Although autograft cancellous bone is considered the gold standard for bone grafting,1 a high level of morbidity is associated with its harvest.2 Many bone graft substitutes have been proposed in an attempt to eliminate the resultant side effects of iliac crest harvest.3,4 Important and variable features of bone graft substitutes include their osteogenic and osteoconductive potentials, injectability, ability to uniformly fill defects, and propensity to remain at the intended site.
Many animal and clinical tests have been conducted to evaluate allograft material, and demineralized bone matrix (DBM) has been found to be an effective bone graft substitute in a variety of osseous defects.5,6 Demineralized bone matrix is composed of insoluble collagen and proteins that are noncollagenous in nature.7,8
Urist and Strates9 isolated bone morphogenetic protein (BMP) and declared it partially responsible for bone cell differentiation and the inductive nature of DBM. They also found that minerals in bone shield the BMPs from signaling bone induction. Therefore, the demineralization process enables the insoluble BMPs to induce bone formation.
Bone morphogenetic protein initiates the cascade of bone formation that takes place at a defect site.8 The bone matrix proteins signal mesenchymal cells to transform into osteoprogenitor cells to produce new bone growth.10 In addition to the inductive effect of the proteins in DBM, the collagen structure plays an osteoconductive role."
The use of calcium sulfate as a bone graft substitute was documented as early as 1 892 by Dreesman.12 A thorough literature review conducted by Peltier4 summarized successful filling of bone defects with calcium sulfate. These studies demonstrated that calcium sulfate resorbs and is well tolerated by tissues. Peltier and Speer13 confirmed that calcium sulfate is an osteoconductive material that allows ingrowth of blood vessels and osteogenic cells.
The purpose of this study was to evaluate an injectable, hard-setting, calcium sulfate-based putty containing DBM (AlloMatrix II, Wright Medical Technology, Ine) for its ability to restore large medullary bone defects. The putty is a mixture of surgical-grade calcium sulfate hemihydrate, DBM, calcium sulfate dihydrate, and hydroxypropylmethylcellulose and hardens approximately 5-10 minutes after mixing.
Rgure 1: Sequential radiographs of a defect treated with putty (top to bottom: preoperatively, immediate postoperatively, and 2, 6, 13, and 26 weeks). Figure 2: Sequential radiographs of a defect treated with autograft (top to bottom: preoperatively, immediately postoperatively, and Z1 6, 1 3, and 26 weeks).
Materials and Methods
Healing of canine defects implanted with a calcium sulfate-based putty with DBM was compared to defects implanted with autogenous bone. Thirteen mongrel dogs were implanted bilaterally in their proximal humeri with the study treatments randomly assigned to each side. The schedule of euthanasia and evaluation was as follows: three dogs at 6 weeks, five dogs at 13 weeks, and five dogs at 26 weeks. At each study interval, healing was characterized using radiographic and histologic methods, in addition to measuring area fraction, modulus of elasticity, and compressive strength of newly formed bone.
1% Secant Modulus of Elasticity (mPa) by Implant Material and Interval
Area Fraction of New Bone by Implant Material and Interval
All DBM and cancellous bone chips used in this study were canine derived.
Under general anesthesia and using an aseptic technique, a cranial approach to the greater tubercle of the left and right humerus was performed. A cylindrical cavity was created bilaterally in the humerus by drilling axially through the greater tubercle into the medullary canal. The cavity measured 13 mm in diameter X 50 mm in length.
The prepared cavity was filled with 6 cc of either AlloMatrix ? Putty or autograft according to a computergenerated randomization schedule. After implantation of the putty or autograft, the supraspinatus tendon was closed over the defect in the greater tubercle, and the wound was closed in layers in a routine fashion.
The putty injected easily into the defects and was intruded into all defect interstices. In contrast, autografttreated defects were filled with bone particles as a bolus and small areas of the defects were under-filled.
All surgeries and animal care were performed in accordance with Institutional Animal Care and Use Committee-approved guidelines, the Guide for Care and Use of Laboratory Animals, H and the regulations of the United States Department of Agriculture Welfare Act.15
Kits intended to produce 7-cc batches of putty were made with the following materials: surgical-grade calcium sulfate hemihydrate, calcium sulfate accelerator, hydroxypropylmethylcellulose, canine DBM, and sterile water for irrigation. The kits were packaged and sterilized with electronic beam radiation.
