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Tsukasa Ohno, Hiroto Suenaga, Aika Yamawaki-Ogata, Kei Kanie, Ryuji Kato, [Koichiro Uto](https://orcid.org/0000-0001-7091-0585), [Mitsuhiro Ebara](https://orcid.org/0000-0002-7906-0350), Hideki Ito, Yuji Narita, Akihiko Usui, Masato Mutsuga

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[Development of novel waxy bone haemostatic agents composed of biodegradable polymers with osteogenic-enhancing peptides in rabbit models](https://mdr.nims.go.jp/datasets/3f08984c-eed4-4f54-a21b-5c8575fb71d7)

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OP-ICVA230171 1..9Cite this article as: Ohno T, Suenaga H, Yamawaki-Ogata A, Kanie K, Kato R, Uto K et al. Development of novel waxy bone haemostatic agents composed of biode-gradable polymers with osteogenic-enhancing peptides in rabbit models. Interdiscip CardioVasc Thorac Surg 2023; doi:10.1093/icvts/ivad170.Development of novel waxy bone haemostatic agents composed ofbiodegradable polymers with osteogenic-enhancing peptides inrabbit modelsTsukasa Ohno a,†, Hiroto Suenagaa,†, Aika Yamawaki-Ogataa, Kei Kanieb,e, Ryuji Katob,c, Koichiro Utod,Mitsuhiro Ebarad, Hideki Ito a, Yuji Narita a,*, Akihiko Usuia and Masato Mutsuga aa Department of Cardiac Surgery, Nagoya University Graduate School of Medicine, Tokai National Higher Education and Research System, Nagoya, Japanb Department of Basic Medicinal Sciences, Graduate School of Pharmaceutical Sciences, Nagoya University, Tokai National Higher Education and Research System,Nagoya, Japanc Division of Micro-Nano Mechatronics, Institute of Nano-Life-Systems, Institutes of Innovation for Future Society, Nagoya University, Tokai National HigherEducation and Research System, Nagoya, Japand Biomaterials Field, Research Center for Functional Materials, National Institute for Materials Science (NIMS), Tsukuba, Japane Department of Biotechnology and Chemistry, Kindai University, Higashi-Hiroshima, Japan* Corresponding author. 65, Tsurumai-cho, Syowa-ku, Nagoya, Aichi 466-8560, Japan. Tel: +81-52-741-2111; fax: +81-52-744-2785; e-mail: ynarita@med.nagoya-u.ac.jp (Y. Narita).Received 8 September 2023; received in revised form 2 October 2023; accepted 30 October 2023†The first two authors contributed equally to this work.THORACICNON-ONCOLOGYVC The Author(s) 2023. Published by Oxford University Press on behalf of the European Association for Cardio-Thoracic Surgery.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), whichpermits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.Interdisciplinary CardioVascular and Thoracic Surgery 2023, 37(5), ivad170 ORIGINAL ARTICLEhttps://doi.org/10.1093/icvts/ivad170 Advance Access publication 31 October 2023Downloaded from https://academic.oup.com/icvts/article/37/5/ivad170/7334459 by guest on 07 December 2023https://orcid.org/0000-0002-9051-4202https://orcid.org/0000-0002-0164-6108https://orcid.org/0000-0002-6067-6833https://orcid.org/0000-0001-9740-2142AbstractOBJECTIVES: The use of bone wax (BW) is controversial for sternal haemostasis because it increases the risk of wound infection andinhibits bone healing. We developed new waxy bone haemostatic agents made from biodegradable polymers containing peptides andevaluated them using rabbit models.METHODS: We designed 2 types of waxy bone haemostatic agents: peptide wax (PW) and non-peptide wax (NPW), which usedpoly(e-caprolactone)-based biodegradable polymers with or without an osteogenesis-enhancing peptide, respectively. Rabbits wererandomly divided into 4 groups based on treatment with BW, NPW, PW or no treatment. In a tibial defect model, the bleedingamount was measured and bone healing was evaluated by micro-computed tomography over 16 weeks. Bone healing in a mediansternotomy model was assessed for 2 weeks using X-ray, micro-computed tomography, histological examination and flexuralstrength testing.RESULTS: The textures of PW and NPW (n = 