Cell Tissue Bank DOI 10.1007/s10561-015-9504-y
Development of an improved bone washing and demineralisation process to produce large demineralised human cancellous bone sponges Mark J. Eagle • Paul Rooney • John N. Kearney
Received: 9 December 2014 / Accepted: 26 February 2015 Ó Springer Science+Business Media Dordrecht 2015
Abstract Shaped demineralised bone matrices (DBM) made from cancellous bone have important uses in orthopaedic and dental procedures, where the properties of the material allow its insertion into confined defects, therefore acting as a void filler and scaffold onto which new bone can form. The sponges are often small in size, \1.0 cm3. In this study, we report on an improved bone washing and demineralisation process that allows production of larger DBM sponges (3.375 or 8.0 cm3) from deceased donor bone. These sponges were taken through a series of warm water washes, some with sonication, centrifugation, 100 % ethanol and two decontamination chemical washes and optimally demineralised using 0.5 N hydrochloric acid under vacuum. Demineralisation was confirmed by quantitative measurement of calcium and qualitatively by compression. Protein and DNA removal was also determined. The DBM sponges were freeze dried before terminal sterilisation with a target dose of 25 kGy gamma irradiation whilst frozen. Samples of the sponges were examined histologically for calcium, collagen and the presence of cells. The data indicated lack of cells, absence of bone marrow and a maximum of 1.5 % residual calcium.
M. J. Eagle (&) P. Rooney J. N. Kearney NHSBT, Tissue Services R&D, 14 Estuary Banks, Speke, Liverpool L24 8RB, UK e-mail: [email protected]; [email protected]
Keywords Demineralised bone sponge Vacuum Protein DNA Allograft
Introduction NHS Blood and Transplant have developed a rapid method of demineralising cancellous bone under vacuum which results in a demineralised bone matrix (DBM) sponge in which, in excess of 99 % of soluble protein, DNA and haemoglobin has been removed and which has proved osteoinductive in an animal model (Eagle et al. 2014). Demineralisation was performed under a vacuum and could be achieved in less than 1 h. Demineralisation of this bone results in a spongy, compressible bone scaffold which can be used as a bone void filler and which could be used in combination with DBM paste/putty or powder to stimulate improved bone healing. There is the potential to use such demineralised bone sponges in the field of dentistry, maxillofacial surgery or in treatment of osteotomy where the demineralised bone can be manipulated into gap-forming defects ( Mulliken et al. 1981, Rosenthal et al. 1999). One potential drawback to the DBM sponges is their size, 1.0 cm 9 1.0 cm 9 1.0 cm (1 cm3) and surgeons have requested larger sponges of dimensions 1.5 cm 9 1.5 cm 9 1.5 cm [3.375 cm3 (3.4 cm3)] and 2.0 cm 9 2.0 cm 9 2.0 cm (8.0 cm3). We have developed a bone washing protocol which is capable of removing 99.9 % of soluble protein, DNA
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and haemoglobin from cortical and cancellous bone using only ethanol as an organic solvent. We have omitted harsher, more volatile organic solvents such as chloroform, ether, acetone etc. Which could have harmful effects on processing staff or could recirculate within a clean room environment (Eagle et al. 2015). This bone is non-cytotoxic, the biomechanical properties have not been affected and it has been used as a scaffold to allow attachment and proliferation of mesenchymal stem cells (Smith et al. 2014). The bone washing process has been used on 1.0 cm3 mineralised and demineralised cubes/sponges, however larger volume cubes contain more marrow components and the published process could not fully remove all marrow and lipid from the larger cubes, therefore, in this report we have improved the bone washing process, incorporating longer incubations in 100 % ethanol under vacuum to fully wash the bone cubes. During demineralisation, the acid removes mineral from bone and exposes bone morphogenetic proteins (BMPs) (Mauney et al. 2005), at the same time the reaction releases CO2 gas. This is not a problem for ground cortical bone powder but is a problem for 3-dimensional cancellous bone cubes, due to the trabecular nature of the tissue, CO2 can get trapped in the porous trabeculae and the bone cube floats; the gas can also prevent the acid from coming into contact with the bone surface and delay demineralisation. We have overcome this problem by demineralising cancellous bone cubes under a vacuum and have used the vacuum system in this report (Eagle et al. 2014).
Materials and methods Tissue Bone was obtained from the knees of three donors, age range 45–63 years. Full ethical consent for use of the tissue in research and development was obtained. Bone washing Knees, including regions of proximal tibia and distal femur, were stored at -80 °C until required. These were allowed to thaw at 4 °C overnight and soft tissue and cortical bone was dissected off leaving the cancellous portion of bone. Cancellous bone from the distal femur was cut into slices of 2.0 cm thick and then
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further cut into appropriately sized cubes with a saggital saw. Up to 12 2.0 cm 9 2.0 cm 9 2.0 cm cubes could be obtained from one donor. Cancellous bone from the proximal tibia was cut into slices of 1.5 cm thick and then further cut into appropriately sized 1.5 cm 9 1.5 cm 9 1.5 cm cubes; up to 24 cubes could be obtained from one donor. These mineralised cubes were then washed according to a protocol adapted from Eagle et al. (2014) that included two 100 % ethanol washes and centrifugations (Table 1). All washes and rinses were performed in warm water (Baxter, sterile water for irrigation, UKF7114) at 56–59 °C (standard bone allograft washing temperature used in our tissue bank) except those in hydrogen peroxide (Sigma, 30 % (w/w) solution (H1009) diluted in sterile water to 3 % solution (v/v)) (Step 10, Table 1) or 100 % ethanol (VWR, AnalaR, L992101) (Steps 11 and 13, Table 1) which were performed at room temperature. Studies have shown that recombinant BMP-2 can withstand exposure to temperatures up to 70 °C (Ohta et al. 2005). Sonication was performed in a Decon F5300b sonicating water bath. All wash solutions were collected; the volumes were measured and samples were then taken for determination of soluble protein, and DNA content. Assessment of bone marrow component removal Wash solutions were collected at each step of the process (Table 1) and the total amounts of soluble protein and DNA were calculated by multiplying the concentration, obtained as described below by the volume of each wash solution. Residual components were calculated by immersing fully washed bone in a container with a volume of pre-heated sterile water equivalent to at least 5 times the weight of bone (vol/ w). The bone was agitated in an orbital shaker (56–59 °C) for 1 h at 200 rpm. The water was removed and replaced with fresh sterile water and the agitation was repeated. Both residual wash solutions were combined for measurement (a preliminary study using 10 consecutive washes demonstrated that no further residual components could be detected after the second wash, therefore no more than two washes were required). Once residual values were ascertained, they were added to the total amounts in each wash solution and the proportions removed at each step were evaluated. The amount removed was expressed as a percentage removal from the bone.
