Platelet concentrate as an additive to bone allografts: a laboratory study using an uniaxial compression test

Platelet concentrate as an additive to bone allografts: a laboratory study using an uniaxial... Cell Tissue Bank https://doi.org/10.1007/s10561-018-9704-3 Platelet concentrate as an additive to bone allografts: a laboratory study using an uniaxial compression test . . . . David Putzer Markus Dobersberger Alex Pizzini Debora Corac ¸a-Huber Christoph Ammann Michael Nogler Received: 30 November 2017 / Accepted: 25 May 2018 The Author(s) 2018 Abstract Chemical cleaning procedures of allo- Keywords Processing allografts  Platelet grafts are destroying viable bone cells and denaturing concentrate  Additive to allografts  Chemical osteoconductive and osteoinductive proteins present cleaning procedures  Platelet concentrate gel in the graft. The aim of the study was to investigate the mechanical differences of chemical cleaned allografts by adding blood, clotted blood; platelet concentrate and platelet gel using a uniaxial compression test. The Introduction allografts were chemically cleaned, dried and stan- dardized according to their grain size distribution. Bone grafts are used to fill bone defects in different Uniaxial compression test was carried out for the four applications of orthopaedic and trauma surgery with groups before and after compacting the allografts. No good long term results (Schreurs et al. 2009). Auto- statistically significant difference was found between grafts are the gold standard in reconstructive surgery; native allografts, allografts mixed with blood, clotted however they are available only in limited quantity. blood, platelet concentrate and platelet concentrate gel They can be obtained from the femoral head during regarding their yield limit after compaction. The total hip arthroplasty or from the iliac crest (Khan et al. authors recommend to chemical clean allografts for 2005; Myeroff and Archdeacon 2011; Nogler et al. large defects, optimize their grain size distribution and 2012). Autografts have optimum osteoconductive, add platelet concentrate or platelet rich plasma for osteoinductive properties and allow osteogenesis as enhancing as well primary stability as well bone they contain surviving cells and osteoinductive pro- teins (BMPs) such as BMP-2 and BMP-7, fibroblast ingrowth. growth factor (FGF), insulin-like growth factor (IGF) and platelet-derived growth factor (PDGF) (Bauer and Muschler 2000; Dimitriou et al. 2011; Brydone et al. D. Putzer (&)  M. Dobersberger  D. Corac¸a-Huber 2010; Parikh 2002). C. Ammann  M. Nogler To compensate for the reduced availability of Department of Orthopaedics – Experimental autografts, allografts or synthetic materials are widely Orthopaedics, Medical University of Innsbruck, Innrain 36, 6020 Innsbruck, Austria used. Allografts have variable osteoinductive and e-mail: david.putzer@i-med.ac.at osteoconductive properties but are lacking viable cells which results in lower osteogenic potential than A. Pizzini autografts (Zimmermann and Moghaddam 2011). Department of Internal Medicine, Medical University of Innsbruck, Anichstrasse 35, 6020 Innsbruck, Austria 123 Cell Tissue Bank Sterilization processing of allografts includes the producing a 3–5 times higher platelet concentrated gel usage of hypotonic solutions, acetone, ethylene oxide, than native plasma contains (Petrungaro 2001). The or gamma irradiation which can eliminate cellular and release of PDGF, IGF, VEGF, PDAF and TGF-b viral particles and therefore reduce the risk of present in the PRP is triggered by the activation of infectious and transmissible diseases (Muller et al. platelets by means of a variety of substances or stimuli 2013). Chemical cleaning processes are also used to such as thrombin, calcium chloride, or collagen (Wang remove the fat content of the allografts enhancing the and Avila 2007). mechanical properties of the allograft (Putzer et al. PRP indeed is widely used in plastic surgery and 2014a; van der Donk et al. 2003; Fosse et al. 2006; jaw reconstruction surgery for enhancing bone and Voor et al. 2004). However, despite modern steriliza- connective tissue growth (Wang and Avila 2007; Marx tion and storage methods, processing of allografts is et al. 1998; Board et al. 2008; Anitua et al. 2004; not completely safe (Zimmermann and Moghaddam Thorwarth et al. 2006). Enriched platelet preparations 2011; Malinin and Temple 2007). have shown a rapid bone healing and regeneration The chemical cleaning as well as gamma irradiation when combined with autologous bone and bone of allografts may destroy the bone cells and denature substitute materials (Anitua et al. 2004; Kim et al. proteins present in the graft and alter osteoconductive 2001). In PRP no cross reactivity, immune reaction or and osteoinductive characteristics, essentially elimi- disease transmission has been observed (Weibrich nating the osteogenic properties and inhibiting the et al. 2001). A reduced healing time (50%) has been bone remodeling process (Keating and McQueen shown in a study of Kassolis and Reynolds (2005). In 2001). animal studies no statistically significant difference During fracture healing and implant ingrowth the was found between cancellous bone graft material recruitment and migration of osteogenic cells are with and without PRP (Butterfield et al. 2005; Jakse essential for bone regeneration (Kark et al. 2006). The et al. 2003). migration of these cells is stimulated by growth factors There is large evidence that by adding blood, as transforming growth factors (TFG), PDGF, IGF, autologous PRP or PLC the bone in-growth is vascular endothelial growth factors (VEGF), platelet enhanced, although antigenity is getting reduced in derived angiogenic factor (PDAF) and FGF (Fiedler some cases (Khan et al. 2005; Anitua et al. 2004; et al. 2002; Mayr-Wohlfart et al. 2002; Martineau et al. Hannink et al. 2009; Baylink et al. 1993; Canalis et al. 2004; Wang and Avila 2007), all of which are released 1989; Canalis 1985; Lozada et al. 2001; Cenni et al. by platelets in response to injury (Martineau et al. 2010; Blair and Flaumenhaft 2009). The aim of the 2004; Weibrich et al. 2002). In addition to growth study was to investigate the mechanical differences of factors (GFs), platelets release numerous other sub- chemical cleaned allografts with known grain size stances (e.g., fibronectin, vitronectin, sphingosine distribution mixed with blood (BL), clotted blood 1-phosphate, etc.