The authors thank the Spanish State Research Agency for financial support through projects PID2020-114019RBI00 and PID2021-125150OB-I00.

All experimental procedures were conducted in accordance with the European Community Guidelines for the protection of animals used for scientific purposes (Directive 2010/63/EU) as implemented in Spanish law (Royal Decree 53/2013) and Generalitat de Catalunya regulations (Decree 214/97). Ethics approval for all animal procedures and handling was obtained from the Ethics Committee for Animal Experimentation of the Vall D&#39;Hebron Institut de Recerca (registration number 72/18 CEEA). For the osteoporotic model, female Sprague Dawley rats with an average weight of 484 g and an average age of 12 weeks were utilized. As for the diabetic model, genetically modified female BBDR (Bio Breeding Diabetes Resistant) rats with an average weight of 730 g and an average age of 12 weeks were employed. All animals were sourced from a commercial supplier. The specific details of the animals, reagents, and equipment utilized in the study are listed in the Table of Materials.
1. Anaesthesia/pharmacology and preparation of animals
<ol>
	<li>Administer presurgical analgesia using buprenorphine at 0.05 mg/kg and meloxicam at 2 mg/kg through subcutaneous injections 10-15 min before starting the surgical procedure.</li>
	<li>Perform intra-surgical anesthesia with inhaled isoflurane: initiate at 5% in fresh air during induction and maintain at 3%. Induce anesthesia in a rat chamber and maintain the isoflurane supply with a conical nose adaptation during surgery.</li>
</ol>
2. Preparation for the surgery
<ol>
	<li>Measure the anesthetized animal&#39;s body temperature with a rectal probe and use an electronically controlled heating pad for thermal support throughout the surgical procedure. Eye ointment must be applied prior to surgery beginning to avoid corneal dryness. 
	NOTE: If necessary, the ointment must be reapplied after checking eye dryness.</li>
	<li>Trim hair with an electric shaver and apply hair removal cream to eliminate any remaining fur.</li>
	<li>Obtain an aseptic surgical field by cleaning the knee skin in a pattern of iodine and 70% (v/v) ethanol using sterile swabs, starting from inside and moving outward the incision line without retracing. Perform a minimum of three consecutive cleaning rotations (iodine-ethanol-iodine).</li>
	<li>Isolate the operating field by positioning a sterile fenestrated&#160;surgical drape over the animal, exposing the leg through the central opening (Figure 1).</li>
</ol>
3. Surgery
<ol>
	<li>Surgical exposure
	<ol>
		<li>Make a full-thickness skin incision of approximately 1 cm in length vertically along the proximal border of the anteromedial face of the tibia, in the metaphysis region to expose the bone (Figure 2).</li>
		<li>Stabilize the leg and pull the skin taut against the underlying bone while making the incision, ensuring a clean incision stays in the correct location. Manage by cleaning the expected light bleeding with a compress soaked in saline solution (Figure 2). 
		NOTE: The rat skin is thin and sagging or loose. Stabilization of the skin is crucial and required.</li>
		<li>Fully detach the tissue from the bone using small periosteal elevators (Figure 3).</li>
		<li>Expose the bone until identification of the insertion of the tibialis cranialis muscle, the gracilis, and the gastrocnemius lateral head muscle&#160;in the posterior border of the medial aspect of the tibia, as a fibrous white tissue firmly adhered to the bone (Figure 3). 
		NOTE: It is important to identify this group of muscular insertions to enable the implant to be placed in a bone region with similar characteristics and stimuli across the entire sample, regardless of the rat&#39;s size.</li>
	</ol>
	</li>
	<li>Drilling process
	<ol>
		<li>Begin the drilling process at the correct region between the proximal tibial crest and the posterior limit of the medial face of the tibia bone, contiguous to the insertion of the tibialis cranialis muscle, the gracilis, and the gastrocnemius lateral head muscle, avoiding any muscle injury.1 
		NOTE: The correct location should be 5 mm &#177; 2 mm from the tibial plateau.</li>
		<li>Drill with a maximum of 150 rpm (rotations/min) under saline solution irrigation at a temperature close to 20 &#176;C with a surgical electric motor with a 20:1 reduction contra-angle. 
