Study of Cell Viability and Etiology of Contamination in Decalcified Bone Allograft: A Pilot Study

Abstract

Background

Bone allografts can elicit immune responses which is correlated with the presence of Human Leukocyte Antigen (HLA) and cellular DNA. It also has risk of causing occult infection arising out of contamination during its processing and storage. The presence of immunogenic materials like cells, cellular remnants and DNA in a decalcified bone allograft during different phases of processing has never been studied. Present study was conducted to explore- the cell viability using routine Hematoxylin and Eosin, presence of DNA using Feulgen staining and etiology of contamination in decalcified bone allograft during procurement, demineralization and ethanol preservation.

Methods

The harvested bones from patients undergoing hemireplacement/THR/TKR were processed to prepare decalcified bone allografts. The samples during procurement (A), HCL treatment (B) and ethanol preservation (C) were sent for histopathological analysis (number of osteocytes in the maximum density field under 40x and the cells demonstrating presence of DNA on feulgen stain) and microbiological assessment (aerobic/anaerobic/fungal cultures).

Results

Histopathological study demonstrated the presence of osteocytes and other cells like bone marrow, adipocytes, endothelial cells in the decal bone allograft. The average number of osteocytes gradually decreased from 55.47, 9.6, 0.86 in sample A, B, C, respectively. Feulgen staining confirmed the presence of DNA in osteocytes and other cells which decreased both qualitatively and quantitatively in subsequent stages of processing. Rate of contamination demonstrated at the procurement was 6.67% (Staphylococcus aureus). After treatment with HCl (demineralisation), 7.14% of non-contaminated allografts were found contaminated (Staphylococcus epidermidis). None of the remaining 13 non-contaminated allografts showed contamination after storage in ethanol. Overall 13% of the patients had positive cultures on microbiological assessment.

Conclusion

The population of osteocytes in the harvested bone reduced significantly after processing with HCl and ethanol preservation. Presence of DNA, demonstrated by using Feulgen staining, was observed in bone marrow cells, adipocytes along with osteocytes which showed quantitative reduction on processing. Hence, antigenicity, conferred by cells and their DNA, reduced significantly after processing of decal bone. Contamination rate of banked decalcified allograft was 13%. Thus, culture and sensitivity tests should be carried out at each step of processing of decal bone allograft.

Introduction

Bonegrafts are required to reconstruct bone defects and augment bone healing [1].The autologous bonegraft is gold standard but with limited availability and additional donor site surgical complications as limiting factors [2]. Allografts can be used as a strut/buttress/to fill up cavities or as an augmentation in combination with autografts but with limited osteoconduction and osteoinduction properties [3]. They can be prepared from bone harvested following hemiarthroplasty/THA (head of femur) /femoral/tibial condyles following TKA or from freshly amputated limbs/cadavers.

Three types of bone allografts are Fresh frozen, Freeze-dried and Demineralised (or Decalcified) bone allografts.

Fresh Allografts are characterized by high resorptive activity and are associated with vigorous inflammatory as well as specific immune response. Due to development of better processing methods and understanding of immune responses, fresh bone allografts are rarely used now [4]. Frozen bone allografts, after harvesting, are stored as a sterile specimen at − 80 °C until transplanted. Freeze drying, where bone is freezed to make it dehydrated, alters the mechanical properties necessitating reconstitution (rehydration) of the graft while implantation. Decalcified/Demineralized bone allograft is promising/cost-effective as processing/preparation requires few chemicals and domestic refrigerator, making it economically viable and large volumes can be prepared/stored in a hospital/centres with limited resources.

Bone allografts can elicit immune responses in the hosts which may be triggered by the bone components, such as cells/collagen/fat/or matrix proteins, and is strongly correlated with the presence of Human Leukocyte Antigen (HLA)—a membrane bound immunological receptor [5]. Also, the cellular DNA may act as a trigger for a strong immune response through the cGAMP synthase enzyme [6]. The risk of occult infection due to contamination of allograft while harvesting/processing despite established tissue banking protocols is real [7, 8].

Present study was conducted to explore the cells/remnant DNA after processing of decalcified bone allograft using Feulgen staining to document the immunogenicity. The potential contamination of decalcified bone allograft, its microbiological culture during different phases (procurement, HCl treatment, ethanol preservation) of processing was analysed.

