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A biocompatibility study of injectable poly(caprolactone-trifumarate) for use as a bone substitute material A biocompatibility study of injectable poly(caprolactone-trifumarate) for use as a bone...
Al-Namnam, N.M.; Kim, K.H.; Chai, W.L.; Ha, K.O.; Siar, C.H.; Ngeow, W.C.
2015-07-03 00:00:00
Frontiers in Life Science, 2015 Vol. 8, No. 3, 215–222, http://dx.doi.org/10.1080/21553769.2015.1051240 A biocompatibility study of injectable poly(caprolactone-trifumarate) for use as a bone substitute material a b c a a a∗ N.M. Al-Namnam , K.H. Kim ,W.L.Chai ,K.O.Ha , C.H. Siar and W.C. Ngeow Department of Oro-Maxillofacial Surgical and Medical Sciences, Faculty of Dentistry, University of Malaya, Kuala Lumpur 50603, b c Malaysia; Department of Physiology, Faculty of Medicine, University of Malaya, Kuala Lumpur 50603, Malaysia; Department of Diagnostic and Integrated Dental Practice, Faculty of Dentistry, University of Malaya, Kuala Lumpur 50603, Malaysia (Received 13 October 2014; accepted 11 May 2015 ) The need for bone graft alternatives has led to the development of numerous bone graft substitutes. Here, the authors have synthesized a biodegradable poly(caprolactone-trifumarate) (PCLTF) polymer solution that could be injected into any bony defect. This polymer solution was synthesized using polycaprolactone-triol and fumaryl chloride (FCl). PCLTF is a multiple-branching, unsaturated and cross-linkable in situ material. The surface microstructure of PCLTF was investigated using a field emission scanning electron microscope. The incorporation of double bonds originating from FCl into the poly(caprolactone) backbone was confirmed in the Fourier transform infrared spectra. The in vitro cytotoxic effects of PCLTF, its leachable extracts and degradation products were evaluated in direct and indirect contact tests against human oral fibroblasts. Cell viability was evaluated using the microculture tetrazolium assay and cytotoxicity evaluations of PCLTF were tested in accordance with ISO 10993-5 standards. The results showed that there was evidence of reasonable cell growth, good cell viability and intact cell morphology after exposure to PCLTF, its extracts and degradation products. There was no evidence of critical cytotoxic effects. Keywords: poly(caprolactone-trifumarate); cross-link; in situ; bone graft substitute; cytotoxicity; fibroblast cells Introduction polylactide–polyglycolide acid (Douglass 2005; Vignoletti The extraction of teeth results in alveolar ridge resorption, et al. 2012). Most of these materials form a scaffold that preserves the socket volume to enable it eventually to be with the buccal aspect of the ridge being affected more than filled with naturally regenerated bone during the healing the crestal region (Pietrokovski & Massler 1967; Devlin process. In general, most bone substitute scaffolds only & Ferguson 1991). A systematic review reported that the have osteoconductive properties, but are not osteoinduc- alveolar ridge undergoes a mean 3.8 mm horizontal and tive on their own. Another common problem with syn- 1.24 mm vertical reduction over 6 months (Lang et al. thetic biomaterial such as calcium phosphate bioceramics, 2012). This amount of bone loss may significantly affect besides its poor mechanical properties, is that it has a slow the amount of bone available for dental implant insertion, biodegradation rate (Julien et al. 2007). However, even which is nowadays performed to replace missing teeth. To when using natural autologous bone grafts, some retention reduce bone loss and preserve bone volume, some authori- of non-vital grafted bone with the formation of connective ties advocate alveolar ridge preservation (ARP) (Zubillaga tissue around it has been reported, which may slow down et al. 2003). bone regeneration (Artzi et al. 2003). Various ARP techniques have been described, using Developing a biocompatible material as a scaffold and either autologous bone or bone substitutes (allogenic, vehicle carrier for growth factors, which is the concept xenogenic and synthetic grafts) with or without non- of tissue engineering, is very much desired. For example, resorbable or resorbable barrier membranes (Douglass in implant dentistry such a material can be used for ARP 2005; Vignoletti et al. 2012). Examples of bone graft (Zubillaga et al. 2003) or to cover exposed implant sur- materials are demineralized freeze-dried bone allograft (DFDBA) with