The present study was primarily financially supported by the foundation Vrienden Diergeneeskunde Universiteit Utrecht; MT has received long-term funding from the Dutch Arthritis Society (LLP22); FV and JM are funded by Eurostars Project E115515 - 3DHIP. IK is a holder of a scholarship from the Faculty of Veterinary Medicine, Kasetsart University, Thailand.

Extracapsular 3D-Printed Implant for Restoring Hip Stability in Canines

<ol>
	<li>King, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Etiopathogenesis+of+canine+hip+dysplasia,+prevalence,+and+genetics.">Etiopathogenesis of canine hip dysplasia, prevalence, and genetics.</a> Vet Clin North Am Small Anim Pract. 47 (4), 753-767 (2017).</li><li>Akis, 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=The+association+of+genetic+polymorphisms+of+bone+formation+genes+with+canine+hip+dysplasia.">The association of genetic polymorphisms of bone formation genes with canine hip dysplasia.</a> Iran J Vet Res. 21 (1), 40-45 (2020).</li><li>Loder, R. T., Todhunter, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+demographics+of+canine+hip+dysplasia+in+the+United+States+and+Canada.">The demographics of canine hip dysplasia in the United States and Canada.</a> J Vet Med. 2017, 1-15 (2017).</li><li>Schachner, E. R., Lopez, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnosis,+prevention,+and+management+of+canine+hip+dysplasia:+a+review.">Diagnosis, prevention, and management of canine hip dysplasia: a review.</a> Vet Med (Auck)l. 6, 181-192 (2015).</li><li>Vezzoni, A., Boiocchi, S., Vezzoni, L., Vanelli, A. B., Bronzo, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Double+pelvic+osteotomy+for+the+treatment+of+hip+dysplasia+in+young+dogs).">Double pelvic osteotomy for the treatment of hip dysplasia in young dogs).</a> Vet Comp Orthop Traumatol. 23 (6), 444-452 (2010).</li><li>Tavola, F., Drudi, D., Vezzoni, L., Vezzoni, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Postoperative+complications+of+double+pelvic+osteotomy+using+specific+plates+in+305+dogs.">Postoperative complications of double pelvic osteotomy using specific plates in 305 dogs.</a> Vet Comp Orthop Traumatol. 35 (1), 47-56 (2022).</li><li>Koch, D. A., Hazewinkel, H. A. W., Nap, R. C., Meij, B. P., Wolvekamp, W. T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Radiographic+evaluation+and+comparison+of+plate+fixation+after+triple+pelvic+osteotomy+in+32+dogs+with+hip+dysplasia.">Radiographic evaluation and comparison of plate fixation after triple pelvic osteotomy in 32 dogs with hip dysplasia.</a> Vet Comp Orthop Traumatol. 06 (01), 09-15 (1993).</li><li>Rose, S. A., Bruecker, K. A., Petersen, S. W., Uddin, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+locking+plate+and+screws+for+triple+pelvic+osteotomy.">Use of locking plate and screws for triple pelvic osteotomy.</a> Vet Surg. 41 (1), 114-120 (2012).</li><li>Remedios, A. M., Fries, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Implant+complications+in+20+triple+pelvic+osteotomies.">Implant complications in 20 triple pelvic osteotomies.</a> Vet Comp Orthop Traumatol. 06 (04), 202-207 (1993).</li><li>Moses, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alternative+surgical+methods+for+treating+juvenile+canine+hip+dysplasia.">Alternative surgical methods for treating juvenile canine hip dysplasia.</a> Aust Vet J. 78 (12), 822-824 (2000).</li><li>Harper, T. A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Femoral+head+and+neck+excision.">Femoral head and neck excision.</a> Vet Clin North Am Small Anim Pract. 47 (4), 885-897 (2017).</li><li>Forster, K. E., 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=Complications+and+owner+assessment+of+canine+total+hip+replacement:+a+multicenter+internet+based+survey.">Complications and owner assessment of canine total hip replacement: a multicenter internet based survey.</a> Vet Surg. 41 (5), 545-550 (2012).</li><li>Volstad, N. J., Schaefer, S. L., Snyder, L. A., Meinen, J. B., Sample, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metallosis+with+pseudotumour+formation:+Long-term+complication+following+cementless+total+hip+replacement+in+a+dog.">Metallosis with pseudotumour formation: Long-term complication following cementless total hip replacement in a dog.</a> Vet Comp Orthop Traumatol. 29 (4), 283-289 (2016).</li><li>Nesser, V. E., Kowaleski, M. P., Boudrieau, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Severe+polyethylene+wear+requiring+revision+total+hip+arthroplasty+in+three+dogs.">Severe polyethylene wear requiring revision total hip arthroplasty in three dogs.</a> Vet Surg. 45 (5), 664-671 (2016).</li><li>Verhoeven, G., Fortrie, R., Van Ryssen, B., Coopman, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Worldwide+screening+for+canine+hip+dysplasia:+where+are+we+now.">Worldwide screening for canine hip dysplasia: where are we now.</a> Vet Surg. 41 (1), 10-19 (2012).</li><li>Willemsen, 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=Patient-specific+3D-printed+shelf+implant+for+the+treatment+of+hip+dysplasia:+Anatomical+and+biomechanical+outcomes+in+a+canine+model.">Patient-specific 3D-printed shelf implant for the treatment of hip dysplasia: Anatomical and biomechanical outcomes in a canine model.</a> J Orthop Res. 40 (5), 1154-1162 (2021).</li><li>Willemsen, 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=Patient-specific+3D-printed+shelf+implant+for+the+treatment+of+hip+dysplasia+tested+in+an+experimental+animal+pilot+in+canines.">Patient-specific 3D-printed shelf implant for the treatment of hip dysplasia tested in an experimental animal pilot in canines.</a> Sci Rep. 12 (1), 3032 (2022).</li><li>Kwananocha, 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=Acetabular+rim+extension+using+a+personalized+titanium+implant+for+treatment+of+hip+dysplasia+in+dogs:+short-term+results.">Acetabular rim extension using a personalized titanium implant for treatment of hip dysplasia in dogs: short-term results.</a> Front Vet Sci. 10, 1160177 (2023).</li><li>Van Der Wal, B. C. H., Sakkers, R. J. B., Meij, B. P., Evers, L. A. M., Weinans, H. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Method of manufacturing an implant. EP3463198B1. , (2021).</li><li>Brainard, B. M., Hofmeister, E. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anesthesia+principles+and+monitoring.">Anesthesia principles and monitoring.</a> Small Animal Surgery. , (2012).</li><li>Martinez-Taboada, F., Redondo, J. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+the+hanging-drop+technique+and+running-drip+method+for+identifying+the+epidural+space+in+dogs.">Comparison of the hanging-drop technique and running-drip method for identifying the epidural space in dogs.</a> Vet Anaesth Analg. 44 (2), 329-336 (2017).</li><li>Johnson, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Approach+to+the+craniodorsal+aspect+of+the+hip+joint+through+a+craniolateral+incision+in+the+dog.">Approach to the craniodorsal aspect of the hip joint through a craniolateral incision in the dog.</a> Piermattei's Atlas of Surgical Approaches to the Bones and Joints of the dog and cat. , (2014).</li><li>Whitelock, R. G., Dyce, J., Houlton, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Repair+of+femoral+trochanteric+osteotomy+in+the+dog.">Repair of femoral trochanteric osteotomy in the dog.</a> J Small Anim Pract. 38 (5), 195-199 (1997).</li><li>Silveira, C. J., Saunders, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Greater+trochanter+osteotomy+as+a+component+of+cementless+total+hip+replacement:+Five+cases+in+four+dogs.">Greater trochanter osteotomy as a component of cementless total hip replacement: Five cases in four dogs.</a> Vet Surg. 51 (2), 303-310 (2022).</li><li>Archibeck, M. J., Rosenberg, A. G., Berger, R. A., Silverton, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trochanteric+osteotomy+and+fixation+during+total+hip+arthroplasty.">Trochanteric osteotomy and fixation during total hip arthroplasty.</a> J Am Acad Orthop Surg. 11 (3), 163-173 (2003).</li><li>Vezzoni, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complications+of+double+and+triple+pelvic+osteotomies.">Complications of double and triple pelvic osteotomies.</a> Complications in Small Animal Surgery. , (2016).</li></ol>

