Poly(α-hydroxy acid) carrier for delivering recombinant human bone morphogenetic protein-2 for bone regeneration

journal of ELSEVIER

controlled release Journal of Controlled Release 39 (1996) 287-304

Poly(c -hydroxy acid) carrier for delivering recombinant human bone morphogenetic protein-2 for bone regeneration J. Hollinger a,*, M. Mayer a, D. Buck a, H. Zegzula a, E. Ron b, j. Smith c, L. Jin c, J. W o z n e y

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a Oregon Health Sciences University 3181 SW Sam Jackson Park Rd L352A, Portland, OR 97201-3098, USA b GelMed, 213 Burlington Road, Bedfi)rd, MA 01730, USA Genetics Institute, One Burtt Rd, Andover, MA 01810, USA

Received 24 February 1995: accepted 6 November 1995

Abstract Autografting is the most common treatment to correct osseous defects, with donor sites of calvarium, iliac crest, and rib used most often [1]. While varying degrees of 'success' have been reported with this therapy, a universal concern is donor site morbidity. Allogeneic bank bone is an alternative to the autograft and while donor site morbidity is not an issue, bank bone has liabilities that make it less effective than the autograft. Therefore, an off-the-shelf product to replace these therapies would improve the quality of care for patients. Towards the development of such a product, we report on two studies that assessed a combination of recombinant human bone morphogenetic protein-2 delivered to osseous wound sites with a poly(c~-hydroxy acid) carrier: poly(lactide-co-glycolide). Results from these studies indicate bone repair can be accomplished with this combination. However, despite a rich therapeutic promise, clinical utility must be amplified. Therefore, the focus of our attention in the future will be the delivery system. Keywords: Poly(c~-hydroxy acid); Recombinant human bone morphogenetic protein-2; Bone repair

1. Introduction A symphony of biochemical mediators in bone repair and regeneration are being identified and expressed through recombinant gene technology. These achievements will culminate in clinical administration of genetically engineered molecules. For example, elderly patients who sustain bone fractures could

* Corresponding author. Division of Plastic and Reconstructive Surgery-L352A, Oregon Health Sciences University, 3181 SW Sam Jackson Park Rd, Portland, OR 97201-3098, USA.

receive recombinant human factors such as bone morphogenetic proteins to improve their ability to regenerate bone. Combined with a delivery system, therapeutic doses of recombinant bone morphogenetic protein could minimize or eliminate the need for massive bone transplants to ablative osseous wounds and developmental deficiencies. Bone morphogenetic proteins (BMPs) promote differentiation of pluripotential cells into cartilageforming and bone-forming cells [2-4] and are pivotal for bone regeneration [2,5,6]. Currently, nine recombinant human (rh) BMPs have been reported (desig-

0168-3659/96/$15.00 © 1996 Elsevier Science B.V. All rights reserved SSD1 0 1 6 8 - 3 6 5 9 ( 9 5 ) 0 0 1 8 3 - 2

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nated rhBMP-1 through rhBMP-9) [7-15]. The BMPs appear to be highly conserved polypeptides with homology across species [2-4]; therefore, applications of the human gene product have been applied successfully to preclinical studies [16-22]. Preclinical studies have validated the efficacy and safety of rhBMP-2 and 7 (also referred to as osteogenic protein-1 or OP-1) [16-23]. However, before rhBMPs are applied to human clinical situations, appropriately tailored delivery systems must be developed. Properties of the delivery systems will depend on physiologic and biofunctional regional needs of bone regeneration (reviewed by Hollinger [24,25]). Poly(c~-hydroxy acids) have a 25 year history of efficacy and safety as sutures and, therefore, are likely choices for delivery system roles. Based on previous data, we chose particulate poly(lactide-co-glycolide) (PLG), a type of poly(~hydroxy acid), combined with either autogenous blood or carboxymethyl cellulose (CMC) as carrier systems [19,21]. The rationale for the autogenous blood and CMC was they would function as a 'binder' for the particulate PLG and aid in localizing the rhBMP-2 at the wound bed. The purpose of the two studies we will report was to regenerate bone in an intraosseous wound model that left untreated would not heal spontaneously. This wound model is known as a criticalsized defect (CSD) [26,27]. The aims for the experimental studies were (1) to prepare CSDs in radii of skeletally mature New Zealand white rabbits and alveolar processes of foxhound dogs and either to treat with (2) several doses of rhBMP-2 combined with PLG and autogenous blood (or CMC), (3) PLG and autogenous blood (or CMC), (4) autograft, or (5) to leave untreated and determine bone regeneration using quantitative radiomorphometry and histomorphometry. At designated times, experimental animals were euthanized and recipient beds were retrieved and radiographed. Following radiography, tissues were prepared for quantitative histology to measure the bone regenerative responses. We tested the hypotheses that the bone regenerative effects of rhBMP-2 with PLG/autogenous blood (or CMC) would be (1) dose- and time-dependent and (2) equal to or greater than the 'gold standard' autograft at the same time point.