or without calcium sulfate, cortico- faces (Chai 2009). These increased demands continue to cancellous porcine bone, bovine-derived hydroxyapatite, fuel interest in improving and developing the performance magnesium-enriched hydroxyapatite, calcium sulfate or of existing medical-grade polymers. medical-grade calcium sulfate hemihydrate, a biologically Researchers are studying biomaterials that are in an active silicate-based glass, collagen sponge and a sponge of injectable form, so that they can be easily implanted in *Corresponding author. Email: ngeowy@um.edu.my © 2015 Taylor & Francis 216 N.M. Al-Namnam et al. vivo and inserted into irregular osseous defects. This pre- the esterification reaction), whereas the use of potassium viously led to the development of the injectable bone carbonate (K CO ) in white PLCTF yields a translucent, 2 3 cement poly(methy-methacrylate) (PMMA) (Temenoff & white colour in the final product. The addition of K CO 2 3 Mikos 2000). However, since this biomaterial is non- shortens drastically the laboratory time needed to prepare biodegradable, it is not suitable for insertion into extraction the PCLTF polymer solution, from 48 h to 12 h. How- sockets as it would interfere with the eventual placement of ever, the application of K CO results in polycondensation 2 3 dental implants. Therefore, a biodegradable cross-linkable between PCL and FCl, which yields a higher molecular- and injectable scaffold would offer a great advantage in weight copolymer compared with the use of triethylamine. that no invasive surgery would be needed for either the As a result, white PCLTF has a longer degradation time application or the removal of the material. than the dark polymer. Weighing up the advantages and An alternative material that can serve this purpose is disadvantages of these two versions of PCLTF, the authors polycaprolactone (PCL), an injectable and biodegradable decided to explore further the full potential of dark PCLTF polymer that is relatively inexpensive and has low toxicity for use as an extraction socket filler, because of its more (Kweon et al. 2003; Patel et al. 2011). This polymer con- favourable degradation time (Muhammad et al. 2012). sists of a highly elastic polyester structure that degrades Before this biomaterial is indicated for clinical use, it is by hydrolytic cleavage of its ester bonds in physiological crucial to ensure that dark PLCTF is not toxic to the body conditions. Its degradation products are either metabo- (Gomes et al. 2001). Therefore, it was the aim of this lized via the tricarboxylic acid (Kreb’s) cycle or eliminated study to investigate the in vitro cytocompatibility of the via direct renal secretion (Woodward et al. 1985). How- biodegradable dark PCLTF. ever, this degradation process can take up to 2 years, which is a significant drawback clinically (Middleton & Materials and methods Tipton 2000). Various modifications, such as the addi- Sample preparation tion of sebacic acid, have been reported to improve its The PCLTF polymer solution was synthesized using PCL- degradation rate (Salgado et al. 2012). Similarly, PCL can triol, FCl and triethylamine (Et3N), as described in a be modified by cross-linking a functional group such as previous study (Chai et al. 2004). First, this mixture was fumarate, resulting in the synthesis of polycaprolactone stirred in an ice bath, and after 2 h, nitrogen gas was fumarate (PCLF) (Wang et al. 2009; Salgado et al. 2012). extracted. The stirring process was continued at room tem- However, this semi-crystalline product needs to be heated perature for a total reaction time of 48 h. This process to a high temperature before it can be injected into any eventually formed a viscous PCLTF polymer solution. bone defect site. As a result, necrosis or damage to the A PCLTF porous scaffold was then fabricated by first native tissue can occur (Jabbari et al. 2005). cross-linking the PCLTF polymer solution with a ben- To overcome these problems, the present research zoyloxide radical, N -vinyl-2-pyrrolidone benzoyl peroxide group has developed a resorbable polymeric solution (NVP-BP) solution, which acted as a cross-linking initia- based on PCL-triol and fumaryl chloride (FCl), named tor. Following the addition of N,N -dimethyl-o-toluidine poly(caprolactone-trifumarate) (PCLTF), which may have (DMT), which served as an accelerator, sodium chloride great potential for use as a bone tissue engineering scaf- (NaCl) salt particles (sieved at 215–325 μm) were added as fold (Chai et al. 2006). FCl is a derivative of fumaric acid the porogen, finally making a porous material (Chai 2004). that is found in the body and is metabolized in the Krebs’s ® The resultant paste was injected into a cylindrical Teflon cycle. PCLTF is an in situ cross-linkable polymer with mould (5 mm in diameter × 2 mm in height), which was multiple branching of unsaturated polyester, and contains then compressed using a glass plate to create a disc of extra non-reacted hydroxyl groups (–OH) that enhance its even thickness. PCLTF cross-linked in situ and solidified hydrophilicity. It has a higher degradation rate than PCL. within approximately 15 min. After cross-linking, the sam- It is a viscous liquid at room temperature and is therefore ples were removed from the moulds. The salt porogen injectable without needing to be heated before application. was leached out by placing the scaffold in double-distilled Furthermore, because of its thick viscosity, migration of water for 4 days, with water changes twice a day to prevent PCLTF from the injection site is prevented during the 15 salt saturation. The resulting porous scaffolds were dried in min curing time. This injectable form of PCLTF has many a desiccator for 1 day and then sterilized in 70% ethanol advantages over other materials as it allows application as overnight. Before being used in the experiments, the scaf- a bone-filling material directly into an irregular bony defect folds were washed three times with phosphate-buffered with minimal pressure. saline (PBS). In a previous study, this group developed two types of PCLTF, namely white and dark PCLTF scaffolds (Chai Characterization of poly(caprolactone-trifumarate) et al. 2004; Muhammad et al. 2012). Structurally, the To characterize PCLTF, Fourier transform infrared (FTIR) two versions of PCLTFs are similar, with the only dif- spectroscopy (OMNIC 8.1 program, Nicolet 6700; Thermo ference being that the opaque, dark PLCTF is produced Scientific, Madison, WI, USA) was employed using an as a result of the use of triethylamine (a catalyst used in Frontiers in Life Science 217 internal reflection element made of diamond as the sample Direct contact cytotoxicity evaluation was preceded holder. The nuclear magnetic resonance ( H-NMR) spectra by seeding the human gingival oral fibroblasts on to the 4 2 of PCL-triol and PCLTF were recorded in a 400 MHz spec- PCLTF discs at a density of 2 × 10 cells/cm in 300 trometer, using tetramethylsilane as the internal reference μl/cm of culture medium in a six-well plate (Jabbari et al. and dimethyl sulfoxide (DMSO) as the solvent. 2005). A total volume of 1600 μl culture medium was The surface microstructure and the morphology of used in each well. Cells plated with the same density but the test samples (scaffolds) were also investigated using without exposure to PCLTF were used as the control. The a field emission scanning electron microscope (FESEM) cells were incubated at 37°C, 95% relative humidity and (low vacuum operating mode; Quanta FEG 250; FEI, Eind- 5% CO . The medium was changed once during the 72 hoven, the Netherlands). The test samples were dehydrated h incubation period. The cells were incubated for 3 h in through a graded series of alcohol solutions, dried in a 300 μl/sample of MTT solution (2 mg/ml of thiazolyl blue critical-point dryer and sputtered with a gold coater. They tetrazolium bromide in PBS) (Sigma, St Louis, MO, USA). were viewed at an accelerating voltage of 10 kV. At the end of the incubation period, the MTT-containing medium was aspirated off and DMSO was added to dis- solve the formazan crystal formed by living cells. An Cytotoxicity tests aliquot of 150 μl of the solution was transferred to a 96- Following approval from the Medical Ethics Commit- well flat-bottomed plate, and the OD was measured with a tee/IRB, Faculty of Dentistry, University of Malaya [DF microplate reader (Biotek ELx808 Absorbance Microplate OS1211/0071(P)], human gingival fibroblasts were har- Reader; Winooski, VT, USA). vested from marginal gingival tissue of extracted teeth, Indirect cytotoxicity evaluations of PCLTF extract taken during routine orthodontic teeth extractions, and cul- were tested in accordance with the ISO 10993-5 standards. tured for the purpose of evaluating the cytotoxic effect of In brief, the EtOH-sterilized discs were immersed in cul- PCLTF. ture medium at a ratio of 1 ml of medium/3 cm