Osseous regeneration in preclinical models using bioabsorbable delivery technology for recombinant...

E L S E V I E R Journal of Controlled Release 36 (1995) 183-195

journal of control led

release

Osseous regeneration in preclinical models using bioabsorbable delivery technology for recombinant human bone morphogenetic

protein 2 (rhBMP-2)

Jennifer L. Smith a,., Lisa Jin a, Thomas Parsons a, Thomas Turek a, Eyal Ron l,a, C. Michael Philbrook l'a, Richard A. Kenley 2 a, Leslie Marden b, Jeffrey Hollinger 3,b,

Mathias P.G. Bostrom c, E. Tomin 4,c, J.M. Lane 4'c a Genetics Institute, 1 Burtt Rd., Andover, MA 01810, USA

b US Army Institute of Dental Research, Walter Reed Army Medical Center, Washington, DC, USA ~ The Hospital for Special Surgery, New York, NY, USA

Received 9 May 1994; accepted 6 February 1995

Abstract

Novel delivery systems for the bone morphogenetic protein rhBMP-2, consisting of poly(D,L-lactide-co-glycolide) porous microspheres as bioabsorbable filling material with either autologous blood clot or carboxymethylcellulose as binding agent, were tested in several preclinical models including a rat calvarial defect model, a rabbit radius segmental defect model, and a rabbit ulna segmental defect model. In the rat calvarial defect model, these novel delivery systems were shown to be comparable to inactivated collagenous bone matrix as a matrix for rhBMP-2 delivery at the appropriate dose. The incidence of union in the rabbit long bone studies exhibited a rhBMP-2 dose-response, achieving an incidence of union of nearly 100% at the higher doses tested. Histological evaluation showed remodelling of newly generated bone, and biomechanical testing found that the healed limbs were as strong as untreated control limbs. These studies demonstrate that it is possible to obtain osseous regeneration of critical-size defects by combining rhBMP-2 with synthetic delivery systems.

Keywords: Bone regeneration; Morphogenetic factor; Growth factor; Orthopedics; Bioabsorbable polymer

1. Introduction

* Corresponding author. 4 Present address: UCLA Medical Center, 10833 Le Conte Ave-

nue, Los Angeles, CA 90024, USA. 3 Present address: Oregon Health Sciences University, Portland,

OR, USA. z Present address: Amylin Pharmaceuticals, Inc., 9373 Towne

Centre Drive, San Diego, CA 92121, USA. l Present address: Focal Interventional Therapies, Inc., 1 Kendall

Square, Bldg 600, Cambridge, MA 02139, USA.

0168-3659/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved S S D I 0 1 6 8 - 3 6 5 9 ( 9 5 ) 0 0 0 4 6 - 1

Bone grafts are pe r fo rmed more than 250 000 t imes annual ly in the Uni ted States by or thopedic , plastic,

and oral surgeons [ 1 ] to treat serious skeletal defects

result ing f rom trauma, birth defects, onco log ic resec-

tions, and disease. Current t reatment predominate ly

uses autogenous bone graft (autograf t ) that is har-

ves ted f rom a donor site such as the il iac crest, Al though highly effect ive, autograft has disadvantages such as

inc idence o f donor site pain, morbidi ty , infect ion and

l imi ted supply o f graft material. A l logene ic (human

cadaver ) bone graft is the most c o m m o n al ternat ive to

184 ZL. Smith et al. /Journal of Controlled Release 36 (1995) 183-195

autograft, but is less effective and bears the risk of immunologic rejection and disease transmission. A thorough review of bone grafts and substitutes is given by Kenley et al. [2].

Bone defects are healed through the processes of osteoinduction and osteoconduction. Osteoinductive materials actively stimulate the formation of new bone, while osteoconductive materials passively provide a matrix upon which new bone may be deposited. While autograft is both osteoinductive and osteoconductive, most products currently approved for use in the United States as bone graft substitutes are only osteoconductive. There is a great medical need for products which replace both the osteoinductive and osteoconductive properties of bone graft.

