The authors would like to thank Biomet 3i for their continued financial support, and particularly Randy Goodman for help in the design and fabrication of the custom parts. Spencer Bell is a recipient of an Industrial Postgraduate Scholarship, provided by the National Sciences and Engineering Research Council of Canada (NSERC). We would also like to thank Dr. John Brunski for his very valuable feedback during manuscript preparation.

1. Implant Design, Fabrication, and Surface Treatment
<ol>
	<li>Manufacture rectangular implants (dimensions 4 mm x 2.5 mm x 1.3 mm; length x width x height) from commercially pure titanium (cpTi). Drill a hole centrally down the long axis of the implant (diameter = 0.7 mm) to facilitate early implant stability within the surgical site and subsequent mechanical testing (Figure 1).</li>
	<li>Treat the upper and lower surfaces of the implant.
	<ol>
		<li>To create two distinct surfaces, use a standard grit-blasting (GB) treatment to create a microtopographically complex surface. Further modify half of the implants by superimposing calcium phosphate (CaP) nanoparticles to create a nanotopographically complex surface.</li>
	</ol>
	</li>
</ol>
Note: Various chemical or mechanical treatments can be applied to create a desired surface topography and/or chemistry, and these will depend upon the nature of the experimental question to be addressed. In the example provided herein, one group of commercially pure titanium (cpTi) implants was subjected to grit-blasting (GB) -&#160;a subtractive process -&#160;to create a complex microtopography. Half of the implants were then further modified by the addition of calcium phosphate (CaP) nanocrystals to create a super imposed nanotopography (GB-DCD).
Note: When viewing the grit-blasted micro-surface, compared to the modified nano-surface, at 10,000X&#160;magnification, there is no obvious difference in surface characteristics. However, when viewed at 100,000X&#160;magnification, differences become quite obvious&#160;(Figure 2). It has been previously shown that such surface changes have profound effects on osteoconduction14.
2. Animal Model and Surgical Procedure
<ol>
	<li>Use young male Wistar rats (200-250 g) for this model. All procedures must be approved by local animal care committees. Allow animals free access to water and rat chow. 
	Note: Wistar rats were selected for this procedure due to prior experience with this strain of rat, although other strains of rat could be employed. Access to food and water can be altered, depending on the nature of the experimental question being addressed.</li>
	 
	<li>Sedate rats using inhalation anesthesia administered through a nose cone: 4% isoflurane in 1 L O2/min&#160;for induction; 2% isoflurane in 1 L nitrous oxide and 0.6 L O2/min for maintenance. Conduct a standard toe-pinch test to ensure effective sedation before continuing with the procedure.</li>
	<li>Administer analgesic pre- and post-operatively through a subcutaneous injection of 0.01-0.05 mg/kg buprenorphine.</li>
	<li>Assign implants by partial randomization and place bilaterally in the distal metaphyses of rat femora. This allows a different implant, one each from a selection of different surface topographies to be compared, in contralateral femora, to optimize statistical analysis.</li>
	<li>Prepare the animals by shaving&#160;and cleaning the antero-lateral aspect of each hind leg with 10% Betadine. To prevent hypothermia, place a warm water circulation pad underneath the anesthetized rat.</li>
	<li>Using a #15 surgical scalpel, make an incision through the skin along the lateral aspect of the thigh to expose the muscle. Expose the distal femur using blunt dissection to deflect the muscle bodies in a minimally invasive manner.</li>
	<li>Scrape away the thin layer of periosteum overlying the femur, using a periosteal elevator, to fully expose the cortical bone for drilling. Care must be taken not to damage the growth plate or articular cartilage of the knee joint during blunt dissection and removal of periosteum.</li>
	<li>Once cleaned and inspected, rotate the femur laterally to expose the anterior aspect of the distal femur (Figure 3A).</li>
	<li>To prepare the surgical site, drill a bicortical rectangular slot down the middle of the bone through both cortices. To avoid overheating the tissue, saline irrigation must be maintained throughout drilling by a surgical assistant. Conduct the drilling in three stages:
	<ol>
		<li>First, drill through the anterior cortex, exposed by dissection, using a 1.3 mm dental burr attached to a dental hand piece to create two holes 2.5 mm apart along the mid line of the femur.</li>
		<li>Next, use a second drill bit (twist 1.3 mm dental burr) to extend these holes through the opposing cortex, resulting in bicortical parallel holes.</li>
		<li>Finally, join the holes using a third custom side-cutting burr in a proximal-distal direction, forming the site for the implant (Figure 3B).</li>
	</ol>
	</li>
	<li>Pass a biodegradable suture through the bone defect using the attached needle and return around the outer femoral cortex.</li>
	<li>Thread the implant over the free end of the suture and guide it into the defect, where it should be pressure-fitted. In this manner, the long axis of the implant should be oriented perpendicular to the long axis of the femur (Figure 3C).</li>
	<li>Tie the suture around the lateral aspect of the femur to provide implant stability during post-operative recovery and early stages of healing. Use the remaining suture to close the muscle tissue, and reoppose the cutaneous tissue using surgical staples (9 mm wound clips).</li>
	<li>Inspect surgical sites for signs of infection, and monitor animals daily for compromised ambulatory ability. Exclude the animals that do not fully recover ambulation, or those that have femoral fractures at sacrifice, from analysis.</li>
</ol>
3. Sample Harvesting
<ol>
	<li>Sacrifice animals at 9 days post-operative by cervical dislocation after CO2 exposure.</li>
	<li>Upon sacrifice, detach femora and clean of soft tissue. Store immediately in 15% sucrose buffer solution to maintain tissue hydration in preparation for mechanical testing (Figure 4A). 
	Note: Specimens are stored in the sucrose buffer solution to maintain tissue hydration during transport between facilities. Specimens will spend approximately 2-3 hr&#160;in solution during preparation for mechanical testing.</li>
	 
