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Scientific Article   |    
Radiographic and Histologic Assessment of Calcium Sulfate in Experimental Animal Models and Clinical Use as a Resorbable Bone-Graft Substitute, a Bone-Graft Expander, and a Method for Local Antibiotic Delivery One Institution’s Experience
Thomas M. Turner, DVM; Robert M. Urban; Steven Gitelis, MD; Ken N. Kuo, MD; Gunnar B.J. Andersson, MD
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Thomas M. Turner, DVM
Robert M. Urban
Steven Gitelis, MD
Ken N. Kuo, MD
Gunnar B.J. Andersson, MD
Department of Orthopedic Surgery, Rush-Presbyterian-St. Luke’s Medical Center, 1653 West Congress Parkway, 1471 Jelke, Chicago, IL 60612

In support of their research or preparation of this manuscript, one or more of the authors received grants or outside funding from Wright Medical Technology. 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 (Wright Medical Technology). 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:S8-18 
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For more than a decade, the radiographic and histologic appearance of a refined calcium sulfate has been studied in various experimental animal models in our laboratory and in clinical applications. This report summarizes our institution’s experience with calcium sulfate as a synthetic bone graft1, a graft expander (the synergistic combination of calcium sulfate with demineralized bone matrix)2-4, and a method for local delivery of antibiotics5-8. Historically, orthopaedic usage of calcium sulfate was popularized by Peltier. Clinically, we have used calcium sulfate to treat numerous osseous lesions and conditions occurring in the axial and appendicular skeleton, including a variety of benign lesions of bone, osseous defects following implant removal, corrective osteotomy sites, spinal fusion sites, graft sites, fracture defects, and osteomyelitic lesions.
Both our research studies and clinical experience have shown consistent osteoconduction, excellent biocompatibility, and complete resorption of calcium sulfate, which was replaced by newly formed bone that ultimately remodeled to be comparable with autogenous bone. The scientific basis for the use of calcium sulfate, the typical radiographic and histologic progression of the implanted material, and the indications and expectations for clinical use are illustrated. Contributions from orthopaedic surgeons from several subspecialties demonstrate the use of calcium sulfate in various applications and anatomic sites.
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Anchor for JumpAnchor for JumpTABLE I:  Amount of Bone in the Defects at Six Weeks in Study of Calcium Sulfate as a Synthetic Bone Graft*
GroupMean† (%)Range (%)
CaSO414.37.9-18.7
Autograft?8.64.7-12.0
No graft?3.71.8-6.1
*Area fraction of bone as determined with scanning electron microscopic image analysis. †p = 0.002 for CaSO4 compared with autograft, and p = 0.001 for CaSO4 compared with no graft.
 
Anchor for JumpAnchor for JumpTABLE II:  Amount of Bone in the Defects at Twenty-four Weeks in Study of Calcium Sulfate as a Synthetic Bone Graft*
GroupMean† (%)Range (%)
CaSO4?9.47.5-11.8
Autograft10.08.4-11.8
*Area fraction of bone as determined with scanning electron microscopic image analysis. †p = 0.277.
 
Anchor for JumpAnchor for JumpTABLE III:  Prevalence of Osseous Fusion Determined from Sagittal Sections in Study of Calcium Sulfate as a Bone-Graft Expander
Group
Autograft and CaSO45 of 5
CaSO4 alone0 of 5
No graft0 of 5
 
Anchor for JumpAnchor for JumpTABLE IV:  Area of Bone Fusion Mass Determined from Transaxial Computed Tomography (Mid-Level) Images in Study of Calcium Sulfate as a Bone-Graft Expander
GroupMean* (%)Range (%)
Autograft91.3 86.0-98.9
Autograft and CaSO478.9; p = 0.683 53.1-91.1
CaSO4 alone32.9; p = 0.00010.7-58.7
No graft34.3; p = 0.000?5.6-64.4
*The p values are for the difference from the group treated with autograft (one-way multivariate analysis of variance [MANOVA]).
 