…) that are important in wound (CB), platelet concentrate (PC) and platelet concen- healing (Wang and Avila 2007). Platelets can be trated gel (PG) using an uniaxial compression test. applied as autologous product to sites of bone injury by either being concentrated in combination with blood plasma [platelet-rich plasma (PRP)] or as a Methods platelet gel that is created by clotting the PRP (Kark et al. 2006). Bone tissue was donated by 5 patients to the local bone PRP, plasma rich in growth factors (PRGF), and bank, from whom informed consent was obtained. In platelet concentrate (PLC) are essentially an increased the preparation of allografts the local bone bank concentration of autologous platelets suspended in a requirements for producing fresh-frozen allografts small amount of plasma after centrifugation (Wang were followed. Cartilage and cortical tissue was and Avila 2007). By centrifugation donors blood is removed and using a bone mill (Spierings Medische separated into platelet poor plasma (PPP), PRP and red Techniek BV, Nijmegen, The Netherlands) allografts blood cells (Marlovits et al. 2004). Prior to applica- sizing 5–10 mm were produced (McNamara 2010). tion, topical bovine thrombin and 10% calcium The allografts were frozen to - 80 C, carefully chloride is added to activate the clotting cascade, mixed to reduce patient specific properties and stored 123 Cell Tissue Bank at - 11 C. A chemical cleaning procedure was used In group PC 4 ml of concentrated platelets from one to remove fat content of the allografts and reduce the donor, who gave his informed consent prior donation, contamination risk (Coraca-Huber et al. 2013). For the were added. The PC was stored at - 4 C. In platelet cleaning procedure a sonicator (40 kHz, 200 W , concentrated gel (PG) in addition to the 4 ml of eff BactoSonic, Bandelin eletronic GmbH & Co. KG, concentrated platelets 666 ll of 1 mol calcium chlo- Berlin, Germany) was used. As washing solutions ride (CaCl ) were added (Marx et al. 1998; Oakley and 700 ml of 1% Triton X-100 (Sigma-Aldrich, Schnell- Kuiper 2006; Camenzind et al. 2000). After 6 min dorf, Germany), 500 ml 3% hydrogen peroxide solu- activation time the samples were used for mechanical tion (Sigma-Aldrich, Schnelldorf, Germany) and a testing. 70% ethanol solution were used. The allografts were All samples were filled into a compaction chamber dried in an incubator (Memmert GmbH & Co. KG, with an internal diameter of 40 mm. A uniaxial Schwabach, Germany) at 37 C. compression test was carried out before and after a Allograft samples were assembled according to standardized compaction procedure resulting in 20 their grain size in proportions specified in Table 1 measurements before and 20 measurements after after being separated using sieves ranging from 0.063 compaction for each of the four groups. In the to 16 mm in correspondence to ASTM C 125 standard standardized compaction procedure a fall hammer (Application time 1 h, Amplitude 10 mm, Haver und (1.45 kg) was dropped 10 times from a height of Bocker, Olde, Germany) (Putzer et al. 2014b). Sam- 18 cm. ples with a mean weight of 8 ± 0.01 g were obtained The uniaxial compression test was carried out with and divided into four groups each containing 20 a 15 mm punch which was lowered with a speed of samples. 1 mm/min into the allografts. Force and displacement In one group (BL) 4 ml blood from the same donor, were measured by the testing machine after reaching a who gave his informed consent prior donation, were preload of 5 N (Zwicki—Line Z 2.5, maximal load 2.5 added. In different studies native allografts showed a kN, 320 kHz sample rate, Zwick GmbH & Co. KG, liquid component of 50% in weight (Putzer et al. Ulm, Deutschland) with a preciseness of ± 0.04 N 2014a). Adding 4 ml of blood approximately com- and ± 2 lm. pensates the liquid component, which was previously A peak analysis was performed on the resulting removed. The blood was stored at - 4 C and was force displacement curves using OriginPro8.5 (Origin obtained from the local tissue bank. In the second Lab Corporation, Northampton, Massachusetts, USA) group (CB) clotting was induced by adding 480 llof (Fig. 1a). In all curves a fitted baseline (50 anchor 1 mol calcium chloride (CaCl ) in addition to the 4 ml points, 1 and 2 derivation method, polynomial blood and mixed thoroughly for 1 min as described by smoothing of order 2) was subtracted to remove the Oakley and Kuiper (2006) and Camenzind et al. logarithmic trend (Fig. 1b). The signal was analyzed (2000). The cleaned allografts were mixed with the for positive local maxima over 100 data points with a clotted blood and after 5 min (6 min activation time) smoothing window size of 10 data points. The yield the samples were used for mechanical testing. limit (YL) was determined as the first local maxima on the force curve (Fig. 1c). The corresponding displace- ment value was used to obtain the density at the yield limit d . The punch displacement value at 5 N was YL Table 1 All samples were reassembled after sieving with used to calculate the initial density of the samples d . specific grain size proportions achieving a total weight of 8 g Previously published data by the authors were used Grain size [mm] Weight [g] as control groups were native allografts (NA) (Putzer et al. 2014a) and allografts with optimized grain size [ 4 5.08 distribution (OG) were evaluated (Putzer et al. 2014b). 4–2 0.86 Measurements of each variable and group were 2–1 0.60 tested for normal distribution using the Kolmogorov– \ 1 1.46 Smirnov Test. Comparison before and after com- Total 8.00 paction were analyzed using the two-tailed T Test for dependent samples. All groups were tested for 123 Cell Tissue Bank Brown Forsythe test was used for group comparisons and Tukey Post-Hoc analysis was used for pairwise comparison. Outliers were defined as values that are greater than 100% and smaller than 10% of the median. They were removed from the data set. In all analysis (SPSS software v.20, IBM, Chicago, IL) a p value of 0.05 was considered statistically significant. Results NA, OG, BL, PC and PG had a normal distribution for all investigated measurement parameters. CB showed a normal distribution except for the yield limit before compaction. IN BL 1 outlier was eliminated for the yield limit before and 1 outlier after compaction. In CB one outlier was eliminated for the initial density, two outliers were eliminated for the density at the yield limit and two for the yield limit when uncom- pacted. After compaction in CB one outlier was eliminated for the yield limit. PC showed two outliers for the initial density when uncompacted. PG