The authors have no conflicts of interest to declare. BM is one of the five inventors of the method of manufacturing an implant to extend the dorsal acetabular rim (2021) of which the patent (EP3463198B1) has been licensed to Rita Leibinger.

Acetabular rim extension using the 3DHIP implant provides advantages over conventional surgical therapies for canine hip dysplasia and has shown promising results to increase coverage of the dysplastic hip joint and reverse hip laxity in short-term follow-up17,18. This publication aimed to showcase the surgical technique with tips and tricks as a surgical manual for implantation of the 3DHIP implant in dogs affected by hip dysplasia.
Selection of candidates for the 3DHIP implant placement-young dogs between 6 and 24 months of age with clinical hip dysplasia marked by hip laxity (FCI grade B-D) with a positive Ortolani subluxation test are adequate candidates. The triradiate acetabular growth plate has to be closed and preferably, no osteoarthritis is present on CT imaging although minor osteophytes up to 2 mm are accepted. Dogs with luxoid hips with near-complete luxation of the femoral head are not accepted for 3DHIP implant placement because of the rapid progression of osteoarthritis, the inability of the femoral head to move into the acetabulum, and expected early conversion to total hip replacement.
There are some critical steps within the surgical technique.
Implant design
Given the individualized design of the 3DHIP implants, a preoperative assessment of the dysplastic hip joint using a CT scan is absolutely mandatory. In addition to the determination of the correct implant size and position of the ventral ilial flange, particularly the amount of acetabular rim extension needed to provide sufficient coverage of the femoral head can be determined.
Surgical approach
A critical step during surgery is sufficient exposure of the dorsal acetabular rim and ventral border of the caudal iliac body for implant placement. The surgical approach to the iliac body and craniodorsal aspects of the hip joint in 3DHIP implantation differs from conventional approaches22. In the presented technique, a trochanteric osteotomy was omitted and a deep gluteal tenotomy was performed while the superficial and middle gluteal muscles were preserved. Hereby, the risk of complications associated with trochanteric osteotomies23,24,25 such as delayed or non-union were avoided and the recovery process was expedited. Additionally, this modified craniodorsal approach can be employed across a variety of ages, breeds, and sizes of dogs without any necessary modifications. Notably, no complications were reported in association with the presented surgical approach.
Correct implant placement
Even though the custom-made 3D-printed hip implant is designed to perfectly fit the unique acetabular anatomy of each dog, imperfect implant placement with 4-5 mm craniocaudal deviation to planning target position was still observed in the first cohort of dogs possibly related to the learning curve with the technique18. The ventral ilial flange of the bone attachment part of the 3DHIP implant allows for more accurate positioning, especially in the dorsoventral direction. However, due to the extracapsular location of the implant, it is still difficult to achieve perfect implant positioning; the inner edge of the acetabulum is obscured by the synovial membrane. Further, osteophyte formation during the lead time of implant production may influence proper implant positioning. To ensure accurate implant positioning according to the preoperative plan, verification using intraoperative fluoroscopy is currently required. It is also expected that with increased experience, the precision of implant positioning will further decrease to below 1-2 mm accurate placement. In the future, guided surgery using 3D-printed surgical drill guides may obviate the need for fluoroscopy.
This technique also has some limitations. Previous short-term results suggest a broad bandwidth of different hip anatomies that can be treated using 3DHIP implants. While the long-term study findings are not yet available, it is advised to consider 3DHIP implantation for dogs that do not exhibit signs of osteoarthritis (OA) or have only a mild degree of OA in their hip joints. The 3DHIP implant placement aims to effectively slow down the progression of hip joint deterioration. Dogs with luxoid hips and moderate to severe hip degeneration as determined in the pre-operative evaluation should be excluded.
Compared to 3DHIP implantation, conventional surgeries to treat canine hip dysplasia such as DPO/TPO present more challenges, especially in a single-stage bilateral procedure and/or in giant dogs due to their invasive nature involving two or three pelvic osteotomies5,6,7,26. Therefore, dogs with bilateral HD can benefit from an acetabular rim extension using a 3DHIP implant; it provides an effective and low invasive single-stage bilateral procedure. In addition, the 3DHIP implantation helps to save valuable time and can prevent the further development of OA that might occur in double-stage bilateral procedures.
To conclude, utilization of the presented 3DHIP implant to extend the dorsal acetabular rim exhibits significant promise as an alternative surgical treatment for hip dysplasia in canines. Especially, the option to offer an effective and low invasive single-stage bilateral procedure for dogs with bilateral hip dysplasia and laxity is an enormous advantage to current alternative treatments. Further monitoring of this new technique in mid- and long-term follow-up is mandatory.

