Repair of rabbit segmental defects with the thrombin peptide, TP508

~~~

ELSEVIER

Journal of Orthopaedic Research

Journal of Orthopaedic Research 22 (2004) 1094-1099

www.elsevier.com/locate/orthres

Repair of rabbit segmental defects with the thrombin peptide, TP508 Michael R. Sheller a,f, Roger S. Crowther b, John H. Kinney Jinping Yang e , Sherry Di Jorio a,f, Tom Breunig d, Darrell H. Carney b3e, James T. Ryaby

c,d,

‘‘ Reseurcli und Developmeni,

OrtlzoLogic Corp, 127’5 W. Washington Street. Tempe, A 2 85281, USA Cl?rj3suhBiotc~clinology.Guheston, TX 77550, USA Luwrence Liwrmore Nutionul Luhorutor): Livermore, CA 94551, USA ‘I Unisersiiy of Culifimiu, Sun Fruncisco, CA 94143, USA ‘ University of’ Tesus Medicd Brunch, Gulveston, T X 7’7555, USA Arizonu Siuie Unicersiij., Tempe, A Z 85287, USA

Abstract The synthetic peptide, TP508 (Chrysalin@),was delivered to rabbit segmental bone defects in biodegradable controlled-release PLGA microspheres to determine its potential efficacy for enhancing healing of non-critically and critically sized segmental defects. Non-critically sized radial defects were created in the forelimbs of New Zealand White rabbits, which were randomized into three treatment groups receiving 10, 50 and 100 pg doses of TP508 in the right radius and control microspheres (without TP508) in the left radius. Torsional testing of the radii at six weeks showed a significant increase in ultimate torque, failure torque, ultimate energy, failure energy, and stiffness when treated with TP508 compared to controls (p < 0.01 for all measures). Thus, TP508 appeared to enhance or accelerate bone growth in these defects. In a second set of experiments, critically sized ulnar defects were created in the forelimbs of New Zealand White rabbits, which were randomized into two groups with each rabbit receiving microspheres with 100 or 200 pg of TP508 into the right ulnar defect and control microspheres (without TP508) alone into the left ulnar defect. Bone healing was evaluated with plain radiographs, synchrotron-based microtomography, and mechanical testing. Radiographs of the rabbit limbs scored by three blinded, independent reviewers demonstrated a significantly higher degree of healing when treated with TP508 than their untreated control limbs (p < 0.05). Three-dimensional synchrotron tomography of a limited number of samples showed that the new bone in TP508-treated samples had a less porous surface appearance and open marrow spaces, suggesting progression of bone remodeling. Torsional testing of the ulnae at nine weeks showed a significant increase in maximum torque and failure energy when treated with TP508 compared to controls (p < 0.01 for both measures). These results suggest that TP508 in a controlled release delivery vehicle has the potential to enhance healing of segmental defects in both critically and non-critically sized defects. 0 2004 Orthopaedic Research Society. Published by Elsevier Ltd. All rights reserved. Kcyvorrl.\

Rabbit model; Ulna; Radius; Segmental bone defect: Thrombin related peptide; Microtomography; Synchrotron radiation: Mechanical

testing

Introduction

Autogenous bone is the de facto standard for bone grafting material; however, there are disadvantages including donor site morbidity and limited graft material availability [25]. Obtaining graft material is espe*Corresponding author. Address: Research and Development, OrthoLogic Corp. 1275 W. Washington Street, Tempe, AZ 85281, USA. Tel.: + 1-602-286-5326;fax: + 1-602-286-2926. E-muil uddwss: [email protected] (J.T. Ryaby).

cially critical in limb-salvage surgery caused by tumor reconstruction or chronic infection because segmental defects of these types often lead to delayed unions or non-unions. Allograft bone is a less attractive material than autograft bone because there is the risk of disease transmission and infection [ 11. In addition, allografts do not contain viable cells and therefore only provide an osteoconductive surface to support re-establishment of viable osteogenic cells, bone formation, and subsequent revascularization. Thus, there is a strong clinical need for alternatives to autografts and allografts.

