Name: surgical bone implantation technique for rat tibia models of diabetes and osteoporosis
Uploaded: 2024-07-05T12:40:00
Description: The rat has long served as a valuable animal model in implant dentistry and orthopedics, particularly in studying the interactions between biomaterials ...

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 Sp...

<ol>
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T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+structural+and+hormonal+basis+of+sex+differences+in+peak+appendicular+bone+strength+in+rats.">The structural and hormonal basis of sex differences in peak appendicular bone strength in rats.</a> J Bone Miner Res. 18 (1), 150-155 (2003).</li><li>Alenezi, A., Galli, S., Atefyekta, S., Andersson, M., Wennerberg, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osseointegration+effects+of+local+release+of+strontium+ranelate+from+implant+surfaces+in+rats.">Osseointegration effects of local release of strontium ranelate from implant surfaces in rats.</a> J Mater Sci Mater Med. 30 (10), 116 (2019).</li><li>Blanc-Sylvestre, N., Bouchard, P., Chaussain, C., Bardet, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pre-clinical+models+in+implant+dentistry:+Past,+present,+future.">Pre-clinical models in implant dentistry: Past, present, future.</a> Biomedicines. 9 (11), 1538 (2021).</li><li>Schliephake, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functionalization+of+titanium+implants+using+a+modular+system+for+binding+and+release+of+VEGF+enhances+bone-implant+contact+in+a+rodent+model.">Functionalization of titanium implants using a modular system for binding and release of VEGF enhances bone-implant contact in a rodent model.</a> J Clin Periodontol. 42 (3), 302-310 (2015).</li><li>Mafra, C. E. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+different+doses+of+synthetic+parathyroid+hormone+(1-34)+on+bone+around+implants:+A+preclinical+rat+model.">Effect of different doses of synthetic parathyroid hormone (1-34) on bone around implants: A preclinical rat model.</a> Braz Dent J. 30 (1), 43-46 (2019).</li><li>Rybaczek, T., Tangl, S., Dobsak, T., Gruber, R., Kuchler, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+parathyroid+hormone+on+osseointegration+in+insulin-treated+diabetic+rats.">The effect of parathyroid hormone on osseointegration in insulin-treated diabetic rats.</a> Implant Dent. 24 (4), 392-396 (2015).</li><li>Zou, G. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+local+delivery+of+bfgf+from+plga+microspheres+on+osseointegration+around+implants+in+diabetic+rats.">Effects of local delivery of bfgf from plga microspheres on osseointegration around implants in diabetic rats.</a> Oral Surg Oral Med Oral Pathol Oral Radiol. 114 (3), 284-289 (2012).</li><li>Zhang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+nerve+growth+factor+on+osseointegration+of+titanium+implants+in+type+2+diabetic+rats.">Effect of nerve growth factor on osseointegration of titanium implants in type 2 diabetic rats.</a> Int J Oral Maxillofac Implants. 31 (5), 1189-1194 (2016).</li><li>Kuchler, U., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intermittent+parathyroid+hormone+fails+to+stimulate+osseointegration+in+diabetic+rats.">Intermittent parathyroid hormone fails to stimulate osseointegration in diabetic rats.</a> Clin Oral Implants Res. 22 (5), 518-523 (2011).</li><li>Hashiguchi, C., Kawamoto, S., Kasai, T., Nishi, Y., Nagaoka, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+an+antidiabetic+drug+on+biomechanical+and+histological+parameters+around+implants+in+type+2+diabetic+rats.">Influence of an antidiabetic drug on biomechanical and histological parameters around implants in type 2 diabetic rats.</a> Implant Dent. 23 (3), 264-269 (2014).</li><li>Han, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sustained+topical+delivery+of+insulin+from+fibrin+gel+loaded+with+poly(lactic-co-glycolic+acid)+microspheres+improves+the+biomechanical+retention+of+titanium+implants+in+type+1+diabetic+rats.">Sustained topical delivery of insulin from fibrin gel loaded with poly(lactic-co-glycolic acid) microspheres improves the biomechanical retention of titanium implants in type 1 diabetic rats.</a> J Oral Maxillofac Surg. 70 (10), 2299-2308 (2012).</li><li>De Molon, R. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+diabetes+mellitus+and+metabolic+control+on+bone+healing+around+osseointegrated+implants:+Removal+torque+and+histomorphometric+analysis+in+rats.">Impact of diabetes mellitus and metabolic control on bone healing around osseointegrated implants: Removal torque and histomorphometric analysis in rats.</a> Clin Oral Implants Res. 24 (7), 831-837 (2013).</li><li>Simon, M. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparative+phenotypic+and+genomic+analysis+of+c57bl/6j+and+c57bl/6n+mouse+strains.">A comparative phenotypic and genomic analysis of c57bl/6j and c57bl/6n mouse strains.</a> Genome Biol. 14 (7), R82 (2013).</li><li>Cirano, F. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+curcumin+on+bone+tissue+in+the+diabetic+rat:+Repair+of+peri-implant+and+critical-sized+defects.">Effect of curcumin on bone tissue in the diabetic rat: Repair of peri-implant and critical-sized defects.</a> Int J Oral Maxillofac Surg. 47 (11), 1495-1503 (2018).</li><li>De Oliveira, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+zoledronic+acid+and+dexamethasone+on+osseointegration+of+endosseous+implants:+Histological+and+histomorphometrical+evaluation+in+rats.">The effects of zoledronic acid and dexamethasone on osseointegration of endosseous implants: Histological and histomorphometrical evaluation in rats.</a> Clin Oral Implants Res. 26 (4), e17-e21 (2015).</li><li>Vandeweghe, S., Coelho, P. G., Vanhove, C., Wennerberg, A., Jimbo, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Utilizing+micro-computed+tomography+to+evaluate+bone+structure+surrounding+dental+implants:+A+comparison+with+histomorphometry.">Utilizing micro-computed tomography to evaluate bone structure surrounding dental implants: A comparison with histomorphometry.</a> J Biomed Mater Res B Appl Biomater. 101 (7), 1259-1266 (2013).</li><li>Kleinert, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+obesity+and+diabetes+mellitus.">Animal models of obesity and diabetes mellitus.</a> Nat Rev Endocrinol. 14 (3), 140-162 (2018).</li><li>Grimm, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advancing+the+3Rs:+Innovation,+implementation,+ethics+and+society.">Advancing the 3Rs: Innovation, implementation, ethics and society.</a> Front Vet Sci. 10, 1185706 (2023).</li><li>Melcher, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+the+periosteum+in+repair+of+wounds+of+the+parietal+bone+of+the+rat.">Role of the periosteum in repair of wounds of the parietal bone of the rat.</a> Arch Oral Biol. 14 (9), 1101-1109 (1969).</li><li>Barrak, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heat+generation+during+guided+and+freehand+implant+site+preparation+at+drilling+speeds+of+1500+and+2000+rpm+at+different+temperatures:+An+in+vitro+study.">Heat generation during guided and freehand implant site preparation at drilling speeds of 1500 and 2000 rpm at different temperatures: An in vitro study.</a> Oral Health Prev Dent. 17 (4), 309-316 (2019).</li><li>Kniha, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+thermal+osteonecrosis+around+implants+in+the+rat+tibia:+Numerical+and+histomorphometric+results+in+context+of+implant+removal.">Effect of thermal osteonecrosis around implants in the rat tibia: Numerical and histomorphometric results in context of implant removal.</a> Sci Rep. 12 (1), 22227 (2022).</li><li>Da Silva, J. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apoptosis+in+bone+defect+of+diabetic+rats+treated+with+low+intensity+laser:+Radiological+and+immunohistochemical+approach.">Apoptosis in bone defect of diabetic rats treated with low intensity laser: Radiological and immunohistochemical approach.</a> International Journal of Morphology. 35, 178-183 (2017).</li><li>Zeller-Plumhoff, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+the+bone+ultrastructure+around+biodegradable+Mg-&#967;Gd+implants+using+small+angle+X-ray+scattering+and+X-ray+diffraction.">Analysis of the bone ultrastructure around biodegradable Mg-&#967;Gd implants using small angle X-ray scattering and X-ray diffraction.</a> Acta Biomater. 101, 637-645 (2020).</li><li>Bruns, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+material+dependency+of+peri-implant+morphology+and+stability+in+healing+bone.">On the material dependency of peri-implant morphology and stability in healing bone.</a> Bioact Mater. 28, 155-166 (2023).</li><li>De Morais, J. 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Experimental in vitro study.</a> Clin Oral Investig. 22 (1), 349-359 (2018).</li><li>Abdel Motagly, M., El Khadem, A., Abdel Rassoul, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+low-speed+drilling+without+irrigation,+versus+convencionaldrilling+with+irrigation+regarding+heat+generation+and+peri-implant+marginal+bone+loss+(randomised+clinical+trial).">Assessment of low-speed drilling without irrigation, versus convencionaldrilling with irrigation regarding heat generation and peri-implant marginal bone loss (randomised clinical trial).</a> Alex Dent J. 46 (2), 33-38 (2021).</li><li>Lelovas, P. P., Xanthos, T. T., Thoma, S. E., Lyritis, G. P., Dontas, I. 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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 ...

