Bone allografts in periodontal therapy

Introduction

An allograft is tissue transferred from a donor to a recipient of the same species but of non-identical genetic composition. The need for an allogeneic source of bone arose because of the surgical requirements for reconstructing large or multiple osseous defects that could not be fulfilled by an autogenous source (^{Mellonig et al., 1976}). Other problems associated with autogenous bone, namely, morbidity accompanying a secondary surgical site for procurement and the possibility of root resorption stimulated the search for an alternative source of bone in periodontics (^{Dragoo and Sullivan, 1973}).

It is often reported that autogenous bone provides the best post-bone grafting results (^{Khan et al., 2000}). This is based on the belief that all of the cells within the autogenous bone graft survive the transplantation process. However, unless the osteocytes are within 1,0 mm or so of a vascular source all cells within the autogenous bone graft will die (^{Schenk et al.,1994}). ^{Heiple et al. (1963)} compared autogenous bone with different types of processed allografts, including mineralized freeze-dried bone allografts (FDBA). One hundred and fifty-two dogs were used to measure all levels of wound healing from several days to one year. They concluded that other than early wound healing at two weeks, there was no difference between autogenous bone and FDBA. Similar results were also demonstrated in radionuclide studies (^{Mellonig et al., 1981a} ; ^{Mellonig et al., 1981b} ; ^{Solheim et al., 2001}).

There are several types of bone allografts used in medicine and dentistry : fresh frozen bone allograft, mineralized freeze-dried bone allograft, and decalcified freeze-dried bone allograft (DFDBA). Fresh frozen bone allograft and FDBA are frequently used in medicine for spinal fusion (^{Ehrler and Vaccaro, 2000}), for craniofacial reconstruction (^{Neigel and Ruzicka, 1996}), and non-union fractures (^{Rosenthal et al., 1999}). Dentistry and in particular periodontics almost exclusively use FDBA or DFDBA as a substitute for autogenous bone. The reader is referred to the following reviews for an in depth discussion of different types of bone replacement grafts : ^{Mellonig et al., 1992} ; ^{Garret, 1996} ; ^{Reynolds and Bowers, 1996} ; ^{Nasr et al., 1999} ; ^{Costantino et al., 2002}.

Procurement of allogeneic bone

Over 500 000 allogeneic bone graft procedures are done annually in the USA. Approximately 100 000 of this number are used in dentistry. The first step in the procurement of allogeneic bone involves the selection of an acceptable donor. The development of exclusionary criteria for donor procurement has provided a significant degree of safety for the transplantation of disease-free allografts of all types. It is calculated that the chance of obtaining a fresh bone allograft containing HIV from a seropositive infected donor, who failed to be excluded by one of the below techniques, is one in 1,67 million (^{Buck et al., 1989}). If fresh frozen the risk is 1 in 8 million (^{Buck and Malinin, 1994}) :

- omission of donors from high-risk groups by medical and social screening. Unless reliable information regarding previous hospitalizations, blood transfusions, serious illnesses, and life-style can be ascertained, the donor must be regarded as unacceptable ;

- HIV antibody and antigen testing ;

- autopsy or biopsies to rule out occult disease, such as carcinoma ;

- special lymph node studies beyond those usually performed at autopsy. Such studies are performed to recognize changes characteristic of early HIV infection and provide another opportunity to exclude individuals with morphologic nodal changes typical of nonspecific infection (e.g., bacterial, viral, parasitic, or fungal, and chronic infection or drug abuse) ;

- blood cultures for bacterial contamination ;

- serologic tests for syphilis and all types of hepatitis ;

- follow-up studies of grafts from the same donor. A third of the bone donors are concomitant donors of vital organs such as heart, kidneys, and liver. With the exception of fresh bone allografts, months usually pass between procurement and the clinical use of a frozen or processed bone allograft. If a vital organ recipient were to be identified as having HIV or another related illness, the bone from the donor would not be released for clinical use.

