JBJS | Bone Induction by BMPs/OPs and Related Family Members in Primates : The Critical Role of Delivery Systems

The Journal of Bone & Joint Surgery, Volume 83, Issue 1 suppl 2

Delivery Systems for the BMPs | April 01, 2001

Bone Induction by BMPs/OPs and Related Family Members in Primates : The Critical Role of Delivery Systems

Ugo Ripamonti, MD, PhD; Lentsha Nathaniel Ramoshebi, PhD; Thato Matsaba, MSc; Jacqueline Tasker, MSc; Jean Crooks, MSc; June Teare, DMT

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Investigation performed at Bone Research Unit, South African Medical Research Council/University of the Witwatersrand, Medical School, Johannesburg, South Africa
Ugo Ripamonti, MD, PhD
Lentsha Nathaniel Ramoshebi, PhD
Thato Matsaba, MSc
Jacqueline Tasker, MSc
Jean Crooks, MSc
June Teare, DMT
Bone Research Unit, MRC/University of the Witwatersrand, 7 York Road, Parktown 2193, Johannesburg, South Africa. E-mail address for U. Ripamonti: 177ripa@chiron.wits.ac.za

In support of their research or preparation of this manuscript, one or more of the authors received grants or outside funding from the South African Medical Research Council and University of the Witwatersrand. The authors choose not to provide The Journal and its readers with information concerning any commercial party and any material in this Work, which relationship may represent a conflict of interest.

The Journal of Bone & Joint Surgery. 2001; 83:S116-S127

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Abstract

Background: In a series of studies in the primate Papio ursinus, we have examined the capacity of bone morphogenetic proteins (BMPs/OPs) delivered in a variety of biomaterial carrier systems to elicit bone formation in heterotopic and orthotopic sites. In this review, we compare the osteoinductive effects of different biomaterial delivery systems that have or have not been pretreated with BMPs/OPs. In particular, we focus on the geometric induction of bone formation by sintered porous hydroxyapatite (SPHA) discs with concavities on their planar surfaces, which elicit bone formation without exogenously applied BMPs/OPs.

Methods: Heterotopic bone formation was examined by bilaterally implanting 100-mg pellets of a collagenous carrier containing BMPs/OPs in the rectus abdominis muscle of the adult baboon. Orthotopic bone formation was examined by implanting 1 g of a collagenous carrier containing BMPs/OPs into two full-thickness critical-sized 25-mm-diameter defects on each side of the calvaria of adult baboons. The BMPs/OPs whose effects were examined included recombinant human osteogenic protein-1 (rhOP-1), recombinant human transforming growth factor-ß1 (rhTGF-ß1), rhTGF-ß2, and porcine platelet derived transforming growth factor-ß1 (pTGF-ß1). Tissue from the rectus abdominis muscle was harvested 30 or 90 days after implantation. Tissue from the orthotopic calvarial model was examined at 1, 3, 6, 9, and 12 months after implantation. To demonstrate the effect of surface geometry on bone induction, hydroxyapatite powders were sintered to form solid discs with a series of concavities on the planar surfaces of the SPHA discs. The discs were either pretreated with exogenous rhOP-1 or not treated with exogenous OP-1. They were then implanted heterotopically or orthotopically into calvarial defects. Bone formation was evaluated histologically in undecalcified sections stained with Goldner’s trichrome stain or 0.1% toluidine blue.

Results: Naturally derived BMPs/OPs or rhOP-1 in a collagenous carrier elicit heterotopic bone formation and the complete healing of 25-mm-diameter critical-sized defects by day 90 following implantation. Binary applications of TGF-ß1 together with rhOP-1 in the collagen carrier induced massive endochondral ossicles in heterotopic sites and bone formation in calvarial defects. pTGF-ß1, rhTGF-ß1, and rhTGF-ß2 are powerful inducers of heterotopic endochondral bone formation but elicit limited bone formation in calvarial defects. SPHA discs pretreated with rhOP-1 elicited extensive bone formation in both heterotopic and orthotopic sites. However, SPHA without rhOP-1 also elicited bone formation in heterotopic and orthotopic sites and complete healing of the calvarial defects.

Conclusion: We have prepared SPHA discs with concavities on their planar surfaces that induce bone formation in heterotopic or orthotopic critical-sized calvarial defects without exogenously applied BMPs/OPs. This biomaterial induces bone formation by intrinsic osteoinductivity regulated by the geometry of the substratum. The incorporation of specific biological activities into biomaterials by manipulating the geometry of the substratum, defined as geometric induction of bone formation, may make it possible to engineer morphogenetic responses for therapeutic osteogenesis in clinical contexts.

Clinical Relevance: We have implemented a clinical trial using naturally derived BMPs/OPs extracted and purified from bovine bone matrices and implanted in craniofacial defects in humans. In addition, the discovery that specific geometric and surface characteristics of sintered hydroxyapatites can induce intrinsic osteoinductivity in primates paves the way for formulation and therapeutic application of porous substrata designed to obtain predictable intrinsic osteoinductivity in clinical contexts.