Anchor for JumpAnchor for JumpTABLE V:  Amount of Bone in the Defects at Six Weeks in Study of Calcium Sulfate Combined with Demineralized Bone Matrix*
GroupMean† (%)Range (%)
CaSO4 and demineralized bone matrix10.5?8.6-15.3
Demineralized bone matrix alone?7.04.2-9.5
*Area fraction of bone as determined with scanning electron microscopic image analysis. †p = 0.018.

Research

Synthetic bone-graft materials are of clinical interest because of the morbidity, potential for disease transmission, and procurement issues associated with autografts and allografts. The purpose of this study was to evaluate healing after the use of calcium sulfate as a synthetic bone graft compared with spontaneous healing with no graft material and healing after use of autogenous bone graft in a large medullary defect model1.

Methods

Graft materials: The grafts consisted of either circular 4.7 3-mm tablets of calcium sulfate dihydrate (CaSO4) or autogenous cancellous bone.
Experimental model: The grafts were implanted into 13 50-mm cylindrical cavities in a bilateral canine humeral model.
Experimental design: In seven animals, fifty tablets of CaSO4 were implanted in the left humerus and 6 cm3 of autograft was implanted in the right humerus; healing was evaluated at six weeks. The same protocol was carried out in another seven animals, but healing was evaluated at twenty-four weeks. In five animals, fifty tablets of CaSO4 were implanted in the left humerus and no graft was implanted in the right humerus; healing was evaluated at six weeks.
Histologic analysis: Undecalcified transverse sections, cut at sequential 1-cm levels and stained with basic fuchsin and toluidine blue, were studied by light microscopy. Similar unstained sections were quantitated with computer analysis of scanning electron microscopic images.

Results

Radiographic assessment (Fig. 1): The nongrafted defects were devoid of new bone, and the autografted defects were filled with new bone. At six weeks after treatment with CaSO4, residual tablet sites were evident; at twenty-four weeks, no tablets were visible.
Histologic assessment (Fig. 2): In the group treated with CaSO4, all defects were filled with new bone and the tablets were replaced with concentric circular lamellae. At six weeks there was residual CaSO4 at the central tablet sites, whereas at twenty-four weeks only minute residual material was seen. In the group treated with autogenous bone, all defects were filled with new bone. At six weeks, there was bone formation on the surfaces of the fragments. At twenty-four weeks, the graft fragments were encompassed in new bone. The defects that were not grafted were not filled with bone; there was minimal new bone, only at the margins of the defect. The amounts of new bone in the three groups at the two time-periods are shown in Tables I and II.

Discussion

The amount of new bone formed in the defects with CaSO4 was approximately four times that in the empty defects with spontaneous healing and equivalent to that found in the defects with autogenous bone. The pattern of bone formation with the CaSO4 tablets suggested guided bone formation. The studied form of CaSO4 was found to provide an osteoconductive substrate with a predictable and consistent rate of resorption and excellent biocompatibility. The canine humeral defect model replicated the time-course of CaSO4 tablet resorption and replacement by bone observed radiographically in our patients.

Clinical Application (Fig. 3)

A twenty-six-year-old woman presented with a large aneurysmal bone cyst of the proximal part of the humerus. The defect was debrided with curettage and was filled with OSTEOSET pellets (Wright Medical Technology, Arlington, Tennessee) without additional graft material. Progressively, over the next three months, the pellets completely resorbed and, concurrently, new bone filled the defect.

Research

In this study, CaSO4 was used as a synthetic bone graft alone and as a graft expander at a 50:50 ratio with autogenous cancellous bone for spinal fusion following dorsal laminectomy in a canine model2.