showed two outliers for the yield limit before compaction. No statistically significant change of the initial density was observed after compaction for BL and PC (Table 2). In NA a statistically significant increase of 22% and in OG a of 34% was observed, while CB showed a statistical significant increase of the initial density by 10% and PG increased its initial density after compaction by 13%. Considering the density at the yield limit before and after compaction BL showed a statistically significant increase of 13% and PG of 14%, while NA showed an increase of 10% and OG of 22% (Table 3). In CB and PC no statistically significant increase of the density at the yield limit could be observed. All groups showed a statistical significant differ- ence when comparing the yield limit before and after Fig. 1 a Force displacement curves were from uniaxial compaction (Table 4). BL and PC showed a * 35% compression test using a testing machine. b In all curves a higher yield limit after compaction, while in the fitted baseline with 50 anchor points (squares) was subtracted to groups with the activation liquid CB and PG the yield remove the logarithmic trend. c The signal was analyzed for limit increased by 15% for CB and 20% for PG. NA positive local maxima. The yield limit (YL) was determined as the first local maxima on the force curve and is indicated by a showed an increase of the yield limit by 80% and OG line in all three graphs of 90%. The uncompacted initial density and uncompacted variance homogeneity. In case of variance homogene- yield limit showed a homogeneity distribution of ity ANOVA was used for group comparisons and variances. All other variables did not show homo- Games Howel Post-Hoc analysis for pairwise com- geneity of variances. parisons. If variance homogeneity was not fulfilled the 123 Cell Tissue Bank Table 2 Mean and standard deviation of the initial density before and after compaction are reported for the four groups under investigation d [g/cm ] Uncompacted Compacted Difference p value NA 0.91 (SD 0.14) 1.17 (SD 0.09) 22.3 \ 0.001 OG 0.39 (SD 0.04) 0.59 (SD 0.07) 33.9 \ 0.001 BL 1.12 (SD 0.08) 1.14 (SD 0.22) 0.695 CB 1.13 (SD 0.09) 1.26 (SD 0.30) 10.3 0.044 PC 1.13 (SD 0.09) 1.14 (SD 0.14) 0.621 PG 1.16 (SD 0.08) 1.34 (SD 0.26) 13.4 0.004 The difference was calculated as a percentage and p value of the T test (comparison before and after compaction) is reported The sample weight was half of the other groups Table 3 Mean and standard deviation of the density at the yield limit before and after compaction are reported for the four groups under investigation d [g/cm ] Uncompacted Compacted Difference p value YL NA 1.45 (SD 0.24) 1.60 (SD 0.21) 10.1 0.008 OG 0.58 (SD 0.19) 0.74 (SD 0.08) 21.7 \ 0.001 BL 1.48 (SD 0.13) 1.71 (SD 0.46) 13.4 0.049 CB 1.48 (SD 0.13) 1.48 (SD 0.30) 0.969 PC 1.53 (SD 0.19) 1.70 (SD 0.35) 0.178 PG 1.53 (SD 0.13) 1.78 (SD 0.45) 14.0 0.031 The difference was calculated as a percentage and p value of the T test (comparison before and after compaction) is reported The sample weight was half of the other groups Table 4 Mean and standard deviation of the yield limit before and after compaction are reported for the four groups under investigation YL [kPa] Uncompacted Compacted Difference p value NA 24 (SD 19) 117 (SD 62) 79.1 \ 0.001 OG 35 (SD 37) 353 (SD 187) 90.1 \ 0.001 BL 33 (SD 30) 90 (SD 28) 36.7 \ 0.001 CB 12 (SD 14) 83 (SD 32) 14.4 \ 0.001 PC 32 (SD 23) 91 (SD 32) 35.2 \ 0.001 PG 16 (SD 11) 78 (SD 34) 20.5 \ 0.001 The difference was calculated as a percentage and p value of the T test (comparison before and after compaction) is reported The OG group showed a statistically significant PG, CB and PC) showed no statistically significant lower initial density and a lower density at the yield difference between each other (p [ 0.376). After limit to all other groups before and after compaction compaction no statistically significance was found (NA, BL, PG, CB and PC) (p B 0.006). NA showed a for all pairwise comparison between NA, BL, CB, PC statistically lower initial density to BL, PG, CB and PC and PG (p [ 0.105) except for a higher initial density (p B 0.006) before compaction. All other groups (BL, 123 Cell Tissue Bank which was found for PG in comparison to BL After compaction initial density was similar between (p = 0.030). all groups PG had the highest initial density after All pairwise comparison between NA, BL, CB, PC compaction, which was statistically different from BL. and PG of the density at the yield limit before and after As PG can be considered as a gel it can be deduced that compaction did not reach statistical significance level the gel may absorb better kinetic energy during the (p [ 0.080). standardized compaction procedure and therefore When considering the yield limit a statistically reduce its volume not as much as BL. significant lower value could be found for CB in When considering the density at the yield limit a comparison to PC (p = 0.027), to NA (p = 0.008), to statistically significant reduction before and after BL (p = 0.016) and to OG (p = 0.003) before com- compaction was found for BL and PG, which was paction. After compaction OG showed a statistical again higher than 10%. Between the five groups under significant higher yield limit in comparison to other investigation no statistically significant difference was groups (p B 0.001). NA showed a statistically higher found. When considering the mean of each group after yield limit in comparison to PG (p = 0.038) after compaction, they are surprisingly similar for BL,CB, compaction. All other pairwise comparisons between PC,PG and NA, which could be an indication for the groups did not reach statistically significance level mixage with blood for BL, CB and NA and mixage (p [ 0.077) as well before as after compaction. with platelets in PC and PG. However no statistically evidence was found for this observation and the samples did not differ from native allografts. Discussion The yield limit was improved in all cases signifi- cantly after compaction. In case of the clotted groups Adding blood, PRP or PLC in allografts has shown in CB and PG the difference before and after compaction different studies to enhance bone ingrowth (Khan et al. was less prominent \ 21% than for the not activated 2005; Anitua et al. 2004; Hannink et al. 2009; Baylink groups BL and PC, where an increase of [ 35% was et al. 1993; Canalis et al. 1989; Canalis 1985; Lozada observed. In the uncompacted group the Yield limit et al. 2001; Cenni et al. 2010; Blair and Flaumenhaft