Modeling Principle

The Beagle Radial Bone Defect Model is a SCI gold-standard large-animal pathological model of weight-bearing long bone defects established by surgical osteotomy to create standardized critical-sized bone defects at the middle segment of bilateral radii. It accurately recapitulates the complete pathological cascade induced by traumatic bone defects, bone loss after curettage of bone tumor lesions and bone defects left by debridement of infection in clinical practice, including destruction of bone marrow tissue in defect area, local acute inflammatory infiltration, insufficient endogenous osteogenic potential, preferential occupation by fibrous connective tissue and nonunion caused by failed spontaneous osseous healing of bone defects. This model makes up for the experimental deficiencies of rodent animals, which have prominent differences from human long bones of extremities in slender long bone diameter, limb weight-bearing biomechanics, long bone remodeling cycle, cortical/cancellous bone ratio and osteogenic differentiation characteristics of bone marrow mesenchymal stem cells.

The anatomical morphology of radius, cortical bone thickness, medullary cancellous bone distribution, physiological weight-bearing load of limbs, homeostasis of bone marrow microenvironment, RANKL/OPG bone remodeling regulatory axis and local injury inflammatory response pathway of adult Beagle dogs are highly homologous to human forearm long bones. The forearm soft tissues are incised layer by layer via surgical operation, and the radial muscles are dissected to expose the middle diaphysis of radius. A bone oscillating saw is adopted to completely resect a fixed length of radial cortical bone and medullary cancellous bone tissue to form a critical bone defect structure without autologous bone bridging, resulting in thorough destruction of the native bone marrow osteogenic microenvironment. In the early stage of defects, a large number of neutrophils and macrophages are recruited and infiltrated, secreting pro-inflammatory factors TNF-α, IL-1β and matrix metalloproteinases MMPs to block the transmission of endogenous osteogenic signals. Fibroblasts proliferate rapidly inside the defect cavity to form fibrous scar tissue occupying the defect space. With the extension of feeding period, only a small amount of sparse callus forms at the defect margin, failing to generate osseous bridging structure connecting both ends of the defect, thus stably presenting the pathological state of long bone nonunion. This model fully recapitulates the classic injury-repair cascade of human long bone defects in extremities: surgical destruction of bone tissue-local inflammatory outbreak-inhibition of endogenous osteogenesis-occupational filling by fibrous tissue-obstacle of osseous healing in critical bone defects.

Bilateral radii of Beagle dogs can be modeled synchronously with stable and controllable weight-bearing mechanics, the defect size can be accurately standardized, the bone repair sequence is stable with low discrete degree of individual data, and there are no spontaneous bone metabolic abnormalities or osteoarthrosis degeneration. Three-stage pathological phenotypes can be clearly divided: acute inflammatory stage of defects, fibrous tissue proliferation and occupation stage, and terminal permanent nonunion stage of bone defects. It serves as a standardized large-animal gold-standard model for translational researches on bone repair scaffold materials, artificial bone graft substitutes, locally sustained-release osteogenic drugs, stem cell composite bone preparations and intervention strategies for fracture nonunion.

Modeling Success Criteria

Macroscopic Skeletal Imaging and Limb Gross Phenotype

Beagle dogs in the sham control group bore weight evenly on limbs with normal walking gait; radial X-ray showed continuous and intact cortical bone with uniform and dense medullary cavity structure, and the gross radial bone was hard with intact skeletal continuity. At Week 12 terminal point, the affected limbs of model group presented slight claudication and reduced weight-bearing performance. Obvious low-density transmission zone could be observed at osteotomy area on radial X-ray, and Micro-CT detection showed extremely low bone mineralization value in defect area. Gross dissection of radial defect cavity revealed that gray-white dense fibrous scar tissues filled the defect space without osseous callus bridging between both ends of defects. The macroscopic phenotype of critical bone defect nonunion was typical with extremely significant statistical difference between groups, which could preliminarily confirm the successful construction of the radial bone defect model.

Quantitative Biochemical Gold-Standard Serum Indexes

The levels of serum inflammatory factors TNF-α and IL-1β in the model group were extremely significantly higher than those in the sham group; the content of bone formation marker OC decreased remarkably while the bone resorption marker CTX-Ⅰ rose slightly compensatorily; RANKL was upregulated and OPG was downregulated with obviously elevated RANKL/OPG ratio; serum calcium ion decreased slightly accompanied by secondary elevation of PTH, which fully matches the biochemical diagnostic characteristics of clinical long bone defects: sustained local inflammatory activation, attenuation of endogenous osteogenic capacity, unbalanced systemic bone remodeling and blocked osseous healing.