Cell Tissue Bank Table 1 Bone wash procedure
Step
Time
1
Sonication in water (56–59 °C)
15 min
2
Rinse (56–59 °C)
5 min
3
Wash and agitation (200 rpm, 56–59 °C)
30 min
4
Centrifugation (1850 g)
15 min
5
Wash and agitation (200 rpm, 56–59 °C)
10 min
6
Centrifugation (1850 g)
15 min
7
Wash and agitation (200 rpm, 56–59 °C)
10 min
8 9
Centrifugation (1850 g) Wash and agitation (200 rpm, 56–59 °C)
15 min 10 min
10
Hydrogen peroxide wash (3 % v/v) with sonication at RT
10 min
11
100 % Ethanol wash with 5 min vacuum, and then agitation at 70 rpm
Overnight
12
Centrifugation (1850 g)
15 min
13
100 % Ethanol wash with vacuum
5 min
14
Centrifugation (1850 g)
15 min
15
70 % Ethanol wash with sonication
10 min
16
Wash and agitation (200 rpm, 56–59 °C)
10 min
17
Wash and agitation (200 rpm, 56–59 °C)
10 min
18
Centrifugation (1850 g)
15 min
Protein removal A standard curve was prepared using bovine serum albumin (Sigma) at a range of concentrations, 100 ll of each concentration was pipetted into a well of a 96-well microplate (Nunclon). 100 ll of every wash solution (from Table 1) and residual soaks were also pipetted into the microplate. To each well, 100 ll of Bradford reagent (Sigma, UK) was added and mixed before incubating at room temperature for 5 min. The solutions were measured on a microplate reader (Elx808, Biotek) at 595 nm against a blank of distilled water and Bradford reagent. A graph of the standard curve was produced from which protein concentrations could be calculated. Absolute protein amounts were calculated taking into account dilution factors and volumes of wash solutions. The values were used to calculate the amount of protein present in each wash solution. Using the residual wash values, the overall percentage protein removal from the bone samples was calculated. DNA removal A standard curve was prepared from calf thymus DNA (Sigma) at a range of concentrations, 100 ll of each concentration was pipetted into a well of a 96-well
black bottomed microplate (Nunclon) 100 ll of every wash solution (from Table 1) and residual soaks were also pipetted into the microplate. To each well, 100 ll of Picogreen reagent was added and the plate was gently shaken to mix and incubated at room temperature for 5 min. The microplate was read on a fluorescence microplate reader (Flx 800, Biotek) with excitation at 485 nm and emission at 528 nm. A graph of the standard curve was produced from which DNA concentrations could be calculated. Absolute DNA amounts were calculated taking into account dilution factors and volumes of wash solutions. The values were used to calculate the amount of DNA present in each wash solution. Using the residual wash values, the overall percentage of DNA removal from the bone samples was calculated. Acid demineralisation of mineralised cancellous cubes Acid demineralisation, at a ratio of 1 g bone: 50 ml 0.5 N HCl, resulted in the continuous production of CO2 which displaced the acid solution within the trabeculae of the cancellous bone thus leading to uneven demineralisation within the graft; and it also caused the grafts to float on the surface of the solution, again leading to incomplete demineralisation. Experiments with
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unsecured cubes and at atmospheric pressure also showed a long time period for demineralisation to take place (not shown). To overcome these problems the cubes were placed inside an acetyl plastic ring surrounded by a polyethylene mesh cage (Fig. 1) presterilised by gamma irradiation. This ensured that the cubes were submerged within the demineralisation chamber. Up to 4 9 8.0 cm3 cubes from a single donor could be placed inside the chamber or 12 9 3.4 cm3 cubes at one time. CO2 was removed from the demineralising cubes by applying a continuous vacuum of 270 mbar to the demineralisation chamber (Fig. 1). This enabled uniform demineralisation throughout each cube. All connections were made with 8 mm outer diameter silicone tubing, and luer lock connectors. A 0.22 lm filter (Sterivex, Millipore, UK) was connected between the pump and chamber. After addition of the acid solution to the chamber, the chamber was closed and the vacuum pump (Dymax 14, Charles Austin Pumps, UK) was started. The level of vacuum was monitored using a vacuum gauge (DVR2, Vacuubrand, UK). Acid was continually stirred at 500 rpm by a 40 mm magnetic stirrer with crosshead, operated by a magnetic stirring plate. 3.4 cm3 sponges were demineralised for 24 h and 8.0 cm3 sponges were demineralised for 48 h. At the end of demineralisation, the acid was poured off and an equal volume of distilled water was added and stirred continuously for 20 min, again with vacuum applied to the chamber. Sequential washes with water, PBS, PBS, water and water were carried out, also with stirring at 500 rpm. The pH was measured to ensure a neutral value before the final water wash was discarded and each DBM sponge was gently squeezed between thumb and index finger to remove excess water. This also served as a manual test
of demineralisation, as any large remaining calcium deposits would prevent the cube from being completely compressed. After compression the cubes were seen to return to their original dimensions. The bone was then stored frozen at -40 °C. Terminal sterilisation The frozen DBM sponges were lyophilised in a freeze drier (VirTis, Advantage) using an established programme for production of freeze-dried bone and subsequently irradiated at 25–40 kGy (target dose 25 kGy, Synergy Health, UK) packed in dry ice at circa -79 °C. Irradiation of lyophilised DBM at low temperature has been reported to maintain the osteoinductive potential of the DBM (Wolfinbarger et al. 2008; Qiu and Connor 2008). Residual calcium determination Three 3 DBM sponges of both sizes from each donor, demineralised for 24 or 48 h were placed in a drying oven and left overnight at 120 °C. The dried sponges were weighed before being placed in individual containers with 10 ml of 12 N HCl (for 3.4 cm3 sponges) or 20 ml (for 8.0 cm3 sponges) to achieve complete hydrolysis. Once dissolved, the acid was diluted by addition of 30 ml (for 3.4 cm3 sponges) or 60 ml (for 8.0 cm3 sponges) of distilled water. Calcium content was measured using an assay kit (Quantichrom, Universal Biologicals, Cambridge, UK) following the manufacturers instructions. Briefly a standard curve was set up via a series of dilutions from the supplied calcium standard. 5 ll duplicate samples of the standards and the diluted hydrolysed DBM sponges of each donor were added to individual wells on a clear 96 well microplate. 200 ll of the assay reagent was then pipetted into each well, with the plate then left at room temperature for 3 min. Absorbance was read on a microplate reader (Elx 808, Biotek, UK) at 595 nm. The percentage weight of calcium to weight of dry bone was then calculated. Histological evaluation
Fig. 1 Apparatus setup of vacuum pump equipment, safety flask and pH probe
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Irradiated and non-irradiated DBM sponges were cut in half diagonally and then fixed in 10 % neutral buffered formalin for at least 2 days. The fixed tissue was then processed according to standard histology
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practice before being embedded in paraffin wax, with the diagonally cut surfaces face down in the wax mould. The wax embedded tissue was then sectioned at 5–10 lm thickness. Sections were stained with a range of stains including H&E for general visualisation, Van Gieson’s for appearance of collagen (Bradbury and Gordon 1990), Alizarin red S for mineral (Stevens 1990) and DAPI (Sigma, UK) for DNA. Sections were examined under an inverted microscope and photographs taken with a digital camera (Coolpix 5000, Nikon, UK). Statistical analysis Data for the calcium assay are represented as means with bars showing 95 % confidence intervals. One way analysis of variance (ANOVA) was used to calculate significant difference between multiple means. Difference between data was considered statistically significant when the p \ 0.05.