Urist [3] demonstrated in 1965 that demineralized bone matrix (DBM; bone extracted with organic sol- vent and hydrochloric acid to remove fats and inorgan- ics) generates osseous tissue when implanted in subcutaneous sites in rats. Later studies [4,5] showed that extracting the DBM with guanidinium hydrochlo- ride produced inactivated collagenous bone matrix (ICBM). Reconstituting the ICBM with the aqueous extract restored the osteoinductive activity, thus dem- onstrating the existence of an extractable, autoinductive bone-forming activity which he termed 'bone morphogenetic protein' (BMP).

More recently, Wang, Wozney, and co-workers have isolated, identified, cloned, and expressed at least seven distinct human BMPs [6-9]. The BMPs are present in bone matrix in trace amounts and probably operate in a complex synergy. It is known, however, that at least one of the individual proteins, rhBMP-2, in the absence of other BMPs, induces bone formation when implanted at extraskeletal sites using ICBM as a cartier [ 10]. In combination with allogeneic ICBM, rhBMP- 2 has been shown to effectively regenerate bone in a variety of animal species and anatomical sites including canine mandible [ 11 ], sheep femur [ 12], rat femur [ 13 ], and rat calvarial [ 14] models. Despite the effect- iveness of ICBM as a substrate in rhBMP-2-induced bone formation, its potential as a commercial product is low due to limited availability, potential immuno- genicity, sterility assurance, and risk of disease tras- mission.

rhBMP-2 combined with an appropriate delivery system has great potential to fill the need for an osteoinductive bone graft substitute. To meet this need, we

have investigated novel bioabsorbable delivery systems for rhBMP-2. These novel delivery systems com- bine poly(D,L-lactide-co-glycolide) porous microspheres (PLGA) with a binding agent to produce a semi-solid paste-like implant material. Kenley et al. reported that such delivery systems using autologous blood clot, hydroxypropyl methylcellulose, or sodium alginate as binding agents successfully regenerated bone in rat calvarial defects [ 15]. The PLGA/blood delivery system has also been shown to regenerate bone in a rat femoral defect [ 16].

In this report we focus on delivery systems for rhBMP-2 consisting of PLGA microparticles with either autologous blood or carboxymethylcellulose (CMC) as binding agent. A series of four preclinical studies was performed to investigate the osteogenic capacity of these delivery systems in combination with rhBMP-2. First, the efficacy of these two formulations in combination with rhBMP-2 was evaluated in the rat calvarial defect model. The defects were treated with either ICBM, PLGA/Blood, or PLGA/CMC implants containing either 0, 10, or 30/~g rhBMP-2 per 0.1 ml of implant.

Second, the PLGA/CMC (0 and 10/zg rhBMP-2 per 0.1 ml implant) and PLGA/Blood ( 10/zg rhBMP- 2/0.1 ml) delivery systems were implanted in a rabbit radius segmental defect model, which provides a larger, weight-bearing orthopedic defect site. Third, the osteogenic response to increasing doses of rhBMP-2 (0, 5, 10, 20, and 40/~g rhBMP-2 per 0.1 ml implant) in the PLGA/blood delivery system was investigated radiographically and histologically in a critical-size rabbit radius segmental defect model. Finally, biomechanical testing was performed in a similar study (PLGA/Blood with 0, 5, 10, 20, or 40/zg rhBMP-2 per 0.1 ml implant) using a critical-size rabbit ulna segmental defect model.

2. Materials and methods

2.1. Materials

rhBMP-2 was produced at Genetics Institute using a Chinese hamster ovary (CHO) cell expression system. Purified ( > 9 8 % ) protein was formulated in either arginine buffer [ 17 ] or glutamate buffer and lyophili- zed. The rhBMP-2 was reconstituted with sterile water for injection just prior to use.

J.L. Smith et aL /Journal of Controlled Release 36 (1995) 183-195 185

Table 1 Study designs and compositions of the rhBMP-2 delivery systems tested

Study design Individual implant composition

Model and defect Implant type rhBMP-2 dose PLGA or ICBM Binding agent rhBMP-2 volume rhBMP-2 concentration volume

(/a,g/0.1 ml) (mg) (ml) (mg/ml)

Rat calvaria 0.1 ml ICBM 0, 10, 30 20 - 0.04 0, 0.25, 0.75 PLGA/blood 0, 10, 30 20 0.06 ml blood 0.015 0, 0.67, 2.0 PLGA/CMC 0, 10, 30 20 2 mg CMC 0.04 0, 0.25, 0.75

