Accepted Manuscript Comparison of four different allogeneic bone grafts for alveolar ridge reconstruction – A preliminary histological and biochemical analysis Tobias Fretwurst, DDS Alexandra Spanou, DDS Katja Nelson, DDS, PhD Martin Wein, MSc Thorsten Steinberg, ScD, PhD Andres Stricker, MD, DDS PII:
S2212-4403(14)00516-1
DOI:
10.1016/j.oooo.2014.05.020
Reference:
OOOO 936
To appear in:
Oral Surgery, Oral Medicine, Oral Pathology and Oral Radiology
Received Date: 10 January 2014 Revised Date:
14 May 2014
Accepted Date: 16 May 2014
Please cite this article as: Fretwurst T, Spanou A, Nelson K, Wein M, Steinberg T, Stricker A, Comparison of four different allogeneic bone grafts for alveolar ridge reconstruction – A preliminary histological and biochemical analysis, Oral Surgery, Oral Medicine, Oral Pathology and Oral Radiology (2014), doi: 10.1016/j.oooo.2014.05.020. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
Comparison of four different allogeneic bone grafts for alveolar ridge reconstruction – A preliminary histological and
RI PT
biochemical analysis
Tobias Fretwurst DDS1*, Alexandra Spanou DDS2*, Katja Nelson DDS, PhD1, Martin Wein MSc3, Thorsten Steinberg ScD, PhD3, Andres Stricker MD, DDS1
SC
*These two authors contributed equally to this work
2
M AN U
¹Department of Oral and Craniomaxillofacial Surgery University Medical Center Freiburg, Hugstetter Straße. 55, D-79106 Freiburg, Germany Private Clinic, Athens, Greece
3
TE D
Department of Oral Biotechnology University Medical Center Freiburg, Hugstetter Straße 55, D-79106 Freiburg, Germany
EP
There are no disclosures related to this study. The authors reported no conflicts of interest related to this study.
AC C
Word count for the abstract: 137 Word count for the manuscript: 2004 Number of tables: 2 Number of figures: 17 Number of supplementary elements: none
Send correspondence and offprints to: Dr. Tobias Fretwurst Department für Zahn-, Mund- und Kieferheilkunde Klinik für Mund-, Kiefer- und Gesichtschirurgie/Plastische Operationen Universitätsklinikum Freiburg, Albert Ludwig Universität Freiburg Hugstetter Str. 55, D-79106, Freiburg, Germany Phone: +49-761-270-47010, Fax: +49-761-270-48770 e-mail: [email protected]
1
ACCEPTED MANUSCRIPT Abstract
Objectives: Allograft material for alveolar ridge reconstruction is quite promising and appears to be as equally successful as bone autograft material.
RI PT
The aim of the present study was to compare four different allogeneic bone grafts in terms of their histologic structure and DNA content prior grafting.
Study Design: Four allograft specimens from different suppliers were
SC
analyzed histologically, and the DNA content was analyzed prior to clinical
M AN U
use of the allografts.
Results: Organic tissue remnants were detected in all of the evaluated samples. In the present samples Adipocytes, fibroblasts, osteocytes and chondrocytes were identified and DNA isolation and purification was possible.
TE D
Conclusion: Demineralized freeze-dried allogeneic bone transplants can stimulate new bone formation and are a viable alternative to bone autograft material. However, the safe utilization of allograft material in regard to our
AC C
EP
findings should be further investigated.
2
ACCEPTED MANUSCRIPT Introduction Reconstruction of the atrophied edentulous alveolar ridge and the subsequent prosthodontic rehabilitation is still a challenge in dental and maxillofacial
RI PT
surgery. The clinically and radiologically measured extent of bone loss guides the surgical procedure. In moderate to extensive bone deficiencies bone grafting is usually performed using materials with various origins (e.g. autograft, allograft, xenograft and alloplastic material) and structure
SC
(cancellous, cortical or combination) [1,2]. Autogenous material represents the
M AN U
gold standard and is harvested from intra- or extraoral sites, but autogenous material isn’t a panacea [3,4]. Limited availability and the need for a second surgical site increases the surgical mobility and duration of anaesthesia [511].
TE D
As an alternative to autogenous material, allogeneic materials have recently become popular. There are several options of allogenic materials available which include: fresh-frozen bone (FFBA), freeze-dried bone (FDBA) and
EP
demineralized freeze-dried bone allograft (DFDBA) of living or deceased donors [12]. The implant survival rate with allograft material is remarkable and
AC C
well over 95 % in published case series [12-17]. New bone formation
of
almost 30 % after a healing period of six months has been described in case reports [13,18] Overall allograft material appears quite promising and almost equally successful as autogenous material [12,13]. There has been limited histologic and molecular data in the literature to confirm the viable incorporation of allograft material [13,19,20]. There is a lack of long term studies regarding the healing of allograft material. The reported
3
ACCEPTED MANUSCRIPT data in previous studies are based on a low evidence level with small sample sizes [13-15,20]. The safety of allograft material has not been evaluated extensively in the literature and allograft material should be considered as an organic material with all the risks of using human tissue. The antigenicity and
RI PT
infectivity of allogeneic materials depends on donor selection and purification of the material like sterilization, lyophilization and irradiation. The transmission of Hepatitis C and HIV after transplantation of allograft material has been
SC
documented [21].
The aim of the present study was to compare four different allogeneic bone
M AN U
grafts for alveolar ridge reconstruction in terms of their histologic appearance and the DNA content prior to clinical application.
Bone Allografts different
commercially
available
allograft
materials
used
for
EP
Four
TE D
Material and methods
augmentation in oral and maxillofacial surgery were examined prior to clinical
AC C
application. The allogeneic grafts are processed using various standard techniques, which, according to the suppliers, provide biological and immunological safety (see Table 1). Specimens from Puros® block allograft (two samples), Allograft CTBA, Osteograft® and Maxgraft® were purchased from the suppliers (sterilized and packed). After removal of the bone block with sterile forceps under a Thermo Typ HS 12 bench (Laminar Flow) each block was divided into two equal pieces using a sterile scalpel. Each half of
4
ACCEPTED MANUSCRIPT the specimen was further processed either histologically or biochemically as detailed below. The allograft material selected for analysis was not used in patient care;
RI PT
therefore, approval from the local institutional review board was waived. Histologic Processing
The specimen was dehydrated in 100 % ethanol and subsequently infiltrated,
Germany)
according
to
the
manufacturer’s
SC
embedded and polymerized in Technovit 9100 (Heraeus Kulzer, Wehrheim, instructions.
After
the
M AN U
polymerization process, samples were cut in 500 µm sections using a rotary diamond saw Secotom - 50 (Stuers, Ballerup, Denmark). The sections were mounted onto opac acrylic-slides (Maertin, Freiburg, Germany) and ground to a final thickness of approximately 60 µm on a rotating grinding plate (Stuers,
TE D
Ballerup, Denmark). All specimens were stained with azure II and pararosaniline (Merck, Darmstadt, Germany).
EP
The slide imaging was performed with an Axio Imager M1 microscope equipped with a digital AxioCam HRc (Carl Zeiss, Göttingen, Germany).
