ScienceDirect. Changes of porous poly( 8-caprolactone) bone grafts resulted from e- beam sterilization process

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1 ScienceDirect ELSEVIER Radiation Physics and Chemistry I ()III-III RadiationPhysics and Chemistry te/radphyschem 1 1 Changes of porous poly( 8-caprolactone) bone grafts resulted from e- beam sterilization process L. Ohiha,b,*, K. Filipczakc, T. Czvikovszkya, T. Cziganya, L. Borbasb a Department of Polymer Engineering, Budapest University of Technology and Economics, H-llll Budapest, Muegyetem Rakpart, Hungary bresearch Center for Biomechanics, Budapest University of Technology and Economics, H-IIII Budapest, Muegyetem Rakpart, Hungary CInstitute of Applied Radiation Chemistry, Technical University of Lodz, Lodz, Poland Abstract The most important mechanical feature of poly(e-caprolactone) (PCL) foams applied in bone tissue engineering as a scaffold, has been investigated as a function of irradiation dose. Radiation is proposed for the sterilizf(tipd of the polymer before the implantation. Polycaprolactone scaffold foams were obtained by combination of compression rnolding and particulate leaching techniques. The porogen was changed in the range 4-6% and the irradiation dose was varied from 2to 1 kgy. Our results show that yield strength is not a function of radiation dose, but is rather influenced by the porosity, while the critical strain is mainly dependent on the dose. All these together mean that the modulus of the elasticity of PCL foams is dependent on both the porosity and the 2 Published by Elsevier Ltd. Keywords: Biomaterials; Mechanical properties; Macroporous polymers; PoIY'caproli[lctone; Scaffold; Bone implants 1 1. Introduction Tissue engineering is a new, emerging interdisciplinary field of biomaterial science aimed at overcoming the current drawbacks and the lack of ddnors for tissue replacements. One of the most recenttasksis to create a porous template, called scaffold, to replace the human bone tissue. The polymer scaffolds uldhave appropriate biocompatibility, and mecha, morphological and degradation characteristics (HUtmacher, 2). The main points related to the scaffold morphology are the size and interconnectivityof the pores. Two types of pores can be differentiated, the micro- and the macropores. The size of the prior one is up to 1 /lm and the latter ranges in 1-!lm. The pores below 1/lm are to improve the gas-, air- and nutritive transport, while the macropores are to give mechanical support and a temporary frame to the bone cells, where they can penetrate and form the bony tissue (Hutmacher, 2). *Corresponding author. Department of Polymer Engineering, Budapest University of Technology and Economics, H-IIII Budapest, Miiegyetem Rakpart, Hungary. Tel.: + 4 ; fax: address:olah@pt.bme.hu (L. Ol:ih). -86X/$ - see front matter ~ 2 Published by Elsevier Ltd. doi: 1.6/j.radphyschem Porous scaffolds can be obtained from any kind of materials. However, the aliphatic polyesters are investigated most frequently. Polylactide (PLLA), polyglycolide (PGA) and poly(s-caprolactone) (PCL) are the most relevant members of this family. PCL is a low melting point (6 DC) linear, resorbable polymer. The slow degradation and resorption kinetics of PCL might limit the application to the field of drug delivery and res orb able sutures; however, this property could be beneficial for bone tissue engineering. As the degradation time exceeds 1 year, the human bone cells have enough time to replace the whole scaffold before its disappearence. The typical load of the scaffolds is the compression as a result of the function of the bone (OJah et ai., 26). Equations to describe the mechanical properties of cellular solids were suggested by Gibson and Ashby (1) and Gibson (2). The relationship is often called as powerlaw relationship, as modulus and strength changes as a power function of porosity. A typical compression curve of such foams can be described by characteristic points and lines (Fig. 1). The modulus of elasticity (Eelasticity),which is the initial slope of the compression curve, characterizes the scaffold in the 6