12 each) were similar to that of BW and achieved a comparable degree of haemostasis.The crevice area of the sternal fracture line in the BW group was significantly larger than that in other groups (n = 10 each). The PW groupdemonstrated the strongest sternal flexural strength (n = 10), with complete tibial healing at 16 weeks. No groups exhibited woundinfection, including osteomyelitis.CONCLUSIONS: Waxy biodegradable haemostatic agents showed satisfactory results in haemostasis and bone healing in rabbit modelsand may be an effective alternative to BW.Keywords: Haemostatic agents • Bone wax • Sternum • Biodegradable polymer • Peptides • BleedingABBREVIATIONSBMPs Bone morphogenetic proteinsBW Bone waxCL e-CaprolactoneDLLA D,L-LactideG0 Storage modulusG00 Loss modulusGPC Gel permeation chromatographyHap HydroxyapatiteHU Hounsfield unitsNPW Non-peptide waxMSCs Mesenchymal stem cellsPCL Poly(e-caprolactone)PDLLA Poly(D,L-lactide)PDLLA-PCL Poly(D,L-lactide-co-e-caprolactone)PW Peptide waxTm Melting temperatureINTRODUCTIONFull median sternotomy, developed by Ormand Julian in 1957, is theconventional procedure used to enter the mediastinum in cardiovas-cular surgery [1]. It is estimated that 10 000 cardiothoracic surgeonsin 6000 centres globally perform >2 million open-heart operationsper year [2]. Inadequate sternal haemostasis leads to poor surgicalvisibility, and haematoma formation also increases the incidence ofbacterial infections and the risk of postoperative mortality andmorbidity [3]. Therefore, careful haemostasis with electric coagulationor haemostatic agents is essential for successful surgery [4].Bone wax (BW) is widely applied for bone haemostasis be-cause it is effective, has a high performance-to-cost ratio and issimple to use. Although BW is an effective haemostatic agent forbone bleeding, it is a non-biodegradable material that is not me-tabolized or resorbed and remains indefinitely at the site of ap-plication [5, 6]. In addition, it has been reported that BWincreases infection rates, interferes with bone healing and resultsin chronic inflammatory reactions and sternal nonunion [6, 7].Hence, BW is not recommended for sternal application and re-ceived a class III recommendation in a guideline for the preven-tion of mediastinitis [8]. An ideal bone haemostatic agent shouldhave the following properties: a waxy texture, so it can be easilypacked into bone marrow; biodegradability and biocompatibility,so it does not impair bone healing; and adequate haemostatic ef-ficacy. In addition, bone haemostatic agents should ideally pro-mote bone regeneration.Poly(e-caprolactone) (PCL) and poly(D,L-lactide) (PDLLA) arewell-known biodegradable polymers that are widely used in bio-medical devices such as bone scaffolds and pins for sternal fixa-tion. Hydroxyapatite (Hap) is also used in medical applicationsbecause its mineral composition resembles that of natural boneand it exhibits good osteoconductivity [9]. Recently, short func-tional peptides have attracted attention for biomedical applica-tions [10, 11]. We previously showed that osteogenic-enhancingpeptides promoted the differentiation of mesenchymal stem cells(MSCs) to osteoblasts [12]. In the present study, we describenovel waxy bone haemostatic agents developed using PCL,PDLLA, Hap and osteogenic-enhancing peptides and evaluatedtheir haemostatic and bone healing capability.MATERIALS AND METHODSAnimals and ethicsSeven- to 12-week-old male Japanese white rabbits (1.26–1.56 kg, Slc: JW/CSK, closed colony) were purchased from JapanSLC, Inc. (Hamamatsu, Shizuoka, Japan) and maintained on a reg-ular chow diet under standard conditions. All animal experimentswere performed in accordance with the Guide for the Care andUse of Laboratory Animals published by the United StatesNational Institutes of Health (NIH publication No. 85-23, revised2011) and were approved by the Animal Care and UseCommittee of Nagoya University (protocol No. 