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Introduction

Introduction | Materials and Methods | Results | Discussion | References

The bone morphogenetic proteins (BMPs/OPs) are macromolecules involved in the formation of bone during embryonic development and the induction of bone formation and regeneration in postnatal osteogenesis. Studies on BMPs/OPs and related gene products of the transforming growth factor-ß (TGF-ß) superfamily have suggested that the emergence in postnatal life of complex tissue morphologies rests on a surprisingly simple and fascinating concept: morphogens involved in embryonic development can be re-deployed for the initiation of postnatal morphogenesis and regeneration^16,17,38. Extensive studies in animal models, particularly nonhuman primates, have made possible the clinical use of both recombinant human osteogenic protein-1 (rhOP-1) and rhBMP-2 for craniofacial and orthopaedic applications in humans^{13,14,16,17,29,60}.

Several recent studies have emphasized that the BMPs/OPs are involved in morphogenesis, axial growth, hard tissue development and repair, and tooth morphogenesis, including but not limited to organs and tissues as different as bone, cartilage, kidney, the periodontal ligament, dentine, the root cementum, and the central and peripheral nervous system including the cerebellum^{14,16,17,28,36,38,56}. The BMPs/OPs are gene products that have pleiotropic functions encompassing the developmental mechanisms of tissues and organs architecturally engineered also by epithelial-mesenchymal interactions, a central mechanism regulating morphogenesis and differentiation in embryonic and postnatal life^14,16,17.

Our unit has used baboon species (Papio ursinus) that share similar if not identical bone physiology and remodeling with humans⁵⁵ to test naturally derived or recombinantly produced BMPs/OPs for craniofacial and periodontal regeneration^23,25,27. In this review, we describe the heterotopic endochondral osteoinductivity of TGF-ß proteins in the primate, as well as the synergistic interactions of TGF-ß1 with rhOP-1, in heterotopic and orthotopic sites of adult baboons^3,37,48.

In addition, we describe the spontaneous and intrinsic osteoinductivity of novel smart sintered hydroxyapatite biomaterial matrices that are helping to elicit morphogenetic responses for therapeutic osteogenesis and tissue engineering in clinical contexts at low doses of rhBMP-2 or rhOP-1. We also discuss the generation of bone by the implantation of smart biomaterials that in their own right can induce a desired and specific morphogenetic response from the host tissues without the addition of exogenously applied BMPs/OPs^32,39,41,43. Specifically, we have developed sintered hydroxyapatites capable of inducing bone formation in adult primates by intrinsic osteoinductivity regulated by the geometry of the substratum, promoting desirable and coordinated responses based on a memory of embryonic developmental events when implanted in heterotopic sites of adult baboons, without exogenously applied BMPs/OPs^39,41,44,45.

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Fig. 1:: Fig. 1 Bone induction by naturally derived bone morphogenetic proteins (BMPs/OPs) implanted in adult primates (Goldner’s trichrome stain). Figs. 1-A and 1-B Orthotopic induction of bone 30 days after implantation of 280 g of baboon osteogenic fractions delivered by allogeneic collagenous matrix in calvarial defects of an adult primate of the genus Papio. Arrows (Fig. 1-B) indicate large osteoid seams populated by contiguous osteoblasts. Fig. 1-C Mineralized bone covered by continuous osteoid seams in a specimen of a patient treated with naturally derived BMPs/OPs implanted in a large mandibular defect at 90 days after implantation. Fig. 1-D Histological appearance of a human mandibular defect treated with an autogenous bone graft 90 days after grafting. Resorption of the graft with absent osteoblastic activity.

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Fig. 2:: Fig 2 Low-power photomicrographs of specimens of calvarial defects of adult baboons implanted with bone morphogenetic proteins (BMPs/OPs). Fig. 2-A Naturally derived BMPs/OPs, 2.5 mg, delivered by the g-irradiated bovine collagenous matrix 90 days after implantation. Figs. 2-B and 2-C g-irradiated human osteogenic protein-1 (hOP-1), 0.5 mg, and Fig. 2-D hOP-1, 2.5 mg, 12 months after implantation in calvarial defects of adult baboons and delivered by xenogeneic bovine collagenous matrix.

