A novel tissue-engineered bone graft composed of silicon-substituted calcium phosphate, autogenous fine particulate bone powder and BMSCs promotes posterolateral spinal fusion in rabbits

Abstract

Background: Autogenous bone graft is the gold standard bone grafting substrate available in spinal fusion because of its osteoconductive, osteogenic, and osteoinductive properties. However, several shortcomings including bleeding, infection, chronic pain, and nerve injury are known to be associated with the procedure. Bone tissue engineering has emerged as an alternative therapeutic strategy for bone grafts. New materials have been developed and tested that can substitute for the autogenous bone grafts used in the spinal fusion. The purpose of this study is to evaluate the role of a novel tissue-engineered bone graft with silicon-substituted calcium phosphate (Si-CaP), autogenous fine particulate bone powder (AFPBP), and bone marrow mesenchymal stem cells (BMSCs) using a rabbit posterolateral lumbar fusion model based on bone tissue engineering principles. The application of this graft can represent a novel choice for autogenous bone to reduce the amount of autogenous bone and promote spinal fusion.

Methods: BMSCs from New Zealand white rabbits were isolated and cultured in vitro. Then, BMSCs were marked by the cell tracker chloromethyl-benzamidodialkylcarbocyanine (CM-Dil). A total of 96 New Zealand White rabbits were randomly divided into four groups: (a) AFPBP, (b) Si-CaP, (c) Si-CaP/AFPBP, (d) Si-CaP/AFPBP/BMSCs.The rabbits underwent bilateral posterolateral spine arthrodesis of the L5-L6 intertransverse processes using different grafts. Spinal fusion and bone formation were evaluated at 4, 8, and 12 weeks after surgery by manual palpation, radiology, micro-computed tomography (micro-CT), histology, and scanning electronic microscopy (SEM).

Results: The rate of fusion by manual palpation was higher in the Si-CaP/AFPBP/BMSCs group than the other groups at 8 weeks. The fusion rates in the Si-CaP/AFPBP/BMSCs and the AFPBP groups both reached 100%, which was higher than the Si-CaP/AFPBP group (62.5%) (P ​> ​0.05) and Si-CaP group (37.5%) (P ​< ​0.05) at 12 weeks. New bone formation was observed in all groups after implantation by radiology and micro-CT. The radiographic and CT scores increased in all groups from 4 to 12 weeks, indicating a time-dependent osteogenetic process. The Si-CaP/AFPBP/BMSCs group showed a larger amount of newly formed bone than the Si-CaP/AFPBP and Si-CaP groups at 12 weeks. Bone formation in the Si-CaP/AFPBP/BMSCs group was similar to the AFPBP group. Histology showed that new bone formation continued and increased along with the degradation and absorption of Si-CaP and AFPBP from 4 to 12 weeks in the Si-CaP, Si-CaP/AFPBP, and Si-CaP/AFPBP/BMSCs groups. At 4 weeks, a higher proportion of bone was detected in the AFPBP group (23.49%) compared with the Si-CaP/AFPBP/BMSCs group (14.66%, P ​< ​0.05). In the Si-CaP/AFPBP/BMSCs group at 8 weeks, the area percentage of new bone formation was 28.56%, which was less than the AFPBP group (33.21%, P ​< ​0.05). No difference in bone volume was observed between the Si-CaP/AFPBP/BMSCs group (44.39%) and AFPBP group (45.06%) at 12 weeks (P ​> ​0.05). At 12 weeks, new trabecular were visible in the Si-CaP/AFPBP/BMSCs group by SEM. CM-Dil-positive cells were observed at all stages. Compared with histological images, BMSCs participate in various stages of osteogenesis by transforming into osteoblasts, chondrocytes, and osteocytes.

Conclusion: This study demonstrated for the first time that Si-CaP/AFPBP/BMSCs is a novel tissue-engineered bone graft with excellent bioactivity, biocompatibility, and biodegradability. The graft could reduce the amount of autogenous bone and promote spinal fusion in a rabbit posterolateral lumbar fusion model, representing a novel alternative to autogenous bone.

The translational potential of this article: The translational potential of this article lies in that this graft will be a novel spinal fusion graft with great potential for clinical applications.

Keywords: Autogenous fine particulate bone powder; Bone marrow mesenchymal stem cells; Bone tissue engineering; Silicate-substituted calcium phosphate; Spinal fusion.

Fig. 1

The fusion rate results by manual palpation in each group at 8 and 12 weeks after the operation. AFPBP, autogenous fine particulate bone powder; BMSCs, bone marrow mesenchymal stem cells; Si-CaP, silicon-substituted calcium phosphate. Data were expressed as the mean ​± ​SEM. n ​= ​8. ∗P ​< ​0.05.

Fig. 2

Spinal fusion evaluated by X-ray (A), 3D-CT (B) and micro-CT (C). AFPBP, autogenous fine particulate bone powder; BMSCs, bone marrow mesenchymal stem cells; Si-CaP, silicon-substituted calcium phosphate.

Fig. 3

The BV/TV, Tb.Th, Conn.D, and Tb.Sp values at 4, 8, and 12 weeks after the operation in each group were evaluated by micro-CT. BV/TV, bone volume/total volume; Tb.Sp, trabecular separation; Tb.Th, trabecular thickness; Conn.D, connectivity density. AFPBP, autogenous fine particulate bone powder; BMSCs, bone marrow mesenchymal stem cells; Si-CaP, silicon-substituted calcium phosphate. Data were expressed as the mean ​± ​SEM. n ​= ​8. ∗P ​< ​0.05.

Fig. 4

Histological sections in each group at 4, 8, and 12 weeks after the operation (yellow arrows: osteoblasts, yellow asterisks: newly formed bone, green arrows: osteoclasts, green asterisks: Si-CaP, blue arrows: AFBFP, white asterisks: chondrocytes).

Fig. 5

Statistical analysis of histological sections of new bone area in each group at 4, 8, and 12 weeks after the operation. AFPBP, autogenous fine particulate bone powder; BMSCs, bone marrow mesenchymal stem cells; Si-CaP, silicon-substituted calcium phosphate. Data were expressed as the mean ​± ​SEM. n ​= ​20 sections from 3 animals. ∗P ​< ​0.05.

Fig. 6

Bone formation was evaluated by scanning electron microscopy (SEM) examination at 12 weeks after the operation. Yellow arrows: trabecular

Fig. 7

CM-Dil-positive cells in the Si-CaP/AFPBP/BMSCs group were observed by fluorescence microscopy at 4, 8, and 12 weeks after the operation compared with HE-stained sections in the same field of view. (A) CM-Dil-positive cells (blue arrows) in soft tissue at 4 weeks; (B) CM-Dil-positive cells (blue arrows) in bone matrix at 4 weeks; (C) CM-Dil-expressing chondrocytes (yellow arrows) in cartilage matrix at 8 weeks; (D) CM-Dil-positive osteocytes (green arrows) in bone matrix at 8weeks; (E) CM-Dil-positive osteocytes (green arrows) in the new bone lacunae at 12 weeks.