		NOTE: Only two drills were required.</li>
		<li>Start with a lance pilot drill (Figure 4) at a depth of 2.4 mm under saline solution irrigation. 
		NOTE: Each drill had a maximum of 10 uses.</li>
		<li>As a second drill, use (Figure 4) a twist design drill at a depth of 2.4 mm with a diameter of 1.6 mm under saline solution. 
		NOTE: Each drill had a maximum of 10 uses.</li>
	</ol>
	</li>
	<li>Implant placement
	<ol>
		<li>Insert the implant with an intermediary piece (Figure 5) attached to the 20:1 reduction contra-angle.</li>
		<li>Before placing the implant, clean the implant from any residual chemical sterilant by rotating it in the contra-angle with simultaneous saline irrigation for 10 s (Figure 5).</li>
		<li>Place the titanium implant (2 mm lengthwise and 1.8 mm in diameter) using the intermediate piece at 20 rpm while monitoring real-time torque value, registering the maximum insertion torque. 
		NOTE: An initial difficulty in inserting the implant is expected due to the difference between the final drill and the implant, as well as the cylindrical profile of the implant; however, this initial difficulty is quickly normalized as soon as the implant penetrates the initial cortical bone.</li>
		<li>Finish implant insertion before it completely passes the cortical bone where it is inserted, that is, the flat medial face of the tibia. 
		NOTE: At the final moment of implant insertion, it is important to leave the implant slightly outside the cortical bone or level with the cortical bone where it is inserted, that is, the flat medial face of the tibia, to ensure primary stability (Figure 6).</li>
	</ol>
	</li>
	<li>Wound closure
	<ol>
		<li>Suture the muscle tissue borders with simple internal sutures using a 4/0 monofilament synthetic resorbable suture (Glyconate) (Figure 7).</li>
		<li>Perform skin closure with an intradermal suture using a 4/0 monofilament synthetic resorbable suture (Glyconate) (Figure 7).</li>
	</ol>
	</li>
</ol>
4. Micro-CT scan
<ol>
	<li>After completing the surgery and still under general anesthesia, conduct a micro-CT scan to confirm proper implant placement.</li>
	<li>Remove the rat from the surgery bed and place it on the scanning bed. Locate the operated leg using the micro-CT live scan mode and center the Field of View on the implant. 
	NOTE: Recommended acquisition parameters are Field of view of 5 mm, spatial resolution of 0.0001 mm3, 50 kV, 200 &#181;A, and acquisition time of 3 min.</li>
	<li>Once the scan is acquired, confirm the correct distance between the proximal aspect of the implant and the surface of the tibial plateau, according to step 5.2.1. 
	NOTE: This value will be helpful for the standardization of the technique (Figure 8).</li>
</ol>
5. Postoperative care
<ol>
	<li>Following imaging acquisition, return the rat to its cage and monitor until full recovery. 
	NOTE: This takes approximately 5-10 min, depending on the animal model. Diabetic rats are expected to remain anesthetized longer and have a longer recovery time due to the metabolic changes associated with diabetes.</li>
	<li>Administer buprenorphine (0.1 mg/kg) every 6-8 h and meloxicam (5 mg/kg) every 24 h, subcutaneously, up to 72 h.</li>
	<li>Suture removal time
	<ol>
		<li>Examine the surgical wound daily for infection, suture integrity, or other issues, and remove suture remnants, if necessary, 15 days post-surgery.</li>
	</ol>
	</li>
</ol>
6. Euthanasia
<ol>
	<li>Euthanize the animals using a CO2 chamber, according to the National Institutes of Health (NIH) guidelines, with a CO2 fill rate of 30%-70% of the chamber volume/min after the implantation time (4 weeks or 12 weeks).</li>
</ol>
7. Postsurgical analysis
<ol>
	<li>For both models (osteoporosis and diabetes), remove the tibia by disarticulation after euthanasia for further analysis. 
	NOTE: Regarding the micro-CT analysis, data allowed for calculating bone-implant contact (BIC). The CT acquisition was conducted using the previously described parameters, and the BIC analysis was performed by dividing the bone area (mm2) by the implant area (mm2) calculated at the cortical region, adapting a protocol already described in the literature18. Representative images are shown for each model, osteoporosis (Figure 9) and diabetes (Figure 10).</li>
</ol>

