GB2469205A

GB2469205A - Bone washing process

Info

Publication number: GB2469205A
Application number: GB1005616A
Authority: GB
Inventors: John N Kearney; Paul Rooney; George Galea; Tom Mcquillan
Original assignee: Common Services Agency
Current assignee: Common Services Agency
Priority date: 2009-04-04
Filing date: 2010-04-01
Publication date: 2010-10-06
Also published as: GB0905859D0; GB201005616D0; GB2469205A8

Abstract

Bone intended for implantation into a recipient is sterilised using low concentrations of peroxide together with peroxyacid. The bone e.g. femur, is centrifuged to remove bone marrow. Washing steps are also included. Up to a 6 log reduction in bacterial spore count may be achieved in short contact times; and up to 15 log reduction in vegetative cells. The exemplified protocol comprises various sonication in water, rinsing, washing, agitation and centrifugation steps, followed by a chemical sterilant wash with 3% v/v hydrogen peroxide and 0.02% peracetic acid with sonication at 56-59°C, then a 70% ethanol wash (v/v) with sonication at RT followed by wash and agitation steps.

Description

BONE WASHING PROCESS

The present invention relates to a process for the sterilisation of bone intended for implantation into a human recipient.

Bone which has been recovered from living patients or from deceased donors has been a source of bone for bone replacement surgery for many years.

Importantly, it is desirable to sterilise the bone to avoid disease transmission, including bacterial and viral infection. It may also be beneficial in reducing any prion load that may exist. Naturally, the bacterial burden in bone from deceased donors is controlled by very strict donor selection procedures but it may still be present. In practice, femoral head bone harvested from living donors during primary hip replacement surgery have been the source of bone allografts for many years. The majority of this bone is issued as fresh frozen bone, with donor blood and bone marrow still present. If the bone was found to be bacteriology-positive, it can also be irradiated. In contrast, bone from deceased donors is processed (grinding, washing and some form of microbial inactivation/disinfection) prior to clinical use.

Processes for the removal of tissue and bone marrow have been disclosed in Lomas et al.; Cell and Tissue Banking (2000) 1: 193-200 and validated by Yates et al.; Cell and Tissue Banking (2005) 6: 277-285. The procedure involves a combination of sonication and shaking in sterile water; chemical treatment and centrifugation. Centrifugation is used to physically remove bone marrow. The authors report removing 98.2% of nucleated cells and 98.7% of soluble protein content from within intact femoral heads. However, such processing does not have the result of sterilisation of the bone. In fact since * S.S *.

S

the process is an open one, there is a concern that the bacterial bio-burden may S ** in fact be increased. The authors also report that many of the known ways to S..

sterilise bone may have deleterious effects on the mechanical and inductive

SS S *

properties of the bone.

A conventional method of sterilising bone has been to subject the bone (either as whole femoral heads or as bone morsels) to gamma irradiation (usually a dose of at least 25 kGy). However, gamma irradiation s not preferred by surgeons working in the field, since it tends to lead to reduction in the mechanical properties of the bone, to the extent that many surgeons will not use irradiated bone in view of its poor mechanical properties.

There therefore exists a need for a method of sterilising bone for implantation, which can successfully remove bone marrow components and reduce or eliminate the bacterial burden from bone without substantially affecting either the biological or biomechanical properties of the tissue. It would be preferred to have a process which could work both on bone harvested from living donors and also deceased donors.

The sterilisation of bone for implantation has previously been attempted using chemical means. WO 0158497 discloses the sterilisation of bone using peracetic acid. Other prior art documents disclose the sterilisation of bone using peroxide, for example WO 04067049 and WO 2005/082432.

However, the present invention is based on the surprising discovery that effective elimination of both vegetative bacterial cells and bacterial spores can be achieved using a synergistic combination of a peroxide and a peroxyacid, without resulting in any significant deterioration in the mechanical and inductive properties of the bone.

It is known to use combinations of peroxides and peroxyacids for purposes of sterilisation of tissue, infected surfaces, medical devices and surgical components (US 2006/0008494). The sterilisation of intestine tissue for * ** *** * * implantation with a mixture of peracetic acid and hydrogen peroxide is disclosed * ** * * * in WO 0032253. However, the treatment involves the use of 1% peracetic acid *** * and 10% hydrogen peroxide at a ratio of 5:1 by weight with contact times of 1 * S hour. There are concerns that such high levels of oxidising agents may cause *5 * * S S * ** damage or lead to cytotoxicity; whilst lower concentrations may be ineffective as sterilisation agents.