Hip dysplasia (HD) in dogs manifests due to a bad fit between the hip socket (acetabulum) and the femoral head resulting in subluxation of the hip joint. It affects mainly young medium- to large-breed dogs, resulting in joint cartilage deterioration, and ultimately, severe osteoarthritis (OA) leading to chronic pain and low quality of life1,2. The overall prevalence of hip dysplasia in dogs is 15.56%, which varies widely based on breed and classification systems3,4.
Apart from lifestyle changes, hip dysplastic dog patients are treated with antiinflammatory and analgesic drugs to control pain and maintain mobility4. In case of hip laxity in young adult dogs, the only surgical resort is double (DPO) or triple pelvic osteotomy (TPO), a procedure involving two or three full cuts of the pelvic bones to expand the coverage of the femoral head. However, complications after osteotomies are common and the progression of OA is still observed5,6,7,8,9. Once severe OA and chronic pain have developed, only high-impact complex surgery like total hip replacement (THR) or salvage femoral head and neck ostectomy (FHO) remain10. However, FHO presents less favorable outcomes in large breed dogs and necessitates prolonged physical therapy for the restoration of limb function11. Furthermore, THR is technically challenging and inherently associated with severe complications12,13,14. Therefore, effective hip dysplasia therapy necessitating only low-impact surgery and with a lower complication risk is required before this end stage is achieved.
The 3-dimensional (3D)-printed personalized hip implant (3DHIP) is a first-of-a-kind treatment for canine hip dysplasia developed with the intention to offer a minimal traumatic dog-specific implant that restores hip joint stability. The technique involves a titanium implant to treat mainly young adult (6 months to 2 years old) dog patients with a dysfunctional hip joint showing hip laxity grade B (borderline) to D (moderate) according to the F&#233;d&#233;ration Cynologique Internationale (FCI)15. After computed tomography (CT) imaging of the dysplastic joint, an implant is designed following the specific anatomy of the hip joint in a personalized manner to extend the dorsal acetabular rim, thereby preventing hip joint subluxation and restoring hip joint stability.
A previous canine cadaver study revealed that the implant enhanced femoral head coverage and demonstrated failure under an impact force of 1,330 &#177; 320 Newtons16. Subsequently, a pilot study with experimental dogs demonstrated enhanced femoral head coverage, reduced hip laxity, and increased weight bearing by force plate analysis. Furthermore, examination of the intervened hips at 6 months post-implantation revealed normal volume and a smooth surface of both the femoral head and acetabulum cartilage, accompanied by joint capsule hypertrophy based on gross and histological assessments17. Upon confirmation of the efficacy and safety of the implant and treatment concept, a clinical investigation was carried out on client-owned dogs suffering from hip dysplasia. The short-term study revealed that the benefits of the 3D-printed acetabular rim extension implant are a personalized, good fit of the implant to the acetabulum restoring hip joint stability, decreased pain-related activities, and a low-impact surgical procedure18. Application of the implant requires access to the ventrocaudal aspect of the iliac body and the craniodorsal aspect of the hip joint. In this paper, we describe our surgical planning and surgical procedure with a modified craniodorsal approach to the hip joint as a manual for implantation of the 3DHIP in dogs affected by hip dysplasia.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>The laborotory for implant design</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>3D Lab</td>
			<td>University Medical Center Utrecht 3D, Utrecht, Netherlands&#160;</td>
			<td></td>
			<td>The laboratory responsible for designing the 3DHIP implant. [https://www.umcutrecht.nl/nl/3d-lab/]</td>
		</tr>
		<tr>
			<td>Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>3-Matic software version 17</td>
			<td>Materialise, Leuven, Belgium</td>
			<td></td>
			<td>CT DICOM data processing</td>
		</tr>
		<tr>
			<td>Materialise Mimics software version 25.1</td>
			<td>Materialise, Leuven, Belgium</td>
			<td></td>
			<td>Software to design the 3DHIP implant on the 3D model of the pelvis</td>
		</tr>
		<tr>
			<td>Implant manufacturer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Amnovis</td>
			<td>Amnovis, Aarschot, Belgium</td>
			<td></td>
			<td>Printing and postprocessing of the 3DHIP implant. [https://www.amnovis.com/]</td>
		</tr>
		<tr>
			<td>Instrument and machine</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>2.4 LeiLOX locking screw titanium</td>
			<td>Rita Leibinger, BW, Germany</td>
			<td>242-224</td>
			<td>Titanium self tapping locking screw 2.4 mm.</td>
		</tr>
		<tr>
			<td>2.7 LeiLOX locking screw titanium</td>
			<td>Rita Leibinger, BW, Germany</td>
			<td>242-227</td>
			<td>Titanium self tapping locking screw 2.7 mm.</td>
		</tr>
		<tr>
			<td>3.5 LeiLOX locking screw titanium</td>
			<td>Rita Leibinger, BW, Germany</td>
			<td>242-235</td>
			<td>Titanium self tapping locking screw 3.5 mm.</td>
		</tr>
		<tr>
			<td>BLUE SEAL 100 x 360 mm</td>
			<td>Interster, Wormerveer, Netherlands</td>
			<td>3FKFB210819</td>
			<td>The transparent sterilization laminate size 100 x 360 mm</td>
		</tr>
		<tr>
			<td>ETHILON 3-0 with FS-1 needle</td>
			<td>Johnson &#38; Johnson Medical GmbH, Norderstedt, Germany</td>
			<td>669H</td>
			<td>Polyamide 6 3-0 (non-absorbable suture material) with 24 mm 3/8c reverse cutting needle using for skin closure</td>
		</tr>
		<tr>
			<td>Fluoroscopy model NZS 229</td>
			<td>Philips, Eindhoven, Netherlands</td>
			<td></td>
			<td>Fluoroscopy</td>
		</tr>
		<tr>
			<td>Foley Catheter 10 fr x 90 cm (36&#34;) with 3 cc Balloon</td>
