JBJS | The Effect of Osteogenic Protein-1 on the Healing of Segmental Bone Defects Treated with Autograft or Allograft Bone

The Journal of Bone & Joint Surgery, Volume 83, Issue 6

Articles | June 01, 2001

The Effect of Osteogenic Protein-1 on the Healing of Segmental Bone Defects Treated with Autograft or Allograft Bone

Samantha L. Salkeld, MS; Laura Popich Patron, BS; Robert L. Barrack, MD; Stephen D. Cook, PhD

Investigation performed at the Department of Orthopaedic Surgery, Tulane University School of Medicine, New Orleans, Louisiana
Samantha L. Salkeld, MS
Laura Popich Patron, BS
Robert L. Barrack, MD
Stephen D. Cook, PhD
Department of Orthopaedic Surgery, Tulane University School of Medicine, 1430 Tulane Avenue, New Orleans, LA 70112. E-mail address for S.D. Cook: scook2@mailhost.tcs.tulane.edu

In support of their research or preparation of this manuscript, one or more of the authors received grants or outside funding from Stryker Biotech. In addition, one or more of the authors received payments or other benefits or a commitment or agreement to provide such benefits from a commercial entity (Stryker Biotech). No commercial entity paid or directed, or agreed to pay or direct, any benefits to any research fund, foundation, educational institution, or other charitable or nonprofit organization with which the authors are affiliated or associated.

The Journal of Bone & Joint Surgery. 2001; 83:803-816

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Abstract

Background: Large amounts of bone graft are frequently used to elicit the healing of bone defects resulting from reconstructive procedures. Autograft and allograft bone are often used, but each has its limitations. Bone morphogenetic proteins (BMPs) improve the healing of segmental bone defects treated with autograft or allograft. The objective of the present study was to determine the effect of implantation of a recombinant osteogenic protein-1 (OP-1) in combination with bone graft on the healing of a critical-sized (2.5-cm) segmental defect in canine ulnae.

Methods: Either autograft bone, allograft bone, osteogenic protein-1 (OP-1) mixed with type-1 bovine collagen, or various combinations of OP-1 and collagen (OP-1 device) mixed with allograft or autograft were implanted in the segmental bone defects. The combinations included 67% bone graft with 33% OP-1 device and 33% bone graft with 67% OP-1 device. The healing of the defects was assessed with radiographic, biomechanical, and histological studies. The animals were killed at twelve weeks postoperatively.

Results: The use of the OP-1 device alone or any combination of autograft or allograft bone and the OP-1 device demonstrated improved healing on radiographic, mechanical, and histological studies compared with that demonstrated after use of autograft or allograft bone alone. The highest radiographic and histological grades and the greatest mechanical strength were achieved with the use of 33% allograft and 67% OP-1 device, although no significant differences were observed among the different groups containing the OP-1 device. At twelve weeks postoperatively, the defects treated with any amount of the OP-1 device obtained greater mechanical strength than that obtained by autograft bone alone.

Conclusions: Major bone defects may be treated with allograft bone combined with the OP-1 device, instead of autograft alone, to avoid complications associated with the use of autograft. The combination of allograft bone and the OP-1 device resulted in optimum healing of the defect, according to the radiographic, mechanical, and histological parameters measured in this study.

Clinical Relevance: The combination of freeze-dried allograft bone with the OP-1 device is an attractive graft material for the treatment of large bone defects. Although similar results were observed when autogenous bone graft was used in combination with the OP-1 device, the results of the present study suggest that allograft, because of its relatively unlimited supply, can be substituted without reduced efficacy. In addition, avoiding the need to harvest autogenous bone eliminates the additional operative time and risk associated with a second surgical procedure.

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Introduction

Introduction | Materials and Methods | Results | Discussion | References

Trauma, infection, or a failed arthroplasty can result in a bone defect that does not heal. Bone harvested locally or from the iliac crest, rib, or fibula is frequently used to elicit healing in such cases^1,2. However, the usefulness of autogenous bone is often limited by the amount that is available, the necessity of a second operation, and the risk of complications at the donor site^3,4. Allograft bone is an attractive alternative to autograft bone because it supports bone formation, its supply is less limited, and large structural restorations are possible. However, allograft bone has only a fraction of the osteoinductive capacity of autograft bone^2,5,6 and a limited capacity to incorporate with host bone^6-10.

Recent research has centered on the use of osteoinductive materials such as bone morphogenetic proteins (BMPs) to aid in the healing of bone. BMPs are a group of proteins that, either alone or in combination with other regulatory molecules, induce new-bone formation^11-19. These proteins are members of the transforming growth factor-b superfamily of proteins involved in the cascade of cellular events of tissue formation and regeneration, including stem-cell commitment, differentiation, and proliferation¹⁴. Recombinant human osteogenic protein-1 (rhOP-1) combined with type-I collagen derived from bone has been shown to elicit the healing of segmental bone defects in animal models^20-25.

The use of osteogenic proteins in the clinical setting is limited by the method of delivery as well as by the containment of both the implant material and the new-bone formation^22,26,27. The development of carrier materials and methods of protein delivery represents an important step in the development of clinically realized solutions for bone regeneration. The use of an OP-1 device consisting of rhOP-1 mixed with type-I collagen in conjunction with autograft or allograft bone offers potential advantages. Containment of the OP-1 device in the site may be enhanced by combining it with the bone-graft material, resulting in greater and better localized new-bone formation. When a structural graft is required or if the volume of the bone defect is large, the use of the OP-1 device alone may not be satisfactory since it has no structural integrity. The purpose of the present study was to determine the efficacy of the OP-1 device combined with allograft or autograft bone in eliciting the healing of critical-sized segmental bone defects. We sought to determine the optimal volume ratio of the OP-1 device to allograft and autograft bone with use of a surgical model in which the OP-1 device alone has been previously evaluated.

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Fig. 1:: Radiographic appearance of a segmental defect treated with the OP-1 device alone. A: Two weeks postoperatively. B: At six weeks postoperatively, new bone fills the defect and extends beyond the original ulnar contours. C: At twelve weeks postoperatively, the defect is filled with new bone. The lateral borders of new bone have begun to coalesce, suggestive of early cortex formation.

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Fig. 2:: A, B, and C: Radiographic appearance of a segmental defect treated with 100% autograft bone. A: Two weeks postoperatively. B: At six weeks postoperatively, the graft has consolidated and some early new bone is visible. C: At twelve weeks postoperatively, the amount of new bone is considerably less than that in defects treated with the OP-1 device. A radiolucent seam is present at the distal host-bone junction. D, E, and F: Radiographic appearance of a segmental defect treated with 67% autograft and 33% OP-1 device. D: Two weeks postoperatively. E: At six weeks postoperatively, osseous bridging of the defect is nearly complete and the graft is well incorporated. F: At twelve weeks postoperatively, the defect is bridged and filled with new bone and is incorporated with the host-bone ends. G, H, and I: Radiographic appearance of a segmental defect treated with 33% autograft and 67% OP-1 device. G: Two weeks postoperatively. H: At six weeks postoperatively, new bone is incorporated with the autograft bone and fills the defect. I: At twelve weeks postoperatively, the defect is filled with new bone incorporated with the host bone, although a dense fragment of autograft bone is discernible at the distal end of the defect.

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Fig. 3:: A, B, and C: Radiographic appearance of a segmental defect treated with 100% allograft bone. A: Two weeks postoperatively. B: At six weeks postoperatively, the graft has consolidated, although little new bone has formed. C: At twelve weeks postoperatively, minimal new-bone formation has occurred and the allograft has been resorbed. D, E, and F: Radiographic appearance of a segmental defect treated with 67% allograft and 33% OP-1 device. D: Two weeks postoperatively. E: At six weeks postoperatively, the bone graft has consolidated and its radiodensity has increased. New bone has formed in and around the graft. F: At twelve weeks postoperatively, the defect is bridged at both ends with new bone that appears to be incorporated with the allograft. G, H, and I: Radiographic appearance of a segmental defect treated with 33% allograft and 67% OP-1 device. G: Two weeks postoperatively. H: At six weeks postoperatively, incorporation of the allograft with new bone is nearly complete and the amount of new-bone formation is greater than that in defects treated with 67% allograft and 33% OP-1 device. I: At twelve weeks, dense bone bridges the defect. The allograft is completely incorporated.

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Fig. 4:: Undecalcified histological section of a segmental defect treated with the OP-1 device alone. At twelve weeks postoperatively, the defect is filled with new bone (NB) that has a dense trabecular and new lamellar structure. The lateral border of the new bone is beginning to remodel toward the original ulnar contours (arrows), although cortices are not yet organized. Extensive new bone is well incorporated with the host bone (H) (basic fuchsin and toluidine blue, ¥1).

