Evaluation of bone regeneration using the rat critical size calvarial defect

Abstract

Animal models that are reliably reproducible, appropriate analogs to the clinical condition they are used to investigate, and that offer minimal morbidity and periprocedural mortality to the subject, are the keystone to the preclinical development of translational technologies. For bone tissue engineering, a number of small animal models exist. Here we describe the protocol for one such model, the rat calvarial defect. This versatile model allows for evaluation of biomaterials and bone tissue engineering approaches within a reproducible, non-load-bearing orthotopic site. Crucial steps for ensuring appropriate experimental control and troubleshooting tips learned through extensive experience with this model are provided. The surgical procedure itself takes ∼30 min to complete, with ∼2 h of perioperative care, and tissue collection is generally performed 4-12 weeks postoperatively. Several analytical techniques are presented, which evaluate the cellular and extracellular matrix components, functionality and mineralization, including histological, mechanical and radiographic methods.

Figure 1

Use of the push-out jig. (A) Photograph of push-out jig. The peak push-out load of the specimens (n = 6) with implanted (B) polymeric and (C) ceramic scaffolds with or without platelet rich plasma and/or bone marrow derived mononuclear cells. Implant type abbreviations denote the scaffold material (polymer, P or ceramic, C) followed by the presence or absence of platelet rich plasma (- or P, respectively) and the presence or absence of mononuclear cells (or M, respectively). * indicates significant difference from material control (p <0.05). Adapted with permission.

Figure 2

Creation of the defect. The bone is exposed and the defect created by incision and retraction of the (A) skin and (B) periosteum (shown with white arrowheads) overlying the calvarium. Note the clear line of the sagittal suture in the bone of the calvarium indicated with a black arrow. A trephine with 8 mm diameter is used to cut the calvarial bone (C) resulting in a scored calvarium (D). * indicates the anterior side of the cranium.

Figure 3

Use of the elevator. The elevator is used to gently lift the bone from the dura, first by (A) lifting the edge then (B) running the elevator under the surface to free any adherent dura. The exposed dura and brain beneath the defect are shown in (C). Additionally, a 4-0 Monocryl suture is seen loosely threaded through the periosteum indicated by the black arrowhead in (C). This technique of beginning the suture before trephination allows for easy apposition of the periosteal layer as it is very thin and delicate.

Figure 4

Scoring guide for extent of bony bridging and union. The gray areas in the circles represent mineralized tissue formed within the defect, which can be used for planar radiographs or microCT datasets. Reprinted with permission.

Figure 5

Representative planar radiographs of defects at 12 weeks from a study looking at release of plasmid DNA encoding BMP-2 from a hydrogel (OPF) with gelatin microparticles (CGMS). The images show specimens implanted with (A) OPF and CGMS, (B) OPF and 10 μg of pDNA in the CGMS phase, (C) CGMS and 10 μg of pDNA in the OPF phase and (D) CGMS and 100 μg of pDNA in OPF phase. Arrowheads indicate the areas of bone growth into the defect.

Figure 6

Histological sections cut coronally through the defect after 12 weeks of implantation. Ceramic (A–B) and polymeric (C–E) scaffolds were used in combination with platelet rich plasma (PRP) and/or bone marrow derived mononuclear cells (bmMNCs). The sections are stained with (A, D) Goldner’s trichrome, (B, E) hematoxylin and eosin and (C) von Kossa/van Gieson and show representative sections from the (A) ceramic scaffold with PRP group, (B) the ceramic scaffold with PRP and bmMNCs, (C) the polymeric scaffold with bmMNCs, (D) the polymeric scaffold with PRP and bmMNCs and (E) the polymeric scaffold alone. Arrows indicate mineralized tissue and arrowheads indicate non-mineralized osteoid. Scale bars for the full size images on the left represent 1 mm, while scale bars for the higher magnifications on the right represent 100 μm. Adapted with permission.

Figure 7

Fluorescent image of a coronally oriented histological section showing temporal fluorochrome labeled mineral deposition. This study implanted titanium meshes seeded with bone marrow derived marrow stromal cells transfected with an adenoviral vector of BMP-2. Red fluorescence is due to alizarin complexone injected at 1 week and green fluorescence is due to calcein injected at 3 weeks postoperatively. Insets (B) and (C) show high magnification (10X) images of the section indicated by the white boxes in the image of the full defect (A) at low magnification (2.5X). Reprinted with permission.