2. Materials and methods 2.1. Rabbit s u r g e ~

Cefazolin was administered intravenously (22 m g / k g body weight) 30 rain before surgery. Anesthesia consisted of 10 ml (100 m g / m l ) ketamine hydrochloride (Vetalar, Park Davis, Morris Plains, NJ), 1 ml xylazine (100 m g / m l ) (Rompun, Mobay Corp., Shawnee, KS), and 5 ml of 0.9% physiologic saline given intramuscularly (IM) at a dose of 0.14 m l / 1 0 0 g body weight. Animals were put in a supine position and a tourniquet was placed around the antecubital fossa to maintain hemostasis during aseptic surgery. A 4-cm incision was prepared longitudinally, superiorly-medially over the radius, and soft tissues were dissected to expose the diaphyseal segment of the radius. A 15-ram ostectomy (4 × the diaphyseal diameter of the radius), including periosteum, was accomplished with a reciprocating saw and copious irrigation of physiologic saline. After the defect was prepared, the designated experimental treatment was inserted (described vide infra) and retained at the site by surrounding soft tissues, which were closed in layers with 3-0 Dexon suture. After assuring for hemostasis, the tourniquet was removed. When the rabbits were sternal and ambulatory they were returned to their individual cages. Each animal received a veterinary analgesic for routine post-operative pain control. 2.2. Rabbit treatments

A total of 48 skeletally mature rabbits (equal numbers male and female) were divided evenly among six treatment groups and one time period. Treatments consisted of: 2.2.1. rhBMP-2

Using a Chinese hamster ovarian (CHO) cell expression system previously described [7,23], rhBMP2 was prepared in a sterile manner as a glycosylated 32 kDa homodimer. The amino acid sequence and carbohydrate sites were determined [14,28] and the highly purified rhBMP-2 ( > 98%) was placed in sterile glass vials, closed with rubber stoppers, and maintained in an 0.5 M arginine and 10 mM histidine buffer at pH 6.5.

J. Hollingeret al. / Journal of ControlledRelease39 (1996) 287-304 2.2.2. PLG The preparation of the particulate PLG (average molecular mass = 40 kDa, lactide to glycolide ratio = 1:1) has been described [29]. As determined by BET surface analysis, the high surface area (0.02-4.0 m2/g) and void volume impart a porous architecture to the particles, thereby allowing controlled, predictable release of rhBMP-2 and bioabsorption of the particles in harmony with new bone formation. 2.2.3. rhBMP-2 / PLG / CMC Known quantities of rhBMP-2 (0, 10, 40, and 80 /xg) were delivered to each recipient bed in combination with 95 mg of PLG and 125 /xl of CMC. 2.2.4. rhBMP-2 / PLG / blood l 0 / x g rhBMP-2, 1.3 ml autogenous blood, and 95 mg of PLG were combined immediately prior to surgery and inserted into the defect (Fig. 1). 2.3. Dog surgeo' 2.3.1. Maxillary cleft preparation Ketamine hydrochloride (20 m g / k g body weight) and benthazine penicillin (1.5 million units) were

