Artificial niche combining elastomeric substrate and platelets guides vascular differentiation of bone marrow mononuclear cells

Abstract

Bone marrow-derived progenitor cells are promising cell sources for vascular tissue engineering. However, conventional bone marrow mesenchymal stem cell expansion and induction strategies require plating on tissue culture plastic, a stiff substrate that may itself influence cell differentiation. Direct scaffold seeding avoids plating on plastic; to the best of our knowledge, there is no report of any scaffold that induces the differentiation of bone marrow mononuclear cells (BMNCs) to vascular cells in vitro. In this study, we hypothesize that an elastomeric scaffold with adsorbed plasma proteins and platelets will induce differentiation of BMNCs to vascular cells and promote vascular tissue formation by combining soft tissue mechanical properties with platelet-mediated tissue repairing signals. To test our hypothesis, we directly seeded rat primary BMNCs in four types of scaffolds: poly(lactide-co-glycolide), elastomeric poly(glycerol sebacate) (PGS), platelet-poor plasma-coated PGS, and PGS coated by plasma supplemented with platelets. After 21 days of culture, osteochondral differentiation of cells in poly(lactide-co-glycolide) was detected, but most of the adhered cells on the surface of all PGS scaffolds expressed calponin-I and α-smooth muscle actin, suggesting smooth muscle differentiation. Cells in PGS scaffolds also produced significant amount of collagen and elastin. Further, plasma coating improves seeding efficiency, and platelet increases proliferation, the number of differentiated cells, and extracellular matrix content. Thus, the artificial niche composed of platelets, plasma, and PGS is promising for artery tissue engineering using BMNCs.

FIG. 1.

Components of the artificial niche: elastomeric PGS scaffolds coated with fibrin and platelets constitute the artificial niche. Bone marrow mononuclear cell adhesion, vascular differentiation, and proliferation are mediated by the elasticity of PGS and biochemical signals from fibrin and platelet. PGS, poly(glycerol sebacate). Color images available online at www.liebertonline.com/tea

FIG. 2.

Scheme of the spinning culture systems. Constructs were secured in stainless steel meshes and fixed near the edge of culture vessels. A magnetic stir bar was used to circulate culture medium (MCDB131 containing 10% fetal bovine serum, 50 mg/L ascorbic acid, and 20 μM L-glutamine).

FIG. 3.

Characterization of scaffolds. (A) SEM micrographs of PGS scaffolds showed high porosity and good interconnectivity between pores. Scale bar=100 μm. (B) Platelet-poor plasma and (C) platelet-poor plasma-coated scaffolds displayed plasma deposition in the interior surfaces. Scale bars=100 μm. (D) Anti–von Willebrand factor immunofluorescent staining of Pl-P-PGS cross sections demonstrate wide distribution of platelets throughout the Pl-P-PGS scaffold. 100×, scale bar=100 μm. (E) Adherent platelets on Pl-P-PGS scaffolds extended multiple processes characteristic of activated platelets. Scale bar=10 μm. (F) Platelet adhesion increased with time and plateaued at 15 min incubation. Pl-P-PGS, PGS scaffolds with platelet supplemented plasma. SEM, scanning electron microscope. Color images available online at www.liebertonline.com/tea

FIG. 4.

Cell capture by PGS scaffolds. (A) and (D) are representative images of PGS scaffolds, (B) and (E) of P-PGS, and (C) and (F) of Pl-P-PGS. After 15 h of rotational cell seeding, constructs were stained with DAPI to observe cells on the scaffold surface (A–C), and cross sections of constructs were stained with H&E to observe cells in the interior of the scaffolds (D–F). Arrows indicate stained cell nucleus (dark purple points) distributed in scaffolds. Scale bars equal 100 μm for images (A–F). (G–I) SEM images showed detailed morphology of cells on the surface of the scaffolds; arrows indicate attached cells. 100×, scale bars equal 20 μm. (J) The number of cells adhered in scaffolds was quantified using crystal violet staining, n=5, p<0.05. P-PGS and Pl-P-PGS scaffolds had significantly higher cell seeding efficiency than bare PGS. H&E, hematoxylin and eosin. Color images available online at www.liebertonline.com/tea

FIG. 5.