0736-0266/$ - see front matter 0 2004 Orthopaedic Research Society. Published by Elsevier Ltd. All rights reserved. doi:lO.1016/j.orthres.2004.03.009

M. R. Sheller et ul. I Journal of' Ortliopueriic Reseurch 22 (2004) 1094- I099

Previous studies on the repair of segmental defects have focused on bone matrix substitutes [8,10,11,13, 20,281. However, these substitute matrices do not perform as well as autograft for several reasons including histochemical responses by the host tissue and a dearth of living cells. Enhancing the osteogenic capabilities of these bone matrix substitutes for the treatment of segmental defects is currently an active area of research. In addition, the use of cytokines and growth factors, such as bone morphogenetic proteins, with and without bone matrix substitutes has great potential for enhancing the bone repair process [2,3,5-7,14,15,19,21,22,26,30]. However, few of these studies have been able to demonstrate acceleration of fracture healing. In the present study, a new investigational orthobiologic synthetic peptide drug, TP508 (Chrysalin@),has been tested for its potential to enhance bone formation in rabbit segmental defects. TP508 is a 23 amino acid peptide representing the natural amino acid sequence of the receptor-binding domain of human thrombin. It has been shown to stimulate healing of bone, with both Simmons et al. and Ryaby et al. reporting that a single injection of TP508 accelerated fracture healing in a rat closed diaphyseal fracture model [23,24]. In these studies with young and aged rats, TP508 significantly increased breaking strength of treated bones relative to controls by an average of approximately 30%. Moreover, increases in Chfal gene expression have been reported in the same model by Wang et a]. [27]. The purpose of the present study was to evaluate if TP508, formulated in biodegradable controlled-release microspheres, could enhance bone formation in larger segmental defects in rabbits. This study examined two different defect models, a non-critically sized radial defect model and a critically sized ulnar defect model. The results of these studies demonstrate that TP508 microspheres significantly increased the rate and extent of bone regeneration in both defect models. Thus, these studies suggest that TP508 may be useful for surgical treatment of non-unions or bone defects.

Material and methods

I095

infection prophylaxis. Water and food were supplied ad libitum. Finally, the animals were euthanized by an overdose of pentobarbital (50 mg/kg). The Institutional Animal Care and Use Committee at The University of Texas Medical Branch approved all experimental protocols. Radial segmentul

defect stuclj

Bilateral non-critically sized defects were created in the radii of each rabbit forelimb by removing 0.5 cm of midshaft diaphyseal bone. In the osteotomy space of the right limb, rabbits (eight animalslgroup) received 10, SO, or 100 pg of TP508 in 10 mg of PLGA microspheres. Rabbits in all groups received 10 mg of control microspheres (without TPSO8) in the left limb. After euthanasia at six weeks post-surgery, repair strength was tested by torsional testing (MTS-858 Minibionix machine, MTS Systems Corporation, Minneapolis, M N ) at an angular velocity of I"/s. LJhw segznental rlej>ct ,rtu&

Bilateral critically sized defects were created in the ulna of each rabbit forelimb by removing 1.5 cm of midshaft diaphyseal bone. In the osteotomy space of the right limb, rabbits (15 animals/group) received either 100 o r 200 pg doses of TP508 in 30 mg of PLGA microspheres. Rabbits in all groups received 30 mg of control microspheres (without TPSO8) in the left limb. Radiographs of the left and right ulnae were taken at 3, 5. 7. and 9 weeks. After euthanasia at nine weeks. bone regenerates from six ulnar defect sites were imaged with synchrotron radiation on the 15 period wiggler beamline (10-2) at the Stanford Synchrotron Radiation Laboratory, Stanford, CA [16]. In addition, repair strength was tested by torsional testing as in the radial segmental defect study. Radiogruphic unulj..sis An analysis of the ulnar segmental defects was performed to quantify the degree of fracture healing at nine weeks. The radiographs were digitally photographed and randomized in a Microsoft PowerPoint file, one slide for each bone. There was no indication of whether the reviewer was grading a treated o r untreated ulna. A visual digital scale (VDS) was depicted next to each radiograph with a scale from 054 to 100Y0 healed. Three reviewers with experience in interpreting radiographs digitally marked on the VDS the degree of healing which was defined as the percentage of new bone bridging the osteotomy defect. Reviewers were instructed to ignore bone outside of the defect site and bone not spanning the defect. Synchrotron tomography imuge rrconstruction Data sets describing the synchrotron beam attenuation through the sample were reconstructed into three-dimensional images of the bone regenerates by Fourier-filtered backprojection. The reconstruction voxels were cubic with an edge length of 23.3 pm. Through histogram analysis. the synchrotron data sets were segmented into voxels identified as bone and voxels identified as non-bone. Thresholding to view only bone voxels results in images that represent only the mineralized tissue [17].

