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β-tricalcium phosphate scaffold for effective bone tissue engineering
Accepted Manuscript Title: Raloxifene microsphere-embedded collagen/chitosan/-tricalcium phosphate scaffold for effective bone tissue engineering Author: Ming-Lei Zhang Ji Cheng Ye-Chen Xiao Ruo-Feng Yin Xu Feng PII: DOI: Reference:
S0378-5173(16)31173-5 http://dx.doi.org/doi:10.1016/j.ijpharm.2016.12.031 IJP 16302
To appear in:
International Journal of Pharmaceutics
Received date: Revised date: Accepted date:
20-9-2016 7-12-2016 13-12-2016
Please cite this article as: Zhang, Ming-Lei, Cheng, Ji, Xiao, Ye-Chen, Yin, RuoFeng, Feng, Xu, Raloxifene microsphere-embedded collagen/chitosan/-tricalcium phosphate scaffold for effective bone tissue engineering.International Journal of Pharmaceutics http://dx.doi.org/10.1016/j.ijpharm.2016.12.031 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Raloxifene microsphere-embedded collagen/chitosan/β-tricalcium phosphate scaffold for effective bone tissue engineering
Ming-Lei Zhang1, Ji Cheng2, Ye-Chen Xiao3, Ruo-Feng Yin1*,Xu Feng4 1
Depatrment of Orthopaedics, China-Japan Union Hospital，Jilin University, China Depatrment Obstetrics and Gynecology, China-Japan Union Hospital, Jilin University, China 3 Department of Biochemistry and Molecular Biology, College of Basic Medical Science, Jilin University, China 2
Corresponding author I: Ruo-Feng Yin, Depatrment of Orthopaedics, China-Japan Union Hospital， Jilin University, 126 Xian-Tai Street，ChangChun, JiLin Province P.R.China Tel&Fax:0086-0431-89876909 Email:[email protected] Corresponding author II: Xu Feng, Department of Spine Surgery, 1st Hospital of Jilin University No. 71 Xinmin St, Changchun P.R.China Tel&Fax: 0086-0431-88782222 Email:[email protected]
Abstract Engineering novel scaffolds that can mimic the functional extracellular matrix (ECM) would be a great achievement in bone tissue engineering. This paper reports the fabrication of novel collagen/chitosan/β-tricalcium phosphate (CCTP) based tissue engineering scaffold. In order to improve the regeneration ability of scaffold, we have embedded raloxifene (RLX)-loaded PLGA microsphere in the CCTP scaffold. The average pore of scaffold was in the range of 150-200 µm with ideal mechanical strength and swelling/degradation characteristics. The release rate of RLX from the microsphere (MS) embedded scaffold was gradual and controlled. Also a significantly enhanced cell proliferation was observed in RLX-MS exposed cell group suggesting that microsphere/scaffold could be an ideal biomaterial for bone tissue engineering. Specifically, RLX-MS showed a significantly higher Alizarin red staining indicating the higher mineralization capacity of this group. Furthermore, a high alkaline phosphatase (ALP) activity for RLX-MS exposed group after 15 days incubation indicates the bone regeneration capacity of MC3T3-E1 cells. Overall, present study showed that RLX-loaded microsphere embedded scaffold has the promising potential for bone tissue engineering applications.
Keywords Bone tissue engineering, raloxifene, scaffold, chitosan, and collagen
Introduction Regeneration of bone disorders which originated from malfunctions, oncologic process, infection or ageing is one of the most challenging task in the clinical practice (Poumarat et al., 1993). Although autologous bone grafting material is available in the market, yet its compatibility with done site and immune reaction limits its application in clinical segment (Walsh et al., 2003). In this regard, bone healing without any complications and side effects would be the most effective strategy. Recently, bone tissue engineering (BTE) has gained increasing attention from researchers across the globe owing to its excellent compatibility and access (Santo et al., 2003). The BTE avoids several disadvantages of traditional treatments such as chronic inflammation, complicated surgical processes and immune rejection. The principle of BTE lies in the combination of knowledge of cells, engineering, and biochemical factors to effectively regenerate the damaged bone tissues. A tissue engineering scaffold is the one which supports the extracellular matrix (ECM) and serves as a place for the proliferation and differentiation of bone cells and tissues (Petite et al., 2000). An ideal scaffold for BTE would possess similar chemical composition of natural bone with additional features such as surface chemistry, good mechanical strength, biocompatibility and bioactivity. In these conditions, the new cells could perform the biological functions of bone (Sun et al., 2013). Therefore, materials intended for scaffold preparation should be biocompatible, biodegradable and non-toxic in nature which could degrade into non-toxic entity. In general, materials originated from biomacromolecules and inorganic constituents could mimic natural ECM and could provide sufficient mechanical strength which will allow the favorable environment for the cell proliferation and tissue regeneration. It is well known that the natural collagen consists of bone minerals contain both organic and inorganic constituents (Yang et al., 2014). Hydroxyapatite (HAP) is one of the most preferred
materials for BTE owing to its chemical similarity with the natural bone and offers excellent biodegradability and biocompatibility (Ruhe et al., 2006; Link et al., 2009). To further increase the osteogenic properties, HaP can be mixed with calcium phosphate (β tricalcium phosphates [TCP]). Additionally, chitosan has been reported as one of the most commonly used scaffolding materials due to its excellent in vivo properties such as non-toxicity, biocompatibility, biodegradability, cell adhesion, and cell proliferation characteristics (Zugravu et al., 2013). Moreover, chitosan is structurally similar to that of glycosaminoglycan’s (GAG) which forms the ECM of body tissues. Many scaffolds have employed chitosan as one of the important material for tissue regeneration (Di Martino et al., 2005). Collagen has been employed owing to its unique features including biodegradability/biocompatibility and cellular affinity and it will provide much needed mechanical strength (Yu et al., 2013). It has been already reported that type I collagen-HaP based scaffold could be immediately degraded by the collagenases and thereby support the proliferation and differentiation of bone forming cells (Nagai et al., 2010; Bauer et al., 2014). Raloxifene (RLX) has been indicated in the treatment of osteoporosis and is a second generation selective estrogen receptor modulator (SERM) (Maximov et al., 2014). The systemic administration of RLX for prolonged period would create unnecessary side effects such as leg cramps, thromboembolism, and hot flushes. One way to control or reduce the associated side effect is to control the release of RLX for longer time period in a gradual manner (Elsheikh et al., 2012). In the present study, polymeric carrier was selected which can provide a constant release of drug while undergo biodegradation. Poly-(DL-lactide-co-glycolide) acid (PLGA), an FDA approved biodegradable and biocompatible polymer was selected in the present study to form the microspheres (Singh et al., 2006).
