Guided bone regeneration with tripolyphosphate cross-linked asymmetric chitosan membrane.

JJOD-2353; No. of Pages 10 journal of dentistry xxx (2014) xxx–xxx

Available online at www.sciencedirect.com

ScienceDirect journal homepage: www.intl.elsevierhealth.com/journals/jden

Guided bone regeneration with tripolyphosphate cross-linked asymmetric chitosan membrane Shiqing Ma a, Zhen Chen a, Feng Qiao a, Yingchun Sun a, Xiaoping Yang b, Xuliang Deng c, Lian Cen d,e, Qing Cai b, Mingyao Wu a, Xu Zhang a,*, Ping Gao a,* a

School and Hospital of Stomatology, Tianjin Medical University, 12 Observatory Road, Tianjin 300070, PR China The Key Laboratory of Beijing City on Preparation and Processing of Novel Polymer, Beijing University of Chemical Technology, Beijing 100029, PR China c Department of Geriatric Dentistry, Peking University School and Hospital of Stomatology, Beijing 100081, PR China d National Tissue Engineering Center of China, No. 68, East Jiang Chuan Road, Shanghai 200241, PR China e School of Chemical Engineering, East China University of Science and Technology, No. 130, Mei Long Road, Shanghai 200237, PR China b

article info

abstract

Article history:

Objectives: The objective of this study was to prepare a novel asymmetric chitosan guided

Received 14 June 2014

bone regeneration (GBR) membrane, which is composed of a dense layer isolating the bone

Received in revised form

defect from the invasion of surrounding connective fibrous tissue and a loose layer which

26 July 2014

can improve cell adhesion and stabilize blood clots, thus guided bone regeneration.

Accepted 26 August 2014

Methods: The chitosan membrane was fabricated through liquid nitrogen quencher com-

Available online xxx

bined with lyophilization and cross-linked by sodium tripolyphosphate (TPP). The physical properties of asymmetric chitosan membrane were measured by scanning electron micro-

Keywords:

scope (SEM), Fourier-transform infrared (FTIR), x-ray diffraction (XRD) and tensile test

Guided bone regeneration

machine. MTT assay and Live/Dead cell staining for MC3T3-E1 osteoblasts cultured on

Asymmetric

the membrane were used to characterize the biocompatibility of the membrane. In animal

Chitosan membrane

experiments, full-thickness and critical sized skull defects were made to evaluate the effect

Sodium tripolyphosphate

of the membrane on bone regeneration. Results: The results of this study indicate that the asymmetric chitosan membrane can be built and cross-linked by TPP to enhance the tensile strength of the membrane. In vitro experiment showed that no significant numbers of dead cells were detected on the chitosan membrane, indicating that the membrane had good biocompatibility. In animal experiments, the chitosan membrane had faster new bone formation, showing the capability to enhance bone regeneration. Conclusions: The chitosan membrane prepared in this study has an asymmetric structure; its tensile strength, biodegradation and biocompatibility fulfil the requirements of guided bone regeneration. Therefore, the asymmetric chitosan membrane is a promising GBR membrane for bone regeneration. Clinical significance: Guided bone regeneration (GBR) is an effective method for healing bone defects caused by periodontitis and implantitis, in which GBR membrane is a key biomaterial. # 2014 Elsevier Ltd. All rights reserved.

* Corresponding authors. Tel.: +86 13 920376897; fax: +86 22 23332122. E-mail addresses: [email protected], [email protected] (X. Zhang), [email protected] (P. Gao). http://dx.doi.org/10.1016/j.jdent.2014.08.015 0300-5712/# 2014 Elsevier Ltd. All rights reserved.

Please cite this article in press as: Ma S, et al. Guided bone regeneration with tripolyphosphate cross-linked asymmetric chitosan membrane. Journal of Dentistry (2014), http://dx.doi.org/10.1016/j.jdent.2014.08.015

JJOD-2353; No. of Pages 10

2

journal of dentistry xxx (2014) xxx–xxx

1.

