Background
Demineralized dentin matrix (DDM) is one of the most acid-insoluble collagenous scaffolds containing minerals and noncollagenous proteins (NCPs) such as bone morphogenetic protein (BMP) and is now commonly used as a bone-graft substitute. Experimental and clinical studies have clearly documented the osteoinductive properties of DDM and its value as an alternative to autogenous bone graft [
1,
2]. In addition to the development of autogenous DDM, several in vivo experimental studies on allogenic DDM have been performed and showed promising results for bone repair without any immunologic responses hindering the osteoinductive and osteoconductive capacities of DDM. Subsequently, several clinical studies have been conducted to evaluate the clinical efficacy and safety of allogenic DDM between biological family members [
3,
4].
However, in addition to the biologic aspects of allogenic DDM applications, the single largest potential disadvantage might be the risk of transmission of viral disease as has been seen throughout the developmental history of Bone Bank [
5,
6]. Even though the biologic use of allogenic DDM has been supported by several experimental and clinical studies, certain viral diseases, such as those caused by human immunodeficiency virus (HIV) and hepatitis B and C viruses may be transmitted through the implantation of human dentin-based products derived from infected donors.
The processing of bone to produce demineralized bone matrix (DBM) entails conditions harsh enough to achieve significant levels of viral inactivation. This allows the pooling process of samples to enhance product quality and effectiveness without increasing, and perhaps even decreasing, the risk of viral transmission [
7]. For using DDM as an allogenic bone-graft substitute, the risk of viral-disease transmission should be reduced or eliminated. The processing procedure for producing DDM includes washing, defatting, demineralization, freeze-drying, and sterilization, which removes unwanted materials from the extracted tooth, such as fat, antigens, and inactivated pathogens, while preserving the valuable minerals, collagen matrix, and non-collagenous proteins leading to rapid bone regeneration and bone remodeling [
8].
An issue for using DDM as an allogenic bone-graft substitute is whether the allogenic dentin from infected patients could be a carrier for transmissible viral diseases and if there is a potential risk of viral transmission, would the processing procedure reduce or eliminate the risk of transmission. In the current study, hepatitis B virus (HBV) was chosen as the target virus since it is the most prevalent viral infection in the Korean population at 3.7%, is present in saliva, semen, vaginal secretions and serum of infected patients, and since even with careful donor-screening and testing procedures in dental clinics, it is still impossible to be completely sure that an extracted tooth is free of viral contamination [
9,
10]. This study was aimed at evaluating viral inactivation by the procedure used to process DDM by measuring HBV DNA in dentin isolated from infected patients both before and after processing. The hypothesis was that dentin obtained from HBV-infected patients could be a carrier of viable HBV to graft recipients through the transplantation and that the processing procedure to generate DDM could completely inactivate or eliminate any HBV present in the dentin matrix.
Discussion
The purpose of the current study was to evaluate the ability of a processing procedure for the generation of DDM to inactivate or eliminate HBV in dentin obtained from chronically infected patients by comparing the HBV DNA levels in processed dentin with those in fresh dentin. It was our hypothesis that the processing procedure would degrade nucleic acids, inactivating HBV and rendering it noninfectious. The viral burden in fresh dentin might be a primary factor for determining the processing method to prevent HBV transmission since the removal of HBV DNA in processed dentin might assure the safety for the allogenic application of dentin. Validation of viral inactivation by the processing procedure could lead to the expanded application of allogenic DDM in the dental field.
Based on the results of the current study, dentin may be a potential carrier of HBV since 55.6% (10/18) of the fresh dentin samples that were stored refrigerated without removal of blood, saliva, or other foreign bodies at the dental clinic tested positive for HBV DNA by qPCR.
Eight of the processed dentin samples among the ten HBV DNA-positive fresh dentin samples (80%) showed complete degradation of nucleic acid of HBV DNA with the reduction being statistically significant. HBV DNA persisted in two of the processed dentin samples among the ten HBV DNA-positive fresh dentin samples (20%). These two corresponded with the fresh dentin samples containing the highest and second highest copy number of HBV DNA 85.42 and 34.4 compared to 1.79 and 4.03, respectively, post processing. A copy number less than 10 is generally considered to be negative for virus even though the actual cut off levels and specificity were not determined in the current experiment.