Based on the background information described so far, in the present study, we described the fabrication of a tricomponent system consists of chitosan, collagen, and β tricalcium phosphates to form a most effective tissue engineering scaffolds (Figure 1). The RLX-loaded PLGA microsphere was grafted on the tricomponent scaffold to provide a localized and controlled release of scaffold. The scaffolds were studied for the RLX release and its characterization. The composite scaffold was studied for its suitability for bone tissue engineering applications in terms of cell attachment, cell viability, mineral formation, ALP activity and alizarin red assay.
Materials and Methods Materials Nano-hydroxyapatite (HAp, particle size: 60 nm) was purchased from Nanjing Emperor Nano Material
Co.,
Ltd.,
China.
Poly(D,L-lactide-co-glycolide)
(PLGA,
Mw:
8000–
15000g/mol,75:25), Raloxifene, Collagens, Chitosan, phosphoric acid, calcium acetate were purchased from Sigma Aldrich, China. Preparation of Raloxifene-loaded PLGA microspheres The RLX-loaded PLGA microsphere was prepared by solvent evaporation technique. Briefly, 500 mg of PLGA was dissolved in 5 ml of dichloro methane (DCM) and to this 50 mg of RLX was added and homogenized for 3 min. Then 5 ml of 5% polyvinyl alcohol (PVA) was added and immediately vortexed for 10 min. The formed emulsion was immediately transferred to 75 ml of 0.5% PVA solution and stirred at constant rate for 30 min. After evaporation of DCM,
drug-loaded microspheres were collected and washed thoroughly with water. The microspheres were then lyophilized and stored in refrigerator. Fabrication of Collagen/Chitosan/β-Tricalcium phosphate scaffold At first, β-Tricalcium phosphate was synthesized as described earlier. Briefly, Phosphoric acid and calcium acetate monohydrate were used. 7 ml of phosphoric acid was dissolved in methanol and 25.6 mg of calcium acetate monohydrate was added to the above solution with vigorous stirring and left undisturbed for 10h. The formed precipitate was washed and sintered at 800°C for 5h. Next, biphasic calcium phosphate (BCP) was prepared by mixing hydroxyapatite (HaP) and βTricalcium phosphate in a weight ratio of 80:20. Now, chitosan and collagen (1%w/v) solution was prepared in 2% acetic acid. The chitosan and collagen was mixed with 2% of β-Tricalcium phosphate in a mechanical mixture for 3h. The solution was then crosslinked with 0.25% glutaraldehyde and sonicated for 45min. The final mixture was carefully transferred to a 6-well plate and kept in -80°C for 8h. The samples were then freeze dried in a freeze dryer and stored at 4°C. Fabrication of microsphere-embedded tissue engineering scaffold The suspension of drug-loaded microsphere was poured on a dried scaffold such that all parts of scaffold is covered and allowed to incubate for 4h. The scaffold was keot at -80°C and then freeze dried using a lyophilizer and stored at 4°C until further use.
Characterization of microspheres The mean particle size of microsphere was determined by Malvern Master Sizer Sirocco 2000 (Malvern Instrument Ltd., Worcestershire, UK). The microspheres were dissolve din water and vortexed and the size was measured. The surface morphology of microsphere was measured using scanning electron microscope (SEM) (LEO 1450 VP, Leo Electron microscopy Ltd., Cambridge, UK). The loading content of RLX in microsphere was determined using HPLC method. For this purpose, RLX-loaded microsphere (RLX-M) was dissolved in ethylacetate and sonicated for 10 min. The organic solution was then centrifuged at 14000 rpm for 15 min. The supernatant was collected and analyzed for amount of drug using HPLC technique. In vitro drug release The in vitro drug release was determined by as reported earlier. Briefly, specified amount of RLX-M and RLX-MS was dissolved in 10 ml of phosphate buffered saline (PBS, pH 7.4) and kept in a shaker maintained at 37°C and 100 rpm. At specified time points, entire sample was centrifuged at high speed and supernatant was collected. The buffer was replaced with an equal amount of fresh buffer. The amount of drug released in the release media was calculated from the HPLC method as mentioned above. Cell culture and biocompatibility assay The pre-osteoblast cells (MC3T3-E1) cells were obtained from Americal Type Culture Collection (ATCC, USA) and cells were grown the in the RPMI growth media supplemented with 10% of FBS and 1% of antibiotic mixture. The cells were maintained at ambient conditions of 37°C with 5% CO2. Prior to the assay, scaffolds were cut into thin slices and UV sterilized for 60 min. The scaffold was incubated in the respective culture for 12h. The cells with a seeding
density of 3×105 cells/well was seeded in the 6-well plate and allowed to attach for 24h. Next day, cells were exposed with free RLX, RLX-M and RLX-MS and incubated for predetermined time points (until 15 days). Control cells were parallelly grown. At each time point, cell viability was evaluated by means of MTT assay. To be brief, cells were exposed with MTT solution and incubated for 4h and then added with DMSO. The absorbance was then read by a microplate reader at 570 nm. Alizarin red staining assay Scaffolds were cut into thin slices and UV sterilized for 60 min. The scaffold was incubated in the respective culture for 12h. Next day, cells in a seeding density of 1×104 cells/well were seeded in a 6-well plate. The wells were respectively treated or exposed with RLX, RLX-M and RLX-MS and incubated for specified time point until 15 days. After each time point, cells were fixed with 4% paraformaldehyde and stained with Alizarin red S staining solution (pH 4.1) for 5 min at room temperature. After the staining protocol, cells were washed with distilled water and photographs were taken using an phase contrast microscope (Nikon ECLIPSE, TS 100, Japan). ALP activity assay ALP activity was evaluated to determine the osteogenic capacity of individual formulations. Scaffolds were cut into thin slices and UV sterilized for 60 min. The scaffold was incubated in the respective culture for 12h. Next day, cells in a seeding density of 1×104 cells/well were seeded in a 6-well plate. The wells were respectively treated or exposed with RLX, RLX-M and RLX-MS and incubated for specified time point until 15 days. ALP assay kit (Sigma-Aldrich, USA) was used. Briefly, individual cell groups were washed carefully thrice with PBS and then 5 ml of PBS containing 20% v/v of nitrophenyl phosphate disodium salt (pNPP reagent) was
added and washed. The samples were incubated for 15 min and 100 µl of supernatant was added in 96-well plate and absorbance was read at 405 nm using a microplate reader. The ALP activity was determined in relation to the protein concentration. The protein concentration in the cell sample was determined by BCA bicinchoninic acid assay test. Statistical analysis All results were represented as mean±standard deviation (SD). Statistical analyses of the data were performed by SPSS Software version 11.5 (SPSS Inc., USA). Statistically significant difference was assigned at the level of p<0.05.