In order to ensure sterile bone, bone allografts are procured in a sterile manner usually within 12 hours of death of the donor. After 12 hours, there is a significant increase in the incidence of bacterial contamination. If bacterially contaminated, the bone is subjected to secondary means of sterilization. The gender of the donor appears to make no difference in the quality of the allograft but age does (^{Schwartz et al., 1998}). Donors younger than 50 years of age are preferred. Irradiation and ethylene oxide are currently used as end stage sterilizing agents. The use of irradiation is controversial (^{Wientroub and Reddi, 1988} ; ^{Munting et al., 1988} ; ^{Conway et al., 1991}). However, it is generally agreed that doses above 2,0 to 2,5 mega rads of gamma irradiation is destructive to new bone formation (^{Forsell, 1993}). Ethylene oxide, a powerful alkylating agent, will have significant deleterious effects on bone induction (^{Zislis et al., 1989}). An ethylene oxide sterilization procedure of sufficient dose to kill spores will render a bone allograft incapable of inducing new bone formation. Furthermore, residual levels of ethylene oxide will cause morphologic changes in fibroblasts (^{Zislis et al., 1989}).

Processing of allogeneic bone

Although the processing of a freeze-dried bone allograft will vary from tissue bank to tissue bank, it will usually include the following steps :

- cortical bone is harvested in a sterile manner. Long bones are the source for mineralized freeze-dried bone allograft (FDBA) and demineralized or decalcified freeze-dried bone allograft (DFDBA). The femoral cortical bone is harvested because it has been found to be less antigenic than cancellous bone (^{Quattlebaum et al., 1988}). Since the bone inductive proteins are located in the bone matrix (^{Urist et al., 1970}) and since cortical bone has more bone matrix than cancellous bone, cortical bone is the material of choice for dental bone allografts ;

- all soft tissue is removed from the bones. The cortical bone is rough cut to a particle size ranging from 500 µm to 5 mm and subjected to repeated washings to remove the bone marrow. This fragmentation increases the efficiency of defatting the cortical bone and subsequent decalcification, if the allograft is to be demineralized ;

- the graft is then immersed in 100 % ethanol for one hour to remove all fat. Fat inhibits osteogenesis (^{Aspenberg and Thoren, 1990}). Viral activity is undetectable within one minute following treatment with 70 % ethyl alcohol (^{Resnick et al.,1986}). This process is usually repeated at various stages during processing ;

- the bones are frozen in liquid nitrogen at - 70 °C for one to two weeks to interrupt the degradation process. During this time, the results from bacterial cultures, serologic tests, and antibody and antigen tests are analyzed. If contamination is found, the bones are discarded or more likely sterilized by irradiation or ethylene oxide ;

- sterile graft material free from infectious agents is now freeze-dried. Freeze-drying is a process in which dehydration is achieved by removing water directly from the frozen state to vapor state and bypassing the liquid phase. This process takes place in a vacuum and is termed sublimation. Freeze-drying removes more than 95 % of the moisture content from the bone. Although freeze-drying kills all cells (the bone lacunae become empty spaces), it has the advantage of facilitating long-term storage and markedly reducing antigenicity. Patients who received multiple periodontal grafts did not develop any anti-HLA antibodies when evaluated by sensitive microcytotoxicity assays (^{Quattlebaum et al., 1988}). However, if the patient developed anti-HLA antibodies from either FDBA or DFDBA, the presence of antibodies does not preclude a satisfactory outcome (^{Friedlaender et al., 1999}) ;

- the cortical bone is ground and sieved to a particle size of approximately 250 to 750 µm. Particle sizes within this range have been shown to promote osteogenesis, whereas particles below 125 µm are quickly engulfed by multinucleated foreign body giant cells (^{Mellonig and Levey, 1984}) ;

- if the bone allograft is to be decalcified, it is immersed in 0,6 N HCl. Demineralization removes the calcium and exposes the bone inductive proteins. This step is not necessary if FDBA is the desired end product. Cortical FDBA has the same amount of bone inductive proteins as DFDBA but the calcium must first be biologically removed over a long period of time before induction can take place. DFDBA forms new bone by osteoinduction (^{Urist and Strates, 1971}). Thus new bone is induced by stimulating the differentiation of host mesenchymal or stem cells into bone forming cells. FDBA forms bone by osteoconduction. The graft acts as a lattice network and passively assists the host in forming new bone ;

- the bone is washed in a sodium phosphate buffer to remove residual acid. Repeated washing with various solutions is frequently done throughout the processing ;

- if the bone is demineralized, it is re-freeze-dried. Some banks arrange the processing steps so that only a single freeze-drying cycle is necessary ;

- vacuum sealing in glass containers protects against contamination and degradation of the material while permitting storage at room temperature for an indefinite period of time.

