RHLC/PLA (group 3)