Radial Defect Histopathological Characteristics

Combined observation of radial defect tissues via HE, Goldner trichrome, Masson and ALP/TRAP double staining can identify sequential pathological changes of radial critical defects:

1. Acute stage at Week 4 after modeling: massive inflammatory cell infiltration in defect cavity, bone marrow stem cells gathered at the edge of defect section, no neocortical bone tissues inside defects and no positive bone mineralization signal in Goldner staining;
2. Middle proliferative stage at Week 8 after modeling: massive fibroblast proliferation inside defect cavity and disordered collagen fiber deposition to form fibrous connective tissue, only a small amount of ALP-positive osteoblasts distributed at defect margin with slightly increased number of TRAP osteoclasts;
3. Terminal nonunion stage at Week 12 after modeling: the defect cavity was completely filled with thick and disorderly arranged fibrous scar tissues without trabecular osseous bridging connecting both ends of defects, only scattered punctate callus existed at defect margin, and the area of Goldner bone mineralization staining decreased significantly. It perfectly recapitulates the graded pathological characteristics of fibrous occupation, insufficient osteogenesis and permanent bone nonunion of human critical long bone defects in extremities.

Core Indexes of Inflammation, Osteogenic Differentiation and Bone Remodeling Pathways

The local TLR4/NF-κB inflammatory pathway of defects in the model group was persistently activated to inhibit the expression of Runx2 core osteogenic transcription factor. The unbalanced RANKL/OPG ratio induced mild activation of osteoclasts, bone marrow mesenchymal stem cells showed differentiation bias toward fibroblasts and the normal osteogenic differentiation process was blocked. The secretion of bone mineralization-related proteins decreased significantly. It accurately conforms to the complete repair pathological mechanism of radial defects constructed by surgical osteotomy: damage of bone tissue defects-local inflammatory storm-Runx2-mediated osteogenic inhibition-proliferation and occupation of fibrous tissue-permanent long bone nonunion, serving as the core academic basis for verifying effective model formation.

Model Advantages

This model is a well-recognized exclusive large-animal gold-standard model for radial critical bone defects constructed by surgical osteotomy in orthopedic tissue engineering SCI field. The 12-week standardized surgical modeling can stably form pathological phenotypes of long bone filled with fibrous scars, insufficient endogenous osteogenesis and permanent bone nonunion highly homologous to humans. The radial anatomical structure, limb weight-bearing biomechanics, osteogenic potential of bone marrow stem cells and bone remodeling cycle of Beagle dogs are highly matched with human forearm long bones. The lesion gradient of defect repair is uniform and mild with low discrete degree of individual intra-group data, and the experimental repeatability is far superior to small experimental animals such as mice, rats and rabbits. The modeling mode adopts pure physical surgical defect construction without interference from exogenous substances such as chemical reagents and hormones, which highly restores the pathological state of long bone defects in extremities left after clinical trauma and debridement. Bilateral radii of a single animal can be modeled synchronously to save the dosage of experimental animals with sufficient bone tissue samples. Multi-dimensional detection and analysis including imaging, biochemistry, pathology and molecules can be carried out simultaneously. It is suitable for preclinical pharmacodynamic and safety evaluation of porous bone repair scaffolds, composite stem cell bone graft materials, locally sustained-release osteogenic drugs and clinical intervention schemes for fracture nonunion. Data obtained from this model are widely recognized by high-impact SCI journals of orthopedics, biomaterials and bone regeneration, which is applicable for National Natural Science Foundation projects, master & doctoral proposal reports, orthopedic graduation theses and translational medical researches on regenerative repair of limb bone defects.

Research Applications

The Beagle Radial Bone Defect Model is mainly applied to analyze basic pathological mechanisms related to bone tissue defects caused by surgical osteotomy, activation of local inflammatory response in defects, inhibited osteogenic differentiation of bone marrow stem cells, massive fibroblast proliferation & collagen deposition, fibrous scar occupation and osseous nonunion of long bone critical defects. It is specially adopted to screen and verify porous bone scaffold materials, stem cell composite bone grafts, locally sustained-release osteogenic small-molecule drugs and natural plant osteogenic active extracts with effects of inhibiting local inflammatory response in defects, improving osteogenic differentiation capacity of bone marrow stem cells, upregulating Runx2 osteogenic transcription signal, reducing abnormal deposition of collagen fibers, promoting nascent trabecular bridging in defect areas and accelerating osseous healing of bone defects. It is widely used for excavation of regenerative targets of limb long bone defects, elucidation of regulatory network between inflammation and osteogenic differentiation, and preclinical in-vivo efficacy verification of various bone repair products in large animals. It acts as an indispensable essential standardized large-animal gold-standard model in the fields of orthopedic tissue engineering, bone regeneration pharmacology and fracture repair intervention.

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