seemed to be higher for BL and PC than for CB and PG 2009). It is therefore a promising additive to chemical although no statistically significant difference cleaned allografts, were growth factors may be washed between groups could be found. After compaction out by the cleaning procedure itself. Our measure- the yield limit of al four groups (BL, PC, CB and PG) ments showed that the yield limit of four different reached similar values and no difference could be prepared allografts did not significantly differ from found. It can be deduced that the liquid phase is each other after compaction and did not differ in absorbed in the spongious allograft material and plays comparison to native allografts. However a statisti- an inferior role on the mechanical properties. OG cally significant difference was found in comparison showed the highest yield limit after compaction, which to dried allografts with optimized grain size distribu- shows the benefit in reducing liquid and fatty content tion. This findings are in accordance with several other for the improving mechanical interlocking of the studies (Putzer et al. 2014a, b; Fosse et al. 2006; Voor particles. NA showed a statistically higher yield limit et al. 2004). in comparison to PG (p = 0.038) after compaction, OG showed a statistically significant lower initial however all other pairwise comparisons between density and a lower density at the yield limit before groups did not reach statistically significance level and after compaction in all cases, as in the preparation (p [ 0.077) as well before as after compaction. This process no liquids were added resulting in a sample indicates that all four mixtures are similar to native weight of 8 g. All samples from the other groups had a bone. Their usage can be recommended, especially the weight of 16 g. In CB and PG the initial density was platelet concentrate gel as it should contain the highest increased by more than 10%, while the other two amount of GF, while having similar mechanical groups, where clotting was not activated, did not show properties to native allografts. a statistically significant difference. The activation of Several studies show, that the fat and liquid content the clotting may have induced a better interlocking of allografts reduce the primary stability of allografts between particles for the uncompacted allografts. (Putzer et al. 2014a; Fosse et al. 2006; Voor et al. 123 Cell Tissue Bank experiment for the particle size distribution was carried out in 2004). However the authors believe that by optimizing the laboratory of IGT (Institut fu¨r Geotechnik und Tunnelbau, the grain size distribution (Putzer et al. 2014b), Innsbruck) and was supported by Stefan Tilg, technical defatting the graft material by an appropriate cleaning assistant. procedure (Coraca-Huber et al. 2013; Wurm et al. 2016) and adding GF using platelet concentrate gel or Author contribution All authors have seen and concur with the contents of the manuscript. All authors have made PRP will enhance primary stability and speed up bone substantial contributions and were involved in the study as growth in. well as the preparation of the manuscript. The authors declare To reduce patient specific properties all samples that the material within the submitted paper has not been and were carefully remixed before usage to reduce any will not be submitted for publication elsewhere, including electronically in the same form, in English or in any other biasing effect. A part of liquids may be lost during the language, without the written consent of the copyright-holder. compaction process, altering the sample composition during the measurements. In our experiments a large Compliance with ethical standards quantity of liquids (50% in weight) were added to compensate for any liquid loss during the measure- Conflict of interest This research did not receive any specific grant from funding agencies in the public, commercial, or not- ments. Bone quality was not assessed radiological by for-profit sectors. the authors, however all sample where previously screened for osteoporosis according to the quality Ethical approval Ethical approval was not required for this guidelines of the local bone bank. study. Informed consent Informed consent was obtained from all human tissue donors. Conclusion Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http:// In conclusion the study shows that there was no creativecommons.org/licenses/by/4.0/), which permits unre- statistically significant difference in the yield limit stricted use, distribution, and reproduction in any medium, between allografts mixed with blood, clotted blood, provided you give appropriate credit to the original platelet concentrate and platelet concentrate gel in author(s) and the source, provide a link to the Creative Com- mons license, and indicate if changes were made. comparison to native allografts. All of them are therefore suitable from a mechanical point of view to be used in bone impaction grafting to enhance bone References remodeling by adding growth factors. From literature it seems that platelet concentrate gel or PRP has the Anitua E, Andia I, Ardanza B, Nurden P, Nurden AT (2004) highest change to speed up bone ingrowth. Adding Autologous platelets as a source of proteins for healing and liquids could decrease primary stability in comparison tissue regeneration. Thromb Haemost 91(1):4–15. https:// to dry allografts an optimum level of liquid content doi.org/10.1160/TH03-07-0440 Bauer TW, Muschler GF (2000) Bone graft materials. An still needs to be defined. The authors recommend to overview of the basic science. Clin Orthop Relat Res chemical clean allografts for large defects, optimize 371:10–27 their grain size distribution and add GF for enhancing Baylink DJ, Finkelman RD, Mohan S (1993) Growth factors to bone ingrowth. All of the findings have to be evaluated stimulate bone formation. J Bone Miner Res 8(Suppl 2):S565–S572. https://doi.org/10.1002/jbmr.5650081326 and tested in an in vivo study for further applicability. 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Injury 42(Suppl 408:302–310 2):S16–S21. https://doi.org/10.1016/j.injury.2011.06.199 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Cell and Tissue Banking Springer Journals

Platelet concentrate as an additive to bone allografts: a laboratory study using an uniaxial compression test

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Abstract

