55. Bone - Osseous Surgery - IV Augmentation B- Allografts                                     

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Quick Search Topics

  1. what is an allograft
  2. allografts and HIV
  3. tissue bank processing and safety of bone allografts
  4. what is FDBA?
  5. what is DFDBA?
  6. allogenic bone matrix gel
  7. different preparations of allograft
  8. Does the origin of the bone make a difference in healing potential
  9. the addition of antibiotics to bone allografts
  10. comparing results of bone allografts with autogenous bone grafts
  11. review bone allografts in periodontal therapy

Discussion Topics

  1. Poulsom R, et al. Allogeneic iliac transplants in rhesus monkeys. A sequential histologic study. J Periodontol 47:187-195, 1976.

  2. Schallhorn RG, Hiatt WH. Human allografts of iliac cancellous bone and marrow in periodontal osseous defects. II. Clinical observation. J Periodontol 43:67-81,1972.

  3. Schrad SC, Tussing GJ. Human allografts of iliac bone and marrow in periodontal osseous defects. J. Periodontol. 57:205-210,1986.

  4. Buck BE, Resnick L, Shal SM, Malinin TI. Human immuno-deficiency virus cultured from bone. Implications for transplantation. Clin Orthop 251:249- , 1990.

  5. Mellonig JT, Prewett AB, Moyer MP. HIV inactivation in a bone allograft. J. Periodontol 63: 979 - 983, 1992

  6. Moreau MF, Gallois Y, Basle MF, Chappard D. Gamma irradiation of human bone allografts alters medullary lipids and releases toxic compounds for osteoblast-like cells. Biomaterials 21:369-376, 2000

  7. Holtzclaw D, Toscano N, Eisenlohr L, Callan D. The safety of bone allografts used in dentistry: a review. J Am Dent Assoc. 2008 Sep;139(9):1192-9.

  1. Sepe W, Bowers G, Lawrence J, et al. Clinical evaluation of freeze-dried bone allografts in periodontal osseous defects - Part II. J. Periodontol. 49:9-14, 1978.

  2. Quattlebaum JB, Mellonig JT, Hensal NF: Antigenicity of freeze-dried cortical bone allograft in human periodontal osseous defects. J. Periodontol. 59:394-397,1988.

  1. Urist MR. Bone formation by autoinduction. Science 150:893, 1965.

  2. Quintero G, et al: A six-month clinical evaluation of decalcified freeze-dried bone allografts in periodontal osseous defects. J. Periodontol. 53:726-730, 1982.

  3. Bowers GM, et al. Histologic evaluation of new attachment apparatus formation in humans. Part II. J. Periodontol. 60:675-682, 1989.

  4. Bowers GM, et al. Histologic evaluation of new attachment apparatus formation in humans. Part III. J. Periodontol. 60:683-693, 1989.

  5. Reynolds M, Bowers G. Fate of demineralized freeze-dried bone allografts in human infrabony defects. J Periodontol 1996; 67: 150-157

  6. Schwartz Z, Mellonig J, et al. Ability of commercial demineralized freeze-dried bone allograft to induce new bone formation. J Periodontol 1996; 67: 918-926.

  7. Schwartz Z, Somers A, et al. Ability of commercial demineralized freeze-dried bone allograft to induce new bone formation is dependent on donor age but not gender. J Periodontol 69:470-478,1998.

  8. Shigeyama Y, D'Errico JA, Stone R, Somerman MJ. Comercially-prepared allograft material has biological activity in vitro. J Periodontol 66:478-487,1995.

  1. Sonis ST, Kaban LB, Glowacki J: Clinical trial of demineralized bone powder in the treatment of periodontal defects. J. Oral Med. 38:117-122, 1983.

  2. Maddox E, Zhan M, Mondy G, Drohan W, Burgess W. Optimizing human demineralized bone matrix for clinical application. Tissue Engineering 6:465-440, 2000.

  3. Francis JR, Brunsvold MA, Prewett AB, Mellonig JT. Clinical evaluation of an allogeneic bone matrix in the treatment of periodontal osseous defects. J Periodontol 66:1074-1079,1995.

  4. Flemmig TF, Ehmke B, et al. Long-term maintenance of alveolar bone gain after implantation of autolyzed, antigen-extracted, allogenic bone in periodontal intraosseous defects. J Periodontol 69:47-53,1998.

  5. Kim C-K, Cho K-S, et al. Periodontal repair in dogs: Effect of allogenic freeze-dried demineralized bone matrix implants on alveolar bone and cementum regeneration. J Periodontol 69:26-33,1998.

  1. Browning ES, Mealey BL, Mellonig JT.Evaluation of a mineralized cancellous bone allograft for the treatment of periodontal osseous defects: 6-month surgical reentry. Int J Perio Rest Dent. 2009 Feb;29(1):41-7.

  1. Rabie A-BM, Dan Z, Samman N. Ultrastructural identification of cells involved in the healing of intramembranous and endochondral bones. Int J Oral Maxillofac Surg 25:383-388,1996.

  2. Rabie A-BM, Ortho C, Chay SH, Wong AMK. Healing of autogenous intramembranous bone in the presence and absence of homologous demineralized imtramembranous bone. Am J Orthod Dentofac Orthop 117:288-297,2000

  1. Mabry T, et al. Freeze-dried bone allografts combined with tetracycline in the treatment of juvenile periodontitis. J. Periodontol. 56:74- , 1985.

  2. Masters L, Mellonig JM, Brunsvold M, Pirkka V. A clinical evaluation of demineralized freeze-dried bone allograft in combination with tetracycline in the treatment of periodontal osseous defects. J Periodontol 1996; 67: 770-781

  3. Petri III WH, Wilson TM. Clinical evaluation of antibiotic-supplemented bone allograft. J Oral Maxillofac Surg 1993; 51:982-985.

  1. Hiatt WH, Schallhorn RG, Aaronian AJ. The induction of new bone and cementum formation. IV. Microscopic evaluation of the periodontium following human bone and marrow allograft, autograft, and non-graft periodontal regenerative procedures. J Periodontol 49:495-517, 1978

  2. Listgarten MA, Rosenberg MM: Histologic study of repair following new attachment procedures in human periodontal lesions. J Periodontol 50:333- ,1979.

  3. Mellonig JT, Bowers GM, Bailey RC: Comparison of bone graft materials Part I. New bone formation with autografts and allografts as determined by Strontium - 85. J. Periodontol. 52:291-296, 1981.

  4. Mellonig JT, Bowers GM, Cotton WR: Comparison of bone graft materials. Part II. New bone formation with autografts and allografts: a histologic evaluation. J Periodontol. 52:297-302, 1981

  5. Sanders JJ, et al: Clinical evaluation of freeze-dried bone allografts in periodontal osseous defects. Part III. Composite freeze-dried bone allografts with and without autogenous bone grafts. J. Periodontol. 54:1-8, 1983

  6. Rummelhart JM, Mellonig, JJ, Gray JL, Towle HJ: A comparison of freeze-dried bone allograft and demineralized freeze-dried bone allograft in human periodontal osseous defects. J. Periodontol. 60:655-663, 1989.

  7. Mellonig JT. Freeze-dried bone allografts in periodontal reconstructive surgery. Dent Clin North Am 35: 505-520. 1991

  8. Position Paper; Tissue Banking of Bone Allografts Used in Periodontal Regeneration. The Committee on Research, Science and Therapy of the American Academy of Periodontology Journal of Periodontology Jun 2001, Vol. 72, No. 6, Pages 834-838: 834-838.

  9. Mellonig JT. Bone allografts in periodontal therapy. Clin Orthop Relat Res. 1996 Mar;(324):116-25

  10. Wang HL, Greenwell H. Periodontal regeneration. J Periodontol. 2005 Sep;76(9):1601-22. (section on allografts only)

What is an allograft? How have they changed since their introduction? What are some of the questions or concerns that patients might have concerning allografts? How are the preparations of different allografts able to address these concerns?

Poulsom 1976             ARTICLE

P: To determine whether there are any differences in the sequential healing events in fresh and frozen allogeneic iliac transplants, and to determine whether the freezing process affects the osteogenic potential of the allograft.

M&M: Four adult rhesus monkeys were used in the experiment, were sacrificed and provided 19 histological specimens for 0, 3, 7, 14, 21, 28, 42, and 56 days post-op. There were 16 graft specimens (some frozen and some fresh) and three control specimens that were treated with surgical curettage only. 30 days prior to experiment, two walled osseous defects were created with a straight fissured bur on distal of premolars and first molars. Wooden toothpick was introduced into each defect to serve as a chronic irritant. Monkeys were sedated and the cores of cancellous bone and marrow from anterior iliac crest were obtained 7 days prior to defect correction. Graft materials were subjected to freezing at -197 degrees Celcius. At time of placement, additional graft material was obtained and used as the fresh graft material.

Still an acute lesion at 30days. Healing would be better than in chronic perio lesion.

R:

- Clinically at 2 weeks the areas appeared to be healed for all groups.

