Internal fixator for use in the mouse

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Internal fixator for use in the mouse

Romano Matth�s1, Stephan M Perren2,3

1 AO Development, Davos, Switzerland2 AO Research Institutes, Davos, Switzerland3 Queensland University of Technology, Brisbane, Australia

KEywORdS:Knockout, bone-frac-ture healing, mouse,flexible osteosynthe-sis, internal fixator

Summar�1 Knockout techniques enable us to expand our knowledge about bone-repair processes. Since they require the use of mice, such studies necessitate thedevelopment of special technologies. Mechano-biological reactions play a deter-mining role in fracture healing, and therefore controlled conditions of stability areessential. Achieving fixation with a low-mass implant avoids uncontrolled inertialloading and enables free ambulation.Aminiaturised version of conventional internal fixation as used in humans has beendeveloped. The method consists of a plate-like internal fixator with locked screws.It permits compression fixation or splinting fixation at selectable levels of flexibil-ity. A guide is used for standardised application of the fixator, the screws and theosteotomy. Locking the screws to the plate enables elevation of the fixator fromthe bone surface; therefore, minimal contact damage to the periosteal blood sup-ply can be expected. The technology is now in use in several laboratories.

Intro�uction

The use of mice is practically a prerequisite for ge-netic studies with knockout technology. Experimen-tal studies of fracture healing require exactly de-fined, reproducible mechanical conditions, whichhave not yet been achieved in mice. An approachto solving these problems using internal-fixatortechnology is reported here.Osteotomy of the mouse femur with subsequent

internal fixation had to meet the following require-ments:• Fracture fixation either by splinting with adjust-able levels of flexibility or by compression platingwith so-called “absolute stability”

• Osteotomy with adjustable gap width• Minimisation of uncontrolled loading during ac-celeration of relatively large fixation devicesduring locomotion

• Unrestricted activity (freeweight bearing)• Minimal damage to the blood supply of soft tis-sues and boneThe methods hitherto available were regarded

as open to improvement with respect to definitionof the mechano-biological circumstances, stand-ardisation and/or simulation of conditions in humanfracture treatment [3, 4, 5, 8, 15].

Materials

The implants, which are plate-like internal fixators[1, 13], aremade ofmedical grade titanium accord-ing to ISO standards [16]. The internal fixator platesconsist of a plate body including holes for applyinglocking head screws [13, 14]. Although the term“plate” is used to describe the shape of the implant,a plate-like implant may function as, for instance,a compression plate or as the “rod” of an internalfixator. The arrangement, design and dimensionsof these holes are shown in Figure 1. Two different

1 Abstracts in German, French and Spanish are printed atthe end of this supplement.

0020–1383/$—see front matter © 2009 Published by Elsevier Ltd.doi:10.1016/j.injury.2009.10.044

Injury, Int. J. Care Injured (2009) 40S4, S103– S109

S104 Romano Matthys, Stephan M Perren

plates were developed to enable the effects ofvarious fixation stabilities to be studied. Either theintact plate, as illustrated in Figure 1, was appliedor a customised plate body with four holes wascut in half, whereby the two segments were fixedto the bone on either side of the osteotomy. The

Figure 2: The flexible internal fixator plate is made ofa plate (Figure 1a) cut transversely into two segmentsthat are bridged with flexible wires. The wire materialand its diameter and length determine the stiffness andstrength.

a

b

Figure 3: Design of the screw: because the diameter ofthe screw head is very small, a conventional screw driverconnection was considered impracticable. Furthermore,precise guidance at insertion was required. Therefore,the drive connection was constructed to consist of a pre-determined breaking shank. At the end of screw tight-ening, when a predetermined torque value is reached,the drive shaft twists off. The square drive connectionis available for removal of the screw at the end of theimplantation period.Figure 3a: Overview: screw and drive shaftFigure 3b: details:a Screw thread outer diameter 0.47 mm, core diameter

0.34 mm, pitch 0.17 mmb Self-tapping tip with two cutting flutesc Conical undersurface of the screw head designed for

locking within the screw holed Square drive connection for removale Predetermined breaking connectionf Drive shaft

Figure 1: The internal fixatorFigure 1a: The internal fixator is similar to a convention-al plate, but its function differs: the screws are lockedwithin the plate holes. The dimensions are reported inmm.Figure 1b: Cross section of the plate. The screw holesare conically recessed with a cylindrical portion to ena-ble locking of the screw heads. The undersurface of theplate is cylindrically undercut with a radius of 2.0 mm.