Introduction

Both periodontitis and peri-implantitis can cause inflammation around the soft tissue as well as progressive bone loss.1 The resulting bone defect surrounding tooth or implant can lead to tooth loss and implant failure.2–4 Recently, guided bone regeneration (GBR) using barrier membranes has been routinely employed in dealing with bone defects and accomplished considerable success in clinical practice.5 GBR membranes, as physical barriers, prevent the surrounding fibrous connective tissue from invading into bone defects and create a space for bone regeneration.6 Therefore, GBR technique is effective in halting bone destruction and promoting new bone formation.7 GBR membranes are made of various nondegradable and degradable materials.8 Expanded polytetrafluoroethylene (ePTFE), a typical nondegradable GBR membrane, has achieved good clinical results;9 however, a second surgical procedure is required to remove the membrane, which creates additional surgical trauma to patients and raises their treatment costs. So far, the GBR membranes made of degradable materials, such as poly-lactic acid (PLA), poly (DL-lactic-co-glycolic acid) (PLGA), collagen, and chitosan have been developed, which avoid the second surgical procedure due to their degradability.10–14 Among them, Bio-Gide1, a commercially available collagen membrane, has already been used in clinical practice and achieved excellent clinical effects. More recent studies have paid much attention to developing GBR membranes with an asymmetric structure including a dense layer and a loose layer.8,15,16 The dense layer of asymmetrical membranes can effectively isolate the bone defect from the invasion of surrounding connective fibrous tissue, while the loose layer can improve adhesion to bone and stabilize blood clots.15 The techniques for fabricating asymmetric membranes mainly include the phase inversion (usually combined with different drying techniques)8,16,17 and electrospinning.18 In general, the phase inversion can be accomplished through solvent vapourization and subsequent immersion precipitation (coagulation),8,16 and liquid nitrogen quencher.19 The difference in the rate of solvent vapourization between the surface and the bulk of polymer solution generates a rich polymer phase and a poor polymer phase, forming a heterogeneous structure which can be stabilized by coagulating solution.16,17 By contrast, the liquid nitrogen quencher can quickly achieve the effects mentioned above, and there is no coagulating solution remaining either. Thus, in this study, we chose the method of liquid nitrogen quencher to prepare asymmetric GBR membranes. Chitosan(poly (1,4-D-glucosamine)), one deacetylated derivative of chitin, is a cationic natural biopolymer, which is easily processed into nanoparticles, nanofibres, gels, scaffolds and membranes.20 Recently, some studies have focused on the membranes made of chitosan for bone tissue regeneration and skin tissue regeneration17,21 due to its biocompatibility, biodegradability, antibacterial ability, and non-toxicity.19,22–24 Up to now, some researchers have already developed asymmetric chitosan membranes for wound healing and guided periodontal tissue regeneration.16,17,25 However, to our knowledge, the effect of asymmetric chitosan membrane on

bone regeneration has not been investigated, lacking the relevant evidence from animal study. In order to fill this gap, we would investigate the effect of asymmetric chitosan membranes on bone regeneration through animal experiments. GBR membranes should be equipped with satisfactory mechanical properties to accomplish the functions of barrier action, space maintenance and clinical manageability.24 However, these requirements may not be fulfilled by using pure chitosan membrane due to its poor mechanical properties.26,27 In general, cross-linking agents such as glutaraldehyde, genipin and sodium tripolyphosphate (TPP) are used to improve the mechanical properties of chitosan membranes.19,28–30 Chitosan can be cross-linked with glutaraldehyde and genipin through the formation of covalent bonds. Glutaraldehyde is limited for the application on biomaterials because of its toxicity.19,31 Genipin is a biocrosslinker but somewhat expensive. In comparison, the anionic groups of TPP react with amino groups of chitosan, producing ionic cross-linking, which is the simplest and mildest one among chitosan cross-linking methods.32–34 The objective of this study was to develop asymmetric chitosan GBR membranes through liquid nitrogen quencher combined with lyophilization. The mechanical properties of the chitosan membranes were enhanced by TPP cross-linking. The hypothesis was that the asymmetric bioabsorbable chitosan membrane can guide bone regeneration. In this study, the morphology, tensile strength, porosity, biodegradation, nutrient permeability, and biocompatibility of the membranes were investigated. The rat skull defect model was established to examine the bone regeneration behaviour of the membrane.