Results and Discussion Characterization of microsphere embedded tissue engineering scaffold The main function of scaffold is to act as a temporary ECM and it should exhibit the similar mechanical properties as that of original ECM. In the study, we have included HaP specifically to improve the mechanical hardness of the prepared scaffold for tissue engineering applications. The compressive stress of CCTP scaffold was observed to be around ~120 kPa. At the same time, swelling abilities of scaffold is important to maintain the 3-dimensional network which can adsorb nutrients and cell growth will be enhanced. The swelling of CCTP scaffold was ~65% indicating the idealness of the tricomponent material combinations. The weight loss of the scaffold was evaluated in order to ensure that ECM could replace the scaffold materials during regeneration. At the end of 15 days, only 45% of scaffold weight (wt) was remained indicating that all the materials are suitable for the scaffold formation. It has been reported that pore size of
fabricated scaffold plays an important role in the cell proliferation and cell migration. The interconnected pores allow the easy proliferation, differentiation, migration and flow of the essential nutrients. There is an ideal pore size as the small pore size will limit the follow the nutrients while a larger pore size eventually will decrease the surface area for cell growth within the scaffold mass. The interconnected pores will help supply the nutrients and at the same time will allow the removal of metabolic waste products. In the present study, CCTP scaffold has an average pore size of ~200 µm which is well-interconnected with the deeper pores and with the adjacent pores (Figure 1). Based on these parameters, it can be assumed that scaffold is suitable for the further applications. This scaffold was used to incorporate with the drug-loaded microspheres (Tan et al., 2011; Tan et al., 2009). PLGA microsphere was prepared to incorporate the RLX and present it in a controlled form. The mean particle size of PLGA microsphere was ~12 µm with a perfect spherical shape (Figure 2). The particles were smooth and spherical in nature. The loading of drug in the PLGA microsphere did not alter or change the shape of the particles. HPLC analysis showed an active drug loading (DL%) of ~11.5% with an encapsulation efficiency of ~93%. The SEM image of microsphere embedded scaffold that microspheres are uniformly distributed in the pores of the scaffold. The incorporation of microsphere in the scaffold did not affect the scaffold morphology neither the pore size. In vitro release of RLX from microsphere and scaffold system The in vitro RLX release from microsphere and microsphere embedded scaffold system was performed in phosphate buffered saline (pH 7.4) in order to simulate the physiological conditions (Figure 3). The results clearly showed a two different release pattern for RLX-M and RLX-MS.
First in case of RLX-M, no initial burst release of RLX was observed indicating that the entire drug was loaded in the core of the microspheres and not on the surface of the carrier. Results clearly showed that the drugs released in a sustained manner from both the system. To be specific, drug release was slower after the microsphere was loaded in the scaffold due to the increase in the path length of the drug from inner atmosphere to the outer release media. At the end of 96h, approximately, ~80% of drug released from RLX-M compared to that of only ~35% from scaffold system. Such a sustained release of drug would benefit the constant exposure of drug to the growing cells and resulting in the effective bone tissue engineering. Drug was expected to release in a multiple stages including (a) drug dissolution (b) lag phase (c) controlled release of drug. In case of scaffold, release of drug is associated with the degradation of scaffold along with the decrease in the polymeric mass. In vitro cell viability and response To evaluate the cell response to the RLX as well as RLX-M and RLX-MS (microsphere embedded scaffold), MTT assay was performed (Figure 4). First, a dose dependent effect was observed in all cases (free RLX, RLX-M, and RLX-MS). It was found that RLX at a dose of 0.1 µg was safe without much cell death whereas 10 µg was very toxic with almost more than 50% of cell death was observed in case of free RLX. The RLX-M and RLX-MS however did not show considerable effect even at highest concentration indicating that the slow release of drug would be beneficial. Most notable of all, RLX-MS showed the high cell viability throughout all the time points indicating the excellent growth conditions of microsphere embedded scaffold. Based on this result it can be expected that at low concentration (0.1µg) of RLX, cell growth will be higher. The results also indicate the good biocompatibility for the microsphere as well as scaffold materials.
In vitro mineralization assay: Alizarin red assay The mineralization study was performed by Alizarin Red staining. This method is one of the functional methods to determine the bone regeneration ability and mineralization capacity of cell in the in vitro study (Figure 5). After each designated study interval, cells were stained with Alizarin red S. As seen, less mineralization of scaffold was observed in RLX treated cells whereas remarkably higher mineralization was noted in RLX-M and RLX-MS indicating the superior cytocompatibility of the tested materials. This result suggests that RLX-MS scaffolds offer excellent biocompatibility for bone regeneration. Especially, higher staining and higher amounts of mineralized nodule-like formations were observed for the cells exposed with RLXMS. Furthermore, mineral content was determined to further evaluate the bone regeneration capacity of each sample. Consistent with the Alizarin red staining, RLX-MS showed the highest mineral deposition (Figure 6). It should be noted that during first 5 days, almost all groups have similar level of calcium deposition however at the end of 15 days; RLX-MS presented the maximum calcium deposition. The biocompatible nature of scaffold materials leads to the proliferation of cells that will lead to the development of finely scaled mineral deposits on the scaffold membrane. Such a mineralized scaffold can improve specific biological functions such as the adhesion, differentiation, and proliferation of pre-osteoblastic cells. Moreover, a slow and consistent release of RLX from microsphere embedded scaffold ensured the maximum mineral formation which is indicative the bone regeneration capacity (Ramirez et al., 1999; Ramtoola et al., 1992).
