Modeling Principle

The Sheep Femoral Defect Model is a standardized weight-bearing long bone critical-sized segmental bone defect pathological model recognized by SCI, which is constructed via mid-femoral osteotomy combined with plate internal fixation. It accurately reproduces the pathological characteristics after high-energy trauma, resection of bone tumor lesions and debridement of osteomyelitis in clinic, including osseous defect of weight-bearing femur, destruction of native bone marrow osteogenic units in medullary cavity, massive loss of periosteal osteoblasts, insufficient spontaneous creeping osteogenesis capacity of bone ends, preferential filling of defect area by fibrous connective tissue, failure of physiological osseous bridging healing, as well as long-term formation of bone nonunion and invalid mechanical support of limbs. It overcomes the experimental deficiencies of rodents and rabbits with small body size, which have huge gaps with human lower limb weight-bearing bones in femoral load bearing, cortical bone thickness, medullary microenvironment, long bone remodeling cycle and osteogenic response under mechanical stimulation.

The anatomical stratification of adult sheep femur, compactness of cortical bone, distribution of medullary cancellous bone, physiological load of lower limbs, activity of periosteal stem cells, dual repair mechanism of intramembranous and endochondral ossification, RANKL/OPG bone metabolic axis and response rule of TGF-β/BMP osteogenic signaling pathway are highly homologous to human femur. Animals are fixed in lateral position under general anesthesia. Subcutaneous tissue, tensor fasciae latae and muscle groups are incised layer by layer via lateral approach, and the middle segment of femoral shaft is exposed by dissection. An oscillating saw is adopted to resect full-thickness cortical bone and medullary tissues of femur with fixed length to create critical segmental bone defects. Locking compression plate and screws are implanted to fix the proximal and distal femur to maintain mechanical stability of limbs, and partial peripheral periosteum is preserved to reduce excessive trauma. In-situ bone marrow stromal cells and vascular networks in defect area are thoroughly destroyed, and the body only relies on a small amount of surviving bone tissue at both ends of the defect to generate bone callus slowly. After organization of blood clot inside the defect cavity, it is rapidly filled by fibroblasts and collagen tissues to form a fibrous scar barrier preventing bone callus from growing across the defect. Only a small number of punctate calluses can be observed during the observation period without continuous trabecular bridging penetrating the defect, stably forming a pathological phenotype of permanent nonunion of weight-bearing femur. This model fully recapitulates the classic repair cascade of human weight-bearing long bone defects: segmental femoral tissue defect – destruction of medullary osteogenic microenvironment – shortage of osteogenic cells – fibrous tissue occupying filling – spontaneous osseous healing disorder of critical defects – weight-bearing bone nonunion.

Sheep have large body size and femoral load close to human lower limbs. The osteotomy length can be precisely set to construct standardized critical defects, and plate internal fixation eliminates limb movement variables. The bone repair cycle is long and stable with low discrete degree of individual data, and there are no spontaneous skeletal metabolic diseases. Three pathological stages can be clearly divided: acute inflammatory blood clot stage of defects, fibrous tissue proliferation and occupation stage, and terminal permanent nonunion stage with restricted callus growth. It serves as a standardized large-animal gold-standard model for translational researches on weight-bearing bone repair scaffolds, absorbable internal fixation materials, stem cell composite bone grafts, locally sustained-release osteogenic drugs and clinical treatment strategies for fracture nonunion.

Modeling Success Criteria

In-vivo Imaging, Limb Function and General Femoral Phenotype

X-ray and Micro-CT of femur in the sham control group showed continuous and intact cortical bone with regular trabecular arrangement, and the animals walked normally with weight bearing on the affected limbs. Micro-CT of the model group at Week 16 showed extremely low BV/TV value in the defect area with defect closure rate less than 10%, only scattered punctate bone callus existed at the defect margin. Gross anatomical observation revealed that the defect cavity was filled with thick gray-white fibrous tissues without trabecular bridging between femoral broken ends, accompanied by slight claudication and reduced weight bearing during walking of the affected limb. The macroscopic phenotype of critical nonunion of weight-bearing femoral defects was typical with extremely significant statistical difference between groups, which could confirm the qualified construction of the sheep femoral defect model.

Quantitative Biochemical Gold-Standard Serum Indexes

The serum levels of TNF-α and IL-1β in the acute phase of the model group were significantly higher than those in the sham group; the expression of bone formation markers OC and ALP was down-regulated in the middle and late observation period, while the bone resorption marker CTX-Ⅰ increased compensatorily; the expression levels of osteogenic proteins TGF-β1 and BMP-2 in local defect tissues decreased obviously, which fully matches the biochemical diagnostic characteristics of clinical weight-bearing long bone defects: local traumatic inflammatory response, decreased endogenous osteogenic activity, excessive proliferation & deposition of fibrous tissue and blocked osseous healing process.