The present invention is based on the discovery that relatively low concentrations of peroxide and peroxyacid can successfully sterilise bone for implantation without significant degradation of the mechanical properties of the bone or other undesirable or harmful effects.

In particular, the present invention provides a process for the sterilisation of bone intended for implantation into a recipient, which comprises: washing the bone with a solution comprising a peroxide and a peroxyacid.

Generally, the sterilisation step is provided as part of an overall process of cleaning the bone ready for use. In particular, the cleaning process may include the removal of remaining tissue and bone marrow, which may harbour contamination by a series of washing, agitation and centrifugation steps, as will be described in more detail. Marrow components (soluble protein, DNA and haemoglobin) may be removed by up to 99.7% of the original load.

The sterilisation solution will generally be an aqueous solution comprising a peroxide and a peroxyacid.

The peroxyacid may be selected from peracetic acid, perpropionic acid, perbenzoic acid etc. Generally, the peroxyacid is used in a concentration of 0.002-0.2% v/v, particularly 0.005-0.1% and especially 0.01-0.05%. Peracetic acid is preferably used, typically at a concentration of 0.01-0.03%.

Various peroxides may be used including hydrogen peroxide, benzoyl peroxide and sodium peroxide. Preferably, hydrogen peroxide is employed, S..

typically at a concentration of 0.05-5% v/v, particularly 0.1-3% v/v.

*** s** * * It has surprisingly been found that employing relatively low concentrations * . * * * of peroxide and peroxyacid, can nevertheless result in very good sterilisation of * the bone. Previously, there were concerns that high concentrations could cause *SS* * I

SI I. S * I I * SI

damage to the bone itself, and the use of low concentrations, particularly of peracetic acid, is surprisingly effective.

The bone may be harvested from either living donors (when the infection status of the donor may be monitored) or from deceased donors, where the bacterial contamination may be high. However, the use of the chemical sterilisation system according to the present invention avoids the need for the use of gamma radiation, so that sterilisation may be carried out without substantial change to the mechanical properties of the bone. The bone may be used as intact bone structures, for example femoral head bone, or may be morseiised (i.e. rendered into small pieces of bone), which may be readily sterilised, and which are useful for filling gaps in the bone structure during reconstruction. The live, host bone normally grows more readily into gaps filled with morselised bone pieces.

The sterilisation process of the present invention is capable of removing up to three log10, up to four log10, up to five log10 and particularly up to six log10 reduction in the bacterial spore count on the bone for a relatively short contact time in the order of 1 -60 minutes, preferably 5 -30 minutes. Sterilisation may achieve up to a 5 log10 and particularly up to a 15 log10 reduction in vegetative cells.

D-values (the time required to reduce bacterial cell numbers by 90%) in the range 20 to 100 seconds, particularly 30 to 60 seconds, may be achieved.

Sterilisation is accomplished without the requirement for irradiation of the tissue.

The bone, when harvested from a living donor, is generally stored at low S...

temperatures, for example -80°C until required. Bone from diseased donors may * ,i * also be used. S.

Before sterilisation, the bone is generally cleaned to remove tissue and *.a bone marrow by a process involving multiple washing and rinsing steps. Each * S..

step generally takes from 5 -45 minutes, usually from 10 -30 minutes. Usually, the washing steps take place at a temperature from 50 -65°C, particularly 56 -59°C.

As a first step, the bone may be sonicated in water to loosen extraneous material. It is then rinsed. There then follows a wash and agitation step carried out using an agitator at 100 -300 rpm. The bone may be centrifuged in the dry state, to remove bone marrow, e.g. by centrifugation at 1500g -2000g.

Typically, there are 3 -5 wash and agitation steps and 3 -5 centrifugation steps.

Once the bone has been effectively cleaned of extraneous material, it is washed with the solution comprising a peroxide and a peroxyacid, as described above. This may be sonicated, if desired.

Following the sterilisation step, the bone may be washed with alcohol e.g. 70% ethanol with sonication at room temperature, to aid in lipid removal and provide a bactericidal/viricidal treatment.

Finally, there may be one or more wash and agitation steps to remove any chemical residues.

Some of the washing steps also contribute to the bactericidal effect of the process. For example, sonication in water has been found to reduce the vegetative cell number. Also, the treatment in alcohol instantaneously kills at least 106 cfu/ml of vegetative cells. The process has been found to be non-cytotoxic and the mechanical strength of the bone is largely unaffected.

