AnexperimentalsetuptouateinnovativetherapyoptionsfortheenhancementofbonehealingusingBMPas

1、 europeanb preiningercellsetandal. materials vol. 23 2012 (pages 262-272)healing potential in a longissnbone1473-2262modelan experimental setup to evaluate innovative therapy options forthe enhancement of bone healing using bmpas a benchmark a pilot studyb. preininger *, h. gerigk , j. bruckner , c.

2、 perka , h. schell , a. ellinghaus , k. schmidt-bleek and g. duda1,2, 1 1 1,2 1 1 1 1,21julius wolff institute and center for musculoskeletal surgery, charité universitätsmedizin berlin,berlin, germany2berlin-brandenburg center for regenerative therapies, charité universitätsmedi

3、zin berlin, berlin, germanyabstractintroductioncritical or delayed bone healing in rat osteotomy (ot) enhancement of bone regeneration in impaired healingmodels is mostly achieved through large defects or situations remains a clinical challenge as a signicantinstability. we aimed to design a rat ot

4、model for impaired number of fractures exhibit some kind of disturbed repairbone healing based on age, gender and parity. the outcome (einhorn, 1995). such delayed healing situations may beshould be controllable through variations of the haematoma attributed to inadequate reduction or instability of

5、xation,in the ot including a bone morphogenetic protein (bmp) insufcient local nutritious supply and/or general health2 guided positive control.of the patient, infection, or simply by the very nature ofusing external xation to stabilise femoral a 2 mm the insulting trauma. in many cases the source r

6、emainsdouble ot in 12 month old, female sprague dawley rats unidentified (giannoudis et al., 2007). under theseafter a minimum of 3 litters healing was characterised circumstances an enhancement of bone healing appearsfollowing in situ haematoma formation (ish-group),mandatory.transplantation of a b

7、mp charged autologous blood clotto date, bone morphogenetic proteins (bmp) (bmp2,(bmp-group) and the articial blood clot only (abc-group) bmp7 and gdf-5 (bmp-14) are the only drugs availableinto the ot-gap. in vivo micro-computer tomography (ct) to enhance bone healing next to traditional techniques

8、scans were performed after 2, 4 and 6 weeks.after 6 weeks which aim to locally supplement bone material throughspecimens underwent histological analyses.either autologous or allogeneous bone product graftingin ct examinations and histological analyses no bony (wildemann et al., 2011). several new dr

9、ugs andbridging was observed in all but one animal in the ish- “bioproducts”, however, are currently in the stage ofgroup. in the bmp group complete bridging was achieved development (chen et al., 2010; harvey et al., 2010; tarein all animals. the abc-group showed less mineralised et al., 2010). in

10、order to be able to evaluate the potentialtissue formation and smaller bridging scores during the of such newly found therapeutic measures, small animalcourse of healing than the ish-group.models are most efcient screening models.in this pilot study we introduce a model for impairedsmall animal mode

11、ls, especially in rats, are wellbone healing taking the major biological risk factors into established to investigate the course of bone healingaccount. we could show that the in situ fracture haematoma (harrison et al., 2003; jager et al., 2007; drosse etis essential for bone regeneration. using bm

12、p as a positive al., 2008; claes et al., 2009; peters et al., 2009). thiscontrol the presented experimental setup can serve to is due to the advantages of small animals regardingevaluate innovative therapeutical concepts in long bone availability, costs and logistic matters. moreover, a wideapplicat

13、ion.range of reagents such as antibodies, molecular probesand growth factors are available for rats so that thekeywords: bone haematoma, bone morphogenetic protein biologic mechanisms underlying the healing process can(bmp), delayed healing model, rat, articial blood clot.be elucidated. by the use o

14、f osteotomy (ot) models incombination with external xator devices the mechanicalenvironment can be controlled and kept standardisedat high accuracy during the trial (strube et al., 2008a).furthermore, the combination of an ot and an externalfixator device provides the advantage of minimisedinteracti

15、on between the implant and the healing processat the bone healing site.currently available, impaired bone healing modelshave been designed using large articial bone and/or softtissue defects or unstable xation conditions factorsthat under clinical circumstances need either a form ofbone and/or soft

16、tissue graft or a change of the xationregime. in addition, these animal models have mostly been*address for correspondence:bernd preiningerjulius wolff institutaugustenburger platz 1, forum 4,d-13353 berlin, germanytelephone number: +4930450659096 conducted using rather juvenile animals. factors rel

17、atedfax number: +4930450559969 to reduced bone mineral density and thereby to highere-mail: bernd.preiningercharite.de fracture risk (demir et al., 2008) as well as to limited bone 262 b preininger et al.healing potential in a long bone modelhealing capacity, like age (meyer et al., 20

18、01; meyer et articial blood clot was implanted into the ot gap (abc,al., 2006; strube et al., 2008b) and gender (david et al., n = 5), while in a third group a bmp-charged articial2006; parker et al., 2007; yamada et al., 2007 strube et blood clot was implanted into the ot gap (positive controlal.,