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introduced IM in the caudal thigh muscle. General inhalational anesthesia was administered and each dog was monitored throughout surgery with a continuous readout electrocardiogram and esophageal thermometer. All surgical procedures were accomplished in the approved aseptic manner. Palatal and mucobuccal mucosa circumscribing and contiguous to the fight maxillary canine to the left maxillary canine were infiltrated with 3.6 ml of 2% lidocaine hydrochloride with 1:100 000 epinephrine. Full-thickness palatal and mucobuccal flaps were raised to visualize maxillary incisors, the palatine process of the maxilla, and alveolar crestal bone. The incisor teeth were extracted and nasoalveolar fistulas were prepared in the bone using a surgical drill. A surgical stent was inserted in the nasoalveolar fistula to ensure patency. Post-surgically, dogs were given 250 mg of phenoxymethyl penicillin PO twice daily for 7 days. An analgesic (buprenorphine HC1, 0.01-0.03 m g / k g , IM) was administered post-operatively as needed. Tissue circumscribing the stent was irrigated twice per week. After 4 months, and 4 weeks prior to treatment with the experimental materials or autograft, ketamine hydrochloride (20 m g / k g body weight) was administered IM in the caudal thigh and stents were removed. The fistulas were irrigated with physiologic saline and patency was verified. 2.3.2. Cleft surgery treatment Preoperative, operative, and post-operative anesthesia, antibiotics and analgesia were managed as described above for the maxillary cleft. Once the surgical site was prepared and draped, soft tissue within the bilateral maxillary clefts was incised and the bony walls of the palatine process of the maxillary complex were freshened to bleeding bone with a surgical drill using copious sterile saline. The designated treatment was administered (described below). Gingival and palatal mucosa were sutured closed in layers with 3-0 Dexon suture. 2.4. Dog treatments

Fig. 1. Bloodwas drawn from the experimental animal, combined with 10 /xg rhBMP-2 and thoroughly mixed between two Leurlock syringes. The mixture was added to the PLG, allowed to clot and inserted into the defect.

A total of 24 skeletally mature foxhound dogs (55-60 lb) were divided evenly between two time periods (2 and 4 months) and among four treatments:

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autograft, untreated cleft, and combinations of PLG/autogenous blood with or without rhBMP-2. Each dog had a different treatment randomly assigned to the fight and left maxillary alveolar cleft. There were six replicates for each experimental treatment at each time period.

2.4.1. rhBMP-2 Preparation of the rhBMP-2 was the same as described for the rabbit. A known amount of buffer containing 200 /xg of rhBMP-2 was combined with 400 mg of PLG particles and autogenous blood (2 cm 3 of venous blood were drawn from the antebrachial part of the cephalic vein into a 5 cm 3 syringe, and 1.1 cm 3 was retained for combination therapies described below). 2.4.2. PLG The preparation procedure was the same as that described for the rabbits. 2.4.3. Combinations: PLG / autogenous blood with and without rhBMP-2 Using double female luer lock connector, syringes containing 1.1 cm 3 of autogenous blood and 100 /zl rhBMP-2 in buffer (200 /xg rhBMP-2) were connected and contents between the two syringes were mixed thoroughly and retained within the 5 cm 3 syringe. The mixture was added immediately to a 120 ml specimen cup containing 400 mg of PLG particles. The combination was mixed thoroughly and allowed to sit 20 min before implantation into the recipient beds. PLG/autogenous blood without rhBMP-2 was added directly to and mixed with 400 mg of PLG particles. Venipuncture and preparation of combination treatments were timed to ensure consistency among treatments. 2.4.4. Graft Following placement in a semi-supine position, the posterior iliac crest was shaved, prepped, and draped for sterile surgery. A rectangular bony cortical flap (attached to tendon and muscle) was prepared and reflected medially and cortico-cancellous bone was obtained. The donor site was closed in layers with Dexon suture. The graft was morselized to a pat6 in a bone mill and kept moist with 0.9% physiologic saline in a gauze sponge until needed.

The skin was closed with 3-0 Dexon suture and sprayed with an antibacterial spray (Topazone, Norden, Lincoln, NE).

2.5. Wound assessments 2.5.1. Radiography For the rabbits, immediately post-operatively, every two weeks in-life, and at the time of euthanasia, experimental sites were radiographed in a standard manner using a dental X-ray (50 kVp, 10 mA, 0.2 s), constant object to film to X-ray cone distance, and ultra-high contrast mammography film (X-OMATL, Kodak, Eastman Kodak, Rochester, NY). In dogs, the experimental sites were retrieved at necropsy and placed in 70% ethanol for 24 h prior to being radiographed at a fixed object to X-ray source distance and angle, in a Minishot Benchtop Cabinet X-ray System (TFI Corp., West Haven, CT), at 25 kVp, 3 mA, and 5 s using X-OMATL film. 2.5.2. Radiomorphometry X-ray films of the experimental sites were assessed in standardized manner by gray level densities of tissues using a Cambridge 970 Image Analysis System (Leica Instruments Ltd., Cambridge, UK) as previously described [27,30]. 2.5.3. Histology / histomorphometry Following euthanasia, experimental sites were recovered en bloc and placed immediately into 70% ethanol, taken to 100% ethanol, embedded in poly(methyl-methacrylate) and histologic sections 4.5 /~m thick were prepared and stained with a modification of the Goldner-Masson trichrome stain and von Kossa stain. Using bright-field light microscopy, Goldner-Masson trichrome-stained sections were examined for cell type and morphology and stromal detail using a Zeiss Axiophot Microscope (Zeiss Instruments Co., Inc., New York, NY). New bone was assessed from the von Kossa-stained specimens using a Leica 970 Image Analysis System (Leica Instruments Ltd., Cambridge, UK) interfaced with a Zeiss Axiophot Microscope. Bony trabeculae were detected as a 'features' composed of a variable number of pixel points, with each pixel point having a finite area value. At least three consecutive serial sections for each treatment were measured in a step-