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Fig. 3:: Fig. 3 Tissue morphogenesis and heterotopic bone induction by transforming growth factor-ß1 (TGF-ß1) in the rectus abdominis muscle of adult baboons 30 days after implantation. Fig. 3-A Bone induction after implantation of 5 g recombinant human (rh) TGF-ß1. Fig. 3-B Synergistic tissue morphogenesis by the combinatorial action of 125 g human osteogenic protein-1 (hOP-1) and 5 g rhTGF-ß1. Fig. 3-C Bone induction by 25 g of hOP-1. Fig. 3-D Heterotopic bone induction by 5 g porcine transforming growth factor-ß1 (pTGF-ß1). Fig. 3-E Heterotopic ossicle induced by the application of 25 g hOP-1 and 5 g pTGF-ß1. Fig. 3-F Juxtaposed heterotopic ossicles induced by a binary application of hOP-1 and human transforming growth factor-ß1 (hTGF-ß1) with classic features of chondrogenesis and osteogenesis as seen in the embryonic growth plate.

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Fig. 4:: Fig. 4 Endochondral bone induction in the rectus abdominis muscle by doses of recombinant human transforming growth factor-ß2 (rhTGF-ß2) 30 and 90 days after implantation. Fig. 4-A rhTGF-ß2, 5 g. Fig. 4-B Islands of chondrogenesis in an ossicle induced by 25 g rhTGF-ß2. Figs. 4-C and 4-D Ossicles generated 90 days after implantation of 1 and 5 g rhTGF-ß2 delivered by allogeneic collagenous matrix in adult baboons. Fig. 4-E Spontaneous and intrinsic bone morphogenesis in a specimen of a sintered porous hydroxyapatite (SPHA) 90 days after implantation in the rectus abdominis muscle. Fig. 4-F Bone induction in heterotopic sites in a specimen of SPHA pretreated with 1 g rhTGF-ß2.

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Fig. 5:: Fig. 5 Low-power photomicrographs of calvarial defects treated by bone morphogenetic proteins (BMPs/OPs) with or without the addition of transforming growth factor-ß1 (TGF-ß1). Fig. 5-A Human osteogenic protein-1 (hOP-1), 100 g, 30 days after implantation. Fig. 5-B Binary application of 100 g hOP-1 and 5 g porcine transforming growth factor-ß1 (pTGF-ß1), 30 days after implantation, showing extensive bone differentiation. Fig. 5-C hOP-1, 20 g, 90 days after implantation. Fig. 5-D hOP-1, 20 g, and pTGF-ß1, 5 g, 90 days after implantation. Fig. 5-E hOP-1, 100 g, 90 days after implantation showing complete regeneration of the calvarial defect. Fig. 5-F hTGF-ß2, 100 g, 90 days after implantation (note limited bone differentiation across the defect but only pericranially).

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Fig. 6:: Fig. 6 Influence of geometry on bone induction and morphogenesis. Fig. 6-A Bone differentiation exclusively in concavities on the planar surfaces of a solid disc of sintered hydroxyapatite implanted in the rectus abdominis muscle of an adult baboon and harvested on day 90. Fig. 6-B Substantial bone induction in a specimen of a sintered porous hydroxyapatite (SPHA) harvested from the rectus abdominis muscle of an adult primate 90 days after implantation. Fig. 6-C In a specimen of SPHA pretreated with 5 g human osteogenic protein-1 (hOP-1), bone morphogenesis is also preferentially initiated in concavities of the substratum.

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Fig. 7:: Fig. 7 Low-power photomicrographs of sintered porous hydroxyapatites (SPHAs) pretreated with doses of human osteogenic protein-1 (hOP-1) and implanted in calvarial defects of adult baboons. Figs. 7-A, 7-B, and 7-C Specimens pretreated with 500 mg hOP-1 on day 30 (Fig. 7-A), 90 (Fig. 7-B), and 365 (Fig. 7-C) after implantation in calvarial defects. Fig. 7-D Bone differentiation throughout the porous interconnected spaces of a sintered hydroxyapatite without exogenously applied hOP-1 as control and harvested 90 days after implantation. (Reprinted with permission from: Ripamonti U, Crooks J, Rueger DC. Induction of bone formation by recombinant osteogenic protein-1 (hOP-1) and sintered porous hydroxyapatite in adult primates. Plastr Reconstr Surg. 2001; in press.)

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Fig. 8:: Fig. 8 Pre-clinical application of implants with osteoinductive geometric configurations. Fig. 8-A A hydroxyapatite-coated titanium implant with a series of concavities prepared on the coated surface is implanted in the edentulous ridge of an adult primate. Fig. 8-B Examination of undecalcified titanium/bone sections shows continuous osseointegration along the geometric surface of the implant as early as 30 and 90 days after surgical insertion.

