Bioengineering strategies to generate vascularized soft tissue grafts with sustained shape

Abstract

Tissue engineering offers the possibility for soft tissue reconstruction and augmentation without autologous grafting or conventional synthetic materials. Two critical challenges have been addressed in a number of recent studies: a biology challenge of angiogenesis and an engineering challenge of shape maintenance. These two challenges are inter-related and are effectively addressed by integrated bioengineering strategies. Recently, several integrated bioengineering strategies have been applied to improve bioengineered adipose tissue grafts, including internalized microchannels, delivery of angiogenic growth factors, tailored biomaterials and transplantation of precursor cells with continuing differentiation potential. Bioengineered soft tissue grafts are only clinically meaningful if they are vascularized, maintain shape and dimensions, and remodel with the host. Ongoing studies have begun to demonstrate the feasibility towards an ultimate goal to generate vascularized soft tissue grafts that maintain anatomically desirable shape and dimensions.

Fig. 1

In vivo implantation of PEG hydrogel. PEG hydrogel was fabricated in four configurations: PEG alone, PEG with bFGF, microchanneled PEG, or both bFGF-adsorbed and microchanneled PEG. (A) PEG hydrogel molded into 6 × 4 mm (diameter × height) cylinder (without either bFGF or microchannels). (B) PEG hydrogel with three microchannels. (C) PEG hydrogel cylinder with adsorbed 0.5 mg/mL bFGF and three microchannels. Following in vivo implantation subcutaneously in the dorsum of immunodeficient mice, the harvested PEG hydrogel samples showed distinct histological features. (A1) PEG hydrogel with microchannels but without bFGF showed host tissue infiltration primarily in the lumen of microchannels, and scarcely in the rest of PEG hydrogel. The infiltrating host tissue includes erythrocyte-filled blood vessels that are lined by endothelial cells (arrow). (A2) VEGF was immunolocalized only to host-derived tissue within the lumen of microchannels, indicating the vascular nature of the infiltrating host tissue. Arrows point to microchannels and the infiltrating host tissue. (B1) PEG hydrogel with bFGF but without microchannels showed apparently random and isolated islands of infiltrating host tissue (arrow). The infiltrating host tissue includes vascular structures with erythrocyte-filled blood vessels that are lined by endothelial cells (arrow). (B2) VEGF was immunolocalized to host-derived tissue within PEG hydrogel (without microchannels). (C1) PEG hydrogel with both microchannels and bFGF showed host tissue infiltration only in the lumen of microchannels, but scarcely in the rest of the PEG hydrogel. The infiltrating host tissue includes vascular structures with erythrocyte-filled blood vessels that are lined by endothelial cells (arrow). (C2) VEGF was immunolocalized only to host-derived tissue within the lumen of microchannels. Since no cells were delivered in any of the PEG hydrogel samples, tissue infiltration following in vivo implantation is derived from the host. Arrows point to microchannels.

Fig. 2

In vivo implantation of bFGF and microchanneled PEG hydrogel loaded with adipogenic cells derived from hMSCs. Diagrams (top row) and corresponding representative photographs at the time of harvest of in vivo samples. (A) PEG hydrogel molded into 6 × 4 mm (width × height) cylinder (without either bFGF or microchannels). (B) PEG hydrogel cylinder with 0.5 mg/mL bFGF and three microchannels, but without the delivery of cells. (C) PEG hydrogel cylinder loaded with 0.5 mg/mL bFGF and three microchannels, in addition to the encapsulation of adipogenic cells that have been derived from human mesenchymal stem cells at a cell seeding density of 3 × 10⁶ cells/mL. Following in vivo implantation subcutaneously in the dorsum of immunodeficient mice, the harvested PEG hydrogel samples showed distinct histological features. (A’) PEG hydrogel cylinder without either microchannels or bFGF showed somewhat transparent appearance. (B’) PEG hydrogel cylinder with both bFGF and three microchannels, but without delivered cells, showed darker color and a total of three openings of microchannels (arrows) that are confirmed to be areas of host cell infiltration histologically. (C’) PEG hydrogel cylinder with both microchannels and bFGF in addition to encapsulated hMSC-derived adipogenic cells showed the opening of microchannels (red color and pointed with arrows) that are confirmed to be areas of host cell infiltration histologically in Fig. 3.

Fig. 3

Histological and immunohistochemical characterization of vascularized adipose tissue from human mesenchymal stem cells. (A) Hematoxylin and eosin staining revealed IF tissue interposing between foam-like space labeled with A for adipose tissue. The presence of adipose tissue is confirmed in (B), showing substantial Oil red O positive staining in PEG hydrogel encapsulating hMSC-derived adipogenic cells, in addition to bFGF and built-in microchannels. In contrast, there is no evidence of adipogenesis in PEG hydrogel with bFGF and built-in microchannels, despite the seeding of hMSCs but without adipogenic differentiation. (C) Positive immunolocalization of VEGF antibody in the IF tissue interposing areas of adipogenesis, indicating the presence of vascular supply. (D) Positive immunolocalization of lectin WGA in the interstitial fibrous tissue interposing areas of adipogenesis, serving as further indication of the presence of vascular endothelial cells.

Fig. 4

A soft hydrogel with internal microchannels fabricated with interlaid strands and interconnecting microchannels. Polyethylene oxide (PEO) was dissolved in methyl ethyl and bioplotted with an input CAD file specifying the geometry of internal microchannels. (A, B) The schematics of the bioplotting technique and the adjustable parameters for the bioplotter. (C) Photomicrograph of the 3D-bioplotted PEO hydrogel with interlaid strands and interconnecting microchannels. (D) Scanning electron microscopy (SEM) image of a bioplotted PEO hydrogel with interlaid strands and interconnecting microchannels.