- Histologically, the epithelium migrated along tooth surface apical to its preoperative position. Chronic inflammation infiltrate found in the connective tissue throughout the first month. By day 42 and 56, connective tissue was back to normal where collagen fiber bundles were becoming organized and maturing. Alveolar bone adjacent to defect showed repair and osteoclastic/osteoblastic remodeling present. At day 7, osteoid seen on the crest and on the wall of marrow spaces. At day 28, few osteoclasts, more osteoblastic activity. Looking at the root surface, by day 21, new cementum, cementoid, and cementoblasts were seen.

Day

Fresh

Frozen

Control

7

Osteoblastic and osteoclastic activity

Osteoblastic and osteoclastic activity

Fibroblastic and endothelial proliferation

14/21

Graft remained non-vital. Found osteoclasts, osteoblasts and bone apposition

New bone formation, osteoblastic activity predominant

CNT organization and some maturation

28

Alveolar crest and graft fragments connected by new bone formation

Very few osteoclasts. Alveolar crest and graft fragments connected

n/a

42/56

A few nonviable graft fragments, new PDL

Very few graft fragments, functionally oriented PDL

CNT was mature and organized. New bone noted, but less than fresh and frozen marrows

BL: Clinically and histologically there was no significant difference in healing of fresh and frozen allogeneic iliac crest transplants. Allogeneic iliac crest transplants induce the formation of a new bone, new cementum, and new PDL fibers. Both fresh and frozen grafts induced osseous regeneration more rapidly than surgical curettage alone.

Schallhorn, 1972         ARTICLE

Purpose: To present clinical observations on 20 patients treated with allografts over a 26 month period.

M&M: A total of 194 sites consisting of furcations, 1-, 2-, 3-walled defects, dehiscence defects, or crestal type defects were treated with cross-matched frozen iliac allografts. Post-operative evaluations were completed from 5-26 months.

Results: For all categories there was an average of 3.07 mm of coronal bony apposition per graft. Graft sites healed rapidly without any rejection phenomena. Comparable exfoliation to autografts was observed when overfill techniques were used. Pocket depths decreased from a range of 6-14mm to 1-3mm. No evidence of root resorption or other pathology seemed to be related to the grafts.

BL: There are several possible explanations explaining this phenomenon.

  1. Lack of rejection may be due to the periodontium being a privileged graft site.

  2. The immunologic competency of the host may be overwhelmed, so it is unable to detect or reject antigenic material that closely approximates itself.

  3. Freezing alters antigenicity of the graft rendering it more compatible.

Schrad & Tussing 1986             ARTICLE

P: To compare bone fill in osseous defects treated with frozen allogenic cancellous iliac bone and marrow grafts to defects treated by flap curettage. antibodies formation, success on max or mand arch & clin effect of soft tss attach level were also evaluated.

M&M: 6 pts w/ bilateral defects. 23 defects were treated w/ iliac bone and 32 defects treated w/ OFD. Initial tx: SRP, occlusal adjustment, endo, antibiotics, hematological studies for leukocyte antigen & blood typing.

R: Overall defect fill was greater in grafted sites than non-grafted (56% vs. 35%). Percentage of crestal resorption was less in grafted sites than non-grafted (3% to 17%). Grafting procedures demonstrated 43.5% compared to 22% in obtaining 50% or better defect fill. As depth of defects increased, only the grafted cases tended to get increase in amount of bone fill. No clinically root detectable resorption was noted at reentry, nor radiographic or clinical evidence of ankylosis at 1 year post-op

Disc: Reduction in % defect fill with increasing defect depth was more predictable for grafted areas than for control sites, which showed twice as much defect fill.

Buck 1990             ARTICLE

P: To determine if HIV could be cultured from bone from donors after they have died and if the virus could survive freezing as well as washing and freeze-drying of bone samples.

M&M: Small fragments of various tissues, including bone and tendons were excised under sterile conditions from autopsy of 5 individuals who died with AIDS. In addition, 1 control homicide victim who had a history of drug abuse, but was HIV (-) was used. 6 bone samples and 5 tendon samples were submitted for viral culture in a fresh state. Bone fragments for subsequent culture were frozen in liquid nitrogen. After being frozen for 4-17 weeks, bone fragments that initially yielded the virus were submitted for culture. The remaining bone, frozen for 18 weeks, was washed, freeze-dried, then cultured with HIV to see the response.

R: HIV was found in serum from all 5 HIV (+) patients. The control case was HIV (-). 3/5 fresh bone cultures were + for HIV. After freezing, the virus could still be isolated from 1/5 bone cultures. If the virus could still be cultured after freezing, HIV was not inactivated by procedures usually employed by tissue banks, such as washing and freeze-drying.

D: HIV resides in bone. Freezing alone does not completely deactivate HIV from infected samples, it does however appear to reduce the viral load to reduce the chance of transmission. Washing and freeze drying the one sample that was positive did not alter its ability to be cultured with HIV. Screening donors for active HIV is crucial. A past publication calculated the risk of using/receiving an allograft from unrecognized early HIV infected donor was calculated to be one in 1.6 million after following the methods/precautions of the tissue bank. The risk of an unrecognized HIV-positive graft containing culturable virus after freezing would be reduced further, 1/1.6millionx1/5 = 1/8 million.

BL: HIV virus can be cultured from bone and in some cases is inactivated by freezing. Screening and testing methods remain the principal safeguards against possible transmission of the virus from an unrecognized infected donor.

Mellonig 1992             ARTICLE

P: To provide direct evidence that the processing of DFDBA would render it free from HIV and safe for human use.

M+M: Part I: Human cortical bone obtained from cadavers - tested to be free of HIV contamination, was spiked with 5.26 X 10 ^9 viral particles (148 μg of viral protein).

Part II: Cortical bone from a patient who died from AIDS.

Bone from both was ground to 90-500 μm, treated with viricidal agent (combination of ethanol and non-ionic detergent) and demineralized in HCl. Control was untreated. All samples cultivated with stimulated peripheral blood lymphocytes and assayed for p24 (core protein), reverse transcriptase, and viral gag gene by polymerase chain reaction (PCR).

Results: Part I: untreated virus-infected bone was positive for HIV replication, treated samples were negative. Part II: virus-infected bone was positive by PCR, replication of viable HIV could not be demonstrated after treatment.

BL: Demineralization and treatment with a virucidal agent inactivates HIV in spiked and infected bone. Authors report that screening of donors place the risk of disease transfer at 1 in 1.67 million. Simply freezing the bone further reduces the risk to 1 in 8 million. The acid decalcification process in combination with certain virucidal agents as used in preparation of decalcified freeze-dried bone allograft destroys HIV, which under extremely rare circumstances may be present in bone allograft.

Cant tell a patient that it is imposible. Quote the odds, if asked. To my knowledge, it has never happened using dental FDBA

Moreau 2000                 ARTICLE

BG: Allograft is an alternative to autograft nowadays when large bone volumes are necessary. Fresh-frozen, freeze-dried, and gamma irradiation are the most common preparation techniques. However, a large amount of lipids is present in the medullary spaces (near 70% in weight for a human femoral head). They are known to strongly influence the biocompatibility of the graft, but the exact changes of lipids upon sterilization and storage process are poorly known.

P: To identify biochemically the effects of gamma irradiation on medullary lipids and to identify the cytotoxicity of gamma-irradiated bank bone with/without lipid on cultures of osteoblast-like cells.

M&M: Bone cores from 8 femoral heads retrieved during prosthesis surgeries for arthritis were prepared with a drilling trephine. Cores underwent de-fatting procedures to remove the non-mineralized organic components that fill the marrow spaces and then were sterilized by gamma radiation (25,000 gray) or kept frozen. Total and peroxidated lipids (generated during gamma radiation) were measured. Bone slices were placed on the surface of the cell layers (Osteoblast-like cells).

R: Biomechanical analysis:

- Weight of bone cores did not significantly differ between gamma or UV-irradiated groups and both irradiations did not alter the amount of lipids in the bone cores

Cytological analysis:

- Delipidated slices were never associated with a surrounding area containing necrotic or dead cells

- Raw slices: UV-sterilized induced a minimal amount of cell death. Gamma-irradiated slices were associated with a considerable amount of cell death.

BL: De-fatting procedures should be added when preparing bone allografts in human bone banks.

Holtzclaw 2008             ARTICLE

Purpose: To review safety aspects of human bone allografts as they apply to the practice of the dentistry.

Discussion

Industry regulation: Federal agencies such US Food and Drug Administration (FDA) retain ultimate authority over regulation of US – based human allograft acquisition, processing and use. Independent no-profit organizations such as the American Association of Tissue Banks (AATB) also help to ensure safe an ethical use of donated human tissues.

FDA Center for Biologic Evaluation and Research (CBER) regulate these procedures under specific parts (1270 and 1271) of the US Code of Federal Regulations (CFR). It also established donor eligibility, good tissue practice and other guidelines to prevent the introduction, transmission and spread of communicable diseases. The Donor Eligibility Final Rule (DEFR) requires all Human Cellular and Tissue based Products (HCT/P) manufactures to screen and test donors for risk factors and clinical evidence of relevant of several diseases and infections such as HIV, HBV, HCV.

The AATB publishes tissue-banking industry standards and offers rigorous accreditation for institutional members as well as a certification program for people working in this field. Membership in the AATB is not legally required 75-80% of tissue – banking agencies follow the voluntary industry standards established. AATB publishes a list of its accredited tissue banks on a quarterly basis, which are accessible online at www.aatb.org.