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TABLE I: Heterotopic Morphogens: Implantation and Number of Primates

Treatment Group	Number of Primates	Implant Doses	Collagenous Matrix	Number of Implants
Naturally derived BMPs/OPs	8	80 mg	100 mg	?16
hOP-1	8	5, 25, and 125 mg	100 mg	128
PTGF-ß1	2	0.5, 1.5, and 5 mg	100 mg	??8
TGF-ß2	4	1, 5, and 25 mg	100 mg	?32
h-OP1/pTGF-ß1	8	25 mg/(0.5, 1.5, and 5 mg)	100 mg	?96
h-OP1/hTGF-ß1	6	25 mg/(0.5, 1.5, and 5 mg)	100 mg	?72

BMPs/OPs, bone morphogenetic proteins/osteogenic proteins; hOP-1, human osteogenic protein-1; PTGF-ß1, porcine transforming growth factor-ß1; TGF-ß2, transforming growth factor-ß2; h-OP1/pTGF-ß1, human osteogenic protein-1/porcine transforming growth factor-ß1; and h-OP1/hTGF-ß1, human osteogenic protein-1/human transforming growth factor-ß1.

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TABLE I: Heterotopic Morphogens: Implantation and Number of Primates

Treatment Group	Number of Primates	Implant Doses	Collagenous Matrix	Number of Implants
Naturally derived BMPs/OPs	8	80 mg	100 mg	?16
hOP-1	8	5, 25, and 125 mg	100 mg	128
PTGF-ß1	2	0.5, 1.5, and 5 mg	100 mg	??8
TGF-ß2	4	1, 5, and 25 mg	100 mg	?32
h-OP1/pTGF-ß1	8	25 mg/(0.5, 1.5, and 5 mg)	100 mg	?96
h-OP1/hTGF-ß1	6	25 mg/(0.5, 1.5, and 5 mg)	100 mg	?72

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TABLE II: Orthotopic Morphogens: Implantation and Number of Primates

Treatment Group	Number of Primates	Implant Doses	Collagenous Matrix	Number of Implants
Naturally derived BMPs/OPs	26	1.8 and 280 mg	1 g	84
	10	280 mg	1 g	40
hOP-1	14	0.1, 0.5, and 2.5 mg	1 g	56
g-irradiated hOP-1	24	0.1, 0.5, and 2.5 mg	1 g	80
hOP-1/pTGF-ß1	?8	100 mg/(5, 15 mg)	1 g	32
		20 mg/5 mg
SPHAs	?4	—	—	16
SPHAs + hOP-1	12	0.1, 0.5, and 2.5 mg	—	48
Coral-derived HA	24	—	—	96
	?8	—	—	32
Coral-derived HA + naturally derived BMPs/OPs	?8	150 mg	—	32

BMPs/OPs, bone morphogenetic proteins/osteogenic proteins; hOP-1, human osteogenic protein-1; hOP-1/pTGF-ß1, human osteogenic protein-1/porcine transforming growth factor-ß1; SPHAs, sintered porous hydroxyapatites; and HA, hydroxyapatite.

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TABLE II: Orthotopic Morphogens: Implantation and Number of Primates

Treatment Group	Number of Primates	Implant Doses	Collagenous Matrix	Number of Implants
Naturally derived BMPs/OPs	26	1.8 and 280 mg	1 g	84
	10	280 mg	1 g	40
hOP-1	14	0.1, 0.5, and 2.5 mg	1 g	56
g-irradiated hOP-1	24	0.1, 0.5, and 2.5 mg	1 g	80
hOP-1/pTGF-ß1	?8	100 mg/(5, 15 mg)	1 g	32
		20 mg/5 mg
SPHAs	?4	—	—	16
SPHAs + hOP-1	12	0.1, 0.5, and 2.5 mg	—	48
Coral-derived HA	24	—	—	96
	?8	—	—	32
Coral-derived HA + naturally derived BMPs/OPs	?8	150 mg	—	32

Materials and Methods

Introduction | Materials and Methods | Results | Discussion | References

Primate Models for Tissue Induction and Morphogenesis

Histomorphometric studies of Papio ursinus iliac crest biopsies showed a remarkable degree of similarity with human biopsies, making the adult baboon ideally suited for the study of comparative bone physiology and repair with relevance to humans⁵⁵. More than one hundred and sixty clinically healthy adult Chacma baboons (Papio ursinus), selected from the primate colony of the Central Animal Services of the University of the Witwatersrand, Johannesburg, were used to test a variety of devices with osteoinductive activity in heterotopic and orthotopic sites (Tables I and II). Criteria for selection, housing conditions, and diet were as described^20,21. Research protocols were approved by the Animal Ethics Screening Committee of the University and were conducted according to the Guidelines for the Care and Use of Experimental Animals prepared by the University and in compliance with the National Code for Animal Use and Research, Education and Diagnosis in South Africa.

Purification of Naturally Derived BMPs/OPs

Naturally derived BMPs/OPs were prepared from baboon or bovine bone and characterized, as previously described^23,25,47,52. Protein fractions obtained from the various steps of purification were tested for osteogenic activity in the subcutaneous space of Long-Evans rats, and the activity was assessed by tissue alkaline phosphatase, calcium content, and histology. Doses of osteogenic protein fractions used for implantation in nonhuman and human primates ranged from 80 g to 2.5 mg and were delivered by collagenous matrix or porous hydroxyapatites as shown in Tables I and II for heterotopic and orthotopic studies, respectively^{4,23-25,27,42,47}.