	<li>To prepare specimens for mechanical testing, trim the bone to the width of the implants using a cylindrical diamond burr attached to a high speed system. The final test specimens consist of two arches of bone attached to each face of the implant (Figure 4B). For arches that fall off during preparation or transportation, assign a mechanical testing value of 0 N. 
	Note: It is important to be very gentle and accurate when trimming the specimens, in order to avoid damaging or prestressing the interface. Bony reparative callus can grow around the long axis of the implant and even into the longitudinal hole. Such excess bone must be removed by trimming specimens to the exact dimensions of the rectangular implant, as it can skew mechanical testing results.</li>
</ol>
4. Mechanical Testing
A custom breakaway mold was designed to pot each specimen, creating a repeatable and accurate method of preparing samples for mechanical testing. The design allows for isolation of a 0.5 mm region of peri-implant bone for a consistent testing zone, while holding the specimen centered and completely horizontal during the potting process, allowing for the application of a force directly perpendicular to the implant surface. See Figure 5 for complete engineering drawings and Figure 6 for the final components.
Note: Conduct all testing using a mechanical testing apparatus, operating at a crosshead speed of 30 mm/min. For a qualitative assessment of residual bone following testing, a dissecting microscope can be used.
<ol>
	<li>Potting of the Specimens and Mechanical Testing
	<ol>
		<li>Remove specimens from the sucrose buffer solution and gently blot dry.</li>
		<li>Position specimen in the custom mold. Slide the pin horizontally through holes in the walls of the mold and through the hole in the middle of the implant. Place the stabilizing plate on the posterior side of the mold to stabilize the implant (Figure 7).</li>
		<li>Fill the base of the mold with flowable dental composite and cure for 60 sec using a high intensity curing light. 
		Note: It is important to choose a composite that does not set with an exothermic reaction, as such heat generated may influence the tissue properties.</li>
		<li>After curing, open the mold and remove the hardened specimen block. Draw a thin black line in permanent marker on the lateral arch for identification purposes.</li>
		<li>Fix a prefabricated replica of the test specimen in a vice, and center the unit on the base of the mechanical testing instrument.</li>
		<li>Secure the specimen into vice and pass a nylon line through the hole in the implant (Figure 8). Attach the loose ends to the center of the moving crosshead. For consistency, always label and test the lateral side first. Repeat the process with the medial arch.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Brunski, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+bone+response+to+biomechanical+loading+at+the+bone-dental+implant+interface.">In vivo bone response to biomechanical loading at the bone-dental implant interface.</a> Adv. Dental Res. 13, 99-119 (1999).</li><li>Brunski, J. B., Glantz, P. -. O., Helms, J. A., Nanci, A., Br&#229;nemark, P. I., Chien, S., Gr&#246;ndahl, H. G., Robinson, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Transfer of mechanical load across the interface. In: The Osseointegration Book. , 209-249 (2005).</li><li>Br&#229;nemark, R., Ohrnell, L. O., Nilsson, P., Thomsen, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomechanical+characterization+of+osseointegration+during+healing:+an+experimental+in+vivo+study+in+the+rat.">Biomechanical characterization of osseointegration during healing: an experimental in vivo study in the rat.</a> Biomaterials. 18 (14), 969-978 (1997).</li><li>It&#228;l&#228;, A., Koort, J., Yl&#228;nen, H. O., Hupa, M., Aro, H. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biologic+significance+of+surface+microroughing+in+bone+incorporation+of+porous+bioactive+glass+implants.">Biologic significance of surface microroughing in bone incorporation of porous bioactive glass implants.</a> J. Biomed. Mater. Res. A. 67 (2), 496-503 (2003).</li><li>Br&#229;nemark, R., Emanuelsson, L., Palmquist, A., Thomsen, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bone+response+to+laser-induced+micro-+and+nano-size+titanium+surface+features.">Bone response to laser-induced micro- and nano-size titanium surface features.</a> Nanomedicine. 7 (2), 220-227 (2011).</li><li>Kato, 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=Bonding+of+Alkali-+and+Heat-Treated+Tantalum+Implants+to+Bone.">Bonding of Alkali- and Heat-Treated Tantalum Implants to Bone.</a> J. Biomed. Mater. Res. 53, 28-35 (2000).</li><li>Hong, L., Xu, H. C., de Groot, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tensile+strength+of+the+interface+between+hydroxyapatite+and+bone.">Tensile strength of the interface between hydroxyapatite and bone.</a> J. Biomed. Mater. 26 (1), 7-18 (1992).</li><li>Currey, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+properties+of+bone+tissues+with+greatly+different+functions.">Mechanical properties of bone tissues with greatly different functions.