Donor Screening: More than 90% of the donor referrals to AATB - accredited tissue banks come from hospitals.

The location of the initial tissue recovery varies (hospital morgue, operating room, coroner’s facility, tissue banks), but the team must use a clean environment and follow established aseptic protocols.

On receipt of donor material FDA regulations require tissue processing facilities to quarantine the harvested tissue and perform further donor screening and testing procedures. The facility conducts a behavioral risk assessment and medical history review by interviewing the donor’s next of kin or other close acquaintances and compares the feedback with available medical records. Then bone allografts are tested for HIV, HBV, HCV, HTLV and syphilis in an FDA – registered and certified lab. Finally the facility samples and tests all recovered tissues for both bacterial and fungal contamination.

Researchers have calculated the risk of harvesting bone from a donor with HIV using these exclusionary methods at one in 1.67 million.

According to the AATB accredited institutions accepted less than 5% of all screened donors in 2008. Although these donor screening programs are successful, the risk of human allograft contamination still exists.

With proper processing human bone allografts for dental purposes routinely achieve a sterility assurance level (SAL) of 1 in one million. If a tissue bank uses the mentioned donor screening process coupled with the act of graft freezing the risk of producing an HIV – contaminated human bone allograft decreases to 1 in 8 million, and if the tissue bank demineralizes the allograft, the risk in 1 in 2.8 billions. Each graft is labeled with a unique identification code that allows to record and track the donor graft to its recipient and vice versa, in the event of HCT/P recall. This records must be kept for ten years, but many tissue banks retain their records indefinitely.

Clinicians who have used recalled allografts should immediately notify the patient and test them for suspected pathogens for a minimum of six months after implantation.

DFDBA= 1:2.8B FDBA= 1:8M

What exactly is FDBA? What is the clinical and histologic result of using FDBA in periodontal infrabony defects?

Sepe 1978             ARTICLE

Clinical evaluation of Freeze-dried bone allografts in periodontal osseous defects part II

Purpose: to reinforce the FDBA have the potential as a grafting material in certain periodontal osseous defects in humans

Materials and methods

Results

BL: FDBA has potential as a grafting material when good surgical protocol is followed.

Quattelbaum 1988                 ARTICLE

In humans, chromosome 6 contains the MHC, which codes for the human lymphocyte antigens (HLA). These antigens are expressed on the cell surface of nearly every nucleated cell in the body and represent the primary stimulus for transplant tissue rejection when HLA mismatches occur between donor and recipient.

P: To determine whether donor-specific anti-HLA antibodies could be detected against FDBA implanted during treatment of periodontal osseous defects in humans.

M&M: 18 pts that needed more than one grafting procedure were included. Pt with previous bone allografting procedures, blood transfusions, or pre-existing HLA antibodies detected were excluded. The FDBA came from one donor of known HLA tissue-type. This bone was cortical bone from tibia, fibula or femur. The pts were tissue-typed, in order to determine the number of HLA mismatches between each pt and the FDBA donor. After a serum sample was taken (sample I), and there were no detectable anti-HLA antibodies, the first allograft procedure was performed. 2 weeks later, a second serum sample (sample II) was obtained and the second allograft procedure was done. 2 weeks after that, sample III was taken. Finally, 3 months (104 days) after the second graft, sample IV was taken. Detection of anti-HLA anti-bodies (IgM class) against known donor HLA was accomplished using a complement dependent microcytotoxicity assay against a panel of 66 reference cells, covering all HLA specificities known to be present in the donor. These were compared to positive and negative control groups.

R: 18 pt underwent a total of 34 allograft procedures. All of the procedures were judged clinically successful with no adverse tissue reactions and no pts had HLA specificities exactly matching those of the donor. At no time could any anti-HLA antibodies be detected in any pt.

BL: FDBA lacks clinically significant antigenicity when used to repair perio defects.

What is DFDBA? What makes it different than FDBA and are there different ways to prepare it? What are the theoretic benefits of using DFDBA and how has this been researched? Is there histologic proof of regeneration with DFDBA? What happens to the DFDBA particles once healing occurs?

Urist 1965                 ARTICLE

P: Review article summarizing 70 experiments that were performed in the long bones of approximately 300 animals to introduce a hypothesis of postfetal osteogenesis by autoinduction. Article provides evidence in favor of the theory of induction gathered from the process of bone formation in the interior of an implant of acellular, devitalized, decalcified bone matrix.

DISC: Wandering histiocytes, foreign body giant cells and inflammatory connective tissue cells are stimulated by degradation products of dead matrix to grow in and repopulate the area of an implant of decalcified bone. There are more histiocytes than any other cell type and may transfer collagenolytic activity to the substrate to cause dissolution of the matrix. The process is followed by new bone formation when one wandering histiocyte (the inductor cell) and one perivascular connective tissue cell (the responding or induced cell) divide and interact. Cellular differentiation results via autoinduction to produce two additional cells, one responding cell and one specialized cell, either an osteoprogentitor or a chondroprogenitor cell.

Bone induction in decalcified matrix raises the question of whether the matrix produces a specific diffusible chemical agent that induces the cells of the host to differentiate into osteoblasts. The author believes that the system is more complex than a simple chemical stimulus and direct cell response. The evidence of these experiments shows that the differentiation of the osteoprogenitor cell is elicited by local alterations in cell metabolic cycles that are not yet characterized.

BL: There is substantial evidence for osteogenesis by autoinduction. Bone formation occurs in extraskeletal implants of decalcified bone matrix and the new osteoblasts are derived, not from the donor tissue, but from proliferating pluripotent, ingrowing stem cells of the host.

Quintero 1982                 ARTICLE

Purpose: To evaluate clinically the osteogenic potential of decalcified freeze- dried bone allografts in the treatment of human periodontal intraosseous defects.

M&M: A 6-month study. 27 defects in 11 patients were grafted with DFDBA. Cortical bone was obtained from the femur of a human donor within 24 hours after death. The tissue was decalcified and then frozen at -197oC for 4 weeks. DFDBA was used in one-, two- and wide three-wall defects. Presurgical management included patient demonstration of effective plaque control, scaling, root planing, prophylaxis and testing the vitality of involved teeth. Measurements were obtained before surgery, at the time of surgery, and at re-entry to document the osseous changes (4-6 months) using a stent and a periodontal probe. Measurements were made from the base of the stent to the CEJ, to the gingival margin and to the base of the pocket. Osseous measurements were made from the base of the stent to the alveolar crest and to the base of the intrasseous defect. Clinical data was supplemented with radiographs and photographs.

Results:

The overall mean osseous regeneration for the 27 defects was 2.4mm or a 65% fill of the defect. The greatest regeneration was obtained in 3-wall defects. Crestal apposition of new bone was noted in 1 two-wall and 2 three-wall defects. Each of these sites demonstrated 1mm of new bone coronal to the original osseous crest. The overall mean increase in clinical attachment was 1.9mm. Loss of attachment was noted in only 2 of the 27 treated defects.

BL: DFDBA has some potential as a graft material in the treatment of periodontal osseous defects. The degree of osseous regeneration is directly proportional to the number of bony walls lining the defect.

Bowers 1989 Part II                 ARTICLE

P: To 1. compare the potential for regeneration in pathologically exposed osseous defects that were subsequently submerged, with or without DFDBA, in a 6-month human histologic study. 2. examine whether DFDBA will enhance amount & frequency of new attach, & if root resorption is a sequela of DFDBA grafting.

M&M: 10 pts had 2 or more max incisiors, canines or premolars that were recommended for ext due to advanced bone loss, deep pockets and radiographic IPx lesions. All teeth were vital and asymptomatic. Teeth flapped, base of calculus notched, debridement, roots severed at highest level of alveolar crest, RP only coronal to calculus reference notch, defects recorded, some sites grafted with DFDBA, coronally positioned flap for root submersion. Measurements from 30 grafted and 13 nongrafted defects in 9 pts were submitted for statistical analysis. Biopsies obtained at 6 months

R: 11 nongrafted defects could not be used due to epithelial breakthrough. SS more new attach apparatus (1.76 mm) in grafted vs non-grafted sites (0.76mm), as well as in the amount of new bone (1.96 mm vs 0.8mm).

More cementum in grafted sites, but difference was NS. Cementum formed more commonly over dentin in grafted sites, and over cementum & dentin in non-grafted sites. Fibers of the PDL were both parallel & perpendicular in non-grafted sites, and more frequently parallel in grafted sites

Sig greater loss of alv crest ht in non-grafted vs grafted sites. No extensive root resorption, ankylosis or pulp death.

Overall there was a higher percentage of regeneration in grafted than nongrafted sites.

D/BL: The authors pointed out that while epithelial exclusion may be necessary for regeneration to occur, it is only one of the several factors, as only limited bone and cementum formation were observed in non-grafted sites that remained submerged. Epithelial exclusion and DFDBA in this study was associated with a greater degree of regeneration and new CTA in the absence of root resorption or ankylosis.

-SS more new attach and new bone formation, and greater chance for regeneration in submerged DFDBA grafted vs non-grafted sites. PDL may be perpendicuclar, parallel or both in the same defect. New CEM can form on old CEM, dentin, or both in the same defect. Greater chance for the regeneration of a CTA in submerged non-grafted than grafted sites.