Preparation of rhOP-1 Devices

Mature rhOP-1 was provided by Creative BioMolecules and Stryker Biotech (Hopkinton, Massachusetts). Stock solutions of hOP-l prepared in 50% ethanol, 0.01% trifluoroacetic acid were aliquoted in doses of 0.1, 0.5, and 2.5 mg hOP-1, combined with 1 g of allogeneic baboon or xenogeneic bovine collagenous matrices, and lyophilized to produce the hOP-1 devices^35,37,47. In some experiments, the devices were sterilized at ambient temperature with g-irradiation using an irradiation dose of approximately 0.3 Mrads/hour for a total of 2.5-3.0 Mrads^47,50. To reduce the xenoantigenic load, the xenogeneic bovine collagenous matrix was additionally treated with 0.1 M acetic acid at 55°C for 1 hour, washed with distilled water, and dried^35,47.

Naturally Derived pTGF-ß1, rhTGF-ß1, and hTGF-ß2

pTGF-ß1 was purified from lyophilized porcine platelets as described³. rhTGF-ß1² was provided by Genentech Inc. (South San Francisco, California)³⁷and hTGF-ß2 was from Genzyme Corporation (Framingham, Massachusetts)⁴⁸. Devices for implantation were prepared by adding 5 g of hTGF-ß1 and 5, 30, and 100 g hTGF-ß1 to 100 mg and 1 g of lyophilized collagenous bone matrix as carrier for the preparation of heterotopic and orthotopic implants, respectively^33,37. Two different carrier matrices were used for the local delivery of hTGF-ß2, as optimal tissue induction is known to be dependent on the combined action of the molecular signal with the complementary substratum, which triggers the bone differentiation cascade³⁰.

Doses of hTGF-ß2 for heterotopic implantation in the rectus abdominis muscle were prepared by adding 1, 5, and 25 g of hTGF-ß2 in 100 l of liquid vehicle (35% ethanol, 0.1% HCl) to 100 mg of allogeneic insoluble collagenous matrix and were lyophilized⁴⁸. In addition to the insoluble collagenous matrix, smart novel biomaterials of sintered hydroxyapatite^32,39(to be described) were used as carriers for hTGF-ß2⁴⁸. Sintered porous hydroxyapatites (SPHAs) were prepared in disc configuration, 1.5 cm in diameter, for heterotopic implantation preloaded with 1, 5, and 25 g of hTGF-ß2. For orthotopic implantation, the discs were 2.5 cm in diameter, preloaded with 10 and 100 g of TGF-ß2. Similarly, 10 and 100 g of hTGF-ß2 were added to 1 g of collagenous bone matrix for orthotopic implantation in the baboon model⁴⁸.

Preparation of SPHAs

In the past several years, our research has focused on devising alternative matrices to deliver the osteogenic activity of BMPs/OPs and we have found that porous hydroxyapatites act optimally as delivery systems for the biological activity of BMPs/OPs in heterotopic and orthotopic sites of adult baboons^23,24. Our unit has devised smart biomaterials made of SPHA capable of intrinsically inducing bone in heterotopic sites when implanted in the rectus abdominis of adult baboons without the addition of exogenously applied BMPs/OPs^{32,39,41,43,45}.

This intrinsic osteoinductivity appears to be dependent on the surface characteristics, geometric configuration, and adsorption of endogenous, circulating, or locally produced BMPs/OPs onto the hydroxyapatite^39,41,43. This has been suggested by several experiments in which porous hydroxyapatites derived from the exoskeleton of corals were implanted intramuscularly in adult baboons^21,26,31 and by the immunolocalization of BMP-3 and OP-1 at the interface of sintered hydroxyapatites in association with invading fibrovascular tissue and responding cells^39,41.

To demonstrate the critical role of substratum geometry on bone induction, slurry preparations of hydroxyapatite powder were sintered to form solid monolithic hydroxyapatite discs with a series of concavities prepared on each planar surface^39,41,45. On one surface, the concavities were 1,600 m in diameter and 800 m in depth; on the opposite surface, they were 800 and 400 m in diameter and depth, respectively³⁹. SPHAs were made by a sponge impregnation method using three starting powders of hydroxyapatite that were then sintered so that porous spaces formed by the coalescence of repetitive sequences of concavities³⁹. For heterotopic implantation, a total of 40 monolithic hydroxyapatite discs (20 mm in diameter and 4 mm thick) and 104 sintered hydroxyapatite rods (20 mm in height and 7 mm in diameter) were sintered and sterilized in an autoclave³⁹. For orthotopic implantation, porous hydroxyapatites were prepared so as to obtain discs 25 mm in diameter for adaptation into surgically created calvarial defects³⁹.

