Variant Purification of an Allogeneic Bone Block.

OBJECTIVE: This short communication reports on a histological analysis of the composition of the commercially available Maxgraft® allogeneic bone block.
MATERIALS AND METHODS: Based on previously published, easily applicable histological methods, blanc samples of the Maxgraft® allogeneic bone block have been decalcified, dehydrated and embedded in paraffin before histological and histochemical staining. Afterwards, the slides were evaluated for their material characteristics, such as the bone matrix structure and other components, including collagen or cells/cell remnants.
RESULTS: The results show that this bone block exhibits a trabecular structure with lamellar sub-organization. Additionally, cellular remnants within the osteocyte lacunae and at the outer trabecular surfaces reside together with remnants of the former inter-trabecular fatty and connective tissue, i.e., collagenous structures and connective tissue cells or cell remnants.
CONCLUSION: Consistent with a previous study on this topic, the data presented here demonstrate that some of the certified purification techniques might not allow for the production of allogeneic materials free of organic cell and tissue components.

Introduction

Numerous allogeneic and xenogeneic bone substitutes of different kinds have been introduced, since these classes of materials could exhibit a favorable regenerative potential based on their “natural” composition and bone tissue structure comparable to autografts (, ). Before their clinical application, the donor tissue must be purified and freed from all immunologically effective components such as the different cell types and matrix proteins (). To accomplish this, a variety of purification techniques with different physical and/or chemical methods have been developed. In this context, most of the material companies have introduced their own purification methods, which follow relevant guidelines, i.e., standards such as the ISO 10993 (, ).

However, before using general histochemical staining methods to detect the inorganic matrix and cellular or organic matrix components, we had previously found wide variations among different commercially available bone blocks, including two allogeneic and three xenogeneic bone blocks (). Furthermore, we found some discrepancies between the manufacturer’s information and the composition of the bone blocks as analyzed subsequently. Altogether, the results obtained in this study revealed that three out of five bone blocks contained organic/cellular remnants.

The aim of this short communication was to analyze the (ultra-) structure of commercially available Maxgraft® allogeneic bone block. Specifically, we performed a structural analysis of the bone block to analyze the (ultra-) structure of the calcified bone matrix and to investigate the presence of other components such as collagen and organic/cellular remnants.

Materials and Methods

Three samples of a commercially available Maxgraft® allogeneic bone block were histologically prepared and examined to evaluate their composition as previously published by our group (). We particularly focused on the detection of possible organic components to establish their purification quality. Additionally, the manufacturer’s information and the study results were compared.

Maxgraft®

Maxgraft® (botiss biomaterials GmbH, Berlin, Germany) is an allogeneic cancellous bone substitute block derived from the bone of femoral heads of living human donors from German, Austrian and Swiss hospitals (). The bone blocks are processed by the Cells + Tissue bank Austria, a certified and audited non-profit organization that is regulated by the Austrian Ministry of Health (, ). The purification of the bone tissue is in accordance with the respective European Directives and the Austrian Tissue Safety Act (, ). This purification process is stated to be “validated by independent institutes” and by the Austrian Health Ministry ().

The purification process, the “C+TBA process”, is described in more detail on the manufacturer’s homepage (). Briefly, it is stated to be a highly secure quality process that is in compliance with the highest quality standards that are employed when inactivating viruses and bacteria (, ). This purification process includes different physical and chemical purification steps (). It is stated that an ultrasonic-based removal of blood, cells and tissue components is applied, which should predominantly remove adipose tissue, as a physical method. Additionally, chemical and oxidative cleaning steps by means of diethyl ether and ethanol at different durations were employed to inactivate both pathogens, such as viruses and bacteria, and also non-collagen proteins (). Furthermore the oxidative purification step should eliminate soluble proteins by denaturation and potential antigens (). Finally, lyophilization and sterilization via gamma irradiation were applied to preserve the natural tissue structure (). No information was given about the composition of the final bone block, so that neither the (ultra-) structure of the bone matrix nor other components, such as bone tissue-specific collagen, are described.

