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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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&#227;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&#227;o Medical</td>
			<td>Ref: AM1702</td>
			<td></td>
		</tr>
		<tr>
			<td>Dental surgery scissors curved and straight</td>
			<td>Ant&#227;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 &#216;1.6 mm</td>
			<td>soadco</td>
			<td>Ref. OS-8001</td>
			<td></td>
		</tr>
		<tr>
			<td>Implants / SCREW OSTEO-PIN &#216;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&#227;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 -&#160; Molt 2-4</td>
			<td>Ant&#227;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&#38;H</td>
			<td>Ref: WS-75 LED G</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical contra-angle handpiece</td>
			<td>W&#38;H</td>
			<td>SN 08877</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical contra-angle handpiece</td>
			<td>W&#38;H</td>
			<td>SN 01309</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical Electric Motor</td>
			<td>WH Implantmed Type: SI-1023</td>
			<td>&#160;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

533 Views.  University of Barcelona. 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.

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

Models of Bone Metastasis

Bone metastases are a common occurrence in several malignancies, including breast, prostate, and lung. Once established in bone, tumors are responsible for significant morbidity and mortality1. Thus, there is a significant need to understand the molecular mechanisms controlling the establishment, growth and activity of tumors in bone. Several in vivo models have been established to study these events and each has specific benefits and limitations. The most commonly used model utilizes intracardiac inoculation of tumor cells directly into the arterial blood supply of athymic (nude) BalbC mice. This procedure can be applied to many different tumor types (including PC-3 prostate cancer, lung carcinoma, and mouse mammary fat pad tumors); however, in this manuscript we will focus on the breast cancer model, MDA-MB-231. In this model we utilize a highly bone-selective clone, originally derived in Dr. Mundy's group in San Antonio2, that has since been transfected for GFP expression and re-cloned by our group3. This clone is a bone metastatic variant with a high rate of osteotropism and very little metastasis to lung, liver, or adrenal glands. While intracardiac injections are most commonly used for studies of bone metastasis2, in certain instances intratibial4 or mammary fat pad injections are more appropriate. Intracardiac injections are typically performed when using human tumor cells with the goal of monitoring later stages of metastasis, specifically the ability of cancer cells to arrest in bone, survive, proliferate, and establish tumors that develop into cancer-induced bone disease. Intratibial injections are performed if focusing on the relationship of cancer cells and bone after a tumor has metastasized to bone, which correlates roughly to established metastatic bone disease. Neither of these models recapitulates early steps in the metastatic process prior to embolism and entry of tumor cells into the circulation. If monitoring primary tumor growth or metastasis from the primary site to bone, then mammary fat pad inoculations are usually preferred; however, very few tumor cell lines will consistently metastasize to bone from the primary site, with 4T1 bone-preferential clones, a mouse mammary carcinoma, being the exception 5,6.
This manuscript details inoculation procedures and highlights key steps in post inoculation analyses. Specifically, it includes cell culture, tumor cell inoculation procedures for intracardiac and intratibial inoculations, as well as brief information regarding weekly monitoring by x-ray, fluorescence and histomorphometric analyses.

Animal models are frequently utilized to study cancer metastasis to bone. In this protocol we will describe two common methods of tumor inoculation for bone metastasis studies and briefly describe some of the analyses utilized to monitor and quantify these models.

Bone metastases are a common occurrence in several malignancies, including breast, prostate, and lung. Once established in bone, tumors are responsible ...

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 ...

Implantation of Inferior Vena Cava Interposition Graft in Mouse Model

Biodegradable scaffolds seeded with bone marrow mononuclear cells (BMCs) are often used for reconstructive surgery to treat congenital cardiac anomalies. The long-term clinical results showed excellent patency rates, however, with significant incidence of stenosis. To investigate the cellular and molecular mechanisms of vascular neotissue formation and prevent stenosis development in tissue engineered vascular grafts (TEVGs), we developed a mouse model of the graft with approximately 1 mm internal diameter. First, the TEVGs were assembled from biodegradable tubular scaffolds fabricated from a polyglycolic acid nonwoven felt mesh coated with &#949;-caprolactone and L-lactide copolymer. The scaffolds were then placed in a lyophilizer, vacuumed for 24 hr, and stored in a desiccator until cell seeding. Second, bone marrow was collected from donor mice and mononuclear cells were isolated by density gradient centrifugation. Third, approximately one million cells were seeded on a scaffold and incubated O/N. Finally, the seeded scaffolds were then implanted as infrarenal vena cava interposition grafts in C57BL/6 mice. The implanted grafts demonstrated excellent patency (&#62;90%) without evidence of thromboembolic complications or aneurysmal formation. This murine model will aid us in understanding and quantifying the cellular and molecular mechanisms of neotissue formation in the TEVG.