AC C
Analysis of the prepared histologic sections was done with analySIS FIVE – software (Soft Imaging System, Münster, Germany). Biochemical Analysis Biochemical analysis was conducted with Osteograft®, Maxgraft® and Puros® specimens. The samples were initially comminuted and snap-frozen in liquid nitrogen. Afterwards a TissueLyser II (Qiagen® GmbH, Hilden) crushed the samples for 60 sec at 30 Hz with a 7 mm stainless steel bead.
5
ACCEPTED MANUSCRIPT After the addition of 200 µl Buffer ATL (without Proteinase K) from the DNeasy Blood & Tissue Kit (Qiagen® GmbH, Hilden) the tissues were further disrupted in the TissueLyser II for 120 seconds at 30 Hz. The processing of the samples continued in a QIAcube® (Qiagen® GmbH, Hilden) with the
RI PT
preinstalled protocol for the DNeasy Blood & Tissue Kit Procedure. For Lysis 25 mg tissue were cut into small pieces and placed in a 1.5 ml microcentrifuge tube and DNA extraction was performed using DNeasy (Qiagen® GmbH,
SC
Hilden) according to the manufacturers protocol. The quantity of the dsDNA from the eluate was subsequently measured with a NanoDrop 1000
M AN U
Spectrophotometer (PEQLAB Biotechnologie GmbH, Erlangen).
Results
TE D
Histologic Evaluation
The low power magnification demonstrated, that the Puros® and Osteograft® presented cortical and cancellous bone structures (Fig. 2a and Fig. 4a),
EP
whereas in the other allogenic bone blocks Mastgraft® and Allograft CTBA only trabecular bone structures were present. (Fig. 1a, 3a and 5a). The use of
AC C
azure II and pararosaniline staining allows the differentiation of bone (red), fibrous tissue (blue) and cell nuclei (blue). Cell and fibrous tissue remnants were visible within the intratrabecular area in low magnification in all grafts (Fig. 1a-5a).
At higher magnification each of the allogeneic bone blocks demonstrated organic material consisting of cells which vary in type and number, cell debris and fibrous tissue between the bone trabeculae of the donor bone graft.
6
ACCEPTED MANUSCRIPT Puros® block allograft contained osteocytes, chondrocytes and adipocytes in many intertrabecular segments of the sample (Fig. 1 and 2). Examination revealed osteocytes and adipocytes in the other allogeneic bone blocks as
RI PT
well (Fig. 3, 4 and 5). Biochemical Analysis (DNA content)
The spectrophotometric measurements showed that the DNA concentrations
SC
varied within the allograft samples. A correlation to the amount of organic substance within one sample cannot be analyzed (Allograft CTBA); however,
M AN U
DNA Analysis demonstrated that DNA still exists in the allografts (Table 2).
Discussion
Various bone grafting materials are used in oral and maxillofacial surgery to
TE D
restore bone defects in the maxilla or mandible. The ideal grafting material is biocompatible, induces no immunologic response, ensures that there is no risk of disease transmission, provides mechanical stability, offers a scaffold
EP
that promotes bone regeneration and is applied easily [22,23]. Obviously an
AC C
ideal bone grafting material does not exist, but autogenous bone graft remains the safest and widely used grafting material although there are some limitations and shortcomings [24]. Recent studies indicate that allografts constitute an alternative to autografts regarding the implant survival rate and new bone formation [25]. Antigenicity and immune response as well as the infectivity of allogenic materials are controversial subjects in literature [20].
7
ACCEPTED MANUSCRIPT The aim of the present study was to analyze four different allogeneic bone grafts in terms of their histologic appearance prior to clinical application. The present study showed that organic matter was present in all samples. The organic material varied in the number of cells, cell remnants and bony
RI PT
trabeculae. The Puros® block allograft contained islands of preserved bone marrow and endochondral ossification within the sample. The presence of a large amount of organic matter may be due to different purification techniques
SC
that vary amongst suppliers. The product insert for the Puros block allograft stated that the block is purified by the Tutoplast® process consisting of
M AN U
delipidization, osmotic and oxidative treatment, solvent dehydration and lowdose gamma irradiation. It remains to be elucidated how this treatment allows a major amount of residual organic material to remain as observed in this study. Organic remnants were found in all allogeneic grafts in this study.
TE D
Despite the challenge of extracting DNA, successful DNA isolation and purification from three allograft samples was possible [26]. Although there are
EP
observations that DNA can cause an immune response, cytosolic DNA can trigger a strong immune response via the enzyme cGAMP synthase (CGAs)
AC C
[27]. Autoantibodies to double strands of DNA play a role in the context of autoimmune diseases but further studies are needed to show if an immune response is initiated by exogenous DNA [28]. Contamination of the samples by human DNA after opening the packaging could be excluded by the sterile procedure that was used to process the allografts examined in this study. Cells and cell residues were found in all four allograft samples. Further studies will show if the cell remnants in the bone graft material are able to
8
ACCEPTED MANUSCRIPT elicit an immune response in the recipient and whether it contributes to an inferior clinical outcome. Canine studies showed a local immune reaction to allograft material [29]. Further murine studies showed that allograft induces the formation of specific CD8+ and CD4- T-cells [30]. But current studies
RI PT
could not detect antibodies in the blood of human individuals which have received allograft material [31]. Peripheral blood cell balance is not altered after allograft application [20]. Inflammatory cells couldn’t be detected next to
SC
the inserted mineralized block allograft [13,19,20,32].
M AN U
The immediate immune response remains unclear. Some authors suggested that interleukins and the white blood cell count, specifically lymphocytes, should be considered as a litmus test for an immune response. However the specificity is limited because leukocytes such as eosinophils, play an important role in both the immune response and in the healing phase of bone
TE D
healing and angiogenesis [20,33,34]. It would be necessary to isolate surface markers in the cell debris so that questions regarding the antigenicity could be
EP
clarified. In this context further studies should focus on the detection of major histocompatibility complex I (MHC-I) and MHC-II to elicit whether allograft
AC C
material could induce a competent immune response or not [35,36]. The antigenicity of bone allograft has been reported to be reduced because the different treatment procedures results in a removal of the cells [20]. The investigated allografts are processed and sterilized using different protocols in order to obtain safety and minimize the risk of transmission of infectious diseases. Surprisingly, the allogeneic bone grafts were not deprived of all genetic information. Further studies are needed to demonstrate the clinical
9
ACCEPTED MANUSCRIPT relevance of the existence of DNA. Especially with the history of transmission of viruses such as Hepatitis C and HIV after transplantation of bone allograft prior to the introduction of a routine screening of the donors for HIV 1 and 2, Hepatitis B, Hepatitis C and Syphilis [21,31,37]. HIV has been observed to
RI PT
reside in the bone marrow, giving obvious implications in the transplantation of allograft material [37]. Nevertheless, the risk of seroconversion after allograft transplantation is considered to be extremely low (HIV: 1 to 8 Million,
SC
Hepatitis C: 1 to 200.000) [12,31,38,39].
M AN U
One prospective study confirms the safety of allograft material according to the standards established in 2006 [40]. Donors are tested for a specific number of temporarily known pathogens [41]. Bone marrow can contain viruses with a long-term viral latency, such as, Epstein-Barr virus (EBV), Cytomegalovirus (CMV) and Human Parvovirus B19 [42, 43], as well as the
TE D
human T-cell leukemia virus 1 [44]. The risk of transmission of CreutzfeldtJakob (CJD) is currently considered as low, because none of the 469 cases of
EP
iatrogenic CJD that have been reported worldwide involved bone allograft [45]. A further study detected JC-Virus DNA by quantitative polymerase chain
AC C
reaction in human bone marrow samples to serve as a reservoir for a progressive multifocal leukoencephalopathy [46]. Viruses within a virus family are able to develop different resistances to chemicals theoretically and the virus types differ in their thermostability [39]. Therefore the modulation of sterilization methods such as the adaption of gamma irradiation are being discussed today [47].