2.

Materials and methods

2.1.

Materials

Chitosan (Mw, 70,000 and 87% deacetylated), ice acetic acid (Mw, 60.05) and ethylenediaminetetraacetic acid (EDTA Mw, 292.24) were purchased from Life Science Products& Services (Shanghai, China). Sodium tripolyphosphate (TPP Mw, 367.86), MTT (3-[4,5-dimethyl-thiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) and Live/Dead cell double staining kit were purchased from Sigma (St. Louis, MO, USA). Dulbecoco’s modified Eagle medium (DMEM), horse serum, penicillin, and streptomycin were all purchased from HyClone (USA). BioGide1 (collagen typesI/III bilayer membrane), commercialized GBR membrane, was purchased from Geistlich Pharma AG (Switzerland). Distilled water (ultrapure grade, 0.05), suggesting that the chitosan membranes possess a good biocompatibility. The osteoblasts stained by AO/EB on the chitosan membrane surfaces were

Fig. 2 – ATR–FTIR spectra of noncross-linked (a) and crosslinked (b) chitosan membrane.

characterized with LSCM (Fig. 8). The representative images of LSCM shows that after 24 h culturing, the osteoblasts on the chitosan membranes maintained sound cell morphology and no significant number of dead cells (red staining) were detected. These results indicate that the osteoblasts on the chitosan membranes presented satisfactory cell viability and the membranes have good cytocompatibility.

3.3.

Bone regeneration behaviour

The images of histological sections contained H&E and MT staining were shown in Figs. 9 and 10. In the control group (no membrane used), fibrous connective tissue invaded into bone defects, which suppressed the formation of new bone (Fig. 9(a)). In contrast, in the membrane groups new bone appeared in the bone stump (Fig. 9(b and c)). At the end of the first month, a small amount of new bone and osteoid formed in the chitosan membrane group and the Bio-Gide1 group (Figs. 9(b1, c1) and 10(b1, c1)); moreover, more newly created bone appeared around bone stump in the both groups after the second month (Figs. 9(b2, c2) and 10(b2, c2)). These results indicate that the chitosan membranes have equal bone regeneration rate to that of Bio-Gide1. Additionally, at the end of third month, in the chitosan membrane group not only new bone formed, but also marrow organ-like structure appeared (Fig. 9(b3)). Also, Figs. 9(b3) and 10(b3) show that chitosan membranes had been degraded by the end of the third month, indicating that the membranes could maintain morphological integrity for two month at least.



6


Fig. 3 – X-ray diffraction patterns of noncross-linked (a) and cross-linked (b) chitosan membrane.

Fig. 4 – Tensile strength of the GBR membranes at dried and wetted condition. The difference in tensile strength between noncross-linked and cross-linked chitosan membrane is statistically significant (* p < 0.05, n = 6), while there is no statistically significant difference between cross-linked chitosan membrane and Bio-GideW (# p > 0.05, n = 6).

Fig. 5 – Weight loss of noncross-linked and cross-linked chitosan membrane during the soaking time (n = 3).

Fig. 6 – Cumulative FITC-BSA permeation profiles through the GBR membranes. The difference in permeability between cross-linked chitosan membrane and Bio-GideW is statistically significant at various intervals (* p < 0.05, n = 3).

Fig. 7 – MTT assay results of rat osteoblasts seeded on chitosan membranes and plate after 1 d, 4 d, 7 d and 10 d attachment periods. There is no statistically significant difference in cell proliferation on the surfaces of chitosan membrane and plate at various intervals ( p > 0.05, n = 3).

4.