Results Bone washing and marrow component removal The 100 % ethanol washes visibly removed lipid from the cubes prior to demineralisation, and allowed the successful decalcification of the cubes to take place (Fig. 2). Analysis of the wash solutions showed mean protein removal of 98.8 % and mean DNA removal of 98.1 % (Table 2). The patterns of component removal for soluble protein from 8.0 and 3.4 cm3 sponges are shown in Figs. 3 and 4 respectively and for DNA removal in Figs. 5 and 6 respectively. Residual calcium determination Residual levels of calcium were measured in three DBM sponges of both sizes from three different donors. 3.4 cm3 sponges were demineralised for 24 h and 8.0 cm3 sponges were demineralised for 48 h. Residual calcium values are shown in Fig. 7. Mean residual calcium levels in 3.4 cm3 sponges were 0.49 % (±0.23 %) of the DBM sponge weight and residual calcium levels in 8.0 cm3 sponges were 0.93 % (±0.27 %) of the DBM sponge weight (Fig. 7). All mean residual calcium values were below
Fig. 2 8.0 cm3 DBM sponges at various stages of production. Washed mineralised cubes on the left, washed and 100 % ethanol treated cubes in the centre and demineralised sponges on the right
1.5 %, statistical analysis of the data with ANOVA showed no significant difference in residual calcium levels in the 3.4 cm3 sponges between donors but a significant difference was observed in 8.0 cm3 sponges between the three donors (p \ 0.01). Histology H&E staining of DBM sponges demonstrated a bony trabecular network with absence of any cells (not shown). Van Gieson’s staining demonstrated intact trabeculae after demineralisation with intense collagen staining and no apparent sign of degradation (Fig. 8a, b). Alizarin red S staining demonstrated absence of calcium (Fig. 8c, d). DAPI staining demonstrated that the process essentially decellularised the tissue with no indication of fluorescent nuclei (Fig. 8e, f). Irradiation had no effect on collagen structure or residual calcium appearance.
Discussion Demineralised cancellous bone sponges are used by surgeons to fill bony defects where, due to the compressibility of the bone and the interporosity of the trabecular scaffold, the grafted bone can be ‘‘pressfitted’’ into small cavities to act as a bone void filler and an osteoconductive environment to allow bone cell attachment and growth. Clinically, DBM sponges have been used to treat osteochondral lesions of the talus and have been reported to cause no complications or immune reactions and have been reported to aid in alleviation of pain and disability (Bleazey and Brigado 2012; Galli et al. 2014). NHS Blood and Transplant have developed a method of producing DBM sponges,
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Cell Tissue Bank Table 2 Summary of overall component removal from different sized cubes Component
Cube size (cm3)
% component removal Donor 1
Protein DNA
Donor 2
Donor 3
Donor mean
Component mean 98.79
8.0
98.15
98.23
98.83
98.40
3.4
99.37
99.09
99.08
99.18
8.0
95.62
97.17
99.65
97.48
3.4
98.57
97.77
99.81
98.72
98.10
Fig. 3 Pattern of mean protein removal from 8.0 cm3 sponges, N = 3, Error bars = 95 % CI
using mineralised cancellous bone cubes as the starting material, which are compressible, non-cytotoxic and are osteoinductive when implanted intramuscularly into mice (Eagle et al. 2014). The procedure implemented to produce these DBM cubes also removes blood, bone marrow components and essentially decellularises the sponge. However, the sponges are 1.0 cm 9 1.0 cm 9 1.0 cm and as such could only be used to fill small bone defects; there is a need to be able to produce a larger DBM sponge. DBM sponges are available commercially but the largest volume sponge this group was able to find was 1.4 cm 9 1.4 cm 9 1.4 cm (2.75 cm3, OsteoSponge, Bacterin, USA). In this report we describe methodology to wash and demineralise DBM sponges with volumes of 3.4 and 8.0 cm3. Using a stringent bone washing process, bone cubes of 1.5 cm 9 1.5 cm 9 1.5 cm and 2.0 cm 9 2.0 cm 9 2.0 cm demonstrated in excess of 98.1 % of DNA and
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soluble protein removal. Histological examination could not detect any cells either in the marrow space or within the bony trabeculae, indicating that the process also decellularises the bone. The washing process used 100 % ethanol, warm water washes and centrifugation to remove marrow components without the need to use volatile organic solvents such as chloroform, ether or acetone (Eagle et al. 2015). Warm water washes were performed at 56–59 °C, our experience of washing mineralised allograft bone at this temperature range indicates no deleterious effect on the clinical outcomes achieved. In addition, 1.0 cm3 demineralised sponges produced using solutions in the same temperature retain an osteoinductive potential (Eagle et al. 2014). We are investigating the possibility of using lipase to aid in the removal of lipid (Zhang et al. 2014). The use of a vacuum during demineralisation has ensured that any CO2 gas, released as the 0.5 N HCl acted on bone mineral, was removed from the cubes/
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Fig. 4 Pattern of mean protein removal from 3.4 cm3 sponges. N = 3, Error bars = 95 % CI
Fig. 5 Pattern of mean DNA removal from 8.0 cm3 sponges. N = 3, Error bars = 95 % CI
sponges allowing acid to continually come into contact with the bone surface. The resultant DBM sponges from three human donors had mean residual calcium levels below 1.5 % of the dry weight of the DBM sponge. Mineralised bone has approximately 28 % calcium by weight (Eagle et al. 2014) and the American Association for Tissue Banks has stated that for a bone to be classed as demineralised it must contain less than 8 % residual calcium by weight (Bethesda 2006). Consequently the bone sponges
produced here can be classed as demineralised. Different sizes of mineralised bone cube required different times of demineralisation, in this report 24 h was found to demineralise all 3.4 cm3 sponges and 48 h was found to demineralise all 8.0 cm3 sponges. Most studies on demineralised bone have reported on ground cortical bone as the starting material and several reports have suggested that cancellous bone contains low levels of BMPs and as such are mostly osteoconductive (Mauney et al. 2005), although a
