Growth factor gradients via microsphere delivery in biopolymer scaffolds for osteochondral tissue engineering

Abstract

Temporally and spatially controlled delivery of growth factors in polymeric scaffolds is crucial for engineering composite tissue structures, such as osteochondral constructs. In the present study, microsphere-mediated growth factor delivery in polymer scaffolds and its impact on osteochondral differentiation of human bone marrow-derived mesenchymal stem cells (hMSCs) was evaluated. Two growth factors, bone morphogenetic protein 2 (rhBMP-2) and insulin-like growth factor I (rhIGF-I), were incorporated as a single concentration gradient or reverse gradient combining two factors in the scaffolds. To assess the gradient making system and the delivery efficiency of polylactic-co-glycolic acid (PLGA) and silk fibroin microspheres, initially an alginate gel was fabricated into a cylinder shape with microspheres incorporated as gradients. Compared to PLGA microspheres, silk microspheres were more efficient in delivering rhBMP-2, probably due to sustained release of the growth factor, while less efficient in delivering rhIGF-I, likely due to loading efficiency. The growth factor gradients formed were shallow, inducing non-gradient trends in hMSC osteochondral differentiation. Aqueous-derived silk porous scaffolds were used to incorporate silk microspheres using the same gradient process. Both growth factors formed deep and linear concentration gradients in the scaffold, as shown by enzyme-linked immunosorbent assay (ELISA). After seeding with hMSCs and culturing for 5 weeks in a medium containing osteogenic and chondrogenic components, hMSCs exhibited osteogenic and chondrogenic differentiation along the concentration gradients of rhBMP-2 in the single gradient of rhBMP-2 and reverse gradient of rhBMP-2/rhIGF-I, but not the rhIGF-I gradient system, confirming that silk microspheres were more efficient in delivering rhBMP-2 than rhIGF-I for hMSCs osteochondrogenesis. This novel silk microsphere/scaffold system offers a new option for the delivery of multiple growth factors with spatial control in a 3D culture environment for both understanding natural tissue growth process and in vitro engineering complex tissue constructs.

Figure 1

Characterization of alginate gel scaffolds. A,B, gradient distribution of alginate and silk microspheres in alginate scaffolds, respectively, as determined by measuring encapsulated HRP activities. The scaffolds were cut into 9 segments after preparation, and each gel segment was dissolved in 20 mM EDTA and HRP content in the released microspheres was determined. Data represent the Ave.± SD (n = 3). Data were fit linear with least-squares linear regression, R² = 0.75 for both A and B. C, photomicrographs of alginate gel scaffold incorporated with silk (a) and PLGA microspheres (b,c) as well as hMSCs. The scaffolds were cultured in a growth medium for three days before imaging. Arrows indicate PLGA microspheres and circles indicate round shaped hMSCs. Bar = 500 μm in a; 200 μm in b.

Figure 2

Transcript levels from hMSCs in alginate gel scaffolds after 3 weeks culture. For all three groups of scaffolds (rhBMP-2, IGF-I, rhBMP-2/IGF-I), one scaffold was sectioned into 11 segments along the direction of growth factor gradient and analyzed. For the dual growth factor scaffold, rhBMP-2 concentration increased from segment 1 to 11, while the rhIGF-I concentration decreased. The results from segment 2, 6, 10 are presented. A, B, Bone makers, collagen type I (Col I) and bone sialoprotein (BSP), respectively. C, hypertrophic chondrocyte maker, collagen type X (Col X). D, Chondrocyte maker, collagen type II (Col II). *** Extremely significant differences between groups (P<0.001). Data represent the Ave.± SD (n = 3–4)

Figure 3

Growth factor gradient in silk microsphere-incorporated silk scaffolds. The scaffold was sectioned into segments and the rhBMP-2 and rhIGF-I content in each segment was quantified by ELISA. Data were fit with least-squares linear regression, R² = 0.92 and 0.97 for A and B, respectively. A, rhBMP-2 gradient. B, IGF-I gradient. Data represent the Ave.± SD (n = 3–4)

Figure 4

hMSC osteochondral differentiation in growth factor-gradient silk scaffolds. For all three groups of scaffolds (rhBMP-2, IGF-I, rhBMP-2/IGF-I), scaffolds were sectioned into 7 segments along the direction of growth factor gradient. The results from segment 1, 3, 5, 7 of rhBMP-2 and rhBMP-2/rhIGF-I-incorporated scaffolds are presented. For the scaffolds containing two growth factors, rhBMP-2 concentration increased from segment 1 to 7, while the rhIGF-I concentration decreased. A, B, Bone makers, collagen type I (Col I) and bone sialoprotein (BSP), respectively. C, calcium deposition as weight percentage per (wet) scaffold segment. D, hypertrophic chondrocyte maker, collagen type X (Col X). E, Chondrocyte maker, collagen type II (Col II). F, formation of cartilage specific extracellular matrix material GAG in a scaffold as weight percentage. *Significant differences between the groups (P<0.05). **Very significant differences between the groups (P<0.01). ***Extremely significant differences between groups (P<0.001). Data are Ave.±SD (n = 3–4).

Figure 5

Histological analysis on silk scaffolds with rhBMP-2/rhIGF-I reverse gradient. A–F, the first scaffold segment (lowest rhBMP-2 and highest rhIGF-I). G–L, the seventh scaffold segment (highest rhBMP-2 and lowest rhIGF-I). A,D,G,J, Hematoxylin and eosin (H&E) staining. B,E,H,K, Alcian blue staining for proteoglycan formation. C,F,I,L, Von Kossa staining for calcium deposition. Scale bars = 200 μm (A–C, G–I) or 50 μm (D–F, J–L)