Methods

Graft materials: The defects were treated with (1) 10 cm3 of autogenous cancellous bone, (2) 5 cm3 of autogenous bone and twenty-five circular 4.7 3-mm tablets of CaSO4, (3) fifty CaSO4 tablets alone, or (4) no graft.
Experimental model: A dorsal laminectomy was performed at the first and second lumbar level and at the fourth and fifth lumbar level in ten dogs. No fat graft was used. CaSO4 tablets and/or bone was placed directly on the dura. The defect was filled above the level of the laminae.
Experimental design: Five dogs were treated with a bone graft at one level and no graft at the other, and five were treated with bone graft and CaSO4 at one level and with CaSO4 alone at the other. All defects were examined at three months.
Spine analysis: Transaxial and sagittal computed tomography scans were performed.
Histologic analysis: Undecalcified sagittal sections were examined.

Results

All animals recovered uneventfully from the surgical procedures with no neurological deficits, and all walked normally within two days. The prevalence of osseous fusion as determined from sagittal sections is shown in Table III. The mean area of the bone fusion mass, quantitated from transaxial computed tomography images at the mid-laminectomy levels, is shown in Table IV.
Histologically, autogenous bone was well incorporated at the laminectomy cut surfaces and throughout the fusion mass, with a well-organized trabecular pattern and a thin neocortex along the dorsal and internal canal surfaces. Sites treated with the CaSO4 and autograft mixture demonstrated a similar pattern of bone development. Defects grafted with CaSO4 tablets alone had irregular areas of bone formation principally at the sites of the tablets and at the periphery of the defect, but centrally there were areas of well-organized fibrous tissue. The laminectomy defects that were not grafted contained only slight bone arising from the cut surface of the lamina, and the entire defect was filled with a highly organized fibrous tissue (Fig. 4).

Discussion

This study indicated that CaSO4, at a 50:50 mixture with autogenous cancellous bone, can be used as a graft extender. In this model, the complete dorsal laminectomy with exposure of the cord and no ancillary internal fixation provided an extreme test for spinal fusion.

Clinical Application

Case 1. A forty-six-year-old man presented with a history of low-back pain and degenerative disc disease with spinal stenosis. A laminectomy and fusion from the third and fourth lumbar level to the fifth lumbar and first sacral level was performed. A combination of OSTEOSET pellets (Wright Medical Technology) and cancellous bone chips was applied bilaterally over the transverse processes. Spinal fixation plates and screws were applied from the fourth lumbar to the first sacral level. A solid fusion was obtained by nine months (Fig. 5).
Case 2. A forty-six-year-old man presented, twelve years after a total hip arthroplasty, with a periprosthetic osteolytic lesion adjacent to a Harris-Galante acetabular component. The cavity was opened and was debrided superiorly with curettage and inferiorly and medially with a high-speed burr through the holes in the acetabular component. The superior defect was treated with autograft, and the medial and inferior defects were filled with OSTEOSET pellets. At seven weeks a majority of the pellets had resorbed, and at three months the pellets were absent, having been replaced by well-organized trabecular bone (Fig. 6).

Research

CaSO4 has shown excellent biocompatibility, and demineralized bone matrix has been used successfully as a graft material in the clinical setting. The purpose of this study was to evaluate the combination of these materials in the treatment of bone defects.

Methods

Graft materials: Either circular 4.7 ¥ 3-mm tablets of CaSO4 and demineralized bone matrix or demineralized bone matrix, supplied frozen, was used.
Experimental model: The grafts were implanted into 13 ¥ 50-mm cylindrical defects in a bilateral canine humeral model.
Experimental design: In seven dogs, 6 cm3 of demineralized bone matrix was implanted in the right humerus and fifty tablets of CaSO4 and demineralized bone matrix were implanted in the left humerus.
Histologic analysis: Undecalcified transverse sections cut at sequential 1-cm levels were stained with basic fuchsin and toluidine blue and studied by light microscopy. Computer analysis of backscattered scanning electron microscopy images of similarly prepared unstained sections was used to quantify the area fraction of new bone, excluding identifiable areas of residual CaSO4 and particles of demineralized bone matrix. The amount of bone on the side treated with the CaSO4 tablets and demineralized bone matrix was compared with that on the side treated with demineralized bone matrix alone. The data were analyzed with paired t tests.