Histological preparation of the bone blocks

Three material samples were initially decalcified in Tris-buffered 10% EDTA (Carl Roth, Karlsruhe, Germany), dehydrated in a series of increasing alcohol concentrations followed by xylol application and embedded in paraffin as previously described (, -). Histological sections of 3-5 μm thickness were obtained using a rotation microtome (Leica RM2255, Wetzlar, Germany).

Three histochemical stains were used, including hematoxylin and eosin (HE), Masson-Goldner’s trichrome and Sirius red and Giemsa. An additional section was stained using a histochemical method to detect tartrate-resistant acid phosphatase (TRAP) to identify osteoclasts. A bone section was used to control the quality of the TRAP staining method.

Histological analysis

The histological analysis of the bone blocks was conducted as previously described (, -). Briefly, the histological bone substitute slides were evaluated for their material characteristics, such as the bone matrix structure and other components, including collagen or cells/cell remnants. This procedure was performed independently by two authors (JL and SG) using a light microscope (Nikon Eclipse 80i, Tokyo, Japan). A Nikon DS-Fi1 digital camera and a DS-L2 digital sight control unit (both: Nikon, Tokyo, Japan) were also used to obtain histological photomicrographs.

Results

The histological analysis revealed that the inorganic bone matrix of the Maxgraft® bone block exhibited a trabecular structure (Figure 1A). Some inorganic bone matrix fragments were detected within the trabecular interspaces (Figure 1A). The inorganic bone matrix also exhibited a lamellar substructure (Figure 1B). Cells or cell remnants were observed within the osteocyte lacunae throughout the inorganic bone matrix (Figure 1B). Only a few lacunae were free of cells or cell remnants (Figure 1B). Furthermore, mononucleate and multinucleate cells were sporadically observed at the trabecular surfaces of the bone blocks (Figure 1C). The multinucleate cells did not express tartrate-resistant acid phosphatase (TRAP) (data not shown). Additionally, fatty tissue-like structures were apparent within the trabecular interspaces (Figure 1D). Also, the remnants of the former intertrabecular connective tissue were occasionally found, comprising both extracellular matrix and cells or cell remnants (Figure 1E).

Figure 1

Histological images of the Maxgraft® allogeneic bone block with a focus on its (ultra-) structure and composition. (A) A cross-section of the bone block illustrates the trabecular structure of the inorganic bone matrix (black asterisks). Fragments of bone matrix (black arrows) can be regularly observed within the interspaces of the trabeculae fragments (Masson Goldner-staining, “total scan”, 100 x magnification). (B) The bone matrix (asterisk) exhibits a lamellar sub-arrangement. In most of the osteocyte lacunae cells or cell remnants (red arrowheads) were found with only a few empty lacunae (green arrowheads). Cells/cell remnants and extracellular matrix are also apparent in the Haversian channels (blue arrow) (Giemsa-staining, 400x magnification, scale bar = 10 µm). (C) At the outer surfaces of the bone matrix (asterisks), cells or cellular remnants are identified (arrows). In addition to mononuclear cells (cyan arrows), multinucleated cells (red arrow) are detected (Giemsa-staining, 400x magnification, scale bar = 10 µm). (D) and (E) Within the trabecular interspaces, fatty-like tissue structures (black arrowheads in D) and connective tissue-like strands include both cells or cellular remnants (cyan arrows in E) and extracellular matrix (yellow asterisk in E) (bone matrix = asterisk) (D: Masson-Goldner-staining, 200x magnification; E = Giemsa-staining, 400x magnification, scale bars = 10 µm).

Discussion

The use of purifying allogeneic or xenogeneic biomaterials from potentially immunogenic components, such as donor cells, bacteria and viruses, is vital for their successful and safe application (). A variety of different bone substitute materials based on both human and animal donor tissue is available. They are processed by different purification methods, which are all guaranteed as safe for clinical application. In this context, manufacturers guarantee that their different purification methods follow guidelines, such as the ones set by the International Organization for Standardization (ISO) or the national and international guidelines that are required for their approval by different ministries of health.

