Modeling Principle

The Sheep Lumbar Vertebral Bone Defect Model is a SCI gold-standard large-animal pathological model of spinal bone defect established by posterior spinal surgical fenestration to create standardized cylindrical cancellous bone lesions inside lumbar vertebral bodies. It accurately recapitulates the complete pathological cascade induced by spinal trauma and artificial bone defects during spinal fusion surgery, including bone marrow loss in local defect area, inflammatory cell infiltration, insufficient osteoblast activity, preferential fibrous connective tissue filling, poor spontaneous osseous healing of bone defects and spinal fusion failure, and compensates for experimental defects of rodent animals with tremendous differences from humans in spinal size, cancellous bone proportion, spinal weight-bearing biomechanics, bone remodeling cycle and local spinal immune microenvironment.

The physiological load of sheep lumbar spine, vertebral cancellous bone ratio, spatial arrangement of trabeculae, osteogenic differentiation potential of bone marrow mesenchymal stem cells, RANKL/OPG bone remodeling regulatory pathway and local spinal inflammatory response mechanism are highly homologous to human thoracolumbar spine. Paravertebral muscles are stripped layer by layer via posterior spinal approach to expose transverse processes and laminae. A cylindrical cancellous bone defect with fixed diameter and depth is drilled vertically into vertebral body through laminar fenestration, and cancellous bone and bone marrow tissues within the lesion are completely removed to destroy the natural osteogenic microenvironment of vertebral body. A large number of neutrophils and macrophages infiltrate in early defect stage, releasing pro-inflammatory factors TNF-α, IL-1β and matrix metalloproteinases MMPs to inhibit endogenous osteogenic signals, and fibrous connective tissue rapidly proliferates and occupies the defect cavity. With prolonged feeding period, only a small amount of scattered callus forms inside the defect, and complete osseous bridging healing cannot be achieved, presenting persistent bone defect filled with fibrous scar tissue to simulate the pathological state of nonunion and poor fusion of clinical surgical spinal bone defects. This model fully recapitulates the classic injury-repair cascade of human vertebral bone defects: cancellous bone and bone marrow destruction-local inflammatory activation-inhibited osteogenic potential-fibrous tissue occupation-obstacle of osseous healing.

Sheep lumbar spine bears weight load similar to human spine with sufficient vertebral volume, highly controllable defect size, long bone remodeling cycle and minimal intra-group individual dispersion, without spontaneous spinal degeneration and osteoporosis. It can clearly distinguish three-stage phenotypes including acute inflammatory stage of defect, fibrous connective tissue proliferative stage and terminal non-union stage of bone defect, serving as a standardized large-animal gold-standard model for translational research on spinal bone regeneration biomaterials, bone graft substitutes, osteogenic drugs and spinal fusion techniques.

Modeling Success Criteria

Macroscopic Spinal Imaging and Gross Vertebral Phenotype

The lumbar vertebral X-ray image of sham control group was dense and uniform with intact cancellous bone without cavities inside vertebral body, and the gross vertebral body was hard with continuous medullary trabeculae. At Week 12 terminal point, circular low-density defect area inside vertebral body could be seen on spinal X-ray of model group, and Micro-CT showed extremely low bone mineralization in defect cavity. Gross vertebral dissection revealed regular cavities filled with gray-white fibrous scar tissue without continuous trabecular bridging both ends of defect. The macroscopic phenotype of critical-sized bone defect nonunion was typical with extremely significant inter-group difference, confirming preliminary successful construction of lumbar vertebral bone defect model.

Quantitative Biochemical Gold-Standard Indexes of Serum

Serum pro-inflammatory factors TNF-α and IL-1β of model group were extremely significantly higher than sham control group; the expression of bone formation marker OC decreased remarkably while bone resorption marker CTX-Ⅰ rose slightly; RANKL was upregulated and OPG downregulated with elevated RANKL/OPG ratio; serum calcium decreased slightly and PTH elevated secondarily, fully matching the core biochemical diagnostic characteristics of post-spinal surgery bone defects: sustained local inflammatory activation, insufficient endogenous osteogenic capacity, unbalanced bone remodeling and blocked osseous healing.