Results

Radiographic assessment (Fig. 7): The defects treated with CaSO4 and demineralized bone matrix were filled with new bone, with a few circular densities corresponding to sites of previous tablets. The density of these defects was comparable with that of native bone, whereas the density of the defects treated with demineralized bone matrix only was less than that of the CaSO4-treated defects and that of native bone.
Histologic analysis (Fig. 8): The defects treated with CaSO4 and demineralized bone matrix had no remaining tablets, trabeculae were mature, and unmineralized osteoid was rare. The appearance was similar to that of the defects treated with autogenous bone graft after twenty-four weeks (Fig. 1). There were few demineralized bone-matrix particles, and residual CaSO4 was scarce. In the defects treated with demineralized bone matrix alone, new bone was found predominantly at the margins and haversian surfaces of the demineralized bone matrix particles. Associated trabeculae were thinner, and a large number of demineralized bone matrix particles remained. The amount of bone in the defects at six weeks in both groups is shown in Table V.

Discussion

Tablets of CaSO4 and demineralized bone matrix demonstrated excellent biocompatibility. Bone developed between the tablets, replacing resorbed tablets with mature bone trabeculae, with little remodeling activity, and without an adverse inflammatory response. The amount of new bone formed following grafting with CaSO4 tablets and demineralized bone matrix was equivalent to the amount of bone formed six weeks following treatment with autogenous bone graft1, twenty-four weeks following treatment with autogenous bone graft1, twenty-four weeks following treatment with CaSO4 tablets1, and in native bone of untreated humeri.

Clinical Application (Fig. 9)

A sixteen-year-old male athlete presented with a large aneurysmal bone cyst involving the ilium and the acetabulum. The defect was curetted and filled with 1800 OSTEOSET pellets (Wright Medical Technology) and an equivalent amount of demineralized bone matrix. After fifteen months, the defect was filled with new bone, with reformation of the subchondral plate of the acetabulum.

Research

The efficacy of calcium sulfate as an osteoconductive medicated bone-graft substitute has been demonstrated in previous canine and clinical studies from our institution5-8. This study was performed to investigate the local and systemic effects of the maximum prescribed dose and 1.8 times the maximum prescribed dose of tobramycin delivered by calcium sulfate tablets implanted in an osseous defect model. The maximum prescribed dose is 10 mg/kg in humans and 20 mg/kg in canines (surface area conversion).

Methods

Tablet resorption and bone response were assessed radiographically and histologically. Local tobramycin levels were measured by medullary aspiration. Systemic tobramycin levels as well as serum chemistry, hematology, and coagulation parameters were also determined.
Graft material: Circular 4.7 3-mm tablets of CaSO4 loaded with 10% tobramycin (by weight) were implanted.
Experimental model: The grafts were implanted into 13 100-mm cylindrical cavities in a bilateral canine humeral model.
Experimental design: With use of a randomization scheme, five dogs received CaSO4 loaded with the maximum prescribed dose of tobramycin (102 tablets [nominally]) and five received CaSO4 loaded with 1.8 times the maximum prescribed dose of tobramycin (184 tablets [nominally]); the defects were assessed at twenty-eight days.

Results

Radiographically, tablets that were visible postoperatively decreased in density and then became undetectable at twenty-eight days, while the medullary defects appeared to fill with new bone (Fig. 10). Histologically, the resorbed tablets were replaced by concentric layers of thin osseous trabeculae (Fig. 11). The two drug dosages exhibited similar local and systemic profiles. Serum chemistry, hematology, and coagulation parameters remained within normal limits throughout the study (Fig. 12).