Generul rxperimental protocol Stut ist ic.s

TP508 was formulated in 20 pm poly(oL-lactic-co-glycolic acid) (PLGA) porous microspheres courtesy of Dr. Antonios Mikos, Rice University, Houston, TX. Microspheres comprising 90% PLGA (5050, MW -63 kDa, Alkermes, Cambridge, MA), 5%) TP508 and 5% PEG (MW 4600, Aldrich, Milwaukee, WI) were manufactured using a double emulsion technique and solvent extraction [12]. A rabbit segmental defect model was used in these studies [3]. Young (5-6 months) male New Zealand White rabbits (2-3.5 kg, Myrtle's Rabbitry, Thompson Station, TN) were first anesthetized with a cocktail of 50 mg/kg ketamine and 8 mg/kg xylazine administered intramuscularly. Surgical procedures to create either ulnar or radial defects were then performed with a rotating oscillating saw under saline irrigation (see details below). After surgery, animals were given buprenorphine (0.05 mg/kg) for pain and enrofloxacin (50 mg/kg) for

Radiographic review data and the data from mechanical testing of rabbit radial and ulnar bones were analyzed by SAS software (Version 8.2, SAS Institute Inc., Cary, NC). Since three reviewers scored the same images, the data were analyzed by Generalized Estimating Equations (GEE, SAS PROC GENMOD). The degree of healing, measured by the Visual Digital Scale (VDS), was adjusted for TP508 dose (100 pg versus 200 pg), active TP508 versus placebo microsphere (right versus left), and reviewer. For mechanical testing data, G E E models with TP508 dose (10/S0/100 pg for radial, and 1001200 pg for ulnar) and active TP508 versus placebo microsphere (right versus left) as covariates were used to account for the natural and partial pairing in the data, The interaction between TP508 dose and other covariates was also explored.

M.R. Sheller et ul. 1 Journul of’Orthopuedic Research 22 (2004) 1094-1099

1096

Results Radial non-critically sized segmental defect study

An initial experiment was conducted to determine the effect of TP508 microspheres on the healing of radial segmental defects. All animals survived the surgery with no significant post-operative events and no adverse reactions to the treatment. All animals returned to weight bearing activity shortly after surgery. As shown in Table 1, radial defects treated with TP508 showed significant differences between untreated controls for ultimate torque, failure torque, ultimate energy, failure energy, and stiffness (p <0.0001, <0.0001,0.0039,0.0021, and 0.0009, respectively). The greatest percent increases in mean over control occurred at the 50 pg level for all measures except stiffness, which was greatest for the 100 pg dose. In addition, radii treated with 10 pg of TP508 yielded significantly higher ultimate torque than those treated with 50 pg of TP508 (p = 0.030). All other dose response effects were equivalent. Ulnar critically sized segmental defect study

Based on effects of TP508 observed in 0.5 cm radial defects, a second set of experiments was carried out using larger critically sized 1.5 cm ulnar defects. All but four animals survived surgery, and there were no significant post-operative events or adverse reactions to the treatment. The remaining animals returned to weight bearing activity although four rabbits were euthanized due to subsequent complications. Radiographic evaluation

Radiographs of the ulnar segmental defect healing process were evaluated both qualitatively and quantitatively. Fig. 1 depicts typical radiographic results after nine weeks of healing for the ulnar critically sized seg-

Fig. 1. Representative radiographs of ulnar defects at nine weeks postsurgery. The left panel represents left ulnar control (A) and 100 pg TP508-treated right ulna (B) from the same animal. The right panel represents left ulnar control (C) and 200 pg TP508-treated right lllndr (D) from the same animal.