31168) on 8March 2019.2 T. Ohno et al. / Interdisciplinary CardioVascular and Thoracic SurgeryDownloaded from https://academic.oup.com/icvts/article/37/5/ivad170/7334459 by guest on 07 December 2023Poly(e-caprolactone)-based biodegradablepolymerTo develop degradable base materials with a soft texture resem-bling that of BW, we selected a random copolymer composed ofD,L-lactide (DLLA), e-caprolactone (CL) and Hap. Four-armedpoly(D,L-lactide-co-e-caprolactone) (PDLLA-PCL) was synthesizedby ring-opening polymerization of DLLA and CL from the termi-nal hydroxyl groups of pentaerythritol using tin octanoate as acatalyst, as described in previous studies [13, 14]. The DLLA/CLratio was 40/60 (mol%). The structure and molecular weight wereestimated by proton nuclear magnetic resonance spectroscopy(JEOL, Tokyo, Japan) and gel permeation chromatography (GPC;JASCO International, Tokyo, Japan). The thermal properties of theobtained polymer were characterized by differential scanningcalorimetry (DSC6100, Seiko Instruments, Chiba, Japan). Themeasurements were conducted from 0 to 120�C at a heating rateof 5�C min-1. The viscoelastic properties of the copolymers wereevaluated using a rheometer (MCR 301, Anton Paar, Tokyo,Japan) with parallel plate geometry (rotating top plate of 10-mmdiameter). Furthermore, equal weights of Hap (50 wt%) andPDLLA-PCL (50 wt%) (in a molten state) were vigorously mixed tocreate a homogeneous PDLLA-PCL/Hap composite. The textureof this PCL-based biodegradable polymer above the meltingtemperature (Tm) was achieved by the use of Hap [13].Osteogenic-enhancing peptidesOsteogenesis-enhancing peptides were determined by in silicoand peptide array screening in a previous study [12]. Briefly, sev-eral homologous sequences of bone morphogenetic proteins(BMPs), including BMP-2, BMP-4, BMP-6 and BMP-7, were iden-tified by in silico screening. Twenty-five candidate peptides wereselected from the homologous regions of 9 consecutive aminoacid sequences. Then, a direct cell assay using a peptide arraywas employed to select the osteogenic-enhancing peptideTLVNSVNSK, which enhanced osteogenic cell-selective prolifera-tion and osteogenic differentiation of MSCs. The TLVNSVNSKpeptide was prepared using a conventional solid-phase chemicalsynthesis method (GL Biochem (Shanghai) Ltd., Shanghai, China)and had a purity of over 90%.Preparation of waxy bone haemostatic agentsWe prepared 2 types of waxy bone haemostatic agents: peptidewax (PW) and non-peptide wax (NPW), which were PCL-basedbiodegradable polymers with or without an osteogenic-enhancing peptide, respectively. PW was obtained by kneadingthe osteogenic-enhancing peptide (TLVNSVNSK) at 0.2% (w/w)into the PCL-based biodegradable polymer. SupplementaryMaterial, Fig. S1A shows the appearance of each haemostaticagent, specifically BW, NPW and PW.All rabbits were anaesthetized using intramuscular ketamine(10 mg/kg) and xylazine (1–3 mg/kg) and maintained on isoflur-ane. All animals received mefenamic acid (25 mg) after surgeryfor analgesia. The animals were returned to the recovery roomafter surgery for postoperative care.Tibia modelTo evaluate the amount of bone marrow bleeding after the use ofhaemostatic agents, a cortical bone defect model was established bycreating bone defects on each tibia. Twenty-four rabbits were ran-domly divided into 4 groups. Twelve tibiae of 6 rabbits per groupwere pierced with a 2-mm drill and treated with or without haemo-static agents. Bleeding from the bone defects was controlled usingNPW, PW, BW or a beeswax-based haemostat (Ethicon, Inc,Somerville, NJ). The sham group was treated without any haemostaticagents. Equal amounts (0.02 g) of each haemostatic agent were ap-plied to cover the tibial holes. Sterilized dry gauze was then appliedto the bone defect for 3 min (Supplementary Material, Fig. S1B), andthe amount of bleeding was measured using a digital scale with aminimum display of 0.01 g (HT-120, A&D Company, Tokyo, Japan).Measurements were rounded off to the second decimal place andexpressed to 2 significant digits. Finally, the surgical wounds wereclosed using 3–0 VICRYLVR (Ethicon, Inc., Somerville, NJ). Sixteen weekspostoperatively, the animals were sacrificed by an overdose of intra-venous potassium chloride under general anaesthesia using intramus-cular ketamine and xylazine, and the tibias were harvested.Sternal modelSternotomy was performed with a circular saw under anaesthesiawith 1–2% isoflurane. Forty rabbits were randomly divided into 4groups based on haemostatic treatment with NPW, PW, BW orno haemostatic agent as a control (n = 10, respectively). Equalamounts (0.2 g) of the haemostatic agents were applied to covereach cut bone surface. The sternum was fixed by 2–0 braid silksuture (Supplementary Material, Fig. S1C), and subcutaneous tis-sue was closed with 4–0 VICRYL (Ethicon, Inc., Somerville, NJ).Two weeks postoperatively, the animals were sacrificed by anoverdose of intravenous potassium chloride under general anaes-thesia using intramuscular ketamine and xylazine, and the sternawere harvested. The sterna of 10 rabbits without sternotomywere harvested as a control group. The enucleated sterna werethen osteotomized at the third and fourth joints; the formerpieces were subjected to mechanical fracture strength testing,and the latter were fixed immediately in 10% phosphate-bufferedformaldehyde for histological analysis.X-ray and micro-computed tomography analysisX-ray and micro-computed tomography (MCT) evaluations wereperformed on all sterna and tibiae. The crevice area on sternal X-rays was quantified by ImageJ software (National Institutes ofHealth, Bethesda, MD, USA). MCT analysis was performed on aSkyScan 1176 Technical Specifications 64 MCT scanner (BRUKER,Kontich, Belgium). MCT images of tibiae were obtained at themiddle of the defective area, and those of the sternum wereobtained at the middle of the third joint. Sternal quality wasquantitatively assessed on MCT scans by analysing Hounsfieldunits (HU) on 10 randomly selected horizontal slices using CT-analyser software version 1.13 (Bruker).Three-point flexural strength testsFresh sternal specimens in each group were used for three-pointflexural strength testing. The test was performed with anTHORACICNON-ONCOLOGY3T. Ohno et al. / Interdisciplinary CardioVascular and Thoracic SurgeryDownloaded from https://academic.oup.com/icvts/article/37/5/ivad170/7334459 by guest on 07 December 2023https://academic.oup.com/icvts/article-lookup/doi/10.1093/icvts/ivad170#supplementary-datahttps://academic.oup.com/icvts/article-lookup/doi/10.1093/icvts/ivad170#supplementary-datahttps://academic.oup.com/icvts/article-lookup/doi/10.1093/icvts/ivad170#supplementary-datahttps://academic.oup.com/icvts/article-lookup/doi/10.1093/icvts/ivad170#supplementary-dataAUTOGRAPH AGS-J 500N (SHIMADZU Corporation, Kyoto, Japan)using a three-point evaluation with a 0.2-mm/s crosshead speed(Supplementary Material, Fig. S1D). The data were analysed byTRAPEZIUM2 data processing software (SHIMADZU Corporation).HistologySpecimens were obtained from the fourth joint of the sternum,washed with saline and fixed with 4% paraformaldehyde (PFA,Fujifilm Wako Pire Chemical Corporation, Osaka, Japan) at 4�Cfor 2 days. They were then decalcified with ethylenediamine tet-raacetic acid (Sigma-Aldrich, St. Louis, MO, USA) for about2 months before embedding in paraffin. Specimens were cut into5-mm-thick horizontal sections and stained with haematoxylin–eosin. All microscopic assessments were performed using an FSX-100 microscope (OLYMPUS, Tokyo, Japan).Statistical analysisStatistical analyses of the bleeding amount, crevice area, HUvalue and flexural strength were performed using Tukey’s multi-ple comparisons test. P < 0.05 was considered to be statisticallysignificant. All statistical analyses were performed using SPSS forWindows, version 24 (SPSS, Chicago, IL, USA). Data are expressedas median and interquartile range (IQR).RESULTSCharacterization of poly(D,L-lactide-co-e-caprolactone)PDLLA-PCL was obtained as a viscous solid, and its number aver-age molecular weight was estimated to be 37100 by GPC cali-brated with a polyethylene glycol standard. GPC also showedthat the copolymer possessed a relatively narrow molecularweight distribution (polydispersity index: weight-average molec-ular weight/number average molecular weight = 1.44). The DLLA/CL ratio in the copolymer was determined by proton nuclearmagnetic resonance to be 63.9/36.1 mol% (SupplementaryMaterial, Fig. S2A). Differential scanning calorimetry showed thatthe copolymer had a Tm of around 37�C, which was lower thanthat of BW (Supplementary Material, Fig. S2B and C). To provideinsight into the texture of the copolymer, we carried out a rheo-logical analysis. For PDLLA-PCL, the loss modulus (G00) was higherthan the storage modulus (G0) at 37�C. The values of G0 and G00for PDLLA-PCL were much higher than those for BW(Supplementary Material, Fig. S2D and E), suggesting that the co-polymer behaves as a viscous liquid above Tm and is stickier thanBW. Hap was then mixed with molten PDLLA-PCL to create awaxy biodegradable polymer. The stickiness of PDLLA-PCL wassignificantly reduced by mixing with Hap, which made the result-ing substance easier to handle, even above Tm.Evaluation of the bleeding amountThe waxy haemostatic agents PW and NPW were malleable andcould be applied to bone surfaces with the fingertips as easily asBW. Bleeding from the bone surfaces was quickly and easily con-trolled in the NPW and PW groups, and the haemostatic actionof these substances showed similar efficacy to that of BW(Fig. 1A). The median amount of bleeding in each group was asfollows: NPW group, 0.03 (IQR 0.02–0.09) g; PW group, 0.04 (IQR0.01–0.05) g; BW group, 0.02 (IQR 0.02–0.07) g; sham group, 0.4(IQR 0.36–0.60) g. The sham group exhibited the greatest amountof bleeding (P < 0.001), with no significant difference between thePW, NPW and BW groups (Fig. 1B).Sternal assessmentNo postoperative sternal infection or mediastinitis was observedin any of the rabbits during the study. Radiograms of the sternumare shown in Supplementary Material, Fig. S3. Sterna treated withBW showed large crevices at the implantation sites. In contrast,those treated with PW and NPW showed adequate coaptationthat was similar to that in the sham group (SupplementaryMaterial, Fig. S3A). The crevice area in the BW group was signifi-cantly larger [median 26.5 (IQR 25.5–28.0)%] than that in anyother group [NPW group, median 11 (IQR 9.1–13.4)%; PW group,median 9.6 (IQR 7.9–10.3)%; sham group, median 10.2 (IQR 9.3–11.0)%; P < 0.001, Supplementary Material, Fig. S3B].MCT images are shown in Fig. 2A. Sternal fractures were ob-served in the BW group. The median HU value was -368.5 (IQR-386.8 to -281.8) HU in the BW group, which was significantlylower than that in any other group (Fig. 2B, P < 0.001). Therewere no significant differences between the other groups [NPWgroup versus PW group versus sham group, median 459 (IQR290.0–827.3) HU versus median 560 (IQR 407.5–673.8) HU versusmedian 440.5 (IQR 165.8–732.5) HU, respectively]. In the three-point flexural strength test, the median result in the BW groupwas 59.1 (IQR 41.1–70.9) N, which indicates significant weaknesscompared to the other groups [NPW group, median 120.9 (IQR84.7–137.7) N; PW group, median 152.1 (IQR 134.9–177.1) N;sham, median 144.7 (IQR 117.9–159.1) N; BW versus NPW,P < 0.05; BW versus PW, P < 0.001; BW versus sham, P < 0.001; BWversus normal, P < 0.05; Fig. 2C]. The strength in the NPW group,median 111.0 (IQR 105.8–121.0) N, was similar to those in thesham and normal groups. Moreover, the PW group exhibited sig-nificantly greater strength than the NPW group (P < 0.05).Histological findingsResults of histological analysis of the sternum are shown