Discussion

In this study, we developed a novel method to fabricate an asymmetric bioabsorbable chitosan GBR membrane, which shows excellent biocompatible and biodegradable characteristics. Currently, the development of asymmetric membranes or scaffolds becomes one of the new trends in the biomedical material field. Mi et al. prepared asymmetric chitosan membranes by dry/wet phase separation as wound dressing.17 Cho et al. developed an asymmetric hydrophilized polycaprolactone nanofibre mesh-embedded poly (glycolic-co-lactic acid) membrane for guided bone regeneration.8 Generally, the dense layer of asymmetric membrane is designed to prevent the fibrous connective tissue from invading into the defect spaces, and the loose layer that directly contacts the bone defect spaces is beneficial for osteoblasts adhesion and blood clots stabilization, thus guiding bone regeneration. The asymmetric structure of chitosan membrane was formed through liquid nitrogen quencher. This process results



7

Fig. 8 – LSCM images of rat osteoblasts attaching to the surface of chitosan membrane after culturing for 24 h. (*, chitosan membrane, (a) T100, (b) T400).

in a temperature gradient between the upper layer and lower layer of chitosan solution in the mould.37 It is proved that the number of crystal nuclei initially forming at a higher temperature is lower than that forming at a lower freezing temperature.38 Therefore, fewer ice crystal nuclei will form in the upper layer of the chitosan solution contacting air and

more ice crystal nuclei in the lower layer contacting the mould. This difference in the number of ice crystal nuclei leads to larger pores on the upper layer(loose layer) and smaller pores on the lower layer (dense layer) after lyophilization (Fig. 1). Chen et al. had prepared asymmetric chitosan membranes for skin tissue engineering using liquid nitrogen

Fig. 9 – Histological sections of rat cranial defect and surrounding cranial tissue, (a) covered without membrane, (b) covered with chitosan membrane and (c) covered with Bio-GideW membrane with different periods. (*, host bone; black arrow, new bone; white arrow, GBR membrane; short arrow, marrow organ-like structure; H&E staining, T40). Please cite this article in press as: Ma S, et al. Guided bone regeneration with tripolyphosphate cross-linked asymmetric chitosan membrane. Journal of Dentistry (2014), http://dx.doi.org/10.1016/j.jdent.2014.08.015


8


Fig. 10 – Histological sections of rat cranial defect and surrounding cranial tissue, (a) covered without membrane, (b) covered with chitosan membrane and (c) covered with Bio-GideW membrane with different periods. (*, host bone; black arrow, new bone; white arrow, GBR membrane; Masson’s trichrome staining, T40).

quencher.19 The similar asymmetric structure of chitosan membranes was obtained in our study. In addition, liquid nitrogen quencher is a time saving and simple method to prepare asymmetric structure compared with phase inversion (solvent vapourization and coagulation).16,19 The mechanical properties of pure chitosan membrane need to be enhanced by cross-linking treatment to satisfy the clinical requirements. In this study, we chose TPP, a mildest ionic cross-linker, to cross-link chitosan membranes. In acidic environment TPP is highly negatively charged, while chitosan is highly positively charged, so that TPP can strongly interact with chitosan.39 In this study, the results of ATR–FTIR suggest that the chitosan membranes were cross-linked by TPP through ionic cross-linking (Fig. 2). The cross-linked chitosan membranes contained more suppressed peaks at 108(2u) and 208(2u) in XRD spectra (Fig. 3), which could be attributed to the modification in the arrangement of molecules in the crystallattice,31 leading to amorphization. Although TPP is usually used to cross-link chitosan to form microspheres under stirring,26 in this study, we found that the tensile strength of chitosan membranes can be enhanced by TPP cross-linking. The tensile strength of the chitosan membranes matched with that of Bio-Gide1 at both dried and wetted states (Fig. 4). The permeation of nutrients and growth factors through GBR membranes is essential for bone regeneration.8 In this