2 IRPc: 41 2 L. Oldh et al. / Radiation Physics and Chemistry I () III-III " ~ ".. dens.~... 8 elasticity II Eelasticity Plateau 1 Strain Edens Densification I I Edens point Fig. I. Compression curve of polymer foams, the yield and densification 1 points are indicated. 1 linear elastic region where all the deformations are reversible. The yield strength (O"elasticity) is the intersection 1 of the slope of Eelasticityand Eplateau' Eplateau is in the connection with the non-reversible demolishment of the 21 cell structure. Beyond the densification point the material is thought as non-porous material, and the modulus con- 2 verges to the value of the bulk modulus. This theory was applied to PCL and PLLA foams by 2 Feijen and co-workers (Hou et ai., 2). They performed compression tests, and their experiments validated the 2 power relation of the modulus and porosity. However, they also indicated that the material constant in case of cubic shape pores is significantly different from these described by Gibson. 1 Porous scaffolds interact with human tissues and, tlierefore, they have to be sterile. The benefits of radiation sterilization are obvious. The changes caused by the radiation on the polymer system have been extensively investigated on non-porous samples. In onem oulearlier papers, radiation-induced cross-linking p.rocess qf PCL was investigated (Filipczak ct ai., 26), andwefqpnd that the cross-linking process is dominatingovc:r the chain scission. The properties of porous scaffolds..have not been investigated after irradiation as widc:l)'ias the bulk polymer. Only 41 recently a paper was publisb.ed cross-linking and chainscission process of porous scaffolds (Plikk et ai., 26). 4 The aim of this paper is to study the changes of compressive characteristic of PCL foams as a function of 4 radiation dose Materials and methods Poly(e-caprolactone) (number-average molecular weight 1 MI1= 8 kda and weight-average molecular weight Mw = 1kDa) was supplied by Sigma-Aldrich Hungary Ltd. Before use it was dried in vacuum at 4 C overnight. Common table salt (sodium chloride) was used as pore- forming agent (porogen). Prior use it was fractionized by a sieving machine; and only the grains in the range 2- f.lm were used, as they are considered the most appropriate pore sizes for human bone cells. Distilled water was utilized for the porogen extraction. The scaffold was prepared by combination of compression molding and particulate leaching technique. The polymer was melted in a Brabender-type internal mixer at 12 C, and then the sodium chloride was added. The mixing process was continued till the equilibrium of the torque was obtained. Small pieces of salt-pcl mixture were created by cutting. The mixture was molded at 12 C and 1 bar pressure for 1 min, later the tool was cooled by water to room temperature. Following the processing steps, the cylindrical samples were immersed in distilled water for I week for total extraction of porogen. After the leaching out, the samples were vacuum dried for 4 days before use.. The morphology of the scaffold cross-section was investigated by scanning electro.n microscopy (SEM; 8L Va JEOL, Japan) of the samples coated with gold. The gel content was obtainyd by a Soxhlet extraction method using analytical grade tetrahydrofuran as the solvent. The extraction was continued for 8 h at 6 C. After the extraction, the samples were dried until constant weight, and the gel content was calculated from gravimetric measurements. The porosity of the sample was determined from weight and volume measurements. We estimated the theoretical weight; afterwards, we calculated the total porosity of specimens as a quotient of the real and theoretical weight. Number of the compressive tests was minimum to investigate the mechanical properties as a function of radiation dose. For compression tests, the specimen was manufactured with a cylindrical shape with 12mm diameter and 2 mm height. Tests were done with a Zwick BZjTNIS universal testing machine at an upper load limit 1 kn or % deformation limit, and at a cross-head speed of I mm min -I. The scope of the experiment was to investigate the dose dependency of the