To improve our knowledge of cellular and molecular neotissue formation, a murine model of the TEVG was recently developed. The grafts were implanted as infrarenal vena cava interposition grafts in C57BL/6 mice. This model achieves similar results to those achieved in our clinical investigation, but over a far shortened time-course.

Biodegradable scaffolds seeded with bone marrow mononuclear cells (BMCs) are often used for reconstructive surgery to treat congenital cardiac anomalies. ...

Surgical Models of Gastroesophageal Reflux with Mice

Multiple surgical procedures have been reported to induce gastroesophageal reflux in animals. Herein, we report three surgical models with mice aiming to induce reflux of gastric contents, duodenal contents or mixed contents. Surgical procedures and general principles have been described in detail. A researcher with surgical experience should be able to grasp the technique after a short period of practice. After surgery, most mice can survive and develop reflux esophagitis similar to that in humans. However, it should be noted that histological differences between mouse and human esophagus are the inherent limitations of these surgical models. If used for research on Barrett&#8217;s esophagus and adenocarcinoma, these procedures may need to be combined with genetic modifications.

This article demonstrates surgical procedures of gastroesophageal reflux with mice. These models are useful tools for research on mechanisms and treatment of gastroesophageal reflux disease and potentially Barrett&#8217;s esophagus and esophageal adenocarcinoma.

Multiple surgical procedures have been reported to induce gastroesophageal reflux in animals. Herein, we report three surgical models with mice aiming to ...

Surgical Training for the Implantation of Neocortical Microelectrode Arrays Using a Formaldehyde-fixed Human Cadaver Model

This protocol describes a procedure to assist surgeons in training for the implantation of microelectrode arrays into the neocortex of the human brain. Recent technological progress has enabled the fabrication of microelectrode arrays that allow recording the activity of multiple individual neurons in the neocortex of the human brain. These arrays have the potential to bring unique insight onto the neuronal correlates of cerebral function in health and disease. Furthermore, the identification and decoding of volitional neuronal activity opens the possibility to establish brain-computer interfaces, and thus might help restore lost neurological functions. The implantation of neocortical microelectrode arrays is an invasive procedure requiring a supra-centimetric craniotomy and the exposure of the cortical surface; thus, the procedure must be performed by an adequately trained neurosurgeon. In order to provide an opportunity for surgical training, we designed a procedure based on a human cadaver model. The use of a formaldehyde-fixed human cadaver bypasses the practical, ethical and financial difficulties of surgical practice on animals (especially non-human primates) while preserving the macroscopic structure of the head, skull, meninges and cerebral surface and allowing realistic, operating room-like positioning and instrumentation. Furthermore, the use of a human cadaver is closer to clinical daily practice than any non-human model. The major drawbacks of the cadaveric simulation are the absence of cerebral pulsation and of blood and cerebrospinal fluid circulation. We suggest that a formaldehyde-fixed human cadaver model is an adequate, practical and cost-effective approach to ensure proper surgical training before implanting microelectrode arrays in the living human neocortex.

We designed a procedure in which a formaldehyde-fixed human cadaver is used to assist neurosurgeons in training for the implantation of microelectrode arrays into the neocortex of the human brain.

This protocol describes a procedure to assist surgeons in training for the implantation of microelectrode arrays into the neocortex of the human brain. ...