Safety of bone allografts

The possible transmission of infectious diseases by the transplantation of contaminated allogeneic tissue is an overriding concern for some clinicians and patients. Viral, bacterial and fungal infections have been transmitted via transplantation of allografts such as bone, skin, cornea, heart valves, whole organs, blood and semen (^{Gottesdiener, 1989} ; ^{Kayaiga et al., 1991}). Human immunodeficiency virus (HIV), hepatitis B virus (HBV), hepatitis C virus (HCV), bacterial infection, and the agent, or « prion », responsible for Creutzfeldt-Jakob Disease (CJD) are the most widely discussed, and forms the basis for concern in the dental and medical communities (^{Eastlund, 1995}). Over the past decades, improvements in donor screening criteria such as excluding potential donors with infection and those with behaviors risky for HIV-1 and hepatitis infection and the introduction of new donor blood tests have greatly reduced the risk of HIV, hepatitis, and have eliminated the risk of CJD. The transfer of disease from freeze-dried bone allografts is of particular concern. Yet, unfounded concerns and skepticism persist.

Laboratory tests for the detection of the AIDS virus are mandatory for the screening of bone allograft donors. An enzyme-linked immunosorbant assay (ELISA) has been used as the standard test to detect the presence of HIV antibody (^{Tomford, 1993a}). The major disadvantage to the ELISA test is that there may be a prolonged period of time between infection with the virus and seroconversion with subsequent development of antibodies to HIV. Antibody to HIV forms in most persons infected with HIV within six months (^{Sandler et al., 1990}). Most individuals will seroconvert within six weeks from time of inoculation. Based on blood bank data, the chance of a patient having a negative ELISA antibody test yet being an HIV viral carrier has been estimated at 1/40 000 to 1/153 000 (^{Cummings et al., 1989} ; ^{Ward et al., 1988}). An additional blood test that is available as a screen for the AIDS virus is the HIV antigen (p24) test. This test is sometimes used as an adjunct to the ELISA antibody test (^{Tomford, 1993a}). Another test that is now used for AIDS screening is based on the polymerase chain reaction (PCR) technology. PCR tests amplify portions of genetic make up of HIV, thereby making the virus more easily detectable (^{Eisenstein, 1990}). The use of the PCR test is extremely accurate in the diagnosis of patients who are HIV carriers and decreases the window of vulnerability to one week or less (^{Wolinsky et al., 1989}). PCR provides the most reliable screen possible.

There are four known cases of HIV transmission from a bone allograft donor. All of these involved unprocessed fresh frozen bone. Fresh frozen bone allografts are not used in dentistry. The first case involved a 23-year old, married female with no risk factors for HIV infection. She underwent a spinal fusion procedure in 1984 using a fresh frozen femoral allograft with no interposed secondary sterilization or chemical treatment. The bone allograft was procured from a 52-year old male undergoing hip arthroplasty secondary to a degenerative joint disease. At the time of surgery, there was a history of the excision of a neck « cyst » and cervical lymphadenopathy was noted. A subsequent history of intravenous drug use was obtained. The donor eventually developed Pneumocystis carinii pneumonia and was diagnosed with AIDS. The recipient of the fresh frozen bone allograft, following a protracted series of episodes of fever, lymphadenopathy, and pulmonary disturbances, was diagnosed with HIV infection. Today, a medical history and lymph node biopsy would have ruled this patient out as a suitable donor.

The other three cases received bone from a single donor (^{Simonds et al., 1992}). The donor was a 22-year old male who died in 1985 after receiving a gunshot wound to the head. This donor had no known risk factors for HIV and tested twice negative for HIV antibody. PCR testing and HIV antigen tests were not available at this time. Retrospectively, this donor was within the « window of vulnerability » or the time from infection to seroconversion. Four organs and 54 other tissues were distributed from the donor. Four recipients of the solid organs all became infected with HIV. Three of the four recipients of fresh frozen bone seroconverted. The fourth received a fresh frozen bone allograft washed free of marrow. Thirty-eight mineralized freeze-dried bone allografts treated with ethanol were distributed to 30 recipients (all non-dental patients). These recipients continue to remain seronegative. Other recipients of dura mater and cornea are also seronegative.