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Fig. 5:: A: Undecalcified histological section of a segmental defect treated with 100% autograft bone. This defect is nearly completely healed. The graft has reorganized, and the cartilage seams (large arrows) between the graft and the host (H) are mineralizing. The specimen failed through the graft during torsional testing (small arrows). B: Histological section of a segmental defect treated with 100% allograft bone. Fibrous tissue spans the defect (arrows). A small piece of allograft (A) is present but is not continuous with the host bone (basic fuchsin and toluidine blue, ¥1).

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Fig. 6:: A: Undecalcified histological section of a segmental defect treated with 67% autograft and 33% OP-1 device that was not mechanically tested because of insufficient rigidity. New bone spans the defect and connects the autograft bone (A). Incompletely mineralized seams (arrows) are present between the graft pieces. B: Undecalcified histological section of a segmental defect treated with 67% allograft and 33% OP-1 device. Extensive new bone (NB) has formed and connects the allograft bone (A) with the host bone (H). The contours of the ulna are not yet restored, and cartilage and osteoid seams (arrows) are present between the host bone and the new bone (basic fuchsin and toluidine blue, ¥1).

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Fig. 7:: A: Undecalcified histological section of a segmental defect treated with 33% autograft and 67% OP-1 device. Extensive new bone (NB) fills the defect and is well incorporated with the host-bone ends (H). Remodeling to the original ulnar contours and incorporation of the autograft bone are nearly complete. B: Undecalcified histological section of a segmental defect treated with 33% allograft and 67% OP-1 device. The section has a similar appearance to segmental defects treated with 33% autograft and 67% OP-1 device. The allograft bone is difficult to detect, although a small dense fragment (A) is incorporating with new bone (NB). New bone has fused to the radius (R) (basic fuchsin and toluidine blue, ¥1).

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TABLE I: Experimental Bone-Graft Treatment Groups

*The OP-1 device consists of recombinant human osteogenic protein-1 (rhOP-1) combined with type-I bovine bone-derived collagen. †100% volume is 3.0 cm³ of implant material.

Treatment Group*	Volume Percentage (Graft:OP-1 Device)†	Amount of OP-1 Device (mg)	Amount of Saline Solution Added for Reconstitution (ml)	No. of Defects
100% allograft	100:0	??0	0	9
67% allograft and 33% OP-1 device	67:33	250	0.08	6
33% allograft and 67% OP-1 device	33:67	500	0.16	6
100% autograft	100:0	??0	0	9
67% autograft and 33% OP-1 device	67:33	250	0.08	6
33% autograft and 67% OP-1 device	33:67	500	0.16	6
100% OP-1 device	0:100	750	0.24	6

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TABLE I: Experimental Bone-Graft Treatment Groups

*The OP-1 device consists of recombinant human osteogenic protein-1 (rhOP-1) combined with type-I bovine bone-derived collagen. †100% volume is 3.0 cm³ of implant material.

Treatment Group*	Volume Percentage (Graft:OP-1 Device)†	Amount of OP-1 Device (mg)	Amount of Saline Solution Added for Reconstitution (ml)	No. of Defects
100% allograft	100:0	??0	0	9
67% allograft and 33% OP-1 device	67:33	250	0.08	6
33% allograft and 67% OP-1 device	33:67	500	0.16	6
100% autograft	100:0	??0	0	9
67% autograft and 33% OP-1 device	67:33	250	0.08	6
33% autograft and 67% OP-1 device	33:67	500	0.16	6
100% OP-1 device	0:100	750	0.24	6

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TABLE II: Radiographic Grading Scale for the Degree of Healing

Description	Grade
No change from immediate postoperative appearance	0
A slight increase in radiodensity distinguishable from the graft	1
Recognizable increase in radiodensity, bridging of one cortex with new-bone formation to the graft	2
Bridging of at least one cortex with material of nonuniform radiodensity, early incorporation of the graft suggested by obscurity of graft borders	3
Defect bridged on both medial and lateral sides with bone of uniform radiodensity, cut ends of the cortex still visible, graft and new bone not easy to differentiate	4
Same as grade 3, with at least one of four cortices obscured by new bone	5
Defect bridged by uniform new bone, cut ends of cortex no longer distinguishable, graft no longer visible	6

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TABLE II: Radiographic Grading Scale for the Degree of Healing

Description	Grade
No change from immediate postoperative appearance	0
A slight increase in radiodensity distinguishable from the graft	1
Recognizable increase in radiodensity, bridging of one cortex with new-bone formation to the graft	2
Bridging of at least one cortex with material of nonuniform radiodensity, early incorporation of the graft suggested by obscurity of graft borders	3
Defect bridged on both medial and lateral sides with bone of uniform radiodensity, cut ends of the cortex still visible, graft and new bone not easy to differentiate	4
Same as grade 3, with at least one of four cortices obscured by new bone	5
Defect bridged by uniform new bone, cut ends of cortex no longer distinguishable, graft no longer visible	6

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TABLE III: Histological Grading Scale for the Degree of Healing

	Description	Grade
Quality of union	No sign of fibrous or other union	0
	Fibrous union	1
	Fibrocartilaginous union or cartilage union	2
	Mineralizing cartilage and bone union	3
	Bone union	4
Cortex development and remodeling	No cortex formed	0
	Densification of new bone along exterior borders	1
	Recognizable formation of both the outer cortex border and the medullary space	2
	Cortices formed but incomplete bridging	3
	Complete formation of cortices with bridging of defect	4
Bone-graft incorporation and new-bone formation
No new bone, all or most of graft visible	Graft material present, no incorporation, and no new-bone formation	0
	Graft present, some incorporation with new-bone formation, and small amount of new bone	1
	Graft present, some incorporation with new-bone formation, and moderate amount of new bone	2
Decreasing graft, increasing new bone	Graft present, some incorporation with new-bone formation continuous with host bone, and early remodeling changes in new bone	3
	Decreased amount of graft (compared with grade 3), good graft incorporation, and ample new bone	4
	Less amount of graft still visible (compared with grade 4), good incorporation of graft and new bone with host and ample new bone	5
No graft visible, extensive new bone	Difficult to differentiate graft from new bone, excellent incorporation, and advanced remodeling of new bone with graft and host	6

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TABLE III: Histological Grading Scale for the Degree of Healing

	Description	Grade
Quality of union	No sign of fibrous or other union	0
	Fibrous union	1
	Fibrocartilaginous union or cartilage union	2
	Mineralizing cartilage and bone union	3
	Bone union	4
Cortex development and remodeling	No cortex formed	0
	Densification of new bone along exterior borders	1
	Recognizable formation of both the outer cortex border and the medullary space	2
	Cortices formed but incomplete bridging	3
	Complete formation of cortices with bridging of defect	4
Bone-graft incorporation and new-bone formation
No new bone, all or most of graft visible	Graft material present, no incorporation, and no new-bone formation	0
	Graft present, some incorporation with new-bone formation, and small amount of new bone	1
	Graft present, some incorporation with new-bone formation, and moderate amount of new bone	2
Decreasing graft, increasing new bone	Graft present, some incorporation with new-bone formation continuous with host bone, and early remodeling changes in new bone	3
	Decreased amount of graft (compared with grade 3), good graft incorporation, and ample new bone	4
	Less amount of graft still visible (compared with grade 4), good incorporation of graft and new bone with host and ample new bone	5
No graft visible, extensive new bone	Difficult to differentiate graft from new bone, excellent incorporation, and advanced remodeling of new bone with graft and host	6

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TABLE IV: Number of Defects Bridged from Cortex to Cortex by New Bone at Twelve Weeks Postoperatively

*Defects deemed rigidly healed on manual palpation and subsequent mechanical testing. †Defects with a mean quality-of-union score of 3.0.

Treatment Group	Radiographic Evidence		Mechanical Evidence*	Histological Evidence†
1 Cortex Bridged	All Cortices Bridged
100% allograft (n = 9)	3	0	2	0
67% allograft and 33% OP-1 device (n = 6)	6	5	5	4
33% allograft and 67% OP-1 device (n = 6)	6	6	6	6
100% autograft (n = 9)	5	4	6	6
67% autograft and 33% OP-1 device (n = 6)	5	5	5	3
33% autograft and 67% OP-1 device (n = 6)	5	5	6	5
100% OP-1 device (n = 6)	5	5	5	4

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TABLE IV: Number of Defects Bridged from Cortex to Cortex by New Bone at Twelve Weeks Postoperatively

*Defects deemed rigidly healed on manual palpation and subsequent mechanical testing. †Defects with a mean quality-of-union score of 3.0.