19、2009) are inherently considered in osteoporosis models group (bmp), n = 5). in vivo micro-computer tomography(kalu, 1991; duque et al., 2009); yet these factors have (ct) scans and x-rays were performed at 2, 4 and 6not been taken into account so far in bone healing models. weeks after surgery to mo

20、nitor bone healing.after 6 weeksthe hypotheses of this work were: i) that in 12-month- the animals were sacriced. femora were harvested andold female, ex-breeder sprague dawley rats, a 2 mm underwent histological analysis. all animal experimentsfemoral ot gap stabilised by an external xator device w

21、ere carried out according to the policies and principleswould not heal within 6 weeks, whereas by the local established by the animalwelfare act. the design of theapplication of bmp within a blood clot bony bridging animal surgeries was critically reviewed and approved bywould be achieved (govender

22、et al., 2002; einhorn et al., the local legal representative (landesamt fürarbeitsschutz,2003; wildemann et al., 2011); ii) that the delivery of an gesundheitsschutz und technische sicherheit, berlin,autologous blood clot would not alter the healing outcome germany: g0428/08).compared to the gr

23、oup in which native haematoma wasallowed to form following the ot.surgical techniquethe aims of this study were: i) to characterise a surgery was performed under general anaesthesia. thedelayed healing model without large bone and/or soft animals received an intraperitoneal injection with 0.3 mg/

24、74;tissue defects or instability, but with a choice of animals kg medetomidin (domitor , pzer, karlsruhe, germany)having formally reduced biological bone healing potential; and 60 mg/kg ketamine (actavis, langenfeld, germany).ii) to set a benchmark within the model by the use of the prior to the ope

25、ration, all animals were weighed and thecurrently solely approved drug therapy for bone healing left hind portion, including the entire leg, was shaveddisorders bmp. in future, newly developed products for and disinfected by using povidone-iodine. all animalsthe enhancement of bone healing can be co

26、mpared to the were subcutaneously injected with an antibiotic (4 mg/kgclinical standard (bmp) within a well-designed animal clindamycin, ratiopharm, ulm, germany). as analgesiamodel at high efciency.materials and methodsin vitro xator testingthe animals received an injection of 20 mg/kg tramadol, gr

27、ünendal aachen, germany) directly prior tothe surgery. the animals were laid on their right side on aheating plate (37 °c) covered with a sterile sheet leavingthe left hind leg uncovered. a longitudinal lateral skin(tramal®in order to check the suitability of the used xator devices in

28、cision was made over the femur. the femur was exposedfor the animal model, the range of loading likely to occur by blunt dissection of the fascia between the quadricepsduring the in vivo experiment was tested in vitro. the and hamstring muscles. a primary stainless steel xatortesting procedure was p

29、erformed as described previously served as drilling guide to ensure correct and reproducible(strube et al., 2008a). in brief: for in vitro mechanical positioning of the k-wires relative to the bone. using thistesting, the xators were mounted on harvested femora drilling guide, the four holes were dr

30、illed consecutivelyof 12-month-old female ex-breeder sprague-dawley rats, with a drill of 0.8 mm diameter (s-11, implantmeds,identical to the ones in in vivo surgery (n = 3). an ot was w&h oral surgery, büromoos, austria), using constantperformed after the xators were mounted on the bones i

31、rrigation (0.9 % saline solution) and followed by screwingto test the stiffness of the xator device bridging the ot in the wires bicortically, still using the guide. the templategap. specimens were examined using titanium k-wires was removed and the cross-linked carbon xator bar wasand cross linked

32、carbon bars mounted at an offset of mounted at an offset of 7.5 mm. the musculature in the7.5 mm (as described in the surgical technique section). middle between the two inner k-wires was then dissectedboth ends of the specimens were embedded into custom- from the bone at a length of approximately 4

33、 mm andmade casting containers with polymethylmethacrylate protected by a wound elevator. the femur was double(technovits 3040, heraeus kulzer, wehrheim, germany). osteotomised (0.3 mm saw blade, s-8r, implantmeds,for rotational stability after embedding, a short pin was w&h oral surgery) in the

34、 centre between the two innerimplanted perpendicular to the bone long axis proximally k-wires using a 2 mm saw guide xed on the k-wires toand distally of the k-wires. the specimens were mounted set a standardised gap size. the bone segment was removedonto a biomechanical testing machine (electroforc

35、e®thereafter. irrigation was performed during cutting totestbench lm1, bose, mn, usa). torsional stiffness prevent thermal damage to the bone and surroundingwas tested at 0.5°/s up to 5° at an axial preload of 0.3 n. tissues and the k-wires were then shortened ush withaxial stiffness

36、was tested at 1 mm / min displacement with the bar. the fascia of the muscle was sutured with 3.0a preload of 0.3 n up to 10 n.absorbable sutures, the skin then closed with 3.0 non-absorbable sutures, which were removed after 2 weeksduring the wound care routine. the animals were closelyin vivo expe

37、rimental designin a total of 17 female ex-breeder sprague-dawley rats monitored during the rst three days after surgery. for(weight: 407 ± 28 g; minimum 3 litters) a midfemoral post-surgery analgesia, the animals received tramal®indouble ot was stabilised with an external xator. one their