Cr: control size pretty small if 11/13 were thrown out…

Bowers 1989 Part III                 ARTICLE

P: To compare healing of intrabony defects with and without DFDBA in humans.

M&M: Data was collected from 12 patients with 32 grafted (DFDBA) and 25 non-grafted defects. Patients had 2 or more maxillary incisors, canines, or premolars recommended for extraction. All teeth had advanced bone loss, deep pockets, and associated IPx intrabony defects with radiographically visible calculus on the root surfaces. All teeth were vital and asymptomatic. All surgical procedures were performed by the same investigator. Gingivoplasty was performed over IPx bony defects, crevicular epithelium was removed. FTFs reflected, base of calculus and level of alveolar crest were marked with round bur. Root surfaces were planed coronally to the calculus reference notch. The base of the defect was debrided but no attempt was made to contact the root surface apical to the calculus reference groove. Measurements were made from alveolar crest to calculus groove and to the base of the defect, CEJ to base of the defect. DFDBA was placed into randomly selected defects. A free gingival graft was sutured over the gingivoplasty wound of grafted and non-grafted sites to enhance wound closure and to retard epithelial migration. Non-grafted defects were treated identical to experimental defects except no graft material was placed. Patients were recalled weekly for 1 month, & monthly until biopsy at 6 months. Histology was done.

R:

A mean new attachment apparatus of 1.21mm from calculus reference notch was observed in grafted defects. There was a mean 1.24 mm new cementum, 0.13 mm new connective tissue attachment, & 1.75 mm of new bone. Junctional epithelium located 1.36 mm coronal to notch. There was significant difference in the amount of new attachment in grafted versus non-grafted defects, new cementum, new connective tissue and new bone formation. New cementum was cellular in nature and was found more frequently over both cementum and dentin in the same defect. Formation of new attachment apparatus was not observed in any of the 25 non-grafted control sites after placement of free gingival graft. No extensive root resorption, ankylosis or pulp death was observed in grafted or non-grafted specimens.

BL:

1. The formation of a new attachment apparatus is possible when intrabony defects are grafted with DFDBA.

2. Significantly more new attachment apparatus, new cementum, new connective tissue, and new bone will form in intrabony defects grafted with DFDBA than in nongrafted defects.

3. There is a greater chance for regeneration of a new attachment apparatus, new cementum, new connective tissue, and new bone in intrabony defects grafted with DFDBA than in nongrafted defects.

4. New cellular cementum may form on dentin, old cementum, or both dentin and old cementum in the same defect.

5. The periodontal ligament is most often oriented perpendicular to the root at 6 months.

6. There is a significantly greater loss of alveolar crest height in non-grafted defects than grafted defects.

7. Free gingival grafts do not enhance the regeneration of a new attachment apparatus, new cementum, new connective tissue, or new bone in non-grafted defects.

8. Extensive root resorption, ankylosis, and pulp death are not common sequelae of grafting intrabony defects with DFDBA.

Reynolds 1996                     ARTICLE

P: To histologically examine the fate of DFDBA used for regeneration in intrabony defects and to compare the amount of new attachment apparatus formation associated w/ presence or absence of particles.

M+M: Histologic data from 12 pts with 32 grafted intrabony defects, teeth deemed for extraction, each pt had 2 or more teeth with defects. Initial treatment given but calculus not removed off of teeth in the study. Surgery performed, defects debrided, base of calculus and level of alveolar crest marked with bur, roots planed from calculus notch coronally, defect type recorded. Graft material placed into defect, FGG sutured over site (to enhance wound closure and retard epith migration). Non-grafted controls had no graft material placed into defect. Postoperative maintenance was performed weekly for the first month, biweekly the second month, and monthly

until biopsy. Block sections taken 6 months post-op. Histo eval and measurements were performed.

R: 72% of the grafted defects exhibited residual DFDBA particles. DFDBA appeared amalgamated within the new viable bone. In subgroup analysis of 14 grafted defects in 5 pt, comparing sites within pts based on the presence/ absence of residual graft material indicated SSD in regenerative response.

Bone particle

present

Bone particle

absent

New attachment

1.72 mm

0.20

New bone

2.33 mm

0.23

New cementum

1.74 mm

0.23

Connective tissue

0.11mm

0.04

BL: 6 months post-op, 72% of the grafted defects exhibited residual DFDBA particles.

Schwartz 1996                 ARTICLE

B: Demineralization of bone graft exposes the bone inductive proteins located in the bone matrix, and, in fact activates the osteoinductive ability of the graft.

P: To determine the ability of commercial DFDBA preparations to induce new bone formation and to test the hypothesis that variability in clinical outcomes could be a function of differences in either DFDBA processing techniques and/or donor characteristics.

M&M: DFDBA with particles sizes ranging from 200-500um was obtained from 6 bone banks (different processing methods). Different batches of DFDBA from the same tissue bank were available in 4/6 tissue banks and a total of 14 batches were available for evaluation. DFDBA from each donor was implanted subcutaneous (gelatin capsules) or intramuscular into 49 mice for 28 and 56 days. Mice were sacrificed and new bone/cartilage formation was histologically evaluated. The surface area of the DFDBA particles was also evaluated by histomorphometric analysis pre and post-op.

R: Regardless of the source, there was no bone or cartilage formed around any of the subcutaneous implants and no resorption of particles was observed (all particles surrounded by connective tissue). For the IM implants, the amount of bone formation varied with the source of the particles. Wide variation (from no bone formation to 40% of bone and/or cartilage) was seen between sources, with variation even occurring among different batches from the same bank. While some samples did not exhibit bone formation up to 2 months, some DFDBA particles induced bone at 1 month and some changes occurred in the quality of the newly formed bone up to 2 months. Particle size and surface area varied significantly among different banks and within the same batch, but did not correlate with the amount of bone formation (the particles differed significantly in size, but were all between 200-500microns).

D: The DFDBA particles used in this study varied greatly in their ability to induce bone at ectopic sites. While particles from some bone banks exhibited osteoinductive properties, samples from other bone banks failed to do so. Moreover, in one bone bank, one of the batches had no osteoinductive activity, while the other batch did. These results suggest that some of the DFDBA used clinically today may have little-to-no osteoinductive capability, although the graft could function in an osteoconductive capacity (hence, the different results from DFDBA studies). Each batch of DFDBA should have its osteoinductive capacity evaluated. Differences in DFDBA samples may be a function of donor age and sex, previous exposure to pathology and/or drug therapy, genetic variation and time to harvest grafts after death. However, the most important reason is most likely tissue collection and preparation (subtle variations in demineralization time, neutralization of the demineralized particles, extraction of bone inductive agents with saline, differences in sterilization).

BL: Wide variations in commercial bone banks preparations of DFDBA exist and the osteoinductive potential can vary considerably between them, as well as between samples from the same tissue bank.

Schwartz 1998                 ARTICLE

Purpose: To test the hypothesis that variability in clinical outcome using DFDBA could be a function of the age and gender of the donor., since in previous studies it was shown that the osteinductive capacity of DFDBA from different donors from the same bank varied considerably.

Materials and methods: Human DFDBA 200-500µm in diameter obtained from a tissue bank. 27 batches from different donors available for evaluation, with ages 16-59 years. 7 female, 20 male. Then, 108 male mice with reduced immune systems had DFDBA in gel capsules implanted into 2 legs, in their muscles. After 56 days implants were recovered and analysed by light microscopy and histologic evaluation was performed. Sections were evaluated for the presence or absence of DFDBA and new bone cartilage.

Results: Not all the batches showed the same ability to induce bone. Five batches had little or no ability to induce bone, 11 demonstrated moderate ability and 7 batches demonstrated good ability to induce bone.

When data was segregated into 3 groups (0-29years, 30-49, 50 +), there was an age-dependent decrease in new bone score. Same difference was observed when cortical bone and marrow where examined separately. No differences were found in bone score according to gender.

Conclusion: Variability exists among the batches of DFDBA. The ability of DFDBA to induce bone is age-dependent. These results are similar to other studies that showed that bone from younger donors is more inductive. Gender had no effect on osteoinduction ability. It appears that DFDBA should be used from donors under 50. Osteogenic potential of each batch should be determined before it is sold.

Shigeyama 95                 ARTICLE

Commercially-prepared allograft material has biological activity in vitro

Purpose: To characterize and compare biological activities of protein extracts prepared from commercially obtained bone graft material (Dimeneralized freeze-dried bone allograft-DFDBA) in vitro vs. extracts prepared from freshly obtained human bone

Materials and methods

Results

BL: Commercially prepared material retained proteins having the capacity to influence cell differentiation in vivo, however, some biological activity that was measured in vitro was lost as a result of tissue processing.

What are some other preparations of demineralized allograft? How are these different and how do they compare to DFDBA?

Sonis 1983                 ARTICLE

P: To report preliminary results on the safety and efficacy of demineralized allogenic bone powder used in the treatment of periodontal defects in 21 pts.

M & M: 16 female & 5 male patients with periodontal disease (mean age: 41 years) were selected for participation. Allogenic bone (femur) was obtained from an organ bank. The cortical portion was isolated, sectioned and processed. 75-450 micron particles were collected and demineralized*. The surgery was performed and the defects were slightly over-filled with the bone powder mixed sterile saline to make a thick paste. No bone perforation was performed prior to graft placement. Antibiotics were prescribed for 7 days. Clinical and radiographic measurements were taken at the baseline and at the post-op visit (ranging between 3-18 months). (Note this material is radiolucent.)