However, the presence of impurities within allogeneic and xenogeneic biomaterials could already be demonstrated by our workgroup and other researchers (, ). In this context, five commercially available allogeneic or xenogeneic bone blocks were analyzed with widely available histological methods, with a focus on (ultra-) structure and detection of cellular or organic matrix components. Additionally, we compared the manufacturer’s description of the material composition with our histological results. Interestingly, the results of our analyses revealed that three out of the five bone blocks contained cells or cell remnants, which suggests that much care has to be given to material processing or the efficacy of the methods used to purify these materials. Based on different purification results, the analyzed bone blocks have been classified into four different groups. These classifications ranged from a complete purification of the bone matrix with a loss of its lamellar structure to materials that contained both bone matrix with its origin lamellar structure and collagenous structures of the bone tissue as well as cells or cellular remnants.

In the present study, the allogeneic Maxgraft® bone block was analyzed using the same histological methods. The results demonstrate that this bone block exhibits a trabecular structure with a lamellar sub-organization. In addition, cellular remnants within the osteocyte lacunae and at the outer trabecular surfaces, i.e. former osteoblasts and osteoclasts, together with remnants of the former inter-trabecular adipose and connective tissue, i.e., collagenous structures and connective tissue cells or cell remnants were detected.

A comparison of the results of our analysis and the manufacturer’s information revealed that the applied purification method preserved the trabecular structure of human donor tissue, including the lamellar sub-organization. However, the manufacturer’s description and our results differed regarding the presence of cellular organic remnants, which should have been eliminated by alternating solvents for rinsing, which means using diethyl ether ethanol (). Obviously, it is not possible to remove all intra- and extra trabecular cellular remnants from the donor tissue, which occurs due to an inadequate access of the solvents into hard mineral structure amongst others.

These results, i.e., the presence of the remaining cells or cellular remnants in the Maxgraft® bone block, could be of great importance for clinical applications of this bone substitute, since some studies assessing the transplantation of xenogeneic tissue have demonstrated that different types of remnants can induce non-physiological pro-inflammatory tissue responses (-). Altogether, our results suggest that the Maxgraft® block should be categorized within group 4, which includes bone blocks with the highest levels of remnants, such as the Puros® Allograft Spongiosa and the OsteoBiol® Sp bone blocks according to a previously published study by our group (). Finally, the results of the present study again show that the use of different purification methods could lead to different bone substitute materials based on their composition, i.e. the occurrence of different cells or cellular remnants. However, the abovementioned techniques are histological findings. Therefore, further clinical studies are needed to investigate into the relevance of cellular remnants for clinical outcomes after the application of this specific material. There are no data in the available literature about the degree of purification that is necessary for safe clinical application of such bone substitutes. In this context, it is important to note that a clinical report has been published by Pruss and colleagues that reports on successful application of different kinds of allogeneic tissue transplants sterilized, also, by a mixture of peracetic acid and ethanol but without presenting any data concerning the tissue reaction ().

However, it has to be stated that histological methodology, which has been described in this paper, can only show the presence of cellular remnants. Within the limit of present methods, the question about the vitality of the remnants cannot be answered by this methodology. Therefore, a potential immunologic response within the human organism needs to be investigated by immunologic methodologies in a clinical setting, as the question arises if the observed cellular remnants have any influence on the integration process of such bone substitute materials and the healing process. To date, the final impact of insufficient purification on the clinical performance of bone substitute materials is still unresolved. However, the process of quality insurance of non-homogenous bone graft materials makes interpretation of etiology of complications difficult and, also, calls into question the validity of interpretation of failure, technique or material.

Conclusion

In the present study, the structure of a commercially available allogeneic bone block was histologically analyzed, with the analysis particularly focused on its composition, including the appearance of cell or tissue remnants. The results demonstrate that this bone block exhibits a trabecular structure with lamellar sub-organization that harbors cellular remnants within the osteocyte lacunae and at the outer trabecular surfaces with remnants of the former inter-trabecular fatty and connective tissue, i.e., collagenous structures and connective tissue cells or cell remnants. In conclusion, clinical application will have to confirm the relevance of the results obtained in this study.