Embodiments of the present invention will now be described by way of example only with reference to the following Figures: Figure 1 shows washed and unwashed femoral heads; * *** * **S* Figure 2 shows MG63 cells added to unwashed bone; * S Figure 3 shows MG63 cells added to washed bone; * ** Figure 4 shows washed femoral head half on the left with unwashed half

S

from the same femoral head on the right. The area between the dashed lines * SSS * * shows where cubes of bone were cut from for testing; ** 0 * * * * *0 Figure 5 shows Young's modulus derived from compressing cubes cut from washed and unwashed donor bone from three individual donors. No significant differences were observed between paired samples using Student's t-test or ANOVA of the three donors; and Figure 6 shows strain at yield derived from compressing cubes cut from washed and unwashed donor bone from a further three donors Example I -Bone washing and sterilisation protocol Femoral head bone is collected from living donors in an operating theatre during primary hip replacement. The bone is put into a polypropylene container, closed and then placed inside another, larger polypropylene container and placed in a freezer on site to await collection.

The femoral head container is then transferred to a -80°C freezer in the tissue bank where it is stored, untouched until release and issue to a surgeon. In this project, the container is removed from the freezer and allowed to thaw in a coidroom overnight. Once thawed, the bone is trimmed of all soft connective tissue and transferred to a fresh sterile polypropylene container. Pre-heated water (350 ml) is added, the container is placed into the sonicating water bath and the protocol in Table 1 is followed. All solutions are used at a volume of 350 ml, centrifugation is performed dry but the bone is placed on a platform with holes in it to collect expelled bone marrow. A fresh container is used after each centrifugation step. *.** * * * ** * * . * * *.* * * .** * * **S* ** * * S S *

Table I -femoral head washing protocol _______________ Time 1 Sonication in water 15 mins (56-59°C) ________ 2 Rinse (56 -59°C) 5 mins 3 Wash and agitation (200 30 mins rpm, 56 -59°C) _________ 4 Centrifugation (1850g) 15 mins Wash and agitation (200 10 mins prn, 56-59°C) _______ 6 Centrifugation (1850g) 15 mins 7 Wash and agitation (200 10 mins rpm, 56-59°C) _______ 8 Centrifugation (185Q9) 15 mins 9 Wash and agitation (200 10 mins rpm, 56 -59°C) _________ Chemical sterilant wash (3% 10 mins v/v/ Hydrogen Peroxide plus 0.02% v/v Peracetic acid) with sonication at 56 -59°C _________ 11 70% Ethanol wash (v/v) with 10 mins sonication at RT __________ 12 Wash and agitation (200 10 mins rpm, 56 -59°C) __________ 13 Wash and agitation (200 10 mins rpm, 56 -59°C) _________ Reagents: Hydrogen peroxide -diluted from a 30% solution Peracetic acid -diluted from a 36 -40% solution with sterile water.

Ethanol -70% denatured ethanol.

Other consumables: Centrifugation containers -polypropylene containers.

Support -made in house from square fibreglass tube and polypropylene *.* . . . circle with holes drilled in it. * *

Agitation is performed in an orbital incubator I * ** ** * Sonication is performed in a heated sonicator. * **** * * **S. ** * * * * *

Example 2 (results)

The following tests were carried out using the washing and sterilisation protocol described in Example 1.

1) Bactericidal activity of washing steps A washing protocol (Table 1) has been developed to clean intact femoral head bone of blood and bone marrow components (Figure 1).

The steps in the protocol (Table 1) were individually investigated for their ability to kill bacterial cells. Potential bactericidal steps were Step 1, Step 3 (and 5, 7 and 9) Step 10 and Step 11. Two bacterial cell types were tested -vegetative cells (Staph. epidermidis) and bacterial spores (Bacillus atrophaeus).

Step 3 (and 5 and 7 and 9) had rio bactericidal effect. Steps 1 and 11 did have bactericidal properties but only on vegetative cells, they had no effect on bacterial spores. Step 10 had bactericidal effects on both vegetative cell and spores.

Step 1 (sonication in water) -reduced vegetative cell number by 2.71 log1.

Step 11 (sonication in 70% ethanol) -instantaneous killing of all cells -at least a 6.0 log10 reduction.

Step 10 (sonication in the peroxide and peroxyacid combination) -reduced spore number by 6 log10. D-values (the time required to reduce bacterial cell numbers by 90%) were calculated and were found to be less than 40 seconds. Therefore in the 10-minute treatment period, there would be a reduction in Bacillus cell number of approximately 15 log10.