</a> J. Biomech. 9 (12), 313-319 (1979).</li><li>Nakamura, T., Yamamuro, T., Higashi, S., Kokubo, T., Itoo, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+glass-ceramic+for+bone+replacement:+evaluation+of+its+bonding+to+bone+tissue.">A new glass-ceramic for bone replacement: evaluation of its bonding to bone tissue.</a> J. Biomed. Mater. Res. 19 (6), 685-698 (1985).</li><li>Hench, L. L., Splinter, R. J., Allen, W. C., Greenlee, T. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bonding+mechanisms+at+the+interface+of+ceramic+prosthetic+materials.">Bonding mechanisms at the interface of ceramic prosthetic materials.</a> J. Biomed. Mater. Res. Symp. 1, 117-141 (1972).</li><li>Edwards, J. T., Brunski, J. B., Higuchi, H. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+and+morphologic+investigation+of+the+tensile+strength+of+a+bone-hydroxyapatite+interface.">Mechanical and morphologic investigation of the tensile strength of a bone-hydroxyapatite interface.</a> J. Biomed. Mater. Res. 36 (4), 454-468 (1997).</li><li>Davies, J. E., Ajami, E., Moineddin, R., Mendes, V. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+roles+of+different+scale+ranges+of+surface+implant+topography+on+the+stability+of+the+bone/implant+interface.">The roles of different scale ranges of surface implant topography on the stability of the bone/implant interface.</a> Biomaterials. 34, 3535-3546 (2013).</li><li>R&#248;nold, H. J., Lyngstadaasb, S. P., Ellingsen, 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=Analysing+the+optimal+value+for+titanium+implant+roughness+in+bone+attachment+using+a+tensile+test.">Analysing the optimal value for titanium implant roughness in bone attachment using a tensile test.</a> Biomaterials. 24, 4559-4564 (2003).</li><li>Mendes, V. C., Moineddin, R., Davies, 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=The+effect+of+discrete+calcium+phosphate+nanocrystals+on+bone-bonding+to+titanium+surfaces.">The effect of discrete calcium phosphate nanocrystals on bone-bonding to titanium surfaces.</a> Biomaterials. 28 (32), 4748-4755 (2007).</li><li>Skedros, J. G., Holmes, J. L., Vajda, E. G., Bloebaum, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cement+lines+of+secondary+osteons+in+human+bone+are+not+mineral+deficient:+new+data+in+a+historical+perspective.">Cement lines of secondary osteons in human bone are not mineral deficient: new data in a historical perspective.</a> Anat Rec. 286, 781-803 (2005).</li><li>McKee, M. D., Nanci, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osteopontin+and+the+bone+remodelling+sequence:+colloidal-gold+immunocytochemistry+of+an+interfacial+extracellular+matrix+protein.">Osteopontin and the bone remodelling sequence: colloidal-gold immunocytochemistry of an interfacial extracellular matrix protein.</a> Ann. N.Y. Acad. Sci. 760, 177-189 (1995).</li><li>Davies, J. E., Hosseini, M. M., Davies, 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=.">.</a> Histodynamics of endosseous wound healing In: Bone Engineering. , 1-14 (2000).</li><li>Welsh, R. P., Pilliar, R. M., Macnab, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surgical+implants.+The+role+of+surface+porosity+in+fixation+to+bone+and+acrylic.">Surgical implants. The role of surface porosity in fixation to bone and acrylic.</a> J. Bone Joint Surg. Am. 53 (5), 963-977 (1971).</li><li>O'Brien, F. J., Taylor, D., Clive, L. T. <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+bone+microstructure+on+the+initiation+and+growth+of+microcracks.">The effect of bone microstructure on the initiation and growth of microcracks.</a> J. Orthop. Res. 23 (2), 475-480 (2005).</li><li>Steflik, , 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=Ultrastructural+analyses+of+the+attachment+(bonding)+zone+between+bone+and+implanted+biomaterials.">Ultrastructural analyses of the attachment (bonding) zone between bone and implanted biomaterials.</a> J. Biomed. Mater. Res. 39 (4), 611-620 (1998).</li><li>Sul, Y. -. T., Johansson, C., Albrektsson, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+in+vivo+method+for+quantifying+the+interfacial+biochemical+bond+strength+of+bone+implants.">A novel in vivo method for quantifying the interfacial biochemical bond strength of bone implants.</a> J. Royal Soc. 7 (42), 81-90 (2010).</li><li>Wong, 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=Effect+of+surface+topography+on+the+osseointegration+of+implant+materials+in+trabecular+bone.">Effect of surface topography on the osseointegration of implant materials in trabecular bone.</a> J. Biomed. Mater. Res. 29 (12), 1567-1575 (1995).</li><li>Gotfredsen, 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=Anchorage+of+titanium+implants+with+different+surface+characteristics:+an+experimental+study+in+rabbits.">Anchorage of titanium implants with different surface characteristics: an experimental study in rabbits.</a> Clin. Implant Dent. Relat. Res. 2 (3), 120-128 (2000).</li><li>Lekholm, U., Zarb, G. A., Albrektsson, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Patient selection and preparation. In: Tissue integrated prostheses. , 199-209 (1985).</li></ol>