Treatment Group	Radiographic Evidence		Mechanical Evidence*	Histological Evidence†
1 Cortex Bridged	All Cortices Bridged
100% allograft (n = 9)	3	0	2	0
67% allograft and 33% OP-1 device (n = 6)	6	5	5	4
33% allograft and 67% OP-1 device (n = 6)	6	6	6	6
100% autograft (n = 9)	5	4	6	6
67% autograft and 33% OP-1 device (n = 6)	5	5	5	3
33% autograft and 67% OP-1 device (n = 6)	5	5	6	5
100% OP-1 device (n = 6)	5	5	5	4

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TABLE V: Radiographic Grade of Ulnar Segmental Defects

*The values are given as the mean and the standard deviation. The radiographic grading scale (Table II) ranges from a score of 0, indicating no change from the immediate postoperative appearance, to a score of 6, indicating a defect bridged by uniform new bone with the cut ends of cortex and graft no longer distinguishable.

Treatment Group	Radiographic Grade*
2 Weeks	4 Weeks	6 Weeks	8 Weeks	12 Weeks
100% allograft (n = 9)	0.1 ± 0.3	0.6 ± 0.5	1.2 ± 0.7	1.7 ± 1.1	1.8 ± 1.1
67% allograft and 33% OP-1 device (n = 6)	0.8 ± 0.4	2.3 ± 0.8	3.0 ± 0.9	3.7 ± 0.8	4.3 ± 1.0
33% allograft and 67% OP-1 device (n = 6)	1.0 ± 0.6	2.5 ± 0.8	4.0 ± 0.6	5.0 ± 0.6	5.3 ± 0.5
100% autograft (n = 9)	0.0 ± 0.0	0.7 ± 0.5	1.4 ± 1.0	2.0 ± 1.2	2.9 ± 1.8
67% autograft and 33% OP-1 device (n = 6)	0.8 ± 0.4	2.0 ± 0.4	3.2 ± 1.2	3.7 ± 1.5	4.2 ± 1.2
33% autograft and 67% OP-1 device (n = 6)	1.2 ± 0.8	2.7 ± 0.8	3.3 ± 0.8	3.8 ± 1.0	4.5 ± 1.2
100% OP-1 device (n = 6)	0.8 ± 0.8	2.3 ± 0.8	3.5 ± 1.2	4.0 ± 1.6	4.3 ± 1.9

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TABLE V: Radiographic Grade of Ulnar Segmental Defects

Treatment Group	Radiographic Grade*
2 Weeks	4 Weeks	6 Weeks	8 Weeks	12 Weeks
100% allograft (n = 9)	0.1 ± 0.3	0.6 ± 0.5	1.2 ± 0.7	1.7 ± 1.1	1.8 ± 1.1
67% allograft and 33% OP-1 device (n = 6)	0.8 ± 0.4	2.3 ± 0.8	3.0 ± 0.9	3.7 ± 0.8	4.3 ± 1.0
33% allograft and 67% OP-1 device (n = 6)	1.0 ± 0.6	2.5 ± 0.8	4.0 ± 0.6	5.0 ± 0.6	5.3 ± 0.5
100% autograft (n = 9)	0.0 ± 0.0	0.7 ± 0.5	1.4 ± 1.0	2.0 ± 1.2	2.9 ± 1.8
67% autograft and 33% OP-1 device (n = 6)	0.8 ± 0.4	2.0 ± 0.4	3.2 ± 1.2	3.7 ± 1.5	4.2 ± 1.2
33% autograft and 67% OP-1 device (n = 6)	1.2 ± 0.8	2.7 ± 0.8	3.3 ± 0.8	3.8 ± 1.0	4.5 ± 1.2
100% OP-1 device (n = 6)	0.8 ± 0.8	2.3 ± 0.8	3.5 ± 1.2	4.0 ± 1.6	4.3 ± 1.9

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TABLE VI: Results of Mechanical Testing*

*The values are given as the mean and the standard deviation. †Defects that were not mechanically tested because of instability on manual palpation were assumed to have a torsional strength and energy-absorbed value equal to zero.

Treatment Group	No. of Segments Tested†	Maximum Torque to Failure (Nm)	Percent of Strength ofIntact Controls	Energy Absorbed to Failure (Nmldeg)
100% allograft (n = 9)	2	0.15 ± 0.30	3.84 ± 7.63	4.33 ± 8.61
67% allograft and 33% OP-1 device (n = 6)	6	1.60 ± 1.43	40.73 ± 36.61	40.77 ± 44.87
33% allograft and 67% OP-1 device (n = 6)	6	3.18 ± 1.68	81.27 ± 42.90	82.13 ± 55.07
100% autograft (n = 9)	6	1.33 ± 1.42	34.08 ± 36.37	32.78 ± 30.22
67% autograft and 33% OP-1 device (n = 6)	6	2.76 ± 1.69	70.53 ± 43.08	69.70 ± 43.42
33% autograft and 67% OP-1 device (n = 6)	6	2.85 ± 1.40	72.70 ± 35.71	75.88 ± 33.58
100% OP-1 device (n = 6)	5	2.74 ± 1.60	69.95 ± 40.85	65.80 ± 38.32

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TABLE VI: Results of Mechanical Testing*

Treatment Group	No. of Segments Tested†	Maximum Torque to Failure (Nm)	Percent of Strength ofIntact Controls	Energy Absorbed to Failure (Nmldeg)
100% allograft (n = 9)	2	0.15 ± 0.30	3.84 ± 7.63	4.33 ± 8.61
67% allograft and 33% OP-1 device (n = 6)	6	1.60 ± 1.43	40.73 ± 36.61	40.77 ± 44.87
33% allograft and 67% OP-1 device (n = 6)	6	3.18 ± 1.68	81.27 ± 42.90	82.13 ± 55.07
100% autograft (n = 9)	6	1.33 ± 1.42	34.08 ± 36.37	32.78 ± 30.22
67% autograft and 33% OP-1 device (n = 6)	6	2.76 ± 1.69	70.53 ± 43.08	69.70 ± 43.42
33% autograft and 67% OP-1 device (n = 6)	6	2.85 ± 1.40	72.70 ± 35.71	75.88 ± 33.58
100% OP-1 device (n = 6)	5	2.74 ± 1.60	69.95 ± 40.85	65.80 ± 38.32

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TABLE VII: Histological Grade of Ulnar Segmental Defects*

*The values are given as the mean and the standard deviation. The histological grading scale is shown in Table III.

Treatment Group	Quality of Union (0 to 4)	Cortex Development and Remodeling (0 to 4)	Bone-Graft Incorporation and New-Bone Formation (0 to 6)	Total Grade (0 to 14)
100% allograft (n = 9)	1.1 ± 0.4	0.1 ± 0.3	0.6 ± 0.9	?1.8 ± 1.5
67% allograft and 33% OP-1 device (n = 6)	2.8 ± 0.7	1.1 ± 0.7	3.3 ± 1.1	?7.3 ± 2.2
33% allograft and 67% OP-1 device (n = 6)	3.9 ± 0.3	1.7 ± 0.9	5.0 ± 1.0	10.7 ± 1.4
100% autograft (n = 9)	2.5 ± 1.0	0.9 ± 0.9	2.3 ± 1.6	?5.7 ± 3.4
67% autograft and 33% OP-1 device (n = 6)	3.2 ± 0.9	0.9 ± 0.7	3.3 ± 1.4	?7.3 ± 2.8
33% autograft and 67% OP-1 device (n = 6)	3.3 ± 1.2	1.4 ± 0.9	3.7 ± 1.7	?8.4 ± 3.5
100% OP-1 device (n = 6)	3.3 ± 1.2	1.2 ± 0.7	3.1 ± 1.2	?7.5 ± 2.9

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TABLE VII: Histological Grade of Ulnar Segmental Defects*

*The values are given as the mean and the standard deviation. The histological grading scale is shown in Table III.

Treatment Group	Quality of Union (0 to 4)	Cortex Development and Remodeling (0 to 4)	Bone-Graft Incorporation and New-Bone Formation (0 to 6)	Total Grade (0 to 14)
100% allograft (n = 9)	1.1 ± 0.4	0.1 ± 0.3	0.6 ± 0.9	?1.8 ± 1.5
67% allograft and 33% OP-1 device (n = 6)	2.8 ± 0.7	1.1 ± 0.7	3.3 ± 1.1	?7.3 ± 2.2
33% allograft and 67% OP-1 device (n = 6)	3.9 ± 0.3	1.7 ± 0.9	5.0 ± 1.0	10.7 ± 1.4
100% autograft (n = 9)	2.5 ± 1.0	0.9 ± 0.9	2.3 ± 1.6	?5.7 ± 3.4
67% autograft and 33% OP-1 device (n = 6)	3.2 ± 0.9	0.9 ± 0.7	3.3 ± 1.4	?7.3 ± 2.8
33% autograft and 67% OP-1 device (n = 6)	3.3 ± 1.2	1.4 ± 0.9	3.7 ± 1.7	?8.4 ± 3.5
100% OP-1 device (n = 6)	3.3 ± 1.2	1.2 ± 0.7	3.1 ± 1.2	?7.5 ± 2.9

Materials and Methods

Introduction | Materials and Methods | Results | Discussion | References

Experimental Design

A total of twenty-four adult male mongrel dogs were used. A bilateral 2.5-cm critical-sized segmental ulnar defect was created in each animal. Seven combinations of the OP-1 device, autograft bone, and allograft bone were implanted into the defects: the OP-1 device alone, autograft bone (100%, 67%, and 33% by volume), and allograft bone (100%, 67%, and 33% by volume), with the remaining graft volume made up by a volume fraction of the OP-1 device. Each one-third of the implant volume was equivalent to 1 cm³ of bone-graft material or 250 mg of the OP-1 device. All sites received a total of 3 cm³ of implanted material. Table I defines each experimental group and the number of samples evaluated.