Characterization of cell proliferation in scaffolds after 10 days of culture. SEM micrographs revealed adherent cells on the surface of the scaffolds: (A) PGS, (B) P-PGS, and (C) Pl-P-PGS scaffolds. Pl-P-PGS constructs exhibited more cells on the surface than the other groups. Inset: higher magnification (1000×). Two distinct cell morphologies are present: spread, spindle-like cells (arrows) and more spherical cells (dotted circles). (D) Macroscopic examination showed that Pl-P-PGS constructs deformed the most. (E) DNA quantification shows significantly increased proliferation in Pl-P-PGS scaffolds compared with P-PGS and uncoated PGS, n=5, *p<0.05, **p<0.01. Color images available online at www.liebertonline.com/tea

FIG. 6.

Construct tissue formation after 21 days of culture. (A) Macroscopic images showed more drastic construct deformation for all constructs than day 10. Ruler ticks=1 mm. (B) Wet weight percentage of biologic tissues in constructs (n=3). (C) Histological evaluation of the constructs (a–c: H&E staining, 40×, scale bars=500 μm. d–f: MTS, 200×, scale bars=50 μm). (D) Cell density of the constructs approximated by nucleus counting of construct cross sections using Nikon Elements software (n=6). Pl-P-PGS group demonstrated significantly higher cell density than the other groups. Values represent mean±standard error, **p<0.01. Analysis performed in triplicate. (E) Immunofluorescent images of anti–von Willebrand factor staining revealed that platelets (red) were still present in Pl-P-PGS constructs after 21 days. DAPI co-staining (blue) labeled cell nuclei and is also nonspecifically adsorbed by PGS. 100×, scale bars=100 μm. MTS, Masson's trichrome; DAPI, 4′,6-diamidino-2-phenylindole. Color images available online at www.liebertonline.com/tea

FIG. 7.

(A–C) H&E, Masson's trichrome, and von Kossa staining of PLGA constructs indicated calcification distributed sparsely in the constructs (dark spots, indicated by arrows). 200×, scale bars=100 μm. (D–F) In contrast, staining for PGS, P-PGS, and Pl-P-PGS showed no calcium deposition. 200×, scale bars=100 μm. (G) Quantification of ALP activity in all groups indicated that cells in PLGA scaffold have significantly higher ALP activity than those in all PGS groups, n=4, **p<0.01. PLGA, poly(lactic-co-glycolic acid); ALP, alkaline phosphate. Color images available online at www.liebertonline.com/tea

FIG. 8.

(A–F) Immunofluorescent staining with DAPI co-stain (blue) showed expression of α-SMA and calponin-I in all constructs. (G–I) Constructs stained without primary antibodies (negative control) demonstrated nonspecific staining of PGS with secondary antibodies and DAPI. Pl-P-PGS constructs had the strongest expression of α-SMA and calponin-I. (J–L) SMCs seeded in PGS scaffolds act as positive control. 200×, scale bars=50 μm. (M) Western blotting confirms α-SMA and calponin-I expression in all groups. β-actin protein levels were measured to show protein loading. α-SMA, α-smooth muscle actin; SMC, smooth muscle cell. Color images available online at www.liebertonline.com/tea

FIG. 9.

Immunofluorescent staining of extracellular matrix proteins in the constructs with DAPI co-stain. (A–F) All groups were positive for collagen III and elastin staining throughout the constructs, including within pores (arrows). Pl-P-PGS constructs (C and F) appeared to express more extracellular matrix protein than the other two groups. (G–I) Constructs stained without primary antibodies (negative control) showed nonspecific staining of PGS with secondary antibodies and DAPI. (J–L) SMCs seeded in PGS scaffolds act as positive control. 200×, scale bars=50 μm. (M and N) Quantification of collagen and insoluble elastin in constructs normalized to construct wet weight. Pl-P-PGS groups had significantly higher protein expression than the other groups. SMC-PGS abbreviated as S-PGS in the figure, n=4. *p<0.05 (PGS vs. S-PGS), **p<0.05 (P-PGS vs. PGS), ***p<0.05 (Pl-P-PGS vs. P-PGS). (O and P) Quantification of collagen and insoluble elastin in constructs normalized by DNA content. The S-PGS group had higher collagen and elastin production than any other group. No significant differences were detected in normalized collagen content among all the bone marrow mononuclear cells groups. However, elastin/DNA in Pl-P-PGS group was significantly higher than the other groups. n=4, **p<0.05 (P-PGS vs. PGS), ***p<0.05 (S-PGS vs. P-PGS). Color images available online at www.liebertonline.com/tea