Both FDBA and DFDBA are viewed as posing less risk for viral transmission than large, unprocessed fresh frozen osteochondral allografts. This perceived reduction in risk is attributed to an expectation that the chemical agents, such as ethanol and hydrochloric acid, used in the processing produce a virucidal effect on tissue borne HIV. The ability of chemical agents to inactivate HIV under clinical and laboratory conditions is well known (^{Aspenberg and Thoren, 1990}). Exposure of 7 to 10 ogs of infectious dose of virus to alcohol at 70 % concentration inactivated HIV below detectable levels within one minute (^{Resnick et al., 1986}). During processing, the allogeneic bone is defated by soaking in a bath containing 100 % ethanol for one hour. It has been demonstrated that ethanol will completely penetrate through cortical bone (5,5 cm x 2,5 cm) within 15 minutes following introduction of this virucidal agent (^{Prewett et al., 1991}). Further evidence of the effectiveness of chemical decontamination is provided by the implantation of the simian immunodeficiency virus (SIV) contaminated bone either processed with ethanol or frozen (^{Cook et al., 1995}). Monkeys receiving non-processed frozen bone allograft, all tested positive for SIV within two weeks. No animal receiving ethanol treated bone became infected with SIV. Furthermore, exposure of HIV to a low pH such as that obtained with hydrochloric acid will inactivate the virus (^{Ongradi et al., 1990}). Strongly acidic solutions have been shown to disrupt the phosphodiester bonds of nucleic acids (^{Vodicka and Hemminki, 1988}). It has been demonstrated that HIV, hepatitis B and C virus, cytomegalic virus, and the polio virus can all be inactivated by the demineralization process used in DFDBA. Reports indicated that freezing and freeze-drying (^{Quinnan et al., 1986}) may also result in a reduction of infectivity of HIV.

Additional evidence that FDBA or DFDBA is safe for human implantation is provided by a study in which bone free from HIV infection and bone obtained from a donor who died of AIDS was evaluated after processing and freeze-drying (^{Mellonig et al., 1992}). Both spiked and infected bone was treated with a virucidal agent and demineralization. Replication of viable HIV could not be demonstrated after treatment. Even if an HIV infected donor could escape detection by the various exclusionary techniques, processing a bone allograft with a virucidal agent such as ethanol, hydrochloric acid, or other suitable chemical agents, will render the processed bone allograft safe for human implantation. The probability that a particular brand of DFDBA contains HIV has been calculated to be one chance in 2,8 billion. Hepatitis A is spread by person-to-person contact and it is highly unlikely to be transmitted in tissues (^{Tomford, 1993a} and ^1993b). In addition, hepatitis A is a mild subclinical illness and is unlikely to result in a chronic carrier state. Patients who have a history of hepatitis A are eliminated as allograft donors. Hepatitis B is almost always transmitted by blood and blood products. Screening for hepatitis B is mandatory. The standard test for hepatitis B is hepatitis B surface antigen (HBsAG). Like HIV there is a « window » of approximately six weeks between infection and antibody development. However, combined with the fact that acute hepatitis B infection is an easily recognizable condition, screening of donors by blood testing history, and physical examination provides a very sensitive evaluation. Ethanol soaks are effective in inactivating hepatitis B virus (^{Kobayashi et al., 1984}). Since 1954, there have no reported cases of hepatitis transmission by a bone allograft. Hepatitis C (HVC) or hepatitis non-A non-B is the chief cause of postransfusion hepatitis (^{Tomford, 1993a}). Screening for HVC is mandatory. The standard test is antibody to hepatitis C virus (anti-HCV). The introduction of PCR in testing for HCV has improved screening methods (^{Cha et al., 1991}). Transmissible HVC has not been detected in any patient receiving a FDBA or DFDBA.