The Primate Heterotopic Model: Implantation Design

The primate heterotopic model has been described in detail^{20,21,24,26,31,37,39,48}. Lyophilized 100-mg pellets of collagenous matrix containing doses of hOP-1, pTGF-ß1, hTGF-ß1, and hTGF-ß2 and discs of SPHAs were implanted bilaterally in ventral intramuscular pouches in the rectus abdominis muscle of the baboon. In separate experiments, relatively low doses of hTGF-ß1 and pTGF-ß1 were added to 5, 25, and 125 g of hOP-1, delivered by 100 mg of collagenous matrix for heterotopic implantation (Tables I)^3,37. Analogous materials were prepared for orthotopic calvarial implantation (II)³. Generated tissues were then harvested from the rectus abdominis muscle 30 or 90 days after implantation (Tables I).

The Primate Orthotopic Model: Implantation Design

The orthotopic calvarial model in the baboon used to test a variety of osteoinductive preparations has been described in detail^{22,24,25,33,35,47}. On each side of the calvaria, two full-thickness critical-sized defects, 25 mm in diameter, were created with a craniotome under saline irrigation²². The defects were implanted with varying doses of naturally derived BMPs/OPs, hOP-1, recombinant or platelet-derived TGF-ß1, and hTGF-ß2 delivered in 1 g of collagenous matrix as carrier (Table II)^{23,25,33,35,47}. In other experiments, naturally derived BMPs/OPs or hOP-1 was delivered by coral-derived²⁴or SPHAs⁴⁹. The doses of the morphogens are illustrated in Table II. Generated tissues and specimen blocks were harvested from the euthanized animals 1, 3, 6, 9, and 12 months after surgical implantation, as illustrated in Table II.

Tissue Processing, Histology, and Morphometric Analyses

Heterotopic and orthotopic specimen blocks were processed as described^{22,23,35,37,47}. Serial undecalcified sections were cut at 4-6 m from undecalcified blocks embedded in a polymethylmethacrylate resin (K-Plast; Medim, Buseck, Germany) with use of a Polycut-S (Reichert, Heidelberg, Germany) with tungsten-carbide blades^{20,22,23,35,37,47}. Undecalcified serial sections were stained free-floating with a Goldner’s trichrome stain or with 0.1% toluidine blue in 30% ethanol. Goldner’s trichrome-stained sections were examined with a research microscope equipped with a calibrated Zeiss Integration Platte II (Oberkochem, Germany) with 100 lattice points for determination by the point-counting technique of mineralized bone, osteoid, and residual collagenous or hydroxyapatite matrix volumes (as a percentage) as described^{22,23,25,33,35,37,39,47,48}.

Results

Introduction | Materials and Methods | Results | Discussion | References

Naturally derived BMPs/OPs purified from baboon or bovine demineralized bone matrices induced endochondral bone at doses of 80 g per implant in the rectus abdominis muscle of adult baboons 30 days after heterotopic implantation. Collagenous matrices reconstituted with 280 g of BMPs/OPs induced copious amounts of mineralized bone in calvarial defects of adult primates as early as day 30 (Figs. 1-A and 1-B). On day 90, bone formation culminated in complete regeneration of the calvarial defect even when the BMPs/OPs were delivered by g-irradiated xenogeneic bovine collagenous matrix (Fig. 2-A).

Naturally derived BMPs/OPs purified after gel filtration chromatography and additional chromatography on heparin-Sepharose also induced bone in large mandibular defects of human patients, treated with 1 mg of BMPs/OPs delivered by 1 g of demineralized human bone matrix (Fig. 1-C), as evaluated by histology 90 days after implantation. Of the seven patients given grafts of autogenous bone harvested from the iliac crest, five showed histological evidence of osteogenesis. The two autogenous bone grafts failed in two patients due to resorption as evaluated radiographically and histologically (Fig. 1-D).

rhOP-1, non-irradiated or g-irradiated and delivered by either baboon or bovine collagenous matrices, induced extensive bone formation in calvarial defects of adult baboons as evaluated 3 and 12 months after implantation (Fig. 2). Doses of 5 and 25 g of hOP-1 also induced endochondral bone formation in the rectus abdominis muscle 30 days after heterotopic implantation (Fig. 3-C).

Contrary to all the results obtained in the rodent bioassay, implantation of rhTGF-ß1 or pTGF-ß1 induced endochondral bone by day 30 when implanted in the rectus abdominis muscle of adult primates (Figs. 3-A and 3-D). The binary applications of hOP-1 with relatively low doses of recombinant or platelet-derived TGF-ß1 induced the generation of large ossicles in the rectus abdominis muscle (Figs. 3-B and 3-E). At the margins of some juxtaposed ossicles, newly formed tissues had grown toward each other, and each ossicle, separated by a layer of intervening fibrous tissue, showed a histological gradient of morphological structures highly suggestive of a rudimentary embryonic growth plate (Fig. 3-F)

rhTGF-b2 also induced endochondral bone formation in the rectus abdominis muscle of adult primates (Fig. 4). Substantial islands of chondrogenesis were seen on day 30 when 25 g of the recombinant factor was implanted (Fig. 4-B). On day 90, large corticalized ossicles that formed in the rectus abdominis muscle of adult baboons were generated (Figs. 4-C and 4-D). Induction of bone was also observed in specimens of SPHA pretreated with doses of hTGF-ß2 (Fig. 4-F). Sintered hydroxyapatites implanted in the rectus abdominis without any morphogen (BMP-2 or OP-1) intrinsically induced bone differentiation on day 90 (Fig. 4-E).