The authors received funding and materials support from Biomet 3i (Palm Beach Gardens, FL, USA). Biomet 3i had no part in the writing of this manuscript or the design of experiments described.

The mechanical testing model presented here provides an improved method to assess the anchorage of bone to candidate implant surfaces, since it allows for accurate perpendicular, or parallel, alignment of the test sample with the axis of disruption force applied; and limits the fracture zone to within half a millimeter of the implant surface. The model is easily incorporated into studies comparing the effectiveness of any range of chemically, or mechanically, modified surfaces; but is not suitable for smooth surfaces as ...

Assessing anchorage of bone to endosseous implant surfaces has been the focus of considerable attention, for which many mechanical testing methods have been described1,2. All such methods impose a force to disrupt the bone/implant model being employed, and can be broadly grouped into shear, generally presented as push-out or pull-out models3,4, reverse torque3,5, and tensile types6,7. Commonly in such tests, either bone8 or implant material (in the case of brittle glasses and ceramics9,10) is fractured and, assuming some form of anchorage has occurred, the bone/implant interface remains (at least partially) intact. Such experimental outcomes mean not only that the force required to cause the fracture (or disruption) of the model is not the force required to separate the bone/implant interface11,12, but also that the complex surface area of the created fracture plane can be refractory to accurate measurement. Nevertheless, such tests can be clinically relevant, since they provide a comparative gauge of the ability of implants of differing surface designs to be anchored in bone. However, it should also be noted that such comparisons are only valid within an experimental model, while comparisons between experimental models are fraught with difficulty since investigators use different animal species exhibiting either lamellar or woven bone; trabecular or cortical bone healing models, and different mechanical test geometries and conditions.
In an effort to derive a measurement of the tensile strength of the bone/implant interface, many investigators have used the nominal surface area of the implant to derive a &#34;tensile strength&#34; value, since tensile strength is measured as force per unit area. This is clearly an approximation given, as explained above, that the bone/implant interface remains intact in many of the disruption tests employed. In addition measuring the surface area of implants, particularly topographically complex surfaces, is limited by the resolution of the measurement technique as discussed by Ronald et al.13&#160;However, as reviewed by Brunski et al.2, when the nominal surface area of an implant is taken into account, apparent differences in &#34;tensile strength&#34; associated with different implant surface designs are negated, suggesting that implant surfaces with higher surface area provide larger areas of bone/implant contact and thus require more force to fracture the model. The implication therefore is that more topographically complex surfaces can increase contact osteogenesis, which results in greater bone implant contact (BIC) and resultant higher disruption values in mechanical tests. Contact osteogenesis is the product of two distinct phenomena: osteoconduction and bone formation. Indeed, we have shown that increases in osteoconduction on topographically complex surfaces can be quantified by measuring the resultant BIC14, and that such surfaces also result in higher mechanical disruption values12.
However, it is salutary to note that peri-implant bone can form by two mechanisms. In contact osteogenesis cells of mesenchymal origin migrate to the implant surface (osteoconduction), differentiate into bone cells, and elaborate de novo bone matrix on the implant surface (bone formation). The first bony matrix elaborated is a mineralized cement line as seen in normal bone remodeling15&#160;(there is much confusion in the literature concerning this mineralized biological structure that is sometimes thought to be un-mineralized1 or is syncretized with all interfaces in bone16&#160;-&#160;for a full discussion on this topic see&#160;Davies and&#160;Hosseini17). Contact osteogenesis is an essential prerequisite for the phenomenon of bone-bonding, but is nonessential for bone ingrowth18. The mineralized cement line of bone is mechanically weaker than the mineralized collagen compartment of bone19. Thus, intuitively, if the interdigitation of cement line matrix with implant nano&#160;features is compared with bone tissue in growth into macro implant features then the mechanical force required to disrupt the former would, reasonably, be expected to be less than the latter, and we have recently demonstrated this experimentally12.
Peri-implant bone can also form by distance osteogenesis. In this case, bone is deposited on the old bone surface and gets progressively closer to the implant surface resulting in an interface comprising amorphous matrix and the remains of osteogenic cells20. In general, distance osteogenesis is associated with smooth, or machined, endosseous implant surfaces and is often seen in cortical bone healing, while microtopographically complex surfaces are associated with contact osteogenesis that is more typical of trabecular bone healing. Tensile test models using smooth implant surfaces and cortical bone healing have been able to test the adhesive properties of this amorphous biological matrix absent of the contact osteogenesis associated with topographically complex surfaces, and have shown that the so called &#8220;biochemical&#8221; bonding that occurs provides a minor component of the &#8220;tensile strength&#8221; values reported with topographically complex surfaces21. On the contrary, using a trabecular bone healing model, Wong et al.22 showed &#8220;an excellent correlation&#8221; between implant surface roughness and push-out failure load, and indicated that chemical bonding indeed played a negligible role in anchorage of bone to the implant surface. While it is probable that both contact and distance osteogenesis occur, to differing degrees, in all endosseous peri-implant healing compartments, microtopographically complex surfaces have shown themselves to be particularly advantageous in trabecular bony healing compartments23. The latter are classified as Class III or Class IV bone in the dental literature24.
