This Article Abstract Full Text (PDF) Free Letters to the Editor: Submit a response Alert me when this article is cited Alert me when Letters to the Editor are posted Alert me if a correction is posted Services E-mail this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Add to My File Cabinet Download to citation manager Reprints and Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by ZEGZULA, H. D. Articles by HOLLINGER, J. O. Search for Related Content PubMed PubMed Citation Articles by ZEGZULA, H. D. Articles by HOLLINGER, J. O. Social Bookmarking             What's this?

Bone Formation with Use of rhBMP-2 (Recombinant Human Bone Morphogenetic Protein-2)*

H. DANIEL ZEGZULA, M.D., DAVID C. BUCK, M.S., JOHN BREKKE, D.D.S., JOHN M. WOZNEY, PH.D. and JEFFREY O. HOLLINGER, D.D.S., PH.D., PORTLAND, OREGON

Investigation performed at Oregon Health Sciences University, Portland

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
We examined the effect of rhBMP-2 (recombinant human bone morphogenetic protein-2), delivered in a porous poly(DL-lactic acid) implant, on bone formation in a critical-sized defect in the radial diaphysis in rabbits. A unilateral segmental defect, twenty millimeters long, was created in the radius in ninety-six skeletally mature New Zealand White rabbits. Forty-eight rabbits were evaluated at four weeks and forty-eight, at eight weeks. Six groups were studied at each time-period. The defect was left empty in one group (control), the defect was filled with an autogenous corticocancellous bone graft in one group, and the defect was filled with a porous poly(DL-lactic acid) implant containing zero, seventeen, thirty-five, or seventy micrograms of rhBMP-2 (one group each). Radiographs of the defects were made every two weeks. The percentage of the total area of the defect that was radiopaque was determined with use of computerized radiomorphometry, and this percentage was used as a quantitative measure of the extent of new-bone formation in the defect. There were time and dose-dependent responses to rhBMP-2 for as long as four weeks; thereafter, the effects of seventeen, thirty-five, and seventy micrograms of rhBMP-2 were independent of dose and time (p 0.05). The defects that had been treated with either thirty-five or seventy micrograms of rhBMP-2 had a significantly greater (p 0.05) area of radiopacity than the defects that had been treated with either zero or seventeen micrograms of rhBMP-2. No significant difference could be found between the defects treated with thirty-five or seventy micrograms of rhBMP-2 and the defects filled with an autogenous graft. Healing and bone formation were examined histologically and histomorphometrically as well. At four weeks, polarized light microscopy revealed remnants of poly(DL-lactic acid) only in the defects that had been filled with an implant containing zero micrograms of rhBMP-2. At eight weeks, regardless of the dose of rhBMP-2, poly(DL-lactic acid) was not visible on histological examination. The presence of multinucleated giant cells was the hallmark of the inflammatory response elicited by poly(DL-lactic acid). At four and eight weeks, macrophages and lymphocytes were also present. The intensity of the cellular response at four weeks suggested an inverse relationship between these cells and the dose of rhBMP-2—that is, there appeared to be more multinucleated giant cells in defects treated with zero micrograms of rhBMP-2 than in defects treated with seventy micrograms of rhBMP-2. At eight weeks, multinucleated giant cells were rare in the defects treated with seventeen, thirty-five, or seventy micrograms of rhBMP-2. Histomorphometric data at four and eight weeks indicated that the amount of bone formation in the defects treated with seventeen, thirty-five, or seventy micrograms of rhBMP-2 was equivalent to the amount in the defects treated with an autogenous graft and was significantly less (p 0.05) in the untreated defects and the defects treated with zero micrograms of rhBMP-2 (p 0.05). By eight weeks, only thirty-five and seventy micrograms of rhBMP-2 had restored cortices and marrow elements. CLINICAL RELEVANCE: It is a clinical challenge to restore bone lost as a result of trauma, pathological processes, oncological resection, or developmental malformations. Autogenous and banked allogenic bone grafts are used routinely to correct bone defects. However, problems associated with these treatments are well known, and a safe, reliable, and convenient alternative is desirable. In the present study, rhBMP-2 delivered in a porous poly(DL-lactic acid) implant promoted bone formation. Therefore, rhBMP-2 in a poly(DL-lactic acid) delivery system may be suitable to elicit bone formation and healing in segmental bone defects.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
The use of various therapeutic measures to induce the formation of bone in bone defects has become common, with autogenous and banked allogenic bone grafts having been effective. Despite the efficacy of these conventional methods, problems associated with their use have led to a search for alternatives^1,22,48. Toward that goal, we evaluated the effect of a porous poly(DL-lactic acid) implant containing rhBMP-2 (recombinant human bone morphogenetic protein-2) on the healing of segmental bone defects.

The availability of rhBMPs has led to much research aimed at gaining an understanding of the functional roles of the BMPs and using this knowledge to develop substitutes for bone grafts. Thirteen BMPs, designated as BMP-1 through BMP-13, have been identified^{6,7,14,26,27,35}. At the time of writing, there were at least forty BMP-like molecules within the TGF-ß (transforming growth factor-ß) superfamily, including TGF-ß1 through TGF-ß5, BMPs, growth/differentiation factors 1 through 10 (a subclass of BMPs³⁶), the inhibins, activins, vegetal-pole-related genes, nodal-related genes, Drosophila genes (such as decapentaplegic and Drosophilia 60A), and glial-derived neurotropic factor^{16,26,27,31,32,39,40,46,54}.

The BMPs and related molecules provide essential cues to the embryo for body-patterning and the development of tissue and organs^26,27,52. However, it is the capacity for BMP to promote osteoblastic differentiation⁵⁴ that is of considerable interest to clinical disciplines involved with bone repair. It is possible that this property could be exploited to engineer bone-graft substitutes containing BMP that could be used instead of autogenous and banked allogenic grafts²¹.