Animal Model

All animals were skeletally mature and weighed between 50 and 64 lb (23 and 29 kg). The animals were evaluated radiographically before the operation to verify skeletal maturity and the absence of osseous abnormalities. The operative procedures and animal care were performed in compliance with the regulations of the National Institutes of Health of the United States Public Health Service and the University’s Animal Care and Use Committee, under the supervision of a licensed veterinarian.

OP-1 Device

The OP-1 device (Creative BioMolecules, Hopkinton, Massachusetts) was supplied sterile for implantation and consisted of 2.5 mg of recombinant human osteogenic protein-1 (rhOP-1) combined with 1 g of highly purified bovine bone-derived type-I collagen^13,28.

The bovine bone-derived type-I collagen carrier does not have cartilage or bone-inductive properties^20,23,29. The rhOP-1 is combined with the collagen by dissolving the protein in 1 ml of 50% acetonitrile/0.1% trifluoroacetic acid and combining it with the appropriate amount of carrier. The final preparation is freeze-dried and sterilized by gamma irradiation. The device is reconstituted with sterile saline solution at the time of surgery. One gram of reconstituted OP-1 device (total rhOP-1 dose, 3.5 mg) has a volume of approximately 4 cm³. The concentration of rhOP-1 in 1g of reconstituted OP-1 device is 0.625 mg/ml.

Allograft Bone

Fresh cancellous allograft bone was harvested aseptically from the distal part of the femur and the proximal part of the tibia of donor animals in the same range of body weight as the study animals. It was cleaned of all soft tissues, morselized into 5-mm cubes, and processed aseptically in sterile saline solution, ethyl alcohol solutions, and a 10% antibiotic solution (Flocillin; penicillin G procaine and benzathine) in an ultrasonic cleaner. Finally, the cubes were washed in sterile saline solution in an ultrasonic cleaner for one hour and then were lyophilized^30,31. The allograft cubes were divided into volume portions required for implantation. No attempt was made to characterize the histocompatibility or antigenicity of the donor or recipient animals.

Autograft Bone

Autograft bone was obtained from the distal part of the femora and/or the proximal part of the tibiae of each animal at the time of surgery. After blunt dissection and elevation of the periosteum, a large-bore bone-biopsy trephine was used to harvest cores of corticocancellous bone graft. Cortical bone and soft tissues were removed, and the autograft bone was morselized into 5-mm cubes.

Surgical Procedure

With use of standard aseptic techniques, surgery was performed with the animals under isoflurane anesthesia. A lateral skin incision was made, and the ulna was exposed. A 2.5-cm segmental osteoperiosteal defect was created in the mid-part of the ulna with use of an oscillating saw. This defect was approximately two to two and one-half times the diameter of the middle of the shaft and represents a critical-sized defect that will not heal spontaneously^2,6,7,32. The radius provided mechanical stability. Internal fixation was not used. The site was irrigated with saline solution to remove bone debris. The graft materials were carefully placed in the defect. The soft tissues were closed in layers, and the procedure was repeated on the contralateral limb. Postoperatively, the animals were allowed to walk ad libitum. The animals were managed for pain, as required, with subcutaneous injections of butorphanol tartrate (Torbutrol) (0.01 mg/kg body weight). Intramuscular antibiotics (Ambi-Pen; penicillin G procaine and benzathine) (1 ml/10 to 20 lb [4.5 to 9 kg] body weight) were administered for four days following surgery.

At the end of the study period, the animals were killed with use of an intravenous overdose of barbiturate (Beuthanasia-D; pentobarbital) (0.45 ml/kg body weight). The ulna and radius were immediately harvested and placed in physiological saline solution during preparation for mechanical testing.

Radiographic Evaluation

Anteroposterior radiographs of the forelimbs were made immediately after surgery, biweekly until eight weeks, and at the time that the animals were killed at twelve weeks. In order to quantify the degree of healing, each radiograph was assigned a numerical score with use of the grading scale described in Table II^20,21,24.

Mechanical Testing

Soft tissues were removed from the defect site. The ulna was cut to a uniform length of 9 cm with the defect site centered in the middle of the test specimen. If the defect was deemed to be completely healed and rigid by manual manipulation, the specimen was tested to failure in torsion on a closed-loop hydraulic testing machine (model 810; MTS Systems, Minneapolis, Minnesota). One centimeter of each bone-segment end was mounted in methylmethacrylate. One end of the test specimen was rigidly fixed while the other was rotated counterclockwise by the actuator at a constant displacement rate of 50 mm/min^20,33,34. Actuator displacement and the resulting load were recorded by means of an analog-to-digital board, a personal computer, and acquisition software (Data Translation, Marlboro, Massachusetts). Maximum torque and energy absorbed to failure were obtained from force-angular displacement curves.

Histological Studies

Tested and untested specimens were processed for histological preparation of undecalcified ground sections. Specimens were fixed by immersion in 10% formalin solution. The specimens were bisected along the long axis of the ulna on a water-cooled diamond saw. Following fixation, the specimens were dehydrated in graduated ethyl alcohol solutions and embedded in methylmethacrylate. Ground sections were obtained by making cuts parallel to the long axis of the ulna into sections approximately 700 to 1000 m thick. Sections were mounted on acrylic slides with cyanoacrylate adhesive, ground by hand to a thickness of 70 to 90 m, and stained with basic fuchsin and toluidine blue. The two most central sections from each specimen were evaluated with a 14-point histological grading scale to determine the quality of the union, the appearance and quality of the cortical and cancellous bone-remodeling, and the degree of bone-graft incorporation and remodeling (Table III).

Statistical Analysis

All mechanical testing data were initially screened for statistical outliers. Data screening was performed by identifying the maximum and minimum values for each variable and calculating the z-score for both values. The z-score for a data point was defined as the difference between the data-point value and the variable mean divided by the standard deviation of the variable mean. A statistical outlier was defined as a data point having a z-score of 3.0 or greater³⁵. After outliers were eliminated, analysis of variance was used to verify that there was no significant effect because of animal-to-animal variation. Thereafter, all data were pooled. Analysis of variance was also used to examine the effects of graft type and graft volume independently and then to examine the effect of treatment group (graft type and volume combined). Significance was defined as a p value of = 0.05. If analysis of variance demonstrated significant effects for a treatment group, multiple paired Student t tests were used to compare the means for individual treatment groups. The Bonferroni correction was utilized in multiple comparisons to correct for the increased probability of finding false significant differences between group means.

Nonparametric data, which included radiographic and histological grading results, were evaluated with the Kruskal-Wallis one-way analysis of variance to examine the effects of treatment group and postoperative time, and a p value was calculated to determine significance³⁶. Kruskal-Wallis multiple comparisons were performed for each implant type at each time-period (for the radiographic grading results only). An alpha level of 0.10 was chosen as the desired overall significance level. All statistical analyses were performed on a personal computer with use of statistical analysis software (BMDP Statistical Software, Los Angeles, California).

Results

Introduction | Materials and Methods | Results | Discussion | References

There were no intraoperative or postoperative complications, and all animals were able to walk within forty-eight hours after surgery. Postoperative radiographs consistently demonstrated segmental defects of uniform size in the middle of the ulnar shaft.

Radiographic Findings

The number of defects continuously bridged by bone from one cortex to an opposite cortex and the number of defects bridged by new bone across all four cortices at twelve weeks postoperatively are shown in Table IV. The mean radiographic scores, indicating the quantity of new-bone formation and the quality of radiographic healing for each treatment group, are given in Table V. All groups treated with the OP-1 device (OP-1 device alone, autograft and OP-1 device, and allograft and OP-1 device) had significantly higher mean radiographic grades than the two groups treated with allograft alone or autograft alone, independent of the time of the radiographic evaluation (p = 0.00001). No significant difference was detected among the mean radiographic grades for the groups treated with any amount of the OP-1 device, with or without bone graft.