Upon return from sterilization, a trial kit was mixed to ensure appropriate handling and setting characteristics. The putty exhibited good handling characteristics, was injectable through a large diameter syringe, and hardened in approximately 7 minutes.
Figure 3: Contact radiographs of autograft-treated defect (right) and putty-treated defect (left) at 6 weeks. Demineralized bone matrix particles are evident in the center of the putty-treated defect (A). At 1 3 weeks, virtually complete defect filling is seen with both materials (B). At 26 weeks, the defects are completely filled, and the bone has remodeled to a normal architecture (C).
The canine DBM was purchased from a veterinarian tissue supply company (Veterinary Transplant Services, Kent, Wash) and had undergone the following processing. Canine bones were cleaned of soft tissue and ground to particle size <1250 mm (sieved). Bone particles were treated in a 0.6-N HCl solution until the pH dropped below 1.0 and were then treated with antibiotics. The bone particles were rinsed with sterile water and phosphate buffer solution, packaged into plastic syringes, and frozen (-800C). This process resulted in canine DBM that was "wet frozen." Prior to putty formulation, the putty was dried to remove free moisture.
Radiographs were taken of the humeri preoperatively, immediately postoperatively, and at 2, 6, 13, and 26 weeks throughout the study. Additionally, high resolution contact radiographs were obtained of the isolated right and left humeri after sacrifice. The bones were sectioned serially in the transverse plane to produce sections for mechanical testing and other sections for histological analysis.
For mechanical testing, a 17-mm thick section was obtained from between the middle and distal section of each humerus. At the time of testing, a cylindrical trabecular specimen (8 mm diameter X 16 mm long) was machined from the specimen block. The prepared specimen was mounted on an Instron 1321 servo-hydraulic mechanical test system (Instron Corporation, Canton, Mass) and subjected to a uniaxial compressive strain at a rate of 0.5 mm/min until failure.
A stress-strain curve was generated, and the 1% secant modulus of elasticity was calculated. Data were analyzed using a paired T-test (SigmaStat, Jandel Scientific, San Rafael, CaIiO and differences were considered significant when P<.05.
Area fraction of new bone in the defects was quantitated from plasticembedded, undecalcified, transverse histological sections stained with basic fuchsin and toluidine blue. Standard point-counting techniques were used. A 12-mm diameter field was identified within the bone defect in each section evaluated. A point-counting grid consisting of 1-mm squares was placed over each field to be analyzed.
The tissue underlying each grid intersection (or point) was recorded as either new bone, residual autograft, residual DBM, residual calcium sulfate, or none of the preceding. An average of 108 intersections were evaluated for each histological field.
Three histological sections (proximal, middle, and distal) were assessed for each humeral defect. The number of intersections in each of the three sections was summed. The area fraction of new bone was calculated as the total number of intersections positive for new bone divided by the total number of possible intersections. The data were entered into SPSS (Version 10), and the significance of the difference in new bone formation between humeri treated with putty and with cancellous autograft was determined using the nonparametric Friedman test. Differences of <.05 were considered significant.
Qualitative assessments of the postmortem contact radiographs of intact bones and cross sections, and the stained histologic sections, were performed to characterize the nature of the new bone and the residual graft material in the defects.
Figure 4: Histological image of putty-treated defect at 6 weeks. Putty and DBM particulate remain in the center of the defect, with abundant new bone formation throughout the defect (left). Demineralized bone matrix particles (D) are surrounded by new woven bone (W). Trace amounts of putty (A) are fully incorporated by new bone (right).
Figure 5: Histological section of autograft-treated defect at 6 weeks. An area of fibrous tissue is present in the center, surrounded by new bone formation (left). Graft remnants (G) are present, as are new trabeculae (T) and regions of cartilage (C) (right).
Figure 6: Histological section of putty-treated defect at 13 weeks. Abundant new bone formation surrounded by marrow is present.
Figure 7: Histological section of autografttreated defect at 1 3 weeks. Abundant new bone formation surrounded by marrow is present.
All animals completed 6, 13, or 26 weeks of experimentation with no surgical complications or adverse reactions. In radiographs of specimens containing the DBM putty, the putty was clearly evident in the postoperative and 2-week period but appeared to have been replaced by bone in the 6-, 13-, and 26- week periods (Figure 1). Radiographs of autograft-treated defects showed a uniform density comparable to surrounding bone throughout the study (Figure 2).