			<td>MILA international inc., Kentucky, USA</td>
			<td>MLIUC1036</td>
			<td>Foley urine catheter size 10 fr</td>
		</tr>
		<tr>
			<td>Foley Catheter 6 fr x 60 cm (24&#34;) with 3 cc Balloon</td>
			<td>MILA international inc., Kentucky, USA</td>
			<td>MLIUC624</td>
			<td>Foley urine catheter size 6 fr</td>
		</tr>
		<tr>
			<td>Foley Catheter 8 fr x 90 cm (36&#34;) with 3 cc Balloon</td>
			<td>MILA international inc., Kentucky, USA</td>
			<td>MLIUC836</td>
			<td>Foley urine catheter size 8 fr</td>
		</tr>
		<tr>
			<td>Ioban 2</td>
			<td>3M, MN, USA</td>
			<td>6640EU</td>
			<td>Iodine-impregnated surgical drape&#160;</td>
		</tr>
		<tr>
			<td>Miele professional G 7826</td>
			<td>Miele Nederland B.V., Vianen, Netherlands</td>
			<td></td>
			<td>The hygienic washing machine&#160;</td>
		</tr>
		<tr>
			<td>MMM sterilizer OB10643</td>
			<td>MMM Group, Planegg, Germany</td>
			<td></td>
			<td>Steam autoclave</td>
		</tr>
		<tr>
			<td>MONOCRYL 2-0 with SH Plus needle</td>
			<td>Johnson &#38; Johnson Medical GmbH, Norderstedt, Germany</td>
			<td>MCP3170H</td>
			<td>Poliglecaprone 25&#160; plus antibacterial 2-0 (absorbable suture material) with 26 mm 1/2c taperpoint needle using for subcutaneous tissue closure</td>
		</tr>
		<tr>
			<td>MONOCRYL 3-0 with SH Plus needle</td>
			<td>Johnson &#38; Johnson Medical GmbH, Norderstedt, Germany</td>
			<td>MCP3160H</td>
			<td>Poliglecaprone 25&#160; plus antibacterial 3-0 (absorbable suture material) with 26 mm 1/2c taperpoint needle using for subcutaneous tissue closure</td>
		</tr>
		<tr>
			<td>PDS 0 with CP needle</td>
			<td>Johnson &#38; Johnson Medical GmbH, Norderstedt, Germany</td>
			<td>PDP485H</td>
			<td>Polydioxanone plus antibacterial 0 (absorbable suture material) with 40 mm 1/2c&#160; reverse cutting needle using for muscle fascia and tendon closure</td>
		</tr>
		<tr>
			<td>PDS 2-0 with CP-1 needle</td>
			<td>Johnson &#38; Johnson Medical GmbH, Norderstedt, Germany</td>
			<td>PDP466H</td>
			<td>Polydioxanone plus antibacterial 2-0 (absorbable suture material) with 36 mm 1/2c reverse cutting needle using for muscle fascia and tendon closure</td>
		</tr>
		<tr>
			<td>ProX DMP320&#160;</td>
			<td>3D systems, South Carolina, USA</td>
			<td></td>
			<td>Direct metal printing machine using selective laser melting technology&#160;</td>
		</tr>
		<tr>
			<td>Medications</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Betadine oplossing</td>
			<td>Mylan B.V., Amstelveen, Netherlands</td>
			<td>RVG 01331&#160;</td>
			<td>Povidone-iodine solution 100 mg/mL (500 mL)</td>
		</tr>
		<tr>
			<td>Betadine shampoo</td>
			<td>Mylan B.V., Amstelveen, Netherlands</td>
			<td>RVG 08943</td>
			<td>Povidone-iodine 75 mg/mL (120 mL)</td>
		</tr>
		<tr>
			<td>Carporal 20 mg</td>
			<td>AST Farma B.V. Oudewater, Netherlands</td>
			<td>REG NL 101766</td>
			<td>Carprofen 20 mg/tablet</td>
		</tr>
		<tr>
			<td>Carporal 40 mg</td>
			<td>AST Farma B.V. Oudewater, Netherlands</td>
			<td>REG NL 115715</td>
			<td>Carprofen 40 mg/tablet</td>
		</tr>
		<tr>
			<td>Carporal 50 mg</td>
			<td>AST Farma B.V. Oudewater, Netherlands</td>
			<td>REG NL 101767</td>
			<td>Carprofen 50 mg/tablet</td>
		</tr>
		<tr>
			<td>Cefazolin Mylan 1 g</td>
			<td>Mylan B.V., Amstelveen, Netherlands</td>
			<td>RVG 16532</td>
			<td>Cefazolin powder 1 g for injection</td>
		</tr>
		<tr>
			<td>Chlorhexidine 0.5% in alcohol 70%&#160;spray</td>
			<td>Orphi Farma BV, Lage Zwaluwe, Netherlands</td>
			<td>8711407672906</td>
			<td>Chlorhexidine 0.5% in alcohol 70%&#160;spray (250 mL)</td>
		</tr>
		<tr>
			<td>Dexdomitor 0.5 mg/mL</td>
			<td>Orion Corporation, Espoo, Finland</td>
			<td>EU/2/02/033/001-002</td>
			<td>Dexmedetomidine hydrochloride 0.5 mg/mL for injection (20 mL)&#160;</td>
		</tr>
		<tr>
			<td>Gabapentin Sandoz 300 mg&#160;</td>
			<td>Sandoz B.V., Almere, Netherlands</td>
			<td>RVG 33681</td>
			<td>Gabapentin 300 mg/capsule&#160;</td>
		</tr>
		<tr>
			<td>GABAPENTINE TEVA 100 mg</td>
			<td>Teva B.V., Haarlem, Netherlands</td>
			<td>RVG 31980</td>
			<td>Gabapentin 100 mg/capsule&#160;</td>
		</tr>
		<tr>
			<td>HiBiScrub</td>
			<td>M&#246;lnlycke Health Care AB., Utrecht, Netherlands</td>
			<td>RVG 10156</td>
			<td>Chlorhexidine digluconate 40 mg/mL (500 mL)</td>
		</tr>
		<tr>
			<td>Insistor 10 mg/mL</td>
			<td>Richter pharma AG, Oostenrijk, Netherlands</td>
			<td>REG NL 121166</td>
			<td>Methadone hydrochloride 10 mg/mL for injection (10 mL)&#160;</td>
		</tr>
		<tr>
			<td>Isoflutek 1000 mg/g</td>
			<td>Laboratorios Karizoo S.A., Barcelona, Spain</td>
			<td>REG NL 118938</td>
			<td>Isoflurane 1000 mg/g (250 mL)&#160;</td>
		</tr>
		<tr>
			<td>Levobupivacaine Fresenius Kabi 2.5 mg/mL</td>
			<td>Fresenius Kabi Nederland b.v., Huis ter Heide, Netherlands</td>
			<td>AWA 0611</td>
			<td>Levobupivacaine 2.5 mg/mL solution for injection (10 mL)</td>
		</tr>
		<tr>
			<td>Morfine HCI CF 10 mg/mL</td>
			<td>Centrafarm B.V., Breda, Netherlands</td>
			<td>RVG 50836</td>
			<td>Morphine hydrochloride 10 mg/mL (1 mL)&#160;</td>
		</tr>
		<tr>
			<td>Narketan 10</td>
			<td>Vetoquinol B.V., Breda, Netherlands</td>
			<td>vm08007/4090</td>
			<td>Ketamine 10 mg/mL (10 mL)</td>
		</tr>
		<tr>
			<td>Propofol 10 mg/mL</td>
			<td>Fresenius Kabi Nederland b.v., Huis ter Heide, Netherlands</td>
			<td>RVG 110627</td>
			<td>Propofol 10 mg/mL emulsion for injection or infusion (50 mL)&#160;</td>
		</tr>
		<tr>
			<td>Rimadyl&#160;</td>
			<td>Zoetis B.V., Capelle a/d Ijssel, Netherlands</td>
			<td>REG NL 10101</td>
			<td>Carprofen 50 mL/mL for injection (20 mL)</td>
		</tr>
		<tr>
			<td>Sufentanil-hameln 50 mcg/mL</td>
			<td>Hameln pharma gmbh, Hameln, Germany</td>
			<td>4260016653249</td>
			<td>Sufentanil citrate 50 mcg/mL for injection</td>
		</tr>
		<tr>
			<td>Trazadone EG 100 mg</td>
			<td>EG (Eurogenerics) NV Heizel, Brussel, Belgium</td>
			<td>BE439607</td>
			<td>Trazadone hydrochloride 100 mg/tablet</td>
		</tr>
	</tbody>
</table>