Studies of animals have validated the efficacy and the safety of rhBMP-2 and rhBMP-7 for bone repair^{5,10,11,15,24,25,30,33,43,44,49,57}. However, a satisfactory delivery system for rhBMP must be developed before it can be used in humans. Some investigators have described a collagen carrier for rhBMP^{10-12,24,43,44,49}, but collagen has a number of clinical disadvantages^13,48. Therefore, poly(DL-lactic acid) was considered as a delivery system for rhBMP-2. Poly(DL-lactic acid) is a poly(-hydroxy acid), a class of biodegradable, biocompatible polymers that has a thirty-year history of clinical safety⁹. Consequently, we developed a combination of rhBMP-2 and poly(DL-lactic acid) that could be used in selected situations as an alternative to either autogenous or banked allogenic bone. The purpose of this study was to examine the effect of rhBMP-2 in a porous poly(DL-lactic acid) implant on the formation of bone in a critical-sized defect (one that does not heal spontaneously) in the radius in rabbits.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 

Experimental Design
A unilateral twenty-millimeter segmental defect was created in the radial diaphysis in ninety-six skeletally mature New Zealand White rabbits. Skeletal maturity was verified by radiographic evidence of the epiphyseal plates. Bone formation was evaluated, at two-week intervals, for four weeks in forty-eight rabbits and for eight weeks in forty-eight rabbits. Six groups of eight animals were studied at each time-period. The defect was left empty in one group (untreated controls), it was filled with an autogenous corticocancellous bone graft in one group, and it was filled with a porous poly(DL-lactic acid) implant containing zero, seventeen, thirty-five, or seventy micrograms of rhBMP-2 in one group each. According to a power analysis, the presence of eight animals in each group indicated that the alpha would be 0.05 and the beta would be 0.10 (that is, a power of 0.90)²⁸.

Operative Procedure
The rabbits were anesthetized with an intramuscular injection, at a dose of 0.65 milliliter per kilogram of body weight, of a solution consisting of ten milliliters of ketamine hydrochloride (Ketaset, 100 milligrams per milliliter) and five milliliters of xylazine (Gemini SA, twenty milligrams per milliliter). When an adequate state of anesthesia had been achieved, an ophthalmic ointment (Lacri-Lube) was placed on the conjunctiva of each eye and an antibiotic (Baytril [enrofloxacin]) was administered. The rabbit was placed in the supine position, and the operative site on either the left or the right front limb was shaved, prepared, and draped for an aseptic operation. A tourniquet was placed around the axillary region, and the operative site was infiltrated with 0.5 milliliter of 2 per cent lidocaine with epinephrine (1:100,000) (Xylocaine, twenty milligrams per milliliter). A superomedial incision, approximately four centimeters long, was made, and the tissues overlying the distal part of the radial diaphysis were dissected. A twenty-millimeter segmental defect in the radius was made with an oscillating saw, under irrigation with 0.9 per cent sterile saline solution, and the designated treatment was carried out. Internal fixation was not necessary because of the fibro-osseous syndesmosis between the ulna and radius. The soft tissues were approximated with interrupted 4-0 sutures (Ethicon, Somerville, New Jersey), and the skin was closed with staples (Precise DS-25; 3M, St. Paul, Minnesota). Immediately after the operation, standardized lateral radiographs were made of the involved limb, and the animals then were returned to their cages. Postoperatively, 0.3 milliliter of buprenorphine hydrochloride (Buprenex, 0.3 milligram per milliliter) was given as needed for pain. Water and food were supplied ad libitum.

Procedure for Placement of the Autogenous Graft
After the twenty-millimeter segment of bone had been removed, the gap was irrigated with sterile physiological saline solution and the segment of corticocancellous bone was repositioned in the defect. Flexor and extensor tendons augmented by soft-tissue closure retained the autogenous graft.

Preparation of the rhBMP-2
The rhBMP-2 was produced by a Chinese Hamster ovarian cell expression system to yield a glycosylated thirty-two-kilodalton homodimer, with use of methods that have been previously described^30,51,56. The highly (more than 98 per cent) purified rhBMP-2 was placed in sterile glass vials in sodium glutamate buffer (five millimeters, pH 4.5). The rhBMP-2 was provided by Genetics Institute (Andover, Massachusetts).

Preparation of the Poly(DL-Lactic Acid) Delivery System
The poly(DL-lactic acid) delivery system was prepared from DL-lactic acid and organic solvents with use of a technique of rapid volume expansion-solvent extraction curing to yield a porous product (Fig. 1). The post-synthesis polymer was manufactured into cylinders (4.7 by twenty millimeters) that were sterilized with gamma irradiation with thirty gray of cobalt-60. The void volume for each cylinder (determined with helium pyknometry) was 90 per cent, and the maximum fluid volume that could be accommodated by this void volume was 0.17 milliliter. The poly(DL-lactic acid) delivery system was provided by THM Biomedical (Duluth, Minnesota) and is manufactured as the product OPLA.

View larger version (150K): [in this window] [in a new window]   Fig. 1 Scanning electron photomicrograph of the biodegradable, porous poly(DL-lactic acid) delivery system. The distance between the two white crosshairs is approximately 170 micrometers (original magnification, x 51).

Preparation of the Poly(DL-Lactic Acid) Implant Containing rhBMP-2
At the time of the operation, a known concentration of rhBMP-2 (zero, seventeen, thirty-five, or seventy micrograms) in 0.17 milliliter of buffer was added under sterile conditions, with use of a syringe, to the porous poly(DL-lactic acid) cylinder (Fig. 2-A) and the cylinder was placed within the defect (Fig. 2-B). The selected volume (0.17 milliliter) was the maximum fluid volume that could be maintained within the void volume of the poly(DL-lactic acid) cylinder implanted in the radial defect. Therefore, a known amount of rhBMP-2 was loaded into the poly(DL-lactic acid) delivery system.

View larger version (129K): [in this window] [in a new window]   Fig. 2-A Photograph showing 0.17 milliliter of buffer containing either zero, seventeen, thirty-five, or seventy micrograms of rhBMP-2 being transferred in a sterile fashion, with use of a syringe, to the poly(DL-lactic acid) delivery system at the time of the operation.

View larger version (136K): [in this window] [in a new window]   Fig. 2-B Photograph showing the poly(DL-lactic acid) cylinder containing rhBMP being implanted within the defect. The triangles indicate the edges of the defect in the radius, and the star indicates the ulna.

Radiography
Standardized radiographs were made immediately postoperatively, every two weeks, and at the time of death (four or eight weeks). The radiographs of the operative site were made at fifty kilovolts peak, ten milliamperes, and 0.2 second, with use of a constant object-to-film-to-x-ray-source distance and ultra-high-contrast mammography film (X-OMATL; Eastman Kodak, Rochester, New York).