mental defect model. From these radiographs, it appeared that the segmental defect elicits bone formation from the periosteum of the nearby radius in both the control and treated ulnae. In the treated bones this seemed to have resulted in various degrees of bridging of the defect from the proximal to distal end and to some extent outside of the defect region. In contrast, the majority of the controls had little or no bridging of the defect. Qualitatively, the best bridging effect was associated with the 100 pg treatments. To better evaluate the degree of defect healing, three reviewers performed blinded analysis of the radiographs from this experiment. TP508-treated limbs had significantly higher VDS scores than those treated by placebo microsphere (p = 0.049) by a margin of 0.17. In addition, left ulnae (placebo-treated) had a mean VDS value of 0.12 while right ulnae (TPSO8-treated) had a mean VDS of 0.45, for a 0.33 increase for the 100 pg TP508 treatment. In contrast, among those in the 200 pg group,

Table 1 Torsional properties of normal and 10, 50 and 100 pg TP508-treated radii at six weeks post-surgery Group

Ultimate torque (N m)

Failure torque (N m)

Ultimate energy (N m deg)

Failure energy (N m deg)

Dose 10 pg Control 10 pg TP508

0.50 k 0.14 0.60 f 0. I4

0.49 2 0. I4 0.57 f 0.16

5.38 f 4.56 6.53 t 3.53

5.87f4.61 7.37 f 3.89

0.046 t 0.008 0.053 f 0.009

Dose 50 p g Control 50 pg TP508

0.3440.13 0.54 2 0.14

0.34k0.13 0.5650.13

2.68 5 I .41 9.47 f 8.55

2.76 f 1.49 11.37f9.26

0.041 fO.010 0.050+_0.011

0.42 2 0.11 0.62 4 0.16

0.40 k 0.1 I 0.61 f 0 . 1 7

5.08 f 3.82 8.67 t 4.56

5.81 t 4 . 1 6 9.17 f 4.31

0.041 f 0.014 0.056f 0.01 1

Stiffness coefficient ( N mldeg)

DOSK100 p g

Control 100 pg TP508

TP508-treated radii showed statistically significant increases over controls for ultimate torque, failure torque, ultimate energy, failure energy, and stiffness for all doses (p < 0.01 for all measures). All values presented as the mean f one standard deviation.

Ad. R. Sheller el ul. 1 Journal of Orthopaedic Research 22 12004) 1094 l O Y Y

1H

Degree of Healing Control

ioovg

H Control

2oovg

100%

80%

60%

IT

T

T

r

I

40% 20%

-

no," /,

Reviewer #l

Reviewer #2

Reviewer #3

Fig. 2. Three blinded reviewers independently scored the radiographs of TP508-treated critically sized segmental defects of rabbit ulna at nine weeks. The images were randomized so that control and treated ulnae were indistinguishable. Bridging the defects was defined as spanning the original defect site, and regenerated bone outside of the defect was considered as not bridging the defect. TP508-treated ulnae were significantly different than control ulnae for both doses (p = 0.049).

left ulnae (placebo-treated) had a mean VDS value of 0.27 while the right ulnae (TP508-treated) had a mean VDS of 0.31, a margin of only 0.04 for the 200 pg TP508 treatment. Fig. 2 depicts the results for all three reviewers. There was a significant between-reviewer difference as well. VDS scores from Reviewer #1 and Reviewer #3 were significantly higher than those from Reviewer #2 @-values <0.0001 and 0.0074, respectively). The interreviewer differences were 13% between Reviewer #1 and Reviewer #2, and 9% between Reviewer #3 and Reviewer #2. Mechanical testing To better determine the degree of healing of the segmental defects, a total of 32 ulnae were separated from their associated radii and prepared for mechanical testing. Of these, six control bones (two at the 100 pg dose and four at the 200 pg dose) could not be tested as they either fractured prior to mechanical testing o r had

I097

no bridging; in contrast, all treated bones had sufficient bridging and strength to be tested. Rather than bias the control values by assigning a measure of zero to the nontestable bones, a conservative approach was taken to include only those controls for which actual torsion measurements were possible. As shown in Table 2, ulnar defects treated with TP508 showed significant increases over untreated controls for maximum torque and failure energy (p = 0.0039 and 0.0003, respectively). Stiffness in TP508-treated ulna also resulted in a nearly significantly increase over control 0, = 0.07). The greatest percent increases in mean over control occurred at the 200 pg level for maximum torque and failure energy. In addition, ulnae treated with 200 pg of TP508 yielded a nearly significantly higher failure energy than those treated with 100 pg of TP508 @ = 0.052). All other dose response effects were equivalent.