compressive characteristics (e.g. modulus, strength), of PCL-based foams in the linear elastic regime. The samples were irradiated with an ELU-6 linear accelerator (IARC, Lodz, Poland). Irradiation was performed by applying pulses of 6 MeV electrons at a frequency of 2 Hz (single pulse duration: 4 f.ls) at room temperature at air environment. The average dose rate, determined by calorimetry, was.kgymin-l. Different doses were applied from 2 kgy up to 1 kgy.. Results and discussion The pore-forming agent content of the scaffolds was varied from 4% up to 6% by weight (wt%) indicated. Although it was possible to obtain scaffolds containing 4% and 6% pores, they had very small resistance to mechanical load, and in these cases mechanical tests could not be performed. The scaffolds with initial 4-2 wt% salt concentrations had better integrity and were further investigated III

3 IRPC: 41 L. Oldh et at. / Radiation Physics and Chemistry I () III~III II The obtained scaffolds were scanned by electron microscope, and the cross-cut morphology was investigated. In Fig. 2(left), a typical scaffold which had /1 initial NaCI/ PCL ratio by weight is shown. The macropores can be observed, and a high amount of micropores is also present in the system. The total pore system has a very high interconnectivity. Scaffold with an initial 4 wt% NaCI is shown in Fig. 2(right). The pores, visibly, do not have very good interconnectivity, and there are significantly less micro pores, than there were in the previous case. Regardless to the differences of the scaffold structure, measurements of residual salt contents indicated that the total porogen content can be leached out through the interconnective pores. In case of very high porosity it seemed that the pores of cubic shape originated from the geometry of the salt crystals as can predicted. In Fig. 2(right), the pore geometry slightly differs from the porogen crystal geometry, and we think that this micropore geometry could be the result of the shear stress. The main requirement for a bone substituent is the mechanical stability, especially the compressive characteristic, as it is the most probable load in the human bones. Based on our own measurements, the typical relationship of mechanical property and porosity is shown in Fig.. The relationship can be expressed 1glO(Y) = C) + C2 x 1glO(I - p), where C1 and C2 are material constants, Y is the investigated compressive strength or modulus, p is the porosity of the sample. This relationship is valid for the modulus of elasticity, for the modulus of plateau, for the yield strength, and also it is thought to be correct foj:'the strength at the densification point and for the modulus of densification. The calculated lines (yield strength, modulus of elasticity) correlate well with the scattered points, the R2 is. or above. All these are parallefwith the existing theory (Gibson and Ashby, 1). Table 1 contains the exact data of modulus of elasticity. It can be seen that at high porosity the radiation dose does not influence significantly the modulus value. We can state that the higher the polymer content, the higher the modulus enhancing effect of ionizing radiation. In the (I) investigated dose range the modulus value was a linear function of the dose at constant porosity. Tablc 2 contains the data of yield strength at different porosity values and different doses. The results indicate that the strength is mainly dependent on the porosity. The strength was increased by the ionizing radiation just as the modulus of elasticity; however, the enhancement was not as significant as before..6.4.h,r~ '"}.2 )'W. '" :;:;,,/ '" -E.. ~.".'!!',FJ!fII -.2.,,~~ -.4 }8'" a" -.6,!I' IO(1-p) fig.i. Compressive modulus of PCL foam as a function of the relative polymer content. 8 Table 1 Modulus of elasticity (MPa) of the PCL scaffold as a function of porosity and as a function of radiation dose Porosity (%) Epla"icity (MPa) Radiation dose 2 kgy kgy kgy. \. \ Fig. 2. SEM pictures on the cross-cut section of scaffold prepared with initial /1 salt/pcl ratio (left), and initial 4/26 salt/pcl ratio (right) kGy 1kGy \.4 \ III