surface Cell culture was performed by expanding the fibrob- area and incubated at 37°C, 95% relative humidity and 5% lasts in Dulbecco’s modified Eagle’s medium (LGC Scien- CO . After a 24 h incubation period, the medium contain- tific, Sigma, St Louis, MO, USA) supplemented with 10% ing the extracts was diluted by 10 × and 100 × dilution (v/v) foetal bovine serum (Euroscience, Paa Austria) and with fresh culture medium for the indirect cytotoxicity test. 1% (v/v) antibiotics comprising of penicillin/streptomycin Human gingival oral fibroblast culture was prepared by and amphotericin (Euroscience, PAA Austria, Fasching, seeding the cells at a concentration of 1 × 10 cells/well Austria) in a humidified atmosphere at 37°C in 5% carbon in a 96-well plate. The cells were cultured to 80–90% dioxide (CO ). The confluent layer of cells obtained was confluency before the test. Once it reached the required designated as the ‘first passage cells’ after the cells reached confluency, the culture medium was replaced with 100 μl 80–90% confluence. Oral fibroblasts with a passage num- of one of three concentrations (undiluted, 10 × and 100 × ber no greater than six were used in experiments. Cell diluted) of the extracts containing the medium, and incu- viability was evaluated using the microculture tetrazolium bated for 24 h, 48 h and 72 h. The cytotoxicity of extracts (MTT) assay. at each dilution was assessed by the MTT assay. Each The optical density (OD) of the MTT assay is theoret- viability measurement was normalized with the control ically proportional to the growth rate and viability of the group, where the fibroblasts were cultured in regular media cells. Thus, the mean cell viability was determined by mea- without any extracts (Zhang et al. 2009). suring the OD of the MTT assay. The percentage of cell The cytotoxicity test of the cross-linked PCLTF degra- viability was determined using the formula from the guide- dation products was carried out by bulk degradation of lines of the International Organization for Standardization PCLTF and exposure of its degradation products to the cul- (ISO) for biological evaluation of medical devices (ISO tured cells. The degradation process was carried out in a 10993-5) (ISO 2009): strong base at 37°C, which accelerated the ester hydroly- sis reaction (Morrison & Boyd 1992; Zhang et al. 2009). Cell viability % = × 100% xc In brief, a PCLTF sample (0.1 g) was placed in 10 ml 1 N where x is the absorbance in the test group (with PCLTF) NaOH solution and secured in a water bath at 37°C for 2 and xc is the absorbance in the control group (without days. The sample was considered to have degraded com- PCLTF). The tests were repeated at least three times to pletely when no solid material was visible in the solution. ensure reproducibility. Based on this reference, the cyto- Then, the pH of the solution was adjusted to 7.4 using 1 toxicity responses were qualitatively rated as severe, mod- N HCl and the solution was filtered through a cellulose erate, slight and non-cytotoxic when the percentage of acetate membrane filter (0.2 μm pore diameter). The degra- cell viability was < 30%, 30–59%, 60–90% and > 90%, dation solution was then diluted in culture medium to 2 × , respectively (Loushine et al. 2011; Mukhtar-Fayyad 2011). 10 × ,50 × and 100 × dilutions. Sterile PBS was diluted Three replicate wells were used for each test and each test by the same factors and used as a negative control (Zhang was repeated three times. et al. 2009). 218 N.M. Al-Namnam et al. −1 Figure 1. Fourier transform infrared spectra of poly(caprolactone-trifumarate) (PCLTF) polymer solution: 1646.05 cm (C = C −1 −1 stretch), 1732 cm (C = O), 3400.35 cm (O–H). Similarly to the test for the extraction products, conflu- ent human gingival oral fibroblasts at a concentration of 1 × 10 cells/well in a 96-well plate were exposed to an aliquot of 100 μl of four different concentrations of degra- dation product-containing medium and incubated for 24 h. The viability of the cells was determined by the MTT assay as described above. Data were analysed using Statistical Package for Social Science (SPSS) statistical software (Version 12.0; SPSS, Chicago, IL, USA). Descriptive statistics were applied where appropriate. The Mann–Whitney U test was per- formed for direct contact tests and the Kruskal–Wallis test for indirect cytotoxicity tests. The significance level was setat95%(p < 0.05). Figure 2. H-Nuclear magnetic resonance