Our purpose has been to focus on the mechanisms of contact osteogenesis and the resultant bone/implant anchorage that can ensue in a trabecular bone healing environment. This anchorage, which is dependent on the topography of the implant surface (see above), can occur at different scale-ranges. On the one hand, only submicron implant features are implicated in bone-bonding -&#160;as described by interdigitation of the bony cement line matrix with such surfaces, and seen on bioactive glasses, ceramics and reticulate metal oxides. On the other, bone tissue (sometimes complete with blood vasculature) can grow into multi-micron, or macro-scale, features of implant surfaces18. Both cases result in a form of bone anchorage to the implant surface, although the mechanisms are clearly different. However, a common failing of the majority of mechanical testing methods referenced above is to align the disruption force in an exactly perpendicular, or parallel plane to that of the implant surface (depending on whether tensile or shear mode is employed). We report herein a method that overcomes this limitation.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr >
			<td >Dulbecco&#8217;s Phosphate Buffer solution (DPBS)</td>
			<td >Gibco Life Technologies, Burlington, ON, Canada</td>
			<td >14190-250</td>
			<td ></td>
		</tr>
		<tr >
			<td >10% neutral buffered formalin solution</td>
			<td >Sigma-Aldrich Co. LLC., Canada</td>
			<td >HT501128-4L</td>
			<td></td>
		</tr>
		<tr >
			<td >Custom-designed rectangular implants (commercially pure titanium; dimensions: 4mm x 2.5mm x 1.3mm with a 0.7mm hole drilled centrally down the long axis)</td>
			<td >Biomet 3i, FL, USA</td>
			<td >N/A</td>
			<td></td>
		</tr>
		<tr >
			<td >Custom-designed breakaway mould</td>
			<td >Biomet 3i, FL, USA</td>
			<td >N/A</td>
			<td></td>
		</tr>
		<tr >
			<td >Isoflurane</td>
			<td >Baxter Internationl Inc.</td>
			<td >N/A</td>
			<td></td>
		</tr>
		<tr >
			<td >Buprenorphine</td>
			<td >Bedford Laboratories</td>
			<td >N/A</td>
			<td></td>
		</tr>
		<tr >
			<td >10% betadine</td>
			<td >Bruce Medical, MA, US</td>
			<td >FR-2200-90</td>
			<td></td>
		</tr>
		<tr >
			<td >Scalpel</td>
			<td >Almedic, Medstore, University of Toronto, Canada</td>
			<td >2586-M36-0100</td>
			<td></td>
		</tr>
		<tr >
			<td >Scalpel blade #15 (sterile)</td>
			<td >Magna, Medstore, University of Toronto, Canada</td>
			<td >2586</td>
			<td></td>
		</tr>
		<tr >
			<td >Periosteal elevator #24G</td>
			<td >Spectrum Surgical, OH, USA</td>
			<td >EX7</td>
			<td></td>
		</tr>
		<tr >
			<td >Forceps</td>
			<td >Almedic, Medstore, University of Toronto, Canada</td>
			<td >7747-A10-108</td>
			<td></td>
		</tr>
		<tr >
			<td >Tissue forceps</td>
			<td >Almedic, Medstore, University of Toronto, Canada</td>
			<td >7722-A10-308</td>
			<td></td>
		</tr>
		<tr >
			<td >Scissors</td>
			<td >Almedic, Medstore, University of Toronto</td>
			<td >7603-A8-240</td>
			<td></td>
		</tr>
		<tr >
			<td >Absorbant Fabric General Purpose Drape (sterile)</td>
			<td >Vitality Medical</td>
			<td >1089</td>
			<td></td>
		</tr>
		<tr >
			<td >Gauze (non-sterile)</td>
			<td >VWR</td>
			<td >89133-260</td>
			<td></td>
		</tr>
		<tr >
			<td >Needles 25G X 5/8&#34; (disposable)</td>
			<td >BD, Canada</td>
			<td >305122</td>
			<td></td>
		</tr>
		<tr >
			<td >Syringes (sterile)</td>
			<td >VWR, Canada</td>
			<td >CABD309653</td>
			<td></td>
		</tr>
		<tr >
			<td >Needle Driver</td>
			<td >Almedic, Medstore, University of Toronto, Canada</td>
			<td >A17-132</td>
			<td></td>
		</tr>
		<tr >
			<td >Dynarex Surgical gloves (sterile)</td>
			<td >Amazon.com</td>
			<td >2475</td>
			<td></td>
		</tr>
		<tr >
			<td >Surgical masks</td>
			<td >Fisherbrand, Medstore, University of Toronto, Canada</td>
			<td >296360759</td>
			<td></td>
		</tr>
		<tr >
			<td >0.9% sterile saline</td>
			<td >House brand, Medstore, University of Toronto, Canada</td>
			<td >1011-L8001</td>
			<td></td>
		</tr>
		<tr >
			<td >Hair clippers</td>
			<td >Remington, US</td>
			<td >N/A</td>
			<td></td>
		</tr>
		<tr >
			<td >4-0 Polysorb</td>
			<td >Syneture</td>
			<td >SL5627G</td>
			<td></td>
		</tr>
		<tr >
			<td >9mm Wound Clips</td>
			<td >Becton Dickinson, MD, USA</td>
			<td >427631</td>
			<td></td>
		</tr>
		<tr >
			<td >ImplantMED DU 900 and WS-75 dental hand piece&#160;</td>
			<td >W&#38;H Dentalwerk, Austria</td>
			<td >DU1000US</td>
			<td></td>
		</tr>
		<tr >
			<td >1.3 mm twist drill</td>
			<td >Brasseler, GA, USA</td>
			<td >203.21.013</td>
			<td></td>
		</tr>
		<tr >
			<td >1.3 mm dental burr&#160;</td>
			<td >Biomet 3i, FL, USA</td>
			<td >custom</td>
			<td></td>
		</tr>
		<tr >
			<td >1.2 mm cylindrical side-cutting burr</td>
			<td >Biomet 3i, FL, USA</td>
			<td >custom</td>
			<td></td>
		</tr>
		<tr >
			<td >Cylindrical diamond burr</td>
			<td >Brasseler, GA, USA</td>
			<td >H1.21.014</td>
			<td></td>
		</tr>
		<tr >
			<td rowspan="2" >High speed dental drilling system</td>
			<td >Handpiece: KaVo Dental Corporation, IL, USA</td>
			<td rowspan="2" >N/A</td>
			<td></td>
		</tr>
		<tr >
			<td >Handpiece Control: DCI International, OR, USA</td>
			<td></td>
		</tr>
		<tr >
			<td >99.5% Ultra Pure sucrose</td>
			<td >BioShop Canada Inc., Burlington, ON, Canada</td>
			<td >57-50-1</td>
			<td></td>
		</tr>
		<tr >
			<td >Flowable dental composite</td>
			<td >Filtek Supreme Ultra Flowable Restorative, 3M ESPE, St Paul, Minnesota, USA</td>
			<td >6033XW</td>
			<td></td>
		</tr>
		<tr >
			<td >Sapphire Plasma Arc high intensity curing light</td>
			<td >Den-Mat Holdings, Santa Maria, CA, USA</td>
			<td >N/A</td>
			<td></td>
		</tr>
		<tr >
			<td >Instron 4301 with 1000 N load cell</td>
			<td >Instron, Norwood, MA, USA</td>
			<td >N/A</td>
			<td></td>
		</tr>
		<tr >
			<td rowspan="2" >Red Wolf 10lb nylon &#64257;shing line</td>
			<td rowspan="2" >Canadian Tire, Canada</td>
			<td rowspan="2" >78-3610-6</td>
			<td></td>
		</tr>
		<tr >
			<td ></td>
		</tr>
		<tr >
			<td >Leica Wild M3Z Stereozoom dissecting microscope</td>
			<td >Leica, Heerbrugg, Switzerland</td>
			<td >N/A</td>
			<td></td>
		</tr>
		<tr >
			<td >QImaging Micropublisher 5.0 RTV digital camera coupled with QCapture 2.90.1 acquisition software</td>
			<td >QImaging, Surrey, BC, Canada</td>
			<td >N/A</td>
			<td></td>
		</tr>
		<tr >
			<td >Electronic digital caliper&#160;</td>
			<td >Fred V. Fowler Company, Inc., Newton, MA, USA</td>
			<td >N/A</td>
			<td></td>
		</tr>
		<tr >
			<td >Mechanical testing instrument</td>
			<td >Instron, Norwood, MA, USA</td>
			<td >N/A</td>
			<td></td>
		</tr>
	</tbody>
</table>