Discussion

As evidenced in this study, CaSO4 is effective as an osteoconductive medicated bone-graft substitute; it achieves a predictable local response and long-term release of the drug over weeks without adverse systemic effects and with undetectable systemic levels after twenty-four hours. The lack of adverse systemic effects was supported by no adverse elevations in serum chemistry parameters or abnormal pathological findings at necropsy. This indicates a safe and effective method for local antibiotic treatment and dead-space management. In a previous study in which tablets containing 2% and 4% tobramycin were used, an increased concentration of the drug resulted in a concurrent local and systemic increase, as might be expected5. This was further accentuated in our study, in which a higher concentration (10%) of tobramycin led to even higher local and systemic levels. A dosage effect was demonstrated in our study, with an increasing number of tablets correlating to increased systemic and local levels. Regardless of the concentration of the drug in the tablets or the number of tablets used, systemic levels dissipated to undetectable limits after twenty-four hours. The present study also indicates that the ability of calcium sulfate to enhance the healing of large medullary defects, as seen in previous studies1, persists even in the presence of high local levels of antibiotics.

Clinical Application

In a forty-three-year-old woman with a Staphylococcus aureus infection of the proximal part of the tibia (Fig. 13, area of involvement highlighted on the preoperative radiograph), the defect was surgically debrided and filled with calcium sulfate pellets loaded with tobramycin. The infection resolved, and the cavity was filled with new bone by thirty-one months (Fig. 13).
Note: Dr. Richard Berger’s contribution of Case 2 to the study of calcium sulfate as a bone-graft expander is greatly appreciated.
TurnerTM, Urban RM, Gitelis S, Infanger S, Berzins A, Hall DJ, Haggard WO,Parr JE. Efficacy of calcium sulfate, a synthetic bone graft material, in healing a large canine medullary defect. Trans Orthop Res Soc,1999;24: 522. 24522  1999 
 
TurnerTM, Urban RM, Andersson GBJ, Lawrence AM, Igloria RV, Haggard WO,Parr JE. Spinal fusion using synthetic bone graft calcium sulfate compared to autogenous bone in a canine model. Trans Soc for Biomaterial,1999;24: 90. 2490  1999 
 
KellyCM, Wilkins RM, Gitelis S, Hartjen C, Watson JT,Kim PT. The use of a surgical grade of calcium sulfate as a bone graft substitute: results of a multicenter trial. Clin Orthop,2001;328: 42-50. 32842  2001 
 
GitelisS, Piasecki P, Turner T, Haggard W, Charters J,Urban R. Use of a calcium sulfate-based bone graft substitute for benign lesions of bone. Orthopedics,2001;24: 162-6. 24162  2001  [PubMed]
 
TurnerTM, Urban RM, Gitelis S, Sumner DR, Haggard WO,Parr JE. Antibiotic delivery from calcium sulfate as a synthetic bone graft in a canine bone defect. Trans Soc for Biomaterial,1998;21: 111. 21111  1998 
 
Turner TM, Urban RM, Gitelis S, Lawrence-Smith AM, Hall DJ, Haggard WO, Parr JE. Delivery of tobramycin using calcium sulfate tablets to graft a large medullary defect: local and systemic effects. Sixth World Biomaterials Congress Transactions. 2000;767. 
 
TurnerTM, Urban RM, Gitelis S, Lawrence-Smith AM, Hall DJ, Haggard WO,Parr JE. Local and systemic effects of tobramycin released from calcium sulfate tablets used to graft a large medullary defect. Trans Orthop Res Soc,2000;25: 213. 25213  2000 
 
Gitelis S, Piasecki P. The treatment of chronic osteomyelitis with a biodegradable antibiotic delivery implant; a clinical study. WPOA 2001 
 