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We hereby declare that there is no conflict of interest regarding this scientific article.

Although the rat is a widely used model for studying osseointegration, it is important to define and describe a reproducible surgical technique for adequately placing implants. Such a technique could serve as a guide for the scientific community. Moreover, the fact that certain diseases, such as osteoporosis and diabetes, alter bone metabolism implies stronger demands for correctly designing surgical procedures. The rat compares favorably with other animal models since it presents the main features of both osteoporosis (...

The common choice of rats as experimental subjects is due to the fact that they are easy to breed and relatively inexpensive compared to larger animal models. The emergence of new procedures, such as the reliable reproduction of a disorder, e.g., osteoporosis or diabetes, makes this model especially useful for analyzing the potential use of treatments and/or the influence of the disease in the biological response to drugs and surgical devices or procedures1,2.
The rat&#39;s bone mass gain occurs mostly during the first 6 months of life, although some researchers believe that the long bone grows constantly for at least a year with a progressive increase in length1. With aging, there is a transition from modeling to remodeling, which does not occur in all cases equally throughout the bones2. Female Sprague Dawley rats grow more slowly than male rats and achieve a lower peak in weight than male rats1. Continuous bone elongation and varied bone remodeling dynamics in rats are factors that have to be taken into account when addressing human health issues; however, it has not yet been possible to find any experimental research that shows either lifelong rat bone development or the species&#39; inability to remodel bone1. If the experimentation starts around 10 months of age, a margin of at least 1 mm from the growth plate of the tibia should be left intact due to this longitudinal bone growth, an issue to be considered in dental implant studies2. Hormones are also a key parameter in bone research since at 8 months of age, male rats were found to have 22% greater bone width and 33% greater breaking strength than females in the tibia3.
The reliable reproduction of a disorder is thus very important in orthopedics and implant dentistry since osseointegration of an orthopedic screw or a dental implant is a complex process that depends on numerous factors influencing the systemic response to the device implantation into the bone. Systemic disorders like osteoporosis and diabetes are known to affect the success rate in orthopedics and implant dentistry, so the reliable reproduction of those disorders in rat models can be applied to explore ways to overcome these limitations.
The rat tibia, due to the easy surgical access, moderate bone volume, and the flat shape on the medial plate, makes it suitable for surgical bone implantation experiments4,5, and it has been used in numerous research studies exploring the effects of implant surface on osseointegration4,5,6. A growing number of studies assess the effects on osseointegration of coatings and substances added to the implant surface in both healthy animals7 and in compromised animals affected by diabetes or osteoporosis8,9,10,11,12,13,14.
The number of implant devices placed in one rat&#39;s tibia is limited and can differ depending on the type of study. Depending on the number of implants or study conditions, the dimensions of the devices must be adapted to minimize surgical trauma. In studies with one implant, a nearly human-size implant can be placed (2.0 mm in diameter and 4 to 5 mm in length), and bi-cortical anchorage can be achieved6,7,15,16. The dimensions of the implants in multi-implant protocols should adopt an appropriate implant size (1.5 mm in diameter and 2.5 mm in length)4,17.
The present study aims to describe a standardized surgical protocol for titanium implant placement on the tibia of two rat models: the osteoporosis and the diabetes rat model. Moreover, this study permits testing the surgical protocol to assess different types of implant surface biofunctionalization and its effect on osseointegration.
A sample of 80 rats was divided into two groups. In group 1, 40 ovariectomized Sprague Dawley females and 5 sham animals were selected, with a mean weight of 484 g and a mean age of 12 weeks. Based on vendor recommendations (see Table of Materials), three months after neutering, the experiment started. This waiting period ensured the disappearance of sex hormones. Osteoporosis was confirmed at the time of surgery based on micro-computed tomography (micro-CT) bone analysis, which reflected an average of 20% bone loss compared to the sham group. Group 2 consisted of 40 BBDR (Bio Breeding Diabetes Resistant) genetically modified Sprague Dawley rats with type II diabetes. The mean weight was 730 g, and the average age was 12 weeks. Prior to surgery, the diabetic status was confirmed with three consecutive days of glucose measurements with results higher than 200 mg/dL. Glucose was measured with a glucometer in 6 h of fasting, and a blood drop was collected by tail puncture.
Grade 3 titanium implants measuring 2 mm in length and 1.8 mm in diameter were used. All implants were sterilized in clean room conditions, by being ultrasonically cleaned in cyclohexane (3 times for 2 min), acetone (once for 1 min), deionized water (3 times for 2 min), ethanol (3 times for 2 min), and acetone (3 times for 2 min) using an ultrasound bath (230 VAC, 50/60 Hz, 360 W). Then, the samples were dried with nitrogen gas, and a nitrogen beam at 0.5 bar was applied directly onto the samples. Prior to implantation, the implants were first soaked in deionized water and then immersed in 70% ethanol (v/v) for 10 min. After this, the implants were transferred to sterile microcentrifuge tubes, and kept under sterile conditions until the surgery.