an improved mechanical testing method to assess bone-implant anchorage

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

All animals increased their ambulatory activity with time following their recovery from surgery. This is important because load has differential effects on topographies of different scale ranges, as we have recently reported12. A representative force/displacement curve for test specimens following mechanical testing is presented in Figure 9A, and the averaged data for each implant surface is presented in Figure 9B. The maximum force value achieved by each specimen was recorded...

Watch this Scientific Journal Video about An Improved Mechanical Testing Method to Assess Bone-implant Anchorage at JoVE.com

An Improved Mechanical Testing Method to Assess Bone-implant Anchorage

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

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

an-improved-mechanical-testing-method-to-assess-bone-implant-anchorage

Research

JoVE Journal

Bioengineering

15.4K Views.  University of Toronto. An improved method to mechanically test bone anchorage to candidate implant surfaces is presented. This method allows for alignment of the disruption force exactly perpendicular, or parallel, to the plane of the implant surface, and provides an accurate means to direct the disruption forces to an exact peri-implant region.

Video: An Improved Mechanical Testing Method to Assess Bone-implant Anchorage

Mechanical Testing of Mouse Carotid Arteries: from Newborn to Adult

The large conducting arteries in vertebrates are composed of a specialized extracellular matrix designed to provide pulse dampening and reduce the work performed by the heart. The mix of matrix proteins determines the passive mechanical properties of the arterial wall1. When the matrix proteins are altered in development, aging, disease or injury, the arterial wall remodels, changing the mechanical properties and leading to subsequent cardiac adaptation2. In normal development, the remodeling leads to a functional cardiac and cardiovascular system optimized for the needs of the adult organism. In disease, the remodeling often leads to a negative feedback cycle that can cause cardiac failure and death. By quantifying passive arterial mechanical properties in development and disease, we can begin to understand the normal remodeling process to recreate it in tissue engineering and the pathological remodeling process to test disease treatments.
	Mice are useful models for studying passive arterial mechanics in development and disease. They have a relatively short lifespan (mature adults by 3 months and aged adults by 2 years), so developmental3 and aging studies4 can be carried out over a limited time course. The advances in mouse genetics provide numerous genotypes and phenotypes to study changes in arterial mechanics with disease progression5 and disease treatment6. Mice can also be manipulated experimentally to study the effects of changes in hemodynamic parameters on the arterial remodeling process7. One drawback of the mouse model, especially for examining young ages, is the size of the arteries. 
	We describe a method for passive mechanical testing of carotid arteries from mice aged 3 days to adult (approximately 90 days). We adapt a commercial myograph system to mount the arteries and perform multiple pressure or axial stretch protocols on each specimen. We discuss suitable protocols for each age, the necessary measurements and provide example data. We also include data analysis strategies for rigorous mechanical characterization of the arteries.

Passive mechanical testing of mouse carotid arteries is described, with special consideration for adapting to different specimen ages. The procedures include determining the in vivo length of the artery, mounting it in a pressure myograph, recording data, measuring the unloaded dimensions and analyzing the resulting data.

The large conducting arteries in vertebrates are composed of a specialized extracellular matrix designed to provide pulse dampening and reduce the work ...

Microfluidic Chip Fabrication and Method to Detect Influenza

Fast and effective diagnostics play an important role in controlling infectious disease by enabling effective patient management and treatment. Here, we present an integrated microfluidic thermoplastic chip with the ability to amplify influenza A virus in patient nasopharyngeal (NP) swabs and aspirates. Upon loading the patient sample, the microfluidic device sequentially carries out on-chip cell lysis, RNA purification and concentration steps within the solid phase extraction (SPE), reverse transcription (RT) and polymerase chain reaction (PCR) in RT-PCR chambers, respectively. End-point detection is performed using an off-chip Bioanalyzer (Agilent Technologies, Santa Clara, CA). For peripherals, we used a single syringe pump to drive reagent and samples, while two thin film heaters were used as the heat sources for the RT and PCR chambers. The chip is designed to be single layer and suitable for high throughput manufacturing to reduce the fabrication time and cost. The microfluidic chip provides a platform to analyze a wide variety of virus and bacteria, limited only by changes in reagent design needed to detect new pathogens of interest.

An integrated microfluidic thermoplastic chip has been developed for use as a molecular diagnostic. The chip performs nucleic acid extraction, reverse transcriptase, and PCR. Methods for fabricating and running the chip are described.

Fast  and effective diagnostics play an important role in controlling infectious  disease by enabling effective patient management and treatment. Here, we ...

Stress-induced Antibiotic Susceptibility Testing on a Chip

We have developed a rapid microfluidic method for antibiotic susceptibility testing in a stress-based environment. Fluid is passed at high speeds over bacteria immobilized on the bottom of a microfluidic channel. In the presence of stress and antibiotic, susceptible strains of bacteria die rapidly. However, resistant bacteria survive these stressful conditions. The hypothesis behind this method is new: stress activation of biochemical pathways, which are targets of antibiotics, can accelerate antibiotic susceptibility testing. As compared to standard antibiotic susceptibility testing methods, the rate-limiting step -&#160;bacterial growth -&#160;is omitted during antibiotic application. The technical implementation of the method is in a combination of standard techniques and innovative approaches. The standard parts of the method include bacterial culture protocols, defining microfluidic channels in polydimethylsiloxane&#160;(PDMS), cell viability monitoring with fluorescence, and batch image processing for bacteria counting. Innovative parts of the method are in the use of culture media flow for mechanical stress application, use of enzymes to damage but not kill the bacteria, and use of microarray substrates for bacterial attachment. The developed platform can be used in antibiotic and nonantibiotic related drug development and testing. As compared to the standard bacterial suspension experiments, the effect of the drug can be turned on and off repeatedly over controlled time periods. Repetitive observation of the same bacterial population is possible over the course of the same experiment.

We have developed a microfluidic platform for rapid antibiotic susceptibility testing. Fluid is passed at high speeds over bacteria immobilized on the bottom of a microfluidic channel. In the presence of stress and antibiotic, susceptible strains of bacteria die rapidly. However, resistant bacteria can survive these stressful conditions.

