This article was downloaded by: [Florida International University] On: 28 December 2014, At: 11:08 Publisher: Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK
Journal of Biomaterials Science, Polymer Edition Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tbsp20
Evaluation of the cytocompatibility hemocompatibility in vivo bone tissue regenerating capability of different PCL blends a
a
b
Andrew R. Padalhin , Nguyen Thuy Ba Linh , Young Ki Min & a
Byong-Taek Lee a
Click for updates
Department of Regenerative Medicine, Institute of Tissue Regeneration, College of Medicine, Soonchunhyang University, Cheonan 330-090, Korea b
Department of Physiology, College of Medicine, Soonchunhyang University, Cheona, Korea Published online: 22 Jan 2014.
To cite this article: Andrew R. Padalhin, Nguyen Thuy Ba Linh, Young Ki Min & Byong-Taek Lee (2014) Evaluation of the cytocompatibility hemocompatibility in vivo bone tissue regenerating capability of different PCL blends, Journal of Biomaterials Science, Polymer Edition, 25:5, 487-503, DOI: 10.1080/09205063.2013.878870 To link to this article: http://dx.doi.org/10.1080/09205063.2013.878870
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content.
Downloaded by [Florida International University] at 11:08 28 December 2014
This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http://www.tandfonline.com/page/termsand-conditions
Journal of Biomaterials Science, Polymer Edition, 2014 Vol. 25, No. 5, 487–503, http://dx.doi.org/10.1080/09205063.2013.878870
Evaluation of the cytocompatibility hemocompatibility in vivo bone tissue regenerating capability of different PCL blends Andrew R. Padalhina, Nguyen Thuy Ba Linha, Young Ki Minb and Byong-Taek Leea*
Downloaded by [Florida International University] at 11:08 28 December 2014
a
Department of Regenerative Medicine, Institute of Tissue Regeneration, College of Medicine, Soonchunhyang University, Cheonan 330-090, Korea; bDepartment of Physiology, College of Medicine, Soonchunhyang University, Cheona, Korea (Received 15 October 2013; accepted 20 December 2013) In this study, the optimized formulations of polycaprolactone (PCL) combined with poly(lactic-co-glycolic acid) (PLGA), gelatin (GEL), and biphasic calcium phosphate (BCP) were analyzed in terms of cytocompatibility with bone-related cells, hemocompatibility, and in vivo bone-regenerating capacity to determine their potentials for bone tissue regeneration. Fiber morphology of PCL/GEL and PCL/BCP electrospun mats considerably differs from that of the PCL membrane. Based on the contact angle analyses, the addition of GEL and PLGA was shown to reduce the hydrophobicity of these membranes. The assessment of in vitro cytocompatibility using MC3T3-E1 cells indicated that all of the membranes were suitable for pre-osteoblast proliferation and adhesion, with PCL/BCP having a significantly higher reading after seven days of incubation. The results of the in vitro hemocompatibility of the different fibrous scaffolds suggest that coagulation and platelet adhesion were higher for hydrophobic membranes (PCL and PCL/PLGA), while hemolysis can be associated with fiber morphology. The potential of the membranes for bone regeneration was determined by analyzing the microCT data and tissue sections of samples implanted in 5 mm sized defects (one and two months). Although all of the membranes were suitable for pre-osteoblast proliferation, in vivo bone regeneration after two months was found to be significantly higher in PCL/BCP (p < 0.001). Keywords: PCL; electrospinning; in vitro cytocompatibility; hemocompatibility; in vivo bone regeneration
1. Introduction Graft transplantation has been typically favored over artificial implants when dealing with tissue regeneration, due to its inherent advantage of enabling the use of similar tissue that has been matched with the recipient form donors. However, using tissue grafts also poses significant disadvantages, namely donor availability, donor site morbidity, graft rejection, and to a lesser degree, donor–recipient pathogen transmission.[1] Numerous studies have been conducted on both naturally occurring and synthetic polymers as surface modifiers and components for different tissue scaffolds that lead to the improved biocompatibility of biomaterials used for regenerative medicine such as bone tissue engineering.[1–3] Among the polymers used in the field of tissue engineering, aliphatic polyesters are widely used due to their biodegradability and biocompatibility.[3,4] Various research *Corresponding author. Email: [email protected] © 2014 Taylor & Francis
Downloaded by [Florida International University] at 11:08 28 December 2014
488
A.R. Padalhin et al.
have been conducted on different methods of fabricating tissue scaffolds using polycaprolactone (PCL). Aside from being a relatively cheaper biocompatible synthetic polymer, PCL has been proven to be a versatile substance capable of forming stable microspheres, porous bodies, and electrospun membranes used as either drug delivery systems or tissue scaffolds. Whether as a monolithic scaffold or as a composite combined with other biocompatible materials, PCL can be considered as a good base material for developing scaffolds for tissue engineering.[5] Subsequent studies on electrospun PCL-based fibrous scaffolds have provided a good background for applying these materials in tissue regeneration studies.[6–12] In this study, three optimized binary PCL mixtures have been tested to define their respective performances in terms of contributing to bone regeneration. The purpose of this study is to compare these binary systems of PCL-based electrospun membranes by taking previously optimized systems fabricated by incorporating a co-polymer, hydrolyzed protein (gelatin (GEL)) and biphasic calcium phosphate (BCP) based powder. Each additive combined with PCL has been determined to improve the biocompatibility of the resulting optimized membranes in vitro; however, comparison between these materials in terms of application for bone tissue regeneration is yet to be established through in vitro and in vivo experimentation. The first optimized membrane is a mixture of copolymers PCL and poly (lactic-co-glycolic acid) (PLGA). An initial study has been conducted for the application of PCL/PLGA blend as a scaffold for skin [7] and for general tissue engineering purposes [8] tested with keratinocytes and fibrous tissue cells (L-929), respectively. The optimized mixture is composed of PCL and GEL and has already been tested for tissue engineering and dermal reconstruction.[9,10] More recently it has also been improved through the loading of hydroxyapatite particles [11] for bone regeneration application. The final optimized membrane composed of PCL and BCP powder was also selected due to its high in vitro performance based on a previous report using L929 cells, although it has not yet been tested in vivo.[12] The in vitro cytocompatibility and bone tissue regeneration potential of the optimized formulations of PCL/PLGA (80:20 ratio), PCL/GEL (50:50 ratio), and PCL/BCP (50% weight:volume) scaffolds were evaluated through proliferation assay, observation of attachment behavior through confocal microscopy, and implantation in 5 mm diameter calvarial defects in rats. An additional parameter, hemocompatibility, was also tested to observe blood reaction upon contact with the tissue scaffolds. The hemocompatibility in terms of hemolysis or break down of red blood cells upon material contact, blood cell attachment on the surface of scaffolds, and platelet adhesion have been taken into consideration, noting that blood would be the primary fluid that will come in contact with implants. The healing process of damaged bone tissue involves the accumulation and stabilization of blood clots and dissolution of hematoma, which in turn provides the factors for initiating chemotaxis for cell-mediated repair.[13–17] 2. Materials and methods 2.1. Materials Materials for fabricating the different electrospun membranes PCL (Mn 80,000), PLGA (85:15, Mw 50,000–75,000) and GEL (from porcine skin) were purchased from SigmaAldrich, USA. BCP powder measuring around 50–100 nm was synthesized through microwave assisted process using Ca (OH)2 (Aldrich Chemical) and H3PO4 (Dongwoo Fine Chemicals, Korea) as per reference.[18] Solvents used to dissolve the polymer
Journal of Biomaterials Science, Polymer Edition
489
Downloaded by [Florida International University] at 11:08 28 December 2014
components, tetrahydrofuran (THF, minimum 99%), and dimethylformamide (DMF, 99%), were also purchased from Sigma–Aldrich, USA. The other two solvents, methyl chloride and 2,2,2-trifluoroethanol (TFE, 99.0%), were procured from SK Chemicals Co., Korea and Fluka, Buchs, Switzerland, respectively. Methylene chloride was purchased from Dae Jung chemicals, Korea. 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) was purchased from GIBCO Carlsbad, CA, USA. Dimethylsulfoxide (DMSO, 99.0%) was obtained from Samchun Pure Chemical Co., Ltd (Pyeongtaek City, South Korea). Minimum essential medium (MEM) was obtained from HyClone (Logan, UT, USA). Pre-osteoblast (MC3T3-E1) mice cells were obtained from the American Type Culture Company, USA. All chemicals and solvents were of analytical reagent grade. 2.2. Fabrication of fibrous membrane scaffolds The scaffolds were fabricated using different PCL blends, as optimized in previous studies [6–12] and using a membrane composed of 12 wt.%/v PCL as the control. The optimized membranes were made by mixing PLGA, GEL, and BCP in separate independent solutions with PCL. For creating PCL/PLGA and PCL/BCP blends, a solution of 12 wt.%/v PCL was dissolved in a solvent containing 40% THF, 40% DMF, and 20% MC. PLGA solution was made by dissolving 10 wt.%/v of PLGA using the same solvent. The PCL/PLGA blend was made by combining 1:4 ratio of PLGA and PCL, while the PCL/BCP blend was made by adding 50 wt.%/wt. of BCP in the 12 wt.%/v PCL solution. The PCL/GEL blend was achieved by combining equal amounts of separate solutions of 12 wt.%/v of PCL and 12 wt.%/v GEL, both dissolved in TFE. Each PCL blend was then electrospun using an electrospinning machine (Electrospinning System, NanoNC, Korea) to create nanofibrous membranes. Each membrane required different parameters due to the varying material compositions. Their respective electrospinning parameters are enumerated as follows (voltage-kV, needle gauge – G, flow rate – ml/h, and tip-collector-distance – cm): PCL – 20 kV, 23 G, 0.5 ml/h, 20 cm; PCL/PLGA – 25 kV, 25 G, 0.5 ml/h, 20 cm; PCL/GEL – 25 kV, 21 G, 0.5 ml/h, 17 cm; and PCL/BCP – 20 kV, 21 G, 2 ml/h, 12 cm. 2.3. Characterization of fibrous membrane scaffolds After fabrication, the dry samples were placed on a scanning electron microscope (SEM) mount and coated with platinum (Cressington 108 Auto). The fiber morphology and average fiber diameter were observed and measured from images taken using SEM microscopy (SEM, JSM-7401F). Energy-dispersive X-ray spectroscopy (EDS) profile was also taken to confirm the presence of elemental components associated with GEL and BCP. The contact angle of each membrane scaffold was also tested using EasyDrop Contact Angle Measuring System (DSA, KRUSS GmbH). 2.4. Cell adhesion, proliferation and viability profiles Celll adhesion, viability, and proliferation were observed using a pre-osteoblast (MC3T3-E1) commercial cell line. Circular samples of each membrane measuring 6 mm in diameter were sterilized using ethanol and UV irradiation, and were conditioned with MEM media containing 10% fetal bovine serum and 5% penicillin streptomycin for 20 min prior to seeding with 1.5 × 104/ml of M3CT3-E1 cells.
490
A.R. Padalhin et al.