Radiomorphometry
The radiographs of the defects were assessed in a standard fashion with use of an image-analysis system (Leica Instruments, Cambridge, England) based on discrimination of gray-level density, as has been previously described^18,29. A standard-sized (twenty-two-by-five-millimeter) measuring frame, generated by the computer, was superimposed over each x-ray film, and the percentage of radiopacity was determined with a computer algorithm that divides the radiopaque area of the defect by the total area of the measuring frame. The technique involves video-camera input of each ultra-high-contrast x-ray film and computer imaging that permits the detection of 4000 gray-level densities. The gray-level densities were standardized among the films with use of a customized software program, eliminating the need for step-wedges. With use of a consistent-input threshold level (that is, a gray-level density setting) for each x-ray film, radiopaque areas within the defect were defined and detected. The radiopacity could be measured reproducibly at a confidence level of 95 per cent or more. A value of 100 per cent radiopacity was recorded when the measuring frame was filled completely with new bone, and the appropriate percentage was recorded when radiopacity incompletely filled the measuring frame. The values were reported as the mean per cent radiopacity (and one standard deviation) for each group.

Histological Analysis
At four and eight weeks postoperatively, the defects and adjacent host bone were obtained en bloc, placed in a series of graded ethanol (70 to 100 per cent), and embedded in polymethylmethacrylate. Undecalcified sections, 4.5 micrometers thick, were prepared and stained with a modification of the Goldner-Masson trichrome stain or the von Kossa stain²⁹. Six longitudinal sections were taken through the center of each defect, and each section included host bone proximal and distal to the defect. Alternate sections were stained with Goldner-Masson trichrome stain or the von Kossa stain and were examined with use of an Axiophot microscope (Carl Zeiss, Thornwood, New York).

Histomorphometry
New bone was detected and measured in the von Kossa-stained histological specimens with use of a Leica 970 image-analysis system (Leica Instruments) interfaced with the Axiophot microscope (Carl Zeiss), as previously described^18,20,33. Briefly, new bone within each defect was measured from three consecutive histological slides at a magnification of 3.25 times. Three contiguous histological fields on each of the three histological slides were measured at this magnification; thus, the value for new bone was based on nine measurements for each animal, and the mean area for each group was determined from seventy-two measurements (nine histological fields in each of eight animals). New bone was measured within a reference frame that was twenty-one by five millimeters, and the area of new bone was determined with the same computer algorithm used to obtain the radiomorphometric data. Furthermore, the location of each histological sampling (in a longitudinal plane) concurred with the recognized anisotropic nature of bone^37,38,45, and the number of histological fields that were measured was based on a determination of nominal values^37,38. The histomorphometric data were reported as the mean area of new-bone formation (in square millimeters) and one standard deviation.

Statistical Analysis
Multiple linear-regression analysis was used in order to test time-dependent and dose-dependent effects of rhBMP-2 on the per cent radiopacity and the area of new-bone formation. The histomorphometric and radiomorphometric data were analyzed with use of multiple analysis of variance to determine whether there was an over-all difference in the per cent radiopacity and the area of new bone among the treatment groups over time. Individual differences among the groups at each time-period were determined with use of the Fisher protected least-significant-difference test for multiple comparisons. Significance was established at p 0.05.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 

Clinical Course
The clinical course was uneventful for all of the animals in the present study. There were no indications of adverse sequelae associated with the experimental procedures.

Radiography
Throughout the eight-week period of study, there was little or no bone formation and a radiolucent nonunion in the untreated defects (control group) (Fig. 3). The defects with an autogenous graft had a radiolucent interface bracketing the graft at four weeks; the interface was less evident by eight weeks, at which time callus was apparent. In the group with a poly(DL-lactic acid) implant with zero micrograms of rhBMP-2, the proximal and distal ends of the defect were slightly radiopaque and the mid-portion was radiolucent at four and eight weeks. At eight weeks, the highest dose (seventy micrograms) of rhBMP-2 had promoted the formation of cortices of bone (Fig. 3). The dramatic effect of rhBMP-2 on the healing of the segmental defect was clearly demonstrated by comparison of radiographs, made at two-week intervals from the day of the operation until eight weeks, of an untreated defect and a defect filled with an implant containing seventy micrograms of rhBMP-2 (Fig. 4). Both of these defects were radiolucent immediately after the operation. The untreated defects remained completely radiolucent over the eight-week period. In contrast, the per cent radiopacity increased progressively in the defect filled with the implant containing seventy micrograms of rhBMP-2, and by eight weeks the healed defect contained cortical and cancellous bone (Fig. 4).

View larger version (114K): [in this window] [in a new window]   Fig. 3 Typical radiographic appearance of the defects in each group at eight weeks. The radiolucency of the untreated critical-sized twenty-millimeter defect (CSD) demonstrates that it did not heal spontaneously. In the defects treated with an autogenous graft, there was a radiolucent junction (triangles) between the graft and host bone. The defect filled with a poly(DL-lactic acid) (PLA) implant containing zero micrograms of rhBMP displayed slight radiopacity. There was a concurrent increase in the radiopacity as the dose of rhBMP-2 increased from seventeen to thirty-five micrograms. The poly(DL-lactic acid) implant containing seventy micrograms of rhBMP-2 appeared to promote the formation not only of trabecular bone but also of new cortical bone (arrows).

View larger version (124K): [in this window] [in a new window]   Fig. 4 Representative set of radiographs depicting the temporal progression in an untreated critical-sized defect and a defect filled with a poly(DL-lactic acid) (PLA) implant containing seventy micrograms of rhBMP-2. Both defects are radiolucent immediately after the operation (Postop. Day 0); the poly(DL-lactic acid) implant is radiolucent and indistinguishable from the background. There was no radiopacity within the untreated defect throughout the eight weeks. In contrast, the defect filled with a poly(DL-lactic acid) implant containing seventy micrograms of rhBMP-2 demonstrated slight radiopacity at two weeks, which had increased by four weeks. A bone-like pattern was noted at six weeks, and the defect was filled with bone by eight weeks.

Radiomorphometry
The highest dose (seventy micrograms) of rhBMP-2 promoted a time-dependent increase in the per cent radiopacity between two and four weeks (p 0.05) (Fig. 5). By the second week, the per cent radiopacity was significantly higher (p 0.05) in the defects treated with an autogenous graft than in the defects in the other groups. At six weeks, we could detect no significant difference in the per cent radiopacity between the untreated defects and the defects treated with an implant containing zero micrograms of rhBMP-2; however, the per cent radiopacity in both of these groups was significantly lower (p 0.05) than that in the other four groups. At eight weeks, the per cent radiopacity in the defects treated with thirty-five or seventy micrograms of rhBMP-2 was significantly higher (p 0.05) than that in the untreated defects and the defects treated with zero micrograms of rhBMP-2.