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Fig. 6 Pattern of mean DNA removal from 3.4 cm3 sponges. N = 3, Error bars = 95 % CI
Fig. 7 Residual calcium values in 3.4 and 8.0 cm3 DBM sponges. Values are expressed as a percentage of the dry weight of demineralised sponges. N = 3, Error bars = 95 % CI
recent study indicated that cancellous bone contains similar levels of BMP2, BMP4 and BMP7 to those found in cortical bone (Shi et al. 2012). In addition, the nature of the porous bony trabeculae could provide a suitable microenvironment for osteoblastic cell attachment and differentiation, again previously reported (Shi et al. 2012; Smith et al. 2014). We have not investigated the osteoinductive potential of these
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larger DBM sponges, however, we have previously demonstrated that 1.0 cm3 cancellous DBM sponges, produced using similar methodology as described here, are osteoinductive when implanted into a rodent model (Eagle et al. 2014). In this report, demineralisation proceeds under closed conditions and each of the reagents and equipment used in this study are Good Manufacturing Practice (GMP) compliant and are
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Fig. 8 Micrographs of DBM sponges stained with Van Gieson’s for visualisation of collagen, Alizarin red for calcium and DAPI to show cell nuclei (3.4 cm3 sponge a, c, e; 8.0 cm3 sponge b, d, f). Scale bars show 500 lm (original magnification 940)
currently used safely in GMP processing of tissue. DBM sponges could be used in combination with mesenchymal stem cells (Shi et al. 2012; Liu et al. 2008) or could be used in combination with DBM pastes or putties. If required, the graft material could also be enhanced with the addition of recombinant human BMPs (rhBMP), such as rhBMP-2 (Elsalanty et al. 2008; Burkus et al. 2002). Use of DBM as a drug delivery system has also been reported (Holt and Grainger 2012; Gruskin et al. 2012) due to the way it slowly degrades as a result of host proteolysis and hydrolysis, releasing its own growth factors and any added therapeutics. One particular area of interest
described is that of antibiotic delivery to reduce surgical site infection, this would be well suited to a DBM sponge allograft (Rhyu et al. 2003). The observation that the DBM sponge is compressible and springs back to its original shape and size when compressed and released would allow the sponge to be shaped and pressed into cavities and provide an immediate natural osteoconductive environment for recipient osteogenesis to occur. In conclusion, this report demonstrates the successful washing and demineralisation of human DBM sponges with a volume up to three times that currently available.
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Cell Tissue Bank Acknowledgments from NHSBT.
We acknowledge support for this work
Conflict of interest The authors declare that they have no conflict of interest.
References Bethesda MD (2006) American association of tissue banks. Standards for tissue banking, (11th ed) American association of tissue banks Bleazey S, Brigado SA (2012) Reconstruction of complex osteochondral lesions of the Talus with cylindrical sponge allograft and particulate juvenile cartilage graft : provisional results with a short-term follow-up. Foot Ankle Spec 5:300–305 Bradbury P, Gordon KC (1990) Connective tissue and stains. In: Bancroft JD, Stevens A (eds) Theory and practice of histological techniques, 3rd edn. Churchill Livingstone, Edinburgh, pp 119–142 Burkus JK, Gornet MF, Dickman CA, Zdeblick TA (2002) Anterior lumbar interbody fusion using rhBMP-2 with tapered interbody cages. J Spinal Disord Tech 15(5):337–349 Eagle MJ, Rooney P, Kearney JN (2014) Optimized demineralization of human cancellous bone by application of a vacuum. J Biomed Res B [e-pub ahead of print] Eagle MJ, Man J, Rooney P, Hogg P, Kearney JN (2015) Assessment of an improved bone washing protocol for deceased donor human bone. Cell Tissue Bank 16(1):83–90 Elsalanty ME, Por YC, Genecov DG, Salyer KE, Wang Q, Barcelo CR, Troxler K, Gendler E, Opperman LA (2008) Recombinant human BMP-2 enhances the effects of materials used for reconstruction of large cranial defects. J Oral Maxillofac Surg 66:277–285 Galli MM, Protzman NM, Bleazey ST, Brigado SA (2014) Role of demineralised allograft subchondral bone in the treatment of shoulder lesions of the Talus: clinical results with two-year follow up. J Foot Ankle Surg [e-pub ahead of print] Girasole G, Muro G, Mintz A, Chertoff J (2013) Transforaminal lumbar interbody fusion rates in patients using a novel titanium implant and demineralised cancellous bone sponge. Int J Spine Surg 7(1):95–100 Gruskin E, Doll BA, Futrell FW, Schmitz JP, Hollinger JO (2012) Demineralized bone matrix in bone repair: history and use. Adv Drug Deliv Rev 64(12):1063–1077 Holt DJ, Grainger DW (2012) Demineralized bone matrix as a vehicle for delivering endogenous and exogenous
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therapeutics in bone repair. Adv Drug Deliv Rev 64(12):1123–1128 Liu G, Sun J, Li Y, Zhou H, Cui L, Liu W, Cao Y (2008) Evaluation of partially demineralized osteoporotic cancellous bone matrix combined with human bone marrow stromal cells for tissue engineering: an in vitro and in vivo study. Calcif Tissue Int 83:176–185 Mauney JR, Jaquiery C, Volloch V, Heberer M, Martin I, Kaplan D (2005) In vitro and in vivo evaluation of differentially demineralized cancellous bone scaffolds combined with human bone marrow stromal cells for tissue engineering. Biomaterials 26:3173–3185 Mulliken JB, Glowaki J, Kaban LB, Folkman J, Murray JE (1981) Use of demineralised allogeneic bone implants for the correction of maxillocraniofacial deformities. Ann Surg 194:366–372 Ohta H, Wakitani S, Tensho K, Horiuchi H, Wakabayashi S, Saito N, Nakamura Y, Nozaki K, Imai Y, Takaoka K (2005) The effects of heat on the biological activity of recombinant human bone morphogenetic protein-2. J Bone Miner Metab 23:420 