Periodontal repair in dogs: effect of allogenic freeze-dried demineralized bone matrix implants on alveolar bone and cementum regeneration.

The objective of this study was to evaluate alveolar bone and cementum regeneration following surgical placement of an allogenic, freeze-dried, demineralized bone matrix (DBM) cortical strip implant. Critical size, supraalveolar periodontal defects were surgically created around the second, third, and fourth mandibular premolar teeth in eight mongrel dogs. Contralateral jaw quadrants in six animals were randomly assigned to receive the DBM implant, or serve as surgical control. Two additional animals received bilateral DBM implants. Flaps were coronally advanced to submerge teeth and implants, and sutured. Three animals were exited from the study due to extensive early wound failure. Remaining animals were sacrificed at 8 weeks postsurgery. Histometric recordings included defect height, bone regeneration/DBM implant height, cementum regeneration height, root resorption, and ankylosis. Large areas of unresorbed DBM exhibiting fragmentation and empty osteocyte lacunae were observed adjacent to new bone formation, or bone formation was observed adjacent to or within the implant, often exhibiting ankylosis. Cementum regeneration appeared enhanced in shelter of the DBM implant. Histometric recordings (mean+/-SD) for DBM and control defects, respectively, were: defect height, 4.8+/-0.2 mm and 4.4+/-0.2 mm; bone regeneration/DBM implant height, 4.0+/-1.3 mm and 1.2+/-0.6 mm; cementum regeneration height, 1.4+/-0.4 mm and 0.7+/-0.2 mm; root resorption, 0.5+/-0.3 mm and 1.2+/-0.3 mm; and ankylosis, 0.5+/-0.2 mm and 0.1+/-0.1 mm without statistically significant differences between experimental conditions (N=3). Within the limitations of this study, the histologic observations suggest that surgical implantation of allogenic, freeze-dried DBM cortical strip implants may have a potential to support cementum regeneration, possibly by providing conditions for guided tissue regeneration, however, alveolar regeneration appears unpredictable.

Induction of bone by a demineralized bone matrix gel: a study in a rat femoral defect model.

Demineralized bone matrix contains osteoinductive factors and stimulates filling of gaps and defects with bone; however, it is difficult to handle by itself and various preparations have been tested. Demineralized bone matrix with a gel consistency now is available for clinical use. We studied, in a femoral segment defect in the rat, the effects of rat demineralized bone matrix gel with and without a ceramic substratum. This preparation is analogous to the human demineralized bone matrix in the same carrier, used clinically for humans. One hundred adult male Fischer rats were divided into 10 experimental groups. Independent variables included the presence or absence of hydroxyapatite ceramic cylinders, the presence of demineralized bone matrix in carrier or carrier alone (glycerol), and the duration of observation (1, 2, and 4 months). Defects filled with the gel alone had significantly higher radiographic scores for host-graft union at 4 months compared with ceramic with the gel, ceramic alone, or carrier alone. Demineralized bone matrix gel significantly increased the total histologic score for host-graft union, whether ceramic was present or not, and a three-way interaction occurred among ceramic, the gel, and time. Demineralized bone matrix gel was an effective inducer of bone formation in this model. An additional substratum was not required; in fact, significantly more bone was formed in the absence of the ceramic cylinder. Neither the gel nor the ceramic were impediments to revascularization of the defect. Host-graft union was enhanced by demineralized bone matrix gel but not by the ceramic cylinder.

An age-related decrease in the concentration of insulin-like growth factor binding protein-5 in human cortical bone.

The skeletal contents of insulin-like growth factor-2 (IGF-II), insulin-like growth factor binding protein-5 (IGFBP-5), and insulin-like growth factor binding protein-3 (IGFBP-3) were determined in duplicate samples of human femoral cortical bone obtained from 64 subjects (44 males and 20 females) between the ages of 20 and 64 years. The results of these quantitative measurements revealed an age-related decrease in the femoral cortical content of IGFBP-5 (r = -0.272, P = 0.031) in the total population. Although the femoral cortical content of IGF-II did not show a similar decrease with age, it could be correlated to the femoral cortical content of IGFBP-5 (r = 0.442, P < 0.001). In contrast, the femoral cortical content of IGFBP-3 did not decrease with age and could not be correlated to the femoral cortical contents of either IGFBP-5 or IGF-II. Comparisons of these results with previous measurements of insulin-like growth factor-1 (IGF-I) and transforming growth factor-beta (TGF-beta), in extracts of the same bones, showed significant cross-correlations between the femoral cortical contents of each of these growth factors and the femoral cortical contents of IGFBP-5 (r = 0.625 for IGF-I versus IGFBP-5, r = 0.554 for TGF-beta versus IGFBP-5, P < 0.001 for each) but not IGFBP-3.(ABSTRACT TRUNCATED AT 250 WORDS)

Simian immunodeficiency virus (human HIV-II) transmission in allograft bone procedures.