Rabbit radius 10.25 PLGA/CMC 0, 10 50 1.7 mg CMC in 0.063 ml 0.013 0 a, 2 a ml 60:40 glycerol/water

PLGA/blood 10 50 0,163 ml blood 0.013 2 a Rabbit radius II 0.3 PLGA/blood 0, 5, 10, 20, 40 60 0.135 ml blood 0.015 0, 1, 2, 4, 8 a ml Rabbit ulna 0.4 ml PLGA/blood 0, 5, 10, 20, 40 80 0.22 ml blood 0.02 0, 1, 2, 4, 8 a

~rhBMP-2 in glutamate buffer

Poly (D,L-lactide-co-glycolide) microparticles (d = 150-500/xm) were prepared from a 50:50 copo- lymer having a molecular weight of 30 000-50 000 by a phase-separation technique, as described elsewhere [ 18,19]. The particles were dried in vacuo, transferred into glass vials, and terminally sterilized by ~/irradia- tion at 25 kGy (Isomedix, Morton Grove, IL) .

Rat ICBM was prepared from long bones of Long- Evans rats as previously described [ 14]. The prepared material was freeze-dried, and stored under vacuum at ambient temperature until use. Carboxymethylcel lu- lose sodium was Aqualon grade 7LFPH. Glycerol was Dow Optim grade.

2.2. Test systems

The rat calvarial defect model [15] Long-Evans rats (28-35 days old, both sexes) were

used ( 12 animals per test group) . Allogeneic blood for implant preparation was obtained from donor rats via cardiac puncture. An 8-mm craniotomy defect was cre- ated using a trephine in a dental handpiece. The calvarial disk was dissected free, avoiding dural perforations and superior sagittal sinus intrusion. The implant material was placed into the defect and soft tissues were closed with skin staples.

Rats were sacrificed by CO2 asphyxiation 21-days post-implantation. Craniotomy sites with 3 -4 mm con- tiguous bone were recovered from the skull. Specimens were radiographed using X - O M A T TL high-contrast

X-ray film (Eastman) in a Minishot benchtop cabinet (TFI Corp.) at 25 kVp, 3Ma, for 5 s. Each roentgen- ogram was assessed for radiopacity within a standard 8-mm diameter circle superimposed over the defect site. A Model 520 Image Analysis system (Leica, Inc.) measured the area of radiopacity within the standard circle.

For histologic determinations coronal sections (4.5 /xm) were cut and stained with Goldner-Masson trichrome stain (for photomicrography and examination of cell and stromal detail) and von Kossa stain. Spec- imens stained with von Kossa were quantitatively assessed for calcified tissue using a standard magnifi- cation and the Leica Image Analysis system interfaced with an Axiophot microscope (Zeiss, Inc.) . The area (mm 2) of calcified tissue within the defect sites was recorded.

The rabbit radius defect model I

New Zealand White Rabbits, male, minimum age 6 months were used (8 animals per test group). A blood sample ( 1 ml) was withdrawn via a marginal ear vein for implant preparation for animals in the P L G A / B l o o d test group. An ostectomy was performed with an oscillating saw, removing approximately 15 mm of the radius shaft, and leaving the periosteum in place. The implant was placed in the defect site, and the soft tissues and skin were closed with sutures.

Radiographs were taken immediately following surgery, and at 2, 4, 6, and 8 weeks post-implantation.

ICBM

rhBMP-2 0 p,g 10 pg 30 ~g

C

PLGA/ Blood

PLGA/ CMC

H

A

~!!ii!iiii!!iiii~:ii: q! I~ ~i:ii~!!iiiiii!!!i '~

D ? ~{! ¸¸¸:ill¸¸¸¸

?!

]

i ,!~i;/~ B

186 J.L. Smith et al. / Journal of Controlled Release 36 (1995) 183-195

Fig. 1. Radiographs of rat calvarial defects treated with: ICBM ( A = 0, B = 10, C = 3 0 / z g rhBMP-2 per 0.1 ml implant); PLGA/b lood ( D = 0, E = 10, F = 30 p.g rhBMP-2); P L G A / C M C (G = 0, H = 10, I = 3 0 / x g rhBMP-2) at 21 days post-implantation.

