Name: Half-segmental Diaphyseal Bone Defect Model in Rats for Evaluating Bone Substitute Performance in Load-bearing Regions (Video) | JoVE Journal
Uploaded: 2025-12-30T13:45:00.000+00:00
Duration: 4 min 32 s
Description: Learn to establish a rat femoral half-segmental defect model for evaluating bone substitute performance under load-bearing conditions without fixation. This protocol enables reproducible in vivo mechanical assessment of implants.

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5 minutes

This study was supported by the Natural Science Foundation of Sichuan Province (2025ZNSFSC0754).

All animal procedures conducted in this study received approval from the Ethical Committee of the West China School of Stomatology, Sichuan University (WCHSIRB-AT-2025-342). Male Sprague-Dawley rats (180 g each) were obtained from a commercial source and randomly assigned to two groups (n = 6 per group) according to the implanted material. The reagents and the equipment used are listed in the Table of Materials.
1. Pre-operative preparation
<ol>
	<li>Surgical items
	<ol>
		<li>Prepare surgical instruments referring to Figure 1A, corresponding to the numbered labels in the figure, in the following order: curved forceps, disposable sterile scalpel, straight scissors, periosteal elevator, irrigation needle, a specific dental diamond bur (see the enlarged view in Figure 1B), high-speed handpiece, vernier caliper, surgical motor, monofilament suture (4-0), and needle holder.</li>
		<li>Prepare 0.9% sterile saline solution for irrigation.</li>
	</ol>
	</li>
	<li>Sterilization and disinfection
	<ol>
		<li>Sterilize surgical instruments that can withstand autoclaving, including curved forceps, scissors, periosteal elevator, dental diamond bur, high-speed handpiece, and needle holder. 
		NOTE: Instruments should be wrapped in medical non-woven fabrics and sealed with autoclave tape for steam sterilization at 121-134 &#176;C for 20-25 min.</li>
		<li>For heat-sensitive instruments, such as vernier calipers and surgical motor, expose them to ultraviolet light for at least 30 min for disinfection. Disposable items, such as the disposable sterile scalpel, irrigation needle, and sutures, should remain sealed in their original sterile packaging until use.</li>
		<li>Ensure the surgical environment is sterile and temperature-controlled. Wipe down the surgical bench and surrounding surfaces with 75% ethanol. Use sterile, medical-grade non-woven fabrics to cover the operating table. 
		CAUTION: Ethanol is flammable; handle away from ignition sources and store in closed containers. Collect ethanol waste in designated chemical waste containers for proper disposal. Wear appropriate personal protective equipment, including gloves, a lab coat, and safety goggles.</li>
	</ol>
	</li>
	<li>Anesthesia
	<ol>
		<li>Weigh the rat and calculate the anesthetic dosage (100 mg/kg of ketamine and 10 mg/kg of xylazine, intraperitoneally).</li>
		<li>Induce anesthesia 20 min before surgery (following institutionally approved protocols) and confirm its depth by checking for the absence of response to toe pinch and the loss of the righting reflex.</li>
		<li>Apply ophthalmic ointment to both eyes to prevent corneal drying during the procedure.</li>
	</ol>
	</li>
</ol>
2. Surgical procedure
<ol>
	<li>Site preparation
	<ol>
		<li>Place the rat in a lateral recumbent position on the sterile surgical bench. Shave the lateral thigh area corresponding to the femur projection and disinfect the skin using 2% iodine tincture, followed by 75% ethanol (Figure 2Ba). 
		CAUTION: Iodine tincture is flammable and irritant. Handle in a well-ventilated area while wearing gloves, a lab coat, and safety goggles. Dispose of used wipes and solutions in designated chemical waste containers according to institutional safety regulations.</li>
		<li>Change into fresh sterile gloves. Drape the surgical site with the sterile fenestrated sheet to maintain asepsis.</li>
	</ol>
	</li>
	<li>Surgical site opening
	<ol>
		<li>Use curved forceps to assist in stabilizing the tissues, holding the muscles, and gently securing the femur throughout the surgical procedure (Figure 2Bb-h).</li>
		<li>Make a 2-3 cm longitudinal skin incision over the lateral aspect of the thigh with a sterile scalpel blade to expose the underlying muscles (Figure 2Bb).</li>
		<li>Identify the rectus femoris and vastus lateralis muscles, and carefully separate them along the visible white fascia (indicated by the yellow arrow in Figure 2Bb) using a straight scissor (Figure 2Bc).</li>
		<li>Locate and expose the muscle attachments on the lateral surface of the femur (Figure 2Bd).</li>
		<li>Make a longitudinal incision along the muscle attachments with a disposable sterile scalpel to gain access to the femoral surface (Figure 2Be). 