View larger version (21K): [in this window] [in a new window]   Fig. 5 Bar graph showing the radiomorphometric data (mean per cent radiopacity and one standard deviation) obtained at two-week intervals. At two weeks, the untreated defects (CSD) and the defects filled with a poly(DL-lactic acid) (PLA) implant containing either zero or seventeen micrograms of rhBMP-2 had an equivalent per cent radiopacity (a), which differed from the percentage for the other groups. The defects treated with an autogenous graft had a significantly higher (p 0.05) per cent radiopacity than the defects in the other groups (b). By four and six weeks, the untreated defects had less radiopacity than the defects in all of the other groups (a), except for the defects treated with a poly(DL-lactic acid) implant containing zero micrograms of rhBMP-2 (a).

Histological Analysis
Little or no new bone formed in the untreated defects. Over the duration of the study, the autogenous graft integrated with the host bone, proximally and distally, through the formation of callus.

Fibrous tissue filled the defects treated with zero micrograms of rhBMP-2. A dose of seventeen, thirty-five, or seventy micrograms of rhBMP-2 produced numerous isotropically oriented trabeculae by four weeks; larger trabeculae and cortices developed by eight weeks in the defects treated with thirty-five or seventy micrograms of rhBMP-2.

At four and eight weeks, the osseous contour stimulated by seventeen micrograms of rhBMP-2 was not as uniform as that promoted by the two higher doses (Fig. 6).

View larger version (126K): [in this window] [in a new window]   Fig. 5 Representative series of photomicrographs of a defect from each group at four and eight weeks, depicting the appearance of the healing bone. A dose of seventeen, thirty-five, or seventy micrograms of rhBMP-2 produced numerous trabeculae by four weeks. By eight weeks, the trabeculae had consolidated and cortices had formed in the defects treated with thirty-five or seventy micrograms of rhBMP-2. The lower dose (seventeen micrograms) of rhBMP-2 produced a less robust osseous response than the higher doses (von Kossa stain; original magnification x 1). CSD = untreated critical-sized defect and PLA = poly(DL-lactic acid).

View larger version (132K): [in this window] [in a new window]   Fig. 7-A Photomicrograph, made at four weeks, of a defect filled with a poly(DL-lactic acid) implant containing thirty-five micrograms of rhBMP-2 (von Kossa stain, x 1.25). There was bone growth into the poly(DL-lactic acid) implants containing seventeen, thirty-five, or seventy micrograms of rhBMP-2. The bone ingrowth was more uniform in the implants containing either of the two higher doses than it was in the implants containing seventeen or zero micrograms of rhBMP-2. Old and new bone are black. The arrows indicate the margin of the host bone. There is a section of host bone and new-bone trabeculae in the inset.

View larger version (75K): [in this window] [in a new window]   Fig. 7-B Higher-magnification photomicrograph of the inset in Fig. 7-A. The arrows indicate the margin of the host bone. Growth of new bone (black) into the porous poly(DL-lactic acid) implant can be seen to the right of the arrows (von Kossa stain, x 10).

View larger version (104K): [in this window] [in a new window]   Fig. 8 Photomicrograph, made at four weeks, of a defect filled with a poly(DL-lactic acid) implant containing zero micrograms of rhBMP-2. The host bone margin is on the left (von Kossa stain, x 1.25). Bar = 0.8 millimeter. The higher magnification (x 5) in Inset A demonstrates an area of lymphocytes (arrows) and the areas where the poly(DL-lactic acid) was solubilized and washed out by solvents during histological preparation (white areas, such as the one with the star). The higher magnification (x 25) in Inset B shows multinucleated giant cells (triangles) associated with poly(DL-lactic acid) (white areas).

View larger version (122K): [in this window] [in a new window]   Fig. 9 Photomicrograph, made at eight weeks, of a defect filled with a poly(DL-lactic acid) implant containing seventy micrograms of rhBMP-2. New bone (black areas) is apparent across the defects, and cortex formation can be seen in the lower portion of the photomicrograph (von Kossa stain, x 1). The higher magnification (x 10) in Inset A demonstrates multinucleated giant cells (triangle) and the voids indicative of areas that had contained poly(DL-lactic acid) (stars). The higher magnification (x 10) in Inset B shows active osteoblasts (arrows).

View larger version (150K): [in this window] [in a new window]   Fig. 10 Photomicrograph, made under polarized light at four weeks, of a defect filled with a poly(DL-lactic acid) implant containing zero micrograms of rhBMP-2. Residual crystalline poly(DL-lactic acid) (arrows) is seen. No residual polymer could be detected with polarized light microscopy at eight weeks. The triangle indicates multinucleated giant cells, and the star indicates a void created when relatively non-crystalline poly(DL-lactic acid) was dissolved during histological processing (von Kossa stain, x 10).

Histomorphometry
Histomorphometric data indicated that there was significantly less (p 0.05) new bone at four and eight weeks in the untreated defects and the defects filled with an implant containing zero micrograms of rhBMP-2 than in the defects in the other groups (Fig. 11). Moreover, there was no difference in the area of new bone measured at four or eight weeks in the untreated defects and the defects treated with zero micrograms of rhBMP-2. There was neither a time-dependent nor a dose-dependent difference in the area of bone in the defects treated with an autogenous graft or the defects treated with seventeen, thirty-five, or seventy micrograms of rhBMP-2. Furthermore, at four and eight weeks the area of new bone in these groups did not differ.

View larger version (24K): [in this window] [in a new window]   Fig. 11 Bar graph showing the histomorphometric data (mean area of new bone and one standard deviation) at four and eight weeks. The quantity of bone measured in the untreated critical-sized defects (CSD) was not different from that in the defects filled with a poly(DL-lactic acid) (PLA) implant containing zero micrograms of rhBMP-2 (a), but it was significantly less (p 0.05) than that in the defects in the other groups.

Statistical Analysis
Because of the similarity among the responses to the higher doses of rhBMP-2, neither a time-dependent nor a dose-dependent relationship could be determined with use of multiple linear-regression analysis. However, two-factor analysis of variance and the Fisher protected least-significant-difference test for multiple comparisons demonstrated that the histomorphometric findings for the untreated defects and the defects treated with zero micrograms of rhBMP-2 were significantly different (p 0.05) from the findings for the defects in the other four groups. No differences in the quantity of new bone at four or eight weeks could be detected among the defects treated with seventeen, thirty-five, or seventy micrograms of rhBMP-2 and the defects treated with an autogenous graft.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The results of the present study show that rhBMP-2 delivered in porous poly(DL-lactic acid) elicits bone formation and healing in segmental bone defects that do not heal spontaneously. Furthermore, there was clear morphological evidence that thirty-five and seventy micrograms of rhBMP-2 promoted the formation of more new cortical bone and thicker trabeculae than did the lower dose (seventeen micrograms).