R: No infection or bone sequestration was reported.

Pre-Surgical Probing Depth (mm)

Post-Operative Probing Depth (mm)

Percentage Difference

1 – 2 walled defects (n=7)

7.8

4.8

51%

2 walled (n=7)

7.3

3.7

49%

Furcations (n=3)

7.0

2.3

67%

2/3 walled (n=2)

7.0

3.0

57%

0/1 walled (n=2)

5.0

3.0

40%

The radiographs showed bone fill in 61% of patients at 4 months post op. The location of the treated defect seemed to be important (In the anterior region: 5/7 defects demonstrated some fill. In the posterior region: 6 /11 defects showed some fill). Mobility was decreased in 11 of 15 patients whose teeth were not splinted.

BL: Cortical bone powder seems to act as an osteo-inductive material with a resulting decrease in both probing depth and mobility. It also seems to produce radiographic improvement in bone height.

Cr. changes in pocket depth were measured from the gingival margin. No re-entry. No histo. No controls.

* Preparation of implant material:

  1. cortical bone ( femur) isolated and sectioned into 4cm-washed with water

  2. 100% ethanol and then ethyl ether for one hour

  3. Pulverized with liquid nitrogen

  4. sieved for 75 to 450 micrometer particles

  5. demineralized – 3 hours in .5M HCL

  6. washed with water at 4degrees C until neutral pH was obtained

  7. dehydrated via 100% ethanol followed by ethyl ether

  8. packaged

  9. irradiated- cathode ray 2Megarads

Standardizing of bone:

Portions of each batch of material were used in a rat bioassay to standardize and document osteogenic activity prior to clinic use. Two subcutaneous pockets were made over the thorax of 28 eight day old male rats; 20mg of test material was implanted. At 7, 14 and 21 days implants were harvested and histology was performed. Formation of cartilage was performed on day 7 and bone on days 14 and 21- this was used as evidence of induced osteogenesis

Effect of Radiation of bone implants on induced osteogenesis at the time of this article was unclear. Results in animals using this material showed an overall 0%-20% in the reduction of osteogenesis potential of the irradiated material. This showed that significant osteogenic activity was retained.

Maddox 2000                 ARTICLE

P: To establish a simple in vitro/in vivo screen of the quality of different lots of DBM powder to induce bone formation to provide predictive value for the performance of the substance in animal models.

M&M:

In vitro assay: calvarial osteoblasts form mice were isolated and grown to confluence (day 0) in 6 well tissue culture plates. The media was changed to induce differentiation and pieces of DBM powder were added to the cultures. At days 5 and 15 cells were examined by light microscopy.

In vivo assay: small discs of 30mg human DBM powder with 20mg/mL fibrin sealant were performed in a 1mL syringe. 17 different lots of DBM were tested. The implants were placed into gluteal muscle pouches (2 per rat). All lots tested in triplicate. At 28 days implants were retrieved; half studied under light microscopy and the other half the calcium content was determined.

Craniotomy implant assay: different lots of DBM were mixed with human fibrinogen (20mg/mL final concentration) and human thrombin (2.5 U/mL final concentration) to form1 x 8mm disks to test in an 8mm trepan defect. After 28 days, implants analyzed by x-ray and histology.

R: By day 15, newly formed bone nodules could be seen in positive cultures. In the mineralization analysis, no lot that scored poorly in vitro did well, yet some lots that did well in vitro did not induce significant mineralized bone. With respect to the 8mm calvarial defect, the lots that did well in the in vitro and in vivo study healed well. Lot 9, which did not score effective, was mixed with an effective lot (lot 17) to see if it had any negative effect, which it did not. Unfortunately, the effective material did not overcome the ineffective material either, the result is the average of the two mixed.

BL: If the material scores poorly in vitro, there is no need to test it in animals. DBM powder that performs well in vitro and in vivo promotes optimal healing of calvarial defects. Osteogenic potential of each lot of DBM should be verified prior to clinical use.

Francis 1995                ARTICLE

Background: Allogenic bone matrix gel (Grafton) is processed from decalcified freeze-dried bone allograft, which extracts the remaining minerals, contaminating lipids, serum proteins and cellular components. These listed materials, when present, may reduce the inherent osteoinductive potential of allografts.

Purpose: To evaluate the effectiveness of an allogenic bone matrix gel (ABM) in the txt of periodontal osseous defects and to compare the bone fill of ABM with DFDBA.

M&M: 11 pts with paired osseous defects ranging from 3-12 mm were randomized to receive ABM or DFDBA. Defects included 1-, 2-, and combination of 1- and 2- walled IPx lesions with PD of at least 5-7 mm. PD, CAL, bone fill and defect resolution were determined at BL and at 6 month re-entry. Bone defect was measured from CEJ or other standardized marking. Standardized XRs & CADIA (computer-assisted densitometric image analysis) used to assess bone density changes. Complete debridement of defects with SRP & intramarrow penetrations done before implantation with either ABM or DFDBA to the most coronal level of the adjacent alveolar process. CPF and perio dressing, 10 days doxycycline, peridex, pain control. Pts seen q 10 days for plaque removal for 1 month, then q 3 months. Re-entry at 6 mos.

Results: NSD b/w groups for baseline measurements. Complete defect resolution was seen in 27% of defects w/ both ABM & DFDBA. Mean defect fill w/ ABM was 69% compared to 77% w/ DFDBA. ABM & DFDBA sites showed similar CAL gains of 2.64mm & 2.36mm respectively w/PD reductions of 3.82mm & 3.55mm respectively. DFDBA had 100% of defects have >50% bone fill whereas ABM had 82% of defects have >50% bone fill.

BL: ABM showed NSD from DFDBA and might have better handling properties.

Flemmig 1998                 ARTICLE

Purpose: To assess the long-term maintenance (over 3 yrs) of alveolar bone gain after implantation of autolyzed, antigen-extracted, allogenic (AAA) bone graft (Type of DFDBA) in periodontal intraosseous defects.

Background: AAA is a type of DFDBA, but also includes the extraction of cell-surface glycoproteins, which represent major antigens responsible for bone allograft reactivity.

M&M: Randomized controlled trial. 14 patients with radiographic signs of interdental periodontal intraosseous defects and probing attachment loss of more than 6 mm on at least 2 teeth were enrolled.

AAA bone was implanted into intraosseous defect of 1 tooth with a fibrinogen covering membrane for stabilization (test); a second tooth with an intraosseous defect was treated by OFD alone (control). All patients had SPT at 3 to 6-month intervals following tx. Clinical measurements were taken prior to surgery, 6 months, and 3 years following surgery. SubG plaque samples at 3y were analyzed by PCR for Actinobacillus actinomycetemcomitans and P. gingivalis.

Results: Of the 14 patients enrolled, 11 patients completed the 6-month and 8 patients the 3-year examination.

 (mm)

6months

3years

 

Test

Control

Test

Control

Bone gain

2.2+0.5

1.2+0.5

2.3+0.7

1.1+ 0.8

CAL

2.3+0.5

0.8+0.3

2.0+0.7

0.8+0.5

From 6 mos to 3y no sign changes in clin meas. At 3 years, Pg was detected in 3 test and 2 control teeth, whereas no Aa was found. Due to the low detection frequency, there was no clear correlation between the maintenance of alveolar bone during SPT and subgingival infection with Pg.

Discussion: 46% of osseous defect fill w/ AAA graft. Range of alveolar bone gain results were highly variable (0.5mm to 4.5mm) indicating low predictability of tx outcome.

Chong-Kwan Kim 1998                 ARTICLE

P: To evaluate alveolar bone and cementum regeneration following surgical placement of an allogenic, freeze-dried, demineralized bone matrix (DBM) cortical strip implant in supraalveolar periodontal defects.

M&M: 8 mongrel dogs were included. Initial preparation consisted of scaling and plaque control to obtain gingival health. Supraalveolar periodontal defects were created around second, third and fourth mandibular premolar teeth in the right and left mandibular jaw quadrants. Following sulcular incisions and elevation of buccal and lingual mucoperiosteal flaps, alveolar bone was removed around the teeth with chisels and water-cooled rotated burs. The defect height from the CEJ to the reduced alveolar crest was 5mm. Following alveolar bone reduction, exposed root surfaces were instrumented and the crowns of the teeth were reduced in height approximately 2mm above CEJ. Long bones, femur and tibiae obtained from the dogs were used to produce DBM implants. Contralateral jaw quadrants in 6 animals were randomly assigned to receive the DBM implant, or serve as surgical control (flap surgery alone). Two additional animals received bilateral DBM implants. DBM strips were carved and press fitted into the furcation and interproximal aspects of the premolar teeth defects. The flaps were coronally advanced to submerge teeth and DBM implants .The animals were sacrificed at 8 weeks post-surgery. Tissue blocks including teeth, bone and soft tissues were removed. Histometric recordings (defect height, bone and cementum regeneration, root resorption and ankylosis) were obtained.