Synchrotron tomography eualuution Three-dimensional synchrotron tomographic renderings uniquely allow the visualization of the texture of the bone surface and a qualitative assessment of the degree of bone remodeling since only mineralized bony tissue is visualized after thresholding the data set. As shown in Fig. 3B, the surface texture of regenerated bone within the defect was not as regular or smooth along its length as normal intact untreated rabbit bone. Examining cross sections of the reconstructed 3-D images showed the development of a marrow space in a representative TP508-treated sample, as shown in Fig. 3C, which was not evident in the untreated samples. Although the process of marrow space development in the TP508treated samples was not yet complete a t nine weeks, the outer shape of the newly formed bone was similar to the shape a normal ulna would have in this region. Fig. 3A, shown for comparison, is a normal, unoperated cross section at the same anatomical location as those used in this study. These sections also showed fusion of the ulnar cortex with the radius during healing of the segmental defect, which does not occur a t the midshaft of an unoperated limb.

Table 2 Torsional properties of normal and 100 and 200 pg TP508-treated ulnae at nine weeks Group

Failure torque (N m)

Energy absorbed (N m deg)

Stiffness (N m/deg)

Control-I00 pg ( n = 5) TP508 @ 100 pg ( n = 7)

0.13 ? 0.09 0.24f0.11

0.77 2 0.79 2.06? 1.11

0.01 7 ? 0.014 0.028 ?r 0.015

Control-200 pg ( n = 5 ) TP508 @ 200 pg ( n = 9)

0.15 f 0.12 0.29 ?r 0.16

1.232 1.11 3.4322.10

0.026f0.012 0.033 2 0.01 3

Six control ulnae that could not be tested were excluded from this analysis, see text. Ulnar defects treated with TP508 showed significant increases over untreated controls for maximum torque and failure energy for both doses 0, = 0.01 for both measures). All values presented are mean f one standard deviation.

I098

M . R. Sheller er ul. I Journal of Orthopedic Research 22 (2004) I09441099

Fig. 3. Synchrotron imaging showing transverse sections of a representative TP508-treated bone (100 pg), The representative TP508treated bone is shown as a mineral-thresholded surface rendering of the synchrotron data in (B). Note the presence of the marrow cavity indicating that bone is being remodeled ( C ) .These cross sections, taken at regular intervals as indicated in (B), may be compared to a cross section of normal bone with no defect (A). The color scheme of the cross sections indicates the mineral concentration in the tissue with yellow being highest (observed primarily in radius farthest away from the regenerating bone in the defect or in the normal ulna), blue somewhat lower, and red and black indicating a total absence of mineral.

Discussion

The use of a single peptide or growth factor to stimulate bone repair is predicated on the assumption that the entire fracture cascade can be activated and subsequent specific regulatory factors would be expressed or upregulated at the proper time. The present results suggest that the use of a controlled release formulation of TPSOX may initiate such a cascade and mimic the early events in bone repair. Immediately after a bone is fractured, there is a localized conversion of prothrombin to active thrombin, which then activates platelets and forms a fibrin clot. Wilner et al. [29] has shown that thrombin is sequestered within the clot where it can later be released, either intact or as partially degraded peptide fragments, as the clot is degraded by proteases released from invading inflammatory cells. The above processes occur within the first few days after the bone has been fractured [4]. Since PLGA microspheres have in vitro release characteristics that allow TP508 to be available for a minimum of four days, this microsphere preparation and its release kinetics may be