J.L. Smith et al. / Journal of Controlled Release 36 (1995) 183-195 187

100-

go.

>- 80" I -

< 70-

< n- I-.-- 50. z ku o 40.

30-

20-

10.

0- ICBM PLGNCMC PLGNBIood

[ [ ] o ~,g • ~o ~,g [ ] 3o.g ]

Fig. 2. Rad iomorphomet r i c data (percent radiopaci ty , expressed as

mean + SD for n = 12) for rat calvarial defects treated with ICBM,

P L G A / b l o o d , and P L G A / C M C with 0, 10, or 30 Ixg rhBMP-2 per

0.1 ml implant at 21 days post- implantat ion. *, s ignif icantly different

f rom other t reatment groups at the same rhBMP-2 dose (p < 0 .05) .

Radiographs were assessed for radiopacity using the system described above.

The rabbit ulna defect model New Zealand White Rabbits, mixed gender, mini-

mum age 6 months, received bilateral implants (10 animals per test group; n = 20 limbs). A blood sample (2 ml) was withdrawn via a marginal ear vein for implant preparation. An ostectomy was performed using a high speed burr, excising approximately 20 mm of the ulna shaft with its periosteum intact. The implant was placed in the defect site, and the soft tissue and skin closed with sutures.

Tlae animals were sacrificed at 8 weeks post-implantation. Radiographs were taken and presence/absence of union was evaluated. The limbs that went to union underwent biomechanical testing on a Burstein torsion tester equipped with a 200 in-oz Leblow torque cell. Specimens were failed in external rotation while acquiring torque and angular displacement at 5 kHz with a Tektronics Model 2230 Digital oscilloscope. Data were analyzed to obtain the stiffness and energy to failure.

2.3. Implant compositions

The rabbit radius defect model H New Zealand White Rabbits, male, minimum age 6

months were used ( 10 animals per test group). A blood sample ( 1.5 ml) was withdrawn via a marginal ear vein for implant preparation. An ostectomy was performed with an oscillating saw, removing approximately 20 mm of the radius shaft and leaving the periosteum in place. The implant was placed in the defect site and the soft tissues and skin were closed with sutures.

Radiographs were taken immediately following surgery, and every two weeks until sacrifice. Five animals in each test group were euthanized at 8 weeks post- implantation, and the remainder of the animals were euthanized at 12 weeks post-implantation. For each ostectomy site, the implant was excised including 0.5 cm of bone on either side. The samples were decalci- fied, embedded in paraffin, sectioned longitudinally, and stained with hematoxylin and eosin.

The radiographs and the histological samples were evaluated for percentage of the defect occupied by bone according to the following scoring scale: (0) not observed, (1) 10-20%, (2) 2040%, (3) 40-60%, (4) 60-80%, (5) 80-100%. The presence or absence of osseous bridging of the defect was also evaluated radiographically.

All rhBMP-2 doses are expressed on a unit volume basis to facilitate comparison between various implant and defect volumes. Individual implant compositions for the four preclinical models are given in Table 1. The PLGA implants were prepared by mixing the microparticles with the binding agent and rhBMP-2. The ICBM implants were prepared by combining the matrix with rhBMP-2 and lyophilizing, rhBMP-2 was in arginine buffer except when noted.

3. Results

Rat calvarial defect model Radiographs taken at 21 days post-implantation are

given in Fig. 1 for ICBM (A-C), PLGA/Blood (D- F), and PLGA/CMC (G-I) delivery systems. For each matrix, representative specimens at 0, 10, and 30/xg rhBMP-2 per 0.1 ml implant are shown. For each treatment, the 0/zg specimen shows minimal radiopacity, which is consistent with previous observations that the 8-mm diameter defect is critical-size in that bone does not spontaneously regenerate. At both the 10 and 30 p~g rhBMP-2 doses, all treatment groups show significant radiopacity.


ICBM PLGA/Blood

Fig. 3. Photomicrographs ( 1.6 X ) of rat calvarial defects treated with ICBM and 0/xg (A) or 10 ~g (B) rhBMP-2, or PLGA/blood and 0/,Lg (C) or 30 p,g (D) rhBMP-2 per 0.1 ml implant. (A and B von Kossa stain, C and D Goldner trichrome stain).