surgical technique of the 3-dimensional-printed personalized hip implant for the treatment of canine hip dysplasia

Hip dysplasia causes major disability in dogs. Treatment options are limited to palliative treatment (e.g., pain relief, physical exercise, lifestyle changes, and weight control) or invasive surgeries such as pelvic osteotomies and total hip arthroplasty. Hence, a strong unmet need exists for an effective and dog-friendly solution that enhances the quality of life of man&#39;s best friend. We fill this treatment gap by offering a minimally traumatic and extraarticular, dog-specific, 3-dimensional-printed, hip implant (3DHIP) that restores hip joint stability. The surgical treatment using a 3DHIP implant is less invasive than osteotomies and can be performed bilaterally in one surgical session. The 3DHIP implant extends the dorsal acetabular rim of the dysplastic hip joint thereby increasing coverage of the femoral head and inhibiting joint subluxation with fast recovery. Sufficient access to the dorsal acetabular rim and ventral border of the iliac body together with optimal fitting and fixation of the implant are key steps for a successful 3DHIP implantation and imply the need for a specific approach. The present article aims to showcase this innovative surgical technique with tips and tricks as a surgical manual for implantation of the 3DHIP implant in dogs affected by hip dysplasia.

Short-term results of acetabular rim extension have previously been published, arising from an ongoing observational study at the Utrecht University Department of Clinical Sciences18. From December 2019 to March 2022, a total of 61 hips from 34 dogs were included in the study. The cohort consisted of 24 males and 10 females, with a median age of 12 months (ranging from 7 to 38 months) and a median body weight of 27.3 kg (ranging from 12 to 86 kg). Seven dogs underwent surgery on a unilateral hip, while twenty dogs underwent bilateral hip surgery in a single session. Additionally, seven dogs received surgery on both hips, conducted in two separate sessions.
The previous study found a significant increase in the Norberg Angle (NA), linear percentage of femoral head coverage (LFO), and percentage of femoral head coverage (PC) immediately after implantation (Table 1). Moreover, the postoperative Ortolani subluxation sign was negative in 96.7% of operated limbs indicating that the acetabular rim extension implant restored hip congruency and diminished laxity of dysplastic hips18. Particularly, the ability to increase coverage of the femoral head without performing any re-directional osteotomy allowed physiological pelvic geometry retainment. The minimally invasive technique resulted in low incidences of complications (4.9%) in the short-term, encouraged early mobilization, and decreased pain related to activity (Table 1).
Furthermore, this technique allowed single-stage bilateral 3DHIP implant placement. Treated limb(s) were weightbearing without pelvic support within 12 to 24 h after surgery. During the 12-month monitoring period, 3 dogs required revision surgery due to either implant failure (2 dogs) or a significant advancement of osteoarthritis (1 dog). Using the presented surgical approach simultaneously with the suggested hip joint movements (abduction, flexion, and external rotation), better exposure of the ventrocaudal aspect of the iliac shaft and craniodorsal aspect of the hip joint was obtained, facilitating 3DHIP implant positioning. In addition, intra-operative fluoroscopy increased the accuracy of implant positioning.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66005/66005fig1.jpg" /> 
Figure 1: Schematic illustrations showing a positive Ortolani subluxation sign counteracted by the 3DHIP implant. (A-D) Positive Ortolani subluxation sign. (A) The dog&#39;s limb is positioned in neutral flexion and adduction, and a force (red arrows) is exerted towards the dorsum of the dog along the femoral axis that causes (B) dorsal subluxation of the dysplastic hip joint. (C) Gradual limb abduction (blue arrow) is performed while maintaining pressure on the femur. (D) Dependent on the acetabular rim deficiency, the subluxated femoral head falls back into the socket (green arrows). (E) The 3DHIP implant is introduced to enhance the stability of the dysplastic hip joint by reinforcing the hip capsule and labrum, which serve as weight-bearing and stabilizing surfaces (purple arrows). (F) Upon magnification of the rectangular area, the internal 1.5 mm offset of the implant is visible in the red circle, which ensures the capsule attachment remains unaffected. This figure was modified from Willemsen et al.17. Abbreviation: 3DHIP = 3-dimensional-printed, hip implant. <a href="https://www.jove.com/files/ftp_upload/66005/66005fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/66005/66005fig2.jpg" /> 
Figure 2: Example of the preoperative hip radiographs used for F&#233;d&#233;ration Cynologique Internationale hip dysplasia classification. Radiographs are taken in the ventro-dorsal hip extended position. From left to right, FCI classifies hip dysplasia into five different categories: A (normal), B (borderline), C (mild hip dysplasia), D (moderate hip dysplasia), and E (severe hip dysplasia). Abbreviation: FCI = F&#233;d&#233;ration Cynologique Internationale. <a href="https://www.jove.com/files/ftp_upload/66005/66005fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/66005/66005fig3.jpg" /> 
Figure 3: Images of hip joint CT examinations illustrating osteophytes of varying sizes. The thickness of all featuring osteophytes is measured in both (A,B) coronal planes and (C) transverse planes at the cranial (white arrowhead) and caudal (red arrowhead) acetabular rim and femoral neck (black arrowhead). Dogs that have femoral neck and/or cranial and caudal acetabular rim osteophytes &#62; 2 mm are excluded. CT examination slice thickness is 5 mm. <a href="https://www.jove.com/files/ftp_upload/66005/66005fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/66005/66005fig4.jpg" /> 
Figure 4: Design process of 3DHIP implant. (A) Segmentation of the region of interest from CT DICOM data. (B) Measurements of the native Norberg angles on the 3D model of the pelvis. (C) Rendering of a 3DHIP implant on the right hip, lateral view. (D) Rendering of bilateral 3DHIP implants, dorsal-ventral view. Abbreviation: 3DHIP = 3-dimensional-printed, hip implant. <a href="https://www.jove.com/files/ftp_upload/66005/66005fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/66005/66005fig5.jpg" /> 
Figure 5: Rendering of a designed 3DHIP implant. (A) Rendered image of the lateral/outer side of the 3DHIP implant. (B) Rendered image of the inner implant surface showing the porous surface allowing bone ingrowth for osseointegration. The bone attachment part (black arrowhead) of rendered implant incorporating 4 locking screw holes and the ventral ilium flange (black arrow) for assisting in correct implant positioning and stabilization. The rim extension part (red arrowhead) of the rendered implant with the internal 1.5 mm offset (red arrow) allowing unhindered joint capsule attachment. (C) Photograph of a titanium 3DHIP implant showcasing 4 screw holes arranged in the sequence for screw insertion. Abbreviation: 3DHIP = 3-dimensional-printed, hip implant. <a href="https://www.jove.com/files/ftp_upload/66005/66005fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/66005/66005fig6.jpg" /> 
Figure 6: Schematic illustration of skin incision. (A) Red oval marks the area in which the skin incision is made. (B) Magnification of red circle in (A). The skin incision is made using a #10-blade centered on the tip of the greater trochanter aiming at the cranial dorsal iliac spine. The incision length is approximately 8-15 cm. In the magnified image, the superficial leaf of fascia latae is incised along the cranial muscle border of the biceps femoris muscle. Orientation: left is cranial, top is dorsal. <a href="https://www.jove.com/files/ftp_upload/66005/66005fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/66005/66005fig7.jpg" /> 
Figure 7: Schematic illustrations and photographs of an embalmed cadaver depicting the surgical approach for 3DHIP implantation. (A-D) Schematic illustrations and&#160;(E-H) photographs of an embalmed cadaver depict the surgical approach for 3DHIP implantation.&#160;(A and E) The red dotted line marks the line of the incision&#160;through the intermuscular septum between the superficial gluteal muscle, middle gluteal muscle, and the tensor fascia latae muscle. (B and F) Red dotted line marks the tenotomy site. The superficial and middle gluteal muscles are retracted dorsally to expose the deep gluteal muscle. Dissecting scissors are used to undermine the deep gluteal muscle near its insertion on the greater trochanter. A tenotomy is performed close (at 0.5-1 cm) to its insertion on the bone. (C and G) Adequate exposure for 3DHIP implant placement requires&#160;freeing the deep gluteal muscle from the joint capsule and lateral surface of the iliac body and partially freeing the iliacus muscle and rectus femoris muscles from the caudoventral border of the ilial shaft (red dotted line). (D and H) The 3DHIP implant is placed outside the capsule of the hip joint. For accuracy and ease of positioning, the ilium flange of the attachment part of the implant is placed under the ventral border of the exposed caudoventral iliac shaft. Orientation: left is cranial, top is dorsal. 1) biceps femoris muscle, 2) tensor fascia latae muscle, 3) fatty triangle, 4) superficial gluteal muscle, 5) middle gluteal muscle, 6) deep gluteal muscle/tendon, 7) vastus lateralis muscle, 8) hip joint capsule, 9) articularis coxae muscle, 10) caudal part of iliac body, 11) rectus femoris muscle, 12) ilium flange of the implant, and 13) rim extension part of the implant. <a href="https://www.jove.com/files/ftp_upload/66005/66005fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/66005/66005fig8.jpg" /> 
Figure 8:&#160;Intraoperative fluoroscopy. After implant positioning and temporary fixation with one locking screw, intraoperative fluoroscopy is performed in (A) lateral and (B) latero-oblique views using a digital image intensifier to assess and compare the positioning of the implant to the preoperative planning. <a href="https://www.jove.com/files/ftp_upload/66005/66005fig8large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 9" class="xfigimg" src="/files/ftp_upload/66005/66005fig9.jpg" /> 
Figure 9:&#160;Examples of postoperative radiographs in three planes and postoperative CT scan after single-stage bilateral 3DHIP implant surgery in one dog. (A) Radiograph ventrodorsal view; (B) radiograph right latero-oblique view; (C) radiograph left latero-oblique view. 3D reconstruction from post-operative CT in lateral view showing the (D) right hip&#160;and (E) dorso-ventral view. (F)&#160;Postoperative CT of both hips in the transverse plane with a slice thickness of 5 mm. The 3DHIP implants were fixed with four locking screws on each side. <a href="https://www.jove.com/files/ftp_upload/66005/66005fig9large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Outcome measurements</td>
			<td>Preoperatively</td>
			<td>Immediated postoperatively</td>
			<td>1.5 months</td>
			<td>3 months</td>
			<td>p-value</td>
		</tr>
		<tr>
			<td>NA (&#9702;)</td>
			<td>87 &#177; 13a</td>
			<td>134 &#177; 19b</td>
			<td>-</td>
			<td>131 &#177; 20b</td>
			<td>&#60;0.001*</td>
		</tr>
		<tr>
			<td>LFO (%)</td>
			<td>22 &#177; 15a</td>
			<td>81 &#177; 16b</td>
			<td>-</td>
			<td>76 &#177; 19b</td>
			<td>&#60;0.001*</td>
		</tr>
		<tr>
			<td>PC (%)</td>
			<td>33 &#177; 17a</td>
			<td>79 &#177; 21b</td>
			<td>-</td>
			<td>77 &#177; 20b</td>
			<td>0.002*</td>
		</tr>
		<tr>
			<td>HCPI (%)</td>
			<td>31.44 &#177; 11.9a</td>
			<td>-</td>
			<td>20.39 &#177; 10.09b</td>
			<td>17.69 &#177; 10.8b</td>
			<td>&#60;0.001**</td>
		</tr>
	</tbody>
</table>
Table 1: Short-term results (Mean &#177; SD) of radiographic measurements using coronal CT and pain-related owner questionnaire using Helsinki Chronic Pain Index in dogs with hip dysplasia that underwent 3DHIP implantation. This table was modified from Kwananocha et al.18. HCPI (%) = 100% &#215; total index score/maximum possible index score of the answered questions. a,bp-value &#60; 0.05 from Bonferroni, p-value* from repeated measure analysis, p-value** from generalized linear mixed model. Abbreviations: NA = Norberg angle; LFO = linear percentage of femoral head overlap; PC = percentage of femoral head coverage; SD = standard deviation; HCPI = Helsinki Chronic Pain Index.
Supplementary Video S1: Direct postoperative weight-bearing allowed with only short leash walks on a slip-resistant floor from the day after surgery.&#160;<a href="https://www.jove.com/files/ftp_upload/66005/Supplementary_video.zip" style="font-size: 14px;" target="_blank">Please click here to download this video.</a>