matched temporally to the natural release of thrombin fragments that trigger the healing process. TP508 may provide significant advantages over the use of recombinant growth factors for repair of segmental bone defects. Typically, a growth factor is present for only a short period of time due to rapid degradation by enzymes present at the fracture site. It is crucial that the bioactive molecule be available at a therapeutic concentration at the desired location for successful bone repair. However, it is difficult to predict which growth factor (or combination of growth factors) and which carrier will satisfy these criteria. Such difficulties have been noted with the use of bone morphogenetic proteins [ls]. Using a peptide that can activate a cascade that results in appropriate subsequent regulation of specific growth and differentiation factors obviates the need for selecting a specific factor or group of factors. For example, TP508 may activate platelets, which are known to secrete cytokines and growth factors such as IL-1, IL-6, TGF-01, FGF, and PDGF [9]. The potential involvement of cells depositing new bone from the radial periosteum nearest to the ulna confounds the interpretation of the radiographic analysis. In this study, it was observed that new bony material appeared to be generated from the periosteum even in control defects that had microspheres alone without TP508. Based on comments from the blinded radiographic reviewers, this made it difficult to score the amount of filling of the defect and may have led to an overestimate of healing in the defects. In spite of this difficulty, defects treated with TP508, were found by blinded analysis to have significantly more bone healing than controls. Since this segmental defect model may be more similar to the clinical situation than are models that employ stripping the periosteum [3] or shielding it from the defect [22], these results are especially encouraging. Visual inspection of transverse slices of synchrotron data in the healing defect regions showed the development of marrow space forming in the regenerating bone. To our knowledge, this is the first study to image the forming marrow space during segmental defect repair. Marrow space development was limited to TP508-treated samples and not evident in untreated samples. It is speculated that the forming of a marrow space may become more pronounced over time and as healing progresses. Thus, it appears that this may represent a more advanced state of healing and remodeling in the TP508-treated defects. In summary, the radiographic, synchrotron, and mechanical data support the conclusion that a controlled release formulation of TP508 enhances and accelerates the healing of critically sized ulnar segmental defects and non-critically sized radial segmental defects in the rabbit. Further work will be necessary to establish a definitive dose response relationship in either model.

M.R. Sheller ei crl. I Journal of Orihopaedic Research 22 (2004) 1094-1099

These results complement our previous work conducted on the effect of TP508 as an injectable stimulator of fracture repair [23]. These data suggest that TP508, formulated in a controlled release vehicle, may be useful in clinical repair of segmental defects and other orthopaedic indications. Acknowledgements

The authors acknowledge NIH SBIR 2R44AR45508 to R.C. for partial support of this work. The following authors wish to acknowledge that through their employment or consulting with OrthoLogic Corp or Chrysalis BioTechnology they have a disclosable financial interest in this project: M.S., R.C., S.D., D.C., and J.R. The authors also acknowledge Larry Muenz and Associates for the helpful discussion and statistical analysis involving GEES. TP508 (Chrysalina) is an Investigational Drug, not approved for human use that was developed at the University of Texas Medical Branch and Chrysalis BioTechnology and licensed for human orthopaedic applications by OrthoLogic Corp. References Bauer TW, Muschler GF. Bone graft materials. Clin Orthop 2000;37 1:10-27. Beck LS, Wong RL, DeGuzman L, et al. Combination of bone marrow and TGF-beta1 augment the healing of critical-sized bone defects. J Pharm Sci 1998;87(11):1379-86. Bostrom M, Lane JM, Tomin E, et al. Use of bone morphogenetic protein-2 in the rabbit ulnar nonunion model. Clin Orthop 1996;327:272-82. Carney DH, Redin W, McCroskey L. Role of high-affinity thrombin receptors in postclotting cellular effects of thrombin. Semin Thromb Hemost 1992;18(1):91-103. Cook SD, Baffes GC, Wolfe MW, et al. Recombinant human osteogenic protein-7 induces healing in a canine long-bone segmental defect model. Clin Orthop 