Downloaded by [Florida International University] at 11:08 28 December 2014
Observation of cell adhesion was carried out by confocal microscopy of seeded membrane samples stained with FITC and DAPI, while live and dead cell analysis was carried out by staining seeded samples with Calcein AM and Ethidium homodimer, one and five days after seeding. Micrographs of each sample were taken using a confocal laser scanning microscope (CLSM, Fluoview 1000, Olympus). Quantification of living and dead cells was performed through computer aided analysis of at least 10 micrographs per sample. The images were analyzed using the accompanying FV10-ASW 3.0 Viewer software and ImageJ. Cell proliferation on each scaffold was measured at 1, 3, 5, and 7 days after seeding by conducting an MTT assay and using an ELISA reader (TECAN 150, Turner Biosystems) at 595 nm. 2.5. Hemocompatibility To determine the blood reaction upon contact with each material, a hemocompatibility test was conducted on each individual rat previously implanted with a specific sample. A minimum amount of anti-coagulated blood, 1–2 ml, was aseptically drawn from each individual rat using a syringe with a 26 gauge needle and containing 1:9 ratio of Citrate-dextrose solution (Sigma). Each optimized material was tested for hemolytic effect, blood cell deposition, and platelet adhesion. The following procedures are based on several studies on blood contacting biomaterials [14–17] with some slight modifications. Hemolysis due to material contact was evaluated by submerging 6.0 mm diameter samples in 1 ml of test solution consisting of 1:10 ratio of anti-coagulated whole blood in sterile PBS. To eliminate hemolysis that might have resulted from the handling of blood samples prior to testing, a negative control was also prepared consisting of the test solution without any submerged material and a positive control was prepared consisting of a similar amount of anti-coagulated blood diluted in distilled water. After 30 and 60 min of exposure to test samples, the test solutions were removed and centrifuged at 3000 rpm for 3 min to pellet out the cellular components of the solution. To determine the amount of free hemoglobin released from hemolyzed erythrocytes, the supernatant was placed in a 96-well plate and read for absorbance at 540 nm. Blood cell deposition on material surface was analyzed by applying 200 μl of anti-coagulated blood on 6.0 mm samples which were then left to stand for 5, 10, and 15 min. After each sampling time, non-adherent blood cells were washed off the membranes using PBS and adherent cells were quantified through hemolysis. Platelet adhesion was observed using platelet rich plasma (PRP). PRP was prepared by centrifugation of 10 ml anti-coagulated blood at 2500 rpm for 5 min and removing the pelleted red blood cells, leaving behind the buffy coat in the middle and the upper supernatant consisting of the plasma. The supernatant was again centrifuged at 1000 rpm for 5 min to remove the remaining RBC component. Taking into consideration that only a very small amount of PRP can be prepared from a very small blood sample, the plasma was no longer removed and the volume of the solution was increased up to 5 ml and the platelets were resuspended through vortex mixing. A similar experimental design based on a previous study [19] was employed to quantify the amount of platelets adhered to the sample membranes. MTT assay was conducted to quantify the number of platelets attached to the membranes after 15 and 30 min of incubation.
Journal of Biomaterials Science, Polymer Edition
491
Downloaded by [Florida International University] at 11:08 28 December 2014
2.6. In vivo tests The in vivo bone regeneration of the different PCL blends was evaluated by implantation on rat calvarial defects. Circular samples of PCL, PCL/PLGA, PCL/GEL, and PCL/BCP measuring 6 mm in diameter were cut out using a paper puncher after which 3–4 layers of each samples were stacked together to form a single implant material. Prior to implantation, each implant material was briefly washed with 70% ethanol and sterilized with ultraviolet radiation on both sides. Sprague Dawley rats were sedated and circular defects measuring 5 mm in diameter were created on both sides of the frontal plate of the skulls. The left side defect was implanted with a sample membrane pre-soaked in phosphate buffered solution, while the right side defect was considered as a control and no implant was placed. Implant samples were extracted after four and eight weeks of recovery. Micro CT data of the formalin fixed samples were recorded and tissue sections were stained with hematoxylin, eosin, and Masson’s trichrome for further analysis. 2.7. Statistical analysis Each experiment was repeated at least three times on different days and data were expressed as the mean ± SD and analyzed through one-way ANOVA.
3. Results and discussion 3.1. Characterization of fibrous membrane scaffolds The material characteristics of the fabricated samples closely resemble those of the original descriptions from previous studies.[7–9,12] Both the fiber morphology and measurements of fiber diameters approximately match those of the former descriptions, with only a marginal difference. Figure 1 shows the SEM micrographs of fibrous membrane scaffolds composed of different PCL blends. Each PCL blend, with the exception of PCL/PLGA, has a distinct fiber morphology. For this experiment, the PCL membrane was established as a control membrane. The PCL (A) and the PCL/PLGA (B) membranes are basically composed of approximately similar diameter fibers ranging from 0.51 to 0.96 μm. On the other hand, membranes consisting of composite materials, PCL/GEL (C) and PCL/BCP (D), both have a highly altered fiber morphology compared to that of the PCL membrane. The PCL/GEL membrane consists of fibers with different sized diameters (0.25–1.03 μm). Incorporation of GEL into the electrospun membrane was confirmed through EDS data, showing the presence of nitrogen within the fiber, which can only be derived from the hydrolyzed proteins. Of the membranes, the PCL/BCP composite membrane has the most altered fiber morphology. Aside from having a wider range of fiber diameter (1.3–7.8 μm), the fibers formed from the solution are highly irregular along their length. Some clumps of the PCL/BCP blend can be seen at random sites, which contribute to the rough overall uneven surface morphology of the fibers. In addition, the incorporation of the BCP powder was confirmed through EDS showing calcium content within the fibers (Figure 1(D1)), which can only be derived from the BCP powder. To determine the hydrophilic properties of each membrane scaffold, the samples were tested for water contact angle. The average of four measurements per sample was used to determine the respective contact angles of each optimized membrane scaffold (Figure 2). Among the optimized scaffolds, the PCL/GEL (C) blend was the most
Downloaded by [Florida International University] at 11:08 28 December 2014
492
A.R. Padalhin et al.
Figure 1. SEM micrographs showing respective fiber morphologies of PCL (A), PCL/PLGA (B), PCL/GEL (C), and PCL/BCP (D) electrospun membranes. EDS data showing presence of nitrogen in electrospun GEL fibers (C1) interspersed with larger PCL fibers with lower nitrogen reading (C2) in PCL/GEL mat. Presence of calcium is also confirmed through EDS profile of PCL/BCP electrospun membranes.
Figure 2. Contact angle measurements of PCL (A), PCL/PLGA (B), PCL/GEL (C), and PCL/ BCP (D) electrospun membranes.
hydrophilic, yielding the lowest contact angle, 80.7 ± 5.77°, while the control membrane composed of 12% PCL (A) was the most hydrophobic, having the highest reading of 97 ± 6.02°. The blends, PCL/BCP (D) and PCL/PLGA (B), were relatively more hydrophilic than the control membrane, but less hydrophilic than the PCL/GEL membrane (91.75 ± 2.81° and 93.53 ± 2.91°, respectively). As expected, PCL/GEL had
Journal of Biomaterials Science, Polymer Edition
493
the lowest contact angle measurement due to hydrophilic property and the ability to absorb and retain the water of the GEL.[20] The addition of a more hydrophilic polymer (PLGA) and BCP powder in the PCL electrospun fibers also reduces the contact angle to a lesser extent by contributing to the limited hydrophilic property.
Downloaded by [Florida International University] at 11:08 28 December 2014
3.2. Cell adhesion, proliferation, and viability profiles In vitro cytocompatibility is an important factor for developing materials suitable for tissue engineering. In this study, PCL optimized membranes combined with PLGA, GEL, and BCP have been tested using MC3T3-E1 cells to chiefly establish suitability of these membranes for bone tissue growth. Confocal microscopy of seeded cells indicates minute differences between the adhesion behavior and viability of MC3T3-E1 cells on different micro-fibrous membrane scaffolds. Cell proliferation on the seeded scaffolds was determined using MTT assay. Quantitative data of cell viability were also gathered from confocal micrographs using seeded cells stained using a live/dead cell kit. Figure 3 shows the cell proliferation profiles of MC3T3-E1 cells seeded on the PCL, PCL/PLGA, PLC/GEL, and PCL/BCP fibrous membrane scaffolds. Results show that among these samples, PCL/BCP, followed by PCL/GEL, shows the greatest cell proliferation within seven days of culture. Statistical analysis indicates that the PCL/PLGA, PCL/GEL, and PCL/BCP electrospun membranes were significantly higher three and seven days after cell seeding (PCL/PLGA and PCL/GEL: p < 0.05; PCL/BCP: p < 0.001). On the fifth day, there is a noticeable lag phase in which no significant difference is observed between the proliferations on all the samples. This is possible considering that all the membranes were fabricated using the optimized formulations for tissue engineering. Figure 4 shows the confocal micrographs of MC3T3-E1 cells seeded on PCL (A1, A2), PCL/PLGA (B1, B2), PCL/GEL (C1, C2), and PCL/BCP (D1, D2) membrane scaffolds after 1–7 days. Twenty-four hours after seeding, the cell adhesion behavior on all four membranes did not show any obvious differences. However, at seven days of incubation it can be clearly seen that the cell attachment on PCL/GEL and PCL/BCP
Figure 3. Cell proliferation of MC3T3-E1 cells seeded on PCL (A), PCL/PLGA (B), PCL/GEL (C), and PCL/BCP (D) electrospun membranes measured up to seven days.
Downloaded by [Florida International University] at 11:08 28 December 2014
494
A.R. Padalhin et al.
Figure 4. Confocal micrographs showing adhesion behavior of MC3T3-E1 cells on PCL (A1–A2), PCL/PLGA (B1–B2), PCL/GEL (C1–C2), and PCL/BCP (D1–D2) electrospun membranes one and seven days after seeding.
membranes was more extensive compared to that of the PCL and PCL/PLGA membranes. In addition to the extensive attachments of the cells seeded on PCL/GEL and PCL/BCP membranes, both membranes also contained more cells compared to the remaining samples. Micrographs of seeded scaffolds stained for live/dead cell assay were also obtained through confocal microscopy. A minimum of 10 images pictures per sample were analyzed and counted for the ratio of living and dead cells. As indicated in the kit manual, all the cells in the samples are stained green and the dead cells are identified through the red stained nucleus. Cell viability for the samples is established by averaging the reading from each micrograph, which is calculated by subtracting the number of dead cells from the total count of cells, dividing the difference by the total count of cells, and then finally by multiplying the quotient by 100. Figure 5 shows the average MC3T3-E1 cell viability count in each sample, one and seven days after seeding and confocal micrographs for stained cells. Dead cells are red and viable cells are stained green. The results of the viability count further supports the cell proliferation data suggesting that at seven days, MC3T3-E1 cells have higher viability in the PCL/BCP electrospun membrane. Based on the results of the in vitro tests, all of the electrospun membranes were suitable for proliferating pre-osteoblast cells, with PCL/BCP membranes having a the significantly higher number of cells by the end of the seven day observation period. The initial observations showing propensity of cells to attach on the PCL/GEL membrane may be related to the aptitude of GEL to absorb fluids, allowing the perfusion of nutrients within the membrane matrix. The hydrophilicity and protein rich surface of the PCL/GEL contribute to cell attachment and proliferation.[9,10,20] Conversely, the PCL/BCP membrane also has the same effect, as described in other previous studies, whereby osteoblasts cells prefer to adhere on rough surfaces and the highly modified surface topography also provides an appropriate substrate for protein adsorption, consequently increasing the potential for cell adhesion [21,22] when in a serum supplemented culture media. In this study, the PCL/BCP membrane possessed a highly altered, uneven surface suitable for osteoblastic cell attachment. This is further exemplified through observing the cell viability and
Downloaded by [Florida International University] at 11:08 28 December 2014
Journal of Biomaterials Science, Polymer Edition
495
Figure 5. Confocal micrographs showing adhesion behavior of MC3T3-E1 cells on PCL (A1–A2), PCL/PLGA (B1–B2), PCL/GEL (C1–C2), and PCL/BCP (D1–D2) electrospun membranes one and seven days after seeding.