OP-1 Device

Traces of radiodense new bone were observed as early as two weeks postoperatively in defects treated with the OP-1 device alone (Fig. 1). From two to six weeks postoperatively, the amount and density of new bone increased. New bone formed within the defect and, in some cases, outside the original ulnar contours into the surrounding soft tissues. Five of the six defects were bridged and filled with new bone at six weeks. By eight weeks, the new bone began to remodel and to incorporate with the adjacent host bone. Bone that had formed outside the defect borders began to lose radiodensity. At twelve weeks, new bone completely bridged five of the six defects and had begun to conform to the original ulnar contours.

The mean radiographic grade for the six defects treated with the OP-1 device alone was influenced by the grade for one defect that demonstrated less new-bone formation than the other five. At twelve weeks postoperatively, the final radiographic grade for this defect was 1 of a possible 6 points. The other five defects treated with the OP-1 device had final radiographic grades of 4 points or greater.

Autograft Implants

Seven of the nine defects treated with autograft bone alone had evidence of new-bone formation at six weeks postoperatively (Fig. 2, A, B, and C). Bridging of the defects with new bone occurred between six and twelve weeks postoperatively. At twelve weeks, four of the nine defects treated with autograft bone alone demonstrated complete bridging of the defect (Table IV).

In contrast to the defects treated with autograft bone alone, five of the six defects treated with 67% autograft and 33% OP-1 device had new-bone formation as early as two weeks postoperatively (Fig. 2, D, E, and F). By eight weeks, the amount of new bone had increased and had begun to incorporate with the host-bone cortices, except in one defect that demonstrated little new-bone formation at any time. By twelve weeks, the new bone had begun to coalesce toward neocortices in five of the six defects. Although the mean radiographic grades at each time-period were greater than those of the defects treated with autograft and allograft bone alone, the differences in the means were not significant.

Similar to the defects treated with 67% autograft and 33% OP-1 device, five of the six defects treated with 33% autograft and 67% OP-1 device had new-bone formation at two weeks postoperatively (Fig. 2, G, H, and I). Five of the six defects were bridged by bone as early as four weeks postoperatively. Graft incorporation and early remodeling changes were apparent at six weeks in all but one defect. At twelve weeks, five of the six defects were completely filled by new bone. The mean twelve-week radiographic grade for the defects treated with 33% autograft and 67% OP-1 device was significantly greater than that for the defects treated with allograft bone alone (alpha = 0.05). No significant difference was detected in the mean radiographic grades for the defects treated with 33% autograft and 67% OP-1 device and those treated with 67% autograft and 33% OP-1 device.

Allograft Implants

Compared with defects treated with any amount of the OP-1 device or with autograft bone alone, defects treated with 100% allograft bone had markedly reduced new-bone formation. Only three of the nine defects treated with allograft bone alone had radiographically evident new-bone formation that bridged the defect at one cortex at twelve weeks postoperatively (Table IV). Substantial resorption of the allograft bone occurred by twelve weeks (Fig. 3, A, B, and C).

In contrast to defects treated with 100% allograft bone, five of the six defects treated with 67% allograft and 33% OP-1 device had evidence of new-bone formation as early as two weeks postoperatively. By twelve weeks, five of the six defects were bridged and filled with new bone (Fig. 3, D, E, and F). No significant differences were detected between the mean radiographic grades of segmental defects treated with 67% allograft and 33% OP-1 device and those treated with 67% autograft and 33% OP-1 device at any time-point.

Defects treated with 33% allograft and 67% OP-1 device had a radiographic appearance similar to that of defects treated with 33% autograft and 67% OP-1 device. At two weeks postoperatively, new-bone formation was observed in five of the six defects treated with 33% allograft and 67% OP-1 device. By six weeks, new bone had bridged all six defects (Fig. 3, G, H, and I). At twelve weeks, bridging and filling of the defects was complete, and the density of the new bone was equivalent to that of the host bone. No significant differences were detected between the mean radiographic grades of defects treated with 33% autograft and 67% OP-1 device and those treated with 33% allograft and 67% OP-1 device at any time-point. At all radiographic evaluation periods, the defects treated with 33% allograft and 67% OP-1 device had a significantly higher mean radiographic grade than defects treated with either 100% allograft or 100% autograft (alpha = 0.10).

Mechanical Findings

The results of mechanical testing are given in Table VI. Segmental defects that were not mechanically tested because of instability on manual palpation were assumed to have a torsional strength equal to zero. In these instances, a value for zero was included for the calculation of the mean group strength and mean energy absorbed to failure. A total of eleven defects (23%) were not mechanically tested because of lack of rigidity. Only two of the nine defects treated with allograft bone alone and six of the nine defects treated with autograft bone alone were firmly united and able to be mechanically tested. In contrast, twenty-nine (97%) of thirty defects treated with any amount of the OP-1 device were firmly united and could be mechanically tested. No statistical outliers were found (all z-scores were less than 3.0).

Torque to Failure

Segmental defects treated with 33% allograft and 67% OP-1 device had the greatest mean torque to failure (Table VI). This represented 81% of the mean torque to failure of previously tested intact canine ulnae²³. The mean torque to failure for defects receiving the OP-1 device alone (70% of the torque to failure of the intact ulnae) was consistent with the results of previous tests of segmental defects in canine ulnae treated with 750 mg of the OP-1 device²⁰. The mean torques to failure for the defects treated with 100% OP-1 device, 33% allograft and 67% OP-1 device, 33% autograft and 67% OP-1 device, and 67% autograft and 33% OP-1 device were significantly greater than that for the defects treated with allograft bone alone (p = 0.001). With the numbers available, no significant differences were detected between the group treated with the OP-1 device alone and the other groups receiving any combination of bone graft and the OP-1 device with respect to the mean torque to failure (p > 0.05).

Defects treated with 100% allograft, 100% autograft, 67% autograft and 33% OP-1 device, and 67% allograft and 33% OP-1 device failed either at the host bone-new bone interface or in the area with the least amount of new-bone formation. Defects treated with the OP-1 device alone, 33% autograft and 67% OP-1 device, and 33% allograft and 67% OP-1 device most frequently failed through the center of the new bone and bone graft within the defect at an oblique angle to the long axis or through the host bone end at an oblique angle to the long axis of the ulna.

Energy Absorbed to Failure

Consistent with the results for the mean torque to failure, the mean energy absorbed prior to failure was greatest for segmental defects treated with 33% allograft and 67% OP-1 device (Table VI). The mean values for energy absorbed to failure for the groups treated with 33% allograft and 67% OP-1 device, 33% autograft and 67% OP-1 device, 67% autograft and 33% OP-1 device, and the OP-1 device alone were significantly greater than the mean energy absorbed to failure for the group treated with allograft alone (p = 0.05).

Histological Findings

The number of defects with histological evidence of osseous bridging is shown in Table IV, and the results of histological grading are given in Table VII. The defects treated with allograft alone or autograft alone demonstrated markedly less new-bone formation compared with any of the defects treated with any amount of the OP-1 device. Although autograft alone stimulated more new-bone formation than did allograft alone, new-bone formation was meager compared with that in the defects treated with the OP-1 device (Table VII). The mean histological grade for the defects treated with 100% allograft was significantly lower than those for all of the other groups (alpha = 0.10). Similar to the radiographic results, the mean histological grade for the defects treated with 33% allograft and 67% OP-1 device was significantly greater than those for both allograft alone and autograft alone (alpha = 0.05).

Quality of Union

The defects treated with 33% allograft and 67% OP-1 device had the highest prevalence of osseous bridging (Table IV) and the highest mean grade for quality of union (Table VII). In contrast, the nine defects treated with allograft alone were filled and bridged with mature fibrous tissue and had the lowest grade for quality of union. All of the other mean grades for quality of union for the defects treated with any combination of the OP-1 device and bone graft were greater than those for the defects treated with autograft or allograft alone. The group treated with 33% allograft and 67% OP-1 device had a significantly greater mean quality-of-union score than did the groups treated with allograft and autograft alone (alpha = 0.05). The mean quality-of-union grades for defects that received any amount of the OP-1 device or autograft alone were significantly greater than that for defects treated with allograft alone (alpha = 0.10).

Bone-Graft Incorporation and New-Bone Formation

Bone-graft incorporation was observed to be dependent on the formation of new bone that bridged the graft fragments and the host bone. Segmental defects treated with the OP-1 device alone had the greatest amount of new-bone formation observed in any of the groups (Fig. 4), which is in accord with the radiographic results. Extensive graft resorption was observed in defects treated with allograft alone. In defects treated with autograft alone, the graft pieces consolidated although cartilage seams were observed at the new bone-host bone junctions and at the bone graft-bone graft junctions (Fig. 5).