10
ACCEPTED MANUSCRIPT Spores and fungi are also remarkable as pathogens. Allografts should be strictly controlled, the sterilization methods should be enhanced continuously and patients should be informed about the risks which are associated with
RI PT
allograft material.
Conclusion
SC
The safe applicability of allogenic bone grafts with regard to the histologic findings within this study should be investigated further, with a focus on the
M AN U
antigenicity of the graft.
Acknowledgement
TE D
The authors reported no conflicts of interest related to this study. We would like to thank Annette Lindner for their excellent assistance in the preparation
AC C
References:
EP
of the histological samples and Dr. John Nelson for his resourceful ideas.
1. von Arx T, Cochran DL, Hermann JS: Lateral ridge augmentation and implant
placement:
an
experimental
study
evaluating
implant
osseointegration in different augmentation materials in the canine mandible. Int J Oral Maxillofac Implants 2001;16(3):343-54.
11
ACCEPTED MANUSCRIPT 2. Netto HD, Olate S, Klüppel L: Analyses of cancellous and cortical interface in autogenous bone grafting. Int J Clin Exp Pathol 2013; 6(8):1532-7.
RI PT
3. Kao ST, Scott, DD: A review of bone substitutes. Oral Maxillofac Surg Clin North Am 2007; 19(4): 513-521.
4. Rodella LF, Favero G, Labanca M: Biomaterials in maxillofacial
M AN U
SC
surgery: membranes and grafts. Int J Biomed Sci 2011; 7(2):81-88.
5. Bauer TW, Muschler GF: Bone graft materials. An overview of the basic science. Clin Orthop Relat Res 2000; 371:10-27.
6. Nkenke, E, Radespiel-Troger M, Wiltfang J: Morbidity of harvesting of
TE D
retromolar bone grafts: a prospective study. Clin Oral Implants Res
EP
2002; 13(5): 514-521.
7. Nkenke E, Schultze-Mosgau S, Radespiel-Troger M: Morbidity of
AC C
harvesting of chin grafts: a prospective study. Clin Oral Implants Res 2001; 12(5):495-502.
8. Nkenke E, Weisbach V, Winckler E: Morbidity of harvesting of bone grafts from the iliac crest for preprosthetic augmentation procedures: a prospective study. Int J Oral Maxillofac Surg 2004; 33(2):157-163.
12
ACCEPTED MANUSCRIPT 9. Raghoebar GM, Louwerse C, Kalk WW: Morbidity of chin bone harvesting. Clin Oral Implants Res 2001; 12(5):503-507. 10. Weibull L, Widmark, G, Ivanoff, CJ: Morbidity after chin bone
RI PT
harvesting--a retrospective long-term follow-up study. Clin Implant Dent Relat Res 2009; 11(2):149-157.
11. Takamoto M, Takechi M, Ohta K: Risk of bacterial contamination of
surgery. Head Face Med 2013; 11(9):3.
SC
bone harvesting devices used for autogenous bone graft in implant
M AN U
12. Acocella A, Bertolai R, Ellis E 3rd: Maxillary alveolar ridge reconstruction with monocortical fresh-frozen bone blocks: a clinical, histological and histomorphometric study. J Craniomaxillofac Surg 2012; 40(6):525-33.
TE D
13. Nissan J, Marilena V, Gross O: Histomorphometric analysis following augmentation of the anterior atrophic maxilla with cancellous bone
EP
block allograft. Int J Oral Maxillofac Implants 2012; 27(1):84-9. 14. Nissan J, Mardinger O, Calderon S: Cancellous bone block allografts
AC C
for the augmentation of the anterior atrophic maxilla. Clin Implant Dent Relat Res 2011; 13(2):104-11.
15. Schlee M, Rothamel D: Ridge augmentation using customized allogenic bone blocks: proof of concept and histological findings. Implant Dent. 2013; 22(3):212-8.
13
ACCEPTED MANUSCRIPT 16. Peleg M, Sawatari Y, Marx RN: Use of corticocancellous allogeneic bone blocks for augmentation of alveolar bone defects. Int J Oral Maxillofac Implants 2010; 25(1):153-62.
RI PT
17. Carinci F, Brunelli G, Franco M: retrospective study on 287 implants installed in resorbed maxillae grafted with fresh frozen allogenous bone. Clin Implant Dent Relat Res 2010; 12(2):91-8.
SC
18. Iasella JM, Greenwell H, Miller RL : Ridge preservation with freezedried bone allograft and a collagen membrane compared to extraction
M AN U
alone for implant site development: a clinical and histologic study in humans. J Periodontol 2003; 74(7):990-9.
19. Boniello R, Gasparini G, D'Amato G: Reconstruction of severe atrophic jaws with Fresh Frized Bone Allografts: clinical histologic and
17(10):1411-8.
TE D
histomorphometric evaluation. Eur Rev Med Pharmacol Sci 2013;
EP
20. Spin Neto R, Felipe Leite C, Pereira LA: Is peripheral blood cell balanced altered by the use of fresh frozen bone block allografts in
AC C
lateral maxillary ridge augmentation? Clin Implant Dent Relat Res 2013; 15(2):262-70.
21. Conrad EU, Gretch DR, Obermeyer KR : Transmission of the hepatitisC virus by tissue transplantation. J Bone Joint Surg Am 1995; 77(2):214-24.
14
ACCEPTED MANUSCRIPT 22. Chaushu G, Mardinger O, Peleg M: Analysis of complications following augmentation with cancellous block allografts. J Periodontol 2010; 81(12):1759-64.
RI PT
23. Rajan GP, Fornaro J, Trentz O: Cancellous allograft versus autologous bone grafting for repair of comminuted distal radius fractures: a prospective, randomized trial. J Trauma 2006; 60(6):1322-9.
SC
24. Gazdag AR, Lane JM, Glaser D: Alternatives to Autogenous Bone Graft: Efficacy and Indications. J Am Acad Orthop Surg 1995; 3(1):1-8.
M AN U
25. Putzier M, Strube P, Funk JF: Allogenic versus autologous cancellous bone in lumbar segmental spondylodesis: a randomized prospective study. Eur Spine J 2009; 18(5):687-695.
26. Holland MM, Cave CA, Holland CA: Development of a quality, high
TE D
throughput DNA analysis procedure for skeletal samples to assist with the identification of victims from the World Trade Center attacks. Croat
EP
Med J 2003; 44(3):264-72.
27. Civril F, Deimling T, de Oliveira Mann CC: Structural mechanism of
AC C
cytosolic DNA sensing by cGAS. Nature 2013; 498(7454):332-7.
28. Eriksson C, Kokkonen H, Johansson M: Autoantibodies predate the onset of systemic lupus erythematosus in northern Sweden. Arthritis Res Ther 2011; 22;13(1):R30. 29. Goldberg VM, Powell A, Shaffer JW: Bone grafting: role of histocompatibility in transplantation. J Orthop Res 1985; 3(4):389-404.