Creutzfeldt-Jakob Disease (CJD) is a slow progressive encephelopathy. It is caused by a prion, a novel self-replication protein, and is the smallest known infectious particle (^{Prusiner et al., 1990}). In the United States and Europe, the estimated crude annual incidence is one case in one million (^{Brown et al., 1992}). The principle routes of disease transmission are through infected neurological tissue such as dura mater, pituitary growth hormone, and cornea (^{Gottesdiener, 1989}). Bone is not known to have ever transmitted CJD and is classified by the World Health Organization as type IV or no infectivity possible. Excluding donors with a past medical history of neurological disease has eliminated the risk of CJD transmission from bone allografts.

Syphilis, caused by the organism Treponema pallidum, is not known to be transmissible in bone or soft tissue. The causative agent is sensitive to antibiotics, heat, and drying. Tissue banks test for syphilis mostly because blood banks have historically tested for syphilis (^{Tomford, 1993b}). The tests generally used include the rapid plasma reagin (RPR), the venereal disease research laboratory (VDRL), and the fluorescent treponemal antibody absorption test (FTA-ABS).

Bacterial infection of 7 % was reported in a series of 303 applications of small freeze-dried bone allografts (^{Tomford et al., 1981}). Twelve of the 21 infections were minor infections characterized by local wound erythema. Therefore, the overall incidence was 3,6 %. The most common isolated organism was Staphylococcus epidermidis. Cultures from only 1/11 patient's who had positive wound cultures showed the same organism that had been cultured postoperatively from the allograft. The 3,6 % infection rate compares favorable with the 3,9 % infection rate reported for autogenous bone grafts (^{Cruse, 1975}).

Clinical studies with FDBA and DFDBA

The objectives of the clinician seeking to regenerate lost periodontal tissues are :

- probing depth reduction ;

- clinical attachment gain ;

- bone fill of the defect as observed radiographically or on re-entry ;

- regeneration of new bone, cementum, and periodontal ligament.

In order for a bone replacement graft to be a viable addition to the armentarium of the clinician, the bone replacement graft must fulfill all of these objectives.

Mineralized freeze-dried bone allograft was introduced to the periodontal community in 1976 (^{Mellonig et al., 1976}). This field test study evaluated FDBA for the treatment of bone defects associated with chronic periodontitis. Eighty-nine clinicians implanted FDBA a total of 1 887 sites. Enough information was retrieved to make some analysis of clinical out-comes in 997 sites treated with FDBA alone or FDBA plus autogenous bone (FDBA + A). Sufficient data as determined by surgical reentry at one-year post grafting was documented to determine predictability in 329 sites treated with FDBA and 176 sites treated with FDBA + A. Complete or greater than 50 % bone fill was obtained in 67 % of sites treated with FDBA and in 78 % of sites treated with FDBA + A. Results for probing depth reduction were similar. FDBA + A appears to be more effective for the treatment of furcation invasions (^{Sanders and Thomas, 1978}). A composite of FDBA and tetracycline in a 4/1 ratio of bone to tetracycline has shown impressive results for the treatment of osseous defects associated with localized chronic periodontitis formerly classified as localized juvenile periodontitis (^{Evans et al., 1989}).

Urist and coworkers over a period of several years demonstrated that demineralization of a cortical bone graft greatly enhances it osteogenic potential (^{Urist et al., 1967} ; ^{Urist et al., 1975}). The work of Urist has been confirmed by others (^{Chalmers et al., 1975} ; ^{Mellonig et al., 1981a} ; ^{Mellonig et al., 1981b}). Demineralization removes calcium and exposes the bone inductive proteins located in the bone matrix (^{Urist and Strates, 1970}). These bone inductive proteins are collectively referred to as bone morphogenetic protein (BMP) (^{Urist and Strates, 1971}). They are composed of a group of polypeptides that have been cloned and sequenced (^{Wozney et al., 1988} ; ^{Wozney, 1998}). The biologic activity of human recombinant BMP is one tenth that of purified BMP derived from human bone matrix (^{Bessho et al., 1999}). The BMPs are well conserved, as there appears to be homology between mammalian species (Sampath and Reddi, 1983). BMP in DFDBA stimulates the formation of new bone by osteoinduction (^{Urist et al., 1970}). FDBA has the same amount of BMP as DFDBA but the mineralized allograft has to first be biologically demineralized to expose the bone inductive proteins and therefore heals by osteoconduction (^{Croteau et al., 1999}). The sequence of bone induction with DFDBA is a cascade. At day 1, there is chemotaxis of undifferentiated fibroblasts and cell attachment to the bone matrix of the DFDBA graft particle. At day 5, there is continued cell proliferation and differentiation of the bone forming cells. The first few days after grafting are critical as the majority of the effect of the BMPs is between 3 and 7 days (^{Okubo et al., 2002}). At day 7, cells synthesize and secrete bone matrix. From day 10 to 12, there is continued vascular invasion, differentiation, mineralization, and bone formation. The end product of grafting a periodontal osseous defect with DFDBA is cortical and cancellous bone and marrow (^{Mellonig et al., 1981b}). Clinically, this is compatible with a type 3 or type 4 bone as described by ^{Brånemark (1984)}.