Qiu QQ, Connor J (2008) Effects of c-irradiation, storage and hydration on osteoinductivity of DBM and DBM/AM composite. J Biomed Res A 87(2):373–379 Rhyu KH, Jung MH, Yoo JJ, Lee MC, Seong SC, Kim HJ (2003) In vitro release of vancomycin from vancomycin-loaded blood coated demineralised bone. Int Orthop 27:53–55 Rosenthal R, Folkman J, Glowacki J (1999) Demineralized bone implants for non-union fractures, bone cysts, and fibrous lesions. Clin Orthop Relat Res 364:61–69 Shi T, Niedzinski JR, Samaniego A, Bogdansky S, Atkinson BL (2012) Adipose-derived stem cells combined with a demineralised cancellous bone substrate for bone regeneration. Tissue Eng Part A 18:1313–1321 Smith CA, Richardson SM, Eagle MJ, Rooney P, Board T, Hoyland JA (2014) The use of a novel bone allograft wash process to generate a biocompatible, mechanically stable and osteoinductive biological scaffold for use in tissue engineering. J Tissue Eng Regen Med [e-pub ahead of print] Stevens A (1990) Pigments and minerals. In: Bancroft JD, Stevens A (eds) Theory and practice of histological techniques, 3rd edn. Churchill Livingstone, Edinburgh, pp 245–267 Wolfinbarger L, Eisenlohr LM, Ruth K (2008) Demineralized bone matrix: maximizing new bone formation for successful bone implantation. In: Pietrzak W (ed) Orthopedic biology and medicine: musculoskeletal tissue regeneration biological materials and methods. Humana Press, Totowa Zhang N, Zhou M, Zhang Y, Wang X, Ma S, Dong L, Yang T, Ma L, Li B (2014) Porcine bone grafts defatted by lipase: efficacy of defatting and assessment of cytocompatibility. Cell Tissue Bank 15:357–367
Development of an improved bone washing and demineralisation process to produce large demineralised human cancellous bone sponges Mark J. Eagle • Paul Rooney • John N. Kearney
Received: 9 December 2014 / Accepted: 26 February 2015 Ó Springer Science+Business Media Dordrecht 2015
Abstract Shaped demineralised bone matrices (DBM) made from cancellous bone have important uses in orthopaedic and dental procedures, where the properties of the material allow its insertion into confined defects, therefore acting as a void filler and scaffold onto which new bone can form. The sponges are often small in size, \1.0 cm3. In this study, we report on an improved bone washing and demineralisation process that allows production of larger DBM sponges (3.375 or 8.0 cm3) from deceased donor bone. These sponges were taken through a series of warm water washes, some with sonication, centrifugation, 100 % ethanol and two decontamination chemical washes and optimally demineralised using 0.5 N hydrochloric acid under vacuum. Demineralisation was confirmed by quantitative measurement of calcium and qualitatively by compression. Protein and DNA removal was also determined. The DBM sponges were freeze dried before terminal sterilisation with a target dose of 25 kGy gamma irradiation whilst frozen. Samples of the sponges were examined histologically for calcium, collagen and the presence of cells. The data indicated lack of cells, absence of bone marrow and a maximum of 1.5 % residual calcium.
M. J. Eagle (&) P. Rooney J. N. Kearney NHSBT, Tissue Services R&D, 14 Estuary Banks, Speke, Liverpool L24 8RB, UK e-mail: [email protected]; [email protected]
Keywords Demineralised bone sponge Vacuum Protein DNA Allograft
Introduction NHS Blood and Transplant have developed a rapid method of demineralising cancellous bone under vacuum which results in a demineralised bone matrix (DBM) sponge in which, in excess of 99 % of soluble protein, DNA and haemoglobin has been removed and which has proved osteoinductive in an animal model (Eagle et al. 2014). Demineralisation was performed under a vacuum and could be achieved in less than 1 h. Demineralisation of this bone results in a spongy, compressible bone scaffold which can be used as a bone void filler and which could be used in combination with DBM paste/putty or powder to stimulate improved bone healing. There is the potential to use such demineralised bone sponges in the field of dentistry, maxillofacial surgery or in treatment of osteotomy where the demineralised bone can be manipulated into gap-forming defects ( Mulliken et al. 1981, Rosenthal et al. 1999). One potential drawback to the DBM sponges is their size, 1.0 cm 9 1.0 cm 9 1.0 cm (1 cm3) and surgeons have requested larger sponges of dimensions 1.5 cm 9 1.5 cm 9 1.5 cm [3.375 cm3 (3.4 cm3)] and 2.0 cm 9 2.0 cm 9 2.0 cm (8.0 cm3). We have developed a bone washing protocol which is capable of removing 99.9 % of soluble protein, DNA
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and haemoglobin from cortical and cancellous bone using only ethanol as an organic solvent. We have omitted harsher, more volatile organic solvents such as chloroform, ether, acetone etc. Which could have harmful effects on processing staff or could recirculate within a clean room environment (Eagle et al. 2015). This bone is non-cytotoxic, the biomechanical properties have not been affected and it has been used as a scaffold to allow attachment and proliferation of mesenchymal stem cells (Smith et al. 2014). The bone washing process has been used on 1.0 cm3 mineralised and demineralised cubes/sponges, however larger volume cubes contain more marrow components and the published process could not fully remove all marrow and lipid from the larger cubes, therefore, in this report we have improved the bone washing process, incorporating longer incubations in 100 % ethanol under vacuum to fully wash the bone cubes. During demineralisation, the acid removes mineral from bone and exposes bone morphogenetic proteins (BMPs) (Mauney et al. 2005), at the same time the reaction releases CO2 gas. This is not a problem for ground cortical bone powder but is a problem for 3-dimensional cancellous bone cubes, due to the trabecular nature of the tissue, CO2 can get trapped in the porous trabeculae and the bone cube floats; the gas can also prevent the acid from coming into contact with the bone surface and delay demineralisation. We have overcome this problem by demineralising cancellous bone cubes under a vacuum and have used the vacuum system in this report (Eagle et al. 2014).
