Osseous Healing Using Injectable Calcium Sulfate-Based Putty for the Delivery of Demineralized Bone Matrix and Cancellous Bone Chips

ABSTRACT

Three formulations of injectable calcium sulfate-based putties containing demineralized bone matrix (DBM), 50% DBM/50% cancellous bone (CB) chips, and 30% DBM/70% CB were studied in canines. Four humeral defects per dog were implanted with one of each of the putty formulations while the fourth defect was left untreated. After 6 weeks, the dogs were euthanized. Radiographs and histology showed that the area fraction of new bone in the defects was greater for the three putty formulations than the untreated defects. The area of residual cancellous bone graft remaining in the defects was <10% in both CB putties. Residual calcium sulfate was not apparent in any of the histological sections. We conclude that fast-resorbing calcium sulfate-based putties are effective delivery means of bone graft materials for the successful restoration of bony defects.

Demineralized bone matrix (DBM) and nondemineralized cancellous bone (CB) matrix are allogenic bone graft substitute materials widely used by orthopedic surgeons for the restoration of bone defects. Demineralized bone matrix and CB products are established osteoinductive and osteoconductive bone graft substitute materials, respectively.^1-4

Demineralized bone matrix is available in various forms, including whole bones, blocks of bone, strips, and particles, but the most common form is particulate, or powder. Demineralized bone matrix particles typically range from 125 to 710 µm in size. Cancellous bone matrix is available in larger particulate sizes, typically between 1 and 10 mm. Efficient delivery of these materials into a defect can be hampered by difficulties in handling the materials, positioning during surgery, and the robustness of the materials.⁵

Calcium sulfate-based putties containing DBM and CB (AlloMatrix Cancellous Putty, Wright Medical Technology, Inc, Arlington, Tenn) were developed to address these concerns. Numerous animal and clinical studies have shown calcium sulfate to be an effective bone graft substitute material when used in a hardened form or as an injectable putty containing DBM.^6-11

Improved handling characteristics, in situ robustness, and improved structural properties make this putty a promising bone graft substitute material. The goal of this study was to determine the effectiveness of AlloMatrix Cancellous Putty in healing an osseous defect.

This canine study evaluated the bone healing response to two formulations of calcium sulfate-based putties containing DBM and CB compared with a calcium sulfate-based putty containing DBM only (AlloMatrix Injectable Putty, Wright Medical Technology, Inc).

The putties comprised a powder blend of surgical-grade calcium sulfate hemihydrate, carboxymethylcellulose, DBM, and a solution of sterile water mixed together at the time of surgery, resulting in a material with putty-like characteristics placed into an osseous defect either manually or with the use of a syringe.

Previous animal studies have shown that the material is biocompatible and its implantation causes little or no stimulation of foreign body giant cell reaction.¹¹ The addition of the larger CB particles increases the stiffness of the putty, allowing it to resist certain loads and offer interoperative structural support, as well as increase the available surface area for cell attachment and proliferation.

Laboratory and surgeon evaluations have shown that putties with CB have the same handling characteristics and robust properties as the putty without CB but with increased stiffness due to the inclusion of CB. This study demonstrated that the resorbing calcium sulfate-based putty is an effective means for delivery of bone graft materials for the successful restoration of bony defects.

Materials and Methods

Study Overview
The bone healing response to two formulations of calcium sulfate putty with DBM and CB was compared with that of calcium sulfate putty with DBM alone and an untreated (empty) defect. Seven male mongrel dogs were used for this study. There were three test materials:

• Putty containing DBM (DBM putty)
  
  
• Putty containing 70% CB/30% DBM (70/30 CB putty)
  
  
• Putty containing 50% CB/50% DBM (50/50 CB putty)

All DBM and CB used in this study were canine derived.

Surgical Procedure
Two osseous defects (proximal and distal) were created in each humerus (bilaterally) using a low speed drill inserted transversely through the lateral cortex and into the cancellous bone of the proximal humeral metaphysis, resulting in a 9 mm diameter × 20 mm deep cavity. Therefore, each canine had four defects.

After preparing the cylindrical defects, 1-1.5 cc of each of the test materials were implanted into one of the defects in each dog according to a computer-generated randomization scheme. A large bore, 20-cc syringe was used to inject the material into the prepared defects. One defect in each dog was left untreated. After injecting materials into the defects, hyaluronic acid membranes were cut to size and then sutured to periosteum for graft containment.

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,¹² and regulations of the United States Department of Agriculture Animal Welfare Act.¹³

Implant Preparation
All powder and solution materials were packaged in glass vials that were terminally sterilized using electronic beam radiation.

Canine DBM and CB were purchased from a veterinarian tissue supply company (Veterinary Transplant Services, Kent, Wash). Canine bones from two dogs 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 (-80°C). This process resulted in canine DBM that was “wet frozen.” Prior to putty formulation, the bone was dried to remove free moisture.

Analytical Methods
Radiographs of the humeri were taken preoperatively, immediately postoperatively, and at 2, 4, and 6 weeks. After 6 weeks, the dogs were euthanized with intravenous pentobarbital, and the humeri were explanted. High-resolution contact radio-graphs of the humeri were taken. The bones were then sectioned perpendicular to the long axis of the cylindrical defect, radiographed, and processed for plastic embedded undecalcified histology.