1994;301:302--12. Cook SD, Baffes GC, Wolfe MW, et al. The effect of recombinant human osteogenic protein-I on healing of large segmental bone defects. J Bone Joint Surg 1994;76-A(6):827-38. Cook SD, Salkeld SL, Brinker MR, et al. Use of osteoinductive biomaterial (rhOP-I) in healing large segmental bone defects. J Orthop Trauma 1998;12(6):407-12. Delloye C, Verhelpen M, D’Hemricourt J, et al. Morphometric and physical investigations of segmental cortical bone autografts and allografts in canine ulnar defects. Clin Orthop 1990;282:27392. Einhorn TA. The cell and molecular biology of fracture healing. Clin Orthop 1998;355S:S7-S21. Gogolewski S, Pineda L, Busing CM. Bone regeneration in segmental defects with resorbable polymeric membranes. Biomaterials 2000;21:2513-20. Grundel RE, Chapman MW, Yee T, Moore DC. Autogeneic bone marrow and porous biphasic calcium phosphate ceramic for

1099

segmental bone defects in the canine ulna. Clin Orthop 1991;266:24458. Hedberg EL, Tang A, Crowther RS, et al. Controlled release of an osteogenic peptide from injectable biodegradable polymeric composites. J Control Re1 2002;84:137-50. Hopp SG, Dahners LE, Gilbert JA. A study of the mechanical strength of long bone defects treated with various bone autograft substitutes. J Orthop Res 1989;7:579-84. Itoh T, Mochizuki M, Nishimura R, et al. Repair of ulnar segmental defect by recombinant human bone morphogenetic protein-2 in dogs. J Vet Med Sci 1998;60(4):451-8. Kato T, Kawaguchi H, Hanada K, et al. Single local injection of recombinant fibroblast growth factor-2 stimulates healing of segmental bone defects in rabbits. J Orthop Res 1998;16:6549. Kinney JH, Nichols MC. X-ray tomographic microscopy (XTM) using synchrotron radiation. Annu Rev Mater Sci 1992;22:121-52. Kinney JH, Haupt DL, Balooch M, et al. Three-dimensional morphometry of the L6 vertebra in the ovariectomized rat model of osteoporosis: biomechanical implications. J Bone Miner Res 2000;15( 10):1981-91, Lane JM. BMPs: Why are they not in everyday use? J Bone Joint Surg 200 l;83-A, S-1(2): 161-2. Mackenzie DJ, Sipe R, Buck D, et al. Recombinant human acidic fibroblast growth factor and fibrin carrier regenerates bone. Plast Reconstr Surg 2001;107(4):989-96. Meinig RP, Buesing CM, Helm J, Gogolewski S. Regeneration of diaphyseal bone defects using resorbable poly(L/DL-lactide) and poly(~-lactide)membranes in the Yucatan pig model. J Orthop Trauma 1997;11(8):551-8. Murakami N, Saito N, Horiuchi H, et al. Repair of segmental defects in rabbit humeri with titanium fiber mesh cylinders containing recombinant human bone morphogenetic protein-2 (rhBMP-2) and a synthetic polymer. J Biomed Mater Res 2002; 62(2):169-74. Ongpipattanakul B, Nguyen T, Zioncheck T F , et al. Development of tricalcium phosphatelamylopectin paste combined with recombinant human transforming growth factor beta 1 as a bone defect filler. J Biomed Mater Res 1997;36:295-305. Ryaby JT, Carney DH, Campbell M, et al. Acceleration of fresh fracture healing with an injectable thrombin peptide in a rat model. Trans ORS 200025:877. Simmons DJ, Yang J, Yang S, et al. Acceleration of rat femoral fracture healing by a synthetic thrombin peptide. In: Dacke C, Danks J, et al., editors. Calcium metabolism: comparative endocrinology Proc Satellite Meeting, San Francisco CA, Nov 30, 1998. Bradley Stoke, Bristol, UK: BioScientifica; 1999. Stevenson S. Enhancement of fracture healing with autogenous and allogeneic bone grafts. Clin Orthop 1998;3553:823946. [26] Teixeira JOC, Urist MR. Bone morphogenetic protein induced repair of compartmentalized segmental diaphyseal defects. Arch Orthop Trauma Surg 1998;117:27-34. [27] Wang H, Convery J, Ryaby JT. TP508, activates genes that assist fracture healing. Paper No: 0234, 48th Annual Meeting of the Orthopaedic Research Society, Dallas, Texas, February 1&13, 2002. [28] Wheeler DL, Stokes KE, Park HM, Hotlinger JO. Evaluation of particular Bioglass@in a rabbit radius ostectomy model. J Biomed Mater Res 1997;35:249-54. [29] Wilner GD, Danitz MP, Mudd MS, et al. Selective immobilization of alpha-thrombin by surface-bound fibrin. J Lab Clin Med 1981;97:403-11. [30] Zegzula HD, Buck DC, Brekke J, et al. Bone formation with use of rhBMP-2. J Bone Joint Surg 1997;79-A(l2):1778-90.