Fig. 2 gives the percent radiopacity for each treatment group as determined by radiomorphometry. Three-factor analysis of variance revealed that both the rhBMP-2 dose and matrix type significantly affected radiopacity. Comparing individual treatment means using Fisher's protected least-significant-difference (PLSD) test showed that defects treated with ICBM/ 0/.Lg rhBMP-2 and ICBM/10/xg rhBMP-2 generated more radiopacity than the PLGA formulations at the same rhBMP-2 doses. There were no differences between treatment groups at the 30/zg rhBMP-2 dose.

Representative histological specimens are shown in Fig. 3. Fig. 3A and 3B show ICBM with 0 and 10/xg rhBMP-2 per 0.1 ml implant doses. Minimal new bone formation is observed for the 0/xg group and residual particles of ICBM are observed. For the 10/xg treatment group, bone bridging has generally occurred across the defect, new marrow is observed and few remnants of ICBM are present. Fig. 3C and 3D show

2

z

1

ICBM P L G N C M C PLGA/Blood

Fig. 4. Histomorphometric data (area of new bone, expressed as mean + SD for n = 12) for rat calvarial defects treated with ICBM, PLGA/blood, and PLGA/CMC with 0, 10, or 30/xg rhBMP-2 per 0.1 ml implant at 21-d post-implantation. *, significantly different from other treatment groups at the same rhBMP-2 dose (p < 0.05).

J.L. Smith et al. / Journal o f Controlled Release 36 (1995) 183-195

PLGA/CMC 0Fg PLGA/CMC 10Fg PLGA/BlooO 10Fg

189

Fig. 5. Radiographs of rabbit radius (I) defects treated with PLGA/CMC 0.1 ml implant taken at 4 and 8 weeks post-implantation.

representative PLGA/Blood specimens from the 0 and 30/xg rhBMP-2 per 0.1 ml implant groups. For the 0 /xg group, numerous PLGA microparticles are observed within scanty, wispy connective tissue stroma. No new bone or cartilage is observed. For the 30/zg dose, regeneration of the inner and outer tables

100 c

90 ~

80-

7 0

40-

30 ~

20-

10 c

[ ] Week 2

F =1 Week 4

[ ] Week 6

[ ] Week 8

PLGA/CMC 0 p.g PLGNCMC 10 p,g PLGA/BIood 10 p,g

Fig. 6. Radiomorphometry data (percent radiopacity, expressed as m e a n + S D for n = 8 ) for rabbit radius (I) defects treated with PLGA/CMC and 0 or 10/xg rhBMP-2 or PLGA/blood and 10/xg rhBMP-2 per 0.1 ml implant. The fraction of animals showing radiographic evidence of union at 8 weeks is given on the 8-week bar. *, significantly different from other test groups at the same time point (p < 0.05).

and 0 or 10/xg rhBMP-2 or PLGA/blood and 10/xg rhBMP-2 per

is observed. PLGA microparticle ghosts are present, populated with multinucleated giant cells which have been shown to be involved in the bioabsorption of PLGA.

Fig. 4 shows the area of new bone formation for each treatment group as quantitated by histomorphom- etry. These data agree with the qualitative observations that little or no new bone is observed at the 0 /zg rhBMP-2 dose, and that significant bone is regenerated at both the 10 and 30/xg doses. Three-factor analysis of variance revealed that both rhBMP-2 dose and matrix type significantly affected new bone generation. Fisher' s PLSD showed that defects treated with ICBM / 10 ~g rhBMP-2 had significantly more bone regeneration than other treatment types at the same dose; there were no differences between treatment groups at either 0 or 30/zg rhBMP-2.

The rabbit radius defect model I Radiographs taken at 4 and 8 weeks post-implanta-

tion (Fig. 5) show the healing of representative implants consisting of PLGA/CMC with 0 or 10/xg rhBMP-2, and PLGA/Blood with 10/xg rhBMP-2. The 0/xg specimen exhibits some bony ingrowth from the ends of the defect, but no bridging of the defect. Both the PLGA/Blood and PLGA/CMC delivery systems with 10 ~g rhBMP-2 per 0.1 ml implant show radi-


A. OFg

2

7

4

B. lOFg

0 S ;

5

~I ~:̧ ̧i! il ! !i

12

~2

Fig. 7. Radiographs of rabbit radius (II) defects treated with PLGA/blood and 0 (A) or 10 ~g (B) rhBMP-2 per 0.1 ml implant taken at 0, 2, 4, 6, 8, and 12 weeks post-implantation.

opacity appearing by 2 weeks, the defect filled in with radiopacity at 4 weeks, and evidence of remodell ing at 8 weeks post-implantation.