Cell Tissue Bank https://doi.org/10.1007/s10561-018-9704-3 Platelet concentrate as an additive to bone allografts: a laboratory study using an uniaxial compression test . . . . David Putzer Markus Dobersberger Alex Pizzini Debora Corac ¸a-Huber Christoph Ammann Michael Nogler Received: 30 November 2017 / Accepted: 25 May 2018 The Author(s) 2018 Abstract Chemical cleaning procedures of allo- Keywords Processing allografts  Platelet grafts are destroying viable bone cells and denaturing concentrate  Additive to allografts  Chemical osteoconductive and osteoinductive proteins present cleaning procedures  Platelet concentrate gel in the graft. The aim of the study was to investigate the mechanical differences of chemical cleaned allografts by adding blood, clotted blood; platelet concentrate and platelet gel using a uniaxial compression test. The Introduction allografts were chemically cleaned, dried and stan- dardized according to their grain size distribution. Bone grafts are used to fill bone defects in different Uniaxial compression test was carried out for the four applications of orthopaedic and trauma surgery with groups before and after compacting the allografts. No good long term results (Schreurs et al. 2009). Auto- statistically significant difference was found between grafts are the gold standard in reconstructive surgery; native allografts, allografts mixed with blood, clotted however they are available only in limited quantity. blood, platelet concentrate and platelet concentrate gel They can be obtained from the femoral head during regarding their yield limit after compaction. The total hip arthroplasty or from the iliac crest (Khan et al. authors recommend to chemical clean allografts for 2005; Myeroff and Archdeacon 2011; Nogler et al. large defects, optimize their grain size distribution and 2012). Autografts have optimum osteoconductive, add platelet concentrate or platelet rich plasma for osteoinductive properties and allow osteogenesis as enhancing as well primary stability as well bone they contain surviving cells and osteoinductive pro- teins (BMPs) such as BMP-2 and BMP-7, fibroblast ingrowth. growth factor (FGF), insulin-like growth factor (IGF) and platelet-derived growth factor (PDGF) (Bauer and Muschler 2000; Dimitriou et al. 2011; Brydone et al. D. Putzer (&)  M. Dobersberger  D. Corac¸a-Huber 2010; Parikh 2002). C. Ammann  M. Nogler To compensate for the reduced availability of Department of Orthopaedics – Experimental autografts, allografts or synthetic materials are widely Orthopaedics, Medical University of Innsbruck, Innrain 36, 6020 Innsbruck, Austria used. Allografts have variable osteoinductive and e-mail: david.putzer@i-med.ac.at osteoconductive properties but are lacking viable cells which results in lower osteogenic potential than A. Pizzini autografts (Zimmermann and Moghaddam 2011). Department of Internal Medicine, Medical University of Innsbruck, Anichstrasse 35, 6020 Innsbruck, Austria 123 Cell Tissue Bank Sterilization processing of allografts includes the producing a 3–5 times higher platelet concentrated gel usage of hypotonic solutions, acetone, ethylene oxide, than native plasma contains (Petrungaro 2001). The or gamma irradiation which can eliminate cellular and release of PDGF, IGF, VEGF, PDAF and TGF-b viral particles and therefore reduce the risk of present in the PRP is triggered by the activation of infectious and transmissible diseases (Muller et al. platelets by means of a variety of substances or stimuli 2013). Chemical cleaning processes are also used to such as thrombin, calcium chloride, or collagen (Wang remove the fat content of the allografts enhancing the and Avila 2007). mechanical properties of the allograft (Putzer et al. PRP indeed is widely used in plastic surgery and 2014a; van der Donk et al. 2003; Fosse et al. 2006; jaw reconstruction surgery for enhancing bone and Voor et al. 2004). However, despite modern steriliza- connective tissue growth (Wang and Avila 2007; Marx tion and storage methods, processing of allografts is et al. 1998; Board et al. 2008; Anitua et al. 2004; not completely safe (Zimmermann and Moghaddam Thorwarth et al. 2006). Enriched platelet preparations 2011; Malinin and Temple 2007). have shown a rapid bone healing and regeneration The chemical cleaning as well as gamma irradiation when combined with autologous bone and bone of allografts may destroy the bone cells and denature substitute materials (Anitua et al. 2004; Kim et al. proteins present in the graft and alter osteoconductive 2001). In PRP no cross reactivity, immune reaction or and osteoinductive characteristics, essentially elimi- disease transmission has been observed (Weibrich nating the osteogenic properties and inhibiting the et al. 2001). A reduced healing time (50%) has been bone remodeling process (Keating and McQueen shown in a study of Kassolis and Reynolds (2005). In 2001). animal studies no statistically significant difference During fracture healing and implant ingrowth the was found between cancellous bone graft material recruitment and migration of osteogenic cells are with and without PRP (Butterfield et al. 2005; Jakse essential for bone regeneration (Kark et al. 2006). The et al. 2003). migration of these cells is stimulated by growth factors There is large evidence that by adding blood, as transforming growth factors (TFG), PDGF, IGF, autologous PRP or PLC the bone in-growth is vascular endothelial growth factors (VEGF), platelet enhanced, although antigenity is getting reduced in derived angiogenic factor (PDAF) and FGF (Fiedler some cases (Khan et al. 2005; Anitua et al. 2004; et al. 2002; Mayr-Wohlfart et al. 2002; Martineau et al. Hannink et al. 2009; Baylink et al. 1993; Canalis et al. 2004; Wang and Avila 2007), all of which are released 1989; Canalis 1985; Lozada et al. 2001; Cenni et al. by platelets in response to injury (Martineau et al. 2010; Blair and Flaumenhaft 2009). The aim of the 2004; Weibrich et al. 2002). In addition to growth study was to investigate the mechanical differences of factors (GFs), platelets release numerous other sub- chemical cleaned allografts with known grain size stances (e.g., fibronectin, vitronectin, sphingosine distribution mixed with blood (BL), clotted blood 1-phosphate, etc.