Read this Scientific Article Protocol about Author Spotlight: A Novel 3D-Printed Titanium Implant for Minimally Invasive Treatment of Hip Dysplasia in Young Dogs at JoVE.com

Author Spotlight: A Novel 3D-Printed Titanium Implant for Minimally Invasive Treatment of Hip Dysplasia in Young Dogs

This work describes a novel surgical technique for extracapsular implantation of a personalized, 3-dimensional-printed, joint-preserving implant. This novel treatment aims to restore hip stability in young adult dogs suffering from hip dysplasia with laxity by uniquely reproducing the anatomical shape of the acetabular rim of the hip joint.

Surgical Technique of the 3-Dimensional-printed Personalized Hip Implant for the Treatment of Canine Hip Dysplasia

Hip dysplasia causes major disability in dogs. Treatment options are limited to palliative treatment (e.g., pain relief, physical exercise, lifestyle ...

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1.5K Views.  Utrecht University. This work describes a novel surgical technique for extracapsular implantation of a personalized, 3-dimensional-printed, joint-preserving implant. This novel treatment aims to restore hip stability in young adult dogs suffering from hip dysplasia with laxity by uniquely reproducing the anatomical shape of the acetabular rim of the hip joint.

Video: Author Spotlight: A Novel 3D-Printed Titanium Implant for Minimally Invasive Treatment of Hip Dysplasia in Young Dogs

A Rapid Technique for the Visualization of Live Immobilized Yeast Cells

We present here a simple, rapid, and extremely flexible technique for the immobilization and visualization of growing yeast cells by epifluorescence microscopy. The technique is equally suited for visualization of static yeast populations, or time courses experiments up to ten hours in length. My microscopy investigates epigenetic inheritance at the silent mating loci in S. cerevisiae. There are two silent mating loci, HML and HMR, which are normally not expressed as they are packaged in heterochromatin. In the sir1 mutant background silencing is weakened such that each locus can either be in the expressed or silenced epigenetic state, so in the population as a whole there is a mix of cells of different epigenetic states for both HML and HMR. My microscopy demonstrated that there is no relationship between the epigenetic state of HML and HMR in an individual cell. sir1 cells stochastically switch epigenetic states, establishing silencing at a previously expressed locus or expressing a previously silenced locus. My time course microscopy tracked individual sir1 cells and their offspring to score the frequency of each of the four possible epigenetic switches, and thus the stability of each of the epigenetic states in sir1 cells. See also  Xu et al., Mol. Cell 2006.

A rapid technique for the visualization of growing immobilized yeast cells, here applied to fluorescent reporters at the silent mating loci HML and HMR

We present here a simple, rapid, and extremely flexible technique for the immobilization and visualization of growing yeast cells by epifluorescence ...

Electroporation of Mycobacteria

High efficiency transformation is a major limitation in the study of mycobacteria. The genus Mycobacterium can be difficult to transform; this is mainly caused by the thick and waxy cell wall, but is compounded by the fact that most molecular techniques have been developed for distantly-related species such as Escherichia coli and Bacillus subtilis. In spite of these obstacles, mycobacterial plasmids have been identified and DNA transformation of many mycobacterial species have now been described. The most successful method for introducing DNA into mycobacteria is electroporation. Many parameters contribute to successful transformation; these include the species/strain, the nature of the transforming DNA, the selectable marker used, the growth medium, and the  conditions for the electroporation pulse. Optimized methods for the transformation of both slow- and fast-grower are detailed here. Transformation efficiencies for different mycobacterial species and with various selectable markers are reported.