R: 3 animals were exited from the study as they exhibited early wound failure exposing implant and root surface. Bone regeneration exhibited variable healing patterns in defects having received the DBM implant. Commonly, the DBM implant remained in an unabsorbed state, exhibiting fragmentation at exterior borders and empty osteocyte lacunae. Newly formed bone was observed between the DBM implant and the root surface. Other specimens presented bone formation and the implant was ankylosed to the tooth. Bone regeneration was limited in control defects. Also, limited cementum regeneration was observed in all DBM and control defects but was more enhanced in DBM defects. Root resorption was common in both groups but the severity of resorption appeared greater in control defects. Ankylosis was more frequent in DBM defects.

Conclusion: There is some evidence of new bone formation in DBM-implanted defects but this regeneration appears unpredictable. Allogenic freeze-dried DBM implants have potential to support cementum regeneration.

What are other preparations of allograft? What makes these graft materials different than the freeze-dried allografts? What are there advantages or disadvantages?

Browning 2009                 ARTICLE

P: To evaluate the effectiveness of mineralized cancellous bone allograft (MBA; Puros) for the management of intrabony defects in patients with chronic periodontitis.

M+M: 22 healthy pts (11M, 11F) with at least one radiographic bone defect of >4mm and a PD >7mm. All subjects underwent initial therapy (OHI, SRP everywhere except study site, occlusal adjustments) and OL <20%. Sx: FTF, debridement, SRP, roots conditioned with TTC 50mg/mL for 1 min, and defect grafted with MBA. Pts given 100mg doxy for 10 days, 800mg Ibuprofen tid, and 0.12% CHX rinse bid. Measurements(including CEJ to alv crest, CEJ to base of defect, and alveolar crest to base of defect) taken at day of sx and final measurements taken at re-entry 6 months post-op.

R: 20 of 22 pts returned for reentry sx. Average bone fill was 4.1 mm, average percent bone fill was 66.8% and average percent defect resolution was 71.5%. Mean residual defect depth was 1.7mm. Mean reduction in PD was 4.8 mm and mean gain in CAL was 4.2 mm.

BL: Mineralized cancellous bone allograft appears to be an effective material for treatment of osseous defects in pts with chronic perio.

CR: No controls, no stent for measurements

Does the origin of the bone make a difference in healing potential?

Rabie 1996                 ARTICLE

P: To identify the cells involved in healing of autogenous intramembranous (IM) and endochondral (EC) bone grafts.

M&M: 36 10x5mm defects created in skulls of 18 rabbits. The defects were filled with IM graft alone (ramus), EC graft alone (tibia), demineralized bone matrix (DBM) alone [harvested from endochondral bone], or combined IM-DBM. Rabbits sacrificed at 7 and 14d post sx, grafts harvested and prepared for cell identification by LM and EM.

R: Control- fibrous CT fill (untreated/not grafted)

IM alone- pre-osteoblasts, osteoblasts, and osteocytes (observed at 14 days) observed with no cartilage intermediate stage

EC alone- chondroblasts and chondrocytes observed

DBM alone- cartilage stage present

IM-DBM- cartilage stage present. At 14 days: osteoblasts, osteocytes and chondrocytes identified as well. This group showed bone formation throughout the whole defect, in contrast to the IM graft alone group, where this occurred only at the interface between the host bone and the bone graft.

D: The best time for harvest of newly formed cartilage devoid of bone is 7-8 days (quoted form another study). Days 18-21 are advantageous for the harvest of bone without cartilage (quoted from same study). Hence, day 7 would allow the detection of cartilage cells if present in the healing of IM bone, and day 14 of both cartilage and bone.

BL: IM bone healed by osteogenic ossification route, and EC by EC ossification route. In presence of DBM (from EC origin), IM bone adopts an EC ossification route. Since IM skips the cartilage phase, it is preferable in cases where earlier healing is desired.

Rabie 2000                 ARTICLE

Purpose: To assess the amount of newly formed bone when using composite demineralized intramembranous (IM) bone matrix DBMIM and IM autogenic bone grafts and to compare it with IM bone alone. To identify the cells involved in healing at histologic and ultrastructural level.

Materials and methods: 42 adult rabbits were used. 42 defects were created in the skulls of the rabbits, 21 grafted with IM bone and 21 with composite IM-DBMIM. In the control group 22 defects were created in 11 rabbits, 11 were left empty and 11 were filled with rabbit skin collagen.

Histologic and ultrastructural analysis were performed on tissues obtained at 1, 2, 3, 4, 5, 6, 7, and 14 days after healing.

10x5 mm, critical size, full thickness cranial defects using templates were created in the parietal bones, periosteum was removed, so that healing is largely attributed to the implant material.

Group I, control: Defects empty at one side, and filled with collagen at the other.

Group II, autogenous IM bone graft: The IM bone graft placed into he defect and fixed in place as described above.

Group III, composite IM- DBMIM bone graft: IM bone graft was sandwiched between two layers of the demineralized IM bone powder that was mixed with whole blood and fixed in place.

Results: All animals remained in excellent health, no signs of infection or other complications.

Group I: Control defects did not exhibit bone formation, were soft in palpation, in contrast with defects filled with bone grafts. Histologically they were filled with fibrous connective tissue.

Group II: Inflammatory cells on days 1, 2. Mesenchymal cells on day 4, preosteoblasts, osteoblasts and osteocytes noted on day 5. Small blood vessels noted on day 5. New bone in the host bone-graft interface seen on days 7 and 14.

Group III: Inflammatory cells also present on days 1, 2 but mesenchymal cells, preosteoblasts, osteoblasts and osteocytes present earlier on day 3. At day 14 most of the DBMIM particles were replaced by bone. There was a gradient in the amount of new bone formed from the host bed to the graft.

Conclusions: The amount of bone formed two weeks after grafting was significantly greater in the composite IM- DBMIM bone grafted defects, 204% more.

Does adding antibiotics improve allografts? Would different allografts react differently to particular antibiotics. If so, why?

Mabry 1985                 ARTICLE

Freeze-Dried bone allografts combined with tetracycline in the treatment of juvenile periodontitis

Purpose: To compare open flap debridement curettage and freeze-dried bone allografts with and without the use of local and systemic tetracycline in treatment of osseous defects associated with Juvenile Periodontitis.

Materials and methods

Results

Conclusions

Masters 1996                 ARTICLE

P: To evaluate the use of demineralized freeze-dried bone allograft reconstituted with 50mg/ml tetracycline hydrochloride in the treatment of intrabony periodontal defects.

M&M: 15 healthy patients (12 F, 3M; age 35-61) with mod-adv perio were treated. Pts had 3 osseous defects with PD >5mm after initial therapy. Each site was randomly assigned to one of the following groups: 1) demineralized freeze-dried bone allograft reconstituted with 50 mg/ml tetracycline (DFDBA+TCN); 2) demineralized freeze-dried bone allograft alone (DFDBA); or 3) debridement only (D). Clinical measurements were taken the day of surgery, 6 months, and 1 year. Standardized radiographs were taken at baseline and 1 year and were evaluated by computer assisted densitometric image analysis (CADIA). Clinical measurements included gingival recession, PD, clinical attachment level, and mobility. Osseous defect measurements were taken at baseline and at the 1 year re-entry.

R: No adverse healing responses occurred. The results showed that all patients had a statistically significant improvement in probing depth and attachment level at 1 year. Osseous measurements showed bone fill of 2.27 mm (51.6%) for the DFDBA+TCN group, 2.20 mm (52.4%) for the DFDBA group, and 1.27 mm (32.8%) for the D group. Defect resolution was 77.3% for the DFDBA+TCN group, 77.9% for the DFDBA group, and 63.8% for the D group. The mean CADIA values were 5.04 for the DFDBA+TCN group, 6.79 for the DFDBA group and 2.78 for the D group. The CADIA values did not correlate with the clinical parameters. Although the grafted groups showed greater bone fill and defect resolution, there was no statistically significant difference in any of the clinical parameters between the treatment groups.

BL: This study suggests that there is no significant benefit from reconstituting the allograft with 50 mg/ml of tetracycline hydrochloride.

Petri 1993                 NO ARTICLE

P: To present the results of the clinical assessment of antibiotic-supplemented bone allograft (ASBA) in the mandibular third molar site

M&M: ASBA was prepared using DFDBA combined with equal volume of purified gelatin powder (Gelfoam) to which was added 1 mg of cephalothin and 1 mg of tobramycin per cubic centimeter of bone-gelatin mix. Twenty-five patient having bony impacted mandibular third molars included. Split-mouth design: ASBA vs no graft. Absence or presence of osteitis (socket pain within 10 days) or infection (swelling, purulence, need of antibiotic therapy) and radiographic evidence of bone height (at 12 months) and density (at 6 months using densitometer) were evaluated

R: There was no incidence of osteitis in either control or ASBA-treated sites. Four control sites showed evidence of infection, all occurring between 2 and 4 weeks postoperatively. At 6 months, bone density was significantly greater in ASBA-treated sites. At 12 months, bone height was significantly greater in ASBA-treated sites.

BL: The use of ASBA appears to be beneficial to patients having third molar surgical removal.

How do the different materials compare to eachother? Compare to autogenous grafts?


Hiatt 1978                 ARTICLE

Purpose: To evaluate histologically healing dynamics following regenerative procedures.