<table><tbody><tr><td>22 G needles+A2:C30</td><td>Terumo</td><td>NN-2238R</td><td></td></tr><tr><td>4/0 monofilament synthetic resorbable suture</td><td>Braun ( MonoSyn)</td><td></td><td></td></tr><tr><td>5 mL, 10 mL syringes</td><td>Braun</td><td>4617100V-02 4606051V</td><td></td></tr><tr><td>Adson forceps</td><td>Ant&amp;atilde;o Medical</td><td>Ref: A586</td><td></td></tr><tr><td>BBDR ( Biobreeding Diabetes Resistant ) Sprague Dawley Rats</td><td>Janvier Labs</td><td></td><td></td></tr><tr><td>Betadine</td><td>Mylan</td><td></td><td></td></tr><tr><td>Buprecare</td><td>Animalcare (UK)</td><td></td><td></td></tr><tr><td>Castroviejo Caliper 0-40 mm 15 cm angled</td><td>UL AMIN Industries</td><td></td><td></td></tr><tr><td>Castroviejo Needle Holder</td><td>Ant&amp;atilde;o Medical</td><td>Ref: AM1702</td><td></td></tr><tr><td>Dental surgery scissors curved and straight</td><td>Ant&amp;atilde;o Medical</td><td>AMA603 / AMA600</td><td></td></tr><tr><td>Electric shaver</td><td>Oster Pro 3000i</td><td>34264482227</td><td></td></tr><tr><td>Extra Fine Graefe Forceps</td><td>F.S.T</td><td>Ref: 11150-10</td><td></td></tr><tr><td>Gauze pads</td><td>COVIDIEN</td><td>441001</td><td></td></tr><tr><td>Glucometer</td><td>Menarini (Italy)</td><td></td><td></td></tr><tr><td>Helicoidal Drill / OSTEO-PIN DRILL &amp;Oslash;1.6 mm</td><td>soadco</td><td>Ref. OS-8001</td><td></td></tr><tr><td>Implants / SCREW OSTEO-PIN &amp;Oslash;1.8 x 2.0 mm</td><td>soadco</td><td>Ref. OS-3</td><td></td></tr><tr><td>Isoflo</td><td>Le Vet Pharma (Netherlands)</td><td></td><td></td></tr><tr><td>Lance pilot drill / Lanceolate Drill (DS)</td><td>soadco</td><td>Ref. 10 02 01 T</td><td></td></tr><tr><td>Latex gloves - Surgical gloves sterile</td><td>Hartmann</td><td>Ref: 9426495</td><td></td></tr><tr><td>Lucas Surgical Curette</td><td>Ant&amp;atilde;o Medical</td><td>Ref: AMA940-3</td><td></td></tr><tr><td>Metacam</td><td>Boehringer Ingelheim(Germany)</td><td></td><td></td></tr><tr><td>Micro forceps straight</td><td>nopa</td><td>Ref: AB 542/12</td><td></td></tr><tr><td>Micro-CT scan( Quantum Fx microCT )</td><td>Perkin Elmer (US)</td><td></td><td></td></tr><tr><td>Osteoporotic Sprague Dawley females Rats</td><td>Janvier Labs</td><td></td><td></td></tr><tr><td>Periosteal elevator -&amp;nbsp; Molt 2-4</td><td>Ant&amp;atilde;o Medical</td><td>Ref: A1564</td><td></td></tr><tr><td>Physiologic solution for Irrigation</td><td>Hygitech</td><td>Ref:10238</td><td></td></tr><tr><td>Scalpel Blade Carbon Steel 15C</td><td>Razor Med</td><td>Ref: 02846</td><td></td></tr><tr><td>Sterile Gauze Swabs</td><td>Alledental</td><td>Ref: 270712</td><td></td></tr><tr><td>Sterile Irrigation system</td><td>Hygitech</td><td>Ref:HY1-110001D</td><td></td></tr><tr><td>Sterile towels (1 piece per animal)</td><td>Dinarex</td><td>4410</td><td></td></tr><tr><td>Surgical contra-angle handpiece</td><td>W&amp;amp;H</td><td>Ref: WS-75 LED G</td><td></td></tr><tr><td>Surgical contra-angle handpiece</td><td>W&amp;amp;H</td><td>SN 08877</td><td></td></tr><tr><td>Surgical contra-angle handpiece</td><td>W&amp;amp;H</td><td>SN 01309</td><td></td></tr><tr><td>Surgical Electric Motor</td><td>WH Implantmed Type: SI-1023</td><td>&amp;nbsp;Ref: 30288000</td><td></td></tr><tr><td>Surgical scalpel handle</td><td>AsaDental</td><td>Ref: 0350-3</td><td></td></tr><tr><td>Towel clamps</td><td>Xelpov surgical</td><td>AF-773-11</td><td></td></tr><tr><td>Ultrasonic device</td><td>J.P. Selecta, Abrera, Spain</td><td></td><td></td></tr></tbody></table>

surgical bone implantation technique for rat tibia models of diabetes and osteoporosis