Standardized Technique of Aortic Valve Re-implantation for Valve-sparing Aortic Root Replacement

Despite the obvious advantages of the preservation of a normal aortic valve during aortic root replacement, the complexity of valve sparing procedures prevents a number of cardiac surgeons from incorporating them into their practice. The aim of this protocol is to describe a simplified and user-friendly technique of an aortic valve-sparing root replacement (VSRR) procedure by re-implantation of the aortic valve. Proper selection of patients and limitations of the technique are discussed.
In 54 consecutive patients, normal appearing aortic valves were re-implanted in a commercially available polyester prosthesis with pre-shaped sinuses by a simplified and standardized technique. Placement of the first row of the proximal suture line, choice of the prosthesis size, and adjustment of the height of the commissures of the patient to the fixed height of the sinus portion of the prosthesis were slightly modified from the reference techniques with the aim of increasing its feasibility for use by other cardiac surgeons. Early mortality and morbidity as well as 5-year survival, freedom from aortic valve reoperation, and freedom from recurrent moderate regurgitation were collected in all patients.
Thirty-day mortality, re-sternotomy for bleeding, re-sternotomy for mediastinitis, and the incidence of stroke were very low, 1.8% for each (1 of 54). No patient required permanent pace-maker implantation. At 5 years, survival, freedom from aortic valve reoperation, and freedom from recurrent moderate regurgitation were 97.5%, 95.2%, and 91.6%, respectively.
Mid-term results of our standardized technique of re-implantation of the aortic valve for valve-sparing aortic root replacement are very good and compare with more complex techniques reported by experienced surgeons. By following the present protocol of the standardized re-implantation technique, a greater number of cardiac surgeons can perform this procedure with comparable good results.

Valve-sparing aortic root replacement has the advantage of preserving the patient&#39;s own aortic valve. The complexity of the reported techniques to date restricts their use to a limited number of cardiac surgeons. This protocol describes step-by-step a standardized technique reproducible by a greater number of cardiac surgeons.

Despite the obvious advantages of the preservation of a normal aortic valve during aortic root replacement, the complexity of valve sparing procedures ...

A Rat Model of Orthotopic Liver Transplantation Using a Novel Magnetic Anastomosis Technique for Suprahepatic Vena Cava Reconstruction

The rat model of orthotopic liver transplantation (OLT) is essential for transplant research. It is a very sophisticated animal model and requires a steep learning curve. The introduction of the cuff technique for anastomosis of the portal vein (PV) and infrahepatic vena cava (IHVC) has significantly simplified the transplant procedure in rats. However, due to the short anterior wall of the recipients' suprahepatic vena cava (SHVC), the cuff technique is very difficult to use for the reconstruction of the SHVC. Most researchers in this field still use the hand-suture technique for SHVC reconstruction, which makes it the bottleneck step in rat orthotopic liver transplantation. The magnetic anastomosis technique (i.e., magnamosis) is a method of connecting two vessels using the attractive force between two magnets. Our recent study has shown that the magnetic anastomosis technique is superior to the hand-suture technique for SHVC reconstruction in rats. In this article, we show a step-by-step protocol for SHVC reconstruction in rats using the novel magnetic anastomosis technique. In this model, the reconstruction of the PV and IHVC was performed by the standard cuff technique, while the reconstruction of the bile duct (BD) was performed by a stent technique. The hepatic re-arterialization was not performed. The magnetic anastomosis technique made SHVC reconstruction much easier and significantly shortened the anphepatic phase. After a reasonable learning curve, even researchers without advanced microsurgical skills can produce reliable and reproducible results using this rat model of OLT.

The reconstruction of the suprahepatic vena cava (SHVC) remains a difficult step in rat orthotopic liver transplantation. In this article, we show a step-by-step protocol for SHVC reconstruction in rats using a novel magnetic anastomosis technique.

The rat model of orthotopic liver transplantation (OLT) is essential for transplant research. It is a very sophisticated animal model and requires a steep ...

Vacuum-Sealed Hot Water Bath Immersion for the Preparation of Anatomical and Surgical Cadaveric Bone Models

Bone models serve many purposes, including improving anatomical understanding, preoperative surgical planning, and intraoperative referencing. Several techniques for the maceration of soft tissues have been described, mainly for forensic analysis. For clinical research and medical use, these methods have been superseded by three-dimensional (3D) printed models, which require substantial equipment and expertise, and are costly. Here, cadaveric sheep vertebral bone was cleaned by vacuum sealing the specimen with commercial dishwashing detergent, immersing in a hot water bath, and subsequently manually removing the soft tissue. This eliminated the disadvantages of the previously existing maceration methods, such as the existence of foul odors, usage of hazardous chemicals, substantial equipment, and high costs. The described technique produced clean, dry samples while maintaining anatomical detail and structure to accurately model the osseous structures that can be useful for preoperative planning and intraoperative referencing. The method is simple, low-cost, and effective for bone model preparation for education and surgical planning in veterinary and human medicine.