		NOTE: When performing sharp dissection (using a scissor or scalpel) for soft tissue separation, take care not to damage the underlying blood vessels and nerves. If unfamiliar with the anatomical structures, use fine-tipped forceps and a periosteal elevator to perform blunt dissection.</li>
		<li>Perform blunt dissection using a periosteal elevator to detach muscle attachments and fully expose the mid-diaphysis of the femur (Figure 2Bf).</li>
		<li>Further separate the femur from the surrounding muscles and hold the midshaft of the femur using curved forceps (Figure 2Bg).</li>
	</ol>
	</li>
	<li>Half-segmental diaphyseal bone defect model establishment
	<ol>
		<li>Attach the flat-ended cylindrical specific dental diamond (Figure 1B) bur to the high-speed handpiece, powered by the surgical motor. Set the surgical motor to 35,000 rpm.</li>
		<li>Under direct visualization, identify the highest point of the femoral midshaft as the anatomical center for defect creation. Drill vertically downward at this point using the bur until a sudden loss of resistance is observed, accompanied by the appearance of blood effusion within the defect, which indicates full cortical penetration and access to the medullary cavity (Figure 2Bh). 
		NOTE: Given inter-animal variability in cortical thickness, operators are advised to familiarize themselves with femoral anatomy and to perform pre-experimental training to standardize this step.</li>
		<li>Enlarge the defect by applying controlled horizontal push-pull motions centered around the initial penetration point. Extend the defect longitudinally (along the femoral axis) and laterally (along the femoral diameter) to gradually form a standardized semi-cylindrical defect measuring 4 mm in length and 1.5 mm in radius (Figure 2Bi, see Figure 2A for the morphology and position of the defect). 
		NOTE: Once the center point is established, use a vernier caliper to assist in stepwise defect enlargement, ensuring uniform dimensions across all specimens. The flat-end cylindrical design of the specialized dental bur matches the semi-cylindrical defect, allowing efficient and reproducible preparation.</li>
		<li>Continuously irrigate the surgical site with sterile saline using the irrigation needle to prevent thermal necrosis during bone cutting.</li>
	</ol>
	</li>
	<li>Bone substitute implantation
	<ol>
		<li>Gently remove bone debris and irrigate the site to clear residual particles.</li>
		<li>Implant non-load-bearing Gelatin methacryloyl (GelMA) hydrogels (see Table of Materials) or load-bearing 3D-printed polymethyl methacrylate (3DP-PMMA) intended for mechanical performance testing into the defect site, ensuring a snug fit (Figure 2Bj). 
		NOTE: The two biomaterials used here were deliberately selected to represent contrasting mechanical and biological properties: GelMA hydrogel serves as a soft, non-load-bearing material with limited osteogenic potential, whereas 3DP-PMMA acts as a rigid, load-bearing scaffold capable of effective stress transmission and supporting bone formation14 (Figure 2A). This contrast highlights the model&#39;s ability to evaluate implants with a wide range of mechanical strength and biological performance, and in practice, the defect model can be applied to test other candidate materials with mechanical properties.</li>
	</ol>
	</li>
	<li>Surgical site closure
	<ol>
		<li>Suture the muscle layers using 4-0 monofilament sutures and a needle holder, taking care to avoid excessive tension (Figure 2Bk).</li>
		<li>Perform skin closure with interrupted sutures and disinfect the surgical site using 2% iodophor solution (Figure 2Bl).</li>
	</ol>
	</li>
</ol>
3. Post-operative care
<ol>
	<li>Transfer the rats onto a disinfected, constant heating pad (37 &#176;C) to allow post-operative recovery.</li>
	<li>Administer appropriate post-operative analgesia (e.g., buprenorphine at 0.03 mg/kg subcutaneously every 12 h for 48-72 h).</li>
	<li>Once the rats regain consciousness, place them in clean cages with soft bedding and easy access to food and water.</li>
	<li>Monitor the surgical site daily for signs of infection, dehiscence, or abnormal swelling. Observe the rats&#39; behavior, weight, and mobility for at least 7 days post-surgery to ensure recovery and assess welfare.</li>
</ol>
4. Sample harvesting and data collection
<ol>
	<li>Sample collection
	<ol>
		<li>Euthanize the rats with carbon dioxide inhalation followed by secondary confirmation (following institutionally approved protocols).</li>
		<li>Harvest the femur specimens at 4 weeks post-implantation with animals initially randomized into two material groups (n = 6 per group) to assess the materials&#39; mechanical performance. 