15
ACCEPTED MANUSCRIPT 30. Horowitz
MC,
Friedlaender
GE:
Induction
of
specific
T-cell
responsiveness to allogeneic bone. J Bone Joint Surg Am 1991; 73(8):1157-68.
RI PT
31. Carlson ER, Marx RE, Buck BE: The potential for HIV transmission through allogeneic bone. A review of risks and safety. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 1995; 80(1):17-23.
evaluation
of
a
mineralized
block
SC
32. Keith JD Jr, Petrungaro P, Leonetti JA: Clinical and histologic allograft:
results from
the
2006; 26(4):321-7.
M AN U
developmental period (2001-2004). Int J Periodontics Restorative Dent
33. Bochner BS, Gleich GJ: What targeting eosinophils has taught us
quiz 26-7.
TE D
about their role in diseases. J Allergy Clin Immunol 2010; 126(1):16-25;
34. Puxeddu I, Berkman N, Nissim Ben Efraim AH: The role of eosinophil
EP
major basic protein in angiogenesis. Allergy 2009; 64(3):368-74. 35. Jindra PT, Tripathi S, Tian C: Tolerance to MHC class II disparate
AC C
allografts through genetic modification of bone marrow. Gene Ther 2013; 20(5):478-86.
36. Takenaka M, Tiriveedhi V, Subramanian V: Antibodies to MHC class II molecules induce autoimmunity: critical role for macrophages in the immunopathogenesis of obliterative airway disease. PLoS One 2012; 7(8):e42370.
16
ACCEPTED MANUSCRIPT 37. Buck BE, Resnick L, Shah SM: Human immunodeficiency virus cultured from bone. Implications for transplantation. Clin Orthop Relat Res 1990; (251):249-53.
RI PT
38. Albert A, Leemrijse T, Druez V: Are bone autografts still necessary in 2006? A three-year retrospective study of bone grafting. Acta Orthop Belg 2006; 72(6):734-40.
SC
39. Pruss A, Kao M, Kiesewetter H: Virus safety of avital bone tissue transplants: evaluation of sterilization steps of spongiosa cuboids using
M AN U
a peracetic acid-methanol mixture. Biologicals 1999; 27(3):195-201. 40. Kappe T, Cakir B, Mattes T: Infections after bone allograft surgery: a prospective study by a hospital bone bank using frozen femoral heads from living donors. Cell Tissue Bank 2010; 11(3):253-9.
TE D
41. Uhlenhaut C, Dorner T, Pauli, G: Effects of lyophilization on the infectivity of enveloped and non-enveloped viruses in bone tissue.
EP
Biomaterials 2005; 26(33):6558-6564. 42. Klein E, Kis LL, Klein G: Epstein-Barr virus infection in humans: from
AC C
harmless to life endangering virus-lymphocyte interactions. Oncogene 2007; 26(9):1297-305.
43. Khaiboullina SF, Maciejewski JP, Crapnell K: Human cytomegalovirus persists in myeloid progenitors and is passed to the myeloid progeny in a latent form. Br J Haematol 2004; 126(3):410-7.
17
ACCEPTED MANUSCRIPT 44. Sanzén L, Carlsson A: Transmission of human T-cell lymphotrophic virus type 1 by a deep-frozen bone allograft. Acta Orthop Scand 2007; 68(1):72-74.
RI PT
45. Hamaguchi T, Sakai K, Noguchi-Shinohara M: Insight into the frequent occurrence of dura mater graft-associated Creutzfeldt-Jakob disease in Japan. J Neurol Neurosurg Psychiatry 2013; 84(10):1171-5.
SC
46. Tan CS, Dezube BJ, Bhargava P: Detection of JC virus DNA and proteins in the bone marrow of HIV-positive and HIV-negative patients:
2009; 199(6):881-8.
M AN U
implications for viral latency and neurotropic transformation J Infect Dis
47. Nguyen H, Morgan DA, Forwood MR: Sterilization of allograft bone: is 25 kGy the gold standard for gamma irradiation? Cell Tissue Bank
AC C
EP
TE D
2007; 8(2):81-91.
18
ACCEPTED MANUSCRIPT Figures Fig. 1 and 2 (a-d): Histological comparison of Puros® block I and II (azure 2 and pararosanillin stain): Overall view, 50 x, 100 x, 200 x and 400 x Magnification
RI PT
Fig. 1 a Overall view Fig. 1 b 50 x Fig. 1 c 400 x
SC
Fig. 1 d 400 x
Fig. 2 a Overall view
M AN U
Fig. 2 b 100 x Fig. 2 c 200 x Fig. 2 d 200 x
Fig. 3 a Overall view
Fig. 3 c 200 x
EP
Fig. 3 b 100 x
TE D
Fig. 3 - 5 (a - c): Histological comparison of Allograft, Osteograft®, and Maxgraft® (azure 2 and pararosanillin stain): Overall view, 50 x, 200 x and 400 x Magnification
AC C
Fig. 4 a Overall view Fig. 4 b 50 x
Fig. 4 c 400 x
Fig. 5 a Overall view Fig. 5 b 50 x Fig. 5 c 200 x
19
ACCEPTED MANUSCRIPT Tables Product name/LOT
Supplier
Purification method
Delipidization,
Puros® block allograft PA I
Lot#11266068
PA II
Lot#12671529
Zimmer Dental
process:
RI PT
Tutoplast®
osmotic
and oxidative treatment, solvent dehydration and
SC
low-dose
gamma
O
Allograft CTBA
Cells
Lot#SAB12-005
Bank Austria
Osteograft®
Tissue chemically
processed,
lyophilized and irradiated
peracetic
acid
DIZG
ethanol sterilization (PES)
M
AC C
EP
Lot#1312100A
TE D
A
M AN U
irradiation
Maxgraft®
Lot#B10-0465
Botiss
chemically
purified,
freeze-dried and gamma irradiated
Table 1: Characteristic data of the examined allograft materials
ACCEPTED MANUSCRIPT Osteograft®
10,2 ng/ ml
Maxgraft®
25,7 ng/ ml
Puros®
56,2 ng/ml
AC C
EP
TE D
M AN U
SC
RI PT
Table 2: DNA Concentrations measured with the NanoDrop 1000 Spectrophotometer
Puros®
Puros® II
ACCEPTED MANUSCRIPT Fig. 1
Fig. 2
1
a
a
Overall view
Overall view
RI PT
2
2
b
b x 50
M AN U
1
SC
1
x 100
2
c
AC C
x 400
EP
TE D
1
d
c x 200
2
2
d x 400
x 200
Fig. 1 and 2 (a-d): Histological comparison of Puros® block I and II (azure 2 and parsoanillin stain): Overall view, 50 x, 100 x, 200 x and 400 x Magnification
Allograft
Osteograft®
Maxgraft®
ACCEPTED MANUSCRIPT Fig. 4
a Overall view
Overall view
M AN U
Overall view
a
SC
a
Fig. 5
RI PT
Fig. 3
b
b
b x 100
x 50
c
AC C
EP
TE D
x 50
x 200
c
c x 400
x 200
Fig. 3 - 5 (a - c): Histological comparison of Allograft, Osteograft®, and Maxgraft® (azure 2 and parsoanillin stain): Overall view, 50 x, 200 x and 400 x Magnification
ACCEPTED MANUSCRIPT Statement of clinical relevance
To the authors’ knowledge this is the first examination of different allograft material for alveolar ridge reconstruction prior to use. This publication may inspire researchers and surgeons who are investigating antigenicity, immune response as well as infectivity of
AC C
EP
TE D
M AN U
SC
RI PT
allogenic materials.