Materials and methods Tissue Bone was obtained from the knees of three donors, age range 45–63 years. Full ethical consent for use of the tissue in research and development was obtained. Bone washing Knees, including regions of proximal tibia and distal femur, were stored at -80 °C until required. These were allowed to thaw at 4 °C overnight and soft tissue and cortical bone was dissected off leaving the cancellous portion of bone. Cancellous bone from the distal femur was cut into slices of 2.0 cm thick and then
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further cut into appropriately sized cubes with a saggital saw. Up to 12 2.0 cm 9 2.0 cm 9 2.0 cm cubes could be obtained from one donor. Cancellous bone from the proximal tibia was cut into slices of 1.5 cm thick and then further cut into appropriately sized 1.5 cm 9 1.5 cm 9 1.5 cm cubes; up to 24 cubes could be obtained from one donor. These mineralised cubes were then washed according to a protocol adapted from Eagle et al. (2014) that included two 100 % ethanol washes and centrifugations (Table 1). All washes and rinses were performed in warm water (Baxter, sterile water for irrigation, UKF7114) at 56–59 °C (standard bone allograft washing temperature used in our tissue bank) except those in hydrogen peroxide (Sigma, 30 % (w/w) solution (H1009) diluted in sterile water to 3 % solution (v/v)) (Step 10, Table 1) or 100 % ethanol (VWR, AnalaR, L992101) (Steps 11 and 13, Table 1) which were performed at room temperature. Studies have shown that recombinant BMP-2 can withstand exposure to temperatures up to 70 °C (Ohta et al. 2005). Sonication was performed in a Decon F5300b sonicating water bath. All wash solutions were collected; the volumes were measured and samples were then taken for determination of soluble protein, and DNA content. Assessment of bone marrow component removal Wash solutions were collected at each step of the process (Table 1) and the total amounts of soluble protein and DNA were calculated by multiplying the concentration, obtained as described below by the volume of each wash solution. Residual components were calculated by immersing fully washed bone in a container with a volume of pre-heated sterile water equivalent to at least 5 times the weight of bone (vol/ w). The bone was agitated in an orbital shaker (56–59 °C) for 1 h at 200 rpm. The water was removed and replaced with fresh sterile water and the agitation was repeated. Both residual wash solutions were combined for measurement (a preliminary study using 10 consecutive washes demonstrated that no further residual components could be detected after the second wash, therefore no more than two washes were required). Once residual values were ascertained, they were added to the total amounts in each wash solution and the proportions removed at each step were evaluated. The amount removed was expressed as a percentage removal from the bone.
Cell Tissue Bank Table 1 Bone wash procedure
Step
Time
1
Sonication in water (56–59 °C)
15 min
2
Rinse (56–59 °C)
5 min
3
Wash and agitation (200 rpm, 56–59 °C)
30 min
4
Centrifugation (1850 g)
15 min
5
Wash and agitation (200 rpm, 56–59 °C)
10 min
6
Centrifugation (1850 g)
15 min
7
Wash and agitation (200 rpm, 56–59 °C)
10 min
8 9
Centrifugation (1850 g) Wash and agitation (200 rpm, 56–59 °C)
15 min 10 min
10
Hydrogen peroxide wash (3 % v/v) with sonication at RT
10 min
11
100 % Ethanol wash with 5 min vacuum, and then agitation at 70 rpm
Overnight
12
Centrifugation (1850 g)
15 min
13
100 % Ethanol wash with vacuum
5 min
14
Centrifugation (1850 g)
15 min
15
70 % Ethanol wash with sonication
10 min
16
Wash and agitation (200 rpm, 56–59 °C)
10 min
17
Wash and agitation (200 rpm, 56–59 °C)
10 min
18
Centrifugation (1850 g)
15 min
Protein removal A standard curve was prepared using bovine serum albumin (Sigma) at a range of concentrations, 100 ll of each concentration was pipetted into a well of a 96-well microplate (Nunclon). 100 ll of every wash solution (from Table 1) and residual soaks were also pipetted into the microplate. To each well, 100 ll of Bradford reagent (Sigma, UK) was added and mixed before incubating at room temperature for 5 min. The solutions were measured on a microplate reader (Elx808, Biotek) at 595 nm against a blank of distilled water and Bradford reagent. A graph of the standard curve was produced from which protein concentrations could be calculated. Absolute protein amounts were calculated taking into account dilution factors and volumes of wash solutions. The values were used to calculate the amount of protein present in each wash solution. Using the residual wash values, the overall percentage protein removal from the bone samples was calculated. DNA removal A standard curve was prepared from calf thymus DNA (Sigma) at a range of concentrations, 100 ll of each concentration was pipetted into a well of a 96-well
black bottomed microplate (Nunclon) 100 ll of every wash solution (from Table 1) and residual soaks were also pipetted into the microplate. To each well, 100 ll of Picogreen reagent was added and the plate was gently shaken to mix and incubated at room temperature for 5 min. The microplate was read on a fluorescence microplate reader (Flx 800, Biotek) with excitation at 485 nm and emission at 528 nm. A graph of the standard curve was produced from which DNA concentrations could be calculated. Absolute DNA amounts were calculated taking into account dilution factors and volumes of wash solutions. The values were used to calculate the amount of DNA present in each wash solution. Using the residual wash values, the overall percentage of DNA removal from the bone samples was calculated. Acid demineralisation of mineralised cancellous cubes Acid demineralisation, at a ratio of 1 g bone: 50 ml 0.5 N HCl, resulted in the continuous production of CO2 which displaced the acid solution within the trabeculae of the cancellous bone thus leading to uneven demineralisation within the graft; and it also caused the grafts to float on the surface of the solution, again leading to incomplete demineralisation. Experiments with