		NOTE: In the GelMA group, three animals developed complete fractures shortly after surgery and were excluded, leaving three specimens available for subsequent analyses.</li>
		<li>Fix the femoral specimens in 4% paraformaldehyde for 48 h at 4 &#176;C. 
		CAUTION: Paraformaldehyde is toxic and should be handled in a certified fume hood. Wear gloves, a lab coat, and safety goggles or a face shield. Collect waste solutions in labeled hazardous chemical waste containers for disposal.</li>
	</ol>
	</li>
	<li>Micro-computed tomography (&#181;CT) evaluation
	<ol>
		<li>Perform &#181;CT scanning on femoral specimens collected at 4 weeks post-implantation with the following scanning parameters: X-ray tube potential, 70 kVp; X-ray intensity, 0.2 mA; filter, AL 0.5 mm; integration time, 1 x 300 ms; and voxel size, 17 &#181;m.</li>
		<li>Perform 3D reconstruction using the predefined scripts provided in the &#181;CT machine software. Generate cross-sectional images of the defect site using the reslice function of dedicated image-processing software. Follow the standard workflows as described in the respective software manuals.</li>
	</ol>
	</li>
	<li>Histological staining
	<ol>
		<li>Decalcify the samples at 4 &#176;C using 12% ethylenediaminetetraacetic acid (EDTA) (pH 7.2) for 6 weeks on a shaker. 
		NOTE: Replace the EDTA solution every 2 days. Verify decalcification progress regularly. Decalcification is complete when the sample is pliable under pressure or passes a needle. 
		CAUTION: Dispose of EDTA solutions according to institutional guidelines for aqueous chemical waste. Contaminated biological material should be treated as biohazardous waste.</li>
		<li>Dehydrate the samples in a graded series of ethanol dilutions and then embed them in paraffin.</li>
		<li>Cut the paraffin-embedded samples sagittally into 6 &#181;m-thick slices. 
		NOTE: Ensure sections include the interface between the implanted material and host tissue.</li>
		<li>Perform hematoxylin-eosin (H&#38;E) and Masson&#39;s trichrome staining using commercially available kits (see Table of Materials). Carry out all procedures strictly according to the manufacturer&#39;s standard protocols without any deviations. Observe defect site healing by histopathology. 
		CAUTION: Dispose of staining solutions and contaminated consumables in accordance with institutional biosafety and hazardous waste disposal protocols.</li>
	</ol>
	</li>
	<li>Immunofluorescent (IF) staining
	<ol>
		<li>Deparaffinize and rehydrate 6 &#181;m-thick paraffin sections using a series of xylene and graded ethanol solutions. 
		CAUTION: Xylene is flammable and toxic. Handle xylene in a fume hood while wearing gloves, a lab coat, and safety goggles. Collect waste solvents separately in chemical waste containers for appropriate disposal.</li>
		<li>Perform antigen retrieval by incubating the slides in citrate buffer (pH 6.0) at 95-100 &#176;C for 15-20 min. Allow the slides to cool naturally to room temperature.</li>
		<li>Wash the sections with phosphate-buffered saline (PBS) for 5 min and treat the sections with 0.1% Triton X-100 in PBS for 20 min to facilitate antibody penetration. Then block non-specific binding sites with 5% bovine serum albumin (BSA) in PBS for 1 h at room temperature. 
		CAUTION: Triton X-100 is an irritant; handle with gloves, a lab coat, and safety goggles in a ventilated area. Dispose of Triton X-100 solutions as chemical waste in designated containers. For all procedures, solutions that have come into contact with tissue sections, such as BSA and PBS, should be regarded as biological waste and must be collected and disposed of in accordance with institutional biosafety protocols.</li>
		<li>Incubate the sections with primary antibodies (anti-Piezo type mechanosensitive ion channel component 1 (Piezo1), diluted 1:200; anti-leptin receptor (LepR), diluted 1:50) in antibody dilution buffer. Apply 50 &#181;L of diluted antibody solution to each section and incubate for 12-18 h at 4 &#176;C in the dark in a humidified chamber.</li>
		<li>Wash the slides with PBS (3 &#215; 5 min), then incubate them with fluorophore-conjugated secondary antibodies (diluted 1:200). Apply 50 &#181;L of diluted antibody solution to each section and incubate for 2 h at room temperature in the dark in a humidified chamber.</li>
		<li>Rinse the slides again in PBS (3 &#215; 5 min), then counterstain nuclei with 4&#39;,6-diamidino-2-phenylindole (DAPI) (diluted 1:50) for 5 min.</li>
		<li>Mount the sections with antifade mounting medium and cover with a coverslip.</li>
		<li>Visualize and capture images using a laser scanning confocal microscope with appropriate filter settings.</li>
	</ol>
	</li>
</ol>