S2212-4403(14)00516-1
DOI:
10.1016/j.oooo.2014.05.020
Reference:
OOOO 936
To appear in:
Oral Surgery, Oral Medicine, Oral Pathology and Oral Radiology
Received Date: 10 January 2014 Revised Date:
14 May 2014
Accepted Date: 16 May 2014
Please cite this article as: Fretwurst T, Spanou A, Nelson K, Wein M, Steinberg T, Stricker A, Comparison of four different allogeneic bone grafts for alveolar ridge reconstruction – A preliminary histological and biochemical analysis, Oral Surgery, Oral Medicine, Oral Pathology and Oral Radiology (2014), doi: 10.1016/j.oooo.2014.05.020. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
Comparison of four different allogeneic bone grafts for alveolar ridge reconstruction – A preliminary histological and
RI PT
biochemical analysis
Tobias Fretwurst DDS1*, Alexandra Spanou DDS2*, Katja Nelson DDS, PhD1, Martin Wein MSc3, Thorsten Steinberg ScD, PhD3, Andres Stricker MD, DDS1
SC
*These two authors contributed equally to this work
2
M AN U
¹Department of Oral and Craniomaxillofacial Surgery University Medical Center Freiburg, Hugstetter Straße. 55, D-79106 Freiburg, Germany Private Clinic, Athens, Greece
3
TE D
Department of Oral Biotechnology University Medical Center Freiburg, Hugstetter Straße 55, D-79106 Freiburg, Germany
EP
There are no disclosures related to this study. The authors reported no conflicts of interest related to this study.
AC C
Word count for the abstract: 137 Word count for the manuscript: 2004 Number of tables: 2 Number of figures: 17 Number of supplementary elements: none
Send correspondence and offprints to: Dr. Tobias Fretwurst Department für Zahn-, Mund- und Kieferheilkunde Klinik für Mund-, Kiefer- und Gesichtschirurgie/Plastische Operationen Universitätsklinikum Freiburg, Albert Ludwig Universität Freiburg Hugstetter Str. 55, D-79106, Freiburg, Germany Phone: +49-761-270-47010, Fax: +49-761-270-48770 e-mail: [email protected]
1
ACCEPTED MANUSCRIPT Abstract
Objectives: Allograft material for alveolar ridge reconstruction is quite promising and appears to be as equally successful as bone autograft material.
RI PT
The aim of the present study was to compare four different allogeneic bone grafts in terms of their histologic structure and DNA content prior grafting.
Study Design: Four allograft specimens from different suppliers were
SC
analyzed histologically, and the DNA content was analyzed prior to clinical
M AN U
use of the allografts.
Results: Organic tissue remnants were detected in all of the evaluated samples. In the present samples Adipocytes, fibroblasts, osteocytes and chondrocytes were identified and DNA isolation and purification was possible.
TE D
Conclusion: Demineralized freeze-dried allogeneic bone transplants can stimulate new bone formation and are a viable alternative to bone autograft material. However, the safe utilization of allograft material in regard to our
AC C
EP
findings should be further investigated.
2
ACCEPTED MANUSCRIPT Introduction Reconstruction of the atrophied edentulous alveolar ridge and the subsequent prosthodontic rehabilitation is still a challenge in dental and maxillofacial
RI PT
surgery. The clinically and radiologically measured extent of bone loss guides the surgical procedure. In moderate to extensive bone deficiencies bone grafting is usually performed using materials with various origins (e.g. autograft, allograft, xenograft and alloplastic material) and structure
SC
(cancellous, cortical or combination) [1,2]. Autogenous material represents the
M AN U
gold standard and is harvested from intra- or extraoral sites, but autogenous material isn’t a panacea [3,4]. Limited availability and the need for a second surgical site increases the surgical mobility and duration of anaesthesia [511].
TE D
As an alternative to autogenous material, allogeneic materials have recently become popular. There are several options of allogenic materials available which include: fresh-frozen bone (FFBA), freeze-dried bone (FDBA) and
EP
demineralized freeze-dried bone allograft (DFDBA) of living or deceased donors [12]. The implant survival rate with allograft material is remarkable and
AC C
well over 95 % in published case series [12-17]. New bone formation
of
almost 30 % after a healing period of six months has been described in case reports [13,18] Overall allograft material appears quite promising and almost equally successful as autogenous material [12,13]. There has been limited histologic and molecular data in the literature to confirm the viable incorporation of allograft material [13,19,20]. There is a lack of long term studies regarding the healing of allograft material. The reported
3
ACCEPTED MANUSCRIPT data in previous studies are based on a low evidence level with small sample sizes [13-15,20]. The safety of allograft material has not been evaluated extensively in the literature and allograft material should be considered as an organic material with all the risks of using human tissue. The antigenicity and
RI PT
infectivity of allogeneic materials depends on donor selection and purification of the material like sterilization, lyophilization and irradiation. The transmission of Hepatitis C and HIV after transplantation of allograft material has been
SC
documented [21].
The aim of the present study was to compare four different allogeneic bone
M AN U
grafts for alveolar ridge reconstruction in terms of their histologic appearance and the DNA content prior to clinical application.
Bone Allografts different
commercially
available
allograft
materials
used
for
EP
Four
TE D
Material and methods
augmentation in oral and maxillofacial surgery were examined prior to clinical
AC C
application. The allogeneic grafts are processed using various standard techniques, which, according to the suppliers, provide biological and immunological safety (see Table 1). Specimens from Puros® block allograft (two samples), Allograft CTBA, Osteograft® and Maxgraft® were purchased from the suppliers (sterilized and packed). After removal of the bone block with sterile forceps under a Thermo Typ HS 12 bench (Laminar Flow) each block was divided into two equal pieces using a sterile scalpel. Each half of
4
ACCEPTED MANUSCRIPT the specimen was further processed either histologically or biochemically as detailed below. The allograft material selected for analysis was not used in patient care;
RI PT
therefore, approval from the local institutional review board was waived. Histologic Processing
The specimen was dehydrated in 100 % ethanol and subsequently infiltrated,
Germany)
according
to
the
manufacturer’s
SC
embedded and polymerized in Technovit 9100 (Heraeus Kulzer, Wehrheim, instructions.
After
the
M AN U
polymerization process, samples were cut in 500 µm sections using a rotary diamond saw Secotom - 50 (Stuers, Ballerup, Denmark). The sections were mounted onto opac acrylic-slides (Maertin, Freiburg, Germany) and ground to a final thickness of approximately 60 µm on a rotating grinding plate (Stuers,
TE D
Ballerup, Denmark). All specimens were stained with azure II and pararosaniline (Merck, Darmstadt, Germany).
EP
The slide imaging was performed with an Axio Imager M1 microscope equipped with a digital AxioCam HRc (Carl Zeiss, Göttingen, Germany).
AC C
Analysis of the prepared histologic sections was done with analySIS FIVE – software (Soft Imaging System, Münster, Germany). Biochemical Analysis Biochemical analysis was conducted with Osteograft®, Maxgraft® and Puros® specimens. The samples were initially comminuted and snap-frozen in liquid nitrogen. Afterwards a TissueLyser II (Qiagen® GmbH, Hilden) crushed the samples for 60 sec at 30 Hz with a 7 mm stainless steel bead.