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unsecured cubes and at atmospheric pressure also showed a long time period for demineralisation to take place (not shown). To overcome these problems the cubes were placed inside an acetyl plastic ring surrounded by a polyethylene mesh cage (Fig. 1) presterilised by gamma irradiation. This ensured that the cubes were submerged within the demineralisation chamber. Up to 4 9 8.0 cm3 cubes from a single donor could be placed inside the chamber or 12 9 3.4 cm3 cubes at one time. CO2 was removed from the demineralising cubes by applying a continuous vacuum of 270 mbar to the demineralisation chamber (Fig. 1). This enabled uniform demineralisation throughout each cube. All connections were made with 8 mm outer diameter silicone tubing, and luer lock connectors. A 0.22 lm filter (Sterivex, Millipore, UK) was connected between the pump and chamber. After addition of the acid solution to the chamber, the chamber was closed and the vacuum pump (Dymax 14, Charles Austin Pumps, UK) was started. The level of vacuum was monitored using a vacuum gauge (DVR2, Vacuubrand, UK). Acid was continually stirred at 500 rpm by a 40 mm magnetic stirrer with crosshead, operated by a magnetic stirring plate. 3.4 cm3 sponges were demineralised for 24 h and 8.0 cm3 sponges were demineralised for 48 h. At the end of demineralisation, the acid was poured off and an equal volume of distilled water was added and stirred continuously for 20 min, again with vacuum applied to the chamber. Sequential washes with water, PBS, PBS, water and water were carried out, also with stirring at 500 rpm. The pH was measured to ensure a neutral value before the final water wash was discarded and each DBM sponge was gently squeezed between thumb and index finger to remove excess water. This also served as a manual test
of demineralisation, as any large remaining calcium deposits would prevent the cube from being completely compressed. After compression the cubes were seen to return to their original dimensions. The bone was then stored frozen at -40 °C. Terminal sterilisation The frozen DBM sponges were lyophilised in a freeze drier (VirTis, Advantage) using an established programme for production of freeze-dried bone and subsequently irradiated at 25–40 kGy (target dose 25 kGy, Synergy Health, UK) packed in dry ice at circa -79 °C. Irradiation of lyophilised DBM at low temperature has been reported to maintain the osteoinductive potential of the DBM (Wolfinbarger et al. 2008; Qiu and Connor 2008). Residual calcium determination Three 3 DBM sponges of both sizes from each donor, demineralised for 24 or 48 h were placed in a drying oven and left overnight at 120 °C. The dried sponges were weighed before being placed in individual containers with 10 ml of 12 N HCl (for 3.4 cm3 sponges) or 20 ml (for 8.0 cm3 sponges) to achieve complete hydrolysis. Once dissolved, the acid was diluted by addition of 30 ml (for 3.4 cm3 sponges) or 60 ml (for 8.0 cm3 sponges) of distilled water. Calcium content was measured using an assay kit (Quantichrom, Universal Biologicals, Cambridge, UK) following the manufacturers instructions. Briefly a standard curve was set up via a series of dilutions from the supplied calcium standard. 5 ll duplicate samples of the standards and the diluted hydrolysed DBM sponges of each donor were added to individual wells on a clear 96 well microplate. 200 ll of the assay reagent was then pipetted into each well, with the plate then left at room temperature for 3 min. Absorbance was read on a microplate reader (Elx 808, Biotek, UK) at 595 nm. The percentage weight of calcium to weight of dry bone was then calculated. Histological evaluation
Fig. 1 Apparatus setup of vacuum pump equipment, safety flask and pH probe
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Irradiated and non-irradiated DBM sponges were cut in half diagonally and then fixed in 10 % neutral buffered formalin for at least 2 days. The fixed tissue was then processed according to standard histology
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practice before being embedded in paraffin wax, with the diagonally cut surfaces face down in the wax mould. The wax embedded tissue was then sectioned at 5–10 lm thickness. Sections were stained with a range of stains including H&E for general visualisation, Van Gieson’s for appearance of collagen (Bradbury and Gordon 1990), Alizarin red S for mineral (Stevens 1990) and DAPI (Sigma, UK) for DNA. Sections were examined under an inverted microscope and photographs taken with a digital camera (Coolpix 5000, Nikon, UK). Statistical analysis Data for the calcium assay are represented as means with bars showing 95 % confidence intervals. One way analysis of variance (ANOVA) was used to calculate significant difference between multiple means. Difference between data was considered statistically significant when the p \ 0.05.
Results Bone washing and marrow component removal The 100 % ethanol washes visibly removed lipid from the cubes prior to demineralisation, and allowed the successful decalcification of the cubes to take place (Fig. 2). Analysis of the wash solutions showed mean protein removal of 98.8 % and mean DNA removal of 98.1 % (Table 2). The patterns of component removal for soluble protein from 8.0 and 3.4 cm3 sponges are shown in Figs. 3 and 4 respectively and for DNA removal in Figs. 5 and 6 respectively. Residual calcium determination Residual levels of calcium were measured in three DBM sponges of both sizes from three different donors. 3.4 cm3 sponges were demineralised for 24 h and 8.0 cm3 sponges were demineralised for 48 h. Residual calcium values are shown in Fig. 7. Mean residual calcium levels in 3.4 cm3 sponges were 0.49 % (±0.23 %) of the DBM sponge weight and residual calcium levels in 8.0 cm3 sponges were 0.93 % (±0.27 %) of the DBM sponge weight (Fig. 7). All mean residual calcium values were below
Fig. 2 8.0 cm3 DBM sponges at various stages of production. Washed mineralised cubes on the left, washed and 100 % ethanol treated cubes in the centre and demineralised sponges on the right
1.5 %, statistical analysis of the data with ANOVA showed no significant difference in residual calcium levels in the 3.4 cm3 sponges between donors but a significant difference was observed in 8.0 cm3 sponges between the three donors (p \ 0.01). Histology H&E staining of DBM sponges demonstrated a bony trabecular network with absence of any cells (not shown). Van Gieson’s staining demonstrated intact trabeculae after demineralisation with intense collagen staining and no apparent sign of degradation (Fig. 8a, b). Alizarin red S staining demonstrated absence of calcium (Fig. 8c, d). DAPI staining demonstrated that the process essentially decellularised the tissue with no indication of fluorescent nuclei (Fig. 8e, f). Irradiation had no effect on collagen structure or residual calcium appearance.
