Hybrid adipogenic implants from adipose stem cells for soft tissue reconstruction in vivo

Abstract

A critical barrier in tissue regeneration is scale-up. Bioengineered adipose tissue implants have been limited to ∼10 mm in diameter. Here, we devised a 40-mm hybrid implant with a cellular layer encapsulating an acellular core. Human adipose-derived stem cells (ASCs) were seeded in alginate. Poly(ethylene)glycol-diacrylate (PEGDA) was photopolymerized into 40-mm-diameter dome-shaped gel. Alginate-ASC suspension was painted onto PEGDA surface. Cultivation of hybrid constructs ex vivo in adipogenic medium for 28 days showed no delamination. Upon 4-week in vivo implantation in athymic rats, hybrid implants well integrated with host subcutaneous tissue and could only be surgically separated. Vascularized adipose tissue regenerated in the thin, painted alginate layer only if ASC-derived adipogenic cells were delivered. Contrastingly, abundant fibrous tissue filled ASC-free alginate layer encapsulating the acellular PEGDA core in control implants. Human-specific peroxisome proliferator-activated receptor-γ (PPAR-γ) was detected in human ASC-seeded implants. Interestingly, rat-specific PPAR-γ was absent in either human ASC-seeded or ASC-free implants. Glycerol content in ASC-delivered implants was significantly greater than that in ASC-free implants. Remarkably, rat-specific platelet/endothelial cell adhesion molecule (PECAM) was detected in both ASC-seeded and ASC-free implants, suggesting anastomosis of vasculature in bioengineered tissue with host blood vessels. Human nuclear staining revealed that a substantial number of adipocytes were of human origin, whereas endothelial cells of vascular wall were of chemaric human and nonhuman (rat host) origins. Together, hybrid implant appears to be a viable scale-up approach with volumetric retention attributable primarily to the acellular biomaterial core, and yet has a biologically viable cellular interface with the host. The present 40-mm soft tissue implant may serve as a biomaterial tissue expander for reconstruction of lumpectomy defects.

FIG. 1.

Fabrication of hybrid soft tissue implant. (A) Human ASCs were culture expanded for 3 weeks. (B) PEGDA hydrogel was photopolymerized in breast shape (diameter 40 mm) without any cells. (C) ASCs were seeded in alginate solution and painted on PEDGA surface to form a hybrid construct. (D) After 4-week ex vitro cultivation, alginate-PEGDA hybrid construct showed no delamination, with or without ASCs. (E) After 4-week in vivo implantation, adipose tissue formed in ASC-seeded alginate later encapsulating an acellular PEGDA core. ASCs, adipose-derived stem cells; PEGDA, poly(ethylene)glycol-diacrylate. Color images available online at www.liebertonline.com/ten.

FIG. 2.

Ex vivo cultivation and in vivo implantation of hybrid implants. (A, B) Light microscopy of ASCs seeded in alginate gel layer encapsulating 40-mm PEGDA core, but without the adipogenic differentiation medium at 2 and 4 weeks showing no adipose lipid-bearing cells. (C, D) ASCs seeded in alginate gel layer encapsulating 40-mm PEGDA core in the adipogenic differentiation medium at 2 and 4 weeks showing intracellular lipid accumulation. Arrows indicate lipid vacuoles in adipocytes. (E) Glycerol content of ASC-seeded hybrid implants after 4-week ex vivo adipogenic differentiation in comparison with ASC-seeded hybrid implants without adipogenic differentiation. *p < 0.01. (F) Live/Dead (Live: green/FITC; Dead: red/rhodamine-RHO) viability assay of ASC-seeded hybrid implant after 4-week ex vivo adipogenic differentiation. Dashed line marking the external surface of alginate layer. Scale bar: 50 μm. (G) Implantation of breast-shaped hybrid implants subcutaneously in the dorsum of athymic rats. (H) Implant shape and dimensions were maintained after 4-week in vivo implantation. Arrows in G and H indicate adipogenic graft at the time of implantation and retrieval, respectively. Color images available online at www.liebertonline.com/ten.

FIG. 3.

Harvested hybrid soft tissue grafts after 4-week in vivo implantation. (A, B) Hybrid implants with ASC-seeded in external alginate layer showing adipose tissue integration with surrounding host tissues (arrows). Angiogenesis (arrow heads) is present in ASC-seeded alginate layer of the hybrid implant. (C) Cross section of the hybrid implant showing apparent soft tissue over a filler core (arrows). (D, E) Control, cell-free hybrid implant showing absence of adipose tissue surrounding filler core. (F) Cross section of control hybrid implant showing absence of tissue coat. Color images available online at www.liebertonline.com/ten.

FIG. 4.

Hematoxylin and eosin staining of in vivo harvested hybrid implants. (A–C) ASC-seeded hybrid implants; (D–F) ASC-free hybrid implants. (A) Adipogenic tissue coat surrounding the acellular core of the hybrid implant in low magnification. Asterisk indicates residue alginate. (B) Higher magnification of box in (A) showing abundant adipocytes (arrows) with blood vessels (arrowheads). (C) Higher magnification of box in (B) showing erythrocyte containing blood vessels lining with endothelial cells (arrowheads). (D) Control, cell-free hybrid implants showing absence of adipose tissue and acellular PEGDA core. Asterisk indicates residue alginate. (E) Fibrous tissue formation was present in the alginate layer without any seeded ASCs. (F) Fibroblast-like cells populated alginate layer with some blood vessels (arrowhead). Asterisk indicates residue alginate. Color images available online at www.liebertonline.com/ten.

FIG. 5.

Masson's trichrome staining of in vivo harvested hybrid implants. (A–C) ASC-seeded alginate layer showing abundant adipocytes with minimal surrounding fibrous tissue. Fibrous tissue surrounds residue alginate particles (*) and over the surface of acellular PEGDA hydrogel. (D–F) Abundant fibrous tissue was present in the alginate layer of control hybrid implants (arrows), with residue alginate particles (*), encapsulating the acellular PEGDA hydrogel core. Color images available online at www.liebertonline.com/ten.

FIG. 6.

Characterization of in vivo harvested hybrid implants. (A) Real-time (RT)-polymerase chain reaction for human peroxisome proliferator-activated receptor-γ (PPAR-γ), rat PPAR-γ, and rat platelet endothelial cell adhesion molecule (PECAM) of control hybrid implants and cell-seeded adipogenic hybrid implants. Human PPAR-γ was expressed in human adipose stem cell (ASC)-seeded hybrid implants. Rat PPAR-γ was not detected in human ASC-seeded hybrid implants, indicating that bioengineered adipose tissue was derived primarily from transplanted human ASCs. Rat-specific PECAM was detected in both acellular and cell-seeded samples, suggesting anastomosis of blood vessels in bioengineered adipose tissue with host vasculature. (B) Glycerol content of ASC-seeded hybrid implants was significantly greater than cell-free specimens (*p < 0.05; n = 4). (C) Human nuclear staining revealed few positive cells, suggesting that cells in alginate layer of the control group without human cell transplantation are host derived. (D) Positive human nuclear staining of the majority of adipogenic cells (red arrows) suggests human origin of transplanted cells, whereas mixed endothelial cell staining (green arrows) reveals a chimera of host and transplanted endothelial cells, indicating anastomosis of blood vessels in bioengineered adipose tissue with host vasculature. Scale bar: 100 μm. Color images available online at www.liebertonline.com/ten.