Animal models for bone tissue engineering and modelling disease

Abstract

Tissue engineering and its clinical application, regenerative medicine, are instructing multiple approaches to aid in replacing bone loss after defects caused by trauma or cancer. In such cases, bone formation can be guided by engineered biodegradable and nonbiodegradable scaffolds with clearly defined architectural and mechanical properties informed by evidence-based research. With the ever-increasing expansion of bone tissue engineering and the pioneering research conducted to date, preclinical models are becoming a necessity to allow the engineered products to be translated to the clinic. In addition to creating smart bone scaffolds to mitigate bone loss, the field of tissue engineering and regenerative medicine is exploring methods to treat primary and secondary bone malignancies by creating models that mimic the clinical disease manifestation. This Review gives an overview of the preclinical testing in animal models used to evaluate bone regeneration concepts. Immunosuppressed rodent models have shown to be successful in mimicking bone malignancy via the implantation of human-derived cancer cells, whereas large animal models, including pigs, sheep and goats, are being used to provide an insight into bone formation and the effectiveness of scaffolds in induced tibial or femoral defects, providing clinically relevant similarity to human cases. Despite the recent progress, the successful translation of bone regeneration concepts from the bench to the bedside is rooted in the efforts of different research groups to standardise and validate the preclinical models for bone tissue engineering approaches.

Keywords: 3D printing; BMPs; Bone defect; Bone metastasis; Bone regeneration; Cancer xenograft; Scaffolds; Tibia segmental defect.

Fig. 1.

Prevalent bone defect models. (A) Calvarial defects are generally created via the introduction of a circular burr hole and the subsequent removal of the resulting bone disk. The surgery is performed in a manner so as to not damage the underlying dura mater. (B) In the segmental bone defect model, a larger and completely penetrating bone defect is generated. A segment of the bone is surgically removed, leaving a large and non-joining wound area (gap) between the bone edges. The gap is usually stabilised with a fixation device and/or filled with a tissue-engineered bone substitute to stimulate bone healing and to study bone formation. (C) In the burr hole, or partial defect model, an incomplete hole is drilled into the side of the bone to create a wounded area. The burr hole usually penetrates the cortical bone and can extend into the underlying cancellous bone or the bone marrow cavity. In this model, usually only one side of the bone is wounded.

Fig. 2.

Surgery and scaffold/rhBMP-7 preparation. (A-G) Surgical generation of a segmental bone defect and implantation of a TE scaffold. To create a 3 cm segmental tibial defect, the bone was exposed and a dynamic compression plate was temporarily fixed with two screws (A). Subsequently, the screw holes were drilled, the defect middle and osteotomy lines were marked (B,C), and the bone segment was removed after osteotomy (D,E). The periosteum was removed 1 cm on the either end of the tibia defect site before the bone fragments were realigned (F) and fixed with plate and screws (G). (H-M) Top (H) and lateral (I) views of a cylindrical medical grade polycaprolactone tricalcium phosphate (mPCL-TCP) scaffold produced via fused deposition. Prior to transplantation, the scaffolds were surface treated with NaOH to render them more hydrophilic, as demonstrated in the scanning electron microscopy images prior to (J, inset) and after (J) NaOH treatment. To load the scaffolds with the recombinant human bone morphogenic protein BMP-7, the lyophilised BMP-7 was mixed with sterile saline and transferred to the inner duct of the scaffold and onto the contact interfaces between the bone and the scaffold (K,L). The BMP-7-augmented scaffolds were then implanted into the segmental tibial defects (M). (N-P) Representative X-ray images showing segmental tibial defects after 3 months of treatment with the scaffold only (N), the scaffold augmented with 1.75 mg rhBMP-7 (O) or the scaffold augmented with 3.5 mg rhBMP-7 (P), showing superior bone regeneration in the scaffolds with increasing amounts of rhBMP-7 loading due to the potent osteoinductive properties of rhBMP-7. Adapted from Cipitria et al. (2013).

Fig. 3.

Tissue-engineered ectopic bone formation for disease model research. (A) Tissue-engineered (TE) bone construct biomaterials consisting of hydrogel-, cell- or scaffold-based systems can be subcutaneously implanted into immunocompromised mouse models in order to create ectopic, humanised bone in a mouse-as-a-bioreactor-style system. (B) Ectopic bone can form through the process of endochondral ossification, whereby the bone is generated from a cartilage intermediate. Safranin O and Toluidine Blue are histochemical dyes that bind to proteoglycans and glycosaminoglycans and stained the cartilage tissue orange-red and purple, respectively, indicating endochondral ossification in TE bone constructs. Haematoxylin and Eosin (H&E) staining of the TE bone showed marrow infiltration into the bone organ, while pentachrome staining showed black nuclei, yellow bone tissue, green hyaline cartilage, dark red bone marrow and bright red unmineralised osteoid. Alcian Blue staining showed cartilage-associated extracellular matrix in blue and bone marrow in pink. Immunohistochemical staining for human-specific vimentin (hsVIM) demonstrated that the cellular components of the newly formed bone, apart from the bone marrow, were of human origin in cell-based TE bone constructs. (C) The humanised TE bone construct (hTEBC) implanted in a mouse model can be used for disease model research. Cancer cells may be introduced into the mouse system following intraosseous injection to study primary bone tumours and direct cancer-bone interactions, whereas intracardiac injection of cancer cells replicates experimental metastasis in the mouse circulation, allowing investigation of cancer cell homing to distant organ sites. Additionally, cancer cells can be injected at the orthotopic site (e.g. mammary fat pad for breast cancer or intraprostatic injection for prostate cancer studies) in order to study spontaneous metastasis from a primary tumour. (D) Histological examination of metastatic breast and prostate cancer cells (M) in a human TE bone construct with newly formed bone (NB) in vivo demonstrated tumour cells residing in the bone marrow (BM) in H&E-stained images. Tartrate-resistant acid phosphatase (TRAP) staining revealed osteoclastic (highlighted by pink staining) breast and prostate cancer metastases in the TE bone. (E) Representative H&E images of patient-derived breast and prostate cancer bone metastases highlight the similarity of the TE bone to the human disease. Adapted from Reinisch et al. (2015) and Martine et al. (2017).