38、drinking water (25 ml/l) for three days. afterwardsgroup received no further treatment, leaving the in situ the animals were seen daily, the wound was monitored,haematoma in the otgap (ish, n = 7). in a second group an and weight was controlled weekly. 263 b preininger et al.healing po

39、tential in a long bone modelfig. 1. the articial blood clot: depicted next to a 1 ml syringe for size comparison (a). this blood clot is theninserted in the osteotomy gap of the femur, stabilised with an external xator (b).blood clot formationa blood clot composed of 200 l autologous peripheralblood

40、 was prepared for each animal at the beginning ofthe surgical procedure. peripheral blood was drawn fromthe saphenous vein from the right shank under generalanaesthesia using a 1 ml syringe prepared with 30 l ofcitrate (buffered sodium citrate (equivalent to 3.2 % sodiumcitrate) 0.109 m, taken from

41、bd vacutainer citrate plusplastic tubes # 363080, bd franklin lakes, nj, usa) toinhibit the immediate coagulation and keep the blood fromclotting. the blood was then lled into the lid of a 1.5 mleppendorf tube (ver international, darmstadt, germany)as a form-giving device for the round shape needed

42、to tinto the ot gap (fig. 1). in the bmp group, rhbmp2 (50g of rhbmp-2 (derived from e. coli; (kubler et al., 1998)per animal (1 mg/ml, ddh2o; prof. sebald, würzburg,germany) was added before clotting was initiated withthe addition of 4 l cacl2·12h2o and 4 l thrombin bothin the abc and in

43、the bmp groups (500 i.e. /ml; baxterdeutschland, unterschleißheim, germany).fig. 2. five regions within a section through the callustissue in the voi (volume of interest) of the ctevaluation have been dened as indicated by the greennumbers. the black square on the right side symbolisesthe xator

44、 bar and the pins mounted on the lateral sideof the femur.in vivo micro computer tomography (ct)bone healing was assessed by in vivo micro computertomography (ct) at 2, 4 and 6 weeks post-operation.the animals were under general anaesthesia during thismeasures included bone volume (bv, mmvolume (cv,

45、 mm ), bv/cv, and tissue mineral density(tmd, mg ha/cm ). bone mineral content (bmc) as a3), total callus33procedure (medetomidin 0.3 mg/kg (domitor , pzer)®product of bv and tmd was calculated. 3d pictures werereconstructed using scanco software.and ketamine 60 mg/kg (actavis) i.p.). all femor

46、a werescanned using quantitative micro-computed tomography(vivact 40, scanco medical, brüttisellen, switzerland).scans were reconstructed at 35 m isotropic resolution.a volume of interest (voi) was dened for the periostealand endosteal callus, excluding the cortical bone based oninner and outer

47、 values for semi-automatic contouring. a 4mm volume of interest (voi) was applied, 2 mm in eachaxial direction starting at the centre slice of the osteotomygap. accordingly, the voi included the 2 mm gap regionand 1 mm in both the proximal and distal directions fromthe borders of the original ot.fur

48、thermore, a score was used to quantify the bridgingof the ot gap by mineralised tissue during the course of theexperiment.therefore, in an imaginary perpendicular planeto the length axis of the xator in the medial region of thegap, mineralised tissue formations bridging the gap wereassessed in 5 sep

49、arate regions (fig. 2). one blinded scorerevaluated bridging with the help of a 3d-reconstruction ofct-imaging. in every separate region, bridging was rated 1for bridging or 0 for non-bridging. the bridging score forevery individual animal represents the sum of the scoresof every region with a maxim

50、um of 5 points.aglobal threshold for fracture callus at 233 mg ha/cm3was manually determined, corresponding to 50 % of themineralisation of intact cortical bone from the tibia. thisvalue was based on visual inspection of ten random singletomographic slices from each animals tibia. outcomehistologica

51、l procedure and analysesthe femora of the ish and the abc group were xedfor 2 days in normal buffered formaldehyde, decalcied 264 b preininger et al.healing potential in a long bone modelfig. 3. 3d reconstructions from ct scans from one representative animal after 2, 4 and 6 weeks of e

52、ach group areshown. in the ish group (left) mineralised tissue was formed predominantly at the endosteal regions.after 6 weeksthe gap had not been bridged; rather a sealing of the medullary canal was observed. in the abc group (middle)callus formation was mainly located between the cortices adjoinin

53、g the gap. neither bridging nor cap-formationwas observed. in the bmp group (right) massive callus formation in the periosteal and intercortical regions wasobserved between week 2 and 4. complete bridging with a reformed medullary canal had occurred during the 6weeks observation period.in edta (42 d

54、ays at 37 °c), dehydrated with alcohol chicago, il, usa) was used. a p value of less than 0.05and xylol, and embedded in parafn. the femora of the was taken to be a signicant difference.bmp group were xed for 2 days in normal bufferedformaldehyde, dehydrated in ethanol, and embeddedin polymethylmethacrylate (technovit1 9100, heraeuskulzer). longitudinal serial sections (4 m) were cut inthe plane of the k-wire ducts for all femora.resultsin vitro testing of the xator devicessections of each animal were stained with movats to