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<li>Altmann, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Promotion+of+osteoblast+differentiation+in+3D+biomaterial+micro-chip+arrays+comprising+fibronectin-coated+poly%28methyl+methacrylate%29+polycarbonate%5BTitle%5D)+AND+%22Biomaterials%22%5BJournal%5D)">Promotion of osteoblast differentiation in 3D biomaterial micro-chip arrays comprising fibronectin-coated poly(methyl methacrylate) polycarbonate.</a> Biomaterials. 32 (34), 8947-8956 (2011).</li>
<li>Bastidas-Coral, A. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Polymethyl+methacrylate+does+not+adversely+affect+the+osteogenic+potential+of+human+adipose+stem+cells+or+primary+osteoblasts%5BTitle%5D)+AND+%22J+Biomed+Mater+Res+B+Appl+Biomater%22%5BJournal%5D)">Polymethyl methacrylate does not adversely affect the osteogenic potential of human adipose stem cells or primary osteoblasts.</a> J Biomed Mater Res B Appl Biomater. 108 (4), 1536-1545 (2020).</li>
<li>Öztürk, Z., Tosun, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Comparison+of+3D+printed+and+conventional+denture+base+materials+in+terms+of+durability+and+performance+characteristics%5BTitle%5D)+AND+%22Sci+Rep%22%5BJournal%5D)">Comparison of 3D printed and conventional denture base materials in terms of durability and performance characteristics.</a> Sci Rep. 15, 18234(2025).</li>
<li>Wu, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Transcriptomic+and+cellular+decoding+of+scaffolds-induced+suture+mesenchyme+regeneration%5BTitle%5D)+AND+%22Int+J+Oral+Sci%22%5BJournal%5D)">Transcriptomic and cellular decoding of scaffolds-induced suture mesenchyme regeneration.</a> Int J Oral Sci. 16 (1), 33(2024).</li>
<li>Kan, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((BMP-dependent%2C+injury-induced+stem+cell+niche+as+a+mechanism+of+heterotopic+ossification%5BTitle%5D)+AND+%22Stem+Cell+Res+Ther%22%5BJournal%5D)">BMP-dependent, injury-induced stem cell niche as a mechanism of heterotopic ossification.</a> Stem Cell Res Ther. 10 (1), 14(2019).</li>
<li>Bao, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((3D+biomimetic+artificial+bone+scaffolds+with+dual-cytokines+spatiotemporal+delivery+for+large+weight-bearing+bone+defect+repair%5BTitle%5D)+AND+%22Sci+Rep%22%5BJournal%5D)">3D biomimetic artificial bone scaffolds with dual-cytokines spatiotemporal delivery for large weight-bearing bone defect repair.</a> Sci Rep. 7 (1), 7814(2017).</li>
</ol>