Material and Methods

The prospective observational study was conducted at a tertiary care center, New Delhi (November 2018–June 2020).Bone samples were harvested in 15 cases from the femoral head of the eligible patients undergoing Hemiarthroplasty/Total hip replacement and femoral/tibial condyles after TKA after obtaining informed written consent. The exclusion criteria to harvest bones were patient having history of malignancy/active infection/autoimmune disorders/taken live vaccine within 4 weeks/positive serology for HIV/HBV/HCV/history of diabetes mellitus/endocrine disorders and narcotics use (persons who report to non-medically administered IV/SC/IM injection of addictive drugs like heroine).

A thorough physical examination (to look for unexplained generalized lymphadenopathy, needle tracks or other signs of parenteral drug abuse, oral thrush, genital ulcers, etc.) of the prospective bone donor was carried out to rule out any active infection in the body. Blood samples were sent for blood grouping, CBC, ESR, CRP, serology for HIV/Hepatitis C/Hepatitis B preoperatively. Decalcified bone allografts were processed under aseptic condition by ‘sterile double jar technique’ developed by Nather [9, 10].

The procured bone was profusely washed with normal saline to remove blood/fluids (Fig. 1) and cleaned of any attached soft tissues (Fig. 2). Bone was then washed with hydrogen peroxide (Fig. 3A, B) followed by an antibiotic solution(1gram Vancomycin/100 ml saline) and finally washed with copious saline.Small piece (of size 1 cm × 1 cm × 1 cm, made using manual osteotome) from the harvested bone was sent (SampleA) for aerobic/anaerobic/fungal cultures, and for histopathological examination and feulgen staining (DNA analysis).

Fig. 1.

Procured bone being washed with normal saline

Fig. 2.

Removal of soft tissue attached to the procured bone using bone nibbler

Fig. 3.

Treatment of procured bone with Hydrogen Peroxide

The harvested bone was then placed in a sterile closed jar which was then put into a slightly larger outer jar and was sealed. The closed, sterile double jar covered with a sterile drape was labelled and was then transferred to the institute’s bone bank. In the bone bank, the procured bone was then put into a sterile glass beaker containing freshly prepared 0.6 N HCl (53 ml of HCl + 947 ml of distilled water) to achieve the decalcification. The bone was kept in the acid solution till it starts floating on the surface of the solution (Fig. 4) which marked the end of the  decalcification.

Fig. 4.

After the end of decalcification process in 0.6 N HCl, femoral head starts floating in the HCl solution

Decalcified bony specimen was then taken out from the jar under all aseptic precautions and was washed with copious volume of distilled water to neutralize the remaining acid. The bone samples (Sample B) were sent for aerobic/anaerobic/fungal culture and histopathology/DNA analysis. The decalcified harvested bone was then preserved in 70–90% ethanol in the domestic refrigerator (Fig. 5). At the completion of 3 months of ethanol preservation, two small bony pieces from the harvested bone were again sent (Sample C) for aerobic/anaerobic/fungal culture and histopathology/DNA analysis. The donor was again followed-up at 6 months post-harvesting for serological (HIV, HBV, HCV) testing and the donors who tested positive, were rejected.Only grafts of seronegative donors were stored for later clinical use.

Fig. 5.

Decalcified bony specimens (preserved in 70% ethanol) kept in domestic refrigerator

The samples were inoculated in appropriate culture media and kept for overnight incubation for aerobic isolation, 72 hrs for anaerobic bacterial isolation and 28 days for fungal isolation. The presence and identification of bacteria & fungus was documented and recorded. Estimation of bioburden (cfu/gm), as done in samples of endotracheal aspirates, urine, etc., was not done as the estimation is not possible in sterile samples like bone/blood/CSF.

For histopathological analysis, 5–7 micron thick sections were cut from the specimen and stained with haematoxylin and eosin stain (H&E) to study organic, inorganic, cellular components, lacunae and number of osteocytes. A trinocular microscope (Nikon Eclipse 80i) was utilized to count the number of osteocytes. They were counted manually under 40x in 3 fields of maximum cell density and then the average was taken. All other cells (hematopoietic bone marrow cells, endothelial cells, adipocytes etc.) demonstrating the presence of DNA on feulgen staining were studied.

Results

4 bone donors were male while 11 were female. The average age was 64 (40–75) years. 9 Patients underwent hemiarthroplasty for fracture neck of femur (n = 8)/fracture intertrochanteric femur (n = 1). 6  Patients of OA knee underwent TKA.

