Surgical Bone Implantation Technique for Rat Tibia Models of Diabetes and Osteoporosis
Summary
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.
Abstract
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.
Introduction
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'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' 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'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 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 sterilized by immersion 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.
Protocol
Representative Results
Discussion
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 (…
Disclosures
The authors have nothing to disclose.
Acknowledgements
The authors thank the Spanish State Research Agency for financial support through projects PID2020-114019RBI00 and PID2021-125150OB-I00.
Materials
| 22 G needles+A2:C30 | Terumo | NN-2238R | |
| 4/0 monofilament synthetic resorbable suture | Braun ( MonoSyn) | ||
| 5 mL, 10 mL syringes | Braun | 4617100V-02 4606051V | |
| Adson forceps | Antão Medical | Ref: A586 | |
| BBDR ( Biobreeding Diabetes Resistant ) Sprague Dawley Rats | Janvier Labs | ||
| Betadine | Mylan | ||
| Buprecare | Animalcare (UK) | ||
| Castroviejo Caliper 0-40 mm 15 cm angled | UL AMIN Industries | ||
| Castroviejo Needle Holder | Antão Medical | Ref: AM1702 | |
| Dental surgery scissors curved and straight | Antão Medical | AMA603 / AMA600 | |
| Electric shaver | Oster Pro 3000i | 34264482227 | |
| Extra Fine Graefe Forceps | F.S.T | Ref: 11150-10 | |
| Gauze pads | COVIDIEN | 441001 | |
| Glucometer | Menarini (Italy) | ||
| Helicoidal Drill / OSTEO-PIN DRILL Ø1.6 mm | soadco | Ref. OS-8001 | |
| Implants / SCREW OSTEO-PIN Ø1.8 x 2.0 mm | soadco | Ref. OS-3 | |
| Isoflo | Le Vet Pharma (Netherlands) | ||
| Lance pilot drill / Lanceolate Drill (DS) | soadco | Ref. 10 02 01 T | |
| Latex gloves – Surgical gloves sterile | Hartmann | Ref: 9426495 | |
| Lucas Surgical Curette | Antão Medical | Ref: AMA940-3 | |
| Metacam | Boehringer Ingelheim(Germany) | ||
| Micro forceps straight | nopa | Ref: AB 542/12 | |
| Micro-CT scan( Quantum Fx microCT ) | Perkin Elmer (US) | ||
| Osteoporotic Sprague Dawley females Rats | Janvier Labs | ||
| Periosteal elevator - Molt 2-4 | Antão Medical | Ref: A1564 | |
| Physiologic solution for Irrigation | Hygitech | Ref:10238 | |
| Scalpel Blade Carbon Steel 15C | Razor Med | Ref: 02846 | |
| Sterile Gauze Swabs | Alledental | Ref: 270712 | |
| Sterile Irrigation system | Hygitech | Ref:HY1-110001D | |
| Sterile towels (1 piece per animal) | Dinarex | 4410 | |
| Surgical contra-angle handpiece | W&H | Ref: WS-75 LED G | |
| Surgical contra-angle handpiece | W&H | SN 08877 | |
| Surgical contra-angle handpiece | W&H | SN 01309 | |
| Surgical Electric Motor | WH Implantmed Type: SI-1023 | Ref: 30288000 | |
| Surgical scalpel handle | AsaDental | Ref: 0350-3 | |
| Towel clamps | Xelpov surgical | AF-773-11 | |
| Ultrasonic device | J.P. Selecta, Abrera, Spain |
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