two plate segments were connected with a pair ofwires made of flexible material (“bridging wires”).These bridging wires were welded to correspondingrecesses in the side walls of the plate segments atthe ends remote from the osteotomy site (Figure2). The flexural characteristics of the fixation weremodifiable using two parallel bridging wires withdifferent diameters and length, and by selectingdifferent materials.The self-tapping screw is shown in Figure 3. The

undersurface of the screw head is conical (20o) andthreaded to enable locking within the plate hole.To ensure precise guidance through the drill guide,the screws are connected to a shaft. There is apredetermined breaking point (∅ 0.35 mm) wherethe shaft connects to the screw head, so that theshaft shears off the screw at an insertion torque of8 mNm, which avoids the difficult handling of a tinydrive connection. The insertion tool consists of asmall 3-jaw chuck. The upper surface of the screwhead is 4-flanged to enable removal. The total massof the implants used is 36 mg.The drill guide (Figure 4) permits precise posi-

tioning of the screws (Figure 4a), correct alignmentof the plate in respect to the longitudinal axis ofthe bone (Figure 4b) and guidance of the saw during

a

b

Internal fixator for use in the mouse S105

the osteotomy (Figure 4c). An additional drill guideat the tip of one handle can be displaced with thecompression nut towards the osteotomy to produceadaptation and compression (Figure 4a).The drill bit (Figure 5a) has a 0.3 mm diameter.

Tapering is not required because of the self tappingscrew tip design. The application of the self tap-ping screw alowsallows to have a smooth preloadin between the screw and the bone interface. Suchpreload has been shown to prevent loosening dueto bone surface resorption at the interface bonescrew [10]. The drill bit is operated by a miniatureelectrical pen drill producing 2500 rpm at a powerof 500 mW (Figure 5a).

A micro Gigli saw (Figure 6) is used to producea transverse osteotomy. To minimise a slip-stickeffect, two or more elements of this saw areconstructed from wires of different diameter. Thethicker wire (∅ 0.07 mm) serves as an axial coreand the thinner wire (∅ 0.04 mm) forms a cuttingspiral with an adjustable pitch around the corewire. By using Gigli saws with different numbersof such pairs of wires, the following gap sizes weregenerated: 0.22, 0.44 and 0.66 mm.All instrumentsand devices, including the electrical drill, can besteam sterilised at 134oC.

a b

c

�

Figure 4a: Drill guide used for application of the internal fixator plate. The four drill holes enable the plate screws tobe applied at predetermined distances. The slots between the inner holes serve as guide for the micro Gigli saw. At thetip of the left arm (see arrow) of the handle, the knurled nut and a displaceable drill guide element are seen. Twistingof the knurled nut achieves adaptation and compression when the left bone fragment connects through a plate screw,with the displaceable drill guide shifted towards the right bone fragment.Figure 4b: Drill guide seen from below. The position of the plate, snugly fitting into a corresponding groove within thedrill guide, undersurface is visualised.Figure 4c: View from below showing the two arms on each side of the bone aligning the drill guide with the plate overthe long axis of the bone. The slots in the arms guide the Gigli wire while cutting the osteotomy.Figure 4�: Ex vivo application in the mouse.The guide containing the plate is placed on top of the lateral surface of the femur. The screws are inserted and, at thisstage in the procedure, the osteotomy is made using the micro Gigli saw.


Metho�

The method uses a miniature set of instrumentsand implants to create an osteotomy and achievestandardised fixation for it (Figures 1−6). Thetechnique is designed to enable flexible internal-fixator splinting with variable gap widths, as wellas compression plating of osteotomy fragments thatare in contact. Special care was taken to design theinstruments, implants and procedures to minimisesurgical damage.To test the performance of screw-head locking

within the plate hole, the plate is fixed at one endin a bench vice. A screw is then inserted into a platehole near the other end of the plate. An increasingload is then applied to the tip of the screw, actingperpendicular to the long axis of the screw, untilfailure occurs, either because the locking mecha-nism failed or the screw deformed.For ex vivo testing an anterolateral surgical ap-

proach is used. The drill guide containing the plateis aligned with the longitudinal axis of the bone,whereby the flat arms of the drill guide help tocentre the plate. The drill guide is held in placewith a suture, which is fixed to the drill guide atone end, encompassing the bone, and its free endis tightened and clamped. The pilot holes are madeusing the 0.3 mm drill bit and the pen drill. Thescrews are inserted and sheared off from the driveshaft. One drive shaft is temporarily left in place to

a

b

Figure 5: Application of the plate screwsFigure 5a: Miniature electrical pen drill. The caseincludes replaceable batteries CR123A.1300mAh at 3 V.The drill bit is operated at 2500 rpm at a power of about500 mW. The drill bit has a diameter of 0.3 mm.Figure 5b: The consecutive steps of application of theplate screws.a Drill bit of 0.30 mm diameter. The shaft (∅ 1 mm)

enables precise guidance within the drill guide.b Screw with drive shaft (∅ 1mm) before insertion.c Screw securely locked within the plate hole.d Final situation, the drive shaft is twisted off the screw

head.