Contact radiographs at 6 weeks showed that residual putty and DBM particulate were clearly present in the putty-filled specimens (Figure 3). The 1 3- and 26- week specimens illustrated nearly complete defect filling for both the putty and autograft-treated specimens.
Histological sections at 6 weeks showed that the center of the defect treated with putty contained residual putty material surrounded by a zone of cellular activity with new bone formation present beyond the zone (Figure 4). New, immature woven bone formation was clearly evident surrounding the DBM particles.
The results with autograft at 6 weeks showed an area of fibrous tissue in the center of the defect with new trabeculae forming around this area (Figure 5). Areas of cartilage were present, as were areas of the original graft tissue and new trabeculae.
At 13 weeks, the histologic picture was more mature. Putty-filled defects had new bone throughout the defect surrounded by marrow (Figure 6). Trace amounts of putty remained. In the autograft specimens, a similar histologic picture was present (Figure 7). Bone was located throughout the defect surrounded by marrow. Small areas of the original graft were present.
The histological results at 26 weeks showed virtually complete filling of both the defect treated with putty (Figure 8) and the defect treated with autograft (Figure 9). New bone remodeling was evident in both groups.
Mechanical testing was completed for the 6-, 13-, and 26- week specimens. For each specimen, a stress-strain curve was generated and the secant modulus at Wo was calculated. The data were analyzed using a T-test. No significant differences between the groups at 6, 13, or 26 weeks were detected (P =.98, .93, and .665, respectively) (Table 1 ).
At the 26-week period, the stressstrain curves exhibited a typical pattern of brittle fracture for trabecular bone under compression. At the 6- and 13week intervals, the specimens did not consistently demonstrate a definitive yield for either the putty- or autografttreated specimens. The mean compressive strength of new bone at 26 weeks was 1.5 mPa (SD= 1.2) for the puttyfilled defects and 1.2 mPa (SD=0.5) for the autograft-treated defects. These values were not significantly different (P=. 655).
The 13- and 26- week results from the bone quantification analysis showed that there was no statistical difference according to a Friedman test when the amount of new bone formed in the defects filled with putty was compared to the defects filled with autograft (Table 2). However, at 6 weeks, the putty-filled defects had a greater amount of new bone formed than the autograft defects, and the difference was statistically significant (P=. OS).
Histologically, at 6 weeks, the puttyand autograft-treated defects contained abundant new trabeculae, many of which had formed around DBM or autograft particles. Foci of fibrous tissue contained autograft particles with adjacent fibrocartilage formation. The majority of the putty had resorbed. Residual putty in the central part of the defect was undergoing resorption by multinucleated, osteoclast-like cells. Small particles of residual putty material were present in macrophages scattered in the intertrabecular spaces. The low amounts of residual putty and graft materials were mostly incorporated into the new bone.
Radiographically, the putty-treated defects had <25% decrease in density at 2 weeks, and the putty was completely resorbed by 6 weeks. Concurrent with putty resorption was replacement by bone, so that at 6 weeks, the density of the defect was comparable to the surrounding medullary bone. The autografted defects maintained a uniform density comparable to surrounding bone throughout the study. At 26 weeks, there was no difference in the radiographic appearance of putty compared to the autograft-treated defects.
In this canine humeral animal model, a calcium sulfate-based putty containing DBM was compared with autograft at 6-, 13-, and 26- week time points using radiographic, histologic, and mechanical testing methods. Both the putty and autograft resulted in complete filling of the defect at the 13- and 26- week time periods. There were no substantial differences in the bone formation or strength of the healed bone resulting from the implantation of AlloMatrix II and autograft.
Figure 8: Histological section of putty-treated defect at 26 weeks. Bone formation throughout the defect is complete and the periphery of the defect is difficult to determine. A few DBM particles remain in the center (A). Demineralized bone matrix particles are completely surrounded by woven and lamellar bone (B).
Figure 9: Histological section of autograft-treated defect at 26 weeks. Bone formation throughout the defect is complete, and the periphery of the defect is difficult to determine (A). An area of cartilage is seen adjacent to newly formed lamellar bone (B).
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1% Secant Modulus of Elasticity (mPa) by Implant Material and Interval
Area Fraction of New Bone by Implant Material and Interval