M&M: Report includes 64 allografts, 15 autografts, 21 non-grafted sites & 6 resorption cases of cases treated over 16 years. 39 graft cases and 21 nongraft cases were removed in block section which included the surrounding periodontium. Also, 40 graft cases where initial repair from grafting was inadequate and was followed by recurrent disease were extracted and sectioned. Postsurgical histologic sections were made at time intervals ranging from 14 days to 81/2 years following surgery.

Results: No adverse immunological reactions could be detected in this study, which involved the placement of human allografts. Histology showed no evidence of cell mediated hypersensitivity. There appeared to be a difference in regenerative repair between graft versus nongraft sites. New bone was seen in 33/39 grafted cases and 7/21 non-grafted. New attachment attempts without grafts were associated with lack of cementogenesis & alveolar bone formation. In contrast, grafted sites (autografts and allografts) demonstrated regeneration of cementum (constant finding), bone and a functionally oriented PDL. Furthermore, only fresh iliac autografts were associated with ankylosis or root resorption.

BL: Findings suggest that grafting enhances osteogenesis & cementogenesis The potential for regeneration of a new attach apparatus including new bone, new cementum & a functionally oriented PDL was demonstrated for both auto & allografts. No adverse immune reaction. Root resorption only noted in cases of fresh iliac crest autograft material.

Listgarten 1979                 ARTICLE

P: To document clinically and histologically the response of human chronic periodontitis lesions treated with curettage and root planing only, or with the use of osseous autografts or allografts with and without root planing.

M&M: 21 pts w/ 25tth that required extractions of premolars or anterior teeth associated with periodontal intraosseous defects, 1-, 2-, 3-wall defects or combination. Photographs, radiographs and mobility were recorded. Flap raised, defect was debrided and the depth of the defect was measured from the base to the alveolar crest (notch on the root surface at the bottom of the defect). The defects were treated as following: 1) curettage and root planing 2) autograft without root planing 3) autograft and root planing 4) allograft without root planing 5) allograft and root planing. Osseous autografts were obtained from adjacent edentulous areas in the same mouth or from tuberosity regions and allografts from the iliac crest. No regular recalls and poor oral hygiene was noticed when patients returned after 6 months to 1 year. Block sections were obtained of the experimental teeth and adjacent alveolar bone and examined.

R:

As compared with non-grafted sites, the grafted sites demonstrated more obvious deposits of new cementum. This was particularly true in the grafted sites which had been planed. Also, grafted sites demonstrated greater bone fill of the defect, whereas non –grafted sites showed no bone fill. At grafted sites the percentage of bone fill ranged from 17 to 61%. Finally, in all groups JE proliferated readily below the alveolar crest, with the epithelium occupying from 52-85% of the distance from the alveolar crest to the base of the original defect.

BL: Bone fill occurs at grafted sites and a long JE is common after treatment.

Mellonig 1981                 ARTICLE

P: to obtain a direct comparison of the bone forming abilities of autogenous osseous coagulum (OC), autogenous bone blend (BB), FDBA, and DFDBA, as determined by the incorporation of a radionuclide, Strontium, into newly formed bone.

M&M: Defects were created in the calvaria of 35 female guinea pigs. The autogenous (BB, OC) graft came from the defect created. Six holes (2.5mm in diameter) were created with round bur in the calvaria of each animal. Osseous coagulum consisted of particulate bony shavings mixed with host’s blood. The remaining autogenous bone shavings were placed in a sterile amalgam capsule with a pestle and triturated for 30 sec to obtain (BB). The allograft materials were obtained from 10 male guinea pigs and processed (FDBA, DFDBA). Both grafts were placed in porous nylon chambers and implanted into the defects. Empty nylon chambers served as the controls. One defect in each animal remained empty and served as a histologic measure of the lesion to heal spontaneously. Strontium chloride was injected intraperitoneally 3 days prior to sacrifice. Animals were killed at 3,7,14,21,28,35 and 42 days. At sacrifice, the nylon chambers and their contents were removed. A small section of the illium was also removed from each animal. An osteogenic index was obtained by dividing cpm/mg for each sample by cpm/mg of ilium. This permitted comparisons to be made among samples with regard to rate of new bone formation evoked as opposed to that which normally occurs in the general skeleton.

R: From days 28-42 the incorporation of Sr. into new bone was greater with DFDBA than the other graft materials or the control. There was little difference in the uptake of Sr between OC and BB. The lowest Sr. incorporation was seen with FDBA and control. The rate of new bone formation in the presence of DFDBA increased very rapidly form day 14 to day 28 and declined thereafter. DFDBA had a more rapid increase in the rate of osteogenesis than OC, BB, FDBA or control.

Disc: results suggested that DFDBA has high osteogenic potential (due to BMPs). Decalcification would release the mineral which insulates the BMP. BMP guides modulation and differentiation of mesenchymal cells into osteoblasts. Agrees with other studies, that new bone formation in the presence of DFDBA began to increase at 14 days w/ peak at 28 days.

BL: DFDBA had the highest osteogenic potential followed by OC and BB, which were about equal to each other, and the least was FDBA which was slightly greater than control group.

Mellonig, 1981(partII)                 ARTICLE

P: To make histologic comparison of new bone formation evoked by DFDBA, FDBA, autogenous osseous coagulum (OC), and autogenous bone blend (BB).

M+M: 35 guinea pigs, according to protocol of part I study- 5 guinea pigs served as donors for the grafts that were placed in defects created in the experimental guinea pigs of same strain. Defects created surgically in calvaria, graft materials were placed in porous nylon chambers and implanted into the defects. Implanted empty nylon chambers served as controls. Animals were sacrificed in groups of 5 at 3, 7, 14, 21, 28, 35, and 42 days. Histo eval was done.

R: Day 3: No new bone was noted by any graft material.

Day 7: only DFDBA showed new bone but was minimal.

All test materials and controls exhibited some degree of new bone formation at day 14 which increased through day 42. The amount of new bone was greater with DFDBA in all time periods. There was only a minimal bone form in the controls.

Mean % new Bone

Days

N

DFDBA

OC

BB

FDBA

Control

3

5

0

0

0

0

0

7

5

.49

0

0

0

0

14

5

19.66

11.04

8. 48

9.71

7.32

21

5

43.66

21.68

21.34

19.43

19.20

28

5

58.31

57.13

42.21

34.52

32.35

35

5

65.06

43.28

43.75

38.98

43.06

42

5

62.14

61.31

42.17

40.34

39.27


BL: DFDBA had the highest osteogenic potential followed by OC and BB, which were about equal to each other. FDBA demonstrated the least osteogenic ability which was slightly greater than control group.

Sanders 1983 (Part III)                 ARTICLE

P: To clinically evaluate and compare FDBA and composite FDBA + autogenous bone graft (ABG) in the treatment of periodontal osseous defects.

M&M: 272 defects in 127 patients were treated with FDBA (100-300µ). 109 defects in 44 patients were treated with combined grafts. Defects were 1, 2, wide 3-wall, combined defects and furcation defects. After initial preparation, clinicians were given opportunity of flap design, SRP, intramarrow penetration (IMP), filling or overfilling defect, closure, dressing, antibitoic regimen and FDBA/ABG of their choice. Radiographs and photos taken before surgery, immediately after and at 1 year re-entry.

Complete bone regeneration or >50% bone regeneration was considered successful.

R: FDBAs : complete bone regen. >50% regen. <50% regen. failed

272 60(22%) 111(41%) 63(23%) 38(14%)

(21 furca)

FDBA/ABGs: complete bn regen >50% regen <50% regen failed

109 36(33%) 51(47%) 12(11%) 10(9%)

(3 furca)

- Successful bone regeneration: FDBA 63% vs. 80% combined graft (SSD)

- Type of defect: combined grafts provided statistically significant improved results than FDBA in 2 wall, combined 1 and 2 walls and in furcation defects.

- >50% pocket reduction: FDBA 65% vs. 83% combined graft (SSD)

- Antobiotics: a significant difference in osseous regeneration was noted when antibiotics were used (85% vs. 38%). (mostly used = erythromycin and tetracycline)

- IMP: associated with higher % of success (66% vs 56%) without IMP. (NSSD)

- Endodontically obturated teeth: defects adjacent to endodontically obturated teeth (total of 18 grafts) showed poorer results when compared to non-endodontically treated teeth(> 50% bone regeneration 33% vs 65%) (SSD).

- Complete wound closure: Complete wound closure showed better success than incomplete closure (> 50% bone regeneration : 66% vs. 39%).(SSD)

BL: When compared with FDBA, composite FDBA/ABG appear to offer significantly improved results in osseous regeneration, PD reduction, treatment of 1, 2-walls, and furcation defects. Complete wound closure and antibiotic coverage enhanced graft success. Endodontically treated teeth (especially with poor obturation) may be a consideration in success or failure of the graft.

Rummelhart 1989                 ARTICLE

Background: The demineralization step of DFDBA preparation is thought to remove the inorganic phase of bone and “expose” BMP, thereby allowing an osseoinductive event where host mesenchymal cells can be differentiated into osteoblasts. Cortical bone may have a greater resident osteogenic potential than cancellous as it has more bone matrix which potentially contains more BMPs.