Figure 6: Custom made micro Gigli saw is used to pro-duce an osteotomy with predetermined gap widths of0.22, 0.44 and 0.66 mm. To prevent displacement ofthe devices during cutting, the Gigli saw must operatesmoothly. The construction cuts the bone with minimaljerking and avoids the slip-stick effect.


maintain the alignment of the drill guide. The Giglisaw is passed snugly around the bone and insertedinto the guiding slots of the drill guide. The wireis then moved back and forth carefully until con-tact with the plate indicates that the osteotomy iscomplete. This procedure is used for pure splintingwith rigid or flexible plates. To achieve a higherdegree of instability, the screws may be loosenedby a controlled amount of twist (eg, ½ turn, whichcorresponds to a displacement of 0.1 mm).

Proce�ure for compression fixation: One bonefragment is fixedwith two screws. In the other frag-ment beneath the compression guide, the two plateholes are left open temporarily. First, the compres-sion guide is opened a predetermined amount usingthe compression nut (Figure 4a). The underlyingbone fragment is connected to the compressionguide with a temporary screw. The osteotomy isthen cut as outlined above. Using the compressionnut and the drill guide, the bone fragment is shiftedtowards the bone fragment that has already beenfixed. Due to the elastic properties of the drillguide handle, adaptation and compression of theosteotomy result. The remaining screws are appliedand the temporary screw and handle are removed.The numerical analysis of the flexural charac-

teristic of the rigid and flexible plates, performedat Queensland University of Technology, Brisbane,was based on the ISO 9585 [17] standard (Figure 7).The results are reported in the technically correctunit for equivalent stiffness as Nmm2, as well as ina more practical unit as Nmm/degree, which canbe more easily interpreted. The bending stiffness

calculated in this analysis was found to be 0.44Nmm/degree or 19.02 Nmm2 for the flexible platewith 0.25 mm diameter bridging wires, and 23.90Nmm/degree or 1026.7 Nmm2 for the rigid plate.

discussion

The method presented here enables the selectionof different degrees of flexibility of fixation and, atthe same time, the creation of different gapwidths.Along with functional loading, these two conditionsdetermine the amount of strain within the repairtissue. The deformation of the repair tissue playsan important role in inducing and allowing tissuedifferentiation [12].To keep the surgical trauma low, conventional

rotating or oscillating saws were not considered astools for creating the osteotomies. Such saws eithercut into adjacent tissues, or stripping of the peri-osteum occurs when the tissues are retracted. Toavoid both these effects, we took advantage of theGigli saw. A conventional Gigli saw has substantiallydifferent static and kinetic friction. To limit themechanical load at the interface between implantsand bone, the Gigli saw had to be constructed toavoid the slip-stick effect. This effect interruptsthe cuttingmovement and requires additional forceto overcome static friction.The technology of radial preload according to

Hyldahl et al [10], was applied to avoid biologicalloosening of the screws. The self-cutting tip of thescrew then opens the pilot hole less than the corediameter of the screwto result in a1−2%misThegoalof developing the new technology for use in micewas to provide an osteotomy model with a fixationthat avoided mechanical artefacts and providedwell-standardised conditions. Absolute stability ordifferent degrees of reversible instability shouldpermit investigation of the effects of fixationstability on fracture healing and nonunions underdifferent genetic conditions achieved by knockouttechnology. A standardised gap width ranging from0.22−1.25 mm serves the same purpose.Minimal mass of the fixation device to avoid un-

controlled loading due to inertia was an unsolvedproblem. The relationship (1:1000) of the massof the new “MouseFix™” implant (36 mg) to themass of the mouse (40 g) corresponds well to therelationship between the mass of a broad femurplate with its screws (65 g) to the average mass ofa human (75 kg).Standardisation of insertion torque of screws was

a precondition for achieving reliable locking: keep-ing the diameter of the predetermined breaking

Figure 7: Model used for the numerical analysis of theplate stiffness, simulating a 4-point bending experiment(based on ISO 9585) that results in a pure bending situa-tion between the inner loading pads. The results are alsoreported in Nm/degree, whereas the definition of thedeflection angle a is illustrated in this figure.