STUDY DESIGN: Infection of rhesus monkeys (Macaca mulatta) with simian immunodeficiency virus (SIV, HIV-II) was used to study disease transmission in allograft bone. Four allograft bone processing techniques--fresh, fresh frozen, double freeze-thaw, and double freeze-thaw with chemical decontamination--were evaluated. OBJECTIVES: To determine if SIV could be transmitted in allograft bone and if processing techniques could be used to eliminate the potential for disease transmission. SUMMARY OF BACKGROUND DATA: Although the risk of HIV transmission in bone allograft was reported to be low, HIV transmission had occurred. In all cases, frozen allograft was used. Donor screening and serologic testing significantly reduced the risk of transmission, although a window of time existed in which an individual was infected but had not seroconverted. Experimental infection of rhesus monkeys with SIV induced a disease syndrome similar to AIDS and provided an ideal model to study disease transmission. METHODS: Corticocancellous cylinders were obtained aseptically from SIV-infected rhesus monkeys. The grafts were randomly placed into one of four processing groups and implanted into noninfected animals. The presence of SIV antibody was monitored by serologic testing. After the monkeys were killed, the graft sites were studied by histology. RESULTS: All animals receiving fresh allograft or allograft bone that had been subjected to either single or double -70 C freeze-thaw cycles became infected with SIV. Animals receiving allograft that had been subjected to a double freeze-thaw cycle and chemical decontamination were disease-free after 26 weeks when the animals were killed. CONCLUSIONS: The results show that SIV (HIV-II) can be transmitted in bone allograft procedures. Although freeze-thaw cycles and lavaging to remove blood elements can reduce the infectivity of a graft, it appears chemical decontamination is necessary to provide a high level of confidence in its safety.

In vivo evaluation of demineralized bone matrix as a bone graft substitute for posterior spinal fusion.

STUDY DESIGN: Posterior lumbar spinal fusion segments were evaluated in 9 adult mongrel dogs 6, 12, and 26 weeks after implantation. Four sites on each animal received implants consisting of demineralized bone matrix alone, demineralized bone matrix with allograft bone, allograft bone alone, and autograft bone. Each unilateral fusion spanned one motion segment with one intervening vertebral level left undisturbed using T13-L7. The fusions were evaluated radiographically, mechanically, and histologically. OBJECTIVE: The purpose of this study was to determine the efficacy of demineralized bone matrix as a bone graft substitute for stable posterior spinal fusion. SUMMARY OF BACKGROUND DATA: Posterior spinal fusion is a procedure commonly performed for spinal stabilization. Increasing the incidence and speed of stable spinal fusion is a primary goal in spinal surgery. Concerns have developed regarding the graft material used to induce bone healing at the fusion site. The advent of osteoinductive materials, such as demineralized bone matrix, may eliminate the need to harvest autograft bone and may circumvent the immunologic response and lower osteogenic potential associated with allograft bone. METHODS: The quality of fusion and new bone formation was evaluated radiographically using plain films, computed tomography, and magnetic resonance imaging. After the dogs were killed, each fusion segment was evaluated mechanically in torsion to determine stiffness and histologically to determine qualitative parameters of new bone formation and remodeling. RESULTS: Radiographic studies showed that autograft bone sites achieved stable fusion by 26 weeks after surgery. Conversely, the demineralized bone matrix alone and with allograft bone demonstrated some new bone formation at 6 and 12 weeks, but did not achieve fusion by 26 weeks. The fusion sites of allograft bone alone showed minimal new bone formation at all time periods. Mechanically, the autograft fusion sites demonstrated torsional stability that was significantly greater than that of all other fusion sites at all time periods. The remaining fusion sites showed equivalent torsional stiffness at all time periods. Histologic analysis confirmed the radiographic and mechanical findings. CONCLUSIONS: The results indicate that demineralized bone matrix alone or with allograft bone is ineffective in achieving stable posterior spinal fusions.