A weaker combination of 0.1% H202/0.02% PAA also produced a 6-log * I reduction in spore number but the D-value was 1.41 minutes and therefore in the * I. 10-minute treatment period, only a 7-log reduction would be achieved. *.

Since vegetative cells are easier to kill than spores, the log reduction in * .1S * I vegetative cells will be at least the same as for spores. I. * * * * * **

Consequently, the killing potential of the entire process for vegetative cells, i.e. Step 1(2.71) plus Step 10 (-15) plus Step 11 (-6) = 23.71 log10. For bacterial spores, the washing process would produce a 15 log10 reduction.

2) Cytotoxicity Small pieces of washed bone, snipped off from the ends and from the centre of washed bone were tested for contact and extract cytotoxicity, as per British Standard 10993-5. Contact cytotoxicity is where a piece of tissue is attached to the centre of a tissue culture dish and cells are added to the dish -if cells grow up to and onto the tissue there is no cytotoxicity, if cells do not grow up to the tissue, or their morphology changes considerably, there may be cytotoxicity. Extract cytotoxicity is where small/minced pieces of tissue are soaked in solution overnight and then the solution is added to a growing culture of cells and cell viability/activity is assessed visually and by a standard MTT assay. Two cell types were used, human skin fibroblasts and MG63 an osteoblastic cell line. Similar data were obtained for both cytotoxicity assays but only contact results are shown below.

Unwashed bone was found to be cytotoxic to MG63 cells (Figure 2), in contrast, no cytotoxicity was observed in washed bone according to the invention (Figure 3).

3) Biomechanical strength.

Frozen femoral heads were cut in half, one half was stored frozen *.S.

I III*

(unwashed) and the other half was washed as per Table 1 (invention). The I..... * I

unwashed halves were then thawed and the two halves were used as paired * II samples (Figure 4). Cubes or rectangles were cut from a central region (as

III

shown) and marked to maintain correct alignment such that compression was

I

always along the long axis of the bone. Each sample was tested for compressive

II S * I S * *S

strength using a universal compressive testing machine (Lloyds Instruments) with a preload of 0.2N, speed 5 mm/mm and stop at 30% deflection using a 5 kN load cell. The mean Young's modulus, load at yield and percentage strain at yield were recorded. Figure 5 shows examples of mean Young's modulus for three donor bones and Figure 6 shows examples of strain at yield for a different three donor bones -there is no significant difference between washed and unwashed bone.

4) Conclusion.

The data presented here indicate that when ferrioral head bone is processed through the entire washing protocol shown in Table 1, the protocol is capable of killing at least 15 log10 of bacterial spores and greater than 23 log10 of vegetative cells. In addition, at the end of the protocol the tissue is not cytotoxic nor does the bone lose any of its structural strength. S... S *

S

S..... * . * .5 * S S 4.

S *..

S 55 * S

S * S S * **

Claims

CLAIMS1. A process for the sterilisation of bone intended for implantation into a recipient, which comprises: washing the bone with a solution comprising a peroxide and peroxyacid.
2. A process according to claim 1, wherein the peroxyacid is selected from peracetic acid, perpropionic acid and perbenzoic acid.
3. A process according to any preceding claim, wherein the peroxyacid is used in a concentration of 0.002 to 0.2%.
4. A process according to claim 3, wherein the peroxyacid is used in a concentration of 0.01 to 005%.
5. A process according to any preceding claim, wherein the peroxide is hydrogen peroxide, benzoyl peroxide or sodium peroxide.
6. A process according to claim 5, wherein hydrogen peroxide is used at a concentration of 0.05 to 5% v/v.
7. A process according to claim 6, wherein hydrogen peroxide is used at a concentration of 0.1 to 3% v/v. * * * *.**
8. A process according to any preceding claim, wherein as a first step the * bone is sonicated in water. * * S.. * *S C. SS * . I...SI I * * S I C'
9. A process according to claim 8, wherein the bone is then washed in an agitator at 100 -300 rpm.
10. A process according to any preceding claim, wherein the bone is centrifuged to remove bone marrow.
11. A process according to any preceding claim, wherein, following washing with peroxide and peroxyacid, the bone is washed with ethanol.
12. A process according to any preceding claim, wherein the washing with peroxide and peroxyacid acid is for 5 to 30 minutes. S.. S..S* S.* S. * . * eS *.* SSS (eIS * S S.., S. S * S *