All the original data and images are included in this paper. The authors declare no conflicts of interest.

Apart from the widely emphasized properties of osteoinductivity, osteointegration, and biocompatibility5, the mechanical responsiveness and load-bearing capacity of bone substitutes are equally vital, particularly for repairing defects in weight-bearing bones22. Conventional assessments of mechanical performance typically rely on in vitro methods, such as universal testing machines, compression testing, or tensile strength analysis23,</...

Under physiological conditions, bone plays a central role in maintaining the structural integrity and mechanical function of the body. It supports body weight, enables movement, and protects internal organs. When bone is damaged due to trauma, tumor resection, or infection, especially in load-bearing regions, the mechanical continuity is interrupted. This disruption often leads to complications such as bone deformity, reduced mobility, limb instability, chronic pain, and even life-threatening issues from severe fractures and hemorrhage1,2. Nowadays, bone substitutes such as bone grafting and biomaterials have been clinically applied to replace severely damaged bone tissue or restore large-scale bone defects. Similarly, for the implanted bone substitutes, their compromised mechanical performance can lead to implant failure, resulting in non-union or even catastrophic fractures at the defect site3,4. To restore both structure and function, bone substitute materials must not only promote bone regeneration but also possess sufficient mechanical strength to withstand physiological loading5.
A critical step prior to clinical translation is the realistic assessment of whether a biomaterial, once implanted in vivo, can meet the mechanical demands required for structural support in load-bearing bone regions. Although many biomaterials have shown promise in vitro or in small, non-load-bearing models, their translation to clinical practice is often hindered by the lack of reliable preclinical models that simulate in vivo mechanical conditions. Currently used animal models for bone defect studies include mandibular defects6, calvarial defects7, drill-hole&#160;defects in long bones8, post-extraction sockets9, and so on. These models primarily evaluate a material&#39;s osteogenic capacity and focus on histological or radiographic regeneration endpoints10,11. However, considering that these sites are either non-load-bearing or mechanically protected, they fail to replicate the dynamic stress conditions encountered in long bones12. Even when mechanical properties are tested by in vitro techniques such as compressive or tensile strength assays, these fail to replicate the dynamic loading and host tissue interactions inherent to in vivo environments13.
A major gap in preclinical research remains the lack of a standardized, reproducible animal model that allows in vivo mechanical assessment of bone substitutes under load-bearing conditions. To address this, the current study established a half-segmental diaphyseal bone defect model in rats. This model provides a stable and reproducible defect site in a load-bearing region, enabling direct evaluation of the mechanical support capacity of bone substitute materials under physiological loading, while concurrently allowing assessment of osteogenic outcomes. Moreover, the model can be applied across various animal species, with defect size and location adjusted according to the specific animal used. Nevertheless, it does not fully replicate the complex biomechanical environment of human bones, and for studies focused solely on non-load-bearing defects, cranial bone healing, or very large structural implants, alternative models may be more appropriate.