Alkaline phosphatase (ALP) activity analysis The bone regeneration ability of individual formulation was further confirmed by ALP activity analysis after respective days of incubation in the standard conditions (Figure 7). Results showed that blank scaffold did not have much effect on the ALP level at different time points. In contrast, cells cultivated with RLX-M and RLX-MS showed significantly higher ALP activity after day 10 and day 15. The results observed in ALP activity analysis was consistent with the results observed for Alizarin red staining and mineral deposition analysis. Conclusion In this study, we have reported the fabrication of novel collagen/chitosan/β-tricalcium phosphate (CCTP) based tissue engineering scaffold. In order to improve the regeneration ability of scaffold, we have embedded raloxifene (RLX)-loaded PLGA microsphere in the CCTP scaffold. The average pore of scaffold was in the range of 150-200 µm with ideal mechanical strength and swelling/degradation characteristics. The release rate of RLX from the microsphere embedded scaffold was gradual and controlled. Also a significantly enhanced cell proliferation was observed in RLX-MS exposed cell group suggesting that microsphere/scaffold could be an ideal biomaterial for bone tissue engineering. Specifically, RLX-MS showed a significantly higher Alizarin red staining indicating the higher mineralization capacity of this group. Furthermore, a high ALP activity for RLX-MS exposed group after 15 days incubation indicates the bone regeneration capacity of MC3T3-E1 cells. Overall, present study showed that RLX-loaded microsphere embedded scaffold has the promising potential for bone tissue engineering applications.
Acknowledgement The research work was supported from the research grant of Jilin University, China. Competing Interests The authors report no conflict of interest in this work. References Bauer, A., Liu, J., Windsor, J., Song, F., Li, B. 2014. Current Development of CollagenBased Biomaterials for Tissue Repair and Regeneration, Soft Materials 12, 359-370. Di Martino, A., Sittinger, M., Risbud, M. 2005. Chitosan: A versatile biopolymer for orthopaedic tissue-engineering. Biomaterials 26, 5983-5990. Elsheikh, M.A., Elnaggar, YS.R., Gohar, E.Y., Abdallah, O.Y. 2012. Nanoemulsion liquid preconcentrates for raloxifene hydrochloride: optimization and in vivo appraisal. Int. J. Nanomed. 7, 3787–3802. Link, D.P., van den Dolder, J., van den Beucken, J.J.J.P., Habraken, W., Soede, A., Boerman, O.C., Mikos, A.G., Jansen, J.A. 2009. Evaluation of an orthotopically implanted calcium phosphate cement containing gelatin microparticles. J. Biomed. Mater. Res. A 90, 372–379. Maximov, P., Lee, T., Jordan, V.C. 2013. The discovery and development of selective estrogen receptor modulators (SERMs) for clinical practice, Curr. Clin. Pharmacol. 8, 135–155. Nagai, N., Kumasaka, N., Kawashima, T., Kaji, H., Nishizawa, M., Abe, T. 2010. Preparation and characterization of collagen microspheres for sustained release of VEGF. J. Mat. Sci. Mat. Med. 21, 1891-1898.
Petite, H., Viateau, V., Bensaid, W., Meunier, A., De Pollak, C., Bourguignon, M., Oudina, K., Sedel, L., Guillemin, G. 2000. Tissue-engineered bone regeneration. Nat. Biotechnol. 18, 959– 963. Poumarat, G., Squire, P., 1993.Comparison of mechanical properties of human, bovine bone and a new processed bone xenograft. Biomaterials 14, 337–340. Ramirez, L., Pastoriza, P., Herrero-Vanrell, R. 1999. J. Microencapsul. 16, 105–115. Ramtoola, Z., Corrigan, O.I., Barrett, C.J. 1992. J. Microencapsul. 9, 415–423. Ruhé, P.Q., Hedberg-Dirk, E.L., Padron, N.T., Spauwen, P.H.M., Jansen, J.A., Mikos, A.G. 2006. Porous poly(dl-lactic-co-glycolic acid)/calcium phosphate cement composite for reconstruction of bone defects. Tissue Eng. 12, 789–800. Santo, V.E., Gomes, M.E., Mano, J.F., Reis, R.L. 2013. Controlled release strategies for bone, cartilage, and osteochondral engineering-part I: recapitulation of native tissue healing and variables for the design of delivery systems. Tissue Eng. B 19, 308–326. Singh, J., Pandit, S., Bramwell, V.W., Alpar, H.O. 2006. Diphtheria toxoid loaded poly(epsilon-caprolactone) nanoparticles as mucosal vaccine delivery systems. Methods 38, 96–105. Sun, X., Kang, Y., Bao, J., Zhang, Y., Yang, Y., Zhou, X. 2013. Modeling vascularized bone regeneration within a porous biodegradable CaP scaffold loaded with growth factors. Biomaterials 34, 4971–4981. Tan, H., Rubin, J.P., Marra, K.G. 2011. Direct synthesis of biodegradable polysaccharide derivative hydrogels through aqueous Diels–Alder chemistry. Macromol. Rapid. Commun. 32, 905–911.
Tan, H., Ramirez, C.M., Miljkovic, N. et al. 2009. Thermosensitive injectable hyaluronic acid hydrogel for adipose tissue engineering, Biomaterials 30, 6844–6853. Yang, M., Shuai, Y., Zhang, C., Chen, Y., Zhu, L., Mao, C., OuYang, H. 2014. Biomimetic nucleation of hydroxyapatite crystals mediated by Antheraea pernyi silk sericin promotes osteogenic differentiation of human bone marrow derived mesenchymal stem cells. Biomacromolecules 15, 1185–1193. Walsh, W.R., Chapman-Sheath, P.J., Cain, S., Debes, J., Bruce, W.J.M., Svehla, M.J., Gillies, R.M., 2003. A resorbable porous ceramic composite bone graft substitute in a rabbit metaphyseal defect model. J. Orthop. Res. 21, 655–661. Yu, X., Wang, L., Peng, F., Jiang, X., Xia, Z., Huang, J et al. 2013. The effect of fresh bone marrow cells on reconstruction of mouse calvarial defect combined with calvarial osteoprogenitor cells and collagen–apatite scaffold. J. Tissue Eng. Regen. Med. 7, 974–983. Zugravu, M.V., Smith, R.A., Reves, B.T., Jennings, J.A., Cooper, J.O., Haggard, W.O. et al. 2013. Physical properties and in vitro evaluation of collagen-chitosan calcium phosphate microparticle-based scaffolds for bone tissue regeneration. J. Biomat. Appl. 28, 566-579.