Greater new-bone formation and bone-graft incorporation were observed in segmental defects treated with 67% bone graft and 33% OP-1 device than in defects treated with allograft or autograft alone (Fig. 6). However, the presence of incompletely mineralized cartilaginous seams and incompletely incorporated bone graft was observed more frequently in the defects treated with 67% bone graft and 33% OP-1 device than in defects treated with 33% bone graft and 67% OP-1 device (Fig. 7).

In the groups treated with 33% bone graft and 67% OP-1 device, new bone spanned the defects and was integrated with the host bone and the bone graft (Fig. 7). The defects treated with 33% allograft and 67% OP-1 device had the most advanced graft incorporation observed in any treatment group (Table VII). The allograft was either completely resorbed and replaced with new bone or occasional dense allograft fragments appeared to be well incorporated with new bone. In defects treated with 33% autograft and 67% OP-1 device, the residual autograft bone appeared to be incorporated with new bone although remodeling of the bone graft was not yet complete. The mean grades for bone-graft incorporation and new-bone formation for defects treated with any amount of the OP-1 device were significantly greater than the mean grade for the group treated with allograft alone (alpha = 0.05). The mean grade for the defects treated with 33% allograft and 67% OP-1 device was significantly greater than that for the group treated with autograft alone (alpha = 0.05).

As was noted previously, one defect treated with 33% autograft and 67% OP-1 device and one defect treated with the OP-1 device alone had relatively little new-bone formation compared with other defects treated with any amount of the OP-1 device. Histological studies verified that the amount of new bone within these defects was reduced and, consequently, incorporation of bone graft and new bone was not as advanced. The presence of incompletely mineralized regions of cartilage and osteoid rather than mature fibrous tissue was noted in both cases. Neither an inflammatory response nor residual OP-1 device was observed in these defects.

Cortex Formation and Remodeling

Complete reformation of mature cortices (a grade of 4 on the histological grading scale) was not observed in any treatment group at twelve weeks postoperatively. However, segmental defects with increased new-bone formation and advanced graft incorporation were more likely to have evidence of early cortex formation and reconstitution of marrow space regardless of treatment group (Fig. 4). The defects treated with 33% allograft and 67% OP-1 device had the greatest mean grade for cortex formation (Table VII). Defects treated with any amount of the OP-1 device had a significantly higher mean grade for cortex formation than did the defects treated with allograft alone (alpha = 0.10).

Discussion

Introduction | Materials and Methods | Results | Discussion | References

Large amounts of bone-graft material are frequently utilized to elicit the healing of bone defects associated with a number of orthopaedic reconstructive procedures, including joint revision, tumor resection, spinal fusion, and treatment of trauma or infection. In cases in which allograft bone is expected to sustain mechanical load, failure may occur as the allograft is being resorbed and subjected to repetitive loading⁹. Allograft bone has little osteoinductive capacity. The addition of osteogenic proteins to allograft and autograft bone induces more rapid and greater new-bone formation for initial stability and assurance of rapid graft incorporation.

The present study demonstrates that the osteoinductive capacity of autograft or allograft bone alone is only a fraction of that of the OP-1 device. The combination of bone graft with the OP-1 device improves the amount and rate of new-bone formation and should increase the prevalence of osseous union in difficult clinical applications. The combination of freeze-dried allograft bone with the OP-1 device appears to optimize the osteoconductive and osteoinductive properties of the heterogeneous graft material. Although autogenous graft in combination with the OP-1 device performed similarly, the complications associated with the donor site can be eliminated with the use of allograft, without reducing the efficacy of the bone graft^3,4.

Characterization of bone-healing with the segmental defect model in the canine ulna was first reported in 1934 by Key, who observed that a segmental bone defect at least one and one-half times the diameter of the ulna would not heal spontaneously³². This model has been used to evaluate the efficacy of bone graft and bone-graft substitutes reproducibly and with minimal complications^6,7,20,37. The healing rate and mechanical strength as well as the radiographic and histological findings associated with the defects that were treated with the OP-1 device alone in the present investigation were consistent with previously reported results^20,23. In a recent series of twenty-eight bilateral nonstabilized segmental defects in canine ulnae treated with the OP-1 device alone, new bone formed in all defects although three defects (11%) had incomplete osseous union at twelve weeks²³.

In the present study, twenty-nine (97%) of thirty defects treated with any amount of the OP-1 device achieved a stable union, as assessed by manual palpation, at twelve weeks. Defects treated with 33% bone graft and 67% OP-1 device achieved the highest rate of stable osseous union (twelve of twelve defects) and had the most advanced healing, both radiographically and histologically, compared with the defects in all of the other treatment groups. In comparison, Heiple et al., in a study of bilateral nonstabilized 2.5-cm defects in canine ulnae that were treated with cancellous autograft bone, reported a union rate of only 25% (four of sixteen) at twelve weeks; however, by six months, the rate of osseous union was 94% (fifteen of sixteen)⁷.

Moore et al., in a study of segmental defects in canine ulnae that were treated with cancellous autograft, reported that 89% of the torsional strength of intact control ulnae was achieved at twenty-four weeks postoperatively³⁸. In our study, at twelve weeks, the torsional strength of the defects treated with any amount of the OP-1 device ranged from 41% to 81% of that of the intact ulnae. The early restoration of mechanical strength is suggestive of a more advanced healing stage because of extensive new-bone formation and advanced remodeling³⁹.

Delloye et al., in a study on the reconstruction of 3.0-cm defects in the canine ulna, demonstrated that cortical allografts, whether fresh, fresh frozen, or demineralized, had a higher rate of peripheral resorption than did autograft controls⁶. In our study, the speed at which the allograft bone resorbed in the group treated with 33% allograft and 67% OP-1 device seemed to accommodate the accelerated rate of new-bone formation. Consistent with the observations of Delloye et al., our study showed that autograft in combination with the OP-1 device remained viable while incorporating with the new bone. Radiographically and histologically, remodeling of the new construct was not as advanced in the defects managed with autograft as it was in the defects treated with the equivalent amount of allograft and the OP-1 device.

All three methods for evaluation of bone-healing gave consistent results in terms of the relative prevalence of osseous union. However, only histological analysis yields the true prevalence of osseous healing. On plain radiographs, it can be difficult or impossible to discern thin fibrous or cartilaginous seams. Mechanically, even in the absence of complete osseous union, a nonunion or fibrocartilaginous union may have substantial strength. Even on manual palpation, a small amount of motion may not be appreciated and the presence of a fibrous or cartilaginous seam may be missed. Histological studies demonstrated the presence of incompletely mineralized seams between new bone and bone graft or between new bone and the host-bone ends in three defects treated with some portion of the OP-1 device that were incompletely healed at twelve weeks despite the formation of new bone.

Radiographically and histologically, defects treated with the OP-1 device alone demonstrated the greatest osteoinductive response. However, the mean histological and radiographic grades for the defects treated with 100% OP-1 device were less than those for the defects treated with 33% bone graft and 67% OP-1 device. This may be attributed, in part, to less advanced remodeling of the new bone. The remodeling of callus involves the resorption of the unorganized bone mass required for initial stability followed by the deposition of bone in a mature lamellar structure. One might expect that the remodeling phase after an extensive proliferation of callus may take longer than the remodeling phase when less osteoinductive factor is used and less extensive callus is produced. Ripamonti et al. examined the dose effect of rhOP-1, bound to a collagen carrier, on the restoration of critical-sized calvarial defects in primates⁴⁰. They found all doses capable of inducing osseous healing; however, the highest dose resulted in new-bone formation that exceeded the calvarial cortices, with a slower rate of overall remodeling than that achieved with lower doses. They concluded that a lower dose of rhOP-1 may be optimal to control the amount of new bone formed and thus the rate of remodeling.

A multitude of processing methods affect the biomechanical properties, osteoinductivity, and biocompatibility of allograft bone^41-44. Cleaned and lyophilized allograft is a form commonly found in bone banks. Because no attempt was made to match histocompatibility, the cleaning process chosen in this study reduced blood and marrow transfer. Antigenicity was further reduced by lyophilization^41,42. After the addition of the OP-1 device to fresh autograft and freeze-dried allograft, both materials behaved similarly. Thus, any deleterious effects upon the allograft from cleaning and freeze-drying seemed to be negated. This is a clinically important finding in that freeze-dried allograft in combination with OP-1 can produce a graft with osteoinductive properties superior to those of autograft or allograft alone.

The size and form of the bone graft controls the incorporation and, to some extent, the osteoinductivity of the graft^2,5,45. The size and shape of bone-graft materials used in the present study were based on a clinically relevant scenario in which a bone graft must fill and maintain a large void volume while acting as a scaffold to host new-bone formation. Once prepared for implantation, the OP-1 device alone can migrate because of its particulate nature. Cancellous cubes of bone graft of the size used in this investigation are commonly used to fill bone voids. Small bone particles or powders, whether allograft or autograft, would be suboptimal to fill a large void since they also could migrate or flow. Small bone chips, alternatively, might revascularize faster, resorb quicker, and perhaps impart an improved osteoinductive response because of greater surface area. Optimization of the size and shape of allograft to use in conjunction with an osteoinductive factor is an area for future research.