Discussion Demineralised cancellous bone sponges are used by surgeons to fill bony defects where, due to the compressibility of the bone and the interporosity of the trabecular scaffold, the grafted bone can be ‘‘pressfitted’’ into small cavities to act as a bone void filler and an osteoconductive environment to allow bone cell attachment and growth. Clinically, DBM sponges have been used to treat osteochondral lesions of the talus and have been reported to cause no complications or immune reactions and have been reported to aid in alleviation of pain and disability (Bleazey and Brigado 2012; Galli et al. 2014). NHS Blood and Transplant have developed a method of producing DBM sponges,
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Cell Tissue Bank Table 2 Summary of overall component removal from different sized cubes Component
Cube size (cm3)
% component removal Donor 1
Protein DNA
Donor 2
Donor 3
Donor mean
Component mean 98.79
8.0
98.15
98.23
98.83
98.40
3.4
99.37
99.09
99.08
99.18
8.0
95.62
97.17
99.65
97.48
3.4
98.57
97.77
99.81
98.72
98.10
Fig. 3 Pattern of mean protein removal from 8.0 cm3 sponges, N = 3, Error bars = 95 % CI
using mineralised cancellous bone cubes as the starting material, which are compressible, non-cytotoxic and are osteoinductive when implanted intramuscularly into mice (Eagle et al. 2014). The procedure implemented to produce these DBM cubes also removes blood, bone marrow components and essentially decellularises the sponge. However, the sponges are 1.0 cm 9 1.0 cm 9 1.0 cm and as such could only be used to fill small bone defects; there is a need to be able to produce a larger DBM sponge. DBM sponges are available commercially but the largest volume sponge this group was able to find was 1.4 cm 9 1.4 cm 9 1.4 cm (2.75 cm3, OsteoSponge, Bacterin, USA). In this report we describe methodology to wash and demineralise DBM sponges with volumes of 3.4 and 8.0 cm3. Using a stringent bone washing process, bone cubes of 1.5 cm 9 1.5 cm 9 1.5 cm and 2.0 cm 9 2.0 cm 9 2.0 cm demonstrated in excess of 98.1 % of DNA and
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soluble protein removal. Histological examination could not detect any cells either in the marrow space or within the bony trabeculae, indicating that the process also decellularises the bone. The washing process used 100 % ethanol, warm water washes and centrifugation to remove marrow components without the need to use volatile organic solvents such as chloroform, ether or acetone (Eagle et al. 2015). Warm water washes were performed at 56–59 °C, our experience of washing mineralised allograft bone at this temperature range indicates no deleterious effect on the clinical outcomes achieved. In addition, 1.0 cm3 demineralised sponges produced using solutions in the same temperature retain an osteoinductive potential (Eagle et al. 2014). We are investigating the possibility of using lipase to aid in the removal of lipid (Zhang et al. 2014). The use of a vacuum during demineralisation has ensured that any CO2 gas, released as the 0.5 N HCl acted on bone mineral, was removed from the cubes/
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Fig. 4 Pattern of mean protein removal from 3.4 cm3 sponges. N = 3, Error bars = 95 % CI
Fig. 5 Pattern of mean DNA removal from 8.0 cm3 sponges. N = 3, Error bars = 95 % CI
sponges allowing acid to continually come into contact with the bone surface. The resultant DBM sponges from three human donors had mean residual calcium levels below 1.5 % of the dry weight of the DBM sponge. Mineralised bone has approximately 28 % calcium by weight (Eagle et al. 2014) and the American Association for Tissue Banks has stated that for a bone to be classed as demineralised it must contain less than 8 % residual calcium by weight (Bethesda 2006). Consequently the bone sponges
produced here can be classed as demineralised. Different sizes of mineralised bone cube required different times of demineralisation, in this report 24 h was found to demineralise all 3.4 cm3 sponges and 48 h was found to demineralise all 8.0 cm3 sponges. Most studies on demineralised bone have reported on ground cortical bone as the starting material and several reports have suggested that cancellous bone contains low levels of BMPs and as such are mostly osteoconductive (Mauney et al. 2005), although a
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Fig. 6 Pattern of mean DNA removal from 3.4 cm3 sponges. N = 3, Error bars = 95 % CI
Fig. 7 Residual calcium values in 3.4 and 8.0 cm3 DBM sponges. Values are expressed as a percentage of the dry weight of demineralised sponges. N = 3, Error bars = 95 % CI
recent study indicated that cancellous bone contains similar levels of BMP2, BMP4 and BMP7 to those found in cortical bone (Shi et al. 2012). In addition, the nature of the porous bony trabeculae could provide a suitable microenvironment for osteoblastic cell attachment and differentiation, again previously reported (Shi et al. 2012; Smith et al. 2014). We have not investigated the osteoinductive potential of these
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larger DBM sponges, however, we have previously demonstrated that 1.0 cm3 cancellous DBM sponges, produced using similar methodology as described here, are osteoinductive when implanted into a rodent model (Eagle et al. 2014). In this report, demineralisation proceeds under closed conditions and each of the reagents and equipment used in this study are Good Manufacturing Practice (GMP) compliant and are
Cell Tissue Bank
Fig. 8 Micrographs of DBM sponges stained with Van Gieson’s for visualisation of collagen, Alizarin red for calcium and DAPI to show cell nuclei (3.4 cm3 sponge a, c, e; 8.0 cm3 sponge b, d, f). Scale bars show 500 lm (original magnification 940)
currently used safely in GMP processing of tissue. DBM sponges could be used in combination with mesenchymal stem cells (Shi et al. 2012; Liu et al. 2008) or could be used in combination with DBM pastes or putties. If required, the graft material could also be enhanced with the addition of recombinant human BMPs (rhBMP), such as rhBMP-2 (Elsalanty et al. 2008; Burkus et al. 2002). Use of DBM as a drug delivery system has also been reported (Holt and Grainger 2012; Gruskin et al. 2012) due to the way it slowly degrades as a result of host proteolysis and hydrolysis, releasing its own growth factors and any added therapeutics. One particular area of interest
described is that of antibiotic delivery to reduce surgical site infection, this would be well suited to a DBM sponge allograft (Rhyu et al. 2003). The observation that the DBM sponge is compressible and springs back to its original shape and size when compressed and released would allow the sponge to be shaped and pressed into cavities and provide an immediate natural osteoconductive environment for recipient osteogenesis to occur. In conclusion, this report demonstrates the successful washing and demineralisation of human DBM sponges with a volume up to three times that currently available.
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Cell Tissue Bank Acknowledgments from NHSBT.
We acknowledge support for this work
Conflict of interest The authors declare that they have no conflict of interest.
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