Figure captions Figure 1: Schematic presentation of microsphere embedded scaffold preparation. The RLX was loaded in the PLGA microsphere which was then embedded in the pores of CCTP scaffold Figure 2: SEM image of blank PLGA microsphere and RLX-loaded PLGA microsphere Figure 3: In vitro drug profile of RLS from RLX-M and RLX-MS. The release study was performed in phosphate buffered saline (pH 7.4). *p<0.05 and **p<0.01 Figure 4: Cell viability assay in MC3T3-E1 pre-osteoblast cells. The cells were exposed with RLX, RLX-M, and RLX-MS, respectively. The cell viability was determined by MTT assay Figure 5: Phase contrast microscope image of cells stained with Alizarin red after 15 days. The cells were exposed with RLX, RLX-M, and RLX-MS, respectively and incubated for 15 days. Figure 6: Calcium deposition analysis after 15 days. The cells were exposed with RLX, RLX-M, and RLX-MS, respectively and incubated for 15 days. **p<0.01 Figure 7: ALP activity analysis of cells exposed with RLX, RLX-M, and RLX-MS, respectively. The cells were then subjected with the ALP kit. **p<0.01
*Graphical Abstract (for review)
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S0378-5173(16)31173-5 http://dx.doi.org/doi:10.1016/j.ijpharm.2016.12.031 IJP 16302
To appear in:
International Journal of Pharmaceutics
Received date: Revised date: Accepted date:
20-9-2016 7-12-2016 13-12-2016
Please cite this article as: Zhang, Ming-Lei, Cheng, Ji, Xiao, Ye-Chen, Yin, RuoFeng, Feng, Xu, Raloxifene microsphere-embedded collagen/chitosan/-tricalcium phosphate scaffold for effective bone tissue engineering.International Journal of Pharmaceutics http://dx.doi.org/10.1016/j.ijpharm.2016.12.031 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Raloxifene microsphere-embedded collagen/chitosan/β-tricalcium phosphate scaffold for effective bone tissue engineering
Ming-Lei Zhang1, Ji Cheng2, Ye-Chen Xiao3, Ruo-Feng Yin1*,Xu Feng4 1
Depatrment of Orthopaedics, China-Japan Union Hospital，Jilin University, China Depatrment Obstetrics and Gynecology, China-Japan Union Hospital, Jilin University, China 3 Department of Biochemistry and Molecular Biology, College of Basic Medical Science, Jilin University, China 2
Corresponding author I: Ruo-Feng Yin, Depatrment of Orthopaedics, China-Japan Union Hospital， Jilin University, 126 Xian-Tai Street，ChangChun, JiLin Province P.R.China Tel&Fax:0086-0431-89876909 Email:[email protected] Corresponding author II: Xu Feng, Department of Spine Surgery, 1st Hospital of Jilin University No. 71 Xinmin St, Changchun P.R.China Tel&Fax: 0086-0431-88782222 Email:[email protected]
Abstract Engineering novel scaffolds that can mimic the functional extracellular matrix (ECM) would be a great achievement in bone tissue engineering. This paper reports the fabrication of novel collagen/chitosan/β-tricalcium phosphate (CCTP) based tissue engineering scaffold. In order to improve the regeneration ability of scaffold, we have embedded raloxifene (RLX)-loaded PLGA microsphere in the CCTP scaffold. The average pore of scaffold was in the range of 150-200 µm with ideal mechanical strength and swelling/degradation characteristics. The release rate of RLX from the microsphere (MS) embedded scaffold was gradual and controlled. Also a significantly enhanced cell proliferation was observed in RLX-MS exposed cell group suggesting that microsphere/scaffold could be an ideal biomaterial for bone tissue engineering. Specifically, RLX-MS showed a significantly higher Alizarin red staining indicating the higher mineralization capacity of this group. Furthermore, a high alkaline phosphatase (ALP) activity for RLX-MS exposed group after 15 days incubation indicates the bone regeneration capacity of MC3T3-E1 cells. Overall, present study showed that RLX-loaded microsphere embedded scaffold has the promising potential for bone tissue engineering applications.