There have been several approaches used beyond standard donor testing and screening procedures in the developmental history of bone banks to prevent viral transmission. The use of H
2O
2 as an oxidizing chemical is used to process bone allografts in effort to eradicate microorganisms and viruses. Viral clearance following a 1 h H
2O
2 treatment verifies that the risk for disease transmission can be greatly reduced or eliminated by greater than six logs sterility assurance level (SAL), except for the porcine parvovirus (PPV) virus [
11]. SAL is the probability that an item will not be sterile after it has been subjected to a validated sterilization process. With a SAL of 10
−6, the odds of an organisms surviving after allograft processing are less than one in one million [
5]. Processed bone allografts obtained from an HIV-infected donor and cleaned with a 30% ethanol solution and rinsed in 100% ethanol prior to lyophilization failed to transmit the virus to the graft recipient, whereas unprocessed bone allografts obtained from the same donor transmitted the virus [
12]. Proprietary solutions, including those used for chemical sterilization with ethylene oxide, may contain particular bactericidal, viricidal, and fungicidal agents but there is no industry-wide standard for their use [
13]. Freeze-drying is a process by which water is removed from the tissue to the point where cellular activity is no longer supported, which may inactivate HIV and HCV and reduce the risk of transmission by infected blood products and bone marrow [
14].
Although the processing of dentin employs steps of washing, defatting, demineralization, freeze-drying, and sterilization with ethylene oxide gas, the majority of studies on viral clearance has been focused on the demineralization process in relation to the development of demineralized freeze-dried bone allograft (DFDBA). In the processing of preparing DFDBA, investigators have demonstrated that exposing allografts to low-pH solutions such as hydrochloric acid inactivates numerous viruses, including HIV, HBV, HCV, cytomegalovirus, and poliovirus [
15‐
17]. Scarborough et al. performed a study to validate the effectiveness of a bone demineralization process with respect to its inactivation of viruses, including HIV, duck hepatitis B virus (a model for human hepatitis B), bovine viral diarrheal virus (a model for human hepatitis C), human cytomegalovirus, and human poliovirus (a model for small non-enveloped viruses such as hepatitis A). The infectivity of all RNA and DNA viruses is reduced more than one-million-fold (10
−6) for all the viruses tested and as much as one-trillion-fold (10
−12) for poliovirus. For example, the probability of HIV survival after bone demineralization is less than 1 in 2.8 billion [
15,
17]. The demineralization method degrades nucleic acids in retrovirus-infected cortical bone and thereby preventing disease transmission through the allotransplantation of DBM powder. The ability of a demineralization procedure to effectively inactivate an infectious retrovirus in systemically infected bone while maintaining the desired osteoinductive properties of powdered DBM appears to provide an additional margin of safety while sustaining optimal allograft efficacy [
18].
The Centers for Disease Control and Prevention (CDC) report that DFDBA materials are widely used in periodontal and dental therapy and that there are no reports of disease transmission during the 30-year history of using freeze-dried bone allografts [
19]. There has also been no report of disease transmission (HIV or hepatitis viruses) using demineralized bone products (FDBA, DFDBA, DBM) [
20].
DDM consists of osteoconductive type 1 collagen and noncollagenous proteins, including osteoinductive bone morphogenetic proteins (BMPs), which stimulate the formation of bone at a defect site similar to that treated with DBM that is washed, demineralized with organic solvents, freeze-dried, and sterilized, resulting in a significant level of viral inactivation as previously mentioned [
1,
21]. The major differences between DDM and DBM are that bone contains viable osteocytes and blood vessels (harversian canals and endothelial cells) while there are no cells or blood vessels in dentin that could be a potential source of transmissible viruses [
22]. Tissues obtained from living donors have lower rates of bacterial contamination than tissues harvested from cadavers at autopsy [
23]. Likewise, because dentins are obtained from patients during dental treatment, the possibility of disease transmission from dentin might be lower than that from a bone allograft. For the described reasons, a dentin allograft might be safer than a bone allograft.
However, while we measured HBV DNA levels in processed dentin, the precise step of washing, defatting, demineralization, or freeze-drying that resulted in the degradation of the virus, either alone or in combination, was not determined. Therefore, more studies to determine the effects of the various steps of dentin processing on the degradation of HBV and other viruses are warranted.
Of note, we currently do not know whether or not the positive HBV DNA in processed dentin was infectious since we did not determine the cutoff levels of HBV DNA in this experiment. Since qPCR techniques may amplify a small segment of degraded HBV DNA in the absence of intact virus, additional studies are necessary to determine whether the detected HBV DNA that remained in the processed dentin samples correlates with infectious virus. Finally, the established validation of viral inactivation procedures, including the harvesting of tissue in a sterile manner, repeated washings, immersion in ethanol, freeze-drying, demineralization, and sterilization, should be confirmed for rendering a safe DDM allograft [
24,
25].
Authors’ contributions
IU, SC, YK, KP, JL, ML, and BK designed the study. IU prepared the grafting material and defined the protocol. YK, JL, and ML were approved by the Seoul National University of Bundang IRB and the Seoul National University Dental Hospital Dankook University Hospital IRB. SC, YK, JL, ML, and BK collected the teeth from the hepatitis B virus patients and analyzed the dental specimens for DNA. SC and KP statistically analyzed the DNA analysis data. IU, SC, YK, KP, JL, ML, and BK prepared the manuscript. IU was responsible for critically revising the manuscript for important intellectual content. All authors read and approved the final manuscript.