morphology. The live dead cell assay showed more living cells attached to the PCL membranes that were with GEL and BCP. This is also evident through the extensive cell attachment visible from the confocal micrographs. GEL has been known to be a good platform for culturing numerous types of cells,[20] while BCP typically serves as a biomimetic component for bone regeneration.[1,2,22,23] 3.3. Hemocompatibility tests Developing hemocompatible tissue scaffolds is crucial for not only blood vessel grafts but also for materials used for bone tissue regeneration. Blood is the principal physiological fluid that comes into contact with bone tissue scaffolds. The formation of hematoma is a naturally occurring process in bone fractures and allows the release of chemotactic elements that signals the migration of cells towards the site of injury. Thus, the effective formation of a blood clot on the surface of tissue scaffolds substantially contributes to the inflammatory phase of the healing process.[24,25] Platelets and their corresponding growth factors have been found to contribute to the healing process and regeneration of injured tissues.[26–29] During the inflammatory phase, breakdown of the clot and surrounding dead tissues occurs, which in turn releases signaling chemicals that guide the migrating repair cells.[30,31] To determine the blood tissue behavior upon contact, hemolysis, coagulation, and platelet adhesion were tested for each optimized PCL blend. Figure 6 shows the free hemoglobin reading after 30 and 60 min of incubating each membrane scaffold with diluted whole blood. Based on the results, the PCL/BCP membrane generated a significantly higher number of hemolyzed cells after 30 and 60 min, while PCL/GEL had the least number of hemolyzed cells (P < 0.001). The rough surface of PCL/BCP fibers, being abrasive against red blood cells, could have possibly contributed to the increased hemolysis. None of the electrospun membranes exceeded the acceptable range of red blood cell breakdown (5%) for implant materials based on ISO 10993-4:2002.
Downloaded by [Florida International University] at 11:08 28 December 2014
496
A.R. Padalhin et al.
Figure 6. Hemocompatibility data based on hemolysis (A), blood cell deposition (B) and platelet adhesion (C) of PCL, PCL/PLGA, PCL/GEL, and PCL/BCP electrospun membranes.
Figure 6 shows the results of blood coagulation tests after 30 and 60 min incubation. Blood cell deposition was significantly higher in the PCL/GEL membrane followed by PCL/BCP, while it was the lowest in the PCL membrane, was the lowest across all observation periods. The absorption of blood fluids, coupled with the dissolution of the majority of the GEL fibers in PCL/GEL, resulted in increased deposition of the blood cell component on the membrane surface. None of the membranes had greater than 1% blood cell deposited on their surface. Finally, membranes were tested for platelet adhesion using PRP from whole blood samples. The number of platelets adhered to the surface of each membrane was estimated using MTT assay (Figure 6). Based on the results, PCL and PCL/PLGA were the most conducive surfaces for platelet attachment. In the case of PCL/GEL, GEL’s absorbing capability and dissolution of GEL component again contributes to the
Downloaded by [Florida International University] at 11:08 28 December 2014
Journal of Biomaterials Science, Polymer Edition
497
attachment of the platelets. Protein in the blood can be adsorbed on calcium phosphate surfaces to some extent, as suggested in previous studies,[32–34] which could then result in platelet adhesion. Platelets adhered on the membrane surfaces were also observed through SEM microscopy (Figure 7), compared to other membranes, and the platelets adhered on PCL/GEL are highly obscured by the melted GEL (Figure 7(C)). Based on the hemocompatibility tests, PCL and PCL/PLGA were the membranes most suitable for forming clots and accumulating blood cells. Although not fully investigated in this paper, numerous studies regarding polymer surfaces have supported that reduction of contact angle and reduced protein adsorption on material surfaces significantly reduces platelet adhesion.[21,35–38] In this study, PCL and PCL/PLGA are characteristically more hydrophobic than PCL/GEL and PCL BCP, making these surfaces more suitable for protein adsorption which could contribute to increased platelet deposition.[36,38–40] In the case of PCL/GEL, capability of GEL to absorb fluids, hydrophilic and dissolution property contributes to reduced platelet attachment. Upon contact of hydrophilic surfaces to whole blood serum, plasma proteins that also include anti-platelet adhesion molecules, are adsorbed resulting to reduced platelet adhesion.[41] In addition to protein adsorption, the dissolution and swelling of the GEL component of the PCL/GEL membrane would enable entrapment and retention of blood cells. However, the GEL in PCL/GEL membrane was not cross-linked thus, it is susceptible to break down and eventual displacement in aqueous conditions resulting to the reduced attachment and deposition of both cells and platelets. Protein in the blood
Figure 7. SEM micrographs showing platelet adhesion on the surfaces of PCL (A), PCL/PLGA (B), PCL/GEL (C), and PCL/BCP (D) electrospun membranes after 30 min of incubation in PRP (-platelets).
498
A.R. Padalhin et al.
can also be adsorbed on calcium phosphate surfaces to some extent as suggested by previous studies [32–34] which could then result to platelet adhesion but to a lesser degree compared to bare polymer surfaces.
Downloaded by [Florida International University] at 11:08 28 December 2014
3.4. In vivo bone regeneration Figure 8 shows the reconstructed 3D images of skull sections implanted with the different membrane samples. Within a month (Figure 8(D1)), PCL/BCP electrospun membrane showed increased regeneration along the margin of the 5 mm diameter defect. This becomes more pronounced two months after implantation, with PCL/BCP (Figure 8(D2)) achieving greater bone regeneration compared with PCL, PCL/PLGA, and PCL/GEL (Figure 8(A2–C2)). 3D analysis of the micro CT data (Figure 9) also confirms a significantly higher percent bone volume when PCL/BCP was implanted for both observation periods. 3D analysis of the micro CT data supports the 3D reconstructed models of the rat calvarias. Little significant difference was observed between all the optimized membranes one month after implantation, with PCL/BCP having the highest reading of percent bone volume (6.12 ± 1.95 mm3). Calculated 3D analysis indicates that bone volume and percent bone volume are significantly higher (p < 0.001) in PCL/BCP (12.12 ± 1.75 mm3) than in PCL (6.85 ± 0.23 mm3), and slightly higher than the PCL/PLGA (9.27 ± 0.85 mm3) and PCL/GEL (9.63 ± 0.45 mm3) membranes, one and two months after implantation. Bone regeneration on the defect sites was visualized using tissue sections stained with hematoxylin and eosin (H and E) and Masson’s trichrome. Figure 10 shows the H and E stained tissue sections one month post implantation. While fibrous tissue formation is prevalent on all implanted samples, it is more pronounced in the PCL and PCL/PLGA samples (Figure 10(A–B)). All tissue sections also show bone regeneration along the margin of the defect. Tissue sections of samples extracted two months after implantation (Figure 11) show markedly improved bone regeneration along the margin of the defects; however, fibrous tissue is persistent within the majority of the implanted
Figure 8. Three-dimensional reconstruction of micro CT data of rat calvaria implanted with PCL (A), PCL/PLGA (B), PCL/GEL (C), and PCL/BCP (D) electrospun membranes showing regeneration after 1 (A1–D1) and 2 (A2–D2) months.
Downloaded by [Florida International University] at 11:08 28 December 2014
Journal of Biomaterials Science, Polymer Edition
499
Figure 9. Calculated tissue volume, bone volume and corresponding percent bone volume indicate significant difference (p > 0.001) in bone tissue formation after two months.
samples. Defects implanted with PCL/GEL membranes (Figure 11(C)) revealed more extensive vascularization among all the other implanted samples. Although defects implanted with PCL/BCP and PCL/PLGA membranes (Figure 11(B) and (D)) still possessed extensive fibrous tissue, these implants also showed dense collagen deposition (Figure 11(B1) and (D1)) distant from that of the defect margin. Masson’s trichome staining was also applied on a tissue section to further characterize tissue formation on implanted membranes. The majority of the samples did have blue stained fibrous collagen (Figure 11(A1–D1)). However, compared to other samples, PCL/PLGA and PCL/BCP showed deposition of dense collagen. Also, compared to PCL/PLGA, PCL/BCP contained larger amounts of dense collagen (stained blue), which is also identified to be new bone, and dense collagen with red pigmentation (Figure 11(D1)). Bone tissue repair is initiated through the migration and differentiation of stem cells and other surrounding tissue.[30,31,42,43] Deposition of dense collagen during callus formation in defect areas is carried out by osteoblast cells, thus scaffolds intended for bone tissue regeneration should primarily be osteoconductive, in addition to being osteoinductive, and be capable of osteointegration.[13,30,31] Interestingly, even though PCL yielded the highest blood cell deposition and relatively lower hemolysis, it simply promoted persistence of fibrous tissue up to two months after implantation. Development of a stable hematoma is crucial for healing in bone fractures. However, prolonged presence of inflammatory agents was found to negatively affect bone regeneration.[25,44] Although not fully observed in the current research, this condition could be associated with the PCL membrane’s susceptibility for activating platelets and contributing to the coagulation cascade, forming a highly stable hematoma. Further study is recommended to investigate this possibility. Although extensive vascularization was prevalent within the defect area, this did not contribute significantly to the overall regeneration of bone tissue on the 5 mm defects when PCL/GEL membranes were used. PCL/BCP still generated significantly higher regeneration among all optimized membranes, possibly due to its potential for reduced blood cell adhesion and osteoconductivity. The combination of BCP and PCL polymer combines the appropriate characteristics of materials for bone tissue regeneration. Hyrophobic polymers have been found to have an affinity for protein adsorption, while calcium phosphate derived powders have long been identified as
A.R. Padalhin et al.
Downloaded by [Florida International University] at 11:08 28 December 2014
500
Figure 10. Tissue sections of one month implant samples of PCL (A), PCL/PLGA (B), PCL/ GEL (C), and PCL/BCP (D) electrospun membranes stained with hematoxilyn and eosin 5 mm rat calvaria defect showing mostly fibrous tissue growth for the majority of the samples and initial bone regeneration along the defect margin.
competent materials for bone healing due to their biodegradation, osteoconductivity, and potentially osteoinductive properties under in vivo conditions. PCL has already been proven to be a suitable material for tissue engineering, while combining it with other polymers further enhances its functionality.[45,46] The addition of BCP, or other calcium phosphate derived components, significantly improves the bone tissue regenerating capability of electrospun PCL.
Downloaded by [Florida International University] at 11:08 28 December 2014
Journal of Biomaterials Science, Polymer Edition
501
Figure 11. Micrographs of tissue sections of implanted samples of PCL (A, A1, A2), PCL/ PLGA (B, B1, B2), PCL/GEL (C, C1, C2), and PCL/BCP (D, D1, D2) electrospun membranes stained with hematoxilyn and eosin, and Masson’s trichome, two months after implantation in critical sized rat calvaria defect (blood vessel – , collagen – ♦, new bone – ).