point of the drive connection within 0.35 +/− 0.02mm ensured a minimal torque value at insertionfor proper locking, and avoided too high a torquewith consequent damage of the locking connection.This performance parameter is being tested duringproduction using a sampling procedure.The use of internal fixator technology [11] ena-

bles pure splinting to be combined with minimalcontact between implant and bone, because theplate body can be locked in a position elevated fromthe bone surface [2]. Applying the same technol-ogy to larger animals and humans [7] has shownthat the blood supply was improved compared toconventional plate fixation where the plate hasextended surface contactwith the bone surface [6].Implant design and application technologies thatpermit optimal blood supply as a positive controlare preconditions for studies of variable degrees ofdamage to the blood supply. With elevated plates,reduced damage to the blood supply of the softtissues and bone can be expected. Furthermore,with elevation of the plate, there is no demandfor exact contouring of the plate, thus keeping thetechnique simple and safe.The use of a range of fixation stabilities permits

investigation of both direct and secondary healingas well as mechanically-induced, delayed healingand nonunion. Producing a mechanically-inducednonunion requires a certain degree of reversibledisplacement to exceed the tolerated deformation(strain) of the repair tissue [11, 12]. This displace-ment could not be realised by simple reductionof the cross section of the plate that bridges theosteotomy: The length of the deformable sectionof the implant is critical. The degree of instabilitydepends on the stiffness of the “flexible” crosssection and of its free (span) length. For a givenflexibility, strength increases with span length be-cause, with a longer span, a thicker bridging wirecan be used, thus restoring the same flexibility withincreased length. “Clinical” experience has shownthat when allowing unrestricted weight bearing,a 0.25 mm diameter wire is the lower limit withrespect to strength.One method of increasing the instability of fixa-

tion is to loosen the screws by a controlled amountof twist. One revolution of the screw head releasesa distance corresponding to the pitch of the screwthread. The resulting mechanical situation may bedescribed as a coupling, with minimal stiffness upto a distance of displacement after which the stiff-ness of coupling changes from loose to tight. Fromthis point on, the stiffness of the implant plays thedeterminant role. The discontinuity of stiffness willhave to be taken into account when using such aprocedure.

The model developed (Figure 8) is expected tofacilitate studies to be conducted with minimaldamage to blood and bone as its internal fixatortechnology enables fixation of the plate away fromthe bone surface. Thus, implant-to-bone contactand, consequently, damage to the periosteal bloodsupply is reduced.Small rodents differ in their quantitative but

less in their qualitative reactions to fracture orosteotomy. They have been shown to exhibit di-rect fracture healing by internal remodelling [9];needless to say that any extrapolation of results tohuman conditions requires care.The numerical analysis shows that a wide range

of stiffness can be achieved by modifying the diam-eter and material of the bridging wires. The rangeof flexibility can be extended when using a longerfree span of the wire (omega shape etc).The advantage of the low cost of the mouse is

partially offset by the fact that the production ofthe implants and instruments requires a high degreeof precision and special machinery.No attempts have been undertaken so far to re-

place the osteotomies in this model by fractures.The study of fractures instead of osteotomies mayrequire the use of intramedullary fixation. Thetechnology lends itself to studies using segmentalgrafts similar to those performed in rats by Hut-zschenreuter [9].

a

b

Figure 8: Aspect of the internal fixator applied to themouse femur.Figure 8a: Plate applied ex vivo to the mouse femur withan osteotomy gap of 0.22 mm.Figure 8b: X-ray showing the plate in place and bridgingthe well-aligned osteotomy.


Conclusions

The demands and solutions are compared as follows:• Fracture fixation with either splinting with ad-justable flexibility or compression plating withso-called “absolute stability” could be achievedas required

• Using the custom-made micro Gigli wires, a gapwidth between 0.22 and 0.66 mm was achieved

• Minimisation of uncontrolled loadingwas achievedby limiting the implant mass to 1:1000 of the ani-mal’s mass

• Unrestricted locomotor activity (freeweight bear-ing) was safely allowed with bridging wires of 0.25mm diameter or more

• Minimal damage to the blood supply has not yetbeen proven quantitatively but comparison tosimilar techniques used in larger animals and inhumans indicates that positive results can be ex-pectedA method has been developed that enables

internal-fixator technology to be applied to themouse femur for studies of fracture healing understandardised mechanical and biological conditions.Studies of fracture healing under genetically altered(knockout) conditions with defined mechanical sta-bility are the goal of the development.The method consists of a miniature set of instru-

ments for reproducible application of an osteotomyand fixation with plates of variable degrees of flex-ibility including compression plating. Ongoing stud-ies at various institutions have exceeded 1500 caseswithout major technical problems being reported.

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Correspondence address:Romano MatthysAO Development InstituteClavadelerstrasse 87270 Davos, SwitzerlandPhone +41 81 414 2461Direct +41 81 414 2598Fax +41 81 414 2285email: [email protected]

This paper has been written entirely by the authors, andhas received no external funding. The authors have nosignificant financial interest or other relationship.

Ackno�legements

We acknowledge the valuable help of ErichZweifel and Peter Däscher who contributed to thedemanding realisation of the miniature implantsand instruments. Roland Steck, Queensland Uni-versity of Technology, performed the calculationsof stiffness. Feedback from Keita Ito, Petra Heiland Ina Groengraft led to valuable improvementsin the techniques of practical application, and JoyBuchanan transformed our “Swiss English” intoproper language.

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Internal fixator for use in the mouse