J. Hollinger et al. / Journal of Controlled Release 39 (1996) 287-304

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Fig. 2. (A,B) Representative in life radiographs of a 0 /xg rhBMP-2/PLG/CMC (A) and a 80 /zg r h B M P - 2 / P L G / C M C (B) treatment groups taken immediately post-surgery, 2 weeks, 4 weeks, 6 weeks and at euthanasia (8 weeks).

J. Hollinger et a l . / Journal of Controlled Release 39 (1996) 287-304

292

wise fashion and new bone formation was quantitated as previously described [27,30].

ences among doses of rhBMP-2 could not be detected. Histologic analysis at 8 weeks revealed the untreated defects and the 0 /xg rhBMP-2 group, generally had incomplete bridging (Fig. 4A and B). As noted above, two 'CSDs' healed by complete bony union (Fig. 5). The 10 and 40 /,tg r h B M P - 2 / P L G / C M C treated defects developed complete bony bridging (Fig. 4C and D), but little evidence of a marrow cavity. The 80 /xg r h B M P - 2 / P L G / C M C and 10 /xg r h B M P - 2 / P L G / b l o o d treated defects developed cortical bone and a central marrow cavity (Fig. 4E and F). The bone contour was more mature in the 80 /xg r h B M P - 2 / P L G / C M C group. There was no evidence of residual PLG or giant cell reaction in any specimen. Histomorphometric analysis at 8 weeks (Fig. 6) revealed new bone formation in all treatment groups. The 40 /zg r h B M P - 2 / P L G / C M C treated defects yielded the most new bone, followed by the 80 /zg r h B M P - 2 / P L G / C M C group. Differences among the remaining treatment groups could not be detected.

3. R e s u l t s

3.1. Rabbits Comparing representative radiographs of rhBMP-2 treated defects with controls (Fig. 2A and B) revealed both a greater amount of radiopacity and a more uniform distribution of radiopacity within the bony wound. Untreated defects and those treated with carrier only, developed less radiopacity that was non-uniform. Two untreated defects proceeded to complete bony union. In-life radiographs revealed measurable radiopacity in all treatment groups (Fig. 3). Defects treated with 10 /xg r h B M P - 2 / P L G / b l o o d had significantly more radiopacity than controls at the 2 week time period. Wounds treated with 0 /xg rhBMP2 / P L G / C M C had significantly less radiopacity than other treatments at all time periods. By 4 weeks, all treatments had significantly more radiopacity than the 2 week measurements. After 4 weeks, radiopacity had 'peaked', and only subtle changes in radiodensity were observed. Radiomorphometric analysis at 6 and 8 weeks revealed similar findings: rhBMP-2 treated groups had significantly more radiopacity than the 0 /xg rhBMP-2 or untreated defects. Differ-

3.2. Dogs It was often difficult to delineate radiopacity of new bone from host bone margins in some X-ray films. Therefore, rather than attempt computerized imaging of radiographs that may have yielded questionable results, radiographs were ranked visually on

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Fig. 3. Rabbit radiomorphometry:results reported as mean + 1 SD; significance established at p _<0.05. aSignificantly different vs. other treatments within time period; bsignificantly different vs. 10 /xg/rhBMP-2/PLG/blood within time period; Csignificantlydifferent vs. all BMP-2 treated groups within time period;dsignificantly different vs. same treatment in previous time period.