▴
4. Conclusion This study has successfully compared the bone regeneration capability of several optimized binary formulations of PCL. Even though all membranes show competitive results regarding in vitro cytocompatibility and hemocompatibility, the incorporation of a calcium phosphate containing compound in the composite PCL/BCP membrane has shown that this is the most effective material for bone regeneration based on the results of the in vivo implantation. This reveals that although all of the membranes were fabricated using optimized formulations and conditions, the incorporation of an osteoconductive component resulting in a composite material was more conducive for bone tissue growth. Further studies are suggested to investigate the possible enhancement of the PCL/BCP for bone tissue engineering. Acknowledgments This work was supported by Mid-career Research Program through NFR grant funded by the MEST (NO 2009-0092808), Republic of Korea and partially supported by Soonchunhyang University Research Fund. The author would also like to thank Rose Ann Franco and Thi-Heip Nguyen for their help in the sample preparation and Kim Shin Woo and Kim Hyoung Suk for facilitating the in vivo experiments.
References [1] Salgado AJ, Coutinho OP, Reis RL. Bone tissue engineering: state of the art and future trends. Macromol. Biosci. 2004;4:743–765.
Downloaded by [Florida International University] at 11:08 28 December 2014
502
A.R. Padalhin et al.
[2] Schroeder JE, Mosheiff R. Tissue engineering approaches for bone repair: concepts and evidence. Injury. 2011;42:609–613. [3] Tian H, Tang Z, Zhuang X, Chen X. Biodegradable synthetic polymers: preparation, functionalization and biomedical application. Prog. Polym. Sci. 2012;37:237–280. [4] Zong C, Xue D, Yuan W, Wang W, Shen D, Tong X, Shi D, Liu L, Zheng Q, Gao C, Wang J. Reconstruction of rat calvarial defects with human mesenchymal stem cells and osteoblastlike cells in poly-lactic-co-glycolic acid scaffolds. Eur Cell Mater. 2010;20:109–120. [5] Woodruff MA, Hutmacher DW. The return of a forgotten polymer – polycaprolactone in the 21st century. Prog. Polym. Sci. 2010;35:1217–1256. [6] Roohani-Esfahani SI, Nouri-Khorasani S, Lu Z, Appleyard R, Zreiqat H. The influence hydroxyapatite nanoparticle shape and size on the properties of biphasic calcium phosphate scaffolds coated with hydroxyapatite–PCL composites. Biomaterials. 2010;31:5498–5509. [7] Franco RA, Nguyen TH, Lee BT. Preparation and characterization of electrospun PCL/ PLGA membranes and chitosan/gelatin hydrogels for skin bioengineering applications. J. Mater. Sci. Mater. Med. 2011;22:2207–2218. [8] Hiep NT, Lee BT. Electro-spinning of PLGA/PCL blends for tissue engineering and their biocompatibility. J. Mater. Sci. Mater. Med. 2010;21:1969–1978. [9] Chong EJ, Phan TT, Lim IJ, Zhang YZ, Bay BH, Ramakrishna S, Lim CT. Evaluation of electrospun PCL/gelatin nanofibrous scaffold for wound healing and layered dermal reconstitution. Acta Biomater. 2007;3:321–330. [10] Zhao P, Jiang H, Pan H, Zhu K, Chen W. Biodegradable fibrous scaffolds composed of gelatin coated poly(ε-caprolactone) prepared by coaxial electrospinning. J. Biomed. Mater. Res. A. 2007;83A:372–382. [11] Ba Linh NT, Min YK, Lee BT. Hybrid hydroxyapatite nanoparticles-loaded PCL/GE blend fibers for bone tissue engineering. J. Biomater. Sci., Polym. Ed. 2013;24:520–538. [12] Nguyen TP, Lee BT. Fabrication and characterization of BCP nano particle loaded PCL fiber and their biocompatibility. Korean J. Mater. Res. 2010;20:392–400. [13] Albrektsson T, Johansson C. Osteoinduction, osteoconduction and osseointegration. Eur Spine J. 2001;10:S96–101. [14] Motlagh D, Allen J, Hoshi R, Yang J, Lui K, Ameer G. Hemocompatibility evaluation of poly(diol citrate) in vitro for vascular tissue engineering. J. Biomed. Mater. Res. A. 2007;82A:907–916. [15] Chenglong L, Dazhi Y, Guoqiang L, Min Q. Corrosion resistance and hemocompatibility of multilayered Ti/TiN-coated surgical AISI 316L stainless steel. Mater. Lett. 2005;59:3813–3819. [16] Zhao ML, Li DJ, Yuan L, Yue YC, Liu H, Sun X. Differences in cytocompatibility and hemocompatibility between carbon nanotubes and nitrogen-doped carbon nanotubes. Carbon. 2011;49:3125–3133. [17] Li J, Zheng W, Zheng Y, Lou X. Cell responses and hemocompatibility of g-HA/PLA composites. Sci. China Life Sci. 2011;54:366–371. [18] Lee BT, Youn MH, Paul RK, Lee KH, Song HY. In situ synthesis of spherical BCP nanopowders by microwave assisted process. Mater. Chem. Phys. 2007;104:249–253. [19] Maekawa Y, Yagi K, Nonomura A, Kuraoku R, Nishiura E, Uchibori E, Takeuchi K. A tetrazolium-based colorimetric assay for metabolic activity of stored blood platelets. Thromb. Res. 2003;109:307–314. [20] Gómez-Guillén MC, Giménez B, López-Caballero ME, Montero MP. Functional and bioactive properties of collagen and gelatin from alternative sources: a review. Food Hydrocoll. 2011;25:1813–1827. [21] Wilson CJ, Clegg RE, Leavesley DI, Pearcy MJ. Mediation of biomaterial–cell interactions by adsorbed proteins: a review. Tissue Eng. 2005;11:1–18. [22] Garrido CA, Lobo SE, Turibio FM, LeGeros RZ. Biphasic calcium phosphate bioceramics for orthopaedic reconstructions: clinical outcomes. Int. J. Biomater. 2011;2011:129727. [23] Anselme K. Osteoblast adhesion on biomaterials. Biomaterials. 2000;21:667–681. [24] Ozaki A, Tsunoda M, Kinoshita S, Saura R. Role of fracture hematoma and periosteum during fracture healing in rats: interaction of fracture hematoma and the periosteum in the initial step of the healing process. J. Orthop. Sci. 2000;5:64–70.
Downloaded by [Florida International University] at 11:08 28 December 2014
Journal of Biomaterials Science, Polymer Edition
503
[25] Kolar P, Schmidt-Bleek K, Schell H, Gaber T, Toben D, Schmidmaier G, Perka C, Buttgereit F, Duda GN. The early fracture hematoma and its potential role in fracture healing. Tissue Eng. Part B: Rev. 2010;16:427–434. [26] Arpornmaeklong P, Kochel M, Depprich R, Kübler NR, Würzler KK. Influence of plateletrich plasma (PRP) on osteogenic differentiation of rat bone marrow stromal cells. An in vitro study. Int. J. Oral Maxillofac. Surg. 2004;33:60–70. [27] Dominiak M, Łysiak-Drwal K, Solski L, Żywicka B, Rybak Z, Gedrange T. Evaluation of healing processes of intraosseous defects with and without guided bone regeneration and platelet rich plasma. An animal study. Ann. Anat. 2012;194:549–555. [28] Jeong Park YJ, Moo Lee YM, Nae Park SN, Yoon Sheen SY, Pyoung Chung CP, Lee SJ. Platelet derived growth factor releasing chitosan sponge for periodontal bone regeneration. Biomaterials. 2000;21:153–159. [29] Marsell R, Einhorn TA. The biology of fracture healing. Injury. 2011;42:551–555. [30] McKibbin B. The Biology of fracture healing in long bones. J. Bone Joint Surg. 1978;60:150–162. [31] Wang K, Zhou C, Hong Y, Zhang X. A review of protein adsorption on bioceramics. Interface focus. 2012;2:259–277. [32] Wang J, Zhang H, Zhu X, Fan H, Fan Y, Zhang X. Dynamic competitive adsorption of bone-related proteins on calcium phosphate ceramic particles with different phase composition and microstructure. J. Biomed. Mater. Res. B: Appl. Biomater. 2013;101B:1069–1077. [33] Zhu XD, Zhang HJ, Fan HS, Li W, Zhang XD. Effect of phase composition and microstructure of calcium phosphate ceramic particles on protein adsorption. Acta Biomater. 2010;6:1536–1541. [34] Chen H, Yuan L, Song W, Wu Z, Li D. Biocompatible polymer materials: role of protein– surface interactions. Prog. Polym. Sci. 2008;33:1059–1087. [35] Comelles J, Estévez M, Martínez E, Samitier J. The role of surface energy of technical polymers in serum protein adsorption and MG-63 cells adhesion. Nanomedicine. 2010;6: 44–51. [36] Marsh RJ, Jones RAL, Sferrazza M. Adsorption and displacement of a globular protein on hydrophilic and hydrophobic surfaces. Colloids Surf., B. 2002;23:31–42. [37] Liu P, Chen Q, Yuan B, Chen M, Wu S, Lin S, Shen J. Facile surface modification of silicone rubber with zwitterionic polymers for improving blood compatibility. Mater. Sci. Eng., C. 2013;33:3865–3874. [38] Li Q, Wang Z, Zhang S, Zheng W, Zhao Q, Zhang J, Wang L, Wang S, Kong D. Functionalization of the surface of electrospun poly(epsilon-caprolactone) mats using zwitterionic poly(carboxybetaine methacrylate) and cell-specific peptide for endothelial progenitor cells capture. Mater. Sci. Eng., C. 2013;33:1646–1653. [39] Zhu A, Lu P, Wu H. Immobilization of poly(ɛ-caprolactone)–poly(ethylene oxide)–poly (ɛ-caprolactone) triblock copolymer on poly(lactide-co-glycolide) surface and dual biofunctional effects. Appl. Surf. Sci. 2007;253:3247–3253. [40] Lee JH, Lee HB. Platelet adhesion onto wettability gradient surfaces in the absence and presence of plasma proteins. J. Biomed. Mater. Res. 1998;41:304–311. [41] Doblaré M, Garcı́a JM, Gómez MJ. Modelling bone tissue fracture and healing: a review. Eng. Fract. Mech. 2004;71:1809–1840. [42] Schmidt-Bleek K, Schell H, Schulz N, Hoff P, Perka C, Buttgereit F, Volk HD, Lienau J, Duda GN. Inflammatory phase of bone healing initiates the regenerative healing cascade. Cell Tissue Res. 2012;347:567–573. [43] Opal SM. Phylogenetic and functional relationships between coagulation and the innate immune response. Crit. Care Med. 2000;28:S77–S80. [44] Baker SC, Rohman G, Southgate J, Cameron NR. The relationship between the mechanical properties and cell behaviour on PLGA and PCL scaffolds for bladder tissue engineering. Biomaterials. 2009;30:1321–1328. [45] Ghasemi-Mobarakeh L, Prabhakaran MP, Morshed M, Nasr-Esfahani MH, Ramakrishna S. Bio-functionalized PCL nanofibrous scaffolds for nerve tissue engineering. Mater. Sci. Eng., C. 2010;30:1129–1136. [46] Sousa I, Mendes A, Bártolo PJ. PCL scaffolds with collagen bioactivator for applications in tissue engineering. Procedia Eng. 2013;59:279–284.