Histopathological Analysis

All samples showed numerous lacunae filled with variable number of osteocytes, which were apparent both on H&E and Feulgen staining. On histopathological study, the average number of osteocytes detected (under 40x) in maximum density area gradually decreased from 55.47 to 9.6 to 0.86 in sample A,B,C, respectively. This signifies the quantitative decrease in osteocytes due to processing of decalcified bone allograft.

Feulgen staining confirmed the presence of DNA in all types of our tissue samples (Sample A, B, C). The staining technique was first standardized using sections from the reactive lymph node (Fig. 6a). Sample A showed characteristics lamellar pattern of bone and purplish-red Feulgen staining in osteocytes, bone marrow cells and endothelial cells in all patients (Fig. 6b). In Sample B, the lacunae were occasionally filled with osteocytes and feulgen staining showed presence of DNA in osteocytes, bone marrow, adipocytes, endothelial cells (Fig. 6c), though staining was much less, quantitatively as well as qualitatively, as compared to Sample A. Sample C showed homogenous bony architecture and lamellar pattern with empty lacunae and marrow spaces (Fig. 6d). Only 7/15 sample C showed remnant of nucleus.

Fig. 6.

FEULGEN STAINING (Magnification 40x): a Control of feulgen stain on a lymph node biopsy. b Feulgen staining after harvesting of bone (numerous osteocytes visible in their respective lacunae along with staining of nuclear material). c Feulgen staining after HCl treatment (lamellar structure preserved; osteocytes and lacunae rarely visible). d Feulgen staining after ethanol preservation (bony tissue homogenized; no cell, lacunae or nuclear material visible)

Microbiological Culture

2/15(13%) of the patients had positive cultures on microbiological assessment. First patient (Case 5) had all the three sample A, B & C, positive for both aerobic and anaerobic cultures but with no growth on fungal culture. The organism isolated was Staphylococcus aureus in aerobic and gram-positive cocci in anaerobic (no species was identified further). Second patient (Case 8) had no growth in sample A but had positive growth in sample B and C in aerobic culture and the organism isolated was Staphylococcus epidermidis.

Thus, contamination demonstrated at the procurement was 6.67% (Staphylococcus aureus).After treatment with HCl (demineralisation), 7.14% of non-contaminated allografts were found contaminated (Staphylococcus epidermidis). None of the remaining 13 non-contaminated allografts showed contamination after storage in ethanol.

Discussion

The process of incorporation of autogenous bonegrafts is characterized by formation of new bone over a necrotic graft bed through the dual processes of resorption and substitution. The host response includes local hematoma formation, inflammatory and/or immune reaction to the graft material, processes of cell proliferation, osteoinduction, migration, differentiation and revascularization, resulting in new bone formation and union between graft and host. In fresh allografts, the processes of incorporation is qualitatively similar to those of autograft, but it occurs more slowly and is accompanied by extensive host response mediated by macrophages and lymphocytes via cell membrane receptors(MHC complex).

To reduce immunogenicity and encourage incorporation, allografts are processed (frozen/freeze dried/demineralised) [11]. Resorption, osteoconduction and osteoinduction proceed more rapidly with processed allografts, although remodelling and revascularisation are inferior as compared to autografts. When an allograft is processed via demineralisation, acid extraction of the graft leaves behind growth factors (BMP etc.), non-collagenous protein, and collagen while removing the mineral phase of the bone. This demineralised bone provides a suitable framework allowing instant permeability to reparative mesenchymal cells and neo-capillaries. The close contact between these cells of the host and the 'exposed' bone matrix induces the former to osteoblastic activity, and in successful implants, the original graft is gradually replaced.

The demineralisation in the preparation of decalcified bone allograft is carried out by 0.6 N HCl. The matrix retains high levels of BMP which is responsible for its osteoinductive property [12, 13]. When this decalcified graft is placed at the fracture site, there is osteoclastic and phagocytic resorption of calcium hydroxyapatite and cellular debris. This makes the graft porous through which neovascularization propagates. In decal bone, resorption of demineralized matrix is faster because of the prior removal of nearly half of minerals in the laboratory. Thus, decal bone provides an easily permeable scaffolding structure that permits creeping substitution [14].

This study used decalcified/demineralised bone allograft which is ethanol preserved and stored in refrigerator. They are economical and large volumes can be stored in a peripheral set up. But the question of contamination and its immunogenicity still remains.