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Anchor for JumpAnchor for JumpTABLE I:  Amount of Bone in the Defects at Six Weeks in Study of Calcium Sulfate as a Synthetic Bone Graft*
GroupMean† (%)Range (%)
CaSO414.37.9-18.7
Autograft?8.64.7-12.0
No graft?3.71.8-6.1
*Area fraction of bone as determined with scanning electron microscopic image analysis. †p = 0.002 for CaSO4 compared with autograft, and p = 0.001 for CaSO4 compared with no graft.
Anchor for JumpAnchor for JumpTABLE II:  Amount of Bone in the Defects at Twenty-four Weeks in Study of Calcium Sulfate as a Synthetic Bone Graft*
GroupMean† (%)Range (%)
CaSO4?9.47.5-11.8
Autograft10.08.4-11.8
*Area fraction of bone as determined with scanning electron microscopic image analysis. †p = 0.277.
Anchor for JumpAnchor for JumpTABLE III:  Prevalence of Osseous Fusion Determined from Sagittal Sections in Study of Calcium Sulfate as a Bone-Graft Expander
Group
Autograft and CaSO45 of 5
CaSO4 alone0 of 5
No graft0 of 5
Anchor for JumpAnchor for JumpTABLE IV:  Area of Bone Fusion Mass Determined from Transaxial Computed Tomography (Mid-Level) Images in Study of Calcium Sulfate as a Bone-Graft Expander
GroupMean* (%)Range (%)
Autograft91.3 86.0-98.9
Autograft and CaSO478.9; p = 0.683 53.1-91.1
CaSO4 alone32.9; p = 0.00010.7-58.7
No graft34.3; p = 0.000?5.6-64.4
*The p values are for the difference from the group treated with autograft (one-way multivariate analysis of variance [MANOVA]).
Anchor for JumpAnchor for JumpTABLE V:  Amount of Bone in the Defects at Six Weeks in Study of Calcium Sulfate Combined with Demineralized Bone Matrix*
GroupMean† (%)Range (%)
CaSO4 and demineralized bone matrix10.5?8.6-15.3
Demineralized bone matrix alone?7.04.2-9.5
*Area fraction of bone as determined with scanning electron microscopic image analysis. †p = 0.018.
TurnerTM, Urban RM, Gitelis S, Infanger S, Berzins A, Hall DJ, Haggard WO,Parr JE. Efficacy of calcium sulfate, a synthetic bone graft material, in healing a large canine medullary defect. Trans Orthop Res Soc,1999;24: 522. 24522  1999 
 
TurnerTM, Urban RM, Andersson GBJ, Lawrence AM, Igloria RV, Haggard WO,Parr JE. Spinal fusion using synthetic bone graft calcium sulfate compared to autogenous bone in a canine model. Trans Soc for Biomaterial,1999;24: 90. 2490  1999 
 
KellyCM, Wilkins RM, Gitelis S, Hartjen C, Watson JT,Kim PT. The use of a surgical grade of calcium sulfate as a bone graft substitute: results of a multicenter trial. Clin Orthop,2001;328: 42-50. 32842  2001 
 
GitelisS, Piasecki P, Turner T, Haggard W, Charters J,Urban R. Use of a calcium sulfate-based bone graft substitute for benign lesions of bone. Orthopedics,2001;24: 162-6. 24162  2001  [PubMed]
 
TurnerTM, Urban RM, Gitelis S, Sumner DR, Haggard WO,Parr JE. Antibiotic delivery from calcium sulfate as a synthetic bone graft in a canine bone defect. Trans Soc for Biomaterial,1998;21: 111. 21111  1998 
 
Turner TM, Urban RM, Gitelis S, Lawrence-Smith AM, Hall DJ, Haggard WO, Parr JE. Delivery of tobramycin using calcium sulfate tablets to graft a large medullary defect: local and systemic effects. Sixth World Biomaterials Congress Transactions. 2000;767. 
 
TurnerTM, Urban RM, Gitelis S, Lawrence-Smith AM, Hall DJ, Haggard WO,Parr JE. Local and systemic effects of tobramycin released from calcium sulfate tablets used to graft a large medullary defect. Trans Orthop Res Soc,2000;25: 213. 25213  2000 
 
Gitelis S, Piasecki P. The treatment of chronic osteomyelitis with a biodegradable antibiotic delivery implant; a clinical study. WPOA 2001 
 
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These activities have been planned and implemented in accordance with the Essential Areas and policies of the Accreditation Council for Continuing Medical Education (ACCME) through the joint sponsorship of the American Academy of Orthopaedic Surgeons and The Journal of Bone and Joint Surgery, Inc. The American Academy of Orthopaedic Surgeons is accredited by the ACCME to provide continuing medical education for physicians.
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