<table><tbody><tr><td>2% Iodophor solution</td><td>Chengdu Jinshan Chemical Reagent Co., Ltd.</td><td>None</td><td></td></tr><tr><td>3D-printed polymethyl methacrylate (3DP-PMMA)</td><td>kindly provided by the College of Polymer Science and Engineering, Sichuan University</td><td>None</td><td></td></tr><tr><td>4% paraformaldehyde</td><td>Biosharp</td><td>BL539A</td><td></td></tr><tr><td>75% Ethanol</td><td>Chengdu Jinshan Chemical Reagent Co., Ltd.</td><td>None</td><td></td></tr><tr><td>Cotton balls</td><td>Haishi Hainuo Group Co.,&amp;nbsp; Ltd.</td><td>None</td><td></td></tr><tr><td>Cotton sticks</td><td>Lakong Medical Devices Co., Ltd.</td><td>None</td><td></td></tr><tr><td>DAPI staining solution</td><td>Beyotime&amp;nbsp;</td><td>Cat#C1005</td><td></td></tr><tr><td>Disposable sterile scalpel</td><td>Hangzhou Huawei Medical Supplies Co., Ltd.</td><td>20100227</td><td></td></tr><tr><td>Gelatin methacryloyl (GelMA) hydrogels</td><td>kindly provided by the College of Polymer Science and Engineering, Sichuan University</td><td>None</td><td></td></tr><tr><td>GelMA 20%</td><td>Cellink</td><td>IKG125000005</td><td></td></tr><tr><td>Goat Anti-Mouse IgG H&amp;amp;L (Alexa Fluor&amp;reg; 594)</td><td>Abcam</td><td>ab150116</td><td></td></tr><tr><td>Goat Anti-Rabbit IgG H&amp;amp;L (Alexa Fluor&amp;reg; 488)</td><td>Abcam</td><td>ab150077</td><td></td></tr><tr><td>Hematoxylin and Eosin Stain kit</td><td>Biosharp</td><td>C1005</td><td></td></tr><tr><td>High-speed handpiece</td><td>Foshan SCS Medical Instrument Co., Ltd.</td><td>9018499000</td><td></td></tr><tr><td>Irrigation needle</td><td>Sichuan New Century Medical Polymer Products Co., Ltd.</td><td>None</td><td></td></tr><tr><td>Masson&amp;rsquo;s Trichrome Stain Kit</td><td>Solarbio</td><td>G1340</td><td></td></tr><tr><td>Medical non-woven fabrics</td><td>Henan Yadu Industrial Co., Ltd.&amp;nbsp;</td><td>None</td><td></td></tr><tr><td>Medical non-woven fabrics</td><td>Henan Yadu Industrial Co., Ltd.&amp;nbsp;</td><td>None</td><td></td></tr><tr><td>Micro-computed tomography (&amp;micro;CT)&amp;nbsp;</td><td>Scanco Medical AG</td><td>&amp;mu;CT 45</td><td></td></tr><tr><td>Mimics 20.0 for cross-sectional images (reslice function)</td><td>Materialise</td><td>None</td><td></td></tr><tr><td>Needle holders</td><td>Chengdu Shifeng Co., Ltd.</td><td>None</td><td></td></tr><tr><td>Ob-R/Leptin Receptor Antibody (B-3)</td><td>Santa Cruz</td><td>sc-8391</td><td></td></tr><tr><td>Olympus Research Grade Whole Slide Scanning System VS200</td><td>Chengdu Knowledge Technology Co., Ltd</td><td>VS200</td><td></td></tr><tr><td>Periosteal elevator</td><td>Chengdu Shifeng Co., Ltd.</td><td>None</td><td></td></tr><tr><td>PIEZO1 Antibody</td><td>Novus</td><td>NBP1-78446</td><td></td></tr><tr><td>Saline solution</td><td>Sichuan Kelun Pharmaceutical Co., Ltd.</td><td>None</td><td></td></tr><tr><td>Scanco medical visualizer software for 3D image reconstruction (predefined scripts)</td><td>Scanco Medical AG</td><td>None</td><td></td></tr><tr><td>Specific dental diamond bur (TF-22/170-021m/845-021m)</td><td>Diantong Dental Materials Company</td><td>None</td><td></td></tr><tr><td>Sprague-Dawley rats&amp;nbsp;</td><td>Byrness Weil Biotech Ltd</td><td>None</td><td></td></tr><tr><td>Straight Scissors</td><td>Chengdu Shifeng Co., Ltd.</td><td>None</td><td></td></tr><tr><td>Surgical Motor</td><td>MARATHON</td><td>N3-140232</td><td></td></tr><tr><td>Surgical sutures (4-0 monofilament)</td><td>Hangzhou Huawei Medical Supplies Co., Ltd.</td><td>None</td><td></td></tr><tr><td>Vernier caliper</td><td>Deli</td><td>DL91150</td><td></td></tr></tbody></table>