Immunogenicity

Incorporation of a graft is influenced by the immunogenicity of the graft [15]. Even a decal bone once transplanted in the recipient would also evoke an immunogenic reaction [16]. Such an immunogenic reaction to a bone allograft is of “low threshold” and may last for a longer period; it is unlike allografting of solid organs where the immunogenic reaction is a sudden surge or “spike.” This antigenicity of bone can be reduced by processing the allograft by cryopreservation/ demineralisation/irradiation, etc.

The number of osteocytes detected (under 40x) in the 15 patient's samples, gradually decreased from an average of 55 per high density field in Sample A to less than 10 in Sample B and to less than 1 in Sample C. Death of osteocytes during decalcification process and storage in ethanol leads to decrease in the number of osteocytes and thereby decrease in the membrane bound immunological receptors, thus reducing immunogenicity, which could otherwise be triggered by the presence of human leukocyte antigen (HLA) on the cell membrane [15]. This is in accordance with a study on immunogenicity of decal bone, in which cellularity (CD4 and CD8 cells) in perigraft area was assessed by fine-needle aspiration cytology and it was concluded that decal bone did not excite an appreciably significant immunological response and partially decalcified allografts are a good substitute of autogenous bone grafts in clinical practice [17].

Also, various authors have described the immunological outcomes of allografting a decal bone over the years (Table 1) none of which have resulted in any local or systemic immune reaction.

Table 1.

Results of allografting decal bone in various studies

S. no	Author	Sample size (n)	Year	Any local/systemic immune reaction
1	Tuli, Srivastava et al. [36]	25	1988	None
2	Goel, Tuli et al. [14]	46	1992	None
3	Jain, Kumar et al. [16]	20	2015	None
4	Gupta, Keshav et al. [22]	42	2016	None

Some studies on other types of bone allografts have shown that some viable cells may persist despite the decontamination process, supporting the literature reports on the presence of anti-HLA antibodies in patients receiving allogeneic bone transplants [18–20].

The cellular DNA also may induce an immune reaction as cytosolic DNA may be the trigger of a strong immune response through the cGAMP synthase enzyme [6]. In our study, purplish-red feulgen staining, demonstrating the DNA content, was observed in all the decal bone allograft samples at the time of harvesting both in osteocytes and in bone marrow cells, which on subsequent processing by HCl was scarcely visible and after the preservation in ethanol was visible in negligible cells.

Although we did not find any study in the literature commenting on the DNA content of a processed decalcified bone, but Coutinho LF et al. (2017) evaluated the samples of fresh frozen bone grafts from three tissue banks in Brazil and demonstrated that light microscopy images from all the bone samples studied, showed presence of osteocyte-like cells in all groups and intense Feulgen staining, demonstrated the presence of DNA in all bone samples, even after tissue processing [21]. Further, the ultrastructural analysis also showed red blood cells in lacunae within the bone tissue.

Therefore, we can say, that although we were able to demonstrate cells and DNA (Feulgen staining) in the processed decalcified bone allograft but as none of the scientists utilizing decal bone allograft in the past has reported any local or systemic immunogenic reaction in the recipient’s body, the ability of these residual cells and nuclear material to provoke immunogenic reaction in recipient’s body is limited and insignificant.

Contamination

Various studies that have used decalcified bone as an allograft have reported post-operative infection as one of the complications [14, 16, 22]. Contamination during harvesting of the graft from donor and graft handling procedure is reported to range from 1 to 37% depending on the source and method used to culture the micro-organism [23–30]. At the time of procurement, we found 1 out of 15 allografts (6.67%) to be contaminated by aerobic species (Staphylococcus aureus). The growth of gram positive cocci in anaerobic culture was identified but specific species was not isolated.No study has reported the contamination rate in a decalcified bone allograft, during harvesting. Although, Chiu CK found the contamination rate of 9.3% in deep frozen/freeze dried allografts, during the time of retrieval of the donor bones (femoral heads) [31]. They found Staphylococcus epidermidis as the main contaminating agent during harvesting followed by Staphylococcus aureus. After the treatment with HCl, only one allograft out of 14 non-contaminated allografts was found to be contaminated. Thus, contamination rate after treatment with HCl was 7.14% and the contaminating species was Staphylococcus epidermidis. Staphylococcus aureus grows within a range of pH 4–10 [32]. It is reported that alcohol treatment does not affect the survival of these organisms [33, 34]. In both of our samples, organism survived the ethanol treatment, hence further study are required to resolve the issue.