Effect of demineralized bone matrix on bone growth within a porous HA material: a histologic and histometric study.

Coralline hydroxyapatite (cHA) is an osteoconductive material currently being used as a bone graft substitute. Created by the hydrothermal conversion of the calcium carbonate skeleton of coral to hydroxyapatite, this material has a porous structure similar to cancellous bone. Addition of demineralized bone matrix (DBM) would conceivably create a composite with both osteoconductive and osteoinductive properties. This pilot study evaluated the healing of rabbit cranial defects that had been filled with cHA or cHA augmented with a DBM gel formed by adding glycerol to the DBM particulate. Data from these were then compared to unfilled defects from a previous study. Results indicated enhancement of new bone formation and an increase in the rate of healing in the defects filled with the cHA-DBM gel composite. Further studies are warranted.

Reduction in perineural scar formation after laminectomy with Polyactive membrane sheets.

STUDY DESIGN: The effectiveness of Polyactive (Osteotech, Inc., Shrewsbury, NJ), an elastomeric segmental copolymer, as a barrier material for the prevention of perineural scar formation was evaluated in eight adult mongrel dogs. Two animals each were killed at 2, 4, 8, and 12 weeks postoperation. OBJECTIVE: This study determined the ability of a Polyactive membrane to prevent perineural scar and compared the results to those obtained using free fat graft and nonimplanted control subjects. SUMMARY OF BACKGROUND DATA: Perineural scar formation after laminectomy presents considerable morbidity in lumbar surgery. To date, a wide variety of materials has been evaluated to prevent scar formation including free fat grafts, mechanical barrier devices, hemostatic agents, and anti-inflammatory drugs. However, all studies indicate scar formation is present, and the search for an effective barrier continues. METHODS: Gross dissection, radiographic studies, including computed tomography and magnetic resonance imaging, and histologic sections were used to evaluate the presence and degree of perineural scarring. If scarring was evident in each of the evaluations, the degree was graded on a 0-3 scale where: 0 = no scarring; 1 = mild scarring; 2 = moderate scarring; and 3 = extensive scar formation. RESULTS: Polyactive membranes were an effective barrier with only minimal scar formation to the dura observed at any time period. Free fat graft was an effective barrier to scar formation at early time periods; however, progressive fat graft degradation with some increased scarring was observed at latter time periods. Nonimplanted control defect sites resulted in moderate to severe scar formation to the dura as early as 2 weeks postimplantation. CONCLUSIONS: The results indicate Polyactive is a reliable and effective means of reducing perineural scar formation. The effectiveness of the Polyactive membrane could be improved markedly if techniques or methods to ensure retention of the membrane in the proper position were developed.

Investigation of an organic delivery system for demineralized bone matrix in a delayed-healing cranial defect model.

The osteogenic potential of demineralized bone matrix (DBM) has been demonstrated in multiple animal models and clinical applications. A particulate form of DBM is generally used to fill defects because it is easily packed into a defect site without operative planning or shaping. One potential disadvantage in the use of a particulate is the migration of particles from the defect site. To stem this, glycerol was added to DBM to improve handling properties. A study was undertaken to compare two forms of DBM with glycerol, with DBM particulate and autograft in a bony defect site. The model chosen consisted of bilateral 8-mm trephine defects created in the parietal skull of 40 rabbits. Animals were sacrificed at 2 and 8 weeks. Results demonstrated both cartilage and bone induction with the three forms of DBM, with 75-90% of the linear width of the defect filled with new bone by 2 weeks. This was similar to the autograft (86%) and significantly greater than the unfilled defects (26%) at 2 weeks. The addition of glycerol to the DBM did not affect the inductive capacity, and produced a response similar to that of DBM particulate alone or autogenous bone.

The biomechanics and histopathology of chemically processed patellar tendon allografts for anterior cruciate ligament replacement.

A study was initiated to examine chemically processed patellar tendon allografts in sheep anterior cruciate ligament repairs, both mechanically and histologically. One group of animals received frozen, untreated allografts, one group received frozen grafts that were processed with a chloroform-methanol solvent extraction technique, and one group received frozen tendons treated with a permeation-enhanced extraction technique. All animals were operated on unilaterally, with the contralateral knee acting as a normal, intact control. Histologic analysis after 2 months of implantation revealed similar enhanced cellular repopulation in both chemically treated ligament allografts compared with the more hypocellular, untreated grafts. At 6 months the chloroform-methanol group demonstrated a more aggressive chronic cellular response with numerous thick-walled vessels relative to the untreated and permeation-enhanced grafts. Mechanical testing after 6 months of implantation showed statistically similar anterior drawer resistance in all grafted knees, yet the two chemically extracted grafts had significantly less stiffness than untreated anterior cruciate ligament grafts. Both treatment groups also tended to be weaker than the untreated allografts. All anterior cruciate ligament reconstructions showed excessive anterior drawer laxity and, regardless of treatment, had lower strength and less stiffness than normal anterior cruciate ligament tissue at the 6-month period.