Several other experimental studies have validated the effectiveness of rhBMP for the stimulation of bone formation^{10,11,19,25,30,33,49}. The importance of the delivery system was noted in all of these studies. The delivery system releases and localizes the BMP, ensuring interaction with mesenchymal cells that can differentiate into osteoblasts. The delivery system also provides instructional guidance as a template to renew osseous contour.

In experimental studies, either rhBMP-2 or rhBMP-7 has been implanted in combination with two classes of delivery systems: collagen-based systems (either bone collagen or demineralized bone matrix treated with guanidine hydrochloride) and poly(-hydroxy acids)^{5,10,11,24,33,49}. Other delivery systems, including inactivated demineralized bone matrix, hydroxyapatite, and hydroxyapatite-tricalcium phosphate, have been used to deliver partially purified BMPs^30,41,42,47. However, a calcium phosphate-based delivery system that will neither biodegrade nor biodegrade in register with bone formation may have limited clinical utility. Moreover, a delivery system prepared from banked demineralized bone matrix has variable properties (inherent with multiple donors), thereby jeopardizing consistent clinical performance. In addition, there are serious risks associated with the transplantation of allogenic and xenogeneic tissues, including infection, immunological rejection, and viral transmission^1,8,34. Consequently, the versatile family of poly(-hydroxy acids), with its well documented thirty-year safety record as a suture material, is a candidate for a delivery system for BMP¹⁹.

The virtues of poly(-hydroxy acid) sutures have been stressed⁹; however, poly(-hydroxy acids) have occasionally provoked unsatisfactory responses when they have been used as fixation devices^2-4. Disparate reports of biocompatibility and toxicity of the poly(-hydroxy acids) have been attributed to variability in the chemistry and syntheses of polymers¹⁷. Moreover, Vert and Garreau mentioned at least nineteen parameters that can affect poly(-hydroxy acids)⁵⁰. Therefore, a highly characterized polymer must be used to ensure the reproducibility, among experiments, of tissue biocompatibility, biodegradation, and delivery of BMP to bone defects.

By employing post-synthesis technologies such as solvent-based casting, volume expansion-solvent extraction curing, solute leaching, reverse evaporation, and emulsification, delivery systems for BMPs and scaffolds for tissue engineering can be custom-tailored for site-specific anatomical properties and wound-healing kinetics^19,23,53. We chose a method of volume expansion-solvent extraction curing to prepare a characterized homopolymer of poly(DL-lactic acid) to deliver selected doses of rhBMP-2.

Despite our efforts to optimize the polymer delivery system, multinucleated giant cells were a component of the wound-healing response. There appeared to be more multinucleated giant cells and lymphocytes in the defects filled with a poly(DL-lactic acid) implant without rhBMP-2 than in the defects filled with an implant with rhBMP-2. Multinucleated giant cells were present throughout the eight weeks of the study. Despite the inflammatory reaction to poly(DL-lactic acid), rhBMP-2 promoted new-bone formation. Moreover, there were no adverse clinical sequelae (such as swelling or the formation of sinus tracts) associated with the implants. Remnants of crystalline poly(DL-lactic acid) could not be detected with fluorescent microscopy at eight weeks. However, some non-crystalline poly(DL-lactic acid) was still present, as indicated by voids that were seen on photomicrographs (Fig. 9). Several factors may be responsible for these observations. The rhBMP-2 and polymer may have influenced the phenotype, quantity, and activity of cells, thereby affecting the environment of the defect, biodegradation of the polymer, recruitment and stimulation of multinucleated giant cells, and formation of bone. For example, there were fewer multinucleated giant cells at eight weeks than at four weeks in the defects filled with a poly(DL-lactic acid) implant containing rhBMP-2. The decreased volume of poly(DL-lactic acid) by eight weeks may be less stimulatory to granulocytes and multinucleated giant cells. However, complete abrogation of the multinucleated-giant-cell response evoked by poly(DL-lactic acid) may not be possible. If the response is transient, which it appears to be, it may have no clinical relevance. Furthermore, we did not observe aseptic draining sinus tracts, which have been reported with use of fixation devices made of poly(DL-lactic acid) or polyglycolic acid^2,4.

We concluded that a porous poly(DL-lactic acid) delivery system with a void volume of 90 per cent or more presents substantially less bulk to the host tissue than do dense poly(DL-lactic acid) fixation devices, is suitable for delivering rhBMP-2 to segmental bone defects, and is effective as a template to renew contour. Persuasive evidence for this last point is the observation that developing bone trabeculae were localized to the defect and were distributed uniformly across the defect and new-cortex formation was evident by eight weeks in the group that had been treated with seventy micrograms of rhBMP-2 (Figs. 8 and 9).

The data from the present study indicate that a porous cylinder of poly(DL-lactic acid) rhBMP-2 promotes bone formation in critical-sized defects. Furthermore, the rhBMP-2 and poly(DL-lactic acid) were clinically convenient to use, biocompatible, and biodegradable, thus increasing the potential therapeutic value of this combination for the stimulation of new-bone formation in segmental defects in a clinical setting.

	Footnotes

 

*One or more of the authors has received or will receive benefits for personal or professional use from a commercial party related directly or indirectly to the subject of this article. Funds were received in total or partial support of the research or clinical study presented in this article. The funding source was Grant R01HD31451 (J. O. H.) from the National Institute of Child Health and Human Development, National Institutes of Health.

Oregon Health Sciences University, 3181 S.W. Sam Jackson Park Road L-352A, Portland, Oregon 97201-3098.

THM Biomedical, 325 South Lake Avenue, Suite 608, Duluth, Minnesota 55802-2397.