We have developed a rapid microfluidic method for antibiotic susceptibility testing in a stress-based environment. Fluid is passed at high speeds over ...

An Improved Method for the Preparation of Type I Collagen From Skin

Soluble type 1 collagen (COL1) is used extensively as an adhesive substrate for cell cultures and as a cellular scaffold for regenerative applications. Clinically, this protein is widely used for cosmetic surgery, dermal injections, bone grafting, and reconstructive surgery. The sources of COL1 for these procedures are commonly nonhuman, which increases the potential for inflammation and rejection as well as xenobiotic disease transmission. In view of this, a method to efficiently and quickly purify COL1 from limited quantities of autologously-derived tissues would circumvent many of these issues; however, standard isolation protocols are lengthy and often require large quantities of collagenous tissues. Here, we demonstrate an efficient COL1 extraction method that reduces the time needed to isolate and purify this protein from about 10 days to less than 3 hr. We chose the dermis as our tissue source because of its availability during many surgical procedures. This method uses traditional extraction buffers combined with forceful agitation and centrifugal filtration to obtain highly-pure, soluble COL1 from small amounts of corium. Briefly, dermal biopsies are washed thoroughly in ice-cold dH2O after removing fat, connective tissue, and hair. The skin samples are stripped of noncollagenous proteins and polysaccharides using 0.5 M sodium acetate and a high speed bench-top homogenizer. Collagen from residual solids is subsequently extracted with a 0.075 M sodium citrate buffer using the homogenizer. These extracts are purified using 100,000 MW cut-off centrifugal filters that yield COL1 preparations of comparable or superior quality to commercial products or those obtained using traditional procedures. We anticipate this method will facilitate the utilization of autologously-derived COL1 for a multitude of research and clinical applications.

Traditional procedures for the isolation of soluble type 1 collagen (COL1) require about 10 days from start to finish because of lengthy buffer incubations and laborious resuspensions of fibrils. Here, we describe a means to purify COL1 from small dermal biopsies in less than 3 hr.

Soluble type 1 collagen (COL1) is used extensively as an adhesive substrate for cell cultures and as a cellular scaffold for regenerative applications. ...

Method and Instrumented Fixture for Femoral Fracture Testing in a Sideways Fall-on-the-Hip Position

Mechanical testing of femora brings valuable insights into understanding the contribution of clinically-measureable variables such as bone mineral density distribution and geometry on the femoral mechanical properties. Currently, there is no standard protocol for mechanical testing of such geometrically complex bones to measure strength, and stiffness. To address this gap we have developed a protocol to test cadaveric femora to fracture and to measure their biomechanical parameters. This protocol describes a set of adaptable fixtures to accommodate the various load magnitudes and directions accounting for possible bone orientations in a fall on the hip configuration, test speed, bone size, and left leg-right leg variations. The femora were prepared for testing by cleaning, cutting, scanning, and potting the distal end and greater trochanter contact surfaces in poly(methyl methacrylate) (PMMA) as presented in a different protocol. The prepared specimens were placed in the testing fixture in a position mimicking a sideways fall on the hip and loaded to fracture. During testing, two load cells measured vertical forces applied to the femoral head and greater trochanter, a six-axis load cell measured forces and moments at the distal femoral shaft, and a displacement sensor measured differential displacement between the femoral head and trochanter contact supports. High speed video cameras were used to synchronously record the sequence of fracture events during testing. The reduction of this data allowed us to characterize the strength, stiffness, and fracture energy for nearly 200 osteoporotic, osteopenic, and normal cadaveric femora for further development of engineering-based diagnostic tools for osteoporosis research.

In this manuscript, we present a protocol to fracture test cadaveric proximal femora in a sideways fall on the hip configuration using instrumented fixtures mounted on a standard servo hydraulic frame. Nine digitized signals comprising forces, moments, and displacement along with two high speed video streams are acquired during testing.

Mechanical testing of femora brings valuable insights into understanding the contribution of clinically-measureable variables such as bone mineral density ...

A Method to Estimate Cadaveric Femur Cortical Strains During Fracture Testing Using Digital Image Correlation

This protocol describes the method using digital image correlation to estimate cortical strain from high speed video images of the cadaveric femoral surface obtained from mechanical testing. This optical method requires a texture of many contrasting fiduciary marks on a solid white background for accurate tracking of surface deformation as loading is applied to the specimen. Immediately prior to testing, the surface of interest in the camera view is painted with a water-based white primer and allowed to dry for several minutes. Then, a black paint is speckled carefully over the white background with special consideration for the even size and shape of the droplets. Illumination is carefully designed and set such that there is optimal contrast of these marks while minimizing reflections through the use of filters. Images were obtained through high speed video capture at up to 12,000 frames/s. The key images prior to and including the fracture event are extracted and deformations are estimated between successive frames in carefully sized interrogation windows over a specified region of interest. These deformations are then used to compute surface strain temporally during the fracture test. The strain data is very useful for identifying fracture initiation within the femur, and for eventual validation of proximal femur fracture strength models derived from Quantitative Computed Tomography-based Finite Element Analysis (QCT/FEA).

In this protocol, the femur surface strains are estimated during fracture testing using the digital image correlation technique. The novelty of the method involves application of a high-contrast stochastic speckle pattern on the femur surface, carefully specified illumination, high speed video capture, and digital image correlation analysis for strain calculations.

This protocol describes the method using digital image correlation to estimate cortical strain from high speed video images of the cadaveric femoral ...

A Performance-testing Platform for a Conduction Micropump with an FR-4 Copper-clad Electrode Plate

Here, a conduction micropump with symmetric planar electrode pairs prepared on flame-retardant glass-reinforced epoxy (FR-4) copper-clad laminate (CCL) is fabricated. It is used to investigate the influence of chamber dimensions on the performance of a conduction micropump and to determine the reliability of the conduction pump when acetone is used as the working fluid. A testing platform is set up to evaluate conduction micropump performance under different conditions. When the chamber height is 0.2 mm, the pump pressure reaches its peak value.