The percent radiopacity as determined by radiomorphometry is given in Fig. 6 for each treatment group. The fraction of limbs evaluated to have formed union at 8 weeks is shown on the 8-week bars. Analysis of variance and Duncan 's Multiple Range Test show that

at all time points, the 0 /~g rhBMP-2 group was less radiopaque than both the treatments containing rhBMP-2. At the 2-week time point, the P L G A / B l o o d group was significantly more radiopaque than the P L G A / C M C group, but the treatments were not different at the later time points. All of the rhBMP-2 treated animals formed osseous union as determined

J.L. Smith et al. /Journal of Controlled Release 36 (1995) 183-195 191

radiographically, while 5/8 of the animals without rhBMP-2 formed union.

The rabbit radius defect model H Fig. 7 shows representative radiographic healing

sequences from 0-12 weeks post-implantation for PLGA/Blood implants with 0 (A) and 10 /xg (B) rhBMP-2 per 0.1 ml implant. The defect treated with 0 ~g rhBMP-2 shows sparse, discontinuous radiopacity at all time points. Radiopacity is evident in the defects treated with 10 and 40/xg rhBMP-2 at 2 weeks, with the entire defect radiopaque at 6 weeks.

Fig. 8 shows the mean percent radiopacity for all treatment groups as determined by the scoring scale described in the Methods section. All doses of rhBMP- 2 seem to be nearly equivalent at the 2-week time point, but at 4, 6, and 8 weeks post-implantation, there appears to be a dose-response, with the most radiopacity occur- ring in the groups with 20 and 40/xg rhBMP-2. Linear regression analysis showed that there is a significant dose-response trend (p < 0.001 ), although analysis of variance and Duncan's MRT showed that, in general, there were only significant differences between the lowest (0 and 5/xg) and the highest (20 and 40/xg) rhBMP-2 doses. Fig. 9 shows the same radiographic data, evaluated only for presence/absence of union. Few limbs treated with 0/xg rhBMP-2 formed union, while slightly more than half of limbs treated with 5 or 10 /~g rhBMP-2 formed union, and all limbs treated with 20 or 40/xg rhBMP-2 formed union by 8 weeks post-implantation.

Representative histological specimens are shown in Fig. 10. Fig. 10A is from a specimen treated with PLGA/Blood and 0 /zg rhBMP-2 per 0.1 ml implant at 12 weeks post-implantation. The ends of the radius have not bridged, although one end of the radius has attached to the ulna. Fig. 10B and 10C show limbs treated with PLGA/Blood and l0 or 40/xg rhBMP-2 per 0.1 ml implant at 8 weeks post-implantation, respectively. The ends of the radius have bridged in the l0/xg implant, and they have fused with the underlying ulna. There is also an indentation over part of the defect site. The 40/zg specimen shows the ends of the radius have bridged and the bone is remodelling to form the marrow space and separate the radius and ulna.

Fig. 11 shows the percentage of the defect occupied by new bone for all treatment groups, as evaluated from the histologic specimens. Similar to the radiographic

80-100%

60-80%

0-

a 40-60% < cc

o 20-40% a.

10-20%

Not detected , = 0 5 10 20

rhBMP-2 Dose (p.g/0.1 mL implant) 40

[ ImlWeak2 []Week4 , ,wee,6 []wee,6

Fig. 8. Radiographic results (percent radiopacity, expressed as mean +_ SD for n = 10) for rabbit radius (II) defects treated with PLGA/blood and 0, 5, 10, 20, or 40 p,g rhBMP-2 per 0.1 ml implant.

scoring, there appears to be an increasing percentage of new bone with increasing rhBMP-2 dose which linear regression analysis confirms is significant (p < 0.01 ) at both 8 and 12 weeks. Analysis of variance and Duncan's MRT showed only a significant difference between the 0/xg and all other doses at the 8-week time point.