Keywords Bone tissue engineering, raloxifene, scaffold, chitosan, and collagen
Introduction Regeneration of bone disorders which originated from malfunctions, oncologic process, infection or ageing is one of the most challenging task in the clinical practice (Poumarat et al., 1993). Although autologous bone grafting material is available in the market, yet its compatibility with done site and immune reaction limits its application in clinical segment (Walsh et al., 2003). In this regard, bone healing without any complications and side effects would be the most effective strategy. Recently, bone tissue engineering (BTE) has gained increasing attention from researchers across the globe owing to its excellent compatibility and access (Santo et al., 2003). The BTE avoids several disadvantages of traditional treatments such as chronic inflammation, complicated surgical processes and immune rejection. The principle of BTE lies in the combination of knowledge of cells, engineering, and biochemical factors to effectively regenerate the damaged bone tissues. A tissue engineering scaffold is the one which supports the extracellular matrix (ECM) and serves as a place for the proliferation and differentiation of bone cells and tissues (Petite et al., 2000). An ideal scaffold for BTE would possess similar chemical composition of natural bone with additional features such as surface chemistry, good mechanical strength, biocompatibility and bioactivity. In these conditions, the new cells could perform the biological functions of bone (Sun et al., 2013). Therefore, materials intended for scaffold preparation should be biocompatible, biodegradable and non-toxic in nature which could degrade into non-toxic entity. In general, materials originated from biomacromolecules and inorganic constituents could mimic natural ECM and could provide sufficient mechanical strength which will allow the favorable environment for the cell proliferation and tissue regeneration. It is well known that the natural collagen consists of bone minerals contain both organic and inorganic constituents (Yang et al., 2014). Hydroxyapatite (HAP) is one of the most preferred
materials for BTE owing to its chemical similarity with the natural bone and offers excellent biodegradability and biocompatibility (Ruhe et al., 2006; Link et al., 2009). To further increase the osteogenic properties, HaP can be mixed with calcium phosphate (β tricalcium phosphates [TCP]). Additionally, chitosan has been reported as one of the most commonly used scaffolding materials due to its excellent in vivo properties such as non-toxicity, biocompatibility, biodegradability, cell adhesion, and cell proliferation characteristics (Zugravu et al., 2013). Moreover, chitosan is structurally similar to that of glycosaminoglycan’s (GAG) which forms the ECM of body tissues. Many scaffolds have employed chitosan as one of the important material for tissue regeneration (Di Martino et al., 2005). Collagen has been employed owing to its unique features including biodegradability/biocompatibility and cellular affinity and it will provide much needed mechanical strength (Yu et al., 2013). It has been already reported that type I collagen-HaP based scaffold could be immediately degraded by the collagenases and thereby support the proliferation and differentiation of bone forming cells (Nagai et al., 2010; Bauer et al., 2014). Raloxifene (RLX) has been indicated in the treatment of osteoporosis and is a second generation selective estrogen receptor modulator (SERM) (Maximov et al., 2014). The systemic administration of RLX for prolonged period would create unnecessary side effects such as leg cramps, thromboembolism, and hot flushes. One way to control or reduce the associated side effect is to control the release of RLX for longer time period in a gradual manner (Elsheikh et al., 2012). In the present study, polymeric carrier was selected which can provide a constant release of drug while undergo biodegradation. Poly-(DL-lactide-co-glycolide) acid (PLGA), an FDA approved biodegradable and biocompatible polymer was selected in the present study to form the microspheres (Singh et al., 2006).
Based on the background information described so far, in the present study, we described the fabrication of a tricomponent system consists of chitosan, collagen, and β tricalcium phosphates to form a most effective tissue engineering scaffolds (Figure 1). The RLX-loaded PLGA microsphere was grafted on the tricomponent scaffold to provide a localized and controlled release of scaffold. The scaffolds were studied for the RLX release and its characterization. The composite scaffold was studied for its suitability for bone tissue engineering applications in terms of cell attachment, cell viability, mineral formation, ALP activity and alizarin red assay.
Materials and Methods Materials Nano-hydroxyapatite (HAp, particle size: 60 nm) was purchased from Nanjing Emperor Nano Material
Co.,
Ltd.,
China.
Poly(D,L-lactide-co-glycolide)
(PLGA,
Mw:
8000–
15000g/mol,75:25), Raloxifene, Collagens, Chitosan, phosphoric acid, calcium acetate were purchased from Sigma Aldrich, China. Preparation of Raloxifene-loaded PLGA microspheres The RLX-loaded PLGA microsphere was prepared by solvent evaporation technique. Briefly, 500 mg of PLGA was dissolved in 5 ml of dichloro methane (DCM) and to this 50 mg of RLX was added and homogenized for 3 min. Then 5 ml of 5% polyvinyl alcohol (PVA) was added and immediately vortexed for 10 min. The formed emulsion was immediately transferred to 75 ml of 0.5% PVA solution and stirred at constant rate for 30 min. After evaporation of DCM,
drug-loaded microspheres were collected and washed thoroughly with water. The microspheres were then lyophilized and stored in refrigerator. Fabrication of Collagen/Chitosan/β-Tricalcium phosphate scaffold At first, β-Tricalcium phosphate was synthesized as described earlier. Briefly, Phosphoric acid and calcium acetate monohydrate were used. 7 ml of phosphoric acid was dissolved in methanol and 25.6 mg of calcium acetate monohydrate was added to the above solution with vigorous stirring and left undisturbed for 10h. The formed precipitate was washed and sintered at 800°C for 5h. Next, biphasic calcium phosphate (BCP) was prepared by mixing hydroxyapatite (HaP) and βTricalcium phosphate in a weight ratio of 80:20. Now, chitosan and collagen (1%w/v) solution was prepared in 2% acetic acid. The chitosan and collagen was mixed with 2% of β-Tricalcium phosphate in a mechanical mixture for 3h. The solution was then crosslinked with 0.25% glutaraldehyde and sonicated for 45min. The final mixture was carefully transferred to a 6-well plate and kept in -80°C for 8h. The samples were then freeze dried in a freeze dryer and stored at 4°C. Fabrication of microsphere-embedded tissue engineering scaffold The suspension of drug-loaded microsphere was poured on a dried scaffold such that all parts of scaffold is covered and allowed to incubate for 4h. The scaffold was keot at -80°C and then freeze dried using a lyophilizer and stored at 4°C until further use.