This paper presents a protocol for the fabrication of a conduction micropump using symmetric planar electrodes on flame-retardant glass-reinforced epoxy (FR-4) copper-clad laminate (CCL) to test the influence of chamber dimensions on the performance of a conduction micropump.

Here, a conduction micropump with symmetric planar electrode pairs prepared on flame-retardant glass-reinforced epoxy (FR-4) copper-clad laminate (CCL) is ...

A Millimeter Scale Flexural Testing System for Measuring the Mechanical Properties of Marine Sponge Spicules

Many load bearing biological structures (LBBSs)&#8212;such as feather rachises and spicules&#8212;are small (&#60;1 mm) but not microscopic. Measuring the flexural behavior of these LBBSs is important for understanding the origins of their remarkable mechanical functions.
We describe a protocol for performing three-point bending tests using a custom-built mechanical testing device that can measure forces ranging from 10-5 to 101 N and displacements ranging from 10-7 to 10-2 m. The primary advantage of this mechanical testing device is that the force and displacement capacities can be easily adjusted for different LBBSs. The device&#39;s operating principle is similar to that of an atomic force microscope. Namely, force is applied to the LBBS by a load point that is attached to the end of a cantilever. The load point displacement is measured by a fiber optic displacement sensor and converted into a force using the measured cantilever stiffness. The device&#39;s force range can be adjusted by using cantilevers of different stiffnesses.
The device&#39;s capabilities are demonstrated by performing three-point bending tests on the skeletal elements of the marine sponge Euplectella aspergillum. The skeletal elements&#8212;known as spicules&#8212;are silica fibers that are approximately 50 &#181;m in diameter. We describe the procedures for calibrating the mechanical testing device, mounting the spicules on a three-point bending fixture with a &#8776;1.3 mm span, and performing a bending test. The force applied to the spicule and its deflection at the location of the applied force are measured.

We present a protocol for performing three-point bending tests on sub-millimeter scale fibers using a custom-built mechanical testing device. The device can measure forces ranging from 20 &#181;N up to 10 N and can therefore accommodate a variety of fiber sizes.

Many load bearing biological structures (LBBSs)&#8212;such as feather rachises and spicules&#8212;are small (&#60;1 mm) but not microscopic. Measuring the ...

Rodent Behavioral Testing to Assess Functional Deficits Caused by Microelectrode Implantation in the Rat Motor Cortex

Medical devices implanted in the brain hold tremendous potential. As part of a Brain Machine Interface (BMI) system, intracortical microelectrodes demonstrate the ability to record action potentials from individual or small groups of neurons. Such recorded signals have successfully been used to allow patients to interface with or control&#160;computers, robotic limbs, and their own limbs. However, previous animal studies have shown that a microelectrode implantation in the brain not only damages the surrounding tissue but can also result in functional deficits. Here, we discuss a series of behavioral tests to quantify potential motor impairments following the implantation of intracortical microelectrodes into the motor cortex of a rat. The methods for open field grid, ladder crossing, and grip strength testing provide valuable information regarding the potential complications resulting from a microelectrode implantation. The results of the behavioral testing are correlated with endpoint histology, providing additional information on the pathological outcomes and impacts of this procedure on the adjacent tissue.

We have shown that a microelectrode implantation in the motor cortex of rats causes immediate and lasting motor deficits. The methods proposed herein outline a microelectrode implantation surgery and three rodent behavioral tasks to elucidate potential changes in the fine or gross motor function due to implantation-caused damage to the motor cortex.

Medical devices implanted in the brain hold tremendous potential. As part of a Brain Machine Interface (BMI) system, intracortical microelectrodes ...

Simultaneous Quantification of Selected Kynurenines Analyzed by Liquid Chromatography-Mass Spectrometry in Medium Collected from Cancer Cell Cultures

The kynurenine pathway and the tryptophan catabolites called kynurenines have received increased attention for their involvement in immune regulation and cancer biology. An in vitro cell culture assay is often used to learn about the contribution of different tryptophan catabolites in a disease mechanism and for testing therapeutic strategies. Cell culture medium that is rich in secreted metabolites and signaling molecules reflects the status of tryptophan metabolism and other cellular events. New protocols for the reliable quantification of multiple kynurenines in the complex cell culture medium are desired to allow for a reliable and quick analysis of multiple samples. This can be accomplished with liquid chromatography coupled with mass spectrometry. This powerful technique is employed in many clinical and research laboratories for the quantification of metabolites and can be used for measuring kynurenines.
Presented here is the use of liquid chromatography coupled with single quadrupole mass spectrometry (LC-SQ) for the simultaneous determination of four kynurenines, i.e., kynurenine, 3-hydroxykynurenine, 3-hydroxyanthranilic, and xanthurenic acid in the medium collected from in vitro cultured cancer cells. SQ detector is simple to use and less expensive compared to other mass spectrometers. In the SQ-MS analysis, multiple ions from the sample are generated and separated according to their specific mass-to-charge ratio (m/z), followed by the detection using a Single Ion Monitoring (SIM) mode.
This paper draws the attention on the advantages of the reported method and indicates some weak points. It is focused on critical elements of LC-SQ analysis including sample preparation along with chromatography and mass spectrometry analysis. The quality control, method calibration conditions and matrix effect issues are also discussed. We described a simple application of 3-nitrotyrosine as one analog standard for all target analytes. As confirmed by experiments with human ovary and breast cancer cells, the proposed LC-SQ method&#160;generates reliable results and can be further applied to other in vitro cellular models.

Described here is a protocol for the determination of four different tryptophan metabolites generated in the kynurenine pathway (kynurenine, 3-hydroxykynurenine, xanthurenic acid, 3-hydroxyanthranilic acid) in the medium collected from cancer cell cultures analyzed by liquid chromatography coupled with a single quadrupole mass spectrometry.

The kynurenine pathway and the tryptophan catabolites called kynurenines have received increased attention for their involvement in immune regulation and ...