The rabbit ulna defect model Fig. 12 shows the percentage of limbs achieving

union for each treatment group at 8 weeks post-implantation. The results appear similar to the previous ,'abbit

0 5 10 40 20

100 ,

• Week 2 n ! [ ] week 4

• week 6

[ ] Week 8

e _

t

rhBMP-2 Dose (p,g/0.1 mL implant)

Fig. 9. Percentage of limbs achieving union as evaluated from radiographs (n = 10) for rabbit radius (II) defects treated with PLGA/ blood and 0, 5, 10, 20, or 40/xg rhBMP-2 per 0.1 ml implant.


B

C Fig. 10. Photomicrographs (3.5 x , hematoxylin and eosin stain) of rabbit radius (II) defects treated with PLGA/blood and 0/xg rhBMP-2 at 12 weeks post-implantation (A), 10/~g ( B ) or 40/xg (C) rhBMP-2 per 0.1 ml implant at 8 weeks post-implantation. The arrows depict the cut ends of the radius. R = radius, U = ulna.

radius model , where the percent union was low for the

0 / x g rhBMP-2 group, modera te for the 5 and 1 0 / z g

groups, and nearly comple te for the 20 and 40 /xg groups. Fig. 13 gives the results o f b iomechan ica l test-

ing for those l imbs which went to union in each group

and for unoperated control l imbs. The stiffness o f

rhBMP-2- induced healed l imbs was significantly

greater than that o f unoperated control l imbs. The 10 /xg/0.1 ml rhBMP-2- t rea ted group had a mean energy

to failure lower than the control; the other groups were

all equiva len t to the control. For both measures o f bio-

mechanica l strength, no dose response was observed.

J.L. Smith et al . / Journal of Controlled Release 36 (1995) 183-195 193

80-100%- • Week8

[ ] week

;~ 60-80%

40-60%

20"40°1"

10*20%

NOt detected 0 5 10 20 40

rhBMP-2 Dose (itg/0.1 mL implant) Fig. 11. Histologic results (percentage of defect occupied by new bone, expressed as mean + SD, n = 5) for rabbit radius (II) defects treated with PLGA/blood and 0, 5, 10, 20, or 40 ixg rhBMP-2 per 0.1 ml implant at 8 and 12 weeks post-implantation.

100

70~ z

,,z, ~ - o ~ 40- Q_

30-

20-

10-

0- 0 5 10 20 40 rhBMP-2 Dose (p.g/0.1 mL implant)

Fig. 12. Radiographic results (percentage of limbs achieving union, n = 20) for rabbit ulna defects treated with PLGA/blood and 0, 5, 10, 20, or 40 /xg rhBMP-2 per 0.1 ml implant at 8 weeks post- implantation.

4. Discussion

The rat calvarial defect model demonstrated that with an appropriate dose of rhBMP-2, the PLGA-based delivery systems can regenerate bone as effectively as inactivated collagenous bone matrix. This is significant because ICBM has been the matrix most commonly used with bone morphogenetic proteins in research, probably because of Urist 's initial experiments with BMP extracted from ICBM. However, as previously discussed, ICBM is not an attractive candidate for a commercial delivery system for rhBMP-2. The PLGA

"0,16 !o.14 ~

i1 ~ 1 ~ 1 ~ 7o.12 5, ' - o l 3

c7 Control 5 16 20 40 rhBMP-2 Dose (p.g/6.1 mL implant)

Fig. 13. Biomechanical results (stiffness and energy to failure expressed as mean + SD, only limbs achieving union were tested) for rabbit ulna defects treated with PLGA/blood and 0, 5, 10, 20, or 40/xg rhBMP-2 per 0.1 ml implant at 8 weeks post-implantation. *, significantly different from control (p < 0.05).

microparticulate systems are potentially attractive delivery systems for rhBMP-2 because the lactide and glycolide polymers are bioabsorbable and have a long record of safe use in medical devices.

At the lower rhBMP-2 dose, superior bone regeneration was observed with ICBM than with PLGA as the delivery matrix. This may be due to a better release profile of rhBMP-2 from the ICBM, a composition and geometry that facilitate cellular infiltration and proliferation, or a combination of both factors. The optimal release profile of rhBMP-2 from an osteoinductive system is not known. It is surely different in different animal species; higher order species generate bone much more slowly than lower order species. The optimal release profile also likely varies with the age of the subject and with the anatomical site of the defect. Bone healing is affected by many factors including vascular- ity and the degree of motion present. For the PLGA- based delivery systems described here, the rhBMP-2 release rate is probably different in the different models, but in all cases is most likely more rapid than the bio- resorption of the PLGA matrix.