Journal of Biomaterials Science, Polymer Edition Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tbsp20
Evaluation of the cytocompatibility hemocompatibility in vivo bone tissue regenerating capability of different PCL blends a
a
b
Andrew R. Padalhin , Nguyen Thuy Ba Linh , Young Ki Min & a
Byong-Taek Lee a
Click for updates
Department of Regenerative Medicine, Institute of Tissue Regeneration, College of Medicine, Soonchunhyang University, Cheonan 330-090, Korea b
Department of Physiology, College of Medicine, Soonchunhyang University, Cheona, Korea Published online: 22 Jan 2014.
To cite this article: Andrew R. Padalhin, Nguyen Thuy Ba Linh, Young Ki Min & Byong-Taek Lee (2014) Evaluation of the cytocompatibility hemocompatibility in vivo bone tissue regenerating capability of different PCL blends, Journal of Biomaterials Science, Polymer Edition, 25:5, 487-503, DOI: 10.1080/09205063.2013.878870 To link to this article: http://dx.doi.org/10.1080/09205063.2013.878870
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content.
Downloaded by [Florida International University] at 11:08 28 December 2014
This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http://www.tandfonline.com/page/termsand-conditions
Journal of Biomaterials Science, Polymer Edition, 2014 Vol. 25, No. 5, 487–503, http://dx.doi.org/10.1080/09205063.2013.878870
Evaluation of the cytocompatibility hemocompatibility in vivo bone tissue regenerating capability of different PCL blends Andrew R. Padalhina, Nguyen Thuy Ba Linha, Young Ki Minb and Byong-Taek Leea*
Downloaded by [Florida International University] at 11:08 28 December 2014
a
Department of Regenerative Medicine, Institute of Tissue Regeneration, College of Medicine, Soonchunhyang University, Cheonan 330-090, Korea; bDepartment of Physiology, College of Medicine, Soonchunhyang University, Cheona, Korea (Received 15 October 2013; accepted 20 December 2013) In this study, the optimized formulations of polycaprolactone (PCL) combined with poly(lactic-co-glycolic acid) (PLGA), gelatin (GEL), and biphasic calcium phosphate (BCP) were analyzed in terms of cytocompatibility with bone-related cells, hemocompatibility, and in vivo bone-regenerating capacity to determine their potentials for bone tissue regeneration. Fiber morphology of PCL/GEL and PCL/BCP electrospun mats considerably differs from that of the PCL membrane. Based on the contact angle analyses, the addition of GEL and PLGA was shown to reduce the hydrophobicity of these membranes. The assessment of in vitro cytocompatibility using MC3T3-E1 cells indicated that all of the membranes were suitable for pre-osteoblast proliferation and adhesion, with PCL/BCP having a significantly higher reading after seven days of incubation. The results of the in vitro hemocompatibility of the different fibrous scaffolds suggest that coagulation and platelet adhesion were higher for hydrophobic membranes (PCL and PCL/PLGA), while hemolysis can be associated with fiber morphology. The potential of the membranes for bone regeneration was determined by analyzing the microCT data and tissue sections of samples implanted in 5 mm sized defects (one and two months). Although all of the membranes were suitable for pre-osteoblast proliferation, in vivo bone regeneration after two months was found to be significantly higher in PCL/BCP (p < 0.001). Keywords: PCL; electrospinning; in vitro cytocompatibility; hemocompatibility; in vivo bone regeneration
1. Introduction Graft transplantation has been typically favored over artificial implants when dealing with tissue regeneration, due to its inherent advantage of enabling the use of similar tissue that has been matched with the recipient form donors. However, using tissue grafts also poses significant disadvantages, namely donor availability, donor site morbidity, graft rejection, and to a lesser degree, donor–recipient pathogen transmission.[1] Numerous studies have been conducted on both naturally occurring and synthetic polymers as surface modifiers and components for different tissue scaffolds that lead to the improved biocompatibility of biomaterials used for regenerative medicine such as bone tissue engineering.[1–3] Among the polymers used in the field of tissue engineering, aliphatic polyesters are widely used due to their biodegradability and biocompatibility.[3,4] Various research *Corresponding author. Email: [email protected] © 2014 Taylor & Francis
Downloaded by [Florida International University] at 11:08 28 December 2014
488
A.R. Padalhin et al.
have been conducted on different methods of fabricating tissue scaffolds using polycaprolactone (PCL). Aside from being a relatively cheaper biocompatible synthetic polymer, PCL has been proven to be a versatile substance capable of forming stable microspheres, porous bodies, and electrospun membranes used as either drug delivery systems or tissue scaffolds. Whether as a monolithic scaffold or as a composite combined with other biocompatible materials, PCL can be considered as a good base material for developing scaffolds for tissue engineering.[5] Subsequent studies on electrospun PCL-based fibrous scaffolds have provided a good background for applying these materials in tissue regeneration studies.[6–12] In this study, three optimized binary PCL mixtures have been tested to define their respective performances in terms of contributing to bone regeneration. The purpose of this study is to compare these binary systems of PCL-based electrospun membranes by taking previously optimized systems fabricated by incorporating a co-polymer, hydrolyzed protein (gelatin (GEL)) and biphasic calcium phosphate (BCP) based powder. Each additive combined with PCL has been determined to improve the biocompatibility of the resulting optimized membranes in vitro; however, comparison between these materials in terms of application for bone tissue regeneration is yet to be established through in vitro and in vivo experimentation. The first optimized membrane is a mixture of copolymers PCL and poly (lactic-co-glycolic acid) (PLGA). An initial study has been conducted for the application of PCL/PLGA blend as a scaffold for skin [7] and for general tissue engineering purposes [8] tested with keratinocytes and fibrous tissue cells (L-929), respectively. The optimized mixture is composed of PCL and GEL and has already been tested for tissue engineering and dermal reconstruction.[9,10] More recently it has also been improved through the loading of hydroxyapatite particles [11] for bone regeneration application. The final optimized membrane composed of PCL and BCP powder was also selected due to its high in vitro performance based on a previous report using L929 cells, although it has not yet been tested in vivo.[12] The in vitro cytocompatibility and bone tissue regeneration potential of the optimized formulations of PCL/PLGA (80:20 ratio), PCL/GEL (50:50 ratio), and PCL/BCP (50% weight:volume) scaffolds were evaluated through proliferation assay, observation of attachment behavior through confocal microscopy, and implantation in 5 mm diameter calvarial defects in rats. An additional parameter, hemocompatibility, was also tested to observe blood reaction upon contact with the tissue scaffolds. The hemocompatibility in terms of hemolysis or break down of red blood cells upon material contact, blood cell attachment on the surface of scaffolds, and platelet adhesion have been taken into consideration, noting that blood would be the primary fluid that will come in contact with implants. The healing process of damaged bone tissue involves the accumulation and stabilization of blood clots and dissolution of hematoma, which in turn provides the factors for initiating chemotaxis for cell-mediated repair.[13–17] 2. Materials and methods 2.1. Materials Materials for fabricating the different electrospun membranes PCL (Mn 80,000), PLGA (85:15, Mw 50,000–75,000) and GEL (from porcine skin) were purchased from SigmaAldrich, USA. BCP powder measuring around 50–100 nm was synthesized through microwave assisted process using Ca (OH)2 (Aldrich Chemical) and H3PO4 (Dongwoo Fine Chemicals, Korea) as per reference.[18] Solvents used to dissolve the polymer
Journal of Biomaterials Science, Polymer Edition
489
Downloaded by [Florida International University] at 11:08 28 December 2014
components, tetrahydrofuran (THF, minimum 99%), and dimethylformamide (DMF, 99%), were also purchased from Sigma–Aldrich, USA. The other two solvents, methyl chloride and 2,2,2-trifluoroethanol (TFE, 99.0%), were procured from SK Chemicals Co., Korea and Fluka, Buchs, Switzerland, respectively. Methylene chloride was purchased from Dae Jung chemicals, Korea. 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) was purchased from GIBCO Carlsbad, CA, USA. Dimethylsulfoxide (DMSO, 99.0%) was obtained from Samchun Pure Chemical Co., Ltd (Pyeongtaek City, South Korea). Minimum essential medium (MEM) was obtained from HyClone (Logan, UT, USA). Pre-osteoblast (MC3T3-E1) mice cells were obtained from the American Type Culture Company, USA. All chemicals and solvents were of analytical reagent grade. 2.2. Fabrication of fibrous membrane scaffolds The scaffolds were fabricated using different PCL blends, as optimized in previous studies [6–12] and using a membrane composed of 12 wt.%/v PCL as the control. The optimized membranes were made by mixing PLGA, GEL, and BCP in separate independent solutions with PCL. For creating PCL/PLGA and PCL/BCP blends, a solution of 12 wt.%/v PCL was dissolved in a solvent containing 40% THF, 40% DMF, and 20% MC. PLGA solution was made by dissolving 10 wt.%/v of PLGA using the same solvent. The PCL/PLGA blend was made by combining 1:4 ratio of PLGA and PCL, while the PCL/BCP blend was made by adding 50 wt.%/wt. of BCP in the 12 wt.%/v PCL solution. The PCL/GEL blend was achieved by combining equal amounts of separate solutions of 12 wt.%/v of PCL and 12 wt.%/v GEL, both dissolved in TFE. Each PCL blend was then electrospun using an electrospinning machine (Electrospinning System, NanoNC, Korea) to create nanofibrous membranes. Each membrane required different parameters due to the varying material compositions. Their respective electrospinning parameters are enumerated as follows (voltage-kV, needle gauge – G, flow rate – ml/h, and tip-collector-distance – cm): PCL – 20 kV, 23 G, 0.5 ml/h, 20 cm; PCL/PLGA – 25 kV, 25 G, 0.5 ml/h, 20 cm; PCL/GEL – 25 kV, 21 G, 0.5 ml/h, 17 cm; and PCL/BCP – 20 kV, 21 G, 2 ml/h, 12 cm. 2.3. Characterization of fibrous membrane scaffolds After fabrication, the dry samples were placed on a scanning electron microscope (SEM) mount and coated with platinum (Cressington 108 Auto). The fiber morphology and average fiber diameter were observed and measured from images taken using SEM microscopy (SEM, JSM-7401F). Energy-dispersive X-ray spectroscopy (EDS) profile was also taken to confirm the presence of elemental components associated with GEL and BCP. The contact angle of each membrane scaffold was also tested using EasyDrop Contact Angle Measuring System (DSA, KRUSS GmbH). 2.4. Cell adhesion, proliferation and viability profiles Celll adhesion, viability, and proliferation were observed using a pre-osteoblast (MC3T3-E1) commercial cell line. Circular samples of each membrane measuring 6 mm in diameter were sterilized using ethanol and UV irradiation, and were conditioned with MEM media containing 10% fetal bovine serum and 5% penicillin streptomycin for 20 min prior to seeding with 1.5 × 104/ml of M3CT3-E1 cells.
490
A.R. Padalhin et al.