Genetics Institute, One Burtt Road, Andover, Massachusetts 01810.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Asselmejer, M. A.; Caspari, R. B.; and Bottenfield, S.: A review of allograft processing and sterilization techniques and their role in transmission of the human immunodeficiency virus. Am. J. Sports Med., 21: 170-175, 1993.[Abstract/Free Full Text]
2. Bergsma, E. J.; Rozema, F. R.; Bos, R. R.; and de Bruijn, W. C.: Foreign body reactions to resorbable poly(L-lactide) bone plates and screws used for the fixation of unstable zygomatic fractures. J. Oral and Maxillofac. Surg., 51: 666-670, 1993.[Medline]
3. Böstman, O. M.: Intense granulomatous inflammatory lesions associated with absorbable internal fixation devices made of polyglycolide in ankle fractures. Clin. Orthop., 278: 193-199, 1992.
4. Böstman, O.; Partio, E.; Hirvensalo, E.; and Rokkanen, P.: Foreign-body reactions to polyglycolide screws. Observations in 24/216 malleolar fracture cases. Acta Orthop. Scandinavica, 63: 173-176, 1992.[Medline]
5. Bostrom, M.; Lane, J. M.; Tomin, E.; Browne, M.; Berberian, W.; Turek, T.; Smith, J.; Wozney, J.; and Schildhauer, T.: Use of bone morphogenetic protein-2 in the rabbit ulnar nonunion model. Clin. Orthop., 327: 272-282, 1996.
6. Celeste, A. J.; Ross, A. J.; Yamaji, N.; and Wozney, J. M.: The molecular cloning of human bone morphogenetic proteins-10, -11, and -12, three new members of the transforming growth factor-beta superfamily [abstract]. J. Bone and Min. Res., 10 (Supplement): 334, 1995.
7. Celeste, A. J.; Song, J. J.; Cox, K.; Rosen, V.; and Wozney, J. M.: Bone morphogenetic protein-9, a new member of the TGF-beta superfamily [abstract]. J. Bone and Min. Res., 9 (Supplement): 136, 1994.
8. Centers for Disease Control: Update: mortality attributable to HIV infection/AIDS among persons aged 25-44 years—United States, 1990 and 1991. Morbid. and Mortal. Weekly Rep., 42: 481-486, 1993.
9. Chu, C. C.: Degradation and biocompatibility of synthetic absorbable suture materials: general biodegradation phenomena and some factors affecting biodegradation. In Biomedical Applications of Synthetic Biodegradable Polymers, pp. 103-128. Edited by J. O. Hollinger. Boca Raton, CRC Press, 1995.
10. Cook, S. D.; Wolfe, M. W.; Salkeld, S. L.; and Rueger, D. C.: Effect of recombinant human osteogenic protein-1 on healing of segmental defects in non-human primates. J. Bone and Joint Surg., 77-A: 734-750, May 1995.[Abstract/Free Full Text]
11. Cook, S. D.; Baffes, G. C.; Wolfe, M. W.; Sampath, T. K.; and Rueger, D. C.: Recombinant human bone morphogenetic protein-7 induces healing in a canine long-bone segmental defect model. Clin. Orthop., 301: 302-312, 1994.
12. Cook, S. D.; Baffes, G. C.; Wolfe, M. W.; Sampath, T. K.; Rueger, D. C.; and Whitecloud, T. S., III: The effect of recombinant human osteogenic protein-1 on healing of large segmental bone defects. J. Bone and Joint Surg., 76-A: 827-838, June 1994.[Abstract/Free Full Text]
13. DeLustro, F.; Dasch, J.; Keefe, J.; and Ellingsworth, L.: Immune responses to allogeneic and xenogeneic implants of collagen and collagen derivatives. Clin. Orthop., 260: 263-279, 1990.
14. Dube, J. L., and Celeste, A. J.: Human bone morphogenetic protein-13, a molecule which is highly related to human bone morphogenetic protein-12 [abstract]. J. Bone and Min. Res., 10 (Supplement): 333, 1995.
15. Gerhart, T. N.; Kirker-Head, C. A.; Kriz, M. J.; Holtrop, M. E.; Hennig, G. E.; Hipp, J.; Schelling, S. H.; and Wang, E.: Healing segmental femoral defects in sheep using recombinant human bone morphogenetic protein. Clin. Orthop., 293: 317-326, 1993.
16. Hogan, B. L.: Bone morphogenetic proteins in development. Curr. Opin. Genet. and Devel., 6: 432-438, 1996.[Medline]
17. Hollinger, J. [editor]: Biomedical Applications of Synthetic Biodegradable Polymers. Boca Raton, CRC Press, 1995.
18. Hollinger, J. O., and Kleinschmidt, J. C.: The critical size defect as an experimental model to test bone repair materials. J. Craniofac. Surg., 1: 60-68, 1990.[Medline]
19. Hollinger, J. O., and Leong, K.: Poly(alpha-hydroxy acids): carriers for bone morphogenetic proteins. Biomaterials, 17: 187-194, 1996.[Medline]
20. Hollinger, J. O., and Schmitz, J. P.: Restoration of bone discontinuities in dogs using a biodegradable implant. J. Oral and Maxillofac. Surg., 45: 594-600, 1987.[Medline]
21. Hollinger, J. O.; Buck, D. C.; and Bruder, S.: Biology of bone healing: its impact on clinical therapy. Unpublished data.
22. Hollinger, J. O.; Brekke, J.; Gruskin, E.; and Lee, D.: Role of bone substitutes. Clin. Orthop., 324: 55-65, 1996.
23. Jarr, E., and Grainger, D.: Release of proteins from biodegradable poly-L-lactide matrices. Unpublished data.
24. Kenley, R.; Marden, L.; Turek, T.; Jin, L.; Ron, E.; and Hollinger, J. O.: Osseous regeneration in the rat calvarium using novel delivery systems for recombinant human bone morphogenetic protein-2 (rhBMP-2). J. Biomed. Mater. Res., 28: 1139-1147, 1994.[Medline]
25. Kenley, R. A.; Yim, K.; Abrams, J.; Ron, E.; Turek, T.; Marden, L. J.; and Hollinger, J. O.: Biotechnology and bone graft substitutes. Pharm. Res., 10: 1393-1401, 1993.[Medline]
26. Kingsley, D. M.: What do BMPs do in mammals? Clues from the mouse short-ear mutation. Trends Genet., 10: 16-21, 1994.[Medline]
27. Kingsley, D. M.: The TGF-beta superfamily: new members, new receptors, and new genetic tests of function in different organisms. Genes and Devel., 8: 133-146, 1994.[Free Full Text]
28. Lieber, R.: Statistical significance and statistical power in hypothesis testing. J. Orthop. Res., 8: 304-309, 1990.[Medline]
29. McKinney, L., and Hollinger, J. O.: A bone regeneration study: transforming growth factor-beta 1 and its delivery. J. Craniofac. Surg., 7: 36-45, 1996.[Medline]
30. Marden, L. J.; Hollinger, J. O.; Chaudhari, A.; Turek, T.; Schaub, R. G.; and Ron, E.: Recombinant human bone morphogenetic protein-2 is superior to demineralized bone matrix in repairing craniotomy defects in rats. J. Biomed. Mater. Res., 28: 1127-1138, 1994.[Medline]