half-segmental diaphyseal bone defect model in rats for evaluating bone substitute performance in load-bearing regions

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

Accurate preclinical evaluation of bone substitute materials demands models that not only support tissue regeneration but also reflect the mechanical challenges of physiological load-bearing conditions. However, most conventional animal bone defect models fall short in this regard. For example, calvarial defect models, though widely used, are created in non-load-bearing regions. Similarly, drill-hole or partial defects in long bones primarily focus on localized healing, lacking sufficient load transfer to assess mechanical support. This study established a standardized half-segmental diaphyseal bone defect model in the rat femur to assess both the mechanical and biological performance of bone substitute materials in a load-bearing context without the use of fixation devices. Using a high-speed handpiece and a specialized cylindrical dental bur, a semi-cylindrical defect measuring 4 mm in length and 1.5 mm in radius was created in the lateral midshaft of the femur. Two representative materials with distinct properties -- Gelatin methacryloyl (GelMA) hydrogel and 3D-printed polymethyl methacrylate (3DP-PMMA) -- were implanted to validate the model&#39;s capacity to differentiate the mechanical properties of materials. Radiographic, histological, and immunofluorescent analyses performed at 4 weeks post-implantation revealed that, due to inadequate mechanical support, GelMA implantation led to malunion with fibrous tissue predominance and limited bone integration. In contrast, 3DP-PMMA facilitated the organization of new bone formation, periosteal continuity, and the recruitment of Piezo type mechanosensitive ion channel component 1-expressing (Piezo1+) and leptin receptor-expressing (LepR+) cells. This model provides a practical and reproducible platform for assessing the load-bearing capacity, osteoinductivity, and osseointegration potential of biomaterials. It can serve as a valuable tool for screening candidate materials for orthopedic applications, such as long bone reconstruction or fracture repair.

In this study, the half-segmental diaphyseal bone defect model in rats was established through drilling a defect measuring 4 mm in length and 1.5 mm in radius at the femoral midshaft with a flat-ended cylindrical specific dental diamond bur (Figure 2A). To demonstrate the model's capability of testing materials' mechanical and biological performance, we set two comparison groups, with defects implanted with either GelMA hydrogels or 3DP-PMMA (Figure 3, <strong c...

This study established a rat femoral half-segmental defect model to evaluate the mechanical and osteogenic performance of bone substitute materials under load-bearing conditions without the use of internal or external fixation.

Accurate preclinical evaluation of bone substitute materials demands models that not only support tissue regeneration but also reflect the mechanical ...