Downloaded by [Florida International University] at 11:08 28 December 2014
Observation of cell adhesion was carried out by confocal microscopy of seeded membrane samples stained with FITC and DAPI, while live and dead cell analysis was carried out by staining seeded samples with Calcein AM and Ethidium homodimer, one and five days after seeding. Micrographs of each sample were taken using a confocal laser scanning microscope (CLSM, Fluoview 1000, Olympus). Quantification of living and dead cells was performed through computer aided analysis of at least 10 micrographs per sample. The images were analyzed using the accompanying FV10-ASW 3.0 Viewer software and ImageJ. Cell proliferation on each scaffold was measured at 1, 3, 5, and 7 days after seeding by conducting an MTT assay and using an ELISA reader (TECAN 150, Turner Biosystems) at 595 nm. 2.5. Hemocompatibility To determine the blood reaction upon contact with each material, a hemocompatibility test was conducted on each individual rat previously implanted with a specific sample. A minimum amount of anti-coagulated blood, 1–2 ml, was aseptically drawn from each individual rat using a syringe with a 26 gauge needle and containing 1:9 ratio of Citrate-dextrose solution (Sigma). Each optimized material was tested for hemolytic effect, blood cell deposition, and platelet adhesion. The following procedures are based on several studies on blood contacting biomaterials [14–17] with some slight modifications. Hemolysis due to material contact was evaluated by submerging 6.0 mm diameter samples in 1 ml of test solution consisting of 1:10 ratio of anti-coagulated whole blood in sterile PBS. To eliminate hemolysis that might have resulted from the handling of blood samples prior to testing, a negative control was also prepared consisting of the test solution without any submerged material and a positive control was prepared consisting of a similar amount of anti-coagulated blood diluted in distilled water. After 30 and 60 min of exposure to test samples, the test solutions were removed and centrifuged at 3000 rpm for 3 min to pellet out the cellular components of the solution. To determine the amount of free hemoglobin released from hemolyzed erythrocytes, the supernatant was placed in a 96-well plate and read for absorbance at 540 nm. Blood cell deposition on material surface was analyzed by applying 200 μl of anti-coagulated blood on 6.0 mm samples which were then left to stand for 5, 10, and 15 min. After each sampling time, non-adherent blood cells were washed off the membranes using PBS and adherent cells were quantified through hemolysis. Platelet adhesion was observed using platelet rich plasma (PRP). PRP was prepared by centrifugation of 10 ml anti-coagulated blood at 2500 rpm for 5 min and removing the pelleted red blood cells, leaving behind the buffy coat in the middle and the upper supernatant consisting of the plasma. The supernatant was again centrifuged at 1000 rpm for 5 min to remove the remaining RBC component. Taking into consideration that only a very small amount of PRP can be prepared from a very small blood sample, the plasma was no longer removed and the volume of the solution was increased up to 5 ml and the platelets were resuspended through vortex mixing. A similar experimental design based on a previous study [19] was employed to quantify the amount of platelets adhered to the sample membranes. MTT assay was conducted to quantify the number of platelets attached to the membranes after 15 and 30 min of incubation.
Journal of Biomaterials Science, Polymer Edition
491
Downloaded by [Florida International University] at 11:08 28 December 2014
2.6. In vivo tests The in vivo bone regeneration of the different PCL blends was evaluated by implantation on rat calvarial defects. Circular samples of PCL, PCL/PLGA, PCL/GEL, and PCL/BCP measuring 6 mm in diameter were cut out using a paper puncher after which 3–4 layers of each samples were stacked together to form a single implant material. Prior to implantation, each implant material was briefly washed with 70% ethanol and sterilized with ultraviolet radiation on both sides. Sprague Dawley rats were sedated and circular defects measuring 5 mm in diameter were created on both sides of the frontal plate of the skulls. The left side defect was implanted with a sample membrane pre-soaked in phosphate buffered solution, while the right side defect was considered as a control and no implant was placed. Implant samples were extracted after four and eight weeks of recovery. Micro CT data of the formalin fixed samples were recorded and tissue sections were stained with hematoxylin, eosin, and Masson’s trichrome for further analysis. 2.7. Statistical analysis Each experiment was repeated at least three times on different days and data were expressed as the mean ± SD and analyzed through one-way ANOVA.
3. Results and discussion 3.1. Characterization of fibrous membrane scaffolds The material characteristics of the fabricated samples closely resemble those of the original descriptions from previous studies.[7–9,12] Both the fiber morphology and measurements of fiber diameters approximately match those of the former descriptions, with only a marginal difference. Figure 1 shows the SEM micrographs of fibrous membrane scaffolds composed of different PCL blends. Each PCL blend, with the exception of PCL/PLGA, has a distinct fiber morphology. For this experiment, the PCL membrane was established as a control membrane. The PCL (A) and the PCL/PLGA (B) membranes are basically composed of approximately similar diameter fibers ranging from 0.51 to 0.96 μm. On the other hand, membranes consisting of composite materials, PCL/GEL (C) and PCL/BCP (D), both have a highly altered fiber morphology compared to that of the PCL membrane. The PCL/GEL membrane consists of fibers with different sized diameters (0.25–1.03 μm). Incorporation of GEL into the electrospun membrane was confirmed through EDS data, showing the presence of nitrogen within the fiber, which can only be derived from the hydrolyzed proteins. Of the membranes, the PCL/BCP composite membrane has the most altered fiber morphology. Aside from having a wider range of fiber diameter (1.3–7.8 μm), the fibers formed from the solution are highly irregular along their length. Some clumps of the PCL/BCP blend can be seen at random sites, which contribute to the rough overall uneven surface morphology of the fibers. In addition, the incorporation of the BCP powder was confirmed through EDS showing calcium content within the fibers (Figure 1(D1)), which can only be derived from the BCP powder. To determine the hydrophilic properties of each membrane scaffold, the samples were tested for water contact angle. The average of four measurements per sample was used to determine the respective contact angles of each optimized membrane scaffold (Figure 2). Among the optimized scaffolds, the PCL/GEL (C) blend was the most
Downloaded by [Florida International University] at 11:08 28 December 2014
492
A.R. Padalhin et al.
Figure 1. SEM micrographs showing respective fiber morphologies of PCL (A), PCL/PLGA (B), PCL/GEL (C), and PCL/BCP (D) electrospun membranes. EDS data showing presence of nitrogen in electrospun GEL fibers (C1) interspersed with larger PCL fibers with lower nitrogen reading (C2) in PCL/GEL mat. Presence of calcium is also confirmed through EDS profile of PCL/BCP electrospun membranes.
Figure 2. Contact angle measurements of PCL (A), PCL/PLGA (B), PCL/GEL (C), and PCL/ BCP (D) electrospun membranes.
hydrophilic, yielding the lowest contact angle, 80.7 ± 5.77°, while the control membrane composed of 12% PCL (A) was the most hydrophobic, having the highest reading of 97 ± 6.02°. The blends, PCL/BCP (D) and PCL/PLGA (B), were relatively more hydrophilic than the control membrane, but less hydrophilic than the PCL/GEL membrane (91.75 ± 2.81° and 93.53 ± 2.91°, respectively). As expected, PCL/GEL had
Journal of Biomaterials Science, Polymer Edition
493
the lowest contact angle measurement due to hydrophilic property and the ability to absorb and retain the water of the GEL.[20] The addition of a more hydrophilic polymer (PLGA) and BCP powder in the PCL electrospun fibers also reduces the contact angle to a lesser extent by contributing to the limited hydrophilic property.
Downloaded by [Florida International University] at 11:08 28 December 2014
3.2. Cell adhesion, proliferation, and viability profiles In vitro cytocompatibility is an important factor for developing materials suitable for tissue engineering. In this study, PCL optimized membranes combined with PLGA, GEL, and BCP have been tested using MC3T3-E1 cells to chiefly establish suitability of these membranes for bone tissue growth. Confocal microscopy of seeded cells indicates minute differences between the adhesion behavior and viability of MC3T3-E1 cells on different micro-fibrous membrane scaffolds. Cell proliferation on the seeded scaffolds was determined using MTT assay. Quantitative data of cell viability were also gathered from confocal micrographs using seeded cells stained using a live/dead cell kit. Figure 3 shows the cell proliferation profiles of MC3T3-E1 cells seeded on the PCL, PCL/PLGA, PLC/GEL, and PCL/BCP fibrous membrane scaffolds. Results show that among these samples, PCL/BCP, followed by PCL/GEL, shows the greatest cell proliferation within seven days of culture. Statistical analysis indicates that the PCL/PLGA, PCL/GEL, and PCL/BCP electrospun membranes were significantly higher three and seven days after cell seeding (PCL/PLGA and PCL/GEL: p < 0.05; PCL/BCP: p < 0.001). On the fifth day, there is a noticeable lag phase in which no significant difference is observed between the proliferations on all the samples. This is possible considering that all the membranes were fabricated using the optimized formulations for tissue engineering. Figure 4 shows the confocal micrographs of MC3T3-E1 cells seeded on PCL (A1, A2), PCL/PLGA (B1, B2), PCL/GEL (C1, C2), and PCL/BCP (D1, D2) membrane scaffolds after 1–7 days. Twenty-four hours after seeding, the cell adhesion behavior on all four membranes did not show any obvious differences. However, at seven days of incubation it can be clearly seen that the cell attachment on PCL/GEL and PCL/BCP
Figure 3. Cell proliferation of MC3T3-E1 cells seeded on PCL (A), PCL/PLGA (B), PCL/GEL (C), and PCL/BCP (D) electrospun membranes measured up to seven days.
Downloaded by [Florida International University] at 11:08 28 December 2014
494
A.R. Padalhin et al.
Figure 4. Confocal micrographs showing adhesion behavior of MC3T3-E1 cells on PCL (A1–A2), PCL/PLGA (B1–B2), PCL/GEL (C1–C2), and PCL/BCP (D1–D2) electrospun membranes one and seven days after seeding.
membranes was more extensive compared to that of the PCL and PCL/PLGA membranes. In addition to the extensive attachments of the cells seeded on PCL/GEL and PCL/BCP membranes, both membranes also contained more cells compared to the remaining samples. Micrographs of seeded scaffolds stained for live/dead cell assay were also obtained through confocal microscopy. A minimum of 10 images pictures per sample were analyzed and counted for the ratio of living and dead cells. As indicated in the kit manual, all the cells in the samples are stained green and the dead cells are identified through the red stained nucleus. Cell viability for the samples is established by averaging the reading from each micrograph, which is calculated by subtracting the number of dead cells from the total count of cells, dividing the difference by the total count of cells, and then finally by multiplying the quotient by 100. Figure 5 shows the average MC3T3-E1 cell viability count in each sample, one and seven days after seeding and confocal micrographs for stained cells. Dead cells are red and viable cells are stained green. The results of the viability count further supports the cell proliferation data suggesting that at seven days, MC3T3-E1 cells have higher viability in the PCL/BCP electrospun membrane. Based on the results of the in vitro tests, all of the electrospun membranes were suitable for proliferating pre-osteoblast cells, with PCL/BCP membranes having a the significantly higher number of cells by the end of the seven day observation period. The initial observations showing propensity of cells to attach on the PCL/GEL membrane may be related to the aptitude of GEL to absorb fluids, allowing the perfusion of nutrients within the membrane matrix. The hydrophilicity and protein rich surface of the PCL/GEL contribute to cell attachment and proliferation.[9,10,20] Conversely, the PCL/BCP membrane also has the same effect, as described in other previous studies, whereby osteoblasts cells prefer to adhere on rough surfaces and the highly modified surface topography also provides an appropriate substrate for protein adsorption, consequently increasing the potential for cell adhesion [21,22] when in a serum supplemented culture media. In this study, the PCL/BCP membrane possessed a highly altered, uneven surface suitable for osteoblastic cell attachment. This is further exemplified through observing the cell viability and
Downloaded by [Florida International University] at 11:08 28 December 2014
Journal of Biomaterials Science, Polymer Edition
495
Figure 5. Confocal micrographs showing adhesion behavior of MC3T3-E1 cells on PCL (A1–A2), PCL/PLGA (B1–B2), PCL/GEL (C1–C2), and PCL/BCP (D1–D2) electrospun membranes one and seven days after seeding.