The rat has long served as a valuable animal model in implant dentistry and orthopedics, particularly in studying the interactions between biomaterials and bone tissue. The rat's tibia is frequently chosen due to its easy surgical access through thin tissue layers (skin and muscle) and the flattened shape of its medial face, facilitating the surgical insertion of intraosseous devices. Additionally, this model enables the induction of specific diseases, mimicking various clinical conditions to assess biological responses to different implant conditions like geometry, surface texture, or biological cues. However, despite its robust cortical structure, certain intraosseous devices may require adaptations in design and size for successful implantation. Therefore, establishing standardized surgical methods for manipulating both soft and hard tissues in the implantation region is essential for ensuring proper implant or screw device placement, particularly in fields like implant dentistry and orthopedics. This study included eighty Sprague Dawley rats divided into two groups based on their respective diseases: Group 1 with osteoporosis and Group 2 with Type 2 Diabetes. Implantations were performed at 4 weeks and 12 weeks, with the same surgeon following a consistent surgical technique. A positive biological response was observed, indicating complete osseointegration of all implants placed. These results validate the success of the surgical protocol, which can be replicated for other studies and serve as a benchmark for the biomaterials community. Notably, osseointegration values remained stable at both 4 weeks and 12 weeks for both disease models, demonstrating a durable integration of the implant over time and emphasizing the establishment of an intimate bone connection as early as 4 weeks.

Surgical phase 
It is important to mention that both animal models used in this study present certain constraints due to the induced diseases. These constraints regarding the manipulation of hard and soft tissues are reflected during the surgical procedure.
In the diabetic model, the rat is larger, making it difficult to stabilize the legs during surgical procedures. This increases the surgical time and, consequently, the anesthesia time, which requires a longer recovery ...

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 rat has long served as a valuable animal model in implant dentistry and orthopedics, particularly in studying the interactions between biomaterials ...

surgical-bone-implantation-technique-for-rat-tibia-models-diabetes

Research

JoVE Journal

Medicine

634 Views.  University of Barcelona. Om de operatie op het verdoofde dier te starten, maakt u een centimeter lange, volledige huidincisie verticaal langs de proximale grens van het anteromediale gezicht van het scheenbeen in het metafysegebied om het bot bloot te leggen. Maak met behulp van kleine periostale liften het weefsel volledig los van het bot. Leg het bot bloot en identificeer de insertie van de tibialis cranialis-spier, de gracilis en de gastrocnemius laterale hoofdspier in de achterst...

Video: Chirurgische botimplantatietechniek voor tibiamodellen bij ratten van diabetes en osteoporose

An Improved Mechanical Testing Method to Assess Bone-implant Anchorage

Recent advances in material science have led to a substantial increase in the topographical complexity of implant surfaces, both on a micro- and a nano-scale. As such, traditional methods of describing implant surfaces -&#160;namely numerical determinants of surface roughness -&#160;are inadequate for predicting in vivo performance. Biomechanical testing provides an accurate and comparative platform to analyze the performance of biomaterial surfaces. An improved mechanical testing method to test the anchorage of bone to candidate implant surfaces is presented. The method is applicable to both early and later stages of healing and can be employed for any range of chemically or mechanically modified surfaces -&#160;but not smooth surfaces. Custom rectangular implants are placed bilaterally in the distal femora of male Wistar rats and collected with the surrounding bone. Test specimens are prepared and potted using a novel breakaway mold and the disruption test is conducted using a mechanical testing machine. This method allows for alignment of the disruption force exactly perpendicular, or parallel, to the plane of the implant surface, and provides an accurate and reproducible means for isolating an exact peri-implant region for testing.

An improved method to mechanically test bone anchorage to candidate implant surfaces is presented. This method allows for alignment of the disruption force exactly perpendicular, or parallel, to the plane of the implant surface, and provides an accurate means to direct the disruption forces to an exact peri-implant region.

Recent advances in material science have led to a substantial increase in the topographical complexity of implant surfaces, both on a micro- and a ...