study, the chitosan membranes show a high permeability due to the hydrophilicity and the porous structure (Fig. 6), which does not affect the inhibition of fibroblasts (10–15 mm in size) migration by the dense layer of the membranes. In addition, in this study the loose layer of the chitosan membranes was designed to face the bone defects, as a physical support structure, which can facilitate cell adhesion, migration, and support the growth of osteoblasts.40 In fact, chitosan membranes could be regarded as an analogue of the extracellular matrix (ECM), as chitosan is structurally similar to glycosaminoglycans (GAG);41 thus the membranes prepared in this study showed satisfactory cytocompatibility (Figs. 7 and 8). Therefore, the asymmetric chitosan membranes can prevent the invasion of fibrous connective tissue as a physical barrier, and support the growth of osteoblasts, but also allow nutrients permeation, thereby promoting the regeneration of bone defects. Rat cranial defect model was chosen to investigate the bone regeneration behaviour of the GBR membranes. The critical size defects were originally defined as ‘‘the smallest size intraosseous wound in a particular bone and species of animal that will not heal spontaneously during the lifetime of the animal’’ by Schmitz and Hollinger.42 In this study, critical size defects with 8 mm in diameter were selected in animal experiments. The newly created bone was observed in the



chitosan membranes groups at the end of 1st, 2nd, and 3rd month (Figs. 9(b) and 10(b)), suggesting that the loose layer of the membranes is osteoconductive, and the dense layer can act as a physical barrier. Furthermore, at the end of third month, the marrow organ-like structure appeared (Fig. 9(b3)); it may contain mesenchymal stem cells and capillary vessel, showing the potential of osteoinduction.43 In clinic practice, it is necessary for GBR membrane to maintain their barrier function for 4–6 week.21 The ideal GBR membranes can accomplish the balance between the rate of membranes degradation and that of tissue regeneration. In other words, the membranes should maintain structure integrity until tissue regeneration is achieved, and then degrade gradually. The animal experiment in this study shows that chitosan membrane maintained entire structure until the end of second month, but the significant degradation happened at the end of third month (Figs. 9 and 10), which was approximate to the tendency of degradation in vitro (Fig. 5). The membranes prepared in this study show satisfactory regeneration effect on bone defects and biodegradability, and thus have the potential for clinical application. Chitosan is a biomaterial with biodegradability, non-toxic, and biocompatibility. Additionally, it is generally accepted that chitosan also has antibacterial ability.44,45 In our animal experiment, almost no inflammatory response occurred in general view, suggesting that the chitosan membrane is capable of inhibiting bacteria. It should be noted that a number of fibrous connective tissue appeared in some bone defects, which could be attributed to the poor marginal fitness of membranes, and thus the fibrous connective tissue can permeate into bone defects along the edge of membranes. The adhesiveness of chitosan membranes to the bone wall should be enhanced in the future study.

5.

Conclusion

We fabricated a novel asymmetric chitosan GBR membrane through liquid nitrogen quencher and TPP cross-linking. The resulting chitosan membrane has asymmetric structure including a loose layer and a dense layer; the former can promote osteoblast adhesion and blood clot stabilization, and the latter one can prevent fibrous tissue infiltrating into the bone defects but allow nutrient permeation, thereby guiding bone regeneration. The animal experiments demonstrate that the structure, tensile strength, biodegradation and biocompatibility of the membrane fulfil the requirements of guided bone regeneration, thus accomplishing efficient regeneration of bone tissue.

Acknowledgments This work was jointly supported by National Science and Technology Support Project Foundation (Grant no.: 2012BAI07B00), Tianjin Research Program of Application Foundation and Advanced Technology (Grant no.: 14JCYBJC29600), National ‘‘973’’ Project Foundation (Grant no.: 2010CB944804), and National Training Programs of Innovation and Entrepreneurship for Undergraduates (Grant no.: 201310062005).