morphology. The live dead cell assay showed more living cells attached to the PCL membranes that were with GEL and BCP. This is also evident through the extensive cell attachment visible from the confocal micrographs. GEL has been known to be a good platform for culturing numerous types of cells,[20] while BCP typically serves as a biomimetic component for bone regeneration.[1,2,22,23] 3.3. Hemocompatibility tests Developing hemocompatible tissue scaffolds is crucial for not only blood vessel grafts but also for materials used for bone tissue regeneration. Blood is the principal physiological fluid that comes into contact with bone tissue scaffolds. The formation of hematoma is a naturally occurring process in bone fractures and allows the release of chemotactic elements that signals the migration of cells towards the site of injury. Thus, the effective formation of a blood clot on the surface of tissue scaffolds substantially contributes to the inflammatory phase of the healing process.[24,25] Platelets and their corresponding growth factors have been found to contribute to the healing process and regeneration of injured tissues.[26–29] During the inflammatory phase, breakdown of the clot and surrounding dead tissues occurs, which in turn releases signaling chemicals that guide the migrating repair cells.[30,31] To determine the blood tissue behavior upon contact, hemolysis, coagulation, and platelet adhesion were tested for each optimized PCL blend. Figure 6 shows the free hemoglobin reading after 30 and 60 min of incubating each membrane scaffold with diluted whole blood. Based on the results, the PCL/BCP membrane generated a significantly higher number of hemolyzed cells after 30 and 60 min, while PCL/GEL had the least number of hemolyzed cells (P < 0.001). The rough surface of PCL/BCP fibers, being abrasive against red blood cells, could have possibly contributed to the increased hemolysis. None of the electrospun membranes exceeded the acceptable range of red blood cell breakdown (5%) for implant materials based on ISO 10993-4:2002.
Downloaded by [Florida International University] at 11:08 28 December 2014
496
A.R. Padalhin et al.
Figure 6. Hemocompatibility data based on hemolysis (A), blood cell deposition (B) and platelet adhesion (C) of PCL, PCL/PLGA, PCL/GEL, and PCL/BCP electrospun membranes.
Figure 6 shows the results of blood coagulation tests after 30 and 60 min incubation. Blood cell deposition was significantly higher in the PCL/GEL membrane followed by PCL/BCP, while it was the lowest in the PCL membrane, was the lowest across all observation periods. The absorption of blood fluids, coupled with the dissolution of the majority of the GEL fibers in PCL/GEL, resulted in increased deposition of the blood cell component on the membrane surface. None of the membranes had greater than 1% blood cell deposited on their surface. Finally, membranes were tested for platelet adhesion using PRP from whole blood samples. The number of platelets adhered to the surface of each membrane was estimated using MTT assay (Figure 6). Based on the results, PCL and PCL/PLGA were the most conducive surfaces for platelet attachment. In the case of PCL/GEL, GEL’s absorbing capability and dissolution of GEL component again contributes to the
Downloaded by [Florida International University] at 11:08 28 December 2014
Journal of Biomaterials Science, Polymer Edition
497
attachment of the platelets. Protein in the blood can be adsorbed on calcium phosphate surfaces to some extent, as suggested in previous studies,[32–34] which could then result in platelet adhesion. Platelets adhered on the membrane surfaces were also observed through SEM microscopy (Figure 7), compared to other membranes, and the platelets adhered on PCL/GEL are highly obscured by the melted GEL (Figure 7(C)). Based on the hemocompatibility tests, PCL and PCL/PLGA were the membranes most suitable for forming clots and accumulating blood cells. Although not fully investigated in this paper, numerous studies regarding polymer surfaces have supported that reduction of contact angle and reduced protein adsorption on material surfaces significantly reduces platelet adhesion.[21,35–38] In this study, PCL and PCL/PLGA are characteristically more hydrophobic than PCL/GEL and PCL BCP, making these surfaces more suitable for protein adsorption which could contribute to increased platelet deposition.[36,38–40] In the case of PCL/GEL, capability of GEL to absorb fluids, hydrophilic and dissolution property contributes to reduced platelet attachment. Upon contact of hydrophilic surfaces to whole blood serum, plasma proteins that also include anti-platelet adhesion molecules, are adsorbed resulting to reduced platelet adhesion.[41] In addition to protein adsorption, the dissolution and swelling of the GEL component of the PCL/GEL membrane would enable entrapment and retention of blood cells. However, the GEL in PCL/GEL membrane was not cross-linked thus, it is susceptible to break down and eventual displacement in aqueous conditions resulting to the reduced attachment and deposition of both cells and platelets. Protein in the blood
Figure 7. SEM micrographs showing platelet adhesion on the surfaces of PCL (A), PCL/PLGA (B), PCL/GEL (C), and PCL/BCP (D) electrospun membranes after 30 min of incubation in PRP (-platelets).
498
A.R. Padalhin et al.
can also be adsorbed on calcium phosphate surfaces to some extent as suggested by previous studies [32–34] which could then result to platelet adhesion but to a lesser degree compared to bare polymer surfaces.
Downloaded by [Florida International University] at 11:08 28 December 2014
3.4. In vivo bone regeneration Figure 8 shows the reconstructed 3D images of skull sections implanted with the different membrane samples. Within a month (Figure 8(D1)), PCL/BCP electrospun membrane showed increased regeneration along the margin of the 5 mm diameter defect. This becomes more pronounced two months after implantation, with PCL/BCP (Figure 8(D2)) achieving greater bone regeneration compared with PCL, PCL/PLGA, and PCL/GEL (Figure 8(A2–C2)). 3D analysis of the micro CT data (Figure 9) also confirms a significantly higher percent bone volume when PCL/BCP was implanted for both observation periods. 3D analysis of the micro CT data supports the 3D reconstructed models of the rat calvarias. Little significant difference was observed between all the optimized membranes one month after implantation, with PCL/BCP having the highest reading of percent bone volume (6.12 ± 1.95 mm3). Calculated 3D analysis indicates that bone volume and percent bone volume are significantly higher (p < 0.001) in PCL/BCP (12.12 ± 1.75 mm3) than in PCL (6.85 ± 0.23 mm3), and slightly higher than the PCL/PLGA (9.27 ± 0.85 mm3) and PCL/GEL (9.63 ± 0.45 mm3) membranes, one and two months after implantation. Bone regeneration on the defect sites was visualized using tissue sections stained with hematoxylin and eosin (H and E) and Masson’s trichrome. Figure 10 shows the H and E stained tissue sections one month post implantation. While fibrous tissue formation is prevalent on all implanted samples, it is more pronounced in the PCL and PCL/PLGA samples (Figure 10(A–B)). All tissue sections also show bone regeneration along the margin of the defect. Tissue sections of samples extracted two months after implantation (Figure 11) show markedly improved bone regeneration along the margin of the defects; however, fibrous tissue is persistent within the majority of the implanted
Figure 8. Three-dimensional reconstruction of micro CT data of rat calvaria implanted with PCL (A), PCL/PLGA (B), PCL/GEL (C), and PCL/BCP (D) electrospun membranes showing regeneration after 1 (A1–D1) and 2 (A2–D2) months.
Downloaded by [Florida International University] at 11:08 28 December 2014
Journal of Biomaterials Science, Polymer Edition
499
Figure 9. Calculated tissue volume, bone volume and corresponding percent bone volume indicate significant difference (p > 0.001) in bone tissue formation after two months.
samples. Defects implanted with PCL/GEL membranes (Figure 11(C)) revealed more extensive vascularization among all the other implanted samples. Although defects implanted with PCL/BCP and PCL/PLGA membranes (Figure 11(B) and (D)) still possessed extensive fibrous tissue, these implants also showed dense collagen deposition (Figure 11(B1) and (D1)) distant from that of the defect margin. Masson’s trichome staining was also applied on a tissue section to further characterize tissue formation on implanted membranes. The majority of the samples did have blue stained fibrous collagen (Figure 11(A1–D1)). However, compared to other samples, PCL/PLGA and PCL/BCP showed deposition of dense collagen. Also, compared to PCL/PLGA, PCL/BCP contained larger amounts of dense collagen (stained blue), which is also identified to be new bone, and dense collagen with red pigmentation (Figure 11(D1)). Bone tissue repair is initiated through the migration and differentiation of stem cells and other surrounding tissue.[30,31,42,43] Deposition of dense collagen during callus formation in defect areas is carried out by osteoblast cells, thus scaffolds intended for bone tissue regeneration should primarily be osteoconductive, in addition to being osteoinductive, and be capable of osteointegration.[13,30,31] Interestingly, even though PCL yielded the highest blood cell deposition and relatively lower hemolysis, it simply promoted persistence of fibrous tissue up to two months after implantation. Development of a stable hematoma is crucial for healing in bone fractures. However, prolonged presence of inflammatory agents was found to negatively affect bone regeneration.[25,44] Although not fully observed in the current research, this condition could be associated with the PCL membrane’s susceptibility for activating platelets and contributing to the coagulation cascade, forming a highly stable hematoma. Further study is recommended to investigate this possibility. Although extensive vascularization was prevalent within the defect area, this did not contribute significantly to the overall regeneration of bone tissue on the 5 mm defects when PCL/GEL membranes were used. PCL/BCP still generated significantly higher regeneration among all optimized membranes, possibly due to its potential for reduced blood cell adhesion and osteoconductivity. The combination of BCP and PCL polymer combines the appropriate characteristics of materials for bone tissue regeneration. Hyrophobic polymers have been found to have an affinity for protein adsorption, while calcium phosphate derived powders have long been identified as
A.R. Padalhin et al.
Downloaded by [Florida International University] at 11:08 28 December 2014
500
Figure 10. Tissue sections of one month implant samples of PCL (A), PCL/PLGA (B), PCL/ GEL (C), and PCL/BCP (D) electrospun membranes stained with hematoxilyn and eosin 5 mm rat calvaria defect showing mostly fibrous tissue growth for the majority of the samples and initial bone regeneration along the defect margin.
competent materials for bone healing due to their biodegradation, osteoconductivity, and potentially osteoinductive properties under in vivo conditions. PCL has already been proven to be a suitable material for tissue engineering, while combining it with other polymers further enhances its functionality.[45,46] The addition of BCP, or other calcium phosphate derived components, significantly improves the bone tissue regenerating capability of electrospun PCL.
Downloaded by [Florida International University] at 11:08 28 December 2014
Journal of Biomaterials Science, Polymer Edition
501
Figure 11. Micrographs of tissue sections of implanted samples of PCL (A, A1, A2), PCL/ PLGA (B, B1, B2), PCL/GEL (C, C1, C2), and PCL/BCP (D, D1, D2) electrospun membranes stained with hematoxilyn and eosin, and Masson’s trichome, two months after implantation in critical sized rat calvaria defect (blood vessel – , collagen – ♦, new bone – ).