Bioengineering

A Syngeneic Orthotopic Osteosarcoma Sprague Dawley Rat Model with Amputation to Control Metastasis Rate

The most recent advance in the treatment of osteosarcoma (OS) occurred in the 1980s when multi-agent chemotherapy was shown to improve overall survival compared to surgery alone. To address this problem, the aim of the study is to refine a lesser-known model of OS in rats with a comprehensive histologic, imaging, biologic, implantation, and amputation surgical approach that prolongs survival. We used an immunocompetent, outbred Sprague-Dawley (SD), syngeneic rat model with implanted UMR106 OS cell line (originating from a SD rat) with orthotopic tibial tumor implants into 3-week-old male and female rats to model pediatric OS. We found that rats develop reproducible primary and metastatic pulmonary tumors, and that limb amputations at 3 weeks post implantation significantly reduce the incidence of pulmonary metastasis and prevent unexpected deaths. Histologically, the primary and metastatic OSs in rats were very similar to human OS. Using immunohistochemistry methods, the study shows that rat OS are infiltrated with macrophages and T cells. A protein expression survey of OS cells reveals that these tumors express ErbB family kinases. Since these kinases are also highly expressed in most human OSs, this rat model could be used to test ErbB pathway inhibitors for therapy.

Here, a syngeneic orthotopic implantation followed by an amputation procedure of the osteosarcoma with spontaneous pulmonary metastasis that can be used for preclinical investigation of metastasis biology and development of novel therapeutics is described.

The most recent advance in the treatment of osteosarcoma (OS) occurred in the 1980s when multi-agent chemotherapy was shown to improve overall survival ...

Cancer Research

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

The mechanical environment around the healing of broken bone is very important as it determines the way the fracture will heal. Over the past decade there has been great clinical interest in improving bone healing by altering the mechanical environment through the fixation stability around the lesion. One constraint of preclinical animal research in this area is the lack of experimental control over the local mechanical environment within a large segmental defect as well as osteotomies as they heal. In this paper we report on the design and use of an external fixator to study the healing of large segmental bone defects or osteotomies. This device not only allows for controlled axial stiffness on the bone lesion as it heals, but it also enables the change of stiffness during the healing process in vivo. The conducted experiments have shown that the fixators were able to maintain a 5 mm femoral defect gap in rats in vivo during unrestricted cage activity for at least 8 weeks. Likewise, we observed no distortion or infections, including pin infections during the entire healing period. These results demonstrate that our newly developed external fixator was able to achieve reproducible and standardized stabilization, and the alteration of the mechanical environment of in vivo rat large bone defects and various size osteotomies. This confirms that the external fixation device is well suited for preclinical research investigations using a rat model in the field of bone regeneration and repair.

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.

The mechanical environment around the healing of broken bone is very important as it determines the way the fracture will heal. Over the past decade there ...

Semiautomated Longitudinal Microcomputed Tomography-based Quantitative Structural Analysis of a Nude Rat Osteoporosis-related Vertebral Fracture Model

Osteoporosis-related vertebral compression fractures (OVCFs) are a common and clinically unmet need with increasing prevalence as the world population ages. Animal OVCF models are essential to the preclinical development of translational tissue engineering strategies. While a number of models currently exist, this protocol describes an optimized method for inducing multiple highly reproducible vertebral defects in a single nude rat. A novel longitudinal semiautomated microcomputed tomography (&#181;CT)-based quantitative structural analysis of the vertebral defects is also detailed. Briefly, rats were imaged at multiple time points post-op. The day 1 scan was reoriented to a standard position, and a standard volume of interest was defined. Subsequent &#181;CT scans of each rat were automatically registered to the day 1 scan so the same volume of interest was then analyzed to assess for new bone formation. This versatile approach can be adapted to a variety of other models where longitudinal imaging-based analysis could benefit from precise 3D semiautomated alignment. Taken together, this protocol describes a readily quantifiable and easily reproducible system for osteoporosis and bone research. The suggested protocol takes 4 months to induce osteoporosis in nude ovariectomized rats and between 2.7 and 4 h to generate, image, and analyze two vertebral defects, depending on tissue size and equipment.

The goal of this protocol is to generate a nude rat osteoporosis-related vertebral compression fracture model that can be longitudinally evaluated in vivo using a semiautomated microcomputed tomography-based quantitative structural analysis.

Osteoporosis-related vertebral compression fractures (OVCFs) are a common and clinically unmet need with increasing prevalence as the world population ...

