Induction of angiogenesis in tissue-engineered scaffolds designed for bone repair: a combined gene therapy-cell transplantation approach

Abstract

One of the fundamental principles underlying tissue engineering approaches is that newly formed tissue must maintain sufficient vascularization to support its growth. Efforts to induce vascular growth into tissue-engineered scaffolds have recently been dedicated to developing novel strategies to deliver specific biological factors that direct the recruitment of endothelial cell (EC) progenitors and their differentiation. The challenge, however, lies in orchestration of the cells, appropriate biological factors, and optimal factor doses. This study reports an approach as a step forward to resolving this dilemma by combining an ex vivo gene transfer strategy and EC transplantation. The utility of this approach was evaluated by using 3D poly(lactide-co-glycolide) (PLAGA) sintered microsphere scaffolds for bone tissue engineering applications. Our goal was achieved by isolation and transfection of adipose-derived stromal cells (ADSCs) with adenovirus encoding the cDNA of VEGF. We demonstrated that the combination of VEGF releasing ADSCs and ECs results in marked vascular growth within PLAGA scaffolds. We thereby delineate the potential of ADSCs to promote vascular growth into biomaterials.

Fig. 1.

Scanning electron micrographs showing ADSC proliferation at days 7 and 14 on PLAGA sintered microsphere scaffolds. (a) Day 7. (Magnification: ×75.) (b) Day 7. (Magnification: ×150.) (c) Day 14. (Magnification: ×75.) (d) Day 14. (Magnification: ×350.)

Fig. 2.

VEGF production by transfected ADSCs. (a) The kinetics of VEGF release by ADSCs cultured on six-well plates. Data are representative of three independent experiments, and all data points are plotted as mean ± SD (n = 5). A single asterisk denotes a significantly (P ≤ 0.05) higher VEGF production, as compared with groups denoted by two asterisks. Two asterisks denote significantly (P ≤ 0.05) higher VEGF production, as compared with groups denoted by three asterisks. Results show that ADSCs transfected at MOI = 100 PFU per cell secreted the maximum level of VEGF as compared with other selected MOIs. The multiway ANOVA assessment did not show a significant interaction between ADSC culture time and VEGF production. (b and c) Immunofluorescent staining of VEGF production by transfected ADSCs (b) and nontransfected ADSCs (negative control) (c) cultured for 10 days on PLAGA sintered microsphere scaffolds. ADSCs were stained with antibody directed against VEGF (green) and nuclei counterstained with DAPI (blue). Images are composed of sections of the scaffolds moving through its thickness at depth increments of 4 μm and a focus depth of 224 μm that are collapsed on each other.

Fig. 3.

EC proliferation on coculture plates containing EC alone, EC plus ADSC, and EC plus transfected ADSC. Data are representative of three independent experiments, and all data points are plotted as mean ± SD (n = 5). A single asterisk denotes a significantly (P ≤ 0.05) higher EC proliferation in an EC+ADSC coculture system as compared with EC alone. Two asterisks denote a significantly (P ≤ 0.05) higher EC proliferation in an EC+transfected ADSC coculture system as compared with EC+ADSC. The multiway ANOVA assessment did not show a significant interaction between EC proliferation and coculture time.

Fig. 4.

Representative histological cross sections of tissue implanted with sintered microsphere scaffolds 21 days after s.c. implantation in SCID mice. (a) Transfected ADSCs. (Scale bar: 100 μm. Magnification: ×10.) (b) Transfected ADSCs. (Scale bar: 10 μm. Magnification ×100.) (c) ADSCs. (Scale bar: 10 μm. Magnification: ×100.) (d) ECs. (Scale bar: 10 μm. Magnification: ×100.) Blood vessels were identified by the presence of luminal structure containing red blood cells.

Fig. 5.

SEM micrographs of blood vessel growth in the 3D PLAGA scaffolds at 21 days after implantation. [Magnification: ×200 (a), ×350 (b), and ×700 (c).]

Fig. 6.

Kinetics of scaffold vascularization at predetermined time points after implantation. For each group, the number of blood vessels was counted in H&E-stained slides. Each group consisted of five animals. All data points were plotted as mean ± SD. A single asterisk denotes a significantly (P ≤ 0.05) higher number of blood vessels as compared with blank scaffolds. Two asterisks denote a significantly (P ≤ 0.05) higher number of blood vessels as compared with scaffolds denoted by a single asterisk. Three asterisks denote a significantly (P ≤ 0.05) higher number of blood vessels as compared with groups denoted by two asterisks. Scaffolds containing EC and transfected ADSC showed the highest number of blood vessels. The multiway ANOVA assessment did not show a significant interaction between implantation time and blood vessel density in the scaffolds.