Osteoblast-oriented differentiation of BMSCs by co-culturing with composite scaffolds constructed using silicon-substituted calcium phosphate, autogenous fine particulate bone powder and alginate in vitro

Abstract

Autogenous bone graft is the best for spinal fusion in clinics, however, lacking sources, bleeding and infection are limited its practice. Seeking alternative materials are urgent for orthopaedic surgeon. Here, we evaluated osteoblast-oriented differentiation of rabbit BMSCs by co-culturing with composite scaffolds constructed using silicon-substituted-CaP-fine particulate bone powder-alginate. Using CCk8-kit, biocompatibility was evaluated by testing BMSCs proliferation; morphology and survival of osteoblasts within scaffolds were observed using EM and HE staining; growth factors and related genes were detected using RT-PCR. HE staining showed spindle-shaped BMSCs after the 3rd passage; EM data showed that uneven surface and longitudinal section were observed with scattered distribution of 5-100 mm interspaces, which leave enough space for BMSCs adhesion and growth. Interestingly, at 14-day culture with HE staining, osteocytes within the scaffolds grew well with regular shape and integrate structure. RT-PCR results showed that expression levels of BMP2, TGF-b and COL-I, ALP, OPN were increased significantly and time-dependently. Collectively, all mentioned effects were more obvious in co-culture BMSCs with scaffolds than those with other components. Immunohistochemistry showed that positive OPN expression was detected at 7-day co-culturing BMSCs with scaffold, rather than other situations. These results suggest that composite scaffolds constructed with Si-CaP-fine particulate bone powder-alginate have a certain degree of biocompatibility and bioactivity to promote osteoblast-oriented BMSCs differentiation.

Keywords: Pathology Section; autogenous fine particulate bone powder; bone marrow stromal cells; silicon-substituted calcium phosphate; spinal fusion; tissue-engineering.

Figure 1. Isolation and cell culture of BMSCs

A. the BMSCs was separated using gredient centrifugation with Ficoll solution (with portion on the bottom of centrifugation tube) and the cloudy layer rich in monocyte (BMSCs) was located at the interface between Ficoll solution and cell suspension (red portion); B. 7-day primary culture of BMSCs (´100) with 50% cellular fusion; C. 9-days primary culture of BMSCs (´100) with 80% of cell fusion; D. the 3-days of the 3^rd passage of continuous culture of BMSCs (´100) with significant cell fusion.

Figure 2. The survival rate and proliferation rate of BMSCs

Top image: showing the survival of BMSCs after isolation procedures and the survival rate is near 99%; bottom figure: the proliferation rate detected by using CCK8 kit, the optical density (OD) at 465 nm was used to quantified BMSCs proliferation and the OD curve was plotted as the function of culture time (day). With this proliferation curve, the BMSCs from the 3rd passage grow slowly within the first 3-days, fast from the 4 to 6 days, and become stable from 7 days.

Figure 3. Morphological Identification of BMSCs under culture condition with HE staining

The representative image showing the P3 BMSCs with HE staining. Arrows present the BMSCs (´100).

Figure 4. Scanning electronmicroscope observation of 7-days co-cultured BMSCs with composite scaffolds

A. co-culturing BMSCs with the scaffold constructed using silicon-substituted calcium phosphate (Si-CaP), autogenous fine particulate bone powder, and sodium alginate; B. co-culturing BMSCs with the scaffold constructed using autogenous fine particulate bone powder and alginate; C. co-culturing BMSCs with the scaffold constructed using Si-CaP and alginate. Blue asterisks present the BMSCs (´1000). The scale bar in (C) is 10 mm and applied to (A) and (B).

Figure 5. The ultrastructure of composite scaffold constructed using Si-CaP, autogenous fine particulate bone powder, and alginate under electromicroscope

A. the surface of the scaffold (´1000): showing rough and uneven surface with thicknesses and irregular shaped sodium alginate (filiform or and mesh) linking Si-CaP and autogenous fine particulate bone powder. 5-15 mm size of interspaces can be seen on the surface of scaffold; B. the interior structure of the scaffold (´1000). White asterisks, yellow asterisks, and white/yellow arrow heads are represented as Si-CaP, bone powder, and interspace, respectively.

Figure 6. HE staining and evaluation of co-culture of BMSCs with composite scaffold constructed using Si-CaP, autogenous fine particulate bone powder, and alginate

Left panel: 1-day after co-culture; Right panel: 14-days after co-culture. These images, especially at 14-days, autogenous fine particulate bone powder and Si-CaP are distributed uniformly, and the bone lacuna (blue arrow heads) and osteocytes within the bone matrix are clearly obseved with an integral structure, the nucleus of osteocytes (white arrow heads) are deep blue stained, indicating osteocytes live well under current experimental condition.

Figure 7. Expression profiles of growth factors (BMP2 and TGF-b) and related genes (ALP, OPN, and Col-I) using real-time RT-PCR

The PCR data were collected from each tested group at different time points during co-culture and averaged data were expressed as mean ± SD, and n = 6. *P < 0.05 and **P < 0.01 vs BMSCs alone. A. BMP-2 expression profiles; B. TGF-1 expression profiles; C. ALP expression profiles; D. OPN expression profiles; E. Col-I expression profiles.

Figure 8. Immunohistochemical analysis of OPN expression co-cultured with BMSCs at one day (D1) and 7 days (D7)

A. BMSCs were co-cultured with composite scaffold constructed using 20 mg Si-CaP, 20 mg fine particulate bone powder, and 3 ml alginate (group 1); B. BMSCs were co-cultured composite scaffold constructed using with 40 mg fine particulate bone powder and 3 ml alginate (group 2); C. BMSCs were co-cultured with composite scaffold constructed using with 40 mg Si-CaP and 3 ml alginate (group 3); D. BMSCs were cultured without scaffold as the control (group 4). The red arrows represent the positive OPN expression.