An Efficient and Reproducible Protocol for Distraction Osteogenesis in a Rat Model Leading to a Functional Regenerated Femur

This protocol describes the use of a newly developed external fixator for distraction osteogenesis in a rat femoral model. Distraction osteogenesis (DO) is a surgical technique leading to bone regeneration after an osteotomy. The osteotomized extremities are moved away from each other by gradual distraction to reach the desired elongation. This procedure is widely used in humans for lower and upper limb lengthening, treatment after a bone nonunion, or the regeneration of a bone defect following surgery for bone tumor excision, as well as in maxillofacial reconstruction. Only a few studies clearly demonstrate the efficiency of their protocol in obtaining a functional regenerated bone, i.e., bone that will support physiological weight-bearing without fracture after removal of the external fixator. Moreover, protocols for DO vary and reproducibility is limited by lack of information, making comparison between studies difficult. The aim of this study was to develop a reproducible protocol comprising an appropriate external fixator design for rat limb lengthening, with a detailed surgical technique that permits physiological weight-bearing by the animal after removal of the external fixator.

This study describes a reproducible and detailed protocol using a newly developed external fixator for distraction osteogenesis (DO) in a femoral rat model which permits physiological weight-bearing by the animal after removal of the external fixator.

This protocol describes the use of a newly developed external fixator for distraction osteogenesis in a rat femoral model. Distraction osteogenesis (DO) ...

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

The use of tissue-engineered bone constructs is an appealing strategy to overcome drawbacks of autografts for the treatment of massive bone defects. As a model organism, the mouse has already been widely used in bone-related research. Large diaphyseal bone defect models in mice, however, are sparse and often use bone fixation which fills the bone marrow cavity and does not provide optimal mechanical stability. The objectives of the current study were to develop a critical-size, segmental, femoral defect in nude mice. A 3.5-mm mid-diaphyseal femoral ostectomy (approximately 25% of the femur length) was performed using a dedicated jig, and was stabilized with an anterior located locking plate and 4 locking screws. The bone defect was subsequently either left empty or filled with a bone substitute (syngenic bone graft or coralline scaffold). Bone healing was monitored noninvasively using radiography and in vivo micro-computed-tomography and was subsequently assessed by ex vivo micro-computed-tomography and undecalcified histology after animal sacrifice, 10 weeks postoperatively. The recovery of all mice was excellent, a full-weight-bearing was observed within one day following the surgical procedure. Furthermore, stable bone fixation and consistent fixation of the implanted materials were achieved in all animals tested throughout the study. When the bone defects were left empty, non-union was consistently obtained. In contrast, when the bone defects were filled with syngenic bone grafts, bone union was always observed. When the bone defects were filled with coralline scaffolds, newly-formed bone was observed in the interface between bone resection edges and the scaffold, as well as within a short distance within the scaffold. 
The present model describes a reproducible critical-size femoral defect stabilized by plate osteosynthesis with low morbidity in mice. The new load-bearing segmental bone defect model could be useful for studying the underlying mechanisms in bone regeneration pertinent to orthopaedic applications.

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.

The use of tissue-engineered bone constructs is an appealing strategy to overcome drawbacks of autografts for the treatment of massive bone defects. As a ...

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

Orthopedic research relies heavily on animal models to study mechanisms of bone healing in vivo as well as investigate the new treatment techniques. Critical-sized segmental defects are challenging to treat clinically, and research efforts could benefit from a reliable, ambulatory small animal model of a segmental femoral defect. In this study, we present an optimized surgical protocol for the consistent and reproducible creation of a 5 mm critical diaphyseal defect in a rat femur stabilized with an external fixator. The diaphyseal ostectomy was performed using a custom jig to place 4 Kirschner wires bicortically, which were stabilized with an adapted external fixator device. An oscillating bone saw was used to create the defect. Either a collagen sponge alone or a collagen sponge soaked in rhBMP-2 was implanted into the defect, and the bone healing was monitored over 12 weeks using radiographs. After 12 weeks, rats were sacrificed, and histological analysis was performed on the excised control and treated femurs. Bone defects containing only collagen sponge resulted in non-union, while rhBMP-2 treatment yielded the formation of a periosteal callous and new bone remodeling. Animals recovered well after implantation, and external fixation proved successful in stabilizing the femoral defects over 12 weeks. This streamlined surgical model could be readily applied to study bone healing and test new orthopedic biomaterials and regenerative therapies in vivo.

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.

Orthopedic research relies heavily on animal models to study mechanisms of bone healing in vivo as well as investigate the new treatment techniques. ...