…) that are important in wound (CB), platelet concentrate (PC) and platelet concen- healing (Wang and Avila 2007). Platelets can be trated gel (PG) using an uniaxial compression test. applied as autologous product to sites of bone injury by either being concentrated in combination with blood plasma [platelet-rich plasma (PRP)] or as a Methods platelet gel that is created by clotting the PRP (Kark et al. 2006). Bone tissue was donated by 5 patients to the local bone PRP, plasma rich in growth factors (PRGF), and bank, from whom informed consent was obtained. In platelet concentrate (PLC) are essentially an increased the preparation of allografts the local bone bank concentration of autologous platelets suspended in a requirements for producing fresh-frozen allografts small amount of plasma after centrifugation (Wang were followed. Cartilage and cortical tissue was and Avila 2007). By centrifugation donors blood is removed and using a bone mill (Spierings Medische separated into platelet poor plasma (PPP), PRP and red Techniek BV, Nijmegen, The Netherlands) allografts blood cells (Marlovits et al. 2004). Prior to applica- sizing 5–10 mm were produced (McNamara 2010). tion, topical bovine thrombin and 10% calcium The allografts were frozen to - 80 C, carefully chloride is added to activate the clotting cascade, mixed to reduce patient specific properties and stored 123 Cell Tissue Bank at - 11 C. A chemical cleaning procedure was used In group PC 4 ml of concentrated platelets from one to remove fat content of the allografts and reduce the donor, who gave his informed consent prior donation, contamination risk (Coraca-Huber et al. 2013). For the were added. The PC was stored at - 4 C. In platelet cleaning procedure a sonicator (40 kHz, 200 W , concentrated gel (PG) in addition to the 4 ml of eff BactoSonic, Bandelin eletronic GmbH & Co. KG, concentrated platelets 666 ll of 1 mol calcium chlo- Berlin, Germany) was used. As washing solutions ride (CaCl ) were added (Marx et al. 1998; Oakley and 700 ml of 1% Triton X-100 (Sigma-Aldrich, Schnell- Kuiper 2006; Camenzind et al. 2000). After 6 min dorf, Germany), 500 ml 3% hydrogen peroxide solu- activation time the samples were used for mechanical tion (Sigma-Aldrich, Schnelldorf, Germany) and a testing. 70% ethanol solution were used. The allografts were All samples were filled into a compaction chamber dried in an incubator (Memmert GmbH & Co. KG, with an internal diameter of 40 mm. A uniaxial Schwabach, Germany) at 37 C. compression test was carried out before and after a Allograft samples were assembled according to standardized compaction procedure resulting in 20 their grain size in proportions specified in Table 1 measurements before and 20 measurements after after being separated using sieves ranging from 0.063 compaction for each of the four groups. In the to 16 mm in correspondence to ASTM C 125 standard standardized compaction procedure a fall hammer (Application time 1 h, Amplitude 10 mm, Haver und (1.45 kg) was dropped 10 times from a height of Bocker, Olde, Germany) (Putzer et al. 2014b). Sam- 18 cm. ples with a mean weight of 8 ± 0.01 g were obtained The uniaxial compression test was carried out with and divided into four groups each containing 20 a 15 mm punch which was lowered with a speed of samples. 1 mm/min into the allografts. Force and displacement In one group (BL) 4 ml blood from the same donor, were measured by the testing machine after reaching a who gave his informed consent prior donation, were preload of 5 N (Zwicki—Line Z 2.5, maximal load 2.5 added. In different studies native allografts showed a kN, 320 kHz sample rate, Zwick GmbH & Co. KG, liquid component of 50% in weight (Putzer et al. Ulm, Deutschland) with a preciseness of ± 0.04 N 2014a). Adding 4 ml of blood approximately com- and ± 2 lm. pensates the liquid component, which was previously A peak analysis was performed on the resulting removed. The blood was stored at - 4 C and was force displacement curves using OriginPro8.5 (Origin obtained from the local tissue bank. In the second Lab Corporation, Northampton, Massachusetts, USA) group (CB) clotting was induced by adding 480 llof (Fig. 1a). In all curves a fitted baseline (50 anchor 1 mol calcium chloride (CaCl ) in addition to the 4 ml points, 1 and 2 derivation method, polynomial blood and mixed thoroughly for 1 min as described by smoothing of order 2) was subtracted to remove the Oakley and Kuiper (2006) and Camenzind et al. logarithmic trend (Fig. 1b). The signal was analyzed (2000). The cleaned allografts were mixed with the for positive local maxima over 100 data points with a clotted blood and after 5 min (6 min activation time) smoothing window size of 10 data points. The yield the samples were used for mechanical testing. limit (YL) was determined as the first local maxima on the force curve (Fig. 1c). The corresponding displace- ment value was used to obtain the density at the yield limit d . The punch displacement value at 5 N was YL Table 1 All samples were reassembled after sieving with used to calculate the initial density of the samples d . specific grain size proportions achieving a total weight of 8 g Previously published data by the authors were used Grain size [mm] Weight [g] as control groups were native allografts (NA) (Putzer et al. 2014a) and allografts with optimized grain size [ 4 5.08 distribution (OG) were evaluated (Putzer et al. 2014b). 4–2 0.86 Measurements of each variable and group were 2–1 0.60 tested for normal distribution using the Kolmogorov– \ 1 1.46 Smirnov Test. Comparison before and after com- Total 8.00 paction were analyzed using the two-tailed T Test for dependent samples. All groups were tested for 123 Cell Tissue Bank Brown Forsythe test was used for group comparisons and Tukey Post-Hoc analysis was used for pairwise comparison. Outliers were defined as values that are greater than 100% and smaller than 10% of the median. They were removed from the data set. In all analysis (SPSS software v.20, IBM, Chicago, IL) a p value of 0.05 was considered statistically significant. Results NA, OG, BL, PC and PG had a normal distribution for all investigated measurement parameters. CB showed a normal distribution except for the yield limit before compaction. IN BL 1 outlier was eliminated for the yield limit before and 1 outlier after compaction. In CB one outlier was eliminated for the initial density, two outliers were eliminated for the density at the yield limit and two for the yield limit when uncom- pacted. After compaction in CB one outlier was eliminated for the yield limit. PC showed two outliers for the initial density when uncompacted. PG showed two outliers for the yield limit before compaction. No statistically significant change of the initial density was observed after compaction for BL and PC (Table 2). In NA a statistically significant increase of 22% and in OG a of 34% was observed, while CB showed a statistical significant increase of the