31. Massagué, J.: The transforming growth factor-beta family. Ann. Rev. Cell Biol., 6: 597-641, 1990.
32. Massagué, J.; Cheifetz, S.; Laiho, M.; Ralph, D. A.; Weis, F. M.; and Zentella, A.: Transforming growth factor-beta. Cancer Surveys, 12: 81-103, 1992.[Medline]
33. Mayer, M.; Hollinger, J.; Ron, E.; and Wozney, J.: Maxillary alveolar cleft repair in dogs using recombinant human bone morphogenetic protein-2 and a polymer carrier. Plast. and Reconstr. Surg., 98: 247-259, 1996.
34. Nemzek, J. A.; Arnoczky, S. P.; and Swenson, C. L.: Retroviral transmission by the transplantation of connective-tissue allografts. An experimental study. J. Bone and Joint Surg., 76-A: 1036-1041, July 1994.[Abstract/Free Full Text]
35. Nguyen, A.-M.; Tran, M.; Oates, T.; Alvares, O.; and Cochran, D. L.: Mitogenic responses of human PDL cells to tissue growth factors [abstract]. J. Dent. Res., 74 (Supplement): 1918, 1995.
36. Nishitoh, H.; Ichijo, H.; Kimura, M.; Matsumoto, T.; Makishima, E.; Yamaguchi, A.; Yamashita, H.; Enomoto, S.; and Miyazono, K.: Identification of type I and type II serine/threonine kinase receptors for growth/differentiation factor-5. J. Biol. Chem., 271: 21345-21352, 1996.[Abstract/Free Full Text]
37. Parfitt, A. M.: Stereologic basis of bone histomorphometry; theory of quantitative microscopy and reconstruction of the third dimension. In Bone Histomorphometry: Techniques and Interpretation, pp. 53-83. Edited by R. R. Recker. Boca Raton, CRC Press, 1983.
38. Parfitt, A. M.; Drezner, M. K.; Glorieux, F. H.; Kanis, J. A.; Malluche, H.; Meunier, P. J.; Ott, S. M.; and Recker, R. R.: Bone histomorphometry: standardization of nomenclature, symbols, and units. Report of the ASBMR Histomorphometry Nomenclature Committee. J. Bone and Min. Res., 2: 595-610, 1987.[Medline]
39. Reddi, A. H.: Regulation of cartilage and bone differentiation by bone morphogenetic proteins. Curr. Opin. Cell Biol., 4: 850-855, 1992.[Medline]
40. Reddi, A. H.: Bone and cartilage differentiation. Curr. Opin. Genet. and Devel., 4: 737-744, 1994.[Medline]
41. Ripamonti, U.; Ma, S. S.; and Reddi, A. H.: Induction of bone in composites of osteogenin and porous hydroxyapatite in baboons. Plast. and Reconstr. Surg., 89: 731-739, 1992.
42. Ripamonti, U.; Ma, S. S.; Cunningham, N. S.; Yeates, L.; and Reddi, A. H.: Reconstruction of the bone—bone marrow organ by osteogenin, a bone morphogenetic protein, and demineralized bone matrix in calvarial defects of adult primates. Plast. and Reconstr. Surg., 91: 27-36, 1993.
43. Sandhu, H. S.; Kanim, L. E.; Kabo, J. M.; Toth, J. M.; Zeegan, E. N.; Liu, D.; Seeger, L. L.; and Dawson, E. G.: Evaluation of rhBMP-2 with an OPLA carrier in a canine posterolateral (transverse process) spinal fusion model. Spine, 20: 2669-2682, 1995.[Medline]
44. Schimandle, J. H.; Boden, S. D.; and Hutton, W. C.: Experimental spinal fusion with recombinant human bone morphogenetic protein-2. Spine, 20: 1326-1337, 1995.[Medline]
45. Schwartz, M. P., and Recker, R. R.: Comparison of surface density and volume of human iliac trabecular bone measured directly and by applied stereology. Calcif. Tissue Internat., 33: 561-565, 1981.[Medline]
46. Sporn, M. B., and Roberts, A. B.: Transforming growth factor-beta: recent progress and new challenges. J. Cell Biol., 119: 1017-1021, 1992.[Free Full Text]
47. Stevenson, S.; Cunningham, N.; Toth, J.; Davy, D.; and Reddi, A. H.: The effect of osteogenin (a bone morphogenetic protein) on the formation of bone in orthotopic segmental defects in rats. J. Bone and Joint Surg., 76-A: 1676-1687, Nov. 1994.[Abstract/Free Full Text]
48. Tomford, W. W.: Current concepts review. Transmission of disease through transplantation of musculoskeletal allografts. J. Bone and Joint Surg., 77-A: 1742-1754, Nov. 1995.[Free Full Text]
49. Toriumi, D. M.; Kotler, H. S.; Luxenberg, D. P.; Holtrop, M. E.; and Wang, E. A.: Mandibular reconstruction with a recombinant bone-inducing factor. Functional, histologic, and biomechanical evaluation. Arch. Otolaryngol.—Head and Neck Surg., 117: 1101-1112, 1991.
50. Vert, M.; Li, S.; and Garreau, H.: New insights on the degradation of bioresorbable polymeric devices based on lactic and glycolic acids. Clin. Mater., 10: 3-8, 1992.[Medline]
51. Wang, E. A.; Rosen, V.; D'Alessandro, J. S.; Bauduy, M.; Cordes, P.; Harada, T.; Israel, D. I.; Hewick, R. M.; Kerns, K. M.; LaPan, P.; Luxenberg, D. P.; McQuaid, D.; Moutsatsos, I. K.; Nove, J.; and Wozney, J. M.: Recombinant human bone morphogenetic protein induces bone formation. Proc. Nat. Acad. Sci., 87: 2220-2224, 1990.[Abstract/Free Full Text]
52. Winnier, G.; Blessing, M.; Labosky, P. A.; and Hogan, B. L.: Bone morphogenetic protein-4 is required for mesoderm formation and patterning in the mouse. Genes and Devel., 9: 2105-2116, 1995.[Abstract/Free Full Text]
53. Wintermantel, E.; Mayer, J.; Blum, J.; Eckert, K. L.; Lüscher, P.; and Mathey, M.: Tissue engineering scaffolds using superstructures. Biomaterials, 17: 83-91, 1996.[Medline]
54. Wozney, J. M.: The bone morphogenetic protein family and osteogenesis. Molec. Reproduct. and Devel., 32: 160-167, 1992.
55. Wozney, J.: Bone morphogenetic proteins and their expression. Cellular and Molecular Biology of Bone, pp. 131-165. Edited by M. Noda. San Diego, Academic Press, 1993.
56. Wozney, J. M.; Rosen, V.; Celeste, A. J.; Mitsock, L. M.; Whitters, M. J.; Kriz, R. W.; Hewick, R. M.; and Wang, E. A.: Novel regulators of bone formation: molecular clones and activities. Science, 242: 1528-1534, 1988.[Abstract/Free Full Text]
57. Yasko, A. W.; Lane, J. M.; Fellinger, E. J.; Rosen, V.; Wozney, J. M.; and Wang, E. A.: The healing of segmental bone defects induced by recombinant human bone morphogenetic protein (rhBMP-2). A radiographic, histological, and biomechanical study in rats. J. Bone and Joint Surg., 74-A: 659-670, June 1992.[Abstract/Free Full Text]

