Bone substitute with osteoinductive biomaterials — Current and future clinical applications

Int. Z Oral Maxillofac. Surg. 1994; 23:413 417 Printed in Denmark. All rights reserved

Copyright © Munksgaard 1994 lntemationalJoumd of

Oral8L MaxillofacialSurgery ISSN 0901-5027

Bone substitute with osteoinductive biomaterialscurrent and future clinical applications

G. Hotz 1, G. Herr 2 1Department of Maxillofacial Surgery, University of Heidelberg; 2Department of Orthopedics, Bone Research Laboratory, University of TObingen, Germany

G. Hotz, G. Herr." Bone substitute with osteoinductive biomaterials - current and future clinical applications. Int. J. Oral Maxillofac. Surg. 1994; 23: 413-417. © Munksgaard, 1994 Abstract. In craniomaxillofacial surgery, possible indications for the use of osteoinductive biomaterials are interposition in intraosseous defects, contour augmentation, and reconstruction of segmental defects. The experimental results in the field of bone morphogenetic protein (BMP) research within the last few years have shown that it is possible to combine osteoinductive proteins with suitable carrier materials to obtain new composite osteoinductive biomaterials. These carrier materials function as slow-delivery systems for BMR By combination of BMP with different carrier materials such as various types of calcium phosphate ceramics, collagen or inactive collagenous bone matrix, and other organic and inorganic carriers, the biomaterial can be adapted to clinical demands in a wide range. In several experimental animal studies, we investigated nine different calcium phosphate ceramics and inactive rat bone matrix for their use as BMP carrier. All materials tested seem to be suitable carriers for BMR The first clinical applications are discussed.

Osteoinductive biomaterials exhibit a new biologic quality. Under the influence of an osteoinductive substance, bone morphogenetic protein (BMP), they can transform primary nonosteogenic cells into osteoblasts. An osteoinductive agent alone, without a carrier, would fail to elicit bone formation. When implanted without a carrier in a heterotropic site, the proteins tend to diffuse too rapidly, before induction can occur. One of the problems in BMP research is how to develop appropriate delivery systems for expression of maximum BMP activity. Delivery systems for intraosseous application should be rapidly degraded and replaced by induced bone. Delivery systems for reconstruction of segmental defects should

sustain the shape and bulk until new bone replaces the carrier. For contour augmentation, we need a delivery system that prevents dislocation and retains the shape and bulk of the reconstruction. Experimental results in the field of BMP research within the last few years have shown that it is possible to combine osteoinductive proteins with suitable carrier materials to obtain new composite osteoinductive biomaterials (Table 1). These carrier materials function as slow-delivery systems for BMP. By the combination of BMP with different carrier materials such as various types of calcium phosphate ceramics, collagen or inactive collagenous bone matrix, and other organic and inor-

Key words: bone substitute; bone induction; bone morphogenetic protein; osteoinductive biomaterial; delivery system; carrier; calcium phosphate ceramic; hydroxyapatite. Accepted for publication 1 January 1994

ganic carriers, the biomaterial can be adapted to a wide range of clinical demands. This study investigated the efficacy of calcium phosphate ceramics of different degradability as carriers for BMR Material and methods

We investigated nine different calcium phosphate ceramics and inactive rat bone matrix (IBM) for application as BMP carriers (Table 2). Ceramic materials were used because of their well-known binding affinity to BMR Different porosities have been tested to clarify the influence of adsorptive surface and pore diameter on BMP coating and bony ingrowth, pBMP was isolated from long bones of pigs under dissociative conditions with GuHC1 and partially purified by gel

414

Hotz and Herr adult rats. The specimens were explanted after 25 days and studied by histologic examination, and the amount of bone formation was quantified by the alkaline phosphatase (AP) activity in the explanted tissues with nitrophenylphosphate as substrate.

Table 1. Delivery systems for osteoinductive biomaterials Anorganic p-Tricalcium phosphate (fl-TCP) Hydroxyapatite (HA) Calcium sulfate (CS) HA+CS

URIST et al. 198424 KAWAMURaet al. 198713 YAMAZAKIet al. 198827 DAMIENet al. 19903

Organic Collagen Fibrin sealant (FS) Glycerol

DEATHERAGE& MILLER 19875 KAWAMURA& URIST 198814 DAMIENet al. 19924

Compound HA/collagen HA+FS HA+ glycerol

TAKAOKAet al. 19882° HOTZ 1991s DAMIENet al. 19924

Results

Table 2. Bone-inductive proteins adsorbed to calcium phosphate ceramics of different pore size Carrier

Origin/porosity

rIBM Frialit Algipore Interpore 200 Osprovit Ceros 80 Endobon Ceros 82 Ceros 82 Ceros 82