5
ACCEPTED MANUSCRIPT After the addition of 200 µl Buffer ATL (without Proteinase K) from the DNeasy Blood & Tissue Kit (Qiagen® GmbH, Hilden) the tissues were further disrupted in the TissueLyser II for 120 seconds at 30 Hz. The processing of the samples continued in a QIAcube® (Qiagen® GmbH, Hilden) with the
RI PT
preinstalled protocol for the DNeasy Blood & Tissue Kit Procedure. For Lysis 25 mg tissue were cut into small pieces and placed in a 1.5 ml microcentrifuge tube and DNA extraction was performed using DNeasy (Qiagen® GmbH,
SC
Hilden) according to the manufacturers protocol. The quantity of the dsDNA from the eluate was subsequently measured with a NanoDrop 1000
M AN U
Spectrophotometer (PEQLAB Biotechnologie GmbH, Erlangen).
Results
TE D
Histologic Evaluation
The low power magnification demonstrated, that the Puros® and Osteograft® presented cortical and cancellous bone structures (Fig. 2a and Fig. 4a),
EP
whereas in the other allogenic bone blocks Mastgraft® and Allograft CTBA only trabecular bone structures were present. (Fig. 1a, 3a and 5a). The use of
AC C
azure II and pararosaniline staining allows the differentiation of bone (red), fibrous tissue (blue) and cell nuclei (blue). Cell and fibrous tissue remnants were visible within the intratrabecular area in low magnification in all grafts (Fig. 1a-5a).
At higher magnification each of the allogeneic bone blocks demonstrated organic material consisting of cells which vary in type and number, cell debris and fibrous tissue between the bone trabeculae of the donor bone graft.
6
ACCEPTED MANUSCRIPT Puros® block allograft contained osteocytes, chondrocytes and adipocytes in many intertrabecular segments of the sample (Fig. 1 and 2). Examination revealed osteocytes and adipocytes in the other allogeneic bone blocks as
RI PT
well (Fig. 3, 4 and 5). Biochemical Analysis (DNA content)
The spectrophotometric measurements showed that the DNA concentrations
SC
varied within the allograft samples. A correlation to the amount of organic substance within one sample cannot be analyzed (Allograft CTBA); however,
M AN U
DNA Analysis demonstrated that DNA still exists in the allografts (Table 2).
Discussion
Various bone grafting materials are used in oral and maxillofacial surgery to
TE D
restore bone defects in the maxilla or mandible. The ideal grafting material is biocompatible, induces no immunologic response, ensures that there is no risk of disease transmission, provides mechanical stability, offers a scaffold
EP
that promotes bone regeneration and is applied easily [22,23]. Obviously an
AC C
ideal bone grafting material does not exist, but autogenous bone graft remains the safest and widely used grafting material although there are some limitations and shortcomings [24]. Recent studies indicate that allografts constitute an alternative to autografts regarding the implant survival rate and new bone formation [25]. Antigenicity and immune response as well as the infectivity of allogenic materials are controversial subjects in literature [20].
7
ACCEPTED MANUSCRIPT The aim of the present study was to analyze four different allogeneic bone grafts in terms of their histologic appearance prior to clinical application. The present study showed that organic matter was present in all samples. The organic material varied in the number of cells, cell remnants and bony
RI PT
trabeculae. The Puros® block allograft contained islands of preserved bone marrow and endochondral ossification within the sample. The presence of a large amount of organic matter may be due to different purification techniques
SC
that vary amongst suppliers. The product insert for the Puros block allograft stated that the block is purified by the Tutoplast® process consisting of
M AN U
delipidization, osmotic and oxidative treatment, solvent dehydration and lowdose gamma irradiation. It remains to be elucidated how this treatment allows a major amount of residual organic material to remain as observed in this study. Organic remnants were found in all allogeneic grafts in this study.
TE D
Despite the challenge of extracting DNA, successful DNA isolation and purification from three allograft samples was possible [26]. Although there are
EP
observations that DNA can cause an immune response, cytosolic DNA can trigger a strong immune response via the enzyme cGAMP synthase (CGAs)
AC C
[27]. Autoantibodies to double strands of DNA play a role in the context of autoimmune diseases but further studies are needed to show if an immune response is initiated by exogenous DNA [28]. Contamination of the samples by human DNA after opening the packaging could be excluded by the sterile procedure that was used to process the allografts examined in this study. Cells and cell residues were found in all four allograft samples. Further studies will show if the cell remnants in the bone graft material are able to
8
ACCEPTED MANUSCRIPT elicit an immune response in the recipient and whether it contributes to an inferior clinical outcome. Canine studies showed a local immune reaction to allograft material [29]. Further murine studies showed that allograft induces the formation of specific CD8+ and CD4- T-cells [30]. But current studies
RI PT
could not detect antibodies in the blood of human individuals which have received allograft material [31]. Peripheral blood cell balance is not altered after allograft application [20]. Inflammatory cells couldn’t be detected next to
SC
the inserted mineralized block allograft [13,19,20,32].
M AN U
The immediate immune response remains unclear. Some authors suggested that interleukins and the white blood cell count, specifically lymphocytes, should be considered as a litmus test for an immune response. However the specificity is limited because leukocytes such as eosinophils, play an important role in both the immune response and in the healing phase of bone
TE D
healing and angiogenesis [20,33,34]. It would be necessary to isolate surface markers in the cell debris so that questions regarding the antigenicity could be
EP
clarified. In this context further studies should focus on the detection of major histocompatibility complex I (MHC-I) and MHC-II to elicit whether allograft
AC C
material could induce a competent immune response or not [35,36]. The antigenicity of bone allograft has been reported to be reduced because the different treatment procedures results in a removal of the cells [20]. The investigated allografts are processed and sterilized using different protocols in order to obtain safety and minimize the risk of transmission of infectious diseases. Surprisingly, the allogeneic bone grafts were not deprived of all genetic information. Further studies are needed to demonstrate the clinical
9
ACCEPTED MANUSCRIPT relevance of the existence of DNA. Especially with the history of transmission of viruses such as Hepatitis C and HIV after transplantation of bone allograft prior to the introduction of a routine screening of the donors for HIV 1 and 2, Hepatitis B, Hepatitis C and Syphilis [21,31,37]. HIV has been observed to
RI PT
reside in the bone marrow, giving obvious implications in the transplantation of allograft material [37]. Nevertheless, the risk of seroconversion after allograft transplantation is considered to be extremely low (HIV: 1 to 8 Million,
SC
Hepatitis C: 1 to 200.000) [12,31,38,39].
M AN U
One prospective study confirms the safety of allograft material according to the standards established in 2006 [40]. Donors are tested for a specific number of temporarily known pathogens [41]. Bone marrow can contain viruses with a long-term viral latency, such as, Epstein-Barr virus (EBV), Cytomegalovirus (CMV) and Human Parvovirus B19 [42, 43], as well as the
TE D
human T-cell leukemia virus 1 [44]. The risk of transmission of CreutzfeldtJakob (CJD) is currently considered as low, because none of the 469 cases of
EP
iatrogenic CJD that have been reported worldwide involved bone allograft [45]. A further study detected JC-Virus DNA by quantitative polymerase chain
AC C
reaction in human bone marrow samples to serve as a reservoir for a progressive multifocal leukoencephalopathy [46]. Viruses within a virus family are able to develop different resistances to chemicals theoretically and the virus types differ in their thermostability [39]. Therefore the modulation of sterilization methods such as the adaption of gamma irradiation are being discussed today [47].