Mycobacterial pathogenic strategies remain poorly understood. The slow growth rate of most species, the impenetrable nature of the cell-wall, and the hazards of working with pathogens make mycobacteria difficult to study and are largely responsible for our poor understanding of these organisms. In this video we will demonstrate the technique of electroporation, which involves subjecting cells to a brief high electrical impulse to allow the entry of DNA. It is the most widely used method for introducing DNA into mycobacterial cells.

High efficiency transformation is a major limitation in the study of mycobacteria. The genus Mycobacterium can be difficult to transform; this is mainly ...

Surgical Induction of Endolymphatic Hydrops by Obliteration of the Endolymphatic Duct

Surgical induction of endolymphatic hydrops (ELH) in the guinea pig by obliteration and obstruction of the endolymphatic duct is a well-accepted animal model of the condition and an important correlate for human Meniere's disease. In 1965, Robert Kimura and Harold Schuknecht first described an intradural approach for obstruction of the endolymphatic duct (Kimura 1965). Although effective, this technique, which requires penetration of the brain's protective covering, incurred an undesirable level of morbidity and mortality in the animal subjects. Consequently, Andrews and Bohmer developed an extradural approach, which predictably produces fewer of the complications associated with central nervous system (CNS) penetration.(Andrews and Bohmer 1989) 
The extradural approach described here first requires a midline incision in the region of the occiput to expose the underlying muscular layer. We operate only on the right side. After appropriate retraction of the overlying tissue, a horizontal incision is made into the musculature of the right occiput to expose the right temporo-occipital suture line. The bone immediately inferio-lateral the suture line (Fig 1) is then drilled with an otologic drill until the sigmoid sinus becomes visible. Medial retraction of the sigmoid sinus reveals the operculum of the endolymphatic duct, which houses the endolymphatic sac. Drilling medial to the operculum into the area of the endolymphatic sac reveals the endolymphatic duct, which is then packed with bone wax to produce obstruction and ultimately ELH. 
In the following weeks, the animal will demonstrate the progressive, fluctuating hearing loss and histologic evidence of ELH.

This video shows how to surgically obstruct the guinea pig's endolymphatic duct to produce endolymphatic hydrops.

Surgical induction of endolymphatic hydrops (ELH) in the guinea pig by obliteration and obstruction of the endolymphatic duct is a well-accepted animal ...

Simple and Efficient Technique for the Preparation of Testicular Cell Suspensions

Mammalian testes are very complex organs that contain over 30 different cell types, including somatic testicular cells and different stages of germline cells. This heterogeneity is an important drawback concerning the study of the bases of mammalian spermatogenesis, as pure or enriched cell populations in certain stages of sperm development are needed for most molecular analyses1.
 Various strategies such as Staput2,3, centrifugal elutriation1, and flow cytometry (FC)4,5 have been employed to obtain enriched or purified testicular cell populations in order to enable differential gene expression studies. 
It is required that cells are in suspension for most enrichment/ purification approaches. Ideally, the cell suspension will be representative of the original tissue, have a high proportion of viable cells and few multinucleates - which tend to form because of the syncytial nature of the seminiferous epithelium6,7 - and lack cell clumps1 . Previous reports had evidenced that testicular cell suspensions prepared by an exclusively mechanical method clumped more easily than trypsinized ones1 . On the other hand, enzymatic treatments with RNAses and/or disaggregating enzymes like trypsin and collagenase lead to specific macromolecules degradation, which is undesirable for certain downstream applications. The ideal process should be as short as possible and involve minimal manipulation, so as to achieve a good preservation of macromolecules of interest such as mRNAs. Current protocols for the preparation of cell suspensions from solid tissues are usually time-consuming, highly operator-dependent, and may selectively damage certain cell types1,8 . 
	The protocol presented here combines the advantages of a highly reproducible and extremely brief mechanical disaggregation with the absence of enzymatic treatment, leading to good quality cell suspensions that can be used for flow cytometric analysis and sorting4, and ulterior gene expression studies9 .

A novel protocol for the mechanical preparation of testicular cell suspensions from rodent material, avoiding enzymes and detergents, is described. The method is very simple, fast, reproducible, and renders good quality cell suspensions, which are suitable for flow sorting and RNA extraction.

Mammalian testes are very complex organs that contain over 30 different cell types, including somatic testicular cells and different stages of germline ...

Autonomously Bioluminescent Mammalian Cells for Continuous and Real-time Monitoring of Cytotoxicity

Mammalian cell-based in vitro assays have been widely employed as alternatives to animal testing for toxicological studies but have been limited due to the high monetary and time costs of parallel sample preparation that are necessitated due to the destructive nature of firefly luciferase-based screening methods. This video describes the utilization of autonomously bioluminescent mammalian cells, which do not require the destructive addition of a luciferin substrate, as an inexpensive and facile method for monitoring the cytotoxic effects of a compound of interest. Mammalian cells stably expressing the full bacterial bioluminescence (luxCDABEfrp) gene cassette autonomously produce an optical signal that peaks at 490 nm without the addition of an expensive and possibly interfering luciferin substrate, excitation by an external energy source, or destruction of the sample that is traditionally performed during optical imaging procedures. This independence from external stimulation places the burden for maintaining the bioluminescent reaction solely on the cell, meaning that the resultant signal is only detected during active metabolism. This characteristic makes the lux-expressing cell line an excellent candidate for use as a biosentinel against cytotoxic effects because changes in bioluminescent production are indicative of adverse effects on cellular growth and metabolism. Similarly, the autonomous nature and lack of required sample destruction permits repeated imaging of the same sample in real-time throughout the period of toxicant exposure and can be performed across multiple samples using existing imaging equipment in an automated fashion.

Mammalian cells expressing the bacterial bioluminescence gene cassette (lux) produce light autonomously. The resulting bioluminescent dynamics upon chemical exposure have been demonstrated to reflect the treatment effects on cellular growth and metabolism, making these cells an inexpensive, continuous, real-time toxicity screening tool that can easily be adapted for high-throughput automation.

Mammalian  cell-based in vitro assays have been  widely employed as alternatives to animal testing for toxicological studies but  have been limited due to ...

