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. 2010 Nov;16(11):3441-8.
doi: 10.1089/ten.TEA.2010.0132. Epub 2010 Jul 23.

Chemically-conjugated bone morphogenetic protein-2 on three-dimensional polycaprolactone scaffolds stimulates osteogenic activity in bone marrow stromal cells

Huina Zhang  1 , Francesco Migneco, Chia-Ying Lin, Scott J Hollister
Affiliations

Chemically-conjugated bone morphogenetic protein-2 on three-dimensional polycaprolactone scaffolds stimulates osteogenic activity in bone marrow stromal cells

Huina Zhang et al. Tissue Eng Part A. 2010 Nov.

Abstract

Poly(ε-caprolactone) (PCL) has received considerable attention in bone tissue engineering. However, the lack of osteoinductive ability of PCL limits its application. The aim of this study was to directly attach bone morphogenetic protein-2 (BMP-2) to PCL scaffolds by a crosslinking conjugation method and to investigate whether the bound BMP-2 maintained bioactivity in vitro. Immunofluorescent staining against BMP-2 and quantitative enzyme-linked immunosorbent assay measurements demonstrated that BMP-2 was successfully immobilized on the PCL three-dimensional scaffold by aminolysis and subsequent chemical conjugation. Conjugation produced much higher immobilization efficiency than the physical adsorption. Conjugated BMP-2 release from the PCL scaffolds was significantly slower than that from BMP-2-adsorbed PCL scaffolds over 15 days, which resulted in more BMP-2 locally retained on the conjugated scaffold. Further, the downstream Smads pathway was upregulated in bone marrow stromal cells cultured on the BMP-2-conjugated PCL scaffolds. Finally, gene expression of osteogenic markers (alkaline phosphatase, osteoclacin, and type I collagen) was upregulated in bone marrow stromal cells cultured on the PCL scaffolds with BMP-2 conjugation, but not on PCL scaffolds after BMP-2 adsorption. Therefore, our finding demonstrated that BMP-2 conjugation on polyester scaffolds is a feasible way to impart scaffolds with osteoinductive capability.

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Figures

FIG. 1.
FIG. 1.
Immunofluorescent staining of bone morphogenetic protein-2 (BMP-2) on the poly(ε-caprolactone) (PCL) films with 5 μg BMP-2 adsorption or conjugation (20 ×). The untreated PCL film served as a negative control. (A) Negative control. (B) PCL films with BMP-2 adsorption. (C) PCL films with BMP-2 conjugation. Color images available online at www.liebertonline.com/ten.
FIG. 2.
FIG. 2.
The immobilized BMP-2 amounts on the PCL scaffolds with BMP-2 adsorption or conjugation were measured by the (A) direct and (B) indirect enzyme-linked immunosorbent assay (ELISA). For the indirect ELISA measurement, the wash buffer was collected three times consecutively, and the BMP-2 in the supernatant was measured. The amount of BMP-2 remaining on the PCL was present as the difference between the original amount and the amount in the wash buffer. The values represent the percentage of the remaining amount on the PCL scaffolds of the original loaded amount (n = 4). *p < 0.05 compared with PCL-adsorbed group.
FIG. 3.
FIG. 3.
The profiles of BMP-2 release from PCL scaffold with 20 μg BMP-2 conjugation or adsorption. The amount of BMP-2 released from scaffolds was determined by ELISA. The values represent the mean ± standard error (n = 4).
FIG. 4.
FIG. 4.
(A) The BMP-2 receptor II expression in the bone marrow stromal cells (BMSCs) was observed by immunofluorescent staining. (B) Western blotting analysis for expression of phosphorylated-smad 1/5/8 expression in rat BMSCs cultured in the PCL scaffold conjugated with 20 μg BMP-2 at 6 h after cell seeding. β-Actin served as a loading control. Lane 1, untreated-PCL; lane 2, PCL conjugated with 5 μg of BMP-2; lane 3, PCL conjugated with 20 μg of BMP-2. (C) Quantification of the p-smad expression in PCL, PCL with 5 μg BMP-2 conjugation, and PCL with 20 μg BMP-2 conjugation. (D) Quantification of the p-smad expression in PCL and PCL with 20 μg BMP-2 adsorption. *p < 0.05 compared with untreated PCL groups. Color images available online at www.liebertonline.com/ten.
FIG. 5.
FIG. 5.
Gene expression of osteogenic markers of BMSCs cultured on the PCL, PCL conjugated, or adsorbed with 20 μg of BMP-2 in the osteogenic medium for 12 days in an orbit shaker. Data were normalized with GAPDH and were expressed as ratio to untreated PCL group (n = 3). *p < 0.05 compared with untreated PCL groups and #p < 0.05 compared to PCL adsorption group.
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