An Intramedullary Locking Nail for Standardized Fixation of Femur Osteotomies to Analyze Normal and Defective Bone Healing in Mice

Bone healing models are essential to the development of new therapeutic strategies for clinical fracture treatment. Furthermore, mouse models are becoming more commonly used in trauma research. They offer a large number of mutant strains and antibodies for the analysis of the molecular mechanisms behind the highly differentiated process of bone healing. To control the biomechanical environment, standardized and well-characterized osteosynthesis techniques are mandatory in mice. Here, we report on the design and use of an intramedullary nail to stabilize open femur osteotomies in mice. The nail, made of medical-grade stainless steel, provides high axial and rotational stiffness. The implant further allows the creation of defined, constant osteotomy gap sizes from 0.00 mm to 2.00 mm. Intramedullary locking nail stabilization of femur osteotomies with gap sizes of 0.00 mm and 0.25 mm result in adequate bone healing through endochondral and intramembranous ossification. Stabilization of femur osteotomies with a gap size of 2.00 mm results in atrophic non-union. Thus, the intramedullary locking nail can be used in healing and non-healing models. A further advantage of the use of the nail compared to other open bone healing models is the possibility to adequately fix bone substitutes and scaffolds in order to study the process of osseous integration. A disadvantage of the use of the intramedullary nail is the more invasive surgical procedure, inherent to all open procedures compared to closed models. A further disadvantage may be the induction of some damage to the intramedullary cavity, inherent to all intramedullary stabilization techniques compared to extramedullary stabilization procedures.

This protocol describes an osteosynthesis technique using an intramedullary locking nail for standardized fixation of femur osteotomies, which can be used to analyze normal and defective bone healing in mice.

Bone healing models are essential to the development of new therapeutic strategies for clinical fracture treatment. Furthermore, mouse models are becoming ...

Protocol for Developing a Femur Osteotomy Model in Wistar Albino Rats

Fracture healing is a physiological process resulting in the regeneration of bone defects by the coordinated action of osteoblasts and osteoclasts. Osteoanabolic drugs have the potential to augment the repair of fractures but have constraints like high costs or undesirable side effects. The bone healing potential of a drug can initially be determined by in vitro studies, but in vivo studies are needed for the final proof of concept. Our objective was to develop a femur osteotomy rodent model that could help researchers understand the development of callus formation following fracture of the shaft of the femur and that could help establish whether a potential drug has bone healing properties. Adult male Wistar albino rats were used after Institutional Animal Ethics Committee clearance. The rodents were anesthetized, and under aseptic conditions, complete transverse fractures at the middle one-third of the shafts of the femurs were created using open osteotomy. The fractures were reduced and internally fixed using intramedullary K-wires, and secondary fracture healing was allowed to take place. After surgery, intraperitoneal analgesics and antibiotics were given for 5 days. Sequential weekly x-rays assessed callus formation. The rats were sacrificed based on radiologically pre-determined time points, and the development of the fracture callus was analyzed radiologically and using immunohistochemistry.

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.

Fracture healing is a physiological process resulting in the regeneration of bone defects by the coordinated action of osteoblasts and osteoclasts. ...

Establishment and Evaluation of a Rat Femoral Box: Cavity Defect Model

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

Severe bone defects or complex fractures can result in serious complications such as nonunion or insufficient bone healing. Tissue engineering, which involves the application of cells, scaffolds, and cytokines, is considered a promising solution for bone regeneration. Consequently, various animal models that simulate bone defects play a crucial role in exploring the therapeutic potential of tissue engineering for bone healing. In this study, we established a box-shaped cortical bone defect model in the mid-femur of rats, which could serve as an ideal model for assessing the function of biomaterials in promoting bone healing. This box-shaped cortical bone defect was drilled using an oral low-speed handpiece and shaped by a lathe needle. Post-operative micro-CT analysis was immediately conducted to confirm the successful establishment of the box-cavity cortical bone defect. The femurs on the operated side of the rats were then harvested at multiple time points post-surgery (0 days, 2 weeks, 4 weeks, and 6 weeks). The healing process of each sample&#39;s defect area was evaluated using micro-CT, hematoxylin and eosin (H&#38;E) staining, and Masson trichrome staining. These results demonstrated a healing pattern consistent with intramembranous ossification, with healing essentially complete by 6 weeks. The categorization of this animal model&#39;s healing process provides an effective in vivo method for investigating novel biomaterials and drugs that target intramembranous ossification during bone tissue defect healing.

Author Spotlight: The Box-Cavity Cortical Approach for Enhanced Evaluation of Biomaterials and Bone Regeneration

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.

Severe bone defects or complex fractures can result in serious complications such as nonunion or insufficient bone healing. Tissue engineering, which ...

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

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