The histologic sections were stained with basic fuchsin and toluidine blue and were examined by light microscopy. The area fraction of new bone and the area fraction of residual cancellous bone graft material in the defects were determined using image analysis software. The data were analyzed using paired sample tests.

Results

Postoperative radiographs revealed all test materials to be well contained in the prepared cavities. Normal wound healing occurred, and there were no postoperative infections. Serial clinical radiographs showed progressive decrease in material density over time.

Immediate postoperative radiographs of the surgical defects in all of the dogs showed that the defects filled with material appeared to have been filled with a homogenous, radio-opaque material. The degree of opacity was greater for the defects filled with material containing CB (ie, 70/30 CB putty had greater opacity than 50/50 putty, which had greater opacity than DBM putty).

Radiographs at weeks 2, 4, and 6 showed a progressive decrease in original material density over time with subsequent densification of the defect area for defects filled with study materials. At 6 weeks, defects treated with the three study materials clearly illustrated the densification (Figures 1 and 2). The empty defect remained radiolucent throughout the 6-week study period (Figure 3).

Figure 1: Six-week radiograph of defect filled with DBM putty (bottom). Figure 2: Six-week radiograph of defect filled with 50/50 CB putty (top) and 70/30 CB putty materials (bottom). Figure 3: Six-week radiograph of empty defect (bottom). Radiolucent void is still present.

The area fraction of new bone in the defects was greater for all three putty formulations compared to the untreated control defects (Table). These differences were significant for both the 50/50 CB putty and the 70/30 CB putty. The mean area fraction of residual bone was <10% for 50/50 CB putty and 70/30 CB putty.

All defects in the DBM-only putty group showed bone growth filling from 70%-100% of the original defect (Figure 4). Samples without complete filling had areas empty of bone in the central portion of the defect. All the defects from the 50/50 CB putty group showed bone growth within the defect with 80%-100% of the original defect filled with bone-like material (Figure 5). In some of the contact radiographs, there appeared to be residual CB particles, typically located in the central portion of the defect.

Figure 4: Contact radiograph of defect filled with DBM putty at 6 weeks. Figure 5: Contact radiograph of defect filled with 50/50 CB putty at 6 weeks. Figure 6: Contact radiograph of defect filled with 70/30 CB putty at 6 weeks. Figure 7: Contact radiograph of empty defect at 6 weeks. New bone formation is present only around the periphery of the defect.

The defects from the 70/30 CB putty group showed similar bone growth within the defect to the 50/50 CB putty group, with 80%-100% of the original defect filled with bone-like material (Figure 6). Also, as with the 50/50 CB putty, some radiographs showed what appeared to be residual CB particles that were typically located in the central portion of the defect.

Generally, the empty defects revealed minimal healing with some samples having marginal bone formation along the circumferential edge of the defect (Figure 7).

The histology results corresponded to the radiographic results. For the DBM-only putty group, histology results showed bone trabeculae forming in association with the residual DBM particles uniformly throughout the entire defect region (Figure 8). The histology results for the 50/50 CB putty (Figure 9) and 70/30 CB putty (Figure 10) groups were similar, with both groups showing new bone trabeculae associated with residual DBM and CB particles and implant material throughout the original defect. There was some residual implant material present in the central region of the defect.

Figure 8: Six-week histological section of DBM putty-filled defect. Spicules of bone and thin trabeculae have formed throughout the defect. Figure 9: Six-week histological section of 50/50 CB putty-filled defect. New thickened bone trabeculae have formed throughout the defect. Bone has formed on both DBM and CB particles.

For the empty defect group, histology results showed sparse, small, thin spicules of new bone at the periphery of the defect region. However, the defect was essentially devoid of any new bone (Figure 11).

Figure 10: Six-week histological section of 70/30 CB putty-filled defect. New thickened bone trabeculae have formed throughout the defect. Bone has formed on both DBM and CB particles. Figure 11: Six-week histological section of an empty defect. A central zone of fibrous tissue is present.

Discussion

New viable bone in the histology slides was in intimate contact with material components of the DBM- only putty, the 50/50 CB putty, and the 70/30 CB putty. Those components include DBM particles, CB particles, and residual calcium sulfate/carboxymethylcellulose material. The histological findings were in agreement with the contact radiograph results.

Conclusion

Demineralized bone matrix-only putty, 50/50 CB putty, and 70/30 CB putty implant materials made using canine allograft bone material were well tolerated with no inflammatory or foreign body response in this canine bone defect model. Minimal new bone growth was shown in the empty defect group, while substantial new bone growth with similar healing rates of new bone were shown for the DBM-only putty, 50/50 CB putty, and 70/30 CB putty groups.

New bone growth was indicated throughout the defects filled with DBM-only putty, 50/50 CB putty, and 70/30 CB. Similar bone healing responses were shown between the 50/50 CB and 70/30 CB putty groups, indicating some degree of flexibility regarding the amount of CB that may be incorporated into the putty while still providing good in vivo performance. AlloMatrix Cancellous Putty contains quantities of cancellous bone within the range investigated in this study.

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Authors

From the *Department of Orthopedic Surgery, Rush-Presbyterian-St. Luke’s Medical Center, Chicago, Ill; and †Wright Medical Technology, Inc, Arlington, Tenn.

This work was supported by Wright Medical Technology, Inc, Arlington, Tenn.