The binary applications of doses of rhOP-1 with relatively low doses of pTGF-ß1 also interacted synergistically to induce large orthotopic ossicles 30 and 90 days after implantation of the morphogens delivered by 1 g of allogeneic baboon collagenous matrix (Fig. 5). Defects treated with 100 g hOP-1 delivered by allogeneic baboon collagenous matrix completely regenerated the calvarial defects as evaluated histologically 90 days after implantation (Fig. 5-D). As opposed to hOP-1, in calvarial defects treated with 100 g of hTGF-ß2, osteogenesis was shown across the defects but bone formation was limited and occurred only pericranially (Fig. 5-F).

To demonstrate the critical role of surface geometry on bone induction, slurry preparations of hydroxyapatite powders were sintered to form solid discs with a series of concavities prepared on each planar surface. Sintered hydroxyapatite discs were then implanted heterotopically in the rectus abdominis muscle of adult baboons without any addition of naturally derived or recombinantly produced BMPs/OPs. Analysis of the specimens harvested on days 30 and 90 from the rectus abdominis revealed that bone differentiation was exclusively initiated in the concavities of the substratum (Fig. 6-A). Heterotopic implantation of SPHAs also induced bone differentiation as shown in Fig. 6-B, representing a specimen harvested 90 days after heterotopic implantation, again without exogenously applied BMPs/OPs .

Sintered hydroxyapatites pretreated with doses of hOP-1 also induced extensive bone differentiation when implanted in calvarial defects of adult primates (Fig. 7). On day 30, discs of hydroxyapatite pretreated with 500 g of hOP-1 induced extensive bone differentiation pericranially and endocranially (Fig 7-A). On days 90 and 365 after implantation, there was remodeling of the newly formed bone (Figs. 7-B and 7-C) with partial resorption of the implanted substratum. Sintered hydroxyapatites implanted without hOP-1 induced considerable bone formation across the entirety of the porous scaffold (Fig. 7-D).

The osteoinductive geometric configuration was tested in pre-clinical trials in primates in which edentulous ridges were created after the extraction of mandibular molars and premolars^41,43,45. Hydroxyapatite-coated titanium implants with a series of concavities prepared on the coated surface but without BMPs of OP-1 were inserted in the edentulous mandibular ridges (Fig. 8-A). Examination of serial undecalcified sections showed continuous osteointegration along the geometric surface configurations of the implants as early as 30 and 90 days after insertion (Fig. 8-B).

Discussion

Introduction | Materials and Methods | Results | Discussion | References

The normal repair and regeneration of bone is a complex process that is temporally and spatially regulated and ordered. The three most important requirements for successful tissue engineering of bone are a suitable extracellular matrix scaffold, capable responding cells, and soluble osteoinductive signals^{15,19,32,36,47}. For a therapeutic perspective, a carrier matrix is required for local delivery of BMPs/OPs to evoke a desired osteogenic response^30,35. Naturally derived and rhBMPs/OPs induce local bone formation when reconstituted with the insoluble collagenous matrix, the inactive residue obtained after dissociative extraction of the bone matrix with 4 M guanidinium-HCl^9,24,25,47. The reconstitution of BMPs/OPs (the soluble signal) with the collagenous matrix (the insoluble substratum) provides a bioassay for bona fide initiators of bone differentiation as well as a delivery system for therapeutic local osteogenesis^30,35,52. These morphogens are thought to interact in a sequence reminiscent of, and in recapitulation of, the cascade of events that occur during embryonic development¹⁹. The concept of using smart biocompatible and bioactive materials for drug delivery and tissue engineering has previously been discussed. These biomaterials have been described as molecular switches that control biological functions by responding smartly to physical, chemical, or biological stimuli. This new technology has been highlighted for the engineering of bone after heterotopic implantation of SPHAs in primates^39,41,57. The underlying mechanism of osteogenesis in these and other bone induction studies is greatly influenced and controlled by the structural geometry of the substratum^{7,8,10,23,39,41,58,59}.