Staphylococcus epidermidis is a Gram-positive bacterium, and is part of the normal skin flora. Such skin contaminants isolated from bone cultures of possible donors can be possibly due to inadequate decontamination of the patients skin pre-operatively and subsequent manipulation of sample during operative procedures. But, sterile cultures at the time of harvesting (Sample A) and presence of contaminants during the processing phase (Sample B and C) was an unusual but an important finding in this patient. The possible explanation could be peri-operative antibiotic coverage to the patient might have eliminated the organism in Sample A or the amount of bone tissue sent for the culture in Sample A might be insufficient and/or unusual delay in transportation of sample vials from operating theatre to the microbiology laboratory. Staphylococcus epidermidis could not be isolated on first culture (Sample A) probably due to any of the above-mentioned reasons. Subsequently the organism probably has survived the decalcification (HCl) and preservation (ethanol), and multiplied and subsequently recovered on cultures of Sample B and Sample C.

The sensitivity and specificity of Nucleic acid amplification technology(NAAT)is superior to culture isolation, hence if NAAT would have been applied to SampleA it probably could have identified the bacteria present in low concentration. Interesting thing to note here is, this patient-who had undergone Total Knee Replacement for osteoarthritis of knee, complained of persistent non-purulent serous discharge from the surgical site in follow-up. Multiple cultures of the discharge did not yield organism on culture, which could be explained by the patient being under coverage of intravenous broad-spectrum antibiotics in post-operative period. Although, after 1 month, patient recovered completely. Staphylococcus epidermidis is not an uncommon organism isolated in bone allograft cultures. L Sims studied the intraoperative cultures of 996 allograft bones and found that 43 (4.3%) had positive cultures and identified Staphylococcus epidermidis as the most common (22%) contaminating agent [35].

Authors have utilized decalcified bone (decal bone) allografts in various procedures like in repairing benign cystic lesions of bone, in repairing lytic lesions of bone, filling large osteoperiosteal gaps and repairing delayed union/atrophic non-union of bones [14, 22, 36, 37]. These authors reported complications arising out of allografting decal bone including post-operative infection (Table 2).

Table 2.

Organism isolated post-operatively after allografting decal bone

S. no.	Author	Sample size (n)	Year	Isolated organism (in post-operative period)
1	Tuli, Srivastava et al. [36]	25	1988	N/A
2	Goel, Tuli et al. [14]	46	1992	E. coli, Kleibsella, Proteus (10.86%)
3	Garg, Dev et al. [37]	16	1997	N/A
4	Jain, Kumar et al. [16]	20	2015	S. aureus (5%)
5	Gupta, Keshav et al. [22]	42	2016	S. aureus (7.14%)

Hence, we recommend that the culture and sensitivity tests should be carried out at each step of processing viz. procurement, after decalcification (HCl)and after ethanol preservation so that any sample of contaminated allograft can be excluded well before clinical use.

Conclusion

The population of osteocytes in the harvested bone reduced significantly after processing with HCl and ethanol. Presence of DNA was also demonstrated by Feulgen staining in bone marrow cells, adipocytes etc. along with osteocytes in these samples which showed quantitative reduction on processing. Hence, antigenicity, conferred by cells and their DNA is reduced significantly after processing of decal bone.

Contamination rate of banked decalcified allograft was found to be13% with isolated species as Staphylococcus epidermidis and Staphylococcus aureus.

This study established a histopathological evidence of the safety of decalcified bone allograft in terms of its antigenicity as done previously by other authors for fresh frozen allografts [21]. Decalcified bone allograft is a feasible option for limited resource countries as its processing and storage requires few chemicals and domestic refrigerator, making it economically viable and large volumes can be prepared/stored in a hospital/centres with limited resources. This would help to fulfil the unmet needs of bone allografts. However, to negate the risk of disease transmission, microbiological cultures must be carried out at each step of processing of decal bone allograft.

Funding

None.

Declarations

Conflict of interest

There is no conflict of interest of above-mentioned authors in this study.

Compliance with Ethical Standards

The authors declare that the above manuscript is in compliance with ethical standards for research, and have no potential sources of conflict of interest associated with its publication.

Ethics Approval

All procedures were in accordance with the ethical standards of the institutional research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards

Informed consent

All participants involved were recruited only from UCMS and GTB Hospital, Delhi, after getting an appropriate ethical clearance from the institutional board and after taking an informed consent. The authors also certify that informed consent was obtained from all the human participants in this study.