in Fig. 3.The injured area in the sham group had a bone structure similarto the normal sternum, with the presence of bone marrow andtrabecular bone. On the other hand, the medullary cavity in theBW group exhibited residual BW, fibrosis and many inflamma-tory cells, including lymphocytes and macrophages. In addition,the cortical bone was surrounded by hyaline cartilage, fibroticconnective tissue and inflammatory cells. Fragmented residualpolymers were observed in the NPW group, while these were al-most absorbed in the PW group. These residual polymers weresurrounded by inflammatory cells and fibroblast-like cells.Fibrous connective tissue, dense connective tissue, hyaline carti-lage and calcified cartilaginous tissue were also present at thecortical bone surgical site in both the NPW and NP groups.Long-term evaluation of tibiaeNone of the groups in the tibia model exhibited wound infection.The NPW, PW and sham groups showed almost complete bone4 T. Ohno et al. / Interdisciplinary CardioVascular and Thoracic SurgeryDownloaded from https://academic.oup.com/icvts/article/37/5/ivad170/7334459 by guest on 07 December 2023https://academic.oup.com/icvts/article-lookup/doi/10.1093/icvts/ivad170#supplementary-datahttps://academic.oup.com/icvts/article-lookup/doi/10.1093/icvts/ivad170#supplementary-datahttps://academic.oup.com/icvts/article-lookup/doi/10.1093/icvts/ivad170#supplementary-datahttps://academic.oup.com/icvts/article-lookup/doi/10.1093/icvts/ivad170#supplementary-datahttps://academic.oup.com/icvts/article-lookup/doi/10.1093/icvts/ivad170#supplementary-datahttps://academic.oup.com/icvts/article-lookup/doi/10.1093/icvts/ivad170#supplementary-datahttps://academic.oup.com/icvts/article-lookup/doi/10.1093/icvts/ivad170#supplementary-datahttps://academic.oup.com/icvts/article-lookup/doi/10.1093/icvts/ivad170#supplementary-datahttps://academic.oup.com/icvts/article-lookup/doi/10.1093/icvts/ivad170#supplementary-dataregeneration at 16 weeks after surgery (Fig. 4), while MCT imagesshowed persistent defects in the BW group.DISCUSSIONPost-sternotomy haemostatic agents should be flexible enoughto evenly cover the rough surface of the bone and also be visco-elastic enough to remain embedded. BW, which is conventionallyapplied, has these properties. Using the biodegradable polymersPDLLA, PCL and Hap, we developed new waxy haemostaticagents that mimicked the texture of BW. These agents softenedaround 37�C and, like BW, could be kneaded with the fingers.Moreover, they could be easily applied to the bone surface andto the bone marrow cavity, which has fine gaps, and showed sta-ble haemostatic ability due to their viscoelastic property. Therewas no significant difference in haemostatic ability between BWand the waxy haemostatic agents, indicating that the latter are aneffective alternative to BW for bone marrow bleeding.Haemostatic agents used for bone marrow bleeding shouldalso avoid inhibiting bone healing. In this study, MCT scans ofthe sternum showed that the area of low HU value surroundingthe divided cortical bone was larger in the BW group than in theother groups. This suggests that the non-absorbed BW remnantimpaired sternal ossification. Additionally, sterna treated with BWshowed significantly lower bone strength than the other 3groups. Sixteen-week tibial observation showed that while BWinhibited bone regeneration, PW and NPW resulted in almostcomplete bone regeneration, indicating that the haemostaticagents did not impair bone healing. An important difference be-tween BW and haemostatic agents is that the latter arebiodegradable.Several biodegradable haemostatic materials for bone marrowhaemostasis have been marketed in the USA. Ostene (BaxterInternational Inc., Chicago, IL, USA), which is a waxy haemostaticagent composed of a high-molecular-weight poly(oxyethylene)/poly(oxypropylene)/poly(oxyethylene) triblock copolymer, wasshown to be absorbed between 24 and 48 h after