Histopathological Characteristics of Femoral Defect Tissues

Sequential pathological evolution law of critical femoral defects can be observed via combined multiple staining of defect tissues with HE, Masson, Goldner and ALP/TRAP:

1. Acute stage at Week 4 after modeling: blood clots accumulated in defect cavity with massive inflammatory cell infiltration, and a small number of ALP-positive osteoblasts could be seen at the edge of defect bone ends;
2. Middle fibrous proliferation stage at Week 10 after modeling: disorderly arranged blue-stained collagen fibers continuously proliferated to fill the defect space, the number of osteoblasts decreased significantly, and almost no new bone mineralization signal was detected by Goldner staining;
3. Terminal stage at Week 16 after modeling: thick collagen fibers formed dense scar tissue occupying the whole defect cavity, no continuous trabecular bridging existed between the proximal and distal femoral broken ends, only trace mineralized matrix existed on the surface of broken ends. It perfectly recapitulates the progressive pathological process from inflammatory infiltration and fibrous proliferation to permanent bone nonunion of human critical weight-bearing femoral defects.

Gold-Standard Indexes for Osteogenic and Inflammatory Pathways

Surgical osteotomy trauma in the model group activated continuous activation of NF-κB inflammatory pathway in the defect area and inhibited the conduction of BMP/TGF-β osteogenic signal axis. Fibroblasts inside the defect cavity were continuously induced to activate and secrete a large amount of collagen fibers to physically block the creeping migration of bone cells toward the defect center. The activity of limited osteoblasts at both ends of the defect was suppressed by the local inflammatory microenvironment, failing to synthesize sufficient mineralized bone matrix to complete osseous bridging repair of the defect. It accurately conforms to the complete pathological mechanism of femoral defects constructed by surgical osteotomy: segmental femoral bone loss – continuous activation of local chronic inflammation – inhibition of osteogenic signaling pathway – growth of fibrous scar occupation – permanent nonunion of critical weight-bearing bone defects, serving as the core academic basis for verifying effective model formation.

Model Advantages

This model is a well-recognized large-animal gold-standard model for sheep critical femoral defects prepared by osteotomy combined with plate fixation in orthopedics and bone biomaterial SCI fields. The 16-week standardized observation cycle can stably reproduce pathological phenotypes of traumatic inflammation, fibrous scar hyperplasia, insufficient endogenous osteogenesis and permanent bone nonunion under weight-bearing environment highly consistent with human lower limb weight-bearing bones. The femoral size, cortical thickness, physiological load, bone repair kinetics and mechanical response law of sheep are highly matched with human femur. The defect size is controllable with mild lesion progression and low intra-group individual differences, and the experimental repeatability is far superior to small animals such as rodents and rabbits. Plate internal fixation is adopted to maintain mechanical stability, with a single variable only retaining the damage of the defect itself, which conforms to the real scenario of internal fixation treatment after clinical fracture defects. A standard defect can be set on the unilateral femur of a single animal with abundant specimen materials, and multi-dimensional evaluation including imaging, gait, biochemistry, pathology and molecules can be carried out simultaneously. It is suitable for preclinical efficacy and safety evaluation of load-bearing bone scaffolds, absorbable internal fixations, stem cell bone repair systems, locally sustained-release bone active factors and therapeutic drugs for fracture nonunion. Experimental data 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 writing, orthopedic graduation theses and translational medical researches on weight-bearing bone defect repair.

Research Applications

The Sheep Femoral Defect Model is mainly used to analyze basic pathological mechanisms including local traumatic inflammation induced by surgical osteotomy removing femoral cortex and medullary tissues, loss of in-situ osteoblasts, inhibition of BMP osteogenic pathway, massive collagen deposition due to fibroblast activation, obstruction of bone callus creeping by fibrous scars, and spontaneous osseous healing disorder of critical weight-bearing femoral defects. It is specially adopted to screen and verify load-bearing porous bone scaffolds, absorbable implant materials, stem cell composite bone grafts, natural plant osteogenic extracts and sustained-release drugs matched with orthopedic implants with functions of inhibiting local inflammatory microenvironment of defects, improving osteogenic differentiation potential of bone marrow stromal cells, up-regulating the expression of BMP/TGF-β osteogenic signals, reducing disordered deposition of collagen fibers, inducing penetrating growth of new trabecular bone in defect areas and realizing osseous repair of weight-bearing femoral defects. It is widely used for excavation of regenerative targets for weight-bearing bone defects, elucidation of regulatory network between inflammation and osteogenic differentiation, as well as preclinical in-vivo efficacy verification of various bone repair medical products in large animals. It is an indispensable essential standardized large-animal gold-standard model in the fields of orthopedic implant development, bone tissue engineering and weight-bearing bone regenerative medicine.

sheep femoral critical segmental bone defect model, weight-bearing long bone defect repair, in-vivo evaluation of bone graft materials, bone regenerative biomaterials, orthopedic implant preclinical research