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Fig. 5. An untreated CSD which spontaneously healed by bony union. Arrows denote host bone margin.

a scale of 1 to 5:1 was no radiopacity across the defect whereas 5 was complete radiopaque bridging. The general trend of radiopacity derived from averaging these scores for each set of six treatment replicates did not reveal differences among treatments and time (Fig. 7A-D). Histologically, at 2 months, three of six untreated clefts progressed to fibrous healing with minimal bone formation, while in the other three sites bone bridging was nearly complete (Fig. 8A). Clefts treated with r h B M P - 2 / P L G responded similarly, with combinations of bone formation and fibrosis (Fig. 8B). New bone was consistent with normal appearing trabeculae and cells. There were no residual PLG particles Sites treated with PLG alone responded poorly with little to no bone formation and connective tissue spanned the host bone margins (Fig. 8C). There was neither residual PLG nor an adverse cellular response. Defects treated with particulate

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Fig. 6. Rabbit histomorphometry: results reported as mean + 1 SD; significance established at p < 0.005. aSignificantly different vs. all treatments except D; bsignificantly different vs. A and B; Cno differences could be established between A, B, E and F.

cortico-cancellous a autograft had substantial bone within the cleft (Fig. 8D). By 4 months, untreated clefts were not signifi-

Fig. 4. (A-F) All photomicrographic sections displayed are 5 × the original magnification at 1.5 × ( v o n Kossa stain). Arrow ( $ ) denotes host bone margin. The new bone may be seen as darkly staining (black) in contrast to the lighter staining connective tissue. (A) CSD, 8 weeks: an incomplete bony bridge has developed with fibrous tissue ( * ) at center. (B) 0 /zg rhBMP-2/PLG/CMC, 8 weeks: new bone has been deposited at wound edges and one side of the defect ( zx). The majority of the defect is filled with fibrous tissue. There is no evidence of residual PLG. (C,D) 10 /xg and 40 p,g rhBMP-2/PLG/CMC, respectively, 8 weeks: an osseous bridge has developed across the ostectomy sites. Panel D reveals signs of marrow cavity development. There is no residual PLG present. (E) 80 /xg rhBMP-2/PLG/CMC, 8 weeks: the healed ostectomy site has remodeled with development of marrow ( * ) and intact cortices restoring original contour. (F) 10 /xg rhBMP-2/PLG/blood, 8 weeks: the ostectomy has healed with robust bony deposition, however, remodeling is incomplete and original contour does not appear to have been restored.

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cantly different than their 2 month counterparts (Fig. 8E). The rhBMP-2/PLG-treated clefts had a prominent bony bridge of consolidating trabeculae and

osseous contour was nearly restored (Fig. 8F). Tissue within the PLG-treated defect was mostly fibrous (Fig. 8G). Similar to the 2 month autograft response,

J. Hollinger et al. / Journal of Controlled Release 39 (1996) 287-304

at 4 months there was a well-contoured osseous bridge with cortices and marrow (Fig. 8H). Histomorphometrically, at 2 months, maxillary alveolar defects treated with autograft had significantly more bone than other treatments ( p < 0.05). At 4 months, the PLG-treated group had significantly less bone than other treatments. There was no difference in the amount of detected bone among the untreated clefts, rhBMP-2/PLG-, or graft-treated clefts (Fig. 9). 3.3. Statistics

Multiple linear regression analysis was used to test time- and dose-dependent effects of rhBMP-2 on radiopacity and bone formation within the CSDs. Histomorphometry and radiomorphometry data were analyzed by two-factor analysis of variance (ANOVA) to determine if there was an overall difference in radiopacity and new bone among treatments over time. Individual differences among treatments at each time period were determined by Fisher's protected least significant difference test for multiple comparisons. Statistical significance was established at p < 0.05.

4. Discussion We prepared intraosseous defects in two different anatomical sites in two different species that should not have healed spontaneously with new bone formation, thus fulfilling the principle of the critical-sized defect (CSD) [26]. Rabbit radius ostectomies, 15 mm in length, and dog alveolar clefts, 10 mm in bony width, were prepared in skeletally mature animals and treated with combinations of PLG/autogenous blood (or CMC) and different doses of rhBMP-2. Negative (untreated CSD) and positive controls (autograft for the dogs, and autogenous blood plus 10

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/~g rhBMP for the rabbit) bracketed the experimental treatments. We anticipated observing a dose- and treatment-dependent bone regeneration response within the recipient beds. Quantitative assessments of bone formation from the rabbit study could not detect a dose-response to the rhBMP at the single 8 week time point. However, the highest dose (80 /zg rhBMP-2) did promote bony healing and an almost completely restored original contour. In the dog study, there was more bone within the alveolar cleft defect treated with the particulate cortico-cancellous graft than with other treatments at 2 months. By 4 months, the quantity of bone associated with PLG/autogenous blood-rhBMP-2 appeared equivalent to the graft. We believe the outcome of the experiments reflects a deficiency in the animal wound models and perhaps, in the timing of the retrieved specimens, rather than in the experimental materials. Despite not being able to validate our hypotheses, valuable lessons were learned. Our discussion will focus on two broad areas: the wound model and delivery system for rhBMP-2. 4.1. Rabbit wound model