10
ACCEPTED MANUSCRIPT Spores and fungi are also remarkable as pathogens. Allografts should be strictly controlled, the sterilization methods should be enhanced continuously and patients should be informed about the risks which are associated with
RI PT
allograft material.
Conclusion
SC
The safe applicability of allogenic bone grafts with regard to the histologic findings within this study should be investigated further, with a focus on the
M AN U
antigenicity of the graft.
Acknowledgement
TE D
The authors reported no conflicts of interest related to this study. We would like to thank Annette Lindner for their excellent assistance in the preparation
AC C
References:
EP
of the histological samples and Dr. John Nelson for his resourceful ideas.
1. von Arx T, Cochran DL, Hermann JS: Lateral ridge augmentation and implant
placement:
an
experimental
study
evaluating
implant
osseointegration in different augmentation materials in the canine mandible. Int J Oral Maxillofac Implants 2001;16(3):343-54.
11
ACCEPTED MANUSCRIPT 2. Netto HD, Olate S, Klüppel L: Analyses of cancellous and cortical interface in autogenous bone grafting. Int J Clin Exp Pathol 2013; 6(8):1532-7.
RI PT
3. Kao ST, Scott, DD: A review of bone substitutes. Oral Maxillofac Surg Clin North Am 2007; 19(4): 513-521.
4. Rodella LF, Favero G, Labanca M: Biomaterials in maxillofacial
M AN U
SC
surgery: membranes and grafts. Int J Biomed Sci 2011; 7(2):81-88.
5. Bauer TW, Muschler GF: Bone graft materials. An overview of the basic science. Clin Orthop Relat Res 2000; 371:10-27.
6. Nkenke, E, Radespiel-Troger M, Wiltfang J: Morbidity of harvesting of
TE D
retromolar bone grafts: a prospective study. Clin Oral Implants Res
EP
2002; 13(5): 514-521.
7. Nkenke E, Schultze-Mosgau S, Radespiel-Troger M: Morbidity of
AC C
harvesting of chin grafts: a prospective study. Clin Oral Implants Res 2001; 12(5):495-502.
8. Nkenke E, Weisbach V, Winckler E: Morbidity of harvesting of bone grafts from the iliac crest for preprosthetic augmentation procedures: a prospective study. Int J Oral Maxillofac Surg 2004; 33(2):157-163.
12
ACCEPTED MANUSCRIPT 9. Raghoebar GM, Louwerse C, Kalk WW: Morbidity of chin bone harvesting. Clin Oral Implants Res 2001; 12(5):503-507. 10. Weibull L, Widmark, G, Ivanoff, CJ: Morbidity after chin bone
RI PT
harvesting--a retrospective long-term follow-up study. Clin Implant Dent Relat Res 2009; 11(2):149-157.
11. Takamoto M, Takechi M, Ohta K: Risk of bacterial contamination of
surgery. Head Face Med 2013; 11(9):3.
SC
bone harvesting devices used for autogenous bone graft in implant
M AN U
12. Acocella A, Bertolai R, Ellis E 3rd: Maxillary alveolar ridge reconstruction with monocortical fresh-frozen bone blocks: a clinical, histological and histomorphometric study. J Craniomaxillofac Surg 2012; 40(6):525-33.
TE D
13. Nissan J, Marilena V, Gross O: Histomorphometric analysis following augmentation of the anterior atrophic maxilla with cancellous bone
EP
block allograft. Int J Oral Maxillofac Implants 2012; 27(1):84-9. 14. Nissan J, Mardinger O, Calderon S: Cancellous bone block allografts
AC C
for the augmentation of the anterior atrophic maxilla. Clin Implant Dent Relat Res 2011; 13(2):104-11.
15. Schlee M, Rothamel D: Ridge augmentation using customized allogenic bone blocks: proof of concept and histological findings. Implant Dent. 2013; 22(3):212-8.
13
ACCEPTED MANUSCRIPT 16. Peleg M, Sawatari Y, Marx RN: Use of corticocancellous allogeneic bone blocks for augmentation of alveolar bone defects. Int J Oral Maxillofac Implants 2010; 25(1):153-62.
RI PT
17. Carinci F, Brunelli G, Franco M: retrospective study on 287 implants installed in resorbed maxillae grafted with fresh frozen allogenous bone. Clin Implant Dent Relat Res 2010; 12(2):91-8.
SC
18. Iasella JM, Greenwell H, Miller RL : Ridge preservation with freezedried bone allograft and a collagen membrane compared to extraction
M AN U
alone for implant site development: a clinical and histologic study in humans. J Periodontol 2003; 74(7):990-9.
19. Boniello R, Gasparini G, D'Amato G: Reconstruction of severe atrophic jaws with Fresh Frized Bone Allografts: clinical histologic and
17(10):1411-8.
TE D
histomorphometric evaluation. Eur Rev Med Pharmacol Sci 2013;
EP
20. Spin Neto R, Felipe Leite C, Pereira LA: Is peripheral blood cell balanced altered by the use of fresh frozen bone block allografts in
AC C
lateral maxillary ridge augmentation? Clin Implant Dent Relat Res 2013; 15(2):262-70.
21. Conrad EU, Gretch DR, Obermeyer KR : Transmission of the hepatitisC virus by tissue transplantation. J Bone Joint Surg Am 1995; 77(2):214-24.
14
ACCEPTED MANUSCRIPT 22. Chaushu G, Mardinger O, Peleg M: Analysis of complications following augmentation with cancellous block allografts. J Periodontol 2010; 81(12):1759-64.
RI PT
23. Rajan GP, Fornaro J, Trentz O: Cancellous allograft versus autologous bone grafting for repair of comminuted distal radius fractures: a prospective, randomized trial. J Trauma 2006; 60(6):1322-9.
SC
24. Gazdag AR, Lane JM, Glaser D: Alternatives to Autogenous Bone Graft: Efficacy and Indications. J Am Acad Orthop Surg 1995; 3(1):1-8.
M AN U
25. Putzier M, Strube P, Funk JF: Allogenic versus autologous cancellous bone in lumbar segmental spondylodesis: a randomized prospective study. Eur Spine J 2009; 18(5):687-695.
26. Holland MM, Cave CA, Holland CA: Development of a quality, high
TE D
throughput DNA analysis procedure for skeletal samples to assist with the identification of victims from the World Trade Center attacks. Croat
EP
Med J 2003; 44(3):264-72.
27. Civril F, Deimling T, de Oliveira Mann CC: Structural mechanism of
AC C
cytosolic DNA sensing by cGAS. Nature 2013; 498(7454):332-7.
28. Eriksson C, Kokkonen H, Johansson M: Autoantibodies predate the onset of systemic lupus erythematosus in northern Sweden. Arthritis Res Ther 2011; 22;13(1):R30. 29. Goldberg VM, Powell A, Shaffer JW: Bone grafting: role of histocompatibility in transplantation. J Orthop Res 1985; 3(4):389-404.
15
ACCEPTED MANUSCRIPT 30. Horowitz
MC,
Friedlaender
GE:
Induction
of
specific
T-cell
responsiveness to allogeneic bone. J Bone Joint Surg Am 1991; 73(8):1157-68.