references

1. Chen FM, Zhang J, Zhang M, An Y, Chen F, Wu ZF. A review on endogenous regenerative technology in periodontal regenerative medicine. Biomaterials 2010;31:7892–927. 2. Pihlstrom BL, Michalowicz BS, Johnson NW. Periodontal diseases. Lancet 2005;366:1809–20. 3. Liu Y, Zheng Y, Ding G, Fang DJ, Zhang CM, Bartold PM, et al. Periodontal ligament stem cell-mediated treatment for periodontitis in miniature swine. Stem Cells 2008;26:1065–73. 4. Park J, Lutz R, Felszeghy E, Wiltfang J, Nkenke E, Neukam FW, et al. The effect on bone regeneration of a liposomal vector to deliver BMP-2 gene to bone grafts in peri-implant bone defects. Biomaterials 2007;28:2772–82. 5. Bashutski JD, Wang HL. Periodontal and endodontic regeneration. Journal of Endodontics 2009;35:321–8. 6. Yang SF, Leong KF, Du ZH, Chua CK. The design of scaffolds for use in tissue engineering. Part 1. Traditional factors. Tissue Engineering 2001;7:679–89. 7. Bunyaratavej P, Wang HL. Collagen membranes: a review. Journal of Periodontology 2001;72:215–29. 8. Cho WJ, Kim JH, Oh SH, Nam HH, Kim JM, Lee JH. Hydrophilized polycaprolactone nanofiber mesh-embedded poly(glycolic-co-lactic acid) membrane for effective guided bone regeneration. Journal of Biomedial Materials Research A 2009;91:400–7. 9. Norowski PA, Bumgardner JD. Biomaterial and antibiotic strategies for peri-implantitis. Journal of Biomedical Materials Research B—Applied Biomaterials 2009;88B:530–43. 10. Jiang T, Nukavarapu SP, Deng M, Jabbarzadeh E, Kofron MD, Doty SB, et al. Chitosan-poly(lactide-co-glycolide) microsphere-based scaffolds for bone tissue engineering: in vitro degradation and in vivo bone regeneration studies. Acta Biomaterialia 2010;6:3457–70. 11. Owen GR, Jackson JK, Chehroudi B, Brunette DM, Burt HM. An in vitro study of plasticized poly(lactic-co-glycolic acid) films as possible guided tissue regeneration membranes: material properties and drug release kinetics. Journal of Biomedical Materials Research A 2010;95A:857–69. 12. Chen TW, Chang SJ, Niu GC-C, Hsu YT, Kuo SM. Alginatecoated chitosan membrane for guided tissue regeneration. Journal of Applied Polymer Science 2006;102:4528–34. 13. Lou CW, Lu CT, Wen SP, Lin CW, Chao CY, Preparation Lin JH. Evaulation of degradable poly(lactic acid) composite dressings. Advanced Materials Research 2010;123:177–80. 14. Chen GP, Ushida T, Tateishi T. Scaffold design for tissue engineering. Macromolecular Bioscience 2002;2:67–77. 15. Oh SH, Kim JH, Kim JM, Lee JH. Asymmetrically porous PLGA/Pluronic F127 membrane for effective guided bone regeneration. Journal of Biomaterials Science—Polymer Edition 2006;17:1375–87. 16. Hong H, Wei J, Liu C. Development of asymmetric gradational-changed porous chitosan membrane for guided periodontal tissue regeneration. Composites B: Engineering 2007;38:311–6. 17. Mi FL, Wu YB, Shyu SS, Chao AC, Lai JY, Su CC. Asymmetric chitosan membranes prepared by dry/wet phase separation: a new type of wound dressing for controlled antibacterial release. Journal of Membrane Science 2003;212:237–54. 18. Fujihara K, Kotaki M, Ramakrishna S. Guided bone regeneration membrane made of polycaprolactone/calcium carbonate composite nano-fibers. Biomaterials 2005;26: 4139–47. 19. Chen KY, Liao WJ, Kuo SM, Tsai FJ, Chen YS, Huang CY, et al. Asymmetric chitosan membrane containing collagenI nanospheres for skin tissue engineering. Biomacromolecules 2009;10:1642–9.