^{Libin et al. (1975)} was the first to report on the periodontal use of cortical and cancellous DFDBA in humans. The three grafted sites responded with 4-10 mm of new bone formation. Particulate cortical DFDBA was evaluated in 27 intraosseous periodontal defects and yielded a mean of 2,4 mm of bone fill as determined at 6-month surgical re-entry procedure (^{Quintero et al., 1982}). In six cases, ^{Werbitt (1987)} showed fill ranging from 75 to 95 % of the original defect. The results of a radiographic analysis of cancellous DFDBA in 16 patients demonstrated a mean bone fill of 1,38 mm, whereas in control sites showed 0,33 mm (^{Pearson et al., 1981}). Another controlled study in 47 periodontal osseous defects demonstrated a mean bone fill of 2,6 mm (65 % defect fill) in sites treated with cortical DFDBA. This was compared to the 1,3 mm (38 % defect fill) in sties treated without DFDBA (^{Mellonig, 1984}). ^{Rummelhart et al. (1989)} compared DFDBA and FDBA in 11 patients with paired sites. No statistical difference in probing depth reduction, clinical attachment gain, or bone fill was reported. This may be reflective of insufficient inductive proteins in the batch of DFDBA that was used or in the types and depths of the grafted lesions (^{Schwartz et al., 1996}).

Human histology

Regeneration of new bone, cementum, and a periodontal ligament on a root surface previously contaminated by bacterial plaque is the ultimate goal and challenge in periodontics. Human histologic analysis is the gold standard for determining the ability of any bone replacement graft material to regenerate the periodontium. A bone replacement graft cannot truly be called a regenerative bone product unless there is clear evidence of probing depth reduction, clinical attachment gain, bone, and regeneration. Regeneration can only be determined through an analysis of human histology. Currently, the most scientifically valid proof of periodontal regeneration is a notch placed in the base of calculus prior to root planing at the time of surgery (^{Bowers et al. (1989)}). Using the histologic marker of a notch in calculus, periodontal regeneration has been identified for autogenous bone (^{Froum et al., 1983}), DFDBA (^{Bowers et al. (1989)}), and anorganic bovine bone (^{Mellonig, 2000}).

In a double blinded, histologic randomized controlled study, ^{Bowers et al. (1989)} presented compelling evidence for the use of DFDBA to treat periodontal bone loss. They compared the healing of intraosseous defects with and without DFDBA. The most apical level of calculus on the root surface served as the histologic reference point to delineate root surfaces exposed to bacterial plaque. Data from 12 patients with 32 grafted and 25 nongrafted defects were analyzed. Block sections were obtained after 6 months of healing. Results indicated that in nongrafted defects, a long junctional epithelium formed along the entire length of the exposed root surface and often apical to the calculus reference notch. The formation of new bone, cementum, and a periodontal ligament was observed when the bony defects were grafted with DFDBA. The periodontal ligament was more frequently oriented perpendicular to the root. Root resorption and ankylosis were not observed in either grafted or nongrafted sites. A later study by ^{Bowers et al. (1991)} investigated the combination of DFDBA with BMP3 to determine if the composite graft would enhance regeneration in intraosseous defects. The most apical level of calculus on the root served as the histologic reference point for histometric measurements. Biopsies (en bloc) obtained from 6 patients with 50 intraosseous defects were analyzed. Mean results indicated that the combination of DFDBA and BMP3 enhanced regeneration when compared to DFDBA alone. The above studies provide the highest evidence available for the use of any bone replacement graft material.