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Introduction | Materials and Methods | Results | Discussion | References

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Gerhart TN; Kirker-Head CA; Kriz MJ; Hipp JA; Rosen V; Schelling S; Wozney JM; and Wang EA: Healing of large mid-femoral segmental defects in sheep using recombinant human bone morphogenetic protein (BMP-2). Trans Orthop Res Soc,1991.16: 172, 16172 1991

Ripamonti U: Delivery systems for bone morphogenetic proteins. A summary of experimental studies in primate models. Ann Chir Gynaecol Suppl,1993.207: 13-24, 20713 1993 [PubMed]

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Sampath TK; Maliakal JC; Hauschka PV; Jones WK; Sasak H; Tucker RF; White KH; Coughlin JE; Tucker MM; Pang RH; and et al: Recombinant human osteogenic protein-1 (hOP-1) induces new bone formation in vivo with a specific activity comparable with natural bovine osteogenic protein and stimulates osteoblast proliferation and differentiation in vitro. J Biol Chem,1992.267: 20352-62, 26720352 1992 [PubMed]

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Malinin TI, Wu NM, Flores A. Freeze-drying of bone for allotransplantation. In: Friedlaender GE, Mankin HJ, Sell KW, editors. Osteochondral allografts: biology, banking, and clinical applications. Boston: Little, Brown; 1983. p 181-92.

Tomford WW; Mankin HJ; Friedlaender GE; Doppelt SH; and Gebhardt MC: Methods of banking bone and cartilage for allograft transplantation. Orthop Clin North Am,1987.18: 241-7, 18241 1987 [PubMed]

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Horowitz M, and Friedlaender GE: Immunologic aspects of bone transplantation. A rationale for future studies. Orthop Clin North Am,1987.18: 227-33, 18227 1987 [PubMed]

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TABLE I: Experimental Bone-Graft Treatment Groups

*The OP-1 device consists of recombinant human osteogenic protein-1 (rhOP-1) combined with type-I bovine bone-derived collagen. †100% volume is 3.0 cm³ of implant material.

Treatment Group*	Volume Percentage (Graft:OP-1 Device)†	Amount of OP-1 Device (mg)	Amount of Saline Solution Added for Reconstitution (ml)	No. of Defects
100% allograft	100:0	??0	0	9
67% allograft and 33% OP-1 device	67:33	250	0.08	6
33% allograft and 67% OP-1 device	33:67	500	0.16	6
100% autograft	100:0	??0	0	9
67% autograft and 33% OP-1 device	67:33	250	0.08	6
33% autograft and 67% OP-1 device	33:67	500	0.16	6
100% OP-1 device	0:100	750	0.24	6

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TABLE I: Experimental Bone-Graft Treatment Groups

*The OP-1 device consists of recombinant human osteogenic protein-1 (rhOP-1) combined with type-I bovine bone-derived collagen. †100% volume is 3.0 cm³ of implant material.

Treatment Group*	Volume Percentage (Graft:OP-1 Device)†	Amount of OP-1 Device (mg)	Amount of Saline Solution Added for Reconstitution (ml)	No. of Defects
100% allograft	100:0	??0	0	9
67% allograft and 33% OP-1 device	67:33	250	0.08	6
33% allograft and 67% OP-1 device	33:67	500	0.16	6
100% autograft	100:0	??0	0	9
67% autograft and 33% OP-1 device	67:33	250	0.08	6
33% autograft and 67% OP-1 device	33:67	500	0.16	6
100% OP-1 device	0:100	750	0.24	6

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TABLE II: Radiographic Grading Scale for the Degree of Healing

Description	Grade
No change from immediate postoperative appearance	0
A slight increase in radiodensity distinguishable from the graft	1
Recognizable increase in radiodensity, bridging of one cortex with new-bone formation to the graft	2
Bridging of at least one cortex with material of nonuniform radiodensity, early incorporation of the graft suggested by obscurity of graft borders	3
Defect bridged on both medial and lateral sides with bone of uniform radiodensity, cut ends of the cortex still visible, graft and new bone not easy to differentiate	4
Same as grade 3, with at least one of four cortices obscured by new bone	5
Defect bridged by uniform new bone, cut ends of cortex no longer distinguishable, graft no longer visible	6

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TABLE II: Radiographic Grading Scale for the Degree of Healing

Description	Grade
No change from immediate postoperative appearance	0
A slight increase in radiodensity distinguishable from the graft	1
Recognizable increase in radiodensity, bridging of one cortex with new-bone formation to the graft	2
Bridging of at least one cortex with material of nonuniform radiodensity, early incorporation of the graft suggested by obscurity of graft borders	3
Defect bridged on both medial and lateral sides with bone of uniform radiodensity, cut ends of the cortex still visible, graft and new bone not easy to differentiate	4
Same as grade 3, with at least one of four cortices obscured by new bone	5
Defect bridged by uniform new bone, cut ends of cortex no longer distinguishable, graft no longer visible	6

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TABLE III: Histological Grading Scale for the Degree of Healing

	Description	Grade
Quality of union	No sign of fibrous or other union	0
	Fibrous union	1
	Fibrocartilaginous union or cartilage union	2
	Mineralizing cartilage and bone union	3
	Bone union	4
Cortex development and remodeling	No cortex formed	0
	Densification of new bone along exterior borders	1
	Recognizable formation of both the outer cortex border and the medullary space	2
	Cortices formed but incomplete bridging	3
	Complete formation of cortices with bridging of defect	4
Bone-graft incorporation and new-bone formation
No new bone, all or most of graft visible	Graft material present, no incorporation, and no new-bone formation	0
	Graft present, some incorporation with new-bone formation, and small amount of new bone	1
	Graft present, some incorporation with new-bone formation, and moderate amount of new bone	2
Decreasing graft, increasing new bone	Graft present, some incorporation with new-bone formation continuous with host bone, and early remodeling changes in new bone	3
	Decreased amount of graft (compared with grade 3), good graft incorporation, and ample new bone	4
	Less amount of graft still visible (compared with grade 4), good incorporation of graft and new bone with host and ample new bone	5
No graft visible, extensive new bone	Difficult to differentiate graft from new bone, excellent incorporation, and advanced remodeling of new bone with graft and host	6

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TABLE III: Histological Grading Scale for the Degree of Healing

	Description	Grade
Quality of union	No sign of fibrous or other union	0
	Fibrous union	1
	Fibrocartilaginous union or cartilage union	2
	Mineralizing cartilage and bone union	3
	Bone union	4
Cortex development and remodeling	No cortex formed	0
	Densification of new bone along exterior borders	1
	Recognizable formation of both the outer cortex border and the medullary space	2
	Cortices formed but incomplete bridging	3
	Complete formation of cortices with bridging of defect	4
Bone-graft incorporation and new-bone formation
No new bone, all or most of graft visible	Graft material present, no incorporation, and no new-bone formation	0
	Graft present, some incorporation with new-bone formation, and small amount of new bone	1
	Graft present, some incorporation with new-bone formation, and moderate amount of new bone	2
Decreasing graft, increasing new bone	Graft present, some incorporation with new-bone formation continuous with host bone, and early remodeling changes in new bone	3
	Decreased amount of graft (compared with grade 3), good graft incorporation, and ample new bone	4
	Less amount of graft still visible (compared with grade 4), good incorporation of graft and new bone with host and ample new bone	5
No graft visible, extensive new bone	Difficult to differentiate graft from new bone, excellent incorporation, and advanced remodeling of new bone with graft and host	6

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TABLE IV: Number of Defects Bridged from Cortex to Cortex by New Bone at Twelve Weeks Postoperatively

*Defects deemed rigidly healed on manual palpation and subsequent mechanical testing. †Defects with a mean quality-of-union score of 3.0.

Treatment Group	Radiographic Evidence		Mechanical Evidence*	Histological Evidence†
1 Cortex Bridged	All Cortices Bridged
100% allograft (n = 9)	3	0	2	0
67% allograft and 33% OP-1 device (n = 6)	6	5	5	4
33% allograft and 67% OP-1 device (n = 6)	6	6	6	6
100% autograft (n = 9)	5	4	6	6
67% autograft and 33% OP-1 device (n = 6)	5	5	5	3
33% autograft and 67% OP-1 device (n = 6)	5	5	6	5
100% OP-1 device (n = 6)	5	5	5	4

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TABLE IV: Number of Defects Bridged from Cortex to Cortex by New Bone at Twelve Weeks Postoperatively

*Defects deemed rigidly healed on manual palpation and subsequent mechanical testing. †Defects with a mean quality-of-union score of 3.0.