RI PT
31. Carlson ER, Marx RE, Buck BE: The potential for HIV transmission through allogeneic bone. A review of risks and safety. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 1995; 80(1):17-23.
evaluation
of
a
mineralized
block
SC
32. Keith JD Jr, Petrungaro P, Leonetti JA: Clinical and histologic allograft:
results from
the
2006; 26(4):321-7.
M AN U
developmental period (2001-2004). Int J Periodontics Restorative Dent
33. Bochner BS, Gleich GJ: What targeting eosinophils has taught us
quiz 26-7.
TE D
about their role in diseases. J Allergy Clin Immunol 2010; 126(1):16-25;
34. Puxeddu I, Berkman N, Nissim Ben Efraim AH: The role of eosinophil
EP
major basic protein in angiogenesis. Allergy 2009; 64(3):368-74. 35. Jindra PT, Tripathi S, Tian C: Tolerance to MHC class II disparate
AC C
allografts through genetic modification of bone marrow. Gene Ther 2013; 20(5):478-86.
36. Takenaka M, Tiriveedhi V, Subramanian V: Antibodies to MHC class II molecules induce autoimmunity: critical role for macrophages in the immunopathogenesis of obliterative airway disease. PLoS One 2012; 7(8):e42370.
16
ACCEPTED MANUSCRIPT 37. Buck BE, Resnick L, Shah SM: Human immunodeficiency virus cultured from bone. Implications for transplantation. Clin Orthop Relat Res 1990; (251):249-53.
RI PT
38. Albert A, Leemrijse T, Druez V: Are bone autografts still necessary in 2006? A three-year retrospective study of bone grafting. Acta Orthop Belg 2006; 72(6):734-40.
SC
39. Pruss A, Kao M, Kiesewetter H: Virus safety of avital bone tissue transplants: evaluation of sterilization steps of spongiosa cuboids using
M AN U
a peracetic acid-methanol mixture. Biologicals 1999; 27(3):195-201. 40. Kappe T, Cakir B, Mattes T: Infections after bone allograft surgery: a prospective study by a hospital bone bank using frozen femoral heads from living donors. Cell Tissue Bank 2010; 11(3):253-9.
TE D
41. Uhlenhaut C, Dorner T, Pauli, G: Effects of lyophilization on the infectivity of enveloped and non-enveloped viruses in bone tissue.
EP
Biomaterials 2005; 26(33):6558-6564. 42. Klein E, Kis LL, Klein G: Epstein-Barr virus infection in humans: from
AC C
harmless to life endangering virus-lymphocyte interactions. Oncogene 2007; 26(9):1297-305.
43. Khaiboullina SF, Maciejewski JP, Crapnell K: Human cytomegalovirus persists in myeloid progenitors and is passed to the myeloid progeny in a latent form. Br J Haematol 2004; 126(3):410-7.
17
ACCEPTED MANUSCRIPT 44. Sanzén L, Carlsson A: Transmission of human T-cell lymphotrophic virus type 1 by a deep-frozen bone allograft. Acta Orthop Scand 2007; 68(1):72-74.
RI PT
45. Hamaguchi T, Sakai K, Noguchi-Shinohara M: Insight into the frequent occurrence of dura mater graft-associated Creutzfeldt-Jakob disease in Japan. J Neurol Neurosurg Psychiatry 2013; 84(10):1171-5.
SC
46. Tan CS, Dezube BJ, Bhargava P: Detection of JC virus DNA and proteins in the bone marrow of HIV-positive and HIV-negative patients:
2009; 199(6):881-8.
M AN U
implications for viral latency and neurotropic transformation J Infect Dis
47. Nguyen H, Morgan DA, Forwood MR: Sterilization of allograft bone: is 25 kGy the gold standard for gamma irradiation? Cell Tissue Bank
AC C
EP
TE D
2007; 8(2):81-91.
18
ACCEPTED MANUSCRIPT Figures Fig. 1 and 2 (a-d): Histological comparison of Puros® block I and II (azure 2 and pararosanillin stain): Overall view, 50 x, 100 x, 200 x and 400 x Magnification
RI PT
Fig. 1 a Overall view Fig. 1 b 50 x Fig. 1 c 400 x
SC
Fig. 1 d 400 x
Fig. 2 a Overall view
M AN U
Fig. 2 b 100 x Fig. 2 c 200 x Fig. 2 d 200 x
Fig. 3 a Overall view
Fig. 3 c 200 x
EP
Fig. 3 b 100 x
TE D
Fig. 3 - 5 (a - c): Histological comparison of Allograft, Osteograft®, and Maxgraft® (azure 2 and pararosanillin stain): Overall view, 50 x, 200 x and 400 x Magnification
AC C
Fig. 4 a Overall view Fig. 4 b 50 x
Fig. 4 c 400 x
Fig. 5 a Overall view Fig. 5 b 50 x Fig. 5 c 200 x
19
ACCEPTED MANUSCRIPT Tables Product name/LOT
Supplier
Purification method
Delipidization,
Puros® block allograft PA I
Lot#11266068
PA II
Lot#12671529
Zimmer Dental
process:
RI PT
Tutoplast®
osmotic
and oxidative treatment, solvent dehydration and
SC
low-dose
gamma
O
Allograft CTBA
Cells
Lot#SAB12-005
Bank Austria
Osteograft®
Tissue chemically
processed,
lyophilized and irradiated
peracetic
acid
DIZG
ethanol sterilization (PES)
M
AC C
EP
Lot#1312100A
TE D
A
M AN U
irradiation
Maxgraft®
Lot#B10-0465
Botiss
chemically
purified,
freeze-dried and gamma irradiated
Table 1: Characteristic data of the examined allograft materials
ACCEPTED MANUSCRIPT Osteograft®
10,2 ng/ ml
Maxgraft®
25,7 ng/ ml
Puros®
56,2 ng/ml
AC C
EP
TE D
M AN U
SC
RI PT
Table 2: DNA Concentrations measured with the NanoDrop 1000 Spectrophotometer
Puros®
Puros® II
ACCEPTED MANUSCRIPT Fig. 1
Fig. 2
1
a
a
Overall view
Overall view
RI PT
2
2
b
b x 50
M AN U
1
SC
1
x 100
2
c
AC C
x 400
EP
TE D
1
d
c x 200
2
2
d x 400
x 200
Fig. 1 and 2 (a-d): Histological comparison of Puros® block I and II (azure 2 and parsoanillin stain): Overall view, 50 x, 100 x, 200 x and 400 x Magnification
Allograft
Osteograft®
Maxgraft®
ACCEPTED MANUSCRIPT Fig. 4
a Overall view
Overall view
M AN U
Overall view
a
SC
a
Fig. 5
RI PT
Fig. 3
b
b
b x 100
x 50
c
AC C
EP
TE D
x 50
x 200
c
c x 400
x 200
Fig. 3 - 5 (a - c): Histological comparison of Allograft, Osteograft®, and Maxgraft® (azure 2 and parsoanillin stain): Overall view, 50 x, 200 x and 400 x Magnification
ACCEPTED MANUSCRIPT Statement of clinical relevance
To the authors’ knowledge this is the first examination of different allograft material for alveolar ridge reconstruction prior to use. This publication may inspire researchers and surgeons who are investigating antigenicity, immune response as well as infectivity of
AC C
EP
TE D
M AN U
SC
RI PT
allogenic materials.