Age-related decreases in insulin-like growth factor-I and transforming growth factor-beta in femoral cortical bone from both men and women: implications for bone loss with aging.

We determined the skeletal content of insulin-like growth factor-I (IGF-I) and transforming growth factor-beta (TGF beta) in human bone as a function of age, using 66 samples of femoral cortical bone obtained from 46 men and 20 women between the ages of 20-64 yr. We found a linear decline in the skeletal content of IGF-I (nanograms per mg protein) with donor age (r = -0.43; P < 0.001) in the total population. The skeletal content of TGF beta also decreased with age (i.e. 1/TGF beta vs. age; r = 0.28; P < 0.02) for the total population. We did not observe any difference in the skeletal growth factor content between male and female donors. IGF-I content, when analyzed by decade divisions of age, showed a reduction between the 20- to 29-yr-old and the 50- to 59-yr-old subjects (P < 0.02). The loss rate of IGF-I was 1.56 ng/mg protein.yr, corresponding to a net loss of 60% of skeletal IGF-I between the ages of 20-60 yr. The loss rate of TGF beta was 0.03 ng/mg protein.yr, corresponding to a net loss of 25% of the skeletal TGF beta between the ages of 20-60 yr.

Demineralized bone matrix. Enhancement of spinal fusion.

A study was conducted to determine the ability of demineralized bone matrix gel to act as an osteoconductive/osteoinductive material to enhance canine spinal fusion. Seven dogs underwent posterior spinal fusion. Four-level fusions were performed with one of four procedures at each level: decortication alone, with gel added, with autograft, or with both gel and autograft. Dogs were killed at 6 weeks and early histologic response was studied. At untreated control sites, little bone formation was evident. Gel-filled sites showed abundant osteoid, with 60% of demineralized particles fused to or surrounded by new bone. Sites filled with autograft had more new bone, but there was more osteoid at gel-treated sites. Autograft augmented with gel showed the most vigorous response, with extensive bridging between demineralized particles, host bone, autograft, and new bone. Significantly less autograft was needed to induce a similar amount of new bone formation when gel was added. Use of the gel as an autograft extender may improve the chance for successful spinal fusion.

Detection of HIV in bone allografts prepared from AIDS autopsy tissue.

Processed bone from an AIDS patient was tested for the presence of the human immunodeficiency virus type 1 (HIV-1). The preliminary procedures used to process bone allografts included removal of adventitious material and two cycles of freeze thawing. Although infectious virus was readily observed in plasma and bone marrow cells taken at autopsy, no infectious virus was detected in processed bone fragments. However, by using the polymerase chain reaction procedure, the presence of proviral HIV DNA could be demonstrated in processed bone allografts from this donor. Whereas the best safeguard against transmission of HIV by allografts is rigorous criteria for the exclusion of seropositive individuals as donors, proper procedures for processing bone allografts can further reduce the possibility of HIV transmission by bone allografts in cases where tissue from an infected donor is collected and processed.

Bone ingrowth into a porous-coated total shoulder component retrieved postmortem.

An uncemented porous-coated humeral component, which functioned in vivo for over 2 1/2 years, was retrieved postmortem and examined histologically. Radiographs of the specimen revealed close approximation to the endosteal cortex medially and laterally. Apposition was limited or absent anteriorly and posteriorly. The overall extent of bone ingrowth was approximately 11% of the available pore volume with over 95% of ingrowth occurring in the medial and lateral quadrants. Bone ingrowth was limited anteriorly and posteriorly to regions in which spot welds were identified radiographically. There were no loose beads or coating failure observed.

HIV inactivation in a bone allograft.