The rational approach to develop a bone wound repair material is to assess that material in a characterized wound. This is the concept behind the critical-sized defect (CSD) [26]. Key was the first to report on a nonhealing intraosseous wound in the ulna of a dog that was 1.5 times the diameter of the diaphysis [31]. Nilson and colleagues used a defect two times the diaphyseal diameter (approximately 25 mm in length) [32]. Enneking et al. reported 40 mm sized defects in fibulae of dogs [33]. All of the defects received treatments; therefore, it is not possible to speculate whether they were CSDs. The algorithm for the rabbit CSD was derived from reports on dog long bone re-sections and sev-

Fig. 7. (A-D: fight and left sides of radiographs correspond to the reader's right and left sides for all panels) (A) There is no evidence of radiopacity at 2 months in CP defects treated with PLG (right side) or untreated (left side). (B) At 2 months, rhBMP-2 with the PLG delivery system (right side) reveals a radiopaque bridge, whereas on the left side, the autogenous graft did not develop bridging. (C) By 4 months, PLG (right side) did not promote a significant radiopaque response. The untreated left side reveals radiopacity across the defect with a radiolucent discontinuity ('7). (D) At 4 months, rhBMP-2 with the PLG delivery system (right side) did not promote a favorable response. The grafted side developed a robust bony union (left).

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Fig. 8. (A-H: all photomicrographic sections displayed are 8 × the original magnification at 1.25 × ( v o n Kossa stain) (A) Untreated CP, 2 months: edges of original host bone margins (based on radiographic comparison with the histologic blocks) are indicated by the arrows ( J, ). The new bone may be seen as darkly staining (black) in contrast to the lighter staining connective tissue (*). (B) rhBMP-2/PLG, 2 months: new bone bridged the gap spanning the CP except for a central zone of fibrous tissue (~). A portion of the tooth root (left side) is evident in the parasagittal section (,L). (C) PLG, 2 months: Connective tissue ('k) spanned the host bone margins. There were neither indications of residual polymer (PLG) nor adverse cellular responses, such as multinucleated glant cells or macrophages. (D) Graft, 2 months: the particulate cortico-cancellous hip autograft promoted substantial bone repair across the defect. Numerous bony trabeculae ( • ) and cortical bone (J,) were apparent. (E) Untreated alveolar cleft, 4 months: histologic responses were essentially the same as the 2 month group with fibrosis ( ~ ) across the defect gap and minimal new bone formation (--*) at the host bone edges ($). (F) rhBMP-2/PLG, 4 months: a prominent bony bridge consisting of consolidating trabeculae spanning the gap. Bony contour was nearly restored. (G) PLG, 4 months: tissue within the defect was fibrous without evidence of PLG particles or adverse cellular reactions. At the right hand margin is a portion of the tooth root ( zx) and the periodontal ligament ( ~ ). (H) Graft, 4 months: consolidation of the trabeculae was evident with development of cortical plates ( • ) , marrow cavities (~r), and good bony contour.

era1 studies on either ulna or radius ostectomies in rabbits [34-42]. However, the literature on rabbit wound models revealed curious variations and pecuDog

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Fig. 9. Dog histomorphometry: results reported as m e a n + 1 SD; significance established at p < 0.05. aSignificantly different vs. other treatments at the same time period; bsignificantly different at 2 and 4 months; Csignificantly different vs. other treatments at 2 months.

liar, unexpected responses. For example, one report described a 6-mm length ostectomy gap (approximately 1.5 × shaft diameter) in rabbits' radii that healed by fibrosis after 150 days. [37]. Using immature animals with bilateral, 20-mm length ostectomies, one group reported no spontaneous bone formation after 9 weeks [34], whereas another group noted bone formation at 13 weeks [39]. The diversity of defect sizes reported (6 mm [37], 10 mm [40,41], 12 mm [35,42], and 20 mm [34,39]), compounded with bilateral or unilateral wounds, lack of identifying skeletal maturity, and assessing at different times, complicated our decision to select a reliable wound model. The work of Massoud et al. noted that 'Use of body weight or such terms as young adult as developmental indicators are inadequate in selecting rabbits for .., experimental studies .... ' Moreover, the authors commented that weight is not a criterion for skeletal maturity, rather, closure of the epiphyseal plates needs to be validated by radiography [43,44]. The distal tibial growth plate closes at about 16 weeks and the proximal at about 25 weeks [43].