To begin the surgery on the anesthetized animal, make a one-centimeter long, full-thickness skin incision vertically along the proximal border of the anteromedial face of the tibia in the metaphysis region to expose the bone. Using small periosteal elevators, fully detach the tissue from the bone. Expose the bone and identify the insertion of the tibialis cranialis muscle, the gracilis, and the gastrocnemius lateral head muscle in the posterior border of the medial aspect of the tibia resembling a fibrous white tissue firmly adhered to the bone.Using a lance pilot drill at a depth of 2.4 millimeters with a maximum speed of 150 rotations per minute, drill the identified area as described previously under saline solution irrigation while avoiding any muscle injury. Perform a second drilling using a 1.6 millimeters diameter twist design drill at a depth of 2.4 millimeters. To place the titanium implant, first insert it with an intermediary piece attached to the 20:1 reduction contra angle.Next, clean the implant from any residual chemical sterilant by rotating it in the contra angle with simultaneous saline irrigation for 10 seconds. Then place the implant while making sure to stop the insertion before it completely passes the cortical bone where it is inserted. That is, the flat medial face of the tibia contiguous to the muscle's insertion.Suture the muscle tissue borders with simple internal sutures using a 4/0 monofilament synthetic resorbable glyconate suture. Finally, perform skin closure with an intradermal suture using the same 4/0 monofilament synthetic resorbable glyconate suture.

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

Research

JoVE Journal

Medicine

Implantation of Radiotelemetry Transmitters Yielding Data on ECG, Heart Rate, Core Body Temperature and Activity in Free-moving Laboratory Mice

The laboratory mouse is the animal species of choice for most biomedical research, in both the academic sphere and the pharmaceutical industry. Mice are a manageable size and relatively easy to house. These factors, together with the availability of a wealth of spontaneous and experimentally induced mutants, make laboratory mice ideally suited to a wide variety of research areas.
In cardiovascular, pharmacological and toxicological research, accurate measurement of parameters relating to the circulatory system of laboratory animals is often required. Determination of heart rate, heart rate variability, and duration of PQ and QT intervals are based on electrocardiogram (ECG) recordings. However, obtaining reliable ECG curves as well as physiological data such as core body temperature in mice can be difficult using conventional measurement techniques, which require connecting sensors and lead wires to a restrained, tethered, or even anaesthetized animal. Data obtained in this fashion must be interpreted with caution, as it is well known that restraining and anesthesia can have a major artifactual influence on physiological parameters1, 2.
Radiotelemetry enables data to be collected from conscious and untethered animals. Measurements can be conducted even in freely moving animals, and without requiring the investigator to be in the proximity of the animal. Thus, known sources of artifacts are avoided, and accurate and reliable measurements are assured. This methodology also reduces interanimal variability, thus reducing the number of animals used, rendering this technology the most humane method of monitoring physiological parameters in laboratory animals3, 4. Constant advancements in data acquisition technology and implant miniaturization mean that it is now possible to record physiological parameters and locomotor activity continuously and in realtime over longer periods such as hours, days or even weeks3, 5.
Here, we describe a surgical technique for implantation of a commercially available telemetry transmitter used for continuous measurements of core body temperature, locomotor activity and biopotential (i.e. onelead ECG), from which heart rate, heart rate variability, and PQ and QT intervals can be established in freeroaming, untethered mice. We also present pre-operative procedures and protocols for post-operative intensive care and pain treatment that improve recovery, well-being and survival rates in implanted mice5, 6.

A surgical technique for implantation of commercially available telemetry transmitters used for continuous measurement of biopotential (one-lead ECG), heart rate, core body temperature and locomotor activity in freely moving mice is shown. Recommendations and protocols for post-operative care and pain relief, improving recovery, well being and survival rate are also presented.

The laboratory mouse is the animal species of choice for most biomedical research, in both the academic sphere and the pharmaceutical industry. Mice are a ...

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. ...

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 ...