initial density by 10% and PG increased its initial density after compaction by 13%. Considering the density at the yield limit before and after compaction BL showed a statistically significant increase of 13% and PG of 14%, while NA showed an increase of 10% and OG of 22% (Table 3). In CB and PC no statistically significant increase of the density at the yield limit could be observed. All groups showed a statistical significant differ- ence when comparing the yield limit before and after Fig. 1 a Force displacement curves were from uniaxial compaction (Table 4). BL and PC showed a * 35% compression test using a testing machine. b In all curves a higher yield limit after compaction, while in the fitted baseline with 50 anchor points (squares) was subtracted to groups with the activation liquid CB and PG the yield remove the logarithmic trend. c The signal was analyzed for limit increased by 15% for CB and 20% for PG. NA positive local maxima. The yield limit (YL) was determined as the first local maxima on the force curve and is indicated by a showed an increase of the yield limit by 80% and OG line in all three graphs of 90%. The uncompacted initial density and uncompacted variance homogeneity. In case of variance homogene- yield limit showed a homogeneity distribution of ity ANOVA was used for group comparisons and variances. All other variables did not show homo- Games Howel Post-Hoc analysis for pairwise com- geneity of variances. parisons. If variance homogeneity was not fulfilled the 123 Cell Tissue Bank Table 2 Mean and standard deviation of the initial density before and after compaction are reported for the four groups under investigation d [g/cm ] Uncompacted Compacted Difference p value NA 0.91 (SD 0.14) 1.17 (SD 0.09) 22.3 \ 0.001 OG 0.39 (SD 0.04) 0.59 (SD 0.07) 33.9 \ 0.001 BL 1.12 (SD 0.08) 1.14 (SD 0.22) 0.695 CB 1.13 (SD 0.09) 1.26 (SD 0.30) 10.3 0.044 PC 1.13 (SD 0.09) 1.14 (SD 0.14) 0.621 PG 1.16 (SD 0.08) 1.34 (SD 0.26) 13.4 0.004 The difference was calculated as a percentage and p value of the T test (comparison before and after compaction) is reported The sample weight was half of the other groups Table 3 Mean and standard deviation of the density at the yield limit before and after compaction are reported for the four groups under investigation d [g/cm ] Uncompacted Compacted Difference p value YL NA 1.45 (SD 0.24) 1.60 (SD 0.21) 10.1 0.008 OG 0.58 (SD 0.19) 0.74 (SD 0.08) 21.7 \ 0.001 BL 1.48 (SD 0.13) 1.71 (SD 0.46) 13.4 0.049 CB 1.48 (SD 0.13) 1.48 (SD 0.30) 0.969 PC 1.53 (SD 0.19) 1.70 (SD 0.35) 0.178 PG 1.53 (SD 0.13) 1.78 (SD 0.45) 14.0 0.031 The difference was calculated as a percentage and p value of the T test (comparison before and after compaction) is reported The sample weight was half of the other groups Table 4 Mean and standard deviation of the yield limit before and after compaction are reported for the four groups under investigation YL [kPa] Uncompacted Compacted Difference p value NA 24 (SD 19) 117 (SD 62) 79.1 \ 0.001 OG 35 (SD 37) 353 (SD 187) 90.1 \ 0.001 BL 33 (SD 30) 90 (SD 28) 36.7 \ 0.001 CB 12 (SD 14) 83 (SD 32) 14.4 \ 0.001 PC 32 (SD 23) 91 (SD 32) 35.2 \ 0.001 PG 16 (SD 11) 78 (SD 34) 20.5 \ 0.001 The difference was calculated as a percentage and p value of the T test (comparison before and after compaction) is reported The OG group showed a statistically significant PG, CB and PC) showed no statistically significant lower initial density and a lower density at the yield difference between each other (p [ 0.376). After limit to all other groups before and after compaction compaction no statistically significance was found (NA, BL, PG, CB and PC) (p B 0.006). NA showed a for all pairwise comparison between NA, BL, CB, PC statistically lower initial density to BL, PG, CB and PC and PG (p [ 0.105) except for a higher initial density (p B 0.006) before compaction. All other groups (BL, 123 Cell Tissue Bank which was found for PG in comparison to BL After compaction initial density was similar between (p = 0.030). all groups PG had the highest initial density after All pairwise comparison between NA, BL, CB, PC compaction, which was statistically different from BL. and PG of the density at the yield limit before and after As PG can be considered as a gel it can be deduced that compaction did not reach statistical significance level the gel may absorb better kinetic energy during the (p [ 0.080). standardized compaction procedure and therefore When considering the yield limit a statistically reduce its volume not as much as BL. significant lower value could be found for CB in When considering the density at the yield limit a comparison to PC (p = 0.027), to NA (p = 0.008), to statistically significant reduction before and after BL (p = 0.016) and to OG (p = 0.003) before com- compaction was found for BL and PG, which was paction. After compaction OG showed a statistical again higher than 10%. Between the five groups under significant higher yield limit in comparison to other investigation no statistically significant difference was groups (p B 0.001). NA showed a statistically higher found. When considering the mean of each group after yield limit in comparison to PG (p = 0.038) after compaction, they are surprisingly similar for BL,CB, compaction. All other pairwise comparisons between PC,PG and NA, which could be an indication for the groups did not reach statistically significance level mixage with blood for BL, CB and NA and mixage (p [ 0.077) as well before as after compaction. with platelets in PC and PG. However no statistically evidence was found for this observation and the samples did not differ from native allografts. Discussion The yield limit was improved in all cases signifi- cantly after compaction. In case of the clotted groups Adding blood, PRP or PLC in allografts has shown in CB and PG the difference before and after compaction different studies to enhance bone ingrowth (Khan et al. was less prominent \ 21% than for the not activated 2005; Anitua et al. 2004; Hannink et al. 2009; Baylink groups BL and PC, where an increase of [ 35% was et al. 1993; Canalis et al. 1989; Canalis 1985; Lozada observed. In the uncompacted group the Yield limit et al. 2001; Cenni et al. 2010; Blair and Flaumenhaft seemed to be higher for BL and PC than for CB and PG 2009). It is therefore a promising additive to chemical although no statistically significant difference cleaned allografts, were growth factors may be washed between groups could be found. After compaction out by the cleaning procedure itself. Our measure- the yield limit