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In a preliminary study in our laboratory (unpublished data), we prepared bilateral, 15-mm length defects in rabbits' radii (4 times the diaphyseal diameter). (Fixation is not required due to the functional support provided by the proximal and distal fusion of the radius to the ulna.) We determined an unacceptable postoperative course: persistent limb retraction by the animal for 7 - 1 4 days (despite analgesics), approximately 10% of one of the operated limbs incurred partial collapse of the interfragment length, and occasional fracture at the metacarpel-ulna-radius junction was observed. Consequently, we followed this investigation with another involving preparation of unilateral, 15-mm length defects. After 3 weeks, we noted an uncomplicated postoperative course. Therefore, the unilateral, 15-mm length ostectomy wound model was selected for the experiment study reported. Cook and workers used bilateral 15-ram osteoperiosteal gaps in rabbits' ulnae to assess whether rhBMP-7 (also known as osteogenic protein-1) would regenerate bone within the gaps [20]. At 8 and 12 weeks post-operatively, control defects (three in number) went on to fibrous union. Cook et al. measured 6 rhBMP-7-treated sites for biomechanical equivalence to a contralateral, unoperated limb. They reported 100 /xg rhBMP-7 produced new bone at 8 weeks that was equivalent functionally to the unoperated control. An additional 23 rabbits received various combinations of nine doses of rhBMP-7 and were evaluated at 8 and 12 weeks by subjective radiography and histology. The Cook group observed that a dose of at least 12.5 /xg was needed for bone formation across the defect. In contrast to the report by Cook's group, we determined with quantitative morphology (radiomorphometry and histomorphometry) that unilateral 15mm sized untreated defects in rabbits' radii may spontaneously progress to bony union over an 8 week period (Fig. 8). The 15-mm sized osteo-periosteal gap is an unacceptable CSD. It is unclear why the (bilateral) ulna model used by Cook's group behaved differently than the (unilateral) radius model we reported. Bilateral, nonfixed limbs may undergo a sufficient degree of biofunctional challenge that mitigates against spontaneous closure unless exogenous BMP is present. In contrast, a unilateral site could be 'favored' and

protected by the contralateral limb, thus ensuring for sufficient stabilization of the operated limb leading to bone regeneration. Development of a product for the regeneration of bone in continuity defects demands a consensus wound model that is reproducible, responds predictably at designated time periods, and will provide clear evidence of efficacy. Moreover, a statistically sufficient number of test sites must be quantitatively assessed. We recommend preparing a unilateral radius defect 20 mm in length. A study recently completed in our laboratory indicates this sized wound will not spontaneously heal by bony union (unpublished data). Moreover, the surgical preparation of the radius has an advantage over the ulna in that less soft tissue dissection is required, there is less post-operative morbidity, and the morphology of the radius is rounder overall than the thin ulna. Importantly, consideration for the experimental animal's welfare warrants unilateral rather than bilateral wounding.

4.2. Dog wound model An alveolar cleft model has been described in the dog by Boyne and colleagues [45] and Marx's group [46]. Marx noted that he was unable to duplicate the Boyne cleft model. Marx et al. determined that untreated clefts fibrosed by 6 months. In our hands, the dog wound model described by Marx and colleagues resulted in some untreated clefts spontaneously healing by 4 months. Therefore, we recommend increasing the bony defect width from 1 to 1.5 cm. We speculate differences in responses between the Marx study and ours may be due to a number of factors. Immature animals treated with a bone regenerative material will respond differently than mature animals due to facial developmental growth in the immature animal. Skeletal growth in the upper face is upward and forward, whereas in the lower face it is in the downward and forward direction as an expanding V [47,48]; therefore, surgical preparation of a cleft in an immature animal may not heal spontaneously due to the upward and forward expansion of the premaxilla. Whereas in a skeletally mature animal, completion of facial development could allow an alveolar cleft < a CSD to close. Marx et al. used '22 adult mongrel dogs weighing