Lumbar Vertebral Histopathological Characteristics

Combined HE, Goldner, Masson and ALP/TRAP double labeling staining of vertebral tissues showed characteristic temporal pathological changes of vertebral bone defects:

1. Acute stage at Week 4 after modeling: massive inflammatory cell infiltration in defect cavity, bone marrow mesenchymal stem cells accumulated at defect margin, no neocartilage tissue inside defect and loss of Goldner bone mineralization staining;
2. Middle proliferative stage at Week 8 after modeling: massive fibroblast proliferation inside defect cavity, disordered collagen fiber deposition to form fibrous connective tissue, only a small amount of ALP-positive osteoblasts distributed at defect margin and slightly increased TRAP osteoclasts;
3. Terminal non-union stage at Week 12 after modeling: defect cavity was completely filled with thick fibrous scar tissue without continuous trabecular osseous bridging, a small amount of scattered callus at defect margin and remarkably reduced intravertebral bone mineralization area, which perfectly recapitulates graded pathological characteristics of human spinal vertebral critical bone defects including fibrous occupation, insufficient osteogenesis and healing disorder.

Core Inflammatory, Osteogenic Differentiation and Bone Remodeling Pathway Indexes

The local vertebral TLR4/NF-κB inflammatory pathway of model group was persistently activated to inhibit the expression of Runx2 osteogenic transcription factor; unbalanced RANKL/OPG induced mild osteoclast activation, bone marrow mesenchymal stem cells showed differentiation bias toward fibroblasts with blocked osteogenic differentiation; secretion of bone mineralization-related proteins decreased significantly. It accurately conforms to the complete pathogenic and repair mechanism induced by laminar fenestration drilling: cancellous bone and bone marrow destruction-local inflammatory storm-inhibited osteogenic differentiation-fibrous tissue occupation-nonunion of vertebral bone defect, serving as core academic criterion for confirming successful modeling.

Model Advantages

This model is a well-recognized exclusive large-animal gold-standard model of laminar fenestration-induced vertebral critical bone defects in spinal regeneration SCI field. Standardized 12-week spinal surgical modeling stably constructs pathological phenotypes of vertebral cancellous bone defects, fibrous scar filling and osseous healing disorder highly homologous to humans. The lumbar spine weight-bearing biomechanics, vertebral cancellous bone microenvironment, osteogenic potential of bone marrow stem cells and bone remodeling cycle of sheep are highly matched with human thoracolumbar spine, with uniform osteogenic repair gradient of defects, extremely low intra-group data dispersion and far higher reproducibility than small animals such as rodents and rabbits. The modeling simulates artificial bone defects during clinical spinal fusion surgery without drug and hormone interference, and accurately recapitulates the core pathological process of postoperative spinal nonunion. Bilateral L3/L4 vertebral bodies of single animal can be modeled synchronously to save experimental animals with sufficient vertebral tissue samples. It is suitable for preclinical efficacy and safety evaluation of bone graft materials, three-dimensional porous scaffolds, local injection osteogenic drugs and matching repair materials for spinal fusion internal fixation. Multi-dimensional detections including imaging, biochemistry, pathology and molecular biology are complete with extremely high clinical transformation credibility and high recognition in high-score SCI journals of spinal surgery, orthopedic tissue engineering and bone regeneration, suitable for National Natural Science Foundation, master/doctor project opening, graduation thesis of spinal surgery/orthopedics and translational medical research of spinal bone regeneration.

Research Applications

The Sheep Lumbar Vertebral Bone Defect Model is corely applied to basic research on local inflammatory activation induced by vertebral cancellous bone and bone marrow destruction, Runx2 osteogenic transcription inhibition, fibrous differentiation bias of bone marrow stem cells, fibrous connective tissue occupation and vertebral osseous healing disorder. It is specially used for screening and evaluating porous bone repair scaffolds, autologous/allogeneic bone graft substitutes, locally sustained-release osteogenic drugs and natural active osteogenic extracts with effects of inhibiting local defect inflammation, promoting osteogenic differentiation of bone marrow stem cells, elevating vertebral bone mineralization, accelerating trabecular bridging, reducing fibrous scar filling and ameliorating osseous healing of spinal defects. It is widely adopted for excavation of regenerative targets of spinal bone defects, elucidation of regulatory network between local vertebral inflammation and osteogenic differentiation, and large-animal preclinical in-vivo verification of spinal bone repair materials and drugs, serving as a scarce and essential standardized large-animal gold-standard model in the fields of spinal surgery, bone tissue engineering and bone regeneration pharmacology.

sheep lumbar vertebral bone defect model, vertebral cancellous bone lesion, spinal bone regeneration repair, bone graft biomaterial preclinical evaluation