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.

half-segmental-diaphyseal-bone-defect-model-rats-for-evaluating-bone

Research

JoVE Journal

Bioengineering

One constraint of preclinical research in the field of bone repair is the lack of experimental control over the local mechanical environment within a healing bone lesion. We report the design and use of an external fixator for bone repair with the ability to change fixator stiffness in vivo.

Adjustable Stiffness, External Fixator for the Rat Femur Osteotomy and Segmental Bone Defect Models

Animal models are frequently employed to mimic serious bone injury in biomedical research. Due to their small size, establishment of stabilized bone lesions in mice are beyond the capabilities of most research groups. Herein, we describe a simple method for establishing and analyzing experimental femoral defects in mice.

A Simple Critical-sized Femoral Defect Model in Mice

Although mouse models are invaluable tools for bone tissue engineering, models of long bone defects are sparse. This need motivated development of the present protocol which uses a locking plate with four screws and a dedicated jig to perform and stabilize a reproducible, femoral, critical-size defect with low morbidity.

Establishment of a Segmental Femoral Critical-size Defect Model in Mice Stabilized by Plate Osteosynthesis

The growth plate is a cartilaginous region in children's long bones where longitudinal growth occurs. When injured, bony tissue can form and impair growth. We describe a rat model of growth plate injury that leads to bony repair tissue, allowing the study of repair mechanisms and growth plate regeneration strategies.

A Rat Tibial Growth Plate Injury Model to Characterize Repair Mechanisms and Evaluate Growth Plate Regeneration Strategies

In vivo mammalian models of critical-sized bone defects are essential for researchers studying healing mechanisms and orthopedic therapies. Here, we introduce a protocol for the creation of reproducible, segmental, femoral defects in rats stabilized using external fixation.

A Reliable and Reproducible Critical-Sized Segmental Femoral Defect Model in Rats Stabilized with a Custom External Fixator

Here we present a surgical protocol in rabbits with the aim to assess bone substitution materials in terms of bone regeneration capacities. By using PEEK cylinders fixed onto rabbit skulls, osteoconduction, osteoinduction, osteogenesis and vasculogenesis induced by the materials may be evaluated either on live or euthanized animals.

Calvarial Model of Bone Augmentation in Rabbit for Assessment of Bone Growth and Neovascularization in Bone Substitution Materials

Here, we present a protocol to iatrogenically fracture the shaft of the femur of Wistar albino rats and follow up on the development of the callus. This femur osteotomy model can help researchers evaluate the process of fracture healing and to study how a drug could influence fracture healing.

Protocol for Developing a Femur Osteotomy Model in Wistar Albino Rats

This protocol establishes a full-thickness cartilage defects (FTCD) model by drilling holes in the femoral trochlear groove of rats and measuring the subsequent pain behavior and histopathological changes.

Development and Evaluation of a Rat Model of Full-Thickness Cartilage Defects

Here, we present a protocol for creating a box-cavity defect in rat femoral diaphysis tissue. This model can assess biomaterial performance under biomechanical stress and explore mechanisms of bone regeneration related to intramembranous osteogenesis.

Creating a Box-Cavity Defect Model in the Cortical Bone of Rat Femora

The placement of implants in a rat model is an essential experimental procedure for clinical research. This study presents a comprehensive surgical protocol for implanting titanium implants into the tibia of rat models with diabetes and osteoporosis.

Surgical Bone Implantation Technique for Rat Tibia Models of Diabetes and Osteoporosis

The study innovatively combines sutures and titanium needles for stable internal fixation in a rat radial fracture model. Fracture healing was assessed via micro-computed tomography (micro-CT), and primary dorsal root ganglion (DRG) neurons were isolated and cultured for further investigation.

Development of a Novel Internal Fixation Model for Rat Radial Fractures: Fracture Healing Assessment and Dorsal Root Ganglion Isolation

Learn to establish a rat femoral half-segmental defect model for evaluating bone substitute performance under load-bearing conditions without fixation. This protocol enables reproducible in vivo mechanical assessment of implants.

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

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