spectra of the synthe- sized poly(caprolactone-trifumarate) (PCLTF) polymer solution: 6.8 ppm (CH = CH). PCL-triol = polycaprolactone-triol. Results and discussion The filling of an extraction socket with natural bone is However, the presence of O–H stretch at a wave num- −1 the result of sequential events that begin with resorption ber of 3366.16 cm indicated that the synthesized PCLTF of the socket walls, followed by recruitment and pro- polymer solution retained some hydroxyl groups (–OH) liferation of osteoprogenitor cells from the surrounding after reaction with FCl, hence giving some degree of tissues, osteoblastic differentiation, matrix formation and hydrophilicity to the PCLTF polymer solution (Figure 1). finally mineralization (Shin et al. 2003). Preservation of These findings were supported by the presence of a the alveolar ridge based on a tissue engineering approach chemical shift value for the C = C–H group at 6.8 ppm rel- usually involves the use of osteoconductive scaffolds. The ative to the tetramethylsilane (TMS) standard, which was H-NMR authors’ research group has developed a three-dimensional not seen in the spectra of PCL-triol obtained by scaffold, which is a polymer solution based on PCL-triol (Figure 2). This proved that the C = C–H group had been and FCl (Chai et al. 2004). The resultant polymer solu- successfully incorporated and a PCLTF polymer solution tion was confirmed by the presence of C = C stretch and had been synthesized. −1 = C–H stretch at 1660–1600 cm and around the region Figure 3 shows an FESEM micrograph of PCLTF −1 3095–3010 cm , respectively, in FTIR spectra (Mahendra porous scaffold. It shows the presence of pores with dif- & Kadier 2012). PCLTF scaffolds fabricated from poly- ferent shapes and sizes, ranging from 100 μm to 300 μm, mer solution were relatively hydrophobic, partly because and the presence of interconnections among the pores. of the presence of long hydrocarbon chains of PCL-triol. The presence of these micropores in a scaffold improves Frontiers in Life Science 219 Figure 3. Field emission scanning electron microscope view of the surface microstructure and morphology of the porous poly(caprolactone-trifumarate) (PCLTF). mass transport and neovasculature formation in addition to assisting cell adhesion. To achieve this, maintaining a bal- Figure 4. Phase-contrast micrograph showing the ance between the optimal pore size for cell migration and spindle-shaped fibroblastic morphology of A) (a) control the specific surface area for cell attachment is essential. and (b) poly(caprolactone-trifumarate) (PCLTF) disc treated In agreement with the previous study (Chai et al. 2006), cells (scale bar = 2 mm); B) extract products after cultivation at FESEM revealed that the PCLTF porous scaffolds con- different time intervals; and C) degradation in different dilutions tained pores of different sizes which were interconnected with cultivation for 24 h. with each other. Furthermore, the weight loss of PCLTF demonstrated a higher degradation rate (30.86 ± 1.15%) than PCL (4.55 ± 0.26%) (Chai et al. 2004; Muhammad characteristics of spindle-shaped fibroblastic cells com- et al. 2012). pared with the controls. This comparably normal morpho- In a clinical situation, when a scaffold is placed in a logical appearance of the cells, examined under phase- dental extraction socket, the biomaterial will come into contrast microscope, was also seen in indirect contact tests contact with the soft tissue (gingiva) before bone regen- (Figure 4). eration takes place in the socket. Because of this, human BP, NVP and DMT, which were used as cross-linking gingival fibroblasts were used to evaluate the cytocompat- reagents, should not increase the toxicity risk of the fab- ibility of PCLTF, its extracts and degradation products in ricated PCLTF as these reagents were either consumed this in vitro study. The effects of a material on cells are indi- completely during the cross-linking reaction (as for BP) cated by changes in cell morphology as well as the viability to form a non-toxic polymer (polyNVP), or present in a of cell growth. Cell viability can be tested using vari- very small amount (as for DMT), where the toxic effects ous biochemical assays, and within these different assays, were negligible (Payne et al. 2002; Sharma et al. 2005; changes in metabolic activity are good indicators of early Patel et al. 2011). Because of this, the cytotoxic effect of cell viability (Davila et