surgery, andunlike BW, it did not inhibit bone healing in animal models [5,15]. In addition, histomorphometric analysis showed thatBoneSeal (Hemostasis, LLC, White Bear Lake, MN, USA), whichconsists of PLA and Hap, resulted in new bone formation in ani-mal experiments [16]. These haemostatic agents consisting of bio-degradable polymers are indeed advantageous for bone healingcompared to BW. However, these studies provided insufficientdata on the flexural strength of healed sterna, including compari-son to intact sterna. Additionally, it was not mentioned whetherthe textures of these materials were similar to that of BW, or ifthese agents accelerated bone regeneration. In this study, wedemonstrated that sterna treated with NPW was significantlystiffer than those treated with BW, and PW resulted in evengreater stiffness than NPW, indicating the efficacy of the addi-tional peptides included in PW. A previous study showing thatthese peptides enhanced osteogenesis indicated that they mayaccelerate bone healing and regeneration; as amino acid sequen-ces of BMPs, they not only enhance the osteogenic differentia-tion of MSCs but also inhibit fibroblast proliferation [12]. Thisevidence demonstrates that the osteogenesis-enhancing peptidesFigure 1: Haemostasis with or without wax in a tibial model. (A) Representative images at the time of haemostasis in each group. (B) The amount of bleeding waslarger in the sham group than in the other groups (n = 12 each). ***P < 0.001 versus sham group assessed by Tukey’s multiple comparisons test.THORACICNON-ONCOLOGY5T. Ohno et al. / Interdisciplinary CardioVascular and Thoracic SurgeryDownloaded from https://academic.oup.com/icvts/article/37/5/ivad170/7334459 by guest on 07 December 2023do not induce abnormal cell proliferation or fibrous scarring butinstead lead to natural bone regeneration, resulting in greaterbone strength.LimitationsThis study has several limitations. First, although the haemostaticagents were completely absorbed at 16 weeks, we did not attemptto optimize the absorption period to promote faster bone healing.Additionally, the amount of these haemostatic agents needed to ob-tain sufficient haemostasis is still unknown. Second, it has beenreported that the incidences of postoperative sternal woundinfection and mediastinitis range from 1.4% to 3.3% [17–19], andthe use of BW is associated with an increased risk of infection [20].We observed no cases of infection with either BW or the new hae-mostatic agents. Theoretically, a biodegradable material should de-crease the risk of surgical site infection, because no foreign materialremains in the implanted site. A study using an animal femur modelshowed that a bioabsorbable phospholipid gel was associated witha significantly lower infection rate than polymethyl methacrylate,non-bioabsorbable material [21]. Third, we did not evaluate the pro-cess of long-term bone healing in the sternum model. However, theflexural strength test 2 weeks postoperatively showed that sternatreated with the haemostatic agents had the same strength as shamand normal sterna, indicating that sternal healing might be achievedFigure 2: Sternum at 2 weeks after surgery. (A) Representative micro-computed tomography images of each group. White arrows indicate the incision line. (B)Hounsfield unit values measured by micro-computed tomography (n = 10 each). (C) Mechanical strength measured by three-point flexural strength testing. The ster-num was weakest in the bone wax group and was stronger in the peptide wax group than in the non-peptide wax group (n = 10 each). *P < 0.05, ***P < 0.001 assessedby Tukey’s multiple comparisons test.6 T. Ohno et al. / Interdisciplinary CardioVascular and Thoracic SurgeryDownloaded from https://academic.oup.com/icvts/article/37/5/ivad170/7334459 by guest on 07 December 2023Figure 3: Haematoxylin–eosin staining of sternal bone in each group at 2 weeks postoperatively. Scale bar = 100 lm. BM: bone marrow; CCT: calcified cartilaginous tis-sue; DCT: dense connective tissue; FBs: fibroblast-like cells; FCT: fibrous connective tissue; FS: fibrotic scarring; HC: hyaline cartilage; ICs: inflammatory cells; RP: resid-ual polymer; TB: trabecular bone; WB: woven bone.Figure 4: Representative images of tibiae at 16 weeks after surgery. The upper panel shows morphological images, and the lower panel shows micro-computed to-mography images. Black and white arrows show the positions of bone defects.THORACICNON-ONCOLOGY7T. Ohno et al. / Interdisciplinary CardioVascular and Thoracic SurgeryDownloaded from https://academic.oup.com/icvts/article/37/5/ivad170/7334459 by guest on 07 December 2023in 2 weeks. Detailed analysis, including that of haemostatic agentdegradation over time, should be performed to better characterizebiomaterials. Fourth, although this study suggests that theosteogenic-enhancing peptides accelerated bone healing, thein vivo pharmacokinetics and pharmacodynamics of these peptidesare unclear. Therefore, the ideal composition of the osteo-enhancing peptides remains to be elucidated. Further investigationsare necessary to resolve these questions prior to clinical application.CONCLUSIONSWe newly developed waxy haemostatic agents using the biode-gradable polymers PDLLA, PCL and Hap. These agents were bio-compatible and were as effective as BW in achieving bonemarrow haemostasis. Furthermore, the haemostatic agent withosteogenic-enhancing peptides increased the strength of thesternum at 2 weeks after surgery.SUPPLEMENTARY MATERIALSupplementary material is available at ICVTS online.ACKNOWLEDGEMENTSThe authors would like to thank the following individuals: KeiichiSato, D.V.M., Ph.D., and Yuya Suzuki, V.D.M., who participated inthe animal experiments and advised us about them during the proj-ect. The authors acknowledge the staff of the Division ofExperimental Animals and the Division of Medical ResearchEngineering, Nagoya University. This study would not have beenpossible without their help. Finally, the authors thank all individualswho answered our questions in interviews and gave us valuable in-formation during this project.FundingThis work was supported by the Japan Society for the Promotion ofScience (JSPS) KAKENHI (grant numbers 17K10729, 20K09124 and20K05227) and by the Japan Agency for Medical Research andDevelopment (AMED) under grant number JP20he0422006j0001.Conflict of interest: none declared.DATA AVAILABILITYData collected for the study will be made available by the corre-sponding author upon reasonable request after publication.Author contributionsTsukasa Ohno: Data curation; Formal analysis; Investigation; Visualization;Writing—original draft. Hiroto Suenaga: Data curation; Investigation;Visualization. Aika Yamawaki-Ogata: Data curation; Formal analysis;Investigation; Visualization; Writing—original draft; Writing—review & editing.Kei Kanie: Data curation; Formal analysis; Investigation; Resources; Writing—original draft. Ryuji Kato: Conceptualization; Investigation; Methodology;Resources. Koichiro Uto: Data curation; Formal analysis; Investigation;Resources; Writing—original draft. Mitsuhiro Ebara: Conceptualization;Investigation; Methodology; Resources. Hideki Ito: Funding acquisition. YujiNarita: Conceptualization; Funding acquisition; Methodology; Project admin-istration; Software; Supervision; Validation; Writing—review & editing. AkihikoUsui: Project administration; Software; Supervision; Validation; Writing—re-view & editing. Masato Mutsuga: Project administration; Software;Supervision; Validation; Writing—review & editing.Reviewer informationInterdisciplinary CardioVascular and Thoracic Surgery thanks Lucio Cagini,Roman Gottardi and the other anonymous reviewer(s) for their contributionto the peer review process of this article.REFERENCES[1] Julian OC, Lopez-Belio M, Dye WS, Javid H, Grove WJ. 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