A major objective of our studies is to develop biomaterials that are specifically designed so that endogenously produced BMPs/OPs are adsorbed onto the implanted matrix or so that their genes are activated by the implantation of these geometrically correct matrices^39,41. The advent of novel synthetic biomimetic biomaterials that can intrinsically evoke the morphogenesis of bone presents a new challenge for the study, design, and development of bioactive materials for tissue engineering of bone in the absence of exogenously applied BMPs/OPs. The versatile nature of these biomimetic biomaterials makes them more practical and more cost effective in their clinical application than devices requiring the combination of both carriers and recombinantly produced BMPs/OPs.

The discovery that specific surface and geometric characteristics of SPHAs can induce bone in heterotopic sites of primates paves the way for the formulation and therapeutic application of smart porous substrata that lead to the formation of predictable tissue types by intrinsic osteoinductivity. We define the incorporation of specific biological activities into sintered hydroxyapatites as geometric induction of bone formation. The geometric osteoinductive configuration may make it possible to elicit therapeutic osteogenesis in clinical contexts. Our studies on the intrinsic osteoinductivity of porous substrata in primates indicate that the bone induction cascade is initiated by endogenously produced BMPs/OPs bound to the surface of the smart concavities of the substratum, with induction of bone as a secondary response. We suggest that the concavities of the substratum are geometric regulators of growth endowed with shape memory, recapitulating events that occur in the normal course of embryonic development and appearing to act as gates that give or withhold permission to grow and differentiate⁴⁴.

Note: This work is supported by the South African Medical Research Council and the University of the Witwatersrand, Johannesburg. The authors thank Creative BioMolecules and Stryker Biotech for continuous supply of hOP-1. For their invaluable help in the past years, they thank Dr. A. Hari Reddi, Barbara van den Heever, Laura Yeates, Dr. Manolis Heliotis, Dr. Carlo Ferretti, and Dr. David Rueger.

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TABLE I: Heterotopic Morphogens: Implantation and Number of Primates

Treatment Group	Number of Primates	Implant Doses	Collagenous Matrix	Number of Implants
Naturally derived BMPs/OPs	8	80 mg	100 mg	?16
hOP-1	8	5, 25, and 125 mg	100 mg	128
PTGF-ß1	2	0.5, 1.5, and 5 mg	100 mg	??8
TGF-ß2	4	1, 5, and 25 mg	100 mg	?32
h-OP1/pTGF-ß1	8	25 mg/(0.5, 1.5, and 5 mg)	100 mg	?96
h-OP1/hTGF-ß1	6	25 mg/(0.5, 1.5, and 5 mg)	100 mg	?72

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TABLE I: Heterotopic Morphogens: Implantation and Number of Primates

Treatment Group	Number of Primates	Implant Doses	Collagenous Matrix	Number of Implants
Naturally derived BMPs/OPs	8	80 mg	100 mg	?16
hOP-1	8	5, 25, and 125 mg	100 mg	128
PTGF-ß1	2	0.5, 1.5, and 5 mg	100 mg	??8
TGF-ß2	4	1, 5, and 25 mg	100 mg	?32
h-OP1/pTGF-ß1	8	25 mg/(0.5, 1.5, and 5 mg)	100 mg	?96
h-OP1/hTGF-ß1	6	25 mg/(0.5, 1.5, and 5 mg)	100 mg	?72

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TABLE II: Orthotopic Morphogens: Implantation and Number of Primates

Treatment Group	Number of Primates	Implant Doses	Collagenous Matrix	Number of Implants
Naturally derived BMPs/OPs	26	1.8 and 280 mg	1 g	84
	10	280 mg	1 g	40
hOP-1	14	0.1, 0.5, and 2.5 mg	1 g	56
g-irradiated hOP-1	24	0.1, 0.5, and 2.5 mg	1 g	80
hOP-1/pTGF-ß1	?8	100 mg/(5, 15 mg)	1 g	32
		20 mg/5 mg
SPHAs	?4	—	—	16
SPHAs + hOP-1	12	0.1, 0.5, and 2.5 mg	—	48
Coral-derived HA	24	—	—	96
	?8	—	—	32
Coral-derived HA + naturally derived BMPs/OPs	?8	150 mg	—	32

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TABLE II: Orthotopic Morphogens: Implantation and Number of Primates

Treatment Group	Number of Primates	Implant Doses	Collagenous Matrix	Number of Implants
Naturally derived BMPs/OPs	26	1.8 and 280 mg	1 g	84
	10	280 mg	1 g	40
hOP-1	14	0.1, 0.5, and 2.5 mg	1 g	56
g-irradiated hOP-1	24	0.1, 0.5, and 2.5 mg	1 g	80
hOP-1/pTGF-ß1	?8	100 mg/(5, 15 mg)	1 g	32
		20 mg/5 mg
SPHAs	?4	—	—	16
SPHAs + hOP-1	12	0.1, 0.5, and 2.5 mg	—	48
Coral-derived HA	24	—	—	96
	?8	—	—	32
Coral-derived HA + naturally derived BMPs/OPs	?8	150 mg	—	32