Purpose: To clinically compare the efficacy of cortical preparations od FDBA and DFDBA in promoting bone repair of human periodontal osseous defects

Materials and methods: 22 osseous defects in nine pts, free of systemic diseases, 31-58 years of age. Initial therapy and clinical measurements (OHI, SRP, occlusal adjustment of needed, PI, PDs, AL, BOP, GI, mobility and standardized radiographs of the defects) were performed.

One defect from each patient was randomly selected to receive FDBA and the remaining defect was grafted with DFDBA. Defects were flapped, SRP to smooth surface, degranulation, intramarrow penetration, placement of graft material, replaced flaps and sutures. Evaluation was made based on standardized radiographs, presurgical and postsurgical soft tissue measurements and osseous measurement were taken at the time of surgery. All sites were re-entered at a minimum of 6 months (mean of 8 months) post surgically.

Results: NSD within both FDBA and DFDBA presurgically in AL, PD, and osseous levels.

Clinical manipulation of the two preparations was similar and was considered excellent. There was a general improvement in both groups. There was visual evidence of continuity of bone within the defect, no evidence of the original particulate graft materials, and sites were firm.

A mean osseous repair of 1.7mm occurred with DFDBA (starting osseous defect 3.3mm) and repair of 2.4mm with FDBA (starting osseous defect 3.7 mm).

55% of the defects were of a one wall configuration.

PDs were reduced by 2.4mm (initial 6.8mm) for DFDBA and by 2.3mm (initial 6.2mm) for the FDBA treated sites.

There was a 59% fill with DFDBA and 66% fill with FDBA, but this difference was not considered statistically significant. There was one defect that showed crestal apposition (DFDBA) and one with no defect fill (FDBA).

DFDBA (11 defects) FDBA (11 defects)

100% repair 2 (18%) 3 (27%)

>50% repair 4 (36%) 7 (64%)

<50% repair 4 (36%) 0 (9%)

Conclusion: 1. FDBA and DFDBA, when placed in predominately one – walled osseous defects, did not display statistically significant differences from each other for any clinical parameter when evaluated at a minimum of 6 months. 2. The use of moth grafts resulted in PD reductions, gingival recession, overall gain in AL and comparable degrees of fill of periodontal bone defects.

Mellonig 1991                 NO ARTICLE

Freeze-dried bone allografts in periodontal reconstructive surgery

Purpose: to discuss the clinical and histological state of the art with regard to undemineralized freeze-dried bone allograft and demineralized freeze-dried bone allograft.

Discussion

FDBA process

  1. Cortical bone is harvested in a sterile manner.

  2. The bone is cut to particle size, ranging from 500m to 5mm.

  3. The material is immersed in 100% ethyl alcohol for 1 hour to remove fat

  4. The bone is frozen at -80 C for 1-2 weeks to interrupt degradation process.

  5. Freeze drying removes more than 95% of water content from the bone

  6. The bone is ground down to particle size 250-750m

  7. The graft material is immersed again in 100% ethyl alcohol

  8. Decalcification with 0.6 N HCL removes calcium, leaves bone matrix, and exposes the bone inductive proteins.

  9. The bone is washed with sodium phosphate buffer to remove residual acid

  10. If the bone is demineralized, it is refreeze dried

  11. Vacuum sealing in glass containers

FDBA

FDBA+Autogenous

Bone fill 50% or greater

67%

78%

PD reduction

69%

79%

FDBA

DFDBA

Conclusion

AAP Position Paper 2001                 NO ARTICLE

Aim is to evaluate current knowledge on bone allograft materials supplied by different bone banks, including knowledge about the efficacy of such material.

Allografts

  1. FDBA-provides an osteoconductive scaffold and elicits resorption when implanted in mesenchymal tissues

  2. DFDBA- also provides osteoconductive surface, in addition to providing osteoinductive factors. Elicitis mesenchymal cell migration, attachment, and osteogenesis when in implanted in well-vascularized bone. It induces endochondral bone formation when implanted into tissues that would otherwise not form bone.

Bone banks adhere to the guidelines of the American Association of Tissue Banks (AATB) with respect to procurement, processing, and sterilization of bone grafts. The AATB advocates excluding collection of bone under the following circumstances:

  1. Donors from high risk- groups, as determined by medical testing and/or behavioral risk assessments.

  2. Donors testing positive for HIV antibody by ELISA.

  3. Autopsy of donor reveals occult disease

  4. Donor bone tests positive for bacterial contamination

  5. Donor and bone test positive for Hep B surface antigen (HBsAG) or Hep C virus (HCV).

  6. Donor tests positive for syphilis

There have been no reports of virus contamination or acquired pathology from DFDBA. Although the banks do not sterilize bone allografts, they do collect and process the bone under sterile conditions. They use ETO (ethylene oxide) and radiation but sometimes may not report this fact on the package insert or labeling. Studies examining the effect of ETO on the ability of DFDBA to induce bone have shows that it can decrease the effectiveness and resorption of the allograft. Some of this may be due to inadequate removal of residuals formed during the sterilization process, or to exposure of the bone graft to temperatures that cause protein denaturation. Thus, sterilization processing may be an important contributor to the variability in DFDBA’s osteoinductive properties. Irradiated bone has been shown to support normal healing, however irradiation of DFDBA reduced bone induction ability by 40% (Zhang et al 1997).

It was found that wide variations in commercial bone bank preparations of DFDBA do exist, including the ability to induce new bone formation, even within the same bank. Which may explain the variability in the clinical response when using FDBA. Clinician must be aware that the donor age may be important. Even though preparations of DFDBA may not be osteoinductive, they may still have potential as a carrier for bioactive components of known activity, like bone morphogenic protein (BMP). When used in particulate form, particle size appears as an important variable in the success of DFDBA as a bone-inductive material.

Particles in the range of 125 to 1,000 microns possess a higher osteogenic potential than do particles below 125 microns. Optimal particle size is between 100-300 microns, this may be due to a combination of surface area and packing density. Very small DFDBA particles elicit a macrophage response and are rapidly resorbed with little or no new bone formation.

Mellonig 1996                 ARTICLE

P: Review bone allografts in periodontal therapy

D: FDBA

- 89 clinicians implanted a total of 997 periodontal defects with FDBA alone and 524 defects with FDBA + Autogenous bone. Surgical reentry and radiographic evaluation at 1 year after surgery were evaluated for predictability in 329 sites with FDBA and 176 sites with FDBA + Autogenous bone. Complete or >50% bone fill was obtained in 67% of FDBA and 78% with FDBA + Autogenous bone

- Animal study has shown that it is safe to use: less humoral and cell-mediated immune response to FDBA in comparison with fresh bone allograft

DFDBA

- Overall bone fill 65%

- Bowers showed significant more new cementum, new PDL, and new bone formed in the bone defects grafted with DFDBA than in non-grafted defects

FDBA vs DFDBA

- Studied in 11 paired periodontal osseous defects. No difference in percent of defect resolution was reported

Allografts vs Synthetic bone grafts

- Some studies suggest only a moderate difference in favor of the allografts when post-treatment clinical parameters are compared

- Major difference is in the histologic results. Allografts heal by regeneration of the periodontium, whereas grafts of synthetic bone heal by encapsulation of the graft particles by CNT

GTR

- A number of case reports indicate that the combination of DFDBA and physical barrier enhances bone fill when compared to physical barrier or DFDBA alone, however some studies did not show any differences

- Long-term (5 years) results of GTR used alone or in combination with a bone graft indicate that the success of GTR is enhanced significantly by the addition of bone graft

GBR

- Both FDBA and DFDBA are used as support for the physical barrier and to provide either a lattice network for osteoconduction, or bone inductive proteins for osteoinduction

- Numerous case reports support the use of FDBA and DFDBA allografts in conjunction with GBR for treatment of dehiscence/fenestration defects at the implant interface

Safety

- The risk of disease transmission has been calculated to be in the range of 1 in 2 million to 1 in 8 million

- Demineralization and treatment with a virucidal agent inactivates HIV in infected bone

Wang HL 2005 Position paper                 ARTICLE

Several types of bone allografts: iliac cancellous bone and marrow, freeze-dried bone allografts, and decalcified freeze-dried bone allografts.

Controlled clinical trials: indicate bone fill ranging from 1.3 to 2.6 mm when freeze-dried bone allografts (FDBA) were used to treat periodontal defects. Human trials: using cortical DFDBA have demonstrated bone fill similar to that achieved with FDBA, ranging from 1.7 to 2.9 mm.

FDBA+ TTC: treating intraosseous defects resulting from juvenile periodontitis.

Reynolds MA et al, 2003 (systematic review): superior gains in bone fill with DFDBA compared to open flap debridement procedures.

Controlled human histologic studies: Regeneration achieved with the grafts was significantly more than that in non-grafted controls.

Pearson GE, 1981: Grafts using decalcified freeze-dried cancellous bone91 have shown less bone fill (mean 1.4 mm). This variation may reflect differences in the amount of bone-inductive proteins in the two tissues ( Urist MR et al; 1973)

Specific molecules with osteogenic activity have been identified. Increased research has been done on delivery systems for these molecules and on the potential for viral transmission. Research has also been done on variability in biological activity associated with human bone. These developments have resulted in an increased focus on developing regenerative therapies using recombinant osteogenic factors in appropriate delivery systems.

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