Rat inactive bone matrix Synthetic dense HA Algae-derived microporous HA Coralline macroporous HA Synthetic macroporous/microporous HA Synthetic macroporous HA Bovine cancellous bone Synthetic macroporous (60%) TCP Synthetic macroporous (80%) TCP Synthetic macroporous (90%) TCP

Induced bone 16/16 16/16 16/16 16/16 16/16 12/16 16/16 16/16 16/16 16/16

The control implants coated with 2 mg of an inactive protein fraction showed no osteogenic activity. All osteoinductive biomaterials tested seemed to be basically suitable carriers for BMP, because incidence of bone formation was high in each group, ranging from 75 to 100% (Table 2). With BMP-coated I B M as standard carrier (100%), only Algipore exhibited an increase in A P activity to 250%. The quantitative activity of Osprovit was 80% and Frialit 20% of that of BMP-coated I B M 6. The quantitative differences in A P activity found among the various calcium phosphate ceramics seemed to stem from their differing specific surface available for protein coating. Implants of BMP-coated I B M formed macroscopically visible ossicles with a shell of lamellar bone and central bone-marrow elements with hematopoietic and fatty tissue (Fig. 1). Implants of BMP-coated hydroxyaparite (HA) granules formed similar ossicles with lamellar-bone and bone-marrow elements. Bone formation took place on the outer and inner surfaces in particular, within the macropores of the ceramic. T h r o u g h o u t this strong peri- and interparticular bone formation, a composite bone tissue had developed. The bone was in direct contact with the surface of the granules, and bone trabeculae connected the granules with each other (Fig. 2).

Discussion

Fig. 1. Undecalcified section of intramuscularly implanted BMP compounded with IBM. Implanted graft forms ossicle with shell of lamellar bone (B), and central bone-marrow elements (BM) with hematopoietic and fatty tissue. At 25 days, there is still bone formation, as indicated by dark-stained osteoid seam (arrow) (Masson Goldner staining, x 10).

filtration on a Sepharose-CL-6B column (90×5 cm), as described previously 1,7,17. Two milligrams of the resulting pBMP fraction was precipitated onto equal volumes (80/A) of nine different calcium phosphate ceramics and inactive bone matrix, as previously de-

scribed 19. The osteogenic activity of these composite implant materials and corresponding controls were examined in vivo in an ectopic bone-formation assay in rats 6. These materials were implanted into abdominal wall muscle pouches of immunodeficient

F o r m a t i o n of n e w , b o n e can occur by three different processes: osteogenesis, osteoconduction, and osteoinduction. Osteogenesis occurs when viable osteoblasts and preosteoblasts are autogeneically transplanted to f o r m new bone. Such cells can be provided by cancellous bone and marrow grafts. Osteoconduction occurs when large segments of cortical bone or allogeneic bone are used to bridge bony defects. These bone transplants serve as a passive scaffold, which is slowly resorbed and replaced by new bone derived from the edges of the defect (creeping substitution). Osteoinduction involves new bone forma-

Osteoinductive biomaterials

Fig. 2. Intramuscularly implanted, BMP-coated macroporous HA (Interpore 200) removed 25 days after implantation. Network of cross-linked bone formation (B) appears on outer and inner surfaces, in particular, within macropores of ceramic (toluidine blue staining, ×25).

tion from osteoprogenitor cells derived from perivascular mesenchymal cells under the influence of BMP 21 23 BMP is used to repair bony defects without the need for autogeneous bone harvesting. The potential applications for human BMP are bony defects caused by trauma, infection, and malignancy, and developmental malformations. Crude demineralized allogeneic bone matrix implants have been used clinically for more than 10 years 1° 12,16.

MULLIKEN et a1.16 have used demineralized implants prepared from cadaver femora to repair human maxillocraniofacial deformities. Nearly 90% of the implant sites that could be physically examined were solid by 3 months. However, without a carrier material, the water-soluble purified protein fractions are incapable of inducing cartilage or bone in vivo in extraskeletal sites. HA granules alone are also incapable of inducing osteogenesis in vivo. However, when os-

teoinductive proteins were adsorbed to the surface and in the pores of calcium phosphate ceramics of different porosity, new bone formation was observed on the surface and in the macropores. The pores of HA merely play the role of a scaffold for new bone formation and certainly do not have osteogenetic ability themselves. This role is sometimes called "osteoconductivity''9. If an osteogenetic protein such as BMP could preserve its biologic properties after being implanted together with HA into living tissue, such a composite material would be considered to be an "osteoinductive biomaterial". In animal experiments, URIST et al. 24 induced new bone formation with a degradable/~-tricalcium phosphate (TCP) delivery system for BMP in muscle pouches. It seems to be possible to create osteoinductive biomaterials for specific clinical demands by the combination of BMP with carrier materials of different chemical composition and different physical properties. Including IBM, we have examined 10 different carrier materials. The spectrum of materials includes easily resorbable organic collagenous bone matrix, resorbable TCP ceramics, Algipore as degradable HA, Interpore, Osprovit Ceros 80 and Endobon as less degradable HA, and Fialit as a nonresorbable HA ceramic. All tested materials seem to be suitable as slow-delivery systems for BMP, because the incidence of bone for-