Freeze-dried bone allografts compared to alloplasts

Both FDBA and DFDBA have been compared to porous particulate hydroxyapatite (PHA), a synthetic or alloplastic graft material (^{Barnett et al., 1989} ; ^{Bowen et al., 1989}). These studies suggest that there is little clinical difference in post-treatment clinical parameters between FDBA, DFDBA and PHA. DFDBA has been compared to bioactive glass in 15 patients with paired intraosseous defects. The use of DFDBA resulted in 2,8 mm of bone fill (63 %) while bioactive glass treated sites resulted in 2,7 mm of bone fill (62 %). While there appears to be little difference in clinical results between freeze-dried bone allograft and synthetic bone graft materials, there is no evidence that any of the synthetic materials result in periodontal regeneration. All of the available histologic evidence indicates that synthetic grafts heal by encapsulation of the graft particles in connective tissue with the formation of a long junctional epithelium (^{Stahl and Froum, 1986} ; ^{Stahl and Froum, 1987} ; ^{Nevins et al., 2000}).

Guided tissue regeneration and DFDBA

Various studies have suggested that a new periodontium is possible if gingival epithelium and connective tissue associated with the soft tissue flap is excluded by a physical barrier from the healing process (^{Nyman et al., 1982}). This allows cells of the periodontal ligament to repopulate the wound. This surgical procedure is called guided tissue regeneration (GTR) (^{Gottlow et al., 1986}). Human histology of the GTR technique indicates that healing is not by regeneration but by new attachment (^{Gottlow et al., 1986}), that is, new cementum and new connective tissue. Thus the combination of DFDBA and GTR is attractive from both a histological and clinical viewpoint, if periodontal regeneration is the desired outcome.

^{Guillemin et al. (1993)} evaluated the use of DFDBA in conjunction with an expanded polytetrafluroethylene (ePTFE) physical barrier specifically designed for the treatment of interproximal intraosseous defects. Fifteen patients with bilateral bone defects participated. Treatment consisted of DFDBA plus ePTFE or DFDBA alone. Both groups showed statistically significant changes compared to baseline but no difference between groups. A companion study using the same materials and methods compared DFDBA plus an ePTFE barrier to the ePTFE barrier alone (^{Gouldin et al., 1996}). This study likewise found no difference between treatments. Similar probing depth reduction, clinical attachment level gains, bone fill, percent bone fill, and defect resolution were found. While these randomized controlled studies suggest that the clinician may use either GTR or DFDBA or the combination treatment, case reports suggest that one-and two-walled defects are best treated with the combination of GTR and DFDBA (^{McGuire, 1992} ; ^{Uyeda et al., 1994} ; ^{Rosen et al., 2000}). Human histology also demonstrates that the combination of DFDBA and a physical barrier in the treatment of intraosseous defects results in regeneration of new bone, cementum, and periodontal ligament (^{Stahl and Froum, 1991} ; ^{Harris, 2000}).

One of the biggest challenges for the clinician that treats periodontal diseases is furcation involvement. The potential of DFDBA combined with a physical barrier in the treatment of molar furcations was studied by ^{Anderegg et al. (1991)}. Fifteen pairs of class II furcation defects in 15 patients comprised the study group. Six months post-treatment, soft and hard tissue measurements were made and compared to baseline. Probing depth reduction and clinical attachment level gain was significantly greater for the combination treatment versus the barrier alone. Likewise, both horizontal and vertical bone fill of the furcation defect was significantly greater with the combination treatment. Furthermore, both long-and short-term clinical case reports favor the use of the combination therapy for furcations (^{McClain and Schallhorn, 2000}). Human histology is also available. Three molars with class II furcation involvement were treated by a combination of DFDBA and a resorbable barrier (^{Harris, 1999}). Histologic evaluation found regeneration in two of the three samples as determined with the use of a calculus reference notch.

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