Isolation of Murine Valve Endothelial Cells

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and surrounded by a monolayer of valve endothelial cells (VECs). VECs play essential roles in establishing the valve structures during embryonic development, and are important for maintaining life-long valve integrity and function. In contrast to a continuous endothelium over the surface of healthy valve leaflets, VEC disruption is commonly observed in malfunctioning valves and is associated with pathological processes that promote valve disease and dysfunction. Despite the clinical relevance, focused studies determining the contribution of VECs to development and disease processes are limited. The isolation of VECs from animal models would allow for cell-specific experimentation. VECs have been isolated from large animal adult models but due to their small population size, fragileness, and lack of specific markers, no reports of VEC isolations in embryos or adult small animal models have been reported. Here we describe a novel method that allows for the direct isolation of VECs from mice at embryonic and adult stages. Utilizing the Tie2-GFP reporter model that labels all endothelial cells with Green Fluorescent Protein (GFP), we have been successful in isolating GFP-positive (and negative) cells from the semilunar and atrioventricular valve regions using fluorescence activated cell sorting (FACS). Isolated GFP-positive VECs are enriched for endothelial markers, including CD31 and von Willebrand Factor (vWF), and retain endothelial cell expression when cultured; while, GFP-negative cells exhibit molecular profiles and cell shapes consistent with VIC phenotypes. The ability to isolate embryonic and adult murine VECs allows for previously unattainable molecular and functional studies to be carried out on a specific valve cell population, which will greatly improve our understanding of valve development and disease mechanisms.

The ability to isolate heart valve endothelial cells (VECs) is critical for understanding mechanisms of valve development, maintenance, and disease. Here we describe the isolation of VECs from embryonic and adult Tie2-GFP mice using FACS that will allow for studies determining the contribution of VECs in developmental and disease processes.

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and ...

Probing High-density Functional Protein Microarrays to Detect Protein-protein Interactions

High-density functional protein microarrays containing ~4,200 recombinant yeast proteins are examined for kinase protein-protein interactions using an affinity purified yeast kinase fusion protein containing a V5-epitope tag for read-out. Purified kinase is obtained through culture of a yeast strain optimized for high copy protein production harboring a plasmid containing a Kinase-V5 fusion construct under a GAL inducible promoter. The yeast is grown in restrictive media with a neutral carbon source for 6 hr followed by induction with 2% galactose. Next, the culture is harvested and kinase is purified using standard affinity chromatographic techniques to obtain a highly purified protein kinase for use in the assay. The purified kinase is diluted with kinase buffer to an appropriate range for the assay and the protein microarrays are blocked prior to hybridization with the protein microarray. After the hybridization, the arrays are probed with monoclonal V5 antibody to identify proteins bound by the kinase-V5 protein. Finally, the arrays are scanned using a standard microarray scanner, and data is extracted for downstream informatics analysis1,2 to determine a high confidence set of protein interactions for downstream validation in vivo.

Using protein microarrays containing nearly the entire S. cerevisiae proteome is probed for rapid unbiased interrogation of thousands of protein-protein interactions in parallel. This method can be utilized for protein-small molecule, posttranslational modification, and other assays in high-throughput.

High-density functional protein microarrays containing ~4,200 recombinant yeast proteins are examined for kinase protein-protein interactions using an ...

Adipo-Clear: A Tissue Clearing Method for Three-Dimensional Imaging of Adipose Tissue

Adipose tissue plays a central role in energy homeostasis and thermoregulation. It is composed of different types of adipocytes, as well as adipocyte precursors, immune cells, fibroblasts, blood vessels, and nerve projections. Although the molecular control of cell type specification and how these cells interact have been increasingly delineated, a more comprehensive understanding of these adipose-resident cells can be achieved by visualizing their distribution and architecture throughout the whole tissue. Existing immunohistochemistry and immunofluorescence approaches to analyze adipose histology rely on thin paraffin-embedded sections. However, thin sections capture only a small portion of tissue; as a result, the conclusions can be biased by what portion of tissue is analyzed. We have therefore developed an adipose tissue clearing technique, Adipo-Clear, to permit comprehensive three-dimensional visualization of molecular and cellular patterns in whole adipose tissues. Adipo-Clear was adapted from iDISCO/iDISCO+, with specific modifications made to completely remove the lipid stored in the tissue while preserving native tissue morphology. In combination with light-sheet fluorescence microscopy, we demonstrate here the use of the Adipo-Clear method to obtain high-resolution volumetric images&#160;of an entire adipose tissue.

Due to the high lipid content, adipose tissue has been challenging to visualize using traditional histological methods. Adipo-Clear is a tissue clearing technique that allows robust labeling and high-resolution volumetric fluorescent imaging of adipose tissue. Here, we describe the methods for sample preparation, pretreatment, staining, clearing, and mounting for imaging.

Adipose tissue plays a central role in energy homeostasis and thermoregulation. It is composed of different types of adipocytes, as well as adipocyte ...

Sample Preparation for Metabolic Profiling using MALDI Mass Spectrometry Imaging

Metabolomics, the study to identify and quantify small molecules and metabolites present in an experimental sample, has emerged as an important tool to investigate the biological activities during development and diseases. Metabolomics approaches&#160;are widely employed in the study of cancer, nutrition/diet, diabetes, and other physiological and pathological conditions involving metabolic processes. An advantageous tool that aids in metabolomic profiling advocated in this paper is matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI). Its ability to detect metabolites in situ without labeling, structural modifications, or other specialized reagents, such as those used in immunostaining, makes MALDI MSI a unique tool in advancing methodologies relevant in the field of metabolomics. An appropriate sample preparation process is critical to yield optimal results and will be the focus of this paper.

The goal of this protocol is to provide detailed guidance on the sample preparation when planning for experiments using MALDI MSI to maximize metabolic and molecular detection in biological samples.

Metabolomics, the study to identify and quantify small molecules and metabolites present in an experimental sample, has emerged as an important tool to ...

The Use of the Patch-Clamp Technique to Study the Thermogenic Capacity of Mitochondria

Mitochondrial thermogenesis (also known as mitochondrial uncoupling) is one of the most promising targets for increasing energy expenditure to combat metabolic syndrome. Thermogenic tissues such as brown and beige fats develop highly specialized mitochondria for heat production. Mitochondria of other tissues, which primarily produce ATP, also convert up to 25% of the total mitochondrial energy production into heat and can, therefore, have a considerable impact on the physiology of the whole body. Mitochondrial thermogenesis is not only essential for maintaining the body temperature, but also prevents diet-induced obesity and reduces the production of reactive oxygen species (ROS) to protect cells from oxidative damage. Since mitochondrial thermogenesis is a key regulator of cellular metabolism, a mechanistic understanding of this fundamental process will help in the development of therapeutic strategies to combat many pathologies associated with mitochondrial dysfunction. Importantly, the precise molecular mechanisms that control acute activation of thermogenesis in mitochondria are poorly defined. This lack of information is largely due to a dearth of methods for the direct measurement of uncoupling proteins. The recent development of patch-clamp methodology applied to mitochondria enabled, for the first time, the direct study of the phenomenon at the origin of mitochondrial thermogenesis, H+ leak through the IMM, and the first biophysical characterization of mitochondrial transporters responsible for it, the uncoupling protein 1 (UCP1), specific of brown and beige fats, and the ADP/ATP transporter (AAC) for all other tissues. This unique approach will provide new insights into the mechanisms that control H+ leak and mitochondrial thermogenesis and how they can be targeted to combat metabolic syndrome. This paper describes the patch-clamp methodology applied to mitochondria to study their thermogenic capacity by directly measuring H+ currents through the IMM.

This method article details the main steps in measuring H+ leak across the inner mitochondrial membrane with the patch-clamp technique, a new approach to study the thermogenic capacity of mitochondria.

Mitochondrial thermogenesis (also known as mitochondrial uncoupling) is one of the most promising targets for increasing energy expenditure to combat ...