of al four groups (BL, PC, CB and PG) ments showed that the yield limit of four different reached similar values and no difference could be prepared allografts did not significantly differ from found. It can be deduced that the liquid phase is each other after compaction and did not differ in absorbed in the spongious allograft material and plays comparison to native allografts. However a statisti- an inferior role on the mechanical properties. OG cally significant difference was found in comparison showed the highest yield limit after compaction, which to dried allografts with optimized grain size distribu- shows the benefit in reducing liquid and fatty content tion. This findings are in accordance with several other for the improving mechanical interlocking of the studies (Putzer et al. 2014a, b; Fosse et al. 2006; Voor particles. NA showed a statistically higher yield limit et al. 2004). in comparison to PG (p = 0.038) after compaction, OG showed a statistically significant lower initial however all other pairwise comparisons between density and a lower density at the yield limit before groups did not reach statistically significance level and after compaction in all cases, as in the preparation (p [ 0.077) as well before as after compaction. This process no liquids were added resulting in a sample indicates that all four mixtures are similar to native weight of 8 g. All samples from the other groups had a bone. Their usage can be recommended, especially the weight of 16 g. In CB and PG the initial density was platelet concentrate gel as it should contain the highest increased by more than 10%, while the other two amount of GF, while having similar mechanical groups, where clotting was not activated, did not show properties to native allografts. a statistically significant difference. The activation of Several studies show, that the fat and liquid content the clotting may have induced a better interlocking of allografts reduce the primary stability of allografts between particles for the uncompacted allografts. (Putzer et al. 2014a; Fosse et al. 2006; Voor et al. 123 Cell Tissue Bank experiment for the particle size distribution was carried out in 2004). However the authors believe that by optimizing the laboratory of IGT (Institut fu¨r Geotechnik und Tunnelbau, the grain size distribution (Putzer et al. 2014b), Innsbruck) and was supported by Stefan Tilg, technical defatting the graft material by an appropriate cleaning assistant. procedure (Coraca-Huber et al. 2013; Wurm et al. 2016) and adding GF using platelet concentrate gel or Author contribution All authors have seen and concur with the contents of the manuscript. All authors have made PRP will enhance primary stability and speed up bone substantial contributions and were involved in the study as growth in. well as the preparation of the manuscript. The authors declare To reduce patient specific properties all samples that the material within the submitted paper has not been and were carefully remixed before usage to reduce any will not be submitted for publication elsewhere, including electronically in the same form, in English or in any other biasing effect. A part of liquids may be lost during the language, without the written consent of the copyright-holder. compaction process, altering the sample composition during the measurements. In our experiments a large Compliance with ethical standards quantity of liquids (50% in weight) were added to compensate for any liquid loss during the measure- Conflict of interest This research did not receive any specific grant from funding agencies in the public, commercial, or not- ments. Bone quality was not assessed radiological by for-profit sectors. the authors, however all sample where previously screened for osteoporosis according to the quality Ethical approval Ethical approval was not required for this guidelines of the local bone bank. study. Informed consent Informed consent was obtained from all human tissue donors. Conclusion Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http:// In conclusion the study shows that there was no creativecommons.org/licenses/by/4.0/), which permits unre- statistically significant difference in the yield limit stricted use, distribution, and reproduction in any medium, between allografts mixed with blood, clotted blood, provided you give appropriate credit to the original platelet concentrate and platelet concentrate gel in author(s) and the source, provide a link to the Creative Com- mons license, and indicate if changes were made. comparison to native allografts. All of them are therefore suitable from a mechanical point of view to be used in bone impaction grafting to enhance bone References remodeling by adding growth factors. From literature it seems that platelet concentrate gel or PRP has the Anitua E, Andia I, Ardanza B, Nurden P, Nurden AT (2004) highest change to speed up bone ingrowth. Adding Autologous platelets as a source of proteins for healing and liquids could decrease primary stability in comparison tissue regeneration. Thromb Haemost 91(1):4–15. https:// to dry allografts an optimum level of liquid content doi.org/10.1160/TH03-07-0440 Bauer TW, Muschler GF (2000) Bone graft materials. An still needs to be defined. The authors recommend to overview of the basic science. Clin Orthop Relat Res chemical clean allografts for large defects, optimize 371:10–27 their grain size distribution and add GF for enhancing Baylink DJ, Finkelman RD, Mohan S (1993) Growth factors to bone ingrowth. All of the findings have to be evaluated stimulate bone formation. J Bone Miner Res 8(Suppl 2):S565–S572. https://doi.org/10.1002/jbmr.5650081326 and tested in an in vivo study for further applicability. Blair P, Flaumenhaft R (2009) Platelet alpha-granules: basic biology and clinical correlates. Blood Rev 23(4):177–189. Acknowledgements Open access funding provided by https://doi.org/10.1016/j.blre.2009.04.001 University of Innsbruck and Medical University of Innsbruck. Board TN, Rooney P, Kay PR (2008) Strain imparted during This study was carried out with internal funds from impaction grafting may contribute to bony incorporation: Experimental Orthopedics, Innsbruck Medical University. an in vitro study of the release of bmp-7 from allograft. David Putzer, PhD, Debora Coraca-Huber, PhD, Christoph J Bone Joint Surg Br 90(6):821–824. https://doi.org/10. 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Journal

Cell and Tissue BankingSpringer Journals

Published: May 31, 2018

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