J. Hollinger et al./ Journal of Controlled Release 39 (1996) 287-304

between 30 and 40 kg.' Validation of skeletal maturity in a mongrel may not be determined accurately by weight. Radiographic evidence of epiphyseal plate closure was not described by Marx. The 24 dogs we reported on were skeletally mature foxhounds (verified by epiphyseal plate closure). Mongrel dogs derived from a nonspecific lineage may not be as physiologically robust as a standard breed (e.g., foxhounds). Marx and colleagues did not indicate whether the dogs had systemic problems that may have contributed to a nonhealing response. While it is more expensive to acquire a genetically pure breed than a mongrel, it is key to the developmental of a bone regenerative material that the animal wound model responds in a predictable, reproducible manner (every mongrel dog is unique). Our recommendations for the alveolar cleft wound model are to use a standard, recognized breed of dog that is skeletally mature (determined radiographically) and to increase the bony width of the cleft defect from 10 mm to 15 mm. 4.3. The delivery system

There are risks in transplanting allogeneic tissues that include infection, immunologic rejection, and viral transmission [49-57]. Moreover, the deficiencies associated with the 'gold standard' bone autograft provide a compelling incentive to develop an effective clinical alternative. Urist identified an acidic glycoprotein endogenous to bone and pivotal for its regeneration and renewal: bone morphogenetic protein (BMP) [58-60] Recombinant technology has enabled the cloning and expressing of human BMPs in sufficient quantities for therapeutic applications in preclinical studieg [2,7,10,13,28]. Preclinical studies with rhBMPs have included craniotomy and long bone repair in rats [ 17,19,21,22] ~',mandibular midbody continuity regeneration in dogs [16], and long bone repair in sheep and dogs [61,62], rabbits [20], and nonhuman primates [63]. Many of these studies have used collagen to deliver rhBMP. Collagen may not be ideal because of its immunologic potential [64-66]. The poly(c~-hydroxy acid) known as po!y(lactide-~co-glycolide) (PLG) may be a suitable carrier (reviewed by Chu [67], Hollinger [68], and Bostman [69]). Poly(c~-hydroxy acids) have

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had an enviable and well-documented safety record as sutures for over 25 years [68]. Recently, some clinical accounts have noted host biocompatibility difficulties when poly(a-hydroxy acids) were used as fixation plates and screws [7073]. However, these reactions may have been due to the quantity of implanted polymer. The design for a delivery system for rhBMP-2 will not require high molecular weight polymer manufactured as a dense, bulk device. A low molecular weight, highly porous delivery system with maximal surface area for biodegradation should not lead to the sequelae reported for fixation devices. Therefore, particulate PLG was selected to deliver rhBMP-2 in combination with either autogenous blood or CMC. PLG was combined with either autogenous blood or CMC with the notion they would bind PLG particles and promote localization of the rhBMP-2. Retention of the rhBMP-2 with this combination was expected, binding was not. Retention followed by slow release as the local hematoma lysed and the PLG biodegraded was expected to promote a sustained, localized concentration of rhBMP-2 enabling cell interaction leading to osteoblast expression. The mass of particles could prevent soft tissue prolapse, and the intraparticle void-volume and interparticle spaces could permit bone ingrowth concurrent with centripetal bone regeneration.

5. Summary The delivery system can be improved. It was not optimal for implanting into osseous wounds: a volume of the particulate implant was dislodged by soft tissue movement and oozing blood from the recipient bed. Consequently, the optimal dose of rhBMP-2 may not have been retained within the recipient beds. Delivery of a therapeutic dose of a drug is pivotal to its success. An improved delivery system must be designed to deploy a therapeutic dose of rhBMP-2. The combinations of rhBMP-2 and PLG with either autogenous blood or CMC did not elicit adverse tissue responses. At the time periods observed, none of the recipient beds had residual PLG particles. Modifications of the animal wound models are warranted to preclude spontaneous bone healing.

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T h e r e are m a n y p o t e n t i a l a p p l i c a t i o n s for the r h B M P s . A d d i t i o n a l p r e c l i n i c a l studies m u s t b e c o m p l e t e d to p e r f e c t the d e l i v e r y o f t h e s e p o t e n t factors.

Acknowledgements In c o n d u c t i n g the r e s e a r c h d e s c r i b e d in this p r o t o col, the i n v e s t i g a t o r s a d h e r e d to the i n s t r u c t i o n s des c r i b e d in ' G u i d e f o r the Care and U s e o f L a b o r a t o r y A n i m a l s ' ( N I H P u b l i c a t i o n s 85-23) as p r o m u l g a t e d b y the C o m m i t t e e o f C a r e a n d U s e o f L a b o r a t o r y A n i m a l s o f the Institute o f L a b o r a t o r y S c i e n c e s , N a t i o n a l A c a d e m y o f S c i e n c e s and N a t i o n a l R e search Council.

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