9


10


20. Jayakumar R, Prabaharan M, Nair SV, Tamura H. Novel chitin and chitosan nanofibers in biomedical applications. Biotechnology Advances 2010;28:142–50. 21. Kuo SM, Chang SJ, Chen TW, Kuan TC. Guided tissue regeneration for using a chitosan membrane: an experimental study in rats. Journal of Biomedial Materials Research A 2006;76:408–15. 22. Ito M, Hidaka Y, Nakajima M, Yagasaki H, Kafrawy AH. Effect of hydroxyapatite content on physical properties and connective tissue reactions to a chitosan–hydroxyapatite composite membrane. Journal of Biomedial Materials Research 1999;45:204–8. 23. Shin SY, Park HN, Kim KH, Lee MH, Choi YS, Park YJ, et al. Biological evaluation of chitosan nanofiber membrane for guided bone regeneration. Journal of Periodontology 2005;76:1778–84. 24. Liu X, Ma L, Mao Z, Gao C. Chitosan-based biomaterials for tissue repair and regeneration. Advances in Polymer Science 2011;244:81–127. 25. Han CM, Zhang LP, Sun JZ, Shi HF, Zhou J, Gao CY. Application of collagen–chitosan/fibrin glue asymmetric scaffolds in skin tissue engineering. Journal of Zhejiang University—Science B 2010;11:524–30. 26. Lin WC, Yu DG, Yang MC. pH-sensitive polyelectrolyte complex gel microspheres composed of chitosan/sodium tripolyphosphate/dextran sulfate: swelling kinetics and drug delivery properties. Colloids and Surfaces B—Biointerfaces 2005;44:143–51. 27. Xu C, Lei C, Meng LY, Wang CN, Song YL. Chitosan as a barrier membrane material in periodontal tissue regeneration. Journal of Biomedical Materials Research B— Applied Biomaterials 2012;100B:1435–43. 28. Monteiro OAC, Airoldi C. Some studies of crosslinking chitosan–glutaraldehyde interaction in a homogeneous system. International Journal of Biological Macromolecules 1999;26:119–28. 29. Risbud MV, Hardikar AA, Bhat SV, Bhonde RR. pH-sensitive freeze-dried chitosan–polyvinyl pyrrolidone hydrogels as controlled release system for antibiotic delivery. Journal of Controlled Release 2000;68:23–30. 30. Chen JP, Chang GY, Chen JK. Electrospun collagen/chitosan nanofibrous membrane as wound dressing. Colloids and Surfaces A—Physicochemical and Engineering Aspects 2008;313:183–8. 31. Bhumkar DR, Pokharkar VB. Studies on effect of pH on cross-linking of chitosan with sodium tripolyphosphate: a technical note. AAPS PharmSciTech 2006;7. Article 50. 32. Liu H, Gao C. Preparation and properties of ionically crosslinked chitosan nanoparticles. Polymers for Advanced Technologies 2009;20:613–9.