Table 3. Osteoinductive bone matrix proteins

Bone morphogenetic protein

Member of TGF-/? superfamily

Calculated mol. mass (kDa) Osteoinductive properties

BMP-1 WOZNEVet al. (1988) 26

-

50

Cartilage induction

BMP-2 (BMP-2A) WOZNEYet al. (1988)a6 WANGet al. (1990)25

+

13

Cartilage and bone induction also by recombinant protein

BMP-3 (osteogenin) WOZNEYet al. (1988) 26

+

15

Cartilage and bone induction

BMP-4 (BMP-2B) WOZNEYet al. (1988)26

+

13

Cartilage and bone (.9)induction

BMP-5 CELESTEet al. (1990)2

+

16

Cartilage and bone (?) induction

BMP-6 CELESTEet al. (1990)2

+

16

Cartilage and bone (.9)induction

BMP-7 (OP1) CELESTEet al. (1990)2

+

16

Cartilage and bone induction

12

Cartilage and bone induction only in presence of TGF-fl

OIF MADISENet al, (1990)is ROSENet al. (1990)28 TGF-fl: transforming growth factor-beta.

41 5

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Hotz and Herr

Fig. 3. A) Profile of 20-year-old woman after orthodontic treatment. B) Postoperative profile after genioplasty and contour augmentation with BMP/HA. C) Biopsy taken 6 months after implantation shows network of cross-linked lamellar bone (B) in direct ceramo-osseous contact with HA particles (toluidine blue staining, ×40).

marion was high in each group, ranging from 75% (Ceros 80) to 100% for all other tested ceramics. In addition to their different biologic degradability, the materials tested showed great differences in their mechanical stability. For unloaded contour augmentation in maxillofacial surgery, a macroporous delivery system can be used. The pores act as a scaffold for new bone formation. For the reconstruction of a loadbearing area, we used dense granules as carrier and spacer. Because of the stability of the carrier, the final shape of " the resulting composite bone tissue can

be predicted. In the last 2 years, we have tested clinically the osteoinductive biomaterial BMP/HA for contour augmentation in man s. In the case of a 20year-old woman, after orthodontic treatment, genioplasty and contour augmentation with BMP/HA bone substitute were done to correct the profile. A biopsy taken 6 months after implantation showed mature lamellar bone with a high binding affinity between the bone and the HA particles (Fig. 3). Our experimental results and preliminary clinical experience may be summarized as follows: calcium phosphate

ceramics are suitable as delivery systems for BMR The osteoinductive biomaterial induces bone formation when placed in the rat muscle model. Ossicle formation with trabecular bone and marrow-cell elements surrounded by lamellar bone was noted at 4 weeks. In addition to their different biologic degradability, the materials tested show great differences in mechanical stability. It seems to be possible to create osteoinductive biomaterials for specific clinical demands by the combination of BMP with carrier materials of different chemical composition and different physical properties: resorbable TCP or Algipore to fill intraosseous defects or bony pockets and furcation defects in periodontology; macroporous delivery systems for reconstruction of unloaded contour augmentations. The pores thereby act as a scaffold for new bone formation. For reconstruction of a load-bearing area, e.g., the augmentation of the severely atrophic alveolar ridge, the final shape of the resulting composite bone tissue can be predicted if BMP is compounded with dense nonresorbable HA granules. The clinical use of hEMP is still in its infancy, but the initial results indicate a potential for wide application if sufficient material can be produced. In craniomaxillofacial surgery, possible indications for the use of osteoinductive biomaterials are interposition in intraosseous defects, contour augmentation, and reconstruction of segmental defects. To be used in these three different fields of clinical application, osteoinductive biomaterials should fulfill quite different requirements with respect to their chemical, biologic, and mechanical properties. At present, the limiting factor is the small amount of purified hEMP available from cadaver bone. By means of recombinant DNA technology, the i~olation and expression of seven different human proteins from highly purified preparations of BMP have been described (Table 3). We are now waiting for the first commercially available recombinant human BMR

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Address:

Gfinter Hotz, MD, DDS, PhD Department of Maxillofacial and Plastic Surgery University Hospital of Heidelberg Im Neuenheimer Feld 400 69120 Heidelberg Germany