The present protocol describes the maceration and cleaning of cadaveric bone with a vacuum-sealed, hot water bath immersion technique. This is a low-cost, safe, and effective method to produce anatomical specimens for surgical planning and medical education as an alternative to three-dimensional (3D) printed models.

Bone models serve many purposes, including improving anatomical understanding, preoperative surgical planning, and intraoperative referencing. Several ...

A Protocol for Constructing a Rat Wound Model of Type 1 Diabetes

A single high dose of streptozotocin injection followed by full-thickness skin excision on the dorsum of rats is a common method for constructing animal models of type 1 diabetic wounds. However, improper manipulation can lead to model instability and high mortality in rats. Unfortunately, there are few existing guidelines on type 1 diabetic wound modeling, and they lack detail and do not present specific reference strategies. Therefore, this protocol details the complete procedure for constructing a type 1 diabetic wound model and analyzes the progression and angiogenic characteristics of the diabetic wounds. Type 1 diabetic wound modeling involves the following steps: preparation of the streptozotocin injection, induction of type 1 diabetes mellitus, and construction of the wound model. The wound area was measured on day 7 and day 14 after wounding, and the skin tissues of the rats were extracted for histopathological and immunofluorescence analysis. The results revealed that type 1 diabetes mellitus induced by 55 mg/kg streptozotocin was associated with lower mortality and a high success rate. The blood glucose levels were relatively stable after 5 weeks of induction. The diabetic wound healing rate was significantly lower than that of normal wounds on day 7 and day 14 (p &#60; 0.05), but both could reach more than 90% on day 14. Compared with the normal group, the epidermal layer closure of diabetic wounds on day 14 was incomplete and had delayed re-epithelialization and significantly lower angiogenesis (p &#60; 0.01). The type 1 diabetic wound model constructed based on this protocol has the characteristics of chronic wound healing, including poor closure, delayed re-epithelialization, and decreased angiogenesis compared to normal rat wounds.

The streptozotocin-induced diabetic wound model in male SD rats is currently the most widely used model for studying wound healing in type I diabetes mellitus. This protocol describes the methods used to construct this model. It also presents and addresses potential challenges and examines the progression and angiogenic characteristics of diabetic wounds.

A single high dose of streptozotocin injection followed by full-thickness skin excision on the dorsum of rats is a common method for constructing animal ...

HE Staining and Micro-CT to Evaluate Murine Trabecular Bone Microarchitecture

Trabecular Bone Microarchitecture Evaluation in an Osteoporosis Mouse Model

Bone microstructure refers to the arrangement and quality of bone tissue at the microscopic level. Understanding the bone microstructure of the skeleton is crucial for gaining insight into the pathophysiology of osteoporosis and improving its treatment. However, handling bone samples can be complex due to their hard and dense properties. Secondly, specialized software makes image processing and analysis difficult. In this protocol, we present a cost-effective and easy-to-use solution for trabecular bone microstructure analysis. Detailed steps and precautions are provided. Micro-CT is a non-destructive three-dimensional (3D) imaging technique that provides high-resolution images of trabecular bone structure. It allows for the objective and quantitative evaluation of bone quality, which is why it is widely regarded as the gold standard method for bone quality assessment. However, histomorphometry remains indispensable as it offers crucial cellular-level parameters, bridging the gap between two-dimensional (2D) and 3D assessments of bone specimens. As for the histologic techniques, we chose to decalcify the bone tissue and then perform traditional paraffin embedding. In summary, combining these two methods can provide more comprehensive and accurate information on bone microstructure.

Author Spotlight: An Economic and Efficient Method for Quantitative Evaluation of Bone Microarchitecture in a Murine Osteoporosis Model 

This protocol presents an economical and efficient method for quantitative evaluation of bone microarchitecture in a mouse model of osteoporosis by combining Hematoxylin-Eosin (HE) staining and micro-computed tomography (Micro-CT) techniques.

Bone microstructure refers to the arrangement and quality of bone tissue at the microscopic level. Understanding the bone microstructure of the skeleton ...