Treatment Group	Radiographic Evidence		Mechanical Evidence*	Histological Evidence†
1 Cortex Bridged	All Cortices Bridged
100% allograft (n = 9)	3	0	2	0
67% allograft and 33% OP-1 device (n = 6)	6	5	5	4
33% allograft and 67% OP-1 device (n = 6)	6	6	6	6
100% autograft (n = 9)	5	4	6	6
67% autograft and 33% OP-1 device (n = 6)	5	5	5	3
33% autograft and 67% OP-1 device (n = 6)	5	5	6	5
100% OP-1 device (n = 6)	5	5	5	4

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TABLE V: Radiographic Grade of Ulnar Segmental Defects

Treatment Group	Radiographic Grade*
2 Weeks	4 Weeks	6 Weeks	8 Weeks	12 Weeks
100% allograft (n = 9)	0.1 ± 0.3	0.6 ± 0.5	1.2 ± 0.7	1.7 ± 1.1	1.8 ± 1.1
67% allograft and 33% OP-1 device (n = 6)	0.8 ± 0.4	2.3 ± 0.8	3.0 ± 0.9	3.7 ± 0.8	4.3 ± 1.0
33% allograft and 67% OP-1 device (n = 6)	1.0 ± 0.6	2.5 ± 0.8	4.0 ± 0.6	5.0 ± 0.6	5.3 ± 0.5
100% autograft (n = 9)	0.0 ± 0.0	0.7 ± 0.5	1.4 ± 1.0	2.0 ± 1.2	2.9 ± 1.8
67% autograft and 33% OP-1 device (n = 6)	0.8 ± 0.4	2.0 ± 0.4	3.2 ± 1.2	3.7 ± 1.5	4.2 ± 1.2
33% autograft and 67% OP-1 device (n = 6)	1.2 ± 0.8	2.7 ± 0.8	3.3 ± 0.8	3.8 ± 1.0	4.5 ± 1.2
100% OP-1 device (n = 6)	0.8 ± 0.8	2.3 ± 0.8	3.5 ± 1.2	4.0 ± 1.6	4.3 ± 1.9

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TABLE V: Radiographic Grade of Ulnar Segmental Defects

Treatment Group	Radiographic Grade*
2 Weeks	4 Weeks	6 Weeks	8 Weeks	12 Weeks
100% allograft (n = 9)	0.1 ± 0.3	0.6 ± 0.5	1.2 ± 0.7	1.7 ± 1.1	1.8 ± 1.1
67% allograft and 33% OP-1 device (n = 6)	0.8 ± 0.4	2.3 ± 0.8	3.0 ± 0.9	3.7 ± 0.8	4.3 ± 1.0
33% allograft and 67% OP-1 device (n = 6)	1.0 ± 0.6	2.5 ± 0.8	4.0 ± 0.6	5.0 ± 0.6	5.3 ± 0.5
100% autograft (n = 9)	0.0 ± 0.0	0.7 ± 0.5	1.4 ± 1.0	2.0 ± 1.2	2.9 ± 1.8
67% autograft and 33% OP-1 device (n = 6)	0.8 ± 0.4	2.0 ± 0.4	3.2 ± 1.2	3.7 ± 1.5	4.2 ± 1.2
33% autograft and 67% OP-1 device (n = 6)	1.2 ± 0.8	2.7 ± 0.8	3.3 ± 0.8	3.8 ± 1.0	4.5 ± 1.2
100% OP-1 device (n = 6)	0.8 ± 0.8	2.3 ± 0.8	3.5 ± 1.2	4.0 ± 1.6	4.3 ± 1.9

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TABLE VI: Results of Mechanical Testing*

Treatment Group	No. of Segments Tested†	Maximum Torque to Failure (Nm)	Percent of Strength ofIntact Controls	Energy Absorbed to Failure (Nmldeg)
100% allograft (n = 9)	2	0.15 ± 0.30	3.84 ± 7.63	4.33 ± 8.61
67% allograft and 33% OP-1 device (n = 6)	6	1.60 ± 1.43	40.73 ± 36.61	40.77 ± 44.87
33% allograft and 67% OP-1 device (n = 6)	6	3.18 ± 1.68	81.27 ± 42.90	82.13 ± 55.07
100% autograft (n = 9)	6	1.33 ± 1.42	34.08 ± 36.37	32.78 ± 30.22
67% autograft and 33% OP-1 device (n = 6)	6	2.76 ± 1.69	70.53 ± 43.08	69.70 ± 43.42
33% autograft and 67% OP-1 device (n = 6)	6	2.85 ± 1.40	72.70 ± 35.71	75.88 ± 33.58
100% OP-1 device (n = 6)	5	2.74 ± 1.60	69.95 ± 40.85	65.80 ± 38.32

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TABLE VI: Results of Mechanical Testing*

Treatment Group	No. of Segments Tested†	Maximum Torque to Failure (Nm)	Percent of Strength ofIntact Controls	Energy Absorbed to Failure (Nmldeg)
100% allograft (n = 9)	2	0.15 ± 0.30	3.84 ± 7.63	4.33 ± 8.61
67% allograft and 33% OP-1 device (n = 6)	6	1.60 ± 1.43	40.73 ± 36.61	40.77 ± 44.87
33% allograft and 67% OP-1 device (n = 6)	6	3.18 ± 1.68	81.27 ± 42.90	82.13 ± 55.07
100% autograft (n = 9)	6	1.33 ± 1.42	34.08 ± 36.37	32.78 ± 30.22
67% autograft and 33% OP-1 device (n = 6)	6	2.76 ± 1.69	70.53 ± 43.08	69.70 ± 43.42
33% autograft and 67% OP-1 device (n = 6)	6	2.85 ± 1.40	72.70 ± 35.71	75.88 ± 33.58
100% OP-1 device (n = 6)	5	2.74 ± 1.60	69.95 ± 40.85	65.80 ± 38.32

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TABLE VII: Histological Grade of Ulnar Segmental Defects*

*The values are given as the mean and the standard deviation. The histological grading scale is shown in Table III.

Treatment Group	Quality of Union (0 to 4)	Cortex Development and Remodeling (0 to 4)	Bone-Graft Incorporation and New-Bone Formation (0 to 6)	Total Grade (0 to 14)
100% allograft (n = 9)	1.1 ± 0.4	0.1 ± 0.3	0.6 ± 0.9	?1.8 ± 1.5
67% allograft and 33% OP-1 device (n = 6)	2.8 ± 0.7	1.1 ± 0.7	3.3 ± 1.1	?7.3 ± 2.2
33% allograft and 67% OP-1 device (n = 6)	3.9 ± 0.3	1.7 ± 0.9	5.0 ± 1.0	10.7 ± 1.4
100% autograft (n = 9)	2.5 ± 1.0	0.9 ± 0.9	2.3 ± 1.6	?5.7 ± 3.4
67% autograft and 33% OP-1 device (n = 6)	3.2 ± 0.9	0.9 ± 0.7	3.3 ± 1.4	?7.3 ± 2.8
33% autograft and 67% OP-1 device (n = 6)	3.3 ± 1.2	1.4 ± 0.9	3.7 ± 1.7	?8.4 ± 3.5
100% OP-1 device (n = 6)	3.3 ± 1.2	1.2 ± 0.7	3.1 ± 1.2	?7.5 ± 2.9

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TABLE VII: Histological Grade of Ulnar Segmental Defects*

*The values are given as the mean and the standard deviation. The histological grading scale is shown in Table III.

Treatment Group	Quality of Union (0 to 4)	Cortex Development and Remodeling (0 to 4)	Bone-Graft Incorporation and New-Bone Formation (0 to 6)	Total Grade (0 to 14)
100% allograft (n = 9)	1.1 ± 0.4	0.1 ± 0.3	0.6 ± 0.9	?1.8 ± 1.5
67% allograft and 33% OP-1 device (n = 6)	2.8 ± 0.7	1.1 ± 0.7	3.3 ± 1.1	?7.3 ± 2.2
33% allograft and 67% OP-1 device (n = 6)	3.9 ± 0.3	1.7 ± 0.9	5.0 ± 1.0	10.7 ± 1.4
100% autograft (n = 9)	2.5 ± 1.0	0.9 ± 0.9	2.3 ± 1.6	?5.7 ± 3.4
67% autograft and 33% OP-1 device (n = 6)	3.2 ± 0.9	0.9 ± 0.7	3.3 ± 1.4	?7.3 ± 2.8
33% autograft and 67% OP-1 device (n = 6)	3.3 ± 1.2	1.4 ± 0.9	3.7 ± 1.7	?8.4 ± 3.5
100% OP-1 device (n = 6)	3.3 ± 1.2	1.2 ± 0.7	3.1 ± 1.2	?7.5 ± 2.9