33. Kafshgari MH, Khorram M, Khodadoost M, Khavari S. Reinforcement of chitosan nanoparticles obtained by an ionic cross-linking process. Iranian Polymer Journal 2011;20:445–56. 34. Piero´g M, Gierszewska-Druz˙yn´ska M, Ostrowska-Czubenko J. Effect of ionic crosslinking agents on swelling behavior of chitosan hydrogel membranes. In: Jaworska MM, editor. Progress on chemistry and application of chitin and its derivatives vol XIV. ˇˇLo´dz´: Polish Chitin Society; 2009. p. 75–82. 2009. 35. Gierszewska-Druz˙yn´ska M, Ostrowska-Czubenko J. The effect of ionic crosslinking on thermal properties of hydrogel chitosan membranes. Progress on chemistry and application of chitin and its derivatives. ˇˇLo´dz´: Polish Chitin Society; 2010: 25–32. 36. Gierszewska-Druz˙yn´ska M, Ostrowska-Czubenko J. Influence of crosslinking process conditions on molecular and supermolecular structure of chitosan hydrogel membrane. Progress on chemistry and application of chitin and its derivatives. ˇˇLo´dz´: Polish Chitin Society; 2011: 15–22. 37. Madihally SV, Matthew HWT. Porous chitosan scaffolds for tissue engineering. Biomaterials 1999;20:1133–42. 38. Mao JS, Zhao LG, de Yao K, Shang QX, Yang GH, Cao YL. Study of novel chitosan–gelatin artificial skin in vitro. Journal of Biomedical Materials Research A 2003;64A:301–8. 39. Hu M, Li Y, Decker EA, Xiao H, McClements DJ. Influence of tripolyphosphate cross-linking on the physical stability and lipase digestibility of chitosan-coated lipid droplets. Journal of Agricultural and Food Chemistry 2010;58:1283–9. 40. O’Brien FJ, Harley BA, Yannas IV, Gibson LJ. The effect of pore size on cell adhesion in collagen-GAG scaffolds. Biomaterials 2005;26:433–41. 41. Yan J, Li X, Liu L, Wang F, Zhu TW, Zhang Q. Potential use of collagen–chitosan–hyaluronan tri-copolymer scaffold for cartilage tissue engineering. Artificial Cells Blood Substitutes and Biotechnology 2006;34:27–39. 42. Schmitz JP, Hollinger JO. The critical size defect as an experimental model for craniomandibulofacial nonunions. Clinical Orthopaedics and Related Research 1986;205:299–308. 43. Fang DJ, Seo BM, Liu Y, Sonoyama W, Yamaza T, Zhang CM, et al. Transplantation of mesenchymal stem cells is an optimal approach for plastic surgery. Stem Cells 2007;25:1021–8. 44. Du YJ, Zhao YQ, Dai SC, Yang B. Preparation of watersoluble chitosan from shrimp shell and its antibacterial activity. Innovative Food Science & Emerging Technologies 2009;10:103–7. 45. No HK, Park NY, Lee SH, Meyers SP. Antibacterial activity of chitosans and chitosan oligomers with different molecular weights. International Journal of Food Microbiology 2002;74: 65–72.


chitosan Membrane Containing Minocycline-loaded Chitosan Nanoparticles for Guided Bone Regeneration.

Bioresorbable zinc hydroxyapatite guided bone regeneration membrane for bone regeneration.

Genipin-Crosslinked Chitosan Gels and Scaffolds for Tissue Engineering and Regeneration of Cartilage and Bone.

Novel bioresorbable strontium hydroxyapatite membrane for guided bone regeneration.

The use of irradiated-crosslinked human collagen membrane in guided tissue regeneration.

The effect of bacterial cellulose membrane compared with collagen membrane on guided bone regeneration.

genipin co-crosslinked beads.

Demineralized Freeze-Dried Bovine Cortical Bone: Its Potential for Guided Bone Regeneration Membrane.

Evaluation of bone formation and membrane degradation in guided bone regeneration using a 4-hexylresorcinol-incorporated silk fabric membrane.

nano-hydroxyapatite membrane for bone regeneration.

Bone regeneration using the principle of guided tissue regeneration.

Mechanisms of guided bone regeneration: a review.

Membranes for the Guided Bone Regeneration.

RGD-conjugated UV-crosslinked chitosan scaffolds inoculated with mesenchymal stem cells for bone tissue engineering.

Horizontal bone augmentation by means of guided bone regeneration.

Morphogenetically-Active Barrier Membrane for Guided Bone Regeneration, Based on Amorphous Polyphosphate.

Silk fibroin and 4-hexylresorcinol incorporation membrane for guided bone regeneration.

Investigating the Potential of Amnion-Based Scaffolds as a Barrier Membrane for Guided Bone Regeneration.

Dimensionally stable and bioactive membrane for guided bone regeneration: An in vitro study.

Chitin-fibroin-hydroxyapatite membrane for guided bone regeneration: micro-computed tomography evaluation in a rat model.

A novel silk fibroin nanofibrous membrane for guided bone regeneration: a study in rat calvarial defects.

Xylan hemicellulose improves chitosan hydrogel for bone tissue regeneration.

Biological Evaluation (In Vitro and In Vivo) of Bilayered Collagenous Coated (Nano Electrospun and Solid Wall) Chitosan Membrane for Periodontal Guided Bone Regeneration.

Exploring the Preparation of Albendazole-Loaded Chitosan-Tripolyphosphate Nanoparticles.