CiteULike

Connotea

Del.icio.us

Technorati

This article has been cited by other articles:

				S. Kokubo, K. Nozaki, S. Fukushima, K. Takahashi, K. Miyata, R. Fujimoto, and S. Yokota Recombinant Human Bone Morphogenetic Protein-2 as an Osteoinductive Biomaterial and a Biodegradable Carrier in a Rabbit Ulnar Defect Model Journal of Bioactive and Compatible Polymers, July 1, 2008; 23(4): 348 - 366. [Abstract] [PDF]

				H. J. Seeherman, K. Azari, S. Bidic, L. Rogers, X. J. Li, J. O. Hollinger, and J. M. Wozney rhBMP-2 Delivered in a Calcium Phosphate Cement Accelerates Bridging of Critical-Sized Defects in Rabbit Radii J. Bone Joint Surg. Am., July 1, 2006; 88(7): 1553 - 1565. [Abstract] [Full Text] [PDF]

				E. Vogelin, N.F. Jones, J.I. Huang, J.H. Brekke, and J.R. Lieberman Healing of a Critical-Sized Defect in the Rat Femur with Use of a Vascularized Periosteal Flap, a Biodegradable Matrix, and Bone Morphogenetic Protein J. Bone Joint Surg. Am., June 1, 2005; 87(6): 1323 - 1331. [Abstract] [Full Text] [PDF]

				R. B. Edwards III, H. J. Seeherman, J. J. Bogdanske, J. Devitt, R. Vanderby Jr., and M. D. Markel Percutaneous Injection of Recombinant Human Bone Morphogenetic Protein-2 in a Calcium Phosphate Paste Accelerates Healing of a Canine Tibial Osteotomy J. Bone Joint Surg. Am., July 1, 2004; 86(7): 1425 - 1438. [Abstract] [Full Text] [PDF]

				D. S. Musgrave, F. H. Fu, and J. Huard Gene Therapy and Tissue Engineering in Orthopaedic Surgery J. Am. Acad. Ortho. Surg., January 1, 2002; 10(1): 6 - 15. [Abstract] [Full Text] [PDF]

				M. L. Bouxsein, T. J. Turek, C. A. Blake, D. D'Augusta, X. Li, M. Stevens, H. J. Seeherman, and J. M. Wozney Recombinant Human Bone Morphogenetic Protein-2 Accelerates Healing in a Rabbit Ulnar Osteotomy Model J. Bone Joint Surg. Am., August 1, 2001; 83(8): 1219 - 1230. [Abstract] [Full Text]

				L. Lu, M. J. Yaszemski, and A. G. Mikos TGF-{beta}1 Release from Biodegradable Polymer Microparticles: Its Effects on Marrow Stromal Osteoblast Function J. Bone Joint Surg. Am., April 1, 2001; 83(90010): S82 - 92. [Abstract] [Full Text] [PDF]

				N. Saito, T. Okada, H. Horiuchi, N. Murakami, J. Takahashi, M. Nawata, H. Ota, S. Miyamoto, K. Nozaki, and K. Takaoka Biodegradable Poly-d,l-Lactic Acid-Polyethylene Glycol Block Copolymers as a BMP Delivery System for Inducing Bone J. Bone Joint Surg. Am., April 1, 2001; 83(90010): S92 - 98. [Abstract] [Full Text] [PDF]

				L. Lu, M. J. Yaszemski, and A. G. Mikos TGF-{beta}1 Release from Biodegradable Polymer Microparticles: Its Effects on Marrow Stromal Osteoblast Function J. Bone Joint Surg. Am., April 1, 2001; 83(1_suppl_2): S82 - S92. [Abstract] [Full Text] [PDF]

				N. Saito, T. Okada, H. Horiuchi, N. Murakami, J. Takahashi, M. Nawata, H. Ota, S. Miyamoto, K. Nozaki, and K. Takaoka Biodegradable Poly-d,l-Lactic Acid-Polyethylene Glycol Block Copolymers as a BMP Delivery System for Inducing Bone J. Bone Joint Surg. Am., April 1, 2001; 83(1_suppl_2): S92 - S98. [Abstract] [Full Text] [PDF]

				J. D. HECKMAN, W. EHLER, B. P. BROOKS, T. B. AUFDEMORTE, C. H. LOHMANN, T. MORGAN, and B. D. BOYAN Bone Morphogenetic Protein But Not Transforming Growth Factor-{beta} Enchances Bone Formation in Canine Diaphyseal Nonunions Implanted with a Biodegradable Composite Polymer*{{dagger}} J. Bone Joint Surg. Am., December 1, 1999; 81(12): 1717 - 29. [Abstract] [Full Text]

This Article Abstract Full Text (PDF) Free Letters to the Editor: Submit a response Alert me when this article is cited Alert me when Letters to the Editor are posted Alert me if a correction is posted Services E-mail this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Add to My File Cabinet Download to citation manager Reprints and Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by ZEGZULA, H. D. Articles by HOLLINGER, J. O. Search for Related Content PubMed PubMed Citation Articles by ZEGZULA, H. D. Articles by HOLLINGER, J. O. Social Bookmarking             What's this?

Stanford University Libraries' HighWire Press (TM) assists in the publication of JBJS Online.
Copyright © 1997 by The Journal of Bone and Joint Surgery, Inc. Online ISSN: 1535-1386.
The Journal of Bone and Joint Surgery®, JBJS®, JB&JS®, and eJBJS® are registered in the U.S. Patent and Trademark Office.