▴
4. Conclusion This study has successfully compared the bone regeneration capability of several optimized binary formulations of PCL. Even though all membranes show competitive results regarding in vitro cytocompatibility and hemocompatibility, the incorporation of a calcium phosphate containing compound in the composite PCL/BCP membrane has shown that this is the most effective material for bone regeneration based on the results of the in vivo implantation. This reveals that although all of the membranes were fabricated using optimized formulations and conditions, the incorporation of an osteoconductive component resulting in a composite material was more conducive for bone tissue growth. Further studies are suggested to investigate the possible enhancement of the PCL/BCP for bone tissue engineering. Acknowledgments This work was supported by Mid-career Research Program through NFR grant funded by the MEST (NO 2009-0092808), Republic of Korea and partially supported by Soonchunhyang University Research Fund. The author would also like to thank Rose Ann Franco and Thi-Heip Nguyen for their help in the sample preparation and Kim Shin Woo and Kim Hyoung Suk for facilitating the in vivo experiments.
References [1] Salgado AJ, Coutinho OP, Reis RL. Bone tissue engineering: state of the art and future trends. Macromol. Biosci. 2004;4:743–765.
Downloaded by [Florida International University] at 11:08 28 December 2014
502
A.R. Padalhin et al.
[2] Schroeder JE, Mosheiff R. Tissue engineering approaches for bone repair: concepts and evidence. Injury. 2011;42:609–613. [3] Tian H, Tang Z, Zhuang X, Chen X. Biodegradable synthetic polymers: preparation, functionalization and biomedical application. Prog. Polym. Sci. 2012;37:237–280. [4] Zong C, Xue D, Yuan W, Wang W, Shen D, Tong X, Shi D, Liu L, Zheng Q, Gao C, Wang J. Reconstruction of rat calvarial defects with human mesenchymal stem cells and osteoblastlike cells in poly-lactic-co-glycolic acid scaffolds. Eur Cell Mater. 2010;20:109–120. [5] Woodruff MA, Hutmacher DW. The return of a forgotten polymer – polycaprolactone in the 21st century. Prog. Polym. Sci. 2010;35:1217–1256. [6] Roohani-Esfahani SI, Nouri-Khorasani S, Lu Z, Appleyard R, Zreiqat H. The influence hydroxyapatite nanoparticle shape and size on the properties of biphasic calcium phosphate scaffolds coated with hydroxyapatite–PCL composites. Biomaterials. 2010;31:5498–5509. [7] Franco RA, Nguyen TH, Lee BT. Preparation and characterization of electrospun PCL/ PLGA membranes and chitosan/gelatin hydrogels for skin bioengineering applications. J. Mater. Sci. Mater. Med. 2011;22:2207–2218. [8] Hiep NT, Lee BT. Electro-spinning of PLGA/PCL blends for tissue engineering and their biocompatibility. J. Mater. Sci. Mater. Med. 2010;21:1969–1978. [9] Chong EJ, Phan TT, Lim IJ, Zhang YZ, Bay BH, Ramakrishna S, Lim CT. Evaluation of electrospun PCL/gelatin nanofibrous scaffold for wound healing and layered dermal reconstitution. Acta Biomater. 2007;3:321–330. [10] Zhao P, Jiang H, Pan H, Zhu K, Chen W. Biodegradable fibrous scaffolds composed of gelatin coated poly(ε-caprolactone) prepared by coaxial electrospinning. J. Biomed. Mater. Res. A. 2007;83A:372–382. [11] Ba Linh NT, Min YK, Lee BT. Hybrid hydroxyapatite nanoparticles-loaded PCL/GE blend fibers for bone tissue engineering. J. Biomater. Sci., Polym. Ed. 2013;24:520–538. [12] Nguyen TP, Lee BT. Fabrication and characterization of BCP nano particle loaded PCL fiber and their biocompatibility. Korean J. Mater. Res. 2010;20:392–400. [13] Albrektsson T, Johansson C. Osteoinduction, osteoconduction and osseointegration. Eur Spine J. 2001;10:S96–101. [14] Motlagh D, Allen J, Hoshi R, Yang J, Lui K, Ameer G. Hemocompatibility evaluation of poly(diol citrate) in vitro for vascular tissue engineering. J. Biomed. Mater. Res. A. 2007;82A:907–916. [15] Chenglong L, Dazhi Y, Guoqiang L, Min Q. Corrosion resistance and hemocompatibility of multilayered Ti/TiN-coated surgical AISI 316L stainless steel. Mater. Lett. 2005;59:3813–3819. [16] Zhao ML, Li DJ, Yuan L, Yue YC, Liu H, Sun X. Differences in cytocompatibility and hemocompatibility between carbon nanotubes and nitrogen-doped carbon nanotubes. Carbon. 2011;49:3125–3133. [17] Li J, Zheng W, Zheng Y, Lou X. Cell responses and hemocompatibility of g-HA/PLA composites. Sci. China Life Sci. 2011;54:366–371. [18] Lee BT, Youn MH, Paul RK, Lee KH, Song HY. In situ synthesis of spherical BCP nanopowders by microwave assisted process. Mater. Chem. Phys. 2007;104:249–253. [19] Maekawa Y, Yagi K, Nonomura A, Kuraoku R, Nishiura E, Uchibori E, Takeuchi K. A tetrazolium-based colorimetric assay for metabolic activity of stored blood platelets. Thromb. Res. 2003;109:307–314. [20] Gómez-Guillén MC, Giménez B, López-Caballero ME, Montero MP. Functional and bioactive properties of collagen and gelatin from alternative sources: a review. Food Hydrocoll. 2011;25:1813–1827. [21] Wilson CJ, Clegg RE, Leavesley DI, Pearcy MJ. Mediation of biomaterial–cell interactions by adsorbed proteins: a review. Tissue Eng. 2005;11:1–18. [22] Garrido CA, Lobo SE, Turibio FM, LeGeros RZ. Biphasic calcium phosphate bioceramics for orthopaedic reconstructions: clinical outcomes. Int. J. Biomater. 2011;2011:129727. [23] Anselme K. Osteoblast adhesion on biomaterials. Biomaterials. 2000;21:667–681. [24] Ozaki A, Tsunoda M, Kinoshita S, Saura R. Role of fracture hematoma and periosteum during fracture healing in rats: interaction of fracture hematoma and the periosteum in the initial step of the healing process. J. Orthop. Sci. 2000;5:64–70.
Downloaded by [Florida International University] at 11:08 28 December 2014
Journal of Biomaterials Science, Polymer Edition
503
[25] Kolar P, Schmidt-Bleek K, Schell H, Gaber T, Toben D, Schmidmaier G, Perka C, Buttgereit F, Duda GN. The early fracture hematoma and its potential role in fracture healing. Tissue Eng. Part B: Rev. 2010;16:427–434. [26] Arpornmaeklong P, Kochel M, Depprich R, Kübler NR, Würzler KK. Influence of plateletrich plasma (PRP) on osteogenic differentiation of rat bone marrow stromal cells. An in vitro study. Int. J. Oral Maxillofac. Surg. 2004;33:60–70. [27] Dominiak M, Łysiak-Drwal K, Solski L, Żywicka B, Rybak Z, Gedrange T. Evaluation of healing processes of intraosseous defects with and without guided bone regeneration and platelet rich plasma. An animal study. Ann. Anat. 2012;194:549–555. [28] Jeong Park YJ, Moo Lee YM, Nae Park SN, Yoon Sheen SY, Pyoung Chung CP, Lee SJ. Platelet derived growth factor releasing chitosan sponge for periodontal bone regeneration. Biomaterials. 2000;21:153–159. [29] Marsell R, Einhorn TA. The biology of fracture healing. Injury. 2011;42:551–555. [30] McKibbin B. The Biology of fracture healing in long bones. J. Bone Joint Surg. 1978;60:150–162. [31] Wang K, Zhou C, Hong Y, Zhang X. A review of protein adsorption on bioceramics. Interface focus. 2012;2:259–277. [32] Wang J, Zhang H, Zhu X, Fan H, Fan Y, Zhang X. Dynamic competitive adsorption of bone-related proteins on calcium phosphate ceramic particles with different phase composition and microstructure. J. Biomed. Mater. Res. B: Appl. Biomater. 2013;101B:1069–1077. [33] Zhu XD, Zhang HJ, Fan HS, Li W, Zhang XD. Effect of phase composition and microstructure of calcium phosphate ceramic particles on protein adsorption. Acta Biomater. 2010;6:1536–1541. [34] Chen H, Yuan L, Song W, Wu Z, Li D. Biocompatible polymer materials: role of protein– surface interactions. Prog. Polym. Sci. 2008;33:1059–1087. [35] Comelles J, Estévez M, Martínez E, Samitier J. The role of surface energy of technical polymers in serum protein adsorption and MG-63 cells adhesion. Nanomedicine. 2010;6: 44–51. [36] Marsh RJ, Jones RAL, Sferrazza M. Adsorption and displacement of a globular protein on hydrophilic and hydrophobic surfaces. Colloids Surf., B. 2002;23:31–42. [37] Liu P, Chen Q, Yuan B, Chen M, Wu S, Lin S, Shen J. Facile surface modification of silicone rubber with zwitterionic polymers for improving blood compatibility. Mater. Sci. Eng., C. 2013;33:3865–3874. [38] Li Q, Wang Z, Zhang S, Zheng W, Zhao Q, Zhang J, Wang L, Wang S, Kong D. Functionalization of the surface of electrospun poly(epsilon-caprolactone) mats using zwitterionic poly(carboxybetaine methacrylate) and cell-specific peptide for endothelial progenitor cells capture. Mater. Sci. Eng., C. 2013;33:1646–1653. [39] Zhu A, Lu P, Wu H. Immobilization of poly(ɛ-caprolactone)–poly(ethylene oxide)–poly (ɛ-caprolactone) triblock copolymer on poly(lactide-co-glycolide) surface and dual biofunctional effects. Appl. Surf. Sci. 2007;253:3247–3253. [40] Lee JH, Lee HB. Platelet adhesion onto wettability gradient surfaces in the absence and presence of plasma proteins. J. Biomed. Mater. Res. 1998;41:304–311. [41] Doblaré M, Garcı́a JM, Gómez MJ. Modelling bone tissue fracture and healing: a review. Eng. Fract. Mech. 2004;71:1809–1840. [42] Schmidt-Bleek K, Schell H, Schulz N, Hoff P, Perka C, Buttgereit F, Volk HD, Lienau J, Duda GN. Inflammatory phase of bone healing initiates the regenerative healing cascade. Cell Tissue Res. 2012;347:567–573. [43] Opal SM. Phylogenetic and functional relationships between coagulation and the innate immune response. Crit. Care Med. 2000;28:S77–S80. [44] Baker SC, Rohman G, Southgate J, Cameron NR. The relationship between the mechanical properties and cell behaviour on PLGA and PCL scaffolds for bladder tissue engineering. Biomaterials. 2009;30:1321–1328. [45] Ghasemi-Mobarakeh L, Prabhakaran MP, Morshed M, Nasr-Esfahani MH, Ramakrishna S. Bio-functionalized PCL nanofibrous scaffolds for nerve tissue engineering. Mater. Sci. Eng., C. 2010;30:1129–1136. [46] Sousa I, Mendes A, Bártolo PJ. PCL scaffolds with collagen bioactivator for applications in tissue engineering. Procedia Eng. 2013;59:279–284.