The use of exclusionary techniques in the procurement of donors for bone allografts greatly reduces chances for disease transmission. Furthermore, treatment of HIV with either chemical agents or strong acids will effectively inactivate the AIDS virus. These data are taken as indirect proof that the risk of obtaining AIDS from a freeze-dried bone allograft is highly remote. The purpose of this study is to obtain direct evidence that the processing of a demineralized freeze-dried bone allograft would render the allograft safe for human use. In Part I, human cortical bone was obtained from a cadaveric source and tested to be free of HIV contamination. The bone was spiked with 5.26 x 10(9) viral particles. This corresponded to 148 micrograms of total viral protein. In Part II, cortical bone was procured from a donor who died of AIDS. In both Parts I and II, the cortical bone was ground to yield particle sizes of 90 to 500 microns. Test samples were treated with a virucidal agent and demineralized in HCl. Control samples were left untreated. All samples were cocultivated with stimulated peripheral blood lymphocytes and assayed for p24 core protein, reverse transcriptase, and viral gag gene by polymerase chain reaction (PCR). In Part I, the HIV spiking experiment, untreated virus infected particulate bone was positive for HIV replication. Treated samples were negative when assayed for HIV. Bone samples in Part II, HIV infected bone, were positive by PCR. Replication of viable HIV could not be demonstrated after treatment. It was concluded that demineralization and treatment with a virucidal agent inactivates HIV in spiked and infected bone.

The use of a new form of allograft bone in implantation or osseointegrated dental implants--a preliminary report.

The use of and early results with a new form of demineralized allograft bone tissue with the immediate placement of a dental implant are described. No occlusive membranes were utilized. GRAFTON Allogeneic Bone Matrix (ABM) is processed from human cortical bone into a thick gel consistency and packaged sterile in disposable syringes. The material was utilized in seven patients with seven implants. New bone formation was noted and, with the exception of two implants with a small amount of thread exposure, complete. It appears that treatment with the bone-grafting material produced clinical results similar to those reported with the use of barrier membranes and may provide an alternative to guided tissue regeneration. Further investigation appears warranted.

Intrahepatic assembly of very low density lipoproteins: immunologic characterization of apolipoprotein B in lipoproteins and hepatic membrane fractions and its intracellular distribution.

Monoclonal antibodies, prepared against rat apoB, were used to examine apoB structure in serum lipoproteins and characterize the forms and localization of apoB in liver membrane fractions and cultured hepatocytes. Of the several antibodies obtained, four, having separate epitopes, were characterized. Western blot analysis showed that three (DB11, F4, and LB14) antibodies recognized both apoBL and apoBS. One antibody (HB41) recognized only apoBL. This antibody showed unusual properties. Competition ELISA assays showed that the epitope recognized by HB41 was more effectively expressed on low density lipoproteins (LDL) compared to very low density lipoproteins (VLDL). In addition, treatment of lipoproteins with detergents and sulfhydryl reducing agents also increased the expression of the HB41 epitope. Since HB41 has been found to inhibit LDL binding to hepatocyte receptors, these data indicate that the HB41 epitope is located on the carboxy-terminal side of the apoBS junction (probably within the LDL receptor binding domain). Western blotting hepatic microsomal subfractions showed that in the rough and smooth microsomes, HB41 recognized only apoBL, while in the Golgi it recognized both apoBL and a protein having a molecular weight slightly smaller. In contrast, Western blotting with a polyclonal antibody known to recognize both apoBL and apoBS showed that, in rough and smooth microsomes, proteins in addition to apoBL and apoBS having molecular weights between 120,000 and 30,000 were recognized. These proteins, likely to be proteolytic fragments of apoB, were barely detectable in the Golgi. Additional biosynthetic studies show that the [35S]methionine-labeled proteins smaller than apoB were immunoprecipitated from the rough microsome subfraction. Pulse-chase experiments show that these are produced with the same kinetics as full-size apoBL and apoBS, indicating that they are not incomplete nascent chains. Finally, immunofluorescence microscopy was used to determine the localization of monoclonal epitopes. ApoB monoclonal antibodies that recognized exclusively apoBL (HB41) and apoBL and apoBS (DB11) produced an immunofluorescence pattern characteristic of the endoplasmic reticulum, but not the Golgi. These data suggest that, in cultured rat hepatocytes, the majority of both molecular weight forms of apoB are localized in the endoplasmic reticulum, the initial site of VLDL assembly. The additional finding that proteolytic fragments of apoB are enriched in the microsomal fraction suggests that if the proteolysis occurs during subcellular fractionation, immature apoB is susceptible to proteolysis.(ABSTRACT TRUNCATED AT 400 WORDS)