In vitro evaluation of a bone morphogenetic protein‑2 nanometer hydroxyapatite collagen scaffold for bone regeneration

Abstract

Scaffold fabrication and biocompatibility are crucial for successful bone tissue engineering. Nanometer hydroxyapatite (nHAP) combined with collagen (COL) is frequently utilized as a suitable osseous scaffold material. Furthermore, growth factors, including bone morphogenetic protein‑2 (BMP‑2), are used to enhance the scaffold properties. The present study used blending and freeze‑drying methods to develop a BMP‑2‑nHAP‑COL scaffold. An ELISA was performed to determine the BMP‑2 release rate from the scaffold. Flow cytometry was used to identify rat bone marrow‑derived mesenchymal stem cells (BMSCs) prior to their combination with the scaffold. Scanning electron microscopy was used to observe the scaffold structure and BMSC morphology following seeding onto the scaffold. BMSCs were also used to assess the biological compatibility of the scaffold in vitro. BMP‑2‑nHAP‑COL and nHAP‑COL scaffolds were assessed alongside the appropriate control groups. Cells were counted to determine early cell adhesion. Cell Counting kit‑8 and alkaline phosphatase assays were used to detect cell proliferation and differentiation, respectively. Gross morphology confirmed that the BMP‑2‑nHAP‑COL scaffold microstructure conformed to the optimal characteristics of a bone tissue engineering scaffold. Furthermore, the BMP‑2‑nHAP‑COL scaffold exhibited no biological toxicity and was demonstrated to promote BMSC adhesion, proliferation and differentiation. The BMP‑2‑nHAP‑COL scaffold had good biocompatibility in vitro, and may therefore be modified further to construct an optimized scaffold for future bone tissue engineering.

Keywords: bone tissue engineering; bone marrow-derived mesenchymal stem cells; nanometer hydroxyapatite; collagen; biocompatibility; bone morphogenetic protein 2.

Figure 1.

BMP-2-nHAP-COL scaffold characterization. (A) The gross morphology of the BMP-2-nHAP-COL scaffold was observed to be white and slightly rough on the surface. (B) Images captured of the BMP-2-nHAP-COL scaffold with a scanning electron microscope (magnification, ×60) revealed a porous, spongy appearance. (C) A large number of nHAP particles attached to the scaffold surface (magnification, ×900). BMP-2, bone morphogenetic protein 2; nHAP, nanometer hydroxyapatite; COL, collagen.

Figure 2.

Cumulative release rate of BMP-2 from BMP-2-nanometer hydroxyapatite-collagen scaffolds over 20 days. BMP-2, bone morphogenetic protein 2.

Figure 3.

BMSC morphology was examined under a light-inverted microscope. (A) Cell morphology was observed following culturing of primary BMSCs for 1 day. BMSCs were mixed with non-adherent red blood cells and exhibited spindle or polygonal morphology (magnification, ×100). (B) BMSCs mixed with fibroblasts and macrophages exhibited round, spindle and polygonal morphology 5 days subsequent to the primary BMSC culture (magnification, ×100). (C) Cultured BMSCs grew well and cell impurity was significantly reduced 1 day following the first passage (magnification, ×200). (D) The density of cultured BMSCs was significantly increased 5 days following the second passage (magnification, ×200). BMSCs, bone marrow-derived mesenchymal stem cells.

Figure 4.

Flow cytometry analysis of surface marker antigens CD29, CD34, CD44 and CD45 in passage 3 bone marrow-derived mesenchymal stem cells. CD, cluster of differentiation; FITC, fluorescein isothiocyanate; PE, phycoerythrin; APC, allophycocyanin.

Figure 5.

BMSCs at 7 days post-inoculation with the bone morphogenetic protein-2-nanometer hydroxyapatite-collagen scaffold. Images were captured with a scanning electron microscope. BMSCs adhered tightly on the scaffold surface via lamellipodia and filopodia. The white arrows point to BMSCs and the black arrow indicates the scaffold. BMSCs, bone marrow-derived mesenchymal stem cells.

Figure 6.

Adhesion rate of bone marrow mesenchymal stem cells cultured with the BMP-2-nHAP-COL and nHAP-COL scaffolds. Data is expressed as the mean ± standard deviation. *P<0.05 vs. control, ^#P<0.05 vs. nHAP-COL group. BMP-2, bone morphogenetic protein 2; nHAP, nanometer hydroxyapatite; COL, collagen.

Figure 7.

Cell Counting kit-8 assay determination of the proliferation of bone marrow mesenchymal stem cells cultured with BMP-2-nHAP-COL and nHAP-COL scaffolds. Data is expressed as the mean ± standard deviation. *P<0.05 vs. control, ^#P<0.05 vs. previous time point of the same group. BMP-2, bone morphogenetic protein 2; nHAP, nanometer hydroxyapatite; COL, collagen.

Figure 8.

ALP activity assay of bone marrow mesenchymal stem cells cultured with the BMP-2-nHAP-COL and nHAP-COL scaffolds. Data is expressed as the mean ± standard deviation. *P<0.05 vs. control, ^#P<0.05 vs. nHAP-COL group. ALP, alkaline phosphatase; BMP-2, bone morphogenetic protein 2; nHAP, nanometer hydroxyapatite; COL, collagen.