Characterization of microspheres The mean particle size of microsphere was determined by Malvern Master Sizer Sirocco 2000 (Malvern Instrument Ltd., Worcestershire, UK). The microspheres were dissolve din water and vortexed and the size was measured. The surface morphology of microsphere was measured using scanning electron microscope (SEM) (LEO 1450 VP, Leo Electron microscopy Ltd., Cambridge, UK). The loading content of RLX in microsphere was determined using HPLC method. For this purpose, RLX-loaded microsphere (RLX-M) was dissolved in ethylacetate and sonicated for 10 min. The organic solution was then centrifuged at 14000 rpm for 15 min. The supernatant was collected and analyzed for amount of drug using HPLC technique. In vitro drug release The in vitro drug release was determined by as reported earlier. Briefly, specified amount of RLX-M and RLX-MS was dissolved in 10 ml of phosphate buffered saline (PBS, pH 7.4) and kept in a shaker maintained at 37°C and 100 rpm. At specified time points, entire sample was centrifuged at high speed and supernatant was collected. The buffer was replaced with an equal amount of fresh buffer. The amount of drug released in the release media was calculated from the HPLC method as mentioned above. Cell culture and biocompatibility assay The pre-osteoblast cells (MC3T3-E1) cells were obtained from Americal Type Culture Collection (ATCC, USA) and cells were grown the in the RPMI growth media supplemented with 10% of FBS and 1% of antibiotic mixture. The cells were maintained at ambient conditions of 37°C with 5% CO2. Prior to the assay, scaffolds were cut into thin slices and UV sterilized for 60 min. The scaffold was incubated in the respective culture for 12h. The cells with a seeding
density of 3×105 cells/well was seeded in the 6-well plate and allowed to attach for 24h. Next day, cells were exposed with free RLX, RLX-M and RLX-MS and incubated for predetermined time points (until 15 days). Control cells were parallelly grown. At each time point, cell viability was evaluated by means of MTT assay. To be brief, cells were exposed with MTT solution and incubated for 4h and then added with DMSO. The absorbance was then read by a microplate reader at 570 nm. Alizarin red staining assay Scaffolds were cut into thin slices and UV sterilized for 60 min. The scaffold was incubated in the respective culture for 12h. Next day, cells in a seeding density of 1×104 cells/well were seeded in a 6-well plate. The wells were respectively treated or exposed with RLX, RLX-M and RLX-MS and incubated for specified time point until 15 days. After each time point, cells were fixed with 4% paraformaldehyde and stained with Alizarin red S staining solution (pH 4.1) for 5 min at room temperature. After the staining protocol, cells were washed with distilled water and photographs were taken using an phase contrast microscope (Nikon ECLIPSE, TS 100, Japan). ALP activity assay ALP activity was evaluated to determine the osteogenic capacity of individual formulations. Scaffolds were cut into thin slices and UV sterilized for 60 min. The scaffold was incubated in the respective culture for 12h. Next day, cells in a seeding density of 1×104 cells/well were seeded in a 6-well plate. The wells were respectively treated or exposed with RLX, RLX-M and RLX-MS and incubated for specified time point until 15 days. ALP assay kit (Sigma-Aldrich, USA) was used. Briefly, individual cell groups were washed carefully thrice with PBS and then 5 ml of PBS containing 20% v/v of nitrophenyl phosphate disodium salt (pNPP reagent) was
added and washed. The samples were incubated for 15 min and 100 µl of supernatant was added in 96-well plate and absorbance was read at 405 nm using a microplate reader. The ALP activity was determined in relation to the protein concentration. The protein concentration in the cell sample was determined by BCA bicinchoninic acid assay test. Statistical analysis All results were represented as mean±standard deviation (SD). Statistical analyses of the data were performed by SPSS Software version 11.5 (SPSS Inc., USA). Statistically significant difference was assigned at the level of p<0.05.
Results and Discussion Characterization of microsphere embedded tissue engineering scaffold The main function of scaffold is to act as a temporary ECM and it should exhibit the similar mechanical properties as that of original ECM. In the study, we have included HaP specifically to improve the mechanical hardness of the prepared scaffold for tissue engineering applications. The compressive stress of CCTP scaffold was observed to be around ~120 kPa. At the same time, swelling abilities of scaffold is important to maintain the 3-dimensional network which can adsorb nutrients and cell growth will be enhanced. The swelling of CCTP scaffold was ~65% indicating the idealness of the tricomponent material combinations. The weight loss of the scaffold was evaluated in order to ensure that ECM could replace the scaffold materials during regeneration. At the end of 15 days, only 45% of scaffold weight (wt) was remained indicating that all the materials are suitable for the scaffold formation. It has been reported that pore size of
fabricated scaffold plays an important role in the cell proliferation and cell migration. The interconnected pores allow the easy proliferation, differentiation, migration and flow of the essential nutrients. There is an ideal pore size as the small pore size will limit the follow the nutrients while a larger pore size eventually will decrease the surface area for cell growth within the scaffold mass. The interconnected pores will help supply the nutrients and at the same time will allow the removal of metabolic waste products. In the present study, CCTP scaffold has an average pore size of ~200 µm which is well-interconnected with the deeper pores and with the adjacent pores (Figure 1). Based on these parameters, it can be assumed that scaffold is suitable for the further applications. This scaffold was used to incorporate with the drug-loaded microspheres (Tan et al., 2011; Tan et al., 2009). PLGA microsphere was prepared to incorporate the RLX and present it in a controlled form. The mean particle size of PLGA microsphere was ~12 µm with a perfect spherical shape (Figure 2). The particles were smooth and spherical in nature. The loading of drug in the PLGA microsphere did not alter or change the shape of the particles. HPLC analysis showed an active drug loading (DL%) of ~11.5% with an encapsulation efficiency of ~93%. The SEM image of microsphere embedded scaffold that microspheres are uniformly distributed in the pores of the scaffold. The incorporation of microsphere in the scaffold did not affect the scaffold morphology neither the pore size. In vitro release of RLX from microsphere and scaffold system The in vitro RLX release from microsphere and microsphere embedded scaffold system was performed in phosphate buffered saline (pH 7.4) in order to simulate the physiological conditions (Figure 3). The results clearly showed a two different release pattern for RLX-M and RLX-MS.
First in case of RLX-M, no initial burst release of RLX was observed indicating that the entire drug was loaded in the core of the microspheres and not on the surface of the carrier. Results clearly showed that the drugs released in a sustained manner from both the system. To be specific, drug release was slower after the microsphere was loaded in the scaffold due to the increase in the path length of the drug from inner atmosphere to the outer release media. At the end of 96h, approximately, ~80% of drug released from RLX-M compared to that of only ~35% from scaffold system. Such a sustained release of drug would benefit the constant exposure of drug to the growing cells and resulting in the effective bone tissue engineering. Drug was expected to release in a multiple stages including (a) drug dissolution (b) lag phase (c) controlled release of drug. In case of scaffold, release of drug is associated with the degradation of scaffold along with the decrease in the polymeric mass. In vitro cell viability and response To evaluate the cell response to the RLX as well as RLX-M and RLX-MS (microsphere embedded scaffold), MTT assay was performed (Figure 4). First, a dose dependent effect was observed in all cases (free RLX, RLX-M, and RLX-MS). It was found that RLX at a dose of 0.1 µg was safe without much cell death whereas 10 µg was very toxic with almost more than 50% of cell death was observed in case of free RLX. The RLX-M and RLX-MS however did not show considerable effect even at highest concentration indicating that the slow release of drug would be beneficial. Most notable of all, RLX-MS showed the high cell viability throughout all the time points indicating the excellent growth conditions of microsphere embedded scaffold. Based on this result it can be expected that at low concentration (0.1µg) of RLX, cell growth will be higher. The results also indicate the good biocompatibility for the microsphere as well as scaffold materials.