al. 1990). In this study, cell viabil- the initiator could be excluded. The cytotoxicity of con- ity was determined using the MTT assay (Loushine et al. cern deals mainly with substances that leach out of the 2011). biomaterial and polymer. These substances often have low molecular weight, and often are additives and initiators For the direct contact test, cell metabolic activity in the MTT assay showed significantly different results between that exhibit varying degrees of physiological activity and the PCLTF group and the control group (p < 0.05). The cell toxicity (Vajrabhaya & Sithisarn 1997). The slightly cell viability of PCLTF scaffold after a 72 h contact test cytotoxic effect may be explained by the presence of salt was found to be 80.8 ± 2% which, according to the ISO particles that remained in the scaffold, similarly to the 10993-5 standard, was considered to be slightly cyto- results reported by Kim et al. (2009). toxic. Therefore, this biomaterial is deemed to be slightly The indirect contact test of extracts evaluated the cytotoxic, but it did not alter the normal morphological effect of the leachable product of PCLTF (Mukhtar-Fayyad 220 N.M. Al-Namnam et al. Figure 6. Results of the indirect contact test of the Figure 5. Results of the indirect contact test of the poly(caprolactone-trifumarate) (PCLTF) degradation prod- poly(caprolactone-trifumarate) (PCLTF) extracts. The mean uct showing the cell viability percentage after its exposure to oral value for the optical density (OD) of the microculture tetrazolium fibroblasts over a period of 24 h. The cell viability percentage assay shows cell viability of the oral fibroblasts after the of the 10 × ,50 × and 100 × diluted degradation products was exposure in the extracts over a period of 24 h, 48 h and 72 h. significantly higher than the 2 × dilution group (p < 0.05). #The mean cell viability of the 10 × , 100 × and control groups was significantly higher than the undiluted group (p < 0.05). PCLTF took months to gradually degrade in PBS. In this *The mean cell viability of the 100 × and control groups was study, an accelerated degradation method was used to com- significant higher than the 10 × group (p < 0.05). The mean cell viability of the control was significantly higher than the pletely break down the networks, yielding the maximum 100 × group (p < 0.05). release of degradation products, by immersing PCLTF in a strong base at 37°C. These conditions accelerate the ester hydrolysis reaction, which is more advantageous than 2011). Following ISO 10993-5 guidelines, the extracts of using a diluted acid to achieve the same result. In addition, PCLTF in this study were obtained by incubating the spec- this reaction is an irreversible process. While these con- imens in culture medium at 37°C in a 5% CO incubator ditions do not exactly replicate the actual changes in the for 24 h. Figure 5 showed that the OD value increased polymer in vivo and may not correlate with actual in vivo gradually over time (24–72 h) in all concentrations of the degradation time, they do simulate the physical changes extracts (p < 0.05). A significantly (p < 0.05) higher OD expected during natural degradation on a shortened time- value, which reflected a higher cell viability, was observed scale. Four different dilution factors were chosen in this in the more diluted group at 24 h, 48 h and 72 h. This study to represent a range of concentrations of the degrada- observation suggests that the toxic effect reduced with dilu- tion product (Zhang et al. 2009). The degradation products tion of the extracts. In addition, the most concentrated level from PCLTF displayed a dose-dependent cytotoxic effect. of extract medium (undiluted extract) had the lowest per- The results showed that the cell viability percentage of centage of cell viability (51.6 ± 4%), which is considered the fibroblasts was the lowest in a 2 × dilution of the moderately cytotoxic according to the ISO 10993-5 stan- degradation products (p < 0.05), which was considered as dard. These results show that the extracts of PCLTF had an having moderate cytotoxicity. However, in a more diluted acceptable degree of biocompatibility, and the most signif- concentration of the degradation products, the cell viabil- icant cytotoxic effect was seen during the first 24 h. With ity percentage increased to 94.1 ± 11%, 96.4 ± 8% and time (at 48–72 h), there was evidence of continuing cell 96.6 ± 17% at 10 ×,50 × and 100 × dilution, respec- growth in all three concentrations of extracts. Although tively (Figure 6), suggesting non-cytotoxic effects in the the cytotoxic effect of the undiluted extracts was moder- diluted conditions. Thus, the cytotoxicity effect gradu- ate initially, in a clinical condition, it may be even lower ally decreased when the concentrations of the degradation as the extracts would be diluted by the surrounding tissue product were diluted. fluid. In addition, during the cross-linking process of the In this study, although the results showed moderate polymer, the constituents continued to integrate into the cytotoxicity of the undiluted extract and degradation prod- network, resulting in less diffusion of leachable products ucts, one should understand that the results obtained in out of the network. Based on the aforementioned factors, in vitro culture conditions are different from those in the although the initial cytotoxicity of the extract was mod- in vivo homeostatic condition. Compared to the in vivo erate, with time, the cytotoxicity is expected to reduce model, in vitro experiments like this one have a lack of gradually (Schmalz 1994; Gomes et al. 2001; Bae et al. defined mechanisms, which has a strong impact on the 2010). precision of toxicity elimination. One example of such a Scaffolds for bone regeneration usually degrade on a mechanism is the presence of the lymphatic system to help relatively long time-scale, but study of their long-term bio- in the elimination of toxic substances (Hartung & Daston compatibility is difficult. The authors noticed that in vitro, 2009; Loushine et al. 2011). Frontiers in Life Science 221 In the previous study, there was evidence of osteoblast Douglass GL. 2005. Alveolar ridge preservation at tooth extrac- tion. J Calif Dent Assoc. 33:223–231. growth, proliferation and differentiation, as well as expres- Gomes M, Reis R, Cunha A, Blitterswijk CA, De Bruijn sion of the osteogenic marker alkaline phosphatase and J. 2001. Cytocompatibility and response of osteoblastic- mineralization on the PCLTF (Chai et al. 2006). Within the like cells to starch-based polymers: effect of several limitations of this study, the direct contact test of PCLTF additives and processing conditions. Biomaterials. 22: showed slight cytotoxicity. However, the indirect contact 1911–1917. Hartung T, Daston G. 2009. Are in vitro tests suitable for tests of the extract and degradation products showed mod- regulatory use? Toxicol Sci. 111:233–237. erate cytotoxicity initially, which reduced when diluted. ISO. 2009. Biological evaluation of medical devices. V. Based on this early encouraging result, the potential of In: Tests for cytotoxicity: In vitro methodsIn: Iso, edi- PCLTF for use as a bone socket filler could be further tor Vol10993-5 Geneva: International Organization for investigated in animal studies. Standardization. Jabbari E, Wang S, Lu L, Gruetzmacher JA, Ameenuddin S, Hefferan TE, Currier BL, Windebank AJ, Yaszemski MJ. 2005. Synthesis, material properties, and biocompatibility Conclusion of a novel self-cross-linkable poly (caprolactone fumarate) The biocompatibility of this injectable scaffold showed that as an injectable tissue engineering scaffold. Biomacro- the cytotoxic effect of PCLTF and its extracts, as well as its molecules. 6:2503–2511. Julien M, Khairoun I, LeGeros RZ, Delplace S, Pilet P, Weiss P, degradation products, were moderate initially, and reduced Daculsi G, Bouler JM, Guicheux J. 2007. Physico-chemical- when diluted as well as with time. Within the limitations mechanical and in vitro biological properties of calcium of the present study, it may be concluded that there was no phosphate cements with doped amorphous calcium phos- critical cytotoxic effect of dark PCLTF for use as a scaf- phates. Biomaterials. 28:956–965. fold for tissue engineering. Further preclinical studies are Kim J, Yaszemski MJ, Lu L. 2009. Three-dimensional porous biodegradable polymeric scaffolds fabricated with required to investigate the use of PCLTF as an injectable biodegradable hydrogel porogens. Tissue Eng Pt C-Meth. scaffold. 15:583–594. Kweon HY, Yoo MK, Park IK, Kim TH, Lee HC, Lee HS, Oh JS, Akaike T, Cho CS. 2003. A novel degradable poly- Disclosure statement caprolactone networks for tissue engineering. Biomaterials. 24:801–808. The authors declare that they have no conflict of interest. Lang NP, Pun L, Lau KY, Li KY, Wong M. 2012. 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