4 IRPC: 41 4 L. Oldh et at. / Radiation Physics and Chemistry I () III-III 1 Table 2 Yield strength (MPa) of the PCL scaffold as a function of porosity and as a function of radiation dose Porosity (%) O"pla"idty(MPa) Radiation dose 2kGy kgy kgy 1kGy 1kGy c: QI 8 c: 2 (,) 6 Qj 2 C> I, I t t I t t t Radiation dose [kgy] 1 Fig. 4. Gel content of the irradiated scaffolds as a function of radiation dose. 1. t: '. (jj '>.. 41 ' t:.q E.E 4 (I) I 2 1 Radiation Dose [kgy] Fig.. The average value of the strain of elasticity as a function of radiation dose. Both the modulus and the strength increased with increasing radiation dose, which is due to the cross-linking of the molecular chains, as the cross-links curb the mobility of the chains. The gel content measurement confirmed our expectations, as above kgy dose we found gel formation in the system (Fig. 4), that exceeded 1% at 1 kgy dose. Gibson and Ashby (1) suggested that the limit of the linear elasticity is a constant value which is not a function of porosity, approximately about. strain value (.% deformation). Our results confirmed the theory; however, it has definitely higher value. We found that the nonirradiated material has yield point at a.:t. strain value, and it is dependent on the dose (Fig. ). The correlation can be expressed by a power law which could have been concluded from the previous two tables as well. The yield strength was not dependent on the irradiation dose, while the modulus was. If the yield strength is constant, and the modulus is changing as a power function of the porosity, the required strain has to change the same way, too. 4. Summary During our researchpcl.based highly porous scaffolds were irradiated; the compressive characteristics were investigated as a function of the dose in the linear-elastic stage. The studied scaffolds indicate that irradiation modifies the modulus and strain behavior of the polymer foams. We found that the strength is not dependent on the dose;..it is mainly dominated by the porosity; while the straino! elasticity is a function of only dose. The modulus was highly dependent on the porosity and also On dose. As the typical strain in the bone is very small, w~think that in our experiments the 1 kgy dose is the most suitable because in the expected strain range it resulted in higher modulus value, bringing closer the synthetic polymer implant to the natural bone. Acknowledgments The authors would like to express their gratefulness to the co-workers of the Institute of Applied Radiation Chemistry (Lodz, Poland) for the help in the irradiation of the samples. L. Olih pays tribute to the Hungarian Chamber of Engineers for their support, and also to the European Commission for a scholarship in the Marie Curie Framework projects. The authors thank to Agnes Safrany (Institute ofisotope of Hungarian Academy of Science) for the help related to the extraction measurements. References Filipczak, K., Wozniak, M., Ulanski, P., Olah, L., Przybytniak, G., Olkowski, R.M., Lewandowska-Szumiel, M., Rosiak, J.M., 26. Poly(e-caprolactone) biomaterial sterilized bye-beam irradiation. Macromol. Biosci. 6 (4), 2-2. Gibson, L.J., 2. Biomechanics of cellular solids. J. Biomech. 8 (), -. Gibson, L.J., Ashby, M.F., 1. Cellular Solids: Structure and Properties. Cambridge University Press, Cambridge, pp

5 IRPC: 41 L. Oldh et al. / Radiation Physics and Chemistry I () III-III Hou, Q., Grijpma, D.W., Feijen, J., 2. Porous polymeric structures for tissue engineering prepared by coagulation compression moulding and salt leaching technique. Biomaterials 24 (II), Hutmacher, D.W., 2. Scaffolds in tissue engineering bone and cartilage. Biomaterials 21 (24),2-24. Olah, L., Filipczak, K., Jaegermann, Z., Sosnowski, S., Ulanski, P., Czigany, T., Borbas, 1., Rosiak, J.M., 26. Synthesis, structural and mechanical properties of porous polymeric scaffolds for bone tissue regeneration based on neat poly( -caprolactone) and its composites with calcium carbonate. Polym. Adv. Technolo., in press. Plikk, P., Odelius, K., Hakkarainen, M., Albertsson, A.C., 26. Finalizing the properties of porous scaffolds of aliphatic polyesters through radiation sterilization. Biomaterials 2 (1),