Half-segmental Diaphyseal Bone Defect Model in Rats for Evaluating Bone Substitute Performance in Load-bearing Regions

We aim to establish a reproducible femoral defect model to evaluate mechanical and osteogenic performance of load-bearing bone materials. The main challenge is the lack of a standardized reproducible load-bearing defect model for realistic in vivo mechanical assessment. To begin place the anesthetized rat in a lateral recumbent position on a sterile surgical bench.

Shave the lateral thigh area aligned with the femur projection and disinfect the skin using 2%iodine tincture, followed by 75%ethanol. Drape the surgical site using a sterile fenestrated sheet to maintain asepsis. Make a two to three centimeter longitudinal skin incision along the lateral aspect of the thigh.

Identify the rectus femoris and vastus lateralis muscles and use a straight scissor to carefully separate the muscles along the visible white fascia. Then with a disposable sterile scalpel, make a longitudinal incision along the muscle attachments to access the femoral surface. Perform blunt dissection using a periosteal elevator to detach the muscle attachments and fully expose the mid diaphysis of the femur.

Drill vertically downward at the defect site using the bur until a sudden loss of resistance is felt. Enlarge the defect by applying controlled horizontal push-pull motions centered around the initial point of penetration. Extend the defect longitudinally along the femoral axis and laterally along the femoral diameter to gradually form a semi cylindrical defect.

Next, insert the load-bearing 3D printed Poly Methyl or PMMA implant intended for mechanical performance testing into the defect site, ensuring a snug fit. Then using a needle holder, suture the muscle layers with 4-0 monofilament sutures, ensuring there is no excessive tension. Finally, close the skin with interrupted sutures and disinfect the surgical site using 2%iota 4 solution.

At four weeks post-operation, micro computed tomography showed that femurs implanted with Gelatin methacryloyl hydrogel exhibited concave defect areas and extensive ectopic ossification within the medullary cavity. In contrast, femurs in the 3D printed PMMA group showed restored external morphology with the defect area supported by aligned new bone and minimal ectopic ossification in the medullary cavity. Hematoxlin and Eosin staining revealed that the Gelatin methacryloyl group contained abundant fibrous tissue within the defect with dark staining ectopic bone nearly occluding the medullary cavity and no visible periosteal formation or cortical bone continuity.

In the 3D printed PMMA group, newly formed bone filled the scaffold void connected with cortical bone at the defect edges and was covered by continuous periosteal bone with no significant ectopic ossification in the marrow. Immunofluorescence staining showed very few Piezo 1 positive and LepR positive cells in regenerated bone in the Gelatin methacryloyl group. In contrast, the 3D printed PMMA group displayed abundant Piezo one positive cells and LepR positive cells in the newly formed bone at the defect site.

Quantitative analysis confirmed that both Piezo 1 positive and LepR positive cell counts were significantly higher in the PMMA group compared to the Gelatin methacryloyl group. Our protocol enables in vivo mechanical evaluation of implants without fixation, offering a simple and reproducible load-bearing defect model.