In both the rat calvarial defect model and the first rabbit radius defect model, the PLGA/Blood and PLGA/CMC delivery systems performed equiva- lently. Autologous blood as a binding agent has the advantage of being always readily available and requir- ing no manufacturing effort. Carboxymethylcellulose, a semi-synthetic carbohydrate, is widely used in the pharmaceutical industry as a tablet binder, suspending

194 J.L. Smith et aL / Journal of Controlled Release 36 (1995) 183-195

and /o r viscosity-increasing agent and coating agent. Although the union rate of rhBMP-2-treated animals

was greater than that of the control animals, 5 /8 of the animals receiving no rhBMP-2 went to union. This suggests that the 15-mm defect is not critical-size, and a larger defect was used in the subsequent models. While the rabbit radius II and rabbit ulna models both used 20-mm defects, the union rates at 8 weeks post- implantation are lower at every rhBMP-2 dose in the ulna model as compared to the radius model: 0 vs. 30% at 0 /xg rhBMP-2; 40-50% vs. 75% at 5 or 10 /xg rhBMP-2; 80% vs. 100% at 20 or 40 /xg rhBMP-2) . This is most likely due not to the defect site, but rather to the periosteum being left in place in the radius model, and being removed with the bone segment in the ulna model.

In the rabbit radius defect model II the radiopacity, percent union, and histologic data all display an apparent rhBMP-2 dose-response. Significant differences could not be determined statistically due to the small dose increments, small number of animals (n = 10 for radiography, n = 5 for histology at each time point) , and large variabili ty as shown by the standard devia- tions. While significant new bone is observed both radiographically and histologically for implants receiving 5 or 10/zg rhBMP-2, complete healing is much more predictable when 20 or 40 /xg rhBMP-2 per 0.1 ml implant is administered.

Qualitatively, healthy normal bone is observed histologically, with remodell ing apparent between the 8 and 12 week timepoints. At 12 weeks post-implantation, the normal cortex and marrow-containing medulla are in the process of forming. In the defects treated with zero or lower doses of rhBMP-2, there is proliferation of bone emanating from the ulna, rather than the cut ends of the radius or throughout the defect. This is most l ikely in response to increased stress on the ulna as the sole supporting member. There is little or no evidence remaining of the PLGA microspheres at either 8 or 12 weeks post-implantation; they have almost entirely been bioabsorbed.

A further test of the efficacy of these rhBMP-2 delivery systems is the biomechanical strength of healed limbs compared to unoperated control limbs. The stiffness of rhBMP-2-induced healed limbs was observed to be significantly higher than that of the control limbs. This is most l ikely due to increased diameter of the ulna from callus formation. The energy to failure was equiv-

alent to the control group for three of the four rhBMP- 2-treated groups; the 10/xg dose had a lower value. No dose-response was observed by either measure of biomechanical strength. These results show that when rhBMP-2 induces osseous bridging of defects, the quality of the healed bone is not significantly different from control limbs.

5. Conclusions

The preclinical studies described in this report demonstrate that rhBMP-2 is capable of inducing osseous regeneration of critical-size defects in combination with bioabsorbable, synthetic delivery systems. The system described, consisting of PLGA microparticles and a binding agent, is one suitable matrix for the delivery of rhBMP-2 to skeletal defects. The rate and incidence of healing of critical-size defects exhibit a rhBMP-2 dose-response. The quality of bone generated, however, is independent of rhBMP-2 dose over the range tested, rhBMP-2 combined with a synthetic, bioabsorbable delivery system has great potential to become the first osteoinductive bone graft substitute.

Acknowledgements

The authors would like to thank Christine Copeman, Shahin Enayati, and Brian Broxup at Bio-Research Laboratories, Ltd., Montreal, Canada, for their careful and efficient execution of the rabbit radius ( I I ) study. We also gratefully acknowledge John Wozney ' s critical reading of this manuscript.

References

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