In vitro mineralization assay: Alizarin red assay The mineralization study was performed by Alizarin Red staining. This method is one of the functional methods to determine the bone regeneration ability and mineralization capacity of cell in the in vitro study (Figure 5). After each designated study interval, cells were stained with Alizarin red S. As seen, less mineralization of scaffold was observed in RLX treated cells whereas remarkably higher mineralization was noted in RLX-M and RLX-MS indicating the superior cytocompatibility of the tested materials. This result suggests that RLX-MS scaffolds offer excellent biocompatibility for bone regeneration. Especially, higher staining and higher amounts of mineralized nodule-like formations were observed for the cells exposed with RLXMS. Furthermore, mineral content was determined to further evaluate the bone regeneration capacity of each sample. Consistent with the Alizarin red staining, RLX-MS showed the highest mineral deposition (Figure 6). It should be noted that during first 5 days, almost all groups have similar level of calcium deposition however at the end of 15 days; RLX-MS presented the maximum calcium deposition. The biocompatible nature of scaffold materials leads to the proliferation of cells that will lead to the development of finely scaled mineral deposits on the scaffold membrane. Such a mineralized scaffold can improve specific biological functions such as the adhesion, differentiation, and proliferation of pre-osteoblastic cells. Moreover, a slow and consistent release of RLX from microsphere embedded scaffold ensured the maximum mineral formation which is indicative the bone regeneration capacity (Ramirez et al., 1999; Ramtoola et al., 1992).
Alkaline phosphatase (ALP) activity analysis The bone regeneration ability of individual formulation was further confirmed by ALP activity analysis after respective days of incubation in the standard conditions (Figure 7). Results showed that blank scaffold did not have much effect on the ALP level at different time points. In contrast, cells cultivated with RLX-M and RLX-MS showed significantly higher ALP activity after day 10 and day 15. The results observed in ALP activity analysis was consistent with the results observed for Alizarin red staining and mineral deposition analysis. Conclusion In this study, we have reported the fabrication of novel collagen/chitosan/β-tricalcium phosphate (CCTP) based tissue engineering scaffold. In order to improve the regeneration ability of scaffold, we have embedded raloxifene (RLX)-loaded PLGA microsphere in the CCTP scaffold. The average pore of scaffold was in the range of 150-200 µm with ideal mechanical strength and swelling/degradation characteristics. The release rate of RLX from the microsphere embedded scaffold was gradual and controlled. Also a significantly enhanced cell proliferation was observed in RLX-MS exposed cell group suggesting that microsphere/scaffold could be an ideal biomaterial for bone tissue engineering. Specifically, RLX-MS showed a significantly higher Alizarin red staining indicating the higher mineralization capacity of this group. Furthermore, a high ALP activity for RLX-MS exposed group after 15 days incubation indicates the bone regeneration capacity of MC3T3-E1 cells. Overall, present study showed that RLX-loaded microsphere embedded scaffold has the promising potential for bone tissue engineering applications.
Acknowledgement The research work was supported from the research grant of Jilin University, China. Competing Interests The authors report no conflict of interest in this work. References Bauer, A., Liu, J., Windsor, J., Song, F., Li, B. 2014. Current Development of CollagenBased Biomaterials for Tissue Repair and Regeneration, Soft Materials 12, 359-370. Di Martino, A., Sittinger, M., Risbud, M. 2005. Chitosan: A versatile biopolymer for orthopaedic tissue-engineering. Biomaterials 26, 5983-5990. Elsheikh, M.A., Elnaggar, YS.R., Gohar, E.Y., Abdallah, O.Y. 2012. Nanoemulsion liquid preconcentrates for raloxifene hydrochloride: optimization and in vivo appraisal. Int. J. Nanomed. 7, 3787–3802. Link, D.P., van den Dolder, J., van den Beucken, J.J.J.P., Habraken, W., Soede, A., Boerman, O.C., Mikos, A.G., Jansen, J.A. 2009. Evaluation of an orthotopically implanted calcium phosphate cement containing gelatin microparticles. J. Biomed. Mater. Res. A 90, 372–379. Maximov, P., Lee, T., Jordan, V.C. 2013. The discovery and development of selective estrogen receptor modulators (SERMs) for clinical practice, Curr. Clin. Pharmacol. 8, 135–155. Nagai, N., Kumasaka, N., Kawashima, T., Kaji, H., Nishizawa, M., Abe, T. 2010. Preparation and characterization of collagen microspheres for sustained release of VEGF. J. Mat. Sci. Mat. Med. 21, 1891-1898.
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Figure captions Figure 1: Schematic presentation of microsphere embedded scaffold preparation. The RLX was loaded in the PLGA microsphere which was then embedded in the pores of CCTP scaffold Figure 2: SEM image of blank PLGA microsphere and RLX-loaded PLGA microsphere Figure 3: In vitro drug profile of RLS from RLX-M and RLX-MS. The release study was performed in phosphate buffered saline (pH 7.4). *p<0.05 and **p<0.01 Figure 4: Cell viability assay in MC3T3-E1 pre-osteoblast cells. The cells were exposed with RLX, RLX-M, and RLX-MS, respectively. The cell viability was determined by MTT assay Figure 5: Phase contrast microscope image of cells stained with Alizarin red after 15 days. The cells were exposed with RLX, RLX-M, and RLX-MS, respectively and incubated for 15 days. Figure 6: Calcium deposition analysis after 15 days. The cells were exposed with RLX, RLX-M, and RLX-MS, respectively and incubated for 15 days. **p<0.01 Figure 7: ALP activity analysis of cells exposed with RLX, RLX-M, and RLX-MS, respectively. The cells were then subjected with the ALP kit. **p<0.01
*Graphical Abstract (for review)
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