Large Animal Models of an In Vivo Bioreactor for Engineering Vascularized Bone

Abstract

Reconstruction of large skeletal defects is challenging due to the requirement for large volumes of donor tissue and the often complex surgical procedures. Tissue engineering has the potential to serve as a new source of tissue for bone reconstruction, but current techniques are often limited in regards to the size and complexity of tissue that can be formed. Building tissue using an in vivo bioreactor approach may enable the production of appropriate amounts of specialized tissue, while reducing issues of donor site morbidity and infection. Large animals are required to screen and optimize new strategies for growing clinically appropriate volumes of tissues in vivo. In this article, we review both ovine and porcine models that serve as models of the technique proposed for clinical engineering of bone tissue in vivo. Recent findings are discussed with these systems, as well as description of next steps required for using these models, to develop clinically applicable tissue engineering applications.

Keywords: bone regeneration; large animal models; ovine model; periosteum; porcine model.

FIG. 1.

A schematic summary of clinical strategy. The complex bone defect is first imaged and then the optimal structure determined to acquire custom design scaffold and tissue chamber shape. A custom-made tissue chamber is filled with biodegradable osteoconductive scaffolding, bioactive growth factors, and cells. The entire mold is then implanted into the patient adjacent to the periosteum of the inner table of the pelvis. After a period of time for tissue generation, the device is harvested to yield a custom fabricated bone flap, which is then surgically transferred to the defect location.

FIG. 2.

Surgical photograph of a bioreactor (1 × 1 × 4 cm) implanted against rib periosteum in a sheep. Bioreactors are implanted on alternating ribs with up to four implanted per animal.

FIG. 3.

A harvested bioreactor (1 × 1 × 4 cm) after removal of generated tissues, a tissue flap with accompanying pedicle generated after 9 weeks of implantation, and a resected mandibular angle (1 × 4 cm) to create a large volume tissue defect for subsequent reconstruction with bioreactor-generated free tissue bone flaps.

FIG. 4.

Completed anastomoses of a free tissue bone flap generated within an in vivo bioreactor used to reconstruct a large ovine mandibular angle defect. Note that the artery anastomosis was completed side to side, and the vein anastomosis was side to end. The lower edge of the tissue flap is located in the upper right corner (partially obscured by the retractor).

FIG. 5.

Periosteum guided pig animal model. (A) Open periosteum area is between green dashed lines, inset shows removed rib segment. (B) PMMA chambers implanted against the periosteum, inset shows empty chamber before implantation. Bioreactors are implanted on alternating ribs with up to two chambers per rib and four ribs used per animal.

FIG. 6.

(A) The exposed periosteal surface (green dashed line) after removal of a 6–8 cm rib segment. (B) Cylindrical shape PMMA molds implanted against periosteum. Examples are images of cylindrical inner PMMA molds with outer (C) cylindrical and (D) rectangular walls. (E) The PMMA mold with rectangular shape typically used in the ovine model. Transparent material around the molds is the “cuff” (ethyl-vinyl-acetate, EVA).

FIG. 7.

Histological cross-section of a bioreactor initially filled with only synthetic biphasic ceramic scaffold (no autologous bone, no exogenous cells, no growth factors) after implantation against the sheep periosteum (bottom) for 9 weeks. Ceramic scaffold appears gray; bone appears dark pink. Greater bone formation occurs closer to the periosteum than the top of the bioreactor.

FIG. 8.

(A) A gross picture of the harvested morcellized bone graft after chamber removal at 4 weeks. (B) A microCT image of the bone graft in A. (C) 3D reconstruction of the regenerated bone volume based on microCT images. (D) A corresponding H&E stained section of image B, scale bar is 1 mm. Purple and dark pink stained cell nuclei and bone, respectively. (E) A higher magnification image from the selected area, showing osteocytes inside the lacuna of the bone matrix. Scale bar is 100 μm.